Multiple cyclin kinase inhibitors promote bile acid-induced apoptosis and autophagy in primary hepatocytes via p53-CD95-dependent signaling

Previously, using primary hepatocytes residing in early G(1) phase, we demonstrated that expression of the cyclin-dependent kinase (CDK) inhibitor protein p21(Cip-1/WAF1/mda6) (p21) enhanced the toxicity of deoxycholic acid (DCA) + MEK1/2 inhibitor. This study examined the mechanisms regulating this apoptotic process. Overexpression of p21 or p27(Kip-1) (p27) enhanced DCA + MEK1/2 inhibitor toxicity in primary hepatocytes that was dependent on expression of acidic sphingomyelinase and CD95. Overexpression of p21 suppressed MDM2, elevated p53 levels, and enhanced CD95, BAX, NOXA, and PUMA expression; knockdown of BAX/NOXA/PUMA reduced CDK inhibitor-stimulated cell killing. Parallel to cell death processes, overexpression of p21 or p27 profoundly enhanced DCA + MEK1/2 inhibitor-induced expression of ATG5 and GRP78/BiP and phosphorylation of PKR-like endoplasmic reticulum kinase (PERK) and eIF2alpha, and it increased the numbers of vesicles containing a transfected LC3-GFP construct. Incubation of cells with 3-methyladenine or knockdown of ATG5 suppressed DCA + MEK1/2 inhibitor-induced LC3-GFP vesicularization and enhanced DCA + MEK1/2 inhibitor-induced toxicity. Expression of dominant negative PERK blocked DCA + MEK1/2 inhibitor-induced expression of ATG5, GRP78/BiP, and eIF2alpha phosphorylation and prevented LC3-GFP vesicularization. Knock-out or knockdown of p53 or CD95 abolished DCA + MEK1/2 inhibitor-induced PERK phosphorylation and prevented LC3-GFP vesicularization. Thus, CDK inhibitors suppress MDM2 levels and enhance p53 expression that facilitates bile acid-induced, ceramide-dependent CD95 activation to induce both apoptosis and autophagy in primary hepatocytes.     

Neratinib kills B-RAF V600E melanoma via ROS-dependent autophagosome formation and death receptor signaling

Melanoma cells expressing mutant B-RAF V600E are susceptible to treatment with the combination of a B-RAF inhibitor and a MEK1/2 inhibitor. We investigated the impact of the ERBB family and MAP4K inhibitor neratinib on the biology of PDX isolates of cutaneous melanoma expressing B-RAF V600E. Neratinib synergized with HDAC inhibitors to kill melanoma cells at their physiologic concentrations. Neratinib activated ATM, AMPK, ULK1, and PERK and inactivated mTORC1/2, ERK1/2, eIF2 alpha, and STAT3. Neratinib increased expression of Beclin1, ATG5, CD95, and FAS-L and decreased levels of multiple toxic BH3 domain proteins, MCL1, BCL-XL, FLIP-s, and ERBB1/2/4. ATG13 S318 phosphorylation and autophagosome formation was dependent upon ATM, and activation of ATM was dependent on reactive oxygen species. Reduced expression of ERBB1/2/4 required autophagosome formation and reduced MCL1/BCL-XL levels required eIF2 alpha phosphorylation. Maximal levels of eIF2 alpha phosphorylation required signaling by ATM-AMPK and autophagosome formation. Knock down of eIF2 alpha, CD95, FAS-L, Beclin1, and ATG5 or over-expression of FLIP-s significantly reduced killing. Combined knock down of Beclin1 and CD95 abolished cell death. Our data demonstrate that PDX melanoma cells expressing B-RAF V600E are susceptible to being killed by neratinib and more so when combined with HDACi.     

