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.     

Cadmium induces cell centrosome amplification via reactive oxygen species as well as endoplasmic reticulum stress pathway

There is evidence that cadmium can initiate carcinogenesis. However, the underlying mechanisms remain unknown. There is also evidence that moderate centrosome amplification can initiate tumorigenesis. The present study investigated whether cadmium could trigger cell centrosome amplification, and examined the underlying molecular mechanisms. We found that cadmium was able to cause cell centrosome amplification at the subtoxic concentrations, in a dose-dependent manner. It could cause centrosome amplification via the signaling of reactive oxygen species (ROS). Proteomic analysis revealed that cadmium caused differential expressions of three proteins, which included HSPA1A which is associated with endoplasmic reticulum (ER) stress. Western blot analysis confirmed that cadmium upregulated HSPA1A. Further analyses showed that cadmium upregulated Bip and decreased the phosphorylation of ASK1 as well as increased the phosphorylation of MKK7 and c-Jun N-terminal kinases (JNK). Knockdown of JNK2 using small interfering RNA inhibited the cadmium-induced centrosome amplification but not the level of ROS. N-acetylcysteine did not inhibit the cadmium-activated ER stress pathway. In conclusion, our results suggest that cadmium can induce cell centrosome amplification via ROS as well as ER stress through the Bip–TRAF2–ASK1–MKK7–JNK signaling route, in parallel. More studies are required to clarify whether centrosome amplification underlies cadmium-induced carcinogenesis.     

Inactivated Sendai virus strain Tianjin induces apoptosis and autophagy through reactive oxygen species production in osteosarcoma MG-63 cells

Sendai virus strain Tianjin, a novel genotype of Sendai virus, has been proven to possess potent antitumor effect on certain cancer cell types although inactivated by ultraviolet (UV). This study was carried out to investigate the in vitro anticancer properties of UV-inactivated Sendai virus strain Tianjin (UV-Tianjin) on human osteosarcoma cells and the underlying molecular mechanism. Our studies demonstrated UV-Tianjin significantly inhibited the viability of human osteosarcoma cell lines and triggered apoptosis through activation of both extrinsic and intrinsic pathways in MG-63 cells. Meanwhile, autophagy occurred in UV-Tianjin-treated cells. Blockade of autophagy with 3-methyladenine remarkably attenuated the inhibition of cell proliferation by UV-Tianjin, suggesting that UV-Tianjin-induced autophagy may be contributing to cell death. Furthermore, UV-Tianjin induced reactive oxygen species (ROS) production, which was involved in the execution of MG-63 cell apoptosis and autophagy, as evidenced by the result that treatment of N-acetyl-L-cysteine, a ROS scavenger, attenuated both apoptosis and autophagy. In addition, inhibition of apoptosis promoted autophagy, whereas suppression of autophagy attenuated apoptosis. Our results suggest that UV-Tianjin triggers apoptosis and autophagic cell death via generation of the ROS in MG-63 cells, which might provide important insights into the effectiveness of novel strategies for osteosarcoma therapy.     

Mycobacterial HBHA induces endoplasmic reticulum stress-mediated apoptosis through the generation of reactive oxygen species and cytosolic Ca2+ in murine macrophage RAW 264.7 cells

Mycobacterial heparin-binding haemagglutinin antigen (HBHA) is a virulence factor that induces apoptosis of macrophages. Endoplasmic reticulum (ER) stress-mediated apoptosis is an important regulatory response that can be utilised to study the pathogenesis of tuberculosis. In the present study, HBHA stimulation induced ER stress sensor molecules in a caspase-dependent manner. Pre-treatment of RAW 264.7 cells with an IκB kinase 2 inhibitor reduced not only C/EBP homology protein expression but also IL-6 and monocyte chemotactic protein-1 (MCP-1) production. BAPTA-AM reduced both ER stress responses and caspase activation and strongly suppressed HBHA-induced IL-6 and MCP-1 production in RAW 264.7 cells. Enhanced reactive oxygen species (ROS) production and elevated cytosolic [Ca²⁺]_i levels were essential for HBHA-induced ER stress responses. Collectively, our data suggest that HBHA induces cytosolic [Ca²⁺]_i, which influences the generation of ROS associated with the production of proinflammatory cytokines. These concerted and complex cellular responses induce ER stress-associated apoptosis during HBHA stimulation in macrophages. These results indicate that the ER stress pathway has an important role in the HBHA-induced apoptosis during mycobacterial infection.     

