Autophagy inhibition enhances ursolic acid-induced apoptosis in PC3 cells

The phosphoinositol 3-kinase/Akt pathway plays a critical role in oncogenesis and the dysregulation of this pathway through loss of PTEN is a particularly common phenomenon in aggressive prostate cancers. Several recent studies have indicated that ursolic acid (UA), a pentacyclic triterpenoid, and its derivatives inhibit the growth of cancer cells by cell cycle arrest and the stimulation of apoptosis. In the present study, we report a novel autophagic response of UA in PTEN-deficient PC3 prostate cancer cells. As one of the major types of programmed cell death, autophagy has been observed in response to several anticancer drugs and demonstrated to be responsible for cell death. UA-induced autophagy in PC3 cells is associated with the reduced cell viability and the enhanced expression of LC3-II, an autophagosome marker in mammals, and monodansylcadaverine incorporation into autolysosomes. Furthermore, we found that UA exhibited anti-proliferative effects characterized by G1 phase arrest and autophagy at an early stage that precedes apoptosis. We also show that UA-induced autophagy in PC3 cells are mediated through the Beclin-1 and Akt/mTOR pathways. Inhibition of autophagy by either 3-methyladenine or Beclin-1/Atg5 small interfering RNA enhanced UA-induced apoptosis. Taken together, our data suggest that autophagy functions as a survival mechanism in PC3 cells against UA-induced apoptosis and a rational for the use of autophagy inhibitors in combination with UA as a novel modality of cancer therapy.     

mTOR is a fine tuning molecule in CDK inhibitors-induced distinct cell death mechanisms via PI3K/AKT/mTOR signaling axis in prostate cancer cells

Purvalanol and roscovitine are cyclin dependent kinase (CDK) inhibitors that induce cell cycle arrest and apoptosis in various cancer cells. We further hypothesized that co-treatment of CDK inhibitors with rapamycin, an mTOR inhibitor, would be an effective combinatory strategy for the inhibition of prostate cancer regard to androgen receptor (AR) status due to inhibition of proliferative pathway, PI3K/AKT/mTOR, and induction of cell death mechanisms. Androgen responsive (AR+), PTEN^?/? LNCaP and androgen independent (AR?), PTEN^+/? DU145 prostate cancer cells were exposed to purvalanol (20 µM) and roscovitine (30 µM) with or without rapamycin for 24 h. Cell viability assay, immunoblotting, flow cytometry and fluorescence microscopy was used to define the effect of CDK inhibitors with or without rapamycin on proliferative pathway and cell death mechanisms in LNCaP and DU145 prostate cancer cells. Co-treatment of rapamycin modulated CDK inhibitors-induced cytotoxicity and apoptosis that CDK inhibitors were more potent to induce cell death in AR (+) LNCaP cells than AR (?) DU145 cells. CDK inhibitors in the presence or absence of rapamycin induced cell death via modulating upstream PI3K/AKT/mTOR signaling pathway in LNCaP cells, exclusively only treatment of purvalanol have strong potential to inhibit both upstream and downstream targets of mTOR in LNCaP and DU145 cells. However, co-treatment of rapamycin with CDK inhibitors protects DU145 cells from apoptosis via induction of autophagy mechanism. We confirmed that purvalanol and roscovitine were strong apoptotic and autophagy inducers that based on regulation of PI3K/AKT/mTOR signaling pathway. Co-treatment of rapamycin with purvalanol and roscovitine exerted different effects on cell survival and death mechanisms in LNCaP and DU145 cell due to their AR receptor status. Our studies show that co-treatment of rapamycin with CDK inhibitors inhibit prostate cancer cell viability more effectively than either agent alone, in part, by targeting the mTOR signaling cascade in AR (+) LNCaP cells. In this point, mTOR is a fine-tuning player in purvalanol and roscovitine-induced apoptosis and autophagy via regulation of PI3K/AKT and the downstream targets, which related with cell proliferation.     