The kinase inhibitor sorafenib induces cell death through a process involving induction of endoplasmic reticulum stress

Sorafenib is a multikinase inhibitor that induces apoptosis in human leukemia and other malignant cells. Recently, we demonstrated that sorafenib diminishes Mcl-1 protein expression by inhibiting translation through a MEK1/2-ERK1/2 signaling-independent mechanism and that this phenomenon plays a key functional role in sorafenib-mediated lethality. Here, we report that inducible expression of constitutively active MEK1 fails to protect cells from sorafenib-mediated lethality, indicating that sorafenib-induced cell death is unrelated to MEK1/2-ERK1/2 pathway inactivation. Notably, treatment with sorafenib induced endoplasmic reticulum (ER) stress in human leukemia cells (U937) manifested by immediate cytosolic-calcium mobilization, GADD153 and GADD34 protein induction, PKR-like ER kinase (PERK) and eukaryotic initiation factor 2alpha (eIF2alpha) phosphorylation, XBP1 splicing, and a general reduction in protein synthesis as assessed by [35S]methionine incorporation. These events were accompanied by pronounced generation of reactive oxygen species through a mechanism dependent upon cytosolic-calcium mobilization and a significant decline in GRP78/Bip protein levels. Interestingly, enforced expression of IRE1alpha markedly reduced sorafenib-mediated apoptosis, whereas knockdown of IRE1alpha or XBP1, disruption of PERK activity, or inhibition of eIF2alpha phosphorylation enhanced sorafenib-mediated lethality. Finally, downregulation of caspase-2 or caspase-4 by small interfering RNA significantly diminished apoptosis induced by sorafenib. Together, these findings demonstrate that ER stress represents a central component of a MEK1/2-ERK1/2-independent cell death program triggered by sorafenib.     

ER stress (PERK/eIF2alpha phosphorylation) mediates the polyglutamine-induced LC3 conversion, an essential step for autophagy formation

Expanded polyglutamine 72 repeat (polyQ72) aggregates induce endoplasmic reticulum (ER) stress-mediated cell death with caspase-12 activation and vesicular formation (autophagy). We examined this relationship and the molecular mechanism of autophagy formation. Rapamycin, a stimulator of autophagy, inhibited the polyQ72-induced cell death with caspase-12 activation. PolyQ72, but not polyQ11, stimulated Atg5-Atg12-Atg16 complex-dependent microtubule-associated protein 1 (MAP1) light chain 3 (LC3) conversion from LC3-I to -II, which plays a key role in autophagy. The eucaryotic translation initiation factor 2 alpha (eIF2alpha) A/A mutation, a knock-in to replace a phosphorylatable Ser51 with Ala51, and dominant-negative PERK inhibited polyQ72-induced LC3 conversion. PolyQ72 as well as ER stress stimulators upregulated Atg12 mRNA and proteins via eIF2alpha phosphorylation. Furthermore, Atg5 deficiency as well as the eIF2alpha A/A mutation increased the number of cells showing polyQ72 aggregates and polyQ72-induced caspase-12 activation. Thus, autophagy formation is a cellular defense mechanism against polyQ72-induced ER-stress-mediated cell death by degrading polyQ72 aggregates, with PERK/eIF2alpha phosphorylation being involved in polyQ72-induced LC3 conversion.     

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

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

The reduced catalase expression in TrkA-induced cells leads to autophagic cell death via ROS accumulation

TrkA receptor activation is a pivotal process for neuronal cell differentiation and survival. However, its overactivation or removal of its ligand NGF tends to cause the cell death. Recently, we demonstrated that TrkA overexpression induces cell death via apoptosis. In this study we also show that the TrkA-mediated cell death is associated with autophagy. TrkA-induced cells revealed an increase of GFP-LC3 punctate formation, development of acidic vesicular organelles (AVO) and formation of autophagosomes, which were eventually blocked by the addition of some autophagy inhibitors such as 3-methyladenine, ammonium chloride or wortmannin. In addition, although expression of autophagy-related proteins such as LC3-II or Beclin-1 was subtly altered during the TrkA-mediated cell death, depletion of ATG5 or Beclin-1 substantially decreased cell death in TrkA-expressing cells. In particular, reactive oxygen species (ROS) were dramatically accumulated in TrkA-induced cells, and the high accumulation of ROS was released by treatment of autophagy inhibitors. Furthermore, addition of an antioxidant N-acetylcysteine promoted the survival of TrkA-expressing cells and suppressed AVO production in cells. We also showed that this ROS accumulation was closely associated with reduction of catalase expression. Taken together, TrkA overexpression causes ROS accumulation via reduced catalase expression, ultimately leading to autophagic cell death.     

