Idarubicin induces mTOR-dependent cytotoxic autophagy in leukemic cells

We investigated if the antileukemic drug idarubicin induces autophagy, a process of programmed cellular self-digestion, in leukemic cell lines and primary leukemic cells. Transmission electron microscopy and acridine orange staining demonstrated the presence of autophagic vesicles and intracellular acidification, respectively, in idarubicin-treated REH leukemic cell line. Idarubicin increased punctuation/aggregation of microtubule-associated light chain 3B (LC3B), enhanced the conversion of LC3B-I to autophagosome-associated LC3B-II in the presence of proteolysis inhibitors, and promoted the degradation of the selective autophagic target p62, thus indicating the increase in autophagic flux. Idarubicin inhibited the phosphorylation of the main autophagy repressor mammalian target of rapamycin (mTOR) and its downstream target p70S6 kinase. The treatment with the mTOR activator leucine prevented idarubicin-mediated autophagy induction. Idarubicin-induced mTOR repression was associated with the activation of the mTOR inhibitor AMP-activated protein kinase and down-regulation of the mTOR activator Akt. The suppression of autophagy by pharmacological inhibitors or LC3B and beclin-1 genetic knockdown rescued REH cells from idarubicin-mediated oxidative stress, mitochondrial depolarization, caspase activation and apoptotic DNA fragmentation. Idarubicin also caused mTOR inhibition and cytotoxic autophagy in K562 leukemic cell line and leukocytes from chronic myeloid leukemia patients, but not healthy controls. By demonstrating mTOR-dependent cytotoxic autophagy in idarubicin-treated leukemic cells, our results warrant caution when considering combining idarubicin with autophagy inhibitors in leukemia therapy.     

Autophagy upregulation by inhibitors of caspase-3 and mTOR enhances radiotherapy in a mouse model of lung cancer

Autophagy has been reported to be increased in irradiated cancer cells resistant to various apoptotic stimuli. We therefore hypothesized that induction of autophagy via mTOR inhibition could enhance radiosensitization in apoptosis-inhibited H460 lung cancer cells in vitro and in a lung cancer xenograft model. To test this hypothesis, combinations of Z-DEVD (caspase-3 inhibitor), RAD001 (mTOR inhibitor) and irradiation were tested in cell and mouse models. The combination of Z-DEVD and RAD001 more potently radiosensitized H460 cells than individual treatment alone. The enhancement in radiation response was not only evident in clonogenic survival assays, but also was demonstrated through markedly reduced tumor growth, cellular proliferation (Ki67 staining), apoptosis (TUNEL staining) and angiogenesis (vWF staining) in vivo. Additionally, upregulation of autophagy as measured by increased GFP-LC3-tagged autophagosome formation accompanied the noted radiosensitization in vitro and in vivo. The greatest induction of autophagy and associated radiation toxicity was exhibited in the tri-modality treatment group. Autophagy marker, LC-3-II, was reduced by 3-methyladenine (3-MA), a known inhibitor of autophagy, but further increased by the addition of lysosomal protease inhibitors (pepstatin A and E64d), demonstrating that there is autophagic induction through type III PI3 kinase during the combined therapy. Knocking down of ATG5 and beclin-1, two essential autophagic molecules, resulted in radiation resistance of lung cancer cells. Our report suggests that combined inhibition of apoptosis and mTOR during radiotherapy is a potential therapeutic strategy to enhance radiation therapy in patients with non-small cell lung cancer.     

