| Background The incidence and mortality of esophageal cancer in China ranks first in the world.90% of patients have been diagnosed at the advanced stage.Chemotherapy is one of the important methods of treatment.However,the commonly used drugs such as cisplatin and oxaliplatin have limited efficacy and serious adverse reactions.The development of chemotherapy drugs is still a hot topic..Studies have found that the content of iron and copper in cancer tissues is higher than other tissues,and the demand for metal ions such as iron,copper and zinc increases when the cancer cells proliferate and metastasize,making the use of chelating agents a new anti-cancer strategy.Dithiocarbamate is a kind of sulfur-containing compound with strong chelating ability to metal ions.Its derivative pyrrolidine dithiocarbamate has anti-hepatocarcinogenic activity.The inhibitory mechanism of dipyridone hydrazone dithiobutyric acid(Dpdtb A)on the growth of gastric cancer cells has been preliminarily recognized,but the relationship between Dpdtb A and the growth inhibition of esophageal cancer cells remains to be explored.Objective To evaluate the effect of Dpdtb A on the growth of human esophageal carcinoma cell lines KYSE 450 and KYSE 150 with high EGFR expression.To investigate the mechanism and determine whether EGFR related molecules are affected.Methods 1.Conventional culture of human esophageal cancer KYSE150 and KYES 450 cells.2.MTT,colony formation and morphological observation were used to evaluate the growth inhibition effect of Dpdtb A on cells and determine the drug concentration for the mechanism study.3.In the mechanism study,Dmso group was used as the control group,and the final concentration was 0.7% by adding Dmso.The experimental group was mainly divided into 5μM group and 10μM group,and Dpdtb A drug was added to make the final concentration 5μM and 10μM,respectively.After 24 hours of drug action,cells were collected for subsequent testing.4.Flow cytometry with PI,Annexin V/FITC and dcfh-da staining was used to detect changes in cell cycle,apoptosis and cell ROS.5.WTS-8 method was used to detect the change of total SOD activity.6.Western Blot analysis of CDK2,BAX,bcl-2,NCOA4,Ferritin,SOD,p53,p-p53,Stub-1,EGFR,p-EGFR,AKT,p-AKT,lc-3,MDM2.7.RT-PCR was used to detect the change of p53 m RNA after Dpdtb A was applied to cells.8.Analysis was performed using SPSS 16.0 and Graphpad Prism 7 software.The experimental data were expressed as mean standard deviation((?)±s).Comparison between groups was performed by one-way analysis of variance,and the test level was α = 0.05.Results 1.Dpdtb A inhibited cell growth,and the IC50 of KYSE 450 cells was 5.50±0.53μM,and that of KYSE 150 cells was 5.00±0.39μM.After 10 days of Dpdtb A treatment with 0.5μM,the number of cell colonies was less than that of Dmso group(p<0.05).After Dpdtb A treatment for 24 hours,the cell density decreased,shriveled,became round and appeared floating.Both 5μM and 10μM Dpdtb A groups had cell survival.2.Dpdtb A induced KYSE 450 and KYES 150 cell cycle S-phase arrest,and the proportion of s-phase cells increased with the increase of drug concentration(p<0.05).CDK2 protein content decreased after Dpdtb A treatment,and the decrease was more obvious with the increase of drug concentration(p<0.05).3.Dpdtb A increased the apoptosis rate of KYSE 450 and KYES 150 cells,and the apoptosis rate increased significantly with the increase of drug concentration(p<0.05).Dpdtb A reduced bcl-2 expression in KYSE 450 cells(p<0.05),BAX expression was not significantly changed(p>0.05),and the BAX/ bcl-2 ratio in 10 M group was higher than that in DMSO group(p<0.05).BAX and bcl-2 expression were decreased in KYSE 150 cells(p<0.05),and the BAX/ bcl-2 ratio in Dpdtb A group was lower than that in DMSO group(p<0.05).4.Dpdtb A induced the increase of ROS in KYSE 450 and KYES 150 cells,and the increase was more obvious with the increase of drug concentration(p<0.05).In KYSE 450 cells,NCOA4 was down-regulated and Ferritin was up-regulated(p<0.05).Total SOD enzyme activity and SOD protein content in cells were decreased(p<0.05).5.After Dpdtb A treated KYSE 450 and KYES 150 cells,the content of EGFR and p-EGFR decreased,and the inhibition was more obvious with the increase of concentration(p<0.05).The expression of p53,the upstream signal molecule of EGFR,and AKT,the downstream signal molecule,were inhibited,and the inhibition was more obvious with the increase of concentration(p<0.05).6.Dpdtb A increased p53 m RNA in KYSE 450 and KYES 150 cells(p<0.05).7.Dpdtb A reduced the MDM2 protein content of KYSE 450 and KYES 150 cells,and the decrease was more obvious with the increase of drug concentration(p<0.05).After the addition of proteasome inhibitor MG132,the protein content of p53 and MDM2 in cells still decreased(p<0.05).8.Dpdtb A induced the decrease of stub-1 and the increase of p-p53 content in KYSE 450 and KYES 150 cells,and the effect became more obvious with the increase of concentration(p<0.05).The addition of autophagy inhibitor 3-ma could weaken the effect of Dpdtb A(p<0.05).9.Dpdtb A increased the content of Lc3 in KYSE 450 and KYES 150 cells,decreased the content of stub-1 and p53,and decreased the content of EGFR,AKT and p-AKT(p<0.05).After the addition of autophagy inhibitor 3-ma and chloroquine,the contents of Stub-1 and p53 increased,and the contents of EGFR,AKT and p-AKT also increased(p<0.05).Conclusion Dpdtb A can inhibit the proliferation of KYSE 450 and KYSE 150 cells in human esophageal cancer.The mechanism may be that Dpdtb A triggers stub1-mediated autophagy degradation of p53 protein and inactivates the p53/EGFR/AKT signaling pathway,promotes the phosphorylation of p53 protein,reduces the intracellular SOD enzyme activity,leads to cell ROS accumulation,induces apoptosis,and blocks cell cycle in S phase. |