Silencing of Bcl-2 expression by small interfering RNA induces autophagic cell death in MCF-7 breast cancer cells

Apoptosis (programmed cell death type I) and autophagy (type II) are crucial mechanisms regulating cell death and homeostasis. The Bcl-2 proto-oncogene is overexpressed in 50-70% of breast cancers, potentially leading to resistance to chemotherapy, radiation and hormone therapy-induced apoptosis. Here, we investigated the role of Bcl-2 in autophagy in breast cancer cells. Silencing of Bcl-2 by siRNA in MCF-7 breast cancer cells downregulated Bcl-2 protein levels (>85%) and led to inhibition of cell growth (71%) colony formation (79%), and cell death (up to 55%) by autophagy but not apoptosis. Induction of autophagy was demonstrated by acridine orange staining, electron microscopy and an accumulation of GFP-LC3-II in autophagosomal membranes in MCF-7 cells transfected with GFP-LC-3(GFP-ATG8). Silencing of Bcl-2 by siRNA also led to induction of LC-3-II, a hallmark of autophagy, ATG5 and Beclin-1 autophagy promoting proteins. Knockdown of ATG5 significantly inhibited Bcl-2 siRNA-induced LC3-II expression, the number of GFP-LC3-II-labeled autophagosome positive cells and autophagic cell death (p < 0.05). Furthermore, doxorubicin at a high dose (IC(95), 1 microM) induced apoptosis but at a low dose (IC(50), 0.07 microM) induced only autophagy and Beclin-1 expression. When combined with Bcl-2 siRNA, doxorubicin (IC(50)) enhanced autophagy as indicated by the increased number cells with GFP-LC3-II-stained autophagosomes (punctuated pattern positive). These results provided the first evidence that targeted silencing of Bcl-2 induces autophagic cell death in MCF-7 breast cancer cells and that Bcl-2 siRNA may be used as a therapeutic strategy alone or in combination with chemotherapy in breast cancer cells that overexpress Bcl-2.     

Targeting autophagy enhances sorafenib lethality for hepatocellular carcinoma via ER stress-related apoptosis

Sorafenib, a potent multikinase inhibitor, has been recognized as the standard systemic treatment for patients with advanced hepatocellular carcinoma (HCC). However, the direct functional mechanism of tumor lethality mediated by sorafenib remains to be fully characterized, and the precise mechanisms of drug resistance are largely unknown. Here, we showed sorafenib induced both apoptosis and autophagy in human HCC cells through a mechanism that involved endoplasmic reticulum (ER) stress and was independent of the MEK1/2-ERK1/2 pathway. Upregulation of IRE1 signals from sorafenib-induced ER stress was critical for the induction of autophagy. Moreover, autophagy activation alleviated the ER stress-induced cell death. Inhibition of autophagy using either pharmacological inhibitors or essential autophagy gene knockdown enhanced cell death in sorafenib treated HCC cell lines. Critically, the combination of sorafenib with the autophagy inhibitor chloroquine produced more pronounced tumor suppression in HCC both in vivo and in vitro. These findings indicated that both ER stress and autophagy were involved in the cell death evoked by sorafenib in HCC cells. The combination of autophagy modulation and molecular targeted therapy is a promising therapeutic strategy in treatment of HCC.     

BIX-01294 induces autophagy-associated cell death via EHMT2/G9a dysfunction and intracellular reactive oxygen species production

We screened a chemical library in MCF-7 cells stably expressing green fluorescent protein (GFP)-conjugated microtubule-associated protein 1 light chain 3 (LC3) (GFP-LC3-MCF-7) using cell-based assay, and identified BIX-01294 (BIX), a selective inhibitor of euchromatic histone-lysine N-methyltransferase 2 (EHMT2), as a strong autophagy inducer. BIX enhanced formation of GFP-LC3 puncta, LC3-II, and free GFP, signifying autophagic activation. Inhibition of these phenomena with chloroquine and increasement in punctate dKeima ratio (550/438) signal indicated that BIX activated autophagic flux. BIX-induced cell death was suppressed by the autophagy inhibitor, 3-methyladenine, or siRNA against BECN1 (VPS30/ATG6), ATG5, and ATG7, but not by caspase inhibitors. Moreover, EHMT2 siRNA augmented GFP-LC3 puncta, LC3-II, free GFP, and cell death, implying that inhibition of EHMT2 caused autophagy-mediated cell death. Treatment with EHMT2 siRNA and BIX accumulated intracellular reactive oxygen species (ROS). BIX augmented mitochondrial superoxide via NADPH oxidase activation. In addition, BIX increased hydrogen peroxide and glutathione redox potential in both cytosol and mitochondria. Treatment with N-acetyl-L-cysteine (NAC) or diphenyleneiodonium chloride (DPI) decreased BIX-induced LC3-II, GFP-LC3 puncta, and cell death, indicating that ROS instigated autophagy-dependent cell death triggered by BIX. We observed that BIX potentiated autophagy-dependent and caspase-independent cell death in estrogen receptor (ESR)-negative SKBr3 and ESR-positive MCF-7 breast cancer cells, HCT116 colon cancer cells, and importantly, in primary human breast and colon cancer cells. Together, the results suggest that BIX induces autophagy-dependent cell death via EHMT2 dysfunction and intracellular ROS accumulation in breast and colon cancer cells, therefore EHMT2 inhibition can be an effective therapeutic strategy for cancer treatment.     

