| Objective:This study aimed to explore the effects of the natural medicinal ingredient α-Hederin on the proliferation,migration and apoptosis of human esophageal squamous cell carcinoma cells,and explore its potential mechanism from the ROS-p38 MAPK pathway,providing theoretical basis for the application of α-Hederin in clinical treatment of esophageal squamous cell carcinoma.Methods:In this study,esophageal squamous cell carcinoma cell KYSE-30 was used as the experimental object for the following steps:(1)The proliferation inhibition rate of KYSE-30 cells treated with α-Hederin at different concentrations(0,20,40,60,80 μM)was measured by tetramethylazazolium salt(MTT)colorimetric method,in order to explore the effect of α-Hederin on the proliferation activity of KYSE-30 cells.According to the results of MTT assay,the IC50 value of α-Hederin treated KYSE-30 cells for 24 h was 21.70 μM,and the subsequent experiments were carried out with 5,10 and 20 μM as low,medium and high dose levels.(2)The effect of α-Hederin on KYSE-30 cell migration was detected by scratch healing assay and transwell assay.(3)Annexin V-FITC/PI double staining flow cytometry was used to detect the effect of various concentrations of α-Hederin on the apoptosis rate of KYSE-30 cells,and Western blot was used to detect the changes of KYSE-30 cells related apoptosis proteins cleaved caspase 3,cleaved PARP,Bcl-2 and Bax.(4)The expression levels of p-ERK,t-ERK,p-JNK,t-JNK,p-p38,t-p38,p-ATF2 and t-ATF2 in KYSE-30 cells were detected by Western blot;KYSE-30 cells were treated with P38 inhibitor SB203580 and α-Hederin.Annexin V-FITC/PI double staining flow cytometry was used to detect the apoptosis rate of KYSE-30 cells,and western blot was used to detect the changes of corresponding P38 MAPK protein and related apoptotic proteins.(5)The changes of ROS in KYSE-30 cells were detected by flow cytometry with reactive oxygen species probe(DCFH-DA);The KYSE-30 cells were treated with ROS inhibitor NAC and α-Hederin.ROS levels were detected by flow cytometry with reactive oxygen species probe,corresponding P38 MAPK protein levels were detected by western blot,and apoptosis rate was detected by Annexin V-FITC/PI double staining flow cytometry.Results:(1)MTT results showed that α-Hederin significantly up-regulated the proliferation inhibition rate of KYSE-30 cells in a concentration and time dependent manner(P < 0.05),suggesting that α-Hederin significantly inhibited the proliferation activity of KYSE-30 cells(2)The results of scratch healing experiment and transwell experiment showed that α-Hederin significantly reduced the migration rate of KYSE-30 cells in a concentration and time dependent manner(P < 0.05),indicating that α-Hederin significantly inhibited the migration ability of KYSE-30 cells.(3)Annexin V-FITC/PI double staining flow cytometry showed that,α-Hederin up-regulated the apoptosis rate of KYSE-30 cells;Western blot showed that,α-Hederin significantly down-regulated the protein level of Bcl-2 in KYSE-30 cells and upregulated the protein expression levels of Bax,cleaved caspase 3 and cleaved PARP.These results indicated that α-Hederin could induce apoptosis of KYSE-30 cells in a dose-dependent manner(P < 0.05).(4)Western blot results showed that α-Hederin up-regulated the expression levels of p-p38 and p-ATF2 in a concentration-dependent manner(all P < 0.05),but had no effect on the expression levels of p-ERK and p-JNK;KYSE-30 cells were treated with SB203580 and α-Hederin,Western blot results showed that SB203580 could reverse the activation of α-Hederin on P38 signaling pathway(P < 0.05).Moreover,the downregulation of Bcl-2 protein level and up-regulation of Bax,cleaved caspase 3,and cleaved PARP protein levels were reversed(P < 0.05).Annexin V-FITC/PI double staining flow cytometry results showed that SB203580 could reverse the up-regulation of apoptosis rate of KYSE-30 cells induced by α-Hederin(P < 0.05).These results indicated that α-Hederin induced apoptosis of KYSE-30 cells by activating p38 signaling pathway.(5)Reactive oxygen flow cytometry results showed that α-Hederin could upregulate ROS levels in a concentration-dependent manner(all P < 0.01);KYSE-30 cells were treated with NAC and α-Hederin,Flow cytometry results showed that NAC could reverse the up-regulation of ROS level by α-Hederin in KYSE-30 cells(P <0.05),Western blot results showed that NAC could reverse the up-regulation of p-p38 protein by α-Hederin(P < 0.05).Annexin V-FITC/PI double staining flow cytometry showed that NAC could reverse the up-regulation of apoptosis rate induced by α-Hederin(P < 0.05).These results indicated that α-Hederin induced apoptosis of KYSE-30 cells through ROS-mediated p38 signaling pathway.Conclusions:α-Hederin can inhibit the proliferation and migration of KYSE-30 cells and promote their apoptosis,the mechanism may be related to the up-regulation of ROSp38 MAPK signal pathway.This study provides a theoretical basis for the application of α-Hederin in clinical treatment of esophageal squamous cell carcinoma. |
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