Anticancer Effect In Vivo And Vitro And Cellular Mechanisms Of Novel 7-Azaisoindigo Derivative

Novel 7-Azaindirubin derivatives(1-9) were synthesized to investigate their anti-tumor effects and possible mechanisms,which will be helpful for developing new anticancer agents with Self-dominated Intellectual Property Right.Among these derivatives,four kinds of derivatives were accepted for good solubility. Four human cancer cell lines including Hela,Eca109,HepG2 and A459 cells were exposed to these compounds.Cells proliferation were detected by MTT assay and the 50% inhibitory concentration(IC₅₀) was calculated;Then No.6 derivative(7-AI-b) was chosed to investigate its effect and mechanism of antitumor.The morphological and nuclei changes in H₂B-GFP-labled HeLa cells were observed by using a live cell system(LCS). A549 cells were stained with Hoechest 33342 and nucleus morphological changes were observed.Cell apoptosis was detected by Annexin-V/PI staining;Releasing of Cytochrome C(Cyto C) from mitochondria to cytosol and activation of caspase-3 was examined by western blot;Intracellular ROS level was detected with DCFH-DA staining;mitochondrial membrane potential was observed with JC-1 staining;ATP content was detected by luciferase assay;intracellular free Ca²⁺ content was detected with Fluo-3 staining;cell cycle was analyzed following PI staining.The formation of autophagic vacuoles was assessed by staining cells with MDC;Expressions of autophagy-related protein LC3 were analysised by western blot;In vivo,the activity of 7-AI-b against human hepatoma carcinoma(Hepes) xenografts was examined.In vitro,7-AI-b exhibited significant inhibition on cancer cell proliferation,especially on A549 cell proliferation in a dose- and time-dependent way.The IC₅₀ of 7-AI-b on different cells were between 28-40μmol/L.Treated with 7-AI-b,it was found that nucleus morphology of cancer cells were changed and the rate of apoptotic cells were significantly increased.7-AI-b could induce release of Cyto C from mitochondria to cytosol and activate caspase-3,which indicated that aopotosis happened via mitochonria-mediated way.Further study showed that 7-AI-b could induce mitochondrial dysfunction:including elevated ROS, overloaded intracellular Ca²⁺,decreased mitochondrial membrane potential and ATP level. 7-AI-b induced G₀/G₁ phase cell cycle arrest which is the same as the indirubin.It enhanced fluorescence intensity of monodansylcadervarine(MDC),as well as elevated expression of autophagy-related protein LC3.Furthermore,our data indicated that 7-AI-b triggered apoptosis through ROS.In vivo,the anticancer activity of 7-AI-b against human hepatoma carcinoma(Hepes) xenografts was comparable to that of 5-Fu and caused no significant adverse effects.These data suggested that 7-AI-b had promising anticancer activity,by showing:In vivo inhibitory effects against human hepatoma cancer xenograft,and in vitro anticancer effects on different cell lines.The mechanism underlying its anticancer effect was related to ROS-induced mitochondrial dysfunction,apoptosis and autophagy induction and G₀/G₁ phase arrest.