BUB1 mediation of caspase-independent mitotic death determines cell fate

The spindle checkpoint that monitors kinetochore-microtubule attachment has been implicated in tumorigenesis; however, the relation between the spindle checkpoint and cell death remains obscure. In BUB1-deficient (but not MAD2-deficient) cells, conditions that activate the spindle checkpoint (i.e., cold shock or treatment with nocodazole, paclitaxel, or 17-AAG) induced DNA fragmentation during early mitosis. This mitotic cell death was independent of caspase activation; therefore, we named it caspase-independent mitotic death (CIMD). CIMD depends on p73, a homologue of p53, but not on p53. CIMD also depends on apoptosis-inducing factor and endonuclease G, which are effectors of caspase-independent cell death. Treatment with nocodazole, paclitaxel, or 17-AAG induced CIMD in cell lines derived from colon tumors with chromosome instability, but not in cells from colon tumors with microsatellite instability. This result was due to low BUB1 expression in the former cell lines. When BUB1 is completely depleted, aneuploidy rather than CIMD occurs. These results suggest that cells prone to substantial chromosome missegregation might be eliminated via CIMD.     

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

The elimination of tumour 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 displying functional defects of caspases. Fenretinide (FenR), a synthetic derivative of retinoic acid, promotes growth inhibition and induces apoptosis in a wide range of tumour 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.     

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.     

Berberine induces autophagic cell death and mitochondrial apoptosis in liver cancer cells: The cellular mechanism

Extensive studies have revealed that berberine, a small molecule derived from Coptidis rhizoma (Huanglian in Chinese) and many other plants, has strong anti‐tumor properties. To better understand berberine‐induced cell death and its underlying mechanisms in cancer, we examined autophagy and apoptosis in the human hepatic carcinoma cell lines HepG2 and MHCC97‐L. The results of this study indicate that berberine can induce both autophagy and apoptosis in hepatocellular carcinoma cells. Berberine‐induced cell death in human hepatic carcinoma cells was diminished in the presence of the cell death inhibitor 3‐methyladenine, or following interference with the essential autophagy gene Atg5. Mechanistic studies showed that berberine may activate mitochondrial apoptosis in HepG2 and MHCC97‐L cells by increasing Bax expression, the formation of permeable transition pores, cytochrome C release to cytosol, and subsequent activation of the caspases 3 and 9 execution pathway. Berberine may also induce autophagic cell death in HepG2 and MHCC97‐L cells through activation of Beclin‐1 and inhibition of the mTOR‐signaling pathway by suppressing the activity of Akt and up‐regulating P38 MAPK signaling. This is the first study to describe the role of Beclin‐1 activation and mTOR inhibition in berberine‐induced autophagic cell death. These results further demonstrate the potential of berberine as a therapeutic agent in the emerging list of cancer therapies with novel mechanisms. J. Cell. Biochem. 111: 1426–1436, 2010. © 2010 Wiley‐Liss, Inc.     

HSpin1, a transmembrane protein interacting with Bcl-2/Bcl-xL,induces a caspase-independent autophagic cell death

The Drosophila spinster (spin) gene product is required for programmed cell death in the nervous and reproductive systems. We have identified a human homologue of the Drosophila spin gene product (HSpin1). HSpin1 bound to Bcl-2 and apoptosis regulator Bcl-X (Bcl-xL), but not to proapoptotic members such as Bcl-2-associated X protein and Bcl-2 homologous antagonist killer, in cells treated with TNF-alpha. Exogenous expression of HSpin1 resulted in the cell death without inducing a release of cytochrome c from mitochondria. Overexpression of Bcl-xL inhibited the HSpin1-induced cell death. Interestingly, a necrosis inhibitor, pyrrolidine dithiocarbomate, but not the pancaspase inhibitors, carbobenzoxy-VAD-fluoromethyl ketone and p35, blocked the HSpin1-induced cell death. HSpin1-induced cell death increases autophagic vacuole and mature form of cathepsin D, suggesting a novel caspase-independent cell death, which is link to autophagy.     

