In-Vitro And Preclinical Trials

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Opioid Growth Factor (OGF)

Published Research About OGF

In-Vitro And Preclinical Trials

In-Vitro And Preclinical Trials

Life Sci. 1992;50(16):1179-87.

An opioid growth factor regulates the replication of microorganisms.

Zagon IS, McLaughlin PJ.

Department of Neuroscience and Anatomy, Pennsylvania State University, M.S. Hershey Medical Center, Hershey 17033.

An opioid growth factor (OGF), [Met5]-enkephalin, interacts with the zeta (zeta) opioid receptor to modulate development of eukaryotes. We have found that [Met5]-enkephalin, an endogenous opioid peptide serves to inhibit the growth of S. aureus. This effect on growth involves cell proliferative events and is under tonic control, since potent opioid antagonists accelerate cell replication. Both the OGF and zeta opioid receptor were associated with these microorganisms. Other opioid receptors (mu, delta and kappa) were not detected. OGF also controlled the growth of other bacteria: P. aeruginosa and S. marcesans. These results indicate that OGF and its receptor, known to be important in the regulation of mammalian development, also function in the growth of simple unicellular organisms. We suggest that the endogenous opioid system related to growth originated billions of years ago.

PMID: 1313136

Brain Res. 1992 Nov 20;596(1-2):149-56.

Ontogeny of zeta (zeta), the opioid growth factor receptor, in the rat brain.

Zagon IS, Gibo DM, McLaughlin PJ.

Department of Neuroscience and Anatomy, Pennsylvania State University College of Medicine, M.S. Hershey Medical Center, Hershey 17033.

Opioid growth factor (OGF), [Met5]enkephalin, serves as an inhibitory influence on the developing nervous system and is especially targeted to cell proliferative events. OGF interacts with the zeta (zeta) opioid receptor to perform its function. Using [3H]-[Met5]enkephalin, the ontogeny of the zeta receptor in the whole brain and cerebellum of rats was explored. Specific and saturable binding was recorded at the earliest time sampled, prenatal day 15 (E15). In the whole brain, binding capacity (Bmax) was two-fold greater at E15 than at E18 and E20. The quantity of zeta receptor appeared to increase in the first postnatal week, reaching a maximum on postnatal day 8. Binding decreased the remainder of the 2nd week and between postnatal days 15 and 25 binding was no longer recorded. In the cerebellum, binding capacity increased from E20 to the 2nd postnatal week, reaching a maximum on postnatal days 8-10. The Bmax of the zeta receptor decreased precipitously on postnatal day 11, being 5.4-fold lower than on postnatal day 10. Between postnatal days 21 and 30, no binding was observed. The binding affinities of the whole brain and cerebellum were 2.3 and 2.7 nM, respectively, and no differences between ages could be detected. Continuous opioid receptor blockade from birth to postnatal day 6 increased body weight, the Bmax of the zeta receptor in the whole brain and cerebellum (but not the Kd), and increased the number of layers of germinal cells in the cerebellum.(ABSTRACT TRUNCATED AT 250 WORDS)

PMID: 1334774

Brain Res Mol Brain Res. 1994 Jan;21(1-2):85-98.

Preproenkephalin mRNA expression in the developing and adult rat brain.

Zagon IS, Isayama T, McLaughlin PJ.

Department of Neuroscience and Anatomy, Pennsylvania State University, M.S. Hershey Medical Center, Hershey 17033.

[Met5]-Enkephalin is derived from the protein precursor, proenkephalin A, which in turn is encoded by the preproenkephalin (PPE) gene. [Met5]-Enkephalin is not only a putative neuromodulatory substance, but also serves as a growth factor (= opioid growth factor, OGF). OGF exerts an inhibitory influence on the developing nervous system and is especially targeted to cell proliferative and differentiative events. This study examined the relationship of PPE mRNA expression to late prenatal and postnatal rat brain development. Northern blot analysis of the whole brain and cerebellum showed that message is present in the fetal nervous system on prenatal day 15 (the earliest timepoint examined), is expressed at relatively similar levels within each tissue during the first 2 postnatal weeks, and reaches adult levels by the beginning of the 3rd postnatal week. In situ hybridization methodology revealed that PPE mRNA was prominent in areas associated with cell generation. Message was found in sites of primary (i.e., ventricular region) and secondary (e.g., external germinal layer of the cerebellum) cellular replication, as well as in discrete foci of cell proliferation (e.g., medullary layer of the cerebellum). PPE mRNA was also present for varying periods of time in postmitotic cells. During development, a number of patterns (decrease, increase, and no perceptible change) of PPE mRNA could be detected in relationship to the fetal/neonatal period. Given the strong evidence (e.g., regulation of cell proliferation and differentiation, temporal and spatial patterns of peptide and zeta opioid receptor) that enkephalin immunoreactivity is associated with proliferating and differentiating neurons and glia, these results suggest that the source of [Met5]-enkephalin is both autocrine and paracrine in nature.

PMID: 8164525

Am J Physiol. 1994 Sep;267(3 Pt 2):R645-52.

Opioid growth factor inhibits DNA synthesis in mouse tongue epithelium in a circadian rhythm-dependent manner.

Zagon IS, Wu Y, McLaughlin PJ.

Department of Neuroscience and Anatomy, Pennsylvania State University College of Medicine, Hershey 17033.

In addition to neuromodulation, endogenous opioids also serve as growth factors. To investigate the involvement of the naturally occurring opioid peptide [Met5]enkephalin [termed opioid growth factor (OGF)] in the renewal of epithelium, adult mice were given systemic injections of OGF (1 mg/kg) and examined 2 h later at 0700 or 1700 h. DNA synthesis in the tongue was investigated using [3H]thymidine and autoradiography. OGF depressed DNA synthesis of the basal epithelial cells in the tip, and dorsal and ventral surfaces of the tongue (42-44% of control levels) only at 0700 h. This decrease in DNA synthesis was blocked by concomitant administration of the opioid antagonist naloxone (10 mg/kg); naloxone alone had no influence on cell replicative processes. Both OGF and its receptor, zeta (zeta), were detected in the stratified squamous epithelium of the ventral and dorsal surfaces of the tongue by immunocytochemistry. Photodensitometric measurements of immunocytochemical preparations revealed almost twofold more OGF and zeta-receptor immunoreactivity at 1700 h than at 0700 h. These results indicate that an endogenous opioid peptide and its receptor are present and govern cellular renewal processes in the tongue and regulate DNA synthesis in a circadian rhythm-dependent fashion.

PMID: 8092307

Brain Res. 1995 Feb 6;671(1):105-11.

Conserved expression of the opioid growth factor, [Met5]enkephalin, and the zeta (zeta) opioid receptor in vertebrate cornea.

Zagon IS, Sassani JW, Allison G, McLaughlin PJ.

Department of Neuroscience and Anatomy, Pennsylvania State University, M.S. Hershey Medical Center, Hershey 17033, USA.

In addition to neuromodulation, endogenous opioids serve as growth factors. The naturally occurring opioid peptide, [Met5]enkephalin, termed opioid growth factor (OGF), has been found to be a potent and tonic inhibitor of processes related to growth and renewal, particularly cell proliferation. OGF mediates its actions through the zeta (zeta) opioid receptor. In order to determine if OGF and/or the zeta receptor are present in human corneal epithelium, immunocytochemistry was utilized. Immunoreactivity with regard to OGF and to the zeta receptor could be detected in the cortical cytoplasm of both basal and suprabasal epithelial cells, but was not associated with the cell nucleus. Investigation of the ubiquity of OGF and zeta receptor in the vertebrate cornea showed that both elements are present in a wide variety of classes of the phylum Chordata, including mammalia, aves, reptilia, amphibia, and osteichthyes. These results suggest that an endogenous opioid system related to growth may have originated as early as 300 million years ago, and that the function of this system in cellular renewal and homeostasis is a requirement of the vertebrate corneal epithelium.

PMID: 7728521

Am J Physiol. 1995 Apr;268(4 Pt 2):R942-50.

Opioid growth factor modulates corneal epithelial outgrowth in tissue culture.

Zagon IS, Sassani JW, McLaughlin PJ.

Department of Neuroscience and Anatomy, Pennsylvania Lions Vision and Research Center, Pennsylvania State University, Milton S. Hershey Medical Center, Hershey 17033, USA.

In addition to neuromodulation, endogenous opioid peptides serve as growth factors. To determine involvement of opioids in the homeostatic renewal and repair of the corneal epithelium, epithelial outgrowths from 3-mm explants of rabbit cornea were investigated. Blockade of opioid-receptor interaction by the potent opioid antagonist naltrexone (NTX) for 7 days significantly increased the extent of outgrowths and the number and labeling index (DNA synthesis) of epithelial cells, relative to control levels. Outgrowths exposed to the opioid growth factor (OGF) [Met5]enkephalin for 7 days were subnormal in extent and labeling index and displayed alterations in architectural pattern. The effects of OGF on epithelial outgrowth were blocked by concomitant exposure to the opioid antagonist naloxone; naloxone alone had no effect on growth at the concentration utilized. NTX and OGF were active in both serum-containing and serum-free cultures. Immunocytochemical investigations showed that both OGF and its opioid receptor zeta (zeta) were present in epithelial cells growing in control media. The results indicate that an endogenous opioid peptide and its receptor are present in mammalian corneal epithelium and serve to modulate cell proliferation, migration, and organization.

PMID: 7733405

Vis Neurosci. 1995 Sep-Oct;12(5):939-50.

Ontogeny of the opioid growth factor, [Met5]-enkephalin, and its binding activity in the rat retina.

Isayama T, Hurst WJ, McLaughlin PJ, Zagon IS.

Department of Neuroscience and Anatomy, Pennsylvania State University, Milton S. Hershey Medical Center, Hershey 17033, USA.

The endogenous opioid peptide [Met5]-enkephalin is a tonically active opioid growth factor (OGF) with an inhibitory action on DNA synthesis in the developing rat retina. In this study, the ontogeny of the spatial and temporal expression of OGF and its binding activity was examined. OGF-like immunoreactivity was detected in the retina at gestation day (E) 20, but not at E18, and was localized to ganglion cell and neuroblast layers; immunochemical reaction was no longer seen in the retina by postnatal day 6. Native OGF was further identified and characterized by high-performance liquid chromatography (HPLC) studies and immunodot assays, which revealed that [Met5]-enkephalin was present in the neonatal, but not adult, rat retina. OGF binding activity was detected as early as E18 using [125I]-[Met5]-enkephalin and in vitro receptor autoradiography. Little OGF binding activity was noted for prenatal retinas, but appreciable activity was observed from birth to postnatal day 4; no OGF binding could be detected after postnatal day 5 or in the adult. These results reveal the transient appearance of the OGF, [Met5]-enkephalin, and its receptor binding activity in the developing mammalian retina, and show that their ontogeny coincides with the timetable of DNA synthesis of retinal neuroblasts.

PMID: 8924417

Am J Physiol. 1996 Jan;270(1 Pt 2):R22-32.

Opioid growth factor-dependent DNA synthesis in the neonatal rat aorta.

Zagon IS, Wu Y, McLaughlin PJ.

Department of Neuroscience and Anatomy, Pennsylvania State University College of Medicine, Hershey 17033, USA.

In addition to neuromodulation, endogenous opioids serve as growth factors in neural and nonneural cells. This study examined the hypothesis that opioids are inhibitory growth factors in vascular development. No circadian rhythm was detected for DNA synthesis in endothelial, smooth muscle, or fibroblast cells in the aorta of 1-day-old rats. Administration of naltrexone (NTX), a potent opioid antagonist, markedly increased the labeling indexes of all three cell types. [Met5]enkephalin, found to be the only opioid peptide to influence DNA synthesis and termed the opioid growth factor (OGF), depressed DNA synthesis in each cell type for 4-6 h in a dose-dependent and receptor-mediated manner. In aortas placed in tissue culture, DNA synthesis was significantly increased by incubation in NTX and decreased by incubation with OGF, Both OGF and its receptor, zeta (zeta), were associated with the cytoplasm of all three cell types in the neonatal aorta. These results indicate that an endogenous opioid peptide (i.e., OGF) and its receptor (i.e., zeta) reside in the developing vascular cells and govern DNA synthesis, with OGF acting directly as a tonic negative regulator of cell generation in the great vessels.

PMID: 8769781

J Invest Dermatol. 1996 Mar;106(3):490-7.

The opioid growth factor, [Met5]-enkephalin, and the zeta opioid receptor are present in human and mouse skin and tonically act to inhibit DNA synthesis in the epidermis.

Zagon IS, Wu Y, McLaughlin PJ.

Department of Neuroscience, The Pennsylvania State University, College of Medicine, Hershey, U.S.A.

Opioid peptides serve as tonically active negative growth factors in neural and non-neural cells, in addition to being neuromodulators. To investigate the involvement of opioids in homeostatic renewal of epithelial cells in the epidermis, mice were given systemic injections of the potent opioid antagonist, naltrexone (NTX) (20 mg/kg). Disruption of opioid-receptor interaction by NTX resulted in an elevation of 42 and 72% in DNA synthesis in skin from the dorsum and plantar surface of the hindfoot, respectively, within 2 h; response to NTX was dependent on the circadian rhythm in each region examined. Injection of the naturally occurring and potent opioid growth factor (OGF), [Met5]-enkephalin, at 1 mg/kg depressed DNA synthesis in the dorsum and plantar surface by 42 and 19%, respectively, within 2 h; the effects of OGF complied with the pattern of circadian rhythm in each area of skin. The decreases in labeling index evoked by OGF were blocked by concomitant administration of the opioid antagonist, naloxone (10 mg/kg); naloxone alone at the dosage utilized had no influence on cell replicative processes. In tissue culture studies, OGF and NTX respectively depressed and elevated DNA synthesis. Both OGF and its receptor, zeta, were detected in all but the cornified layer of the epidermis in murine skin from the dorsum, plantar surface, pinnae, and tail. In addition, both peptide and receptor were observed in basal and suprabasal cells of the human epidermis. These results lead to the suggestion that an endogenous opioid peptide and its receptor are present and govern cellular renewal processes in the skin in a direct manner, regulating DNA synthesis in a tonically inhibitory, circadian rhythm-dependent fashion.

PMID: 8648182

Vis Neurosci. 1996 Jul-Aug;13(4):695-704.

Ontogeny of preproenkephalin mRNA expression in the rat retina.

Isayama T, McLaughlin PJ, Zagon IS.

Department of Neuroscience and Anatomy, Milton S. Hershey Medical Center, Pennsylvania State University, College of Medicine, Hershey 17033, USA.

Endogenous opioid systems (i.e. opioid peptides and opioid receptors) modulate developmental events in the neonatal mammalian retina. In the present study, the mRNA encoding preproenkephalin A (PPE), the prohormone for the opioid growth factor (OGF), [Met5]-enkephalin, was studied in the developing and the adult retinas of rats. Northern analysis indicated the presence of a 1.4-kb message in the developing and adult retinas corresponding to rat PPE mRNA. Quantitation showed that PPE message was present on postnatal day 1 at 5% of the adult level, and increased during development until the adult quantity was reached by postnatal day 27. In situ hybridization experiments first detected the presence of PPE mRNA in retinal tissues during late gestation. In late prenatal and neonatal retinas, PPE message was associated with areas of the developing retina containing proliferating neuroblasts and postmitotic cells. Later in development, message appeared to be located primarily within the inner retina, with abundant PPE mRNA associated with putative horizontal cells of the inner nuclear layer (INL). The adult retina showed a similar pattern of PPE gene expression in the cells of the INL. These findings document that the gene expression in the retina for PPE begins in the fetus, continues during retinal development, and coincides with the presence of a PPE mRNA derivative ([Met5]-enkephalin) that regulates DNA synthesis during retinal ontogeny. Our results are also the first to show the presence of PPE message in the adult mammalian retina, suggesting transcription of an opioid gene in the mature visual system.

PMID: 8870226

Am J Physiol. 1996 Jul;271(1 Pt 2):R115-21.

Identification and characterization of zeta-opioid receptor in human colon cancer.

Hytrek SD, Smith JP, McGarrity TJ, McLaughlin PJ, Lang CM, Zagon IS.

Department of Comparative Medicine, Pennsylvania State University College of Medicine, Hershey 17033, USA.

