Ouabain activates signaling pathways associated with cell death in human neuroblastoma

Cardiotonic steroids (CTS) like ouabain are not only specific inhibitors of the sodium pump (Na⁺,K⁺-ATPase), they also can influence various cytosolic signaling events in a hormone-like manner. In the neuroblastoma cell line SH-SY5Y ouabain triggers multiple signaling pathways. Within 30 min of incubation with 1 or 10 μM ouabain, SH-SY5Y cells generate reactive oxygen species to a level approximately 50% above control and show a modest but significant elevation in cytosolic [Ca²⁺] of about 25%. After 6 h of exposure, ouabain stimulates a series of anti-apoptotic actions in SH-SY5Y cells, including concentration-dependent phosphorylation of Erk1/2, Akt, and Bad. Nevertheless, at the same time this CTS also induces a series of events that inhibit retinoic acid-induced neuritogenesis and promote cell death. Both of these latter phenomena are possibly associated with the observed ouabain-induced reduction in the abundance of the anti-apoptotic proteins Bcl-XL and Bcl-2. In addition, ouabain treatment results in cytochrome c release into the cytosol and induces activation of caspase 3, events that point towards the stimulation of apoptotic pathways that are probably enhanced by the stimulation of p53 phosphorylation at Ser15 also observed in this study. These pathways may eventually lead to cell death: treatment with 10 nM ouabain results in a 20% decrease in cell number after 4 days of incubation and treatment with 1 μM ouabain decreases cells number by about 75%. The results obtained here emphasize the importance of further research in order to elucidate the various signalling cascades triggered by ouabain and possibly other CTS that are used in the treatment of heart failure and to identify their primary receptor(s).     

Ouabain activates signaling pathways associated with cell death in human neuroblastoma

Cardiotonic steroids (CTS) like ouabain are not only specific inhibitors of the sodium pump (Na(+),K(+)-ATPase), they also can influence various cytosolic signaling events in a hormone-like manner. In the neuroblastoma cell line SH-SY5Y ouabain triggers multiple signaling pathways. Within 30 min of incubation with 1 or 10 microM ouabain, SH-SY5Y cells generate reactive oxygen species to a level approximately 50% above control and show a modest but significant elevation in cytosolic [Ca(2+)] of about 25%. After 6 h of exposure, ouabain stimulates a series of anti-apoptotic actions in SH-SY5Y cells, including concentration-dependent phosphorylation of Erk1/2, Akt, and Bad. Nevertheless, at the same time this CTS also induces a series of events that inhibit retinoic acid-induced neuritogenesis and promote cell death. Both of these latter phenomena are possibly associated with the observed ouabain-induced reduction in the abundance of the anti-apoptotic proteins Bcl-XL and Bcl-2. In addition, ouabain treatment results in cytochrome c release into the cytosol and induces activation of caspase 3, events that point towards the stimulation of apoptotic pathways that are probably enhanced by the stimulation of p53 phosphorylation at Ser15 also observed in this study. These pathways may eventually lead to cell death: treatment with 10 nM ouabain results in a 20% decrease in cell number after 4 days of incubation and treatment with 1 microM ouabain decreases cells number by about 75%. The results obtained here emphasize the importance of further research in order to elucidate the various signalling cascades triggered by ouabain and possibly other CTS that are used in the treatment of heart failure and to identify their primary receptor(s).     

Conformational diseases and ER stress-mediated cell death: apoptotic cell death and autophagic cell death

The expanded polyglutamine (polyQ) tracts observed in autosomal dominant neurodegenerative disorders have the tendency to form intracellular aggregates, thus enhancing apoptotic cell death and the formation of autophagic vesicles. PolyQ accumulation inhibits the ER-associated degradation system (ERAD) resulting in reduced retrotranslocation from the ER and increased accumulation of misfolded proteins in the lumen of ER. Autophagy is an early cellular defense mechanism associated with ER stress, but prolonged ER stress may induce autophagic cell death, with destruction of cellular components and apoptotic cell death. Endoplasmic reticulum (ER) stress may be the key signal for both of these events.     

