Studies On The Mechanism Of2,2-dimethylbenzopyran Derivatives-induced Cytotoxicity In Hela Cell

2,2-dimethylbenzopyran derivatives including natural products and designed structures, were reported to present several biological activities, such as potassium channel activating, antitumor, antibacterial and antioxidant properties. Chemists are thus interested in large collections of products based on2,2-dimethylbenzopyran template that possess greater diversity and incorporate optimized pharmacological properties into their structures. In the present study, a series of newly synthetic2,2-dimethylbenzopyran derivatives were evaluated for the cytotoxicity on human cervical carcinoma HeLa cells, and the mechanism of their cytotoxic effect was further investigated.At first, seven2,2-dimethylbenzopyran derivatives (compound1-7) with different substitutions at6position were synthesized and their cytotoxic effects on HeLa cell were assessed by WST-8assay. A preliminary screening of these compounds showed that1-((3,S,4R)-4-(2-ethoxy-4-methyl-1H-pyrrol-1-yl)-3-hydroxy-2,2-dimethylchroman-6-yl)-3-phenylurea (C6) had the highest cytotoxicity (IC50of138μM) and significantly inhibited HeLa cell proliferation after36h. In an effort to understand the cytotoxic mechanism of C6, we examined its effect on apoptosis and cell cycle distribution. Our results showed that C6induced marked changes in apoptotic morphology and significantly increased early apoptosis of HeLa cell after48h by using Annexin V-FITC/PI dual staining assay. This apoptotic induction was associated with an increase in Bax expression, a decrease in Bcl-2expression, release of cytochrome c and subsequent activation of caspase-9and-3, which indicated that C6induced apoptosis via caspase-and mitochondria-dependent pathway. By DNA content analysis and [3H]thymidine incorporation assay, C6was found to induce G1phase arrest of cell cycle, accompanied by a decrease in the S phase to prevent DNA synthesis after only24h of treatment. In addition, C6caused significant DNA damage, as detected by the alkaline comet assay.Since the cytotoxicity of C6is not high enough to act as an anticancer candidate, another group of2,2-dimethylbenzopyran derivatives (compound8-14) with different substitutions at4position were synthesized to screen for reagent with higher activity against HeLa cell. The results of WST-8assay showed that (3R,4S)-2,2-dimethyl-6-nitro-4-(4-(3-(trifluoromethyl)phenyl)piperazin-1-yl)chroman-3-ol (C13) significantly increase the cytotoxicity against HeLa cell with IC50value of17μM. With morphologic observation and Annexin V-FITC/PI dual staining assay, it is confirmed that C13induced apoptosis in HeLa cell. This apoptotic induction was associated with an increase in Bax expression, a decrease in Bcl-2expression, loss of mitochondrial membrane potential, release of cytochrome c to cytosol and subsequent activation of caspase-9and-3, suggesting that C13induced apoptosis via mitochondrial pathway. Furthermore, the procaspase-8expression level was decreased, which indicates that the death receptor pathway is also involved. One of the important reasons to induce apoptosis is DNA damage. The alkaline comet assay indicated that C13caused significant DNA damage after12h, and the p53expression was increased, which in turn up-regulated p21, the target gene of p53, after24h treatment. DNA content analysis showed that C13arrested HeLa cell at G1phase to inhibit the transition from G1to S phase. ROS was one among the most important reasons to cause DNA damage and correlated with p53. However, ROS was not involved in the apoptosis induced by C13as our data showed. In addition, C13was found to inhibit the efflux of rhodamine123, a substrate of the multidrug resistance protein P-gp (P-glycoprotein). The effect of C13on P-gp and multidrug resistance of tumor cell needs to be further investigated.