FPTB, a novel CA-4 derivative, induces cell apoptosis of human chondrosarcoma cells through mitochondrial dysfunction and endoplasmic reticulum stress pathways

Chondrosarcoma is a malignant primary bone tumor that responds poorly to both chemotherapy and radiation therapy. The aim of this study was to elucidate the mechanism of the novel Combretastatin A-4 derivative, 2-(furanyl)-5-(pyrrolidinyl)-1-(3,4,5-trimethoxybenzyl)benzoimidazole (FPTB)-induced human chondrosarcoma cells apoptosis. FPTB induced cell apoptosis in human chondrosarcoma cell line but not primary chondrocytes. FPTB induced up-regulation of Bax and Bak, down-regulation of Bcl-2 and Bcl-XL and dysfunction of mitochondria in chondrosarcoma. FPTB also triggered endoplasmic reticulum (ER) stress, as indicated by changes in cytosol-calcium levels. We found that FPTB increased glucose-regulated proteins (GRP)78 but not GRP94 expression. In addition, treatment of cells with FPTB induced calpain expression and activity. Transfection of cells with GRP78 or calpain siRNA reduced FPTB-mediated cell apoptosis. Therefore, FPTB-induced apoptosis in chondrosarcoma cells through the mitochondria dysfunction and involves caspase-9 and caspase-3-mediated mechanism. FPTB also induced cell death mediated by increasing ER stress, GPR78 activation, and Ca(2+) release, which subsequently triggers calpain, caspase-12 and caspase-3 activity, resulting in apoptosis.     

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.     

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.     

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.     

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.     

Sarsasapogenin induces apoptosis via the reactive oxygen species-mediated mitochondrial pathway and ER stress pathway in HeLa cells

Sarsasapogenin is a sapogenin from the Chinese medical herb Anemarrhena asphodeloides Bunge. In the present study, we revealed that sarsasapogenin exhibited antitumor activity by inducing apoptosis in vitro as determined by Hoechst staining analysis and double staining of Annexin V-FITC/PI. In addition, cell cycle arrest in G2/M phase was observed in sarsasapogenin-treated HeLa cells. Moreover, the results revealed that perturbations in the mitochondrial membrane were associated with the deregulation of the Bax/Bcl-2 ratio which led to the upregulation of cytochrome c, followed by activation of caspases. Meanwhile, treatment of sarsasapogenin also activated Unfolded Protein Response (UPR) signaling pathways and these changes were accompanied by increased expression of CHOP. Salubrinal (Sal), a selective inhibitor of endoplasmic reticulum (ER) stress, partially abrogated the sarsasapogenin-related cell death. Furthermore, sarsasapogenin provoked the generation of reactive oxygen species, while the antioxidant N-acetyl cysteine (NAC) effectively blocked the activation of ER stress and apoptosis, suggesting that sarsasapogenin-induced reactive oxygen species is an early event that triggers ER stress mitochondrial apoptotic pathways. Taken together, the results demonstrate that sarsasapogenin exerts its antitumor activity through both reactive oxygen species (ROS)-mediate mitochondrial dysfunction and ER stress cell death.     

The novel phloroglucinol derivative BFP induces apoptosis of glioma cancer through reactive oxygen species and endoplasmic reticulum stress pathways

Prenyl-phloroglucinol derivatives from hop plants have been shown to have anticancer activities. This study is the first to investigate the anticancer effects of the new phloroglucinol derivative (2,4-bis(4-fluorophenylacetyl)phloroglucinol; BFP). BFP induced cell death and anti-proliferation in three glioma, U251, U87 and C6 cells, but not in primary human astrocytes. BFP-induced concentration-dependently cell death in glioma cells was determined by MTT and SRB assay. Moreover, BFP-induced apoptotic cell death in glioma cells was measured by Hochest 33258 staining and fluorescence-activated cell sorter (FACS) of propidine iodine (PI) analysis. Treatment of U251 human glioma cells with BFP was also found to induce reactive oxygen species (ROS) generation, which was detected by a fluorescence dye used FACS analysis. Treatment of BFP also increased a number of signature endoplasmic reticulum (ER) stress markers glucose-regulated protein (GRP)-78, GRP-94, IRE1, phosphorylation of eukaryotic initiation factor-2α (eIF-2α) and up-regulation of CAAT/enhancer-binding protein homologous protein (CHOP). Moreover, treatment of BFP also increased the down-stream caspase activation, such as pro-caspase-7 and pro-caspase-12 degradation, suggesting the induction of ER stress. Furthermore, BFP also induced caspase-9 and caspase-3 activation as well as up-regulation of cleaved PARP expression. Treatment of antioxidants, or pre-transfection of cells with GRP78 or CHOP siRNA reduced BFP-mediated apoptotic-related protein expression. Taken together, the present study provides evidences to support that ROS generation, GRP78 and CHOP activation are mediating the BFP-induced human glioma cell apoptosis.     

