Caspase-independent Cell Death Mechanism Research Articles

Unraveling the intriguing interplay: Exploring the role of lncRNAs in caspase-independent cell death.

Cell death plays a crucial role in various physiological and pathological processes. Until recently, programmed cell death was mainly attributed to caspase-dependent apoptosis. However, emerging evidence suggests that caspase-independent cell death (CICD) mechanisms also contribute significantly to cellular demise. We and others have reported and functionally characterized numerous long noncoding RNAs (lncRNAs) that modulate caspase-dependent apoptotic pathways potentially in a pathway-dependent manner. However, the interplay between lncRNAs and CICD pathways has not been comprehensively documented. One major reason for this is that most CICD pathways have been recently discovered with some being partially characterized at the molecular level. In this review, we discuss the emerging evidence that implicates specific lncRNAs in the regulation and execution of CICD. We summarize the diverse mechanisms through which lncRNAs modulate different forms of CICD, including ferroptosis, necroptosis, cuproptosis, and others. Furthermore, we highlight the intricate regulatory networks involving lncRNAs, protein-coding genes, and signaling pathways that orchestrate CICD in health and disease. Understanding the molecular mechanisms and functional implications of lncRNAs in CICD may unravel novel therapeutic targets and diagnostic tools for various diseases, paving the way for innovative strategies in disease management and personalized medicine. This article is categorized under: RNA in Disease and Development > RNA in Disease.

Mixed lineage kinase domain-like pseudokinase: Conventional (necroptosis) and unconventional (necroptosis-independent) functions and features.

Mixed lineage kinase domain-like pseudokinase (MLKL) is the terminal and indispensable mediator of necroptosis. Necroptosis, also known as programmed cell necrosis, is a caspase-independent cell death mechanism involved in various pathologic and inflammatory processes. Triggering necroptosis could be an alternative approach in treating apoptosis-resistant cancer cells to prevent recurrent disease. In addition to its function in necroptosis, MLKL plays a role as a regulator in many cellular processes independent of necroptosis. A better understanding of the intracellular function of MLKL and its role in various diseases and pathologic conditions is needed to enable discovery of new targeted therapies. Various necroptosis-dependent and independent functions of MLKL are reviewed in this chapter, with a focus on functions of MLKL in necroptosis, autophagy, inflammation, tissue regeneration, and endosomal trafficking.

Abstract 3008: Induction of necroptosis through caspase-independent mechanisms in MCF-7 breast cancer cells following HDAC inhibition

Abstract Cell proliferation and cell death are essential for maintaining homeostatic balance in eukaryotic organisms. Many diseases develop when cellular homeostasis is disturbed. Currently, several anti-cancer drugs are known to induce apoptosis through activation of intrinsic or extrinsic pathways. However, many types of cancer cells have acquired resistance due to quick adaptation to resist pro-apoptotic mechanisms. Therefore, simultaneous induction of other forms of cell deaths such as necroptosis, autophagy, etc., in addition to apoptosis, is becoming an attractive strategy for controlling tumor growth and metastasis. For this purpose, we have explored the role of XIAP in caspase-independent mechanisms of cell death during HDAC (Histone Deacetylase) inhibition. Our experiments with HDAC inhibitor SAHA (Suberoylanilide hydroxamic acid) have revealed that the anti-apoptotic biomarkers, including survivin and XIAP, exhibit important regulatory functions for inducing necroptosis in cancer cells. Interestingly, many of the signal adaptors of apoptosis and necroptosis are known to show dual functions during drug-induced cancer cell death. For example, the formation of the survivin-XIAP complex prevents XIAP from undergoing proteasomal degradation and polyubiquitination, thereby stabilizing XIAP in cancer cells. When the survivin level is increased, it is also known to stop the inhibition of XIAP by Smac. In addition, since XIAP can inhibit caspase -9 and the executioner caspases -3 and -7, the apoptosis mechanisms will be blocked, and cell proliferation will continue to promote cell survival and tumorigenesis. Our western blot data revealed that the caspase -9, -3, -7, and -8 levels were significantly downregulated in MCF-7 breast cancer cells following SAHA treatment. Furthermore, SAHA treatment also increased the levels of necroptosis inducers such as phospho-RIP3 and MLKL in MCF-7 cells. However, treatment of the same MCF-7 cells with other drugs such as RG-7388, an MDM2 inhibitor, was able to induce apoptosis through activation of caspases. Our current results show that MCF-7 cells may switch to the necroptosis pathway due to the inhibition of caspases and increased levels of survivin and XIAP. (This research was supported by the Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida). Citation Format: Umamaheswari Natarajan, Grace Waldron, Thiagarajan Venkatesan, Appu Rathinavelu. Induction of necroptosis through caspase-independent mechanisms in MCF-7 breast cancer cells following HDAC inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3008.

