Effects Of Propofol On The Proliferation,Invasion And Migration Of Ovarian Cancer SKOV3 Cells And Related Molecular Mechanisms

The overall mortality rate of the malignant tumors is only lower than that of cardiovascular and cerebrovascular diseases and keeps increasing in last several decades.Global data showed that the average number of deaths due to tumor is about 7 million per year.Moreover,past studies have demonstrated that all kinds of traumas and drugs during the operation and operation per se in the treatment of malignant tumors may lead to the disruption of the homeostasis of tumor microenvironment and through which affect the outcome of operation and consequently the prognosis of the patients.While some studies reported that specific interventions made by anesthesiologists could reduce the recurrence and metastasis of tumor,more and more studies showed that anesthetics and stress reaction could aggravate the aggressive behaviors of tumor cells through the influence of multiple pathways.These influences mainly include two aspects,the direct influences,which are usually resulted from the anesthetic or stress reaction on the apoptosis,proliferation of.tumor cell,and invasive/migration ability of carcinoma,and the indirect influences that are usually resulted from the imbalance of immune function during the period of anesthesia.In addition,anesthesia technique,stress injury and special treatment could induce the suppression of immune function,which will do an indirect action on tumor recurrence and metastasis.Ovarian cancer belongs to a kind of gynecologic tumor,which has a higher degree of malignancy and was considered as a serious threat to women’s life and health.Multiple cellular signaling pathways along with different molecular mechanisms have been shown to engaged in the development and progression of ovarian cancer,such as activity of ERK1/2 signaling,expression of MMPx,VEGF,and E-cadhesion.Most patients are asymptomatic at the early stage of ovarian cancer,which make it hard to be diagnosed till the cancer is already advanced and therefore lost the chance of surgery.Under inferior prognosis,worse overall survival rate(less than 5 years)and higher rate of recurrence and metastasis,it is hard to cure ovarian cancer.But many factors have changed,such as:the prevention,treatment and diagnosis of ovarian cancer,which is related with the development of gynecology and obstetrics.It could be attributed to the screening for high-risk factors and combined application of multiple diagnostic methods,leading to more patients were given surgical opportunities.Over the years,researchers have do many explores on the molecular mechanisms and multidisciplinary combination therapy,necessitating deeply understand on the process of diagnosis and treatment.While most scholars pay more attention to the period before and after the operation,but limited attention to the perioperative anesthesia,Anesthesia is a well known important step in the radical operation of ovarian cancer,which demands more for anesthesiologists,not only focusing on the past studies such as stress regulation,selections of anesthesia methods,changes of internal environment under laparoscopy,airway mechanical changes,and postoperative analgesia,but active exploration for the new ideas to reduce the invasion and metastasis of ovarian cancer during anesthesia intervention.Propofol is widely used drugs during anesthesia of surgery,and other treatments outside the operating room as well.Considering anesthesia specialists have paid more attention to the studies related with sedation,inflammation,immune damages,and their intensive interaction with oncologists,it becomes imperative to switch gears and determine the relationship between propofol and the progression of tumors,which will be able to improve the current experimental strategies,accumulate the theoretical data,and consequently promote the overall research of anesthesiology and oncology.Although the current research is mainly based on in vitro experiments,the conclusions what have been drawn and the data what have been accumulated are significant and predictive,indicating the research of anesthesiology and tumor cell biological activity has been kicked off.Propofol has distinct effects on the biological activities of different types of tumors,and the effects of propofol on the tumor cell behaviors are still controversial.Propofol was reported to promote the proliferation,inhibit migration and apoptosis for some tumor cells,but was also reported to play a complete opposite role in others.To the best of my knowledge regarding the ovarian cancer,the impact of propofol on the biological activity of the cultured cells is barely reported.Listed below includes three parts of this study.Throughout this study,ovarian cancer SKOV3 cells were cultured in vitro and randomly divided into 5 groups according to the needs of the experimental design,including control group,fat emulsion group,low concentration propofol group,and two high concentration propofol groups.Firstly,the cell viability,the rate of apoptosis and the migration and invasion of cells were observed.Experimental methods used in this part included CCK8 assay,which was used to observe the effect of propofol on cell viability in ovarian