Effects Of Fusion Protein TAT-OSBP-MKK6(E) On Cisplatin-resistant Ovarian Cancer Cells And Its Mechanism

BackgroundOvarian cancer is the highest mortality rate of gynecological malignancies,a serious threat to women’s life and health,high degree of malignancy,onset of concealment,late onset,easy to transfer,poor prognosis is the characteristics of ovarian cancer.About 70% of patients are already advanced(FIGO grade Ⅲ-Ⅳ).Tumor cytoreductive surgery and cisplatin-based combination chemotherapy is an important means of advanced treatment of advanced ovarian epithelial cancer,the purpose of tumor cytoreductive surgery is to maximize the scope and excision of tumor tissue,but postoperative Residual lesions still can not be avoided,and become an important factor in the recurrence of ovarian cancer.So the application of chemotherapy to ovarian cancer treatment effect is greatly improved,the quality of life of patients improved significantly,but the overall view,the survival rate of patients within 5 years is still hovering around 30%,ovarian cancer resistance to chemotherapy drugs is leading to treatment failure The main reason.At present,ovarian epithelial cancer is mainly based on cisplatin-based chemotherapy,but the efficacy of cisplatin due to cancer cells or acquired(drug-induced)to obtain resistance and weakened.Therefore,the reversal of ovarian cancer resistance and ovarian cancer drug resistance in-depth study is particularly important.At present,molecular targeted therapy has gradually become a new tumor treatment strategy,and made a certain research results.In this study,the ovarian cancer specific binding peptide(OSBP)was successfully screened by whole cell differential subtraction,and the fusion protein was successfully obtained by prokaryotic expression.(TAT)-OSBP-mitogenactivated protein kinase 6 mutant(E),MKK6(E)]},TAT-OSBP-MKK6(E),a targeted fusion peptide targeting the ovarian cancer cell with strong penetrating ability.In vivo experiments show that it can target the apoptosis of ovarian cancer HO8910 cells and enhance the anti-tumor activity of paclitaxel effect.This study was to investigate the effect of TATOSBP-MKK6(E)on cisplatin-resistant human ovarian cancer cell line SKOV3 / DDP in human ovarian cancer cisplatin-resistant cell line SKOV3 / DDP.The inhibitory effect of growth and its possible mechanism,and combined with cisplatin in SKOV3 / DDP cells to observe the combined anti-tumor effect of the two,to explore the new target to kill ovarian cancer cells to provide experimental data,Reversing ovarian cancer cisplatin resistance to provide a theoretical basis.ObjectiveSKOV3 / DDP cells were used to study the apoptosis of cisplatin cells in human epithelial ovarian cancer.To investigate the inhibitory effect of TAT-OSBP-MKK6(E)alone or in combination with cisplatin on human cisplatin-resistant ovarian cancer cell line SKOV3 / DDP and its possible molecular mechanism.Methods(1)MTT assay was used to detect the inhibitory effect of fusion protein TAT-OSBPMKK6(E)on the growth of cisplatin-resistant ovarian cancer cell line SKOV3 / DDP.The effect of fusion protein TAT-OSBP-MKK6(E)on invasion and metastasis of cisplatinresistant ovarian cancer cell line SKOV3/DDP was detected by transwell method;The expression of P38 MAPK protein was detected by Western blot when different concentrations of TAT-OSBP-MKK6(E)were used to treat cisplatin-resistant ovarian cancer cell lines SKOV3 / DDP.(2)MTT assay was used to detect whether the anti-tumor effect of fusion protein TATOSBP-MKK6(E)combined with cisplatin on cisplatin-resistant ovarian cancer cell line SKOV3/DDP was enhanced.The morphological changes of TAT-OSBP-MKK6(E)combined with cisplatin on SKOV3/DDP cells were observed under light microscope.The effect of TAT-OSBP-MKK6(E)combined with cisplatin on the apoptosis rate of SKOV3/DDP was detected by flow cytometry.The expression of Cleaved caspase-3 protein was analyzed by Western blot.(3)The MDC stain marker TAT-OSBP-MKKO(E)induces the autophagy in SKOV3 / DDP;Western blot analysis of the expression of LC3-II protein.The use of autophagic specificinhibitors to detect the action in the SKOV3/DDP with the TAT-OSBP-MKK6(E).(4)The expression of P38 protein and LC3-II protein was detected by western blot when the P38 MAPK signal pathway was blocked by SB230580(P38MAPK signal pathway specific inhibitor).The survival rate of SKOV3 / DDP cells was detected by MTT assay.Results 1.Inhibitory effect of fusion protein TAT-OSBP-MKK6(E)on cisplatin-resistant ovarian cancer cell line SKOV3 / DDPSKOV3 / DDP cells were treated with TAT-OSBP-MKK6(E)at 2.5,5,10,20,40 and 80 μmol / ml for 48 h,and the proliferation inhibition was observed in a concentrationdependent manner.Compared with the control group,the TAT-OSBP-MKK6(E)treatment group was statistically significant(P <0.01),and the IC50 was 26.94μmol / m L.2.Transwell was used to detect the invasion and metastasis of cisplatin-resistant ovarian cancer cell line SKOV3 / DDP by TAT-OSBP-MKK6(E)The cells were treated with TAT-OSBP-MKK6(E)at different concentrations(0,10,20 and 40 μmol / ml)for 48 h,and the number of transmembrane cells in each treated group was(85 ± 4),(70 ± 5),(54 ± 6)and(32 ± 2).With the increase of TAT-OSBP-MKK6(E)concentration,the inhibitory effect on SKOV3 / DDP was more obvious.Different concentrations of TAT-OSBP-MKK6(E)treatment group were statistically significant(P <0.05)compared with the control group.