| Cancer has always been a difficult problem to be solved in the world.The occurrence of cancer is related to many factors.Living habits,eating habits and social environment can become the inducing factors of cancer.By 2020,new cases of colorectal cancer account for 10% of 36 cancer diseases.In China,new cancer cases and their deaths account for 24% and 30% of the world.Among them,the number of deaths from gastrointestinal cancer accounts for 45% of the total deaths,and the number of patients with colorectal cancer ranks second.The occurrence of colorectal cancer can be divided into four steps: polyp,neoplasia,epithelial dysplasia and canceration,so the occurrence of colorectal cancer takes a long time.At present,preventive measures have been used for colorectal cancer screening,but many clinical cases show that when cancer is diagnosed,colorectal cancer has developed to an advanced stage,and it is difficult to clean up the cancerous tissue even through surgical treatment.In the face of this situation,chemotherapy will be used in clinic,but many clinical chemotherapeutic drugs show serious side effects.Therefore,the development of new and efficient chemotherapeutic drugs has become an urgent need.Naphthyridine compounds can be divided into 1,5-naphthyridine,1,6-naphthyridine,1,7-naphthyridine,1,8-naphthyridine,2,6-naphthyridine and 2,7-naphthyridine according to their different positions of nitrogen atoms.Among them,1,8-naphthyridine derivatives are favored by researchers because of their antibacterial,anti-inflammatory,anti-cancer and other biological activities.Due to its good anticancer effect,based on the structure of 1,8-naphthyridine,teacher Yan Shengjiao’s team developed a series of 1,3-diazo(1,2a)-1,8-naphthyridine derivatives.Among many 1,8-naphthyridine derivatives,we found that 6-p-methylbenzoyl-11-methoxy-1,2,3,4-tetrahydrobenzo[g][1,3]diazaro[1,2-a][1,8]naphthyridine,which is named 3u,has good biological activity in many tumor cells.However,the mechanism of 3u in colorectal cancer is still unclear.Based on the above reasons,the work of this study is to explore the mechanism of compound 3u on colorectal cancer.Firstly,through the morphological changes,we found that compound 3u had an effect on colorectal cancer cells HCT116 and HT29.We found apoptotic bodies in both cell lines,and the number of apoptotic bodies gradually increased with the increase of time and drug concentration.It is worth noting that in HT29 cell line,we found that apoptotic bodies appeared in the early stage of drug treatment,but bubble blowing appeared in the late stage of drug treatment,which is a typical feature of pyroptotis.Then we studied the specific mechanism of compound 3u in HCT116 and HT29 cell lines.Firstly,we detected the expression of proteins related to apoptosis signaling pathway by extracting total proteins from HCT116 cells;At the same time,the protein of mitochondria was extracted and the protein expression in mitochondria related apoptosis signal pathway was detected.The cell death mode of 3u in HCT116 cell line was analyzed by flow cytometry and protein expression.We also proved that compound 3u can induce cell death in HCT116 cell line by activating death receptor apoptosis signal.In this apoptosis signal pathway,compound 3u can regulate death receptor DR5 and inhibit the expression of anti-apoptosis related proteins c-FLIP and XIAP.Finally,through the establishment of tumor bearing mouse model,the experiment proved that 3u could also inhibit the growth of HCT116 tumor at the evaluation level in vivo.c-FLIP is the main anti-apoptotic protein and the main factor of drug resistance of cancer cells.3u can inhibit the expression of anti-apoptosis related protein c-FLIP when treating HCT116 cells.The expression of c-FLIP in colorectal cancer cells HT29 was significantly higher than that in HCT116.Therefore,we treated HT29 cells with 3u.3u can induce apoptosis of HT29 cells,but the time of apoptosis is longer than that of HCT116 cells.Therefore,we detected that this may be related to the high expression of c-FLIP.By detecting the expression of apoptosis signal pathway related proteins,compound 3u activates apoptosis protein caspase-8/3 by up regulate the expression of DR5 and down regulate the expression of c-FLIP and XIAP proteins in HT29 cell line,and finally activates death receptor related apoptosis pathway.In addition,by Annexin V/PI staining,we found that some HT29 cells appeared pyroptotis in the late stage of apoptosis.Finally,combined with Rcoglamide to treat colorectal cancer HT29 cells for Western blot,it was found that 3u and Rcoglamide inhibited 3u induced HT29 cell apoptosis,and the expression of DR5 was inhibited.Therefore,3u induced HT29 cell apoptosis was achieved by up regulating DR5.In conclusion,this study found that 1,8-naphthidine derivatives had significant inhibitory effects on colorectal cancer HCT116 and HT29 cell lines.3u induced apoptosis of tumor cells HCT116 and HT29 by up-regulating DR5 death receptor and down-regulating the expression of anti-apoptotic proteins c-FLIP and XIAP.The results suggest that compound 3u has great potential in the treatment of colorectal cancer,and provides a new idea for the development of anticancer drugs with targeted regulation of death receptors and targeted inhibition of anti-apoptotic proteins c-FLIP and XIAP. |
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