Autophagic Death Research Articles

Multiple myeloma proteostasis can be targeted via translation initiation factor eIF4E.

Intensive protein synthesis is a unique and differential trait of the multiple myeloma (MM) cells. Previously we showed that tetraspanin overexpression in MM cell lines attenuated mTOR and PI3K cascades, induced protein synthesis, activated unfolded protein response (UPR), and caused autophagic death, all suggesting breach of proteostasis. Here we assessed the role of translation initiation in the tetraspanin‑induced MM cell death with emphasis on eIF4E translation initiation factor. We showed tetraspanins attenuated peIF4E and its targets [c‑Myc, cyclin D1 (cycD1)]; eIF4E attenuation was Akt-dependent. eIF4E inhibition in MM cells [bone marrow (BM), lines] by siRNA and/or the anti‑viral drug and competitive eIF4E inhibitor ribavirin (RBV) deleteriously affected MM cells in a similar manner to the overexpression of tetraspanins. Furthermore, combined application of RBV and velcade had a synergistic anti‑MM effect. Our results demonstrate that breach of proteostasis via eIF4E inhibition is an attractive therapeutic approach that may be relatively easily achieved by employing RBV, making this strategy readily translatable into the clinic.

Acute exposure to ZnO nanoparticles induces autophagic immune cell death

The increasing risk of incidental exposure to nanomaterials has led to mounting concerns regarding nanotoxicity. Zinc oxide nanoparticles (ZnO NPs) are produced in large quantities and have come under scrutiny due to their capacity to cause cytotoxicity in vitro and potential to cause harm in vivo. Recent evidence has indicated that ZnO NPs promote autophagy in cells; however, the signaling pathways and the role of ion release inducing toxicity remain unclear. In this study, we report that ZnO NPs are immunotoxic to primary and immortalized immune cells. Importantly, such immunotoxicity is observed in mice in vivo, since death of splenocytes is seen after intranasal exposure to ZnO NPs. We determined that ZnO NPs release free Zn2+ that can be taken up by immune cells, resulting in cell death. Inhibiting free Zn2+ ions in solution with EDTA or their uptake with CaCl2 abrogates ZnO NP-induced cell death. ZnO NP-mediated immune cell death was associated with increased levels of intracellular reactive oxygen species (ROS). ZnO NP death was not due to apoptosis, necroptosis or pyroptosis. Exposure of immune cells to ZnO NPs resulted in autophagic death and increased levels of LC3A, an essential component of autophagic vacuoles. Accordingly, ZnO NP-mediated upregulation of LC3A and induction of immune cell death were inhibited by blocking autophagy and ROS production. We conclude that release of Zn2+ from ZnO NPs triggers the production of excessive intracellular ROS, resulting in autophagic death of immune cells. Our findings suggest that exposure to ZnO NPs has the potential to impact host immunity.

Autophagy and lysosomal related protein expression patterns in human glioblastoma

Glioblastoma cells are resistant to apoptotic stimuli with autophagic death prevailing under cytotoxic stress. Autophagy interfering agents may represent a new strategy to test in combination with chemo-radiation. We investigated the patterns of expression of autophagy related proteins (LC3A, LC3B, p62, Beclin 1, ULK1 and ULK2) in a series of patients treated with post-operative radiotherapy. Experiments with glioblastoma cell lines (T98 and U87) were also performed to assess autophagic response under conditions simulating the adverse intratumoral environment. Glioblastomas showed cytoplasmic overexpression of autophagic proteins in a varying extent, so that cases could be grouped into low and high expression groups. 10/23, 5/23, 13/23, 5/23, 8/23 and 9/23 cases examined showed extensive expression of LC3A, LC3B, Beclin 1, Ulk 1, Ulk 2 and p62, respectively. Lysosomal markers Cathepsin D and LAMP2a, as well as the lyososomal biogenesis transcription factor TFEB were frequently overexpressed in glioblastomas (10/23, 11/23, and 10/23 cases, respectively). TFEB was directly linked with PTEN, Cathepsin D, HIF1α, LC3B, Beclin 1 and p62 expression. PTEN was also significantly related with LC3B but not LC3A expression, in both immunohistochemistry and gene expression analysis. Confocal microscopy in T98 and U87 cell lines showed distinct identity of LC3A and LC3B autophagosomes. The previously reported stone-like structure (SLS) pattern of LC3 expression was related with prognosis. SLS were inducible in glioblastoma cell lines under exposure to acidic conditions and 2DG mediated glucose antagonism. The present study provides the basis for autophagic characterization of human glioblastoma for further translational studies and targeted therapy trials.

