Inhibitory Effects And Mechanisms Of Methionine Enkephalin (MENK) On Malignant Biological Behavior Of Gastric Cancer Through Opioid Receptor (OGFr)

Object: Gastric cancer(GC)is a malignant gastrointestinal tumor originated from the gastric mucosa epithelium.Gastric cancer is one of the most common malignant tumors in the world.The incidence of gastric cancer ranks the first among all kinds of malignant tumors in China,and the mortality of gastric cancer ranks the third in the world.At present,surgical operation is still the main treatment method for gastric cancer.However,the overall treatment mode of gastric cancer has changed significantly,from the surgical treatment model based on anatomy to the integrated treatment model,combined standardized surgery and perioperative adjuvant therapy,based on the anatomy,oncobiology and immunology.Methionine enkephalin(MENK)is the endogenous ligand of opioid receptors.It is an endogenous opioid,derived from prohormone and proenkephalin produced by adrenal gland and has the amino acid sequence of Tyr-Gly-Gly-Phe-Met,without drug dependence and species specificity.Early studies of MENK was focused on its role as an analgesic and neurotransmitter.The researches of MENK on immune regulation had developed rapidly until the early 1980 s,when Wybran showed a link between the nervous system and the immune system.Opioid receptors are expressed in many immune cells,including T lymphocytes,monocytes(macrophages),natural killer cells(NK),neutrophils,eosinophils,basophils,and Dendritic cells(DC),and also expressed in some tumor cells,such as pancreatic cancer cells,presented on the nuclear membrane surface,and colorectal cancer cells.Studies have shown that MENK,at an appropriate concentration,has a significant effect on immune regulation when combined with the immune cell opioid receptor,which can act as a messenger of endogenous immune system regulation,playing a bridge role between the immune system and the central nervous system,In addition,a series of studies in vivo and in vitro have shown that MENK,not only has a role as an immunomodulatory messenger,but also has a directly inhibited function on tumor growth binding to opioid receptors of cancer cells.The previous studies of our research group have confirmed that MENK can inhibit the proliferationof human melanoma cancer cells in vitro by inducing apoptosis and blocking the cell cycle at G0/G1 phase.The unique effection ways of MENK,which could inhibit tumor growth directly and inhibit tumor growth by regulating immune response,provides an absolute advantage for it to become a new anticancer drug.So,does MENK have the same inhibitory effect on gastric cancer? How does it directly inhibit tumor growth? What is the specific mechanism of action? The aim of this study was to elucidate the effects of MENK on human gastric cancer cells in vitro and in vivo and its potential molecular mechanism.Methods: 1.In vitro,qRT-PCR was used to detect the expression and expression difference of OGFr in several gastric cancer cell lines(AGS,BGC823,SGC7901,HGC27,MGC803,MKN45),and the cell lines with relatively high expression of OGFr were selected for subsequent experimental detection.In vitro,human gastric cancer cells HGC27 and SGC7901 were treated with MENK at different concentrations(0,0.5,1,2,5,10,12.5 mg/mL)for 24,48,72,96 hours,respectively.At the same time,human normal gastric mucosal epithelial cells GES-1 were treated with MENK at the same concentration or 24,48 hours.Then the inhibitory effect of MENK was detected with cck-8 kit.Appropriate action time and dose of MENK were selected for subsequent functional experiments of gastric cancer cells.2.In vitro,we used 5 mg /mL MENK treated on HGC27 and SGC7901 cells for48 h,and the following test was performed: compared with the control group,morphological changes of GC cells were observed by optical microscope.The proliferation of GC cells was detected by clonal formation assay.Cell cycle and apoptosis of GC cells were detected by flow cytometry.Apoptosis morphology was observed by Hoechst 33258 staining.Transwell and cell wound scratch assay were used to detect the migration and invasion of GC cells.After that,mRNA and protein in control group and MENK treatment group of the two GC cells were extracted.The mRNA relative expression of OGFr,Ki67,Bax,BCL-2,MMP-2 and MMP-9 were detected by qRT-PCR,and the protein expression of OGFr,Bax,and BCL-2 were detected by Western blot.3.In vivo,subcutaneous xenograft models of human gastric cancer in nude micewere established.SGC7901 cells were inoculated subcutaneously into the right head and neck region of mice and the mice were randomly separated into two groups:MENK group(5,8,10 mg(14)2 days)and the control group(normal saline).The vital signs of nude mice were observed daily,and body weights and the tumor volumes were monitored dynamically.After 22 days,the mice were sacrificed for cervical dislocation,and the tumors were removed and weighed.Paraffin-embedded sections of the tumors were prepared and the following tests were performed: histopathology was detected by hematoxylin and eosin(HE),the expression of OGFr and Ki67 in tumors were detected by immunohistochemistry(IHC),and the apoptosis of tumors were detected by TUNEL assay.Meanwhile,IHC was used to detect the expression levels of tumor related macrophages(TAMs)surface markers F4/80,CD64,CD206 and secretory cytokines TNF-? and IL-10 in tumors.In addition,a nude mice model of tail vein metastasis was established,and the mice were randomly separated into two groups: MENK group(8 mg/mL,Once every two days)and the control group(normal saline).After 2 months of treatment,the mice were sacrificed for cervical dislocation,and the lungs