Reversing Drug Resistance Of Cisplatin By SCD40L And17-AGG In Epithelial Ovarian Cancer Cells In Vitro

Objective: Ovarian cancer is the most common gynecologicalmalignant tumor, due to its early symptoms concealed and its late stage whenmost patients diagnosed, it is becoming the leading cause of death. In theprocess of clinical treatment, the platinum-resistance makes the ovarian cancerpatients with poor prognosis and high mortality, so reversing the resistance ofovarian cancer and enhancing sensitivity to the platinum is becoming a hotspot. Studies have shown that in the presence of CD40expression of tumorcells, sCD40L can inhibit the proliferation of tumor cells directly, adjust therole of tumor cells growth and induce cell apoptosis.17-AAG is a kind ofHsp90inhibitor, having strong anti-tumor effects. The anti-tumor activity of17-AAG has been found in breast cancer, leukemia and prostate cancer, whichis playing an important role in the induction of apoptosis and inhibiting tumorinvasion and cells resistance. The report is less about sCD40L and17-AAG oninhibiting the proliferation of ovarian cancer cells. Preliminary study haveshown that sCD40L and17-AAG can inhibit the proliferation of ovariancancer cells in a dose-dependent way. sCD40L combined with cisplatin couldinduce apoptosis of SKOV3cells by down-regulating survivin mRNA andup-regulating caspase-3mRNA and caspase-9mRNA. sCD40L combinedwith cisplatin could reverse the drug resistance of SKOV3/DDP cells throughdown-regulation of MDR1mRNA, LRP mRNA, GST-π mRNA and survivinmRNA. However, the inhibition effects of sCD40L on SKOV3andSKOV3/DDP cells have no significant difference. The inhibition effect of17-AAG on SKOV3/DDP cells was stronger than that in SKOV3cells. Here isto further explore the possible mechanisms of sCD40L and17-AAG inducingapoptosis and reversing drug-resistance in ovarian cancer cells line SKOV3and SKOV3/DDP by western-blot. Methods:1The proliferation inhibition effects of sCD40L, cisplatin and sCD40Lcombined with cisplatin on ovarian cancer cells SKOV3and SKOV3/DDPwere tested by MTT, and also the IC50value and reversal fold werecalculated.2Western-blot was used to analyze the expressions of GST-π, LRP,P53, bcl-2and survivin protein in SKOV3and SKOV3/DDP cells treated withcisplatin, sCD40L and sCD40L combined with cisplatin.3The proliferation inhibition effects of17-AAG, cisplatin and17-AAGcombined with cisplatin on ovarian cancer cells SKOV3and SKOV3/DDPwere tested by MTT, and also the IC50value and reversal fold werecalculated.4Western-blot was used to analyze the expression level of GST-π,LRP, P53, bcl-2and survivin protein in SKOV3and SKOV3/DDP cellstreated with cisplatin,17-AAG and17-AAG combined with cisplatin.Results:1The MTT results showed that the different concentrations of sCD40Lhas significant proliferation inhibition effects on SKOV3and SKOV3/DDPcells. With the increase of sCD40L concentration and time prolonged, theproliferation inhibition effects on SKOV3and SKOV3/DDP cells increased ina time-and dose-dependent way. The IC50of sCD40L on SKOV3andSKOV3/DDP cells were2.26μg/ml and2.33μg/ml respectively. Differentconcentrations of cisplatin also have significant proliferation inhibition effectson SKOV3and SKOV3/DDP cells. The IC50of cisplatin on SKOV3andSKOV3/DDP cells were7.34μg/ml and27.06μg/ml respectively. Theresistance ratio of DDP in SKOV3/DDP cells was3.69. With the increase ofdrug concentration, the proliferation inhibition effects of sCD40L combinedwith cisplatin on SKOV3and SKOV3/DDP cells also increased. The IC50of1μ g/ml and2μ g/ml sCD40L combined with cisplatin on SKOV3cell were1.40μ g/ml and1.05μ g/ml, the enhancement ratios were5.24and6.99respectively. The