Inhibition of mTOR-Dependent Autophagy Sensitizes Leukemic Cells to Cytarabine-Induced Apoptotic Death

The present study investigated the role of autophagy, a cellular self-digestion process, in the cytotoxicity of antileukemic drug cytarabine towards human leukemic cell lines (REH, HL-60, MOLT-4) and peripheral blood mononuclear cells from leukemic patients. The induction of autophagy was confirmed by acridine orange staining of intracellular acidic vesicles, electron microscopy visualization of autophagic vacuoles, as well as by the increase in autophagic proteolysis and autophagic flux, demonstrated by immunoblot analysis of p62 downregulation and LC3-I conversion to autophagosome-associated LC3-II in the presence of proteolysis inhibitors, respectively. Moreover, the expression of autophagy-related genes Atg4, Atg5 and Atg7 was stimulated by cytarabine in REH cells. Cytarabine reduced the phosphorylation of the major negative regulator of autophagy, mammalian target of rapamycin (mTOR), and its downstream target p70S6 kinase in REH cells, which was associated with downregulation of mTOR activator Akt and activation of extracellular signal- regulated kinase. Cytarabine had no effect on the activation of mTOR inhibitor AMP-activated protein kinase. Leucine, an mTOR activator, reduced both cytarabine-induced autophagy and cytotoxicity. Accordingly, pharmacological downregulation of autophagy with bafilomycin A1 and chloroquine, or RNA interference-mediated knockdown of LC3β or p62, markedly increased oxidative stress, mitochondrial depolarization, caspase activation and subsequent DNA fragmentation and apoptotic death in cytarabine-treated REH cells. Cytarabine also induced mTOR-dependent cytoprotective autophagy in HL-60 and MOLT-4 leukemic cell lines, as well as primary leukemic cells, but not normal leukocytes. These data suggest that the therapeutic efficiency of cytarabine in leukemic patients could be increased by the inhibition of the mTOR-dependent autophagic response.     

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.     

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 triggered by magnolol derivative negatively regulates angiogenesis

Angiogenesis has a key role in the tumor progression and metastasis; targeting endothelial cell proliferation has emerged as a promising therapeutic strategy for the prevention of cancer. Previous studies have revealed a complex association between the process of angiogenesis and autophagy and its outcome on tumorigenesis. Autophagy, also known as type-II cell death, has been identified as an alternative way of cell killing in apoptotic-resistant cancer cells. However, its involvement in chemoresistance and tumor promotion is also well known. In this study, we used a derivate of natural product magnolol (Ery5), a potent autophagy inducer, to study the association between the autophagy and angiogenesis in both in vitro and in vivo model system. We found that the robust autophagy triggered by Ery5, inhibited angiogenesis and caused cell death independent of the apoptosis in human umbilical cord vein endothelial cells and PC-3 cells. Ery5 induced autophagy effectively inhibited cell proliferation, migration, invasion and tube formation. We further demonstrated that Ery5-mediated autophagy and subsequent inhibition of angiogenesis was reversed when autophagy was inhibited through 3-methyl adenine and knocking down of key autophagy proteins ATG7 and microtubule-associated protein light chain 3. While evaluating the negative regulation of autophagy on angiogenesis, it was interesting to find that angiogenic environment produced by the treatment of VEGF and CoCl2 remarkably downregulated the autophagy and autophagic cell death induced by Ery5. These studies, while disclosing the vital role of autophagy in the regulation of angiogenesis, also suggest that the potent modulators of autophagy can lead to the development of effective therapeutics in apoptosis-resistant cancer.     

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.     

Raloxifene Induces Autophagy-Dependent Cell Death in Breast Cancer Cells via the Activation of AMP-Activated Protein Kinase