Autophagy upregulation by inhibitors of caspase-3 and mTOR enhances radiotherapy in a mouse model of lung cancer

Autophagy has been reported to be increased in irradiated cancer cells resistant to various apoptotic stimuli. We therefore hypothesized that induction of autophagy via mTOR inhibition enhances radiosensitization in apoptosis-inhibited H460 lung cancer cells in vitro and in a lung cancer xenograft model. To test this hypothesis, combinations of Z-DEVD (caspase-3 inhibitor), RAD001 (mTOR inhibitor) and irradiation were tested in cell and mouse models. The combination of Z-DEVD and RAD001 more potently radiosensitized H460 cells than individual treatment alone. The enhancement in radiation response was not only evident in clonogenic survival assays, but also was demonstrated through markedly reduced tumor growth, cellular proliferation (Ki67 staining), apoptosis (TUNEL staining), and angiogenesis (vWF staining) in vivo. Additionally, upregulation of autophagy as measured by increased GFP-LC3-tagged autophagosome formation accompanied the noted radiosensitization in vitro and in vivo. The greatest induction of autophagy and associated radiation toxicity was exhibited in the tri-modality treatment group. Autophagy marker, LC-3-II, was reduced by 3-methyladenine (3-MA), a known inhibitor of autophagy, but further increased by the addition of lysosomal protease inhibitors (pepstatin A and E64d), demonstrating that there is autophagic induction through type III PI3 kinase during the combined therapy. Knocking down of ATG5 and beclin-1, two essential autophagic molecules, resulted in radiation resistance of lung cancer cells. Our report suggests that combined inhibition of apoptosis and mTOR during radiotherapy is a potential therapeutic strategy to enhance radiation therapy in patients with non-small cell lung cancer.     

Inhibitory effects of the HDAC inhibitor valproic acid on prostate cancer growth are enhanced by simultaneous application of the mTOR inhibitor RAD001

AimsTo analyze the combined impact of the histone deacetylase (HDAC) inhibitor valproic acid (VPA) and the mammalian target of rapamycin (mTOR) inhibitor RAD001 on prostate cancer cell growth.Main methodsPC-3, DU-145 and LNCaP cells were treated with RAD001, VPA or with an RAD001–VPA combination for 3 or 5 days. Tumor cell growth, cell cycle progression and cell cycle regulating proteins were then investigated by MTT assay, flow cytometry and Western blotting, respectively. Effects of drug treatment on cell signaling pathways were determined.Key findingsSeparate application of RAD001 or VPA distinctly reduced tumor cell growth and impaired cell cycle progression. Significant additive effects were evoked when both drugs were used in concert. Particularly, the cell cycle regulating proteins cdk1, cdk2, cdk4 and cyclin B were reduced, whereas p21 and p27 were enhanced by the RAD001–VPA combination. Signaling analysis revealed deactivation of EGFr, ERK1/2 and p70S6k. Phosphorylation of Akt was diminished in DU-145 but elevated in PC-3 and LNCaP cells.SignificanceThe RAD001–VPA combination exerted profound antitumor properties on a panel of prostate cancer cell lines. Therefore, simultaneous blockage of HDAC and mTOR related pathways should be considered when designing novel therapeutic strategies for treating prostate carcinoma.     

RAD001 (Everolimus) induces autophagy in acute lymphoblastic leukemia

The elimination of tumor cells by apoptosis is the main mechanism of action of chemotherapeutic drugs used in current treatment protocols of acute lymphoblastic leukemia (ALL). Despite the rapid cytoreduction achieved, serious acute and late complications are frequent, and resistance to chemotherapy develops. During the past decade, new strategies to kill cancer cells by non-apoptotic mechanisms have flourished and many mediators of alternate cell death pathways have been identified. In the present study we have evaluated the efficacy of an mTOR inhibitor, RAD001 (Everolimus), to induce autophagy in an in vivo model of childhood ALL. In particular we found that RAD001 increased Beclin 1 expression, the conversion of the soluble form of microtubule-associated protein 1 light chain 3 (LC3) to the autophagic vesicle-associated form LC3-II and the occurence of lysosomes/autophagosomes. Focal degradation of cytoplasmic areas sequestered by autophagic structures was demonstrated by electron microscopy. This effect was associated with massive reduction of leukemic mass and a strong survival advantage for mice treated with RAD001. The discovery of alternative pathways involved in cell death execution and the role that it plays in leukemia suggest mTOR inhibitors should be included in future chemotherapy protocols of ALL.     

