Study On Antitumor Effects Of Vitamin C And Vitamin C Combined With Ddp, As₂o₃ On Lung Cancer Cell Line A549

Objective To study the inhibition, apoptosis and underlying mechanism of proliferation inhibition and apotosis mediated by Vitamin C, Vitamin C with DDP , Vitamin C with Arsenic Trioxide to lung carcinoma.Methods A549 cell lines were cultured in vitro,and incubated with Vitamin C.The cell viability was measured by growth curve and clonogentic assay, Flow cytometry were used to detect apoptosis and analyze cell cycle. The expression of Caspase-3 mRNA,Survivin mRNA, MRP1mRNA, MDR1mRNA were measured by reverse transcription-polymerase chain reaction(RT-PCR). The expression of Caspase-3protein,Survivin protein were measured by Western blot.Results 1.growth curve :Vitamin C inhibited the growth of A549 cell lines in dose-time-depedented maners(P<0.05), however,Vit C in low doses(40ug/ml) could not inhibit that growth(P>0.05). DDP inhibited the growth of A549 cell lines in dose-time- depedented maners(P<0.05), DDP in low doses(0.4ug/ml) can not inhibited the growth of A549 (P>0.05) ,but can signifacntly inhibited the growth of A549 with Vit C(400ug/ml) (P<0.05). AS₂O₃ inhibited the growth of A549 cell lines in dose-time-depedented maners(P<0.05), AS₂O₃ in low doses(0.04ug/ml) can not inhibited the growth of A549(P>0.05) ,but can signifacntly inhibited the growth of A549 with Vit C(400ug/ml)(P<0.05).2.clonogentic assay: Vitamin C inhibited the colony of A549 cell lines in dose-time- depedented maners(P<0.05), however,Vit C in low doses(40ug/ml) could not inhibit colony (P>0.05). DDP inhibited the colony of A549 cell lines in dose-time-depedented maners (P<0.05), DDP in low doses(0.4ug/ml) can not inhibited the colony of A549 (P>0.05) , but can signifacntly inhibited the colony of A549 with Vit C(400ug/ml) (P<0.05). AS₂O₃ inhibited the colony of A549 cell lines in dose-time-depedented maners(P<0.05), AS₂O₃ in low doses(0.04ug/ml) can not inhibited the colony of A549(P>0.05) ,but can signifacntly inhibited the colony of A549 with Vit C(400ug/ml)(P<0.05).3. Flow cytometry: cells major stagnation was blocked in the G1 and S phase and the apoptotic rate increased after incubated with Vit C(400μg/ml)6h,12h,24h,48h. More cells stayed in G1 phase and got to apoptosis when incubated with Vit C combined with DDP,compared with they were incubated with DDP singly. More cells stayed in G1 phase and got to apoptosis when incubated with Vit C combined with AS₂O₃,compared with they were incubated with AS₂O₃ singly.4. RT-PCR: The expression of Caspase-3mRNA and MDR1mRNA were up-regulated by Vit C; The expression of Survivin mRNA and MRP1mRNA were unchanged after A549 incubated with Vit C(400μg/ml)6h,12h,24h,48h;5. Western blot: The expression of Caspase-3 protein were up-regulated by Vit C; The expression of Survivin protein were unchanged after A549 incubated with Vit C(400μg/ml)6h,12h,24h,48h;Conclusion This in vitro study proved that Vitamin C combined with AS₂O₃or DDP could clearly synergistic inhibit the lung cancer A549 cell proliferation and trigger apoptosis. That synergistic mechanism might be to extend the cell cycle and increase the expression of Caspase-3mRNA to promote apoptosis of tumor cells.