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.     

Apoptosis induced by all-trans retinoic acid in N-acetylglucosaminyltransferase V repressed human hepatocarcinoma cells is mediated through endoplasmic reticulum stress

We previously demonstrated that endoplasmic reticulum (ER) stress was triggered in human hepatocarcinoma 7721 cells transfected with antisense cDNA of N-acetylglucosaminyltransferase V (GnT-V-AS/7721) which were more susceptible to apoptosis induced by all-trans retinoic acid (ATRA). In the present study, we report that ATRA-induced apoptosis in GnT-V-AS/7721 cells is mediated through ER stress. We show here that ER stress is enhanced in GnT-V-AS/7721 cells with 80 microM ATRA treatment for 24 h, which is evidenced by the increase of GRP78/Bip, C/EBP-homologous protein-10 (CHOP, also known as GADD153) and spliced XBP1. Additionally, activation of caspase-12, caspase-9, and -3 was detected, and apoptosis morphology was observed in GnT-V-AS/7721 cells with ATRA treatment. These results suggest that ATRA enhances the ER stress triggered in GnT-V-AS/7721 cells, which represents a novel mechanism of ATRA to induce apoptosis. We further observed that GnT-V was significantly repressed and the structure of N-glycans was changed in GnT-V-AS/7721 cells with 80 microM ATRA treatment for 24 h, suggesting that repression of GnT-V by ATRA causes the enhanced ER stress and ER stress-mediated apoptosis in GnT-V-AS/7721 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.     

Endoplasmic reticulum stress,autophagic and apoptotic cell death,and immune activation by a natural triterpenoid in human prostate cancer cells

Though the current therapies are effective at clearing an early stage prostate cancer, they often fail to treat late-stage metastatic disease. We aimed to investigate the molecular mechanisms underlying the anticancer effects of a natural triterpenoid, ganoderic acid DM (GA-DM), on two human prostate cancer cell lines: the androgen-independent prostate carcinoma (PC-3), and androgen-sensitive prostate adenocarcinoma (LNCaP). Cell viability assay showed that GA-DM was relatively more toxic to LNCaP cells than to PC-3 cells (IC₅₀s ranged 45-55 µM for PC-3, and 20-25 µM for LNCaP), which may have occurred due to differential expression of p53. Hoechst DNA staining confirmed detectable nuclear fragmentation in both cell lines irrespective of the p53 status. GA-DM treatment decreased Bcl-2 proteins while it upregulated apoptotic Bax and autophagic Beclin-1, Atg5, and LC-3 molecules, and caused an induction of both early and late events of apoptotic cell death. Biochemical analyses of GA-DM-treated prostate cancer cells demonstrated that caspase-3 cleavage was notable in GA-DM-treated PC-3 cells. Interestingly, GA-DM treatment altered cell cycle progression in the S phase with a significant growth arrest in the G2 checkpoint and enhanced CD4 ⁺ T cell recognition of prostate tumor cells. Mechanistic study of GA-DM-treated prostate cancer cells further demonstrated that calpain activation and endoplasmic reticulum stress contributed to cell death. These findings suggest that GA-DM is a candidate for future drug design for prostate cancer as it activates multiple pathways of cell death and immune recognition.     

Icariside II enhances cisplatin-induced apoptosis by promoting endoplasmic reticulum stress signalling in non-small cell lung cancer cells

Although cisplatin is the most effective first-line drug in the management of advanced non-small cell lung cancer (NSCLC), drug resistance remains a major clinical challenge. There is increasing evidence that icariside II (IS) exhibits antitumour activity in a variety of cancers. In the current study, we investigated the anticancer effects of icariside II combined with cisplatin and elucidated the underlying mechanism in NSCLC. Here, we showed that cotreatment with IS and cisplatin inhibited cell proliferation and induced cellular apoptosis. Using mRNA sequencing (mRNA-seq), we identified differentially expressed genes (DEGs) in which there was an enrichment in PERK-mediated unfolded protein response (UPR) signalling. The western blot results revealed that IS activated endoplasmic reticulum (ER) stress, including three branches of UPR signalling, PERK, IRE1 and ATF6, and the downstream PERK-eIF2α-ATF4-CHOP pathway, thus potentiating the apoptosis induced by cisplatin. In addition, the combination of IS with cisplatin significantly reduced xenograft tumour growth in C57BL/6 and BALB/c nude mice in vivo. Notably, the combination therapy displayed no evident toxicity. Taken together, IS enhances cisplatin-induced apoptosis partially by promoting ER stress signalling in NSCLC, suggesting that combination treatment with IS and cisplatin is a novel potential therapeutic strategy for NSCLC.     

