Glucolipotoxicity in INS-1E cells is counteracted by carnitine palmitoyltransferase 1 over-expression

Effects of non-esterified fatty acids (FAs) are accentuated when applied together with elevated glucose through preferential use of glucose as fuel, which leads to decreased oxidation of FAs. We examined how over-expression of the mitochondrial FA transporter carnitine palmitoyltransferase 1 (CPT1) affects glucose-stimulated insulin secretion (GSIS), apoptosis and ER stress in INS-1E cells cultured in the presence of elevated levels of glucose and palmitate. INS-1E cells were infected with Tet-ON regulated adenovirus containing CPT1 and cultured for 48 h in the presence of 0.5 mM palmitate and 20 mM glucose. Over-expressing CPT1 lowered basal insulin secretion in a dose-dependent manner thereby improving GSIS from INS-1E cells. Also, apoptosis was alleviated and ER-stress markers p-eIF2α and CHOP were decreased in cells over-expressing CPT1. We conclude that regulated over-expression of CPT1 is beneficial for glucolipotoxic beta-cells.     

Rottlerin induces pro-apoptotic endoplasmic reticulum stress through the protein kinase C-δ-independent pathway in human colon cancer cells

Rottlerin, a compound reported to be a PKC δ-selective inhibitor, has been shown to induce growth arrest or apoptosis of human cancer cell lines. In our study, rottlerin dose-dependently induced apoptotic cell death in colon carcinoma cells. Treatment of HT29 human colon carcinoma cells with rottlerin was found to induce a number of signature ER stress markers; phosphorylation of eukaryotic initiation factor-2α (eIF-2α), ER stress-specific XBP1 splicing, and up-regulation of glucose-regulated protein (GRP)-78 and CCAAT/enhancer-binding protein-homologous protein (CHOP). However, suppression of PKC δ expression by siRNA or overexpression of WT-PKC δ and DN-PKC δ did not abrogate the rottlerin-mediated induction of CHOP. These results suggest that rottlerin induces up-regulation of CHOP via PKC δ-independent pathway. Furthermore, down-regulation of CHOP expression using CHOP siRNA attenuated rottlerin-induced apoptosis. Taken together, the present study thus provides strong evidence to support an important role of ER stress response in mediating the rottlerin-induced apoptosis.     

The double-strand RNA-dependent protein kinase PKR plays a significant role in a sustained ER stress-induced apoptosis

Sustained ER stress leads to apoptosis. However, the exact mechanism still remains to be elucidated. Here, we demonstrate that the double strand RNA-dependent protein kinase (PKR) is involved in the ER stress-mediated signaling pathway. ER stress rapidly activated PKR, inducing the phosphorylation of eIF2alpha, followed by the activation of the ATF4/CHOP pathway. ER-stress-mediated eIF2alpha/ATF4/CHOP signaling and associated cell death was markedly reduced by PKR knockdown. We also found that PKR activation was mediated by PACT, the expression of which was elevated by ER-stress. These results indicate that the ER-stress-mediated eIF2alpha/ATF4/CHOP/cell death pathway is, to some degree, dependent on PACT-mediated PKR activation apart from the PERK pathway.     

Involvement of up-regulation of PUMA in non-steroidal anti-inflammatory drug-induced apoptosis

NSAIDs such as celecoxib induce apoptosis in cancer cells. Although this apoptotic effect is involved in the anti-tumor activity associated with such drugs, the mechanism by which this occurs is not fully understood. We report here that various NSAIDs, including celecoxib, up-regulate PUMA, a Bcl-2 family protein with potent apoptosis-inducing activity, in human gastric carcinoma cell line, accompanying the induction of apoptosis. Experiments using siRNA and an intracellular Ca(2+) chelator revealed that Ca(2+)-dependent up-regulation of ATF4 and CHOP is involved in this up-regulation of PUMA. The siRNA for PUMA inhibited the celecoxib-induced activation and translocation of Bax, release of cytochrome c into the cytosol and induction of apoptosis, suggesting that PUMA plays an important role in celecoxib-induced mitochondrial dysfunction and the resulting apoptosis.     

