Dominant-negative Caspase-9 Research Articles

GZ17-6.02 and Pemetrexed Interact to Kill Osimertinib-Resistant NSCLC Cells That Express Mutant ERBB1 Proteins.

We determined the molecular mechanisms by which the novel therapeutic GZ17-6.02 killed non-small cell lung cancer (NSCLC) cells. Erlotinib, afatinib, and osimertinib interacted with GZ17-6.02 to kill NSCLC cells expressing mutant EGFR proteins. GZ17-6.02 did not interact with any EGFR inhibitor to kill osimertinib-resistant cells. GZ17-6.02 interacted with the thymidylate synthase inhibitor pemetrexed to kill NSCLC cells expressing mutant ERBB1 proteins or mutant RAS proteins or cells that were resistant to EGFR inhibitors. The drugs interacted to activate ATM, the AMPK, and ULK1 and inactivate mTORC1, mTORC2, ERK1/2, AKT, eIF2α; and c-SRC. Knockdown of ATM or AMPKα1 prevented ULK1 activation. The drugs interacted to cause autophagosome formation followed by flux, which was significantly reduced by knockdown of ATM, AMPKα1, and eIF2α, or by expression of an activated mTOR protein. Knockdown of Beclin1, ATG5, or [BAX + BAK] partially though significantly reduced drug combination lethality as did expression of activated mTOR/AKT/MEK1 or over-expression of BCL-XL. Expression of dominant negative caspase 9 weakly reduced killing. The drug combination reduced the expression of HDAC2 and HDAC3, which correlated with lower PD-L1, IDO1, and ODC levels and increased MHCA expression. Collectively, our data support consideration of combining GZ17-6.02 and pemetrexed in osimertinib-resistant NSCLC.

Caspase-9: A Multimodal Therapeutic Target With Diverse Cellular Expression in Human Disease.

Caspase-9, a cysteine-aspartic protease known for its role as an initiator of intrinsic apoptosis, regulates physiological cell death and pathological tissue degeneration. Its nonapoptotic functions, including regulation of cellular differentiation/maturation, innate immunity, mitochondrial homeostasis, and autophagy, reveal a multimodal landscape of caspase-9 functions in health and disease. Recent work has demonstrated that caspase-9 can drive neurovascular injury through nonapoptotic endothelial cell dysfunction. CASP9 polymorphisms have been linked with various cancers, neurological disorders, autoimmune pathologies and lumbar disc disease. Clinical reports suggest alterations in caspase-9 expression, activity or function may be associated with acute and chronic neurodegeneration, retinal neuropathy, slow-channel myasthenic syndrome, lumbar disc disease, cardiomyopathies, atherosclerosis and autoimmune disease. Healthy tissues maintain caspase-9 activity at low basal levels, rendering supraphysiological caspase-9 activation a tractable target for therapeutic interventions. Strategies for selective inhibition of caspase-9 include dominant negative caspase-9 mutants and pharmacological inhibitors derived from the XIAP protein, whose Bir3 domain is an endogenous highly selective caspase-9 inhibitor. However, the mechanistic implications of caspase-9 expression and activation remain indeterminate in many pathologies. By assembling clinical reports of caspase-9 genetics, signaling and cellular localization in human tissues, this review identifies gaps between experimental and clinical studies on caspase-9, and presents opportunities for further investigations to examine the consequences of caspase activity in human disease.

Induction of Apoptosis in Lung Cancer Cells by Viburnum grandiflorum via Mitochondrial Pathway.

BackgroundLung cancer is one of the leading causes of mortality and morbidity. Viburnum grandiflorum is a medicinal herb known for its wide spectrum of pharmacological activities, but its anti-cancer properties against lung cancer cells have not been previously investigated. The present study elucidated the antitumor effect and associated mechanism of methanol extract of Viburnum grandiflorum extract (VGE) against lung cancer cells.Material/MethodsThe viability of H1650, HCC827, and H1299 cells was measured using MTT assay. Apoptosis and cell cycle progression were determined by flow cytometry using annexin-V/PI and JC-1 stains, respectively. The Lipofectamine Plus reagent (Invitrogen) was used for transfection of caspase-9 plasmid to H1650 and H1299 cells.ResultsThe results showed decreased H1650, HCC827, and H1299 cell viability by VGE, which occurred in a concentration- and time-dependent manner. The VGE treatment significantly increased the rate of apoptosis in H1650 (P<0.05) and H1299 (P<0.02) cells at 48 and 72 h. Treatment of H1650 and H1299 cells with 10 μM of VGE significantly enhanced the number of cells in sub-G1 phase. The VGE treatment cleaved pro-caspase-8/-9 and-3 in H1650 and HCC827 cells at 72 h. The VGE treatment of H1650 and HCC827 cells reduced Mcl-1 protein expression. Treatment of H1650 and HCC827 cells with VGE markedly decreased the level of p-Akt. However, dominant-negative caspase-9 (caspase-9 dN) plasmid transfection prevented the viability-inhibitory effect of VGE on H1650 and HCC827 cells. Treatment of H1650 and HCC827 cells with VGE increased levels of cytochrome c in the cytosol.ConclusionsVGE inhibited lung carcinoma cell viability by apoptosis activation through a caspase-dependent pathway. Therefore, VGE is a potent anti-cancer agent against lung cancer cells.