Reticulon-1C acts as a molecular switch between endoplasmic reticulum stress and genotoxic cell death pathway in human neuroblastoma cells

Damage or stress in many organelles may trigger apoptosis by several not yet fully elucidated mechanisms. A cell death pathway is induced by endoplasmic reticulum (ER) stress elicited by the unfolded protein response and/or by aberrant Ca(2+) signalling. Reticulon-1C (RTN-1C) belongs to the reticulon family, neuroendocrine-specific proteins localized primarily on the ER membrane. In the present study, we demonstrate that RTN-1C is able to modulate, in a mutually exclusive way, the cellular sensitivity to different apoptosis pathways in human neuroblastoma cells. In fact, the increase of RTN-1C protein levels per se results in ER stress-induced cell death, mediated by an increase of cytosolic Ca(2+), and significantly sensitizes cells to different ER stress inducers. In line with these findings, the reduction of RTN-1C, by antisense DNA expression, reduced the sensitivity to ER-stressors. In the presence of high RTN-1C levels, genotoxic drugs become ineffective as a consequence of the cytoplasm translocation of p53 protein, while the silencing of endogenous RTN-1C results in the potentiation of the genotoxic drugs action. These data indicate that RTN-1C is able to modulate the cellular sensitivity to different apoptotic pathways representing a promising molecular target for new drug development.     

Chrysin leads to cell death in endometriosis by regulation of endoplasmic reticulum stress and cytosolic calcium level

Chrysin is a natural compound derived from honey, propolis, or passion flowers and has many functional roles, such as antiinflammatory and antiangiogenesis effects. Although endometriosis is a benign gynecological disease, there is a need to identify the pathology and develop a therapy for endometriosis. Elucidating the biological mechanism of chrysin on endometriosis will improve the understanding of endometriosis. In this study, we confirmed the apoptotic effects of chrysin in human endometriotic cells using End1/E6E7 (endocervix-derived endometriotic cells) and VK2/E6E7 (vaginal mucosa-derived epithelial endometriotic cells). The results showed that chrysin suppressed the proliferation of endometriosis and induced programmed cell death through changing the cell cycle proportion and increasing the cytosolic calcium level and generation of reactive oxygen species. In addition, chrysin activated endoplasmic reticulum (ER) stress by stimulating the unfolded protein response proteins, especially the 78-kDa glucose-regulated protein–PRKR-like ER kinase (PERK)–eukaryotic translation initiation factor 2α (eIF2α) pathway in both endometriotic cell lines. Furthermore, chrysin inactivated the intracellular phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB, also known as AKT) signaling pathway in a dose-dependent manner. Collectively, the results of this study indicated that chrysin induced programmed cell death by activating the ER stress response and inactivating the PI3K signaling pathways in human endometriotic cells.     

Involvement of mitochondria in endoplasmic reticulum stress-induced apoptotic cell death pathway triggered by the prion peptide PrP(106-126)

Prion disorders are progressive neurodegenerative diseases characterized by extensive neuronal loss and by the accumulation of the pathogenic form of prion protein, designated PrP^Sc. Recently, we have shown that PrP_106–126 induces endoplasmic reticulum (ER) stress, leading to mitochondrial cytochrome c release, caspase 3 activation and apoptotic death. In order to further clarify the role of mitochondria in ER stress-mediated apoptotic pathway triggered by the PrP peptide, we investigated the effects of PrP_106–126 on the Ntera2 human teratocarcinoma cell line that had been depleted of their mitochondrial DNA, termed NT2 ρ0 cells, characterized by the absence of functional mitochondria, as well as on the parental NT2 ρ+ cells. In this study, we show that PrP_106–126 induces ER stress in both cell lines, given that ER Ca²⁺ content is low, glucose-regulated protein 78 levels are increased and caspase 4 is activated. Furthermore, in parental NT2 ρ+ cells, PrP_106–126-activated caspase 9 and 3, induced poly (ADP-ribose) polymerase cleavage and increased the number of apoptotic cells. Dantrolene was shown to protect NT2 ρ+ from PrP_106–126-induced cell death, demonstrating the involvement of Ca²⁺ release through ER ryanodine receptors. However, in PrP_106–126-treated NT2 ρ0 cells, apoptosis was not able to proceed. These results demonstrate that functional mitochondria are required for cell death as a result of ER stress triggered by the PrP peptide, and further elucidate the molecular mechanisms involved in the neuronal loss that occurs in prion disorders.     

