The Role Of Oxytocin On Stress-induced Depression-like Behaviors And Cancer Metastasis And Underlying Mechanisms

Section ⅠIntranasal oxytocin administration improves depression-like behaviors in adult rats that experienced neonatal maternal deprivationBackgroundOxytocin(OXT),a hypothalamic neuropeptide,stimulates milk ejection and uterine contraction at parturition in mammals and has been implicated in the regulation of social behaviors in various rodent species.The synthetic OXT facilitates pro-social behavior and prevents social avoidance.Early interactions with peers enhance oxytocin receptor(OXTR)levels in central nervous sysytem.High levels of pup licking/grooming(LG)show significantly higher OXTR levels in the medial preoptic area.In contrast,the offspring of Low LG dams have reduced OXTR binding during the postpartum period.Moreover,a number of human studies have found significant correlations between peripheral levels of endogenous OXT and behavior.High levels of plasma OXT have been related to trust and trustworthiness,positive physical contact with a partner,and lower levels of anxiety in patients with depression.On the contrary,low levels of plasma OXT have been found in patients with depression,autism and schizophrenia.Exposures to various types of early life stress can enhance anxiety and depression-like behaviours.Adverse family social environments are strongly associated with the development of depression.Neonatal maternal deprivation(NMD)by daily separation of the whole litter from the dam for up to 3 h during the first 2 weeks of life is considered as an established and extensively used rodent model for early social life stress.A number of studies have demonstrated that NMD may lead to depression-and anxiety-like behaviors later in life.However,to date the role of OXT has not been investigated in NMD.Intranasal administration of OXT is used extensively in clinical investigations for its easy and non-invasive delivery method and its putative rapid and direct access route to the brain.Animal studies revealed that intranasal delivery can easily bypass the blood-brain barrier(BBB).The peripheral olfactory route connecting the nasal passages with the olfactory bulbs and rostral brain regions has been proved to be a possible pathway.Furthermore,a peripheral trigeminal system connecting the nasal passages with brainstem and spinalcord regions has also been involved.Both routes provide rapid entry to the CNS.It has been proved that intranasal administration of OXT markedly increases the levels of OXT in the dorsal hippocampus and amygdala of rats and mice,with peak levels occurring 30-60 min after administration.A growing literature has shown that intranasal administration of OXT influences social information processing.Intranasal administration of OXT might be a promising new adjunctive therapy for mental disorders.In this study,we examined if intranasal OXT would reverse the adverse behavioral effects of NMD in adult male rats.ObjectiveThis study assessed the effects of intranasal administration of OXT on the depressive-like behaviors and hippocampal neurogenesis in adult rats by NMD.Methods1.The establishment of NMD animal model:Experiments were performed on male Sprague-Dawley rats.In brief,pups for the NMD group were removed from their dam and home cage,and placed individually in new paper cages bedded with woodchips for 3 h at 30℃ in a incubator,and then returned to their home cage and dam.NMD was performed during 9:00-12:00 hours daily from postnatal days(PND)2-15,and then the pups were left with their dam undisturbed until weaning on PND 22.The day of birth was defined PND1.Pups for the control group were not exposed to handling and were maintained in their maternity cage with the dam.On the weaning day,male pups in each litter were randomly selected and placed in each cage(n=10 each group),and female pups were excluded from this study.2.Behavioral changes were analyzed in the forced swimming test(FST)and open-field test.3.The OXT levels in plasma and brain were measured by ELISA.4.Intranasal administration of OXT:NMD(3 h/day for 14 days)adult male rats were administrated by atomization inhalation of OXT(O.lmg/kg/d for 14 days).5.The adult hippocampal neurogenesis was investigated by immunohistochemical staining of Ki67 and BrdU incorporation assay.Results1.NMD resulted significant depression-like behaviors,as indicated by the decrease in physical activity and emotional reactivity in novel environment in two-month-old animals.Notably,the OXT levels in plasma,hypothalamus and hippocampus were decreased in these animals.2.Remarkably,intranasal administration of OXT alleviated the depressive-like behaviors in NMD rats.3.NMD dramatically reduced the number of Ki67+ cells in the dentate gyrus(DG)of hippocampus.Intranasal administration of OXT for 14 consecutive days significantly increased the number of Ki67+ cells in DG area.Moreover,daily OXT administration for 14 days significantly increased the number of BrdU+ cells in DG area.These results suggest that intranasal administration of OXT rescued hippocampal long-term plasticity impaired by NMD stress in rats by promoting hippocampal neurogenesis.Conclusions1.NMD induces the depressive-like behaviors in adult rat.2.The intranasal administration of OXT alleviates the depressive-like behaviors in NMD adult rat.3.The intranasal administration of OXT induced beneficial effects are probably mediated by boosting the