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

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

The Combination of RAD001 and MK-2206 Exerts Synergistic Cytotoxic Effects against PTEN Mutant Gastric Cancer Cells: Involvement of MAPK-Dependent Autophagic,but Not Apoptotic Cell Death Pathway

In the current study, we showed that the combination of mammalian target of rapamycin (mTOR) inhibitor RAD001 (everolimus) and Akt inhibitor MK-2206 exerted synergistic cytotoxic effects against low-phosphatase and tensin homolog (PTEN) gastric cancer cells (HGC-27 and SNU-601 lines). In HGC-27 cells, RAD001 and MK-2206 synergistically induced G1/S cell cycle arrest, growth inhibition, cell death but not apoptosis. RAD001 and MK-2206 synergistically induced light chain 3B (LC3B) and beclin-1 expression, two important autophagy indicators. Meanwhile, the autophagy inhibitor 3-methyladenine (3-MA) and chloroquine inhibited the cytotoxic effects by RAD001 and MK-2206, suggesting that autophagic, but not apoptotic cell death was important for the cytotoxic effects by the co-administration. We observed that the combination of RAD001 and MK-2206 exerted enhanced effects on Akt/mTOR inhibition, cyclin D1 down-regulation and ERK/MAPK(extracellular signal-regulated kinase/mitogen-activated protein kinases) activation. Intriguingly, MEK/ERK inhibitors PD98059 and U0126 suppressed RAD001 plus MK-2206-induced beclin-1 expression, autophagy induction and cytotoxicity in HGC-27 cells. In conclusion, these results suggested that the synergistic anti-gastric cancer cells ability by RAD001 and MK-2206 involves ERK-dependent autophagic cell death pathway.     

Insulin receptor substrate-1 prevents autophagy-dependent cell death caused by oxidative stress in mouse NIH/3T3 cells

Background

Insulin receptor substrate (IRS)-1 is associated with tumorigenesis; its levels are elevated in several human cancers. IRS-1 protein binds to several oncogene proteins. Oxidative stress and reactive oxygen species (ROS) are involved in the initiation and progression of cancers. Cancer cells produce greater levels of ROS than normal cells do because of increased metabolic stresses. However, excessive production of ROS kills cancer cells. Autophagy usually serves as a survival mechanism in response to stress conditions, but excessive induction of autophagy results in cell death. In addition to inducing necrosis and apoptosis, ROS induces autophagic cell death. ROS inactivates IRS-1 mediated signaling and reduces intracellular IRS-1 concentrations. Thus, there is a complex relationship between IRS-1, ROS, autophagy, and cancer. It is not fully understood how cancer cells grow rapidly and survive in the presence of high ROS levels.

Methods and results

In this study, we established mouse NIH/3T3 cells that overexpressed IRS-1, so mimicking cancers with increased IRS-1 expression levels; we found that the IRS-1 overexpressing cells grow more rapidly than control cells do. Treatment of cells with glucose oxidase (GO) provided a continuous source of ROS; low dosages of GO promoted cell growth, while high doses induced cell death. Evidence for GO induced autophagy includes increased levels of isoform B-II microtubule-associated protein 1 light chain 3 (LC3), aggregation of green fluorescence protein-tagged LC3, and increased numbers of autophagic vacuoles in cells. Overexpression of IRS-1 resulted in inhibition of basal autophagy, and reduced oxidative stress-induced autophagy and cell death. ROS decreased the mammalian target of rapamycin (mTOR)/p70 ribosomal protein S6 kinase signaling, while overexpression of IRS-1 attenuated this inhibition. Knockdown of autophagy-related gene 5 inhibited basal autophagy and diminished oxidative stress-induced autophagy and cell death.

Conclusion

Our results suggest that overexpression of IRS-1 promotes cells growth, inhibits basal autophagy, reduces oxidative stress-induced autophagy, and diminishes oxidative stress-mediated autophagy-dependent cell death. ROS-mediated autophagy may occur via inhibition of IRS-1/phosphatidylinositol 3-kinase/mTOR signaling. Our data afford a plausible explanation for IRS-1 involvement in tumor initiation and progression.     

