Cell death in fetal oocytes: Many players for multiple pathways

We devised a short-term culture system allowing us to define novel characteristics of programmed cell death (PCD) of fetal oocytes and to underscore new aspects of this process. Mouse fetal oocytes cultured in conditions allowing meiotic progression underwent apoptotic degeneration as revealed by TUNEL staining, DNA ladder, Annexin V binding, PARP cleavage and, usually, caspase activation. TEM observations show, however, recurrent atypical apoptotic morphologies characterized by the absence of chromatin margination and nuclear fragmentation; oocytes with autophagic and necrotic features are also observed. Moreover, under the fluorescence microscope a subpopulation of TUNEL⁺ oocytes appear morphologically healthy and do not show detectable caspase activity. Finally, caspase inhibitors are able to slow down, but not to abolish, oocyte cell death, whereas calpain inhibitor I significantly reduces the number of TUNEL⁺ oocytes after 4 days of culture, and rapamycin (mTOR inhibitor) increases such numbers both at day 3 and 4. These observations together with results showing expression in cultured oocytes undergoing cell death of apoptosis inducing factor and Beclin 1, two important players of caspase-independent and autophagic cell death, respectively, demonstrate that fetal oocytes posses and are able to activate several players of various forms of cell death. However, causal correlation among different cell death pathways in such oocytes remains to be determined and stimuli causing the activation of these pathways in vitro and in vivo also clarified.

Addendum to: Lobascio AM, Klinger FG, Scaldaferri ML, Farini D, De Felici M. Analysis of programmed cell death in mouse fetal oocytes. Reproduction 2007; 134:241-52.     

T cell receptor ligation triggers novel nonapoptotic cell death pathways that are Fas-independent or Fas-dependent

Short-term culture of activated T cells with IL-2 renders them highly susceptible to apoptotic death triggered by TCR cross-linking. Activation-induced apoptosis is contingent upon caspase activation and this is mediated primarily by Fas/Fas ligand (FasL) interactions that, in turn, are optimized by p38 mitogen-activated protein kinase (MAPK)-regulated signals. Although T cells from mice bearing mutations in Fas (lpr) or FasL (gld) are more resistant to activation-induced cell death (AICD) than normal T cells, a significant proportion of CD8(+) T cells and to a lesser extent CD4(+) T cells from mutant mice die after TCR religation. Little is known about this Fas-independent death process. In this study, we demonstrate that AICD in lpr and gld CD4(+) and CD8(+) T cells occurs predominantly by a novel mechanism that is TNF-alpha-, caspase-, and p38 MAPK-independent and has morphologic features more consistent with oncosis/primary necrosis than apoptosis. A related Fas- and caspase-independent, nonapoptotic death process is revealed in wild-type (WT) CD8(+) T cell blasts following TCR ligation and treatment with caspase inhibitors, the p38 MAPK inhibitor, SB203580, or neutralizing anti-FasL mAb. In parallel studies with WT CD4(+) T cells, two minor pathways leading to nonapoptotic, caspase-independent AICD were identified, one contingent upon Fas ligation and p38 MAPK activation and the other Fas- and p38 MAPK-independent. These data indicate that TCR ligation can activate nonapoptotic death programs in WT CD8(+) and CD8(+) T blasts that normally are masked by Fas-mediated caspase activation. Selective use of potentially proinflammatory oncotic death programs by activated lpr and gld T cells may be an etiologic factor in autosensitization.     

Abrin induces HeLa cell apoptosis by cytochrome c release and caspase activation

We identified apoptosis as being a significant mechanism of toxicity following the exposure of HeLa cell cultures to abrin holotoxin, which is in addition to its inhibition of protein biosynthesis by N-glycosidase activity. The treatment of HeLa cell cultures with abrin resulted in apoptotic cell death, as characterized by morphological and biochemical changes, i.e., cell shrinkage, internucleosomal DNA fragmentation, the occurrence of hypodiploid DNA, chromatin condensation, nuclear breakdown, DNA single strand breaks by TUNEL assay, and phosphatidylserine (PS) externalization. This apoptotic cell death was accompanied by caspase-9 and caspase-3 activation, as indicated by the cleavage of caspase substrates, which was preceded by mitochondrial cytochrome c release. The broad-spectrum caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVADfmk), prevented abrin-triggered caspase activation and partially abolished apoptotic cell death, but did not affect mitochondrial cytochrome c release. These results suggest that the release of mitochondrial cytochrome c, and the sequential caspase-9 and caspase-3 activations are important events in the signal transduction pathway of abrin-induced apoptotic cell death in the HeLa cell line.     

