The Effect Of Hypoxia-inducible Factor-1α On Epithelial-Mesenchymal Transition In Human Epithelial Ovarian Cancer

Objective：Ovarian cancer is the most lethal tumour among gynaecolo-gical malignancies,The5-year survival rate is only30%⁴0%. The poorprognosis are mainly related to its rapidly growth and spread, the occurrenceof occult metastasis within the peritoneal cavity and the fact that they are oftendetected at an advanced stage in most patients contributing to low overall curerates. In recent years, Epithelial-mesenchymal transition, Mesenchymal-epithelial transition, the crosstalk between microenvironment and tumor cellsare considered to be closely related to tumor invasion and metastasis. EMTdissolved epithelial cell-cell adhesion and enhance the ability of activity, thentransfers by breakthrough the basement membrane. Hypoxic is one of thebasic features in a solid cancer microenvironment, it exists during the wholetime of solid tumors in progress and treatment. This article will detect theprogress of EMT induced by HIF-1α under hypoxia condition and then theeffect of invasion and metastatic in ovarian cancer cell. Futher explorewhether it can reverse EMT and change the ablity of invasion and metastaticthrough blocking the calcium signal pathway. So the aim of this article is todiscuss the singal transduction of EMT and invasion of ovarian cancer on amolecular level under the condition of hypoxia in order to provide experientalfoundation for new treatment strategies.Methods:The human ovarian serous cancer cell line SKOV-3andmucinous cancer cell line3AO were cultured in RPMI-1640with10%fetalbovine serum and1%penicillin-streptomycin at37C in5%CO₂in air. Cellswere applied to the experiment when they entered the logarithmic phase.1Inverted phase contrast microscope: Observe the changes of SKOV-3and3AO cells' morphology, under normoxic and hypoxia conditions,respectively. 2MTT assay: MTT assay was used to determine cell proliferation. The cellswere inoculateed in96-well plates at a density of5×10⁴/mL. Cells weretreated for different times(12h,24h,48h)with various concentrations(25,50,100,150,200μmol/L) of CoCl₂with six replicate wells for eachconcentration. We choose0μmol/L of CoCl₂as the control group. Analysethe proliferation of SKOV-3and3AO cells by the MTT method.3Western blot analysis: SKOV-3Cells were treated for24hours withdifferent concentrations(25,50,100,150,200μmol/L) of CoCl₂.Then wemeasured the protein expression of HIF-1α protein in different CoC12concentration.3AO Cells were treated for different time(12h,24h,48h)with50μmol/L)of CoCl₂.Then we measured the protein expression ofHIF-1α protein in different times.4Western blot analysis: We measured the protein expression of HIF-1α,Twist,E-cadherin and N-cadherin protein in different experimental groups.Cells were grouped as follows:(1) Control group:cells were only culturedin RPMI-1640supplemented with10%heat-inactivated FBS (2) CoCl₂group: SKOV-3cells were treated with100μmol/L CoCl₂for24hoursï¼›3AO cells were treated with50μmol/L CoCl₂for24hours (3) VPL group:cells were treated with200μmol/L VPL;(4) CoCl₂+VPL group：after cellswere treated with200μmol/L VPL for30min. CoCl₂100μmol/L was addedto the the culture supernatants. The proteins were extracted from SKOV-3and3AO cells24h later after treated.5Transwell invasion assay: Applicated with24-well Transwell invasionchamber for SKOV-3and3AO cells invasiveness test. With100μmol/Land50μmol/L concentrations of CoCl₂were acting on the SKOV-3and3AO cells, add equal amount of PBS as a control group, chemotacticagents, continuously culture for24hours, remove the Transwell cultureafter the invasion of the small rooms to4%paraformaldehyde ice,hematoxylin, high-powered microscope, counting on the back of themembrane-penetrating cells, compared with the control group to detectcell motility and invasion of changes. 6All statistical analysis were performed with SPSS13.0statistical softwarepackage.Results：1Hypoxia involved in conversion of epithelial cells to mesenchymalcells.In this study, we used chemical reagents CoCl₂to establish ahypoxia cell model in vitro to observe the cell morphological changes byinverted phase contrast microscope, The proliferation activity of the cellswas obviously inhibited, Some cells pseudopods out, like polygons. Cellswere disarranged and loosed connection. Whereas the morphology ofcells in control group still presented epithelioid phenotype, adhesionbetween cells is more closely.2The MTT results showed that all the concentrations of CoC12couldmarkedly recede the multiplication of SKOV-3and3AO, after we treatedSKOV-3cells in different time (12h,24h,48h) and with CoC12indifferent concentration(25,50,100,150,200μmol/L),There wassignificant difference in the proliferation of SKOV-3,3AO cells whencompared with the control group.