Anti-autophagic Bcl-2: Not just an innocent bystander

Bcl-2, originally identified as a universal inhibitor of apoptotic cell death, has since been implicated in suppressing autophagy, the cell's quality control mechanism. Our recent study demonstrates that the anti-autophagic aspect of Bcl-2 can function as a promoter of oncogenic growth, independently of its role in apoptosis signaling. It is likely that the increase in Bcl-2 often seen in breast and other cancers might render cells error-prone by blunting autophagy, while concomitantly keeping damaged cells alive. The outcome of such a 'double hit' of Bcl-2 may synergistically promote tumor growth and increase the chance of cancer development and drug resistance.     

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.     

Autophagy Provides Nutrients but Can Lead to Chop-dependent Induction of Bim to Sensitize Growth Factor–deprived Cells to Apoptosis

Tissue homeostasis is controlled by the availability of growth factors, which sustain exogenous nutrient uptake and prevent apoptosis. Although autophagy can provide an alternate intracellular nutrient source to support essential basal metabolism of apoptosis-resistant growth factor–withdrawn cells, antiapoptotic Bcl-2 family proteins can suppress autophagy in some settings. Thus, the role of autophagy and interactions between autophagy and apoptosis in growth factor–withdrawn cells expressing Bcl-2 or Bcl-xL were unclear. Here we show autophagy was rapidly induced in hematopoietic cells upon growth factor withdrawal regardless of Bcl-2 or Bcl-xL expression and led to increased mitochondrial lipid oxidation. Deficiency in autophagy-essential gene expression, however, did not lead to metabolic catastrophe and rapid death of growth factor–deprived cells. Rather, inhibition of autophagy enhanced survival of cells with moderate Bcl-2 expression for greater than 1 wk, indicating that autophagy promoted cell death in this time frame. Cell death was not autophagic, but apoptotic, and relied on Chop-dependent induction of the proapoptotic Bcl-2 family protein Bim. Therefore, although ultimately important, autophagy-derived nutrients appear initially nonessential after growth factor withdrawal. Instead, autophagy promotes tissue homeostasis by sensitizing cells to apoptosis to ensure only the most apoptosis-resistant cells survive long-term using autophagy-derived nutrients when growth factor deprived.     

Downregulation of autophagy by Bcl-2 promotes MCF7 breast cancer cell growth independent of its inhibition of apoptosis

The anti-apoptotic Bcl-2 protein, which confers oncogenic transformation and drug resistance in most human cancers, including breast cancer, has recently been shown to effectively counteract autophagy by directly targeting Beclin1, an essential autophagy mediator and tumor suppressor. However, it remains unknown whether autophagy inhibition contributes to Bcl-2-mediated oncogenesis. Here, by using a loss-of-function mutagenesis study, we show that Bcl-2-mediated antagonism of autophagy has a critical role in enhancing the tumorigenic properties of MCF7 breast cancer cells independent of its anti-apoptosis activity. A Bcl-2 mutant defective in apoptosis inhibition but competent for autophagy suppression promotes MCF7 breast cancer cell growth in vitro and in vivo as efficiently as wild-type Bcl-2. The growth-promoting activity of this Bcl-2 mutant is strongly correlated with its suppression of Beclin1-dependent autophagy, leading to sustained p62 expression and increased DNA damage in xenograft tumors, which may directly contribute to tumorigenesis. Thus, the anti-autophagic property of Bcl-2 is a key feature of Bcl-2-mediated oncogenesis and may in some contexts, serve as an attractive target for breast and other cancer therapies.     

