Research On Mechanisms Of Apoptosis In Human Colon Cancer Cells Induced By Benzimidazole Acridine Derivative

TRAIL(TNF-related apoptosis-inducing ligand) is an important member of the tumor necrosis factor gene families, which owns selectivity to kill tumor cells, without apparent toxicity on normal cells. Evidence is accumulating that down-regulation or mutation of DRsmight allow malignant cells avoid destruction by the immune system and can give rise to drug resistacne. Therefore, agents that increase the levels of DRs in cancer cells should be investigated.Acridine and its derivatives are types of polycyclic aromatic compounds with π-conjugated structures that possess the ability to intercalate into DNA, subsequently inhibiting topoisomerases, resulting in anticancer effects. Substituted benzimidazole derivatives exhibit various bioactivities, including anti-inflammatory, anti-bacterial, and anti-carcinogenic activities, whose structure can bind with DNA minor groove. In line with the better DNA binding to interfere with the cancer cell replication to achieve the purpose of antitumor, our lab designed and synthesized a series of compounds connected acridine with benzimidazole through different connection, and replaced with different substituents on acridine compounds. Among which, compound 8M showed good inhibitory effect for different cancer cell lines. However, the antitumor activity and mechanism of action of 8M in colon cancer cell line is not clear. In this study, we selected two kinds of colon cancer cell lines, HCT116 and SW480, as the research objects. By determined by MTT experiments confirmed that the 8Mowned good inhibitory effect of these two kinds of colon cancer cell lines. Better inhibited HCT116 cells were selected for the further study of the mechanisms of inhibitory action of 8M. Flow cytometryand DAPI assays demonstrated that 8M exhibited potential antitumor activityby inducing apoptosis in colon cancer cells.Western blot experiments demonstrated that 8M can up-regulate the expression of pro-apoptotic protein DR5, down-regulate the expression of anti-apoptotic protein Bcl-2, and induce the truncation of caspases. Results of QRT-PCR further indicated that 8M can up-regulate the expression of DR4, DR5, and the JNK substrate transcription factor c-Fos. We examined the effects of 8M on the activation of MAPK pathways using western blot.Experimental results show that 8M activated the p38 MAPK and JNK. siRNA knockout experiments in combination with inhibitor experiments confirmed that JNK1 played an important role in 8M-induced apoptosis. MAPK is one of the two downstream substrate of ROS(Reactive oxygen species). Dyeing experiments by fluorochrome stain H2-DCFDA and JC-1 show that 8M can induce ROS production and mitochondrial membrane depolarization. Experiments of ROS scavengers demonstrate ROS plays an important role in 8M-induced apoptosis,phosphorylation of JNK, and expression of apoptotic proteins. Summing up the above, our data provided sufficient evidence that 8M-induced apoptosis in HCT116 cells was strongly associated with activation of the ROS-JNK1 pathway. Besides, we have preliminarily studied the biological mechanisms of Lapatinib and 8M in BT-474, MDA-MB-468 and K562.