(708 ± 15),(683 ± 14),(141 ± 15)and(113 ± 18),respectively,with the concentration of TAT-OSBP-MKK6(E)in the treatment group And its inhibition of SKOV3 / DDP was more obvious.Compared with the control group,the TATOSBP-MKK6(E)treatment group was statistically significant(P <0.05).3.Fusion protein TAT-OSBP-MKK6(E)on the expression of P38 MAPK protein in cisplatin-resistant ovarian cancer cell line SKOV3 / DDPTAT-OSBP-MKK6(E)could significantly activate the phosphorylation level in SKOV3 / DDP cells at concentrations of(0-40)μmol / ml,and the more obvious the phosphorylation level was,and the more obvious the P38 MAPK protein The total does not change.4.Fusion protein TAT-OSBP-MKK6(E)increased the sensitivity of cisplatin-resistant ovarian cancer cell line SKOV3 / DDP to cisplatinThe inhibitory rate of TAT-OSBP-MKK6(E)alone on SKOV3 / DDP cells was(13.06 ± 1.089)%;The inhibition rate of SKOV3 / DDP cells was(19.02 ± 2.227)%,and TAT-OSBPMKK6(E)(5μmol / ml)+ cisplatin(20μg / ml)could significantly increase the survival rate of SKOV3 /DDP.The survival rate of DDP cells was(54.03 ± 2.382)%.The results showed that cisplatin combined with TAT-OSBP-MKK6(E)could significantly inhibit the proliferation of SKOV3 / DDP cells,the number of SKOV3 / DDP cells was significantly decreased,the cell morphology changed,and the cells appeared as Bright dots.5.The fusion protein TAT-OSBP-MKK6(E)increased the apoptosis rate of cisplatinresistant ovarian cancer cell line SKOV3 / DDP20 μg / ml of cisplatin,5 μmol / ml of TAT-OSBP-MKK6(E)combined with SKOV3 / DDP cells for 24 h,and the apoptotic rate was measured by flow cytometry.The results show ed that 20μg / ml cisplatin could not cause apoptosis in SKOV3 / DDP cells compared with th e control group,and 5μmol / ml TAT-OSBP-MKK6(E)could also increase the apoptosis rate And the apoptosis rate was significantly increased by cisplatin combined with TAT-OSBP-M KK6(E)(P <0.05).Compared with the control group and the 20μg / ml cisplatin group,cispla-tin combined with TAT-OSBP-MKK6(E)and the cisplatin group were significantly higher t-han those in the cisplatin group and the fusion protein group The expression of Cleaved Casp-ase-3 protein in the 5μmol / ml TAT-OSBP-MKK6(E)group was also reduced.6.Fusion protein TAT-OSBP-MKK6(E)induced cisplatin ovarian cancer cell line SKO V3 / DDP autophagy20 μg / ml cisplatin,5 μmol / ml TAT-OSBP-MKK6(E)combined group showed higher fluorescence intensity and more MDC-labeled cells.Western blot analysis LC3-I was graduall y transformed into LC3-II when 20μg / ml cisplatin,5μmol / ml TAT-OSBP-MKK6(E)alone or in combination with cisplatin-resistant ovarian cancer,LC3-II The highest protein concent ration,while the autophagy occurred in the expression of the iconic protein P62 is gradually w eakened.7.Fusion protein TAT-OSBP-MKK6(E)induced apoptosis of cisplatin ovarian cancer cell line SKOV3 / DDP cells autophagic cell deathWhen the cells were treated with chloroquine and TAT-OSBP-MKK6(E),the aggregation of LC3-II and P62 was significantly increased with TAT-OSBP-MKK6(E)alone,indicating that CQ could effectively block autophagy The accumulation of proteins in autophagate.The results of the MTT assay further show that inhibition of autophagy significantly reduced the inhibition of TAT-OSBP-MKK6(E)on SKOV3 / DDP cells.8.The fusion protein TAT-OSBP-MKK6(E)induced the anti-tumor effect of autophagic sensitized cisplatin by activating the P38 MAPK pathwayThe expression of P38 protein was decreased by SB203580.After p38 protein expression decreased,TAT-OSBP-MKK6(E)induced LC3-I to LC3-II transformation was significantly inhibited.The number of cell death in different treatment groups was further measured by MTT assay.The results showed that TAT-OSBP-MKK6(E)combined with cisplatin treated cisplatin ovarian cancer cell SKOV3 / DDP could significantly increase cell death.The inhibition of the P38 MAPK signaling pathway clearly blocks this sensitization effect.ConclusionThe fusion protein TAT-OSBP-MKK6(E)can induce the autophagic cell death by activating the P38 MAPK signaling pathway,thereby inhibiting the proliferation,invasion and metastasis of human ovarian cancer cisplatin-resistant cell SKOV3 / DDP cells Cisplatin sensitivity.To a certain extent can reverse cisplatin resistance.It is suggested that the fusion protein TAT-OSBP-MKK6(E)is expected to be a new chemotherapeutic agent in the treatment of cisplatin in ovarian cancer and provide a new therapeutic mode for the treatment of ovarian cancer.