Additional evidence of the trypanocidal action of (-)-elatol on amastigote forms through the involvement of reactive oxygen species.

Chagas’ disease, a vector-transmitted infectious disease, is caused by the protozoa parasite Trypanosoma cruzi. Drugs that are currently available for the treatment of this disease are unsatisfactory, making the search for new chemotherapeutic agents a priority. We recently described the trypanocidal action of (−)-elatol, extracted from the macroalga Laurencia dendroidea. However, nothing has been described about the mechanism of action of this compound on amastigotes that are involved in the chronic phase of Chagas’ disease. The goal of the present study was to evaluate the effect of (−)-elatol on the formation of superoxide anions (O2•−), DNA fragmentation, and autophagy in amastigotes of T. cruzi to elucidate the possible mechanism of the trypanocidal action of (−)-elatol. Treatment of the amastigotes with (−)-elatol increased the formation of O2•− at all concentrations of (−)-elatol assayed compared with untreated parasites. Increased fluorescence was observed in parasites treated with (−)-elatol, indicating DNA fragmentation and the formation of autophagic compartments. The results suggest that the trypanocidal action of (−)-elatol might involve the induction of the autophagic and apoptotic death pathways triggered by an imbalance of the parasite’s redox metabolism.

Dying neurons in thalamus of asphyxiated term newborns and rats are autophagic.

Neonatal hypoxic-ischemic encephalopathy (HIE) still carries a high burden by its mortality and long-term neurological morbidity in survivors. Apart from hypothermia, there is no acknowledged therapy for HIE, reflecting the lack of mechanistic understanding of its pathophysiology. (Macro)autophagy, a physiological intracellular process of lysosomal degradation, has been proposed to be excessively activated in excitotoxic conditions such as HIE. The present study examines whether neuronal autophagy in the thalamus of asphyxiated human newborns or P7 rats is enhanced and related to neuronal death processes. Neuronal autophagy and cell death were evaluated in the thalamus (frequently injured in severe HIE) of both human newborns who died after severe HIE (n = 5) and P7 hypoxic-ischemic rats (Rice-Vannuci model). Autophagic (LC3, p62), lysosomal (LAMP1, cathepsins), and cell death (TUNEL, caspase-3) markers were studied by immunohistochemistry in human and rat brain sections, and by additional methods in rats (immunoblotting, histochemistry, and electron microscopy). Following severe perinatal asphyxia in both humans and rats, thalamic neurons displayed up to 10-fold (p < 0.001) higher numbers of autophagosomes and lysosomes, implying an enhanced autophagic flux. The highly autophagic neurons presented strong features of apoptosis. These findings were confirmed and elucidated in more detail in rats. These results show for the first time that autophagy is enhanced in severe HIE in dying thalamic neurons of human newborns, as in rats. Experimental neuroprotective strategies targeting autophagy could thus be a promising lead to follow for the development of future therapeutic approaches.

Inhibition of autophagy via activation of PI3K/Akt pathway contributes to the protection of ginsenoside Rb1 against neuronal death caused by ischemic insults.