Expression of death receptor 4 induces caspase-independent cell death in MMS-treated yeast

DR4, a tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor, is a key element in the extrinsic pathway of TRAIL/TRAIL receptor-related apoptosis that exerts a preferential toxic effect against tumor cells. However, TRAIL and DR4 are expressed in various normal cells, and recent studies indicate that DR4 has a number of non-apoptotic functions. In this study, we evaluated the effects of human DR4 expression in yeast to determine the function of DR4 in normal cells. The expression of DR4 in yeast caused G1 arrest, which resulted in transient growth inhibition. Moreover, treatment of DR4-expressing yeast with a DNA damaging agent, MMS, elicited drastic, and sustained cell growth inhibition accompanied with massive apoptotic cell death. Further analysis revealed that cell death in the presence of DNA damage and DR4 expression was not dependent on the yeast caspase, YCA1. Taken together, these results indicate that DR4 triggers caspase-independent programmed cell death during the response of normal cells to DNA damage.     

Larotrectinib induces autophagic cell death through AMPK/mTOR signalling in colon cancer

Larotrectinib (Lar) is a highly selective and potent small‐molecule inhibitor used in patients with tropomyosin receptor kinase (TRK) fusion‐positive cancers, including colon cancer. However, the underlying molecular mechanisms specifically in patients with colon cancer have not yet been explored. Our data showed that Lar significantly suppressed proliferation and migration of colon cancer cells. In addition, Lar suppressed the epithelial–mesenchymal transition (EMT) process, as evidenced by elevation in E‐cadherin (E‐cad), and downregulation of vimentin and matrix metalloproteinase (MMP) 2/9 expression. Furthermore, Lar was found to activate autophagic flux, in which Lar increased the ratio between LC3II/LC3I and decreased the expression of p62 in colon cancer cells. More importantly, Lar also increased AMPK phosphorylation and suppressed mTOR phosphorylation in colon cancer cells. However, when we silenced AMPK in colon cancer cells, Lar‐induced accumulation of autolysomes as well as Lar‐induced suppression of the EMT process were significantly diminished. An in vivo assay also confirmed that tumour volume and weight decreased in Lar‐treated mice than in control mice. Taken together, this study suggests that Lar significantly suppresses colon cancer proliferation and migration by activating AMPK/mTOR‐mediated autophagic cell death.     

Oxidative stress induces autophagic cell death independent of apoptosis in transformed and cancer cells

Autophagy is a self-digestion process that degrades intracellular structures in response to stresses leading to cell survival. When autophagy is prolonged, this could lead to cell death. Generation of reactive oxygen species (ROS) through oxidative stress causes cell death. The role of autophagy in oxidative stress-induced cell death is unknown. In this study, we report that two ROS-generating agents, hydrogen peroxide (H(2)O(2)) and 2-methoxyestradiol (2-ME), induced autophagy in the transformed cell line HEK293 and the cancer cell lines U87 and HeLa. Blocking this autophagy response using inhibitor 3-methyladenine or small interfering RNAs against autophagy genes, beclin-1, atg-5 and atg-7 inhibited H(2)O(2) or 2-ME-induced cell death. H(2)O(2) and 2-ME also induced apoptosis but blocking apoptosis using the caspase inhibitor zVAD-fmk (benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone) failed to inhibit autophagy and cell death suggesting that autophagy-induced cell death occurred independent of apoptosis. Blocking ROS production induced by H(2)O(2) or 2-ME through overexpression of manganese-superoxide dismutase or using ROS scavenger 4,5-dihydroxy-1,3-benzene disulfonic acid-disodium salt decreased autophagy and cell death. Blocking autophagy did not affect H(2)O(2)- or 2-ME-induced ROS generation, suggesting that ROS generation occurs upstream of autophagy. In contrast, H(2)O(2) or 2-ME failed to significantly increase autophagy in mouse astrocytes. Taken together, ROS induced autophagic cell death in transformed and cancer cells but failed to induce autophagic cell death in non-transformed cells.     

