| Objective:To investigate the effect of carvacrol(CV)on aerobic glycolysis of ovarian cancer cells,and the molecular mechanism of carvacrol affecting aerobic glycolysis of ovarian cancer through HIF-1a,and provide new strategies and ideas for the treatment of ovarian cancer.Method:In this study,after using different concentrations of carvacrol to treat ovarian cancer cells SKOV3 and HO8910 for 48 h,MTT method and colorimetric method were used to detect the effect of carvacrol on the proliferation of ovarian cancer and the half maximal inhibitory concentration(IC50)and the content of glucose and lactic acid in cell culture medium.The expression of aerobic glycolysis-related molecules HIF-1a,HK2 and LDHA were detected from mRNA and protein levels by qRT-PCR and Western blot.Screening of ovarian cancer cell lines with lowly-expressed of HIF-1a to establish stable ovarian cancer cell lines that overexpress HIF-1a.After overexpression of HIF-1a,the effects of carvacrol treatment on ovarian cancer cell proliferation,glucose uptake,lactate production and aerobic glycolysis related molecules were detected by MTT method,glucose and lactate colorimetric detection method,and Western blot method.Results:1.This study used 0mg / L,50 mg / L,100 mg / L,150 mg / L,200 mg / L carvacrol at different concentrations to treat two ovarian cancer cell lines,SKOV3 and HO8910,and the cell growth inhibition rate was measured by MTT method after 48 h.The results showed that carvacrol had an inhibitory effect on the growth of SKOV3 and HO8910 cells,and the respective cell proliferation rates were: 100,65.32 ± 3.13,55.34 ± 2.02,45.03 ± 2.42,33.17 ± 4.23% and 100,68.41 ± 4.29,53.61 ± 3.55,45.12 ± 2.64,35.38 ± 2.78%,with the increase of carvacrol concentration,cell proliferation was gradually inhibited,and obtained the half maximal inhibitory concentration(IC50)was 120 mg / L for later study.2.The two ovarian cancer cell lines were treated in the same way,and the glucose uptake and lactate production were detected by colorimetry.The results showed that in SKOV3 and HO8910 cells,the glucose uptake of the two cells after 48 hours of carvacrol treatment were: 14.09 ± 0.32,12.32 ± 0.21,11.11 ± 0.24,10.20 ± 0.34,8.93 ± 0.17 mM / 106 cells and 8.32 ± 1.23,7.83 ± 1.11,6.61 ± 1.31,5.94 ± 1.22,4.98 ± 1.01 mM / 106 cells,after 48 hours of carvacrol treatment,the extracellular lactic acid contents were 17.54 ± 1.11,14.96 ± 1.32,13.32 ± 1.28,12.19 ± 1.19,9.68 ± 1.21 mM / 106 cells and 14.94 ± 0.93,13.21 ± 1.14,12.12 ± 1.19,10.10 ± 1.34,8.29 ± 1.32 mM / 106 cells,with the increase of carvacrol concentration,glucose intake and extracellular lactic acid content are gradually suppressed(P <0.05).3.After SKOV3 and HO8910 ovarian cancer cells were treated with carvacrol for 48 hours,qRT-PCR and Western blot techniques were used to detect the changes in mRNA and protein levels of aerobic glycolysis-related molecules HIF-1a,HK2 and LDHA in ovarian cancer cells.It was found that carvacrol significantly inhibited the expression levels of HIF-1a,HK2 and LDHA in ovarian cancer cells(P <0.05).4.We used qRT-PCR and Western blot techniques to screen the HIF-1a lowly-expressed ovarian cancer cell line HO8910,and constructed stable overexpressed HIF-1a cell line(HO8910 / HIF-1a)and blank control cell line HO8910 / control).Over-expression of HIF-1a changed the cells.After treatment with carvacrol,MTT method found that carvacrol can inhibit the proliferation of ovarian cancer cells,overexpression of HIF-1a can significantly promote cell proliferation and overexpression of HIF-1a has an antagonistic effect on carvacrol inhibiting the proliferation of ovarian cancer cells(P <0.05).Colorimetric method was used to test glucose uptake and lactic acid production.It was found that carvacrol can inhibit glucose uptake and lactic acid production in ovarian cancer cells,overexpression of HIF-1a can significantly promote cell glucose uptake and lactic acid production,and overexpression of HIF-1a has an antagonistic effect on carvacrol's ability to inhibit glucose uptake and lactic acid production in ovarian cancer cells(P <0.05).5.Stable overexpression of HIF-1a cells treated with carvacrol,Western blot method was used to detect: overexpression of HIF-1a can increase the expression of HK2,LDHA,carvacrol can inhibit the expression of HK2 and LDHA,and overexpression of HIF-1a can significantly weaken the inhibitory effect of carvacrol on HK2 and LDHA(P <0.05).Conclusion:1.Carvacrol can inhibit aerobic glycolysis of ovarian cancer cells2.Carvacrol can inhibit aerobic glycolysis of ovarian cancer cells by down-regulating HIF-1a expression... |
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