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In the natural killer (NK) cells, δ-opiate receptor (DOR) and μ-opioid receptor (MOR) interact in a feedback manner to regulate cytolytic function with an unknown mechanism. Using RNK16 cells, a rat NK cell line, we show that MOR and DOR monomer and dimer proteins existed in these cells and that chronic treatment with a receptor antagonist reduced protein levels of the targeted receptor but increased levels of opposing receptor monomer and homodimer. The opposing receptor-enhancing effects of MOR and DOR antagonists were abolished following receptor gene knockdown by siRNA. Ethanol treatment increased MOR and DOR heterodimers while it decreased the cellular levels of MOR and DOR monomers and homodimers. The opioid receptor homodimerization was associated with an increased receptor binding, and heterodimerization was associated with a decreased receptor binding and the production of cytotoxic factors. Similarly, in vivo, opioid receptor dimerization, ligand binding of receptors, and cell function in immune cells were promoted by chronic treatment with an opiate antagonist but suppressed by chronic ethanol feeding. Additionally, a combined treatment of an MOR antagonist and a DOR agonist was able to reverse the immune suppressive effect of ethanol and reduce the growth and progression of mammary tumors in rats. These data identify a role of receptor dimerization in the mechanism of DOR and MOR feedback interaction in NK cells, and they further elucidate the potential for the use of a combined opioid antagonist and agonist therapy for the treatment of immune incompetence and cancer and alcohol-related diseases.

Prolonged subjection to unstable work or lighting schedules, particularly in rotating shift-workers, is associated with an increased risk of immune-related diseases, including several cancers. Consequences of chronic circadian disruption may also extend to the innate immune system to promote cancer growth, as NK cell function is modulated by circadian mechanisms and plays a key role in lysis of tumor cells. To determine if NK cell function is disrupted by a model of human shift-work and jet-lag, Fischer (344) rats were exposed to either a standard 12:12 light-dark cycle or a chronic shift-lag paradigm consisting of 10 repeated 6-h photic advances occurring every 2 d, followed by 5–7 d of constant darkness. This model resulted in considerable circadian disruption, as assessed by circadian running-wheel activity. NK cells were enriched from control and shifted animals, and gene, protein, and cytolytic activity assays were performed. Chronic shift-lag altered the circadian expression of clock genes, Per2 and Bmal1, and cytolytic factors, perforin and granzyme B, as well as the cytokine, IFN-γ. These alterations were correlated with suppressed circadian expression of NK cytolytic activity. Further, chronic shift-lag attenuated NK cell cytolytic activity under stimulated in vivo conditions, and promoted lung tumor growth following i.v. injection of MADB106 tumor cells. Together, these findings suggest chronic circadian disruption promotes tumor growth by altering the circadian rhythms of NK cell function.

It is becoming increasingly clear that stressful life events can affect cancer growth and metastasis by modulating nervous, endocrine, and immune systems. The purpose of this review is to briefly describe the process by which stress may potentiate carcinogenesis and how reducing body stress may prevent cancer growth and progression. The opioid peptide β-endorphin plays a critical role in bringing the stress axis to a state of homeostasis. We have recently shown that enhancement of endogenous levels of β-endorphin in the hypothalamus via β-endorphin neuron transplantation suppresses stress response, promotes immune function, and reduces the incidence of cancer in rat models of prostate and breast cancers. The cancer-preventive effect of β-endorphin is mediated through the suppression of sympathetic neuronal function, which results in increased peripheral natural killer cell and macrophage activities, elevated levels of anti-inflammatory cytokines, and reduced levels of inflammatory cytokines. β-endorphin inhibition of tumor progression also involves alteration in the tumor microenvironment, possibly because of suppression of catecholamine and inflammatory cytokine production, which are known to alter DNA repair, cell-matrix attachments, angiogenic process, and epithelial–mesenchymal transition. Thus, β-endorphin cell therapy may offer some therapeutic value in cancer prevention. Cancer Res; 72(4); 836–40. ©2012 AACR.

