The excitation wavelength was 485 nm and the emission wavelength was 565 nm. Immunofluorescent microscopy of H2AX foci Cells were fixed with paraformaldehyde for 30min and washed 3 times with PBS. foci in hepatoma cells, and the resveratrol and cisplatin combined treatment results in much more H2AX foci formation than either resveratrol or cisplatin treatment alone. Furthermore, our studies show that over-expression of ASCT2 can attenuate cisplatin-induced ROS production, H2AX foci formation and apoptosis in human hepatoma cells. Collectively, our studies suggest resveratrol may sensitize human hepatoma cells to cisplatin chemotherapy via glutamine metabolism inhibition. H2AX immunofluorescent staining by IOD/Area of C3A and SMCC7721 cells. (G) Picture of H2AX immunofluorescent staining of C3A and SMCC7721 cells. (H) Western blott assays were performed to determine the expression levels of mitochondria and kytoplasm cytochrome c, caspase-9 and cleaved caspase-3 in C3A and SMCC7721 cells. -Actin was used as a loading control. *P 0.05, **P 0.01, ***P 0.0001. Resveratrol increases ROS production in human hepatoma cell lines We Rabbit Polyclonal to PFKFB1/4 performed flow cytometric analysis to measure ROS production in cisplatin treated C3A and SMCC7721 cells with or without reveratrol treatment for 24 hours. The results showed that resveratrol and Azelaic acid cisplatin combined treatment markedly increases ROS production in C3A and SMCC7721 cells compared with those cells treated with resveratrol or cisplatin alone (Fig. 2D, E). These results suggest that increasing of ROS production may be a key role played by resveratrol to enhance cisplatin toxicity. Resveratrol increases DNA damage in human hepatoma cell lines Given that DNA damage is the major cause of cisplatin-induced toxicity, we hypothesized that resveratrol may enhance H2AX-induced DNA damage To test this hypothesis, we performed H2AX foci assay to examine whether RV treatment enhances DNA damage in CDDP treated human hepatoma cells. The Azelaic acid results showed that resveratrol and cisplatin combined treatment results in more H2AX foci formation than resveratrol and cisplatin treatment alone (Fig. 2F, G). CDDP-induced DNA damage will ultimately trigger apoptotic pathways (16). In mitochondrial apoptotic pathways, ROS and DNA activate bcl-2 to promote cytochrome c release. To address this issue, western blot analyses were performed to determine the expression level of mitochondria and kytoplasm cytochrome c, caspase-9 and activated caspase-3. The results show that RV treatment has significant effect on the expression of mitochondria and kytoplasm cytochrome c, caspase-9 and activated caspase-3 (Fig. 2H). These results support the hypothesis that the effect of resveratrol enhancing cisplatin toxicity may be related to increasing ROS-induced DNA damage. Resveratrol decreases the absorption of glutamine by reducing the expression of ASCT2 and improved the anti-tumor activity of cisplatin To determine the Azelaic acid role of glutamine metabolism in RV-mediated CDDP chemosensitization, we transfect pcDNA3.1-ASCT2 into C3A and SMCC7721 cells. Western blot results indicate that the transfected ASCT2 eukaryotic expression vector increases ASCT2 expression in C3A and SMCC7721 cells compared to normal cell and empty vector (Fig. 3A, B). After transfection of ASCT2 expression vectors in C3A and SMCC7721 cells, glutamine metabolism, ROS production, DNA damage and expression of apoptosis-regulating proteins were significantly attenuated (Fig. 4ACG). Moreover, after the recovery expression of ASCT2, the synergistic effect and apoptosis induced effects of resveratrol were lost to cisplatin (Fig. 3CCF). These results prove that ASCT2 is the molecular target of resveratrol. By down-regulating the expression of ASCT2 resulting in inhibiting glutamine metabolism of human hepatoma cell lines, resveratrol improves the sensitivity of tumor cells to cisplatin. Open in a separate window Fig. 3 Enhanced expression of ASCT2 inhibited the synergistic effect of resveratrol on the toxicity of cisplatin on C3A and SMCC7721 cells. (A) C3A cells were transfected with empty vector pcDNA3.1 or pcDNA3.1 + ASCT2. Transfection efficiency is confirmed by Western blot assay. (B) SMCC7721 cells were transfected with empty vector pcDNA3.1 or pcDNA3.1+ASCT2. Transfection.