Raloxifene is a selective estrogen receptor modulator (SERM) that binds to the estrogen receptor (ER), and exhibits potent anti-tumor and autophagy-inducing effects in breast cancer cells. However, the mechanism of raloxifene-induced cell death and autophagy is not well-established. So, we analyzed mechanism underlying death and autophagy induced by raloxifene in MCF-7 breast cancer cells.Treatment with raloxifene significantly induced death in MCF-7 cells. Raloxifene accumulated GFP-LC3 puncta and increased the level of autophagic marker proteins, such as LC3-II, BECN1, and ATG12-ATG5 conjugates, indicating activated autophagy. Raloxifene also increased autophagic flux indicators, the cleavage of GFP from GFP-LC3 and only red fluorescence-positive puncta in mRFP-GFP-LC3-expressing cells. An autophagy inhibitor, 3-methyladenine (3-MA), suppressed the level of LC3-II and blocked the formation of GFP-LC3 puncta. Moreover, siRNA targeting BECN1 markedly reversed cell death and the level of LC3-II increased by raloxifene. Besides, raloxifene-induced cell death was not related to cleavage of caspases-7, -9, and PARP. These results indicate that raloxifene activates autophagy-dependent cell death but not apoptosis. Interestingly, raloxifene decreased the level of intracellular adenosine triphosphate (ATP) and activated the AMPK/ULK1 pathway. However it was not suppressed the AKT/mTOR pathway. Addition of ATP decreased the phosphorylation of AMPK as well as the accumulation of LC3-II, finally attenuating raloxifene-induced cell death.Our current study demonstrates that raloxifene induces autophagy via the activation of AMPK by sensing decreases in ATP, and that the overactivation of autophagy promotes cell death and thereby mediates the anti-cancer effects of raloxifene in breast cancer cells.     

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.     

Blue light emitting diode induces apoptosis in lymphoid cells by stimulating autophagy

The present study was performed to examine the induction of apoptotic cell death and autophagy by blue LED irradiation, and the contribution of autophagy to apoptosis in B cell lymphoma A20 and RAMOS cells exposed to blue LED. Irradiation with blue LED reduced cell viability and induced apoptotic cell death, as indicated by exposure of phosphatidylserine on the plasma outside membrane and fragmentation of DNA. Furthermore, the mitochondrial membrane potential increased, and apoptotic proteins (PARP, caspase 3, Bax, and bcl-2) were observed. In addition, the level of intracellular superoxide anion (O₂⁻) gradually increased. Interestingly the formation of autophagosomes and level of LC3-II were increased in blue LED-irradiated A20 and RAMOS cells, but inhibited after pretreatment with 3-methyladenine (3-MA), widely used as an autophagy inhibitor. Inhibition of the autophagic process by pretreatment with 3-MA blocked blue LED irradiation-induced caspase-3 activation. Moreover, a significant reduction of both the early and late phases of apoptosis after transfection with ATG5 and beclin 1 siRNAs was shown by the annexin V/PI staining, indicating a crucial role of autophagy in blue LED-induced apoptosis in cells. Additionally, the survival rate of mice irradiated with blue LED after injection with A20 cells increased compared to the control group. Our data demonstrate that blue LED irradiation induces apoptosis via the mitochondrial-mediated pathway, in conjunction with autophagy. Further studies are needed to elucidate the precise mechanism of blue LED-induced immune cell death.     

Resveratrol depletes mitochondrial DNA and inhibition of autophagy enhances resveratrol-induced caspase activation

We recently demonstrated that resveratrol induces caspase-dependent apoptosis in multiple cancer cell types. Whether apoptosis is also regulated by other cell death mechanisms such as autophagy is not clearly defined. Here we show that inhibition of autophagy enhanced resveratrol-induced caspase activation and apoptosis. Resveratrol inhibited colony formation and cell proliferation in multiple cancer cell types. Resveratrol treatment induced accumulation of LC3-II, which is a key marker for autophagy. Pretreatment with 3-methyladenine (3-MA), an autophagy inhibitor, increased resveratrol-mediated caspase activation and cell death in breast and colon cancer cells. Inhibition of autophagy by silencing key autophagy regulators such as ATG5 and Beclin-1 enhanced resveratrol-induced caspase activation. Mechanistic analysis revealed that Beclin-1 did not interact with proapoptotic proteins Bax and Bak; however, Beclin-1 was found to interact with p53 in the cytosol and mitochondria upon resveratrol treatment. Importantly, resveratrol depleted ATPase 8 gene, and thus, reduced mitochondrial DNA (mtDNA) content, suggesting that resveratrol induces damage to mtDNA causing accumulation of dysfunctional mitochondria triggering autophagy induction. Together, our findings indicate that induction of autophagy during resveratrol-induced apoptosis is an adaptive response.     

