SLPI suppresses hepatocellular carcinoma progression via endoplasmic reticulum stress induced apoptosis

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. Secretory leukocyte protease inhibitor (SLPI) has been reported to function as a regulatory factor in several cancers. However, its biological functions and underlying mechanisms in HCC remain to be uncovered. Here, we aimed to explore the effect of SLPI in HCC. In our study, we found that the mRNA and protein expression levels of SLPI were significantly down-regulated in HCC tissues and hepatoma cell lines and low level of SLPI predicted worse survival in our HCC cohorts. In term of function, silencing of SLPI markedly promoted whereas overexpression SLPI suppressed proliferation, migration and invasion capabilities of HCC cells in vitro, and ectopic expression of SLPI inhibited the tumorigenicity of HCC cells in vivo. Mechanistic studies demonstrated that SLPI played a protective role in HCC progression via activating endoplasmic reticulum stress (ER stress)-mediated apoptosis of hepatoma cells, which could be regulated by MAPK signaling pathways. In summary, our findings highlight that SLPI could serve as a potential prognostic biomarker and putative tumor suppressor by enhancing ER stress-induced apoptosis in HCC cells mediated by MAPK signaling pathways, which provides new insights into promising therapeutic targets for HCC treatment.     

Isoproterenol instigates cardiomyocyte apoptosis and heart failure via AMPK inactivation-mediated endoplasmic reticulum stress

A prolonged or excessive adrenergic activation leads to myocyte loss and heart dysfunction; however, how it contributes to heart failure remains poorly defined. Here we show that isoproterenol (ISO) induced aberrant endoplasmic reticulum (ER) stress and apoptotic cell death, which was inhibited by activating the AMP-activated protein kinase (AMPK) in vitro and in vivo. Persistent ISO stimulation suppressed the AMPK phosphorylation and function, resulting in enhanced ER stress and the subsequent cell apoptosis in cardiomyocytes in vitro and in vivo. AMPK activation decreased the aberrant ER stress, apoptosis, and brain natriuretic peptide (BNP) release in ISO-treated cardiomyocytes, which was blocked by AMPK inhibitor Compound C. Importantly, increased ER stress and apoptosis were observed in ISO-treated cardiomyocytes isolated from AMPKα2^?/? mice. Inhibition of ER stress attenuated the apoptosis but failed to reverse AMPK inhibition in ISO-treated cardiomyocytes. Moreover, metformin administration activated AMPK and reduced both ER stress and apoptosis in ISO-induced rat heart failure in vivo. We conclude that ISO, via AMPK inactivation, causes aberrant ER stress, cardiomyocyte injury, BNP release, apoptosis, and hence heart failure in vivo, all of which are inhibited by AMPK activation.     

Chelerythrine induces apoptosis via ROS‐mediated endoplasmic reticulum stress and STAT3 pathways in human renal cell carcinoma

Renal cell carcinoma (RCC) is a heterogeneous histological disease and it is one of the most common kidney cancer. The treatment of RCC has been improved for the past few years, but its mortality still remains high. Chelerythrine (CHE) is a natural benzo[c]phenanthridine alkaloid and a widely used broad‐range protein kinase C inhibitor which has anti‐cancer effect on various types of human cancer cells. However, its effect on RCC has not been fully elucidated. In this study, we evaluated the effect and mechanism of CHE on RCC cells. Our study showed that CHE induced colony formation inhibition and G2/M cell cycle arrest in a dose‐dependent manner in RCC cells. In addition, CHE increased cellular ROS level, leading to endoplasmic reticulum (ER) stress, inactivating STAT3 activities and inducing apoptosis in RCC cells which were suppressed by NAC, a special ROS inhibitor. We further found that both knockdown of ATF4 protein and overexpression of STAT3 protein could reduce CHE‐induced apoptosis in Caki cells. These results demonstrated that the apoptosis induced by CHE was mediated by ROS‐caused ER stress and STAT3 inactivation. Collectively, our studies provided support for CHE as a potential new therapeutic agent for the management of RCC.     