Opioid growth factor (OGF, [Met5]enkephalin) inhibits the growth of human colon cancer in nude mice in a receptor-mediated fashion. Ligand binding assays using HT-29 human colon cancer tissue and [3H][Met5]enkephalin were performed to characterize the receptor responsible for the growth-regulatory effects of OGF in colon cancer. Specific and saturable binding was detected, and Scatchard analysis revealed that the data were consistent for a single binding site with a binding affinity of 15.4 +/- 2.0 nM and a binding capacity of 364.8 +/- 25.7 fmol/mg protein. Subcellular fractionation studies revealed that binding was restricted to the nuclear fraction. Competition experiments showed that cold [Met5]enkephalin was the most effective ligand at displacing [3H][Met5]enkephalin. Binding to radiolabeled [Met5]enkephalin also was detected in colon cancers obtained from surgical resections. The function, pharmacological and biochemical characteristics, distribution, and subcellular location of this OGF receptor in human colon cancer are consistent with the zeta-opioid receptor.

PMID: 8760211

Am J Physiol. 1996 Sep;271(3 Pt 2):R511-8.

Opioid growth factor tonically inhibits human colon cancer cell proliferation in tissue culture.

Zagon IS, Hytrek SD, McLaughlin PJ.

Department of Neuroscience and Anatomy, Pennsylvania State University, College of Medicine, Hershey 17033, USA.

Native opioid peptides serve as growth factors in a number of normal and neoplastic cells and tissues, including the prevention and delayed growth of human colon cancer xenografts in nude mice. This study examined the hypothesis that opioids exert a direct inhibitory influence on tumor cell growth by the use of a tissue culture model. The naturally occurring pentapeptide [Met5]enkephalin depressed growth of HT-29 human colon cancer cells from 17 to 41% at 12-72 h after administration of 10(-6)M concentration; consistent with previously defined nomenclature, this peptide was termed opioid growth factor (OGF). OGF action exhibited a dose-response relationship, was reversible and not cytotoxic, and was opioid receptor mediated. Growth inhibition by OGF was not dependent on serum, and was noted in the two other human colon cancer cell lines examined WiDr and COLO 205. This peptide continually repressed growth because an increase in cell number was noted when cells were exposed to the potent opioid antagonist naltrexone or an antibody to OGF. Both OGF and its receptor, zeta (zeta), were found in colon cancer cells by immunocytochemistry, and receptor binding assays revealed a nuclear-associated receptor with a dissociation constant of 8.9 nM and a maximum binding capacity of 43 fmol/mg of protein. OGF was produced and secreted by the tumor cells. These results lead to the suggestion that OGF has a direct, tonic, inhibitory action on the growth of human colon cancer cells and contribute to our understanding of the mechanisms underlying the marked antitumor effect of this peptide in nude mice inoculated with human colon cancer cells.

PMID: 8853370

Am J Physiol. 1996 Sep;271(3 Pt 2):R780-6.

Opioid growth factor ([Met5]enkephalin) prevents the incidence and retards the growth of human colon cancer.

Zagon IS, Hytrek SD, Lang CM, Smith JP, McGarrity TJ, Wu Y, McLaughlin PJ.

Department of Neuroscience and Anatomy, Pennsylvania State University, College of Medicine, Hershey 17033, USA.

Endogenous opioid peptides serve as growth factors in normal and neoplastic cells and tissues, and both opioids and their receptors have been identified in human colon cancer. This study examined the hypothesis that opioids serve to modulate the growth of human colon cancer. Daily administration of the native opioid growth factor (OGF), [Met5]enkephalin, at dosages of 0.5, 5, or 25 mg/kg prevented the occurrence of human colon cancer HT-29 xenografts in nude mice. More than 80% of the mice receiving OGF beginning at the time of tumor cell inoculation did not exhibit neoplasias within 3 wk, in comparison with a tumor incidence of 93% in control subjects. Even 7 wk after cancer cell inoculation, 57% of the mice given OGF did not display a tumor. OGF delayed tumor appearance and growth in animals developing colon cancer with respect to the control group. The suppressive effects of OGF on oncogenicity were opioid receptor mediated. OGF and its receptor, zeta (zeta), were detected in transplanted human HT-29 colon tumors. Surgical specimens of human colon cancers also contained OGF. These results show that a naturally occurring opioid peptide acts as a potent negative regulator of human gastrointestinal cancer and may suggest pathways for tumor etiology, progression, treatment, and prophylaxis.

PMID: 8853403

Cancer Lett. 1997 Jan 30;112(2):167-75.

Opioid growth factor (OGF) inhibits human pancreatic cancer transplanted into nude mice.

Zagon IS, Hytrek SD, Smith JP, McLaughlin PJ.

Department of Neuroscience and Anatomy, Pennsylvania State University, Milton S. Hershey Medical Center, Hershey 17033, USA. iszl$@psuvm.psu.edu

Nude mice inoculated with human pancreatic cancer (BxPC-3) cells and receiving 5 mg/kg of opioid growth factor ([Met5]enkephalin; OGF) three times daily exhibited a marked retardation in tumorigenicity compared to animals injected with sterile water (controls). OGF-treated animals had a delay of 43% in initial tumor appearance compared to control subjects (10.6 days). At the time when all of the control mice had tumors, 62% of the mice in the OGF group had no signs of neoplasia. Tumor tissue excised from mice after 30 days was assayed for levels of [Met5]enkephalin and zeta opioid receptors. Tumor tissue levels of [Met5]enkephalin were 24-fold greater in OGF-treated mice than controls, but plasma levels of OGF were 8.6-fold lower in animals receiving OGF. Specific and saturable binding of radiolabeled [Met5]enkephalin to nuclear homogenates of pancreatic tumor tissue was recorded, with a binding affinity (Kd) of 10 nM and a binding capacity (Bmax) of 46.8 fmol/mg protein. Binding capacity, but not affinity, of [3H-Met5]enkephalin was reduced by 58% of control levels in tumor tissue from mice of the OGF group. OGF and the zeta (zeta) opioid receptor were detected in human pancreatic tumor cells by immuno-cytochemistry. These results demonstrate that an endogenous opioid and its receptor are present in human pancreatic cancer, and act as a negative regulator of tumorigenesis in vivo.

PMID: 9066724

Laryngoscope. 1997 Mar;107(3):335-9.

Expression of the opioid growth factor, [Met5]-enkephalin, and the zeta opioid receptor in head and neck squamous cell carcinoma.

Levin RJ, Wu Y, McLaughlin PJ, Zagon IS.

Division of Otolaryngology/Head and Neck Surgery, The Pennsylvania State University College of Medicine, The Milton S. Hershey Medical Center, Hershey 17033, U.S.A.

Despite the prevalence of cancers of the head and neck, survival rates have not changed in the past few decades. Recent work has implicated peptide growth factors and their receptors in the genesis and progression of head and neck squamous cell carcinoma. Opioid growth factor (OGF, [Met5]-enkephalin) is a tonically active, autocrine and/or paracrine produced, inhibitory factor that influences the growth of normal and abnormal cells and tissues. This peptide interacts with the zeta (zeta) opioid receptor to modulate cellular proliferation, migration, and survival. Both OGF and the zeta receptor are present in mammalian tongue epithelium and skin, and modulate DNA synthesis. In the present study we examined the presence and distribution of OGF and the zeta opioid receptor in the head and neck squamous cell carcinomas from seven individuals. All specimens expressed this growth factor and its receptor regardless of tumor stage, location, and histologic grade. Immunoreactivity for both OGF and the zeta receptor were associated with the cytoplasm but not the nucleus in cells of each of these carcinomas. Our findings that a potent negative growth regulator and its receptor are present in head and neck squamous cell carcinoma lead us to suggest that OGF may modulate the growth of these types of cancers.

PMID: 9121309

Am J Physiol. 1997 Apr;272(4 Pt 2):R1094-104.

Opioid growth factor is present in human and mouse gastrointestinal tract and inhibits DNA synthesis.

Zagon IS, Wu Y, McLaughlin PJ.

Department of Neuroscience and Anatomy, The Pennsylvania State University, College of Medicine, Hershey 17033, USA.

Native opioid peptides serve as growth factors in a number of normal and neoplastic cells and tissues. This study investigated the influence of opioids on circadian rhythm-dependent DNA synthesis in mouse esophagus during homeostatic renewal. In contrast to a labeling index (LI) of 24.0% at 0630 and 5.5% at 1600, disruption of opioid-receptor interaction by the potent opioid antagonist naltrexone hydrochloride (NTX; 10 mg/kg) in mice resulted in an elevation of 49% in DNA synthesis of esophageal epithelial cells at 1600, but had no effect at 0630. Mice subjected to [Met5]enkephalin (1 mg/kg) had an LI that was decreased 23% from control levels at 0630, but was unaffected at 1600. This decrease in DNA synthesis was blocked by concomitant administration of naloxone (10 mg/kg); naloxone alone had no influence on cell replicative processes. In tissue culture studies, NTX and OGF markedly increased and decreased, respectively, the LI from control values. Both opioid growth factor (OGF) and its receptor, zeta, were detected in all but the cornified layer of mouse esophageal epithelium and in the epithelial cells of the stomach and small and large intestines. In addition, both peptide and receptor were observed in the basal and suprabasal cells of human esophageal epithelium. These results indicate that an endogenous opioid peptide (OGF) and its receptor (zeta) reside in gastrointestinal epithelium and play a role in cellular renewal processes in a tonically inhibitory, direct, and circadian rhythm-dependent fashion.

PMID: 9140007

Brain Res. 1997 Jun 6;759(1):92-102.

Homeostasis of ocular surface epithelium in the rat is regulated by opioid growth factor.

Zagon IS, Sassani JW, Kane ER, McLaughlin PJ.

Department of Neuroscience and Anatomy, Pennsylvania State University, Milton S. Hershey Medical Center, Hershey 17033, USA.

Endogenous opioid peptides serve as growth factors in developing, renewing, and neoplastic cells and tissues. This study examined the hypothesis that opioids serve to modulate the homeostatic renewal of ocular surface epithelium in the rat. DNA synthesis in the epithelium of the central (CC) and peripheral (PC) cornea, limbus (LM), and conjunctiva (CN) was investigated using adult male rats. Animals received an injection of opioid growth factor (OGF), [Met5]-enkephalin, OGF and naloxone (NAL), NAL alone, naltrexone (NTX), or an equivalent volume of sterile water (CO) and sacrificed 4 h later (i.e. 16:00 h). [3H]thymidine was administered 1 h before sacrifice. With the exception of NTX (20 mg/kg), all compounds were given at 10 mg/kg. Examination of 5 time points over an 18-h period revealed no variation in DNA synthesis within a region of ocular surface basal epithelium (BE). OGF depressed DNA synthesis of the BE by 25, 48, and 50% in the PC, LM, and CN, respectively; little labeling was recorded in the BE of the CC. Exposure to OGF-NAL or NAL alone did not alter DNA synthesis of the BE. Complete blockade of OGF-zeta receptor interaction by administration of the potent opioid antagonist, NTX, increased the number of epithelial cells in the PC, LM, and CN undergoing DNA synthesis by 30 to 72%. The effects of OGF and NTX on DNA synthesis of BE also were observed in an organ culture setting. Utilizing immunocytochemistry, OGF and its receptor zeta were associated with both the basal and the suprabasal cells of the ocular surface epithelium. These results indicate that an endogenous opioid peptide, OGF, and its receptor are present and govern homeostatic cellular renewal processes in ocular surface epithelium. OGF regulates DNA synthesis in a direct manner, and does so by a tonic, inhibitory, and receptor-mediated mechanism.

PMID: 9219867

Dev Dyn. 1998 Apr;211(4):327-37.

Ontogeny of the opioid growth factor, [Met5]-enkephalin, preproenkephalin gene expression, and the zeta opioid receptor in the developing and adult aorta of rat.

Wu Y, McLaughlin PJ, Zagon IS.

Department of Neuroscience and Anatomy, The Milton S. Hershey Medical Center, The Pennsylvania State University, Hershey 17033-0850, USA.

Opioid peptides are known to play a role in the function of the mammalian cardiovascular system in the newborn. To learn about mechanisms underlying the morphogenesis of the developing vasculature and the principles contributing to the organization and maintenance of adult blood vessels, we have investigated the expression of the opioid growth factor (OGF), [Met5]-enkephalin, and its receptor, zeta. Moreover, gene expression for preproenkephalin mRNA, which encodes OGF, was studied to determine the source(s) of this inhibitory growth factor. By using immunocytochemistry, both OGF and the zeta opioid receptor were detected at embryonic day (E) 16 in the mesenchymal cells of the aortic wall. Staining appeared to be abundant in endothelial cells, smooth muscle cells, and fibroblasts at E20 and in the neonate. Immunoreactivity was noted to decrease progressively from day 5 to 10, but by weaning (day 21) and continuing into adulthood intense staining for both the peptide and receptor were observed. Preproenkephalin mRNA was detected throughout the aortic wall at E16, and the number of silver grains increased up today. Message was progressively reduced at days 5, 10, and 21, but signal in the adult aorta was comparable to that observed at day 5. These results indicate that components related to an endogenous opioid system regulating growth are present in the embryo, display a distinct spatial and temporal pattern of ontogeny, and persist into adulthood. In addition, these data indicate that OGF is an autocrine produced growth factor that is related to the emergence of vascular architecture and the maintenance of homeostasis in blood vessels.

PMID: 9566952

Brain Res. 1998 May 4;792(1):72-8.

The autocrine derivation of the opioid growth factor, [Met5]-enkephalin, in ocular surface epithelium.

Zagon IS, Sassani JW, Wu Y, McLaughlin PJ.

Department of Neuroscience and Anatomy, The Pennsylvania State University, The Milton S. Hershey Medical Center, 500 University Drive, Hershey, PA 17033, USA.

Endogenous opioid peptides serve as growth factors in developing, renewing, healing, and neoplastic cells and tissues. A native opioid peptide, [Met5]-enkephalin, termed opioid growth factor (OGF), has been discovered to regulate DNA synthesis in the epithelium of the ocular surface. OGF and its receptor zeta have been localized in both the basal and suprabasal cells of the epithelium. This study examined the hypothesis that OGF is an autocrine growth factor. Using probe for preproenkephalin (PPE) mRNA that encodes OGF, and in situ hybridization techniques, silver grains related to PPE mRNA were detected in both basal and suprabasal cells of the central and peripheral cornea, limbus, and conjunctiva. No distinct regional differences in the presence or location of message, as reflected by the density and distribution of PPE mRNA signal, were noted. These results demonstrate that a growth factor known to serve as a tonic, inhibitory, and receptor-mediated influence on the epithelium of the ocular surface is derived in an autocrine manner, thereby permitting local control of homeostatic cellular replication. Copyright 1998 Elsevier Science B.V.

PMID: 9593829

Brain Res. 1998 Jul 6;798(1-2):254-60.

Re-epithelialization of the rat cornea is accelerated by blockade of opioid receptors.

Zagon IS, Sassani JW, McLaughlin PJ.

Departments of Neuroscience and Anatomy, The Pennsylvania State University, College of Medicine, 500 University Drive, Hershey, PA 17033, USA. This e-mail address is being protected from spambots. You need JavaScript enabled to view it

A native opioid peptide, [Met5]-enkephalin, termed opioid growth factor (OGF), serves as a constitutively expressed and autocrine produced inhibitory molecule related to developing, neoplastic, renewing, and healing tissues. The present study was designed to examine the effects of interfering with opioid-receptor interaction during re-epithelialization of the cornea in the rat using both systemic injections and topical applications of the potent opioid antagonist naltrexone (NTX). A 4 mm diameter epithelial defect was made in the center of the rat cornea. NTX injected twice daily or applied as eyedrops four times daily significantly accelerated re-epithelialization compared to controls. Beginning as early as 8 h after wounding, both the systemic and topical NTX treatment groups had defects that were approximately 10% to 67% smaller than control abrasions at the time points examined. Similarly, the rate of healing for the NTX groups was 4.7- and 2.8-fold greater than controls for systemic and topical paradigms, respectively. The incidence of complete re-epithelialization in animals given systemic administration of NTX was markedly accelerated in comparison to control rats; however, differences in incidence of repair between NTX and control groups receiving topical application were not observed. These results show that native opioid peptides function in wound healing, and exert a tonically inhibitory influence at the receptor level on repair of corneal epithelial injuries. Copyright 1998 Elsevier Science B.V. All rights reserved.

PMID: 9666142

Brain Res. 1998 Aug 24;803(1-2):61-8.

Re-epithelialization of the rabbit cornea is regulated by opioid growth factor.

Zagon IS, Sassani JW, McLaughlin PJ.

