The Study Of Regulations And Mechanisms Of Methionine Enkephalin (MENK) On Lung Cancer Biological Behaviors

Object: In recent 10 years,immunotherapy has become a breakthrough in the treatment of lung cancer.The mechanism of immune checkpoint inhibitors(ICIs)is to wake up the blocked lymphocytes in the immune TME and allow tumor-specific T cells to remain activated and kill tumor cells.Currently,a variety of ICIs have been approved for the treatment in patients with advanced lung cancer for the satisfactory clinical results.Although some patients may achieve a "cure" result after received immunotherapy of ICIs,the objective response rate of ICIs treatment for patients with lung cancer is only about 20-40% due to various reasons that decrease the activity of immune cells,and the anti-tumor immunity will be blocked again.Thus,therapies that can create an immunogenic environment within tumors that otherwise are immune suppressed or immunologically barren have the potential to stimulate the anti-tumor immune response,so as to enhance the efficacy of the anti-PD-1 treatment for patients received immunotherapy and even to overcome the drug resistance.Methionine encephalin(MENK)is a kind of pentpeptide derived from prohormone and proenkephalin produced by adrenal gland,and consists of five amino acids: Tyr-GlyGly-Phe-Met.As an endogenous opioid peptide of opioid receptors without drug dependence and species specificity,MENK acts as a significant mediator connecting the nervous and the immune system.Opioid receptors are expressed in several immune cells including T lymphocytes,macrophages,natural killer(NK)cells,and dendritic cells(DCs),under an appropriate concentration,MENK can bind to opioid receptors in immune cells,so as to exert significant effect of immunomodulatory and enhance anti-tumor immune activity.In addition,opioid receptors are expressed in many tumor cells,such as pancreatic cancer cells,intestinal cancer cells and gastric cancer cells,etc.Studies in in vitro have shown that MENK could inhibit growth of some cancers directly through binding to opioid receptors in cancer cells.Therefore,the unique character of MENK in antitumor activity via inhibiting tumor growth directly and regulating immune response to enhance antitumor efficacy,endows the agent with an absolute advantage to become a new anticancer drug in lung cancer treatment.One previous literature has demonstrated that lung cancer cells such as A549 and H1975 cells also express OGFr and morphine can inhibit the growth of tumor cells through this receptor.Therefore,we suppose that MENK may have an inhibitory effect on the growth of lung cancer cells through binding to the opioid receptors in lung cancer cells,and may regulate the antitumor immune function through different mechanisms.Thereby,we will verify and explore it through in vivo and in vitro experiments.Methods: 1.In vitro,human lung cancer cell lines A549 and H1975,and mouse lung cancer cell line Lewis were administrated with MENK at different concentrations(0,1,2.5,5,7.5,10,12.5 mg/m L)for 24,48,72 and 96 hours,respectively.At the same time,human normal bronchial epithelial cell line Beas-2B cells were treated with MENK at the above concentrations for 24 and 48 hours.Then the inhibitory effect of MENK on cancer cells was detected with CCK-8 kit at the timepoints of 24,48,72 and 96 hours after administration respectively.Finally,we used 6 mg /m L MENK treated the three lung cancer cell lines for 48 h as the appropriate action time and dose to the subsequent experiments: observing morphological changes of lung cells with optical microscope;detecting proliferation of lung cancer cells with clonal formation assay;detecting cell cycle and apoptosis of lung cancer cells by flow cytometry;observing apoptosis morphology used Hoechst 33258 staining;assessing the alteration of abilities in migration and invasion of lung cancer cells with wound scratch-and transwell invasion test;the expression of calreticulin(CRT)and high mobility group box 1(HMGB1)were detected by immunofluorescence(IF).The relative mRNA expression of OGFr and natural killer group2,member D ligands(NKG2DLs)including MICA,MICB,ULBP1,H60,and REA-1were evaluated by qRT-PCR,and relative protein expression levels of OGFr were assessed by Western blot.The expression of MICA,MICB and ULBP1 on the surface of A549 cells was detected by flow cytometry.2.Signaling pathway identification,using the effective sequence of OGFR-si RNA to package the lentivirus to knockdown the expression of OGFr in human lung cancer cell lines.Then lung cancer cells were divided into the following three groups: si-NC group,si-NC+MENK group,and si-OGFr+MENK group.Then we detected the expression of the Wnt/?-catenin pathway-related factors including cyclin D1,c-myc,?