STAT3 activates the anti-apoptotic form of caspase 9 in oncovirus-infected B lymphocytes

The cancer-causing Epstein-Barr virus (EBV) activates the transcription factor STAT3 upon infecting B-lymphocytes. STAT3 then activates caspase 7 to degrade cellular claspin, resulting in impaired Chk1 phosphorylation. This blockade of ATR-Chk1 signaling allows EBV-transformed cells to proliferate despite DNA lesions from virus-induced replication stress. In addressing the mechanism of caspase 7 activation, we now report that in newly-infected B-cells, STAT3 transcriptionally activates the initiator caspase, caspase 9. Caspase 9 then activates caspase 7 to impair phosphorylation of Chk1 at S345. Importantly, although cleaved products of caspase 9 are detectable in infected cells, there is simultaneous increase in the alternatively-spliced dominant-negative form of caspase 9 – and – expression of dominant-negative caspase 9 is abrogated when STAT3 activation is impaired. Thus EBV, via STAT3, activates caspase 9 but also shifts the balance of transcripts towards its dominant-negative form to allow activation of caspase 7 while avoiding death of EBV-infected cells.

Valproate augments Niraparib killing of tumor cells

ABSTRACTPARP1 inhibitors are approved therapeutic agents in ovarian carcinomas, and have clinical activity in some breast cancers. As a single agent, niraparib killed ovarian and mammary tumor cells via an ATM-AMPK-ULK1 pathway which resulted in mTOR inactivation and the formation of autophagosomes, temporally followed by autolysosome formation. In parallel, niraparib activated a CD95-FADD-caspase 8 pathway, and collectively these signals caused tumor cell death that was suppressed by knock down of Beclin1, ATG5, CD95, FADD or AIF; or by expression of c-FLIP-s, BCL-XL or dominant negative caspase 9. The HDAC inhibitors AR42 and sodium valproate enhanced niraparib lethality in a greater than additive fashion. HDAC inhibitors enhanced niraparib lethality by increasing activation of the ATM-AMPK-ULK1-autophagy and CD95-FADD-caspase 8 pathways. Knock down of eIF2α, ATM, AMPKα, ULK1, Beclin1 or ATG5 reduced tumor cell killing by the niraparib plus HDAC inhibitor combination. Blockade of either caspase 9 function or that of cathepsin B partially prevented cell death. As a single agent niraparib delayed tumor growth, but did not significantly alter the tumor control rate. Tumors previously exposed to niraparib had activated the ERK1/2 and AKT-mTOR pathways that correlated with increased plasma levels of IL-8, MIF, EGF, uPA and IL-12. Collectively our findings argue that the addition of HDAC inhibitors to niraparib enhances the anti-cancer activity of the PARP1 inhibitor niraparib.

The irreversible ERBB1/2/4 inhibitor neratinib interacts with the PARP1 inhibitor niraparib to kill ovarian cancer cells

ABSTRACTThe irreversible ERBB1/2/4 inhibitor neratinib has been shown to rapidly down-regulate the expression of ERBB1/2/4 as well as the levels of c-MET, PDGFRα and mutant RAS proteins via autophagic degradation. Neratinib interacted in an additive to synergistic fashion with the approved PARP1 inhibitor niraparib to kill ovarian cancer cells. Neratinib and niraparib caused the ATM-dependent activation of AMPK which in turn was required to cause mTOR inactivation, ULK-1 activation and ATG13 phosphorylation. The drug combination initially increased autophagosome levels followed later by autolysosome levels. Preventing autophagosome formation by expressing activated mTOR or knocking down of Beclin1, or knock down of the autolysosome protein cathepsin B, reduced drug combination lethality. The drug combination caused an endoplasmic reticulum stress response as judged by enhanced eIF2α phosphorylation that was responsible for reducing MCL-1 and BCL-XL levels and increasing ATG5 and Beclin1 expression. Knock down of BIM, but not of BAX or BAK, reduced cell killing. Expression of activated MEK1 prevented the drug combination increasing BIM expression and reduced cell killing. Downstream of the mitochondrion, drug lethality was partially reduced by knock down of AIF, but expression of dominant negative caspase 9 was not protective. Our data demonstrate that neratinib and niraparib interact to kill ovarian cancer cells through convergent DNA damage and endoplasmic reticulum stress signaling. Cell killing required the induction of autophagy and was cathepsin B and AIF -dependent, and effector caspase independent.