hippocampal neurogenesis.Section IIOxytocin involves in chronic stress-evoked melanoma metastasis via-arrestin 2-mediated ERK signaling pathwayBackground:Melanoma is the most deadly form of skin cancer and its incidence is rising worldwide.Presently,there are no effective therapies to prevent melanoma metastasis,with interferon-alpha,the only Food and Drug Administration(FDA)-approved melanoma adjuvant therapy,conferring a meager 5-year overall survival benefit of 1%-3%.Despite recent advancements in immunotherapies,the majority of patients with metastatic melanoma still succumb to their disease and the mean survival is only 23 months.The poor clinical prognosis and lack of curative therapies for patients with advanced melanoma necessitate the development and implementation of novel approaches that prevent metastatic relapse of this cancer.A growing literature documents that stress can influence cancer progression.The survival rate of patients with cancer decreased if they were made to be socially isolated or separated from their spouse.In contrast,social support can improve the outcome of patients suffering from cancer.Chronic stress acts as a sort of fertilizer,significantly accelerating breast cancer progression in animal models.They discovered a 30-fold increase in cancer spread throughout the bodies of stressed mice compared to those that were not stressed.The chronic stress promotes EGFR inhibitor resistance in the treatment of non-small cell lung cancer,and causes cancer cells to escape from the primary tumor and colonize distant organs.Some researchers found that stress also increased melanoma lung metastasis by suppressing the immune system.However,so far,there is no rational explanations for the poor outcomes of stress-associated melanoma patients.Oxytocin(OXT),as a neuro hormone produced by the hypothalamus,plays a vital role in laboring induction and lactation.Emerging evidences suggest that OXT also regulates human emotions,social cognition,social behaviors and stress-related disorders.It is well known that hormone OXT also belongs to stress hormones and is released in response to several stress stimuli.After exposed to repeated restraint stress,the concentrations of OXT in plasma was significantly increased.But so far whether OXT is involved in the stress-induced promotion in cancer progression is still unknown.OXT acts as a grow regulator,through the activation of a specific G-coupled transmembrane receptor,the oxytocin receptor(OXTR).Although OXT contributes to tumorigenesis,the functions of OXT in malignant melanoma are unclear,and whether OXT signal could be a target for melanoma therapeutics is still unknown.Objectives:The aim of this study was to identify whether chronic stress speeds up lung metastasis of melanoma in mice.If so,we will investigate the effects of exogenous OXT on melanoma cells migration and invasion in vitro,and lung metastasis of melanoma in vivo.Next,we will further observe the molecular target that OXT promotes the melanoma cells migration and invasion.According to the above results,we will test whether OXTR or beta-arrestin 2 knockdown abolishes the lung metastasis of melanoma stimulated by chronic stress.The findings of present study will shed some light on the mechanism of the poorer outcomes of the stress-related promotion of melanoma metastasis and provide a potential therapeutic target for melanoma.Methods:1.Tissue and plasma collection of malignant melanoma(MM):A total of 15 pairs of MM tissues and adjacent normal tissues were collected from Shandong Cancer Hospital Affiliated to Shandong University after written informed consents were obtained.All specimens were confirmed as MM by pathological diagnosis.All patients involved in this study received no radiation therapy or chemotherapy before surgical resection.The study included 7 females and 8 males,ranging in age from 41 to 71 years with a mean age of 61 years.All tissues were snap frozen in liquid nitrogen,and then stored at-80℃ before use.Three benign melanocytic nevi were used to test the expression of OXTR by immunohistochemistry.2.Cell culture:The human melanoma cancer cell line A375 cells were cultured in DMEM and the murine B16 cells were cultured in RPMI 1640.3.Xenograft murine model:Male C57/B6 mice(6-8 week of age)were housed in pathogen-free animal cages under a 12-h light/dark cycle with free access to food and water.For orthotopic mouse tumor model,5×105B16 melanoma cells were injected s.c.into right hind flank of 6-week-old male C57BL/6 mice to develop melanoma.After one week of inoculation,mice with approximate tumor size were divided into two groups and administered intraperitoneally with normal saline or OXT(0.1 mg/kg/d)for 2 weeks.The tumor volume was calculated according to the formula(length × width)/2.Three weeks after tumor inoculation,they were then sacrificed for evaluating primary tumor size and weight.For the chronic stress model,mice are securely restrained in a movement-restricted space for 2 hours daily during the duration of the experiment.In all experiments,5×105 B16 cells were injected via tail vein(to study metastasis)in mice 1 week after the restraint stress procedure began.After 16 days,the animals were killed by cervical dislocation,and the numbers of tumor nodules of lung metastases were noted by researchers blinded to the groups.4.Plasmid construction and transfection:The OXTR shRNA(shOXTR)or β-arrestin 2 shRNA(shβ-arr2)plasmid was transfected