Study Of Anti Tumor Mechanism Of Mifepristone On Endometrial Carcinoma And Cervical Cancer

ObjectiveThe purpose of this study was to investigate the effect and molecular mechanism of mifepristone(MIF)as a treatment drug for endometrial carcinoma and cervical cancer,explore a new therapeutic strategy.Endometrial carcinoma and cervical cancer are common malignancies in gynecology,the incidence has been in a younger trend in recent years.The treatment of endometrial carcinoma patients with expectant reproductive function or old age who cannot tolerate operation and chemotherapy drug resistance is facing difficulties.Early diagnosis of cervical cancer patients and timely treatment,such as surgery,can achieve better prognosis.However,the loss of surgical opportunities and the emergence of radiotherapy and chemotherapy resistance in the treatment process of patients with advanced cervical cancer are still the main causes of treatment failure and poor prognosis.Understanding the pathogenesis and development of cancer can fundamentally improve the prevention,diagnosis and treatment of these cancers.Mifepristone,a progesterone and glucocorticoid receptor antagonist,has been proven effective and safe for termination of pregnancy.The mechanism of termination of pregnancy with mifepristone has been studied at molecular and genetic levels.Previous studies have confirmed that mifepristone interferes with many signal pathways through the regulation of extracellular factors in cell metabolism,such as VEGF signal pathway,adhesion signal pathway and apoptosis pathway.Over the past two decades,numerous studies have found that the key to pregnancy success is the proliferation,migration and invasion of villous trophoblasts that are strikingly similar to the characteristics of cancerous cells.At present,the research on mifepristone has been extended from termination of pregnancy to oncology,and it has been found that mifepristone has a universal inhibitory effect on the proliferation and migration of cancer cells.However,the specific mechanism of the anticancer effect of mifepristone has not been fully clarified.FAK factor,FAK-PI3K/AKT signaling pathway exist in many types of cells and play an important role in various biological processes of the cells.Exosomes are extracellular vesicles secreted by cells,can reflect the physiological function of cells and play an important role in the occurrence,development,migration and metastasis of tumors.Several studies have confirmed the central role of exosomes in tumor metastasis.The purpose of this study was to investigate the effect and molecular mechanism of mifepristone as a treatment drug for endometrial carcinoma and cervical cancer,and explore the role of FAK and FAK-PI3K/AKT signaling pathway and exosomes in proliferation and migration of cancer cell.The study was divided into two parts:1.Mifepristone affected the proliferation and apoptosis of endometrial carcinoma HHUA cells by regulating FAK factor and FAK-PI3K/AKT signaling pathway;2.Mifepristone inhibits the migration of cervical cancer HeLa cells by up-regulating ISG15 to inhibit the secretion of exosomes.Methods1.Part one(1)Culture of human endometrial carcinoma HHUA cells line in vitro.HHUA cells were treated with DMEM containing different concentrations of mifepristone.HHUA cells treated with 25?50?75 and 100 ?mol/L mifepristone were named Mifepristone group.HHUA cells co-transfected with pcDNA3.1-PI3K and pcDNA3.1-AKT overexpression vectors were treated with 100 ?mol/L mifepristone and named Mifepristone + PI3K/AKT group.(2)mRNA expression of FAK,Caspase-3,PI3K and AKT genes was detected by RT-PCR in HHUA cells.(3)Protein expression of FAK,p-FAK,PI3K,p-PI3K,AKT,p-AKT and Caspase-3 was performed by Western blot.(4)Proliferation of HHUA cells under the action of different mifepristone concentration was conducted by MTT assay.(5)Transwell was used to detect cells migration and invasion.(6)Apoptosis detection was performed by flow cytometry.(7)Animal experiment(effect of mifepristone on the growth of HHUA cell line xenografts in nude mice).2.Part two(1)Culture of human cervical cancer HeLa cells line in vitro.(2)Cell viability and migration assay was conducted in HeLa cells treated by MIF by CCK-8 kit,the best time and optimal concentration of mifepristone were selected.