| Objective: Ovarian cancer is one of the female reproductive organmalignant cancers, among them, the most common one is epithelial ovariancancer (EOC), due to the occult incidence and lack of early diagnosis, thedisease is not diagnosed until advanced stages in most patients, which occursabdominal and distant metastasis, the mortality of Epithelial ovarian cancer isin the first place among gynecological malignancies, which seriously affectsthe health and lives of women. Currently, the radical cytoreductive surgeryand platinum-based combination chemotherapy are the main treatments forovarian cancer. Because the tumor cells become resistant to chemotherapydrugs, treatment effect is seriously affected, and5-year survival rate is stillhovering in30%-40%. Therefore, it is significant to explore the molecularmechanisms and overcoming strategies of chemotherapy resistance, improvethe clinical efficacy of the chemotherapy drugs and reduce the mortality rateof epithelial ovarian cancer patientsIn recent years, studies have shown that Epithelial-mesenchymal transition(EMT) is closely related to tumor chemotherapy resistance. EMT refers to theprocess that epithelial phenotype cells transform into mesenchymal phenotypecells in particular physiological and pathological circumstances. Meanwhile,the polarity and connection of epithelial cells are lost, the cytoskeletonrearranges, abilities of migration, invasion and anti-apoptotic increase,accompanying with the expression changes of the epithelial and mesenchymalmarkers.Tumor chemotherapy resistance is a key factor in cancer chemotherapyfailure and relapse. Platinum-based chemotherapy acts as the first-linetreatment of epithelial ovarian cancer, it often failures due to acquired resistance caused by continuing chemotherapy. More and more evidencesshow that EMT not only plays an important role in tumor progression andmetastasis, but also involves in tumor resistance and recurrence, whichprotects tumor cells from extracellular signal, and facilitates the escape ofapoptosis. Phosphoinositol-3-kinase/serine-threonine protein kinase B(PI3K/Akt) signaling pathway is an important intracellular signalingtransduction pathway, which plays important biological functions in tumorprogression, metastasis, chemotherapy resistance and so on. However, thecorrelation of epithelial ovarian cancer chemotherapy resistance and EMT isstill limited at present, therefore, the investigations of internal link betweenovarian cancer chemotherapy resistance and EMT, as well as the relatedsignaling transduction pathways, are significant to deeply understand theovarian cancer chemoresistance mechanisms and explore the new interventiontargets.In this study, we observed the differences of morphology, migration, theexpression levels of EMT molecular markers and PI3K/Akt signaling pathwaymolecule between epithelial ovarian cancer cell line and ovarian cancerresistant strain cultured in vitro, and aimed at investigating whether epithelialovarian cancer cell occurred EMT and its molecular mechanisms, furthermore,we studied whether the PI3K/Akt pathway inhibitor LY294002could reversethe EMT phenotype of ovarian resistant strain cell and enhance its sensitivityto cisplatin, intending to provide new ideas for the mechanisms and treatmentstrategies of epithelial ovarian cancer chemoresistance.Methods:The human ovarian serous cystadenocarcinoma cell lineSKOV-3and cisplatin-resistant cell line SKOV-3/DDP were routinely culturedin RPMI-1640culture solution which contained10%fetal bovine serum underhumidified5%CO2atmosphere, at37°C.1MTT assay: SKOV-3and SKOV-3/DDP cells in the logarithmic phasewere digested into cell suspension, whose adjusted cell concentration was5×104/ml, and it was inoculated in96-well culture plates100ul per well. Whenthe cells had already adhered, we treated the both cell lines with different concentrations of DDP (0,0.625,1.25,2.5,5,10ug/ml) for24,48h,0ug/ml waschose as the Ctrol group. The both cells growth inhibition of DDP wasmeasured with four Methyl Thiazolyl Tetrazolium (MTT), in order todetermine the resistance index of the resistant cell.2Light microscope