| Object:In our previous study,we found that combined application of triptolide(TP)and DDP can promote the apoptosis of human ovarian cancer cells and produce synergistic anti-cancer effects.TP is the main active ingredient of Tripterygium wilfordii Hook.F.(GTW),which is not yet applied to clinical trials.Tripterygium wilfordii has anti-inflammatory and immunosuppressive effects.Does it have anti-tumor effect similar to that of TP? On the basis of previous studies,the effect of GTW on apoptosis and cell cycle of SKOV3/DDP cells and the relationship with PI3K/AKT/NF-kB signaling pathway were discussed in this study,so as to provide a laboratory basis for the application of GTW to advanced drug resistant ovarian cancer.Methods:(1)Logarithmic growth phase cells were randomly divided into the following groups: Group RPMI 1640,Group 10 μg/ml DDP,Group 50 μg/ml GTW,Group 800μg/ml GTW,Group 3200 μg/ml GTW,Group 10 μg/ml DDP +50μg/ml GTW,group10μg/ml DDP +800μg/ml GTW,group 10μg/ml DDP +3200μg/ml GTW.After treatment for 24 h,the morphology of the cells was observed under inverted phase contrast microscope and fluorescent inverted microscope.(2)The logarithmic growth phase cells were randomly divided into the above eight groups.After 24 hours of drug action,the average apoptosis rate was calculated using Hochest 33258 staining method.(3)Logarithmic growth phase cells were randomly divided into the above eight groups.After 24 hours of drug treatment,flow cytometry was used to detect cell cycle changes.(4)Logarithmic growth phase cells were randomly divided into the above eight groups.After 24 hours of drug treatment,PI3K/AKT/NF-kB signaling pathway-related factors PI3 K,T-AKT,and P-AKT(ser473)were detected by Western blot.Expression changes of Ik Bα,NF-kB,and Bcl-2.Results:(1)After 24 hours of drug treatment.Compared with the blank control group and the DDP group,some apoptosis-like changes were observed in the drug group: cell shrinkage,pseudopodia disappeared,cells changed from short shuttle to round,and nuclear chromatin condensed.The necrotic cells were seen floating off the culture medium;there was no significant difference between the DDP group and the blank control group;the proportion of apoptosis-like changes in the cells of the single drug group and the drug combination group was increased compared to the blank control group,and the drug The higher the concentration,the greater the proportion;the proportion of apoptosis in the drug combination group was higher than that of the corresponding single drug group.(2)After 24 hours of drug treatment,the results were observed under an inverted fluorescent microscope.The cells in the blank control group and DDP group were in good condition.The pseudopods were clear,and a small number of dense and contaminated cells were seen in light and even staining.The cells in the drug group became small and stained in the nucleus.Highly concentrated,agglutinated,and visible nuclei cleave into fragments,resulting in apoptotic bodies;compared with the blank control group,the number of densely stained cells in the single drug group and the drug combination group increases significantly,and increases with increasing drug concentration;drug combination The group stained cells were more than the corresponding single drug group.(3)The apoptosis rate of group 10 g/ml DDP(3.33 + 0.61)and 50 mu g/ml GTW group(4.27 + 0.66)had no significant difference compared with that of blank control group(2.20 + 0.20)(p>0.05).Apoptosis rate(9.67 + 0.70)and 3200 mu g/ml GTW group apoptosis rate(17.60 + 1.20)in 800 u g/ml GTW group were different from that in blank control group significantly(p<0.001),the apoptosis rate of the single drug group was concentration dependent(p<0.05),the apoptosis rate(18.27 +1.01)in the 10 g/ml DDP +50 g/ml GTW group was(24.20 + 0.80)and 10 micron apoptotic rate(38.33 + 0.50)in the group of DDP +800 mu g/ml GTW group(38.33+ 0.50),compared with the apoptosis rate in the blank control group,the apoptosis rate was obvious The apoptotic rate of drug combination group was all concentrationdependent.The apoptotic rate of each combination group was significantly higher than that of the corresponding single drug group(p<0.001).(4)Single GTW and GTW combined with DDP can cause S phase arrest in SKOV3/DDP resistant ovarian cancer cells.The proportion of cells in the S phase of the 10 μg/ml DDP group(24.24±2.40)was not significantly different from that of the blank control group(19.34±2.83)(p>0.05),the proportion of the S phase cells in the50 μg/ml GTW group(37.23±0.97),800 μg./ml GTW group(38.86±1.71),3200μg/ml GTW group(43.40±2.09),10 μg/ml DDP +50 μg/ml GTW group(51.51+0.82),10 μg/ml DDP + 800 μg/ml GTW group(53.10+ 0.48)and 10μg/ml DDP+3200μg/ml GTW group(59.52+2.34)were significantly higher than those in the blank control group(p<0.001);the proportion of cells in the S phase of the drug combination group was significantly higher than that of the corresponding single drug group.Elevated(p<0.001);the proportion of cells in each S phase increased in a concentration-dependent manner(p<0.05).(5)The expression of PI3 K,T-Akt and IKBa proteins in all groups was consistent,with no significant difference;the expression of P-Akt(ser473),NF-kB,and Bcl-2 protein increased with the concentration of GTW,showing a decreasing trend overall(p<0.05).The expression of GTW and DDP combined group was significantly lower than that of the corresponding GTW alone group(p<0.05).Among them,P-Akt(ser473)protein was in the 800 μg/ml GTW group,3200 μg/ml GTW group,10 μg/ml DDP + 50 μg/ml GTW group,10 μg/ml DDP + 800 μg/ml GTW group,10 μg/ml DDP + 3200 μg/ml The relative expression levels of ml GTW group were lower than that of blank control group(p<0.05),and the relative expression of drug group histone protein was significantly lower compared with the corresponding single drug group(p<0.01);NF-kB protein was at 800 μg/ml GTW Group,3200μg/ml GTW group,10μg/ml DDP+50μg/ml GTW group,10μg/ml DDP+800μg/ml GTW group,10μg/ml DDP+3200μg/ml GTW group were lower than the blank control group.p<0.05),in which the relative expression of drug-associated histones was decreased compared with the corresponding single drug group(p<0.05);Bcl-2 protein was in the 10 μg/ml DDP group,10 μg/ml DDP + 50 μg/ml GTW group,The relative expression of 10μg/ml DDP +800μg/ml GTW group and 10μg/mlDDP +3200μg/ml GTW group was lower than that of blank control group(p<0.05),among them,800μg/ml GTW group and 10μg/ml DDP + 800μg/ml The relative expression of GTW in GTW group and 3200μg/ml GTW group was significantly lower than that in 10μg/ml DDP +3200μg/ml GTW(P<0.01).Conclusions:1.GTW inhibits the growth of human resistant ovarian cancer SKOV3/DDP cells,induces apoptosis,and induces apoptosis in a concentration-dependent manner.2.GTW can synergistically induce DDP-induced apoptosis in human ovarian cancer SKOV3/DDP cells and increase the sensitivity of SKOV3/DDP cisplatin in a concentration-dependent manner.3.GTW can synergistic DDP by blocking human resistant ovarian cancer SKOV3/DDP cell cycle in the S phase,thereby inducing apoptosis.4.The mechanism of apoptosis induced by GTW may be related to inhibition of PI3K/AKT/NF-kB signaling pathway and down-regulation of P-AKT,NF-kB and BCL-2 expression. |
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