Anti-tumor Activity And Mechanism Of 1,2,3-triazole Derivative 1988 On Gastrointestinal Cancer Cells

ObjectivesGastrointestinal cancer is one of the common malignancies in China;more than one million new cases of gastric cancer,colon cancer and esophageal cancer were diagnosed each year,accounting for about 40% of the total number of new gastrointestinal cancers of the world.Even though in recent years quality of life and living standards significantly have been improved but still incidence of malignancies of the digestive system is increasing which could be due to bad dietary habit and living habits.Due to the lack of compliance and obvious symptoms,most patients at the early stage lost the opportunity of surgery however advanced patients are mainly treated with chemotherapy.Hence,the five-year survival rate is very low due to the drug resistance.Therefore,the development of anti-tumor drugs with low side effects and high efficiency is essential for the treatment of gastrointestinal cancers.Among them,1,2,3-triazole heterocycle is one of the most common choice of medicine in anti-cancers’ drugs research,and it has also been widely used in anti-cancer treatment,especially in drug-resistant cancers.Current study is jointly performed with Beijing University of Traditional Chinese Medicine,which has synthesized a variety of 1,2,3-triazole derivatives and verified their effectiveness in prostate,breast and other cancers.The triazole derivative 1988(Compound 1988)showed vigorous anti-tumor activity.Therefore,in current study we selected for the follow-up and detailed analysis.The cells lines of human gastric adenocarcinoma SGC-7901 cells,human esophageal cancer EC-9706 cells,human colon cancer HT-29 cells,and normal gastric epithelial GES-1 cells were treated with compound 1988 and 5-fluoropyrimidine(5-FU)respectively,and then the anti-proliferative activity of the compound was evaluated by CCK8.Based on these findings,functional and phenotypic studies were further performed.Moreover,Western blot and immunofluorescence were used to detect the changes of related proteins of Wnt/β-catenin signaling pathway and tubulin.Finally,through the above results were speculated the specific treatment mechanism of compound 1988,which provided an experimental basis for the clinical treatment of malignancies of the digestive tract in the future.Materials and Methods1.Compound 1988 and GES-1 cells,SGC-7901 cells,EC-9706 cells,HT-29 cells were cultured together.Cytotoxicity experiment was used to compare the effects of the compound 1988 on the proliferative activity of SGC-7901 and HT-29 cell lines which were more sensitive to the compound 1988.Moreover,the plate cloning experiment was performed to evaluate the proliferation ability of SGC-7901 and HT-29 cell lines in vitro.2.SGC-7901 and HT-29 cells were co-cultured with compound 1988,using DAPI fluorescent staining to detect the number and morphological changes of nucleus of SGC-7901 and HT-29 cell.3.SGC-7901 and HT-29 cells were treated with compound 1988 and detected the changes in the migration ability of the two cells by wound healing experiment and transwell test.4.SGC-7901 cells and HT-29 cells were co-cultured with compound 1988,and the cell cycle changes were detected by flow cytometry.5.SGC-7901 cells and HT-29 cells were co-cultured with compound 1988,and the changes of tubulin polymerization during mitosis were detected by immunofluorescence assay.6.SGC-7901 cells and HT-29 cells were co-cultured with compound 1988 to study its effect on cycle,apoptosis and Wnt/β-catenin pathways.Western blot was used to detect the expression levels of apoptosis-related proteins cleaved Caspase 9,cleaved PARP in SGC-7901 and HT-29 cells by Western blot;the expression of G2/M-phase related proteins Cyclin B1 and CDK1;and the changes of Wnt/β-catenin pathway-related proteins Axin2 and β-catenin.Results1.After the treatment of compound 1988 and 5-FU on SGC-7901,HT-29,EC-7901 and GES-1 cells respectively,the anti-proliferation ability of compound1988 was obviously better than that of 5-FU.And compound 1988 had a weaker effect on the proliferative potential with GES-1 cells.Furthermore,the results of CCK8 experiment showed that the cell activity of SGC-7901 and HT-29 was significantly inhibited with the increasing of concentration.The IC50 at 24 h was123.20±12.10,77.75±8.74,151.90±13.62,respectively(n M).The inhibitory effect on EC-9706 cells was not obvious in 24 h.2.Further,the results of colony formation assay showed that with treatment of compound 1988,the ability of in vitro proliferation was decreased in a concentration-dependent manner,and the difference was statistically significant(P<0.001).3.After the treatment of compound 1988 on SGC-7901 and HT-29 cells,the migration ability of SGC-7901 cells was significantly inhibited(P<0.001);while there was no significant difference in HT-29 cells(not shown).4.After the treatment of compound 1988 on SGC-7901 and HT-29 cells,the tubulin polymerization ability of two cells was significantly inhibited in a concentration-dependent manner.5.Compound 1988 was incubated with SGC-7901 and HT-29 cells.Compared with the blank control group,as the treatment concentration increased,the proportion of cells in the G2/M phase increased,and the proportion of cells in the S phase decreased correspondingly.Further analysis of the G2/M phase related proteins Cyclin B and CDK1 showed significantly down-regulation as compared with the blank control group(P<0.05).6.After the treatment of compound 1988 on SGC-7901 and HT-29 cells,the numbers of both cells decreased in a dose-dependent manner.Among them,the nucleus morphology of SGC-7901 cells changed significantly(pycnosis and nuclear fragmentation increased significantly).Furthermore,the expression of apoptosis-related proteins Cleaved Caspase 9 and Cleaved PARP was significantly higher in a concentration-dependent manner than those of the control group(P<0.05).7.Compound 1988 was incubated with SGC-7901 and HT-29 cells.The expression of wnt/β-catenin pathway related protein Axin 2 was up-regulated compared with the blank control group,while the expression of β-catenin was down-regulated compared with the blank control group.When the compound 1988 concentration reached 0.2 μM,the expression of Axin 2 and β-catenin was significantly up-regulated and down-regulated respectively(P<0.001).Conclusions1,2,3-triazole derivatives 1988 inhibits the proliferation,migration,invasion,cell cycle and promotes cell apoptosis of colon cancer HT-29 cells and gastric cancer SGC-7901 cells by regulating the polymerization of intracellular tubulin and the activity of Wnt/β-catenin signaling pathway.Therefore,this compound may be considered as a potential anti-tumor agent.