Abstract
ObjectiveTo investigate the role of endoplasmic reticulum (ER) stress-mediated CRT/ERp57 complex expression underlying the mechanism of resistance to doxorubicin (DOX) in endometrial carcinoma (EC) in vivo and in vitro.MethodsThe expression of CRT, ERp57, p-PERK, eIF2α, p-eIF2α in EC patients and EC cells was detected by Western blots and by immunofluorescence assay. MTT assay was used to determine the LC50 of EC cells to DOX and cell viability. Apoptosis was assayed using flow cytometer. The protein expression of PERK, cleaved-caspase-8, p-eIF2α and CHOP were detected by Western blot, and the expression of VAMP-1, SNAP23 and PERK was knockdown by siRNA and/or shRNA. The expression of CRT/ERp57 complex was detected by flow cytometry. In addition, the expression of eIF2α and p-eIF2α was detected by Western blot analysis after drug-resistant EC cells were transfected with lentivirus overexpressing CRT, treated with GADD34 inhibitor and ES stress inducer. MTT assay was used to detect the phagocytic activity of T cells induced by maturation of dendritic cells in drug-resistant EC cells.ResultsThe expression of CRT, ERp57, p-PERK and p-eIF2α was significantly decreased in the drug-resistant patients in EC patients. The IC50 of the drug-resistant EC cells was 10 times higher than that of the wild type cells. In the drug-resistant EC cells the expression of CRT, ERp57, p-PERK, p-eIF2α, caspase-8 and CHOP was significantly lower than in the wild type cells. After treatment with DOX, CRT and ER stress-related proteins p-PERK, p-eIF2α, caspase-8 and apoptosis were significantly increased in wild-type EC cells, but not in drug-resistant EC cells. The increased expressions led to inhibition of cell growth and apoptosis. The knockdown of PERK gene and addition of DOX resulted in significant decrease of cleaved-caspase 8 and p-eIF2α in sensitive EC cells. The expression of CRT/ERp57 in sensitive EC cells was further significantly decreased by blocking VAMP and SNAP23. In addition, transfection with CRT overexpressing lentivirus and addition of GADD34 inhibitor and ER stress inducer in drug-resistant EC cells revealed a significant increase in the expression of CRT/ERp57 complex and p-eIF2α when DOX was added simultaneously, which promoted the maturation and chemotaxis of T lymphocytes to phagocytose drug-resistant EC cells.ConclusionDOX can induce the death of tumor cells by ER stress-mediated CRT/ERp57 expression in EC cells. Induction of ER stress in drug-resistant EC cells up-regulates the membrane expression of CRT/ERp57, enhances phagocytosis, induces immunogenic apoptosisand sensitizes the cells to DOX.
Highlights
Endometrial carcinoma (EC) is a common malignant cancer in the female reproductive systems that occurs in the endometrial epithelium of genital tract
In the drug-resistant EC cells the expression of CRT, ERp57, p-PERK, p-eIF2α, caspase-8 and CHOP was significantly lower than in the wild type cells
Transfection with CRT overexpressing lentivirus and addition of GADD34 inhibitor and endoplasmic reticulum (ER) stress inducer in drug-resistant EC cells revealed a significant increase in the expression of CRT/ ERp57 complex and p-eIF2α when DOX was added simultaneously, which promoted the maturation and chemotaxis of T lymphocytes to phagocytose drug-resistant EC cells
Summary
Endometrial carcinoma (EC) is a common malignant cancer in the female reproductive systems that occurs in the endometrial epithelium of genital tract. Studies have shown that due to unknown reasons or diseases that cause calcium imbalance or disorder in processing and transport of proteins, endoplasmic reticulum (ER) becomes dysfunctions and could result in ER stress and apoptosis, apoptosis resistance is an important characteristic of drug resistance [2,3,4]. Study found that the expression of CRT is regulated by ERP57 (a member of disulfide isomerase family) [9]. CHOP is called growth arrest and DNA damage-inducible protein 153 (GADD153). It is an ER-specific stress transconductor and belongs to the CCAAT/enhancer-binding protein (C/EBP) transcription factor family [11]. The PERK-eIF2α pathway selectively upregulates ATF4, which activate CHOP and other genes in amino acid metabolism, redox reaction and transport, leading to significantly increase in the expression of CHOP [12]
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