| BackgroundEndometrial cancer is one of the common causes of death of gynecological malignant tumors.Endometrial cancer has two types according to its molecular and clinical features.Type I endometrial cancer accounts for 80-90%,which include common endometrial adenocarcinoma,frequently occurr in premenopausal women and is associated with exposure to excess estrogen.It is usually early diagnosed and has a good prognosis.In contrast,type II endometrial cancer,which accounts for about 10-20%,is mostly serous and occurs in the atrophic endometrium of older women,independent of hormones.Patients with this histological subtype have a far more aggressive clinical process than type I endometrial cancer,even in the minority who are diagnosed with an early disease.Chemotherapy is the most common treatment options for patients with type II or type I advanced endometrial cancer.Cisplatin is the most common chemotherapeutic drug to treat cancer in clinic.But its resistance is the main cause of its treatment failure.Multi-drug resistance is a major problem in chemotherapy,with 30-80%of cancer patients developing resistance to chemotherapy drugs.Therefore,it is essential to deal with drug resistance to provide optimal treatment.In the mechanism of m μLti-drug resistance in many tumors,the ATP driven transmembrane transportion of structure and function is mostly non-specific cell inhibitors,thus limiting drug’ s therapeutic concentration in cells.Overexpression of membrane transporters of atp-binding box(ABC)superfamily members in tumor cells is also known as MDR regulatory factors through atp-dependent efflux mechanism,including p-glycoprotein(Pgp),mμLti-drug resistance related protein 1(MRP1)and breast cancer resistance protein(BCRP).Various causes of resistance can act simultaneously to increase resistance in a multifactorial manner.Many oxidative substances,such as reactive oxygen species(ROS),are metabolites of organisms in aerobic environments.Studies have shown that ROS plays a signal transduction role in such processes as cell proliferation,differentiation and apoptosis.Studies have found that ROS plays an important role in the development and progression of tumors.The damage of ROS to DNA and proteins is one of the main reasons for the occurrence and development of tumors.The damage of ROS to DNA and proteins can be manifested as damage to tumor suppressor genes and activation of proto-oncogenes to forming malignant transformation of cells.Therefore,ROS is a potential carcinogen.On the other hand,tumor cells often show significant increases in ROS levels.Oxidative stress is a cellular condition caused by excessive production of intracellular ROS and/or damage to the function of the cellular antioxidant defense system.The enhancement of tumor cell oxidative stress can increase abnormal DNA and protein and blood supply of tumor cells,accelerate the development and metastasis of tumor,and strengthen the resistance of tumor cells to chemotherapy drugs.Emodin is a traditional Chinese medicine(TCM)one of the main active ingredients of rhubarb,and aiso a kind of tyrosine kinase inhibitors.Modern pharmacology study found that Emodin not only has anti-inflammatory,anti-cancer effect,and also can inhibit the proliferation of tumor cells in different system,which inducing tumor cell apoptosis,controling the transfer of tumor cells,having collaborative effet with other anticancer chemotherapy drugs,changing the surrounding environment of the tumor cells at the same time,reducing the intake of nutrients,destroying the cell membrane,and achieving the tumor cell death in the end.Emodin has a good synergistic effect with anti-tumor drugs and radiotherapy and chemotherapy currently used in clinical practice.In view of its anti-tumor effect and low toxicity,Emodin is a potential effective drug for clinical tumor treatment.This study is the first to explore whether Emodin alone or combined with cisplatin has anti-tumor effect on two endometrial cancer cells and whether the drug enhances the chemotherapeutic effect of cisplatin and its mechanism.The further proves in vivo experiments of tumor-bearing nude mice provide a new idea for the research and development of alternative therapy for endometrial cancer.Part 1:The effect of Emodin on the anti-tumor effect of endometrial cancer cells,the level of intracellular ROS and the expression of drug-resistant genes Objective:1.To test the