Marchantin M: a novel inhibitor of proteasome induces autophagic cell death in prostate cancer cells

We previously reported that marchantin M (Mar) is an active agent to induce apoptosis in human prostate cancer (PCa), but the molecular mechanisms of action remain largely unknown. Here, we demonstrate that Mar potently inhibited chymotrypsin-like and peptidyl-glutamyl peptide-hydrolyzing activities of 20S proteasome both in in vitro and intracellular systems and significantly induced the accumulation of polyubiquitinated proteins in PCa cells. The computational modeling analysis suggested that Mar non-covalently bound to active sites of proteasome β5 and β1 subunits, resulting in a non-competitive inhibition. Proteasome inhibition by Mar subsequently resulted in endoplasmic reticulum (ER) stress, as evidenced by elevated glucose-regulated protein 78 and CHOP, increased phospho-eukaryotic translation initiation factor 2α (eIF_2α), splicing of X-box-binding protein-1 and dilation of the ER. However, Mar-mediated cell death was not completely impaired by a pan inhibitor of caspases. Further studies revealed that the Mar-induced cell death was greatly associated with the activation of autophagy, as indicated by the significant induction of microtubule-associated protein-1 light chain-3 beta (LC3B) expression and conversion. Electron microscopic and green fluorescent protein-tagged LC3B analyses further demonstrated the ability of autophagy induction by Mar. Time kinetic studies revealed that Mar induced a rapid and highly sustained processing of LC3B in treated cells and simultaneously decreased the expression of p62/SQSTM1. Pharmacological blockade or knockdown of LC3B and Atg5 attenuated Mar-mediated cell death. The autophagic response triggered by Mar required the activation of RNA-dependent protein kinase-like ER kinase/eIF_2α and suppression of the phosphatidylinositol-3 kinase/Akt/mammalian target of rapamycin axis via preventing activation and expression of Akt. Our results identified a novel mechanism for the cytotoxic effect of Mar, which strengthens it as a potential agent in cancer chemotherapy.     

Autophagonizer, a novel synthetic small molecule, induces autophagic cell death

Autophagy is an apoptosis-independent mechanism of cell death that protects the cell from environmental imbalances and infection by pathogens. We identified a novel small molecule, 2-(3-Benzyl-4-oxo-3,4,5,6,7,8-hexahydro-benzo[4,5]thieno[2,3-d]pyrimidin-2-ylsulfanylmethyl)-oxazole-4-carboxylic acid (2-pyrrolidin-1-yl-ethyl)-amide (referred as autophagonizer), using high-content cell-based screening and the autophagosome marker EGFP-LC3. Autophagonizer inhibited growth and induced cell death in the human tumor cell lines MCF7, HeLa, HCT116, A549, AGS, and HT1080 via a caspase-independent pathway. Conversion of cytosolic LC3-I to autophagosome-associated LC3-II was greatly enhanced by autophagonizer treatment. Transmission electron microscopy and acridine orange staining revealed increased autophagy in the cytoplasm of autophagonizer-treated cells. In conclusion, autophagonizer is a novel autophagy inducer with unique structure, which induces autophagic cell death in the human tumor cell lines.     

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.     

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.     

A novel Bcl-XL inhibitor Z36 that induces autophagic cell death in Hela cells

Inhibition of Bcl2 family proteins Bcl-X_L and Bcl-2 represents a promising drug development strategy for cancer treatment by triggering apoptosis in cancer cells. Here we report a novel Bcl-XL inhibitor, Z36, which unexpectedly induces only autophagic cell death, but not apoptosis. This special property distinguishes Z36 from other previously reported Bcl-X_L and Bcl-2 inhibitors that induce cancer cell death mainly through apoptosis, and makes Z36 an attractive molecular tool for studying the cellular regulation of autophagic cell death and apoptosis.     

Saikosaponin-d,a novel SERCA inhibitor,induces autophagic cell death in apoptosis-defective cells

Autophagy is an important cellular process that controls cells in a normal homeostatic state by recycling nutrients to maintain cellular energy levels for cell survival via the turnover of proteins and damaged organelles. However, persistent activation of autophagy can lead to excessive depletion of cellular organelles and essential proteins, leading to caspase-independent autophagic cell death. As such, inducing cell death through this autophagic mechanism could be an alternative approach to the treatment of cancers. Recently, we have identified a novel autophagic inducer, saikosaponin-d (Ssd), from a medicinal plant that induces autophagy in various types of cancer cells through the formation of autophagosomes as measured by GFP-LC3 puncta formation. By computational virtual docking analysis, biochemical assays and advanced live-cell imaging techniques, Ssd was shown to increase cytosolic calcium level via direct inhibition of sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase pump, leading to autophagy induction through the activation of the Ca²⁺/calmodulin-dependent kinase kinase–AMP-activated protein kinase–mammalian target of rapamycin pathway. In addition, Ssd treatment causes the disruption of calcium homeostasis, which induces endoplasmic reticulum stress as well as the unfolded protein responses pathway. Ssd also proved to be a potent cytotoxic agent in apoptosis-defective or apoptosis-resistant mouse embryonic fibroblast cells, which either lack caspases 3, 7 or 8 or had the Bax-Bak double knockout. These results provide a detailed understanding of the mechanism of action of Ssd, as a novel autophagic inducer, which has the potential of being developed into an anti-cancer agent for targeting apoptosis-resistant cancer cells.     