Department of Neuroscience and Anatomy, The Pennsylvania State University, College of Medicine, 500 University Drive, Hershey, PA 17033, USA. This e-mail address is being protected from spambots. You need JavaScript enabled to view it

An endogenous opioid peptide, [Met5]-enkephalin, termed opioid growth factor (OGF) is a tonically active, autocrine-produced inhibitory molecule related to developing, neoplastic, renewing and healing tissues. The present investigation was designed to examine the role of OGF on corneal epithelial wound closure in the rabbit under in vitro and in vivo conditions. A 10-mm diameter epithelial defect was made in the center of the rabbit cornea, and the size of the defect, number of specimens with complete re-epithelialization, and rate of wound closure were evaluated using topical fluorescein and morphometric analysis. In organ culture, the influence of a complete opioid receptor blockade by naltrexone (NTX) showed an acceleration in re-epithelialization compared to controls. The action of excessive agonist (OGF) application revealed that exposure of wounded epithelium to OGF delayed wound closure under in vitro conditions, and did so in a receptor-mediated fashion. The modulatory capability of opioids on wound healing in vivo was explored by examining the effects of opioid peptide-receptor disruption using topical application of NTX, and enhanced healing of the abraded rabbit cornea was noted. The presence and location of OGF and the zeta (zeta) receptor in the normal and injured rabbit corneal epithelium were ascertained by immunocytochemistry, and both OGF and the zeta receptor were detected in basal and suprabasal epithelial cells. These results show that an opioid peptide, OGF, plays a direct role in the repair of injury to the corneal epithelium in the rabbit and acts as a receptor-mediated and constitutively expressed inhibitory molecule. Copyright 1998 Published by Elsevier Science B.V.

PMID: 9729280

Int J Oncol. 1999 Feb;14(2):373-80.

Human neuroblastoma cell growth in tissue culture is regulated by opioid growth factor.

McLaughlin PJ, Zagon IS, Skitzki J.

Department of Neuroscience and Anatomy, The Pennsylvania State University, College of Medicine, Hershey, PA 17033, USA.

Human neuroblastoma is one of the most common solid tumors in infants and children and represents about 10% of all childhood cancers. Nearly 70% of the patients present with disseminated disease and the long-term prognosis remains poor for this group despite advances in diagnosis and therapy. In this study, we discovered that an endogenous opioid peptide, [Met5]-enkephalin, inhibited the growth of human neuroblastoma SK-N-SH in vitro; in view of this pentapeptide's action it has been termed opioid growth factor (OGF). OGF was found to be constitutively expressed and tonically capable of suppressing cell replication, and its effects were opioid receptor mediated. Growth inhibition was dose-related, reversible, not cytotoxic, and independent of serum. Immunocytochemical studies detected both OGF and its related receptor, zeta, in the cytoplasm of log-phase cells. Pharmacological binding assays revealed specific and saturable binding with a one-site model of kinetics; this high-affinity opioid receptor was identified as zeta. These data suggest that a native opioid peptide, OGF, interacts with a novel opioid receptor, zeta, to arrest the growth of human neuroblastoma.

PMID: 9917516

Int J Oncol. 1999 Mar;14(3):577-84.

Human pancreatic cancer cell proliferation in tissue culture is tonically inhibited by opioid growth factor.

Zagon IS, Smith JP, McLaughlin PJ.

Department of Neuroscience and Anatomy, H-109, The Pennsylvania State University, The M.S. Hershey Medical Center, Hershey, PA 17033, USA.

Pancreatic adenocarcinoma is a fatal malignancy that ranks as the fourth most common cause of cancer-related mortality in the United States. The median survival after diagnosis is 3-6 months, with a 5-year survival rate of 3% or less. In spite of treatment efforts of surgery, radiation, and chemotherapy, the survival rate remains unchanged. In this study, we discovered that an endogenous opioid peptide, [Met5]-enkephalin, inhibited the growth of human pancreatic cancers in vitro; in view of this pentapeptide's action it has been termed opioid growth factor (OGF). OGF was found to be constitutively expressed, autocrine produced, and tonically capable of suppressing cell replication in an opioid receptor mediated manner. Growth inhibition was dose-related, reversible, not cytotoxic, and independent of serum. All 4 pancreatic cancer cell lines examined, representative of poor to well-differentiated neoplasias, exhibited growth regulation by OGF. Immunocytochemical studies detected both OGF and its related receptor, zeta, in the cytoplasm of log phase cells. Radioimmunoassays revealed that OGF was produced and secreted by the cells. These data suggest that a native opioid peptide, OGF, interacts with a novel opioid receptor, zeta, to arrest the growth of human pancreatic cancer.

PMID: 10024694

Int J Oncol. 1999 May;14(5):991-8.

Regulation of human head and neck squamous cell carcinoma growth in tissue culture by opioid growth factor.

McLaughlin PJ, Levin RJ, Zagon IS.

Department of Neuroscience and Anatomy, H109, Milton S. Hershey Medical Center, Hershey, PA l7033, USA.

Squamous cell carcinoma of the head and neck (SCCHN) is the sixth most common malignancy worldwide. Approximately half of the patients afflicted die within 5 years of diagnosis, and surviving patients may be left with severe esthetic and functional compromise. In this study, we discovered that an endogenous opioid peptide, [Met5]-enkephalin, inhibited the growth of human SCCHN in vitro; in view of this pentapeptide's action it has been termed opioid growth factor (OGF). OGF was found to be a constitutively expressed, receptor-mediated growth inhibitory agent that appears to be autocrine produced and secreted. Growth regulation was dose-related, reversible, cytostatic, and independent of serum. All 6 human SCCHN cell lines examined exhibited growth modulation by OGF. Blockade of peptide-receptor interaction by opioid antagonists (naltrexone), or addition of antibody to OGF, resulted in substantial increases in cell number compared to control levels, showing the tonic nature of OGF-zeta activity. Immunocytochemical studies detected both OGF and its related receptor, zeta, in these cells, correlating with earlier findings of peptide and receptor in specimens of SCCHN obtained at surgery. These data suggest that a native opioid peptide, OGF, interacts with a novel opioid receptor, zeta, to tonically arrest the growth of human SCCHN.

PMID: 10200353

Brain Res. 1999 Aug 28;839(2):313-22.

Opioid growth factor and organ development in rat and human embryos.

Zagon IS, Wu Y, McLaughlin PJ.

Department of Neuroscience and Anatomy, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey 17033, USA. This e-mail address is being protected from spambots. You need JavaScript enabled to view it

In addition to neurotransmission, the native opioid peptide, [Met5]enkephalin, is a tonically active inhibitory growth molecule that is termed opioid growth factor (OGF). OGF interacts with the zeta (zeta) opioid receptor to influence cell proliferation and tissue organization. We now identify OGF and the zeta receptor in embryonic derivatives including ectoderm, mesoderm, and endoderm of the rat on gestation day 20. Messenger RNA for preproenkephalin (PPE), the precursor of OGF, was detected in the developing cells, suggesting an autocrine production of this peptide. Acute exposure of the pregnant female to OGF resulted in a decrease in DNA synthesis in cells of organs representing all three germ layers, and did so in a receptor-mediated fashion. The influence of OGF was direct, as evidenced in organ culture studies. Blockade of endogenous opioid interaction using naltrexone (NTX) produced an increase in DNA synthesis, indicating the constitutive and functional nature of opioid activity on growth during prenatal life. Human fetal cells contained OGF and the zeta receptor. These data support the hypothesis that endogenous opioid modulation of organ development is a fundamental principle of mammalian embryogenesis, and that OGF has a profound influence on ontogeny. Irregularities in the role of opioids as growth regulators in relationship to the more than 500,000 newborns suffering from birth defects each year in the US needs to be examined.

PMID: 10519055

J Urol. 1999 Dec;162(6):2186-91.

Human renal cell cancer proliferation in tissue culture is tonically inhibited by opioid growth factor.

Bisignani GJ, McLaughlin PJ, Ordille SD, Beltz MS, Jarowenko MV, Zagon IS.

Department of Surgery, The Pennsylvania State University College of Medicine, Hershey 17033, USA.

PURPOSE: Peptide growth factors alter cellular events by binding to specific receptors. One group of peptides, the endogenous opioids, is important in the growth of normal and neoplastic tissue. [Met5]enkephalin, also termed opioid growth factor (OGF), is a tonically active inhibitory factor that interacts with the OGF receptor, OGFr, formerly identified as Greek zeta (zeta) and appears to be autocrine produced by human cancer cells. This study examined the hypothesis that OGF directly inhibits proliferation of renal cell carcinoma in tissue culture. MATERIALS AND METHODS: Human renal cancer cells (Caki-2) were grown using routine tissue culture techniques. A variety of natural and synthetic opioids including OGF, opioid antagonists, and opioid antibodies were added to renal cancer cell cultures to determine role of these peptides in renal cell carcinoma. The experiments were repeated in serum-free media, and with 4 other human renal cancer cell lines: Caki-2, A498, SN12C, and ACHN. Immunocytochemistry was performed to examine the presence of OGF and its receptor. RESULTS: OGF was the most potent opioid peptide to influence human renal cell carcinoma. OGF depressed growth within 12 hours of treatment, with cell numbers subnormal by up to 48% of control levels. OGF action was receptor mediated, reversible, not cytotoxic, neutralized by antibodies to the peptide, and detected in the human renal cell carcinoma lines examined. OGF appeared to be autocrine produced and secreted, and was constitutively expressed. Both OGF and its receptor were detected in these cells. CONCLUSION: OGF tonically inhibits renal cancer cell proliferation in tissue culture, and may play a role in the pathogenesis and management of human renal cell cancer.

PMID: 10569617

Brain Res. 1999 Dec 4;849(1-2):147-54.

Cloning, sequencing, expression and function of a cDNA encoding a receptor for the opioid growth factor, [Met(5)]enkephalin.

Zagon IS, Verderame MF, Allen SS, McLaughlin PJ.

Department of Neuroscience, The Pennsylvania State University, College of Medicine, 500 University Drive, Hershey, PA, USA. This e-mail address is being protected from spambots. You need JavaScript enabled to view it

The native opioid growth factor (OGF), [Met(5)]enkephalin, is a tonic inhibitory peptide that modulates cell proliferation and tissue organization during development, cancer, cellular renewal, wound healing and angiogenesis. OGF action is mediated by a receptor mechanism. We have cloned and sequenced a 2.1-kilobase (kb) cDNA for a receptor to OGF (OGFr). The open reading frame was found to encode a protein of 580 amino acids, and eight imperfect repeats of nine amino acids each were a prominent feature. The protein encoded by this cDNA exhibited the pharmacological, temporal and spatial characteristics of the OGFr. Functional studies using antisense technology demonstrated an enhancement in cell growth. The molecular organization of the OGFr has no homology to classical opioid receptors. These results provide molecular validity for the interaction of OGF and OGFr in the regulation of growth processes.

PMID: 10592296

Invest Ophthalmol Vis Sci. 2000 Jan;41(1):73-81.

Reepithelialization of the human cornea is regulated by endogenous opioids.

Zagon IS, Sassani JW, McLaughlin PJ.

Department of Neuroscience and Anatomy, The Pennsylvania State University College of Medicine, Hershey, USA. This e-mail address is being protected from spambots. You need JavaScript enabled to view it

PURPOSE: To determine the influence of endogenous opioid modulation on reepithelialization of the human cornea. METHODS: Eight-millimeter-diameter epithelial defects were created with a trephine and mechanical scraping in the center of human corneas. Resurfacing was studied in organ culture. The size of the defect, the number of specimens with complete reepithelialization, and rate of closure were evaluated using topical fluorescein and morphometric analysis. The influence of opioid receptor blockade was studied using the potent and long-acting opioid antagonist, naltrexone (NTX; 10(-6) M), and the effects of excess (10(-6) M) opioid growth factor (OGF), [Met5]enkephalin, also were determined. The modulatory activity of NTX and OGF on DNA synthesis was evaluated by monitoring the labeling index (LI) using radioactive thymidine. The presence and location of OGF and its receptor (OGFr) were ascertained by immunocytochemistry 1 hour and 24 hours after abrasion. RESULTS: NTX accelerated the wound-healing process, with 21% to 89% less defect than controls observed from 24 to 96 hours. At 72 hours, 62% of the subjects in the NTX group had complete closure of the corneal defects, in contrast to only 19% of the control specimens. All epithelial abrasions were resurfaced in the NTX group between 96 and 120 hours, whereas all controls were not closed until 168 hours. The rate of healing in the NTX group was 1.06 mm2/h compared to a rate of 0.68 mm2/h in the control group. OGF delayed corneal wound healing, with 24% to 260% more defect recorded than in control specimens at day 7. The healing rate of the OGF group was 0.42 mm2/h compared to 0.82 mm2/h for control subjects. The corneal epithelium adjacent to the wound had an LI that was 152% greater than control specimens, whereas OGF decreased the LI of this region by 75%. OGF and OGFr were detected in the epithelium bordering the damaged region at 1 hour, and both peptide and receptor were noted in the regenerating epithelium at 24 hours. CONCLUSIONS: These results indicate that an endogenous opioid is present and functions as a tonically active, receptor-mediated, negative growth factor during reepithelialization of the abraded human cornea.

PMID: 10634604

Int J Mol Med. 2000 Jan;5(1):77-84.

Identification and characterization of opioid growth factor receptor in human pancreatic adenocarcinoma.

Zagon IS, Smith JP, Conter R, McLaughlin PJ.

Department of Neuroscience and Anatomy, H-109, The Pennsylvania State University, The M.S. Hershey Medical Center, Hershey, PA 17033, USA.

Pancreatic cancer is the fourth most common cancer-related mortality in the United States, and the ninth most common cause of death from cancer worldwide. The opioid growth factor (OGF), [Met5]-enkephalin, inhibits the growth of human pancreatic adenocarcinoma in vitro and in vivo, and acts in a receptor-mediated fashion. Ligand binding assays using PANC-1 human pancreatic tumor cells and [3H]-[Met5]-enkephalin were performed to identify and characterize the receptor responsible for the growth-regulatory effects of OGF in pancreatic cancer. Specific and saturable binding was detected, and a Scatchard analysis revealed that the data were consistent for a single binding site with a binding affinity of 1.2+/-0.3 nM and a binding capacity of 36.4+/-4.1 fmol/mg protein. Subcellular fractionation studies showed that binding was restricted to the nuclear fraction. Competition experiments revealed that cold [Met5]-enkephalin was the most effective ligand at displacing [3H]-[Met5]-enkephalin; ligands for mu, delta, and kappa opioid receptors exhibited little or no competition. Binding was detected in 3 other human pancreatic tumor cell lines. Receptor number in xenografts of Capan-1 was decreased 8.6-fold compared to the same cells grown in tissue culture. Binding to radiolabeled [Met5]-enkephalin was detected in pancreatic cancers obtained from surgical resections. Binding capacity, but not binding affinity, was 7.1-fold greater in normal pancreatic tissues than in pancreatic neoplasia. The function, pharmacological and biochemical characteristics, distribution, and subcellular location of OGF binding in human pancreatic cancer were consistent with the OGF receptor (OGFr). In addition, human pancreatic cancer appears to have a low number of receptors for OGF, having the net effect of diminishing control of cellular replicative events.

PMID: 10601579

Int J Mol Med. 2000 Feb;5(2):191-6.

The opioid growth factor receptor in human head and neck squamous cell carcinoma.

McLaughlin PJ, Levin RJ, Zagon IS.

Department of Neuroscience and Anatomy, H109, The Pennsylvania State University, The M.S. Hershey Medical Center, Hershey, Pennsylvania 17033-0850, USA.

Carcinoma of the head and neck is the sixth leading cause of cancer in the world, and the third most common neoplasia in developing countries. More than 90% of head and neck cancers are squamous cell carcinomas (SCCHN). Approximately half of the patients afflicted die within 5 years of diagnosis and survival rates for cancer of the upper aero-digestive tract have not changed in 25 years. The opioid growth factor (OGF), ¿Met5-enkephalin, inhibits the growth of SCCHN in vitro and in vivo, and acts in a receptor-mediated fashion. Receptor binding assays using CAL-27 human SCCHN cells in culture and ¿3H-¿Met5-enkephalin were employed to identify and characterize the receptor responsible for the growth-regulatory effects of OGF. Specific and saturable binding was recorded, and Scatchard analysis showed that the data were consistent for a single binding site with a binding affinity (Kd) of 5.0+/-0.9 nM and maximal binding capacity (Bmax) of 47.5+/-1.7 fmol/mg protein. Subcellular fractionation studies determined that the optimal binding occurred with the nuclear fraction. Competition experiments demonstrated that cold ¿Met5-enkephalin was at least 7-fold greater than ligands selective for classical opioid receptors. Binding was detected in 4 other SCCHN cell lines. Receptor number in xenografts of CAL-27 was decreased almost 5-fold compared to the same cells grown in vitro. Binding to radiolabeled ¿Met5-enkephalin was recorded in SCCHN obtained from surgical resections. The function, pharmacological and biochemical characteristics, distribution and subcellular location of OGF binding in human SCCHN were consonant with the OGF receptor (OGFr).