effect of Emodin on growth and apoptosis of Ishikawa cells and HEC-IB cells in endometrial cancer.2.To test the changes of ROS levels in the Ishikawa cells and HEC-IB cells of endometrial carcinoma after the treatment of Emodin and cisplatin alone or in combination.3.To explore the effect of Emodin on the expression of drug-resistant genes in the Ishikawa and HEC-IB cells of endometrial cancer.Methods:The growth inhibition rate of two endometrial cancer cells(Ishikawa and HEC-IB cells)was detected by using the CCK-8 kit,and the IC50 concentration of the two drugs was calculated.The growth and proliferation of Ishikawa and HEC-IB cells were detected by treatment of IC50 concentration of Emodin and cisplatin alone and in combination.The apoptosis level of Ishikawa cells and HEC-IB cells was detected by Annexin V/propidide double staining,and the expression level of apoptosis-related protein caspase-3 was detected by Western blot.The intracellular ROS levels in the Ishikawa cells and HEC-IB cells were detected by DCFH-DA,and the expression level of NADPH oxidase was detected by Western blot.The changes of ROS levels in the Ishikawa cells and HEC-IB cells were detected by DCFH-DA.The expressions of drug-resistant genes P-gp,MRP1 and BCRP in Ishikawa cells and HEC-IB cells were detected by RT-PCR,and the expressions of drug-resistant genes Pgp,MRP1 and BCRP proteins were detected by Western blot.The expressions of drug-resistant genes P-gp,MRP1 and BCRP in Ishikawa cells and HEC-IB cells were detected by RT-PCR after H202 pretreatment with Emodin and cisplatin alone and combined with the two drugs.The expression changes of drug-resistant genes Pgp,MRP1 and BCRP in Ishikawa cells and HEC-IB cells before and after siRNA transfection were detected by RT-PCR,and the apoptosis levels of Ishikawa cells and HEC-IB cells after siRNA transfection were detected by Annexin V/propidide double staining.Results:1.Effects of Emodin and cisplatin on growth inhibition and proliferation of Ishikawa and HEC-IB cells:The growth inhibition effect of Emodin and cisplatin on Ishikawa and HEC-IB cells was detected by cck-8 kit.The results showed that Emodin and cisplatin significantly inhibited cell growth,and the IC50 concentration of Emodin and cisplatin was Emodin with a concentration of 10 mm/L and cisplatin with a concentration of 1 u g/mL.Application of IC50 concentration of Emodin and cisplatin treatment and the cell proliferation rate showed that the treatment of Emodin and cisplatin inhibit the Ishikawa cells and the level of HEC-IB cell proliferation,and has the time dependence of this inhibitory ability.The combination of the two drugs application is especially significant inhibition,especially for Ishikawa cells inhibitory effect is more apparent.2.The effect of Emodin and cisplatin on the apoptosis of Ishikawa cells and HEC-IB cells:Annexin V/propidide double staining was used to test the apoptosis level of Ishikawa cells and HEC-IB cells after the treatment of Emodin and cisplatin.The resμLts showed that Emodin and cisplatin significantly promoted the apoptosis of Ishikawa and HEC-IB cells,especially when the two drugs were combined.The expression levels of the apoptosis-related protein caspase-3 in Ishikawa cells and HEC-IB cells were detected by Western blot.The results showed that the expression level of caspase-3 was significantly increased and the increase was more significant when the two drugs were combined.And the combined application showed more significant effect on the apoptosis of Ishikawa cells.3.Emodin and cisplatin can reduce ROS levels in endometrial cancer cells:DCFH-DA method was used to test the treatment of Emodin and cisplatin on the intracellular ROS levels of Ishikawa cells and HEC-IB cells and the res μLt was decreased.The expression level of NADPH oxidase was detected by Western blot.The resμLts showed that the combination of Emodin and cisplatin significantly inhibited the expression of NADPH oxidase.There was no significant difference between the two types of cells.4.Effects of H202 on ROS levels in endometrial cancer cells after Emodin and cisplatin treatment:The changes of ROS levels in the Ishikawa cells and HEC-IB cells after H202 pretreatment with Emodin and cisplatin were detected by DCFH-DA method.The results showed that after H202 treatment,the decrease of ROS levels resulted from the combination of Emodin and cisplatin was also increased.There