Anti-Myeloma Activity of Akt Inhibition Is Linked to the Activation Status of PI3K/Akt and MEK/ERK Pathway

The PI3K/Akt/mTOR signal transduction pathway plays a central role in multiple myeloma (MM) disease progression and development of therapeutic resistance. mTORC1 inhibitors have shown limited efficacy in the clinic, largely attributed to the reactivation of Akt due to rapamycin induced mTORC2 activity. Here, we present promising anti-myeloma activity of MK-2206, a novel allosteric pan-Akt inhibitor, in MM cell lines and patient cells. MK-2206 was able to induce cytotoxicity and inhibit proliferation in all MM cell lines tested, albeit with significant heterogeneity that was highly dependent on basal pAkt levels. MK-2206 was able to inhibit proliferation of MM cells even when cultured with marrow stromal cells or tumor promoting cytokines. The induction of cytotoxicity was due to apoptosis, which at least partially was mediated by caspases. MK-2206 inhibited pAkt and its down-stream targets and up-regulated pErk in MM cells. Using MK-2206 in combination with rapamycin (mTORC1 inhibitor), {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (PI3K inhibitor), or U0126 (MEK1/2 inhibitor), we show that Erk- mediated downstream activation of PI3K/Akt pathway results in resistance to Akt inhibition. These provide the basis for clinical evaluation of MK-2206 alone or in combination in MM and potential use of baseline pAkt and pErk as biomarkers for patient selection.     

Honokiol inhibits in vitro and in vivo growth of oral squamous cell carcinoma through induction of apoptosis,cell cycle arrest and autophagy

Honokiol, an active natural product derived from Magnolia officinalis, exerted anticancer effects through a variety of mechanisms on multiple types of cancers. In this study, the molecular mechanisms of honokiol in suppressing the human oral squamous cell carcinoma (OSCC) cells were evaluated. Treatment of two OSCC cell lines with honokiol resulted in reducing the cell proliferation and arresting the cell cycle at G1 stage which was correlated with the down‐regulation of Cdk2 and Cdk4 and the up‐regulation of cell cycle suppressors, p21 and p27. In addition, the caspase‐dependent programmed cell death was substantially detected, and the autophagy was induced as the autophagosome formation and autophagic flux proceeded. Modulation of autophagy by autophagic inducer, rapamycin or inhibitors, 3‐MA or bafilomycin, potentiated the honokiol‐mediated anti‐OSCC effects where honokiol exerted multiple actions in suppression of MAPK pathway and regulation of Akt/mTOR or AMPK pathways. As compared to clinical therapeutic agent, 5‐FU, honokiol exhibited more potent activity against OSCC cells and synergistically enhanced the cytotoxic effect of 5‐FU. Furthermore, orally administrated honokiol exerted effective antitumour activity in vivo in OSCC‐xenografted mice. Thus, this study revealed that honokiol could be a promising candidate in preventing human OSCCs.     

LncRNA MEG3 contributes to adenosine-induced cytotoxicity in hepatoma HepG2 cells by downregulated ILF3 and autophagy inhibition via regulation PI3K-AKT-mTOR and beclin-1 signaling pathway