Necroptosis and Prostate Cancer: Molecular Mechanisms and Therapeutic Potential.

Necroptosis is a programmed form of necrosis characterized by mitochondrial alterations and plasma membrane permeabilization resulting in the release of cytoplasmic content into extracellular space, and leading to inflammatory reactions. Besides its critical role in viral defense mechanisms and inflammatory diseases, necroptosis plays pivotal functions in the drug response of tumors, including prostate cancer. Necroptosis is mainly governed by kinase enzymes, including RIP1, RIP3, and MLKL, and conversely to apoptosis, is a caspase-independent mechanism of cell death. Numerous compounds induce necroptosis in prostate cancer models, including (i) compounds of natural origin, (ii) synthetic and semisynthetic small molecules, and (iii) selenium and selenium-based nanoparticles. Here, we overview the molecular mechanisms underlying necroptosis and discuss the possible implications of drugs inducing necroptosis for prostate cancer therapy.

Beyond Pseudo-natural Products: Sequential Ugi/Pictet-Spengler Reactions Leading to Steroidal Pyrazinoisoquinolines That Trigger Caspase-Independent Death in HepG2 Cells.

In this work, we describe how stereochemically complex polycyclic compounds can be generated by applying a synthetic sequence comprising an intramolecular Ugi reaction followed by a Pictet-Spengler cyclization on steroid-derived scaffolds. The resulting compounds, which combine a fragment derived from a natural product and a scaffold not found in nature. are both structurally distinct and globally similar to natural products at the same time, and interrogate an alternative region of the chemical space. One of the new compounds showed significant antiproliferative activity on HepG2 cells through a caspase-independent cell-death mechanism, an appealing feature when new antitumor compounds are searched.

Physiopathology of the Permeability Transition Pore: Molecular Mechanisms in Human Pathology.

Mitochondrial permeability transition (MPT) is the sudden loss in the permeability of the inner mitochondrial membrane (IMM) to low-molecular-weight solutes. Due to osmotic forces, MPT is paralleled by a massive influx of water into the mitochondrial matrix, eventually leading to the structural collapse of the organelle. Thus, MPT can initiate outer-mitochondrial-membrane permeabilization (MOMP), promoting the activation of the apoptotic caspase cascade and caspase-independent cell-death mechanisms. The induction of MPT is mostly dependent on mitochondrial reactive oxygen species (ROS) and Ca2+, but is also dependent on the metabolic stage of the affected cell and signaling events. Therefore, since its discovery in the late 1970s, the role of MPT in human pathology has been heavily investigated. Here, we summarize the most significant findings corroborating a role for MPT in the etiology of a spectrum of human diseases, including diseases characterized by acute or chronic loss of adult cells and those characterized by neoplastic initiation.

Staphylococcus aureus small colony variants impair host immunity by activating host cell glycolysis and inducing necroptosis.

Staphylococcus aureus small colony variants (SCVs) are frequently associated with chronic infection, yet they lack expression of many virulence determinants associated with the pathogenicity of wild-type strains. We found that both wild-type S. aureus and a ΔhemB SCV prototype potently activate glycolysis in host cells. Glycolysis and the generation of mitochondrial reactive oxygen species were sufficient to induce necroptosis, a caspase-independent mechanism of host cell death that failed to eradicate S. aureus and instead promoted ΔhemB SCV pathogenicity. To support ongoing glycolytic activity, the ΔhemB SCV induced over a 100-fold increase in the expression of fumC, which encodes an enzyme that catalyses the degradatin of fumarate, an inhibitor of glycolysis. Consistent with fumC-dependent depletion of local fumarate, the ΔhemB SCV failed to elicit trained immunity and protection from a secondary infectious challenge in the skin. The reliance of the S. aureus SCV population on glycolysis accounts for much of its role in the pathogenesis of S. aureus skin infection.