Activation of an alternative death receptor-induced signaling pathway in human hepatocytes under caspase arrest

Caspases are thought to be essential in execution of death receptor-induced apoptosis. However, recent findings suggest the existence of alternative pathways independent of caspases. We provide further evidence for such signaling in hepatocytes. RESULTS: Death receptor-induced activation of caspases and apoptosis in primary murine hepatocytes was completely blocked in presence of 1.5 microM N-benzyloxycarbonyl-Val-Ala-Asp-(O-methyl)fluoromethylketone (zVAD-fmk). Whereas the same concentration of the inhibitor was sufficient to block TNF receptor 1-, CD95- or TRAIL receptor 1/-2-induced activation of caspases in primary human hepatocytes or HepG2 cells, complete prevention apoptotic cell death needed almost 100 microM zVAD-fmk. Under caspase-inhibitory but non-protective conditions, i.e. at 1.5 microM zVAD-fmk, various serine protease inhibitors prevented apoptosis-like cell death. Neither sole arrest of caspases nor inhibition of serine proteases alone protected human hepatocytes. CONCLUSION: Human but not murine hepatocytes bear the potential to activate a permissive, serine protease inhibitor-sensitive alternative death signaling pathway under caspase-inhibitory conditions.     

Targeting autophagy enhances sorafenib lethality for hepatocellular carcinoma via ER stress-related apoptosis

Sorafenib, a potent multikinase inhibitor, has been recognized as the standard systemic treatment for patients with advanced hepatocellular carcinoma (HCC). However, the direct functional mechanism of tumor lethality mediated by sorafenib remains to be fully characterized, and the precise mechanisms of drug resistance are largely unknown. Here, we showed sorafenib induced both apoptosis and autophagy in human HCC cells through a mechanism that involved endoplasmic reticulum (ER) stress and was independent of the MEK1/2-ERK1/2 pathway. Upregulation of IRE1 signals from sorafenib-induced ER stress was critical for the induction of autophagy. Moreover, autophagy activation alleviated the ER stress-induced cell death. Inhibition of autophagy using either pharmacological inhibitors or essential autophagy gene knockdown enhanced cell death in sorafenib treated HCC cell lines. Critically, the combination of sorafenib with the autophagy inhibitor chloroquine produced more pronounced tumor suppression in HCC both in vivo and in vitro. These findings indicated that both ER stress and autophagy were involved in the cell death evoked by sorafenib in HCC cells. The combination of autophagy modulation and molecular targeted therapy is a promising therapeutic strategy in treatment of HCC.     

Protein kinase RNA-like ER kinase/eukaryotic translation initiation factor 2α pathway attenuates tumor necrosis factor alpha-induced apoptosis in nucleus pulposus cells by activating autophagy

