| Objective:To investigate the effect of Triptolide and Paclitaxel on the apoptosis ofdrug-resistant ovarian cancer cell(COC1/DDP cells) in vitro and its possiblemechanism. this result can provide experimental basis for treating cisplatin-resistantovarian cancer and expand the usable range of TP.Methods:1、Cultivated COC1/DDP cell with0.5%DDP,10%FBS and90%RPMI-1640complete and putted it in constant temperature incubator with the condition of37℃、5%CO2and saturated humidity. When the cell is logarithmic growth phase,webegin to test. the whole experiment was divided into6groups, the control group、paclitaxel group、a specific inhibitor of PI3K group(LY294002)、the TP group、theTP combined with paclitaxel group，the LY294002combined with paclitaxel group.2、 using the microculture tetrazolium (MTT) assay to detect effects ofexperimental group on the proliferation of COC1/DDP cells after24h、48h、72h.thenwork out their inhibition rate in every group.3、Observe cell apoptotic morphology by Ordinary light microscope and AO/EBfluorescent dye staining after24h in each group.4、Annexin V-FITC/PI double staining method detect the influence of cellapoptosis rate.5、western blot was used to detect the protein Expression of AKT、P-AKT、GSK3β、P-GSK3β in COC1/DDP cells.Results:1. Different effects of the drugs on the proliferation of COC1/DDP cells.The cells were treated with different ways of intervention, after24,48,72h,detect the cell proliferation.according to the following order: paclitaxel group、aspecific inhibitor of PI3K group(LY294002)、the TP group、the TP combined withpaclitaxel group and the LY294002combined with paclitaxel group.after24hours,the inhibition rate of cells are:39.33±3.567%、6.52±1.229%、45.61±2.645%、49.45±2.75%、56.09±3.131%； after48hours, the inhibition rate of cells are:50.74±2.763%、18.52±1.145%、57.05±2.491%、66.33±3.471%、72.85±1.803%；after72hours, the inhibition rate of cells are:63.34±7.712%、32.787±2.726%、71.08±3.484%、86.33±3.583%、89.60±2.284%.The results showed the inhibition rateof combined group is higher than that of the single drug group(P<0.05),the inhibitionrate of TP combined with paclitaxel group is higher than the LY294002combinedwith paclitaxel group (P<0.05).in the single drug group, the inhibition rate is that: TPgroup> paclitaxel group> LY294002group(P<0.05). The inhibition rate of the groupsshowed time dependence.2、After using different drugs, observe the changes of COC1/DDP cells.(1) Observe of the morphology of apoptotic cells under light microscope afterusing drugs.In the control group,the proliferation of COC1/DDP cells developed well.underordinary microscope,the cell nucleus was oval,their chromatin is rich,and had goodrefraction.using the drug in every group for24hours, under ordinary microscope,thenumber of cells appeared varying degrees of reduction, and found a lot of cellsapoptosis,such as cytoplasmic vacuolization, nuclear pyknosis, fragmentation,dissolution. The results showed the apoptosis rate of combined group is higher thanthat of the single drug group,the apoptosis rate of TP combined with paclitaxel groupis higher than the LY294002combined with paclitaxel group.in the single drug group,the apoptosis rate is that: TP group> paclitaxel group> LY294002group.(2) AO/EB staining to observe the morphology of cell apoptosis after using drug.In the control group,the COC1/DDP cells were stained by AO/EB,we can see themorphology of cells showing green fluorescence under the fluorescence microscope.using drugs for24hours, After AO/EB staining under fluorescence microscope.wecould see that：in paclitaxel group,the cells appeared apoptotic changes, their Nuclearchromatin has green fluorescence, but showed pyknosis or bead-like shape.in theLY294002group,the changes of cells were similar with the paclitaxel group, but thenumber was less than paclitaxel group.in TP group, most cell chromatin was pycnosisor bead-like shape,it had orange fluorescence,there were a lot of cells appearing apoptotic changes,only a few normal cells.in the LY294002combined withpaclitaxel group, Most of these cells were apoptotic changes, a few were lateapoptosis, still a few visible normal cells, the number of apoptotic cells is more thanTP group. in the TP combined with paclitaxel group, Cells almost appeared early andlate apoptotic changes, only few normal cells, the number of apoptotic cells are morethan LY294002combined with paclitaxel group.the changes in each group is similarwith the results under general light microscope.3. Annexin V-FITC/PI double staining method detect the influence of cellapoptosis rate after using drug.After using drug for24hours, the cells were giving Annexin V-FITC/PI doublestaining, flow cytometry showed apoptosis rate in every group is that：paclitaxelgroup（14.90±1.253）%、LY294002group（11.20±1.682）%、TP group（39.0±3.816）%、LY294002combined with paclitaxel group（27.67±1.834）%、TP combined withpaclitaxel group（51.13±3.325）、the control group（5.87±1.815）%. The results showedthe apoptosis rate of combined group is higher than that of the single drug group,theapoptosis rate of TP combined with paclitaxel group is higher than the LY294002combined with paclitaxel group.in the single drug group, the apoptosis rate is that:TP group> paclitaxel group> LY294002group.in the control group,there were also afew apoptotic changes, it was caused by the technical operation error.the results werestatistically signficant.4. the protein Expression of AKT、P-AKT、 GSK3β、P-GSK3β in COC1/DDPcells after using drug.PI3K/AKT/GSK3β is an important signal pathway of apoptosis and cellgrowth,so after using drug for24hours, we use western blot to detect the proteinExpression of AKT、P-AKT、 GSK3β、P-GSK3β in COC1/DDP cells. the resultshowed after24hours, the expression of AKT and GSK3β protein in everyexperimental group, did not produce any changes.but the expression of P-AKT andP-GSK3βprotein showed a decreasing trend from the control group to TP combinedwith paclitaxel group.Conclusion:1. paclitaxel、LY294002、TP、LY294002combined with paclitaxel and TP combined with paclitaxel have inhibitory effects on the proliferation of COC1/DDPcells in vitro. It showed time dependence. the proliferation inhibition of combinationgroup is greater than its corresponding single drug group. in each single group, theproliferation inhibition of TP is the biggest, but the LY294002is the smallest. in thecombined group, the proliferation inhibition of TP combined with paclitaxel group ishigher than the LY294002combined with paclitaxel group.2.When the specific inhibitor of PI3K(LY294002) uses alone, the apoptosis ofCOC1/DDP cells is（11.20±1.682）%,but while TP do it, the result is （39.0±3.816）%, the apoptosis effect of TP is more obvious than LY294002.3. When LY294002or TP cooperate with paclitaxel, they all can induce theapoptosis of COC1/DDP cells, but the effect of TP is more obvious.4. paclitaxel、LY294002、TP、LY294002combined with paclitaxel and TPcombined with paclitaxel which all can decrease the protein Expression of P-AKTand P-GSK3β.the combination groups decrease most obviously.this results show thatTP is similar to LY294002.The mechanism is that TP can inhibit the PI3K/AKT/GSK3βsignaling pathway, through that,it can induce the apoptosis ofCOC1/DDP cells when combined with paclitaxel. |
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