Apigetrin induces extrinsic apoptosis,autophagy and G2/M phase cell cycle arrest through PI3K/AKT/mTOR pathway in AGS human gastric cancer cell

Apigetrin is a flavonoid glycoside phytonutrient derived from fruits and vegetables that is well known for a variety of biological activities such as antioxidant and anti-inflammatory activities. In the current study, we determined the effect of apigetrin on AGS gastric cancer cell. Apigetrin reduced cancer cell proliferation and induced G2/M phase cell cycle arrest by regulating cyclin B1, cdc25c and cdk1 protein expression in AGS cell. Apigetrin treatment caused apoptotic cell death in AGS cells, characterized by the accumulation of apoptosis portion, cleavage of caspase-3 and poly ADP-ribose polymerase (PARP). Apigetrin-treated cells increased the expression of extrinsic apoptosis pathway proteins and mRNA. However, intrinsic apoptosis pathway related proteins were not altered. In addition, AGS cells treated with apigetrin increased autophagic cell death, featured by the formation of autophagic vacuole and acidic vesicular organelles. Autophagy marker proteins, such as LC3B-II and beclin-1, were increased, and p62, an autophagy flux marker protein, was also increased by endoplasmic reticulum stress. Also, the phosphorylation of PI3K/AKT/mTOR pathway proteins and its downstream targets in apigetrin-treated AGS cells was identified to be decreased. Taken together, these data suggest that apigetrin-treated AGS cells induced G2/M phase cell cycle arrest, extrinsic apoptosis and autophagic cell death through PI3K/AKT/mTOR pathway, which can lead to the inhibition of gastric cancer development. Thus, our findings strongly indicate that apigetrin is a basic natural derived compound that could be used as a nutrient source with potential anticancer activities against gastric cancer.     

Mammalian target of rapamycin regulates isoliquiritigenin-induced autophagic and apoptotic cell death in adenoid cystic carcinoma cells

Previous studies, including those from our laboratory, have demonstrated that isoliquiritigenin (ISL), a flavonoid isolated from licorice, is a promising cancer chemotherapeutic agent. However the mechanisms underlying its anticancer effects are still far from clear. We now show, for the first time, that ISL triggers the mammalian target of rapamycin (mTOR)-dependent autophagic and apoptotic cell death in adenoid cystic carcinoma (ACC). Exposure of both ACC-2 and ACC-M cells to ISL resulted in several specific features for autophagy, including the appearance of membranous vacuoles, formation of acidic vesicular organelles, punctate pattern of LC3 immunostaining, and an increase in autophagic flux. Moreover, ISL treatment also resulted in significantly increased apoptosis in ACC cells. The ISL-mediated autophagic and apoptotic cell death were obviously attenuated by transfection with dominant negative Atg5 (DN-Atg5^K130R) plasmids or treatment with 3-methyladenine(3-MA). In additon, the data also revealed that the autophagic and apoptotic cell death induced by ISL occurred through a mTOR-dependent pathway. More importantly, the xenograft model using ACC-M cells provided further evidence of the occurrence of ISL-induced autophagy and apoptosis in vivo, correlating with the suppresson of mTOR activation as well as up-regulation of Atg5 expression. Taken together, these findings in our study suggest that induction of mTOR-dependent autophagic and apoptotic cell death may be an important mechanism in cancer chemotherapy by ISL.     

PIM-2 is an independent regulator of chondrocyte survival and autophagy in the epiphyseal growth plate

The overall goal of the investigation was to examine the activity and role of the PIM serine/threonine protein kinases in the growth plate. We showed for the first time that PIM-2 was highly expressed in epiphyseal chondrocytes and that the kinase was required for critical activities linked to cell survival. These activities were independent of those mediated by Akt-1. It was noted that PIM-2 protected chondrocytes from rapamycin sensitized (TOR inhibited) cell death. Since inhibition of mTOR caused autophagy, we examined the autophagic response of PIM-2 silenced cells. We showed that PIM-2 promoted expression and organization of autophagic proteins LC3, and Beclin-1 and enhanced lysosomal acidification. At the same time, PIM-2 modulated the activity of a key regulator of apoptosis, BAD. Since BAD inhibition and Beclin-1 expression activated autophagy, it is likely that induction of the autophagic pathway would serve to inhibit apoptosis and preserve the life of the terminally differentiated chondrocyte. We conclude that PIM-2 regulates a new intermediate stage in the differentiation pathway, the induction of autophagy.     

