| BackgroundOvarian cancer(OC)is very common in perimenopausal women,with unknown causes and lack of early clinical symptoms.The incidence rate of ovarian cancer ranks third in all gynecological tumors,second only to cervical cancer and endometrial cancer,but its mortality rate is the first.Due to the small size,concealed location,and lack of typical symptoms and signs of the ovaries located deep in the pelvic cavity,early screening and diagnosis are very difficult.Therefore,the onset of ovarian cancer is hidden,and three-quarters of ovarian cancer patients are already in the advanced stage at the time of diagnosis.In China,the annual incidence rate and mortality of ovarian cancer show an upward trend.The five-year survival rate of ovarian cancer patients is 41.8%,and 50%～70%of patients relapse within two years after treatment.At present,the treatment for ovarian cancer is mainly surgery and platinum based chemotherapy.Despite the continuous improvement of surgical skills and chemotherapy programs,the five-year survival rate of ovarian cancer is still rising slowly.Sevoflurane is a volatile anesthetic commonly used in surgical procedures,including tumor resection.Compared with intravenous anesthetics,sevoflurane has the advantages of fast recovery and predictable risk.Sevoflurane has been used in inhalation anesthesia for over 20 years in clinical practice and has been tested in numerous studies,with good safety and effectiveness confirmed.Evidence of in vitro growth suggests that sevoflurane may play a role in the development of tumors,and it exhibits different effects in different tumors,even in different types of cells of the same tumor.Given the significant harm of ovarian cancer in female diseases and the widespread use of the anesthetic sevoflurane in ovarian cancer surgical treatment,in this study,we investigated the effects of sevoflurane on ovarian cancer cells and its related molecular mechanisms through in vitro and in vivo experiments.Part Ⅰ effects of sevoflurane on proliferation,metastasis and apoptosis of ovarian cancer cellsObjective:Sevoflurane was used to treat ovarian cancer cells,and the effects of sevoflurane on proliferation,metastasis,apoptosis and tumor related signal pathways of ovarian cancer cells were studied through various cell function experiments.Methods:Ovarian cancer cells SKOV3 and OVCAR3 were treated with different concentrations of sevoflurane.The viability of SKOV3 and OVCAR3 cells treated with sevoflurane at different concentrations was detected by CCK8 assay.The IC50 values were calculated.The subsequent experiments were performed at IC50*60%concentration.After treated with sevoflurane,the clonogenic ability,cell cycle,cell migration and invasion of SKOV3 and OVCAR3 cells were detected by colony formation assay,cell cycle assay and Western blot,flow cytometry and Western blot,scratch healing assay and transwell assay,the activity of MMP9 secreted by ovarian cancer cells was detected by gelatin zymography assay,and the expression levels of PI3K/AKT signaling pathway protein markers were detected by Western blot.Results:Sevoflurane could inhibit the growth of SKOV3 and OVCAR3 cells in a dose-dependent manner.Sevoflurane treatment of ovarian cancer cells significantly inhibited the colony formation ability of ovarian cancer cells,cells were arrested in G0/G1 phase,and the expression levels of cyclin D1 and CDK4 were significantly reduced.Moreover,the proportion of apoptosis increased,the expression of anti-apoptotic protein Bcl-2 decreased,the expression of pro-apoptotic proteins Bax and Active-Caspase3 up-regulated,and the cell migration and invasion decreased in ovarian cancer cells,and the activity of MMP9 protein,which plays an important role in the invasion process,was inhibited by sevoflurane.In addition,the phosphorylation levels of p-AKT and p-mTOR were significantly decreased in ovarian cancer cells,and the expression of p70S6k protein,which is associated with proliferation,was down-regulated.Conclusion:Sevoflurane can inhibit the viability of SKOV3 and OVCAR3 cells in a dose-dependent manner.Moreover,sevoflurane inhibits the malignant biological behavior of ovarian cancer cells by inhibiting the activation of PI3K/AKT signaling pathway.Part Ⅱ The mechanism of sevoflurane inhibiting malignant behaviors of ovarian cancer cells by down-regulating STC1 expressionObjective:Transcriptome sequencing was performed to screen out the gene STC1 that changes significantly in cells after sevoflurane treatment,and experiments were used to further clarify how sevoflurane regulates the expression of STC1 to inhibit ovarian cancer.Methods:Ovarian cancer cells were treated with sevoflurane.Total RNA was collected and analyzed by transcriptome sequencing.Cluster analysis was performed based on the sequencing results to select genes that were co-up-and down-regulated in the two strains of cells,which were validated using QPCR.Immunohistochemistry was used to detect the expression level of STC1 in ovarian cancer tissue microarrays and its correlation with clinicopathology was statistically analyzed.The effects of sevoflurane on proliferation,apoptosis,migration and invasion of