Cellular loss induced by tumor necrosis factor alpha (TNF-α) contributes to the pathogenesis of intervertebral disc (IVD) degeneration. Cellular stress induced by TNF-α activates several processes to restore cell homeostasis. These processes include autophagy, endoplasmic reticulum stress, and related unfolded protein response (UPR). However, the effect and mechanism of UPR and autophagy regulated by TNF-α in IVD degeneration (IDD) remain unclear. The effect of autophagy on biological changes in nucleus pulposus cells (NPCs) also remains elusive. In this study, rat NPCs were cultured with TNF-α in the presence or absence of the UPR or autophagy pathway small-interfering RNAs. The associated genes and proteins were evaluated through immunofluorescence staining, quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analyses to monitor UPR and autophagy signaling and identify the regulatory mechanism of autophagy by the UPR pathway. Trypan blue exclusion assay, cell flow cytometry, terminal deoxynucleotidyl transferase dUTP nick end labeling staining, qRT-PCR, and western blot analyses were performed to examine the apoptosis of NPCs. The results showed that the acute exposure of TNF-α induced the apoptosis of rat NPCs and activated the protein kinase RNA-like ER kinase/eukaryotic translation initiation factor 2α (PERK/eIF2α) pathway of UPR and initiated autophagy. Silencing the PERK/eIF2α pathway or inhibiting autophagy enhanced the apoptosis of NPCs. Interference of the PERK/eIF2α pathway suppressed the autophagy of rat NPCs under TNF-α stimulation. Taken together, the PERK/eIF2α pathway reinforces the survival of NPCs under TNF-α stimulation by activating autophagy. Therefore, PERK/eIF2α-dependent autophagy could be a novel biological therapeutic target for IDD.     

PERK integrates autophagy and oxidative stress responses to promote survival during extracellular matrix detachment

Mammary epithelial cells (MECs) detached from the extracellular matrix (ECM) produce deleterious reactive oxygen species (ROS) and induce autophagy to survive. The coordination of such opposing responses likely dictates whether epithelial cells survive ECM detachment or undergo anoikis. Here, we demonstrate that the endoplasmic reticulum kinase PERK facilitates survival of ECM-detached cells by concomitantly promoting autophagy, ATP production, and an antioxidant response. Loss-of-function studies show that ECM detachment activates a canonical PERK-eukaryotic translation initiation factor 2α (eIF2α)-ATF4-CHOP pathway that coordinately induces the autophagy regulators ATG6 and ATG8, sustains ATP levels, and reduces ROS levels to delay anoikis. Inducible activation of an Fv2E-ΔNPERK chimera by persistent activation of autophagy and reduction of ROS results in lumen-filled mammary epithelial acini. Finally, luminal P-PERK and LC3 levels are reduced in PERK-deficient mammary glands, whereas they are increased in human breast ductal carcinoma in situ (DCIS) versus normal breast tissues. We propose that the normal proautophagic and antioxidant PERK functions may be hijacked to promote the survival of ECM-detached tumor cells in DCIS lesions.     

CD47 deficiency confers cell and tissue radioprotection by activation of autophagy

Accidental or therapeutic exposure to ionizing radiation has severe physiological consequences and can result in cell death. We previously demonstrated that deficiency or blockade of the ubiquitously expressed receptor CD47 results in remarkable cell and tissue protection against ischemic and radiation stress. Antagonists of CD47 or its ligand THBS1/thrombospondin 1 enhance cell survival and preserve their proliferative capacity. However the signaling pathways that mediate this cell-autonomous radioprotection are unclear. We now report a marked increase in autophagy in irradiated T-cells and endothelial cells lacking CD47. Irradiated T cells lacking CD47 exhibit significant increases in formation of autophagosomes comprising double-membrane vesicles visualized by electron microscopy and numbers of MAP1LC3A/B⁺ puncta. Moreover, we observed significant increases in BECN1, ATG5, ATG7 and a reduction in SQSTM1/p62 expression relative to irradiated wild-type T cells. We observed similar increases in autophagy gene expression in mice resulting from blockade of CD47 in combination with total body radiation. Pharmacological or siRNA-mediated inhibition of autophagy selectively sensitized CD47-deficient cells to radiation, indicating that enhanced autophagy is necessary for the prosurvival response to CD47 blockade. Moreover, re-expression of CD47 in CD47-deficient T cells sensitized these cells to death by ionizing radiation and reversed the increase in autophagic flux associated with survival. This study indicates that CD47 deficiency confers cell survival through the activation of autophagic flux and identifies CD47 blockade as a pharmacological route to modulate autophagy for protecting tissue from radiation injury.     