Neurobehavioral stress has been shown to promote tumor growth and progression and dampen the immune system. In this study, we investigated whether inhibiting stress hormone production could inhibit the development of mammary carcinoma and metastasis in a rat model of breast carcinogenesis. To enhance β-endorphin (BEP), the endogenous opioid polypeptide that boosts immune activity and decreases stress, we generated BEP neurons by in vitro differentiation from fetal neuronal stem cells and transplanted them into the hypothalami of rats subjected to breast carcinogenesis. BEP-transplanted rats displayed a reduction in mammary tumor incidence, growth, malignancy rate, and metastasis compared with cortical cells–transplanted rats. BEP neuron transplants also reduced inflammation and epithelial to mesenchymal transition in the tumor tissues. In addition, BEP neuron transplants increased peripheral natural killer (NK) cell and macrophage activities, elevated plasma levels of antiinflammatory cytokines, and reduced plasma levels of inflammatory cytokines. Antimetastatic effects along with stimulation of NK cells and macrophages could be reversed by treatment with the opiate antagonist naloxone, the β-receptor agonist metaproterenol, or the nicotine acetylcholine receptor antagonist methyllycaconitine. Together, our findings establish a protective role for BEP against the growth and metastasis of mammary tumor cells by altering autonomic nervous system activities that enhance innate immune function. Cancer Res; 71(19); 6282–91. ©2011 AACR.

A novel pyranoside mimetic compound, DMBO (2-(2,6-difluorophenyl)-5-(4-methoxyphenyl)-1-oxa-3-azaspiro[5.5]undecane), was designed and synthesized. The sugar mimicking behavior of DMBO was addressed by its ability to bind several growth factors/cytokines such as vascular endothelial growth factor (VEGF), heparin-binding epidermal growth factor-like growth factor (HB-EGF), and tumor necrosis factor (TNF)-α as demonstrated by the recently developed surface plasmon resonance assay. DMBO exhibited strong anti-proliferation activity in vitro against tumor cells including a highly metastatic murine osteosarcoma cell line LM8G7 that secretes VEGF as well as two human ovarian cell lines, OVSAHO and SKOV-3, which secrete TNF-α and HB-EGF respectively. Furthermore, DMBO inhibited the metastatic activity to the mouse liver of LM8G7 cells injected from a lateral tail vein, and affected the heparan-degrading activity of LM8G7 cells. Here, we report that DMBO acts as a human heparanase inhibitor in vitro possibly as a substrate mimetic. DMBO also inhibited the migration and invasion of LM8G7 cells and angiogenic events such as endothelial cell proliferation, migration and capillary tube-like formation in vitro. More prominently, the administration of DMBO with heparin resulted in synergistic anti-tumor effects in mouse model of osteosarcoma. These preclinical data shows the potential anti-cancer effects of DMBO.

In recent years, experimental studies suggest that impaired hypothalamic-pituitary-adrenal (HPA) axis modulates colonic motor function and pathologies. However, little is known about the functional role of beta-endorphin (BEP) neural populations, which control HPA function, on colonic function in response to inflammation or injury of colon. Based on our previous published work showing that transplantation of BEP neurons in the hypothalamus suppress tumor growth and development in numerous animal models including prostate, breast and liver cancer, we determined whether transplantation of BEP neurons into the hypothalamus suppress preneoplastic and neoplastic lesions and modulate colonic proinflammatory cytokines and epithelial-mesenchymal transition (EMT) by enhancing innate immune response. Colon cancer (injury) was induced in Sprague-Dawley male rats with BEP or control cell transplants by injecting 1,2-dimethylhydrazine (DMH) for 16 weeks. Our results reveled that control groups developed tumors at 100% incidence, while BEP transplanted animals developed tumor only at 30% rate. The mean volume of tumor in colon was smaller in BEP neurons transplanted rats than in control cells transplanted rats. Furthermore, histological results revealed that BEP neurons transplanted animals had lesser histopathological lesions such as aberrant crypt foci (ACF) and adenocarcinoma development in the colon than the control groups. In addition, immunohistochemical and western blot analyses of colon showed that DMH treatment increased expression of Ki-67, TNF-α and NF-κB more in cortical cells transplanted animals than in BEP cell-treated groups. Also, splenic NK cells cytolytic proteins such as perforin, granzyme B and IFN-γ levels in BEP neurons transplanted rats were higher when compared to control groups. Interestingly, BEP neurons transplantation repressed the expression of Snail and Twist, transcription factors linked to EMT, and concomitantly maintained E-cadherin, an epithelial cell marker expression. These data suggest that the antitumor action of BEP neuron transplants are due to the suppression of inflammatory milieu and EMT as well as the activation of NK cells mediated innate immune responses. These data also identify the potential use of BEP cell therapy to combat tumor growth and progression. (Supported by R37 AA08757) Citation Format: Dipak K. Sarkar, Sengottuvelan Murugan, Yatee Dave, Ankush Rakhit. Hypothalamic beta-endorphin neuron transplants modulate colonic proinflammatory cytokines and epithelial-mesenchymal transition factors and suppress preneoplastic and neoplastic lesions in 1,2-dimethylhydrazine-induced colon cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2971. doi:10.1158/1538-7445.AM2014-2971