Cold Atmospheric Plasma induces accumulation of lysosomes and caspase-independent cell death in U373MG glioblastoma multiforme cells

Room temperature Cold Atmospheric Plasma (CAP) has shown promising efficacy for the treatment of cancer but the exact mechanisms of action remain unclear. Both apoptosis and necrosis have been implicated as the mode of cell death in various cancer cells. We have previously demonstrated a caspase-independent mechanism of cell death in p53-mutated glioblastoma multiforme (GBM) cells exposed to plasma. The purpose of this study was to elucidate the molecular mechanisms involved in caspase-independent cell death induced by plasma treatment. We demonstrate that plasma induces rapid cell death in GBM cells, independent of caspases. Accumulation of vesicles was observed in plasma treated cells that stained positive with acridine orange. Western immunoblotting confirmed that autophagy is not activated following plasma treatment. Acridine orange intensity correlates closely with the lysosomal marker Lyso TrackerTM Deep Red. Further investigation using isosurface visualisation of confocal imaging confirmed that lysosomal accumulation occurs in plasma treated cells. The accumulation of lysosomes was associated with concomitant cell death following plasma treatment. In conclusion, we observed rapid accumulation of acidic vesicles and cell death following CAP treatment in GBM cells. We found no evidence that either apoptosis or autophagy, however, determined that a rapid accumulation of late stage endosomes/lysosomes precedes membrane permeabilisation, mitochondrial membrane depolarisation and caspase independent cell death.

Abstract 4715: Combination of Tacedinaline and EHMT2 inhibition increases breast cancer cell death involving BIRC5 repression and GADD45A induction

Abstract The genomes of cancer cells are different from those of normal cells. These differences include genetic and epigenetic alterations, such as variations of DNA methylation and histone modifications, which cause differential gene expression, increased cell growth/migration capacity, and may induce resistance to anticancer therapy. Thus, epigenetic modulation may have a chance to correct abovementioned cancer cell’s aberrations in gene expression and cell behaviors, resulting in eradication of primary and/or suppression of refractory cancers. Tacedinaline (CI-994) and UNC0638 are specific inhibitors of class I histone deacetylase (HDAC) and histone methyltransferase EHMT2 (G9a), which cause an increase of histone acetylation and a decrease of histone H3 dimethylation at lysine 9, respectively. Here we examined the therapeutic effect of CI-994 and UNC0638 combination on the triple-negative (MDA-MB-231) and estrogen receptor-positive (MCF-7) breast cancer cell lines. The results showed that the combination of CI-994 and UNC0638 was able to induce more severe cell death than either agent alone. Although the sub-G1 fraction and Annexin V-positive cells were increased, CI-994 and UNC0638 did not significantly induce the activities of caspases 3, 8 and 9, suggesting the involvement of non-canonical and caspase-independent cell death mechanism. Instead, CI-994 suppressed the expression of BIRC5 (Survivin, a member of the inhibitor of apoptosis protein) and, in combination with UNC0638, induced the expression of GADD45A (involved in stress-induced apoptosis). Mechanistically, combination of CI-994 and UNC0638 resulted in the changes of epigenetic marks, such as acetylation and methylation of histone H3, at the loci of BIRC5 and GADD45A, which were correlated with their altered gene expressions. Genetic knockdown of BIRC5 expression in MCF-7 decreased cell viability, supporting the pro-survival role of BIRC5 in breast cancer cells. Importantly, the targets of CI-994 (HDAC1, HDAC2) and UNC0638 (EHMT2), as well as BIRC5 were overexpressed in breast cancer specimens, especially those of triple-negative breast cancer (TNBC), based on the analyses of The Cancer Genome Atlas dataset. Meanwhile, the expression of GADD45A was downregulated. BIRC5 upregulation and GADD45A downregulation in breast cancer were associated with patient’s overall survival. These results suggest that Tacedinaline and UNC0638, or targeting the class I HDAC and EHMT2, may be a potential strategy to treat breast cancer including TNBC. Citation Format: Pei-Yi Chu, Ji-Ching Lai, Ming-Feng Hou, Chang-Shen Lin. Combination of Tacedinaline and EHMT2 inhibition increases breast cancer cell death involving BIRC5 repression and GADD45A induction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4715.