(3)The invasive and metastatic ability of each group(the control group?the mifepristone group?the GW4869 group?the mifepristone combined GW4869 group)was detected by scratch test.(4)Apoptosis detection was performed by flow cytometry.(5)Extraction of exocrine.(6)Targeted RT-PCR arrays were performed to measure the expression of genes associated with migration function of the cells.(7)Two marker proteins from the exosomes CD63?CD9 and ISG15 expression level was examined in MIF treated HeLa cells by Western blot.Results1.Part one(1)Mifepristone reduced the proliferation capacity of HHUA cells and significantly inhibit the migration and invasion of HHUA cells.This study examined the effects of different concentrations of mifepristone on HHUA cells proliferation by MTT assay.The cell growth inhibition rate was gradually increased with the increase of mifepristone concentration.When the concentration of mifepristone was at 100?mol/L,cell growth inhibition rate was the highest and the results were concentration dependent.At the same time,the effect of mifepristone on the growth of HHUA cells transplanted tumor in nude mice was observed,and the results showed that mifepristone can inhibit the growth of tumor.The effect of mifepristone on the migration and invasion of HHUA cells was detected by Transwell,and the results showed that the migration and invasion of HHUA cells with concentration of 50-100?mol/L mifepristone decreased significantly(P<0.01),and the results were concentration dependent.(2)Mifepristone promoted apoptosis of HHUA cells.The effect of mifepristone on HHUA cell apoptosis was detected by flow cytometry.The results showed that mifepristone promoted HHUA cell apoptosis in a concentration dependent manner.The apoptosis percentage of HHUA cells was significantly increased after 25-100 ?mol/L mifepristone treatment(P<0.01)compared with the control group(0?mol/L mifepristone treatment).We respectively applied RT-PCR and western blot to detect mRNA and protein expression of HHUA cell apoptosis related gene Caspase-3,results showed that under the effect of concentration of 50-100?mol/L of mifepristone Caspase-3 mRNA and protein levels of the HHUA cells were significantly higher(P<0.01).The above results showed that with the increase of action concentrations,mifepristone for the promotion of apoptosis HHUA also increases accordingly.(3)Mifepristone inhibited the activity of FAK factor and the PI3K/AKT signaling pathway in HHUA cells.The mRNA and protein content of FAK in HHUA cells under the action of mifepristone and phosphorylated mucinous kinase(p-FAK)were detected by RT-PCR and western blot.The results showed that when HHUA cells were treated with 50-100?mol/L mifepristone,the relative expression of FAK mRNA and protein and the protein expression of p-FAK were significantly lower than those of the HHUA cells in the control group(0?mol/L mifepristone treatment)(p<0.01)Mifepristone inhibits the expression of FAK mRNA and protein in a concentration dependent manner at this range.These results suggest that mifepristone inhibits the activity of FAK and its phosphorylation pathways in HHUA cells at high concentration.The protein expressions of PI3K,p-PI3K,AKT and p-AKT in the cells of mifepristone group and mifepristone group combined with PI3K/AKT group were detected rby western blot.The differences in apoptosis and proliferation were detected by MTT and flow cytometry.The results showed that the expressions of PI3K,p-PI3K,AKT and p-AKT in the HHUA cells treated with mifepristone combined with PI3K/AKT group were significantly increased,with statistically significant differences(p<0.01).The proliferation inhibition rate and apoptosis rate of HHUA cells in this group were significantly reduced(P<0.01),and the above results further indicated that mifepristone may promote the apoptosis of HHUA cells by inhibiting the activity of PI3 K/AKT signaling pathway.2.Part two(1)Mifepristone inhibited the growth and migration of HeLa cells and promotes apoptosis.We examined the effects of mifepristone on the activity of HeLa cells in cervical cancer by CCK-8.The results showed that the activity of HeLa cells treated with 1?10?50?100?200?g/mL mifepristone decreased compared with the control