Hypoxia triggers endothelial endoplasmic reticulum stress and apoptosis via induction of VLDL receptor

Endothelial cells express very low density lipoprotein receptor (VLDLr). Beyond the function as peripheral lipoprotein receptor, other roles of VLDLr in endothelial cells have not been completely unraveled. In the present study, human umbilical vein endothelial cells were subjected to hypoxia, and VLDLr expression, endoplasmic reticulum (ER) stress, and apoptosis were assessed. Hypoxia triggered endothelial ER stress and apoptosis, and induced VLDLr expression. Silencing or stabilization of HIF-1α reduced and enhanced VLDLr expression, respectively. HIF-1α affected vldlr promoter activity by interacting with a hypoxia-responsive element (HRE). Knockdown or overexpression of VLDLr alleviated and exacerbated hypoxia-induced ER stress and apoptosis, respectively. Thus, hypoxia induces VLDLr expression through the interaction of HIF-1α with HRE at the vldlr promoter. VLDLr then mediates ER stress and apoptosis.     

Cellular responses to endoplasmic reticulum stress and apoptosis

The endoplasmic reticulum (ER) is the cell organelle where secretory and membrane proteins are synthesized and folded. Correctly folded proteins exit the ER and are transported to the Golgi and other destinations within the cell, but proteins that fail to fold properly—misfolded proteins—are retained in the ER and their accumulation may constitute a form of stress to the cell—ER stress. Several signaling pathways, collectively known as unfolded protein response (UPR), have evolved to detect the accumulation of misfolded proteins in the ER and activate a cellular response that attempts to maintain homeostasis and a normal flux of proteins in the ER. In certain severe situations of ER stress, however, the protective mechanisms activated by the UPR are not sufficient to restore normal ER function and cells die by apoptosis. Most research on the UPR used yeast or mammalian model systems and only recently Drosophila has emerged as a system to study the molecular and cellular mechanisms of the UPR. Here, we review recent advances in Drosophila UPR research, in the broad context of mammalian and yeast literature.     

Integrating the mechanisms of apoptosis induced by endoplasmic reticulum stress

The ability to respond to perturbations in endoplasmic reticulum (ER) function is a fundamentally important property of all cells, but ER stress can also lead to apoptosis. In settings of chronic ER stress, the associated apoptosis may contribute to pathophysiological processes involved in a number of prevalent diseases, including neurodegenerative diseases, diabetes, atherosclerosis and renal disease. The molecular mechanisms linking ER stress to apoptosis are the topic of this review, with emphases on relevance to pathophysiology and integration and complementation among the various apoptotic pathways induced by ER stress.     

Trichosanthin induced apoptosis in HL-60 cells via mitochondrial and endoplasmic reticulum stress signaling pathways

Trichosanthin (TCS), a traditional Chinese medicine, exerts antitumor activities by inducing apoptosis in many different tumor cell lines. However, the mechanisms remain obscure. The present study focused on various caspase pathways that may be involved in TCS-induced apoptosis in leukemia HL-60 cells. Key caspases in both intrinsic and extrinsic pathways including caspase-8, -9 and -3 were activated upon TCS treatment. Additionally, TCS treatment induced upregulation of BiP and CHOP and also activated caspase-4, which for the first time strongly supported the involvement of endoplasmic reticulum stress pathway in TCS-induced apoptosis. Interestingly, although caspase-8 was activated, Fas/Fas ligand pathway was not involved as evidenced by a lack of induction of Fas or Fas ligand and a lack of inhibitory effect of anti-Fas blocking antibody on TCS-induced apoptosis. Instead, caspase-8 was activated in a caspase-9 and -4 dependent manner. The involvement of mitochondria was demonstrated by the reduction of mitochondrial membrane potential and release of cytochrome c and Smac besides the activation of caspase-9. Further investigation confirmed that caspase-3 was the major executioner caspase downstream to caspase-9, -4 and -8. Taken together, our results suggested that TCS-induced apoptosis in HL-60 cells was mainly mediated by mitochondrial and ER stress signaling pathways via caspase-3.     

