Tamoxifen and AEBS ligands induced apoptosis and autophagy in breast cancer cells through the stimulation of sterol accumulation

Tamoxifen (Tx) interacts with high affinity to the microsomal antiestrogen binding site (AEBS) which is a hetero-oligomeric complex involved in cholesterol metabolism. We established that Tx and other AEBS ligands induce breast cancer cell differentiation, apoptosis and autophagy through the induction of sterol accumulation. We determined that cell death is sterol- and ROS-dependent and is prevented by the antioxidant vitamin E. Macroautophagy is characterized by the accumulation of autophagic vacuoles, an increase in the expression of Beclin 1 and the stimulation of autophagic flux. We established that macroautophagy is sterol-dependent and is associated with cell survival rather than cytotoxicity, since blockage of macroautophagy sensitizes cells to AEBS ligands. These results show that the accumulation of sterols by AEBS ligands in MCF-7 cells induces both apoptosis and macroautophagy. Collectively, these data support a therapeutic potential for selective AEBS ligands in breast cancer management and reveal a mechanism that explains the induction of autophagy in MCF-7 cells by Tx and other selective estrogen receptor modulators. Moreover these data give pharmacological clues to improve the apoptotic efficacy of AEBS ligands.     

Ceramide-mediated macroautophagy involves inhibition of protein kinase B and up-regulation of beclin 1

The sphingolipid ceramide is involved in the cellular stress response. Here we demonstrate that ceramide controls macroautophagy, a major lysosomal catabolic pathway. Exogenous C(2)-ceramide stimulates macroautophagy (proteolysis and accumulation of autophagic vacuoles) in the human colon cancer HT-29 cells by increasing the endogenous pool of long chain ceramides as demonstrated by the use of the ceramide synthase inhibitor fumonisin B(1). Ceramide reverted the interleukin 13-dependent inhibition of macroautophagy by interfering with the activation of protein kinase B. In addition, C(2)-ceramide stimulated the expression of the autophagy gene product beclin 1. Ceramide is also the mediator of the tamoxifen-dependent accumulation of autophagic vacuoles in the human breast cancer MCF-7 cells. Monodansylcadaverine staining and electron microscopy showed that this accumulation was abrogated by myriocin, an inhibitor of de novo synthesis ceramide. The tamoxifen-dependent accumulation of vacuoles was mimicked by 1-phenyl-2-decanoylamino-3-morpholino-1-propanol, an inhibitor of glucosylceramide synthase. 1-Phenyl-2-decanoylamino-3-morpholino-1-propanol, tamoxifen, and C(2)-ceramide stimulated the expression of beclin 1, whereas myriocin antagonized the tamoxifen-dependent up-regulation. Tamoxifen and C(2)-ceramide interfere with the activation of protein kinase B, whereas myriocin relieved the inhibitory effect of tamoxifen. In conclusion, the control of macroautophagy by ceramide provides a novel function for this lipid mediator in a cell process with major biological outcomes.     

Corilagin inhibits breast cancer growth via reactive oxygen species‐dependent apoptosis and autophagy

