Study On Antitumor Effects Of As₂O₃ Combined With Vitamin C On Resistant Human Lung Cancer

Objective:To study the effect of arsenic trioxide(As₂O₃) , AS₂O₃with Vitamin C on drug resistant lung carcinoma cell A549 ( A549/R cell ) and the subcutaneously transplanted tumor of drug resistant human lung tumors in nude mice. Further explore the underlying mechanism and synergy, and provide a theoretical basis for arsenic trioxide-based clinical treatment program. Methods:1. A549/R cell lines were cultured in vitro,and incubated with As₂O₃ and compared 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. 2. The subcutaneously transplanted tumor models of drug resistant human lung tumors in nude mice were established. Inject different concentrations of As₂O₃ and compared with Vitamin C, rendering the tumor growth curve, and calculated the tumor inhibition.3. Stripping nude mice tumors, hematoxylin-eosin (HE) staining, observed under light microscope to evaluate the pathological changes of tumor. Immunohistochemical staining of tumor tissue,Observed the expression of Survivin, Caspase-3 protein. 4. Extract total RNA and protein in tumor, reverse transcription-polymerase chain reaction (RT-PCR) detection of Survivin mRNA, Caspase-3 mRNA and VEGF mRNA expression, Western blot detection of Survivin protein, Caspase-3 protein, and VEGF protein expression. Results:1. Growth curve : As₂O₃ inhibited the growth of A549/R cell lines in dose-time-depedented maners(P<0.05), As₂O₃ in low doses(0.04μg/ml,0.4μg/ml) can not inhibited the growth of A549/R (P>0.05) ,but can signifacntly inhibited the growth of A549/R with Vit C (400μg/ml) (P<0.05). 2. Clonogentic assay: As₂O₃ inhibited the colony of A549/R cell lines in dose-time-depedented maners(P<0.05), As₂O₃ in low doses (0.04μg/ml) can not inhibited the colony of A549/R (P>0.05), but can signifacntly inhibited the colony of A549/R with Vit C(400μg/ml)(P<0.05). 3. Flow cytometry: More cells stayed in G₁ phase and got to apoptosis when incubated with AS₂O₃ combined with Vit C, compared with they were incubated with As₂O₃ singly. 4. Inhibitory rate and tumor growth curve: 3mg/kg As₂O₃ group can effectively inhibit tumor growth, 1.5mg/kg As₂O₃ combined with vitamin C group can significantly inhibited. 5. Pathological and immunohistochemical staining: 3mg/kg As₂O₃ group make tissue necrosis, and increase apoptosis. 3mg/kg As₂O₃ and the combine group Caspase-3 protein positive rate increased, Survivin protein positive rate reduce. 6. RT-PCR result: The expression of Caspase-3mRNA were up-regulated by 3mg/kg As₂O₃, and 1.5mg/kg As₂O₃ combine with Vit C group. And down-regulated the expression of Survivin mRNA and VEGF mRNA. Compared with the others,combination group has significant differences. 7. Western blot result: The expression of Caspase-3 protein were up-regulated by 3mg/kg As₂O₃, and 1.5mg/kg As₂O₃ combine with Vit C group. And down-regulate the expression of Survivin protein and VEGF protein. Compared with the others,combination group has significant differences. Conclusion:This study proved that As₂O₃ combined with Vitamin C,could clearly synergistic inhibit the drug resistant lung carcinoma cell A549/R (A549/R cell)and subcutaneously transplanted tumor proliferation and trigger apoptosis. That synergistic mechanism might be to extend the cell cycle and increase the expression of Caspase-3, down-regulate Survivin and VEGF , and promote apoptosis of tumor cells, inhibit the vascular of tumor.