The Study Of The Effect Of Drug-resistant Ovairan Cancer Cell Line By Triptolide And Paclitaxei In Vitro And Its Mechanism

Objective:This experiment is about study the effect of Triptolide and Paclitaxel ondrug-resistant ovarian cancer cell in vitro and its possible mechanism.Methods:1、The growth inhibiting rate of COC1/DDP cells after treated by Paclitaxel, wasdetermined by MTT assay:we set blank control group, different concentrations ofpaclitaxel:0.39,0.78,1.56,3.13,6.25,12.5,25,50μg/ml, zero group. We used MTT assayto detect the absorbance (OD) of every hole cells.And we can know the proliferationof COC1/DDP cells after treated by Paclitaxel after24hours.We can simultaneouslycalculate the cell’s inhibition ratio.2、The growth inhibiting rate of COC1/DDP cells treated by Paclitaxel combinedwith TP was determined by MTT assay after24,48,72hours: we set blank controlgroup, Paclitaxel group（3.13ug/ml）, low concentration TP group（3ng/ml）, moderateconcentration TP（10ng/ml）, high concentration TP group（50ng/ml), Paclitaxelcombined with low concentration TP group, Paclitaxel combined with moderateconcentration TP group, Paclitaxel combined with high concentration TP group, zerogroup. We also used MTT assay to detect the absorbance (OD) of every hole cells andcalculate the cell’s inhibition ratio after24,48,72hours.Then we used the Jin’s formulato calculate the two-drug combination index(q),and analyzed the effect of the two drugs incombination.E (AB) is the inhibition of the two drugs in combination,EA,EB is theinhibition rate of each drug used alone.when q=0.85~1.15, the performance of therole of two drugs is additive;q>1.15, the role of the two drugs in combination issynergistic;q <0.85, the role of two drugs is antagonistic.3、The ultrastructural changes of COC1/DDP cells treated by Paclitaxel combinedwith TP by Transmission Electron Microscope after24hours: the experiment wasdivided into four groups: blank control group, moderate concentration TP（10ng/ml）,Paclitaxel group （3.13ug/ml）,Paclitaxel combined with moderateconcentration TP group. Transmission Electron Microscope was used to observe the ultrastructural changes of COC1/DDP cells in each experimental group after24hours.4、The influence of cell cycle and cell apoptosis rate of COC1/DDP cells treatedby Paclitaxel combined with TP by flow cytometry after24hours: the experiment wasdivided into four groups:blank control group,moderate concentration TP（10ng/ml）,Paclitaxel group （3.13ug/ml）,Paclitaxel combined with moderateconcentration TP group.We used flow cytometry to detect the proportion of cell cycleand cell apoptosis rate of COC1/DDP cells.5、The expression of Caspsase-7protein on COC1/DDP cells after treated byPaclitaxel combined with TP by Immunohistochemistry after24hours: the experimentwas divided into four groups:blank control group, moderate concentration TP（10ng/ml）,Paclitaxel group （3.13ug/ml）,Paclitaxel combined with moderateconcentration TP group. Centrifugation, fixed cells, preparation of paraffin blocksaccording to the conventional method. The Immunohistochemical was used to detectthe expression of caspsase-7protein in each group.Results:1、The growth inhibiting rate of COC1/DDP cells after treated by Paclitaxel:Different concentrations of paclitaxel can significantly inhibit the proliferationof COC1/DDP cells,and the inhibition enhanced with the increasing of paclitaxelconcentration, in a dose-dependent manner(P<0.05).2、The growth inhibiting rate of COC1/DDP cells treated by Paclitaxel combinedwith TP was determined by MTT assay:The inhibitory effect of triptolide combined with paclitaxel on COC1/DDP cellswas higher than their respective single used group.And the inhibition enhanced withthe increasing of the concentration of TP, in a dose-time dependent manner(P<0.05).The combination index of the combined treatment groups on COC1/DDP cells were1.16,1.23,1.17after24hours.After48hours, the combination index were0.96,1.02,1.11;and there were0.93,0.95,1.11after72hours.According to the Gin’sformula,we can speculate: the performance of the role of the two drugs incombination was synergistic after24hours; the role of the two drugs in combinationwas additive after48and72hours. 3、The ultrastructural changes of COC1/DDP cells treated by Paclitaxel combinedwith TP by Transmission Electron Microscope:By Transmission Electron Microscope,we can see COC1/DDP cells in blankcontrol group were normal morphology, the nucleus were oval,and rich inchromatin.In TP group, Paclitaxel group and Paclitaxel combined with TP group, we cansee apoptotic kind changes:the microvilli of cell membrane surface disappeared,mitochondria increased, the endoplasmic reticulum expanded, membrane shrinked,karyopyknosis, nuclear fragmentation and nucleus dissolved.4、The influence of cell cycle and cell apoptosis rate of COC1/DDP cells treatedby Paclitaxel combined with TP by flow cytometry:The COC1/DDP cells apoptosis rates of TP group,Paclitaxel group and Paclitaxelcombined with TP group was higher than blank control group（P<0.05）.The apopticratio of Paclitaxel combined with TP group was obviously higher than those only byTriptolide or Paclitaxel（P<0.05）.The apoptic ratio of TP group was higher thanPaclitaxel group.The proportion of cells in G1phase of TP group was the highest（P<0.05）.Compared with TP group and blank control group,the proportion of cellsin G2/M phase of COC1/DDP cells increased obviously in Paclitaxel group andPaclitaxel combined with Triptolide group（P<0.05）.Among them, the proportion ofcells in G2/M phase of Paclitaxel group was the highest of all（P<0.05）.So we canspeculate: Triptolide induced apoptosis may be related to cell cycle arrest in G1phase,while Paclitaxel and triptolide combined with paclitaxel induced apoptosis may berelated to cell cycle arrest in G2/M phase.5、The expression of Caspsase-7protein on COC1/DDP cells after treated byPaclitaxel combined with TP by Immunohistochemistry:In blank control group,we found part of COC1/DDP cells cytoplasm wereyellow by Immunohistochemistry. But most of of COC1/DDP cells cytoplasm weretan, brown in TP group,Paclitaxel group and Paclitaxel combined with TP group.Theexpression of Caspsase-7protein of COC1/DDP cells in each treatment group washigher than the blank control group (P<0.05). The expression of Caspsase-7protein ofCOC1/DDP cells in TP group was higher than Paclitaxel group (P<0.05).Theexpression of Caspsase-7protein of the combined treatment group was higher than its respective treatment group (P<0.05).Conclusion:1、Different concentrations of paclitaxel COC1/DDP cells can significantlyinhibit the proliferation and the inhibition enhanced with the increasing ofconcentration of paclitaxel, with a dose-dependent relationship.2、The inhibitory effect of triptolide combined with paclitaxel COC1/DDP cellswas higher than their respectively single-used, in a dose-time-dependent manner.Thecombined effect of the two drugs was synergistic/additive.3、Triptolide combined with paclitaxel all can promote COC1/DDP apoptosis,they can promote apoptosis-like changes in COC1/DDP cells,such as nuclearcondensation, fragmentation, dissolution and so on.The rate of apoptosis of thecombined treatment group was higher than their respectively single-used.Arrestingcell cycle in G2/M phase, affecting DNA synthesis and mitosis may be the possiblemechanisms of triptolide combined with paclitaxel-induced apoptosis in COC1/DDPcells4、The expression of caspase-7protein in Triptolide combined with paclitaxelgroup was higher than their respective single-drug group.Triptolide combined withpaclitaxel promoted the COC1/DDP mechanism of apoptosis,may be with therelevant of up-regulated expression of caspase-7protein.