WIN55,212-2 induces caspase-independent apoptosis on human glioblastoma cells by regulating HSP70, p53 and Cathepsin D

Despite the standard approaches to treat the highly aggressive and invasive glioblastoma (GBM), it remains incurable. In this sense, cannabinoids highlight as a promising tool, because this tumor overexpresses CB1 and/or CB2 receptors and being, therefore, can be susceptible to cannabinoids treatment. Thus, this work investigated the action of the cannabinoid agonist WIN55-212-2 on GBM cell lines and non-malignant cell lines, in vitro and in vivo. WIN was selectively cytotoxic to GBM cells. These presented blebbing and nuclear alterations in addition to cell shrinkage and chromatin condensation. WIN also significantly inhibited the migration of GAMG and U251 cells. Finally, the data also showed that the antitumor effects of WIN are exerted, at least to some extent, by the expression of p53 and increased cathepsin D in addition to the decreased expression of HSP70.This data can indicate caspase-independent cell death mechanism. In addition, WIN decreased tumoral perimeter as well as caused a reduction the blood vessels in this area, without causing lysis, hemorrhage or blood clotting. So, the findings herein presented reinforce the usefulness of cannabinoids as a candidate for further evaluation in treatment in glioblastoma treatment.

PDGFR and IGF-1R Inhibitors Induce a G2/M Arrest and Subsequent Cell Death in Human Glioblastoma Cell Lines.

Glioblastomas are highly resistant to radiation and chemotherapy. Currently, there are no effective therapies for this type of tumor. Signaling mechanisms initiated by PDGFR and IGF-1R are important in glioblastoma, and inhibition of the signal transduction pathways initiated by these receptors could be a useful alternative strategy for glioblastoma treatment. We have studied the effects of the PDGFR inhibitor JNJ-10198409 (JNJ) and the IGF-1R inhibitor picropodophyllin (PPP) in glioblastoma cell lines as well as in primary cultures derived from patients affected by this type of tumor. JNJ and PPP treatment blocked PDGFR and IGF-1R signaling respectively and reduced Akt and Erk 1/2 phosphorylation. Both inhibitors diminished cell proliferation, inducing a G2/M block of the cell cycle. Cell death induced by JNJ was caspase-dependent, Annexin-V positive and caused PARP cleavage, especially in T98 cells, suggesting an apoptotic mechanism. However, cell death induced by PPP was not completely inhibited by caspase inhibitors in all cell lines apart from LN-229 cells, indicating a caspase-independent mechanism. Several inhibitors targeted against different cell death pathways could not block this caspase-independent component, which may be a non-programmed necrotic mechanism. Apoptotic arrays performed in T98 and LN-229 cells upon JNJ and PPP treatment revealed that procaspase 3 levels were augmented by both drugs in T98 cells and only by JNJ in LN229-cells. Furthermore, XIAP and survivin levels were much higher in LN-229 cells than in T98 cells, revealing that LN-229 cells are more susceptible to undergo caspase-independent cell death mechanisms. JNJ and PPP combination was more effective than each treatment alone.