Adenosine is a promising cytotoxic reagent for tumors, long noncoding RNA (lncRNA) maternally expressed gene 3 (MEG3) has been indicated to play critical roles in tumorigenesis, ILF3 has been recognized as a MEG3-binding protein, however, the roles of adenosine and MEG3 on hepatoma are still ambiguous. To clarify the effects of MEG3 on the adenosine-induced cytotoxicity in hepatoma, MEG3 and ILF3 lentivirus were transduced into human hepatoma HepG2 cells to stimulate overexpression of MEG3 (OE MEG3) and overexpression of ILF3 (OE ILF3), furthermore, ILF3 small interfering RNA (siRNA) was also applied to downregulate the expression of ILF3. In this study, autophagy was markedly inhibited by low concentration of adenosine, which present by not only inhibited transformation from LC3-I to LC3-II and autophagosomes formation, but also the elevation of mTOR and reduction of beclin-1 proteins. Furthermore, low concentration of adenosine also exerted marked cytotoxicity representing induced cell apoptosis together with reductions of cell viability and migration, which were also markedly enhanced by OE MEG3. Novelly and excitingly, adenosine markedly stimulated MEG3 expression, OE MEG3 markedly decreased the ILF3 expression in HepG2 cells, and the adenosine-induced autophagy inhibition, together with the ratio of p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR were also boosted by OE MEG3. More interestingly, OE ILF3 increased autophagy, whereas downregulated ILF3, especially in the case of adenosine, led to marked autophagy inhibition by decreasing beclin-1. The present study demonstrates autophagy inhibition is involved in the adenosine-induced cytotoxicity in HepG2 cells, the cytotoxicity can be synergized by OE MEG3 via downregulated ILF3 to activate PI3K/Akt/mTOR and inactivate the beclin-1 signaling pathway. In conclusion, MEG3 and inhibition of autophagy might be potential targets for augmenting adenosine-induced cytotoxicity in hepatoma.     

Molecular analysis of sunitinib resistant renal cell carcinoma cells after sequential treatment with RAD001 (everolimus) or sorafenib

Sequential application of target drugs is standard procedure after renal cell carcinoma (RCC) patients develop resistance. To optimize the sequence, antitumour effects of the mTOR inhibitor RAD001 or the tyrosine kinase inhibitor (TKI) sorafenib on RCC cells with acquired resistance to the TKI sunitinib was evaluated. RCC cells were exposed to 1 μM sunitinib for 24 hrs (as control) and for 8 weeks (to induce resistance) and then switched to RAD001 (5 nM) or sorafenib (5 μM) for a further 8 weeks. Tumour cell growth, cell cycle progression, cell cycle regulating proteins and intracellular signalling were then investigated. Short‐term application of sunitinib (24 hrs) induced cell growth blockade with accumulation in the G2/M phase. RCC cells became resistant to sunitinib after 8 weeks, demonstrated by accelerated cell growth along with enhanced cdk1, cdk2, loss of p27, activation of Akt, Rictor and Raptor. Switching to sorafenib only slightly reduced growth of the sunitinib resistant RCC cells and molecular analysis indicated distinct cross‐resistance. In contrast, full response was achieved when the cancer cells were treated with RAD001. p19 and p27 strongly increased, phosphorylated Akt, Rictor and Raptor decreased and the tumour cells accumulated in G0/G1. It is concluded that an mTOR‐inhibitor for second‐line therapy could be the strategy of choice after first‐line sunitinib failure.     

AMP-activated protein kinase (AMPK)/Ulk1-dependent autophagic pathway contributes to C6 ceramide-induced cytotoxic effects in cultured colorectal cancer HT-29 cells

Colorectal cancer is the second leading cause of cancer-related deaths. Drug resistance and/or off-target toxicity against normal cells limit the effectiveness of current chemotherapies for the treatment of colorectal cancer. In the current study, we studied the potential cytotoxic effects of short-chain and cell-permeable C6 ceramide in cultured colorectal cancer HT-29 cells and focused on the underlying mechanisms. We observed that C6 ceramide-induced HT-29 cell death and growth inhibition in a dose- and time-dependent manner. However, no significant apoptosis was observed in C6 ceramide-treated HT-29 cells. Our data support that autophagy contributed to C6 ceramide-induced cytotoxic effects, as autophagy inhibitors, 3-methyladenine (3-MA) and hydroxychloroquine, inhibited C6 ceramide’s effect; however, autophagy activators, everolimus (RAD001) and temsirolimus, mimicked C6 ceramide effects and induced HT-29 cell death. Further, we indentified that AMP-activated protein kinase (AMPK)/Ulk1 signaling was required for autophagy induction by C6 ceramide, and AMPK silencing by a specific short hairpin RNA suppressed C6 ceramide-induced autophagy and cytotoxic effects. Reversely, forced activation of AMPK by its activator AICAR or by genetic manipulation caused autophagic death in HT-29 cells, which was inhibited by 3-MA. Our results suggest that autophagy, but not apoptosis, is a major contributor for C6 ceramide-induced cytotoxic effects in HT-29 cells, and activation of AMPK/Ulk1 is required for the process.     

