Esophageal cancer stem cells reduce hypoxia-induced apoptosis by inhibiting the GRP78-perk-eIF2α-ATF4-CHOP pathway .

BACKGROUND

Due to the abnormal angiogenesis, cancer stem cells (CSCs) in esophageal cancer (EC) have the characteristics of a hypoxic microenvironment. However, they can resist hypoxia-induced apoptosis. the molecular mechanism underlying the resistance of esophageal CSCs to hypoxia-induced apoptosis is currently unclear. Therefore, this study will investigate the molecular mechanism based on CHOP-mediated endoplasmic reticulum stress.

METHODS

CD44CD24 cells in EC9706 cells were screened by fluorescence-activated cell sorting (FACS). To clarify which apoptosis pathway esophageal CSCs resist hypoxia-induced cell apoptosis through, the effects of hypoxia on apoptosis were detected by nuclear staining, flow cytometry, and JC-1 reagent, the effects of hypoxia on the expression of apoptosis-related proteins were detected by western blotting (WB) assay and quantitative polymerase chain reaction (qPCR) assay. To clarify the mechanisms of CD44CD24 cells resistance to hypoxia-induced apoptosis is achieved by inhibiting the activation of endoplasmic reticulum stress (ERS) pathway, silenced and cell lines of EC9706 cells and overexpressed and cell lines of CD44CD24 cells were constructed, the effects of hypoxia on apoptosis, cell cycle, and mitochondrial membrane potential were detected by flow cytometry and JC-1 reagent. WB assay and qPCR assay were used to detect the expressions of apoptosis-related proteins and ERS-related proteins.

RESULTS

Hypoxia significantly induce apoptosis and cycle arrest of EC9706 cells (P<0.05), but did not affect apoptosis and cycle of CD44CD24 cells (P>0.05). Hypoxia considerably induced the activation of mitochondrial and ERS apoptosis pathways in EC9706 cells (P<0.05), but did not affect Fas receptor apoptosis pathways (P>0.05). The three apoptosis pathways were not affected by hypoxia in CD44CD24 cells (P>0.05). Silencing the and gene inhibited hypoxia-induced apoptosis of EC9706 cells (P<0.05). and overexpression promoted hypoxia-induced apoptosis of CD44CD24 cells (P<0.05), whereas mitochondrial membrane permeability inhibitors inhibited hypoxia-induced apoptosis of CD44CD24 cells overexpressed gene.

CONCLUSIONS

CD44CD24 tumor stem cells in EC resist to hypoxia-induced apoptosis by the inhibition of ERS-mediated mitochondrial apoptosis pathway, which suggested that ERS pathway can serve as a potential target for reducing EC treatment resistance in clinical treatment.