| Objective: Ovarian cancer is one of the gynecological malignancies. Thepatient with early ovarian carcinoma has no specific symptoms,The majorityof patients with ovarian carcinoma are at advanced stage when they werediagnosed. To date, due to chemoresistance and postoperative recurrence, the5-year survival rate is still about30%,it is a serious threat to women’shealth.The invasion and metastasis of ovarian carcinoma is the main factorthat affects survival rate, the invasion and metastasis of ovarian carcinoma is acomplicated process,but its specific mechanism of action is unknown. Studiesfound that about90%of human ovarian tumors originated from the ovariansurface epithelium (OSE), therefore, it is of great significance to study themolecular mechanism of the invasion and metastasis of epithelial ovariancancer, for the prevention and treatment of this disease. At present, themolecular mechanism of tumor metastasis is always one of the hot topics inthe study of tumor.More and more morphology and molecular evidence indicated thatEpithelial-mesenchymal transition (EMT) plays an important role in tumorinvasion and metastasis, the latest research indicates that, the cancer cellsundergo EMT to be able to obtain the stem cells characteristics. EMT is aprocess that in certain conditions the epithelial cell layers lose adhesion andcell polarity, cytoskeleton rearrangement, simultaneously acquire somecharacteristics of mesenchymal to be able to move among the cellularstroma,at the same time, epithelial and stromal cell markers express change. The mostimportant landmark of EMT is the decrease or loss of E-cadherin and theincrease of N-cadherin. EMT is a fundamental physiological and pathologicalphenomena, involved in embryonic development, organ formation and tumor metastasis. More and more studies have shown that EMT in tumor invasionand metastasis plays an important role, which has become the hot spot in theresearch of tumor. However, little is known about the signaling transductionpathway and specific mechanism, probe into the causes of EMT, and exploresthe signal transduction pathways and transcription factors that control theoccurrence of EMT, to finding new targets for cancer treatment provided newclues. Study confirmed that multiple growth factors in the tumormicroenvironment can induce EMT, TGF-β1is the key factors, which inducesEMT.So we set out to determine the effects of TGF-β1on the EMT, invasionand metastasis of SKOV-3and3AO cells cultured in vitro. Meanwhile, thesignaling transduction pathways of which might had been led by TGF-β1onthe EMT, invasion and metastasis of SKOV-3and3AO cells were studied.Our experiment was designed to provide a new viewpoint and a definedstrategy for preventing and treating epithelial ovarian cancer.Methods: The human ovarian serous cancer cell line SKOV-3andmucinous cancer cell line3AO were cultured in RPMI1640, culture solutioncontaining10%heat-inactivated FBS, under a humidified5%CO2atmosphere，at37℃, cells were applied to the experiment when they enteredthe logarithmic phase.1. We stimulated SKOV-3and3AO cell with TGF-β1at differentconcentrations (0、1、2、5、10ng／ml) in different time courses (24h、48h),and used light microscope to observe the cell morphological changes.2. MTT method: MTT method was used to determine cell proliferation.The cultures were initiated in96-well plates at a density of3×104/mL.Cellswere treated for different times(24h、48h)with various concentrations(0、1、2、5、10ng／ml) of TGF-β1. We choose0ng／ml as the control group. At lastMTT method detected the proliferation of cell growth.3. Transwell invasion assay: Applicated with24-well Transwell invasionchamber for SKOV-3and3AO cell invasiveness test. Removed Transwellinvasion chamber of different experimental groups after culture48h continuously, fixed with paraformal dehyde, after hematoxylin staining,counted cell on the back of transmembrane membrane with high-poweredmicroscope.Compared with the control group to detect cell motility andinvasivness of the change. Cells were grouped as follows:(1) Controlgroup:cells were only cultured in RPMI-1640supplemented with10%heat-inactivated FBS (2) TGF-β1group: cells were treated with TGF-β1(1、2、5、10ng／ml)(3) LY294002group: cells were treated with10μmol/LLY294002;(4) LY294002+TGF-β1group：after cells were treated with10μmol/L LY294002for30min. TGF-β1(10ng／ml) was added to the theculture supernatants.4. Western blot analysis: We measured the protein expression of p-Akt(Thr308)、Twist、E-cadherin and N-cadherin protein in different experimentalgroups. Cells were grouped as follows:(1) Control group:cells were onlycultured in RPMI-1640supplemented with10%heat-inactivated FBS (2)TGF-β1group: cells were treated with TGF-β110ng／ml (3) LY294002group: cells were treated with10μmol/L LY294002;(4) LY294002+TGF-β1group：after cells were treated with10μmol/L LY294002for30min. TGF-β110ng／ml was added to the the culture supernatants. The proteins wereextracted from SKOV-3and3AO cells48h later after treated.5. All statistical analysis were performed with SPSS13.0statisticalsoftware package.Results:1. We treated SKOV-3and3AO cells with TGF-β1in differentconcentration and observed the cell morphological changes under invertedphase contrast microscope. SKOV-3and3AO cell has the morphology change,some cells showed fusiform shape, the partial cells stretch out the pseudopod,the arrangement of the cells disorderly, the intercellular space connection arelooser.2. MTT method results: the proliferation rates of SKOV-3and3AO cellsshowed no significant deference after teated with TGF-β1.There was nosignificant difference in the proliferation of SKOV-3,3AO cells when compared with the control group.