| Background and purpose:According to the global data in 2020,gastric cancer ranks fifth in morbidity and fourth in mortality among all cancers in the world,and facing serious prognosis and drug resistance.Salinomycin(SAL)is a broad-spectrum antimicrobial agent with anti-tumor properties,especially the synergistic effect of salinomycin therapy combined with other chemotherapy drugs or radiotherapy is worth exploring.At present,how SAL affects the main mechanisms of proliferation,migration,invasion and apoptosis of cisplatin-resistant gastric cancer cells,and how to reverse the radiotherapy resistance and multi-drug resistance of cancer cells is still in the exploratory stage.Whether salinomycin can have a better sensitization effect on cisplatin-resistant gastric cancer cells has not been reported yet.Based on this,in this study,salinomycin(SAL)and cisplatin(DDP)were combined to treat Human gastric mucosal epithelial cells GES-1、gastric cancer cells SGC-7901 and cisplatin-resistant gastric cancer cells SGC-7901/DDP to determine whether salinomycin can improve the sensitivity of SGC-7901/DDP to cisplatin chemotherapy in vitro.At the same time,to preliminarily explore the molecular mechanism of the combined action of salinomycin and cisplatin and the sensitization mechanism of salinomycin on cisplatin-resistant gastric cancer cells,so as to provide a theoretical basis for the improvement of cisplatin resistance and new treatments for gastric cancer,and provide a new idea for clinical application.Methods : 1.CCK-8 method to detect the effect of SAL on SGC-7901,SGC-7901/DDP cell viability,and to screen the optimal concentration of SAL and DDP as a single agent and combined application.2.CCK-8,Transwell and cell scratch experiments were used to detect the effects of SAL and DDP as a single agent and their combined application on the proliferation、invasion and migration of SGC-7901 and SGC-7901/DDP,and CCK-8 method was also used to detect the effect of SAL on the proliferation of GES-1 cells.3.Flow cytometry was used to detect the effects of SAL and DDP as a single agent and their combined application on cell cycle and apoptosis of SGC-7901 and SGC-7901/DDP.4.Western blotting and immunohistochemical experiments to detect the effects of SAL and DDP as a single agent and their combined application on the expression of SGC-7901 and SGC-7901/DDP cell apoptosis-related factors Bcl-2,Bax,Caspase 3,Caspase 9,Fas-L,NF-κBp65,and explore the possible mechanism of SAL inducing apoptosis.5.Western blot experiments were performed to detect the expression of the Wnt/ β-catenin pathway effectors p-GSK-3β(Ser9)、 β-catenin and target genes Axin2 and CCND1 and EMT proteins E-cadherin,N-cadherin and Vimentin after SGC-7901 and SGC-7901/DDP cells were treated by SAL,SAL combined with Wnt-C59;immunofluorescence was used to detect the nuclear localization of β-catenin in SGC-7901 and SGC-7901/DDP cells treated with SAL and SAL combined with Wnt-C59;and explore the chemosensitization mechanism of SAL on SGC-7901 and SGC-7901/DDP cell.Results:1.The CCK-8 method screened out that SAL SAL-8 μM and DDP-3 μg/m L acted for 24 h as the best concentration and time.2.The inhibition rate of SAL-8 μM on GES-1 cells at 24 h was not significantly different from that of the control group(0 μM)(P>0.05);compared with the control group,SAL and DDP treatment of SGC-7901 and SGC-7901/DDP cells for 24 h can respectively reduce the proliferation(P<0.01)、invasion(P<0.01)and migration ability(P<0.01)of the two kinds of cells,and significantly increase the apoptosis rate(P<0.01);in SGC-7901 cells,compared with the SAL group,the DDP group and the double-drug group significantly reduced the proliferation ability(P<0.05)、invasion ability(P<0.05)、and migration ability(P<0.05)of SGC-7901 cells and significantly increased the apoptosis rate(P<0.05);in SGC-7901/DDP cells,compared with the DDP group,the SAL group and the double-drug group significantly reduced the proliferation ability(P<0.05)、invasion ability(P<0.05)、migration ability(P<0.05)of