CCL2 protects prostate cancer PC3 cells from autophagic death via phosphatidylinositol 3-kinase/AKT-dependent survivin up-regulation

Resistance to cell death is a hallmark of cancer. Autophagy is a survival mechanism activated in response to nutrient deprivation; however, excessive autophagy will ultimately induce cell death in a nonapoptotic manner. The present study demonstrates that CCL2 protects prostate cancer PC3 cells from autophagic death, allowing prolonged survival in serum-free conditions. Upon serum starvation, CCL2 induced survivin up-regulation in PC3, DU 145, and C4-2B prostate cancer cells. Both cell survival and survivin expression were stunted in CCL2-stimulated PC3 cells when treated either with the phosphatidylinositol 3-kinase inhibitor LY294002 (2 mum) or the Akt-specific inhibitor-X (Akti-X; 2.5 mum). Furthermore, CCL2 significantly reduced light chain 3-II (LC3-II) in serum-starved PC3; in contrast, treatment with LY294002 or Akti-X reversed the effect of CCL2 on LC3-II levels, suggesting that CCL2 signaling limits autophagy in these cells. Upon serum deprivation, the analysis of LC3 localization by immunofluorescence revealed a remarkable reduction in LC3 punctate after CCL2 stimulation. CCL2 treatment also resulted in a higher sustained mTORC1 activity as measured by an increase in phospho-p70S6 kinase (Thr(389)). Rapamycin, an inducer of autophagy, both down-regulated survivin and decreased PC3 cell viability in serum-deprived conditions. Treatment with CCL2, however, allowed cells to partially resist rapamycin-induced death, which correlated with survivin protein levels. In two stable transfectants expressing survivin-specific short hairpin RNA, generated from PC3, survivin protein levels controlled both cell viability and LC3 localization in response to CCL2 treatment. Altogether, these findings indicate that CCL2 protects prostate cancer PC3 cells from autophagic death via the phosphatidylinositol 3-kinase/Akt/survivin pathway and reveal survivin as a critical molecule in this survival mechanism.     

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.     

Silencing of Bcl-2 expression by small interfering RNA induces autophagic cell death in MCF-7 breast cancer cells

Apoptosis (programmed cell death type I) and autophagy (type II) are crucial mechanisms regulating cell death and homeostasis. The Bcl-2 proto-oncogene is overexpressed in 50-70% of breast cancers, potentially leading to resistance to chemotherapy, radiation and hormone therapy-induced apoptosis. Here, we investigated the role of Bcl-2 in autophagy in breast cancer cells. Silencing of Bcl-2 by siRNA in MCF-7 breast cancer cells downregulated Bcl-2 protein levels (>85%) and led to inhibition of cell growth (71%) colony formation (79%), and cell death (up to 55%) by autophagy but not apoptosis. Induction of autophagy was demonstrated by acridine orange staining, electron microscopy and an accumulation of GFP-LC3-II in autophagosomal membranes in MCF-7 cells transfected with GFP-LC-3(GFP-ATG8). Silencing of Bcl-2 by siRNA also led to induction of LC-3-II, a hallmark of autophagy, ATG5 and Beclin-1 autophagy promoting proteins. Knockdown of ATG5 significantly inhibited Bcl-2 siRNA-induced LC3-II expression, the number of GFP-LC3-II-labeled autophagosome positive cells and autophagic cell death (p < 0.05). Furthermore, doxorubicin at a high dose (IC(95), 1 microM) induced apoptosis but at a low dose (IC(50), 0.07 microM) induced only autophagy and Beclin-1 expression. When combined with Bcl-2 siRNA, doxorubicin (IC(50)) enhanced autophagy as indicated by the increased number cells with GFP-LC3-II-stained autophagosomes (punctuated pattern positive). These results provided the first evidence that targeted silencing of Bcl-2 induces autophagic cell death in MCF-7 breast cancer cells and that Bcl-2 siRNA may be used as a therapeutic strategy alone or in combination with chemotherapy in breast cancer cells that overexpress Bcl-2.     