香菇多糖注射液联合CHOP化疗治疗弥漫性大B细胞淋巴瘤的疗效及对Bcl-6、Ki-67、VEGF、β2-MG表达的影响

目的:探讨香菇多糖注射液联合CHOP化疗治疗弥漫性大B细胞淋巴瘤的疗效及对B细胞淋巴瘤因子-6(Bcl-6)、Ki-67抗原(Ki-67)、内皮细胞生长因子(VEGF)、β2微球蛋白(β2-MG)表达的影响。方法:选取2010年1月~2011年12月我院收治的64例弥漫性大B细胞淋巴瘤患者作为研究对象,按治疗方式进行分组,采用香菇多糖注射液联合CHOP化疗治疗的患者为观察组,单纯采用CHOP化疗治疗的患者为对照组。比较两组患者短期疗效、治疗前后Bcl-6、Ki-67、VEGF、β2-MG的表达情况、不良反应的发生情况及生存情况。结果:治疗后,观察组治疗总有效率显著高于对照组[84.38%(27/32)vs.53.13(17/32)](P0.05);肿瘤组织Bcl-6阳性表达率高于对照组[84.38%(27/32)vs.62.50%(20/32)](P0.05),Ki-67、VEGF、β2-MG阳性表达率均低于对照组[31.25%(10/32)vs.59.38%(19/32),28.13%(9/32)vs.56.25%(18/32),18.75%(6/32)vs.40.63%(13/32)](P0.05);不良反应总发生率低于对照组[21.88%(7/32)vs.46.88%(15/32)](P0.05);第5年随访时存活率高于对照组[59.38%(19/32)vs.34.38%(11/32)](P0.05)。结论:香菇多糖注射液联合CHOP化疗方案治疗弥漫性大B细胞淋巴瘤可有效提高近期疗效、生存率及生存质量,且安全性高,可能与其改善机体肿瘤组织中Bcl-6、Ki-67、VEGF、β2-MG的表达有关。     

Cellular Inhibitor of Apoptosis Protein-1 (cIAP1) Plays a Critical Role in β-Cell Survival under Endoplasmic Reticulum Stress: PROMOTING UBIQUITINATION AND DEGRADATION OF C/EBP HOMOLOGOUS PROTEIN (CHOP)

Lipotoxicity in pancreatic β-cells, arising from excess free fatty acid-induced endoplasmic reticulum (ER) stress response, has been recognized as a key pathogenic factor causing loss of β-cell mass and contributing to type 2 diabetes. However, how the adaptive ER stress response causes cell death remains enigmatic. We report herein a critical role of cellular inhibitor of apoptosis protein-1 (cIAP1) in controlling β-cell survival under ER stress. While both palmitate and palmitoleate induced an overt ER stress response, lipotoxicity was only observed in β-cells exposed to palmitate but not palmitoleate. Interestingly, cells treated with palmitoleate exerted a sustainable level of cIAP1, whereas the protein quickly degraded following palmitate treatment. Enforced overexpression of cIAP1 prevented palmitate-induced cell death. In contrast, siRNA-mediated knockdown of cIAP1 in β-cells or knock-out of cIap1 in mouse embryonic fibroblasts not only increased palmitate-induced apoptosis, but also committed cells to death in response to the nontoxic palmitoleate treatment. Of importance, we found that cIAP1 functions as an E3 ubiquitin ligase promoting ubiquitination and degradation of C/EBP homologous protein (CHOP), a key mediator of ER stress-induced cell death. These findings define a novel mechanism for β-cell survival under ER stress and help to identify targets for therapeutic intervention against lipotoxicity in β-cells.     

Inhibition of CHOP accentuates the apoptotic effect of α-mangostin from the mangosteen fruit (Garcinia mangostana) in 22Rv1 prostate cancer cells

The mangosteen (Garcinia mangostana) fruit has been a popular food in Southeast Asia for centuries and is increasing in popularity in Western countries. We identified α-Mangostin as a primary phytochemical modulating ER stress proteins in prostate cancer cells and propose that α-Mangostin is responsible for exerting a biological effect in prostate cancer cells. Two human prostate cancer cell lines, 22Rv1 and LNCaP, and prostate epithelial cells procured from two patients undergoing radical prostatectomy were treated with α-Mangostin and evaluated by RT-PCR, Western blot, fluorescent microscopy and siRNA transfection to evaluate ER stress. Next, we evaluated α-Mangostin for microsomal stability, pharmacokinetic parameters, and anti-cancer activity in nude mice. α-Mangostin significantly upregulated ER stress markers in prostate cancer cells. Interestingly, α-Mangostin did not promote ER stress in prostate epithelial cells (PrECs) from prostate cancer patients. CHOP knockdown enhanced α-Mangostin-induced apoptosis in prostate cancer cells. α-Mangostin significantly suppressed tumor growth in a xenograft tumor model without obvious toxicity. Our study suggests that α-Mangostin is not the only active constituent from the mangosteen fruit requiring further work to understand the complex chemical composition of the mangosteen.