Abstract 2690: Sorafenib/regorafenib and phosphatidyl inositol 3 kinase/thymoma viral proto-oncogene inhibition interact to kill tumor cells

Abstract The present study aimed to determine whether the multikinase inhibitors, sorafenib/regorafenib, worked in conjunction with PI3K/AKT inhibitors to enhance tumor cell death. It has been noted that combination of sorafenib/regorafenib with a PI3K inhibitor acetic acid (PX-866) resulted in tumor cell death, in a greater than additive fashion, in liver, colorectal, lung, breast, kidney and brain cancer cells. Similar data was obtained using AKT inhibitors perifosine and MK2206. Furthermore, even cells lacking PTEN remained as sensitive to this combinational approach as cells expressing PTEN. PX-866 treatment abolished AKT/GSK3 phosphorylation, with tumor cell death correlating with reduced activity of AKT and mTOR. Expression of activated AKT and to a lesser extent activated mTOR reduced drug combination lethality. Expression of B-cell lymphoma-extra large or dominant negative caspase 9, but not cellular FLICE-inhibitory protein short, protected cells from the drug combination. Treatment of cells with PX-866 increased protein levels of p62, LAMP2 and LC3/LC3II which correlated with the resulting increase in LC3-GFP vesicle numbers. Exposure of PX-866 treated cells to sorafenib reduced p62 and LAMP2 levels, decreased the ratio of LC3 to LC3II and reduced LC3-GFP vesicle levels. Knockdown of Beclin1 or ATG5 suppressed drug toxicity by ∼40%. In vivo, combination of either sorafenib and PX-866 or regorafenib and MK2206 cooperated to suppress the growth of established HuH7 and HCT116 tumors, respectively. Collectively our data demonstrates that the combination of sorafenib family kinase inhibitors with inhibitors of the PI3K/AKT pathway kills tumor cells in vitro and in vivo. Citation Format: Larry A. Booth, Nichola A. Cruickshanks, G B. Sajithlal, Hossein A. Hamed, Seyedmehrad Tavallai, J Syed, Steven Grant, Andrew Poklepovic, Paul Dent. Sorafenib/regorafenib and phosphatidyl inositol 3 kinase/thymoma viral proto-oncogene inhibition interact to kill tumor cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2690. doi:10.1158/1538-7445.AM2014-2690

Sorafenib/regorafenib and lapatinib interact to kill CNS tumor cells.

The present studies were to determine whether the multi-kinase inhibitor sorafenib or its derivative regorafenib interacted with the ERBB1/ERBB2 inhibitor lapatinib to kill CNS tumor cells. In multiple CNS tumor cell types sorafenib and lapatinib interacted in a greater than additive fashion to cause tumor cell death. Tumor cells lacking PTEN, and anoikis or lapatinib resistant cells were as sensitive to the drug combination as cells expressing PTEN or parental cells, respectively. Similar data were obtained using regorafenib. Treatment of brain cancer cells with [sorafenib + lapatinib] enhanced radiation toxicity. The drug combination increased the numbers of LC3-GFP vesicles; this correlated with a reduction in endogenous LC3II, and p62 and LAMP2 degradation. Knock down of Beclin1 or ATG5 significantly suppressed drug combination lethality. Expression of c-FLIP-s, BCL-XL, or dominant negative caspase 9 reduced drug combination toxicity; knock down of FADD or CD95 was protective. Expression of both activated AKT and activated MEK1 or activated mTOR was required to strongly suppress drug combination lethality. As both lapatinib and sorafenib are FDA approved agents, our data argue for further determination as to whether lapatinib and sorafenib is a useful glioblastoma therapy.

PDE5 inhibitors enhance the lethality of standard of care chemotherapy in pediatric CNS tumor cells

We determined whether clinically relevant phosphodiesterase 5 (PDE5) inhibitors interacted with clinically relevant chemotherapies to kill medulloblastoma cells. In medulloblastoma cells PDE5 inhibitors interacted in a greater than additive fashion with vincristine/etoposide/cisplatin to cause cell death. Knockdown of PDE5 expression recapitulated the combination effects of PDE5 inhibitor drugs with chemotherapy drugs. Expression of dominant negative caspase 9 did not significantly inhibit chemotherapy lethality but did significantly reduce enhanced killing in combination with the PDE5 inhibitor sildenafil. Overexpression of BCL-XL and c-FLIP-s suppressed individual and combination drug toxicities. Knockdown of CD95 or FADD suppressed drug combination toxicity. Treatment with PDE5 inhibitors and chemotherapy drugs promoted autophagy which was maximal at ~12 h post-treatment, and in a cell type-dependent manner knockdown of Beclin1 or ATG5 either suppressed or enhanced drug combination lethality. PDE5 inhibitors enhanced the induction of chemotherapy-induced DNA damage in a nitric oxide synthase-dependent fashion. In conclusion, our data demonstrate that the combination of PDE5 inhibitors with standard of care chemotherapy agents for medulloblastoma represents a possible novel modality for future treatment of this disease.