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     

Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy

Apoptosis and autophagy are both tightly regulated biological processes that play a central role in tissue homeostasis, development, and disease. The anti-apoptotic protein, Bcl-2, interacts with the evolutionarily conserved autophagy protein, Beclin 1. However, little is known about the functional significance of this interaction. Here, we show that wild-type Bcl-2 antiapoptotic proteins, but not Beclin 1 binding defective mutants of Bcl-2, inhibit Beclin 1-dependent autophagy in yeast and mammalian cells and that cardiac Bcl-2 transgenic expression inhibits autophagy in mouse heart muscle. Furthermore, Beclin 1 mutants that cannot bind to Bcl-2 induce more autophagy than wild-type Beclin 1 and, unlike wild-type Beclin 1, promote cell death. Thus, Bcl-2 not only functions as an antiapoptotic protein, but also as an antiautophagy protein via its inhibitory interaction with Beclin 1. This antiautophagy function of Bcl-2 may help maintain autophagy at levels that are compatible with cell survival, rather than cell death.     

Vitamin K2 induces autophagy and apoptosis simultaneously in leukemia cells

Vitamin K2 (menaquinone-4: VK2) is a potent inducer for apoptosis in leukemia cells in vitro. HL-60bcl-2 cells, which are derived from a stable transfectant clone of the human bcl-2 gene into the HL-60 leukemia cell line, show 5-fold greater expression of the Bcl-2 protein compared with HL-60neo cells, a control clone transfected with vector alone. VK2 induces apoptosis in HL-60neo cells, whereas HL-60bcl-2 cells are resistant to apoptosis induction by VK2 but show inhibition of cell growth along with an increase of cytoplasmic vacuoles during exposure to VK2. Electron microscopy revealed formation of autophagosomes and autolysosomes in HL-60bcl-2 cells after exposure to VK2. An increase of acid vesicular organelles (AVOs) detected by acridine orange staining for lysosomes as well as conversion of LC3B-I into LC3B-II by immunoblotting and an increased punctuated pattern of cytoplasmic LC3B by fluorescent immunostaining all supported induction of enhanced autophagy in response to VK2 in HL-60bcl-2 cells. However, during shorter exposure to VK2, the formation of autophagosomes was also prominent in HL-60neo cells although nuclear chromatin condensations and nuclear fragments were also observed at the same time. These findings indicated the mixed morphologic features of apoptosis and autophagy. Inhibition of autophagy by either addition of 3-methyladenine, siRNA for Atg7, or Tet-off Atg5 system all resulted in attenuation of VK2-incuded cell death, indicating autophagy-mediated cell death in response to VK2. These data demonstrate that autophagy and apoptosis can be simultaneously induced by VK2. However, autophagy becomes prominent when the cells are protected from rapid apoptotic death by a high expression level of Bcl-2.     

The Bcl-2 Homology Domain 3 Mimetic Gossypol Induces Both Beclin 1-dependent and Beclin 1-independent Cytoprotective Autophagy in Cancer Cells

Gossypol, a natural Bcl-2 homology domain 3 mimetic compound isolated from cottonseeds, is currently being evaluated in clinical trials. Here, we provide evidence that gossypol induces autophagy followed by apoptotic cell death in both the MCF-7 human breast adenocarcinoma and HeLa cell lines. We first show that knockdown of the Bcl-2 homology domain 3-only protein Beclin 1 reduces gossypol-induced autophagy in MCF-7 cells, but not in HeLa cells. Gossypol inhibits the interaction between Beclin 1 and Bcl-2 (B-cell leukemia/lymphoma 2), antagonizes the inhibition of autophagy by Bcl-2, and hence stimulates autophagy. We then show that knockdown of Vps34 reduces gossypol-induced autophagy in both cell lines, and consistent with this, the phosphatidylinositol 3-phosphate-binding protein WIPI-1 is recruited to autophagosomal membranes. Further, Atg5 knockdown also reduces gossypol-mediated autophagy. We conclude that gossypol induces autophagy in both a canonical and a noncanonical manner. Notably, we found that gossypol-mediated apoptotic cell death was potentiated by treatment with the autophagy inhibitor wortmannin or with small interfering RNA against essential autophagy genes (Vps34, Beclin 1, and Atg5). Our findings support the notion that gossypol-induced autophagy is cytoprotective and not part of the cell death process induced by this compound.     