according to the manufacturer’s instructions.5.Western blot or immunocytochemical analysis was applied to detect the expressions of OXTR,MMP2,VEGF,β-arrestin 1,β-arrestin 2,Erk or p-ErK.6.Gelatin zymography analysis:The effects of OXT on the gelatinolytic activities of MMP-2 were examined by gelatin zymography.7.Cell proliferation studies:The proliferation of B16 and A375 cells was assessed by Cell Counting Kit-8 assay according to the protocol recommended by the manufacturer.8.Wound-healing assay:The effects of different concentrations of OXT(0,10-9,10-8,10-7,10-6M)on cell migration were tested using an in vitro scratch assay.The width of the scratch was microscopically monitored at various time points and quantified in terms of the difference between the original width of the wound and the width after cell migration.The percentage of wound closure[(original width-width after cell migration)/original width]was calculated.The width of the wound was measured using Image-Pro Plus 6.0.9.Transwell migration assay:The effects of different concentrations of OXT(0,10-9,10-8,10-7,10-6M)on cell migration were teste using a complementary transwell migration assay.The migration assays were incubated for 24 hours at 37℃ in 5%CO2,and then the cells were stained with crystal violet.Cells on the undersides of the filters were observed under a microscope at a magnification of 200 x and counted.10.In vitro HUVEC tube formation assay:The in vitro tube formation assay was performed to observe the effects of OXT on angiogenesis.Results:1.Chronic stress promoted melanoma pulmonary metastasis and shortened survival in mice.Of note,stress largely elevated the level of plasma OXT,a neuro hormone produced by the hypothalamus.2.The expression of OXTRs was observed in malignant melanoma,which was positively correlated with the degree of malignancy.3.The activation of OXTRs dramatically promoted migration,invasion and angiogenesis but not proliferation of melanoma cells in vitro.The primary tumors did not seem to be affected by exogenous OXT and grew similarly in both groups of mice.However,the OXT-treated animals showed significantly more lung metastases than did the control group.4.Exogenous OXT stimulated either the production of VEGF and MMP-2 or the migration and invasion via the-arrestin 2-dependent ERK1/2 activation.5.Strikingly,OXTR or beta-arrestin 2 knockdown markedly abrogated the melanoma pulmonary metastasis evoked by chronic stress in mice.The survival rate of mice also was improved.In parallel with this,the down-regulation of either OXTR or β-arrestin 2 expression significantly diminished phosphorylated ERK1/2 and suppressed the production of MMP-2 and VEGF in lung metastasis samples.Conclusions1.The chronic stress hormone-OXT promotes migration via OXTR signaling by aβ-arrestin 2-dependent mechanism.2.OXT is involved in stress-evoked lung metastasis,suggesting that OXT,a novel pro-metastasis factor,is a potential therapeutic target for melanoma.Section ⅢOxytocin inhibits ovarian cancer metastasis by repressing the expression of MMP-2 and VEGFBackground:Based on 2010-2014 cases and deaths,the number of new cases of ovarian cancer was 11.7 per 100,000 women per year and the number of deaths was 7.4 per 100,000 women per year.More and more literatures reported that breastfeeding is associated with a decreased risk of ovarian cancer.A case-control study in southern China revealed that prolonged lactation reduced ovarian cancer risk.Harvard Center for Cancer Prevention also determined that women who breastfeed for more than one year accompanied with a decrease in ovarian cancer risk compared with those who never breastfeed.Notably,a NewEngland study with 563 participants also demonstrated that ever breastfeeding caused a reduction of ovarian cancer risk.It is well known that oxytocin(OXT)is released during breastfeeding.Therefore,we propose OXT is involved in breastfeeding-associated protect against ovarian cancer.Interestingly,a group from other lab had demonstrated that intraperitoneal administration of OXT resulted in the reduction of intraperitoneal dissemination of ovarian carcinoma cells in mice.Although,as reported,OXT inhibited the proliferation,migration and invasion of ovarian cancer,its underlying mechanisms had not fully elucidated.Objectives:To evaluate the role of OXT,a hypothalamic nonapetide in the proliferation,migration and invasion of ovarian cancer cells and its underlying mechanisms.Methods:1.The proliferation of ovarian cancer cells was assessed by Cell Counting Kit-8 assay.2.The migration and invasion ability was evaluated by wound healing assay and Transwell assay,respectively.3.The MMP-2 and VEGF of protein of cancer cells was examined by Western blotting assay.4.The effects of OXT on the gelatinolytic activities of MMP-2 were examined by gelatin zymography.5.Angiogenesis of human umbilical vein endothelial cells was assessed in vitro using a tube formation assay.Results:1.OXT markedly inhibited the proliferation,and critically migration and invasion of human ovarian cancer cells SKOV3 and A2780.2.Strikingly,OXT inhibited the migration and invasion of human ovarian cancer cells by repressing the expressions of MMP-2 and VEGF.3.Moreover,OXT inhibited vascular endothelial cell tube formation by reducing the VEGF production from ovarian cancer cells.Conclusion:OXT inhibited ovarian cancer metastasis and angiogenesis by downregulating the expressions of MMP-2 and VEGF.