Triggering caspase-independent cell death to combat cancer

Caspase-mediated apoptosis is a major hindrance to tumour growth and metastasis. Accordingly, defects in signalling pathways leading to the activation of caspases are common in tumours. Moreover, many tumour cells can unexpectedly survive the activation of caspases. As a result, caspase-independent cell death programmes are gaining increasing interest among cancer researchers. The heterogeneity of cancer cells with respect to their sensitivity to various death stimuli further emphasizes the need for additional death pathways in the therapeutic control of cell death. An understanding of the molecular control of alternative death pathways is beginning to emerge, being comparable with that of the molecular anatomy of apoptosis at the time of the discovery of caspases less than a decade ago. Here, newly discovered triggers and molecular regulators of alternative cell death programmes are reviewed and their potential in future cancer therapy is discussed.     

Homocysteine-thiolactone induces caspase-independent vascular endothelial cell death with apoptotic features

Objective. Cell death is generally classified into two large categories: apoptosis, which represents active, physiological programmed cell death, and necrosis, which represents passive cell death without underlying regulatory mechanisms. Apoptosis plays an important role in tissue homeostasis and its role in endothelium integrity can be influenced by the functional status of endothelial cells. Homocysteine, a sulfated amino-acid product of methionine demethylation, is an independent risk factor for vascular disease (arterial and venous thombosis). Our goal was to investigate the thiol-derivatives effect on the endothelial cell apoptosis. Methods. Three parameters were measured: mitochondrial membrane potential using DiOC₆(3) as the probe, DEVDase activation, and phosphatidylserine exposure on the cell surface with fluorosceinated annexin V labeling which allows apoptosis to be distinguished from necrosis. Results. Homocysteine-thiolactone induced endothelial cell apoptosis in a concentration-dependent manner (range: 50–200 M), independently of the caspase pathway. Only homocysteine-thiolactone, among the thiol derivatives tested, induced apoptosis. Apoptosis was not influenced by the serum concentration in culture medium, suggesting that the observed apoptotic process could occur in vivo. None of the inhibitors used (e.g., leupeptin, fumosinin Bl, catalase, or z-VAD-fmk) was able to prevent homocysteine-induced apoptosis of vascular endothelial cells. Conclusion. The apoptosis of vascular endothelial cells induced by high concentration of homocysteine-thiolactone might be one step atherosclerotic cardiovascular disease, and contribute to its complication.     

Inhibition of NFkappaB induces caspase-independent cell death in human T lymphocytes

Nuclear factor kappaB (NFkappaB) regulates the expression of various genes essential for cell survival. Here we demonstrate that suppression of NFkappaB nuclear import with SN50 peptide carrying the nuclear localization sequence (NLS) of the NFkappaB p50 subunit induces apoptosis in human peripheral blood T lymphocytes (T-PBL), which can be blocked with the pan-caspase inhibitor Z-VAD.fmk. However, even when caspase function is blocked, the addition of SN50 induces irreversible cell loss due to the reduction in the mitochondrial transmembrane potential (DeltaPsim) followed by disruption of the cell membrane, hallmarks of necrosis. These observations demonstrate that although inhibition of NFkappaB nuclear translocation by SN50 peptide can induce caspase-dependent apoptosis in T-PBL, cell death may still proceed in the absence of functional caspase activity. The availability of downstream caspases appears to determine the mode of cell death in NFkappaB defective cells.     

Clostridium difficile toxin B induces autophagic cell death in colonocytes

Toxin B (TcdB) is a major pathogenic factor of Clostridum difficile. However, the mechanism by which TcdB exerts its cytotoxic action in host cells is still not completely known. Herein, we report for the first time that TcdB induced autophagic cell death in cultured human colonocytes. The induction of autophagy was demonstrated by the increased levels of LC3‐II, formation of LC3⁺ autophagosomes, accumulation of acidic vesicular organelles and reduced levels of the autophagic substrate p62/SQSTM1. TcdB‐induced autophagy was also accompanied by the repression of phosphoinositide 3‐kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) complex 1 activity. Functionally, pharmacological inhibition of autophagy by wortmannin or chloroquine or knockdown of autophagy‐related genes Beclin 1, Atg5 and Atg7 attenuated TcdB‐induced cell death in colonocytes. Genetic ablation of Atg5, a gene required for autophagosome formation, also mitigated the cytotoxic effect of TcdB. In conclusion, our study demonstrated that autophagy serves as a pro‐death mechanism mediating the cytotoxic action of TcdB in colonocytes. This discovery suggested that blockade of autophagy might be a novel therapeutic strategy for C. difficile infection.     