The novel BH-3 mimetic apogossypolone induces Beclin-1- and ROS-mediated autophagy in human hepatocellular cells

Apogossypolone (ApoG2), a novel derivative of gossypol, exhibits superior antitumor activity in Bcl-2 transgenic mice, and induces autophagy in several cancer cells. However, the detailed mechanisms are not well known. In the present study, we showed that ApoG2 induced autophagy through Beclin-1- and reactive oxygen species (ROS)-dependent manners in human hepatocellular carcinoma (HCC) cells. Incubating the HCC cell with ApoG2 abrogated the interaction of Beclin-1 and Bcl-2/xL, stimulated ROS generation, increased phosphorylation of ERK and JNK, and HMGB1 translocation from the nucleus to cytoplasm while suppressing mTOR. Moreover, inhibition of the ROS-mediated autophagy by antioxidant N-acetyl-cysteine (NAC) potentiates ApoG2-induced apoptosis and cell killing. Our results show that ApoG2 induced protective autophagy in HCC cells, partly due to ROS generation, suggesting that antioxidant may serve as a potential chemosensitizer to enhance cancer cell death through blocking ApoG2-stimulated autophagy. Our novel insights may facilitate the rational design of clinical trials for Bcl-2-targeted cancer therapy.     

Hypoxia signals autophagy in tumor cells via AMPK activity, independent of HIF-1, BNIP3, and BNIP3L

Macroautophagy (called autophagy hereafter) is a catabolic process activated by various types of stress, most notably by nutrient deprivation. The autophagic degradation of intracellular macromolecules provides metabolic support for the cell; however, this physiological process can also initiate a form of cell death (type 2 programmed cell death). Here we report that oxygen deprivation can activate the autophagic pathway in human cancer cell lines. We observed that hypoxia induced distinct cellular changes characteristic of autophagy such as an increase in cytoplasmic acidic vesicles, and processing and cellular localization of microtubule-associated protein-1 light chain 3. Oxygen deprivation-induced autophagy did not require nutrient deprivation, hypoxia-inducible factor-1 (HIF-1) activity, or expression of the HIF-1 target gene BNIP3 (Bcl-2 adenovirus E1a nineteen kilodalton interacting protein 3) or BNIP3L (BNIP3 like protein). Hypoxia-induced autophagy involved the activity of 5'-AMP-activated protein kinase (AMPK). Finally, we determined that cells lacking the autophagy gene ATG5 were unable to activate the autophagic machinery in hypoxia, had decreased oxygen consumption and increased glucose uptake under hypoxia, had increased survival in hypoxic environments, and exhibited accelerated growth as xenografted tumors. Together, these findings suggest that the autophagic degradation of cellular macromolecules contributes to the energetic balance governed by AMPK, and that suppression of autophagy in transformed cells can increase both resistance to hypoxic stress and tumorigenicity.     

Polyphyllin VII Induces an Autophagic Cell Death by Activation of the JNK Pathway and Inhibition of PI3K/AKT/mTOR Pathway in HepG2 Cells

Polyphyllin VII (PP7), a pennogenyl saponin isolated from Rhizoma Paridis, exhibited strong anticancer activities in various cancer types. Previous studies found that PP7 induced apoptotic cell death in human hepatoblastoma cancer (HepG2) cells. In the present study, we investigated whether PP7 could induce autophagy and its role in PP7-induced cell death, and elucidated its mechanisms. PP7 induced a robust autophagy in HepG2 cells as demonstrated by the conversion of LC3B-I to LC3B-II, degradation of P62, formation of punctate LC3-positive structures, and autophagic vacuoles tested by western blot analysis or InCell 2000 confocal microscope. Inhibition of autophagy by treating cells with autophagy inhibitor (chloroquine) abolished the cell death caused by PP7, indicating that PP7 induced an autophagic cell death in HepG2 cells. C-Jun N-terminal kinase (JNK) was activated after treatment with PP7 and pretreatment with SP600125, a JNK inhibitor, reversed PP7-induced autophagy and cell death, suggesting that JNK plays a critical role in autophagy caused by PP7. Furthermore, our study demonstrated that PP7 increased the phosphorylation of AMPK and Bcl-2, and inhibited the phosphorylation of PI3K, AKT and mTOR, suggesting their roles in the PP7-induced autophagy. This is the first report that PP7 induces an autophagic cell death in HepG2 cells via inhibition of PI3K/AKT/mTOR, and activation of JNK pathway, which induces phosphorylation of Bcl-2 and dissociation of Beclin-1 from Beclin-1/Bcl-2 complex, leading to induction of autophagy.     