Pazopanib and HDAC inhibitors interact to kill sarcoma cells

The present studies were to determine whether the multi-kinase inhibitor pazopanib interacted with histone deacetylase inhibitors (HDACI: valproate, vorinostat) to kill sarcoma cells. In multiple sarcoma cell lines, at clinically achievable doses, pazopanib and HDACI interacted in an additive to greater than additive fashion to cause tumor cell death. The drug combination increased the numbers of LC3-GFP and LC3-RFP vesicles. Knockdown of Beclin1 or ATG5 significantly suppressed drug combination lethality. Expression of c-FLIP-s, and to a lesser extent BCL-XL or dominant negative caspase 9 reduced drug combination toxicity; knock down of FADD or CD95 was protective. Expression of both activated AKT and activated MEK1 was required to strongly suppress drug combination lethality. The drug combination inactivated mTOR and expression of activated mTOR strongly suppressed drug combination lethality. Treatment of animals carrying sarcoma tumors with pazopanib and valproate resulted in a greater than additive reduction in tumor volume compared with either drug individually. As both pazopanib and HDACIs are FDA-approved agents, our data argue for further determination as to whether this drug combination is a useful sarcoma therapy in the clinic.

Sorafenib/Regorafenib and Phosphatidyl Inositol 3 Kinase/Thymoma Viral Proto-Oncogene Inhibition Interact to Kill Tumor Cells

The present studies were undertaken to determine whether the multikinase inhibitors sorafenib/regorafenib cooperated with clinically relevant , phosphatidyl inositol 3 kinase (PI3K)-thymoma viral proto-oncogene (AKT) inhibitors to kill tumor cells. In liver, colorectal, lung, breast, kidney, and brain cancer cells, at clinically achievable doses, sorafenib/regorafenib and the PI3K inhibitor acetic acid (1S,4E,10R,11R,13S,14R)-[4-diallylaminomethylene-6-hydroxy-1-methoxymethyl-10,13-dimethyl-3,7,17-trioxo-1,3,4,7,10,11,12,13,14,15,16,17-dodecahydro-2-oxa-cyclopenta[a]phenanthren-11-yl ester (PX-866) cooperated in a greater than additive fashion to kill tumor cells. Cells lacking phosphatase and tensin homolog were as sensitive to the drug combination as cells expressing the protein. Similar data were obtained using the AKT inhibitors perifosine and 8-[4-(1-aminocyclobutyl)phenyl]-9-phenyl-1,2,4-triazolo[3,4-f] [1,6]naphthyridin-3(2H)-one hydrochloride (MK2206). PX-866 treatment abolished AKT/glycogen synthase kinase 3 (GSK3) phosphorylation, and cell killing correlated with reduced activity of AKT and mammalian target of rapamycin (mTOR). Expression of activated AKT and to a lesser extent activated mTOR reduced drug combination lethality. Expression of B-cell lymphoma-extra large or dominant negative caspase 9, but not cellular FLICE (FADD-like IL-1b-converting enzyme)-inhibitory protein short, protected cells from the drug combination. Treatment of cells with PX-866 increased protein levels of p62, lysosome-associated membrane protein 2 (LAMP2), and microtubule-associated protein light chain (LC) 3 and LC3II that correlated with a large increase in LC3-green fluorescent protein (GFP) vesicle numbers. Exposure of PX-866 treated cells to sorafenib reduced p62 and LAMP2 levels, decreased the ratio of LC3 to LC3II, and reduced LC3-GFP vesicle levels. Knockdown of Beclin1 or autophagy-related 5 suppressed drug toxicity by ∼40%. In vivo, sorafenib and PX-866 or regorafenib and MK2206 cooperated to suppress the growth of established HuH7 and HCT116 tumors, respectively. Collectively our data demonstrate that the combination of sorafenib family kinase inhibitors with inhibitors of the PI3K/AKT pathway kills tumor cells in vitro and in vivo.

Abstract 3346: A20/TNFAIP3, a novel target of histone deacetylase inhibitor-induced NF-κB activation, functionally disables the extrinsic apoptotic pathway in human leukemia cells.

