Opioid Growth Factor Receptor Axis Research Articles

Biology of the Opioid Growth Factor – Opioid Growth Factor Receptor Axis: Bench to Bedside and Back

The Opioid Growth Factor – Opioid Growth Factor Receptor axis was identified nearly 30 years ago in our laboratory when we demonstrated that an endogenous opioid pentapeptide had inhibitory growth activity following injection into normal animals. In addition to validating this action for the peptide, identification, characterization and cloning of the specific receptor related to the opioid growth factor, chemically termed [Met5]-enkephalin, revealed that the receptor was unique in its biochemical and molecular structure, but had pharmacological properties similar to other opioid receptors. Mechanisms associated with binding of the opioid growth factor to its receptor located on the outer nuclear envelop and the transport into the nucleus were identified. The selectively of the peptide for the receptor was demonstrated in normal and cancer cell lines. This uniqueness of the peptide-receptor interaction was evidenced by the duration of receptor blockade in that intermittent blockade resulted in decreased cell replication and complete blockade resulted in accelerated proliferation. Investigations on the mechanism of action, as well as the dysregulation of the Opioid Growth Factor – Opioid Growth Factor Receptor axis in human pathology, have resulted in bench to bedside and bedside to bench discoveries. Receptor blockade by naltrexone has been used clinically for treatment of fatigue and other immune deficiencies in many autoimmune disorders. The research has gone from bench to bedside and has returned to the bench for additional investigation of mechanisms driving the action of the axis and resultant function following receptor blockade.

Blockade of OGFr delays the onset and reduces the severity of diabetic ocular surface complications.

The opioid growth factor (OGF)-OGF receptor (OGFr) pathway is present in the ocular surface and functions to maintain homeostasis of the epithelium. The OGF-OGFr pathway has been reported to be dysregulated in diabetic individuals and animal models, and is reflected in elevations of the inhibitory growth factor, OGF, chemically termed [Met5]-enkephalin. Recently, our laboratory reported elevated levels of OGF and OGFr in the serum and corneal epithelium of type 1 diabetic rats, suggesting that dysregulation of the OGF-OGFr axis may lead to dry eye, abnormal corneal surface sensitivity, and delayed re-epithelialization. Blockade of OGF-OGFr pathway using naltrexone, a potent opioid receptor antagonist, reverses dry eye symptoms and restores corneal surface sensitivity in diabetic rats when used as a therapy. Based on the evidence that both OGF and OGFr are elevated in type 1 diabetic rats, this study examined whether systemic or topical naltrexone treatment initiated at the time of induction of hyperglycemia could protect against the development of diabetic ocular surface complications. Diabetic male Sprague-Dawley rats treated systemically or topically with naltrexone had a delayed onset of dry eye and altered corneal surface sensitivity, and an improved healing rate for corneal wounds, that were comparable to non-diabetic rats. Serum levels of OGF were normal for rats receiving systemic naltrexone, and OGF tissue levels were normal for type 1 diabetic rats receiving twice daily naltrexone drops. OGFr levels remained elevated. These data support the role of the OGF-OGFr axis in regulation of ocular surface complications, and suggest that naltrexone therapy may be beneficial for pre-diabetic and early diabetic individuals.

Opioid growth factor – opioid growth factor receptor axis inhibits proliferation of triple negative breast cancer

Triple negative breast cancer (TNBC) represents approximately 15% of the newly diagnosed cancers worldwide and is characterized by tissue lacking in estrogen, progesterone and human epidermal growth factor receptors. TNBC disproportionately affects younger women and women of colour, and new treatments are needed. The opioid growth factor (OGF) - opioid growth factor receptor (OGFr) axis is a determinant of cell proliferation in neoplasia, and OGF is an endogenously produced pentapeptide that inhibits cell replication by interacting with OGFr and upregulating cyclin-dependent inhibitory kinase pathways thus reducing DNA synthesis. In these studies we investigated the presence and function of the OGF-OGFr axis in two human TNBC cell lines, as well as in breast cancer cell lines containing hormonal receptors. TNBC cell lines MDA-MD-231 and BT-20, as well as human breast cancer cells SK-BR-3 and MCF-7, were examined for the presence of pentapeptide and receptors, as well as their response to OGF. Specificity of peptide and receptor was confirmed by antibody neutralization and molecular studies to knockdown classical receptor protein. The requirement for protein transcription and translation and RNA transcription were investigated. Growth of TNBC cells in the presence of OGF and standard of care chemotherapeutic agent paclitaxel was evaluated to determine both efficacy and protective effects against toxicity. OGF treatment inhibited TNBC cells in a dosage related, receptor mediated, and reversible manner. OGF was the specific endogenous opioid to inhibit cell proliferation, and this was mediated by p21 cyclin dependent inhibitory kinase pathways, and required protein and RNA synthesis. OGFr was the specific receptor involved; both peptide and receptor were detected in all four cell lines. OGF treatment inhibited growth of all cancer cell lines evaluated, and reduced cell death in cultures exposed to paclitaxel. The OGF-OGFr axis is present and functioning in TNBC cell lines, and provides a novel biological pathway as potential therapy.