group,when the concentration reached 50?g/mL the activity of HeLa cells decreased significantly(P<0.01).When the HeLa cells were treated with 50?g/mL mifepristone at 12?24?36?60 hours,the activity decreased compared with the control group,when the action time reached 36 hours the activity of HeLa cells decreased significantly(P<0.01).Mifepristone inhibited HeLa cells growth in a time-and concentration-dependent manner.We examined the effect of mifepristone on migration and apoptosis of HeLa cells by flow cytometry and wound healing test,at the same time,the levels of Caspase-3 protein in the cells of each group were detected by western blot.The results showed that the apoptosis level of HeLa cells in mifepristone group(50?g/mL mifepristone treated 36 hours)was significantly increased compared with the control group(P<0.01),the migration ability was significantly reduced,and the level of Caspase-3 was significantly increased(P<0.01),indicating that mifepristone could significantly inhibit the migration of HeLa cells and promote apoptosis.(2)Mifepristone inhibited the exosomes secretion of the cervical cancer cellsCD63 and CD9 in exosomes of the control group and mifepristone group were detected by protein western blot.The results showed that the expressions of CD63 and CD9 of the HeLa exosomes treated with mifepristone were significantly reduced(P<0.01),indicating that mifepristone inhibited the secretion of the exosomes.(3)Inhibiting the secretion of exosomes can enhance inhibitory effect of mifepristone on the migration ability of HeLa cells.GW4869 is an exosomes inhibitor,The activity,apoptosis and migration of HeLa cells in the control group(no mifepristone treatment),the mifepristone group(50?g/mL mifepristone culture for 36 hours),the GW4869 group(GW4869 10 ?M culture for 48h),mifepristone combined GW4869 group were detected by CCK-8,flow cytometry and wound healing test.The results showed that the activity of HeLa cell in mifepristone combined GW4869 group was significantly decreased(p<0.01),apoptosis was significantly enhanced(p<0.01),and migration ability was significantly reduced.The results indicated that inhibiting exosomes could enhance inhibitory effect of mifepristone on the migration ability of HeLa cells.(4)Mifepristone inhibited the exocrine secretion by upregulating interferon stimulating gene 15(ISG15).In order to study the molecular mechanism of mifepristone inhibiting exosomes,we first used targeted gene array to screen out ISG15,which was highly enriched in HeLa cells with mifepristone treatment,the ISG15 level of HeLa cells treated with mifepristone at different concentrations was detected by western blot.The results showed that the expression level of ISG15 increased with the increase of mifepristone concentration(P<0.01).Then,the expression of CD63 and CD9 in the HeLa cells of the control group(without drug treatment)and IFN group(16 hours of IFN 1000U/ml culture)was detected by western blot,the results showed that HeLa cells significantly reduced the secretion of exosomes under the effect of IFN(P<0.01).This indicated that mifepristone inhibits the secretion of exosomes by up-regulating the ISG15.Conclusion1.Mifepristone can effectively inhibit the proliferation,migration and migration of endometrial carcinoma HHUA cells and promote apoptosis.The effects of mifepristone on proliferation,migration and apoptosis of HHUA cells showed concentration dependence.The molecular mechanism may be related to the inhibition of FAK and PI3K/AKT signaling pathway and its phosphorylation process.Mifepristone inhibits the growth of transplanted HHUA cells in nude mice.These results provide the theoretical basis for the treatment of endometrial carcinoma with mifepristone.2.Mifepristone can inhibit the secretion of exosomes of cervical cancer HeLa cells,and at the same time,it can enhance the inhibitory effect of mifepristone on the migration of cervical cancer HeLa cells.The molecular mechanism of mifepristone to treat cervical cancer HeLa cells by inhibiting the secretion of exosomes may be to up-regulate the expression of ISG15.This provides a theoretical basis for the treatment of cervical cancer with mifepristone and a theoretical basis for the regulation of exosecretion in the treatment of gynecological tumors.