The Effects And Underlying Mechanisms Of The Parthenolide On The Proliferation And Sensitivity Of Drug-resistant Gastric Cancer Cells Sgc-7901/ddp

BackgroundChemotherapy is the main treatment for advanced gastric cancer and adjuvant post-operative gastric cancer.However,multidrug resistance(MDR)often leads to the failure of chemotherapy.MDR is a distinct phenomenon enabling tumor cells to simultaneously develop cross-resistance to multiple structurally dissimilar anticancer drugs.Gastric cancer exhibits a strong MDR phenotype,resulting in a response rate to chemotherapy less than 50%.The 5-year survival rate in advanced gastric cancer is<30%,which is possibly associated with the development of MDR,too.Therefore,approaches to intervene or reverse the MDR in gastric cancer are required to improve the efficacy of chemotherapy and the survival rate of patients with gastric cancer.Parthenolide is a sesquiterpene lactone extracted from Feverfew(Tanacetum parthenium),which is primarily used for the treatment of fever,arthritis and migraine.Previous studies have demonstrated that the parthenolide inhibits the growth and induces the apoptosis of various tumor cell types in vitro.Evidence suggests that the parthenolide induces anti-tumor effects primarily by targeting nuclear factor-KB(NF-κB),producing reactive oxygen species and activating c-Jun N-terminal kinase.Furthermore,previous studies have revealed that the parthenolide can enhance breast cancer,non-small cell lung cancer,colorectal cancer,liver cancer resistant cell and malignant melanoma cell sensitivity to chemotherapy.However,the underlying mechanisms remain unclear.In the present study,the effects and underlying mechanisms of the parthenolide on the sensitivity of drug-resistant gastric cancer cells SGC-7901/DDP to cisplatin(DDP)were investigated,in order to provide a theoretical basis for the clinical application of the parthenolide.ObjectiveTo observe the effects of the parthenolide on proliferation and drug sensitivity of human gastric cancer cell line SGC-7901/DDP and explore its related mechanism.Methods1.MTT assay was used to detect the inhibitory effect of the parthenolide on drug resistance of gastric cancer cells and the effect of drug-resistant cells on cisplatin resistance.Firstly,screen the appropriate concentration of parthenolide.SGC-7901/DDP cells were treated with different concentrations of parthenolide(0μmol/l,2.5μmol/l,5pμmol/l,10μmol/l,12.5μmol/l,15μmol/I),then the proliferation inhibition rate was detected by MTT assay at 24h,48h and 72h respectively.The appropriate concentration of the parthenolide are 5μmol/l and 10μmol/1.At the same time,in order to observe the sensitization effect of SGC-7901/DDP gastric cancer cell lines on cisplatin,we screen the appropriate concentration of cisplatin.SGC-7901/DDP cells were treated with different concentrations of cisplatin(μg/ml,1.25μg/ml,2.5μg/ml,5μg/ml,10μg/ml,15μg/ml),then the proliferation inhibition rate was detected by MTT assay at 24h,48h and 72h respectively,too.The appropriate concentration of the cisplatinare 1.25μg/ml and 2.5μg/ml.According to the screening results,SGC-7901/DDP cells were treated with the Combination of drugs(①5 μmol/1 parthenolide+1.25μg/ml cisplatin,②5μmol/l parthenolide +2.5pg/ml cisplatin,③ 10μmol/l parthenolide +1.25μg/ml cisplatin,④10μmo1/l parthenolide + 2.5μg/ml cisplatin),The combined inhibition rate was detected after 24h by MTT.The cooperative effect is judged by the Zhengjun Jin Method.The formula is as follows:q=E(AB)/(EA+EB-EAXEB).EAB is the inhibition rate of combination treatment,EA is the inhibition rate of treatment A and EB is the inhibition rate of treatment B.When q<0.85,the two drugs antagonized each other.When q is between 0.85 and 1.15,the two drugs are simply added together.When q>1.15,the two drugs have synergistic effect.2.The effect of the parthenolide on cell cycle was detected by flow cytometry.The treatment of SGC-7901/DDP cells after 48h with different concentrations of the parthenolide,Annexin V-Fitc kit was used to stain the cells and flow cytometry was used to detect cell cycle distribution.3.Using flow cytometry technology to detect the effect of the