PMID: 10639600

Brain Res. 2000 Feb 21;856(1-2):75-83.

Cloning, sequencing, chromosomal location, and function of cDNAs encoding an opioid growth factor receptor (OGFr) in humans.

Zagon IS, Verderame MF, Allen SS, McLaughlin PJ.

Department of Neuroscience, H-109, The Milton S. Hershey Medical Center, The Pennsylvania State University, College of Medicine, 500 University Drive, Hershey PA, USA. This e-mail address is being protected from spambots. You need JavaScript enabled to view it

The native opioid growth factor (OGF), [Met(5)]-enkephalin, is a tonic inhibitory peptide that modulates cell proliferation and tissue organization during development, cancer, cellular renewal, wound healing, and angiogenesis. OGF action is mediated by a receptor mechanism. We have cloned and sequenced cDNAs encoding multiple spliced forms of a human OGF receptor. The open reading frame in the longest cDNA was found to encode a protein of 697 amino acids, and 8 imperfect repeats of 20 amino acids each were a prominent feature. Altogether, five alternatively spliced forms were observed. The cDNA hybridized to mRNA from a variety of normal and neoplastic cells and tissues. Functional studies using antisense oligonucleotides to OGFr demonstrated an enhancement in cell growth. Fluorescent in situ hybridization (FISH) experiments showed the chromosomal location to be 20q13.3. This OGF receptor has no homology to classical opioid receptors. These results provide molecular validity for the interaction of OGF and OGF receptor in the regulation of growth processes in humans.

PMID: 10677613

Cell Prolif. 2000 Apr;33(2):63-73.

The opioid growth factor, [Met5]-enkephalin, inhibits DNA synthesis during recornification of mouse tail skin.

Wilson RP, McLaughlin PJ, Lang CM, Zagon IS.

Department of Comparative Medicine, College of Medicine, The Pennsylvania State University, Hershey 17033-2390, USA. This e-mail address is being protected from spambots. You need JavaScript enabled to view it

Opioid peptides serve as tonically active negative growth regulators in renewing and regenerating epithelia. To examine the involvement of opioids in renewal of the stratum corneum after tape stripping of tail skin, C57BL/6 J mice were given systemic injections of the potent opioid antagonist, naltrexone (NTX, 20 mg/kg i.p.) following injury. Blockade of opioid-receptor interaction by NTX for 4 h resulted in an elevation of 36-66% in basal cell DNA synthesis measured 24 h after injury. Injection of the endogenous opioid peptide, [Met5]-enkephalin (OGF, 10 mg/kg i.p.) 4 h before termination, suppressed radiolabelled thymidine incorporation in the basal cell layer by 37-46% at 24 h after wounding. The magnitude of the effects on DNA synthesis of OGF, but not NTX, depended on the timing of administration with respect to injury. OGF maximally depressed basal cell labelling (72%) when given 16 h after tape stripping. Concomitant administration of naloxone (10 mg/kg) with OGF blocked the inhibition of DNA synthesis; naloxone alone at the dosage utilized had no effect on cell labelling. Both OGF and its receptor, OGFr, were detected by immunocytochemistry in the basal and suprabasal cell layers, but not the cornified layer of tape stripped and uninjured tail skin. These results indicate: (a) a native opioid peptide and its receptor are expressed in epidermal cells of injured and uninjured mouse tail skin; (b) removal of the stratum corneum by tape stripping does not disrupt the function of the endogenous opioid growth system; (c) the proliferative response to wounding of the tail is tonically inhibited by the receptor-mediated action of an endogenous opioid peptide; and (d) DNA synthesis by basal cells can be elevated by disrupting opioid peptide receptor interactions.

PMID: 10845251

Pancreas. 2000 Aug;21(2):158-64.

Elevated levels of opioid growth factor in the plasma of patients with pancreatic cancer.

Smith JP, Conter RL, Demers TM, McLaughlin PJ, Zagon IS.

Department of Medicine, The Pennsylvania State University College of Medicine, Hershey 17033, USA. This e-mail address is being protected from spambots. You need JavaScript enabled to view it

Opioid growth factor (OGF, [Met5]-enkephalin) is an endogenous peptide that regulates the growth of human pancreatic cancer. To evaluate whether human subjects with pancreatic cancer have alterations in plasma levels of OGF, fasting blood samples were obtained from 15 patients with histologically confirmed pancreatic adenocarcinoma. Forty-five subjects with other malignancies, 20 patients with acute pancreatitis, and 30 aged-matched patients without cancer served as control populations. Individuals with pancreatic cancer had OGF values, as determined by radioimmunoassay, that were up to 7.3-fold greater than control subjects. No differences were found between OGF values obtained from patients with other malignancies, acute pancreatitis, or subjects without cancer. The sensitivity and specificity of OGF for pancreatic cancer were greater than either CA 19-9 or CEA. These data indicate that pancreatic cancer is associated with a marked increase in plasma OGF levels and suggest that this peptide may serve as a useful diagnostic tool in the screening for this disease.

PMID: 10975710

Int J Oncol. 2000 Nov;17(5):1053-61.

Opioid growth factor regulates the cell cycle of human neoplasias.

Zagon IS, Roesener CD, Verderame MF, Ohlsson-Wilhelm BM, Levin RJ, McLaughlin PJ.

Department of Neuroscience and Anatomy, H-109, The Pennsylvania State University, College of Medicine, Hershey, PA 17033, USA. This e-mail address is being protected from spambots. You need JavaScript enabled to view it

The native opioid growth factor (OGF), [Met5]-enkephalin, is a tonic inhibitory peptide that modulates cell proliferation and migration, as well as tissue organization, during development, cancer, homeostatic cellular renewal, wound healing, and angiogenesis. OGF action is mediated by the OGF receptor (OGFr). To investigate the target of OGF as to cell proliferation, the effects of excess OGF, and a deprivation of OGF-OGFr interaction by an opioid antagonist, naltrexone (NTX), were examined in 3 human cancer cell lines: pancreatic (BxPC-3), colon (HT-29), and head and neck (CAL-27). OGF exposure decreased growth, DNA synthesis, and mitosis, and increased the doubling time from control levels. FACS analysis revealed a marked increase in cells in the G0/G1 phase and compensatory reduction in cells in S and G2/M phases. Consistent with this observation, the percentage of labeled mitosis (PLM) analysis showed a notable increase in the time of the G0/G1 phase. Receptor blockade with NTX increased the rate of growth, length of DNA synthesis and mitotic phases, and decreased doubling time from control values. FACS analysis indicated an increase in the proportion of cells in S and G2/M phases, and a decrease in the number of cells in the G0/G1 phase. PLM evaluation demonstrated a shortening of the length of the S and G2 phases in the 3 cell lines, and decreases in the M and G0/G1 phases in some cancers. These results indicate that OGF action is directed at the G0/G1 phase, but interruption of OGF-OGFr interfacing has widespread repercussions on the cell cycle. The data on blockade of OGF-OGFr during log phase growth suggest a requisite escorting of the growth peptide and its receptor through the cell cycle.

PMID: 11029512

Brain Res Mol Brain Res. 2000 Dec 8;84(1-2):106-14.

Molecular characterization and distribution of the opioid growth factor receptor (OGFr) in mouse.

Zagon IS, Verderame MF, Zimmer WE, McLaughlin PJ.

The native opioid growth factor (OGF), [Met(5)]-enkephalin, is a tonic inhibitory peptide that modulates cell proliferation and tissue organization during development, cancer, cellular renewal, wound healing, and angiogenesis. OGF action is mediated by a receptor mechanism. The receptor for OGF, OGFr, has been cloned and sequenced in humans and rats. Using primers based on the rat OGFr cDNA, and a mouse embryo expressed sequence tag, the full-length 2.1 kb mouse OGFr cDNA was sequenced. The open reading frame was found to encode a protein of 634 amino acids, and 14 imperfect repeats of 9 amino acids each were a prominent feature. The molecular weight of OGFr was calculated as 70679, and the isoelectric point was 4.5. Northern blot analysis revealed a 2.1 kb OGFr mRNA transcript in adult mouse brain, heart, lung, liver, kidney, and triceps surae muscle. The amino acids for mouse and rat OGFr were 93% similar and 91% identical, but the mouse and human shared only a 70% similarity and a 58% identity. These results emphasize the molecular validity of OGFr, and explain the interaction of OGF with respect to normal and abnormal growth in mouse cells and tissues.

PMID: 11113537

Pharmacol Biochem Behav. 2002 Jan-Feb;71(1-2):171-81.

Chronic exposure to the opioid growth factor, [Met5]-enkephalin, during pregnancy: maternal and preweaning effects.

McLaughlin PJ, Wylie JD, Bloom G, Griffith JW, Zagon IS.

Department of Neuroscience and Anatomy, The Milton S. Hershey Medical Center, The Pennsylvania State University, H-109, 500 University Drive, Hershey, PA 17033, USA. This e-mail address is being protected from spambots. You need JavaScript enabled to view it

The opioid peptide, [Met(5)]-enkephalin (termed opioid growth factor, OGF), is an autocrine growth factor that serves as a constitutively active inhibitory agent. OGF crosses the placenta and depresses DNA synthesis in the fetus. The role of OGF in pregnancy and parturition, and the influence exerted on prenatal and neonatal features of the offspring, were studied in rats. Females received daily injections of 10 mg/kg OGF throughout gestation; all offspring were cross-fostered to lactating noninjected dams at birth. No effects on the length of gestation, course of pregnancy, behavior of the pregnant dam, maternal weight gain, or food and water intake throughout gestation were recorded in OGF-treated mothers. Moreover, nociceptive response in these females was not altered by chronic OGF exposure, and no signs of physical dependence or withdrawal could be observed following a challenge by the opioid antagonist naloxone. Litter size and the number of live births per litter of OGF-treated mothers were reduced by 25% from control subjects and a fourfold increase in stillborns was noted for mothers receiving OGF compared to control levels. Histopathologic analysis confirmed the stillborns to have died in utero. OGF-exposed neonates were normal in body weight and crown-to-rump length, but these pups were observed to be lethargic and cyanotic, and had subnormal weights of many organs. Body weights of 10-, 15-, and 21-day-old OGF-exposed rats were reduced 11-27% from control levels. Wet and dry organ weights of the rats maternally subjected to OGF were decreased from control values in six of the eight organs evaluated at 10 days. At weaning, some organs were subnormal in weight. These data lead us to hypothesize that a native opioid peptide-OGF-is integral to certain aspects of maternal, neonatal, and postnatal well-being, and that disruptions in this opioid peptide have serious repercussions on the course of pregnancy and fetal outcome.

PMID: 11812520

Brain Res Brain Res Rev. 2002 Feb;38(3):351-76.

The biology of the opioid growth factor receptor (OGFr).

Zagon IS, Verderame MF, McLaughlin PJ.

Department of Neuroscience and Anatomy, H-109, The Milton S. Hershey Medical Center, The Pennsylvania State University, College of Medicine, 500 University Drive, Hershey, PA 17033, USA. This e-mail address is being protected from spambots. You need JavaScript enabled to view it

Opioid peptides act as growth factors in neural and non-neural cells and tissues, in addition to serving for neurotransmission/neuromodulation in the nervous system. The native opioid growth factor (OGF), [Met(5)]-enkephalin, is a tonic inhibitory peptide that plays a role in cell proliferation and tissue organization during development, cancer, cellular renewal, wound healing, and angiogenesis. OGF action is mediated by a receptor mechanism. Assays with radiolabeled OGF have detected specific and saturable binding, with a one-site model of kinetics. Subcellular fractionation studies show that the receptor for OGF (OGFr) is an integral membrane protein associated with the nucleus. Using antibodies generated to a binding fragment of OGFr, this receptor has been cloned and sequenced in human, rat, and mouse. OGFr is distinguished by containing a series of imperfect repeats. The molecular and protein structure of OGFr have no resemblance to that of classical opioid receptors, and have no significant homologies to known domains or functional motifs with the exception of a bipartite nuclear localization signal. Immunoelectron microscopy and immunocytochemistry investigations, including co-localization studies, have detected OGFr on the outer nuclear envelope where it interfaces with OGF. The peptide-receptor complex associates with karyopherin, translocates through the nuclear pore, and can be observed in the inner nuclear matrix and at the periphery of heterochromatin of the nucleus. Signal transduction for modulation of DNA activity is dependent on the presence of an appropriate confirmation of peptide and receptor. This report reviews the history of OGF-OGFr, examines emerging insights into the mechanisms of action of opioid peptide-receptor interfacing, and discusses the clinical significance of these observations.

PMID: 11890982

J Vasc Surg. 2002 Mar;35(3):532-8.

Differential effects of vascular growth factors on arterial and venous angiogenesis.

Blebea J, Vu JH, Assadnia S, McLaughlin PJ, Atnip RG, Zagon IS.

Department of Surgery, The Pennsylvania State University College of Medicine, Hershey 17033-0850, USA. This e-mail address is being protected from spambots. You need JavaScript enabled to view it

OBJECTIVE: Angiogenesis, the development of new blood vessels, has become an area of increased interest for both scientific and clinical application purposes. Proangiogenic agents, such as vascular endothelial growth factor (VEGF) and naltrexone, have been shown to effectively induce new blood vessel growth. Other growth factors, such as the endogenous opioid growth factor (OGF; [Met(5)]-enkephalin) and retinoic acid, are inhibitors of angiogenesis. The differential effects on veins and arteries, however, by any vascular growth factor, have not previously been investigated. METHODS: The chick chorioallantoic membrane (CAM) assay was used for the in vivo quantitation of angiogenesis. After 3 days of incubation, fertilized chick embryos were explanted, and a 3.2-mm methylcellulose disk containing either the known angiogenic stimulators VEGF (0.2 microg, 1.0 microg) or naltrexone (0.1 microg, 5.0 microg), or the angiogenic inhibitors OGF (1.0 microg, 5.0 microg) or retinoic acid (1.0 microg) was placed onto the CAM surface. An equal volume of distilled water served as a control. After 2 days of growth, the CAM arteries and veins were identified, and images were obtained with a digital camera. Quantitative analysis of angiogenesis was performed on a 100-mm(2) area surrounding the applied disk, and the number and length of the veins and arteries were measured. RESULTS: The angiogenic stimulators VEGF and naltrexone markedly increased both the total number and length of all blood vessels as compared with control values. The mean length of blood vessels decreased, suggesting the induction of new vessel growth. VEGF and naltrexone proportionately increased vein and arterial angiogenesis, maintaining artery/vein ratios for vessel number and length that were unchanged compared with controls. The angiogenic inhibitors, OGF and retinoic acid, notably decreased the total number and length of blood vessels in the CAM preparations. However, these compounds had a disproportionately greater inhibitory effect on arterial angiogenesis as reflected in decreased artery/vein ratios for vessel number and length. CONCLUSIONS: The angiogenic stimulators VEGF and naltrexone induce development of veins and arteries in a proportional manner. In contrast, the angiogenic inhibitors OGF and retinoic acid demonstrated a greater inhibitory effect on arterial as compared with venous angiogenesis. Such differential effects on angiogenesis may be important in both defining mechanisms of action and designing therapeutic interventions.

PMID: 11877704

J Vasc Surg. 2003 Mar;37(3):636-43.

Opioid growth factor inhibits intimal hyperplasia in balloon-injured rat carotid artery.

Zagon IS, Essis FM Jr, Verderame MF, Healy DA, Atnip RG, McLaughlin PJ.

Department of Neuroscience and Anatomy, H-109, The Pennsylvania State University College of Medicine, M.S. Hershey Medical Center, 500 University Drive, Hershey, PA 17033, USA. This e-mail address is being protected from spambots. You need JavaScript enabled to view it

OBJECTIVE: The endogenous opioid [Met(5)]-enkephalin (opioid growth factor [OGF]) is a tonically active, receptor-mediated inhibitory growth peptide in developing and adult vasculature. This study was designed to determine the role of OGF in neointimal hyperplasia. METHODS: The carotid artery in adult male Sprague-Dawley rats was denuded with balloon catheterization. OGF (10 mg/kg), the opioid antagonist naltrexone (NTX; 30 mg/kg), or saline solution (0.2 mL) was injected intraperitoneally daily for 28 days into the rats, and restenosis of the carotid artery was examined with morphometric analysis using Optimas software. Proliferation of the neointima and media was measured by radioactive thymidine incorporation over 3 hours. The presence of OGF and its receptor, OGFr, were examined with immunofluorescence microscopy. RESULTS: OGF depressed DNA synthesis in the intima and media from 16% to 78% of control levels in the first 2 weeks after deendothelialization, whereas NTX exposure elevated DNA synthesis by 21% to 89%. OGF action was receptor-mediated. In the month after injury the thickness of the intima in OGF-treated rats was decreased by 18% to 31% from control values, whereas intimal thickness was increased in the NTX group by 10% to 31%. Luminal area was almost 25% greater than control values in the OGF group, but was reduced 17% by NTX. OGF and the OGF receptor were detected in the carotid artery with immunohistochemistry. CONCLUSIONS: These results demonstrate for the first time that a native opioid system modulates repair of vascular injury. OGF is a constitutively active peptide that has a receptor-mediated action in the negative regulation of neointimal growth, a major cause of restenosis.