Differential induction of autophagy in caspase-3/7 down-regulating and Bcl-2 overexpressing recombinant CHO cells subjected to sodium butyrate treatment

Previous research showed that co-down-regulation of caspase-3/7 in rCHO cells, unlike Bcl-2 overexpression, did not effectively block apoptotic cell death induced by 3mM sodium butyrate (NaBu). Here, it is found that the control of autophagy is also related to this different response to NaBu treatment. With NaBu treatment, co-down-regulation of caspase-3/7 enhanced autophagy induction, whereas Bcl-2 overexpression delayed onset of autophagy induction in a Beclin-1 independent manner. The blockage of autophagy showed a detrimental effect on cell viability even in the Bcl-2 overexpressing cells, which suggests the importance of autophagy control for successful anti-cell death engineering of rCHO cells.     

Protection by Bcl-2 against apoptotic but not autophagic cell death after photodynamic therapy

Photodynamic therapy (PDT) induces apoptosis in many cell types. Recent reports identified autophagy as an alternative cell-death process following PDT. Here we investigated the occurrence of autophagy after PDT with the photosensitizer Pc 4 in human cancer cells that are deficient in the pro-apoptotic factor Bax (human prostate cancer DU145) or the apoptosis mediator caspase-3 (human breast cancer MCF-7v) and in apoptosis-competent cells (MCF-7c3 stably overexpressing human pro-caspase-3 and Chinese hamster ovary CHO 5A100). Further, each cell line was also studied with and without stably overexpressed Bcl-2. By electron microscopy and immunoblot analysis, autophagy was observed in all cells studied, whether or not they were capable of typical apoptosis or overexpressed Bcl-2. Bcl-2 overexpression protected against PDT-induced apoptosis and loss of clonogenicity in apoptosis-competent cells (MCF-7c3 and CHO); however, it did not protect against the development of autophagy or against loss of clonogenicity in apoptosis-deficient cells (MCF-7v and DU145). The results show that autophagy may be the dominant cell death pathway following PDT in cells that are incapable of undergoing normal apoptosis. In such cells, Bcl-2 does not protect against autophagic death.     

1-Methyl-4-Phenylpyridinium-Induced Cell Death via Autophagy Through a Bcl-2/Beclin 1 Complex-Dependent Pathway

Several lines of evidence suggest that the mechanism underlying drug-induced neuronal apoptosis is initiated by the increased production of reactive oxygen species (ROS). 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a neurotoxin, has been shown to initiate an apoptotic cascade by increasing ROS in the dopaminergic neurons of the substantia nigra, leading to the morphological and physiological features associated with Parkinson’s disease. Recently, it has been reported that autophagy, a type of programmed cell death independent of the apoptotic cascade, also plays a role in neuronal damage. Although autophagy is negatively regulated by the mammalian target of rapamycin receptor (mTOR), there is some evidence showing a novel function for the anti-apoptotic protein Bcl-2. Bcl-2 is proposed to play a role in negatively regulating autophagy by blocking an essential protein in the signaling pathway, Beclin 1. Nevertheless, it is unclear whether autophagy is also correlated with apoptotic signaling in 1-methyl-4-phenylpyridinium (MPP⁺) toxicity. Therefore, we hypothesized that the MPP⁺ toxicity generally associated with initiating the apoptotic signaling cascade also increases an autophagic phenotype in neuronal cells. Using the SK-N-SH dopaminergic cell lines, we demonstrate that MPP⁺ increases the expression of microtubule-associated protein light chain 3 (LC3-II), an autophagosome membrane marker and the mTOR signaling pathway, and Beclin 1 while decreasing the Bcl-2 levels. Moreover, these expressions correlate with a decreased binding ratio between Bcl-2 and Beclin 1, in effect limiting the regulation of the downstream autophagic markers, such as LC3-II. Our results indicate that MPP⁺ can induce autophagy in SK-N-SH cells by decreasing the Bcl-2/Beclin 1 complex.     