parthenolide on apoptosis.The SGC-7901/DDP cells were treated with different concentrations of the parthenolide for 48h,Annexin V-Fitc kit was used to stain the cells and flow cytometry was used to detect the cell apoptosis.4.DAPI staining was used to detect the effect of the parthenolide on apoptosis,too.The SGC-7901/DDP cells were treated with different concentrations of the parthenolide for 48h,DAPI was used to stain the cells,then Cell apoptosis was detected by fluorescence microscope.5.Using Western blot method to detect the effect of the parthenolide on the expression of related proteins,for instance Bax,Bcl-2,Bcl-xl,P53,Caspase-3,Capase-9,Cleaved caspase-3,Cleaved caspase-9,CylinDl,P21,Stat3,P-Stat3;The SGC-7901/DDP cells were treated with different concentrations of parthenolide for 48h,then the cells were collected.The total protein of the cells was extracted and quantified.The protein was separated by electrophoresis,further transfereed to PVDF membrane.Add the Bax,Bcl-2,Bcl-xl,P53,Caspase-3,Capase-9,Cleaved caspase-3,Cleaved caspase-9,cylinDl,P21,Stat3,P-Stat3 antibody respectively on the PVDF membrane,then add ECL luminous fluid,finally the membrane was detected by ultravioletimaging system,Image software was used for quantitative analysis.6.The effect of the parthenolide on the migration ability of gastric cancer cells was investigated by Scratch migration assay.The SGC-7901/DDP cells were treated with different concentrations of parthenolide,Measure the width of the scratches at 24h,48h and 72h respectively.The mobility was calculated as follows:(Distance of migration/scratch width)x 100%.7.Transwell chamber was used to detect the effect of the parthenolide on invasion ability of the gastric cancer cells.The SGC-7901/DDP cells were treated with different concentrations of parthenolide for 48h,The crystal violetwas used to stain the cells,Count the number of the penetrating cells under the microscope.Results1.The parthenolide has a significant inhibitory effect on the proliferation of gastric cancer SGC-7901/DDP cells,and the inhibitory effect was in a time-and concentration-dependent manner.There is synergistic effect of the proliferation inhibition between the parthenolide and Cisplatin.2.The results of flow cytometry showed that there was a decrease in the s-phase and G2/M phase cells after the treatment of the parthenolide,and the G0/G1 phase cells increased,and the difference was statistically significant(P<0.05).3.The results of flow cytometry showed that the early apoptosis was significantly increased after treatment,and the difference with the control group was significant(P<0.05).4.The results of DAPI staining also showed that after 48h,SGC-7901/DDP cells showed obvious apoptosis,And as the concentration increased,the apoptosis increased.Compared with the control group,there were significant differences.5.Using Westernblot method to detect the effect ofthe parthenolide on the expression of related proteins,Bax,Bcl-2,Bcl-xl,P53,Caspase-3,Capase-9,Cleaved caspase-3,Cleaved caspase-9,CylinDl,P21,Stat3,P-Stat3.Results show that,the expression of Bax and P53 was up-regulated,the expression of Bcl-xl and Bcl-2 was down-regulated,Caspase-3 activation fragments and Caspase-9 activation fragments increased,the expression of P21 was up-regulated,while the expression of CylinDl was down-regulated.Meanwhile,the activation of P-Stat3 was significantly inhibited.6.The results of the scratch migration assay showed that the migration ability of SGC-7901/DDP cells was significantly inhibited by the parthenolide.7.The results of Transwell chamber experiment showed that the invasion ability of SGC-7901/DDP cells could be inhibited by the parthenolide.ConclusionsThe present study demonstrated that the parthenolide induces SGC-7901/DDP apoptosis,inhibits SGC-7901/DDP proliferation,arrests cells in G1 phase,and enhances the drug sensitivity of the cells to DDP.The underlying mechanisms may be associated with inhibition of the Stat3 signaling pathway and regulation of the downstream apoptotic protein and cyclin expression levels.In addition,the study revealed that the parthenolide can inhibit SGC7901/DDP cell migration and invasion.