Mammalian target of rapamycin regulates isoliquiritigenin-induced autophagic and apoptotic cell death in adenoid cystic carcinoma cells

Previous studies, including those from our laboratory, have demonstrated that isoliquiritigenin (ISL), a flavonoid isolated from licorice, is a promising cancer chemotherapeutic agent. However the mechanisms underlying its anticancer effects are still far from clear. We now show, for the first time, that ISL triggers the mammalian target of rapamycin (mTOR)-dependent autophagic and apoptotic cell death in adenoid cystic carcinoma (ACC). Exposure of both ACC-2 and ACC-M cells to ISL resulted in several specific features for autophagy, including the appearance of membranous vacuoles, formation of acidic vesicular organelles, punctate pattern of LC3 immunostaining, and an increase in autophagic flux. Moreover, ISL treatment also resulted in significantly increased apoptosis in ACC cells. The ISL-mediated autophagic and apoptotic cell death were obviously attenuated by transfection with dominant negative Atg5 (DN-Atg5^K130R) plasmids or treatment with 3-methyladenine(3-MA). In additon, the data also revealed that the autophagic and apoptotic cell death induced by ISL occurred through a mTOR-dependent pathway. More importantly, the xenograft model using ACC-M cells provided further evidence of the occurrence of ISL-induced autophagy and apoptosis in vivo, correlating with the suppresson of mTOR activation as well as up-regulation of Atg5 expression. Taken together, these findings in our study suggest that induction of mTOR-dependent autophagic and apoptotic cell death may be an important mechanism in cancer chemotherapy by ISL.     

Niclosamide induces mitochondria fragmentation and promotes both apoptotic and autophagic cell death

Mitochondrial dynamics not only involves mitochondrial morphology but also mitochondrial biogenesis, mitochondrial distribution, and cell death. To identify specific regulators to mitochondria dynamics, we screened a chemical library and identified niclosamide as a potent inducer of mitochondria fission. Niclosamide promoted mitochondrial fragmentation but this was blocked by down-regulation of Drp1. Niclosamide treatment resulted in the disruption of mitochondria membrane potential and reduction of ATP levels. Moreover, niclosamide led to apoptotic cell death by caspase-3 activation. Interestingly, niclosamide also increased autophagic activity. Inhibition of autophagy suppressed niclosamide-induced cell death. Therefore, our findings suggest that niclosamide induces mitochondria fragmentation and may contribute to apoptotic and autophagic cell death.     

Involvement of N-acetylcysteine-sensitive pathways in ricin-induced apoptotic cell death in U937 cells

We have found that the antioxidant N-acetylcysteine (NAC) strongly inhibited ricin-induced apoptotic cell death in U937 cells (human myeloid leukemia), as judged by cytotoxicity, nuclear morphological change, and DNA fragmentation. Consistent with these observations, a significant depletion of cellular glutathione was observed in ricin-treated cells, and NAC prevented the decrease in cellular glutathione. On the other hand, among the caspase inhibitors tested, Z-Asp-CH2-DCB, which inhibited ricin cytotoxicity, also suppressed ricin-mediated glutathione depletion, while NAC did not affect the generation of caspase-3 like activity in ricin-treated cells. These results suggest that glutathione loss takes place downstream from caspase activation during the ricin-induced apoptotic process. Treatment with a specific inhibitor of glutathione biosynthesis, buthionine sulfoximine (BSO) failed to induce apoptosis, and had no effect on the overall extent of ricin-induced apoptosis, even though the glutathione level was decreased to less than 5% of the control level. However, NAC still protected against ricin-induced apoptosis in the BSO-treated cells. We conclude that glutathione loss is one of several apoptotic changes caused by ricin, but is not a sufficient factor for the progress of apoptosis. NAC may prevent ricin-induced apoptosis through maintaining an intracellular reducing condition by acting as a thiol supplier.     