Corilagin is a component of Phyllanthus urinaria extract and has been found of possessing anti‐inflammatory, anti‐oxidative, and anti‐tumour properties in clinic treatments. However, the underlying mechanisms in anti‐cancer particularly of its induction of cell death in human breast cancer remain undefined. Our research found that corilagin‐induced apoptotic and autophagic cell death depending on reactive oxygen species (ROS) in human breast cancer cell, and it occurred in human breast cancer cell (MCF‐7) only comparing with normal cells. The expression of procaspase‐8, procaspase‐3, PARP, Bcl‐2 and procaspase‐9 was down‐regulated while caspase‐8, cleaved PARP, caspase‐9 and Bax were up‐regulated after corilagin treatment, indicating apoptosis mediated by extrinsic and mitochondrial pathways occurred in MCF‐7 cell. Meanwhile, autophagy mediated by suppressing Akt/mTOR/p70S6K pathway was detected with an increase in autophagic vacuoles and LC3‐II conversion. More significantly, inhibition of autophagy by chloroquine diphosphate salt (CQ) remarkably enhanced apoptosis, while the caspase inhibitor z‐VAD‐fmk failed in affecting autophagy, suggesting that corilagin‐induced autophagy functioned as a survival mechanism in MCF‐7 cells. In addition, corilagin induced intracellular reactive oxygen species (ROS) generation, when reduced by ROS scavenger NAC, apoptosis and autophagy were both down‐regulated. Nevertheless, in SK‐BR3 cell which expressed RIP3, necroptosis inhibitor Nec‐1 could not alleviate cell death induced by corilagin, indicating necroptosis was not triggered. Subcutaneous tumour growth in nude mice was attenuated by corilagin, consisting with the results in vitro. These results imply that corilagin inhibits cancer cell proliferation through inducing apoptosis and autophagy which regulated by ROS release.     

Inhibiting ROS-TFE3-dependent autophagy enhances the therapeutic response to metformin in breast cancer

Autophagy modulation is a potential therapeutic strategy for breast cancer, and a previous study indicated that metformin exhibits significant anti-carcinogenic activity. However, the ability of metformin to induce autophagy and its role in breast cancer cell death remains unclear. In this study, we exposed MCF-7 cells to different concentrations of metformin (2.5, 5, and 10?mM) for 48?h, and metformin-induced significant apoptosis in the MCF-7 cells. The expression levels of CL-PARP (poly(ADP-ribose) polymerase 1) and the ratio of BAX to BCL-2 were significantly increased. In addition to apoptosis, we showed that metformin increased autophagic flux in MCF-7 cells, as evidenced by the upregulation of LC3-II and downregulation of P62/SQSTM1. Moreover, pharmacological or genetic blocking of autophagy increased metformin-induced apoptosis, indicating a cytoprotective role of autophagy in metformin-treated MCF-7 cells. Mechanistically, metformin-induced TFE3^(Ser321) dephosphorylation activated TFE3 nuclear translocation and increased of TFE3 reporter activity, which contributed to lysosomal biogenesis and the expression of autophagy-related genes and, subsequently, initiated autophagy in MCF-7 cells. Importantly, we found that metformin triggered the generation of reactive oxygen species (ROS) in MCF-7 cells. Furthermore, N-acetyl-l-cysteine (NAC), a ROS scavenger, abrogated the effects of metformin on TFE3-dependent autophagy. Notably, TFE3 expression positively correlated with breast cancer development and poor prognosis in patients. Taken together, these data demonstrate that blocking ROS-TFE3-dependent autophagy to enhance the activity of metformin warrants further attention as a treatment strategy for breast cancer.     

Macroautophagy-generated increase of lysosomal amyloid β-protein mediates oxidant-induced apoptosis of cultured neuroblastoma cells

Increasing evidence suggests the toxicity of intracellular amyloid β-protein (Aβ) to neurons, as well as the involvement of oxidative stress in Alzheimer disease (AD). Here we show that normobaric hyperoxia (exposure of cells to 40% oxygen for five days), and consequent activation of macroautophagy and accumulation of Aβ within lysosomes, induced apoptosis in differentiated SH-SY5Y neuroblastoma cells. Cells under hyperoxia showed: (1) increased numbers of autophagic vacuoles that contained amyloid precursor protein (APP) as well as Aβ monomers and oligomers, (2) increased reactive oxygen species production, and (3) enhanced apoptosis. Oxidant-induced apoptosis positively correlated with cellular Aβ production, being the highest in cells that were stably transfected with APP Swedish KM670/671NL double mutation. Inhibition of γ-secretase, prior and/or in parallel to hyperoxia, suggested that the increase of lysosomal Aβ resulted mainly from its autophagic uptake, but also from APP processing within autophagic vacuoles. The oxidative stress-mediated effects were prevented by macroautophagy inhibition using 3-methyladenine or ATG5 downregulation. Our results suggest that upregulation of macroautophagy and resulting lysosomal Aβ accumulation are essential for oxidant-induced apoptosis in cultured neuroblastoma cells and provide additional support for the interactive role of oxidative stress and the lysosomal system in AD-related neurodegeneration.     