eEF-2 kinase,another meddler in the “Yin and Yang” of Akt–mediated cell fate?

Eukaryotic elongation factor-2 (eEF-2) kinase, also known as calmodulin-dependent protein kinase III, is a unique calcium/calmodulin-dependent enzyme. eEF-2 kinase can act as a negative regulator of protein synthesis and a positive regulator of autophagy under environmental or metabolic stresses. Akt, a key downstream effector of the PI3K signaling pathway that regulates cell survival and proliferation, is an attractive therapeutic target for anticancer treatment. Akt inhibition leads to activation of both apoptosis, type I programmed cell death and autophagy, a cellular degradation process via lysosomal machinery (also termed type II programmed cell death). However, the underlying mechanisms that dictate functional relationship between autophagy and apoptosis in response to Akt inhibition remain to be delineated. Our recent study demonstrated that inhibition of eEF-2 kinase can suppress autophagy but promote apoptosis in tumor cells subjected to Akt inhibition, indicating a role of eEF-2 kinase as a controller in the crosstalk between autophagy and apoptosis. Furthermore, inhibition of eEF-2 kinase can reinforce the efficacy of a novel Akt inhibitor, MK-2206, against human glioma. These findings may help optimize the use of Akt inhibitors in the treatment of cancer and other diseases.     

eEF-2 kinase: Another meddler in the “yin and yang” of Akt-mediated cell fate?

Eukaryotic elongation factor-2 (eEF-2) kinase, also known as calmodulin-dependent protein kinase III, is a unique calcium/calmodulin-dependent enzyme. eEF-2 kinase can act as a negative regulator of protein synthesis and a positive regulator of autophagy under environmental or metabolic stresses. Akt, a key downstream effector of the PI3K signaling pathway that regulates cell survival and proliferation, is an attractive therapeutic target for anticancer treatment. Akt inhibition leads to activation of both apoptosis, type I programmed cell death and autophagy, a cellular degradation process via lysosomal machinery (also termed type II programmed cell death). However, the underlying mechanisms that dictate functional relationship between autophagy and apoptosis in response to Akt inhibition remain to be delineated. Our recent study demonstrated that inhibition of eEF-2 kinase can suppress autophagy but promote apoptosis in tumor cells subjected to Akt inhibition, indicating a role of eEF-2 kinase as a controller in the crosstalk between autophagy and apoptosis. Furthermore, inhibition of eEF-2 kinase can reinforce the efficacy of a novel Akt inhibitor, MK-2206, against human glioma. These findings may help optimize the use of Akt inhibitors in the treatment of cancer and other diseases.     

