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Avarol induces apoptosis in pancreatic ductal adenocarcinoma cells by activating PERK-eIF2?-CHOP signaling.

ABSTRACT: Avarol is a sesquiterpenoid hydroquinone with potent cytotoxicity. Although resolving endoplasmic reticulum (ER) stress is essential for intracellular homeostasis, erratic or excessive ER stress can lead to apoptosis. Here, we reported that avarol selectively induces cell death in pancreatic ductal adenocarcinomas (PDAC), which are difficult to treat owing to the availability of few chemotherapeutic agents. Analyses of the molecular mechanisms of avarol-induced apoptosis indicated upregulation of ER stress marker BiP and ER stress-dependent apoptosis inducer CHOP in PDAC cells but not in normal cells, suggesting that avarol selectively induces ER stress responses. We also showed that avarol activated the PERK-eIF2? pathway but did not affect the IRE1 and ATF6 pathways. Moreover, CHOP downregulation was significantly suppressed by avarol-induced apoptosis. Thus, the PERK-eIF2?-CHOP signaling pathway may be a novel molecular mechanism of avarol-induced apoptosis. The present data indicate that avarol has potential as a chemotherapeutic agent for PDAC and induces apoptosis by activating the PERK-eIF2? pathway.

SUBMITTER: Namba T

PROVIDER: S-EPMC4413216 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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Avarol induces apoptosis in pancreatic ductal adenocarcinoma cells by activating PERK-eIF2α-CHOP signaling.

Namba Takushi T Kodama Rika R

Marine drugs 20150416 4

Avarol is a sesquiterpenoid hydroquinone with potent cytotoxicity. Although resolving endoplasmic reticulum (ER) stress is essential for intracellular homeostasis, erratic or excessive ER stress can lead to apoptosis. Here, we reported that avarol selectively induces cell death in pancreatic ductal adenocarcinomas (PDAC), which are difficult to treat owing to the availability of few chemotherapeutic agents. Analyses of the molecular mechanisms of avarol-induced apoptosis indicated upregulation o ...[more]

PMID: 25894488

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Project description:BackgroundGemcitabine (GEM)-based chemotherapy is the first-line option for pancreatic ductal adenocarcinoma (PDAC). However, the development of drug resistance limits its efficacy, and the specific mechanisms remain largely unknown. RUNX1, a key transcription factor in hematopoiesis, also involved in the malignant progression of PDAC, but was unclear in the chemoresistance of PDAC.MethodsComparative analysis was performed to screen GEM-resistance related genes using our single-cell RNA sequencing(scRNA-seq) data and two public RNA-sequencing datasets (GSE223463, GSE183795) for PDAC. The expression of RUNX1 in PDAC tissues was detected by qRT-PCR, immunohistochemistry (IHC) and western blot. The clinical significance of RUNX1 in PDAC was determined by single-or multivariate analysis and survival analysis. We constructed the stably expressing cell lines with shRUNX1 and RUNX1, and successfully established GEM-resistant cell line. The role of RUNX1 in GEM resistance was determined by CCK8 assay, plate colony formation assay and apoptosis analysis in vitro and in vivo. To explore the mechanism, we performed bioinformatic analysis using the scRNA-seq data to screen for the endoplasm reticulum (ER) stress signaling that was indispensable for RUNX1 in GEM resistance. We observed the cell morphology in ER stress by transmission electron microscopy and validated RUNX1 in gemcitabine resistance depended on the BiP/PERK/eIF2α pathway by in vitro and in vivo oncogenic experiments, using ER stress inhibitor(4-PBA) and PERK inhibitor (GSK2606414). The correlation between RUNX1 and BiP expression was assessed using the scRNA-seq data and TCGA dataset, and validated by RT-PCR, immunostaining and western blot. The mechanism of RUNX1 regulation of BiP was confirmed by ChIP-PCR and dual luciferase assay. Finally, the effect of RUNX1 inhibitor on PDAC was conducted in vivo mouse models, including subcutaneous xenograft and patient-derived xenograft (PDX) mouse models.ResultsRUNX1 was aberrant high expressed in PDAC and closely associated with GEM resistance. Silencing of RUNX1 could attenuate resistance in GEM-resistant cell line, and its inhibitor Ro5-3335 displayed an enhanced effect in inhibiting tumor growth, combined with GEM treatment, in PDX mouse models and GEM-resistant xenografts. In detail, forced expression of RUNX1 in PDAC cells suppressed apoptosis induced by GEM exposure, which was reversed by the ER stress inhibitor 4-PBA and PERK phosphorylation inhibitor GSK2606414. RUNX1 modulation of ER stress signaling mediated GEM resistance was supported by the analysis of scRNA-seq data. Consistently, silencing of RUNX1 strongly inhibited the GEM-induced activation of BiP and PERK/eIF2α signaling, one of the major pathways involved in ER stress. It was identified that RUNX1 directly bound to the promoter region of BiP, a primary ER stress sensor, and stimulated BiP expression to enhance the reserve capacity for cell adaptation, which in turn facilitated GEM resistance in PDAC cells.ConclusionsThis study identifies RUNX1 as a predictive biomarker for response to GEM-based chemotherapy. RUNX1 inhibition may represent an effective strategy for overcoming GEM resistance in PDAC cells.

Project description:BackgroundDue 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.MethodsCD44+CD24- 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 CD44+CD24- cells resistance to hypoxia-induced apoptosis is achieved by inhibiting the activation of endoplasmic reticulum stress (ERS) pathway, silenced CHOP and PERK cell lines of EC9706 cells and overexpressed CHOP and PERK cell lines of CD44+CD24- 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.ResultsHypoxia significantly induce apoptosis and cycle arrest of EC9706 cells (P<0.05), but did not affect apoptosis and cycle of CD44+CD24- 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 CD44+CD24- cells (P>0.05). Silencing the CHOP and PERK gene inhibited hypoxia-induced apoptosis of EC9706 cells (P<0.05). CHOP and PERK overexpression promoted hypoxia-induced apoptosis of CD44+CD24- cells (P<0.05), whereas mitochondrial membrane permeability inhibitors inhibited hypoxia-induced apoptosis of CD44+CD24- cells overexpressed CHOP gene.ConclusionsCD44+CD24- 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.

Dataset Information

Avarol induces apoptosis in pancreatic ductal adenocarcinoma cells by activating PERK-eIF2?-CHOP signaling.

Publications

Avarol induces apoptosis in pancreatic ductal adenocarcinoma cells by activating PERK-eIF2α-CHOP signaling.

Similar Datasets

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