Abstract The zinc finger protein A20/TNFAIP3 is a novel regulator of the pro-survival NF-κB signaling pathway. TNFα induces A20 via an NF-κB-dependent process to inhibit NF-κB, thus acting as a negative feedback regulator. Unlike the classic inhibitor IκBα, which sequesters NF-κB in the cytoplasm, A20 inactivates NF-κB by altering ubiquitination and proteasomal degradation of the receptor-interacting protein (RIP) in the TNF-R1-recruiting complex containing the FAS-associated death domain (FADD). Recently, we and others have found that histone deacetylase inhibitors (HDACIs) trigger sustained NF-κB activation, representing a compensatory cytoprotective response blunting the anti-cancer activity of these agents. Here, we identify, for the first time, A20 as a downstream target of HDACI-mediated NF-κB activation, which plays an important role in antagonizing HDACI lethality by preventing activation of the extrinsic apoptotic pathway. First, like TNFα and PMA, HDACIs (e.g., vorinostat and LBH-589) robustly increased A20 protein expression in various human leukemia cells (e.g., U937, MV4-11, and MOLM-13). Second, qPCR demonstrated increased mRNA levels after HDACI exposure. A20 expression occurred 16 h after HDACI exposure, similar to induction of p21, an NF-κB-dependent gene implicated in HDACI-induced differentiation. Moreover, A20 induction occurred (16 h) later than IκBα S32/S36 and RelA S536 phosphorylation (&amp;lt; 4h), suggesting an NF-κB activation-dependent process. Indeed, the IKK inhibitor Bay 11-7092 clearly diminished HDACI-induced A20 expression and transfection with an IκBα-super repressor (IκBα-SR) almost completely blocked this event. Third, prevention of A20 induction by shRNA dramatically increased (4 - 5-fold) sustained NF-κB activation by TNFα. In contrast, A20 shRNA failed to increase NF-κB activation by HDACI s, but did prolong this response from 8 h to 28 h. Fourth, disruption of A20 induction by shRNA sensitized leukemic cells to HDACIs, manifested by sharply diminished viability and increased apoptosis. The increased HDACI sensitivity of cells expressing A20 shRNA was associated with marked activation of the extrinsic pathway, reflected by increased caspase 8 cleavage and enzyme activity using IETD-pNA as substrate. Conversely, doxycycline-induced A20 expression significantly blocked HDACI-mediated caspase 8 and PARP cleavage in a tet-inducible system. Last, dominant-negative caspase 8 largely diminished HDACI lethality. Together, these findings indicate that HDACIs induce A20 expression through an NF-κB-dependent mechanism, and that A20 induction plays a significant functional role in limiting HDACI anti-leukemia activity by preventing extrinsic apoptotic pathway activation. They also raise the possibility that A20 may represent a therapeutic target for improving HDACI activity. Citation Format: Liang Zhou, Shuang Chen, Hui Lin, Xin-Yan Pei, Mandy M. Aust, Mohamed Rahmani, Roberto Rosato, Yun Dai, Steven Grant. A20/TNFAIP3, a novel target of histone deacetylase inhibitor-induced NF-κB activation, functionally disables the extrinsic apoptotic pathway in human leukemia cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3346. doi:10.1158/1538-7445.AM2013-3346

Resveratrol Sensitizes Acute Myelogenous Leukemia Cells to Histone Deacetylase Inhibitors through Reactive Oxygen Species-Mediated Activation of the Extrinsic Apoptotic Pathway

Histone deacetylase inhibitors (HDACIs) activate the prosurvival nuclear factor-κB (NF-κB) pathway by hyperacetylating RelA/p65, whereas the chemopreventive agent resveratrol inhibits NF-κB by activating the class III histone deacetylase Sirt1. Interactions between resveratrol and pan-HDACIs (vorinostat and panobinostat) were examined in human acute myelogenous leukemia (AML) cells. Pharmacologically achievable resveratrol concentrations (25-50 μM) synergistically potentiated HDACI lethality in AML cell lines and primary AML blasts. Resveratrol antagonized RelA acetylation and NF-κB activation in HDACI-treated cells. However, short hairpin RNA Sirt1 knockdown failed to modify HDACI sensitivity, which suggests that factors other than or in addition to Sirt1 activation contribute to resveratrol/HDACI interactions. These interactions were associated with death receptor 5 (DR5) up-regulation and caspase-8 activation, whereas cells expressing dominant-negative caspase-8 were substantially protected from resveratrol/HDACI treatment, which suggests a significant functional role for the extrinsic apoptotic pathway in lethality. Exposure to resveratrol with HDACI induced sustained reactive oxygen species (ROS) generation, which was accompanied by increased levels of DNA double-strand breaks, as reflected in γH2A.X and comet assays. The free radical scavenger Mn(III)tetrakis(4-benzoic acid)porphyrin chloride blocked ROS generation, DR5 up-regulation, caspase-8 activation, DNA damage, and apoptosis, which indicates a primary role for oxidative injury in lethality. Analyses of cell-cycle progression and 5-ethynyl-2'-deoxyuridine incorporation through flow cytometry revealed that resveratrol induced S-phase accumulation; this effect was abrogated by HDACI coadministration, which suggests that cells undergoing DNA synthesis may be particularly vulnerable to HDACI lethality. Collectively, these findings indicate that resveratrol interacts synergistically with HDACIs in AML cells through multiple ROS-dependent actions, including death receptor up-regulation, extrinsic apoptotic pathway activation, and DNA damage induction. They also raise the possibility that S-phase cells may be particularly susceptible to these actions.