Jaspine B induces nonapoptotic cell death in gastric cancer cells independently of its inhibition of ceramide synthase

Sphingolipids (SLs) have been extensively investigated in biomedical research due to their role as bioactive molecules in cells. Here, we describe the effect of a SL analog, jaspine B (JB), a cyclic anhydrophytosphingosine found in marine sponges, on the gastric cancer cell line, HGC-27. JB induced alterations in the sphingolipidome, mainly the accumulation of dihydrosphingosine, sphingosine, and their phosphorylated forms due to inhibition of ceramide synthases. Moreover, JB provoked atypical cell death in HGC-27 cells, characterized by the formation of cytoplasmic vacuoles in a time and dose-dependent manner. Vacuoles appeared to originate from macropinocytosis and triggered cytoplasmic disruption. The pan-caspase inhibitor, z-VAD, did not alter either cytotoxicity or vacuole formation, suggesting that JB activates a caspase-independent cell death mechanism. The autophagy inhibitor, wortmannin, did not decrease JB-stimulated LC3-II accumulation. In addition, cell vacuolation induced by JB was characterized by single-membrane vacuoles, which are different from double-membrane autophagosomes. These findings suggest that JB-induced cell vacuolation is not related to autophagy and it is also independent of its action on SL metabolism.

Non-thermal atmospheric plasma induces ROS-independent cell death in U373MG glioma cells and augments the cytotoxicity of temozolomide.

Background:Non-thermal atmospheric plasma (NTAP) is an ionised gas produced under high voltage that can generate short-lived chemically active species and induce a cytotoxic insult in cancer cells. Cell-specific resistance to NTAP-mediated cytotoxicity has been reported in the literature. The aim of this study was to determine whether resistance against NTAP could be overcome using the human glioma cell line U373MG.Methods:Non-thermal atmospheric plasma was generated using a Dielectric Barrier Device (DBD) system with a maximum voltage output of 120 kV at 50 Hz. The viability of U373MG GBM cells and HeLa cervical carcinoma cells was determined using morphology, flow cytometry and cytotoxicity assays. Fluorescent probes and inhibitors were used to determine the mechanisms of cytotoxicity of cells exposed to the plasma field. Combinational therapy with temozolomide (TMZ) and multi-dose treatments were explored as mechanisms to overcome resistance to NTAP.Results:Non-thermal atmospheric plasma decreased cell viability in a dose (time)-dependent manner. U373MG cells were shown to be resistant to NTAP treatment when compared with HeLa cells, and the levels of intracellular reactive oxygen species (ROS) quantified in U373MG cells were much lower than in HeLa cells following exposure to the plasma field. Reactive oxygen species inhibitor N-acetyl cysteine (NAC) only alleviated the cytotoxic effects in HeLa cells and not in the relatively NTAP-resistant cell line U373MG. Longer exposures to NTAP induced a cell death independent of ROS, JNK and caspases in U373MG. The relative resistance of U373MG cells to NTAP could be overcome when used in combination with low concentrations of the GBM chemotherapy TMZ or exposure to multiple doses.Conclusions:For the very first time, we report that NTAP induces an ROS-, JNK- and caspase-independent mechanism of cell death in the U373MG GBM cell line that can be greatly enhanced when used in combination with low doses of TMZ. Further refinement of the technology may facilitate localised activation of cytotoxicity against GBM when used in combination with new and existing chemotherapeutic regimens.

Abstract 5363: Navitoclax (Nav) and BMN 673 yield cytotoxicity with lower doses than used for single agents in high-grade serous ovarian cancer (HGSOC)