Enhancement of autophagy is a potential modality for tumors refractory to radiotherapy

Radiotherapy is a well-established treatment for cancer. However, the existence of radioresistant cells is one of the major obstacles in radiotherapy. In order to understand the mechanism of cellular radioresistance and develop more effective radiotherapy, we have established clinically relevant radioresistant (CRR) cell lines, which continue to proliferate under daily exposure to 2 Gray (Gy) of X-rays for >30 days. X-ray irradiation significantly induced autophagic cells in parental cells, which was exiguous in CRR cells, suggesting that autophagic cell death is involved in cellular radiosensitivity. An autophagy inducer, rapamycin sensitized CRR cells to the level of parental cells and suppressed cell growth. An autophagy inhibitor, 3-methyladenine induced radioresistance of parental cells. Furthermore, inhibition of autophagy by knockdown of Beclin-1 made parental cells radioresistant to acute radiation. These suggest that the suppression of autophagic cell death but not apoptosis is mainly involved in cellular radioresistance. Therefore, the enhancement of autophagy may have a considerable impact on the treatment of radioresistant tumor.     

Inhibition of Orai1‐mediated Ca2+ entry enhances chemosensitivity of HepG2 hepatocarcinoma cells to 5‐fluorouracil

Increasing evidence supports that activation of store‐operated Ca²⁺ entry (SOCE) is implicated in the chemoresistance of cancer cells subjected to chemotherapy. However, the molecular mechanisms underlying chemoresistance are not well understood. In this study, we aim to investigate whether 5‐FU induces hepatocarcinoma cell death through regulating Ca²⁺‐dependent autophagy. [Ca²⁺]_i was measured using fura2/AM dye. Protein expression was determined by Western blotting and immunohistochemistry. We found that 5‐fluorouracil (5‐FU) induced autophagic cell death in HepG2 hepatocarcinoma cells by inhibiting PI3K/AKT/mTOR pathway. Orai1 expression was obviously elevated in hepatocarcinoma tissues. 5‐FU treatment decreased SOCE and Orai1 expressions, but had no effects on Stim1 and TRPC1 expressions. Knockdown of Orai1 or pharmacological inhibition of SOCE enhanced 5‐FU‐induced inhibition of PI3K/AKT/mTOR pathway and potentiated 5‐FU‐activated autophagic cell death. On the contrary, ectopic overexpression of Orai1 antagonizes 5‐FU‐induced autophagy and cell death. Our findings provide convincing evidence to show that Orai1 expression is increased in hepatocarcinoma tissues. 5‐FU can induce autophagic cell death in HepG2 hepatocarcinoma cells through inhibition of SOCE via decreasing Orai1 expression. These findings suggest that Orai1 expression is a predictor of 5‐FU sensitivity for hepatocarcinoma treatment and blockade of Orai1‐mediated Ca²⁺ entry may be a promising strategy to sensitize hepatocarcinoma cells to 5‐FU treatment.     

Recombinant human arginase induced caspase-dependent apoptosis and autophagy in non-Hodgkin's lymphoma cells

Arginase, an arginine-degrading enzyme, has gained increased attention recently as a new experimental therapeutics for a variety of malignant solid cancers. In this study, we found that recombinant human arginase (rhArg) could induce remarkable growth inhibition, cell cycle arrest, and caspase-dependent apoptosis in Raji and Daudi non-Hodgkin''s lymphoma (NHL) cells through arginine deprivation. Interestingly, rhArg-treatment resulted in the appearance of autophagosomes and upregulation of microtubule-associated protein light chain 3 II, indicating that rhArg induced autophagy in lymphoma cells. Further study suggested that mammalian target of rapamycin/S6k signaling pathway may be involved in rhArg-induced autophagy in NHL cells. Moreover, blocking autophagy using pharmacological inhibitors (3-methyladenine and chloroquine) or genetic approaches (small interfering RNA targeting autophagy-related gene 5 and Beclin-1) enhanced the cell killing effect of rhArg. These results demonstrated that rhArg has a potent anti-lymphoma activity, which could be improved by in combination with autophagic inhibitors, suggesting that rhArg, either alone or in combination with autophagic inhibitors, could be a potential novel therapeutics for the treatment of NHL.     