PMID: 12618704

Brain Res. 2003 Mar 28;967(1-2):37-47.

Immunoelectron microscopic localization of the opioid growth factor receptor (OGFr) and OGF in the cornea.

Zagon IS, Ruth TB, Leure-duPree AE, Sassani JW, McLaughlin PJ.

Department of Neuroscience and Anatomy, H109, The Milton S. Hershey Medical Center, 500 University Drive, PA 17033, USA. This e-mail address is being protected from spambots. You need JavaScript enabled to view it

This study was conducted to determine the cellular and subcellular location(s) of the opioid growth factor receptor (OGFr), and the opioid growth factor (OGF), [Met(5)]-enkephalin, in the corneal epithelium. Laser scanning confocal microscopy analysis revealed that both OGFr and OGF were colocalized in the paranuclear cytoplasm and cell nuclei in basal, as well as suprabasal, cells of adult rat corneal epithelium. Using a postembedding immunogold procedure for immunoelectron microscopy that included embedding in Unicryl, both single- and double-face labeling studies were performed. Immunogold labeling of OGFr was detected on the outer nuclear envelope, in the paranuclear cytoplasm proximal to the nuclear envelope, perpendicular to the nuclear envelope in a putative nuclear pore complex, and within the nucleus adjacent to heterochromatin. Immunoreactivity for OGF was noted in locations similar to that for OGFr. In addition, aggregates of staining for OGF were found throughout the cytoplasm, including subjacent to the plasma membrane. Double labeling experiments revealed that complexes of OGF-OGFr were colocalized on the outer nuclear envelope, in the paranuclear cytoplasm, extending across the nuclear pore complex, and in the nucleus. Anti-OGFr IgG by itself, but not anti-OGF IgG alone, was associated with the outer nuclear envelope, and uncomplexed OGF immunoreactivity was detected in the cytoplasm in dual labeling experiments. These results based on complementary approaches of confocal microscopy and immunoelectron microscopy, suggest that: (i) OGFr resides on the outer nuclear envelope, (ii) OGF interacts with OGFr at the outer nuclear envelope, (iii) the colocalized receptor and peptide translocates between the cytoplasm and the nucleus at the nuclear pore, and (iv) signal transduction for modulation of cell proliferation necessitates a peptide-receptor complex that interfaces with chromatin in the nucleus.

PMID: 12650964

Neuropeptides. 2003 Apr;37(2):79-88.

Opioids and the apoptotic pathway in human cancer cells.

Zagon IS, McLaughlin PJ.

Department of Neuroscience and Anatomy, The Milton S. Hershey Medical Center, The Pennsylvania State University, College of Medicine, 500 University Drive, H-109, Hershey, PA 17033, USA. This e-mail address is being protected from spambots. You need JavaScript enabled to view it

This study was designed to examine the role of opioids in cell survival, with an emphasis on the mechanism of opioid growth factor (OGF, [Met(5)]-enkephalin)-dependent growth inhibition. Using three human cancer cell lines: MIA PaCa-2 pancreatic adenocarcinoma, HT-29 colon adenocarcinoma, and CAL-27 squamous cell carcinoma of the head and neck, and OGF and the opioid antagonist naltrexone (NTX) at a dosage (10(-6)M) selected because it is known to repress or increase, respectively, cell replication, the effects on apoptosis (TUNEL, Annexin V) and necrosis (trypan blue) were investigated on days 2, 5, and 7 of exposure. In addition, the influence of a variety of other natural and synthetic opioids on apoptosis and necrosis was examined at a dosage of 10(-6)M. OGF, NTX, naloxone, [D-Pen(2,5)]-enkephalin, [Leu(5)]-enkephalin, dynorphin A1-8, beta-endorphin, endomorphin-1 and -2, and methadone at concentrations of 10(-6)M did not alter cell viability of any cancer cell line. Exposure of cultures to [D-Ala(2),MePhe(4),Glycol(5)]-enkephalin (DAMGO), morphine, or etorphine at 10(-6)M significantly increased the number of adherent cells positively stained for TUNEL and Annexin V, as well as the number of necrotic cells in the supernatant, from control levels at all time points studied. The effects of DAMGO, morphine, and etorphine on apoptosis/necrosis were not fully blocked by concomitant administration of naloxone. Despite the increase in cell death in some opioid-treated groups, the number of apoptotic and necrotic adherent cells, and the number of necrotic cells in the supernatant, was no more than 1-2% of the total cell population. These results indicate that the inhibitory (OGF) or stimulatory (NTX) action on cell growth in tissue culture is not due to alterations in apoptotic or necrotic pathways. Moreover, although some opioids increased cell death, and dose-effect relationships need to be established, this activity was not of great magnitude and supports the previously reported lack of growth inhibition of many of these compounds.

PMID: 12747939

Cancer. 2003 Apr 1;97(7):1701-10.

Defects in the opioid growth factor receptor in human squamous cell carcinoma of the head and neck.

McLaughlin PJ, Stack BC Jr, Levin RJ, Fedok F, Zagon IS.

Department of Neuroscience and Anatomy, Division of Otolaryngology, The Milton S. Hershey Medical Center, The Pennsylvania State University, Hershey, Pennsylvania 17033, USA. This e-mail address is being protected from spambots. You need JavaScript enabled to view it

BACKGROUND: The endogenous opioid peptide, [Met(5)]-enkephalin, termed opioid growth factor (OGF), interacts with its receptor (OGFr) to play a role as a constitutively expressed inhibitory growth factor in the proliferation of epithelial cells. This study compared protein and gene expression of OGFr in surgical specimens of human squamous cell carcinoma of the head and neck (SCCHN) with normal epithelium. METHODS: Tissues from 64 patients with SCCHN and from 49 patients undergoing uvulapalatoplasty or tonsillectomy were utilized. Binding affinity and capacity were assessed by receptor binding assays and the levels of OGFr protein were determined by quantitative Western blot analysis. Immunohistochemistry assessed the presence and distribution of OGFr. Levels of OGFr mRNA were quantitated by Northern blot analysis. Protein and gene expressions of OGFr also were evaluated in the margins of SCCHN. RESULTS: Binding analyses indicated nearly ninefold fewer OGFr binding sites in tumor tissue in comparison with normal samples. The OGFr protein levels were reduced fivefold in tumor tissues relative to normal epithelium. Values of OGFr mRNA were comparable in tumors and normal epithelium. Tumor margins had intermediate levels of protein and binding, but OGFr mRNA values were similar to those of normal specimens. CONCLUSIONS: These data demonstrate that OGFr is defective in SCCHN and that translation/posttranslation of OGFr protein, but not transcriptional levels of the OGFr gene, is involved. In addition, the attenuated levels of OGFr binding capacity may serve as a marker for SCCHN. These subnormal levels of OGFr may be responsible in part for tumor progression, diminishing the interaction of OGF with OGFr that aids in stabilizing cell replication by an inhibitory mechanism. Gene therapy to reinstate OGFr and/or function could provide a useful treatment for inhibiting tumor progression. Copyright 2003 American Cancer Society. DOI 10.1002/cncr.11237

PMID: 12655527

Curr Eye Res. 2003 May;26(5):249-62.

Opioid growth factor modulation of corneal epithelium: uppers and downers.

Sassani JW, Zagon IS, McLaughlin PJ.

Department of Ophthalmology, Penn State's Milton S. Hershey Medical Center, 500 University Drive, Hershey, PA 17033, USA. This e-mail address is being protected from spambots. You need JavaScript enabled to view it

PURPOSE: This paper presents a concise review of the role of the intrinsic opioid growth regulation system (IOGRS) in homeostasis and wound repair of the corneal epithelium. METHODS: The article is a summary of published research on the topic. RESULTS: The native opioid peptide, [Met(5)]-enkephalin, also termed opioid growth factor (OGF), has been demonstrated to be present in a wide variety of classes of the phylum. Chordata, and acts as an autocrine/paracrine produced, tonically active, receptor specific, negative growth modulating factor in homeostatic (uninjured) corneal epithelium in humans and non-primates. Similarly, OGF acts to down-regulate epithelial cell division and migration of corneal epithelium in the closing of corneal epithelial abrasions. Such repair can be up-regulated (hastened) in non-diabetic animals by treatment with exogenous administration of the potent opioid antagonist, naltrexone (NTX). The system also is functional in diabetic animals and can be manipulated to restore epithelial wound healing rates to normal. CONCLUSIONS: The IOGRS plays a vital role in supporting corneal epithelial homeostasis, and in modulating closure of epithelial wounds. The system should provide opportunities for novel therapies especially for corneal epithelial wound healing disorders.

PMID: 12854052

Cancer Lett. 2003 Sep 25;199(2):209-17.

Opioid growth factor (OGF) inhibits the progression of human squamous cell carcinoma of the head and neck transplanted into nude mice.

McLaughlin PJ, Levin RJ, Zagon IS.

Departments of Neuroscience and Anatomy, The Milton S Hershey Medical Center, The Pennsylvania State University, 500 University Drive, Room C3727, Hershey, PA 17033-0850, USA. This e-mail address is being protected from spambots. You need JavaScript enabled to view it

Opioid growth factor (OGF) interacts with the OGF receptor (OGFr) and serves as a native inhibitory growth factor. OGF and OGFr are present in squamous cell carcinoma of the head and neck (SCCHN), and OGF represses the replication of SCCHN in tissue culture. In this study, OGF-treated nude mice with xenografts of SCCHN displayed delays in tumor appearance and had reduced tumor size compared to controls. OGF activity was receptor-mediated. Opioid-receptor blockade by the potent opioid antagonist, naltrexone, stimulated tumorigenic processes. Both OGF and OGFr were detected in the tumors by immunohistochemistry, and OGFr was characterized by receptor binding analysis. These results indicate that the OGF-OGFr axis functions in vivo, OGF is a constitutively active molecule, and OGF modulation of SCCHN may have clinical application.

PMID: 12969794

Neuropeptides. 2003 Oct;37(5):290-7.

The expression and function of the OGF-OGFr axis - a tonically active negative regulator of growth - in COS cells.

Zagon IS, Verderame MF, McLaughlin PJ.

Department of Neuroscience and Anatomy, The Milton S. Hershey Medical Centre, College of Medicine, The Pennsylvania State University, 500 University Drive, Room C3729, Hershey, PA 17033, USA. This e-mail address is being protected from spambots. You need JavaScript enabled to view it

This study was designed to examine the presence and role of the opioid growth factor (OGF, [Met(5)]-enkephalin) and the OGF receptor (OGFr) in COS-7 cells; these cells lack classical opioid receptors. Preproenkephalin mRNA, which encodes OGF, was detected by Northern blot analysis, and OGFr mRNA was recorded by RT-PCR. Receptor binding analysis showed specific and saturable binding (K(d)=3.5nm, B(max)=44fmol/mg protein) for OGFr in the nuclear fraction. Both OGF and OGFr were recorded in COS-7 cells by immunocytochemistry. Addition of OGF to log-phase COS-7 cultures depressed growth by 41.6% from control levels, whereas opioid-receptor blockade by the opioid antagonist, naltrexone, increased the number of cells by 29.8% from control values. The effect of OGF was receptor mediated. Exposure to a wide variety of synthetic and natural opioid peptides, including those selective for micro, delta, and kappa opioid receptors, showed that only OGF had an effect. Treatment with antisense OGFr oligonucleotides increased the number of cells by over 2-fold compared to wild-type cultures of COS-7 cells and preparations receiving scrambled oligonucleotides. These results indicate that the OGF-OGFr axis is present and functions in COS-7 cells, and in the absence of classical opioid receptors.

PMID: 14607106

Int J Oncol. 2004 Jan;24(1):227-32.

Opioid growth factor inhibition of a human squamous cell carcinoma of the head and neck in nude mice: dependency on the route of administration.

McLaughlin PJ, Stack BC Jr, Braine KM, Ruda JD, Zagon IS.

Department of Neuroscience and Anatomy, H109, The Penn State University College of Medicine, Hershey, PA 17033, USA. This e-mail address is being protected from spambots. You need JavaScript enabled to view it

Opioid growth factor (OGF), [Met5]-enkephalin, interacts with the OGF receptor (OGFr) to inhibit the growth of human squamous cell carcinoma of the head and neck (SCCHN) in vitro. Administration of OGF by daily intraperitoneal injection (i.p.) to animals with xenografts of CAL-27, a poorly differentiated SCCHN, is known to repress tumorigenic events. In this study, the ubiquity of OGF action on SCCHN was investigated by examination of OGF activity on SCC-1 tumors; this human cell line is well-differentiated and highly invasive. Mice receiving daily i.p. injections (10 mg/kg) of OGF had more than a 3-day delay in tumor appearance, and decreases in tumor volume ranging from 51 to 64% in comparison to controls throughout the experimental period. Receptor binding analysis for OGFr showed that binding capacity (Bmax) was 2.2-fold greater than control values, but binding affinity (Kd) was comparable. Plasma OGF levels did not vary between OGF and control groups. Mice receiving OGF by continuous infusion using minipumps, or by daily intratumoral injection, had characteristics of tumorigenicity similar to their corresponding control animals, although the OGF levels in mice receiving the OGF by minipump were elevated 18-fold greater than the control group. These data indicate that: i) the inhibitory action of OGF may be ubiquitous for SCCHN, ii) OGF treatment alters the characteristics of the OGF receptor but not of plasma OGF levels, and iii) the magnitude of effects of OGF on SCCHN is dependent on the route of administration.

PMID: 14654962

Brain Res Bull. 2004 Mar 1;63(1):57-63.

Gene expression of OGFr in the developing and adult rat brain and cerebellum.

Zagon IS, McLaughlin PJ.

Department of Neural and Behavioral Sciences, The Milton S. Hershey Medical Center, The Pennsylvania State University, 500 University Drive, Room C3729, Hershey, PA 17033, USA. This e-mail address is being protected from spambots. You need JavaScript enabled to view it

The native opioid peptide, [Met5]-enkephalin (termed opioid growth factor (OGF)), is a tonically active negative growth factor targeted to cell proliferation in the developing nervous system. OGF action is mediated by the OGF receptor (OGFr). The present study investigates gene expression of OGFr in the developing and adult brain and cerebellum of the rat using Northern blot analysis and normalization to GAPDH. OGFr was detected in whole brain at embryonic day 20 and birth, and was at least twofold greater than neonatal levels during the first week of life. From postnatal day 15 onwards to adulthood, levels of OGFr mRNA in the whole brain were detectable but less than those at birth. OGFr mRNA in cerebellum was found on embryonic day 20, and remained relatively constant until postnatal day 12 when a sharp increase was recorded. In the third week of life and continuing into adulthood, cerebellar OGFr mRNA was detected at levels comparable to those in postnatal week 1. These results show that message for OGFr is developmentally regulated prior to and after birth, is ubiquitously expressed during development, and is present in the adult brain and cerebellum even though OGF receptor binding is not recorded.

PMID: 15121239

Int J Oncol. 2004 Jun;24(6):1443-8.

Opioid growth factor (OGF) inhibits anchorage-independent growth in human cancer cells.

Zagon IS, McLaughlin PJ.

Department of Neural and Behavioral Sciences, H-109, The Milton S. Hershey Medical Center, Hershey, PA 17033, USA. This e-mail address is being protected from spambots. You need JavaScript enabled to view it

Opioid growth factor (OGF) is a native endogenous opioid peptide ([Met5]-enkephalin) that interacts with the OGF receptor (OGFr), and serves as a tonically active negative growth factor in neoplasia. To inquire whether OGF modulates anchorage-independent growth, HT-29 human colon cancer cells were grown in soft agar and subjected to this peptide. In contrast to controls, HT-29 cells exposed to OGF had 57% fewer colonies, and these colonies were reduced in area by 75%. The changes induced by OGF were abolished by concomitant treatment with naloxone, indicating a receptor-mediated mechanism for peptide activity. Continuous blockade of opioid-receptor interactions with the potent and long-acting opioid antagonist, naltrexone (NTX), revealed an increase of 81 and 49% in the number and area, respectively, of colonies compared to control levels. These data suggest that OGF is tonically active in neoplastic cells growing in soft agar medium. HT-29 cells studied under anchorage-independent conditions were not influenced in growth by a variety of natural and synthetic opioids, including those selective for micro, delta, and kappa opioid receptors. Similar effects on anchorage-independent growth by OGF and NTX observed for HT-29 cells were recorded in pancreatic adenocarcinoma cells (Mia PaCa-2, Panc-1) and squamous cell carcinoma of the head and neck (CAL-27). These results using anchorage-independent conditions are consistent with previous data showing that OGF can markedly influence tumor growth in xenografts, and suggest that clonogenic assays can be utilized as indicators of tumorigenicity when tumor transplantation experiments are restricted.