observation: We selected the SKOV-3andSKOV-3/DDP cells in logarithmic phase, as well as the both cells dealed withDDP, to observe the cell morphology changes by the inverted microscope.3Cell scratch experiment: SKOV-3and SKOV-3/DDP cells selected inlogarithmic phase were digested to vaccinate in6well plates. When Cellswere grown to80％-90％confluence and formed a monolayer covering thesurface of the entire well, we drawed a vertical straight in the centre of eachwell, and washed it to remove the floating cells by PBS, the plates were addedlow concentration serum culture solution and placed in37°C,5%CO2incubator, For each well, at least six pictures were taken with microscoperespectively at0and24hour after scratch, at the same time,the cell count wasdone within24hours.The nonrecoverd scratch area was calculated byPhotoshop programe software to determine the ratio of scratch healing areaand the rate of cell proliferation.4Western bloting: We detected the protein expressions of E-cadherin,Vimentin and p-Akt (Thr308) of SKOV-3and SKOV-3/DDP cells inlogarithmic phase, as well as SKOV-3/DDP cell in each experiment group.SKOV-3/DDP cells were grouped as follows:①Ctrol group: cells were onlycultured in RPMI-1640culture solution supplemented with10%fetal bovineserum for24h;②DDP group: SKOV-3/DDP cells were treated with2.5ug/mlDDP for24h;③LY294002group: SKOV-3/DDP cells were treated with10umol/L LY294002for24h;④LY294002+DDP group: After SKOV-3/DDPcells were treated with LY294002for30min, DDP was added to the culturesupernatants for24h.5Flow cytometry: Flow cytometry marked with propidium iodide (PI) wasused to detecte whether LY294002could enhance the sensitivity of SKOV-3/DDP cells to cisplatin. The experiment groups were the same as those of Weste- rn bloting.6All statistical figures were analysed by SPSS13.0statistical softwarepackage.Results:1Results of MTT: SKOV-3and SKOV-3/DDP cells were treated withdifferent concentrations of cisplatin, as the concentration increased and timeextended, the inhibition rates of the two cells were significantly higher thanthose in the Ctrol groups, the differences were statistically significant(P<0.05). SKOV-3and SKOV-3/DDP cells were treated with the sameconcentration of cisplatin, with time prolonging, the former inhibition rate wassignificantly higher than the latter, and the differences were statisticallysignificant (P<0.05).It showed that the sensitivity of SKOV-3cell to DDP washigher than that of SKOV-3/DDP cell.2Results of cell morphology observation: SKOV-3cells were mostly oval,cobblestone-like, the cells connected tightly and gathered into a sheet growthwhich showed epithelioid cell morphology; the resistant SKOV-3/DDP cellsshowed fusiform shape, lost cell polarity, arranged in disorder, the cellsscattered and stretched out pseudopodia in formation of elongated spindle cellmorphology which performanced the interstitial cell morphology. We treatedSKOV-3cells with2.5ug/ml DDP for24h, the cells morphology had changedsignificantly, the majority of which became round and apoptosis, whose cellmembrane shrinked, volume became generally smaller and adherent abilitysignificantly weakened. After SKOV-3/DDP cells were treated with20ug/mlDDP for24h, the round cells began to generally increase, some cells occurredapoptosis, cell membrane began to shrink, and its volume became smaller.3Results of cell scratch experiment：The cells continued to culture for24hafter scratch, with the microscope, we obversed that scratch healing area ofSKOV-3/DDP cell (45.90%) was more than that of SKOV-3cells (13.93%)(P<0.05), while there was no significantly increase in cell proliferationrate(P>0.05), the above prompted that the migration ability of resistantSKOV-3/DDP cell was more enhanced than that of SKOV-3cells. 4Results of Western blotting:(1) Under basal condition, the expression levels of E-cadherin inSKOV-3/DDP and SKOV-3cell were respectively (0.005±0.002) and (0.764±0.017), those of Vimentin were respectively (0.687±0.013) and(0.007±0.001), and those of p-Akt (Thr308)were respectively(0.749±0.021)and (0.375±0.014); the results showed that the expression levels of E-cadherinin SKOV-3/DDP cell was significantly lower than that of E-cadherin inSKOV-3cell (P<0.05); while the expression levels of Vimentin, p-Akt(Thr308) in SKOV-3/DDP cell were significantly higher than those ofSKOV-3cell (P<0.05).