Lethal autophagy is a pathway leading to neuronal death caused by transient global ischemia. In this study, we examined the effect of Ginsenoside Rb1 (GRb1) on ischemia/reperfusion-induced autophagic neuronal death and investigated the role of PI3K/Akt. Ischemic neuronal death in vitro was induced by using oxygen glucose deprivation (OGD) in SH-SY5Y cells, and transient global ischemia was produced by using two vessels occlusion in rats. Cellular viability of SH-SY5Y cells was assessed by MTT assay, and CA1 neuronal death was evaluated by Hematoxylin-eosin staining. Autophagic vacuoles were detected by using both fluorescent microscopy in combination with acridine orange (AO) and Monodansylcadaverine (MDC) staining and transmission electronic microscopy. Protein levels of LC3II, Beclin1, total Akt and phosphor-Akt at Ser473 were examined by western blotting analysis. GRb1 inhibited both OGD and transient ischemia-induced neuronal death and mitigated OGD-induced autophagic vacuoles in SH-SY5Y cells. By contrast, PI3K inhibitor LY294002 counteracted the protection of GRb1 against neuronal death caused by either OGD or transient ischemia. LY294002 not only mitigated the up-regulated protein level of phosphor Akt at Ser473 caused by GRb1, but also reversed the inhibitory effect of GRb1 on OGD and transient ischemia-induced elevation in protein levels of LC3II and Beclin1.

Antitumor effect of soybean lectin mediated through reactive oxygen species-dependent pathway

AimsThe present study evaluated the potential role of soybean lectin's (SBL) anticancer effect in vitro in different cancer cell lines and the therapeutic effectiveness in vivo in Dalton's lymphoma (DL) bearing mice model. Main methodsThe effect of SBL on cell growth and viability was measured using MTT assay in different cancer cells in vitro. Apoptosis, autophagic cell death, DNA-damaging potential and reactive oxygen species (ROS) were analyzed in HeLa cells. The in vivo efficacy of SBL was demonstrated in Dalton's lymphoma (DL) bearing mice. Key findingsSBL demonstrated clear, strong antiproliferative activity without affecting normal cells; however, heat denaturation of SBL diminished the antiproliferative efficacy of molecule as demonstrated by MTT assay. A sharp 74.51±3.5% and 82.95±5.8% inhibition of tumor cell proliferation in DL mice occurred when SBL was administered at a dosage of 1 and 2mg/kg body weight (i.p.), respectively, for ten days with the induction of autophagic and apoptotic cell death. An in vitro investigation revealed that SBL-mediated autophagy, apoptosis and DNA damage in HeLa cells were inflicted through the generation of ROS in a dose-dependent manner. Interestingly, pre-treating HeLa cells with N-acetylcysteine (NAC), a typical ROS scavenger, led to a noticeable reduction in SBL-induced autophagy, apoptosis and DNA-damaging activities, suggesting that SBL's antitumor potential was governed by ROS activation. SignificanceIn this study, we evaluated the apoptotic, autophagic death, and DNA-damaging effects of SBL in cancer cells, which may have the potential to be used as a phyto-derived protein for cancer therapy.

Apoptotic death of prostate cancer cells by a gonadotropin-releasing hormone-II antagonist.

Gonadotropin-releasing hormone-I (GnRH-I) has attracted strong attention as a hormonal therapeutic tool, particularly for androgen-dependent prostate cancer patients. However, the androgen-independency of the cancer in advanced stages has spurred researchers to look for new medical treatments. In previous reports, we developed the GnRH-II antagonist Trp-1 to inhibit proliferation and stimulate the autophagic death of various prostate cancer cells, including androgen-independent cells. We further screened many GnRH-II antagonists to identify molecules with higher efficiency. Here, we investigated the effect of SN09-2 on the growth of PC3 prostate cancer cells. SN09-2 reduced the growth of prostate cancer cells but had no effect on cells derived from other tissues. Compared with Trp-1, SN09-2 conspicuously inhibited prostate cancer cell growth, even at low concentrations. SN09-2-induced PC3 cell growth inhibition was associated with decreased membrane potential in mitochondria where the antagonist was accumulated, and increased mitochondrial and cytosolic reactive oxygen species. SN09-2 induced lactate dehydrogenase release into the media and annexin V-staining on the PC3 cell surface, suggesting that the antagonist stimulated prostate cancer cell death by activating apoptotic signaling pathways. Furthermore, cytochrome c release from mitochondria to the cytosol and caspase-3 activation occurred in a concentration- and time-dependent manner. SN09-2 also inhibited the growth of PC3 cells xenotransplanted into nude mice. These results demonstrate that SN09-2 directly induces mitochondrial dysfunction and the consequent ROS generation, leading to not only growth inhibition but also apoptosis of prostate cancer cells.