and livers were dissected to observe the number of surface tumor metastases.4.Signal pathway study: Further,OGFr was silenced to observe whether the inhibitory effect of MENK on GC cells was weakened or disappeared.Using siRNA liposome transient method to silence the expression of OGFr,the silencing effect was detected by qRT-PCR,and the effective sequences were screened out.The effective sequence of OGFr-siRNA was encapsulated into lentivirus to construct a stable OGFr-silenced human gastric cancer cell line.After that,the cells were divided into three groups,including si-NC group,si-NC+MENK group,and si-OGFr+MENK group.The effects of MENK on the formation and apoptosis of GC cells was routinely detected.The mRNA levels of Bax,BCL-2,caspase-3,PARP,Ki67,Cyclin D1,c-myc,survivin,MMP-2 and MMP-9 were detected by qRT-PCR.The protein expression levels of Bax,BCL-2,caspase-3,PARP,PI3 K,AKT,p-AKT,mTOR were detected by Western Blot.Results: 1.In vitro,OGFr was prevalent expression in the most of gastric cancer cell lines,HGC27 and SGC7901 cells with relatively high OGFr expression were selectedfor follow-up experiments.In vitro,the cell proliferation of HGC27 and SGC7901 cells were decreased by MENK in a dose-and time-dependent manner.MENK had no significant effect on normal human gastric epithelial cell line(GES-1).After treatment with MENK,the appearance of the cells changed from polygonal,cobblestone-like cells into a fusiform-like,weakly adherent cellular morphology and an accompanying reduction in cell numbers,the number of colonies of the cells decreased significantly.MENK blocked the cell cycle of the two cells at G0/G1 phase,induced the apoptosis of the cells,inhibited the migration and invasion of the cells.MENK could up-regulate OGFr expression in HGC27 and SGC7901 cells.After treatment with MENK,mRNA levels of Ki67,BCL-2,MMP-9 were down-regulated,mRNA level of Bax was up-regulated,and MMP-2 was not significantly changed.Meanwhile,MENK up-regulated Bax protein expression and down-regulated bcl-2protein expression.2.MENK could inhibit the growth of GC subcutaneous xenografts in nude mice.After MENK treatment,the tumor sizes in MENK treated group were significantly smaller which was consistent with the tumor weights from MENK treated group.As measurement of toxicity,MENK treatment did not affect body weight during the experiment.The result of the HE analysis showed that post MENK treatment the tumor elicited large areas of central necrosis accompanied by apoptosis,hyperchromatic nuclei,while the morphology of tumor in the control group was regular and compact,with a few punctate or scattered necrotic regions among the tumor parenchyma.These results demonstrated that MENK could inhibit the tumor growth of SGC7901 in xenografts nude mice by inducing the apoptosis,which was further confirmed by TUNEL analysis.In addition,MENK has an obvious inhibitory effect on hepatic metastasis of GC in the caudal vein model.The effect on lung metastasis should be determined by increasing the sample size and repeating the experiment further.Immunohistochemistry results showed that SGC7901 tumor in xenograft nude mice treated with MENK had an increased expression of OGFr.And MENK down-regulated the expression of Ki67,which was consistent with the results of in vitro experiments.3.In vivo,MENK exerted an anti-GC activity through inducing TAMs from M2 to M1-type.The percentage of TAMs expressing CD64 in the MENK treated group increased significantly,the percentage of CD206 positive cells reduced in the MENK treated group.And the level of M1-related cytokines TNF-? in MENK treated group increased significantly,while the level of M2-related cytokines IL-10 attenuated in MENK group.These data indicated that there was a high level of infiltration of TAMs in xenograft tumors in MENK treated group and MENK may inhibit GC by enhancing the anti-tumor activity of TAMs though inducing the transformation of TAMs from M2 to M1 phenotype.4.After the silencing of OGFr,the inhibitory effect of MENK on the proliferation of HGC27 and SGC7901 cells were significantly weakened,and the effect of MENK on inducing apoptosis of GC cells was reversed.MENK can reduce the mRNA expression levels of the cell cycle related genes Ki67,cyclin D1,and c-myc in GC cells,and this effect is significantly weakened after the silencing of OGFr.MENK down-regulated the mRNA and protein levels of the apoptosis-related gene BCL-2,up-regulated the mRNA and protein levels of Bax and Caspase-3,and down-regulated the protein expressions of PARP.MENK can significantly reduce the mRNA levels of surviving.In addition,MENK could down-regulate the expression of PI3 K,p-AKT and mTOR proteins in the PI3K/AKT/mTOR pathway.All these effect was significantly reduced after OGFr silencing.Conclusions: 1.Both in vivo and in vitro experiments have confirmed that MENK has a significant inhibitory effect on gastric cancer at the appropriate dose.2.MENK exerted an anti-GC activity through inhibiting cell cycle,inducing apoptosis,inhibiting cell migration and invasion.MENK may inhibit GC by enhancing the anti-tumor activity of TAMs though inducing the transformation of TAMs from M2 to M1 phenotype.3.There are different expressions of OGFr in most human GC cell lines.MENK could up-regulate OGFr expression in GC cells.MENK triggers the anti-tumor mechanism by binding to OGFr.4.MENK could inhibit the proliferation of human GC cells by arresting cell cycle at G0/G1 phase though reducing the expression of cyclin D1?c-myc.5.MENK has anticancer activity in human GC cells by inducing apoptosisthough the BCL-2/Bax/caspase-3/PARP signaling pathway.6.MENK could inhibit gastric cancer though regulating the PI3K/AKT/mTOR signaling pathway.