(P<0.05).3Using western blotting to dectect protein result of the two kinds of cells.(1) The expression of HIF-1α protein in skov-3cells after being treated bydifferent concentrations(0,25,50,100,150,200μmol/L) of CoC12for24hours were (0.228±0.017,0.606±0.011,0.936±0.007,1.207±0.18,0.795±0.008,0.445±0.013) respectively. Compared experimental groupwith control group, There were statistically significant difference(P<0.05), And the highest expression of HIF-1α protein was the100μmol/L group.(2) After being treated with50μmol/L of CoCl₂in3AO cells at differenthours(0,12h,24h,48h), each group of HIF-1α protein expression,respectively:(0.322±0.012,0.534±0.007,1.299±0.017,0.825±0.011).Compared experimental group with control group, There werestatistically significant difference(P<0.05). And hypoxia24h group ofHIF-1α expression were the highest. 4Using Western blotting to test the expression of HIF-1α, Twist,E-cadherin,N-cadherin in all experimental groups.(1) HIF-1α protein expression of SKOV-3is significantly higher in hypoxicenvironment(1.195±0.011) than in normoxic condition(0.244±0.007).After intervention by the VPL(0.685±0.013), the expression is greatlyreduced.(P<0.01).3AO's HIF-1α protein expression in hypoxicenvironment(1.453±0.017) was higher than normoxic condition(0.317±0.009),(P<0.01); and expression decreased by VPL's intervention(0.709±0.012),(P<0.01). Compared to the control group, there was nosignificant difference in expression of HIF-1α protein(0.237±0.005,0.295±0.026) in SKOV-3and3AO cells which were being treated withVPL in normoxic condition(P>0.05).(2) SKOV-3and3AO cells in a hypoxic state By the CoCl₂'s treatment.Twist protein level of SKOV-3cells after hypoxia(1.263±0.010)wassignificantly higher than that before(0.231±0.012).(P<0.01); And twistprotein levels greatly reduced(0.584±0.012)after intervention by the VPL.(P<0.01); Twist protein level of3AO cells after hypoxia(1.107±0.008)issignificantly higher than before hypoxia(0.203±0.014)(P<0.01);Andtwist protein levels greatly reduced after intervention by the VPL(0.604±0.018).(P<0.05). But there is no difference in expression of Twistprotein between VPL group (0.227±0.021,0.193±0.011) and controlgroup in SKOV-3and3AO.(3) In normoxic and hypoxic environment, the relative value of E-cadherinprotein in SKOV-3cells is(0.965±0.011,0.226±0.009)(p<0.01). afterinterventioned by VPL,the level of E-cadherin protein is significantlyhigher (0.365±0.011)(p<0.05)than before.The Relative value ofE-cadherin protein in3AO cells is (0.795±0.012,0.163±0.005)(p<0.01).E-cadherin protein levels is markedly elevated (0.359±0.003)(p<0.01)after intervention by the VPL. Compared to the control group, there wasno significant difference in expression of Twist protein(0.872±0.021,0.789±0.008) in SKOV-3and3AO cells which were being treated with VPL in normoxic condition(P>0.05).(4) In normal and hypoxic environment, the relative value of N-cadherinprotein in SKOV-3cells is(0.294±0.007,0.954±0.013)(p<0.01). Afterinterventioned by VPL,the level of N-cadherin protein is significantlylower (0.527±0.016)(p<0.05)than before. The Relative value ofN-cadherin protein in3AO cells is (0.176±0.009,0.866±0.013)(p<0.01).N-cadherin protein levels is markedly elevated(0.594±0.021)(p<0.05)after intervention by the VPL. But the N-cadherin protein level is nodifference between normal environment and interventioned by VPL innormoxic condition in (0.304±0.008,0.164±0.007) in SKOV-3and3AO.(P>0.05)5After treated with CoCl², the ability of invasion and metastasis ofSKOV-3and3AO cells (108±17,131±20)(P<0.01) were significantlymore increased than in normoxic condition (23±5,16±4)(P<0.01);however interventd by VPL, the ability of invasion and metastasis weredeclined obviously(53±7,49±11) in the hypoxic environment. There wasno remarkable difference in ability of invasion and metastasis of twokinds cells whether added VPL or not (P>0.05).Conclusion：1The Hypoxic environment of SKOV-3and3AO in vitro can be createdby CoC1²to induce HIF-1's protein expression.2In the Hypoxic environment, The high level expression of HIF-1α proteincan promote Twist protein's expression, induce cell's EMT and regulatethe expression of marker protein (E-cadherin protein's downregulationand N-cadherin protein's upregulation), and then enhance the ability ofinvasion and metastasis.3There is a relationship between hypoxic signal transduction in tumourcells and the calcium signaling of second messenger. Blocking thecalcium signal pathway may reverse epithelial Mesenchymal transitionand then decline invasion and metastasis of SKOV3and3AO cells byinducing the down-regulation HIF-1α expression. 4Hypoxia, Calcium signaling were related with the development andinvasive ability of ovarian cancer.