Induction of autophagy by proteasome inhibitor is associated with proliferative arrest in colon cancer cells

The ubiquitin-proteasome system (UPS) and lysosome-dependent macroautophagy (autophagy) are two major intracellular pathways for protein degradation. Blockade of UPS by proteasome inhibitors has been shown to activate autophagy. Recent evidence also suggests that proteasome inhibitors may inhibit cancer growth. In this study, the effect of a proteasome inhibitor MG-132 on the proliferation and autophagy of cultured colon cancer cells (HT-29) was elucidated. Results showed that MG-132 inhibited HT-29 cell proliferation and induced G₂/M cell cycle arrest which was associated with the formation of LC3⁺ autophagic vacuoles and the accumulation of acidic vesicular organelles. MG-132 also increased the protein expression of LC3-I and -II in a time-dependent manner. In this connection, 3-methyladenine, a Class III phosphoinositide 3-kinase inhibitor, significantly abolished the formation of LC3⁺ autophagic vacuoles and the expression of LC3-II but not LC3-I induced by MG-132. Taken together, this study demonstrates that inhibition of proteasome in colon cancer cells lowers cell proliferation and activates autophagy. This discovery may shed a new light on the novel function of proteasome in the regulation of autophagy and proliferation in colon cancer cells.     

Regulation of autophagy by sphingosine kinase 1 and its role in cell survival during nutrient starvation

The sphingolipid ceramide induces macroautophagy (here called autophagy) and cell death with autophagic features in cancer cells. Here we show that overexpression of sphingosine kinase 1 (SK1), an enzyme responsible for the production of sphingosine 1-phosphate (S1P), in MCF-7 cells stimulates autophagy by increasing the formation of LC3-positive autophagosomes and the rate of proteolysis sensitive to the autophagy inhibitor 3-methyladenine. Autophagy was blocked in the presence of dimethylsphingosine, an inhibitor of SK activity, and in cells expressing a catalytically inactive form of SK1. In SK1(wt)-overexpressing cells, however, autophagy was not sensitive to fumonisin B1, an inhibitor of ceramide synthase. In contrast to ceramide-induced autophagy, SK1(S1P)-induced autophagy is characterized by (i) the inhibition of mammalian target of rapamycin signaling independently of the Akt/protein kinase B signaling arm and (ii) the lack of robust accumulation of the autophagy protein Beclin 1. In addition, nutrient starvation induced both the stimulation of autophagy and SK activity. Knocking down the expression of the autophagy protein Atg7 or that of SK1 by siRNA abolished starvation-induced autophagy and increased cell death with apoptotic hallmarks. In conclusion, these results show that SK1(S1P)-induced autophagy protects cells from death with apoptotic features during nutrient starvation.     

Bufalin induces autophagy-mediated cell death in human colon cancer cells through reactive oxygen species generation and JNK activation

Colorectal cancer is the second most common cause of cancer death in the world and about half of the patients with colorectal cancer require adjuvant therapy after surgical resection. Therefore, the eradication of cancer cells via chemotherapy constitutes a viable approach to treating patients with colorectal cancer. In this study, the effects of bufalin isolated from a traditional Chinese medicine were evaluated and characterized in HT-29 and Caco-2 human colon cancer cells. Contrary to its well-documented apoptosis-promoting activity in other cancer cells, bufalin did not cause caspase-dependent cell death in colon cancer cells, as indicated by the absence of significant early apoptosis as well as poly(ADP-ribose) polymerase and caspase-3 cleavage. Instead, bufalin activated an autophagy pathway, as characterized by the accumulation of LC3-II and the stimulation of autophagic flux. The induction of autophagy by bufalin was linked to the generation of reactive oxygen species (ROS). ROS activated autophagy via the c-Jun NH₂-terminal kinase (JNK). JNK activation increased expression of ATG5 and Beclin-1. ROS antioxidants (N-acetylcysteine and vitamin C), the JNK-specific inhibitor SP600125, and JNK2 siRNA attenuated bufalin-induced autophagy. Our findings unveil a novel mechanism of drug action by bufalin in colon cancer cells and open up the possibility of treating colorectal cancer through a ROS-dependent autophagy pathway.     

