| Background and Objective Esophageal cancer is one of the most common malignancies,as well as one of the most fatal cause of cancer-related mortality worldwide.According to histological types,esophageal cancer is mainly divided into esophageal squamous cell carcinoma(ESCC)and esophageal adenocarcinoma(EAC).In Asian,especially in China,more than 90% of esophageal cancer patients are esophageal squamous cell carcinoma.Chemotherapy plays an important role in the comprehensive treatment of ESCC,However,some tumor cells in the tumor tissue will develop resistance to chemotherapeutic drugs with different but similar chemical structures and therapeutic mechanisms during chemotherapy,also known as multi-drug resistance(MDR),ultimately leads to treatment failure and tumor recurrence.Therefore,it is of great significance to study the specific molecular mechanism of multidrug resistance of tumors and find a reversal agent with low toxicity,high efficiency and wide range of anti-tumor multi-drug resistance.Many flavonoids have been proven to have anticancer activity both in animal and cellular model systems.Because of their advantages of high efficiency,low toxicity and easy availability,they have attracted extensive attention from scholars both in China and overseas.Luteolin is one of the most common natural flavonoids with potential for tumor prevention and treatment.In recent years,it has been found that luteolin inhibits the cell development,metastasis and angiogenesis of tumors through various mechanisms.At the same time,luteolin can reverse the multidrug resistance of various tumor cells and increase the drug sensitivity of tumor resistance cells,and it can also ameliorate the cytotoxicity that various chemotherapy drugs can cause.In conclusion,luteolin can be used as adjunctive therapy to reverse the multidrug resistance process of tumor cells.In our preliminary work,the human esophageal squamous cell paclitaxel multidrug resistance cell EC1/PTX has been successfully established and identified.High-throughput sequencing and previous studies have found that ESCC multidrug resistance cell express higher resistance-related protein P-gp,BCRP,MRP1,and focal adhesion kinase(FAK)and phosphorylated FAK than parental cell.At the same time,the acyl-inositol 3-kinase(PI3K)/protein kinase B(Akt)signaling pathway may be involved in the multidrug resistance process of ESCC cells.Activation of FAK promotes epithelial-mesenchymal transition(EMT)in tumor cells,which is involved in tumor invasion and metastasis.Then Erb B2 kinase can be recruited by FAK,and they can activite Src kinase,which regulate PI3 K / AKT,MAPK and other signaling pathways,thereby promoting tumor invasion,metastasis and chemotherapy resistance.To further investigate whether the FAK/Src/PI3K/Akt signaling pathway is involved in the multidrug resistance process of ESCC cells,and whether luteolin can inhibit this signaling pathway to reverses the multidrug resistance of ESCC cells or not.Therefore,we firstly explore the efficacy of luteolin on multidrug resistance of ESCC through cell morphology,biology and drug sensitivity experiments,and further expound the molecular mechanism of luteolin in reversing multidrug resistance of ESCC cells.In this study,we investigate the efficacy of luteolin in reversing the multidrug resistance of ESCC cells and its molecular mechanism,and identify its potential therapeutic targets for multidrug resistance in ESCC cells and provide experimental data and theoretical support for luteolin as an novel reversal agent for ESCC multidrug resistance cells.Methods1 Effects of luteolin on the morphology and biological behavior of ESCC multidrug resistance cells Different doses of luteolin were used to treat EC1/PTX cells.After 48 h,the morphology of the cells were observed by inverted microscope.The cell proliferation and colony formation ability were detected by CCK-8 method and plate cloning assay.Flow cytometry was used to detect cell cycle and apoptosis.Cell scratching test and Transwell chamber were employed to examine cell migration and invasion ability.Microsphere formation assay detected the ability of cells to form globules.Western blot was used to detect the expression changes of apoptosis,EMT and stem cell-associated protein in cells.2 Effects of luteolin on drug sensitivity of ESCC multidrug resistance cells Luteolin(10μM)pretreated EC1/PTX cells,then different doses of chemotherapy drugs paclitaxel,cisplatin,doxorubicin and pentafluorouraci were added to cells,CCK-8 method detected cell proliferation and calculated the IC50 of chemotherapy drugs.At the same time,luteolin or