Silencing of Bcl-2 expression by small interfering RNA induces autophagic cell death in MCF-7 breast cancer cells

Apoptosis (programmed cell death type I) and autophagy (type II) are crucial mechanisms regulating cell death and homeostasis. The Bcl-2 proto-oncogene is overexpressed in 50-70% of breast cancers, potentially leading to resistance to chemotherapy, radiation and hormone therapy induced apoptosis. In this study, we investigated the role of Bcl-2 in autophagy in breast cancer cells. Silencing of Bcl-2 by siRNA in MCF-7 breast cancer cells downregulated Bcl-2 protein levels (>85%) and led to inhibition of cell growth (71%) colony formation (79%), and cell death (up to 55%) by autophagy but not apoptosis. Induction of autophagy was demonstrated by acridine orange staining, electron microscopy and an accumulation of GFP-LC3-II in preautopghagosomal and autophagosomal membranes in MCF-7 cells transfected with GFP-LC-3(GFP-ATG8). Silencing of Bcl-2 by siRNA also led to induction of LC-3-II, a hallmark of autophagy, ATG5 and Beclin-1 autophagy promoting proteins. Knockdown of ATG5 significantly inhibited Bcl-2 siRNA-induced LC3-II expression and the number of GFP-LC3-II-labeled autophagosome (punctuated pattern) positive cells and autophagic cell death (p     

Hypoxia induces autophagic cell death in apoptosis-competent cells through a mechanism involving BNIP3

Hypoxia (lack of oxygen) is a physiological stress often associated with solid tumors. Hypoxia correlates with poor prognosis since hypoxic regions within tumors are considered apoptosis-resistant. Autophagy (cellular “self digestion”) has been associated with hypoxia during cardiac ischemia and metabolic stress as a survival mechanism. However, although autophagy is best characterized as a survival response, it can also function as a mechanism of programmed cell death. Our results show that autophagic cell death is induced by hypoxia in cancer cells with intact apoptotic machinery. We have analyzed two glioma cell lines (U87, U373), two breast cancer cell lines (MDA-MB-231, ZR75) and one embryonic cell line (HEK293) for cell death response in hypoxia (     

Hypoxia induces autophagic cell death in apoptosis-competent cells through a mechanism involving BNIP3

Hypoxia (lack of oxygen) is a physiological stress often associated with solid tumors. Hypoxia correlates with poor prognosis since hypoxic regions within tumors are considered apoptosisresistant. Autophagy (cellular "self digestion") has been associated with hypoxia during cardiac ischemia and metabolic stress as a survival mechanism. However, although autophagy is best characterized as a survival response, it can also function as a mechanism of programmed cell death. Our results show that autophagic cell death is induced by hypoxia in cancer cells with intact apoptotic machinery. We have analyzed two glioma cell lines (U87, U373), two breast cancer cell lines (MDA-MB-231, ZR75) and one embryonic cell line (HEK293) for cell death response in hypoxia (<1% O(2)). Under normoxic conditions, all five cell lines undergo etoposide-induced apoptosis whereas hypoxia fails to induce these apoptotic responses. All five cell lines induce an autophagic response and undergo cell death in hypoxia. Hypoxia-induced cell death was reduced upon treatment with the autophagy inhibitor 3-methyladenine, but not with the caspase inhibitor z-VAD-fmk. By knocking down the autophagy proteins Beclin-1 or ATG5, hypoxia-induced cell death was also reduced. The pro-cell death Bcl-2 family member BNIP3 (Bcl-2/adenovirus E1B 19kDainteracting protein 3) is upregulated during hypoxia and is known to induce autophagy and cell death. We found that BNIP3 overexpression induced autophagy, while expression of BNIP3 siRNA or a dominant-negative form of BNIP3 reduced hypoxia-induced autophagy. Taken together, these results suggest that prolonged hypoxia induces autophagic cell death in apoptosis-competent cells, through a mechanism involving BNIP3.     

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.     

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.     

Glucosamine induces autophagic cell death through the stimulation of ER stress in human glioma cancer cells

Autophagy can promote cell survival or death, but the molecular basis of its dual role in cancer is not well understood. Here, we report that glucosamine induces autophagic cell death through the stimulation of endoplasmic reticulum (ER) stress in U87MG human glioma cancer cells. Treatment with glucosamine reduced cell viability and increased the expression of LC3 II and GFP-LC3 fluorescence puncta, which are indicative of autophagic cell death. The glucosamine-mediated suppression of cell viability was reversed by treatment with an autophagy inhibitor, 3-MA, and interfering RNA against Atg5. Glucosamine-induced ER stress was manifested by the induction of BiP, IRE1α, and phospho-eIF2α expression. Chemical chaperon 4-PBA reduced ER stress and thereby inhibited glucosamine-induced autophagic cell death. Taken together, our data suggest that glucosamine induces autophagic cell death by inducing ER stress in U87MG glioma cancer cells and provide new insight into the potential anticancer properties of glucosamine.     

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.     

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.