Targeting the opioid growth factor: Opioid growth factor receptor axis for treatment of human ovarian cancer

The opioid growth factor (OGF) - opioid growth factor receptor (OGFr) axis is a biological pathway that is present in human ovarian cancer cells and tissues. OGF, chemically termed [Met(5)]-enkephalin, is an endogenous opioid peptide that interfaces with OGFr to delay cells moving through the cell cycle by upregulation of cyclin-dependent inhibitory kinase pathways. OGF inhibitory activity is dose dependent, receptor mediated, reversible, protein and RNA dependent, but not related to apoptosis or necrosis. The OGF-OGFr axis can be targeted for treatment of human ovarian cancer by (i) administration of exogenous OGF, (ii) genetic manipulation to over-express OGFr and (iii) use of low dosages of naltrexone, an opioid antagonist, which stimulates production of OGF and OGFr for subsequent interaction following blockade of the receptor. The OGF-OGFr axis may be a feasible target for treatment of cancer of the ovary (i) in a prophylactic fashion, (ii) following cytoreduction or (iii) in conjunction with standard chemotherapy for additive effectiveness. In summary, preclinical data support the transition of these novel therapies for treatment of human ovarian cancer from the bench to bedside to provide additional targets for treatment of this devastating disease.

The opioid growth factor–opioid growth factor receptor axis: Homeostatic regulator of cell proliferation and its implications for health and disease

The opioid growth factor (OGF), chemically termed [Met5]-enkephalin, is an endogenous opioid peptide that interacts with the OGF receptor (OGFr) to delay the G1/S interface of the cell cycle by modulating cyclin-dependent inhibitory kinase (CKI) pathways. The OGF–OGFr axis is a tonically active, inhibitory pathway that is an important regulator during homeostasis and re-epithelialization, and plays a role in the onset and progression of autoimmune diseases and cancer. Modulation of the OGF–OGFr axis can be accomplished by a variety of pharmacological and molecular approaches including use of intermittent or continuous exposure to the opioid antagonist naltrexone, genetic manipulation of OGFr expression, and antibody neutralization of OGF. Clinically, OGF is a biological therapy that has potential application for treatment of cancer. Currently, naltrexone at low dosages is being evaluated for treatment of autoimmune diseases such as Crohn's and multiple sclerosis. High dosages of naltrexone are effective in reversing dry eye and accelerating the repair of corneal abrasions in normal and diabetic rats; these studies are under investigation in the clinical setting. Naltrexone also enhances full-thickness wound closure in animal models of Type 1 or Type 2 diabetes, and translation of this knowledge to the clinic is planned. In summary, understanding the OGF–OGFr axis as a homeostatic regulator of proliferation has substantial implications for maintaining human health and treatment of disease.

Expression of the opioid growth factor–opioid growth factor receptor axis in human ovarian cancer

ObjectiveThe opioid growth factor (OGF) and its receptor (OGFr), serve as inhibitory axis regulating cell proliferation in normal cells and cancer. We investigated the presence and relative expression of OGF and OGFr in normal human ovarian surface epithelial (HOSE) cells, benign ovarian cysts, and ovarian cancers. MethodsSurgical samples of 16 patients with ovarian cancer and 27 patients with ovarian benign cysts were obtained intraoperatively. HOSE were collected by scraping the surface of normal ovaries of 10 post menopausal women undergoing hysterectomy and oophorectomy. Semiquantitative immunohistochemistry was used to assess the presence, distribution, and levels of OGF and OGFr. Receptor binding assays measured binding capacity and affinity of OGFr for radiolabeled OGF. ResultsOGF and OGFr were present in HOSE cells, ovarian cysts, and ovarian cancers. Compared to HOSE cells, OGF and OGFr protein levels were reduced 29% and 34% (p<0.001), respectively, in ovarian cysts, and decreased 58% and 48% (p<0.001), respectively, in ovarian cancers. Binding assays revealed 5.4 fold fewer OGFr binding sites in cancers than cysts (p<0.05). Levels of OGF and OGFr were comparable in primary, metastatic, or recurrent ovarian cancers. ConclusionWe have shown that a native opioid pathway, the OGF–OGFr axis, is present in human ovarian cancer. Importantly, the expression of OGF and OGFr is diminished in human ovarian cancer. As OGF and OGFr normally function in maintaining cell proliferation, therapy to harness OGF/OGFr function could provide a useful biologic-based treatment for human ovarian cancer.