cancer SKOV3 group,the effect of propofol on apoptosis in SKOV3 group was observed by flow cytometry.The effects of propofol on cell migration and invasion in ovarian cancer SKOV3 group were observed by scratch test and Transwell test,respectively.All data were statistically analyzed by using the software mentioned in material method.Secondly,to investigate the mechanism that propofol changing the biological activity of SKOV3 cells.The phosphorylation of ERK1/2 along with the total protein expression of ERK1/2,MMP-2,MMP-9 in each group of SKOV3 cells were detected by Western blot,and the protein expression of each group were statistically analyzed.Our findings from siRNA technology suggested the effects of Propofol on the aggressive behavior of SKOV3 cells is dependent on ERK1/2 expression and demonstrated ERK1/2-MMP2/9 signaling is key in propofol increased proliferation,invasion and migration of SKOV3 cells.Moreover,other alternative signaling including VEGF、TGF-β1、E-Cadherin expression may also be involved in this process.Thirdly,VEGF,E-Cadherin(before and after using TGF-β1)in each group of SKOV3 cells were detected by Western blot technique,and the protein expression of each group was statistically analyzed.The results of the first、second and the third parts are discussed and analyzed to determine the effect of propofol on the biological activity of ovarian cancer SKOV3 cells and elucidate the related mechanisms.Graphpad prime 5.0 was used to analyze all the data and assess the statistic significance of every indicator,and their relative changes.P value less than 0.05 was considered as significant difference between different groups.PART I Effects of propofol on the apoptosis、proliferation、invasion and migration of ovarian cancer SKOV3 cellsObjectiveTo determine the effects of propofol on the apoptosis、proliferation、migration and invasion activity of ovarian cancer SKOV3 cell lines using human ovarian cancer cultured cell model.MethodsThrough culturing human ovarian cancer SKOV3 cell lines in vitro,the confluent cells were randomly divided into control group(group C)、fat emulsion group(group F)、25μmol/L propofol group(group P25)、50μmol/L propofol group(group P50)、100μmol/L propofol group(group P100).CCK8 assay was used to detect the cell proliferation activity among all groups at 24h、48h、72h;Flow cytometry was used to detect cell apoptosis rate at 24h among all groups;Moreover,Wound scratch assay and Transwell migration/invasion assay were used to detect invasion and migration of ovarian cancer SKOV3 cell lines at 24h among all groups,respectively.Results:1、The results of CCK8 assay showed that the differences of OD values(450nm)at 24h、48h、72h among all groups upon the treatment of propofol were significant(Ftime=1610,P<0,001;Fconcentration=180.5,P<0.001;Finteraction=4.0,P<0.001),while there is no significant differences of OD values(450nm)among group C、group F、group P25(P>0.05)at 24h、48h、72h.Moreover,OD values(450nm)at 24h、48h、72h from group P50 and group P100 were respectively and significantly higher than group C、group F、group P25(P<0.001),respectively,and the OD values(450nm)at every time at 24h、48h、72h from group P100 was significantly higher than group P50(P<0.01);2、Flow cytometry results showed that the percentage of cells apoptosis at 24h was significantly different among group C、group F、group P25、group P50 and group P100(F=277.60,P<0.001),while no significant difference for cells apoptotic rates at 24h among group C、group F、group P25(P>0.05).Moreover,cell apoptosis rate at 24h from group P50 and group P100 was and significantly lower than group C、group F、group P25(P<0.01),respectively,and cell apoptosis rate 24h from group P100 was significantly lower than group P50(P<0.01);3、Wound scratch assay showed that the cell migration rate at 24 hour was significantly different among all groups(F=664.3,P<0.001),while there is no significant difference for the cell migration rate at 24 hour among group C、group F、group P25(P>0.05).Moreover,the cell migration rate at 24 hour from group P50 and group P100 was significantly higher than group C、group F、group P25(P<0.01).The cell migration rate at 24 hour from group P100 has no difference from group P50(P>0.05);4、Transwell migration assay showed that the cell migration number at 24 hour was significantly different among all groups(F=254.7,P<0.001),while the cell migration number at 24 hour didn’t exhibit significant difference among group C、group F、group P25(P>0.05).Moreover,the cell migration number at 24 hour from group P50 and group P100 was significantly higher than group C、group F、group P25(P<0.01).The cell migration number at 24 hour from group P100 is significantly more than group P50(P<0.05);5、Transwell invasion assay showed that the cell invasion number at 24 hour was significantly different among group C、group F、group P25、group P50 and group P100(F=160.0,P<0.001),while there is no significant difference for the cell invasion number at 24 hour among the different among group C、group F、group P25(P>0.05).Moreover,the cell migration number at 24 hour from group P50 and group P100 was significantly more than group C、group F、group P25(P<0.01).The cell invasion number of group P100 was respectively and significantly more than group P50(P<0.05).Conclusion:Higher