A defect in cytosolic carboxypeptidase 1 (Nna1) causes autophagy in Purkinje cell degeneration mouse brain

Ceramide is a sphingolipid bioactive molecule that induces apoptosis and other forms of cell death, and triggers macroautophagy (referred to below as autophagy). Like amino acid starvation, ceramide triggers autophagy by interfering with the mTOR-signaling pathway, and by dissociating the Beclin 1:Bcl-2 complex in a c-Jun N-terminal kinase 1 (JNK1)-mediated Bcl-2 phosphorylation-dependent manner. Dissociation of the Beclin 1:Bcl-2 complex, and the subsequent stimulation of autophagy have been observed in various contexts in which the cellular level of long-chain ceramides was increased. It is notable that the conversion of short-chain ceramides (C₂-ceramide and C₆-ceramide) into long-chain ceramide via the activity of ceramide synthase is required to trigger autophagy. The dissociation of the Beclin 1:Bcl-2 complex has also been observed in response to tamoxifen and PDMP (an inhibitor of the enzyme that converts ceramide to glucosylceramide), drugs that increase the intracellular level of long-chain ceramides. However, and in contrast to starvation, overexpression of Bcl-2 does not blunt ceramide-induced autophagy. Whether this autophagy that is unchecked by forced dissociation of the Beclin 1:Bcl-2 complex is related to the ability of ceramide to trigger cell death remains an open question. More generally, the question of whether ceramide-induced autophagy is a dedicated cell death mechanism deserves closer scrutiny.     

Vitamin K2 induces autophagy and apoptosis simultaneously in leukemia cells

Vitamin K2 (menaquinone-4: VK2) is a potent inducer for apoptosis in leukemia cells in vitro. HL-60bcl-2 cells, which are derived from a stable transfectant clone of the human bcl-2 gene into the HL-60 leukemia cell line, show 5-fold greater expression of the Bcl-2 protein compared with HL-60neo cells, a control clone transfected with vector alone. VK2 induces apoptosis in HL-60neo cells, whereas HL-60bcl-2 cells are resistant to apoptosis induction by VK2 but show inhibition of cell growth along with an increase of cytoplasmic vacuoles during exposure to VK2. Electron microscopy revealed formation of autophagosomes and autolysosomes in HL-60bcl-2 cells after exposure to VK2. An increase of acid vesicular organelles (AVOs) detected by acridine orange staining for lysosomes as well as conversion of LC3B-I into LC3B-II by immunonoblotting and an increased punctuated pattern of cytoplasmic LC3B by fluorescent immunostaining all supported induction of enhanced autophagy in response to VK2 in HL-60bcl-2 cells. However, during shorter exposure to VK2, the formation of autophagosomes was also prominent in HL-60neo cells although nuclear chromatin condensations and nuclear fragments were also observed at the same time. These findings indicated the mixed morphologic features of apoptosis and autophagy. Inhibition of autophagy by either addition of 3-methyladenine, siRNA for Atg7, or Tet-off Atg5 system all resulted in attenuation of VK2-incuded cell death, indicating autophagy-mediated cell death in response to VK2. These data demonstrate that autophagy and apoptosis can be simultaneously induced by VK2. However, autophagy becomes prominent when the cells are protected from rapid apoptotic death by a high expression level of Bcl-2.     