Crohn disease ATG16L1 polymorphism increases susceptibility to infection with Helicobacter pylori in humans

Autophagy plays key roles both in host defense against bacterial infection and in tumor biology. Helicobacter pylori (H. pylori) infection causes chronic gastritis and is the single most important risk factor for the development of gastric cancer in humans. Its vacuolating cytotoxin (VacA) promotes gastric colonization and is associated with more severe disease. Acute exposure to VacA initially triggers host autophagy to mitigate the effects of the toxin in epithelial cells. Recently, we demonstrated that chronic exposure to VacA leads to the formation of defective autophagosomes that lack CTSD/cathepsin D and have reduced catalytic activity. Disrupted autophagy results in accumulation of reactive oxygen species and SQSTM1/p62 both in vitro and in vivo in biopsy samples from patients infected with VacA (+) but not VacA (-) strains. We also determined that the Crohn disease susceptibility polymorphism in the essential autophagy gene ATG16L1 increases susceptibility to H. pylori infection. Furthermore, peripheral blood monocytes from individuals with the ATG16L1 risk variant show impaired autophagic responses to VacA exposure. This is the first study to identify both a host autophagy susceptibility gene for H. pylori infection and to define the mechanism by which the autophagy pathway is affected following H. pylori infection. Collectively, these findings highlight the synergistic effects of host and bacterial autophagy factors on H. pylori pathogenesis and the potential for subsequent cancer susceptibility.     

A selective autophagy pathway takes an unconventional route

Selective autophagy transports specific cytoplasmic materials into lysosomes/vacuoles. In the case of macroautophagy the selectivity is mediated by receptors, which usually link the cargos to the machinery that sequesters them into the forming autophagosome. In our recent work, we found that fission yeast Nbr1, a homolog of the mammalian macroautophagy receptor NBR1, acts together with an unconventional autophagy-associated cargo sequestration apparatus, the endosomal sorting complexes required for transport (ESCRTs), to deliver 2 hydrolytic enzymes from the cytosol to the vacuole lumen. In this pathway, which we term the Nbr1-mediated vacuolar targeting (NVT) pathway, soluble cargos transit through the multi-vesicular body (MVB), rather than the autophagosome, on their way to the vacuole. Our findings reveal a novel mode of action of macroautophagy receptors and broaden our understanding of ESCRT-mediated autophagy.     

Involvement of autophagy in NK cell development and function

Natural killer (NK) cells are the prototypical members of the recently identified family of innate lymphoid cells (ILCs). Thanks to their cytotoxic and secretory functions, NK cells play a key role in the immune response to cells experiencing various forms of stress, including viral infection and malignant transformation. Autophagy is a highly conserved network of degradative pathways that participate in the maintenance of cellular and organismal homeostasis as they promote adaptation to adverse microenvironmental conditions. The relevance of autophagy in the development and functionality of cellular components of the adaptive immune system is well established. Conversely, whether autophagy also plays an important role in the biology of ILC populations such as NK cells has long remained elusive. Recent experimental evidence shows that ablating Atg5 (autophagy-related 5, an essential component of the autophagic machinery) in NK cells and other specific ILC populations results in progressive mitochondrial damage, reactive oxygen species (ROS) overgeneration, and regulated cell death, hence interrupting ILC development. Moreover, disrupting the interaction of ATG7 with phosphorylated FOXO1 (forkhead box O1) in the cytosol of immature NK cells prevents autophagic responses that are essential for NK cell maturation. These findings suggest that activating autophagy may support the maturation of NK cells and other ILCs that manifest antiviral and anticancer activity.     