PMID: 15138586

Anat Rec A Discov Mol Cell Evol Biol. 2005 Jan;282(1):24-37.

Nucleocytoplasmic distribution of opioid growth factor and its receptor in tongue epithelium.

Zagon IS, Ruth TB, McLaughlin PJ.

Department of Neural and Behavioral Sciences, Pennsylvania State University, College of Medicine, 500 University Drive, Rm. C3727, Hershey, PA 17033, USA. This e-mail address is being protected from spambots. You need JavaScript enabled to view it

The subcellular distributions of the opioid growth factor (OGF), [Met(5)]-enkephalin, and opioid growth factor receptor (OGFr) in the epithelium of the rat tongue were determined in order to reveal structure-function relationships. Laser scanning confocal microscopic analysis showed that both OGF and OGFr were colocalized in the paranuclear cytoplasm and in the nuclei of keratinocytes in the stratum basale. Using immunoelectron microscopy and postembedding techniques, double labeling experiments disclosed that complexes of OGF-OGFr were colocalized on the outer nuclear envelope, in the paranuclear cytoplasm, perpendicular to the nuclear envelope in a putative nuclear pore complex, and in the nucleus adjacent to heterochromatin. Anti-OGF IgG alone was detected in the cytoplasm, and anti-OGFr IgG alone was associated with the outer nuclear envelope. Study of chronic treatment with the opioid antagonist, naltrexone (NTX), which blocks opioid-receptor binding, revealed the presence of OGFr immunoreactivity alone in the cytoplasm and the nucleus; some OGF-OGFr complexes were also observed. Colocalization of OGFr and karyopherin (importin) beta was recorded in the cytoplasm and nucleus. These results in tongue epithelium are the first to suggest that OGFr resides on the outer nuclear envelope, where OGF interacts with OGFr; that the OGF-OGFr complex translocates between cytoplasm and nucleus at the nuclear pore; and that the nuclear localization signal of OGFr interacts with karyopherin beta for nuclear transport. These novel data also indicate that signal transduction for cell proliferation appears to involve an OGF-OGFr complex that interfaces with chromatin in the nucleus. Moreover, the unique finding that OGFr was found in the cytoplasm and nucleus in NTX-treated specimens may suggest that NTX-OGFr complexes have the same pathway as OGF-OGFr. (c) 2004 Wiley-Liss, Inc.

PMID: 15584033

Int J Oncol. 2005 Mar;26(3):809-16.

Enhanced growth inhibition of squamous cell carcinoma of the head and neck by combination therapy of paclitaxel and opioid growth factor.

McLaughlin PJ, Jaglowski JR, Verderame MF, Stack BC, Leure-Dupree AE, Zagon IS.

Department of Neural and Behavioral Sciences, H109, The Milton S. Hershey Medical Center, Hershey, PA 17033, USA. This e-mail address is being protected from spambots. You need JavaScript enabled to view it

This study evaluated the effects of a combination of opioid growth factor (OGF) and paclitaxel on squamous cell carcinoma of the head and neck (SCCHN) using a tissue culture model of human SCCHN. The combination of OGF and paclitaxel was markedly inhibitory to SCCHN proliferation, reducing growth from control levels by 48 to 69% within 48 h. OGF in combination with carboplatin also depressed cell growth. The effect of a combination of OGF and paclitaxel or carboplatin on SCCHN growth was greater than either of the individual compounds. The efficacy of OGF, but not paclitaxel, was mediated by a naloxone-sensitive receptor and completely reversible. OGF, but no other endogenous or exogenous opioid, altered replication of SCCHN. OGF and paclitaxel depressed DNA synthesis, whereas only paclitaxel induced apoptosis. The combination of OGF and paclitaxel also had a supra-additive effect on the growth of another SCCHN, CAL-27, indicating the ubiquity of the combined drug activity. These data suggest that the combination of a biotherapy (OGF) and chemotherapy (paclitaxel and carboplatin) may provide an enhanced antitumor effect with respect to SCCHN.

PMID: 15703840

Cornea. 2005 Jul;24(5):614-9.

Particle-mediated gene transfer of opioid growth factor receptor cDNA regulates cell proliferation of the corneal epithelium.

Zagon IS, Sassani JW, Verderame MF, McLaughlin PJ.

Department of Neural and Behavioral Sciences, The Pennsylvania State University College of Medicine, Hershey, 17033, USA. This e-mail address is being protected from spambots. You need JavaScript enabled to view it

PURPOSE: This study was designed to determine at the molecular level whether interactions between the opioid growth factor (OGF) and OGF receptor (OGFr) play a role in regulating DNA synthesis in the homeostasis of the corneal epithelium. METHODS: The plasmid pcDNA3.1+OGFr-HA, carrying the rat OGFr cDNA epitope-tagged with a C-terminal hemagglutinin (HA), or the empty-vector (pcDNA3.1+), was delivered twice by the Helios Gene Gun System at 300 psi to the cornea of anesthetized rats. The contralateral (untreated) cornea served as the naive specimen. BrdU was used to determine whether the recombinant OGFr was effective in regulating DNA synthesis in the rat peripheral corneal epithelium. RESULTS: Within 18 hours of transfection, positive HA staining was apparent in both the basal and suprabasal layers (efficiency > 90% of the cells) throughout the central and peripheral cornea. Quantitative immunohistochemistry with rhodamine-conjugated anti-OGFr antibodies revealed twofold more OGFr expression in the central and peripheral epithelium of transfected corneas relative to naive corneas. The number of BrdU-positive basal cells in the peripheral epithelium of the transfected cornea was one-third of that in the naive cornea. CONCLUSIONS: These data demonstrate the direct role of the OGF-OGFr system in determining cellular renewal in the mammalian corneal epithelium. Moreover, the successful establishment of a novel delivery system of cDNAs to the ocular surface suggests a therapeutic role for gene therapy in the eye.

PMID: 15968171

Cancer Chemother Pharmacol. 2005 Jul;56(1):97-104. Epub 2005 Mar 25.

Opioid growth factor enhances tumor growth inhibition and increases the survival of paclitaxel-treated mice with squamous cell carcinoma of the head and neck.

Jaglowski JR, Zagon IS, Stack BC Jr, Verderame MF, Leure-duPree AE, Manning JD, McLaughlin PJ.

Department of Neural and Behavioral Sciences H109, The Milton S. Hershey Medical Center, Pennsylvania State University, Hershey, PA 17033, USA.

Paclitaxel is used as a single agent, and in combination with other drugs, as a standard of care in the treatment of squamous cell carcinoma of the head and neck (SCCHN). However, the use of paclitaxel for therapy of SCCHN may be accompanied by serious side effects. Paclitaxel is a known cytotoxic inhibitor of cell proliferation that acts by stabilizing microtubules and inducing apoptosis. Opioid growth factor (OGF), [Met(5)]-enkephalin, is an endogenous peptide that has tonically active inhibitory effects on the growth of SCCHN in vitro and in vivo. OGF action is rapid, reversible, mediated by the nuclear-associated OGF receptor (OGFr), and is not cytotoxic (nor apoptotic related). The present study was designed to examine whether a combination of chemotherapy with paclitaxel and biotherapy with OGF is more effective than either agent alone in inhibiting tumor growth. Moreover, focus was placed on whether there are changes in the side effects known to occur with paclitaxel alone, following this combined therapy. Human SCC-1 cells, derived from a well differentiated SCCHN, were transplanted into athymic mice. The mice were randomized to receive intraperitoneal (i.p.) injections of sterile saline (controls), OGF (10 mg/kg, daily), paclitaxel (8 mg/kg, every other day), or both paclitaxel (8 mg/kg, every other day) and OGF (10 mg/kg, daily) beginning on the day of tumor inoculation. OGF, but not paclitaxel, delayed measurable and visible tumor appearance of mice with SCCHN. Treatment with paclitaxel, but not with other agents, had a marked effect on the body weights. Survival only was reduced in the paclitaxel group, with an average life span of 34.3+/-3.1 days recorded, in comparison to the 50-day survival (date of termination) for all other groups. Beginning after week 4 of tumor inoculation and drug treatment, the tumor weight of the paclitaxel/OGF group was significantly reduced from the control, OGF, and paclitaxel-exposed mice. The OGFr number of the SCCHN tumors was 2.1-fold greater in the animals exposed to OGF or paclitaxel, and elevated 38% in the paclitaxel/OGF group; significant differences from the control group were found for the OGF and paclitaxel groups. These data suggest that combined chemotherapy (i.e., paclitaxel) and biotherapy (OGF) provides a valuable alternative to the standard of care for SCCHN patients.

PMID: 15791460

Neuropeptides. 2005 Oct;39(5):495-505. Epub 2005 Sep 15.

Opioids and differentiation in human cancer cells.

Zagon IS, McLaughlin PJ.

Department of Neural and Behavioral Sciences, The Pennsylvania State University, College of Medicine, M.S. Hershey Medical Center, 500 University Drive, H109, Hershey, PA 17033, United States. This e-mail address is being protected from spambots. You need JavaScript enabled to view it

This study was designed to examine the role of opioids on cell differentiation, with an emphasis on the mechanism of opioid growth factor (OGF, [Met5]-enkephalin)-dependent growth inhibition. Three human cancer cell lines (SK-N-SH neuroblastoma and SCC-1 and CAL-27 squamous cell carcinoma of the head and neck), along with OGF and the opioid antagonist naltrexone (NTX) at a dosage (10(-6) M) known to repress or increase, respectively, cell replication, were utilized. The effects on differentiation (neurite formation, process lengths, betaIII-tubulin, involucrin) were investigated in cells exposed to OGF or NTX for up to 6 days. In addition, the influence of a variety of other natural and synthetic opioids on differentiation was examined. OGF, NTX, naloxone, [D-Pen2,5]-enkephalin, dynorphin A1-8, beta-endorphin, endomorphin-1 and -2, [D-Ala2, MePhe4, Glycol5]-enkephalin (DAMGO), morphine, and U69,593 at concentrations of 10(-6) M did not alter cell differentiation of any cancer cell line. In NTX-treated SK-N-SH cells, cellular area was increased 23%, and nuclear area was decreased 17%, from control levels; no changes in cell or nuclear area were recorded in OGF-exposed cells. F-actin concentration was increased 40% from control values in SK-N-SH cells subjected to NTX, whereas alpha-tubulin was decreased 53% in OGF-treated cells. These results indicate that the inhibitory or stimulatory actions of OGF and NTX, respectively, on cell growth in tissue culture are not due to alterations in differentiation pathways. However, exposure to OGF and NTX modified some aspects of cell structure, but this was independent of differentiation. The absence of effects on cancer cell differentiation by a variety of other opioids supports the previously reported lack of growth effects of these compounds.

PMID: 16169076

Cancer Chemother Pharmacol. 2005 Nov;56(5):510-20. Epub 2005 Jun 10.

Combination chemotherapy with gemcitabine and biotherapy with opioid growth factor (OGF) enhances the growth inhibition of pancreatic adenocarcinoma.

Zagon IS, Jaglowski JR, Verderame MF, Smith JP, Leure-Dupree AE, McLaughlin PJ.

Department of Neural and Behavioral Sciences, H109, Milton S. Hershey Medical Center, The Pennsylvania State University, Hershey, 17033, USA. This e-mail address is being protected from spambots. You need JavaScript enabled to view it

Gemcitabine is the standard of care for advanced pancreatic neoplasia, and exerts its effect through inhibition of DNA synthesis. However, gemcitabine has limited survival benefits. Opioid growth factor (OGF) is an autocrine-produced peptide that interacts with the nuclear receptor, OGFr, to inhibit cell proliferation but is not cytotoxic or apoptotic. The present study was designed to examine whether a combination of chemotherapy with gemcitabine and biotherapy with OGF is more effective than either agent alone in inhibiting pancreatic cancer growth in vitro and in vivo. The combination of OGF (10(-6) M) and gemcitabine (10(-8) M) reduced MIA PaCa-2 cell number from control levels by 46% within 48 h, and resulted in a growth inhibition greater than that of the individual compounds. OGF in combination with 5-fluorouracil also depressed cell growth more than either agent alone. The action of OGF, but not gemcitabine, was mediated by a naloxone-sensitive receptor, and was completely reversible. OGF, but no other endogenous or exogenous opioids, altered pancreatic cancer growth in tissue culture. The combination of OGF and gemcitabine also repressed the growth of another pancreatic cancer cell line, PANC-1. MIA PaCa-2 cells transplanted into athymic mice received 10 mg/kg OGF daily, 120 mg/kg gemcitabine every 3 days; 10 mg/kg OGF daily and 120 mg/kg gemcitabine every 3rd day, or 0.1 ml of sterile saline daily. Tumor incidence, and latency times to tumor appearance, of mice receiving combined therapy with OGF and gemcitabine, were significantly decreased from those of the control, OGF, and gemcitabine groups. Tumor volumes in the OGF, gemcitabine, and OGF/gemcitabine groups were markedly decreased from controls beginning on days 14, 12, and 8, respectively, after tumor cell inoculation. Tumor weight and tumor volume were reduced from control levels by 36-85% in the OGF and/or gemcitabine groups on day 45 (date of termination), and the group of mice exposed to a combination of OGF and gemcitabine had decreases in tumor size of 70% and 63% from the OGF or the gemcitabine alone groups, respectively. This preclinical evidence shows that combined chemotherapy (e.g. gemcitabine) and biotherapy (OGF) provides an enhanced therapeutic benefit for pancreatic cancer.

PMID: 15947928

Int J Oncol. 2006 Jun;28(6):1577-83.

Progression of squamous cell carcinoma of the head and neck is associated with down-regulation of the opioid growth factor receptor.

McLaughlin PJ, Zagon IS.

Department of Neural & Behavioral Sciences, H109, The Penn State University College of Medicine, Hershey, PA 17033, USA. This e-mail address is being protected from spambots. You need JavaScript enabled to view it

Opioid growth factor (OGF) is a native opioid peptide ([Met5]-enkephalin) that interacts with the OGF receptor (OGFr). OGF serves as a tonically active negative growth factor in neoplasia, and the OGF-OGFr axis contributes to the maintenance of an equilibrium in cell proliferation by targeting the cyclin-dependent inhibitory kinase pathway. To inquire whether the expression of OGFr is related to tumor progression, cell lines of human squamous cell carcinoma of the head and neck (SCCHN) were transplanted into nude mice, and small, medium, and large tumors were assessed for OGFr by receptor binding assays and quantitative immunohistochemistry, and for gene expression of OGFr mRNA. Large tumors had a reduction of 3- to 7-fold in OGFr binding sites relative to small tumors, and medium size tumors showed a progressive diminishment in OGF receptors that ranged between that of small and large neoplasias. Tumors with xenografts of three different cell lines of SCCHN, representing poorly- and well-differentiated cancers, exhibited similar results. Quantitative densitometric immunohistochemistry revealed data comparable to receptor binding assays. Receptor affinity and the gene expression of OGFr mRNA were unchanged in tumors of different sizes. These data demonstrate that OGFr is reduced in SCCHN with tumor progression and that translation/posttranslation of OGFr protein, but not transcriptional levels of the OGFr gene, is (are) involved. The attenuated levels of OGFr binding capacity may serve as a marker of SCCHN. These subnormal levels of OGFr may diminish the efficacy of the OGF-OGFr axis in maintaining cell proliferative activity, and contribute to more active cell replication. Gene therapy to reinstate more OGFr, and thus enhance OGFr function, could serve as a useful treatment for inhibiting tumor progression.

PMID: 16685459

Int J Oncol. 2006 Aug;29(2):489-94.

Opioid growth factor receptor is unaltered with the progression of human pancreatic and colon cancers.

Zagon IS, McLaughlin PJ.

