Abstract
HuR (ELAVL1), a RNA-binding protein, plays a key role in posttranscriptional regulation of multidrug resistance (MDR)-related genes. Among various HuR-regulated oncogenic transcripts, the activation of galectin-3/β-catenin survival pathway is critical to induce transcription of cyclin D1, P-glycoprotein (P-gp) and/or multidrug resistance-associated proteins (MRPs). In this study, we aim to elucidate the HuR-regulating pathways related to epirubicin-mediated resistance in human colorectal carcinoma cells. The effects and mechanisms of epirubicin treatment on the expressions of upstream survival signals (e.g., β-catenin) and downstream MDR transporters (e.g., P-gp) and anti-apoptotic pathways (e.g., Bcl-2) were assessed with or without HuR knockdown (siHuR) or overexpression (overHuR; ectopic HuR or pcDNA3/HA-HuR). Our results showed that siHuR decreased transcriptional expressions of galectin-3, β-catenin, cyclin D1, Bcl-2, P-gp, MRP1, and MRP2 in epirubicin-treated colon cancer cells. Consistently, the co-treatment of epirubicin and siHuR diminished the expressions of galectin-3, ß-catenin, c-Myc, P-gp and MRP1. HuR silencing enhanced the intracellular accumulation of epirubicin in colon cancer cells. On the other hand, overHuR abolished such effects. Furthermore, siHuR significantly intensified epirubicin-mediated apoptosis via increasing reactive oxygen species and thus promoted the cytotoxic effect of epirubicin. The combined treatments of siHuR and epirubicin significantly reduced the expression of Bcl-2, but increased the expression of Bax, as well as activity and expression levels of caspase-3 and -9. In contrast, overHuR abrogated these effects. Our findings provide insight into the mechanisms by which siHuR potentiated epirubicin-induced cytotoxicity via inhibiting galectin-3/β-catenin signaling, suppressing MDR transporters and provoking apoptosis. To our best knowledge, this is an innovative investigation linking the post-transcriptional control by HuR silencing to survival signaling repression, efflux transporter reversal and apoptosis induction. Our study thus provides a powerful regimen for circumventing MDR in colon cancer cells.
Highlights
The mRNA-binding protein HuR acts by stabilizing AU-rich elements (AREs) in the 3’ untranslated regions (3’-UTR) of mRNA [1,2] and mediates posttranscriptional upregulation of key survival or growth-related genes by increasing both mRNA stability and/or protein translation [3,4]
The cell viability decreased about 20% when cells were transfected with small interfering RNA targeting the HuR mRNA (siHuR) at 75 nM (Fig 1A, lower right panel) compared to control group
We found that Epi or siHuR treatment for 24 h decreased the cellular mRNA levels of galectin-3 and ß-catenin (Fig 4C)
Summary
The mRNA-binding protein HuR (human antigen R, ELAVL1) acts by stabilizing AU-rich elements (AREs) in the 3’ untranslated regions (3’-UTR) of mRNA [1,2] and mediates posttranscriptional upregulation of key survival or growth-related genes by increasing both mRNA stability and/or protein translation [3,4]. The development of multidrug resistance (MDR) to conventional chemotherapy causes treatment failure in various cancers. Numerous studies have indicated that cytoplasmic accumulation of HuR has a link to MDR of cancer cells acquired after chemotherapy and causes poor prognosis of survival in various cancers [7,8,9]. Suppression of the cytoplasmic accumulation of HuR during the treatment of antineoplastic therapeutics may be a potential approach for reversing drug resistance [7,10]. HuR acts by binding to the 3’-UTR of many Bcl-2 family members and HuR silencing causes unstable transcript of Bcl-2 and inhibits Bcl-2 protein expression, triggering apoptosis and inhibiting brain glioma cell growth [12]
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