Nitric oxide： orchestrating hypoxia regulation through mitochondrial respiration and the endoplasmic reticulum stress response

Mitochondria have long been considered to be the powerhouse of the living cell, generating energy in the form of the molecule ATP via the process of oxidative phosphorylation. In the past 20 years, it has been recognised that they also play an important role in the implementation of apoptosis, or programmed cell death. More recently it has become evident that mitochondria also participate in the orchestration of cellular defence responses. At physiological concentrations, the gaseous molecule nitric oxide (NO) inhibits the mitochondrial enzyme cytochrome c oxidase (complex Ⅳ) in competition with oxygen. This interaction underlies the mitochondrial actions of NO, which range from the physiological regulation of cell respiration, through mitochondrial signalling, to the development of “metabolic hypoxia”-a situation in which, although oxygen is available, the cell is unable to utilise it.     

Reactive oxygen species-mediated endoplasmic reticulum stress and mitochondrial dysfunction contribute to polydatin-induced apoptosis in human nasopharyngeal carcinoma CNE cells

Previous studies revealed that polydatin, a natural small compound, possessed protective effect against ischemia/reperfusion injury and inflammation. However, the action and molecular mechanism of its potent anti-cancer activity remain poorly understood. In the present study, polydatin significantly killed several human tumor cell lines in a dose- and time-dependent manner. The compound also dose-dependently caused mitochondrial apoptosis in human nasopharyngeal carcinoma CNE cells. In addition, polydatin triggered endoplasmic reticulum (ER) stress and down-regulated the phosphorylation of Akt in CNE cells, while knock-down of CCAAT/enhancer-binding protein homologous protein (CHOP) dramatically abrogated the inactivation of Akt and reversed the pro-apoptotic effect of polydatin. Furthermore, polydatin provoked the generation of reactive oxygen species in CNE cells, while the antioxidant N-acetyl cysteine almost completely blocked the activation of ER stress and apoptosis, suggesting polydatin-induced reactive oxygen species is an early event that triggers ER stress mitochondrial apoptotic pathways in CNE cells. Taken together, these findings strongly suggest that polydatin might be a promising anti-tumor drug and our data provide the molecular theoretical basis for clinical application of polydatin.     

Methylglyoxal induces cell death through endoplasmic reticulum stress‐associated ROS production and mitochondrial dysfunction

Diabetic retinopathy (DR) and age‐related macular degeneration (AMD) are two important leading causes of acquired blindness in developed countries. As accumulation of advanced glycation end products (AGEs) in retinal pigment epithelial (RPE) cells plays an important role in both DR and AMD, and the methylglyoxal (MGO) within the AGEs exerts irreversible effects on protein structure and function, it is crucial to understand the underlying mechanism of MGO‐induced RPE cell death. Using ARPE‐19 as the cell model, this study revealed that MGO induces RPE cell death through a caspase‐independent manner, which relying on reactive oxygen species (ROS) formation, mitochondrial membrane potential (MMP) loss, intracellular calcium elevation and endoplasmic reticulum (ER) stress response. Suppression of ROS generation can reverse the MGO‐induced ROS production, MMP loss, intracellular calcium increase and cell death. Moreover, store‐operated calcium channel inhibitors MRS1845 and YM‐58483, but not the inositol 1,4,5‐trisphosphate (IP3) receptor inhibitor xestospongin C, can block MGO‐induced ROS production, MMP loss and sustained intracellular calcium increase in ARPE‐19 cells. Lastly, inhibition of ER stress by salubrinal and 4‐PBA can reduce the MGO‐induced intracellular events and cell death. Therefore, our data indicate that MGO can decrease RPE cell viability, resulting from the ER stress‐dependent intracellular ROS production, MMP loss and increased intracellular calcium increase. As MGO is one of the components of drusen in AMD and is the AGEs adduct in DR, this study could provide a valuable insight into the molecular pathogenesis and therapeutic intervention of AMD and DR.     