Department of Neural and Behavioral Sciences, H109, The Penn State University College of Medicine, Hershey, PA 17033, USA. This e-mail address is being protected from spambots. You need JavaScript enabled to view it

Opioid growth factor (OGF) is an endogenous opioid peptide ([Met(5)]-enkephalin) that interacts with the OGF receptor (OGFr). OGF serves as a constitutively expressed and tonically active negative growth factor in neoplasia, and the OGF-OGFr axis contributes to the maintenance of an equilibrium in cell replication by targeting cyclin-dependent inhibitory kinase pathways. In a previous study, OGFr binding activity was found to decrease in concert with progression of human squamous cell carcinoma of the head and neck (SCCHN). To investigate the relationship of OGFr to advancement of human pancreatic and colon cancers, tumor cells were transplanted into nude mice, and small, medium, and large neoplasias were assessed for OGFr number and affinity by receptor binding analysis, and for gene expression of OGFr mRNA by Northern blot analysis. In addition, OGF levels were monitored in plasma. OGFr binding affinity and capacity, as well as transcriptional activity of OGFr, were not influenced by the size or state of differentiation of pancreatic or colon tumors. Plasma levels of OGF were 3.5- to 7.9-fold less in animals with pancreatic or colon cancers than in nude mice not receiving xenografts, and no differences in OGF values were recorded between small and large tumors. These data on human pancreatic and colon cancers, along with information in earlier studies on SCCHN, indicate that alterations in the OGF receptor are dependent on tumor type and that the integrity of the OGF-OGFr axis insofar as tumorigenesis needs to be evaluated for each type of neoplasm. This information will be relevant in the design of therapeutic modalities, the diagnosis and prognosis of neoplasia, as well as understanding of the processes and mechanisms of carcinogenesis.

PMID: 16820893

Int J Mol Med. 2007 Mar;19(3):421-8.

Overexpression of the opioid growth factor receptor downregulates cell proliferation of human squamous carcinoma cells of the head and neck.

McLaughlin PJ, Verderame MF, Hankins JL, Zagon IS.

Department of Neural and Behavioral Sciences, H109, The Milton S. Hershey Medical Center, The Pennsylvania State University College of Medicine, Hershey, PA 17033, USA. This e-mail address is being protected from spambots. You need JavaScript enabled to view it

The opioid growth factor (OGF) is a constitutively expressed negative growth regulator whose action is mediated by the OGF receptor (OGFr). The OGF-OGFr axis tonically regulates the growth of human squamous cell carcinoma of the head and neck (SCCHN). To examine the repercussions of amplifying OGFr in SCCHN, constructs were prepared to overexpress OGFr in SCC-1 cells; six clonal lines were examined. OGFr binding assays of clonal cells revealed a 2.4- to 8.4-fold increase in binding capacity compared to wild-type (WT) and empty vector (EV) controls; binding affinity was comparable in all groups. OGFr protein expression, as measured by quantitative immunohistochemistry and Western blotting, was increased in clonal cell lines compared to controls. Under standard growth conditions the cell number of the OGFr clonal lines was reduced by 11 to 68% from the WT group, and doubling times were 7 to 67% longer. Addition of exogenous OGF further reduced (8 to 37%) cell growth of the clonal lines. Depletion of endogenous OGF with antibodies to this peptide increased growth 2-fold in cells amplifying OGFr relative to increases of 32 and 34% for the WT and EV groups, respectively. DNA synthesis of cells overexpressing OGFr was reduced from the WT group by 46 to 75%. These data indicate that the OGF receptor is integral to cell replication of SCCHN, and support treatment modalities that amplify OGFr in order to decrease the growth of these neoplasias.

PMID: 17273790

Int J Oncol. 2007 Apr;30(4):775-83.

Overexpression of the opioid growth factor receptor potentiates growth inhibition in human pancreatic cancer cells.

Zagon IS, Verderame MF, Hankins J, McLaughlin PJ.

Department of Neural and Behavioral Sciences, The Pennsylvania State University College of Medicine, H109, The Milton S. Hershey Medical Center, Hershey, PA 17033, USA. This e-mail address is being protected from spambots. You need JavaScript enabled to view it

The opioid growth factor (OGF), [Met5]-enkephalin, and OGF receptor (OGFr) form an inhibitory axis regulating the growth of human pancreatic cancer. This study examined whether overexpression of OGFr decreases the growth of pancreatic cells in vitro. MIA PaCa-2 cells were transfected with OGFr cDNA, and six clonal lines were examined for protein expression and function. OGFr binding assays revealed a 2.3- to 5.6-fold increase in binding capacity from wild-type (WT) and empty vector (EV) controls; binding affinity was comparable in all groups. OGFr protein expression, as measured by immunohistochemistry and Western blotting, was enhanced in clonal cell lines compared to controls. Doubling times of OGFr clonal lines were 47-91% longer than in the WT/EV groups for all but one clonal line. DNA synthesis of cells overexpressing OGFr was diminished from the WT/EV groups by 28-52%. Addition of exogenous OGF further reduced (14-31%) the cell growth of clonal lines, and the effects of exogenous OGF were receptor-mediated. Exposure of cells overexpressing OGFr to naltrexone increased the cell number by up to 9.4-fold. OGF was identified as the only opioid peptide to depress cell replication in the transfected cell lines. Neutralization of endogenous OGF with antibodies to this peptide elevated the cell number in clonal cell lines. These data identify OGFr at the molecular level as integral to regulating the cell replication of human pancreatic cancer, and support treatment modalities that amplify OGFr in order to decrease the growth of these neoplasias.

PMID: 17332915

Cancer Res. 2007 Nov 1;67(21):10511-8.

The opioid growth factor (OGF)-OGF receptor axis uses the p16 pathway to inhibit head and neck cancer.

Cheng F, Zagon IS, Verderame MF, McLaughlin PJ.

Department of Neural and Behavioral Sciences, The Pennsylvania State University College of Medicine, Hershey, PA 17033, USA.

Head and neck squamous cell carcinoma (HNSCC) represents 5.5% of malignancies worldwide, with approximately 30,000 new cases and approximately 11,000 deaths reported in the United States annually. The opioid growth factor (OGF; [Met(5)]-enkephalin) and the OGF receptor (OGFr) form an endogenous growth regulating system; the OGF-OGFr axis influences the G(0)-G(1) phase of the cell cycle in HNSCC. Cells treated with small interfering RNA (siRNA) for OGFr no longer responded to the growth inhibitory effects of OGF or the growth stimulatory effects of naltrexone, indicating that these activities are entirely mediated by OGFr. In this investigation, we examined the precise target of OGF in the cell cycle. Using SCC-1 cells, OGF decreased the phosphorylation of retinoblastoma protein. This change was correlated with reduced Cdk4, but not Cdk2, kinase activity. OGF treatment increased cyclin-dependent kinase inhibitor p16 protein expression. Importantly, p16 complexed with Cdk4 was increased by OGF treatment at all time points, consistent with the hypothesis that OGF mediated growth inhibition through p16. Blockade of OGF-OGFr interactions with naloxone abolished the increased expression of p16 protein by OGF. Inhibition of p16 (INK4a) activation by p16-specific siRNA blocked OGF's repressive action on proliferation of SCC-1, CAL-27, and SCC-4 HNSCC cells. These data are the first to reveal that the target of cell proliferative inhibitory action of OGF in human HNSCC is a cyclin-dependent kinase inhibitory pathway, and this may be useful in the diagnosis and treatment of HNSCC.

PMID: 17974995

Neuropeptides. 2007 Dec;41(6):441-52. Epub 2007 Oct 1.

Opioids and migration, chemotaxis, invasion, and adhesion of human cancer cells.

Zagon IS, Rahn KA, McLaughlin PJ.

Department of Neural and Behavioral Sciences, The Pennsylvania State University, College of Medicine, Hershey, PA 17033, United States. This e-mail address is being protected from spambots. You need JavaScript enabled to view it

This study was designed to examine the role of opioids on cell migration, chemotaxis, invasion, and adhesion, with an emphasis on whether the opioid growth factor (OGF, [Met(5)]-enkephalin) or the opioid antagonist naltrexone (NTX) impacts any or all of these processes. Drug concentrations of OGF and NTX known to depress or stimulate, respectively, cell proliferation and growth were analyzed. Three different human cancers (pancreatic, colon, and squamous cell carcinoma of the head and neck), represented by seven different cancer cell lines (PANC-1, MIA PaCa-2, BxPC-3, CAL-27, SCC-1, HCT-116, and HT-29), were evaluated. In addition, the influence of a variety of other natural and synthetic opioids on cell motility, invasion, and adhesion was assessed. Positive and negative controls were included for comparison. OGF and NTX at concentrations of 10(-4) to 10(-6)M, and dynorphin A1-8, beta-endorphin, endomorphin-1, endomorphin-2, leucine enkephalin, [D-Pen(2,5)]-enkephalin (DPDPE), [D-Ala(2), MePhe(4), Glycol(5)]-enkephalin (DAMGO), morphine, and U69,593 at concentrations of 10(-6)M, did not alter cell migration, chemotaxis, or invasion of any cancer cell line. OGF and NTX at a concentration of 10(-6)M, and incubation for 24 or 72h, did not change adhesion of these cancer cells to collagen I, collagen IV, fibronectin, laminin, or vitronectin. Moreover, all other opioids tested at 10(-6)M concentrations and for 24h had no effect on adhesion. These results indicate that the inhibitory or stimulatory actions of OGF and NTX, respectively, on cell replication and growth are independent of cell migration, chemotaxis, invasion, and adhesive properties. Moreover, a variety of other exogenous and endogenous opioids, many specific for the micro, delta, or kappa opioid receptors, also did not alter these biological processes, consonant with previous observations of a lack of effects of these compounds and their receptors on the biology of cancer cells.

PMID: 17910895

Mol Cancer. 2008 Jan 11;7:5.

The OGF-OGFr axis utilizes the p21 pathway to restrict progression of human pancreatic cancer.

Cheng F, McLaughlin PJ, Verderame MF, Zagon IS.

Department of Neural and Behavioral Sciences, The Pennsylvania State University College of Medicine, Hershey, PA, USA. This e-mail address is being protected from spambots. You need JavaScript enabled to view it

BACKGROUND: Pancreatic cancer is the 4th leading cause of death from cancer in the U.S. The opioid growth factor (OGF; [Met5]-enkephalin) and the OGF receptor form an inhibitory growth regulatory system involved in the pathogenesis and treatment of pancreatic cancer. The OGF-OGFr axis influences the G0/G1 phase of the cell cycle. In this investigation, we elucidate the pathway of OGF in the cell cycle. RESULTS: Using BxPC-3 cells, OGF decreased phosphorylation of retinoblastoma (Rb) protein without changing total Rb. This change was correlated with reduced cyclin-dependent kinase protein (Cdk) 2 kinase activity, but not total Cdk2. OGF treatment increased cyclin-dependent kinase inhibitor (CKI) p21 protein expression in comparison to controls, as well levels of p21 complexed with Cdk2. Naloxone abolished the increased expression of p21 protein by OGF, suggesting a receptor-mediated activity. p21 specific siRNAs blocked OGF's repressive action on proliferation in BxPC-3, PANC-1, and Capan-2 cells; cells transfected with negative control siRNA had no alteration in p21 expression, and therefore were inhibited by OGF. CONCLUSION: These data are the first to reveal that the target of cell proliferative inhibitory action of OGF in human pancreatic cancer is a p21 CKI pathway, expanding strategies for diagnosis and treatment of these neoplasias.

PMCID: PMC2253554

PMID: 18190706

Int J Oncol. 2008 Aug;33(2):317-23.

Prevention and delay in progression of human pancreatic cancer by stable overexpression of the opioid growth factor receptor.

Zagon IS, Kreiner S, Heslop JJ, Conway AB, Morgan CR, McLaughlin PJ.

Department of Neural and Behavioral Sciences, The Pennsylvania State University College of Medicine, Hershey, PA 17033, USA. This e-mail address is being protected from spambots. You need JavaScript enabled to view it

This study examined overexpression of the opioid growth factor receptor (OGFr) in pancreatic cancer cells and phenotypic changes in tumorigenicity. Tumors of MIA PaCa-2 cells transfected with OGFr cDNA (OGFr-1) had 3.3 times more OGFr than empty vector (EV) neoplasias, and 4.3 times more OGFr than tumors from wild-type (WT) mice. No differences in OGFr binding were detected between tumors of EV and WT animals. Tumor incidence in OGFr-1 animals was reduced by up to 50% from EV mice. Latency times for OGFr-1 tumor expression were increased 30%, tumor volume was decreased 70%, and DNA synthesis was reduced 24% relative to EV mice. Exogenous OGF reduced OGFr-1 tumor volume up to 55% compared to OGFr-1 mice given vehicle. These data support OGFr gene function as a regulator of cell proliferation that impacts on tumorigenic expression, and suggest that molecular and pharmacological manipulation of OGFr may prevent or delay human pancreatic cancer.

PMID: 18636152

Exp Biol Med (Maywood). 2008 Aug;233(8):968-79. Epub 2008 May 14.

Imiquimod upregulates the opioid growth factor receptor to inhibit cell proliferation independent of immune function.

Zagon IS, Donahue RN, Rogosnitzky M, McLaughlin PJ.

Department of Neural and Behavioral Sciences, H109, The M.S. Hershey Medical Center, 500 University Drive, Rm. C3729, Hershey, PA 17033, USA. This e-mail address is being protected from spambots. You need JavaScript enabled to view it

The imidazoquinoline compounds imiquimod and resiquimod are low-molecular-weight immune response modifiers that have potent anti-viral and anti-tumor properties. The mechanism by which they exert their effects remains unclear. Using pancreatic and colorectal cancer cells, as well as squamous carcinoma cells of the head and neck in tissue culture, which eliminated the immune system and toll-like receptors, we show that the imidazoquinolines upregulate the Opioid Growth Factor receptor (OGFr), which in turn stimulates the interaction of the OGF-OGFr axis. This native, tonically active inhibitory pathway regulates cell proliferation by modulating cyclin dependent kinase inhibitors, resulting in a retardation of cells at the G(1)-S interface of the cell cycle. Neutralization of OGF or knockdown of OGFr by siRNA technology eliminates the inhibitory effects of imidazoquinolines on cell replication. This exciting new knowledge of the mechanism of imidazoquinolines has important physiological relevance, and allows strategies to be developed for the use of these agents that will enhance effectiveness as well as attenuate side-effects.

PMID: 18480416

Thyroid. 2008 Nov;18(11):1165-70.

Expression of opioid growth factor (OGF)-OGF receptor (OGFr) axis in human nonmedullary thyroid cancer.

Goldenberg D, Zagon IS, Fedok F, Crist HS, McLaughlin PJ.

Division of Otolaryngology, Department of Surgery, H091, College of Medicine, The Penn State University, Hershey, Pennsylvania 17033, USA. This e-mail address is being protected from spambots. You need JavaScript enabled to view it

BACKGROUND: Although thyroid cancers are readily treatable with surgery and radioactive iodine, there are problems in managing recurring, as well as locally advanced, thyroid cancer. The opioid growth factor (OGF) and its receptor, OGF receptor (OGFr), form a tonically active, autocrine-paracrine loop that serves to inhibit cell proliferation in a wide variety of normal and abnormal cells and tissues. In the present study we examined the presence and distribution of OGF and OGFr in nonmedullary thyroid cancer, including papillary, follicular, and anaplastic, as well as thyroid tissue from patients with nonmalignant disease. METHODS: Patient samples of thyroid cancers and goiter were collected at the time of resection and processed for immunohistochemistry of OGF and OGFr, as well as pharmacological binding assays for OGFr. RESULTS: Both peptide and receptor were detected in the cytoplasm and nucleus of all nonmedullary thyroid cancers, as well as in goiter. Specific and saturable binding of OGFr was found in all thyroid samples. CONCLUSIONS: The finding that a potent negative growth regulator and its receptor are present in nonmedullary thyroid cancers and thyroid tissues from patients with nonmalignant disease lead us to suggest that the OGF-OGFr axis serves as a regulator of cell proliferation in these tissues. Moreover, modulation of this biological system may be used to treat progression of nonmedullary thyroid neoplasias.

PMID: 19014324

Mol Biol Cell. 2009 Jan;20(1):319-27. Epub 2008 Oct 15.

The OGF-OGFr axis utilizes the p16INK4a and p21WAF1/CIP1 pathways to restrict normal cell proliferation.

Cheng F, McLaughlin PJ, Verderame MF, Zagon IS.

Department of Neural and Behavioral Sciences, and Department of Medicine, The Pennsylvania State University College of Medicine, Hershey, PA 17033, USA.

