Experimental Study Of The Role Of Targeted EGFRvâ…¢ CH12 MAb Combined With Cisplatin On Human Ovarian Epithelial Carcinoma In Vitro

Epithelial ovarian cancer accounts for 90% of all ovarian cancer and is a leading cause of cancer death among women.Approximately 60-70% cases are diagnosed normally present at a late stage.The standard of care for these patients is surgical resection followed by systemic platinum-based chemotherapy. However, despite advances in chemotherapeutic strategies, the drug resistance result in most patients ultimately recurrence and 5-year survival rates of only 20-30%. Novel biologically targeted therapies that interfere with specific molecular pathways affecting cancer evolution have been developed as potential treatment.EGFR vlll is the most common mutated version of EGFR. EGFR and EGFR vlll are found in many solid tumors such as GBM, NNSCL,breast cancer,prostate cancer, ovarian cancer and so on,which are closed related to the very poor prognosis. As a result epidermal Growth Factor Receptor variantⅢ(EGFRvⅢ) has been implicated in cancer initiation and development and it has been an important therapeutic target.which can specifically recognize Chimerical CH12 derived from mouse monoclone antibody 12H23 by lymphocyte hybridoma techniques showed almost the same affinity to EGFRvⅢas 12H23 which can specifically recognize over- expressed EGFR and EGFR vⅢ.Futhermore, CH12 is the promising monoclone antibody confirmed by the recent study has published that CH12 can inhibit the cells growth and the size of EFGR vlll subcutaneous hepatocacinoma cell xenograft. Our study aims at whether the targeted EGFR variat III monoclone antibody CH12 contributes to the synergistic inhibitory effect combined with cisplatin on ovarian adenocarcinoma cell line.Deeply study to analysis and interpret this phenomena through the alteration of cells cycle and cell apotosis by using fluorescence-activated cell sorter. PartⅠThe expression of EGFRvⅢin epithelial ovarian neoplasms cell lines[Objective] To investigate the expression of EGFRvlll in epithelial ovarian neoplasms cell lines SK-OV-3 and Caov-3 separately on the protein and mRNA lever. [Methods] Semi-quantitative reverse transcriptionase polymerase chain reaction(RT-PCR)and Western blot analysis was used to detect mRNA and protein expression of EGFRvⅢ, respectively. [Results] Both of the mRNA and protein expression of EGFRvⅢwere detected. [Conclusions] The monoclone antibody CH12 can put into use the study of treatment targeted EGFRvⅢin ovarian adenocarcinoma in vitro.PartⅡThe affinity of ovarian cancer cell lines with EGFRVⅢmonoclone antibody CH12[Objective] To detect the binding affinity of EGFRvⅢantibody with epithelial ovarian neoplasms cell lines SK-OV-3 and Caov-3 [Methods] By the application of flowrescence activated cell sorter to demonstrate the binding affinity of CH12 antibody to ovarian cancer cell lines. [Results] The results showed that CH12 retained the slightly binding activity to ovarian cancer cell [Conclusions] The monoclone antibody CH12 can put into use the study of treatment targeted EGFRvⅢin ovarian adenocarcinoma in vitro.PartⅢ: The therapeutic effect of CH12 combined with cisplatin in ovarian cancer in vitro[Objective] To investigate the treatment for CH12 monoclone antibod y targeted EGFRvⅢcombined with cisplatin in ovarian cancer in vitro. [Meth ods] SK-OV-3 cells were respectively incubated with cisplatin of six different concentrations (0.1μg/ml,0.2μg/ml,0.3μg/ml,lug/ml,2ug/ml,3ug/ml).,CH12 of six different concentrations(20ug/ml,40ug/ml,80ug/ml,160ug/ml,320ug/ml,40 Oug/ml) and the combination group(2ug/ml cisplatin with different CH12).Cel Is proliferation were detected by CCK-8 assay.Based on the equation of comb ination index were counted to analyze the synergistic,adductive,or antagonistic effect.Furthermore the inhibition rate of Caov-3 incubated with the same six concertration cisplatin were detected. [Results] Cisplatin at 0.1,0.2,0.3,1,2,3u g/ml display the cell proliferation inhibition rate (0.59±2.14)%,(1.35±2.12)%,(2.0 5±2.13)%,(17.19±6.09)%,(23.60±3.47)%,(50.82±6.99)%,on SK-OV-3 and (7.12±3. 9)%, (13.36±4.72)%, (24.17±1.99)%,(65.44±6.02)%, (69.71±4.15)%,(7 7.43±5.98)% on Caov-3 cell line, respectively(P<0.05).The IC25 for cisplatin in SK-OV3 and Caov3 is 2ug/ml,0.3ug/ml.The corresponding inhibition rate (18.07±13.85)%,(25.53±9.09)%,(36.11±11.66)%,(40.51±2.96)%,(44.59±2.51)%,(39.99±3.0 4)% were induced by CH12 mAb at the dissimilar oncentration 20.40,80,160,3 20,400ug/ml.Furthermore,With combination of the different concentration(20,4 0,80,260,320ug/ml) of CH12 mAb and 2ug/ml cisplatin could take comparativel y apparent changes than either agent alone as (59.26±2.01)%,(63.63±4.47)%,(6 8.31±1.96)%,(73.58±3.09)%,(75.10±6.92)%(p<0.05). The combination index was 0.6872 when the cell inhibition rate reached 50% incubated either agent alon e or combined.CI<1 means the two agents combined have the synergestic t herapeutic effect. [Conclusions] Cisplatin resistance were much stronger in SK-OV-3 than Caov-3 which IC25 were 2ug/ml and 0.3ug/ml,respectively.The SK-OV-3 cells inculated with 40ug/ml CH12 antibody plus 2ug/ml cisplatin can emer ge in the synergistic therapeutic effect. As far as the cisplatin-resistance of ov arian cancer cells are concerned,CH12 antibody is a proming antibody in clin ical application. PartⅣA flow cytometric analysis of cycle and apotosis in SK-OV-3 treated with CH12 mAb and/or cisplatin[Objective] To explore the cell cycle and apotosis of SK-OV-3 cells after treatment with CH12 mAb or cisplatin used alone or the combination. [Methords] AnnexinV-FITC and Propidium iodide(PI) were used to stain SK-OV-3 cells and flow cytometry was used to determine the cell cycle and apotosis after the 48h treatment of CH12 mAb or/and cisplatin. [Rseults] After the treatment of 48 hour,the cell mumber in S phase treated with 80ug/ml CH12 mAb or 2ug/ml cisplatin used alone or the combination were respectively (33.78±1.39)%,(43.56±1.57)%, (55.03±3.56)%.There was no statistical significance vs the different group(P>0.05).The cell apotosis rate were (4.67±1.54)%, (14.36±1.98)%, (37.91±5.43)% which had significant changes (P<0.05) [Conclusions] Compared with single drug group,the two drugs combination primarily inducing apotosis,have also synergic co-inhibition on cell cycle of SK-OV-3 in S phase.