The Study Of The Effect And Mechanism Of Cepharanthine Hydrochloride On Human Ovarian Cancer

As the highest mortality of gynecological malignant tumor,ovarian cancer is difficult to be perceived at the early time,so tumors often have invaded in the adjacent organs with the symptoms of invasion and metastasis in pelvic and abdominal cavity when the patient visits a doctor.As an important treatment for ovarian cancer,its clinical chemotherapy mainly includes platinum and taxane-based chemotherapy.Initially about 80%of patients show effective treatment,but most of patients maybe encounter the multidrug resistance?MDR?of tumors to make serious influence of treatment effect.Due to the characteristics as being easy for invasion,metastasis and the appearance of MDR,ovarian cancer has been the worst clinnical prognosis of gynecological cancers and the five-year survival rate is less than 45%in the case of adequate treatment.Therefore,the research to find more effective drugs,especially to find the drugs which are not only inhibit tumor growth but also inhibit tumor invasion or MDR,has great value.The cepharanthine hydrochloride?CH?is a semi-synthetic compound obtained by salting the cepharanthine?CEP?extracted from Stephania cepharantha Hayata.Several studies have shown that CEP can exhibit direct antitumor activity in certain types of tumors and can inhibit the invasion and metastasis of certain tumor cells.Besides,it is confirmed by research CEP can effectively enhance the sensitivity of tumor cells to chemotherapeutic drugs,yet the effect of CH or CEP on ovarian cancer has not been reported at home and abroad.In order to explore the effect of CH on ovarian cancer,A2780/Taxol cell line which is the epithelial-derived ovarian cancer with MDR phenotype and its parental A2780 cell line are selected as models.A variety of experimental techniques are adopted to studythe effect and mechanism of CH on the proliferation,apoptosis,migration,invasion and MDR of ovarian cancer for expanding the possible clinical indications of CH and providing theoretical and experimental basis for its further development.Based on the relevance of the content,this study can be divided into the following two parts:Part one:The effect and mechanism of Cepharanthine Hydrochloride on the proliferation and invasion of ovarian cancer cellsPurpose:Taking A2780 cells as the subject,this part is aimed at studying the effect of CH on proliferation,apoptosis,migration and invasion of A2780 cells and its related mechanisms.Methods:A2780 cells were treated with different concentrations of CH and the proliferation of A2780 cells was detected by the MTT assay at 72 h and the IC₅₀ was determined.The MTT assay was used to detect the proliferation inhibitory of CH on A2780 cells at 24,48,72 and 96 h.The effect of CH on the apoptosis of A2780 cells was detected by the flow cytometry using Annexin?-FITC/PI double staining.The effect of CH on the migration ability of A2780 cells was detected by the wound healing assay.The effect of CH on the invasion ability of A2780 cells was detected by the transwell assay.The expression of Bcl-2,MMP-2 and MMP-9 mRNA in A2780 cells was detected by RT-qPCR assay.Results:1.The MTT results showed that the inhibition rate of CH on A2780 cells was increased with the increase of concentration?1.25,2.5,5,10,20,40 and 80?M?,and the IC₅₀ of A2780 cells for 72 h was 30.21±1.24?M.2.After A2780 cells were treated with different concentrations of CH?2,4,8?M?for 48 h,the late apoptosis of A2780 cells was increased in a concentration-dependent manner compared with the control group?8.5±1.7%,12.0±1.0%,14.3±2.1%v.s.2.9±0.7%,p<0.01?by flow cytometry assay.3.After A2780 cells were treated with 8?M CH for 24 and 48 h,the result of wound healing assay showed that wound healing rate was significantly lower than that of the control group?3.8±2.5%v.s.33.3±5.7%,7.1±1.8%v.s.43.5±1.2%,p<0.01?.4.After A2780 cells were treated with different concentrations of CH?2,4,8?M?for 48 h,the number of cells penetrating the matrixgel membrane into the lower chamber was decreased in a concentration-dependent manner compared with the control group?79.6±3.6,68.2±3.1,68.2±3.1 v.s.101.4±4.9?.The invasion inhibition rate of A2780 cells was increased in a concentration-dependent manner?21.5±3.6%,32.7±3.1%,43.0±2.6%,p<0.01?.5.After A2780 cells were treated with different concentrations of CH?2,4,8?M?for 48 h,the expression of Bcl-2,MMP-2 and MMP-9 mRNA was decreased in a concentration-dependent manner by RT-qPCR assay.Conclusions:In this part,it is disclosed that by the