Mitochondrial-targeted active Akt protects SH-SY5Y neuroblastoma cells from staurosporine-induced apoptotic cell death

Akt is a serine/threonine protein kinase that plays a vital role in promoting cellular survival. Predominantly cytosolic, upon stimulation with growth-factors or stress, active Akt translocates into mitochondria, but the functions of Akt in mitochondria are not yet fully understood. Mitochondria play a central role in apoptotic pathways and given Akt's functions in the cytoplasm, Akt in mitochondria may help preserve mitochondrial integrity during cellular stress. To test if the translocation of Akt into mitochondria is neuroprotective, adenoviral vectors expressing a constitutively active Akt, Ad-HA-Akt (DD), and a constitutively active Akt with a mitochondrial targeting signal, Ad-Mito-HA-Akt (DD), were generated. Human SH-SY5Y neuroblastoma cells expressing the adenoviral constructs were treated with staurosporine to initiate intrinsic apoptotic cell death and several aspects of the mitochondrial apoptotic pathway were evaluated. Expression of active Akt targeted to mitochondria was found to be sufficient to significantly reduce staurosporine-induced activation of caspase-3 and caspase-9, the release of cytochrome c from mitochondria, and Bax oligomerization at mitochondria. These findings demonstrate that intramitochondrial active Akt results in efficient protection against apoptotic signaling.     

Autophagy induction and autophagic cell death in effector T cells

The population size of the T cells is tightly regulated. The T cell number drastically increases in response to their specific antigens. Upon antigen clearance, the T cell number decreases over time. Apoptosis, also called type I programmed cell death, plays an important role in eliminating T cells. The role of autophagic cell death, also called type II programmed cell death, is unclear in T cells. Our recent work demonstrated that autophagy is induced in both Th1 and Th2 cells. Both TCR signaling and IL-2 increase autophagy in T cells, and JNK MAP kinases are required for the induction of autophagy in T cells, whereas caspases and mTOR inhibit autophagy in T cells. Autophagy is required for mediating growth factor withdrawal-dependent cell death in T cells. Here, we hypothesize that autophagic cell death plays an important role in T cell homeostasis.     

Paclitaxel resistance is associated with switch from apoptotic to autophagic cell death in MCF-7 breast cancer cells

Taxanes remain first line chemotherapy in management of metastatic breast cancer and have a key role in epithelial ovarian cancer, with increasingly common use of weekly paclitaxel dosing regimens. However, their clinical utility is limited by the development of chemoresistance. To address this, we modelled in vitro paclitaxel resistance in MCF-7 cells. We show that at clinically relevant drug doses, emerging paclitaxel resistance is associated with profound changes in cell death responses and a switch from apoptosis to autophagy as the principal mechanism of drug-induced cytotoxicity. This was characterised by a complete absence of caspase-mediated apoptotic cell death (using the pan-caspase-inhibitor Z-VAD) in paclitaxel-resistant MCF-7TaxR cells, compared with parent MCF-7 or MDA-MB-231 cell lines on paclitaxel challenge, downregulation of caspase-7, caspase-9 and BCl2-interacting mediator of cell death (BIM) expression. Silencing with small interfering RNA to BIM in MCF-7 parental cells was sufficient to confer paclitaxel resistance, inferring the significance in downregulation of this protein in contributing to the resistant phenotype of the MCF-7TaxR cell line. Conversely, there was an increased autophagic response in the MCF-7TaxR cell line with reduced phospho-mTOR and relative resistance to the mTOR inhibitors rapamycin and RAD001. In conclusion, we show for the first time that paclitaxel resistance is associated with profound changes in cell death response with deletion of multiple apoptotic factors balanced by upregulation of the autophagic pathway and collateral sensitivity to platinum.     