Berberine-induced apoptotic and autophagic death of HepG2 cells requires AMPK activation.

BackgroundHepatocellular carcinoma (HCC), the primary liver cancer, is one of the most malignant human tumors with extremely poor prognosis. The aim of this study was to investigate the anti-cancer effect of berberine in a human hepatocellular carcinoma cell line (HepG2), and to study the underlying mechanisms by focusing on the AMP-activated protein kinase (AMPK) signaling cascade.ResultsWe found that berberine induced both apoptotic and autophagic death of HepG2 cells, which was associated with a significant activation of AMPK and an increased expression of the inactive form of acetyl-CoA carboxylase (ACC). Inhibition of AMPK by RNA interference (RNAi) or by its inhibitor compound C suppressed berberine-induced caspase-3 cleavage, apoptosis and autophagy in HepG2 cells, while AICAR, the AMPK activator, possessed strong cytotoxic effects. In HepG2 cells, mammalian target of rapamycin complex 1 (mTORC1) activation was important for cell survival, and berberine inhibited mTORC1 via AMPK activation.ConclusionsTogether, these results suggested that berberine-induced both apoptotic and autophagic death requires AMPK activation in HepG2 cells.

Tougu Xiaotong capsule promotes chondrocyte autophagy by regulating the Atg12/LC3 conjugation systems

We have previously reported that Tougu Xiaotong capsule (TXC) inhibits tidemark replication and cartilage degradation by regulating chondrocyte autophagy in vivo. Autophagy, a cell protective mechanism for maintaining cellular homeostasis, has been shown to be a constitutively active and protective process for chondrocyte survival. However, it remains unclear whether TXC promotes chondrocyte autophagy by regulating the autophagy-related (Atg)12/microtubule-associated protein 1 light chain 3 (LC3) conjugation systems. Thus, in the present study, we investigated the effects of TXC on primary chondrocytes treated with cobalt chloride (CoCl2). We found that CoCl2 induced a decrease in chondrocyte viability and the autophagosome formation of chondrocytes, indicating that CoCl2 induced autophagic death in a dose- and time-dependent manner. To determine the effects of TXC on CoCl2-exposed chondrocytes, we assessed cell viability by MTT assay. Our results revealed that TXC enhanced the viability of CoCl2-exposed chondrocytes. To gain insight into the mechanisms responsible for the enhancing effects of TXC on CoCl2-exposed chondrocytes, the expression of Atg genes was assessed in chondrocytes exposed to CoCl2 and treated with or without TXC. The results revealed that the expression of beclin 1, Atg3, Atg5, Atg7, Atg10, Atg12 and LC3 II/LC3 I in the chondrocytes treated with TXC increased, compared to that in the untreated chondrocytes. In addition, ultrastructural analysis indicated that treated chondrocytes contained more autophagosomes than the untreated cells, suggesting that TXC increased the formation of autophagosomes in the chondrocytes to clear the CoCl2-induced autophagic death. Therefore, these data suggest that TXC is a potential therapeutic agent for the reduction of cartilage degradation that occurs in osteoarthritis.

Protection of ischemic postconditioning against neuronal apoptosis induced by transient focal ischemia is associated with attenuation of NF-κB/p65 activation.