PON3 is upregulated in cancer tissues and protects against mitochondrial superoxide-mediated cell death

To achieve malignancy, cancer cells convert numerous signaling pathways, with evasion from cell death being a characteristic hallmark. The cell death machinery represents an anti-cancer target demanding constant identification of tumor-specific signaling molecules. Control of mitochondrial radical formation, particularly superoxide interconnects cell death signals with appropriate mechanistic execution. Superoxide is potentially damaging, but also triggers mitochondrial cytochrome c release. While paraoxonase (PON) enzymes are known to protect against cardiovascular diseases, recent data revealed that PON2 attenuated mitochondrial radical formation and execution of cell death. Another family member, PON3, is poorly investigated. Using various cell culture systems and knockout mice, here we addressed its potential role in cancer. PON3 is found overexpressed in various human tumors and diminishes mitochondrial superoxide formation. It directly interacts with coenzyme Q10 and presumably acts by sequestering ubisemiquinone, leading to enhanced cell death resistance. Localized to the endoplasmic reticulum (ER) and mitochondria, PON3 abrogates apoptosis in response to DNA damage or intrinsic but not extrinsic stimulation. Moreover, PON3 impaired ER stress-induced apoptotic MAPK signaling and CHOP induction. Therefore, our study reveals the mechanism underlying PON3's anti-oxidative effect and demonstrates a previously unanticipated function in tumor cell development. We suggest PONs represent a novel class of enzymes crucially controlling mitochondrial radical generation and cell death.     

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.     

Suppressive effects of 4-phenylbutyrate on the aggregation of Pael receptors and endoplasmic reticulum stress

Endoplasmic reticulum (ER) stress is defined as an accumulation of unfolded proteins in the endoplasmic reticulum. 4-phenylbutyrate (4-PBA) has been demonstrated to promote the normal trafficking of the DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) mutant from the ER to the plasma membrane and to restore activity. We have reported that 4-PBA protected against cerebral ischemic injury and ER stress-induced neuronal cell death. In this study, we revealed that 4-PBA possesses chemical chaperone activity in vitro, which prevents the aggregation of denatured alpha-lactalbumin and bovine serum albumin (BSA). Furthermore, we investigated the effects of 4-PBA on the accumulation of Parkin-associated endothelin receptor-like receptor (Pael-R) pathologically relevant to the loss of dopaminergic neurons in autosomal recessive juvenile parkinsonism (AR-JP). Interestingly, 4-PBA restored the normal expression of Pael-R protein and suppressed ER stress induced by the overexpression of Pael-R. In addition, we showed that 4-PBA attenuated the activation of ER stress-induced signal transduction pathways and subsequent neuronal cell death. Moreover, 4-PBA restored the viability of yeasts that fail to induce an ER stress response under ER stress conditions. These results suggest that 4-PBA suppresses ER stress by directly reducing the amount of misfolded protein, including Pael-R accumulated in the ER.     

Inhibition of cardiac oxidative and endoplasmic reticulum stress-mediated apoptosis by curcumin treatment contributes to protection against acute myocarditis

Abstract

Curcumin is used anecdotally as an herb in traditional Indian and Chinese medicine. In the present study, the effects and possible mechanism of curcumin in experimental autoimmune myocarditis (EAM) rats were further investigated. They were divided randomly into a treatment and vehicle group, and orally administrated curcumin (50 mg/kg/day) and 1% gum arabic, respectively, for 3 weeks after myosin injection. The results showed that curcumin significantly suppressed the myocardial protein expression of inducible nitric oxide synthase (iNOS) and the catalytic subunit of nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase. In addition, curcumin significantly decreased myocardial endoplasmic reticulum (ER) stress signaling proteins and improved cardiac function. Furthermore, curcumin significantly decreased the key regulators or inducers of apoptosis. In summary, our results indicate that curcumin has the potential to protect EAM by modulating cardiac oxidative and ER stress-mediated apoptosis, and provides a novel therapeutic strategy for autoimmune myocarditis.     