Induction of apoptotic but not autophagic cell death by Cinnamomum cassia extracts on human oral cancer cells

Cinnamomum cassia has been widely studied in different fields to reveal its antidiabetic, antidepressive, antiviral, anti-inflammatory, antiosteoporotic, and anticancer effects. Its antimalignant activities have been explored in lung cancer, breast cancer, colorectal cancer, and even oral cancer, but the detailed signaling mechanism and effects of this plant on animal models need to be clarified. In the current study, C. cassia extract (CCE) was used to investigate the antitumorigenesis mechanism in vitro and in vivo. The major constituents of CCE used in this study were coumarin, cinnamic acid, and cinnamic aldehyde. CCE reduced the viability, number, and colony formation of human oral cancer cells, and induced their apoptosis. Caspase-3 activation, Bcl-2 reduction, and phosphatidylserine inversion were involved in CCE-stimulated apoptosis. CCE also enhanced the expression of autophagic markers, including acidic vesicular organelle, microtubule-associated protein 1 light chain 3-I, autophagy-related protein 14, rubicon, and p62. The combined treatment of CCE and caspase inhibitor significantly restored mitochondrial membrane potential (Δ ψ _m) and cell viability. However, the combined treatment of CCE and autophagy inhibitor further reduced the cell viability indicating that autophagy might be a survival pathway of CCE-treated SASVO3 cells. In contrast, CCE treatment for 12 days did not adversely affect SASVO3 tumor-bearing nude mice. CCE also elicited dose-dependent effects on the decrease in tumor volume, tumor weight, and Ki-67 expression. These results suggested that CCE showed the potential for the complementary treatment of oral caner.     

Dual induction of apoptotic and autophagic cell death by targeting survivin in head neck squamous cell carcinoma

Survivin is ubiquitously expressed in patients with head neck squamous cell carcinoma (HNSCC) and is associated with poor survival and chemotherapy resistance. Sepantronium bromide (YM155) is a selective survivin suppressant that exhibits potent antitumor activities by inducing apoptosis and autophagy in various types of cancer. However, the curative effects and underlying mechanisms of YM155 in HNSCC remain unclear. This study showed that survivin overexpression positively correlated with p-S6, p-Rb and LAMP2 but negatively correlated with the autophagic marker LC3 in human HNSCC tissues. In vitro studies revealed that YM155 triggered apoptosis of HNSCC cells in mitochondria and death receptor-dependent manner. The treatment also significantly enhanced autophagy by upregulating Beclin1, which led to cell death. YM155 not only downregulated the expression of survivin but also remarkably suppressed the activation of the mTOR signaling pathway in vitro and in vivo. YM155 displayed potent antitumor activities in both CAL27 xenograft and transgenic HNSCC mice models by delaying tumor onset and suppressing tumor growth. Furthermore, YM155 combined with docetaxel promoted tumor regression better than either treatment alone without causing considerable body weight loss in the HNSCC xenograft models. Overall, targeting survivin by YM155 can benefit HNSCC therapy by increasing apoptotic and autophagic cell death, and suppressing prosurvival pathways.Head and neck squamous cell carcinoma (HNSCC), which occurs in the oral cavity, oropharynx, larynx and hypopharynx, is the sixth most common malignancy worldwide.¹ It affects 600 000 new patients each year, which accounts for over 90% of head and neck cancers.^{2, 3} The current preferred therapy for HNSCC is combined surgery, radiotherapy, chemotherapy and biotherapy; however, the 5-year survival rate is still <50%, and the long-term survival rate has only marginally improved.^{4, 5, 6} As an important hallmark of head and neck cancer, apoptosis resistance restricts the efficacy of traditional therapies.⁷ Survivin (also called BIRC5) inhibits apoptosis-related proteins, regulates cell division, relates to stress response and promotes tumor-associated angiogenesis in HNSCC.⁸ Survivin is also associated with high-grade and advanced HNSCC, poor survival, high recurrence rate and chemotherapy and radiation resistance. Therefore, targeting survivin is promisingly beneficial for head and neck cancer therapies.Sepantronium bromide (YM155) is a small imidazolium-based compound (1-(2-methoxyethyl)-2-methyl-4,9-dioxo-3-(pyrazin-2-ylmethyl)-4,9-dihydro-1H-naphtho[2,3-d]imidazolium bromide) that selectively suppresses survivin expression⁹ and displays potent anticancer activities against various types of cancer.^{10, 11, 12} Previous researches have focused on that YM155 induced the apoptosis by downregulating survivin in cancer cells.^{10, 13, 14} Recent studies including ours have demonstrated YM155 also triggered autophagy in cancer cells.^{15, 16, 17} Macroautophagy or autophagy is considered to be another type of programmed cell death wherein proteins are degraded by autophagosomes and lysosomes.¹⁸ Autophagy also has an important role in tumorigenesis.¹⁹ Autophagy shares several regulatory systems and common pathways with apoptosis; thus, autophagy is closely linked with apoptosis. Beclin1 (ATG6), an autophagy-specific gene that is essential for autophagosome induction and elongation, interacts with several apoptosis-related genes, such as bcl-2, bcl-xl and survivin.²⁰ Therefore, YM155 may not only induce the apoptosis but also affect the autophagy in HNSCC.The present study investigated the antitumor effects of YM155 on HNSCC in vitro and in vivo through dual induction of apoptotic and autophagic cell death. Although it specifically suppressed the expression of survivin, we here proved YM155 also targeted the mTOR signaling pathway, which was the principal regulator of cancer cell survival and autophagy. Most importantly, in an inducible tissue-specific spontaneous HNSCC mouse model with 100% penetrance by the combined deletion of Tgfbr1 and Pten (Tgfbr1/Pten 2cKO) in the oral mucosa²¹ with ubiquitous activation of the Akt/mTOR/survivin pathway,²² YM155 exerted significant therapeutic effects by delaying tumor onset and suppressing tumor growth. This finding coincided with the xenograft results. Finally, the effects of YM155 when combined with traditional chemotherapeutic agent were also determined.     