A mechanism for synergy with combined mTOR and PI3 kinase inhibitors

Dysregulation of the mammalian target of rapamycin (mTOR) signaling has been found in many human cancers, particularly those with loss of the tumor suppressor PTEN. However, mTORC1 inhibitors such as temsirolimus have only modest activity when used alone and may induce acquired resistance by activating upstream mTORC2 and Akt. Other tumors that do not depend upon PI3K/Akt/mTOR signaling for survival are primarily resistant. This study tested the hypothesis that the limited clinical efficacy of temsirolimus is due to a compensatory increase in survival signaling pathways downstream of Akt as well as an incomplete block of 4E-BP1-controlled proliferative processes downstream of mTOR. We explored the addition of a PI3K inhibitor to temsirolimus and identified the mechanism of combinatorial synergy. Proliferation assays revealed that BEZ235 (dual PI3K/mTOR inhibitor) or ZSTK474 (pan PI3K inhibitor) combined with temsirolimus synergistically inhibited cell growth compared to cells treated with any of the agents alone. Co-treatment resulted in G0/G1 cell cycle arrest and up-regulation of p27. Cell death occurred through massive autophagy and subsequent apoptosis. While molecular profiling revealed that, in most cases, sensitivity to temsirolimus alone was most marked in cells with high basal phospho-Akt resulting from PTEN inactivation, combining a PI3K inhibitor with temsirolimus prevented compensatory Akt phosphorylation and synergistically enhanced cell death regardless of PTEN status. Another molecular correlate of synergy was the finding that temsirolimus treatment alone blocks downstream S6 kinase signaling, but not 4E-BP1. Adding BEZ235 completely abrogated 4E-BP1 phosphorylation. We conclude that the addition of a PI3K inhibitor overcomes cellular resistance to mTORC1 inhibitors regardless of PTEN status, and thus substantially expands the molecular phenotype of tumors likely to respond.     

A functionalized single-walled carbon nanotube-induced autophagic cell death in human lung cells through Akt�CTSC2-mTOR signaling

Nanoparticles are now emerging as a novel class of autophagy activators. Functionalized single-walled carbon nanotubes (f-SWCNTs) are valuable nanomaterials in many industries. This article is designed to assess the autophagic response for f-SWCNTs exposure in vitro and in vivo. A few types of f-SWCNTs were screened in human lung adenocarcinoma A549 cells for the autophagic response and related pathways in vitro. Formation of autophagosomes and LC3-II upregulation were confirmed on the basis of electron microscopy and LC3 western blotting for COOH-CNT, but not for PABS-CNT and PEG-CNT. MTT assay showed marked increase in cell viability, when COOH-CNT was added to cells in the presence of autophagy inhibitor 3MA, ATG6 or TSC2 siRNA. Consistent with the involvement of the Akt–TSC1/2–mTOR pathway, the phosphorylation levels of mTOR, mTOR''s substrate S6 and Akt were shown significantly decreased in A549 cells on treatment with COOH-CNT using western blotting. What''s more, autophagy inhibitor 3MA significantly reduced the lung edema in vivo. In a word, COOH-CNT induced autophagic cell death in A549 cells through the AKT–TSC2–mTOR pathway and caused acute lung injury in vivo. Inhibition of autophagy significantly reduced COOH-CNT-induced autophagic cell death and ameliorated acute lung injury in mice, suggesting a potential remedy to address the growing concerns on the safety of nanomaterials.     

GD2 ganglioside-binding antibody 14G2a and specific aurora A kinase inhibitor MK-5108 induce autophagy in IMR-32 neuroblastoma cells

The process of autophagy and its role in survival of human neuroblastoma cell cultures was studied upon addition of an anti-GD2 ganglioside (GD2) 14G2a mouse monoclonal antibody (14G2a mAb) and an aurora A kinase specific inhibitor, MK-5108. It was recently shown that combination of these agents significantly potentiates cytotoxicity against IMR-32 and CHP-134 neuroblastoma cells in vitro, as compared to the inhibitor used alone. In this study we gained mechanistic insights on autophagy in the observed cytotoxic effects exerted by both agents using cytotoxicity assays, RT-qPCR, immunoblotting, and autophagy detection methods. Enhancement of the autophagy process in the 14G2a mAb- and MK-5108-treated IMR-32 cells was documented by assessing autophagic flux. Application of a lysosomotropic agent—chloroquine (CQ) affected the 14G2a mAb- and MK-5108-stimulated autophagic flux. It is our conclusion that the 14G2a mAb (40 μg/ml) and MK-5108 inhibitor (0.1 μM) induce autophagy in IMR-32 cells. Moreover, the combinatorial treatment of IMR-32 cells with the 14G2a mAb and CQ significantly potentiates cytotoxic effect, as compared to CQ used alone. Most importantly, we showed that interfering with autophagy at its early and late step augments the 14G2a mAb-induced apoptosis, therefore we can conclude that inhibition of autophagy is the primary mechanism of the CQ-mediated sensitization to the 14G2a mAb-induced apoptosis. Although, there was no virtual stimulation of autophagy in the 14G2a mAb-treated CHP-134 neuroblastoma cells, we were able to show that PHLDA1 protein positively regulates autophagy and this process exists in a mutually exclusive manner with apoptosis in PHLDA1-silenced CHP-134 cells.     

