The Effect Of γ-secretase Inhibitors On Chemosensitivity In Platinum-resistant Ovarian Cancer Cell Line

Objective: Ovarian cancer is a kind of highly malignant gynecologiccancer and remains the most deadly gynecologic malignancy, the5-yearsurvival rate is only20-30%.Due to the nonspecific and commonlymisinterpreted symptoms associated with the disease,it is known as the silentkiller. Surgery, combined with Platinum-based chemotherapy, is the maintreatment method for ovarian cancer.However the problem of drug-resistance,which is the most common cause of tumor recurrence and metastasis,immediate impacts on treatment effect and outcome of ovarian cancerpatients.Therefore it is essential and become hotspot to detect the mechanismof drug-resistance,overcome and reverse drug-resistance. Improving thecurrent response to chemotherapy is key not only for achieving a betteroutcome clinically,but also for allowing to have a better quality of life duringtreatment.It is widely accepted that epithelial cells can acquire mesenchymalphenotype by a complex processes of epithelial–mesenchymal transition.Recent studies have shown that the process of EMT is not only important onconferring increased motility and invasion to cancer cells but also cancer stemcells(CSCs) characteristics, which contributes to increased cell viability anddrug-resistance.Therefore,the discovery of precise mechanisms that governsthe acquisition of EMT phenotype in cancer cells would likely be usefuldevising targeted therapeutic agents in Combination with conventionaltherapeutics for improving the treatment of human malignancies.It has been well known that Notch signaling pathway plays importantroles in maintaining the balance involved in cell proliferation, apoptosis anddifferentiation.The Notch gene is abnormally activated in many humanmalignancies including cervical cancer,ovarian cancer, colon cancer,pancreatic cancer and so on,which suggests that Notch signaling pathway is criticallyassociated with the development and progression of tumors.Emergingevidence suggest Notch-1could play a key role in the regulation of EMT andCSC phenotype during the development and progression of tumors andcontribute to chemoresistant.Notch signaling pathway is activated via theactivity of γ-secretase which became a target in cancer therapy to overcomedrug-resistance.GSIs have been tested for anti-tumor effect.The GSIs inhibitscell growth and could induce apoptosis as well as sensitize cells to chemo-therapy.However,the effects of GSIs on drug-resistance and its molecularmechanisms in ovarian cancer is not fully understood.In this study we explored the involvement of the Notch pathway in theregulation of EMT-like phenotype in SKOV3/DDP cell, and further invest-tigated whether DAPT could enhance the response to DDP in SKOV3/DDPcell,which may be a novel strategy in ovarian cancer therapy to achieve abetter outcome.Methods: The human ovarian serous cancer cell line SKOV-3andplatinum-resistant ovarian serous cancer cell line SKOV3/DDP were culturedin RPMI1640containing10%FBS,100units/ml penicillin,100units/mlstreptomycin.All cells were maintained in a5%CO2-humidified atmosphereat37℃.the two cells were applied to the experiment after they entered thelogarithmic phase.Cells were used in the experiment after they entered thelogarithmic phase.All treatments were repeated for at least3times in eachexperiment.1MTT assaySKOV-3and SKOV3/DDP cells(5×103) were seeded in96-well plates.The cells were then incubated in200μl of medium with DDP (0.0,0.625,1.25,2.5,5.0,10.0μg/ml) after the cells adhered to the plate wall for48h tocalculate the drug resistant index.SKOV3/DDP cells (5×103) were seeded in96-well plates and subsequently were treated with25,50,and75μmol/L DAPTfor24,48,and72h to detect the effects of DAPT.Control cells were treatedwith0.35%DMSO in culture medium. SKOV3/DDP cell were divided into four groups: Control group (0.35%DMSO), DDP group (2.5μg/ml), DAPTgroup(50μmol/L), DAPT+DDP group(2.5μg/ml+50μmol/L).Follow treatmentfor24h,analysis of inhibitory rates was carried out using MTT to detectwhether DAPT could sensitize platinum-resistant ovarian cancer cell tocisplatin.2SKOV3/DDPcells were treated with25,50, and75μmol/L DAPT