research CH can inhibit the proliferation of ovarian cancer A2780 cells,and its inhibitory effect has the dependence of concentration and time.CH shows no significant impact on early apoptosis and promote late apoptosis of A2780 cells in a dose-dependent manner which may be related to the expression of Bcl-2.CH can inhibit the migration and invasion of A2780 cells,and its mechanism may be related to the expression of MMP-2 and MMP-9.Part two:The effect and mechanism of Cepharanthine Hydrochloride on reversing tumor multidrug resistancePurpose:Taking ovarian cancer resistant cell A2780/Taxol and its parental A2780 cell as the subjects,this part is aimed at investigating the reversal effect of CH on MDR of A2780/Taxol cells and its related mechanism.Methods:The MTT assay was used to detect the proliferation inhibition rate of A2780/Taxol cells and A2780 cells at different concentrations of paclitaxol,and IC₅₀ and was calculated.The MTT assay was used to detect proliferation inhibition of the paclitaxel alone or combination with CH.The effect of CH on the Rho-123accumulation in A2780/Taxol cells and A2780 cells was detected by flow cytometry.The effect of CH on the expression of MDR1 mRNA in A2780/Taxol cells was detected by RT-qPCR.The effect of CH on the expression of P-gp,total Akt expression and phosphorylated Akt in A2780/Taxol cells was detected by Western blot.The expression of P-gp was detected by Western blot in A2780/Taxol cells treated by CH alone or in combination with the specific PI3K/Akt pathway inhibitor LY294002,and the expression of P-gp in A2780/Taxol cells reflects the effect of CH on PI3K/Akt pathway in A2780/Taxol cells.Results:1.The IC₅₀ of paclitaxel in ovarian cancer-resistant A2780/Taxol cells and sensitive A2780 cells was 1475.0±22.6 and 39.5±2.8 ng/ml for 72 h respectively.The drug resistance index of A2780/Taxol cells was 37.3.2.The result of Rhodamine-123?Rho-123?accumulation experiments showed that the accumulation of Rho-123 in A2780/Taxol cells was significantly higher than that in A2780 cells?25.1±3.1%v.s.2.6±1.1%,p<0.01?.3.The result of RT-qPCR showed that the m RNA expression of MDR1 in A2780/Taxol cells was significantly higher than that in A2780 cells.4.The gradient concentration of paclitaxel alone or with different concentrations?2,4,8?M?CH co-acting on A2780/Taxol cells for 72 h,the MTT assay showed that the IC₅₀ of paclitaxel combined with CH group decreased compared with paclitaxel alone group?688.0±7.9,478.3±9.6,185.7±6.5 v.s.1475.0±22.6 ng/ml,p<0.01?,and the reversal index of CH was increased in a concentration-dependent manner?2.1,3.1 and 7.9?.5.After A2780/Taxol cells were treated with different concentrations of CH?2,4,8?M?for 2 h,the accumulation of Rho-123 in CH-treatment group was significantly higher than that in untreated group?13.8±2.2%,17.5±1.0%,23.1±2.1%v.s.2.6±1.1%,p<0.01?.6.TheexpressionofMDR1mRNAwasdecreasedina concentration-dependent manner after A2780 cells were treated with different concentrations of CH?2,4,8?M?for 48 h.7.The expression of P-gp and and phosphorylated Akt was decreased in a concentration-dependent manner,and the expression of total Akt protein was unchanged after A2780 cells were treated with different concentrations of CH?2,4,8?M?for 48 h.8.The down-regulation of P-gp expression was significantly enhanced compared with CH alone or LY294002 alone when CH and PI3K/Akt pathway-specific inhibitor LY294002 were used together.Conclusions:In this part,it is known that CH can reverse the MDR of ovarian cancer-resistant A2780/Taxol cells in a concentration-dependent manner,and its sensitivity to paclitaxel is greatly increased.CH can inhibit the P-gp function of A2780/Taxol cells in a concentration-dependent manner.CH-induced expression of MDR1 mRNA,P-gp and p-Akt decreased in a dose-dependent manner in A2780/Taxol cells.The mechanism of CH may reverse ovarian cancer resistance is the inhibition of phosphorylation of Akt can reduce the activity of PI3K/Akt signaling pathway and then inhibit the expression of P-gp.Conclusions for full thesis:1.CH can significantly inhibit the proliferation,migration and invasion of ovarian cancer cells,and its mechanism may be related to the expression of Bcl-2,MMP-2 and MMP-9 which are the key regulators of apoptosis and migration.2.CH reverses the multidrug resistance of ovarian cancer by inhibiting the expression and function of P-gp.3.CH inhibits the activity of PI3K/Akt signaling pathway by inhibiting the phosphorylation of Akt,thus achieving its effect of reversing P-gp-mediated ovarian cancer resistance.