Dual functions of autophagy in the response of breast tumor cells to radiation: cytoprotective autophagy with radiation alone and cytotoxic autophagy in radiosensitization by vitamin D 3

In MCF-7 breast tumor cells, ionizing radiation promoted autophagy that was cytoprotective; pharmacological or genetic interference with autophagy induced by radiation resulted in growth suppression and/or cell killing (primarily by apoptosis). The hormonally active form of vitamin D, 1,25D 3, also promoted autophagy in irradiated MCF-7 cells, sensitized the cells to radiation and suppressed the proliferative recovery that occurs after radiation alone. 1,25D 3 enhanced radiosensitivity and promoted autophagy in MCF-7 cells that overexpress Her-2/neu as well as in p53 mutant Hs578t breast tumor cells. In contrast, 1,25D 3 failed to alter radiosensitivity or promote autophagy in the BT474 breast tumor cell line with low-level expression of the vitamin D receptor. Enhancement of MCF-7 cell sensitivity to radiation by 1,25D 3 was not attenuated by a genetic block to autophagy due largely to the promotion of apoptosis via the collateral suppression of protective autophagy. However, MCF-7 cells were protected from the combination of 1,25D 3 with radiation using a concentration of chloroquine that produced minimal sensitization to radiation alone. The current studies are consistent with the premise that while autophagy mediates a cytoprotective function in irradiated breast tumor cells, promotion of autophagy can also confer radiosensitivity by vitamin D (1,25D 3). As both cytoprotective and cytotoxic autophagy can apparently be expressed in the same experimental system in response to radiation, this type of model could be utilized to distinguish biochemical, molecular and/or functional differences in these dual functions of autophagy.     

Autophagy protects breast cancer cells from epirubicin-induced apoptosis and facilitates epirubicin-resistance development

Epirubicin (EPI) is one of the most effective drugs against cancer. But the acquired resistance of cancer cells to EPI is becoming a major obstacle for successful cancer therapy. Recently, some studies have revealed that macroautophagy (here referred to as autophagy) may protect the cancer cell from anticancer drug-induced death, so autophagy might be related to the development of drug resistance to these reagents. However, the relationship between autophagy and drug resistance has yet to be defined. Our study showed that EPI induced autophagy in human breast cancer MCF-7 cells. And the EPI-induced autophagy protected MCF-7 cells from EPI-induced apoptosis. Furthermore, autophagy was elevated in EPI-resistant MCF-7 cells (MCF-7er cells), and inhibition of autophagy restored the sensitivity of MCF-7er cells to EPI. Therefore, autophagy is a prosurvival factor and has a role in the development of EPI-acquired resistance in EPI-treated MCF-7 cells. Also, this finding indicates that the use of clinically applicable autophagy inhibitors might be one of the important strategies for breast cancer therapy.     

Autophagy protects breast cancer cells from epirubicin-induced apoptosis and facilitates epirubicin-resistance development

Epirubicin (EPI) is one of the most effective drugs against cancer. But the acquired resistance of cancer cells to EPI is becoming a major obstacle for successful cancer therapy. Recently, some studies have revealed that macroautophagy (here referred to as autophagy) may protect the cancer cell from anticancer drug-induced death, so autophagy might be related to the development of drug resistance to these reagents. However, the relationship between autophagy and drug resistance has yet to be defined. Our study showed that EPI induced autophagy in human breast cancer MCF-7 cells. And the EPI-induced autophagy protected MCF-7 cells from EPI-induced apoptosis. Furthermore, autophagy was elevated in EPI-resistant MCF-7 cells (MCF-7er cells), and inhibition of autophagy restored the sensitivity of MCF-7er cells to EPI. Therefore, autophagy is a prosurvival factor and has a role in the development of EPI-acquired resistance in EPI-treated MCF-7 cells. Also, this finding indicates that the use of clinically applicable autophagy inhibitors might be one of the important strategies for breast cancer therapy.     

