| Objective: Epithelial ovarian cancer (EOC) is the most commonmalignant tumor of ovary, and it is hard to be diagnosed in early phase and ofhigh recurrence rate as well as death rate. The conventional chemotherapy ofovarian cancer is based on platinum, but about70-80%patients haveeventually developed chemoresistance, resulting in the recurrence andmetastasis of tumor, which are the primary reasons for the treatment failure ofovarian cancer and death of patients. Therefore, the five-year survival ratehovers around30%. Consequently, to explore the invasion and metastasismechanisms of chemotherapy-resistant tumor and to find effective targetingpoints are the urgent needs of EOC patients to promote survival andameliorate poor prognosis.Recently, several studies have demonstrated that epithelial-mesenchymaltransition (EMT) plays critical roles not only in embryonic development andwound healing but also in invasion and metastasis of tumors. EMT refers tothe process of epithelial cells losing the features of epithelial phenotype so asto transition to cells of mesenchymal phenotype under specific physiologicalor pathologic conditions. EMT would lead to cell polarity missing, declinationof adhesion among cells, enhancement of athletic ability, cellular morphologyextension and rising of anti-apoptotic abilities so as to accelerate invasion andmetastasis of tumors. EMT is linked to many kinds of cell factors,transcription factors and signaling pathways. EMT is implicated in varioustypes of signaling pathways, including Notch, Wnt, Hedgehog and so on; inaddition, with up-regulation of transcription factors Snail and Slug and so on,down-regulation of epithelial phenotype marker E-cadherin, as well as up-regulation of vimentin of mesenchymal phenotype markers. Moreover,EMT-derived tumor cells can acquire stem cell-like characteristics such asself-renewal. It may be relevant to the chemoresistance.Many years of research have shown that overexpression and activation ofNotch have been described in a multitude of solid tumors and hematologicalmalignancies. And it is also associated with the occurrence and developmentof tumors. Recent researches have revealed that the activation of Notchsignaling pathway could induce EMT of various tumors to accelerate themetastasis and recurrence of the tumor. γ-secretase is the key link in activationof Notch signaling pathways, and becomes the focus in research of the tumortreatment.This experiment cultures human ovarian serous cystic gland cancer cellsSKOV3and their cisplatin resistant strains SKOV3/DDP in vitro so as to testthe differences of the relative quantity of Notch1mRNA. And further studythe relative expression status of E-cadherin and vimentin as well as thechanging of their migrating and transmembrane abilities after the treatment ofγ-secretase inhibitor DAPT on the SKOV3/DDP cells. It aims to describe frommolecular level whether DAPT could inhibit the Notch signaling pathways soas to inhibit the invasion and metastasis abilities of EOC of chemoresistance.And thus provide experimental evidences for treating strategies of the chemoresistance EOC.Methods: The RPMI-1640culturing substrate containing10%fetalcalf serum had been taken to conventionally culture human ovarian serouscystic gland cancer cell line SKOV3and their cisplatin-resistant cell lineSKOV3/DDP in incubators of constant temperature at37℃and of5%CO2ofsaturated. Cells in logarithmic phase were selected out for conducting theexperiments.1The inverted microscope was applied for observing the shapes andtaking photos of the SKOV3and SKOV3/DDP cells.2Real-time fluorescence quantification PCR (PT-PCR): It had beentaken to test the Notch1mRNA relative expression levels of the SKOV3and SKOV3/DDP cells, and the CT results were analyzed and treated with2-△△C tmethods.3Method of methyl thiazolyl tetrazolium (MTT): The SKOV3/DDP wastreated with DAPT of different concentrations (25,50, and75μmol/L)) fordifferent lengths of time (24h,48h, and72h) so as to be treated with0.375%DMSO which was the solvent of DAPT to act as the control group. The MTTmethod was used to test the influences of DAPT on the growth inhibitor rateof cells, through which the most appropriate condensation and time of thepharmaceutical effect were screened out.4Western blotting: It was used to test the relative expression levels of theepithelial phenotype marker