Modulation of the opioid growth factor ([Met5]‐enkephalin)–opioid growth factor receptor axis: Novel therapies for squamous cell carcinoma of the head and neck

The opioid growth factor (OGF)-OGF receptor (OGFr) axis is a constitutively expressed biologic pathway regulating cell proliferation of squamous cell carcinoma of the head and neck (SCCHN). This study investigated modulation of the OGF-OGFr system by (1) exogenous OGF, (2) upregulation of OGFr using imiquimod, or (3) intermittent opioid receptor blockade with a low dose of naltrexone on progression of established SCCHN. Nude mice with visible human SCCHN SCC-1 tumors received (1) OGF or low-dose naltrexone either 1, 3, or 7 times/week or (2) imiquimod 1 or 3 times/week. Tumor growth and DNA synthesis were monitored. OGF and low-dose naltrexone increased the latency from visible to measurable tumors up to 1.6-fold. OGF, low-dose naltrexone, and imiquimod treatment markedly reduced tumor volume and weight, and decreased DNA synthesis in tumors. Modulation of the native OGF-OGFr regulatory network in SCCHN represents a novel nontoxic and highly efficacious approach for treatment of SCCHN.

Inhibition of DNA synthesis in mouse epidermis by topical imiquimod is dependent on opioid receptors

The imidazoquinolines are immune response modifiers that have potent antiviral and antitumor properties. The mechanism by which they exert their effects on cell replication has been investigated in vitro and is related to the upregulation of the opioid growth factor receptor (OGFr) and modulation of opioid growth factor (OGF; [Met(5)]-enkephalin). The OGF-OGFr axis regulates cell proliferative events through a cyclin-dependent kinase inhibitory pathway. The present study examined the mechanism whereby imiquimod repressed cell proliferation in vivo. Using a nude mouse model that has a compromised T-cell immune system, as well as C57BL/6 mice with an intact immune system, the effects of topical imiquimod (Aldara(®)) on DNA synthesis of basal epithelial cells in skin were examined. Imiquimod's effects on DNA synthesis were detected 24 h after application, and could be observed for one week after a single treatment. The magnitude of change in DNA synthesis following imiquimod was similar for one, three or six applications. Naloxone, an opioid antagonist, blocked the inhibitory effect of imiquimod. Imiquimod in combination with OGF or a low dose of naltrexone (LDN; known to upregulate the OGF-OGFr axis) had no greater inhibitory response on DNA synthesis than either OGF or LDN alone. Both OGF and OGFr were upregulated in basal epithelium after imiquimod treatment. Both nude and C57BL/6 mice exhibited the same repressive action of imiquimod on epithelial DNA synthesis. Imiquimod was neither an opioid agonist nor antagonist using nociceptive testing, and did not induce apoptosis or necrosis. Exposure to imiquimod was found to depress DNA synthesis in cells located in distant epithelium from day 3 and lasted until day 5. These results suggest that the target of imiquimod on DNA synthesis is dependent on an opioid receptor-mediated pathway, and infers that imiquimod is reliant on the OGF-OGFr axis for modulating cell proliferation.

T lymphocyte proliferation is suppressed by the opioid growth factor ([Met 5]-enkephalin)–opioid growth factor receptor axis: Implication for the treatment of autoimmune diseases