Calcium entry via TRPC6 mediates albumin overload-induced endoplasmic reticulum stress and apoptosis in podocytes

Albumin, which is the most abundant component of urine proteins, exerts injurious effects on renal cells in chronic kidney diseases. However, the toxicity of albumin to podocytes is not well elucidated. Here, we show that a high concentration of albumin triggers intracellular calcium ([Ca²⁺]_i) increase through mechanisms involving the intracellular calcium store release and extracellular calcium influx in conditionally immortalized podocytes. The canonical transient receptor potential-6 (TRPC6) channel, which is associated with a subset of familial forms of focal segmental glomerulosclerosis (FSGS) and several acquired proteinuric kidney diseases, was shown to be one of the important Ca²⁺ permeable ion channels in podocytes. Therefore we explored the role of TRPC6 on albumin-induced functional and structural changes in podocytes. It was found that albumin-induced increase in [Ca²⁺]_i was blocked by TRPC6 siRNA or SKF-96365, a blocker of TRP cation channels. Long-term albumin exposure caused an up-regulation of TRPC6 expression in podocytes, which was inhibited by TRPC6 siRNA. Additionally, the inhibition of TRPC6 prevented the F-actin cytoskeleton disruption that is induced by albumin overload. Moreover, albumin overload induced expression of the endoplasmic reticulum (ER) stress protein GRP78, led to caspase-12 activation and ultimately podocyte apoptosis, all of which were abolished by the knockdown of TRPC6 using TRPC6 siRNA. These results support the view that albumin overload may induce ER stress and the subsequent apoptosis in podocytes via TRPC6-mediated Ca²⁺ entry.     

Choline kinase inhibition induces exacerbated endoplasmic reticulum stress and triggers apoptosis via CHOP in cancer cells

Endoplasmic reticulum (ER) is a central organelle in eukaryotic cells that regulates protein synthesis and maturation. Perturbation of ER functions leads to ER stress, which has been previously associated with a broad variety of diseases. ER stress is generally regarded as compensatory, but prolonged ER stress has been involved in apoptosis induced by several cytotoxic agents. Choline kinase α (ChoKα), the first enzyme in the Kennedy pathway, is responsible for the generation of phosphorylcholine (PCho) that ultimately renders phosphatidylcholine. ChoKα overexpression and high PCho levels have been detected in several cancer types. Inhibition of ChoKα has demonstrated antiproliferative and antitumor properties; however, the mechanisms underlying these activities remain poorly understood. Here, we demonstrate that ChoKα inhibitors (ChoKIs), MN58b and RSM932A, induce cell death in cancer cells (T47D, MCF7, MDA-MB231, SW620 and H460), through the prolonged activation of ER stress response. Evidence of ChoKIs-induced ER stress includes enhanced production of glucose-regulated protein, 78 kDa (GRP78), protein disulfide isomerase, IRE1α, CHOP, CCAAT/enhancer-binding protein beta (C/EBPβ) and TRB3. Although partial reduction of ChoKα levels by small interfering RNA was not sufficient to increase the production of ER stress proteins, silencing of ChoKα levels also show a decrease in CHOP overproduction induced by ChoKIs, which suggests that ER stress induction is due to a change in ChoKα protein folding after binding to ChoKIs. Silencing of CHOP expression leads to a reduction in C/EBPβ, ATF3 and GRP78 protein levels and abrogates apoptosis in tumor cells after treatment with ChoKIs, suggesting that CHOP maintains ER stress responses and triggers the pro-apoptotic signal. Consistent with the differential effect of ChoKIs in cancer and primary cells previously described, ChoKIs only promoted a transient and moderated ER stress response in the non-tumorogenic cells MCF10A. In conclusion, pharmacological inhibition of ChoKα induces cancer cell death through a mechanism that involves the activation of exaggerated and persistent ER stress supported by CHOP overproduction.     

Palmitic acid induces human osteoblast-like Saos-2 cell apoptosis via endoplasmic reticulum stress and autophagy