Sorafenib and HDAC inhibitors synergize to kill CNS tumor cells

The present studies were designed to determine whether the multi-kinase inhibitor sorafenib (Nexavar) interacted with histone deacetylase inhibitors to kill glioblastoma and medulloblastoma cells. In a dose-dependent fashion sorafenib lethality was enhanced in multiple genetically disparate primary human glioblastoma isolates by the HDAC inhibitor sodium valproate (Depakote). Drug exposure reduced phosphorylation of p70 S6K and of mTOR. Similar data to that with valproate were also obtained using the HDAC inhibitor vorinostat (Zolinza). Sorafenib and valproate also interacted to kill medulloblastoma and PNET cell lines. Treatment with sorafenib and HDAC inhibitors radio-sensitized both GBM and medulloblastoma cell lines. Knock down of death receptor (CD95) expression protected GBM cells from the drug combination, as did overexpression of c-FLIP-s, BCL-XL and dominant negative caspase 9. Knock down of PDGFRα recapitulated the effect of sorafenib in combination with HDAC inhibitors. Collectively, our data demonstrate that the combination of sorafenib and HDAC inhibitors kills through activation of the extrinsic pathway, and could represent a useful approach to treat CNS-derived tumors.

Abstract 4649: Nbk/Bik induced by flavopiridol switches GX15-070-induced autophagy to apoptosis in human multiple myeloma cells

Abstract Combining the BH3 mimetic GX15-070 (GX, Obatoclax) with the CDK inhibitor flavopiridol (FP, Alvocidib) leads to striking in vitro and in vivo activity against human multiple myeloma (MM) cells, including those resistant to conventional or novel agents. Synergism is associated with both down-regulation of anti-apoptotic proteins (e.g., Mcl-1 and Bcl-xL) and induction of BH3-only proteins e.g., Bim and Bik (Bcl-2-interacting killer). While Mcl-1, Bcl-2, or Bcl-xL over-expression partially attenuates FP/GX lethality, knock-down of either Bim or Bik almost completely blocks apoptosis, suggesting a critical role for BH3-only proteins in this setting. Here, we demonstrate that in contrast to Bim, which directly triggers apoptosis, Bik promotes apoptosis by regulating autophagy. Prevention of Bik induction by shRNA failed to block release of Bak from Mcl-1, arguing against its direct action in apoptosis. Sub-toxic concentrations (500-750 nM) of GX released beclin 1 from Bcl-2 and induced autophagy (e.g., LC3 conversion and autophagosome formation), events occurring as early as 6 hr and sustained for 48 hr, but unaccompanied by marked apoptosis. Confocal microscopy revealed that in both drug-naïve and bortezomib-resistant MM cells, co-exposure to GX/FP strikingly increased autophagosomes extensively packed in the cytoplasm, accompanied by robust apoptosis. This phenomenon was associated with marked Bik up-regulation induced by FP +/− GX. Whereas Bim was up-regulated and predominantly localized to the mitochondria, Bik was induced and exclusively localized to the ER after FP/GX treatment. Autophagy includes initiation, elongation, autophagosome formation, and maturation to autolysosomes for cargo degradation. Interestingly, Bik expression occurred subsequent to autophagy induction and was diminished by beclin 1 or ATG5 shRNA, suggesting that Bik is induced during autophagy and negatively regulates later stages responsible for cargo clearance. Notably, whereas Bim shRNA diminished apoptosis but not autophagy induced by FP/GX, Bik shRNA blocked both processes. Moreover, beclin 1 or ATG5 shRNA significantly reduced apoptosis induced by FP/GX, whereas dominant-negative caspase-9 failed to prevent autophagy. Finally, overexpression of Bcl-xL, but not Bcl-2 or Mcl-1, largely attenuated Bik expression and autophagy induced by FP/GX. Co-immunoprecipitation showed that FP/GX-induced Bik bound to Bcl-xL, rather than to Bcl-2 or Mcl-1. Together, these findings suggest a model where in the presence of FP, Bik is expressed after autophagy induction by GX, and in turn interrupts later stages of autophagy, thus interfering with the ability of cells to remove cargo-loaded autophagosomes. Consequently, autophagosomes accumulate in cells co-exposed to FP/GX, leading to robust apoptosis. Thus, in this setting, Bik, acts as a switch that transforms autophagy into apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4649. doi:1538-7445.AM2012-4649