The Bcl-2-Beclin 1 interaction in (-)-gossypol-induced autophagy versus apoptosis in prostate cancer cells

Bcl-2 is a key dual regulator of autophagy and apoptosis, but how the level of Bcl-2 influences the cellular decision between autophagy and apoptosis is unclear. The natural BH3-mimetic (-)-gossypol preferentially induces autophagy in androgen-independent (AI) prostate cancer cells that have high levels of Bcl-2 and are resistant to apoptosis, whereas apoptosis is preferentially induced in androgen-dependent or -independent cells with low Bcl-2. (-)-Gossypol induces autophagy via blocking Bcl-2-Beclin 1 interaction at the endoplasmic reticulum (ER), together with downregulating Bcl-2, upregulating Beclin 1 and activating the autophagic pathway. Furthermore, (-)-gossypol-induced autophagy is Beclin 1- and Atg5-dependent. These results provide new insights into the mode of cell death induced by Bcl-2 inhibitors, which could facilitate the rational design of clinical trials by selecting patients who are most likely to benefit from the Bcl-2-targeted molecular therapy.     

Cyclin-dependent kinase inhibitors,roscovitine and purvalanol,induce apoptosis and autophagy related to unfolded protein response in HeLa cervical cancer cells

Roscovitine (Rosc) and purvalanol (Pur) are competitive inhibitors of cyclin-dependent kinases (CDKs) by targeting their ATP-binding pockets. Both drugs are shown to be effective to decrease cell viability and dysregulate the ratio of pro- and anti-apoptotic Bcl-2 family members, which finally led to apoptotic cell death in different cancer cell lines in vitro. It was well established that Bcl-2 family members have distinct roles in the regulation of other cellular processes such as endoplasmic reticulum (ER) stress. The induction of ER stress has been shown to play critical role in cell death/survival decision via autophagy or apoptosis. In this study, our aim was to investigate the molecular targets of CDK inhibitors on ER stress mechanism related to distinct cell death types in time-dependent manner in HeLa cervical cancer cells. Our results showed that Rosc and Pur decreased the cell viability, cell growth and colony formation, induced ER stress-mediated autophagy or apoptosis in time-dependent manner. Thus, we conclude that exposure of cells to CDK inhibitors induces unfolded protein response and ER stress leading to autophagy and apoptosis processes in HeLa cervical cancer cells.     

Induction of cytoprotective autophagy in PC-12 cells by cadmium

Laboratory data have demonstrated that cadmium (Cd) may induce neuronal apoptosis. However, little is known about the role of autophagy in neurons. In this study, cell viability decreased in a dose- and time-dependent manner after treatment with Cd in PC-12 cells. As cells were exposed to Cd, the levels of LC3-II proteins became elevated, specific punctate distribution of endogenous LC3-II increased, and numerous autophagosomes appeared, which suggest that Cd induced a high level of autophagy. In the late stages of autophagy, an increase in the apoptosis ratio was observed. Likewise, pre-treatment with chloroquine (an autophagic inhibitor) and rapamycin (an autophagic inducer) resulted in an increased and decreased percentage of apoptosis in contrast to other Cd-treated groups, respectively. The results indicate that autophagy delayed apoptosis in Cd-treated PC-12 cells. Furthermore, co-treatment of cells with chloroquine reduced autophagy and cell activity. However, rapamycin had an opposite effect on autophagy and cell activity. Moreover, class III PI3 K/beclin-1/Bcl-2 signaling pathways served a function in Cd-induced autophagy. The findings suggest that Cd can induce cytoprotective autophagy by activating class III PI3 K/beclin-1/Bcl-2 signaling pathways. In sum, this study strongly suggests that autophagy may serve a positive function in the reduction of Cd-induced cytotoxicity.