Resveratrol protects astrocytes against traumatic brain injury through inhibiting apoptotic and autophagic cell death

Traumatic brain injury (TBI) is often caused by accidents that damage the brain. TBI can induce glutamate excitotoxicity and lead to neuronal and glial cell death. In this study, we investigated the mechanism of cell death during the secondary damage caused by TBI in vivo and in vitro, as well as the protective effect of resveratrol (RV). Here we report that glycogen synthase kinase-3β (GSK-3β) activation and microtubule-associated protein light chain 3 processing were induced in rat brains exposed to TBI. In the in vitro TBI model, apoptotic and autophagic cell death were induced through glutamate-mediated GSK-3β activation in normal CTX TNA2 astrocytes. The GSK-3β inhibitor SB216763 or transfection of GSK-3β small-interfering RNA increases cell survival. By contrast, overexpression of GSK-3β enhanced glutamate excitotoxicity. Administration of RV reduced cell death in CTX TNA2 astrocytes by suppressing reactive oxygen species (ROS)-mediated GSK-3β activation, the mechanism by which RV also exerted protective effects in vivo. Mitochondrial damages, including the opening of mitochondrial permeability transition pore (MPTP) and mitochondrial depolarization, were induced by glutamate through the ROS/GSK-3β pathway. Moreover, cyclosporine A, an MPTP inhibitor, suppressed mitochondrial damage and the percentages of cells undergoing autophagy and apoptosis and thereby increased cell survival. Taken together, our results demonstrated that cell death occurring after TBI is induced through the ROS/GSK-3β/mitochondria signaling pathway and that administration of RV can increase cell survival by suppressing GSK-3β-mediated autophagy and apoptosis. Therefore, the results indicated that resveratrol may serve as a potential therapeutic agent in the treatment of TBI.     