Silibinin-induced endoplasmic reticulum stress and mitochondrial dysfunction suppress growth of endometriotic lesions

Silibinin is a flavonolignan extracted from milk thistle, which has been used for treating liver disorders, various cancers, and gynecological diseases. However, attempts for treating endometriosis with silibinin are lacking. In this study, we observed that silibinin exerts antiproliferative and apoptotic effects on human endometriotic cell lines VK2/E6E7 and End1/E6E7. We also identified that silibinin-induced oxidative stress and lipid peroxidation in human endometriotic cells. Moreover, we observed upregulation of calcium concentration in the cytosol and mitochondrial matrix, which resulted in mitochondrial dysfunction. Furthermore, induction of endoplasmic reticulum stress signals with rapid mitogen-activated protein kinase (MAPK) pathway signaling resulted in apoptosis of both cells. Using an animal model mimicking the retrograde menstruation hypothesis, we verified the effects of silibinin on reducing endometriotic lesions by inhibiting the expression of inflammatory cytokines in mice. Silibinin might be used as a novel therapeutic agent or supplement for inhibiting progression of endometriosis in vitro and in vivo.     

Involvement of endoplasmic reticulum stress in Docetaxel-induced JNK-dependent apoptosis of human melanoma

Our previous studies revealed that Docetaxel-induced apoptosis of melanoma cells is entirely dependent on activation of the JNK signalling pathway. Here, we show that Docetaxel-induced apoptosis is mediated by induction of ER stress. This was shown by Docetaxel-induced activation of proteins involved in ER stress signalling namely GRP78, ATF6, IRE1α, and PERK/eIF2α. Knockdown of IRE1α by siRNA markedly inhibited Docetaxel-induced JNK activation and downstream targets of JNK indicating that activation of IRE1α was upstream of activation of the JNK. Co-immunoprecipitation experiments showed that activation of JNK is due to activation of ASK1 through formation of an IRE1α-TRAF2-ASK1 complex. ER stress mediated activation of the JNK pathway is downstream of activation of PKCδ in that downregulation of PKCδ expression using specific PKCδ siRNA significantly inhibited Docetaxel-induced activation of IRE1α and the JNK pathway. These findings provide new insights to understand the mode of action of taxanes in treatment of human melanoma.     

Spatiotemporal autophagic degradation of oxidatively damaged organelles after photodynamic stress is amplified by mitochondrial reactive oxygen species

Although reactive oxygen species (ROS) have been reported to evoke different autophagic pathways, how ROS or their secondary products modulate the selective clearance of oxidatively damaged organelles is less explored. To investigate the signaling role of ROS and the impact of their compartmentalization in autophagy pathways, we used murine fibrosarcoma L929 cells overexpressing different antioxidant enzymes targeted to the cytosol or mitochondria and subjected them to photodynamic (PD) stress with the endoplasmic reticulum (ER)-associated photosensitizer hypericin. We show that following apical ROS-mediated damage to the ER, predominantly cells overexpressing mitochondria-associated glutathione peroxidase 4 (GPX4) and manganese superoxide dismutase (SOD2) displayed attenuated kinetics of autophagosome formation and overall cell death, as detected by computerized time-lapse microscopy. Consistent with a primary ER photodamage, kinetics and colocalization studies revealed that photogenerated ROS induced an initial reticulophagy, followed by morphological changes in the mitochondrial network that preceded clearance of mitochondria by mitophagy. Overexpression of cytosolic and mitochondria-associated GPX4 retained the tubular mitochondrial network in response to PD stress and concomitantly blocked the progression toward mitophagy. Preventing the formation of phospholipid hydroperoxides and H₂O₂ in the cytosol as well as in the mitochondria significantly reduced cardiolipin peroxidation and apoptosis. All together, these results show that in response to apical ER photodamage ROS propagate to mitochondria, which in turn amplify ROS production, thereby contributing to two antagonizing processes, mitophagy and apoptosis.