CD47 deficiency confers cell and tissue radioprotection by activation of autophagy

Accidental or therapeutic exposure to ionizing radiation has severe physiological consequences and can result in cell death. We previously demonstrated that deficiency or blockade of the ubiquitously expressed receptor CD47 results in remarkable cell and tissue protection against ischemic and radiation stress. Antagonists of CD47 or its ligand THBS1/thrombospondin 1 enhance cell survival and preserve their proliferative capacity. However the signaling pathways that mediate this cell-autonomous radioprotection are unclear. We now report a marked increase in autophagy in irradiated T-cells and endothelial cells lacking CD47. Irradiated T cells lacking CD47 exhibit significant increases in formation of autophagosomes comprising double-membrane vesicles visualized by electron microscopy and numbers of MAP1LC3A/B⁺ puncta. Moreover, we observed significant increases in BECN1, ATG5, ATG7 and a reduction in SQSTM1/p62 expression relative to irradiated wild-type T cells. We observed similar increases in autophagy gene expression in mice resulting from blockade of CD47 in combination with total body radiation. Pharmacological or siRNA-mediated inhibition of autophagy selectively sensitized CD47-deficient cells to radiation, indicating that enhanced autophagy is necessary for the prosurvival response to CD47 blockade. Moreover, re-expression of CD47 in CD47-deficient T cells sensitized these cells to death by ionizing radiation and reversed the increase in autophagic flux associated with survival. This study indicates that CD47 deficiency confers cell survival through the activation of autophagic flux and identifies CD47 blockade as a pharmacological route to modulate autophagy for protecting tissue from radiation injury.     

Silencing of Bcl-2 expression by small interfering RNA induces autophagic cell death in MCF-7 breast cancer cells

Apoptosis (programmed cell death type I) and autophagy (type II) are crucial mechanisms regulating cell death and homeostasis. The Bcl-2 proto-oncogene is overexpressed in 50-70% of breast cancers, potentially leading to resistance to chemotherapy, radiation and hormone therapy induced apoptosis. In this study, we investigated the role of Bcl-2 in autophagy in breast cancer cells. Silencing of Bcl-2 by siRNA in MCF-7 breast cancer cells downregulated Bcl-2 protein levels (>85%) and led to inhibition of cell growth (71%) colony formation (79%), and cell death (up to 55%) by autophagy but not apoptosis. Induction of autophagy was demonstrated by acridine orange staining, electron microscopy and an accumulation of GFP-LC3-II in preautopghagosomal and autophagosomal membranes in MCF-7 cells transfected with GFP-LC-3(GFP-ATG8). Silencing of Bcl-2 by siRNA also led to induction of LC-3-II, a hallmark of autophagy, ATG5 and Beclin-1 autophagy promoting proteins. Knockdown of ATG5 significantly inhibited Bcl-2 siRNA-induced LC3-II expression and the number of GFP-LC3-II-labeled autophagosome (punctuated pattern) positive cells and autophagic cell death (p     

The multi-kinase inhibitor lenvatinib interacts with the HDAC inhibitor entinostat to kill liver cancer cells

Prior studies from our group have combined the multi-kinase inhibitor sorafenib with HDAC inhibitors in GI tumor cells that resulted in the trials NCT02349867 and NCT01075113. The multi-kinase inhibitor lenvatinib, for the treatment of liver cancer, has fewer negative sequelae than sorafenib. We determined the mechanisms by which lenvatinib interacted with the HDAC inhibitor entinostat to kill hepatoma cells. Lenvatinib and entinostat interacted in an additive to greater-than-additive fashion to kill liver cancer cells. The drugs inactivated mTORC1 and mTORC2 and interacted to further increase the phosphorylation of ATM, ATG13 and eIF2α. Elevated eIF2α phosphorylation was responsible for reduced MCL-1 and BCL-XL expression and for increased Beclin1 and ATG5 expression. Over-expression of BCL-XL or knock down of Beclin1 or ATG5, significantly reduced killing. The drugs synergized to elevate ROS production; activation of ATM was ROS-dependent. ATM activation was required for enhanced phosphorylation of γH2AX, eIF2α and ATG13 S318. The drug combination reduced histone deacetylase protein expression which required autophagy. Knock down of HDACs1/2/3 prevented the lenvatinib and entinostat combination from regulating PD-L1 and MHCA expression. Collectively, our data demonstrate that lenvatinib and entinostat interact to kill liver cancer cells via ROS-dependent activation of ATM and inactivation of eIF2α, resulting in greater levels of toxic autophagosome formation and reduced expression of protective mitochondrial proteins.