Background and PurposeAccumulating evidences have demonstrated that nuclear factor κB/p65 plays a protective role in the protection of ischemic preconditioning and detrimental role in lethal ischemia-induced programmed cell death including apoptosis and autophagic death. However, its role in the protection of ischemic postconditioning is still unclear.MethodsRat MCAO model was used to produce transient focal ischemia. The procedure of ischemic postconditioning consisted of three cycles of 30 seconds reperfusion/reocclusion of MCA. The volume of cerebral infarction was measured by TTC staining and neuronal apoptosis was detected by TUNEL staining. Western blotting was used to analyze the changes in protein levels of Caspase-3, NF-κB/p65, phosphor- NF-κB/p65, IκBα, phosphor- IκBα, Noxa, Bim and Bax between rats treated with and without ischemic postconditioning. Laser scanning confocal microscopy was used to examine the distribution of NF-κB/p65 and Noxa.ResultsIschemic postconditioning made transient focal ischemia-induced infarct volume decrease obviously from 38.6%±5.8% to 23.5%±4.3%, and apoptosis rate reduce significantly from 46.5%±6.2 to 29.6%±5.3% at reperfusion 24 h following 2 h focal cerebral ischemia. Western blotting analysis showed that ischemic postconditioning suppressed markedly the reduction of NF-κB/p65 in cytoplasm, but elevated its content in nucleus either at reperfusion 6 h or 24 h. Moreover, the decrease of IκBα and the increase of phosphorylated IκBα and phosphorylated NF-κB/p65 at indicated reperfusion time were reversed by ischemic postconditioning. Correspondingly, proapoptotic proteins Caspase-3, cleaved Caspase-3, Noxa, Bim and Bax were all mitigated significantly by ischemic postconditioning. Confocal microscopy revealed that ischemic postconditioning not only attenuated ischemia-induced translocation of NF-κB/p65 from neuronal cytoplasm to nucleus, but also inhibited the abnormal expression of proapoptotic protein Noxa within neurons.ConclusionsWe demonstrated in this study that the protection of ischemic postconditioning on neuronal apoptosis caused by transient focal ischemia is associated with attenuation of the activation of NF-κB/p65 in neurons.

OP0014 Thermal enhancement sensitises gastric cancer stem cells to chemotherapy through ROS-modulation-induced autophagic death

Background Peritoneal metastasis in gastric cancer is hypothesised to result from residual cancer stem cells (CSCs), which play an important part in the comprehensive treatment of gastric tumour. Hyperthermia could enhance the anti-cancer effects of chemotherapy, but the precise biochemical mechanisms of the synergistic antitumour effect of chemotherapy plus hyperthermia are unclear. In this study, we investigated whether hyperthermic enhancement sensitises gastric CSCs to chemotherapy through ROS-modulation-induced autophagic death. Methods Using CD44 as a marker, we isolated CSCs by fluorescence-activated cell sorting in a MNK-45 human gastric cancer line or tumours from a NOD/SCID mouse xenograft model. After treatment with chemotherapy plus hyperthermia, the proportion change in the CD44-positive MNK-45 cell subpopulation was observed in CD44-positive MNK-45 cells or tumour cells from the mouse model. Tumorigenic ability was studied, autophagy-related gene Beclin1 and LC3 expression were evaluated, ROS production was measured, and HSP multiplex concentrations were determined in CD44-positive MNK-45 cells from the mouse model. Findings After treatment with chemotherapy plus hyperthermia, the proportion of CD44-positive MNK-45 cells significantly decreased, CD44-positive MNK-45 tumour growth was suppressed, and inhibition of tumour growth was closely associated with the decreased proportion of CD44-positive MNK-45 cells. Chemotherapy plus hyperthermia significantly increased the expression of Beclin1 and LC3 and inhibited HSP expression in tumours from the mouse xenograft model. CD44-positive MNK-45 gastric cancer cells showed a significantly higher level of ROS after compared with before hyperthermia and chemotherapy, and the decreased proportion of CD44-positive MNK-45 cells was almost completely consistent with increased expression of Beclin1 and LC3 and suppression of HSP expression after chemotherapy plus hyperthermia treatment in tumours from the mouse xenograft model. Interpretation The addition of hyperthermia can eliminate the resistance of gastric CSCs to chemotherapy. Mild thermal enhancement sensitises gastric CSCs to chemotherapy through ROS-modulation-induced autophagic death.

