Inhibition of Cancer Cell Growth by GRP78 siRNA Lipoplex <i>via</i> Activation of Unfolded Protein Response

Abstract

Proteasome inhibitors are a novel class of molecular-targeted anti-cancer drugs that suppress the degradation of malfolded proteins, trigger endoplasmic reticulum (ER) stress, and activate apoptosis signals. Glucose-regulated protein 78 (GRP78), a major ER chaperone, is one of the most important molecules for transduction of unfolded protein response (UPR) signals. In accordance with past findings that expression of GRP78 is elevated in cancer cells and helps to resist stress-induced apoptosis, GRP78 knockdown could be effective in anticancer therapy. We tested this hypothesis and found that transfection of small interfering RNA (siRNA) targeting GRP78 inhibited the growth of RENCA renal carcinoma cells, in association with elevated gene expression of UPR downstream signaling molecules (CHOP, EDEM1, and ERdj4 mRNA). In addition, the combinatorial effect of GRP78 siRNA with ER stress inducers (tunicamycin, MG132, and 2-deoxyglucose) on survival was measured. Combination of GRP78 siRNA and the ER stress inducers more extensively reduced cell viability than combination with scrambled siRNA. Besides RENCA, B16BL6 melanoma cells were also shown to be sensitive to GRP78 siRNA. These results suggest that GRP78 knockdown might be an effective strategy for cancer therapy targeting UPR-induced apoptosis.Key words glucose-regulated protein 78; small interfering RNA; apoptosis; unfolded protein response; endoplasmic reticulum stress; cancer cellThe endoplasmic reticulum (ER) is responsible for protein synthesis, conformational maturation, and quality control of correctly folded proteins. In cancer cells, protein synthesis and by-product disposal are elevated, therefore, disruption of ER functions is believed to be a promising approach for cancer therapy. Proteasome inhibitors, which are a novel class of molecular-targeted anti-cancer drugs, suppress the degradation of malfolded proteins and trigger ER stress, which leads to the activation of the unfolded protein response (UPR) and subsequent apoptotic signals.1,2) Bortezomib was the first approved proteasome inhibitor, and is used for the treatment of relapsed multiple myeloma 3) and mantle cell lymphoma. 4) A second generation of proteasome inhibitors are now under development to improve drug efficacy and compliance. 5)Molecular chaperones are key factors involved in unfolded protein accumulation. Glucose-regulated protein 78 (GRP78) is known as a stress-inducible ER chaperone protein and serves as an ER stress signal regulator.6) Under unstressed conditions, GRP78 associates with ER transmembrane sensor proteins, such as protein kinase RNA-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme-1 (IRE1) and activating transcription factor 6 (ATF6), 7,8) and these interactions maintain them in an inactive form. However, accumulation of unfolded proteins results in the detachment of GRP78 from the sensor proteins and the subsequent activation of the unfolded protein response (UPR), which results in general translational attenuation, up-regulation of chaperones a...

MicroRNA-30a-5p Inhibits the Growth of Renal Cell Carcinoma by Modulating GRP78 Expression

“…GRP78, a marker of ER stress, has been reported to be up-regulated in RCC and to be involved in the growth of RENCA renal carcinoma cells [23, 24, 30]. To explore whether the anti-tumor function of miR-30a-5p in ccRCC is mediated by modulating the expression of GRP78, we initially analyzed the GRP78 expression pattern in ccRCC tissues and RCC cell lines.…”

GRP78 silencing enhances hyperoxia-induced alveolar epithelial cell apoptosis via CHOP pathway

“…in ER-stress mediated apoptosis (35). Also, Matsumoto et al reported that overexpression of Bcl-2 blocked CHOP-induced apoptosis (36). On the other hand, we treated A549 cells with the CHOP siRNA under hyperoxia, the results showed the expression of Bcl-2 increased, while the expression of Bax decreased, which further supported our hypothesis on the role of GRP78-CHOP-Bcl-2/Bax pathway in ER related apoptosis of lung epithelial cells under hyperoxia.…”

Protective effects of circulating microvesicles derived from myocardial ischemic rats on apoptosis of cardiomyocytes in myocardial ischemia/reperfusion injury

“…ER stress refers to a condition in which normal ER function is impaired, leading to accumulation of unfolded or mis-folded proteins in the ER lumen in myocardial I/R injury. In the case of severe or prolonged ER stress, its responses exceed handling ability of GRP 78 and result in the activation of ER stress related apoptosis signaling pathways [ 24 ]. Caspase 12 is required for apoptosis induced specifically by ER stress.…”