concentration of propofol promotes the invasion and migration of ovarian cancer SKOV3 cell lines,and enhances its proliferation activity as well,which is positively related with the treatment time;The lower concentration of propofol have no considerable effects on proliferation、invasion and migration activity of ovarian cancer SKOV3 cell lines.PART Ⅱ The molecular mechanism of propofol on the proliferation、migration and invasion of ovarian cancer SKOV3 cells by ERK1/2-MMP2/9Objective:To elucidate the molecular mechanism of propofol enhancing the apoptosis,proliferation、migration and invasion activity of ovarian cancer SKOV3 cell lines using cultured cell lines.Material and Methods:Western Blot was used to detect the phosphorylation ERK1/2 at early stage,and the expression of ERK1/2、VEGF、MMP-2、MMP-9 and E-cadherin(before and after the intervention of TGF-β1)in ovarian cancer SKOV3 cell lines at the time points indicated;siRNA technology was used to determine the effects of propofol on the ERK1/2-MMP2/9 signaling activity.Results:1.Western Blot showed that the relative expression amount at 24 hour about ERK1/2、MMP-2、MMP-9 was significantly different among all groups(FERK1/2=226.31,P<0.001;FMMP-2=383.2,P<0.001;FMMP-9=215.1,P<0.001);Moreover,phosphorylation of ERK1/2 was significantly increased upon the treatment of propofol at 10min and 30min,as compared with that of the control group and fat emulsion group(F=568.9,P<0.001);.ERK1/2 phosphorylation level was related with the concentration of propofol and its treated time.Higher concentration and longer treatment induced more phosphorylation of ERK1/2 in SKOV3 cells;2.The relative expression amount at 24 hour about ERK1/2、MMP-2、MMP-9 wasn’t significantly different among group F、group C and group P25(P>0.05);3.The relative expression amount at 24 hour about ERK1/2、MMP-2、MMP-9 from group P50 and group P100 was significantly higher than group C、group F、group P25(P<0.01);4.The relative expression amount at 24 hour about ERK1/2 from group P100 was significantly higher than group P50(P<0.01),but the relative expression amount at 24 hour about MMP-2、MMP-9 wasn’t significantly different between group P50 and group P100(P>0.05);5.ERKsiRNA substantially decreases the expression of ERK1/2 as compared with that of control(non-targeted)siRNA,indicating ERK1/2 was efficiently silenced in SKOV3 cells.The relative expression amount of MMP-2 and MMP-9 substantially increased in control siRNA treated SKOV3 cells upon the treatment of propofol,which is similar with that of non-treated SKOV3 cells;In contrast,ERKsiRNA treatment partially reduces the ability of Propofol to increase the expression of MMP-2 and MMP-9 in SKOV3 cells,suggesting the effects of Propofol on the aggressive behavior of SKOV3 cells is dependent on ERK1/2 expression.These data demonstrated ERK1/2-MMP2/9 signaling is key in propofol increased proliferation,invasion and migration of SKOV3 cells;6.But there are still difference among five groups after ERKsiRNA about the proliferation,migration and invasion of ovarian cancer SKOV3 cells,suggesting other ways may exist that influencing cell biological activities.Conclusion:Application of Propofol enhances the expression of ERK1/2、MMP2/9 at ovarian cancer SKOV3 cell lines;Propofol increased MMP-2/9 is through up-regulating expression of ERK1/2,indicating the key function of ERK1/2-MMP2/9 signaling axis in this process.The lower concentration of propofol has no considerable effects on the expression of ERK1/2,MMP-2/9PART Ⅲ Propofol affects the proliferation、migration and invasion of ovarian cancer SKOV3 cells by modifying the expression of VEGF and E-cadhesionObjective:To elucidate the molecular mechanism of propofol beyond the ERK1/2-MMP signaling enhances the proliferation、migration and invasion activity of ovarian cancer SKOV3 cell lines using cultured cell lines in vitro.Material and Methods:Western Blot was used to detect the expression of VEGF、E-cadherin(before and after the intervention of TGF-β1)at protein level among all groups at 24 hour in ovarian cancer SKOV3 cell lines.Results:1.Western Blot showed that the relative expression of VEGF and E-cadherin(after TGFβ-1 intervention)at 24 hour was significantly different among all groups(FVEGF=293.3,P<0.001;FE-cad+T=203.1,P<0.001),but the relative expression of E-cadherin(begore TGFβ-1 intervention)wasn’t significantly different among all groups(FE-cad=5.961,P>0.05);2.The relative expression of VEGF and E-cadherin(after TGFβ-1 intervention)at 24 hour wasn’t significantly among group C group F and group P25(P>0.05);3.The relative expression of VEGF at 24 hour from group P50 and group P100 was respectively and significantly higher than group C、group F、group P25,but the relative expression of E-cadherin at 24 hour(after TGFβ-1 intervention)was respectively and significantly lower than that(P<0.01);4.The relative expression of VEGF and E-cadherin at 24 hour(after TGFβ-1 intervention)wasn’t significantly different between group P50 and group P100(P>0.05).Conclusion:Propofol enhances the expression of VEGF at ovarian cancer SKOV3 cell lines and promotes TGF-β1 down-regulated E-cadherin expression;The lower concentration of propofol have no considerable effects on the expression of VEGF and E-cadherin.