Apoptosis And Mechanism Study Of EGCG Induced Human Uterine Cervix Cancer HeLa Cells

Objective: Uterine cervix cancer is one of common malignant tumour and seriou threaten national femme healthy. Chemotherapy elevated remission rate of uterine cervix cancer in middle-advanced stage, but the curative effect of part patients wasn't satisfactory because of secondary effect of chemotherapy drug and drug fast of tumour cell. To investigate mechanism of Epigallocatechin-3-gallate (EGCG) induced apoptosis of uterine cervix cancer HeLa cells and provid experimental information for development and search new drug.Methods: MTT method detected 50% inhibiting concentration (IC50) that EGCG inhibted HeLa cellular growth. The growth of HeLa cell inhibited by EGCG was observed through the cellular growth curve, colony formation and light microscope. The cellular apoptosis of EGCG induced HeLa cells were checked by Hochest33258 staining, flow cytometry (FCM) and DNA gel electrophoresis. The expression of Caspase-3 gene and protein was respectively analyzed by RT-PCR, immuocytochemistry and Western- blotting. Meanwhile, the expression of active Caspase-3 protein and spallation of PRAP-1 protein was detected by Western- blotting. In addition, the expression alteration of PRAP-1 protein between nucleus and endochylema of HeLa cells treated with EGCG was observed by immuocytochemistry.Results: 1. IC50 that EGCG inhibted the growth of HeLa cells was 36.82 mg·L^-1. 2. The results of cellular growth curve, colony formation and light microscope showed that EGCG depressed growth of HeLa cell was increased with it concentration increased and treatment time extended (n=3, P<0.05). 3. The result of Hochest 33258 staining displayed that the typical morphology characteristic of cell apoptosis example nuclear fragmentation and karyopyknosis occurred in HeLa cell. The number of cellular apoptosis increased with EGCG concentration increased (n=3, P<0.05).4. The result of flow cytometry (FCM) displayed that the apoptosis rate of HeLa cell gradually increase with EGCG concentration increased and EGCG induced HeLa cell apoptosis for concentration dependent it (n=3, P<0.05). 5. The result of DNA gel electrophoresis showed that DNA ladder strap was detect in HeLa cell treated by EGCG and it is more obviously with its concentration increased. 6. The results of RT-PCR discovered that the expression of Caspase-3 gene in HeLa cells obviously degraded with EGCG concentration increased (P<0.05). 7. The western-blotting results exhibited that expression of Caspase-3 protein in HeLa cells obviously increased with EGCG concentration increased (P < 0.05) and EGCG induced expression of Caspase-3 protein for concentration dependent it. 8. The results of immuocytochemistry exhibited that the expression of Caspase-3 protein lay in cytoplasm and EGCG induced expression of Caspase-3 protein with its concentration increased (P<0.05). 9. The western-blotting results showed the expression of active Caspase-3 protein and spallation of PRAP-1 protein was increased by EGCG , depend on its concentration, in HeLa cells (P<0.05). 10. The results of immuocytochemistry discovered that the expression of PARP-1 protein was in nucleus and endochylema of HeLa cells. With EGCG concentration increased, the expression of PARP-1 protein was up-regulated in endochylema and down-regulated in nucleus (P<0.05).Conclusion:1. EGCG inhibited the growth of uterine cervix cancer HeLa cells to depend its concentration.2. EGCG can induce HeLa cellular apoptosis in vitro.3. EGCG depressed the expression of Caspase-3 gene and protein in HeLa cells to accelerate the spallation of PRAP-1 protein and to provoke the cellular apoptosis and inhibited the growth of HeLa cells.