for48h,Controlcells(SKOV-3and SKOV3/DDP cells) were treated with0.35%DMSO inculture medium.Fluorescence microscope was used to observe morphologicalchanges of the cells.3real time-PCRSKOV3/DDP cells were treated with25,50,and75μmol/L DAPT for48h，Control cells(SKOV-3and SKOV3/DDP cells) were treated with0.35%DMSO in culture medium. Total RNA of each group was extracted from thecells using Trizol reagent.Single-stranded cDNA was synthesized according tothe instruction of inverse transcription kit (TransGen).PCR amplification wasdone on the ABI7300system using2×ultraSYBR Mixture(CWBIO).4FCMSKOV3/DDP cell were divided into four groups: Control group (0.35%DMSO), DDP group (2.5μg/ml), DAPT group (50μmol/L), DAPT+DDPgroup (2.5μg/ml+50μmol/L). Follow treatment for24h, analysis of apoptoticcells was carried out using AnnexinV-FITC/PI to detect whether DAPT couldsensitize platinum-resistant ovarian cancer cell to cisplatin.5Statistical analysis was performed using SPSS13.0software.Data were expressed as the mean±SD and compared using the t-test andANOVA.A P-value of <0.05was considered statistically significant.Results:1The MTT results showed that following DDP-treatment for48h, inhi-bitory effect of SKOV3cell group was higher than SKOV3/DDP cell group atthe same concentration.Significant differences were achieved between theSKOV3cell group and SKOV3/DDP cell group at the different DDP- treatment concentration (P<0.05),except for0.625μg/ml DAPT-treatmentconcentration(P>0.05).And an increase in the treatment concentration enha-nced the inhibitory effect difference between the two cell group.IC50value ofDDP for SKOV-3and SKOV3/DDP cells at48h was2.95μg/ml and14.02μg/ml,respectively. Resistance index of SKOV3/DDP cell was4.75.Thetreatment of SKOV3/DDP cell for24,48,72h with25,50,and75μmol/L DAPTresulted in cell growth inhibition in a dose-time dependent manner,at sametime of different concentrations group P<0.05,at same concentration ofdifferent time group P<0.05.The inhibitory rates of DDP group、DAPT groupand DDP+DAPT group was （9.13±1.14）,（20.43±0.67）and（36.22±0.99），respectively.The inhibitory rates of single treatment was significant lowercompared to combination treatment (P<0.05),q value was1.31>1.15by kimformula.This sayed combination GSI and DDP possessed synegistic effect.2Under Fluorescence microscope, the morphology of SKOV3in controlgroup exerted epithelial cobblestone phenotype with tightened intercellularjunction,however the morphology of SKOV3/DDP in control group exertedelongated spindle-shaped phenotype(mesenchymal phenotype)， cells wasdispersed and losed cell-cell junction.After various concentration of DAPTtreatment,the morphology of SKOV3/DDP cell changed from elongatedspindle-shaped phenotype to short spindle-shaped phenotype,and part of thecells were oval.The change was more pronounced with75μmol/L of DAPTtreatment.3Real time-PCR data indicated that before DAPT treatment onSKOV3/DDP cell, the relative expression of notch-1and Vimentin mRNA wassignificant higher compared to SKOV3cell and E-Cadherin mRNA wasalmost unexpression and significant lower compared to SKOV3cell(P<0.05),respectively.After various concentration of DAPT treatment on SKOV3/DDPcell,the relative expression of E-Cadherin mRNA was up-regulated,howeverthe relative expression of Vimentin mRNA was down-regulated.Comparedwith control group, the change of E-Cadherin and Vimentin mRNA expressionwere significant (P<0.05), respectively. 4FCM results suggested that apoptopic rstes of Control group,DDPgroup, DAPT group and DDP+DAPT group was(7.33±0.59),(9.93±0.71),(24.33±1.36) and (50.10±1.92), respectively.Compared with control group，theapoptosis of treatment group were significant (P<0.05), respectively.Theapoptosis of single treatment was significant lower compared to combinationtreatment (P<0.05), respectively.Conclusion:1DAPT could inhibit SKOV3/DDP cell growth in a dose-time manner.2The notch-1pathway was activated in SKOV3/DDP cell that acquiredEMT-like phenotype during the process of DDP-induced in SKOV3cell,DAPT could induce mesenchymal-epithelial transition(MET) by blocking thenotch-1pathway with morphologic and molecular phenotype in SKOV3/DDPcell.3DAPT could sensitize SKOV3/DDP cell to DDP therapy.