Inhibitory Effect Of Mogroside V On Colorectal Cancer Cells And Its Molecular Mechanism

Siraitia grosvenorii is one of the first plants in China to be included in the"medicinal and edible" category.It is mainly grown in Guangxi province and often used to moisten throat and relieve cough.Mogroside V(MGV)is a triterpenoid saponins in Siraitia grosvenorii.It has been approved as a sweetener in food and has many biological activities,such as anti-inflammation,hypoglycemia and antioxidation.Colorectal cancer is considered to be one of the malignant tumors of digestive tract with high morbidity and mortality in the world.Studies have shown that MGV has a certain preventive effect on lung cancer,pancreatic cancer and cervical cancer.However,there are few studies on the efficacy and molecular mechanism of MGV in preventing colorectal cancer.In this study,colorectal cancer cell HCT116 was used as the cell model to investigate the inhibitory effect and molecular mechanism of MGV on colorectal cancer.It has been found that MGV has no significant cytotoxicity to normal human umbilical vein endothelial cell HUVEC,but has significant cytotoxicity to human colorectal cancer cell HCT116.Flow cytometry showed that 58.30%of HCT116 cells were blocked in G1 phase by 100 μg/mL MGV,which increased the percentage of cells by 9.80%compared with the control group.The intervention of MGV also inhibited the migration ability of HCT116 cells.The results of Western Blot showed that the MGV significantly activated the phosphorylation of JNK and the protein expression of p21,and the expression levels were up-regulated by 1.50 times and 3.69 times,respectively.The treatment also upregulated the expression of p16,p18 and p27,inhibited the expression of Cyclin D1 and Cyclin E1,and inhibited the phosphorylation of CDK2 and CDK6.These results indicated that the MGV could inhibit the proliferation of colorectal cancer cells by regulating cell cycle.The JNK inhibitor SP600125 was used to interfere with the JNK activity induced by MGV in HCT116 cells.The results showed that after inhibiting JNK activity in HCT116 cells,the activation of p16 and p18 was reduced by 62.96%and 68.59%,and the inhibition of Cyclin D1 and p-CDK6 was decreased by 75.13%and 64.37%.These evidences confirmed that the inhibition of the proliferation of HCT116 cells by MGV might be caused by targeting JNK signal regulation.The apoptosis of HCT116 cells also occurred after the treatment of MGV.Flow cytometry further confirmed that the treatment of MGV promoted the late apoptosis and death of HCT116 cells.After 100 μg/mL MGV treatment,the number of late apoptosis cells and dead cells increased by 1.20%and 10.80%,respectively,while the number of living cells decreased by 14.00%.Western Blot analysis showed that the intervention of MGV significantly activated mitochondria-dependent apoptosis,which was manifested in the up-regulation of Bax/Bcl-2 ratio,promotion of Cyt-c protein expression,and stimulation of Caspase family protein activation.PFT-α,a p53 inhibitor was used to interfere with p53 transcriptional activity induced by MGV in HCT116 cells.It was found that the apoptosis induced by MGV was weakened in HCT116 cells.Western Blot results showed that after inhibiting the transcriptional activity of p53 in HCT116 cells,the increase of Bax/Bcl-2 ratio induced by MGV and the activation of Cyt-c were reduced by 64.10%and 48.90%.These evidences confirmed that the promotion of apoptosis of HCT116 cells by MGV might be caused by the targeted regulation of p53 signal.Finally,high-throughput sequencing was used to investigate the effect of MGV on RNA level of HCT116 cells,and 88 genes with significant changes were found.GO analysis and KEGG analysis showed that these genes were mainly enriched in the signaling pathways of colorectal cancer,MAPK,apoptosis,cell cycle and p53.The protein interaction network prediction of these genes showed that the interaction network centered on p53,p16 and p18 was formed.However,the downstream AP-1 of JNK can bind to multiple promoter sites of p16 and p18,so we believe that p53 and JNK may play a key role in the regulation of HCT116 cells by MGV.RT-qRCR was used to verify the mRNA expression of some differential genes,and the results were consistent with the transcription profile.Conclusions:MGV inhibited the proliferation and motility of HCT116 cells,which may be targeted to JNK signal,thus enhancing the expression of CDKIs,inhibiting the activation of CDKs and Cyclins,and finally blocking the cell cycle.MGV could promote mitochondria-dependent apoptosis in HCT116 cells,possibly by targeting the p53 signaling pathway.Transcriptome data showed that 88 genes showed significant changes in HCT116 cells under the intervention of MGV,and these differential genes were mainly concentrated in p53,MAPK,apoptosis,cell cycle and other signaling pathways.