Salinomycin induces cell death with autophagy through activation of endoplasmic reticulum stress in human cancer cells

Salinomycin is perhaps the first promising compound that was discovered through high throughput screening in cancer stem cells. This novel agent can selectively eliminate breast and other cancer stem cells, though the mechanism of action remains unclear. In this study, we found that salinomycin induced autophagy in human non-small cell lung cancer (NSCLC) cells. Furthermore, we demonstrated that salinomycin stimulated endoplasmic reticulum stress and mediated autophagy via the ATF4-DDIT3/CHOP-TRIB3-AKT1-MTOR axis. Moreover, we found that the autophagy induced by salinomycin played a prosurvival role in human NSCLC cells and attenuated the apoptotic cascade. We also showed that salinomycin triggered more apoptosis and less autophagy in A549 cells in which CDH1 expression was inhibited, suggesting that the inhibition of autophagy might represent a promising strategy to target cancer stem cells. In conclusion, these findings provide evidence that combination treatment with salinomycin and pharmacological autophagy inhibitors will be an effective therapeutic strategy for eliminating cancer cells as well as cancer stem cells.     

内质网应激偶联炎症反应与慢性病发病机制

内质网是合成细胞内分泌蛋白和膜蛋白并进行蛋白折叠的主要细胞器。新近研究证明,当内质网蛋白质合成与折叠的负担增加、非折叠或错误折叠蛋白质堆积,可激活内质网的几组特定信号转导通路,将这些应激信号传递到细胞浆和细胞核,引起未/错误折叠蛋白反应。这对维持细胞动态平衡和生物体的发育具有重要意义。更为重要的是,未/错误折叠蛋白反应能够与细胞内炎症反应信号转导通路偶联,是非感染性致病原引发炎症反应的主要原因。因此,内质网应激-未/错误折叠蛋白反应-炎症反应在特定的细胞发生偶联是许多炎症疾病的发病机制。本文综述该领域的研究进展,并介绍了内质网应激信号和炎症反应偶联参与一些慢性病发病的分子细胞机制。这些研究不仅加深人们对这些慢性病发病机制的了解,也有助于对调节内质网应激-炎症反应的药物的研发。     

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.     

Sodium selenite induces apoptosis by ROS-mediated endoplasmic reticulum stress and mitochondrial dysfunction in human acute promyelocytic leukemia NB4 cells

Introduction  In this study, we delineated the apoptotic signaling pathways activated by sodium selenite in NB4 cells. Materials and methods  NB4 cells were treated with 20 μM sodium selenite for different times. The activation of caspases and ER stress markers, ROS levels, mitochondrial membrane potential and cell apoptosis induced by sodium selenite were analyzed by immunoblotting analysis, DCF fluorescence and flow cytometric respectively. siRNA was used to detect the effect of GADD153 on selenite-induced cell apoptosis. Conclusions  Sodium selenite-induced reactive oxygen species generation is an early event that triggers endoplasmic reticulum stress mitochondrial apoptotic pathways in NB4 cells.     

IGL-1 solution reduces endoplasmic reticulum stress and apoptosis in rat liver transplantation

Injury due to cold ischemia reperfusion (I/R) is a major cause of primary graft non-function following liver transplantation. We postulated that I/R-induced cellular damage during liver transplantation might affect the secretory pathway, particularly at the endoplasmic reticulum (ER). We examined the involvement of ER stress in organ preservation, and compared cold storage in University of Wisconsin (UW) solution and in Institute Georges Lopez-1 (IGL-1) solution. In one group of rats, livers were preserved in UW solution for 8 h at 4 °C, and then orthotopic liver transplantation was performed according to Kamada''s cuff technique. In another group, livers were preserved in IGL-1 solution. The effect of each preservation solution on the induction of ER stress, hepatic injury, mitochondrial damage and cell death was evaluated. As expected, we found increased ER stress after liver transplantation. IGL-1 solution significantly attenuated ER damage by reducing the activation of three pathways of unfolded protein response and their effector molecules caspase-12, C/EBP homologous protein-10, X-box-binding protein 1, tumor necrosis factor-associated factor 2 and eukaryotic translation initiation factor 2. This attenuation of ER stress was associated with a reduction in hepatic injury and cell death. Our results show that IGL-1 solution may be a useful means to circumvent excessive ER stress reactions associated with liver transplantation, and may optimize graft quality.     