Autophagy for cancer therapy through inhibition of pro-apoptotic proteins and mammalian target of rapamycin signaling

Autophagy is an alternative cell death pathway that is induced by mammalian target of rapamycin (mTOR) inhibitors and up-regulated when apoptosis is defective. We investigated radiation-induced autophagy in the presence or absence of Bax/Bak with or without an mTOR inhibitor, Rad001. Two isogenic cell lines, wild type (WT) and Bak/Bak(-/-) mouse embryonic fibroblasts and tumor cell lines were used for this study. Irradiated Bak/Bak(-/-) cells had a decrease of Akt/mTOR signaling and a significant increase of pro-autophagic proteins ATG5-ATG12 COMPLEX and Beclin-1. These molecular events resulted in an up-regulation of autophagy. Bax/Bak(-/-) cells were defective in undergoing apoptosis but were more radiosensitive than the WT cells in autophagy. Both autophagy and sensitization of Bak/Bax(-/-) cells were further enhanced in the presence of Rad001. In contrast, inhibitors of autophagy rendered the Bak/Bax(-/-) cells radioresistant, whereas overexpression of ATG5 and Beclin-1 made the WT cells radiosensitive. When this novel concept of radiosensitization was tested in cancer models, small interfering RNAs against Bak/Bax also led to increased autophagy and sensitization of human breast and lung cancer cells to gamma radiation, which was further enhanced by Rad001. This is the first report to demonstrate that inhibition of pro-apoptotic proteins and induction of autophagy sensitizes cancer cells to therapy. Therapeutically targeting this novel pathway may yield significant benefits for cancer patients.     

Inhibition of the mammalian target of rapamycin promotes cyclic AMP-induced differentiation of NG108-15 cells

To clarify the involvement of autophagy in neuronal differentiation, the effect of rapamycin, an mTOR complex inhibitor, on the dibutyryl cAMP (dbcAMP)-induced differentiation of NG108-15 cells was examined. Treatment of NG108-15 cells with 1 mM dbcAMP resulted in induction of differentiation, including neurite outgrowth and varicosity formation, enhanced voltage-sensitive Ca²⁺ channel activity and expression of microtubule-associated protein 2, and these effects involved phosphorylation of cAMP-response element binding protein (CREB) and extracellular signal regulated kinase (ERK). Simultaneous application of dbcAMP and rapamycin synergistically increased and accelerated differentiation. mTOR or raptor silencing with siRNA had a similar effect to rapamycin. Rapamycin and silencing of mTOR or raptor evoked autophagy, while blockade of autophagy by addition of 3-methyladenine or beclin 1 or Atg5 silencing prevented the potentiation of differentiation. Silencing of rictor also evokes autophagy, at a level 55% of that induced by raptor silencing and enhancement of differentiation is proportional. Rapamycin also caused increased ATP generation and cell cycle arrest in G₀/G₁ phase, but had no effect on CREB and ERK phosphorylation. dbcAMP also induced ATP generation, but not autophagy or cell cycle arrest. These results suggest that the increased autophagy, ATP generation and cell cycle arrest caused by mTOR inhibition promotes the dbcAMP-induced differentiation of NG108-15 cells.     