Palmitic acid (PA) is the most common saturated long-chain fatty acid in food that causes cell apoptosis. However, little is known about the molecular mechanisms of PA toxicity. In this study, we explore the effects of PA on proliferation and apoptosis in human osteoblast-like Saos-2 cells and uncover the signaling pathways involved in the process. Our study showed that endoplasmic reticulum (ER) stress and autophagy are involved in PA-induced Saos-2 cell apoptosis. We found that PA inhibited the viability of Saos-2 cells in a dose- and time-dependent manner. At the same time, PA induced the expression of ER stress marker genes (glucose-regulated protein 78 (GRP78) and CCAAT/enhancer binding protein homologous protein (CHOP)), altered autophagy-related gene expression (microtubule-associated protein 1 light chain 3 (LC3), ATG5, p62, and Beclin), promoted apoptosis-related gene expression (Caspase 3 and BAX), and affected autophagic flux. Inhibiting ER stress with 4-PBA diminished the PA-induced cell apoptosis, activated autophagy, and increased the expression of Caspase 3 and BAX. Inhibiting autophagy with 3-MA attenuated the PA and ER stress-induced cell apoptosis and the apoptosis-related gene expression (Caspase 3 and BAX), but seemed to have no obvious effects on ER stress, although the CHOP expression was downregulated. Taken together, our results suggest that PA-induced Saos-2 cell apoptosis is activated via ER stress and autophagy, and the activation of autophagy depends on the ER stress during this process.     

黄芪注射液对阿霉素性心肌细胞凋亡、内质网应激与缝隙连接蛋白表达的影响

目的：研究黄芪注射液对阿霉素（ADR）所致心肌病大鼠心肌细胞凋亡、内质网应激与缝隙连接蛋白表达的影响。方法：36只Wistar雄性大鼠随机分为3组（n=12）：对照组、ADR组及黄芪注射液组。对照组腹腔注射0.9% Nacl （10 ml/kg体重）;ADR组腹腔注射ADR 2 mg/kg体重;黄芪注射液组在每次腹腔注射ADR 2 mg/kg体重的同时,注射黄芪注射液10 g/kg体重,每周注射1次,共注射3次。实验第7周末,3组大鼠行心脏彩超检测左室舒张末期内径、左室收缩末期内径及左室射血分数;处死大鼠后取左心室组织行HE、Masson、醋酸铀及柠檬酸铅染色,于光镜及透射电镜下观察心肌病理及超微结构改变;采用TUNEL法检测大鼠心肌细胞凋亡,用免疫组化技术检测大鼠心肌细胞缝隙连接蛋白Cx43及p-Cx43表达,采用real time PCR检测大鼠心肌细胞内质网应激伴侣蛋白Grp78,ATF-4及CHOP表达。结果：与对照组比较,ADR组大鼠LVEDD、LVESD增大,LVEF减少;心肌纤维排列紊乱,心肌纤维间质水肿,大量淋巴细胞浸润;线粒体肿胀、破坏,呈空泡样;心肌细胞凋亡数明显增多（P<0.01）;内质网应激相关蛋白Grp78、ATF-4及CHOP表达明显增高（P<0.01）;缝隙连接蛋白Cx43表达减少,而p-Cx43表达增多。与ADR组比较,黄芪注射液组大鼠LVEDD、LVESD减少,LVEF增加;心肌病理及超微结构明显改善,同时心肌细胞凋亡数明显减少（P<0.01）;内质网应激伴侣蛋白Grp78、ATF-4及CHOP表达明显减少（P<0.01）;缝隙连接蛋白Cx43表达增多,而p-Cx43减少。结论：黄芪注射液可有效改善阿霉素导致的心肌损伤,其机制可能与黄芪注射液抑制ADR诱导的内质网应激及缝隙连接蛋白磷酸化有关。     

Protein disulfide isomerase inhibits endoplasmic reticulum stress response and apoptosis via its oxidoreductase activity in colorectal cancer

Protein disulfide isomerase (PDI), a principal endoplasmic reticulum resident oxidoreductase chaperone, is known to play a role in malignancies. This study aims to explore the molecular mechanism by which PDI regulates endoplasmic reticulum stress and the apoptosis signaling pathway in colorectal cancer (CRC). We determined the expression of PDI in CRC tissues and adjacent normal tissues. Gain- and loss- of function assays were conducted to evaluate the effects of PDI on oxidative stress, endoplasmic reticulum stress, and apoptosis in CRC cells, as reflected by hydrogen peroxide (H₂O₂) level and the expression of related proteins. PDI protein expression was upregulated in CRC tissues. Small molecule inhibitor of PDI or PDI knockdown reduced CRC cell viability and induced apoptosis. Overexpression of wild-type PDI augmented the viability of CRC cells and inhibited endoplasmic reticulum stress response and apoptosis. Small molecule inhibitor of PDI or PDI knockdown increased intracellular H₂O₂ level and activated apoptosis signaling pathway, which could be reversed by wild-type PDI restoration. Moreover, the catalytic active site of C-terminal of PDI was found to be indispensable for the regulatory effects of PDI on H₂O₂ levels, apoptosis and cell viability in CRC cells. Collectively, PDI inhibits endoplasmic reticulum stress and apoptosis of CRC cells through its oxidoreductase activity, thereby promoting the malignancy of CRC.     