Interferon-gamma induces reactive oxygen species and endoplasmic reticulum stress at the hepatic apoptosis

Interferon-gamma (IFN-gamma) induces cell-cycle arrest and p53-independent apoptosis in primary cultured hepatocytes. However, the detailed mechanism, including regulating molecules, is still unclear. In this study, we found that IFN-gamma induced generation of reactive oxygen species (ROS) in primary hepatocytes and that pyrrolidinedithiocarbamate (PDTC), an anti-oxidant reagent, completely suppressed IFN-gamma-induced hepatic apoptosis. PDTC blocked apoptosis downstream from IRF-1 and upstream from caspase activation, suggesting that the generation of ROS occurred between these stages. However, IFN-gamma also induced the generation of ROS in IRF-1-deficient hepatocytes, cells insensitive to IFN-gamma-induced apoptosis. Moreover, a general cyclooxygenase (COX) inhibitor, indomethacin (but not the cyclooxygenase 2-specific inhibitor, NS-398) also inhibited the apoptosis without blocking the generation of ROS. Both PDTC and indomethacin also blocked IFN-gamma-induced release of cytochrome c from mitochondria. These results suggest that ROS are not the only or sufficient mediators of IFN-gamma-induced hepatic apoptosis. In contrast, we also found that IFN-gamma induced endoplasmic reticulum (ER) stress proteins, CHOP/GADD153 and caspase 12, in wild-type primary hepatocytes, but induced only caspase 12 and not CHOP/GADD153 protein in IRF-1-deficient hepatocytes. These results suggest that IFN-gamma induces ER stress in primary hepatocytes. Both the ROS and ER stress induced by IFN-gamma may be complementary mediators that induce apoptosis in primary hepatocytes.     

疱疹病毒与内质网应激

内质网是分泌型蛋白和膜蛋白折叠及翻译后修饰的主要场所.病毒感染所引起的宿主细胞内环境的改变可使细胞或病毒的未折叠和/或错误折叠蛋白在内质网中大量聚集,使内质网处于生理功能紊乱的应激状态.为了缓解这种应激压力,细胞会启动未折叠蛋白反应(UPR),并通过一系列分子的信号转导维持内质网稳态;同时病毒也会通过对UPR的精密调控...     

Structural and functional link between the mitochondrial network and the endoplasmic reticulum

Mitochondrial and endoplasmic reticulum (ER) networks are fundamental for the maintenance of cellular homeostasis and for determination of cell fate under stress conditions. Recent structural and functional studies revealed the interaction of these networks. These zones of close contact between ER and mitochondria called MAM (mitochondria associated membranes) support communication between the two organelles including bioenergetics and cell survival. The existence of macromolecular complexes in these contact sites has also been revealed. In this contribution, we will review: (i) the ER and mitochondria structure and their dynamics, (ii) the basic principles of ER mitochondrial Ca²⁺ transport, (iii) the physiological/pathological role of this cross-talk.     