Platinum‐based combination chemotherapy triggers cancer cell death through induction of BNIP3 and ROS,but not autophagy

These days, cancer can still not be effectively cured because cancer cells readily develop resistance to anticancer drugs. Therefore, an effective combination of drugs with different mechanisms to prevent drug resistance has become a very important issue. Furthermore, the BH3‐only protein BNIP3 is involved in both apoptotic and autophagic cell death. In this study, lung cancer cells were treated with a chemotherapy drug alone or in combination to identify the role of BNIP3 and autophagy in combination chemotherapy for treating cancer. Our data revealed that various combinational treatments of two drugs could increase cancer cell death and cisplatin in combination with rapamycin or LBH589, which triggered the cell cycle arrest at the S phase. Cells with autophagosome and pEGFP‐LC3 puncta increased when treated with drugs. To confirm the role of autophagy, cancer cells were pre‐treated with the autophagy inhibitor 3‐methyladenine (3‐MA). 3‐MA sensitized cancer cells to chemotherapy drug treatments. These results suggest that autophagy may be responsible for cell survival in combination chemotherapy for lung cancer. Moreover, BNIP3 was induced and localized in mitochondria when cells were treated with drugs. The transfection of a dominant negative transmembrane deletion construct of BNIP3 (BNIP3ΔTM) and treatment of a reactive oxygen species (ROS) inhibitor suppressed chemo drug‐induced cell death. These results indicate that BNIP3 and ROS may be involved in combination chemo drug‐induced cell death. However, chemo drug‐induced autophagy may protect cancer cells from drug cytotoxicity. As a result, inhibiting autophagy may improve the effects of combination chemotherapy when treating lung cancer.     

Rapamycin and Interleukin-1β Impair Brain-derived Neurotrophic Factor-dependent Neuron Survival by Modulating Autophagy

The mammalian target of rapamycin (mTOR) pathway has multiple important physiological functions, including regulation of protein synthesis, cell growth, autophagy, and synaptic plasticity. Activation of mTOR is necessary for the many beneficial effects of brain-derived neurotrophic factor (BDNF), including dendritic translation and memory formation in the hippocampus. At present, however, the role of mTOR in BDNF''s support of survival is not clear. We report that mTOR activation is necessary for BDNF-dependent survival of primary rat hippocampal neurons, as either mTOR inhibition by rapamycin or genetic manipulation of the downstream molecule p70S6K specifically blocked BDNF rescue. Surprisingly, however, BDNF did not promote neuron survival by up-regulating mTOR-dependent protein synthesis or through mTOR-dependent suppression of caspase-3 activation. Instead, activated mTOR was responsible for BDNF''s suppression of autophagic flux. shRNA against the autophagic machinery Atg7 or Atg5 prolonged the survival of neurons co-treated with BDNF and rapamycin, suggesting that suppression of mTOR in BDNF-treated cells resulted in excessive autophagy. Finally, acting as a physiological analog of rapamycin, IL-1β impaired BDNF signaling by way of inhibiting mTOR activation as follows: the cytokine induced caspase-independent neuronal death and accelerated autophagic flux in BDNF-treated cells. These findings reveal a novel mechanism of BDNF neuroprotection; BDNF not only prevents apoptosis through inhibiting caspase activation but also promotes neuron survival through modulation of autophagy. This protection mechanism is vulnerable under chronic inflammation, which deregulates autophagy through impairing mTOR signaling. These results may be relevant to age-related changes observed in neurodegenerative diseases.     

Roles of autophagy in cetuximab-mediated cancer therapy against EGFR

Cetuximab is an epidermal growth factor receptor (EGFR)-blocking antibody that is approved to treat several types of solid cancers in patients. We recently showed that cetuximab can induce autophagy in cancer cells by both inhibiting the class I phosphatidylinositol 3-kinase (PtdIns3K)/Akt/mammalian target of rapamycin (mTOR) pathway and activating the class III PtdIns3K (hVps34)/beclin 1 pathway. In the current study, we investigated the relationship between cetuximab-induced autophagy and apoptosis and the biological roles of autophagy in cetuximab-mediated cancer therapy. We found that cetuximab induced autophagy in cancer cells that show strong or weak induction of apoptosis after cetuximab treatment but not in those that show only cytostatic growth inhibition. Inhibition of cetuximab-induced apoptosis by a caspase inhibitor prevented the induction of autophagy. Conversely, inhibition of cetuximab-induced autophagy by silencing the expression of autophagy-related genes (Atg) or treating the cancer cells with lysosomal inhibitors enhanced the cetuximab-induced apoptosis, suggesting that autophagy was a protective cellular response to cetuximab treatment. On the other hand, cotreatment of cancer cells with cetuximab and the mTOR inhibitor rapamycin resulted in an Atg-dependent and lysosomal inhibition-sensitive death of cancer cells that show only growth inhibition or weak apoptosis after cetuximab treatment, indicating that cell death may be achieved by activating the autophagy pathway in these cells. Together, our findings may guide the development of novel clinical strategies for sensitizing cancer cells to EGFR-targeted therapy.Key words: EGFR, cetuximab, autophagy, apoptosis, cancer therapy     

