Inhibition of endoplasmic reticulum chaperone protein glucose-regulated protein 78 potentiates anti-angiogenic therapy in renal cell carcinoma through inactivation of the PERK/eIF2α pathway.

Tumor microenvironments are characterized by decreased oxygen and nutrition due to the rapid and progressive nature of tumors and also stresses induced by several anti-tumor therapies. These intense cell stressors trigger a protective cell survival mechanism heralded by the unfolded protein response (UPR). The UPR is induced by an accumulation of unfolded proteins in the endoplasmic reticulum (ER) following cell starvation. Although the ER stress response is implicated in cytoprotection, its precise role during anti-angiogenic therapy remains unclear. One of the major proteins involved in ER stress is glucose-regulated protein 78 (GRP78), which binds to unfolded proteins and dissociates from membrane-bound ER stress sensors. To determine the role of ER stress responses during anti-angiogenic therapy and the potential role of GRP78 in combined therapy in renal cell carcinoma (RCC), we used GRP78 overexpressing or knockdown RCC cells under hypoxic or hypoglycemic conditions in vitro and in animal models treated with sunitinib. Here, we report that GRP78 plays a crucial role in protecting RCC cells from hypoxic and hypoglycemic stress induced by anti-angiogenic therapy. Knockdown of GRP78 using siRNA inhibited cancer cell survival and induced apoptosis in RCC cells in vitro and also resulted in ER stress-induced apoptosis and hypoxic/hypoglycemic stress-induced apoptosis by inactivating the PERK/eIF-2α pathway. Finally, GRP78 knockdown showed potent suppression of tumor growth and enhanced the antitumor effect of sunitinib in RCC xenografts. Our findings suggest that GRP78 may serve as a novel therapeutic target in combination with anti-angiogenic therapy for the management of RCC.

Terpestacin inhibits tumor angiogenesis by targeting UQCRB of mitochondrial complex III and suppressing hypoxia-induced reactive oxygen species production and cellular oxygen sensing.

Cellular oxygen sensing is required for hypoxia-inducible factor-1alpha stabilization, which is important for tumor cell survival, proliferation, and angiogenesis. Here we find that terpestacin, a small molecule previously identified in a screen of microbial extracts, binds to the 13.4-kDa subunit (UQCRB) of mitochondrial Complex III, resulting in inhibition of hypoxia-induced reactive oxygen species generation. Consequently, such inhibition blocks hypoxia-inducible factor activation and tumor angiogenesis in vivo, without inhibiting mitochondrial respiration. Overexpression of UQCRB or its suppression using RNA interference demonstrates that it plays a crucial role in the oxygen sensing mechanism that regulates responses to hypoxia. These findings provide a novel molecular basis of terpestacin targeting UQCRB of Complex III in selective suppression of tumor progression.

Effect of selective cyclooxygenase-2 inhibitor meloxicam on liver fibrosis in rats with ligated common bile ducts.

AIM: Cholestasis triggers fibrogenesis in the liver. Hepatic cyclooxygenase-2 (COX-2) expression increases in various chronic liver diseases caused either by viruses or toxins. We hypothesized that selective COX-2 inhibitor meloxicam could suppress inflammation and fibrogenesis in a rat model of cholestasis induced by bile duct ligation (BDL). METHODS: Forty-three Sprague-Dawley rats were assigned to one of four treatment groups (sham-operation, BDL, daily meloxicam injections following BDL, and daily meloxicam injection without BDL). Liver histopathology was analyzed with hematoxylin-eosin and Masson's trichrome staining. The expression of alpha-smooth muscle actin (alpha-SMA), transforming growth factor-beta1 (TGF-beta1), and COX-2 were measured with immunohistochemical staining. The levels of COX-2, TGF-beta1, and matrix metalloproteinase-9 (MMP-9) production were measured with the Western blot method and an enzyme immunoassay. RESULTS: Meloxicam treatment attenuated the expression of alpha-SMA, TGF-beta1, and COX-2 in rats that were treated with BDL for 3 weeks. This was associated with a marked reduction in collagen accumulation and histological improvement. In addition, meloxicam treatment was found to downregulate the levels of hepatic COX-2, TGF-beta1, and MMP-9 production. CONCLUSION: Cholestasis in BDL rats induces hepatic COX-2 expression. Selective COX-2 inhibitor meloxicam reduces BDL-induced hepatic fibrosis, and this is associated with reduced hepatic TGF-beta1 expression as well as decreased cyclooxygenase activity in the liver.

Accumulation of beta-catenin protein, mutations in exon-3 of the beta-catenin gene and a loss of heterozygosity of 5q22 in solid pseudopapillary tumor of the pancreas.

BACKGROUND: Solid pseudopapillary tumors (SPT) of the pancreas are neoplasms with a low malignant potential. The molecular events contributing to the pathogenesis of SPTs are still unknown. OBJECTIVES: This study was intended to help better understand the early steps of human SPT development. METHODS: We microdissected 20 SPTs and normal pancreatic tissue. In addition, we examined the DNA from each SPT for mutations in exon-3 of beta-catenin and loss of heterozygosity (LOH) on 9 chromosome arms using 10 microsatellite markers. Immunohistochemical staining for beta-catenin was performed. RESULTS: Activating mutations between codons 32 and 37 of beta-catenin exon-3 were present in 16 cases (80%). Allelic loss on chromosome 5q22.1 was present in 10 cases (55.5%), while no allelic loss was found on chromosomes 1p, 6q, 9p, 9q, 11p, 11q, 17p, or 22q. Nuclear accumulation of beta-catenin was found in 20 cases (100%). CONCLUSION: Mutations in exon-3 of the beta-catenin gene, nuclear accumulation of beta-catenin, and LOH on chromosome 5q22.1 in SPT tissue suggest that these mutations are involved in SPT tumorigenesis.