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.     

Deficiency in the mitochondrial apoptotic pathway reveals the toxic potential of autophagy under ER stress conditions

Endoplasmic reticulum (ER) stress-induced cell death is normally associated with activation of the mitochondrial apoptotic pathway, which is characterized by CYCS (cytochrome c, somatic) release, apoptosome formation, and caspase activation, resulting in cell death. In this study, we demonstrate that under conditions of ER stress cells devoid of CASP9/caspase-9 or BAX and BAK1, and therefore defective in the mitochondrial apoptotic pathway, still undergo a delayed form of cell death associated with the activation of caspases, therefore revealing the existence of an alternative stress-induced caspase activation pathway. We identified CASP8/caspase-8 as the apical protease in this caspase cascade, and found that knockdown of either of the key autophagic genes, ATG5 or ATG7, impacted on CASP8 activation and cell death induction, highlighting the crucial role of autophagy in the activation of this novel ER stress-induced death pathway. In line with this, we identified a protein complex composed of ATG5, FADD, and pro-CASP8 whose assembly coincides with caspase activation and cell death induction. Together, our results reveal the toxic potential of autophagy in cells undergoing ER stress that are defective in the mitochondrial apoptotic pathway, and suggest a model in which the autophagosome functions as a platform facilitating pro-CASP8 activation. Chemoresistance, a common problem in the treatment of cancer, is frequently caused by the downregulation of key mitochondrial death effector proteins. Alternate stress-induced apoptotic pathways, such as the one described here, may become of particular relevance for tackling the problem of chemoresistance in cancer cells.     

Caspases and neuronal development

Recent developments have shown that inappropriate activation of apoptotic pathways contributes to many neurodegenerative diseases. The basic mechanisms that underlie apoptosis in neurodegenerative diseases are uncertain, although they likely represent the subversion of normal developmental programs. Several types of neuronal cell death have been reported, including autophagic and caspase-independent cell death. In this review we consider evidence for the participation of apoptotic caspases in neuronal development, and examine the hypothesis that differentiating neurons undergo stage-specific alterations in apoptosis sensitivity that may be due to caspase regulation. In addition, we present data supporting this hypothesis.     

Deficiency in the mitochondrial apoptotic pathway reveals the toxic potential of autophagy under ER stress conditions

Endoplasmic reticulum (ER) stress-induced cell death is normally associated with activation of the mitochondrial apoptotic pathway, which is characterized by CYCS (cytochrome c, somatic) release, apoptosome formation, and caspase activation, resulting in cell death. In this study, we demonstrate that under conditions of ER stress cells devoid of CASP9/caspase-9 or BAX and BAK1, and therefore defective in the mitochondrial apoptotic pathway, still undergo a delayed form of cell death associated with the activation of caspases, therefore revealing the existence of an alternative stress-induced caspase activation pathway. We identified CASP8/caspase-8 as the apical protease in this caspase cascade, and found that knockdown of either of the key autophagic genes, ATG5 or ATG7, impacted on CASP8 activation and cell death induction, highlighting the crucial role of autophagy in the activation of this novel ER stress-induced death pathway. In line with this, we identified a protein complex composed of ATG5, FADD, and pro-CASP8 whose assembly coincides with caspase activation and cell death induction. Together, our results reveal the toxic potential of autophagy in cells undergoing ER stress that are defective in the mitochondrial apoptotic pathway, and suggest a model in which the autophagosome functions as a platform facilitating pro-CASP8 activation. Chemoresistance, a common problem in the treatment of cancer, is frequently caused by the downregulation of key mitochondrial death effector proteins. Alternate stress-induced apoptotic pathways, such as the one described here, may become of particular relevance for tackling the problem of chemoresistance in cancer cells.     