Roles of autophagy in cetuximab-mediated cancer therapy against EGFR

Cetuximab is an epidermal growth factor receptor (EGFR)-blocking antibody that is approved to treat several types of solid cancers in patients. We recently showed that cetuximab can induce autophagy in cancer cells by both inhibiting the class I phosphatidylinositol 3-kinase (PtdIns3K)/Akt/mammalian target of rapamycin (mTOR) pathway and activating the class III PtdIns3K (hVps34)/beclin 1 pathway. In the current study, we investigated the relationship between cetuximab-induced autophagy and apoptosis and the biological roles of autophagy in cetuximab-mediated cancer therapy. We found that cetuximab induced autophagy in cancer cells that show strong or weak induction of apoptosis after cetuximab treatment but not in those that show only cytostatic growth inhibition. Inhibition of cetuximab-induced apoptosis by a caspase inhibitor prevented the induction of autophagy. Conversely, inhibition of cetuximab-induced autophagy by silencing the expression of autophagy-related genes (Atg) or treating the cancer cells with lysosomal inhibitors enhanced the cetuximab-induced apoptosis, suggesting that autophagy was a protective cellular response to cetuximab treatment. On the other hand, cotreatment of cancer cells with cetuximab and the mTOR inhibitor rapamycin resulted in an Atg-dependent and lysosomal inhibition-sensitive death of cancer cells that show only growth inhibition or weak apoptosis after cetuximab treatment, indicating that cell death may be achieved by activating the autophagy pathway in these cells. Together, our findings may guide the development of novel clinical strategies for sensitizing cancer cells to EGFR-targeted therapy.     

Mitochondrial ROS generation for regulation of autophagic pathways in cancer

Mitochondria, the main source of reactive oxygen species (ROS), are required for cell survival; yet also orchestrate programmed cell death (PCD), referring to apoptosis and autophagy. Autophagy is an evolutionarily conserved lysosomal degradation process implicated in a wide range of pathological processes, most notably cancer. Accumulating evidence has recently revealed that mitochondria may generate massive ROS that play the essential role for autophagy regulation, and thus sealing the fate of cancer cell. In this review, we summarize mitochondrial function and ROS generation, and also highlight ROS-modulated core autophagic pathways involved in ATG4–ATG8/LC3, Beclin-1, p53, PTEN, PI3K–Akt–mTOR and MAPK signaling in cancer. Therefore, a better understanding of the intricate relationships between mitochondrial ROS and autophagy may ultimately allow cancer biologists to harness mitochondrial ROS-mediated autophagic pathways for cancer drug discovery.     

Propofol protects the autophagic cell death induced by the ischemia/reperfusion injury in rats

Autophagy has been implicated in cardiac cell death during ischemia/reperfusion (I/R). In this study we investigated how propofol, an antioxidant widely used for anesthesia, affects the autophagic cell death induced by the myocardial I/R injury. The infarction size in the myocardium was dramatically reduced in rats treated with propofol during I/R compared with untreated rats. A large number of autophagic vacuoles were observed in the cardiomyocytes of I/R-injured rats but rarely in I/R-injured rats treated with propofol. While LC3-II formation, an autophagy marker, was up-regulated in the I/R-injured myocardium, it was significantly down-regulated in the myocardial tissues of I/R-injured and propofol-treated rats. Moreover, propofol inhibited the I/R-induced expression of Beclin-1, and it accelerated phosphorylation of mTOR during I/R and Beclin-1/Bcl-2 interaction in cells, which indicates that it facilitates the inhibitory pathway of autophagy. These data suggest that propofol protects the autophagic cell death induced by the myocardial I/R injury.     

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.