The Cytotoxic Effect Of Geldanamycin Combination With Cisplatin On Human Ovarian Cancer Cell Line SKOV3

Objective:To investigate the cytotoxic effects of geldanamycin on the proliferation of human ovarian cancer SKOV3 cells.Moreover,and to check the cytotoxic effects of geldanamycin combined with cisplatin(DDP) on human ovarian cancer SKOV3 cells.Furthermore,to analyze the possible mechanisms of GA inhibiting the proliferation and inducing apoptosis to SKOV3 cells.Methods:1 Human ovarian cancer SKOV3 cells were incubated with different concentrations of geldanamycin in vitro.The effect of geldanamycin on the proliferation of SKOV3 cells was measured by MTT colorimetric method.2 Microscopy was used to observe the cells changes in morphology and flow cytometry was used to detected cell cycle and apoptosis after treated by geldanamycin.3 The expressions of Akt and Raf-1 protein of SKOV3 cell treated with geldanamycin for 48h were analyzed by flow cytometry and immunocytochemistory method.4 MTT colorimetric method was performed to evaluate the effect of Geldanamycin combined with DDP on SKOV3 cells. Jin's formula was performed to determine whether the combination of geldanamycin with DDP could produce synergistic cytotoxic effect or not.More than 0.85 of the interaction index was thought to be as a synergic effect,while less than 0.85 was thought to be as antagonism.Results:1 The proliferation of SKOV3 cells could be inhibited by the different concentrations of geldanamycin in a time- and dose- dependent way.The highest inhibition rate reached (74.72±3.16)%and IC50 was 496.57nmol/L after SKOV3 cell were treated GA for 48h.2 After treated with geldanamycin for 48h,cells cycle was arrested in G₂/M phase.3 SKOV3 cells untreated with geldanamycin were fusiform,diamond and satiation by microscopy,while the cells treated with geldanamycin were shrinked and broken,became irregular in shape,spreading in a thin and long pseudopodium in both end points,vacuolus could also be seen in some cells cytoplasm,floating and cast-off cells increased.Giemsa staining showed that the SKOV3 cells untreated with geldanamycin were fusiform,diamond,and they were satiation,furthermore,the cell nucleus staining were uniform and clear,no floating cells.But SKOV3 cells treated with geldanamycin became rounding,and the cytoplasm was concentrated,the staining cell nucleus was uneven,there were also caryocinesis phenomenon,showing the typical apoptotic morphological changes. 4 The expressions of Akt and raf-1 proteins were tested by immunocytochemistory and flow cytometry.The expressions of Akt and raf-1 proteins decreased significantly with the increasing of the concentrations of geldanamycin(P＜0.05).5 MTT results showed that Geldanamycin combined with DDP could inhibit the proliferation of SKOV3 cells.20,40, 200,400nmol/L geldanamycin combined with DDP produced synergic cytotoxic effects to SKOV3 cells,and these synergic cytotoxic effects were further be testified by interaction index from Jin's formula.Conclusions:1 Geldanamycin could induce cytotoxic effects on the proliferation of human ovarian cancer SKOV3 cells in a time-and dose- dependent manner.2 Geldanamycin could cause cells cycle arrested in G₂/M phase as well as apoptosis in SKOV3 cells.3 The cytotoxic effects of geldanamycin to inhibit the proliferation and induce apoptosis to SKOV3 cells were partly due to down-regulating the expression of Akt and raf-1.4 Geldanamycin combined with DDP could inhibit the proliferation of SKOV3 cells and produce synergic cytotoxic effects to SKOV3 cells.