-catenin,apoptosis pathway-related factors including Bax,Bcl-2,caspase-3,and epithelial-mesenchymal transition(EMT)-related markers including E-cadherin,N-cadherin,Vimentin and MMP-2 in vitro experiments by qRT-PCR and Western Blot.3.In vivo,subcutaneous xenograft tumor model of human lung cancer was conducted in nude mice by injecting A549 cell into the right hind of nude mice.After tumor development,the tumor bearing nude mice were randomly divided into MENK treated group(5 mg,10 mg/ time,every other day)and normal saline group(equal volume of NS,every other day).The basic vital signs of nude mice were observed daily,and both the body weight and tumor size of all the mice were recorded every two days since they received the treatment at first time.We killed the mice through cervical dislocation about three weeks post treatment,then got the tumors out to prepare follow-up experiments.The involved tests are hematoxylin and eosin(HE)staining,immunohistochemistry(IHC)and TUNEL assay to detect histopathology,OGFR and Ki67 expression in tissues,and tumor cells apoptosis in tissues,respectively.4.In vivo,a mouse model of lung cancer was established to further define the changes of immune cells.Lewis cells were injected subcutaneously to the C57BL/6 mice under the right armpit.After tumor formation,the mice were randomly assigned into two groups: the MENK group(10 mg/day,every other day)and the negative control group(equal volume of NS,every other day)for 3 weeks.After that,the mice were sacrificed,splenic and tumor samples were obtained to detect the proportion of CD8+T cells,CD4+T cells,natural killer(NK)cells,antigen presenting cells(DCs),and the proportion of NK cells expressing Gz B and IFN-? by flow cytometry.Then subcutaneous tumors were removed and weighed.Tumor paraffin-embedded sections were prepared for HE detection of histopathology,IHC detection of OGFR,Ki-67,Gz B,IFN-?,IL-10,IL-15,etc.,immunofluorescence(IF)detection of CD8,CD4,etc.,and immunogenic cell death markers of CRT and HMGB1.Results: 1.In vitro,a significant growth inhibition of A549,H1975 and Lewis cells under a concentration-dependent and time-dependent manner could be observed in MENK group.However,MENK had no inhibitory effect on human normal bronchial epithelial cell line(Beas-2B).In vitro,MENK blocked the cell cycle of the lung cancer cells at G0/G1 phase;induced apoptosis of lung cancer cells;and decreased the rate of clonal formation,migration and invasion of lung cancer cells.MENK could up-regulate the expression of OGFR.IF assay suggested that MENK could increase the expression of CRT and the release of HMGB1 on the surface of lung cancer cells.The expression of NKG2 DLs on cancer cell cerface were also upregulated after MENK administration.2.MENK could reduce the expression of Wnt/?-catenin pathway-related factors cyclin D1? c-myc??-catenin in both mRNA and protein levels;MENK regulated the expression of Bcl-2/Bax/caspase-3 pathway-related factors Bcl-2,Bax,and caspase-3/Cleaved caspase-3,with up-regulation of Bax and cleaved caspase-3 levels and downregulation of Bcl-2 expression,but no significant change in caspase-3;In addition,MENK could regulate the expression of EMT-related markers.In details,MENK decreased the expression of N-cadherin,Vimentin,MMP-2,while increased E-cadherin expression in both of mRNA and protein levels.All these effects were significantly canceled after the knockdown of OGFr.3.MENK could suppress the growth of subcutaneous xenograft tumor in both BABL/c nude mice and C57BL/6 mice and induce the apoptosis of xenograft tumor cells.IHC results showed that MENK could enhance the expression of OGFR protein and decrease the expression of Ki-67 protein in tumor tissues.The TUNEL staining indicated that MENK could induce apoptosis of tumor cells4.In vivo,Results of flow cytometry showed that MENK could increase the percentages of DCs,NK cells,CD8+T cells,and CD4+T cells in both of tumor tissue and spleen.Additionally,MENK can increase the proportion of NKG2D-positive NK cells and CD8+T cell subsets.Among them,the proportion of NKG2 D positive cells in NK cells increased significantly.At the same time,the function detection of NK cells suggested that MENK could promote the percentage of NK cells expressing Gz B and IFN-? significantly.MENK could inhibit lung cancer by enhancing the immunogenicity and remodeling the distribution of immune cells in the tumor microenvironment.IF results indicated that MENK treatment enhanced the expression of CRT and HMGB1 proteins in TME.In the MENK treated tissue,the proportion of CD8+,CD4+ T cells,NK cells,M1-type macrophages and DCs were enhanced obviously,while the proportion of myeloid inhibitory cells and M2-type macrophages were decreased significantly,compared to that in control group.IHC results indicated that the expression levels of IL-15,IL-21,TNF-?,Gz B and IFN-? were significantly increased,while the expression levels of IL-10 and TGF-?1 were significantly decreased in the tumor tissues of mice.The expression of the cyclin D1,c-myc,?-catenin proteins detected by IHC in tumor tissues were also promoted obviously in MENK group.Conclusions: 1.It indicated that MENK could suppress the proliferation of lung cancer significantly at an appropriate dose both in vivo and in vitro.2.MENK exhibited an antitumor activity for lung cancer by blocking cell cycle,inducing apoptosis,inhibiting cell migration and invasion.MENK might improve the antitumor immune activity by remodeling the distribution of immune cells within TME and enhancing the immunogenicity of cancer cells and its own immunoregulation.3.MENK could upregulate the expression of OGFr in lung cancer cells and initiate downstream signaling pathway after binding to OGFr.The anti-tumor mechanisms involved regulating the NKG2DLs-NKG2 D signaling pathway,Bcl-2/Bax/caspase-3signaling pathway,Wnt/?-catenin pathway and EMT process.