| Objective: Epithelial ovarian cancer (EOC) is the most commonsubtypes of ovarian cancer and the most lethal of the gynecologicmalignancies. It is rarely diagnosed at an early stage because the disease islack of specific symptoms. Approximately75%of ovarian cancers arediagnosed at advanced stages (stages III–IV), when tumors have alreadyspread beyond the ovaries. While the patients accept the recent normalizedtreatment consisting of cytoreductive surgery and systemic chemotherapy withcarboplatin and paclitaxel, the five-year survival for EOC is only30-40%.Metastasis and chemoresistance are two key points which make EOC adevastating disease.The epithelial-mesenchymal transition(EMT) is a highly conservedcellular program, in which epithelial cells lose their epithelial characteristicsand acquire the properties of mesenchymal cells. This important process wasinitially recognized during embryonic development and organogenesis andemerging data suggests it is a critical piece of cancer invasion and metastasis.Furthermore, recent evidence suggests that cells that undergo EMT acquirestem cell-like properties and resist conventional chemotherapy. Therefore,targeting EMT by some technologies and strategies may be a promisingapproach for the treatment of cancer, while it is dependent on the furtherstudies of EMT molecular modulation and tumorigenesis.Transforming growth factorβ (TGFβ) is a secreted polypeptide that isoverexpressed in a variety of cancer tissues. As a major inducer of EMT, TGF-βsignaling is involved in induction and maintenance of EMT in an autocrineand/or paracrine way. Studies demonstrated that TGF-β cooperates with stemcell pathways like Notch，Wnt/β-catenin，Ras，Hedgehog to induce a full EMT phenotype，which suggests a correlation between EMT and cancer stemcells. While the nature of such signaling cross-talk is highly complex andcontext-dependent, the existence and the specific form of it in EOC need to befurther explored.In this study, the EOC cell line SKOV3cells were cultured and wesought to determine the effect of Notch signaling during TGF-β1-inducedEMT in EOC cells and investigate molecular mechanisms of Notch activationby TGF-β1.Methods: In this study, the EOC cell line SKOV3cells wereconventionally cultured. For all experiments, cells in logarithmic growthphase were serum-starved for24h before treatment with10ng/mL humanTGF-β1,10uM DAPT or both for72h.1The morphological changes of SKOV3cells were observed underinverted microscope.2Western blotting was applied to examine the expression of epithelialphenotype marker E-cadherin and mesenchymal marker vimentin andN-cadherin at the protein level. The activation of Notch signaling andNotch-ligand Jagged1expression were also examined by Western blotting.3The change of migratory ability was evaluated by using a woundhealing assay. TGF-β1, DAPT, or both were administered48h before thewound was generated with a uniform scractch when cells formed a confluentlayer. Images were obtained at the beginning and at the24h time point afterscratching to monitor the cell migration for the closure of the wound.4The mRNA expression of Jagged1was examined by quantitativeRT-PCR after48h and72h of TGF-β1treatment.5All statistical figures were analysed by SPSS13.0statistical softwarepackage.Results:1The resulting cells underwent apparent changes in morphologycompatible with EMT after72h of10ng/ml TGF-β1treatment. The phenotypicchanges observed include conversion from a cobble-stone-like shape to a spindle-like appearance and increased formation of pseudopodia and thesecells became disassociated from their neighboring. SKOV3cells retainedepithelial morphology when exposed to DAPT or combination of TGF-β1and DAPT.2Western blotting showed that the expression of epithelial phenotypemarker E-cadherin in TGF-β1group(0.632±0.129) was obviously less thanthat in control group(1.000±0.000) and the difference was statisticallysignificant(P<0.05). However, the expression of mesenchymal markervimentin and N-cadherin in TGF-β1group(3.067±2.394、1.319±0.243)washigher than that in control group(1.000±0.0001.000±0.001), and both hadstatistically significant(P<0.05). When DAPT was administered, theTGF-β1-induced expression change of E-cadherin, vimentin and N-cadherinwas abrogated, and the differences between the combined group(0.961±0.224、1.548±1.015、1.181±0.223) and control group were not statistically significant.We also found that the expression of NICD was significantly increased inTGF-β1-treated cells(1.453±0.379) compared with control cells(1.000±0.002),a statistically significant difference(P<0.05). However, DAPT decreasedTGF-β1-induced NICD expression(0.540±0.318), and the difference betweenthe combined group and control group was not statistically significant. Inaddition，Western blotting showed that Jagged1protein level was obviouslyenhanced by TGF-β1, and the difference between TGF-β1group(1.514±0.151)and control group(1.000±0.003) was statistically significant(P<0.05).3The wound healing assay revealed that the wounded area of SKOV3cell monolayer healed slowly in control cells at24h, while in TGF-β1-treatedcells, the wounded gap was nearly re-covered, and there was statisticalsignificance of recovered area between TGF-β1group[(24980.18±1171.20）um2] and control group[(19758.01±2534.50) um2](P<0.05). Blockage ofNotch pathway by DAPT can ablate TGF-β1–stimulated cellular migration,and the recovered area in combined group[(17703.40±1519.27) um2] wassignificantly less than that in TGF-β1group and control group(P<0.05).4We measured mRNA expression of Jagged1by quantitative RT-PCR. In compared to control cells, cells treated with TGF-β1for48h and72hincreased mRNA expression of Jagged1by1.74-fold and1.91-fold,respectively.Conclusion:These results from the present study demonstrate a link betweenNotch and TGF-β signaling during EMT in epithelial ovarian cancer. TGF-β1probably active Notch signaling by increased expression of Jagged1and theactived Notch signaling mediates the TGF-β1-induced EMT. |
PDF Full Text Request