[Effects of green tea extract on expression of human papillomavirus type 16 oncoproteins-induced hypoxia-inducible factor-1alpha and vascular endothelial growth factor in human cervical carcinoma cells].
Maneno muhimu
Kikemikali
OBJECTIVE
To investigate the effects of green tea extract (GTE) and its major component (-)-epigallocatechin-3-gallate (EGCG) on the human papillomavirus (HPV) type 16 oncoproteins (E6 and E7)-induced expression of hypoxia-inducible factor-1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) in human cervical carcinoma cells.
METHODS
Human cervical carcinoma cells of the line C-33A were divided to 4 groups to be transiently transfected with the plasmid HA-pSG5-HPV-16E6 containing the HPV type 16 oncoprotein E6, HA-pSG5-HPV-16 E7, or empty plasmid HA-pSG5, or just exposed to the transfection reagent Lipofectamine 2000 as mock transfection control group. Western blotting and ELISA were used to detect the protein expression of HIF-1alpha and the protein expression of VEGF 18, 24, and 48 h after the transfection. Twenty-four hours after the transfection, the transfected-cells were treated with GTE (40 microg/ml) or EGCG (50 micromol/L) for 8, 16, and 24 h respectively, or treated with GTE of the concentrations of 20, 40, and 80 microg/ml or EGCG of the concentrations of 25, 50, and 100 micromol/L respectively for 16 h. The HIF-1alpha and VEGF protein levels were analyzed by Western blotting and ELISA respectively and the HIF-1alpha and VEGF mRNA levels were determined with real-time PCR.
RESULTS
Twenty-four hours after transfection, the HIF-1alpha protein expression of the 16E6 and 16E7 groups were significantly decreased, and the VEGF protein levels of the 16E6 and 16E7 groups were (870+/-66) and (1487+/-51) pg/ml respectively, both significantly higher than that of the empty plasmid group [(366+/-65) pg/ml, both P<0.01]. Treated by 40 microg/ml of GTE or 50 micromol/L of EGCG for 16 h, (1) the HIF-1alpha protein levels of the 16E6 and 16E7 groups were significantly decreased; (2) the VEGF protein levels of the 16E6 group were (476+/-34) and (477+/-65) pg/ml respectively, and the VEGF mRNA levels of the 16E6 group were 1.208+/-0.196 and 1.174+/-0.208 respectively, all significantly lower than those of the untreated group [(870+/-66) pg/ml and 1.805+/-0.081 respectively, all P<0.01]; and (3) the VEGF protein levels of the 16E7 group were (649+/-55) and (514+/-37) pg/ml respectively, and the VEGF mRNA levels of the 16E7 group were 1.442+/-0.136 and 1.399+/-0.064 respectively, all significantly lower than those of untreated group [(1487+/-51) pg/ml and 2.123+/-0.120 respectively, all P<0.01]. The 80 microg/ml of GTE or 100 micromol/L of EGCG showed much stronger effects on the inhibition of VEGF protein expression than 40 microg/ml of GTE or 50 micromol/L of EGCG (both P<0.01).
CONCLUSIONS
GTE and EGCG can remarkably inhibit the HPV-16 oncoproteins-induced expression of HIF-1alpha protein, and VEGF protein and mRNA in human cervical carcinoma cells. Moreover, GTE and EGCG decrease the VEGF protein expression dose-dependently.