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.     

A novel diquinolonium displays preclinical anti-cancer activity and induces caspase-independent cell death

Quinolines are a class of chemical compounds with emerging anti-cancer properties. Here, we tested the activity of series of quinolines and quinoline-like molecules for anti-cancer activity and identified a novel diquinoline, 1-methyl-2-[3-(1-methyl-1,2-dihydroquinolin-2-yliden)prop-1-enyl]quinolinium iodide (Q²). Q² induced cell death in leukemia, myeloma, and solid tumor cell lines with LD50s in the low to submicromolar range. Moreover, Q² induced cell death in primary acute myeloid leukemia (AML) cells preferentially over normal hematopoietic cells. In a mouse model of leukemia, Q² delayed tumor growth. Mechanistically, Q² induced cell death through caspase independent mechanisms. By electron microscopy, Q² increased cytoplasmic vacuolization and mitochondrial swelling. Potentially consistent with the induction of autophagic cell death, Q² treatment led to a punctate distribution of LC3 and increased MDC staining. Thus, Q² is a novel quinolinium with preclinical activity in malignancies such as leukemia and myeloma and warrants further investigation.     

Nano neodymium oxide induces massive vacuolization and autophagic cell death in non-small cell lung cancer NCI-H460 cells

Neodymium, a rare earth element, was known to exhibit cytotoxic effects and induce apoptosis in certain cancer cells. Here we show that nano-sized neodymium oxide (Nano Nd2O3) induced massive vacuolization and cell death in non-small cell lung cancer NCI-H460 cells at micromolar equivalent concentration range. Cell death elicited by Nano Nd2O3 was not due to apoptosis and caspases were not involved. Electron microscopy and acridine orange staining revealed extensive autophagy in the cytoplasm of the cells treated by Nano Nd2O3. Autophagy induced by Nano Nd2O3 was accompanied by S-phase cell cycle arrest, mild disruption of mitochondrial membrane potential, and inhibition of proteasome activity. Bafilomycin A1, but not 3-MA, induced apoptosis while inhibiting autophagy. Our results revealed a novel biological function for Nano Nd2O3 and may have implications for the therapy of non-small cell lung cancer.     

CD47 ligation induces caspase-independent cell death in chronic lymphocytic leukemia.

Thrombospondin forms a 'molecular bridge' between phagocytic and apoptotic cells through interaction with alphavbeta3/CD36. We report here that engagement of CD47, a newly described thrombospondin receptor, by immobilized monoclonal antibody against CD47 or by thrombospondin induced in all B-cell chronic lymphocytic leukemia clones the cytoplasmic features of apoptosis (cell shrinkage, decrease in mitochondrial transmembrane potential and phosphatidylserine externalization) without the nuclear features (chromatin condensation, appearance of single-stranded DNA, DNA fragmentation and cleavage of poly ADP-ribose polymerase). These cytoplasmic events of apoptosis were not prevented by the addition of caspase inhibitor z-VAD-fmk, or by the presence of survival factors (such as interleukin-4 and gamma interferon) or cell activation. Morphological studies confirmed the integrity of the nucleus and showed swelling of the mitochondria. This caspase-independent death pathway may be relevant to the development of alternate therapeutic strategies in chronic lymphocytic leukemia, which remains an incurable disease.     

Endostatin induces autophagic cell death in EAhy926 human endothelial cells

Endostatin, a proteolytic fragment of collagen XVIII, is a potent inhibitor of angiogenesis and suppresses neovascularization and tumor growth. However, the inhibitory mechanism of endostatin in human endothelial cells has not been characterized yet. Electron microscopic analysis revealed that endostatin induced formation of numerous autophagic vacuoles in endothelial in 6 to 24 h after treatment. Moreover, there was only a 2- to 3-fold increase in intracellular reactive oxygen species after endostatin treatment. Endostatin-induced cell death was not prevented by antioxidants (vitamin C, vitamin E, or propyl gallate) or caspase inhibitors, suggesting that the increase of oxidative stress or the activation of caspases may not be the crucial factors in the anti-angiogenic mechanism of endostatin. However, the cytotoxicity of endostatin was significantly reduced by 3-methyladenine (a specific inhibitor of autophagy) and serine and cysteine lysosomal protease inhibitors (leupeptin and aprotinin). Taken together, these results suggest that in human endothelial cells: (1) endostatin predominantly causes autophagic, rather than apoptotic, cell death, (2) endostatin-induced autophagic cell death occurs in the absence of caspase activation and through an oxidative-independent pathway, and (3) endostatin-induced "autophagic cell death" or "type 2 physiological cell death" is regulated by serine and cysteine lysosomal proteases.     