Restriction of Food Intake Prevents Postinfarction Heart Failure by Enhancing Autophagy in the Surviving Cardiomyocytes

We investigated the effect of restriction of food intake, a potent inducer of autophagy, on postinfarction cardiac remodeling and dysfunction. Myocardial infarction was induced in mice by left coronary artery ligation. At 1 week after infarction, mice were randomly divided into four groups: the control group was fed ad libitum (100%); the food restriction (FR) groups were fed 80%, 60%, or 40% of the mean amount of food consumed by the control mice. After 2 weeks on the respective diets, left ventricular dilatation and hypofunction were apparent in the control group, but both parameters were significantly mitigated in the FR groups, with the 60% FR group showing the strongest therapeutic effect. Cardiomyocyte autophagy was strongly activated in the FR groups, as indicated by up-regulation of microtubule-associated protein 1 light chain 3-II, autophagosome formation, and myocardial ATP content. Chloroquine, an autophagy inhibitor, completely canceled the therapeutic effect of FR. This negative effect was associated with reduced activation of AMP-activated protein kinase and of ULK1 (a homolog of yeast Atg1), both of which were enhanced in hearts from the FR group. In vitro, the AMP-activated protein kinase inhibitor compound C suppressed glucose depletion-induced autophagy in cardiomyocytes, but did not influence activity of chloroquine. Our findings imply that a dietary protocol with FR could be a preventive strategy against postinfarction heart failure.

Mechanism of the Susceptibility of Remodeled Pulmonary Vessels to Drug‐Induced Cell Killing

BackgroundPulmonary arterial hypertension remains a devastating disease without a cure. The major complication of this disease is the abnormal growth of vascular cells, resulting in pulmonary vascular remodeling. Thus, agents, which affect the remodeled vessels by killing unwanted cells, should improve treatment strategies. The present study reports that antitumor drugs selectively kill vascular cells in remodeled pulmonary vessels in rat models of pulmonary hypertension.Methods and ResultsAfter developing pulmonary vascular remodeling in chronic hypoxia or chronic hypoxia/SU‐5416 models, rats were injected with antitumor drugs including proteasome inhibitors (bortezomib and MG‐132) and daunorubicin. Within 1 to 3 days, these agents reduced the media and intima thickness of remodeled pulmonary vascular walls, but not the thickness of normal pulmonary vessels. These drugs also promoted apoptotic and autophagic death of vascular cells in the remodeled vessels, but not in normal vessels. We provide evidence that the upregulation of annexin A1, leading to GATA4‐dependent downregulation of Bcl‐xL, is a mechanism for specific apoptotic killing, and for the role of parkin in defining specificity of autophagic killing of remodeled vascular cells. The reversal of pulmonary vascular remodeling increased the capacity of vasodilators to reduce pulmonary arterial pressure.ConclusionsThese results suggest that antitumor drugs can specifically kill cells in remodeled pulmonary vascular walls and may be useful for improving the efficacy of current therapeutic strategies to treat pulmonary arterial hypertension.

Cholesterol induces autophagic and apoptotic death in gastric carcinoma cells

Despite conflicting results, there is evidence to suggest an inverse link between total body cholesterol levels and the risk of certain malignancies. Based on previous reports, this phenomenon appears to vary with cancer site, and, in particular, more consistent data on inverse relations was reported in the risk of gastric cancer. In the current study, the effect of cholesterol on gastric cancer cell viability was examined using an invitro cell culture system. Addition of cholesterol in culture medium resulted in reduced viability and clonogenicity of SNU601, SNU638 and SNU216 gastric cancer cells by induction of both autophagic and apoptotic death. Transient inactivation of ERK1/2 was linked to reduction of cholesterol-mediated cell viability, and tumor necrosis factor‑related apoptosis-inducing ligand receptor2 (TRAIL‑R2/DR5) was also involved in cell death signaling. In conclusion, these results imply that cholesterol can act as a signal regulator to modulate cell viability and that proper cellular cholesterol levels may be advantageous to suppress growth of gastric carcinomas.