RAD001 (everolimus) inhibits tumour growth in xenograft models of human hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide and highly resistant to available chemotherapies. Mammalian target of rapamycin (mTOR) functions to regulate protein translation, angiogenesis and cell cycle progression in many cancers including HCC. In the present study, subcutaneous patient-derived HCC xenografts were used to study the effects of an mTOR inhibitor, RAD001 (everolimus), on tumour growth, apoptosis and angiogenesis. We report that oral administration of RAD001 to mice bearing patient-derived HCC xenografts resulted in a dose-dependent inhibition of tumour growth. RAD001-induced growth suppression was associated with inactivation of downstream targets of mTOR, reduction in VEGF expression and microvessel density, inhibition of cell proliferation, up-regulation of p27^Kip1 and down-regulation of p21^Cip1/Waf1, Cdk-6, Cdk-2, Cdk-4, cdc-25C, cyclin B1 and c-Myc. Our data indicate that the mTOR pathway plays an important role in angiogenesis, cell cycle progression and proliferation of liver cancer cells. Our study provides a strong rationale for clinical investigation of mTOR inhibitor RAD001 in patients with HCC.     

ERK-mediated autophagy promotes inactivated Sendai virus (HVJ-E)-induced apoptosis in HeLa cells in an Atg3-dependent manner

Background

Apoptosis and autophagy are known to play important roles in cancer development. It has been reported that HVJ-E induces apoptosis in cancer cells, thereby inhibiting the development of tumors. To define the mechanism by which HVJ-E induces cell death, we examined whether HVJ-E activates autophagic and apoptotic signaling pathways in HeLa cells.

Methods

Cells were treated with chloroquine (CQ) and rapamycin to determine whether autophagy is involved in HVJ-E-induced apoptosis. Treatment with the ERK inhibitor, U0126, was used to determine whether autophagy and apoptosis are mediated by the ERK pathway. Activators of the PI3K/Akt/mTOR/p70S6K pathway, 740 Y-P and SC79, were used to characterize its role in HVJ-E-induced autophagy. siRNA against Atg3 was used to knock down the protein and determine whether it plays a role in HVJ-E-induced apoptosis in HeLa cells.

Results

We found that HVJ-E infection inhibited cell viability and induced apoptosis through the mitochondrial pathway, as evidenced by the expression of caspase proteins. This process was promoted by rapamycin treatment and inhibited by CQ treatment. HVJ-E-induced autophagy was further blocked by 740 Y-P, SC79, and U0126, indicating that both the ERK- and the PI3K/Akt/mTOR/p70S6K-pathways were involved. Finally, autophagy-mediated apoptosis induced by HVJ-E was inhibited by siRNA-mediated Atg3 knockdown.

Conclusion

In HeLa cells, HVJ-E infection triggered autophagy through the PI3K/Akt/mTOR/p70S6K pathway in an ERK1/2-dependent manner, and the induction of autophagy promoted apoptosis in an Atg3-dependent manner.