IC50of1μ g/ml and2μ g/ml sCD40L combined with cisplatin on SKOV3/DDP cells were6.11μg/ml and2.95μg/ml, the reversalratios were4.43and9.17respectively. The q values of1μ g/ml sCD40Lcombined with cisplatin on SKOV3cells and SKOV3/DDP cells were0.92and1.01. The q values of2μ g/ml sCD40L combined with cisplatin onSKOV3cells and SKOV3/DDP cells were0.97and1.02. All calculationsare0.85≤q≤1.15, so sCD40L combined with cisplatin had an additiveinhibition effect on SKOV3cells and SKOV3/DDP cells.2The western-blot results showed that the expressions of bcl-2andsurvivin protein in SKOV3cells treated with sCD40L and cisplatin for48hhad no significant difference compared with the control (P>0.05). Expressionsof survivin and bcl-2protein in SKOV3cells were significantly higher thanthat of SKOV3/DDP cells (P<0.05). Moreover, there was no change ofsurvivin and bcl-2protein in SKOV3and SKOV3/DDP cells after treated withsCD40L and cisplatin (P>0.05). In the group of2μ g/mlsCD40L combinedwith cisplatin, the expressions of GST-π, LRP and p53protein significantlydecreased compared with the control and DDP alone, sCD40L alonegroup(P<0.05). The decreasing differential value of GST-π, LRP protein inSKOV3/DDP cells treated with2μg/mlsCD40L combined with cisplatin wassignificantly higher than that in SKOV3cells (P<0.05), while there was nosignificant difference for the decreasing differential value of p53protein inSKOV3and SKOV3/DDP cells(P>0.05).3The MTT results showed that the different concentrations of17-AAGhas significant proliferation inhibition effects on SKOV3and SKOV3/DDPcells. With the increase of17-AAG concentration and time prolonged, theproliferation inhibition effects on SKOV3and SKOV3/DDP cells increased ina time-and dose-dependent way. The inhibition effect of17-AAG onSKOV3/DDP cells was stronger than that on SKOV3cells(P<0.05).The IC50value of17-AAG in SKOV3and SKOV3/DDP cell were18.39μ g/ml and7.35μ g/ml respectively. The IC50value of17-AAG combined with cisplatinin SKOV3and SKOV3/DDP cells were2.34μ g/ml and1.24μ g/mlrespectively, the reversal fold was3.14and5.92respectively. The q values of 17-AAG combined with cisplatin for SKOV3and SKOV3/DDP cells were0.95and1.01respectively. All calculations are0.85≤q≤1.15which showedthat17-AAG combined with cisplatin play an additive inhibition effect onSKOV3and SKOV3/DDP cells.4The western-blot results showed that the expressions of GST-π andLRP protein expression in SKOV3/DDP cells were significantly higher thanthose in SKOV3cells (P<0.05), while the expression of bcl-2protein inSKOV3cells was significantly lower than that in SKOV3/DDP cells (P<0.05).The expressions of GST-π, LRP and bcl-2protein have no significantdifference in SKOV3and SKOV3/DDP cells after treated with17-AAG,cisplatin, and17-AAG combined with cisplatin (P>0.05). The expression ofsurvivin and P53proteins in17-AAG combined with cisplatin groupdecreased significantly compared with17-AAG alone, cisplatin alone and thecontrol in both cells respectively(P<0.05),but the decreasing differential valueof survivin and P53protein were not significantly different in SKOV3andSKOV3/DDP cells (P>0.05).Conclusions:1sCD40L combined with cisplatin had significant proliferation inhibitioneffects on ovarian cancer cells SKOV3and SKOV3/DDP. sCD40L can reversethe resistance of cisplatin in ovarian cancer cells by down-regulating theexpressions of GST-π, LRP and p53mRNA and protein.217-AAG combined with cisplatin had significant proliferationinhibition effects on ovarian cancer cells SKOV3and SKOV3/DDP.17-AAGcan promote cell apoptosis and reverse the resistance of cisplatin in ovariancancer cells through decreasing the expressions of survivin and p53mRNAand protein.