| Objective: To study the inhibition effect of resveratrol derivatives combined with Caveolin-1 on human gastric cancer SGC7901 cell line in vitro, investigate whether or not enhancement intracellular accumulation of caveolin-1 mutants on resveratrol endocytosis, and whether or not energy required for This process.Methods: Plasmids pcDNA3.1/ NT-GFP-CAV1 (WT-CAV1 gene), CAM1 (CAV1 gene absence 81-101 amino acids Scaffold-domain), CAM2 (CAV1 gene absence 143-156 amino acids as lipid-binding domain), GFP control and CAV1-BLOCK-iTTM Lentiviral RNAi were transfected into SGC7901 cells respectively. Then we use resveratrol and resveratrol derivatives to treat the cell groups. The growth inhibition of SGC7901 cell groups were measured by MTT assay. The Phase distribution of cell cycle and apoptosis ratios were analyzed by flow cytometry (FCM). Spontaneous apoptosis of cells was detected by AO/EB. Detected concentrations of drug in the cytoplasm by HPLC after treated with resveratrol and resveratrol derivatives for some time. The proteins localization and expression of Caveolin-1 and ATPase was assessed by indirect immunofluorescence. The proteins expression of caveolin-1 and ATPase was determined by Western Blot.Results:MTT assay and the flow cytometry analysis showed that resveratrol derivatives induced SGC7901 cells apoptosis in both concentration- and time-dependent way, which 3,4',5-tetranm-ethoxystib highest activity, resveratrol dansyl again, but stronger than RES. Expression of caveolin-1 could enhance its induction of apoptosis. The FCM showed a significant increase of S cell population after the treatment of above 50μM 3,4',5-tetranm-ethoxystib for 48h or more in comparison with the control. AO / EB showed that SGC7901 group mainly to VA, the expression of CAV1 group had significantly increased apoptosis, and mainly to NVA. High performance liquid chromatography (HPLC) demonstrated that intra-cellular drug concentration of RES, DES, 3,4',5-tetranm-ethoxystib is increased about 1~2 fold in cells stably expressing CAV1 or CAM1 (a scaffolding domain 81-101AA-defective CAV1 mutant) compared to the untransduced SGC7901 cell line or after transduction with the green fluorescent protein (GFP) control vector. The increased intra-cellular transport of drug was abolished in cells stably expressing CAM2 (a cholesterol shuttle domain 143-156AA-defective CAV1 mutant) or RNAi. The dansyl- resveratrol fluorochrome probe was synthesised to show the endocytosis and disposition of resveratrol.The results demonstrated that over-expression caveolin-1 and RES displayed the similar distribution in CAV1 group.While in CAM2 group, the co-localization pattern could not be observed. The same co-localization of caveolin-1, ATPase and RES could be observed. ATPase level over with Caveolin-1 expression increased, a positive correlation. Western blot analysis showed that Res derivatives induced caveolin-1 and ATPase expression respectively. Over-expression of Caveolin-1 can enhance this induction.Conclusion:1.Resveratrol derivatives can significantly induced SGC7901 cells apoptosis in both concentration- and time-dependent way, which 3,4',5-tetranm-ethoxystib highest activity, resveratrol dansyl again, but stronger than RES.2.We observed that over-expression Caveolin-1 can increase the cytotoxic and inhuction apoptosis effect of resveratrol derivatives on SGC7901 was associated with lipid-binding domain of Caveolin-1 gene improving the resveratrol derivatives endocytosis and advancing the drug accumulation in tumor cells by active transport. |