E-cadherin and mesenchymal phenotype markervimentin of the SKOV3/DDP cells treated with different concentrations ofDAPT (0,25,50, and75μmol/L) and the SKOV3cells with no treatment.5Wound healing assay: The SKOV3/DDP cells were culturedconventionally so as to be integrated to form monolayer. The synchronizationwas conducted by being dealt with serum free medium for24hours, differentinterferences (experimental group:50μmol/L DAPT; control group:0.25%DMSO) were added according to the group of experiments. A “—” shapedscratch was lined within the center of the bottom of the culturing board.Photos were taken by inverted microscope (40×) at0hour and24hoursrespectively since the treatment of DAPT, so as to calculate the cover area ofthe cells within24hours to evaluate the motility potential of the cells.6Transwell assay: The50μmol/L DAPT was taken to have effect on theSKOV3/DDP so as to add0.25%DMSO to act as the control group. After24hours of consecutive culturing, the transwell chambers were taken out andfixed with95%alcohol. After being dyed with crystal violet, photos weretaken on it with microscope. The cells transferred to the lower chamber of themicroporous membrane were calculated to assess the transferring andinvading abilities of the cells.7The SPSS16.0software was applied for conducting statistical analysis. Results:1SKOV3cells and SKOV3/DDP cells: As was observed under theinverted microscope, the SKOV3cells were in the shape of oval, round orsquare and arranged like flagstones. The connection among the cells wasrather close, showing typical epithelioid cell morphology. The SKOV3/DDPcells are in the shape of long spindle with rather loose arrangement of cellsand not close connection among the cells, displaying mesenchymal cellsforms. The result of the Western blotting test revealed that the E-cadherinrelative expression amount of the SKOV3/DDP was lower than that of theSKOV3cells (0.054±0.012vs.0.718±0.125), while the relative expressionamount of Vimentin was higher than that of the SKOV3cells (3.232±0.267vs0.517±0.059), and the distinction was statistically significant (P＜0.01);moreover, the RT-PCR result showed that the Notch1mRNA relativeexpression quantity of the SKOV3/DDP cells was3.952times of that of theSKOV3cells.2The result of MTT showed that with the increase of the DAPTconcentration and the prolonging of the functioning time, the growthinhibiting rate of the SKOV3/DPP cells would be significantly raised and thedistinctions were statistically significant (all were P＜0.01).3The western blotting result showed that after48hours since the DAPTof various concentrations (25,50, and75μmol/L) had effect on theSKOV3/DDP cells, the relative expression of its epithelial marker E-cadherinhad significantly increased compared to the control group (0.207±0.020,0.295±0.011,0.429±0.024vs.0.054±0.012）（P＜0.01）and it was raised withthe increasing of the concentration of DAPT, and the distinctions among allthe groups were obvious (P＜0.01); furthermore, the relative expression of themesenchymal marker Vimentin had significantly reduced compared to thecontrol group (2.502±0.170,1.873±0.177,1.624±0.057vs.3.232±0.266)(P＜0.01). Nevertheless, there was no statistical significance between the groupstreated with DAPT of50μmol/L and75μmol/L (P＞0.05).4The result of wound healing conducted after24hours since the DAPTof50μmol/L of concentration having effect on the SKOV3/DDP cells showed that the cover area of the scratches in the treating group were significantlysmaller than those of the control group (14.31±3.59VS52.91±10.59)(P＜0.01); and the results of transwell assay showed that the number of thetransmembrane cells in the treating group was significantly less than that inthe control group (13±2vs.37±2in the transwell migration assay while9±1vs.27±3in the transwell invasion assay). All the distinctions were statisticallysignificant (P＜0.01).Conclusion: During the chemoresistance process the SKOV3/DDPcells obtained the EMT phenotype, and DAPT, one of the γ-secretaseinhibitors could target on the Notch signaling pathway to inverse the EMT andinhibit the proliferation, adhesion and invasion and metastasis abilities ofhuman oval cancer cis-platinum resistance cells., which was possible tobecome a new choice for treating the reoccurrence and metastasis ofchemoresistance EOC. |
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