Abstract
Heat shock proteins (HSPs) are a defined set of genes response to a variety of external stimuli and their products are mainly molecular chaperones that are crucially involved in the maintenance of the proper functions of a large range of intracellular proteins. The stress proteins are usually sub-classified a number of HSPs families according to their molecular weight. Among them, the HSP70s is one of the most important HSPs families and highly associated with tumor transformation because of its anti-apoptotic function. However, the detailed mechanisms of HSP70s in gene regulation and anti-apoptosis are not fully understood. First of all, we examined gene regulation of HSP70s in geldanamycin (GA, a potent anti-cancer drug)-treated human non-small cell lung cancer H460 cells. In this study, we showed that the HSP70-1/2 are the major inducible forms under GA treatment using LC-ESI-MS/MS and Northern blotting analysis. Transactivation of HSP70-1/2 was further determined by electrophoretic mobility-shift assay using heat shock element as a probe. The signaling pathway mediators involved in HSP70-1/2 transactivation were screened by the kinase inhibitor scanning technique. The protein kinase C (PKC)-specific inhibitor Ro-31-8425 and the Ca2+-dependent PKC inhibitor Gö-6976 diminished GA-induced HSP70-1/2, suggesting an involvement of the PKC in the process. Moreover, GA treatment causes a transient increase of intracellular Ca2+. Chelating intracellular Ca2+ with BAPTA-AM or depletion of intracellular Ca2+ store with A23187 or thapsigargin significantly decreased GA-transactivated HSP70-1/2 expression. Taken together, our results demonstrated that GA induces specific HSP70-1/2 isoforms expression in H460 cells through signaling pathway mediated by Ca2+ and PKC. On the other hand, the chaperone glucose-regulated protein 78 (GRP78), a member of HSP70s, protects cells from cytotoxicity induced by DNA damage or endoplasmic reticulum (ER) stress. In this study, we showed that GRP78 is a major inducible protein in H460 cells treated with ER stress inducers including A23187 and thapsigargin. 4-[2-aminoethyl]-benzenesulfonyl fluoride hydrochloride (AEBSF), an inhibitor of serine protease, diminished GRP78 induction, enhanced mitochondrial permeability and augmented apoptosis in H460 cells during ER stress. Simultaneously, AEBSF promoted Raf-1 degradation and decreased phosphorylation level of Raf-1 at Ser338 and/or Tyr340 during ER stress. His-tag affinity pull-down assay and subcellular fractionation results also demonstrated that GRP78 associateed and colocalized with Raf-1 on the outer membrane of mitochondria. Treatment of cells with ER stress inducers inactivated Bcl-XL/Bcl-2-associated death promoter (BAD) by phosphorylation at Ser75, which is a phosphorylation site by Raf-1. Nevertheless, AEBSF attenuated phosphorylation of BAD leading to cytochrome c release from mitochondria. Additionally, overexpression of GRP78 and/or Raf-1 protected cells from ER stress-induced apoptosis. These results indicate that GRP78 may chaperone Raf-1 to maintain mitochondrial permeability and thus protects cells from ER stress-induced apoptosis. Taken together, we discovered that molecular mechanisms of HSP70s in gene regulation and anti-apoptotic function. These findings might provide a possible pharmacological manipulation for cancer exposed to chemotherapy.