Enhanced Susceptibility to 5-Fluorouracil in Human Colon Cancer Cells by Silencing of GRP78

摘要

Background: Glucose-regulated protein (GRP78), also known as immunoglobulin heavy chain binding protein and as heat shock 70 kDa protein 5, is present in the endoplasmic reticulum (ER) membrane. GRP78 is generally expressed at low concentrations, but is increased by physiological stress. GRP78 is thought to protect against tissue or organ damage under pathological conditions, such as neurotoxic stress, myocardial infarction, or arteriosclerosis. In addition, in tumors, GRP78 expression is much higher than in normal tissues. Furthermore, high levels of GRP78 expression have been shown to increase the risk of malignancy and metastasis in prostate and colon cancer. Because both anticancer drugs and down-regulation of GRP78 expression inhibit cancer progression and growth, we hypothesized that down-regulation of GRP78 expression might lead to enhanced susceptibility of cancer cells to cytotoxic action of 5-fluorouracil (5-FU). Materials and Methods: GRP78 expression was suppressed in LoVo colon cancer cells by utilizing small-interfering RNA (si-GRP78), and the cells were subsequently used to study the antiproliferative and anticancer effects of 5-FU treatment. The signaling pathways responsible for the increase of LoVo cell susceptibility to 5-FU treatment after exposure to GRP78 siRNA were determined by western blot. Results: GRP78 silencing significantly inhibited cell viability and increased apoptosis of LoVo cells. Furthermore, combined treatment with 5-FU and GRP78 siRNA for 12 h reduced cell viability, and increased apoptosis and generation reactive oxygen species more strongly than either of the two treatments applied separately. In order to examine the role of ER stress in increased susceptibility of LoVo cells to 5-FU after pretreatment with GRP78 siRNA, we analyzed expression levels of ER stress marker proteins, such as phosphorylated protein kinase-like endoplasmic reticulum kinase (PERK), phosphorylated eukaryotic initiation factor 2 alpha (eIF2 alpha), activating transcription factor 4 (ATF4), phosphorylated inositol-requiring enzyme 1 alpha (IRE1 alpha), phosphorylated p38, and C/EBP homologous protein (CHOP). Treatment with 5-FU alone increased the expression of ER stress marker proteins, whereas combined exposure to both 5-FU and GRP78 siRNA led to an even stronger effect on these markers. Similar to the pattern of modulation of ER stress protein expression, the levels of apoptosis-related proteins were also more strongly affected by combined exposure to 5-FU and GRP78 siRNA than by single treatments. In particular, expression of Bcl-2-associated X protein (BAX), cleaved caspase-3, and cleaved poly (ADP-ribose) polymerase 1 (PARP1) were increased, whereas the expression of B-cell lymphoma 2 (BCL2) was reduced by these treatments. Conclusion: GRP78 silencing and incubation with 5-FU have synergistic effects on the inhibition of LoVo colon cancer cell growth via the induction of ER stress-dependent apoptosis.