Paraptosis‐like cell death in Wistar rat granulosa cells

Follicular atresia, a common process present in all mammals, involves apoptotic and autophagic cell death. However, the participation of paraptosis, a type of caspase‐independent cell death, during follicular atresia is unknown. This study found swollen endoplasmic reticulum in the granulosa cells of adult Wistar rats. Calnexin was used as a marker of the endoplasmic reticulum at the ultrastructural and optical levels. The cells with swelling of the endoplasmic reticulum were negative to the TUNEL assay and active caspase‐3 immunodetection, indicating that this swelling is not part of any apoptotic or autophagic process. Additionally, immunodetection of the CHOP protein was used as a marker of endoplasmic reticulum stress, and this confirmed the presence of the paraptosis process. These data suggest that paraptosis‐like cell death is associated with the death of granulosa cells during follicular atresia in adult Wistar rats.     

zVAD-induced autophagic cell death requires c-Src-dependent ERK and JNK activation and reactive oxygen species generation

The treatment of L929 fibrosarcoma cells with zVAD has been shown to induce necroptosis. However, whether autophagy is involved or not in this event remains controversial. In this study, we re-examined the role of autophagy in zVAD-induced cell death in L929 cells and further elucidated the signaling pathways triggered by caspase inhibition and contributing to autophagic death. First, we found that zVAD can stimulate LC3-II formation, autophagosome and autolysosome formation, and ROS accumulation. Antioxidants, beclin 1 or Atg5 silencing, and class III PtdIns3K inhibitors all effectively blocked ROS production and cell death, suggesting ROS accumulation downstream of autophagy contributes to cell necrosis. zVAD also stimulated PARP activation, and the PARP inhibitor DPQ can reduce zVAD-induced cell death, but did not affect ROS production, suggesting the increased ROS leads to PARP activation and cell death. Notably, our data also indicated the involvement of Src-dependent JNK and ERK in zVAD-induced ROS production and autophagic death. We found caspase 8 is associated with c-Src at the resting state, and upon zVAD treatment this association was decreased and accompanied by c-Src activation. In conclusion, we confirm the autophagic death in zVAD-treated L929 cells, and define a new molecular pathway in which Src-dependent ERK and JNK activation can link a signal from caspase inhibition to autophagy, which in turn induce ROS production and PARP activation, eventually leading to necroptosis. Thus, in addition to initiating proteolytic activity for cell apoptosis, inactivated caspase 8 also functions as a signaling molecule for autophagic death.     

Autophagic and apoptotic features during programmed cell death in the fat body of the tobacco hornworm (Manduca sexta)

Two major pathways of programmed cell death (PCD)--the apoptotic and the autophagic cell death--were investigated in the decomposition process of the larval fat body during the 5th larval stage of Manduca sexta. Several basic aspects of apoptotic and autophagic cell death were analyzed by morphological and biochemical methods in order to disclose whether these mechanisms do have shared common regulatory steps. Morphological examination revealed the definite autophagic wave started on day 4 followed by DNA fragmentation as demonstrated by agarose gel electrophoresis and TUNEL assay. By the end of the wandering period the cells were filled with autophagic vacuoles and protein granules of heterophagic origin and the vast majority of the nuclei were TUNEL-positive. No evidence was found of other aspects of apoptosis, e.g. activation of executioner caspases. Close correlation was disclosed between the onset of autophagy and the nuclear accumulation of the ubiquitin-proteasome system.     