The C-terminal moiety of HIV-1 Vpr induces cell death via a caspase-independent mitochondrial pathway

Previous biochemical studies suggested that HIV-1-encoded Vpr may kill cells through an effect on the adenine nucleotide translocase (ANT), thereby causing mitochondrial membrane permeabilization (MMP). Here, we show that Vpr fails to activate caspases in conditions in which it induces cell killing. The knock-out of essential caspase-activators (Apaf-1 or caspase-9) or the knock-out of a mitochondrial caspase-independent death effector (AIF) does not abolish Vpr-mediated killing. In contrast, the cytotoxic effects of Vpr are reduced by transfection-enforced overexpression of two MMP-inhibitors, namely the endogenous protein Bcl-2 or the cytomegalovirus-encoded ANT-targeted protein vMIA. Vpr, which can elicit MMP through a direct effect on mitochondria, and HIV-1-Env, which causes MMP through an indirect pathway, exhibit additive (but not synergic) cytotoxic effects. In conclusion, it appears that Vpr induces apoptosis through a caspase-independent mitochondrial pathway.     

Hyperthermia in combination with oxidative stress induces autophagic cell death in HT-29 colon cancer cells

The purpose of this study was to evaluate the mechanism of ROS-induced hyperthermic cell death in a colon cancer cell line. HT-29 colon cancer cells were exposed to heat (43 degrees C) in the presence of tert-butyl hydroperoxide (t-BOOH). t-BOOH combined with hyperthermia significantly decreased cell viability as compared with t-BOOH or hyperthermia alone. This decrease in cell numbers was associated with retardation in the S phase transit and not through apoptosis. Cell death was noted to be accompanied by specific features characteristic of autophagy: the presence of cytoplasmic autophagic vacuoles; autophagosome membrane association of microtubule-associated protein light chain 3; accumulation of acidic vesicular organelles; and increased incorporation of MDC in the autophagosome. Thermal sensitization through modulation of cellular ROS may represent a novel approach to increase the efficacy of hyperthermia as an anticancer modality.     

Gossypol induces apoptosis in human PC-3 prostate cancer cells by modulating caspase-dependent and caspase-independent cell death pathways

The rate of gossypol-induced apoptosis does not correlate very well with the same dose of gossypol-induced cell growth inhibition, indicating an anti-proliferative effect of gossypol. Using a co-immunoprecipitation assay, it was observed that the level of Bcl-X(L) protein bound to Bax was clearly lower than that of Bcl-2 protein at 5 micro M of gossypol treatment, and the level of Bim protein bound to Bcl-X(L) was lowered at 20 micro M of gossypol treatment for 24 h, implicating that gossypol inhibits the heterodimerization of Bcl-X(L) with Bax and Bim. Gossypol-induced apoptosis is partly suppressed by as low as 0.5 micro M, but not abolished by as high as 50 micro M of a broad range caspase inhibitor, Z-VAD-FMK, suggesting that gossypol-induced apoptosis is both caspase-dependent and -independent. Furthermore, the release of apoptosis inducing factor (AIF), which triggers caspase-independent apoptosis, from mitochondria to cytosol was observed in PC-3 cells exposed to gossypol treatment. In conclusion, gossypol inhibits the proliferation and induces apoptosis in PC-3 cells. Gossypol-induced apoptosis is, at least, through inhibiting the heterodimerization of Bcl-X(L)/Bcl-2 with pro-apoptosis molecules, followed by a caspase-dependent and -independent process which involves the release of AIF from the mitochondria to cytosol.