Abstract Background: The apoptosis inhibitor Bcl-xL is upregulated in recurrent ovarian cancer (OvCa). Modest clinical activity with Nav, a Bcl-2/Bcl-xL inhibitor, was observed in hematologic malignancies, although significant thrombocytopenia has been a challenge to its development. BMN 673 is a PARP inhibitor (PARPi) and is more potent at a molar level than other PARPi in preclinical studies. We observed a decrease in Bim and Bcl-2 protein expression in OvCa tissue after 1 cycle of olaparib (PARPi) and carboplatin compared to pretreatment levels; this change correlated with duration of response in BRCA1/2 mutation-related breast and OvCa patients (r2 = -0.86, p<0.05). We hypothesize the combination of BMN 673 with Nav will yield preclinical synergy at or below clinically attainable concentrations. Methods: We examined cell injury, DNA damage, and apoptosis by XTT, comet assay, and caspase-3 activity in two HGSOC cell lines, CAOV3 and OVCAR8. We examined a dose range based on clinically achievable concentrations of Nav (1-8 μM) and BMN 673 (25-100 nM) and calculated IC50 concentrations for cytotoxicity. 2 μM Nav and 50 nM BMN 673 were selected to examine alone and in combination measures of DNA damage and apoptosis. Controls were cells with no treatment for comet assay, and with DMSO vehicle control for caspase-3 activity. The combination index (CI) was calculated to evaluate synergism. All experiments were performed in at least 3 replicates in all cell lines. Results are presented as mean ± SEM. Result: Nav yielded cytotoxicity in CAOV3 and OVCAR8 cells with IC50 2.67 μM and 4.39 μM, respectively. BMN 673 100 nM monotherapy yielded up to 40% cell injury in CAOV3 and 20% in OVCAR8 cells. The combination of BMN 673 50 nM and Nav 2 μM resulted in more than 80% and 30% cell injury in CAOV3 and OVCAR8, respectively. The CI indicates there is synergism in cell injury between the two drugs at or below clinically attainable concentrations. BMN 673 alone and with Nav augmented% DNA in tail in an additive fashion, over control or Nav alone in both cell lines. The effect with the combination v. Nav alone was significant in OVCAR8 (12.74% +/- 0.38 v. 9.49% +/- 0.55, p<0.05); the change was not significant in CAOV3. Nav alone caused greater caspase-3 activity than seen in control or BMN 673-treated CAOV3 cells (p<0.05), but the difference was not significant in OVCAR8. No additive caspase-3 activity was seen with the combination. Additional HGSOC cell lines are under evaluation. Annexin V/PI flow cytometric analysis and cell cycle analysis will be used to examine both caspase-dependent and caspase-independent mechanisms of cell death. Conclusion: Our preliminary results suggest that the combination of PARP inhibition and Bcl2/Bcl-xL neutralization may be cooperative in yielding better results than either drug alone in HGSOC. Further experiments are currently underway to evaluate this novel drug combination. Citation Format: Takuhei Yokoyama, Nicolas Gordon, Minshu Yu, Elise C. Kohn, Jung-Min Lee. Navitoclax (Nav) and BMN 673 yield cytotoxicity with lower doses than used for single agents in high-grade serous ovarian cancer (HGSOC). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5363. doi:10.1158/1538-7445.AM2015-5363

Cucurbitacin-I inhibits Aurora kinase A, Aurora kinase B and survivin, induces defects in cell cycle progression and promotes ABT-737-induced cell death in a caspase-independent manner in malignant human glioma cells

Because STAT signaling is commonly activated in malignant gliomas as a result of constitutive EGFR activation, strategies for inhibiting the EGFR/JAK/STAT cascade are of significant interest. We, therefore, treated a panel of established glioma cell lines, including EGFR overexpressors, and primary cultures derived from patients diagnosed with glioblastoma with the JAK/STAT inhibitor cucurbitacin-I. Treatment with cucurbitacin-I depleted p-STAT3, p-STAT5, p-JAK1 and p-JAK2 levels, inhibited cell proliferation, and induced G2/M accumulation, DNA endoreduplication, and multipolar mitotic spindles. Longer exposure to cucurbitacin-I significantly reduced the number of viable cells and this decrease in viability was associated with cell death, as confirmed by an increase in the subG1 fraction. Our data also demonstrated that cucurbitacin-I strikingly downregulated Aurora kinase A, Aurora kinase B and survivin. We then searched for agents that exhibited a synergistic effect on cell death in combination with cucurbitacin-I. We found that cotreatment with cucurbitacin-I significantly increased Bcl-2/Bcl-xL family member antagonist ABT-737-induced cell death regardless of EGFR/PTEN/p53 status of malignant human glioma cell lines. Although >50% of the cucurbitacin-I plus ABT-737 treated cells were annexin V and propidium iodide positive, PARP cleavage or caspase activation was not observed. Pretreatment of z-VAD-fmk, a pan caspase inhibitor did not inhibit cell death, suggesting a caspase-independent mechanism of cell death. Genetic inhibition of Aurora kinase A or Aurora kinase B or survivin by RNA interference also sensitized glioma cells to ABT-737, suggesting a link between STAT activation and Aurora kinases in malignant gliomas.