The multi-step mechanism and biological role of noncanonical autophagy targeting Streptococcus pneumoniae during the early stages of infection

ABSTRACT

Multiple autophagic processes are triggered in response to bacterial infection as the host attempts to eliminate intracellular invaders. However, it is still unclear how the mechanisms contributing to canonical macroautophagy/autophagy, including xenophagy, coordinate with the more recently described features that are characteristic of noncanonical autophagy. Recently, we revealed that infection with Streptococcus pneumoniae can trigger the formation of RB1CC1/FIP200-independent LC3-associated phagosome-like vacuoles (PcLVs) that contain the pneumococci at an early stage of infection. We also found that interactions of SQSTM1/p62 with the ATG16L1 WD domain are essential for PcLV formation. Intriguingly, PcLVs were required for the subsequent generation of bactericidal autophagic vacuoles (PcAVs). Furthermore, we also identified LC3-delocalized SQSTM1-positive PcLVs as intracellular intermediates that link PcLVs and PcAVs. These findings reveal a novel multi-step mechanism that contributes to xenophagy of the critical S. pneumoniae respiratory pathogen.     

Effect of IGF-1 on the balance between autophagy of dysfunctional mitochondria and apoptosis

Mutations in mitochondrial DNA (mtDNA) cause excessive production of mitochondrial reactive oxygen species (ROS) and shorten animal life span. We examined the mechanisms responsible for removal of mitochondria with deleterious mtDNA mutations by autophagy. Incubation of primary cells and cell lines in the absence of serum promotes autophagy of mitochondria with deleterious mtDNA mutations but spares their normal counterparts. The effect of serum withdrawal on the autophagy of dysfunctional mitochondria is prevented by the addition of IGF-1. As a result of the elimination of mitochondria with deleterious mutations, excessive ROS production, characteristic of dysfunctional mitochondria, is greatly reduced. Mitochondrial autophagy shares a common mechanism with mitochondrial-induced cell apoptosis, including mitochondrial transition pore formation and increased ROS production.     

NF-kappaB activation represses tumor necrosis factor-alpha-induced autophagy

Activation of NF-kappaB and autophagy are two processes involved in the regulation of cell death, but the possible cross-talk between these two signaling pathways is largely unknown. Here, we show that NF-kappaB activation mediates repression of autophagy in tumor necrosis factor-alpha (TNFalpha)-treated Ewing sarcoma cells. This repression is associated with an NF-kappaB-dependent activation of the autophagy inhibitor mTOR. In contrast, in cells lacking NF-kappaB activation, TNFalpha treatment up-regulates the expression of the autophagy-promoting protein Beclin 1 and subsequently induces the accumulation of autophagic vacuoles. Both of these responses are dependent on reactive oxygen species (ROS) production and can be mimicked in NF-kappaB-competent cells by the addition of H2O2. Small interfering RNA-mediated knockdown of beclin 1 and atg7 expression, two autophagy-related genes, reduced TNFalpha- and reactive oxygen species-induced apoptosis in cells lacking NF-kappaB activation and in NF-kappaB-competent cells, respectively. These findings demonstrate that autophagy may amplify apoptosis when associated with a death signaling pathway. They are also evidence that inhibition of autophagy is a novel mechanism of the antiapoptotic function of NF-kappaB activation. We suggest that stimulation of autophagy may be a potential way bypassing the resistance of cancer cells to anti-cancer agents that activate NF-kappaB.     