SGC-7901/DDP and significantly increased the apoptosis rate(P<0.05).3.In SGC-7901 and SGC-7901/DDP cells,the ratio of G0/G1 phase cells in the DDP treatment group was significantly higher than that in the control and SAL groups(P<0.01);at the same time,the ratio of S phase cells in the SAL treatment group was significantly higher than that in the control group and DDP group(P<0.01).4.Compared with the control group,after SAL and DDP were treated for 24 h,the expression of Caspase 3,Caspase 9,and Bax in SGC-7901 and SGC-7901/DDP were significantly increased(P<0.01),and the expression of Bcl-2 was significantly reduced(P<0.01);compared with the single-agent group,the double-drug combination group has a greater amount of expression change(P<0.01),In SGC-7901/DDP cells,compared with the DDP group,the SAL group and the double-drug group significantly increased the expression levels of Caspase 3,Caspase 9,Bax(P<0.01).5.In SGC-7901 cells,compared with the control group,SAL and DDP treatments increased the expressions of Fas-L、NF-κBp65 in the cytoplasm,but the expressions of Fas-L、NF-κBp65in the cytoplasm were more obvious in the DDP group,and there is no obvious synergistic effect in the combined use of the two drugs;In SGC-7901/DDP cells,SAL and DDP treatments increased the expression of Fas-L、NF-κBp65 in the cytoplasm,and the combination of the two drugs showed a synergistic effect.6.In SGC-7901/DDP and SGC-7901 cells,compared with the control group,the expressions of N-cadherin、Vimentin、p-GSK-3β(Ser9)、 β-catenin 、Axin2、and CCND1 proteins in the SAL treatment group were decreased(P<0.01),while the expression of E-cadherin was increased(P<0.01),the nuclear migration of β-catenin was increased(P<0.01);compared with the SAL group,the expressions of N-cadherin、Vimentin、p-GSK-3β(Ser9)、 β-catenin 、Axin2、and CCND1 in the two gastric cancer cells in the SAL combined with Wnt-C59 treatment group were further down-regulated(P<0.01),and the content of E-cadherin further up-regulation(P<0.01),the nucleus migration of β-catenin further increased.7.Compared with SGC-7901,N-cadherin、Vimentin、p-GSK-3β(Ser9)、 β-catenin 、Axin2、and CCND1 down-regulation and the up-regulation of E-cadherin and the degree of nuclear outward migration of β-catenin are both enhanced in SAL treatment group and SAL combined with Wnt-C59 treatment group of SGC-7901/DDP resistant strain.Conclusion:1.SAL-8 μM has no obvious inhibitory effect on human gastric mucosal epithelial cells GES-1 cells after 24 hours,but it can inhibit the proliferation of gastric cancer cells SGC-7901 and SGC-7901/DDP.2.Both SAL and DDP can inhibit the proliferation,invasion,migration and induce apoptosis of SGC-7901 and SGC-7901/DDP cells.SAL can regulate and enhance the inhibitory effect of DDP on the proliferation,invasion and migration of gastric cancer cells SGC-7901 and cisplatin-resistant cells SGC-7901/DDP and induce apoptosis,the combination of two drugs has a synergistic effect;unlike SGC-7901,SAL and the combination of two drugs are stronger than DDP in inhibiting migration of SGC-7901/DDP and inducing apoptosis.3.DDP can cause gastric cancer cell cycle arrest in G0/G1 phase,and SAL can cause gastric cancer cell cycle arrest in S phase.The two drugs can act on different cycle time points to exert cell arrest.4.Through the Fas-L pathway,SAL and DDP up-regulate the expression of apoptosis-related factors Caspase 3、Caspase 9、Bax,Fas-L,and down-regulate the expression of Bcl-2,while inhibiting the expression of nuclear NF-κBp65 to regulate gastric cancer cells SGC-7901 and SGC-7901/DDP apoptosis,and only in SGC7901/DDP,the two drugs have obvious synergistic effects.5.SAL inhibits the EMT process by down-regulating the expression of Wnt/ β-catenin pathway effectors p-GSK-3β(Ser9)、 β-catenin and target proteins Axin2 and CCND1,thereby inhibiting the invasion and migration of tumor cells and increasing the sensitivity of tumor cells to DDP,reversing drug resistance of drug-resistant cells. |
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