Caspases function in autophagic programmed cell death in Drosophila

Self-digestion of cytoplasmic components is the hallmark of autophagic programmed cell death. This auto-degradation appears to be distinct from what occurs in apoptotic cells that are engulfed and digested by phagocytes. Although much is known about apoptosis, far less is known about the mechanisms that regulate autophagic cell death. Here we show that autophagic cell death is regulated by steroid activation of caspases in Drosophila salivary glands. Salivary glands exhibit some morphological changes that are similar to apoptotic cells, including fragmentation of the cytoplasm, but do not appear to use phagocytes in their degradation. Changes in the levels and localization of filamentous Actin, alpha-Tubulin, alpha-Spectrin and nuclear Lamins precede salivary gland destruction, and coincide with increased levels of active Caspase 3 and a cleaved form of nuclear Lamin. Mutations in the steroid-regulated genes beta FTZ-F1, E93, BR-C and E74A that prevent salivary gland cell death possess altered levels and localization of filamentous Actin, alpha-Tubulin, alpha-Spectrin, nuclear Lamins and active Caspase 3. Inhibition of caspases, by expression of either the caspase inhibitor p35 or a dominant-negative form of the initiator caspase Dronc, is sufficient to inhibit salivary gland cell death, and prevent changes in nuclear Lamins and alpha-Tubulin, but not to prevent the reorganization of filamentous Actin. These studies suggest that aspects of the cytoskeleton may be required for changes in dying salivary glands. Furthermore, caspases are not only used during apoptosis, but also function in the regulation of autophagic cell death.     

Differential involvement of initiator caspases in apoptotic volume decrease and potassium efflux during Fas- and UV-induced cell death.

Caspase activation and apoptotic volume decrease are fundamental features of programmed cell death; however, the relationship between these components is not well understood. Here we provide biochemical and genetic evidence for the differential involvement of initiator caspases in the apoptotic volume decrease during both intrinsic and extrinsic activation of apoptosis. Apoptosis induction in Jurkat T lymphocytes by Fas receptor engagement (intrinsic) or ultraviolet (UV)-C radiation (extrinsic) triggered the loss of cell volume, which was restricted to cells with diminished intracellular K(+) ions. These characteristics kinetically coincided with the proteolytic processing and activation of both initiator and effector caspases. Although the polycaspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone completely inhibited the Fas-mediated apoptotic volume decrease and K(+) efflux, it was much less effective in preventing these processes during UV-induced cell death under conditions whereby caspase activities and DNA degradation were blocked. To define the roles of specific initiator caspases, we utilized Jurkat cells genetically deficient in caspase-8 or stably transfected with a dominant-negative mutant of caspase-9. The results show that the activation of caspase-8, but not caspase-9, is necessary for Fas-induced apoptosis. Conversely, caspase-9, but not caspase-8, is important for UV-mediated shrunken morphology and apoptosis progression. Together, these findings indicate that cell shrinkage and K(+) efflux during apoptosis are tightly coupled, but are differentially regulated by either caspase-8 or caspase-9 depending on specific pathways of cell death.     

Death of CD4(+) T-cell lines caused by human immunodeficiency virus type 1 does not depend on caspases or apoptosis

A critical aspect of AIDS pathogenesis that remains unclear is the mechanism by which human immunodeficiency virus type 1 (HIV-1) induces death in CD4(+) T lymphocytes. A better understanding of the death process occurring in infected cells may provide valuable insight into the viral component responsible for cytopathicity. This would aid the design of preventive treatments against the rapid decline of CD4(+) T cells that results in AIDS. Previously, apoptotic cell death has been reported in HIV-1 infections in cultured T cells, and it has been suggested that this could affect both infected and uninfected cells. To evaluate the mechanism of this effect, we have studied HIV-1-induced cell death extensively by infecting several T-cell lines and assessing the level of apoptosis by using various biochemical and flow cytometric assays. Contrary to the prevailing view that apoptosis plays a prominent role in HIV-1-mediated T-cell death, we found that Jurkat and H9 cells dying from HIV-1 infection fail to exhibit the collective hallmarks of apoptosis. Among the parameters investigated, Annexin V display, caspase activity and cleavage of caspase substrates, TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) signal, and APO2.7 display were detected at low to negligible levels. Neither peptide caspase inhibitors nor the antiapoptotic proteins Bcl-x(L) or v-FLIP could prevent cell death in HIV-1-infected cultures. Furthermore, Jurkat cell lines deficient in RIP, caspase-8, or FADD were as susceptible as wild-type Jurkat cells to HIV-1 cytopathicity. These results suggest that the primary mode of cytopathicity by laboratory-adapted molecular clones of HIV-1 in cultured cell lines is not via apoptosis. Rather, cell death occurs most likely via a necrotic or lytic form of death independent of caspase activation in directly infected cells.     

