Screening And Mechanism Of Nitrogen-Containing Heterocyclic Compounds Induced-gastric Cancer Cell Senescence

BACKGROUND: In recent years,cellular senescence is considered as a new approach in the treatment of tumors by limiting the proliferation of cancer cells.Mounting studies have shown that traditional anti-tumor drugs can inhibit the proliferation of many tumor cells by inducing cellular senescence,but only a few studies on anti-tumor drugs that induced gastric cancer cell senescence.Due to the limitations of the use of traditional drugs and the continuous understanding of the molecular biology of tumors,chemical synthesis of novel small molecule compounds have become one of the research foci in the field of global biomedicine.Therefore,novel small molecule compounds are expected to show therapeutic effects on gastric cancer by inducing cellular senescence,which provide a reliable theoretical basis for the development and synthesis of antitumor drugs.METHODS: MTT assays were used to screen compounds that could significantly inhibit the proliferation of gastric cancer cells and further verified the effect on inhibiting the proliferation of gastric cancer cells.The effects of compounds on cell cycle and apoptosis of gastric cancer were studied by flow cytometry.In the study of cell senescence characteristics,firstly,X-β-Gal staining assay was used to detect intracellular β-galactosidase activity.BrdU incorporation assay was used to detect DNA synthesis.DAPI staining and immunofluorescence assay were used to detect changes in nuclear heterochromatin structure.To elucidate the mechanism of the compounds induced senescence,we primarily used Western blot to test the changes in the level of proteins in relevant signaling pathways.The anti-tumor activity of the compounds screened out in the subcutaneous tumor-bearing mice was verified.Hematoxylin-eosin staining and immunohistochemistry were used to examine the histopathological features of the tumors and the expression of related protein levels.RESULTS: In this study,we firstly selected 4,5-diphenyl-2-methyl picolinate(DMP)from 48 compounds to inhibit the proliferation of gastric cancer cells.DMP showed significant cytotoxic effects on MKN-28 and MKN-45 cell lines in a dose-dependent manner.The cells treated with DMP were significantly blocked at the G2/M phase,compared with the control group(P<0.05 or P<0.01).DMP induced cell senescence in gastric cancer cells.In gastric cancer cells treated with DMP,the activity of senescence-associated β-galactosidase(SA-β-gal)was significantly increased;BrdU incorporation was significantly reduced,while SAHF was formed.To investigate the mechanism of DMP-induced cell senescence,we found that DMP cause distinct DNA damage(P<0.05 or P<0.01)and up-regulate the aging-related signaling pathway proteins p38,p-p38,p-p53,p21,p16 levels.In addition,high dosage of DMP significantly inhibited tumor growth in tumor-bearing mice and upregulated p53 and p21.CONCLUSIONS: 4,5-diphenyl-2-methyl picolinate can inhibit the proliferation of MKN-28 and MKN-45 gastric cancer cells in vitro.The activated p53/p21 signaling pathway mainly induces the senescence of gastric cancer cells and shows significant inhibitory effect on the growth of tumors in vivo.Furthermore,4,5-diphenyl-2-methyl picolinate has no obvious toxic effects on the growth and organs of mice.