Mitochondrial apoptosis-inducing factor is involved in doxorubicin-induced toxicity on H9c2 cardiomyoblasts

The cardiotoxicity induced by the anti-cancer doxorubicin involves increased oxidative stress, disruption of calcium homeostasis and activation of cardiomyocyte death. Nevertheless, antioxidants and caspase inhibitors often show little efficacy in preventing cell death. We hypothesize that a caspase-independent cell death mechanism with the release of the apoptosis-inducing factor from mitochondria is involved in doxorubicin toxicity. To test the hypothesis, H9c2 cardiomyoblasts were used as model for cardiac cells. Our results demonstrate that z-VAD-fmk, a pan-caspase inhibitor, does not prevent doxorubicin toxicity in this cell line. Doxorubicin treatment results in AIF translocation to the nuclei, as confirmed by Western Blotting of cell fractions and confocal microscopy. Also, doxorubicin treatment of H9c2 cardiomyoblasts resulted in the appearance of 50kbp DNA fragments, a hallmark of apoptosis-inducing factor nuclear effects. Apoptosis-inducing factor knockdown using a small-interfering RNA approach in H9c2 cells resulted in a reduction of doxorubicin toxicity, including decreased p53 activation and poly-ADP-ribose-polymerase cleavage. Among the proteases that could be responsible for apoptosis-inducing factor cleavage, doxorubicin decreased calpain activity but increased cathepsin B activation, with inhibition of the latter partly decreasing doxorubicin toxicity. Altogether, the results support that apoptosis-inducing factor release is involved in doxorubicin-induced H9c2 cell death, which explains the limited ability of caspase inhibitors to prevent toxicity.

Neuronal cell death and regeneration in diseases associated with advanced glycation end-products accumulation.

Recent studies have shown that the glycation-associated damage is not limited to patients with diabetes. Because of their association with oxidative stress, advanced glycation end-products (AGEs) have also been implicated in many neurodegenerative diseases, such as Huntington disease, amyotrophic lateral sclerosis, and Alzheimer disease (Yan et al., 1996; Ma and Nicholson, 2004). As in other age-dependent neurodegenerative diseases of the brain, oxidative stress-associated age-dependent pathogenic processes are not unexpected in glaucoma because this disease is also more common in the elderly (Tezel et al. 2007). Growing evidence indicates that neuronal abnormalities including neuronal cell death are associated with the pathogenesis of early diabetic retinopathy. Our previous studies of human retinas indicate that mitochondrial- and caspase-dependent cell death pathways are associated with retinal neuronal cell degeneration in patients with diabetes (Oshitari et al., 2008). Brain-derived neurotrophic factor, neurotrophin-4 (NT-4), vascular endothelial growth factor (VEGF)120, VEGF164, taurine-conjugated ursodeoxycholic acid and citicoline had been studied and showed a survival effect on damaged retinal neurons induced by diabetic stress (Oshitari et al. 2003, 2010). It was also found that NT-4 had the best neuroprotective and regenerative effect under high glucose conditions (Oshitari et al, 2010). Earlier study indicated that the maximum rescue ratio of caspase-1, -3, -8, and -9 inhibitors in cultured retinas was 60% in damaged retinal ganglion cells (RGCs) (Oshitari and Adachi-Usami, 2003). Thus, at least 40% of neuronal cell death in damaged RGCs in cultured retinas should be related to caspase-independent cell death mechanisms. However, no reports have focused on caspase-independent cell death pathways under diabetic stress including AGEs exposure. Apoptosis-inducing factor (AIF) was the first mitochondrial protein shown to mediate cell death independent of caspase (Susin et al., 1996, 1999). It was initially characterized as a mitochondrial protein confined in the intermembrane space of healthy cells. In healthy cells, AIF is a mitochondrial flavin adenine dinucleotide (FAD)-dependent oxidoreductase that plays roles in redox control and oxidative phosphorylation (Modjtahedi et al., 2006).