Amino acid starvation induced autophagic cell death in PC-12 cells: Evidence for activation of caspase-3 but not calpain-1

While the apoptotic and necrotic cell death pathways have been well studied, there lacks a comprehensive understanding of the molecular events involving autophagic cell death. We examined the potential roles of the apoptosis-linked caspase-3 and the necrosis/apoptosis-linked calpain-1 after autophagy induction under prolonged amino acid (AA) starvation conditions in PC-12 cells. Autophagy induction was observed as early as three hours following amino acid withdrawal. Cell death, measured by lactate dehydrogenase (LDH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays occurred within 24 h following starvation and was accompanied by an upregulation in caspase-3 activity but not calpain-1. The cell death that occurred following AA starvation was significantly alleviated by treatment with the autophagy inhibitor 3-methyl adenine but not with the broad spectrum caspase inhibitors. Thus, this study demonstrates that 3-methyladenine-sensitive autophagic cell death due to AA starvation in PC-12 cells is mechanistically and biochemically similar to, yet distinct from, classic caspase dependent apoptosis. Shankar Sadasivan and Anu Waghray have contributed equally to this work.     

Celastrol inhibits growth and induces apoptotic cell death in melanoma cells via the activation ROS-dependent mitochondrial pathway and the suppression of PI3K/AKT signaling

Celastrol has been reported to possess anticancer effects in various cancers; however, the precise mechanism underlying ROS-mediated mitochondria-dependent apoptotic cell death triggered by celastrol treatment in melanoma cells remains unknown. We showed that celastrol effectively induced apoptotic cell death and inhibited tumor growth using tissue culture and in vivo models of B16 melanoma. In addition to apoptotic cell death in B16 cells, several apoptotic events such as PARP cleavage and activation of caspase were confirmed. Pretreatment with caspase inhibitor modestly attenuated the celastrol-induced increase in PARP cleavage and sub-G1 cell population, implying that caspases play a partial role in celastrol-induced apoptosis. Moreover, ROS generation was detected following celastrol treatment. Blocking of ROS accumulation with ROS scavengers resulted in inhibition of celastrol-induced Bcl-2 family-mediated apoptosis, indicating that celastrol-induced apoptosis involves ROS generation as well as an increase in the Bax/Bcl-2 ratio leading to release of cytochrome c and AIF. Importantly, silencing of AIF by transfection of siAIF into cells remarkably attenuated celastrol-induced apoptotic cell death. Moreover, celastrol inhibited the activation of PI3K/AKT/mTOR signaling cascade in B16 cells. Our data reveal that celastrol inhibits growth and induces apoptosis in melanoma cells via the activation of ROS-mediated caspase-dependent and -independent pathways and the suppression of PI3K/AKT signaling.     

Active ras triggers death in glioblastoma cells through hyperstimulation of macropinocytosis

Expression of activated Ras in glioblastoma cells induces accumulation of large phase-lucent cytoplasmic vacuoles, followed by cell death. This was previously described as autophagic cell death. However, unlike autophagosomes, the Ras-induced vacuoles are not bounded by a double membrane and do not sequester organelles or cytoplasm. Moreover, they are not acidic and do not contain the autophagosomal membrane protein LC3-II. Here we show that the vacuoles are enlarged macropinosomes. They rapidly incorporate extracellular fluid-phase tracers but do not sequester transferrin or the endosomal protein EEA1. Ultimately, the cells expressing activated Ras detach from the substratum and rupture, coincident with the displacement of cytoplasm with huge macropinosome-derived vacuoles. These changes are accompanied by caspase activation, but the broad-spectrum caspase inhibitor carbobenzoxy-Val-Ala-Asp-fluoromethylketone does not prevent cell death. Moreover, the majority of degenerating cells do not exhibit chromatin condensation typical of apoptosis. These observations provide evidence for a necrosis-like form of cell death initiated by dysregulation of macropinocytosis, which we have dubbed "methuosis." An activated form of the Rac1 GTPase induces a similar form of cell death, suggesting that Ras acts through Rac-dependent signaling pathways to hyperstimulate macropinocytosis in glioblastoma. Further study of these signaling pathways may lead to the identification of other chemical and physiologic triggers for this unusual form of cell death.     