p53-dependent and -independent pathways of apoptotic cell death in sepsis

Sepsis induces extensive apoptosis of lymphocytes, which may be responsible for the profound immune suppression of the disorder. Two potential pathways of sepsis-induced lymphocyte apoptosis, Fas and p53, were investigated. Lymphocyte apoptosis was evaluated 20-22 h after sepsis by annexin V or DNA nick-end labeling. Fas receptor-deficient mice had no protection against sepsis-induced apoptosis in thymocytes or splenocytes. p53 knockout mice (p53-/-) had complete protection against thymocyte apoptosis but, surprisingly, had no protection in splenocytes. p53-/- mice had no improvement in sepsis survival compared with appropriately matched control mice with sepsis. We conclude that both p53-dependent and p53-independent pathways of cell death exist in sepsis. This differential apoptotic response of thymocytes vs splenocytes in p53-/- mice suggests that either the cellular response or the death-inducing signal is cell-type specific in sepsis. The fact that p53-/- lymphocytes of an identical subtype (CD8-CD4+) were protected in thymi but not in spleens indicates that cell susceptibility to apoptosis differs depending upon other unidentified factors.     

Fenretinide induces autophagic cell death in caspase-defective breast cancer cells

The elimination of tumor cells by apoptosis is the main mechanism of action of chemotherapeutic drugs. More recently, autophagic cell death has been shown to trigger a nonapoptotic cell death program in cancer cells displaying functional defects of caspases. Fenretinide (FenR), a synthetic derivative of retinoic acid, promotes growth inhibition and induces apoptosis in a wide range of tumor cell types. The present study was designed to evaluate the ability of fenretinide to induce caspase-independent cell death and to this aim we used the human mammary carcinoma cell line MCF-7, lacking functional caspase-3 activity. We demonstrated that in these cells fenretinide is able to trigger an autophagic cell death pathway. In particular we found that fenretinide treatment resulted in the increase in Beclin 1 expression, the conversion of the soluble form of LC3 to the autophagic vesicle-associated form LC3-II and its shift from diffuse to punctate staining and finally the increase in lysosomes/autophagosomes. By contrast, caspase-3 reconstituted MCF-7 cell line showed apoptotic cell death features in response to fenretinide treatment. These data strongly suggest that fenretinide does not invariably elicit an apoptotic response but it is able to induce autophagy when apoptotic pathway is deregulated. The understanding of the molecular mechanisms involved in fenretinide action is important for the future design of therapies employing this retinoid in breast cancer treatment.     

Selenium activates p53 and p38 pathways and induces caspase-independent cell death in cervical cancer cells

The mechanisms of sodium selenite-induced cell death in cervical carcinoma cells were studied during 24 h of exposure in the HeLa Hep-2 cell line. Selenite at the employed concentrations of 5 and 50 μmol/L produced time- and dose-dependent suppression of DNA synthesis and induced DNA damage which resulted in phosphorylation of histone H2A.X. These effects were influenced by pretreatment of cells with the SOD/catalase mimetic MnTMPyP or glutathione-depleting buthionine sulfoximine, suggesting the significant role of selenite-generated oxidative stress. Following the DNA damage, selenite activated p53-dependent pathway as evidenced by the appearance of phosphorylated p53 and accumulation of p21 in the treated cells. Concomitantly, selenite activated p38 pathway but its effect on JNK was very weak. p53- and p38-dependent signaling led to the accumulation of Bax protein, which was preventable by specific inhibitors of p38 (SB 203580) and p53 (Pifithrin-α). Mitochondria in selenite-treated cells changed their dynamics (shape and localization) and released AIF and Smac/Diablo, which initiated caspase-independent apoptosis as confirmed by the caspase-3 activity assay and the low effect of caspase inhibitors z-DEVD-fmk and z-VAD-fmk on cell death. We conclude that selenite induces caspase-independent apoptosis in cervical carcinoma cells mostly by oxidative stress-mediated activation of p53 and p38 pathways, but other selenite-mediated effects, in particular mitochondria-specific ones, are also involved.