Anti-tumor Activity And Mechanism Research Of 9-Substituted FL118 Derivatives

Recent studies have shown that FL118 was a camptothecin derivative with high antitumor activity.FL118 not only showed excellent anticancer activity in human colon cancer and head and neck cancer,but also was very sensitive to cancer cell lines resistant to clinically used camptothecin derivatives(Irinotecan and Topotecan).However,the FL118,as a potential anti-tumor candidate,showed poor solubility in both the organic phase and the aqueous phase,and researchers have improved their solubility and bioavailability by means of formulation.In order to explore more efficient and low-toxic FL118 derivatives and improve their bioavailability,our lab have designed and synthesized a series of 9-substituted FL118 derivatives.Through the preliminary screening of anti-tumor activity in vitro and the screening of anti-tumor activity in nude mice,it was found that the new derivatives(9-Q10,9-Q18and 9-Q20)showed superior anti-tumor activity.Researches on the biological properties and anti-tumor mechanisms of 9-Q10,9-Q18 and 9-Q20 will be conducted in this paper.Objective:On the one hand,the cytotoxicity and cellular uptake of FL118 and9-Q20 in 2D and 3D different cell models in vitro were evaluated to predict the improvement of the absorption in vivo of the 9-substituted FL118 derivative compared with raw FL118.On the other hand,the effects of FL118,9-Q10,9-Q18 and 9-Q20on proliferation,metastasis and apoptosis of HCT116 cells were investigated by cell and molecular biology methods to explore its anti-tumor mechanism,and provide experimental basis for the subsequent development of new derivatives.Methods:Firstly,the anti-proliferation activity of FL118,9-Q10,9-Q18 and9-Q20 against HCT116,MCF-7,HepG2,HeLa and A549 cell lines was evaluated by MTT assay.Secondly,3D Caco-2 cell model and HPLC analysis method for FL118and 9-Q20 content determination were established and optimized;the 2D and 3D cell models were used to investigate the effects of FL118 and 9-Q20 concentration,incubation time,temperature and transporter inhibitor on cellular uptake,and to investigate the differential compound uptake in different cell lines.Finally,MTT assay,cell scratch assay,Hoechst 33258 fluorescence staining,DCFH-DA probe method and caspases activity kit assay were used to investigate the effect of FL118,9-Q10,9-Q18and 9-Q20 on the proliferation,migration ability and apoptosis of HCT116 cells;the levels of apoptosis-related proteins such as XIAP,Mcl-1,Survivin,Akt,p-Akt,Cyt-c,Bcl-2 and Bax were detected in HCT116 cells by western blot to study the mechanism of FL118,9-Q10,9-Q18 and 9-Q20 inducing apoptosis of HCT116 cells.Results:(1)The 3D Caco-2 cell model was successfully developed and optimized.The Caco-2 cells cultured at a seeding density of 2.5×10~5 cells/mL and an incubation time of 72 h have formed relatively tight and uniform spheroids.And the cell viability was more than 90%.The 72 h MTT assay showed that FL118 and 9-Q18effectively inhibited the proliferation of these five cell lines at nM level;FL118 and9-Q20 both had superior anti-proliferation effect on HCT116 than HepG2 cells.The 8h MTT assay showed that no significant cytotoxicity of FL118 and 9-Q20(0.1-1?M)was observed in 2D and 3D cell models,and the cell viability in 3D cell models was greater.(2)The uptake of FL118 and 9-Q20 in 2D and 3D cell models was time and concentration dependent pattern,and the uptake of 9-Q20 was more than FL118 in 2D and 3D cell models.At the early time of incubation,the uptake rate of FL118 and9-Q20 were faster in 2D Caco-2 cell model.The uptake of FL118 and 9-Q20 in 3D Caco-2 cell model were significantly improved over time.The accumulation of 9-Q20in 2D and 3D Caco-2 cell models were decreased by P-gp and BCRP,which indicated that the cellular uptake of 9-Q20 was related to P-gp and BCRP(efflux pump proteins).Verapamil(P-gp inhibitor)and Iressa(BCRP inhibitor)more significantly increased the uptake of 9-Q20 in 3D Caco-2 cell model.In contrast,P-gp and BCRP had no effect on the accumulation of FL118 in 2D and 3D Caco-2 cell models,which indicated that the cellular uptake of FL118 was independent of P-gp and BCRP.Although the uptake of FL118 and 9-Q20 in HepG2 cells was greater than in HCT116cells,FL118 and 9-Q20 were more effective against HCT116 cells than HepG2 cells.(3)After incubation of FL118,9-Q10,9-Q18 and 9-Q20 with HCT116 cells,nucleus shrank at different degrees,intracellular ROS levels and Caspase-3,Caspase-8 and Caspase-9 activities were increased.These results indicated that FL118 and 9-Q10,9-Q18 and 9-Q20 induced apoptosis of HCT16 cells.In addition,cell scratch assay showed that FL118 and 9-Q10,9-Q18 and 9-Q20 inhibited the migration of HCT116cells.Western blot analysis showed that FL118 and 9-Q10,9-Q18 and 9-Q20 all decreased the expression of XIAP,Mcl-1 and Survivin in HCT116 cells.Both FL118and 9-Q18 reduced the expression of p-Akt,Akt and Bcl-2 in HCT116 cells and increased the expression of apoptosis-related factors Cyt-c and Bax in the mitochondrial pathway.Conclusions:The absorption of 9-Q20 was superior to FL118 in 2D and 3D cell models,which indicated that the 9-position modified FL118 derivatives in this study were favorable for biofilm absorption and transport.The 3D cell models provided more potency information on the cytotoxicity and the process of cellular uptake of FL118 and 9-Q20,which objectively reflected the drug sensitivity and resistance in vivo compared with 2D cell models.The cellular uptake of 9-Q20 was related to P-gp and BCRP efflux pump proteins,while the cellular uptake of FL118 was independent of P-gp and BCRP.9-Q18 could effectively inhibit the proliferation and migration of HCT116 cells,and could decreased the expression of XIAP,Mcl-1,Survivin,p-Akt and Akt,which were proved to induce apoptosis by inhibiting PI3K/Akt/Survivin signaling pathway.In addition,9-Q18 reduced the expression of Bcl-2,increased the expression of Bax and Cyt-c,and activated Caspase3/8/9,suggestting that 9-Q18might induce apoptosis by participating in the mitochondrial pathway.