Opioid growth factor (OGF) is an endogenous opioid peptide ([Met(5)]enkephalin) that interacts with the OGF receptor (OGFr) and serves as a tonically active negative growth factor in cell proliferation of normal cells. To clarify the mechanism by which OGF inhibits cell replication in normal cells, we investigated the effect of the OGF-OGFr axis on cell cycle activity in human umbilical vein endothelial cells (HUVECs) and human epidermal keratinocytes (NHEKs). OGF markedly depressed cell proliferation of both cell lines by up to 40% of sterile water controls. Peptide treatment induced cyclin-dependent kinase inhibitor (CKI) p16(INK4a) protein expression and p21(WAF1/CIP1) protein expression in HUVECs and NHEKs, but had no effect on p15, p18, p19, or p27 protein expression in either cell type. Inhibition of either p16(INK4a) or p21(WAF1/CIP1) activation by specific siRNAs blocked OGF inhibitory action. Human dermal fibroblasts and mesenchymal stem cells also showed a similar dependence of OGF action on p16(INK4a) and p21(WAF1/CIP1). Collectively, these results indicate that both p16(INK4a) and p21(WAF1/CIP1) are required for the OGF-OGFr axis to inhibit cell proliferation in normal cells.

PMCID: PMC2613082

PMID: 18923142

Exp Biol Med (Maywood). 2009 May;234(5):532-41. Epub 2009 Feb 25.

Dependence on nuclear localization signals of the opioid growth factor receptor in the regulation of cell proliferation.

Cheng F, McLaughlin PJ, Verderame MF, Zagon IS.

Department of Neural and Behavioral Sciences, The Pennsylvania State University College of Medicine, H109, The Milton S. Hershey Medical Center, 500 University Drive, Room C3729, Hershey, PA 17033, USA.

The opioid growth factor receptor (OGFr) mediates the inhibitory action of OGF on cell replication of normal and neoplastic cells. The spatiotemporal course of OGFr nucleocytoplasmic trafficking was determined with a probe of full-length OGFr fused to enhanced green fluorescent protein (eGFP). Translation of OGFr required 8.5 hours, and transit into the nucleus required 8 hours; OGFr remained in the nucleus for 8 days. OGFr was initially expressed on the outer nuclear envelope, transited to the paranuclear cytoplasm, and into the nucleus. Transport through the nuclear pore was elucidated by mutation of the nuclear localization signal (NLS) sequences in full-length OGFr. Mutation of each NLS reduced nuclear localization by 5%-50%, whereas simultaneous mutation of NLS383-386 and NLS456-460 abolished OGFr-eGFP nuclear localization in 80% of the cells. To determine whether intact NLSs are important for the inhibition of cell proliferation, DNA synthesis was monitored with BrdU. Wild-type OGFr-eGFP-transfected cells had 20% BrdU-positive cells, whereas cells with simultaneous mutation of all three NLS sites had a 70% labeling index. These results indicate that the regulation of cell proliferation by the OGF-OGFr axis is dependent on nucleocytoplasmic translocation and reliant on the integrity of two NLSs in OGFr to interact with transport receptors.

PMID: 19244545

Am J Physiol Regul Integr Comp Physiol. 2009 Jun;296(6):R1716-25. Epub 2009 Mar 18.

Cell proliferation of human ovarian cancer is regulated by the opioid growth factor-opioid growth factor receptor axis.

Donahue RN, McLaughlin PJ, Zagon IS.

Department of Neural and Behavioral Sciences, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, USA.

Ovarian cancer is the leading cause of death from gynecological malignancies. Understanding the biology of these tumors, as well as treatment modalities, has been challenging. The opioid growth factor (OGF; [Met(5)]-enkephalin) and the OGF receptor (OGFr) form an endogenous growth-regulating pathway in homeostasis and neoplasia. In this investigation, we examined the relationship of the OGF-OGFr axis to ovarian cancer, and defined its presence, function, and mechanisms. Using OVCAR-3 and SKOV-3 ovarian cancer cell lines, we found that OGF and OGFr were present and functional. Exogenous OGF was observed to have a dose-dependent, serum-independent, reversible, and receptor-mediated inhibitory action on cell proliferation that was dependent on RNA and protein synthesis. The repressive effect of OGF on cell proliferation also was observed in SW626, CAOV-3, and HEY ovarian cancer cell lines. Endogenous OGF was found to be constitutively produced and tonically active on cell replicative activities, with neutralization of this peptide accelerating cell proliferation. Silencing of OGFr using siRNA technology stimulated cell replication, documenting its integral role. The mechanism of OGF-OGFr action on DNA synthesis was related to the cyclin-dependent kinase inhibitory pathway because knockdown of p16 or p21 in OVCAR-3 cells, and p21 in SKOV-3 cells, eliminated OGF's inhibitory effect on growth. These data are the first to report that the OGF-OGFr system is a native biological regulator of cell proliferation in human ovarian cancer. This information will be important in designing treatment strategies for this deadly disease.

PMID: 19297547

Brain Res Bull. 2009 Aug 14. [Epub ahead of print]

Diabetic keratopathy and treatment by modulation of the opioid growth factor (OGF)-OGF receptor (OGFr) axis with naltrexone: A review.

McLaughlin PJ, Sassani JW, Klocek MS, Zagon IS.

Department of Neural & Behavioral Sciences, The Pennsylvania State University College of Medicine, Hershey, PA, United States.

The opioid growth factor (OGF)-OGF receptors (OGFr) axis plays an important role in the homeostasis and re-epithelialization of the mammalian cornea. This tonically active growth regulatory inhibitory pathway is involved in cell replication, and the endogenous neuropeptide OGF targets cyclin-dependent kinase inhibitors, p16 and/or p21. Blockade of OGF-OGFr interfacing by systemic or topical administration of opioid antagonists such as naltrexone (NTX) results in accelerated DNA synthesis, cell replication, and tissue repair. Molecular manipulation of OGFr using sense constructs delayed corneal re-epithelialization, whereas antisense constructs accelerated repair of the corneal surface. Corneal keratopathy, a significant complication of diabetes mellitus, is manifested by delays in corneal re-epithelialization following surgery, injury, or disease. Tissue culture studies have shown that addition of NTX stimulates DNA synthesis and explant outgrowth of rabbit corneal epithelium, whereas OGF depresses DNA synthesis and explant outgrowth in a receptor-mediated manner. NTX accelerated corneal re-epithelialization in organ cultures of human and rabbit cornea. Systemic application of NTX to the abraded corneas of rats, and topical administration of NTX to the injured rabbit ocular surface, increased re-epithelialization. Systemic injections or topical administration of NTX facilitates re-epithelialization of the cornea in diabetic rats. Given the vital role of the corneal epithelium in maintaining vision, the frequency of corneal complications related to diabetes (diabetic keratopathy), and the problems occurring in diabetic individuals postoperatively (e.g., vitrectomy), and that conventional therapies such as artificial tears and bandage contact lenses often fail, topical application of NTX merits clinical consideration.

PMID: 19683562

Am J Physiol Regul Integr Comp Physiol. 2009 Sep;297(3):R844-52. Epub 2009 Jul 15.

Passive diffusion of naltrexone into human and animal cells and upregulation of cell proliferation.

Cheng F, McLaughlin PJ, Banks WA, Zagon IS.

Department of Neural and Behavioral Sciences, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, USA.

Naltrexone (NTX) is a potent opioid antagonist that promotes cell proliferation by upregulating DNA synthesis through displacement of the tonically active inhibitory peptide, opioid growth factor (OGF) from its receptor (OGFr). To investigate how NTX enters cells, NTX was fluorescently labeled [1-(N)-fluoresceinyl NTX thiosemicarbazone; FNTX] to study its uptake by living cultured cells. When human head and neck squamous cell carcinoma cell line (SCC-1) was incubated with FNTX for as little as 1 min, cells displayed nuclear and cytoplasmic staining of FNTX as determined by fluorescent deconvolution microscopy, with enrichment of fluorescent signal in the nucleus and nucleolus. The same temporal-spatial distribution of FNTX was detected in a human pancreatic cancer cell line (MIA PaCa-2), African green monkey kidney cell line (COS-7), and human mesenchymal stem cells (hMSCs). FNTX remained in cells for as long as 48 h. FNTX was internalized in SCC-1 cells when incubation occurred at 4 degrees C, with the signal being comparable to that recorded at 37 degrees C. A 100-fold excess of NTX or a variety of other opioid ligands did not alter the temporal-spatial distribution of FNTX. Neither fluorescein-labeled dextran nor fluorescein alone entered the cells. To study the effect of FNTX on DNA synthesis, cells incubated with FNTX at concentrations ranging from 10(-5) to 10(-8) M had a 5-bromo-2'-deoxyuridine index that was 39-82% greater than for vehicle-treated cells and was comparable to that of unlabeled NTX (37-70%). Taken together, these results suggested that NTX enters cells by passive diffusion in a nonsaturable manner.

PMID: 19605761

Am J Physiol Regul Integr Comp Physiol. 2009 Oct;297(4):R1154-61. Epub 2009 Aug 12.

Opioid growth factor-opioid growth factor receptor axis is a physiological determinant of cell proliferation in diverse human cancers.

Zagon IS, Donahue RN, McLaughlin PJ.

Department of Neural and Behavioral Sciences, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania, USA. This e-mail address is being protected from spambots. You need JavaScript enabled to view it

The opioid growth factor (OGF) regulates cell proliferation of human cancer cells through the cyclin-dependent kinase inhibitory pathway, with mediation of this action by the OGF receptor (OGFr). The ubiquity of the OGF-OGFr axis in human cancer is unknown. We used 31 human cancer cell lines, representative of more than 90% of neoplasias occurring in humans, and found that OGF and OGFr were detected in the cytoplasm and nucleus by immunohistochemistry. The addition of OGF to cultures depressed cell number up to 41%, whereas naltrexone (NTX) increased cell proliferation by up to 44%, a total of 85% in the modulating capacity for the OGF-OGFr axis. Neutralization of OGF by specific antibodies led to a marked increase in cell number. Knockdown of OGFr by OGFr-siRNA resulted in a significant increase in the number of cells, even in the face of the addition of exogenous OGF. The cultures to which NTX was added and subjected to OGFr-siRNA were similar to those with OGF-siRNA alone. The OGF-OGFr axis, a physiological determinant of cell-proliferative activity, is a ubiquitous feature of human cancer cells. The identification of this native biological system in neoplasia may be important in understanding the pathophysiology of neoplasia, and in designing treatment modalities that utilize the body's own chemistry.

PMID: 19675283

BMC Cancer. 2009 Oct 18;9:369.

Growth inhibition of thyroid follicular cell-derived cancers by the opioid growth factor (OGF) - opioid growth factor receptor (OGFr) axis.

McLaughlin PJ, Zagon IS, Park SS, Conway A, Donahue RN, Goldenberg D.

Department of Neural and Behavioral Sciences, The Milton S, Hershey Medical Center, The Pennsylvania State University, Hershey, Pennsylvania, USA. This e-mail address is being protected from spambots. You need JavaScript enabled to view it

BACKGROUND: Carcinoma of the thyroid gland is an uncommon cancer, but the most frequent malignancy of the endocrine system. Most thyroid cancers are derived from the follicular cell. Follicular carcinoma (FTC) is considered more malignant than papillary thyroid carcinoma (PTC), and anaplastic thyroid cancer (ATC) is one of the most lethal human cancers. Opioid Growth Factor (OGF; chemical term - [Met5]-enkephalin) and its receptor, OGFr, form an inhibitory axis regulating cell proliferation. Both the peptide and receptor have been detected in a wide variety of cancers, and OGF is currently used clinically as a biotherapy for some non-thyroid neoplasias. This study addressed the question of whether the OGF-OGFr axis is present and functional in human thyroid follicular cell - derived cancer. METHODS: Utilizing human ATC (KAT-18), PTC (KTC-1), and FTC (WRO 82-1) cell lines, immunohistochemistry was employed to ascertain the presence and location of OGF and OGFr. The growth characteristics in the presence of OGF or the opioid antagonist naltrexone (NTX), and the specificity of opioid peptides for proliferation of ATC, were established in KAT-18 cells. Dependence on peptide and receptor were investigated using neutralization studies with antibodies and siRNA experiments, respectively. The mechanism of peptide action on DNA synthesis and cell survival was ascertained. The ubiquity of the OGF-OGFr axis in thyroid follicular cell-derived cancer was assessed in KTC-1 (PTC) and WRO 82-1 (FTC) tumor cells. RESULTS: OGF and OGFr were present in KAT-18 cells. Concentrations of 10-6 M OGF inhibited cell replication up to 30%, whereas NTX increased cell growth up to 35% relative to cultures treated with sterile water. OGF treatment reduced cell number by as much as 38% in KAT-18 ATC in a dose-dependent and receptor-mediated manner. OGF antibodies neutralized the inhibitory effects of OGF, and siRNA knockdown of OGFr negated growth inhibition by OGF. Cell survival was not altered by OGF, but DNA synthesis as recorded by BrdU incorporation was depressed by 28% in OGF-treated cultures compared to those exposed to sterile water. The OGF-OGFr axis was detected and functional in PTC (KTC-1) and FTC (WRO 82-1) cell lines. CONCLUSION: These data suggest that OGF and OGFr are present in follicular-derived thyroid cancers, and that OGF serves in a tonically active inhibitory manner to maintain homeostasis of cell proliferation. These results may provide a biotherapeutic strategy in the treatment of these cancers.

PMCID: PMC2770570

PMID: 19835629

Brain Res. 2009 Nov 18. [Epub ahead of print]

Opioid growth factor suppresses expression of experimental autoimmune encephalomyelitis.

Zagon IS, Rahn KA, Bonneau RH, Turel AP, McLaughlin PJ.

Department of Neural and Behavioral Sciences, The Pennsylvania State University College of Medicine, Hershey, PA, USA.

Naltrexone, an opioid antagonist, has been shown to modulate expression of experimental autoimmune encephalomyelitis (EAE), an animal model of MS, suggesting that endogenous opioids are inhibitory trophic factors in EAE. In the present study, we investigated the effects of one native opioid peptide, opioid growth factor ([Met(5)]-enkephalin), on the onset and progression of EAE. C57Bl/6 mice injected with myelin oligodendrocyte glycoprotein (MOG) received daily injections of 10 mg/kg OGF (MOG+OGF) or saline (MOG+Vehicle). Over 60% of the MOG+OGF animals did not exhibit behavioral signs of disease (EAE) in contrast to 100% of the mice in the MOG+Vehicle group. The severity and disease indices of EAE in the OGF-treated mice were markedly reduced from MOG+Vehicle cohorts. By day 30, 60% of MOG+OGF mice had a remission, relative to 4% in the MOG+Vehicle group. MOG-injected mice receiving OGF had significant reductions in activated astrocytes and damaged neurons compared to MOG+Vehicle animals. Unlike MOG+Vehicle and MOG+OGF mice with behavioral signs of disease, MOG+OGF animals without manifestation of disease had no lumbar spinal cord demyelination. Both OGF and OGF receptor were detected in splenic-derived T lymphocytes by immunohistochemistry. OGF treatment decreased both DNA synthesis and cell proliferation in comparison to vehicle-treated T cell lymphocyte cultures. These results indicate that an endogenous opioid, OGF, inhibits the onset and progression of EAE, and suggest that clinical studies on the use of OGF treatment for MS are merited.

PMID: 19931226

Am J Physiol Regul Integr Comp Physiol. 2009 Nov 18. [Epub ahead of print]

The Opioid Growth Factor - Opioid Growth Factor Receptor Axis Regulates Cell Proliferation of Human Hepatocellular Cancer.

Avella DM, Kimchi ET, Donahue RN, Tagaram HR, McLaughlin PJ, Staveley-O'Carroll KF, Zagon IS.

The Pennsylvania State Univesity, College of Medicine.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer deaths worldwide, with a mortality rate approximately its incidence. Understanding the biology of these tumors, as well as treatment modalities, has been challenging. The opioid growth factor (OGF; [Met(5)]-enkephalin) and the OGF receptor (OGFr) form an endogenous growth regulating pathway in homeostasis and in neoplasia. In this investigation we examined the relationship of the OGF-OGFr axis in HCC, and define its presence, function, and mechanism. Using SK-HEP-1, Hep G2, and Hep 3B human HCC cell lines, we found that OGF and OGFr were present and functional. Exogenous OGF was observed to have a dose-dependent, reversible, and receptor-mediated inhibitory action on cell proliferation. Endogenous OGF was found to be constitutively produced and tonically active on cell replicative activities, with neutralization of this peptide accelerating cell proliferation. Silencing of OGFr using siRNA stimulated cell replication, even when exogenous OGF was added to the cultures, documenting its importance in mediating OGF activity. The mechanism of OGF-OGFr action on cell number was related to inhibition of DNA synthesis, and not to apoptotic or necrotic pathways. Both OGF and OGFr were detected in surgical specimens of HCC, and no quantitative differences were recorded in peptide or receptor between pathological and normal specimens. These data are the first to report that the OGF-OGFr system is a native biological regulator of cell proliferation in HCC. The findings may provide important insight in designing treatment strategies for this deadly disease. Key words: Hepatocellular Carcinoma, Cell Proliferation, Tissue Culture, siRNA.

PMID: 19923357

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