Combined apoptosis and autophagy,the process that eliminates the oocytes of atretic follicles in immature rats

We studied the alterations of dying oocytes in 1–28 days old rats using TUNEL method, immunolocalizations of active caspase 3, lamp1, localization of acid phosphatase, and DAPI staining. All procedures were performed in adjacent sections of each oocyte. In most dying oocytes exist simultaneously features of apoptosis as active caspase 3 and DNA breaks, and a large increase of lamp1 and acid phosphatase characteristic of autophagy. Large clumps of compact chromatin and membrane blebbing were absent. Electron microscope observations demonstrated the presence of small clear vesicles and autophagolysosomes. All these features indicate that a large number of oocytes are eliminated by a process sharing features of apoptosis and autophagy. In dying oocytes of new born rats the markers of apoptosis predominate over those of autophagy. However, fragmentation and apoptotic bodies were not found. These features suggest that in different cytophysiological conditions the processes of cell death may be differently modulated.     

Cell proliferation and apoptosis in prostate cancer: significance in disease progression and therapy

Recent biochemical and genetic studies have substantially increased our understanding of death signal transduction pathways, making it clear however, that apoptosis is not a single-lane, one-way street. Rather, multiple parallel pathways have been identified. For instance, analysis of bcl-2, bax, p53, and caspase knockout mice while establishing distinct roles for each of these apoptotic players, they also provided valuable information for the design of specific inhibitors of apoptosis. Thus blocking one pathway, as in caspase knockout mice, what we observe is not a complete suppression of apoptosis but rather a delay in apoptosis induction (Hakem et al., 1998; Kuida et al., 1998). In view of nature's means of ensuring activation of a compensatory apoptotic response, when one pathway fails in developing prostate cancer therapeutic interventions, the challenge remains to further dissect individual apoptotic pathways. Advances in our understanding of the integrated functions governing prostate cell proliferation and cell death, clearly suggest that effective prostate cancer therapies are not only molecularly targeted, but that are also customized to take into account the delicate balance of opposing growth influences in the ageing gland. In this review we discuss the evidence on the significance of molecular deregulation of the key players of this growth equilibrium, apoptosis and cell proliferation in prostate cancer progression, and the clinical implications of changes in the apoptotic response in disease detection and therapy.     

PARP and RIP 1 are required for autophagy induced by 11'-deoxyverticillin A, which precedes caspase-dependent apoptosis

The epipolythiodioxopiperazines (ETPs) are fungal secondary metabolites proven to trigger both apoptotic and necrotic cell death of tumor cells. However, the underlying mechanism of their regulatory role in macroautophagy and the interplay between autophagy and apoptosis initiated by the ETPs, remain unexplored. In the current work, we found that 11'-deoxyverticillin A (C42), a member of the ETPs, induces autophagosome formation, accumulation of microtubule-associated protein 1 light chain 3-II (LC3-II ) and degradation of sequestosome 1 (SQSTM1/p62). In addition, the LC3-II accrual and p62 degradation occur prior to caspase activation and coincide with PARP activation. Inhibition of autophagy by either chemical inhibitors or by RNA interference single knockdown of essential autophagic genes partially reduces the cell death and the cleavage of both caspase 3 and PARP. Necrostatin-1, a specific inhibitor of necroptosis, inhibits both the augmentation of LC3-II and the cleavage of caspase 3, which was confirmed by depletion of receptor-interacting protein 1 (RIP-1), a crucial necrostatin-1-targeted adaptor kinase mediating cell death and survival. Moreover, inhibition of PARP by either chemical inhibitors or RNA interference provides obvious protection for cell viability and suppresses the LC3-II accretion caused by C42 treatment. Interestingly, double silencing of LC3 and p62 completely suppressed PARP cleavage and concurrently and maximally augmented the PAR formation induced by C42. Collectively, we have demonstrated that C42 enhances the cellular autophagic process, which requires both PARP and RIP-1 participation, preceding and possibly augmenting, the caspase-dependent apoptotic cell death.