was no significant difference between the two types of cells.5.Effects of Emodin and cisplatin on the expression of drug-resistant genes in endometrial cancer cells:The expressions of drug-resistant genes P-gp,MRP1 and BCRP in Ishikawa cells and HEC-IB cells were detected by RT-PCR after Emodin and cisplatin treatment,and the expressions of drug-resistant genes P-gp,MRP1 and BCRP proteins were detected by Western blot.The resμLts showed that:The expression of drug-resistant genes P-gp,MRP1 and BCRP and the protein expression were significantly decreased,especially when the two drugs were combined,and the inhibition of P-gp and BCRP protein levels in Ishikawa cells was more obvious.6.Effects of H2O2 on the expression of drug-resistant genes in endometrial cancer cells:The expressions of drug-resistant genes P-gp,MRP1 and BCRP in Ishikawa cells and HEC-IB cells after pretreatment with Emodin and cisplatin were detected by RT-PCR,and the results showed that the expressions of drug-resistant genes P-gp,MRP1 and BCRP in both cells were higher than before.7.Effects of inhibiting the expression of drug-resistant genes on the apoptosis of endometrial cancer cells:P-gp,MRP1 and BCRP in Ishikawa cells and HEC-IB cells were transfected with siRNA,and the changes of genes were detected by RT-PCR.The resμLts showed that the levels of P-gp,MRP1 and BCRP were significantly lower than those before transfection.The level of apoptosis in both Ishikawa and HEC-IB cells after siRNA transfection was detected by Annexin V/propidide double staining.The resμLts showed that the down-r egμLat i on of the gene expression significantly increased the level of apoptosis in Ishikawa and HEC-IB cells.The apoptosis of the Ishikawa cells in the siRNA-P-gp group and the HEC-IB cells in the siRNA-MRP1 group was more significant.Conclusion:1.Emodin can enhance the growth inhibition and apoptosis induction effect of cisplatin on the Ishikawa cells and HEC-IB cells of endometrial carcinoma;It can enhance the effect of cisplatin in reducing the level of ROS in the Ishikawa cells and HEC-IB cells of endometrial carcinoma.2.Emodin combined with cisplatin can significantly reduce the expressions of drug-resistant genes P-gp,MRP1 and BCRP in the Ishikawa cells and HEC-IB cells of endometrial cancer and increase the sensitivity of cisplatin to chemotherapy.3.The combination of Emodin and cisplatin showed more significant growth inhibition,apoptosis-promoting and inhibition of drug-resistant gene expression in Ishikawa cells.Part Ⅱ:Study on the therapeutic effect of Emodin in a murine xenograft endometrial cancer modelObjective:A murine xenograft endometrial cancer model was constructed to investigate the anti-tumor effection of Emodin in vivo.Methods:After the treatment of Emodin and cisplatin,endometrial cancer Ishikawa cells and HEC-IB cells were prepared into 1 x 105 cell suspension and subcutaneously injected into the right front leg of the xenograft mice.A xenograft model of endometrial carcinoma in nude mice was established.The group was divided into four groups:control group,Emodin group,cisplatin group,Emodin and cisplatin group.After 40 days of inoculation,a vernier caliper was used to measure the length and diameter of subcutaneous xenograft tumors in each group and to calculate the tumor volume.The survival time of the mice was recorded and calculated.Results:1.Establishment of murine xenograft endometrial cancer model:Ishikawa cells and HEC-IB cells were inoculated into the right front leg of mice,and the nodμLes were plasticity and coμLd be touched in the inoc μ Lation site.And the tumor formation rate was 100%.The general condition of the tumor-bearing nude mice was good,and the subcutaneous transplanted tumor grew rapidly.After 40 days of inoculation,the nude mice showed good tolerance to the drug.2.Effects of Emodin and cisplatin on the growth of tumor in murine xenograft endometrial cancer model:Compared with the untreated control group,Emodin or cisplatin alone treatment group could reduce the tumor volume of nude mice,but the survival time of nude mice was prolonged.These effects were more significant in the two drug combined administration group.Conclusion:Emodin inhibited the growth of tumor of the murine xenograft endometrial cancer model,and Emodin had a synergistic effect on the inhibition of tumor growth by cisplatin. |
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