Sequential treatment with betulinic acid followed by 5-fluorouracil shows synergistic cytotoxic activity in ovarian cancer cells.

Betulinic acid selectively inhibits the growth of ovarian carcinoma cell lines without affecting the normal cells. In the present study, the effect of 5-fluorouracil (5-FU) and betulinic acid (BA) combination on ovarian carcinoma cells was studied. The results demonstrated that ovarian carcinoma cells on concurrent or 5-FU followed by BA treatment show increased Sub-G1 cell population, increased rate of cell apoptosis and morphological changes in mitochondrial membrane. In OVCAR 432 cells treatment with sequential combination of 5-FU and BA increased the Sub-G1 cell population to 51.3% and growth inhibition rate of > 72%. However, exposure to BA before 5-FU treatment caused a decrease in rate of inhibition to < 35%. Treatment with combination of 5 μM of 5-FU and 1 μM of BA for 48 h, led to an induction of apoptosis in 79.7% and induced morphological changes in OVCAR 432 cells. The Western blot results showed high concentration of cytochrome c in the cell cytosol after 24 h of 5-FU and BA combination treatment. Treatment of BA-responsive RMS-13 cells with 5-FU and BA combination resulted in inhibition of GLI1, GLI2, PTCH1, and IGF2 genes. In addition, we found a significant reduction in hedgehog activity of RMS-13 cells after 5-FU and BA combination treatment by means of a hedgehog-responsive reporter assay. Therefore, 5-FU and BA combination can be a promising regimen for the treatment of ovarian carcinoma.