Coupling endoplasmic reticulum stress to the cell death program in mouse melanoma cells: effect of curcumin

The microenvironment of cancerous cells includes endoplasmic reticulum (ER) stress the resistance to which is required for the survival and growth of tumors. Acute ER stress triggers the induction of a family of ER stress proteins that promotes survival and/or growth of the cancer cells, and also confers resistance to radiation and chemotherapy. Prolonged or severe ER stress, however, may ultimately overwhelm the cellular protective mechanisms, triggering cell death through specific programmed cell death (pcd) pathways. Thus, downregulation of the protective stress proteins may offer a new therapeutic approach to cancer treatment. In this regard, recent reports have demonstrated the roles of the phytochemical curcumin in the inhibition of proteasomal activity and triggering the accumulation of cytosolic Ca²⁺ by inhibiting the Ca²⁺-ATPase pump, both of which enhance ER stress. Using a mouse melanoma cell line, we investigated the possibility that curcumin may trigger ER stress leading to programmed cell death. Our studies demonstrate that curcumin triggers ER stress and the activation of specific cell death pathways that feature caspase cleavage and activation, p23 cleavage, and downregulation of the anti-apoptotic Mcl-1 protein.     

Pro‐apoptotic effect of haem oxygenase‐1 in human colorectal carcinoma cells via endoplasmic reticular stress

Several biological effects of haem oxygenase (HO)‐1, including anti‐inflammatory, antiapoptotic and antioxidative properties were reported; however, the role of HO‐1 in apoptosis is still unclear. In the presence of stimulation by cobalt protoporphyrin (CoPP), an HO‐1 inducer, apoptotic characteristics were observed, including DNA laddering, hypodiploid cells, and cleavages of caspase (Casp)‐3 and poly(ADP) ribose polymerase (PARP) proteins in human colon carcinoma COLO205, HCT‐15, LOVO and HT‐29 cells in serum‐free (SF) conditions with increased HO‐1, but not heat shock protein 70 (HSP70) or HSP90. The addition of 10% foetal bovine serum (FBS) or 1% bovine serum albumin accordingly inhibited CoPP‐induced apoptosis and HO‐1 protein expression in human colon cancer cells. CoPP‐induced apoptosis of colon cancer cells was prevented by the addition of the pan‐caspase inhibitor, Z‐VAD‐FMK (VAD), and the Casp‐3 inhibitor, Z‐DEVD‐FMK (DEVD). N‐Acetyl cysteine inhibited reactive oxygen species‐generated H₂O₂‐induced cell death with reduced intracellular peroxide production, but did not affect CoPP‐induced apoptosis in human colorectal carcinoma (CRC) cells. Two CoPP analogs, ferric protoporphyrin and tin protoporphyrin, did not affect the viability of human CRC cells or HO‐1 expression by those cells, and knockdown of HO‐1 protein expression by HO‐1 small interfering (si)RNA reversed the cytotoxic effect elicited by CoPP. Furthermore, the carbon monoxide (CO) donor, CORM, but not FeSO₄ or biliverdin, induced DNA ladders, and cleavage of Casp‐3 and PARP proteins in human CRC cells. Increased phosphorylated levels of the endoplasmic reticular (ER) stress proteins, protein kinase R‐like ER kinase (PERK), and eukaryotic initiation factor 2α (eIF2α) by CORM and CoPP were identified, and the addition of the PERK inhibitor, GSK2606414, inhibited CORM‐ and CoPP‐induced apoptosis. Increased GRP78 level and formation of the HO‐1/GRP78 complex were detected in CORM‐ and CoPP‐treated human CRC cells. A pro‐apoptotic role of HO‐1 against the viability of human CRC cells via induction of CO and ER stress was firstly demonstrated herein.     

Changes in endoplasmic reticulum stress proteins and aldolase A in cells exposed to dopamine

In Parkinson's disease, oxidative stress is implicated in protein misfolding and aggregation, which may activate the unfolded protein response by the endoplasmic reticulum (ER). Dopamine (DA) can initiate oxidative stress via H₂O₂ formation by DA metabolism and by oxidation into DA quinone. We have previously shown that DA quinone induces oxidative protein modification, mitochondrial dysfunction in vitro, and dopaminergic cell toxicity in vivo and in vitro . In this study, we used cysteine- and lysine-reactive fluorescent dyes with 2D difference in-gel electrophoresis, mass spectrometry, and peptide mass fingerprint analysis to identify proteins in PC12 cell mitochondrial-enriched fractions that were altered in abundance following DA exposure (150 μM, 16 h). Quantitative changes in proteins labeled with fluorescent dyes indicated increases in a subset of proteins after DA exposure: calreticulin, ERp29, ERp99, Grp58, Grp78, Grp94 and Orp150 (149–260%), and decreased levels of aldolase A (39–42%). Changes in levels of several proteins detected by 2D difference in-gel electrophoresis were confirmed by western blot. Using this unbiased proteomics approach, our findings demonstrated that in PC12 cells, DA exposure leads to a cellular response indicative of ER stress prior to the onset of cell death, providing a potential link between DA and the unfolded protein response in the pathogenesis of Parkinson's disease.