(2) The expression levels of E-cadherin, Vimentin and p-Akt(Thr308)protein in SKOV-3/DDP cell each exprement group:The expression of E-cadherin in SKOV-3/DDP cell each exprement group,DDP group (0.004±0.001) was lower than that in LY294002group(0.617±0.016) and LY294002+DDP group (0.285±0.011)(P<0.05).The expression of Vimentin in SKOV-3/DDP cell each exprement group,DDP group (0.824±0.012) was higher than that in Ctrol group (0.653±0.009),LY294002group (0.419±0.014) and LY294002+DDP group (0.609±0.100),(P<0.05).The expression of p-Akt(Thr308) in SKOV-3/DDP cell each exprementgroup, DDP group (0.943±0.017) was higher than that in Ctrol group(0.748±0.013), LY294002group (0.431±0.008) and LY294002+DDP group(0.648±0.015),(P<0.05).Compared with the Ctrol group, the expression of E-cadherin inSKOV-3/DDP cell DDP group did not obviously changed, but the expressionlevels of Vimentin, p-Akt (Thr308) increased; after SKOV-3/DDP cell wastreated with LY294002, the expression level of E-cadherin obviously elevated,those of Vimentin, p-Akt(Thr308) significantly decreased; while thecombination of LY294002with DDP could increased the expression level ofE-cadherin, and reduced those of Vimentin, p-Akt(Thr308), when comparedwith DDP group. 5Results of Flow cytometry:(1) The apoptosis rate of SKOV-3/DDP cell each exprement group: DDPgroup (5.70±0.17), LY294002group (5.73±0.42) and LY294002+DDP group(11.86±2.04) were all higer than that of Ctrol group (0.97±0.11)(P<0.05),moreover, the apoptosis rate of LY294002+DDP group was obviously higherthan those of DDP group and LY294002group, the difference was statisticallysignificant (P<0.05); however, there was no statistical significance betweenDDP group and LY294002group (P>0.05).(2) The SKOV-3/DDP cell numbers in G0/G1phase, DDP group(33.10±0.26), LY294002group (31.70±0.35) and LY294002+DDP group(29.93±0.17) were all lower than Ctrol group (40.70±0.62)(P<0.05);LY294002+DDP group (29.93±0.17) was lower than DDP group (33.10±0.26),the difference was statistically significant (P<0.05); while there was nosignificant difference between LY294002+DDP group (29.93±0.17) andLY294002group (31.70±0.35)(P＞0.05); the SKOV-3/DDP cell numbers in Sphase, DDP group (56.20±1.03), LY294002group (51.80±0.43) andLY294002+DDP group (50.13±1.42) were all significantly higher than Ctrolgroup (39.10±0.58)(P<0.05); the SKOV-3/DDP cell numbers in G2/M phase,DDP group (10.70±0.10) was significantly lower than LY294002group(16.50±0.12), LY294002+DDP group (19.94±0.45) and Ctrol group(20.20±0.41), the differences were statistically significant (P<0.05), whilethere was no statistical difference between LY294002+DDP group(19.94±0.45) and Ctrol group (20.20±0.41)（P＞0.05）.Conclusion:1The chemotherapy resistance of SKOV-3/DDP cell was significant.2Compared with SKOV-3cell, SKOV-3/DDP resistant cells exhibited EMTphenomenon, the cell morphology was transformed into that of mesenchymalcell, the migration ability was strengthened, the expression of epithelial cellmarker E-cadherin downregulated and that of mesenchymal cell markerVimentin upregulated, meanwhile, the expression level of PI3K/Akt signalingpathway effector protein p-Akt upregulated. PI3K/Akt signaling pathway inhibitor LY294002could decrease the phosphorylation levels of Akt，downregulate the expression level of Vimentin, and upregulate that ofE-cadherin in SKOV-3/DDP cell. It showed that the cisplatin-resistance ofSKOV-3/DDP might be related to the abnormal activation of PI3K/Aktsignaling pathway and the cells EMT experience, PI3K/Akt signaling pathwayinhibitor LY294002could partially block the EMT of SKOV-3/DDP cell.3PI3K/Akt signaling pathway inhibitor LY294002could enhance thesensitivity of SKOV-3/DDP cell to cisplatin, LY294002alone or combinatedwith cisplatin might positively regulated the cell cycle progression ofSKOV-3/DDP, and promot the cell apoptosis. |
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