paclitaxel alone and the two drugs were applied to EC1/PTX cells,and the total apoptosis rate of cells was detected by flow cytometry.3 Effects of FAK/Src/PI3K/Akt signaling pathway in ESCC multidrug resistance cells Western blot was used to verify the expression levels of key proteins in the FAK/Src/PI3K/Akt pathway in EC1/PTX and EC1.After blocking this pathway by molecule-specific inhibitors,western blot detected the expression changes of key proteins in the FAK/Src/PI3K/Akt pathway and resistance-related proteins in ESCC multidrug resistance cells.4 Effects of luteolin on FAK/Src/PI3K/Akt signaling pathway in ESCC multidrug resistant cells Western blot was used to detect the effects of different concentrations of luteolin on the expression changes of key proteins in FAK/Src/PI3K/Akt pathway and drug resistance-related proteins in EC1/PTX cells.5 Statistical assay The experimental data was analyzed and performed using statistical software SPSS 21.0.Independent sample t-test was used to compare the mean of the two groups of patients with normal distribution,and one-way ANOVA was used for comparison between groups.P value less than 0.05 was considered to be statistical significance.Results1 Effects of luteolin on multidrug resistance of esophageal squamous carcinoma cells1.1 Morphological observation showed that the control group cells were evenly arranged,tightly connected,polygonal,morphology normal,edges clear and the medium was turbid.After the treatment of luteolin,most of the cells lost their normal morphology,the intercellular connections disappeared,the edges were blurred,the cells showed shrinkage and agglomerated,and the suspended dead cells increased.1.2 The results of biological behavioral experiments showed that compared with the control group,different concentrations of luteolin could inhibit the proliferation and colony formation ability of EC1/PTX cells,arrest the cell cycle,induce apoptosis,and promote the expression of pro-apoptotic protein Bax and inhibit the expression of anti-apoptotic protein Bcl-2.In addition,luteolin inhibited cell migration,invasion and globization,and inhibited TGF-β1-induced EMT and stem cell performance in cells.Western blot assay indicated that luteolin reduced the expression levels of mesenchymal markers Snail,N-cadherin and MMP-2 proteins and stem cell markers SOX-2,CD44 and CD133 in the cells.1.3 The results of drug sensitivity experiments indicated that compared with the untreated group,the IC50 values of paclitaxel,cisplatin,doxorubicin and pentafluorouracil in EC1/PTX cells were reduced by 1.5,2.7,1.9 and 3.0 times respectively,and the difference was statistically significant(P<0.05).At the same time,the results of flow cytometry showed that the total apoptotic rate of luteolin combined with paclitaxel was higher than that of luteolin and paclitaxel alone groups(P<0.05).2 Molecular mechanism of luteolin on multidrug resistance in esophageal squamous cell carcinoma2.1 Effects of FAK/Src/PI3K/Akt signaling pathway in ESCC multidrug resistance cells Western blot assay verified that the expression levels of Akt,p-Akt(Ser473),p-Src(Tyr416)and Erb B2 were up-regulated in EC1/PTX cells compared with EC1(P<0.05).After Y15 and LY294002 blocked the FAK/Src/PI3K/Akt signaling pathway,Western blot assay indicated that compared with the control group,the expression levels of total FAK and p-FAK proteins were down-regulated and the expression levels of downstream proteins Erb B2,p-Src(Tyr416),Akt,p-Akt(Ser473)and the drug resistance proteins P-gp,BCRP and MRP1 in the Y15-treated groups were significantly down-regulated in a dose-dependent manner(P<0.05),but the levels of total Src protein didn’t change.At the same time,the expression levels of Akt,p-Akt(Ser473)and drug resistance proteins P-gp,BCRP and MRP1 in LY294002-treated groups were significantly down-regulated in a dose-dependent manner(P<0.05).2.2 Effects of luteolin on FAK/Src/PI3K/Akt signaling pathway in ESCC multidrug resistance cells Western blot assay indicated that compared with the control group,the expression levels of FAK,p-FAK(Tyr397),Erb B2,Src,p-Src(Tyr416)and Akt,p-Akt(Ser473),and the drug resistance proteins P-gp,BCRP and MRP1 in the luteolin-treated groups were down-regulated in a dose-dependent manner(P<0.05).Conclusions1 Luteolin can inhibit proliferation,migration,invasion and EMT ability as well as globing ability and stem cell performance,arrest cycle,induce apoptosis and increace drug sensitivity of ESCC multidrug resistance cells.2 Luteolin can reverse the multidrug resistance of ESCC cells by inhibiting the FAK/Src/PI3K/Akt signaling pathway. |
