Molecular Mechanisms Of Cell Proliferation Inhibition And Anti-tumor Immunity Of Melatonin In Gastric Cancer

Melatonin（N-acetyl-5-methoxy tryptamine）is an indole neuroendocrine hormone synthesized and secreted mainly by the pineal gland.In addition to the pineal gland,it could be synthesized by other tissues and organs,such as the striatum,retina,spleen,liver and gastrointestinal tract etc.As a hydrophobic small molecule,melatonin affects cells with many types which it can combine with membrane receptors on the cell membrane to release second intracellular signal molecule,or directly enter the cell and combine with nucleus receptors as transcription factors.Melatonin has a variety of physiological functions,such as regulation of circadian rhythm,inhibition of reproductive system development and maturation,regulation of bone growth,inhibition of tumor development,regulation of immunity,free radical scavenging,and antioxidant.Melatonin has been proved to inhibit various carcinomas including the gastric cancer.There are many different hypotheses to elucidate melatonin anticancer effect including promoting cellular apoptosis,inhibiting cell growth,regulating anti-cancer immunity,scavenging free radical,inhibiting estrogen competitively,etc.But its underlying mechanisms have remained elusive.In order to elucidate the effect of melatonin on gastric cancer cells and its molecular mechanism,three kinds of human gastric cancer cell lines AGS,SGC-7901 and MGC803 were selected and treated with melatonin to analyze the differences of levels of proteins and phosphorylated proteins used proteome and microarray technology.Several proteins related to cell proliferation were selected and tested in these cells.We constructed melatonin receptors knockout cell lines to analyze their roles in proliferation with melatonin.We also analyzed immunity affection of melatonin to gastric cancer cells by detecting the changes of cytokines.In the first part of this study,we mainly used high-throughput screening technology,proteomic and microarray,to analyze the possible site of action of melatonin.The results of the mass spectrometry analysis of the differentially expressed proteins can be classified into three groups.The first group was cell metabolism related enzymes,Such as lactate dehydrogenase B,citric acid dehydrogenase,phosphate isomerase ect.The second group was cell oxidoreductase,such as glutathione S-transferase,antioxidant protein Peroxiredoxin-6,superoxide dismutase and so on.The third group was molecular chaperone and cytoskeleton,such as:HSP70 protein 1,molecular chaperone protein TCP1 complex,keratin 8,proteasome alpha subunit.The difference protein phosphorylation analysis in phosphorylation chip showed that 50 protein phosphorylation up-regulated and 15 protein phosphorylation level down after melatonin.Online DAVID signal pathway enrichment analysis showed that several signaling pathways were involved in the effect of melatonin on gastric cancer cells,such as:MDM2/p53,Jak1/STAT3,MEK/cJun etc.These results provide the research direction of anti-gastric cancer mechanism of melatonin.The second part of this study focuses on the effect of melatonin on the proliferation of gastric cancer cells.In this study,we found the effect of melatonin on proliferation inhibition by cell cycle arrest in G1/S phase.The effect of this method was to inhibit the AKT/MDM2/CDC25A pathway in gastric cancer cells.And then we used CRISPR/Cas9 gene editing method to knock out the membrane and nuclear receptors of melatonin and analyzed the effect of different receptors in cell proliferation by melatonin inhibition.We constructed the AGS-KOM cell line of MTNR1A and MTNR1B receptors knocked out,the AGS-KOA cell line of RORαnuclear receptor was knocked out,knocked out the AGS-KOB cell line of RORβnuclear receptor knocked out and AGS-KOC cell line of knocked down the RORγknocked out.The proliferation of different receptor deficient cell lines was analyzed by ECIS dynamic cell analyzer,and it was found that the fast response of gastric cancer cells to melatonin was mainly made by membrane receptors and RORαnuclear receptor.In the third part of this study,we investigated the immune mechanism of melatonin against gastric cancer cells by the analysis of secretion of cytokines related the local immunosuppressive microenvironment in gastric cancer cells after melatonin on the.In this study,we compared the effects of melatonin on the changes of VEGF,TGF-β1,IL-10 and IFN-γin human gastric cancer cell culture supernatants before and after melatonin treatment.In the three kinds of gastric cancer cells treated with melatonin,the changes of VEGF and IFN-γwere consistent which the VEGF was down regulated and the IFN-γwas up-regulated.Downregulation of VEGF can play a role in inhibiting the angiogenesis of tumor,and also can change the microenvironment of tumor to inhibit MDSC.Up regulation of IFN-γis helpful to improve the production of anti-tumor immunity.While TGF-β1,IL-10 changes were different:TGF-β1 is down regulated in MGC803 and SGC-7901and up regulated in AGS;IL-10 was down in AGS and SGC-7901and up in MGC803.The gastric cancer cells have the difference in the expression of these two factors and the effect on tumor immunity should be further analyzed in more expressions or in vivo.In conclusion,this study screened differences of the expression and the phosphorylation of gastric cancer cell proteins in melatonin,confirmed that melatonin induced gastric cancer cell cycle arrest in G₁/S phase,caused cell proliferation inhibition by AKT/MDM2/CDC25A pathway,constructed melatonin receptors knockout models of gastric cancer cells which proliferation analyzed by ECIS dynamic cell analyzer showed that membrane receptors and RORαnuclear receptor was mainly receptors in the fast response of gastric cancer cells to melatonin,discussed the immune related cytokines VEGF,TGF-β1,IL-10 and IFN-γsecreted by gastric cancer cells in melatonin,which provided the basis for the further research of molecular mechanism and antitumor immunity of melatonin against gastric cancer.