Polyphenon E®, a standardized green tea extract, induces endoplasmic reticulum stress, leading to death of immortalized PNT1a cells by anoikis and tumorigenic PC3 by necroptosis

Increasing doses of Polyphenon E®, a standardized green tea extract, were given to PNT1a and PC3 prostate epithelial cells mimicking initial and advanced stages of prostate cancer (PCa), respectively. Cell death occurred in both cell lines, with PNT1a being more sensitive [half-maximal inhibitory concentration (IC50) = 35 μg/ml] than PC3 (IC50 = 145 μg/ml) to Polyphenon E®. Cell cycle arrest occurred at G0/G1 checkpoint for PNT1a, and G2/M for PC3 cells. Endoplasmic reticulum stress (ERS) and unfolded protein response (UPR) occurred in both cell lines, with each exhibiting different timing in response to Polyphenon E®. Autophagy was transiently activated in PNT1a cells within 12 h after treatment as a survival response to overcome ERS; then activation of caspases and cleavage of poly (ADP ribose) polymerase 1 occurred, committing cells to anoikis death. Polyphenon E® induced severe ERS in PC3 cells, causing a dramatic enlargement of the ER; persistent activation of UPR produced strong upregulation of GADD153/CHOP, a key protein of ERS-mediated cell death. Thereafter, GADD153/CHOP activated Puma, a BH3-only protein, committing cells to necroptosis, a programmed caspase-independent mechanism of cell death. Our results provide a foundation for the identification of novel targets and strategies aimed at sensitizing apoptosis-resistant cells to alternative death pathways.

Hypertonic stress and cell death. Focus on “Multiple cell death pathways are independently activated by lethal hypertonicity in renal epithelial cells”

Programmed cell death has been synonymous with caspase-mediated apoptosis for many years, until the recent discovery of caspase-independent mechanisms of cell death. Programmed necrosis is a form of caspase-independent cell death and is currently intensively studied as mechanism of kidney cell injury. The inner core of the kidney, known as the renal medulla, is hypertonic due to a specialized counter current arrangement of tubules, vascular elements and transporters. This regional hypertonicity is critical for body fluid osmoregulation as it provides the osmotic driving force for urinary concentration. The hypertonic environment of the kidney medulla can also promote cell death, if the physiologic cell protective mechanisms are impaired (4). Hypertonic cell stress can induce several pathways that lead to cell death and the distinction between hypertonicity-induced apoptosis and necrosis is becoming increasingly complex.

Role of the c subunit of the FO ATP synthase in mitochondrial permeability transition

The term “mitochondrial permeability transition” (MPT) refers to an abrupt increase in the permeability of the inner mitochondrial membrane to low molecular weight solutes. Due to osmotic forces, MPT is paralleled by a massive influx of water into the mitochondrial matrix, eventually leading to the structural collapse of the organelle. Thus, MPT can initiate mitochondrial outer membrane permeabilization (MOMP), promoting the activation of the apoptotic caspase cascade as well as of caspase-independent cell death mechanisms. MPT appears to be mediated by the opening of the so-called “permeability transition pore complex” (PTPC), a poorly characterized and versatile supramolecular entity assembled at the junctions between the inner and outer mitochondrial membranes. In spite of considerable experimental efforts, the precise molecular composition of the PTPC remains obscure and only one of its constituents, cyclophilin D (CYPD), has been ascribed with a crucial role in the regulation of cell death. Conversely, the results of genetic experiments indicate that other major components of the PTPC, such as voltage-dependent anion channel (VDAC) and adenine nucleotide translocase (ANT), are dispensable for MPT-driven MOMP. Here, we demonstrate that the c subunit of the FO ATP synthase is required for MPT, mitochondrial fragmentation and cell death as induced by cytosolic calcium overload and oxidative stress in both glycolytic and respiratory cell models. Our results strongly suggest that, similar to CYPD, the c subunit of the FO ATP synthase constitutes a critical component of the PTPC.