Glucose-starved Cells Do Not Engage in Prosurvival Autophagy

In response to nutrient shortage or organelle damage, cells undergo macroautophagy. Starvation of glucose, an essential nutrient, is thought to promote autophagy in mammalian cells. We thus aimed to determine the role of autophagy in cell death induced by glucose deprivation. Glucose withdrawal induces cell death that can occur by apoptosis (in Bax, Bak-deficient mouse embryonic fibroblasts or HeLa cells) or by necrosis (in Rh4 rhabdomyosarcoma cells). Inhibition of autophagy by chemical or genetic means by using 3-methyladenine, chloroquine, a dominant negative form of ATG4B or silencing Beclin-1, Atg7, or p62 indicated that macroautophagy does not protect cells undergoing necrosis or apoptosis upon glucose deprivation. Moreover, glucose deprivation did not induce autophagic flux in any of the four cell lines analyzed, even though mTOR was inhibited. Indeed, glucose deprivation inhibited basal autophagic flux. In contrast, the glycolytic inhibitor 2-deoxyglucose induced prosurvival autophagy. Further analyses indicated that in the absence of glucose, autophagic flux induced by other stimuli is inhibited. These data suggest that the role of autophagy in response to nutrient starvation should be reconsidered.     

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     

ATG7 deficiency suppresses apoptosis and cell death induced by lysosomal photodamage

Photodynamic therapy (PDT) involves photosensitizing agents that, in the presence of oxygen and light, initiate formation of cytotoxic reactive oxygen species (ROS). PDT commonly induces both apoptosis and autophagy. Previous studies with murine hepatoma 1c1c7 cells indicated that loss of autophagy-related protein 7 (ATG7) inhibited autophagy and enhanced the cytotoxicity of photosensitizers that mediate photodamage to mitochondria or the endoplasmic reticulum. In this study, we examined two photosensitizing agents that target lysosomes: the chlorin NPe6 and the palladium bacteriopheophorbide WST11. Irradiation of wild-type 1c1c7 cultures loaded with either photosensitizer induced apoptosis and autophagy, with a blockage of autophagic flux. An ATG7- or ATG5-deficiency suppressed the induction of autophagy in PDT protocols using either photosensitizer. Whereas ATG5-deficient cells were quantitatively similar to wild-type cultures in their response to NPe6 and WST11 PDT, an ATG7-deficiency suppressed the apoptotic response (as monitored by analyses of chromatin condensation and procaspase-3/7 activation) and increased the LD₅₀ light dose by > 5-fold (as monitored by colony-forming assays). An ATG7-deficiency did not prevent immediate lysosomal photodamage, as indicated by loss of the lysosomal pH gradient. However, unlike wild-type and ATG5-deficient cells, the lysosomes of ATG7-deficient cells recovered this gradient within 4 h of irradiation, and never underwent permeabilization (monitored as release of endocytosed 10-kDa dextran polymers). We propose that the efficacy of lysosomal photosensitizers is in part due to both promotion of autophagic stress and suppression of autophagic prosurvival functions. In addition, an effect of ATG7 unrelated to autophagy appears to modulate lysosomal photodamage.     

ATG7 deficiency suppresses apoptosis and cell death induced by lysosomal photodamage

Photodynamic therapy (PDT) involves photosensitizing agents that, in the presence of oxygen and light, initiate formation of cytotoxic reactive oxygen species (ROS). PDT commonly induces both apoptosis and autophagy. Previous studies with murine hepatoma 1c1c7 cells indicated that loss of autophagy-related protein 7 (ATG7) inhibited autophagy and enhanced the cytotoxicity of photosensitizers that mediate photodamage to mitochondria or the endoplasmic reticulum. In this study, we examined two photosensitizing agents that target lysosomes: the chlorin NPe6 and the palladium bacteriopheophorbide WST11. Irradiation of wild-type 1c1c7 cultures loaded with either photosensitizer induced apoptosis and autophagy, with a blockage of autophagic flux. An ATG7- or ATG5-deficiency suppressed the induction of autophagy in PDT protocols using either photosensitizer. Whereas ATG5-deficient cells were quantitatively similar to wild-type cultures in their response to NPe6 and WST11 PDT, an ATG7-deficiency suppressed the apoptotic response (as monitored by analyses of chromatin condensation and procaspase-3/7 activation) and increased the LD 50 light dose by > 5-fold (as monitored by colony-forming assays). An ATG7-deficiency did not prevent immediate lysosomal photodamage, as indicated by loss of the lysosomal pH gradient. However, unlike wild-type and ATG5-deficient cells, the lysosomes of ATG7-deficient cells recovered this gradient within 4 h of irradiation, and never underwent permeabilization (monitored as release of endocytosed 10-kDa dextran polymers). We propose that the efficacy of lysosomal photosensitizers is in part due to both promotion of autophagic stress and suppression of autophagic prosurvival functions. In addition, an effect of ATG7 unrelated to autophagy appears to modulate lysosomal photodamage.     