ERp16, an endoplasmic reticulum-resident thiol-disulfide oxidoreductase: biochemical properties and role in apoptosis induced by endoplasmic reticulum stress

We have characterized the properties and putative role of a mammalian thioredoxin-like protein, ERp16 (previously designated ERp18, ERp19, or hTLP19). The predicted amino acid sequence of the 172-residue human protein contains an NH(2)-terminal signal peptide, a thioredoxin-like domain with an active site motif (CGAC), and a COOH-terminal endoplasmic reticulum (ER) retention sequence (EDEL). Analyses indicated that the mature protein (comprising 146 residues) is generated by cleavage of the 26-residue signal peptide and is localized in the lumen of the ER. Biochemical experiments with the recombinant mature protein revealed it to be a thioldisulfide oxidoreductase. Its redox potential was about -165 mV; its active site cysteine residue Cys(66) was nucleophilic with a pK(a) value of approximately 6.6; it catalyzed the formation, reduction, and isomerization of disulfide bonds, with the unusual CGAC active site motif being responsible for these activities; and it existed as a dimer and underwent a redox-dependent conformational change. The observations that the redox potential of ERp16 (-165 mV) was within the range of that of the ER (-135 to -185 mV) and that ERp16 catalyzed disulfide isomerization of scrambled ribonuclease A suggest a role for ERp16 in protein disulfide isomerization in the ER. Expression of ERp16 in HeLa cells inhibited the induction of apoptosis by agents that elicit ER stress, including brefeldin A, tunicamycin, and dithiothreitol. In contrast, expression of a catalytically inactive mutant of ERp16 potentiated such apoptosis, as did depletion of ERp16 by RNA interference. Our results suggest that ERp16 mediates disulfide bond formation in the ER and plays an important role in cellular defense against prolonged ER stress.     

BRD7 mediates hyperglycaemia‐induced myocardial apoptosis via endoplasmic reticulum stress signalling pathway

Bromodomain‐containing protein 7 (BRD7) is a tumour suppressor that is known to regulate many pathological processes including cell growth, apoptosis and cell cycle. Endoplasmic reticulum (ER) stress‐induced apoptosis plays a key role in diabetic cardiomyopathy (DCM). However, the molecular mechanism of hyperglycaemia‐induced myocardial apoptosis is still unclear. We intended to determine the role of BRD7 in high glucose (HG)‐induced apoptosis of cardiomyocytes. In vivo, we established a type 1 diabetic rat model by injecting a high‐dose streptozotocin (STZ), and lentivirus‐mediated short hairpin RNA (shRNA) was used to inhibit BRD7 expression. Rats with DCM exhibited severe myocardial remodelling, fibrosis, left ventricular dysfunction and myocardial apoptosis. The expression of BRD7 was up‐regulated in the heart of diabetic rats, and inhibition of BRD7 had beneficial effects against diabetes‐induced heart damage. In vitro, H9c2 cardiomyoblasts was used to investigate the mechanism of BRD7 in HG‐induced apoptosis. Treating H9c2 cardiomyoblasts with HG elevated the level of BRD7 via activation of extracellular signal‐regulated kinase 1/2 (ERK1/2) and increased ER stress‐induced apoptosis by detecting spliced/active X‐box binding protein 1 (XBP‐1s) and C/EBP homologous protein (CHOP). Furthermore, down‐regulation of BRD7 attenuated HG‐induced expression of CHOP via inhibiting nuclear translocation of XBP‐1s without affecting the total expression of XBP‐1s. In conclusion, inhibition of BRD7 appeared to protect against hyperglycaemia‐induced cardiomyocyte apoptosis by inhibiting ER stress signalling pathway.