Shikonin selectively induces apoptosis in human prostate cancer cells through the endoplasmic reticulum stress and mitochondrial apoptotic pathway

Background

Despite the recent progress in screening and therapy, a majority of prostate cancer cases eventually attain hormone refractory and chemo-resistant attributes. Conventional chemotherapeutic strategies are effective at very high doses for only palliative management of these prostate cancers. Therefore chemo-sensitization of prostate cancer cells could be a promising strategy for increasing efficacy of the conventional chemotherapeutic agents in prostate cancer patients. Recent studies have indicated that the chemo-preventive natural agents restore the pro-apoptotic protein expression and induce endoplasmic reticulum stress (ER stress) leading to the inhibition of cellular proliferation and activation of the mitochondrial apoptosis in prostate cancer cells. Therefore reprogramming ER stress-mitochondrial dependent apoptosis could be a potential approach for management of hormone refractory chemoresistant prostate cancers. We aimed to study the effects of the natural naphthoquinone Shikonin in human prostate cancer cells.

Results

The results indicated that Shikonin induces apoptosis in prostate cancer cells through the dual induction of the endoplasmic reticulum stress and mitochondrial dysfunction. Shikonin induced ROS generation and activated ER stress and calpain activity. Moreover, addition of antioxidants attenuated these effects. Shikonin also induced the mitochondrial apoptotic pathway mediated through the enhanced expression of the pro-apoptotic Bax and inhibition of Bcl-2, disruption of the mitochondrial membrane potential (MMP) followed by the activation of caspase-9, caspase-3, and PARP cleavage.

Conclusion

The results suggest that shikonin could be useful in the therapeutic management of hormone refractory prostate cancers due to its modulation of the pro-apoptotic ER stress and mitochondrial apoptotic pathways.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-015-0127-1) contains supplementary material, which is available to authorized users.     

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.     

氧化应激对原代培养乳鼠心房肌细胞中内质网应激及凋亡因子的影响

目的：研究氧化应激对原代培养乳鼠心房肌细胞凋亡、内质网应激及凋亡因子的影响。方法：实验分2组：对照组、氧化应激组。原代培养乳鼠心房肌细胞,氧化应激组在培养的原代心房肌细胞中加入终浓度为100μmol/L的H₂O₂培养2 h,检测氧化和抗氧化指标超氧化物歧化酶（SOD）活力、丙二醛（MDA）及还原型谷胱甘肽（GSH）含量;检测细胞凋亡、细胞GRP78、GRP94及chop、bax、bcl-2 mRNA表达。结果：与对照组相比较,氧化应激组心房肌细胞SOD活力和GSH含量下降、MDA含量增加（P < 0.01）,细胞凋亡增加（P < 0.01）,细胞GRP78、GRP94、chop、bax mRNA表达增加、bcl-2 mRNA表达减少（P < 0.01）。结论：氧化应激反应可能介导内质网应激反应并激活促凋亡因子表达,抑制抗凋亡因子表达,引起心房肌细胞凋亡增加。这可能与心房纤颤的发生有一定关联性。     

Methylseleninic acid induces apoptosis of human bladder cancer cells through the ROS-mediated mitochondrial pathway

As the most common selenium derivative, methylseleninic acid (MSA) has attracted wide attention. Its apoptotic induction ability and the possible molecular mechanism in human bladder cancer (BC) J82 and T24 cells were investigated in the present study. We found that the survival of J82 and T24 cells were inhibited in a dose-dependent manner after MSA treatment. Propidium iodide (PI) staining and Annexin V-fluorescein isothiocyanate/PI double staining clarified that MSA stocked cells at G₂/M phase and caused apoptosis in J82 and T24 cells. Further, typical morphological features of apoptotic cells were also observed. Accumulation of reactive oxygen species (ROS) and loss of mitochondrial membrane potential were also detected by dichlorodihydrofluorescein diacetate and Rhodamin123 staining. Meanwhile, pretreatment with N-acetylcysteine, an ROS scavenging agent, found that the apoptosis of BC cells induced by MSA was related to the production of ROS. Western blot analysis results showed that MSA interrupted Bax/Bcl-2 balance, stimulated cytochrome c release into the cytoplasm, activated caspase-9 and caspase-3, and finally induced the apoptosis of the BC cells. These findings demonstrated that MSA was able to induce apoptosis in J82 and T24 cells through ROS-mediated mitochondrial apoptosis.