Therapeutic potential of sepantronium bromide YM155 in gemcitabine-resistant human urothelial carcinoma cells

Survivin is overexpressed in transitional cell carcinoma (TCC), the most common type of bladder cancer. Previous reports demonstrated that knockdown of survivin by siRNA induced apoptosis of TCC cells. The present study evaluated the therapeutic effects of sepantronium bromide (YM155), a novel small molecule survivin inhibitor under clinical trials, on TCC cells in vitro. BFTC905, a grade III TCC cell line derived from a patient of blackfoot disease in Taiwan, was the most gemcitabine-resistant cell line when compared to BFTC909, TSGH8301 and T24 in cytotoxicity assay, resulting from upregulation of securin and bcl-2 after gemcitabine treatment. YM155 caused potent concentration‑dependent cytotoxicity in 4 TCC cell lines (IC50s ≤20 nM), but exhibited no cytotoxicity in survivin-null primary human urothelial cells. For BFTC905 cells, addition of gemcitabine and/or cisplatin, the standard TCC chemotherapy regimen, to YM155 did not exert additive cytotoxic effects. Molecular analyses indicated that YM155 inhibited the proliferation of BFTC905 cells by increasing p27kip1, suppressing Ki-67, and inducing quiescence. In addition, YM155 elicited apoptosis manifested with DNA fragmentation through suppressing the expression of survivin, securin and bcl-2. Furthermore, YM155 induced autophagy in BFTC905 cells as autophagic inhibitor, 3-methyladenine, attenuated YM155-induced LC3B-II levels and reversed the cytotoxicity of YM155. mTOR inhibitors sirolimus and everolimus did not increase YM155-induced expression of LC3B-II nor augment YM155-induced cytotoxicity. These results indicate that YM155 exerts its lethal effect on BFTC905 cells via apoptotic and autophagic death pathways and suggest that YM155 may be a potential drug for the therapy of gemcitabine-resistant bladder cancer.

The Synergistic Effect of Everolimus and Chloroquine on Endothelial Cell Number Reduction Is Paralleled by Increased Apoptosis and Reduced Autophagy Occurrence

Endothelial Progenitor Cells (EPCs), a minor subpopulation of the mononuclear cell fraction in peripheral blood, play a critical role in cancer development as they contribute to angiogenesis-mediated pathological neovascularization. In response to tumor cytokines, including VEGF, EPCs mobilize from the bone marrow into the peripheral circulation and move to the tumor bed where they incorporate into sprouting neovessels. In the present study, we evaluated the effects of everolimus (Afinitor, Novartis), a rapamycin analogue, alone or in combination with chloroquine, a 4-alkylamino substituted quinoline family member, one of the autophagy inhibitors, on EPCs biological functions. We found that either everolimus or chloroquine induce growth inhibition on EPCs in a dose-dependent manner after 72 h from the beginning of incubation. The combined administration of the two drugs to EPC was synergistic in inducing growth inhibition; in details, the maximal pharmacological synergism between everolimus and chloroquine in inducing growth inhibition on EPCs cells was recorded when chloroquine was administered 24 h before everolimus. Moreover, we have studied the mechanisms of cell death induced by the two agents alone or in combination on EPCs and we have found that the synergistic effect of combination on EPC growth inhibition was paralleled by increased apoptosis induction and reduced autophagy. These effects occurred together with biochemical features that are typical of reduced autophagic death such as increased co-immunoprecipitation between Beclin 1 and Bcl-2. Chloroquine antagonized the inhibition of the activity of Akt→4EBP1 axis mediated by everolimus and at the same time it blocked the feed-back activation of Erk-1/2 induced by RAD in EPCs. These data suggest a new strategy in order to block angiogenesis in tumours in which this process plays a key role in both the sustainment and spreading of cancer cells.