Opioid peptides function as immunomodulatory molecules. Reports have linked the opioid growth factor (OGF), [Met 5]-enkephalin, and its receptor OGFr to autoimmune diseases. OGF repressed the incidence and magnitude of myelin oligodendrocyte-induced experimental autoimmune encephalomyelitis in mice. Given the extensive connection between the immune system and autoimmune diseases, the present study was conducted to examine the relationship of the OGF–OGFr axis and T lymphocyte proliferation. Splenic-derived mouse lymphocytes were stimulated with phytohemagglutin (PHA). All non-stimulated and PHA-stimulated T lymphocytes had immunoreactivity for OGF-like enkephalin and OGFr. OGF markedly suppressed T lymphocyte number in a dose-dependent manner. However, PHA-stimulated T lymphocytes were not altered in cell number by a variety of natural and synthetic opioid-related compounds, some specific for μ, δ, and κ opioid receptors. Persistent blockade of opioid receptors with the general opioid antagonist naltrexone (NTX), as well as antibody neutralization of OGF-like peptides, had no effect on cell number. Non-stimulated T lymphocytes exhibited no change in cell number when subjected to OGF or NTX. Treatment of T lymphocytes with siRNAs for μ, δ, or κ opioid receptors did not affect cell number, and the addition of OGF to these siRNA-exposed cultures depressed the population of cells. T lymphocytes treated with OGFr siRNA also had a comparable number of cells to control cultures, but the addition of OGF did not alter cell number. DNA synthesis in PHA-stimulated T lymphocytes exposed to OGF was markedly decreased from PHA-stimulated cultures receiving vehicle, but the number of cells undergoing apoptosis or necrosis in these cultures was similar to control levels. T lymphocytes subjected to siRNA for p16 and/or p21 had a comparable number of cells compared to controls, and treatment with OGF did not depress cell number in preparations transfected with both p16 and p21 siRNA. These data reveal that the OGF–OGFr axis is present in T lymphocytes and is capable of suppressing cell proliferation. However, T lymphocytes are not dependent on the regulation of cell proliferation by this system. The results showing that the OGF–OGFr axis is an immunosuppressant, offers explanation for reports that autoimmune diseases can be modulated by this system.

B lymphocyte proliferation is suppressed by the opioid growth factor–opioid growth factor receptor axis: Implication for the treatment of autoimmune diseases

Endogenous opioids are known to repress the incidence and progression of autoimmune diseases. One native opioid peptide, [Met 5]-enkephalin, termed the opioid gowth factor (OGF), interacts with the OGF receptor (OGFr) to suppress the expression of experimental autoimmune encephalomyelitis. The present study examined the role of the OGF-OGFr axis in the regulation of B lymphocyte proliferation. Murine B lymphocytes were stimulated with lipopolysaccharide. Both OGF and OGFr were present in all B lymphocytes. OGF had a dose-dependent effect on growth, with cell number inhibited by up to 43% at 72 h; no other synthetic or native opioid altered cell proliferation. Exogenous OGF depressed cell number in cultures treated with siRNAs for the classical opioid receptors, MOR (μ), DOR (δ), and KOR (κ), however this peptide had no effect in preparations exposed to siRNA for OGFr. The decrease in cell number by exogenous OGF was dependent on p16 or p21 cyclin-dependent inhibitory kinase pathways. Exposure to the opioid antagonist, naltrexone, did not change cell number from control levels. These results suggest that the OGF-OGFr axis is present and functional in B lymphocytes, but this system is not an autocrine regulator of cell proliferation. Thus, at least exogenous OGF and perhaps endogenous OGF by paracrine/endocrine sources, can be an immunosuppressant. Modulation of the OGF-OGFr axis may be a novel paradigm for the treatment of autoimmune diseases.

Regulation of Tenon's Capsule Fibroblast Cell Proliferation by the Opioid Growth Factor and the Opioid Growth Factor Receptor Axis

Glaucoma filtration surgery often fails because of the fibrotic reaction from Tenon's capsule fibroblasts (TCFs). This study examined whether the interaction of the opioid growth factor (OGF) [Met(5)]-enkephalin with its receptor (OGFr) is a regulator of TCF proliferation. The presence of OGF and its receptor (OGFr) was determined in rabbit TCFs (RTCFs) by immunocytochemistry. The kinetics of OGFr were established in receptor binding assays. The ability of OGF to inhibit RTCF proliferation was assessed with dose-response, receptor mediation, and reversibility studies. Dependence on OGF and OGFr was ascertained by antibody neutralization and siRNA studies, respectively. The mechanism of action of the OGF-OGFr axis on survival (apoptosis, necrosis) and DNA synthesis of RTCFs was elucidated. OGF and OGFr were detected in RTCF cells, and specific and saturable binding to OGFr was recorded. Exogenous OGF had a dose-dependent, reversible, and receptor-mediated inhibitory effect on cell proliferation. Endogenous OGF was found to be constitutively produced and tonically active in cell replication, with neutralization of this peptide causing acceleration of cell proliferation. The silencing of OGFr by using siRNA technology stimulated cell replication, validating OGFr's integral role. The mechanism of OGF-OGFr action was not related to cell survival, but rather to DNA synthesis-specifically, the cyclin-dependent kinase inhibitory pathway. Knockdown of p16 or p21 eliminated OGF's inhibitory effect on growth. The OGF-OGFr system is a native biological regulator of cell proliferation in RTCFs and may offer a means of improving the success of glaucoma filtration surgery in a safe and nontoxic manner.

The Opioid Growth Factor (OGF)–OGF Receptor Axis Uses the p16 Pathway to Inhibit Head and Neck Cancer

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.