Abstract 4706: Resveratrol potentiates the lethality of HDAC inhibitors in acute myelogenous leukemia cells

Abstract Previous studies from our group and others have shown that interference with NF-κB RelA acetylation/activation significantly potentiates the activity of histone deacetylase inhibitors (HDACIs) in human leukemia and other transformed cells (Dai et al., Mol Cell Biol 25; 5429,2005; Dai et al., J Biol Chem 286:34036, 2011). The natural polyphenol resveratrol (RSV) is an activator of the class III HDAC SIRT1. Recently, preclinical evidence of RSV activity in leukemia has been described. In addition, clinical trials have established that RSV is very well tolerated in humans, with plasma concentrations &amp;gt; 250 μM achievable. Based upon these considerations, we hypothesized that a SIRT1 agonist, by deacetylating RelA, might enhance the antileukemic activity of clinically relevant HDACIs. To test this possibility, the impact of RSV on HDACI antileukemic activity was investigated. Co-administration (24 hr) of RSV (50 μM) with LBH589 (15 nM) or vorinostat (1.5 μM) synergistically induced mitochondrial injury (cytochrome c and AIF release), caspase activation, and apoptosis in AML cell lines (e.g., U937 cells). Of note, caspase 8 cleavage/activation was particularly pronounced after co-treatment, suggesting a potentially important functional role for the extrinsic apoptotic pathway in RSV/HDACI interactions. Similar interactions were also observed in AML cell lines bearing FLT3-ITD (e.g., MV4-11) and in primary AML blasts. These events were associated with deacetylation of RelA accompanied by inactivation of NF-κB, increased ROS formation, a marked increase in DNA damage (reflected by αH2A.X formation, an indicator of double strand DNA breaks), and abrogation of RSV-mediated S phase arrest. Genetic knockdown of SIRT1 by shRNA resulted in increased RelA acetylation and NF-κB activity, consistent with SIRT1 deacetylase actions. However, SIRT1 knockdown increased rather than decreased HDACI lethality, suggesting the contribution of NF-κB independent RSV actions to cell death. On the other hand, cells expressing dominant-negative caspase 8 were substantially protected from HDACI and HDACI/RSV lethality. Finally, co-administration of the antioxidant TBAP sharply reduced ROS generation, diminished caspase 8 activation and γH2AX formation, and prevented cell death in cells exposed to HDACI/RSV. Together, these findings indicate that clinically relevant concentrations of RSV significantly increase HDACI antileukemic activity through a process involving induction of oxidative injury (ROS), promotion of DNA damage, engagement of the extrinsic caspase cascade, potentiation of mitochondrial injury, and interruption of cell cycle regulation, culminating in apoptosis. They also imply that factors other than RelA deacetylation by RSV are primarily responsible for the enhanced antileukemic activity of this regimen. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4706. doi:1538-7445.AM2012-4706

Sildenafil increases chemotherapeutic efficacy of doxorubicin in prostate cancer and ameliorates cardiac dysfunction

We have shown that the potent phosphodiesterase-5 (PDE-5) inhibitor sildenafil (Viagra) induces a powerful effect on reduction of infarct size following ischemia/reperfusion injury and improvement of left ventricular dysfunction in the failing heart after myocardial infarction or doxorubicin (DOX) treatment. In the present study, we further investigated the potential effects of sildenafil on improving antitumor efficacy of DOX in prostate cancer. Cotreatment with sildenafil enhanced DOX-induced apoptosis in PC-3 and DU145 prostate cancer cells, which was mediated by enhanced generation of reactive oxygen species, up-regulation of caspase-3 and caspase-9 activities, reduced expression of Bcl-xL, and phosphorylation of Bad. Overexpression of Bcl-xL or dominant negative caspase 9 attenuated the synergistic effect of sildenafil and DOX on prostate cancer cell killing. Furthermore, treatment with sildenafil and DOX in mice bearing prostate tumor xenografts resulted in significant inhibition of tumor growth. The reduced tumor size was associated with amplified apoptotic cell death and increased expression of activated caspase 3. Doppler echocardiography showed that sildenafil treatment ameliorated DOX-induced left ventricular dysfunction. In conclusion, these results provide provocative evidence that sildenafil is both a powerful sensitizer of DOX-induced killing of prostate cancer while providing concurrent cardioprotective benefit.

