Research On Betulinic Acid Inhanced Anti-Tumor Drugs Induce U266 Cells Apoptosis And Its Mechanism

Objective: To study betulinic acid inhanced anti-tumor drugs induce U266 cells apoptosis and its mechanism. Methods: U266 cells were treated with different concentrations of betulinic acid(20ug/ml, 40ug/ml, 60ug/ml, 80ug/ml), bortezomib(10nmol/ml, 80nmol/ml, 320nmol/ml), thalidomide(10ug/ml, 50ug/ml, 100ug/ml) and melphalanindividually, and betulinic acid combined with different concentrations of bortezomib, melphalan and thalidomide. The inhibitory rate of cell proliferation were detected with MTT method;detection of different concentration of thalidomide, bortezomib, melphalan alone medication and respectively and combined medication of betulinic acid groups of the apoptosis rate in every set was detected with Annexin V/PI flow cytometry; expression level of survivin, Cyto-C, Bcl-2, Bax were detected with Real-time PCR;western blot was used to detetctprotein expression of Survivin, Cyto-C, Bcl-2, Bax.Results: The proliferation inhibition rate in combination drug groups is significantly higher than that of single drug group(p<0.05); Flow cytometry shows that multiple myeloma U266 cells was treated with both betulinic acid and bortezomib combined of, then 10nmol/ml, 80nmol/ml, 320nmol/ml cell apoptosis rates were(36.94±0.04)%、(56.73±0.08)%、(78.67±0.03)%; similarly, apoptosis rates were(27.32±0.02)%、(46.16±0.03)%、(70.17±0.08)% and(37.43±0.07)%、(51.78±0.02)%、(74.19±0.05)% in betulinic acid and thalidomide combination groups and, betulinic acid and melphalan combination groups respectively, apoptosis rate of each group was significantly different(P < 0.05); RT-PCR and Western Blot results showed that after multiple myeloma U266 cellswere treated with bbetulinic acid combination drug therapy, the expression level of cyto-C,Bax is remarkablely increased(p<0.05). Conclusion: Betulinic acid combinated with bortezomib, thalidomide and melphalan can induce the apoptosis of U266 cells. This may be associated with down-regulatizon of Survivin and Bcl-2 expression, and up-regulation of cyto-c and Bax expression(p<0.05).