We have recently identified that a set of hormone secreting nerve cells in the hypothalamus, called beta-endorphin (BEP) neurons, have the ability to prevent the growth, progression and metastasis of mammary and prostate tumors in laboratory animals. How these neuronal cells prevent tumor cell growth and progression is not apparent. In order to better understand the mechanism by which endorphin neurons control tumor growth, we determined the growth and clearance of breast cancer derived MADB106 cells in Fisher rats, which are immune intact, and in athymic Nude rats, which are T cells deficient. Adult female Fischer rats or Nude rats were transplanted with either BEP neurons or cortical neurons (control) in the hypothalamus for 4 weeks and then inoculated with MADB106 cells in the jugular vein or under the skin in the back. These animals were sacrificed after 6 weeks to determine the volume of the tumor at the site of inoculation for determining the growth of tumors and the number of other tumors at the distant sites for determining tumor metastasis. Tumors were also collected for histopathology. Blood samples were collected and used for measurements of cytokines and immune cells. In Fischer rats, after tumor inoculation, animals with control cell transplants had tumor colonization in the lung and some on the skin at the site of cell injection. However, BEP neurons treated Fischer rats had no tumor colonization in the body. In the Nude rats, tumor colonization occurred both at the site of injection and at the distant site under the skin as well as in the lung of control transplanted rats. In BEP neurons transplanted Nude rats, tumor colonization only occurred at the site of inoculation. The mean volume of tumor at the inoculation site was smaller in BEP neurons transplanted rats than in control cells transplanted rats. In BEP neurons transplanted rats, corticosterone and inflammatory cytokines levels were lower, whereas anti-inflammatory cytokines and chemokines levels were higher. In addition, NK cell and macrophage cell numbers and their cytolytic functions in peripheral blood of BEP neurons transplanted rats were elevated. These data suggest that the antitumor action of BEP neuron transplants is due to the activation of primarily NK cells and macrophages and also in lesser extent T cells. The data also identify the potential use of BEP cell therapy for cancer prevention. (Supported by R37 AA08757) Citation Format: Dipak K. Sarkar, Changqing Zhang, Sengottuvelan Murugan, Nadka Boyadjieva, María Ortigüela. Beta-endorphin neuron transplants suppress mammary tumor growth and progression: role of immune cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 183. doi:10.1158/1538-7445.AM2013-183

Prostate cancer remains the second leading cause of cancer death in men. While some of the causes of prostate neoplasia are subject of intensive research, the influence of genetic predisposition as well as diet and other environmental factors are not well studied. Recently it has been shown that prenatal exposure to ethanol makes the offspring more susceptible to the development of childhood cancers. However, it is not known whether prenatal ethanol induces fetal programming for prostate neoplasia. In order to test this, we fed pregnant rats a liquid diet containing alcohol, some were pair-fed with isocaloric liquid diet, or other were fed ad libitum with rat chow between gestational days 7 and 21. A group of male offspring rats were sacrificed at 7 to 8 months of age and their prostates were removed for histopathology. Another group of male offspring rats were treated with N-Nitroso-N-methylurea (NMU) and testosterone to induce prostate neoplasia. After the tissue was processed, the ventral, lateral, and dorsal parts of the epithelium were viewed under a microscope to determine whether the tissue exhibited normal histology, or abnormalities in the form of atypia, low-grade, or high-grade prostatic intraepithelial neoplasia (PIN). Additionally, levels of various cellular proteins associated with prostate pathology were determined by immunohistochemistry. The prostates of most (77%) of the alcohol exposed animals, but not ad lib-fed or pair-fed animals, demonstrated atypical hyperplasia, atypia-dysplasia characterized by nuclear stratification, and epithelial evagination with intense inflammatory infiltration in the ventral prostate gland. Furthermore, a significant number of in utero alcohol exposed animals developed high-grade PIN, whereas control animals developed hyperplasia and low grade PIN during adulthood in response to NMU and testosterone treatments. We also found increased levels of estradiol receptor (ER)-alpha, decreased levels of androgen receptor (AR) in the dorsal and ventral prostate, and low levels of circulating testosterone in alcohol-fed offspring who were not treated with carcinogens. In addition, these rats showed a decreased level of E-cadherin and increased Ki-67 in the ventral prostate. Fetal alcohol exposed rats treated with carcinogens showed increased levels of Ki-67 with concomitant decreased levels of PTEN and Nkx3.1 in the ventral prostate epithelium. The protein data corroborates well with the increased neoplastic transformation of the ventral prostatic cells in fetal alcohol exposed rats. These results suggest for the first time that prenatal ethanol exposure induces pre-malignant lesions in prostatic cells and increases the susceptibility toprostate cancer. (Supported by NIH Grants AA11591 and R37 AA08757) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 537. doi:1538-7445.AM2012-537