(P>0.05).3.Transwell invasion assay results:cultured cells in Transwell chamberafter48hours, observed each field of vision under the microscope,trans-membrane cell number of the SKOV-3cells after teated with TGF-β1（1、2、5、10ng／ml）was (42±5)、(53±3)、(84±9)、(114±11),in the controlgroup trans-membrane cell number was(29±3), in the group of TGF-β1（10ng／ml）/LY294002trans-membrane cell number was(37±5),in the group ofLY294002trans-membrane cell number was (18±3). Trans-membrane cellnumber of the3AO cells after teated with TGF-β1（1、2、5、10ng／ml）was(47±6)、(56±5)、(86±7)、(129±9),in the control group trans-membrane cellnumber was(21±3), in the group of TGF-β1（10ng／ml）/LY294002trans-membrane cell number was(54±7),in the group of LY294002trans-membrane cell number was (12±3).We treated SKOV-3and3AO cells with TGF-β1in differentconcentration, trans-membrane cell more than the control group（P<0.05）,and increased with increasing concentration.It showed that the motility andinvasive ability of SKOV-3and3AO cells was greatly enhanced.We treated SKOV-3and3AO cells with LY294002in differentconcentration, trans-membrane cell less than the control group（P<0.05）.Itshowed that the motility and invasive ability of SKOV-3and3AO cells wasgreatly weakened.4. Results of Western-blotting:(1) While Western blot analysis documented the expression ofp-Akt(Thr308) in SKOV-3increased greatly in TGF-β1group（0.9405±0.002）compared to those in control group（0.8265±0.097）（P<0.05）, and inLY294002group （0.3689±0.001） and LY294002+TGF-β1group（0.6518±0.116）were significantly lower than that in TGF-β1group (P<0.05).And for3AO significant differences were observed in the expressions ofp-Akt (Thr308) among TGF-β1group（0.3534±0.003）, LY294002group（0.1919±0.001）and LY294002+FSH group（0.1993±0.001）when comparedwith Control group（0.2901±0.003）（P<0.05）. TGF-β1group increased greatly compared with those in control group（P<0.05）, and in LY294002group andLY294002+TGF-β1group were significantly lower than that in TGF-β1group(P<0.05).(2) While Western blot analysis documented the expression of Twist andN-cadherin in SKOV-3increased greatly in TGF-β1group, Twist（1.1311±0.001）and N-cadherin（0.8708±0.001）compared to those in Controlgroup Twist（0.7731±0.147）and N-cadherin（0.3841±0.032）（P<0.05）,andin LY294002group Twis（t0.4584±0.006）and N-cadherin（0.2946±0.006）,andLY294002+TGF-β1group Twist （0.6002±0.007） and N-cadherin（0.4299±0.001）were significantly lower than that in TGF-β1group (P<0.05).And for3AO significant differences were observed in the expressions of Twistand N-cadherin among TGF-β1group Twist（1.0901±0.005）and N-cadherin（0.8156±0.003）, LY294002group Twist（0.3846±0.004）and N-cadherin（0.2316±0.007）,and LY294002+TGF-β1group Twist（0.4822±0.003）andN-cadherin （0.4002±0.001） when compared with Control group Twist（0.5506±0.018）and N-cadherin（0.3590±0.036）,（P<0.05）. TGF-β1groupincreased greatly compared with those in control group（P<0.05）, and inLY294002group and LY294002+TGF-β1group were significantly lowerthan that in TGF-β1group (P<0.05).While Western blot analysis documented the expression of E-cadherin inSKOV-3weakened greatly in TGF-β1group（0.4084±0.006） compared tothose in Control group（0.8414±0.019）（P<0.05）,and in LY294002group（0.7852±0.006）and LY294002+TGF-β1group（0.6471±0.005）weresignificantly higher than that in TGF-β1group (P<0.05). And for3AOsignificant differences were observed in the expressions of E-cadherinamong TGF-β1group（0.3186±0.005）, LY294002group（0.5604±0.002）and LY294002+TGF-β1group（0.4709±0.001）when compared with Controlgroup（0.6361±0.037）（P<0.05）. TGF-β1group weakened greatly comparedwith those in control group（P<0.05）, and in LY294002group and LY294002+TGF-β1group were significantly higher than that in TGF-β1group (P<0.05).Conclusion: 1. the proliferation rates of SKOV-3and3AO cells showed no significantdeference after teated with TGF-β1.2. TGF-β1could induce SKOV-3and3AO cells morphological alteration,it increased the expression of the transcription factor Twist and mesenchymalmarker N-cadherin,and decreased the epithelial marker E-cadherin.Simultaneously, SKOV-3and3AO cells obtainsed the characteristics ofmesenchymal, which enhanced the motility and invasive ability.3. TGF-β1acts on SKOV-3and3AO cells could excite PI3K/Aktpathway,thus p-Akt protein expression increase. Inducing Twist proteinexpression increase,promoted cell invasion ability.4. PI3K/Akt inhibitor LY294002acts on SKOV-3and3AO cells, p-Aktactivity is inhibited, Twist protein expression is reduced, then inducing theprocess of EMT reversed.reducing the invasive ability of tumor cells. |
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