Apigeninidin induces apoptosis through activation of Bak and Bax and subsequent mediation of mitochondrial damage in human promyelocytic leukemia HL-60 cells

Treatment of human promyelocytic leukemia HL-60 cells with apigeninidin could induce cytotoxicity (IC₅₀ = ～80 μM), along with apoptotic sub-G₁ cells, TUNEL-positive apoptotic DNA fragmentation, activation of the multidomain pro-apoptotic Bcl-2 proteins (Bak and Bax), mitochondrial membrane potential (Δψ_m) loss, release of mitochondrial cytochrome c and AIF into the cytoplasm, activation of caspase-9, -3, -8, and -7, and cleavage of PARP and lamin B. These induced apoptotic events were accompanied by decrease of Bcl-2 level and increase of Bak and Bax levels. Apigeninidin-induced sub-G₁ cells and activation of Bak and Bax were also detected in human acute leukemia Jurkat T cells, but not in Jurkat T cells overexpressing Bcl-2. Pretreatment of HL-60 cells with the pan-caspase inhibitor z-VAD-fmk reduced significantly apigeninidin-induced sub-G₁ cells and caspase cascade activation, whereas it failed to suppress Bak and Bax activations, Δψ_m loss, and release of mitochondrial cytochrome c and AIF. None of FADD and caspase-8 deficiencies affected the sensitivity of Jurkat T cells to apigeninidin-induced cytotoxicity. These results demonstrated that apigeninidin-induced apoptosis was mediated by activation of Bak and Bax, mitochondrial damage and resultant release of not only cytochrome c, causing caspase cascade activation, but also caspase-independent death effector AIF in HL-60 cells.     

Induction of autophagic cell death in human ovarian carcinoma cells by Antrodia salmonea through increased reactive oxygen species generation

We reported in our previously executed studies that the fermented culture broth of Antrodia salmonea (AS), a mushroom used in Taiwanese folk medicine induced reactive oxygen species (ROS)-mediated apoptosis in human ovarian carcinoma cells. In this study, we studied the anticancer efficacies of AS (0–240 μg/ml) by examining the key molecular events implicated in cell death associated with autophagy in SKOV-3 and A2780 human ovarian carcinoma cells and clarified the fundamental molecular mechanisms. Treatment of ovarian carcinoma cells with AS-induced autophagic cell death mediated by increased microtubule-associated protein LC3-II, GFP-LC3 puncta, and acidic vesicular organelle (AVO) formation. These events are linked with the activation of p62/SQSTM1, the inhibition of ATG4B, the expression of ATG7, and the dysregulation of Beclin-1/Bcl-2 (i.e., B-cell lymphoma 2). N-acetylcysteine inhibited AS-induced ROS generation, which in turn constricted AS-induced LC3 conversion, AVO formation, and ATG4B inhibition, indicating ROS-mediated autophagy cell death. In addition, the 3-methyladenine (3-MA) or chloroquine (CQ)-induced autophagy inhibition decreased AS-induced apoptosis. Additionally, apoptosis inhibition by Z-VAD-FMK, a pan-caspase inhibitor, substantially suppressed AS-induced autophagy. Furthermore, AS-inhibited HER-2/ neu and PI3K/AKT signaling pathways which were reversed by autophagy inhibitors 3-MA and CQ. Thus, A. salmonea is a potential chemopreventive agent that is capable of activating ROS-mediated autophagic cell death in ovarian carcinoma cells.     