     

Clostridium difficile toxin B induces autophagic cell death in colonocytes

Toxin B (TcdB) is a major pathogenic factor of Clostridum difficile. However, the mechanism by which TcdB exerts its cytotoxic action in host cells is still not completely known. Herein, we report for the first time that TcdB induced autophagic cell death in cultured human colonocytes. The induction of autophagy was demonstrated by the increased levels of LC3‐II, formation of LC3⁺ autophagosomes, accumulation of acidic vesicular organelles and reduced levels of the autophagic substrate p62/SQSTM1. TcdB‐induced autophagy was also accompanied by the repression of phosphoinositide 3‐kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) complex 1 activity. Functionally, pharmacological inhibition of autophagy by wortmannin or chloroquine or knockdown of autophagy‐related genes Beclin 1, Atg5 and Atg7 attenuated TcdB‐induced cell death in colonocytes. Genetic ablation of Atg5, a gene required for autophagosome formation, also mitigated the cytotoxic effect of TcdB. In conclusion, our study demonstrated that autophagy serves as a pro‐death mechanism mediating the cytotoxic action of TcdB in colonocytes. This discovery suggested that blockade of autophagy might be a novel therapeutic strategy for C. difficile infection.     

The EphB2 tumor suppressor induces autophagic cell death via concomitant activation of the ERK1/2 and PI3K pathways

EphB2 is a tyrosine kinase receptor that has been shown to be a tumor suppressor gene in various cancers. However the mechanisms of this function are unknown. We report that EphB2 induces a form of cell death that does not involve the formation of apoptotic bodies or nuclear fragmentation and is instead accompanied by extensive vacuolization. Transmission electron microscopy demonstrates cytoplasmic vacuoles in EphB2-overexpressing cells that resembled autophagosomes. Using an EYFP-LC3 fusion protein and immunoblotting, we detected LC3 aggregation and conversion from form I to form II, both hallmarks of autophagy, in EphB2-transfected cells. Silencing of the autophagy regulating genes ATG5 or ATG7 using shRNAs, strongly prevented EphB2-induced cell death, further confirming its autophagic nature. EphB2 expression results in mitochondrial depolarization and translocation of cytochrome c from the mitochondria to the cytosol. Mapping of signaling pathways revealed novel information about the mechanisms of action of EphB2. We demonstrated that the MAPK pathway is important in the pro-death action of EphB2, through ERK1/2 phosphorylation and inhibition of this pathway using PD98059 counters EphB2-driven cell death. In addition, we found that inhibition of class III PI3K pathway, using the autophagy inhibitor 3MA, but not class I PI3K inhibition using LY294002, also effectively blocks EphB2-induced cell death. Finally, EphB2 expression inactivates Akt, which is a known inhibitor of autophagy. In conclusion, the EphB2 receptor induces an autophagic cell death that is mediated through the ERK1/2 and PI3K/Akt pathways.     

Crosstalk between Bak/Bax and mTOR signaling regulates radiation-induced autophagy

Bax and Bak, act as a gateway for caspase-mediated cell death. mTOR, an Akt downstream effector, plays a critical role in cell proliferation, growth and survival. The inhibition of mTOR induces autophagy, whereas apoptosis is a minor cell death mechanism in irradiated solid tumors. We explored possible alternative pathways for cell death induced by radiation in Bax/Bak-/- double knockout (DKO) MEF cells and wild-type cells, and we compared the cell survival: the Bax/Bak-/- cells were more radiosensitive than the wild-type cells. The irradiated cells displayed an increase in the pro-autophagic proteins ATG5-ATG12 and Beclin-1. These results are surprising in the fact that the inhibition of apoptosis resulted in increasing radiosensitivity; indicating that perhaps autophagy is the cornerstone in the cell radiation sensitivity regulation. Furthermore, irradiation upregulates autophagic programmed cell death in cells that are unable to undergo Bax/Bak-mediated apoptosis. We hypothesize the presence of a phosphatase-possibly PTEN, an Akt/mTOR negative regulator that can be inhibited by Bax/Bak. This fits with our hypothesis of Bax/Bak as a downregulator of autophagy. We are currently conducting experiments to explore the relationship between apoptosis and autophagy. Future directions in research include strategies targeting Bax/Bak in cancer xenografts and exploring novel radiosensitizers targeting autophagy pathways.