Dihydroartemisinin Induces Apoptosis by a Bak-Dependent Intrinsic Pathway

The sesquiterpene lactone dihydroartemisinin (DHA), a semisynthetic derivative of the herbal antimalaria drug artemisinin, is cytotoxic to human tumor cells. Treatment of Jurkat T-lymphoma cells with DHA induced a breakdown of the mitochondrial transmembrane potential, release of cytochrome c, activation of caspases, and DNA fragmentation indicative of apoptosis induction. Although the absence of FADD or caspase-8 did not alter apoptosis rates in Jurkat cells, overexpression of dominant-negative caspase-9 or of antiapoptotic Bcl-xL or Bcl-2 largely decreased the cytotoxicity of DHA, demonstrating a role of the intrinsic death pathway. The proapoptotic Bcl-2 effector protein Bak and the Bcl-2 homology domain 3-only protein NOXA turned out to be important mediators of DHA-induced apoptosis in Jurkat cells. DHA treatment triggered the expression of NOXA and the activation of Bak. Furthermore, DHA-induced apoptosis was completely abrogated by loss of Bak and largely reduced in cells with siRNA-mediated downregulation of Bak or NOXA. Proapoptotic signaling of DHA also involved the formation of reactive oxygen species and membrane oxidation. Pretreatment with the lipophilic radical scavenger vitamin E or the hydrophilic radical scavengers glutathione and N-acetylcysteine reduced DHA-induced membrane oxidation and apoptosis, respectively. Oxidative changes also occurred in cells with disruption of the mitochondrial death pathway, suggesting a role of reactive oxygen species and oxidative membrane changes in death signaling upstream of the mitochondria. Interestingly, DHA increased the cytotoxic action of ionizing radiation and of the death receptor agonist tumor necrosis factor-related apoptosis-inducing ligand in Jurkat cells, suggesting a potential benefit of DHA in combined treatment strategies.

Lysosomal-Mitochondrial Axis in Zoledronic Acid-induced Apoptosis in Human Follicular Lymphoma Cells

Bisphosphonates (BPs) are potent inhibitors of osteoclast function, widely used to treat excessive bone resorption associated with bone metastases, that also have anti-tumor activity. Zoledronic acid (ZOL) represents a potential chemotherapeutic agent for the treatment of cancer. ZOL is the most potent nitrogen-containing BPs, and it inhibits cell growth and induces apoptosis in a variety of cancer cells. Recently we demonstrated that accumulation of isopentenyl pyrophosphate and the consequent formation of a new type of ATP analog (ApppI) after mevalonate pathway inhibition by nitrogen-containing BPs strongly correlates with ZOL-induced cell death in cancer cells in vitro. In this study we show that ZOL-induced apoptosis in HF28RA human follicular lymphoma cells occurs exclusively via the mitochondrial pathway, involves lysosomes, and is dependent on mevalonate pathway inhibition. To define the exact signaling pathway connecting them, we used modified HF28RA cell lines overexpressing either BclXL or dominant-negative caspase-9. In both mutant cells, mitochondrial and lysosomal membrane permeabilization (MMP and LMP) were totally prevented, indicating signaling between lysosomes and mitochondria and, additionally, an amplification loop for MMP and/or LMP regulated by caspase-9 in association with farnesyl pyrophosphate synthetase inhibition. Additionally, the lysosomal pathway in ZOL-induced apoptosis plays an additional/amplification role of the intrinsic pathway independently of caspase-3 activation. Moreover, we show a potential regulation by Bcl-XL and caspase-9 on cell cycle regulators of S-phase. Our findings provide a molecular basis for new strategies concomitantly targeting cell death pathways from multiple sites.

Human chorionic gonadotropin (hCG) interacts with lovastatin and ionizing radiation to modulate prostate cancer cell viability in vivo

The ability of human chorionic gonadotropin (hCG) to modify prostate carcinoma viability in vitro and in vivo when combined with the HMG CoA reductase inhibitor lovastatin and ionizing radiation was investigated. Treatment of PC-3 cells in vitro with hCG caused a modest increase in numbers of non-viable cells within 96h. Treatment of cells with hCG followed by exposure to the HMG CoA reductase inhibitor lovastatin suppressed AKT phosphorylation and enhanced the cytotoxic effects of hCG. The cytotoxic effects of hCG were blocked by expression of BCL-XL and dominant negative caspase 9. Treatment of mice bearing PC-3 flank tumors with lovastatin and hCG significantly reduced tumor volume within 7 days; this was also reflected in decreased ex vivo colony survival of the cells which correlated with increased cleavage of pro-caspase 3 and reduced Ki67 immuno-reactivity. In vitro, treatment of PC-3 cells with hCG followed by exposure to ionizing radiation enhanced the cytotoxic effects of hCG, that was further enhanced by lovastatin. In vivo, hCG radiosensitized PC-3 tumors and significantly enhanced the lethality of hCG and lovastatin treatment. Collectively, our findings argue that treatment of PC-3 prostate cancer tumors with hCG, lovastatin and radiation represents a potential novel therapeutic approach.