The Anticancer Agent Di-2-pyridylketone 4,4-Dimethyl-3-thiosemicarbazone (Dp44mT) Overcomes Prosurvival Autophagy by Two Mechanisms: PERSISTENT INDUCTION OF AUTOPHAGOSOME SYNTHESIS AND IMPAIRMENT OF LYSOSOMAL INTEGRITY

Autophagy functions as a survival mechanism during cellular stress and contributes to resistance against anticancer agents. The selective antitumor and antimetastatic chelator di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) causes lysosomal membrane permeabilization and cell death. Considering the integral role of lysosomes in autophagy and cell death, it was important to assess the effect of Dp44mT on autophagy to further understand its mechanism of action. Notably, Dp44mT affected autophagy by two mechanisms. First, concurrent with its antiproliferative activity, Dp44mT increased the expression of the classical autophagic marker LC3-II as a result of induced autophagosome synthesis. Second, this effect was supplemented by a reduction in autophagosome degradation as shown by the accumulation of the autophagic substrate and receptor p62. Conversely, the classical iron chelator desferrioxamine induced autophagosome accumulation only by inhibiting autophagosome degradation. The formation of redox-active iron or copper Dp44mT complexes was critical for its dual effect on autophagy. The cytoprotective antioxidant N-acetylcysteine inhibited Dp44mT-induced autophagosome synthesis and p62 accumulation. Importantly, Dp44mT inhibited autophagosome degradation via lysosomal disruption. This effect prevented the fusion of lysosomes with autophagosomes to form autolysosomes, which is crucial for the completion of the autophagic process. The antiproliferative activity of Dp44mT was suppressed by Beclin1 and ATG5 silencing, indicating the role of persistent autophagosome synthesis in Dp44mT-induced cell death. These studies demonstrate that Dp44mT can overcome the prosurvival activity of autophagy in cancer cells by utilizing this process to potentiate cell death.     

Cardiotoxin III induces c-jun N-terminal kinase-dependent apoptosis in HL-60 human leukaemia cells

Cardiotoxin III (CTX III), a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom, has been reported to have anticancer activity. The molecular effects of CTX III on HL-60 cells were dissected in the present study. We found that the antiproliferative action of CTX III on HL-60 cells was mediated through apoptosis, as characterized by an increase of sub G1 population, DNA fragmentation and poly(ADP-ribose) polymerase (PARP) cleavage. Upregulation of Bax, downregulation of Bcl-2, the release of mitochondrial cytochrome c to cytosol and the activations of capase-9 and -3 were noted, while CTX III had no appreciable effect on the levels of Bcl-X(L) and Bad proteins. Moreover, c-Jun N-terminal kinase (JNK) was activated shortly after CTX III treatment in HL-60 cells. Consistently, the SP600125 compound, an anthrapyrazolone inhibitor of JNK, suppressed apoptosis induced by CTX III. As expected, this JNK inhibitor also attenuated the modulation of Bax and Bcl-2, as well as the cytosolic appearance of cytochrome c and the activation of caspase-3 and caspase-9 that induced by CTX III. These findings suggest that CTX III can induce apoptosis in HL-60 cells via the mitochondrial caspase cascade and the activation of JNK is critical for the initiation of the apoptotic death of HL-60 cells.     

Lapatinib induces autophagy, apoptosis and megakaryocytic differentiation in chronic myelogenous leukemia K562 cells

Lapatinib is an oral, small-molecule, dual tyrosine kinase inhibitor of epidermal growth factor receptors (EGFR, or ErbB/Her) in solid tumors. Little is known about the effect of lapatinib on leukemia. Using human chronic myelogenous leukemia (CML) K562 cells as an experimental model, we found that lapatinib simultaneously induced morphological changes resembling apoptosis, autophagy, and megakaryocytic differentiation. Lapatinib-induced apoptosis was accompanied by a decrease in mitochondrial transmembrane potential and was attenuated by the pancaspase inhibitor z-VAD-fmk, indicating a mitochondria-mediated and caspase-dependent pathway. Lapatinib-induced autophagic cell death was verified by LC3-II conversion, and upregulation of Beclin-1. Further, autophagy inhibitor 3-methyladenine as well as autophagy-related proteins Beclin-1 (ATG6), ATG7, and ATG5 shRNA knockdown rescued the cells from lapatinib-induced growth inhibition. A moderate number of lapatinib-treated K562 cells exhibited features of megakaryocytic differentiation. In summary, lapatinib inhibited viability and induced multiple cellular events including apoptosis, autophagic cell death, and megakaryocytic differentiation in human CML K562 cells. This distinct activity of lapatinib against CML cells suggests potential for lapatinib as a therapeutic agent for treatment of CML. Further validation of lapatinib activity in vivo is warranted.