NBCn1 and NHE1 expression and activity in ΔNErbB2 receptor-expressing MCF-7 breast cancer cells: Contributions to pHi regulation and chemotherapy resistance

Altered pH-regulatory ion transport is characteristic of many cancers; however, the mechanisms and consequences are poorly understood. Here, we investigate how a truncated, constitutively active ErbB2 receptor (ΔNErbB2) common in breast cancer impacts on the Na⁺/H⁺-exchanger NHE1 and the Na⁺,HCO₃⁻-cotransporter NBCn1 in MCF-7 human breast cancer cells and address the roles of these transporters in chemotherapy resistance. Upon ΔNErbB2 expression, mRNA and protein levels of NBCn1, yet not of NHE1, increased several-fold, and the localization of both transporters was altered paralleling extensive morphological changes. The rate of pH_i recovery after acid loading increased by 50% upon ΔNErbB2 expression. Knockdown and pharmacological inhibition confirmed the involvement of both NHE1 and NBCn1 in acid extrusion. NHE1 inhibition or knockdown sensitized ΔNErbB2-expressing cells to cisplatin-induced programmed cell death (PCD) in a caspase-, cathepsin-, and reactive oxygen species-dependent manner. NHE1 inhibition augmented cisplatin-induced caspase activity and lysosomal membrane permeability followed by cysteine cathepsin release. In contrast, NBCn1 inhibition attenuated cathepsin release and had no net effect on viability. These findings warrant studies of NHE1 as a potential target in breast cancer and demonstrate that in spite of their similar transport functions, NHE1 and NBCn1 serve different functions in MCF-7 cells.     

Role of non-canonical Beclin 1-independent autophagy in cell death induced by resveratrol in human breast cancer cells

Resveratrol, a polyphenol found in grapes and other fruit and vegetables, is a powerful chemopreventive and chemotherapeutic molecule potentially of interest for the treatment of breast cancer. The human breast cancer cell line MCF-7, which is devoid of caspase-3 activity, is refractory to apoptotic cell death after incubation with resveratrol. Here we show that resveratrol arrests cell proliferation, triggers death and decreases the number of colonies of cells that are sensitive to caspase-3-dependent apoptosis (MCF-7 casp-3) and also those that are unresponsive to it (MCF-7vc). We demonstrate that resveratrol (i) acts via multiple pathways to trigger cell death, (ii) induces caspase-dependent and caspase-independent cell death in MCF-7 casp-3 cells, (iii) induces only caspase-independent cell death in MCF-7vc cells and (iv) stimulates macroautophagy. Using BECN1 and hVPS34 (human vacuolar protein sorting 34) small interfering RNAs, we demonstrate that resveratrol activates Beclin 1-independent autophagy in both cell lines, whereas cell death via this uncommon form of autophagy occurs only in MCF-7vc cells. We also show that this variant form of autophagic cell death is blocked by the expression of caspase-3, but not by its enzymatic activity. In conclusion, this study reveals that non-canonical autophagy induced by resveratrol can act as a caspase-independent cell death mechanism in breast cancer cells.     

Autophagy delays apoptotic death in breast cancer cells following DNA damage

Early signaling in camptothecin-treated MCF-7 cells followed an intrinsic pathway, but death was delayed and late events exhibited few hallmarks of apoptosis. BH3-only proteins, such as Noxa, Puma and BimEL, were activated and localized to mitochondrial sites within 24 h following drug exposure. However, caspase activity was low and death was unaffected by caspase inhibition. Transmission electron micrographs showed the presence of large vacuoles in drug-treated cells. An autophagic survival response has been attributed to MCF-7 cells following nutrient starvation or exposure to tamoxifen. Here, we show that autophagy also plays an important role in the delayed DNA damage response. Confocal microscopy revealed colocalization of mitochondria with large autophagic vacuoles and inhibitors of autophagy increased mitochondrial depolarization and caspase-9 activity, and accelerated cell death. Furthermore, downregulation of autophagy proteins, Beclin 1 and Atg7, unmasked a caspase-dependent, apoptotic response to DNA damage. We propose that a post-mitochondrial caspase cascade is delayed as a result of early disposal of damaged mitochondria within autophagosomes. Our data also suggest that the use of autophagy as a means of delaying apoptosis or prolonging survival may be characteristic of noninvasive breast tumor cells. These studies underscore a potential role for autophagy inhibitors in combination with conventional chemotherapeutic drugs in early breast cancer therapy.