The functions and regulations of DAPK in cancer metastasis

Metastasis is responsible for most cancer deaths but it remains a poorly understood process. Recent evidence has emerged that death-associated protein kinase (DAPK) is a candidate of metastasis suppressor. DAPK downregulation or inactivation has been observed in a number of metastatic cancers through epigenetic, transcriptional, post-transcriptional, or post-translational mechanism. In certain cases, DAPK downregulation correlates with metastatic recurrence. Animal studies further show that DAPK impedes both early-stage and late-stage metastatic process, which suggests that DAPK possesses multiple mechanisms to suppress metastasis. Cell-based studies revealed that DAPK mediates several types of cell death, including apoptosis, autophagic death and necrosis, depending on death stimuli and cell context. DAPK also regulates cytoskeleton proteins to mediate death-associated cell morphological alterations and to inhibit cell motility. Besides tumor cells, DAPK can influence on stromal cells to regulate their survival and functions. These effects likely all contribute to the metastasis suppressive role of DAPK. The detail molecular mechanisms of these anti-metastatic effects of DAPK are reviewed in this article.

Caspase-like activity during aging and cell death in the toxic dinoflagellate Karenia brevis

The observation of caspase-like activity during cell death has provided a new framework for understanding the evolutionary and ecological contexts of programmed cell death in phytoplankton. However, additional roles for this caspase-like activity, the enzymes responsible, and the targets of this enzyme activity in phytoplankton remain largely undefined. In the present study, the role of caspase-like activity in aging and ROS-mediated cell death were investigated and death programs both dependent on and independent of caspase-like activity were observed in the toxic dinoflagellate, Karenia brevis. The dual use of in situ caspase 3/7 and TUNEL staining identified previously undescribed death-associated morphotypes in K. brevis. In silico motif analysis identified several enzymes with predicted caspase-like activity in the K. brevis transcriptome, although bona fide caspases are absent. Lastly, computational prediction of downstream caspase substrates, using sequence context and predicted secondary structure, identified proteins involved in a wide range of biological processes including regulation of protein turnover, cell cycle progression, lipid metabolism, coenzyme metabolism, apoptotic and autophagic death. To confirm the computational predictions, a short peptide was designed around the predicated caspase cleavage site in a predicted novel K. brevis caspase 3/7-like target, S-adenosylmethionine synthetase (KbAdoMetS). Cleavage of the peptide substrate with recombinant caspase 3 enzyme was determined by MALDI-TOF MS, confirming that KbAdoMetS is indeed a bona fide caspase substrate. These data identify the involvement of caspase-like activity in both aging and cell death in K. brevis and identify novel executioner enzymes and downstream targets that may be important for bloom termination.

Interplay between autophagy and programmed cell death in mammalian neural stem cells

Mammalian neural stem cells (NSCs) are of particular interest because of their role in brain development and function. Recent findings suggest the intimate involvement of programmed cell death (PCD) in the turnover of NSCs. However, the underlying mechanisms of PCD are largely unknown. Although apoptosis is the best-defined form of PCD, accumulating evidence has revealed a wide spectrum of PCD encompassing apoptosis, autophagic cell death (ACD) and necrosis. This mini-review aims to illustrate a unique regulation of PCD in NSCs. The results of our recent studies on autophagic death of adult hippocampal neural stem (HCN) cells are also discussed. HCN cell death following insulin withdrawal clearly provides a reliable model that can be used to analyze the molecular mechanisms of ACD in the larger context of PCD. More research efforts are needed to increase our understanding of the molecular basis of NSC turnover under degenerating conditions, such as aging, stress and neurological diseases. Efforts aimed at protecting and harnessing endogenous NSCs will offer novel opportunities for the development of new therapeutic strategies for neuropathologies. [BMB Reports 2013; 46(8): 383-390]