ovarian cancer cells were examined by CCK8 rescue assay,flow cytometry and transwell rescue assay.The effect of overexpression of STC1 on PI3K/AKT signaling pathway after sevoflurane treatment was examined by Western blot assay.In vivo tumor formation assay in nude mice was performed to detect the effect of sevoflurane and STC1 overexpression on the tumor formation ability of ovarian cancer cells.Finally,the expression level of STC1 was detected by immunohistochemistry in nude mouse tumors.Results:Transcriptome sequencing analysis of ovarian cancer cells treated with sevoflurane showed that 259 genes were significantly up-regulated and 117 genes were significantly down-regulated in SKOV3 cells,and 338 genes were significantly up-regulated and 205 genes were significantly down-regulated in OVCAR3 cells.Cluster analysis revealed 26 co-upregulated genes and 10 co-downregulated genes in both strains of cells.Six oncogenes with significant expression differences were selected for subsequent Q-PCR validation,and the results showed that the STC1 gene was significantly down-regulated.Western blot experiments confirmed the inhibitory effect of STC1 protein expression by sevoflurane in ovarian cancer cells.The immunohistochemical results showed that STC1 protein expression was localized in the nucleus and cytoplasm of ovarian cancer tissue cells.STC1 was darker stained and the proportion of positive cells was higher in ovarian cancer tissue cells;in ovarian cancer adjacent tissue cells,STC1 protein was lighter stained and the proportion of positive cells was lower.Pathological analysis showed that STC1 expression correlated with age(P=0.033),and STC1 expression correlated with pathological grade(P=0.037),but not with tumor size(P=0.14)in ovarian cancer.In ovarian cancer SKOV3 and OVCAR3 cells,overexpression of STC1 significantly increased the proliferation ability,decreased the apoptosis ability,and significantly increased the migration and invasion ability of the cells,and partially relieved the inhibitory effect of sevoflurane on the proliferation,migration and invasion of ovarian cancer cells and the promoting effect of apoptosis.Overexpression of STC1 can activate AKT signaling pathway and downstream key protein expression,and partially relieve the inhibitory effect of sevoflurane on AKT pathway.Sevoflurane significantly inhibited tumor formation in nude mice,while tumors in STC1 overexpression group were significantly larger than those in control group,and the tumor inhibition effect of sevoflurane was partially alleviated due to STC1 overexpression,and the trend of experimental results was consistent with the trend of cellular experimental results.The immunohistochemical results showed that STC1 expression level in the tumors of nude mice treated with sevoflurane was significantly decreased,and STC1 level in the STC1 overexpression group was recovered after sevoflurane treatment,and the STC1 level in the overexpression group was the highest.Conclusion:Sevoflurane inhibits the activation of PI3K/AKT/mTOR signaling pathway by regulating the expression of STC1,thus inhibiting the malignant biological behavior of ovarian cancer cells and the growth of nude mice tumors.Part Ⅲ Sevoflurane inhibits the malignant progression of ovarian cancer cells by regulating STC1/ITGB6 axisObjective:The protein ITGB6 interacting with STC1 was selected through the immune coprecipitation experiment,and the indispensable role of ITGB6 in sevoflurane inhibiting the malignant behavior of ovarian cancer cells by regulating the expression of STC1 was further clarified through the experiment.Methods:GEPIA website was used to analyze the expression relationship between STC1 and ITGB6,and co-immunoprecipitation assay was used to detect the relationship between STC1 and ITGB6.The expression of ITGB6 in ovarian cancer cells and tissues was detected by Western blot and immunohistochemical experiments.Western blot experiments were performed to examine the effect of overexpression of STC1 along with knockdown of ITGB6 on the AKT/mTOR signaling pathway.CCK8,flow cytometry and transwell rescue assays were used to detect the effect of sevoflurane/STC1/ITGB6 on cell proliferation,apoptosis,migration and invasion.Results:Co-immunoprecipitation experiments revealed that STC1 could bind ITGB6.ITGB6 is highly expressed in ovarian cancer cells and tissues.STC1 overexpression can activate AKT signaling pathway,knockdown of ITGB6 can inhibit AKT signaling pathway and corresponding protein activation,while overexpression of STC1 combined with knockdown of ITGB6 can partially alleviate the activation of AKT signaling pathway by just overexpression of STC1.Adding knockdown ITGB6 to the sevoflurane+STC1 overexpression group partially relieved the promoting effect of STC1 on ovarian cancer proliferation,migration,and invasion,and partially relieved the inhibitory effect of STC1 on ovarian cancer apoptosis.Conclusion:Sevoflurane inhibits the activation of PI3K/AKT/mTOR signaling pathway by regulating the expression of STC1/ITGB6,thus inhibiting the malignant biological behavior of ovarian cancer cells. |
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