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1.  Vorinostat and sorafenib increase ER stress, autophagy and apoptosis via ceramide-dependent CD95 and PERK activation 
Cancer biology & therapy  2008;7(10):1648-1662.
We recently noted that low doses of sorafenib and vorinostat interact in a synergistic fashion to kill carcinoma cells by activating CD95, and this drug combination is entering phase I trials. The present studies mechanistically extended our initial observations. Low doses of sorafenib and vorinostat, but not the individual agents, caused an acidic sphingomyelinase and fumonisin B1-dependent increase in CD95 surface levels and CD95 association with caspase 8. Knock down of CD95 or FADD expression reduced sorafenib/vorinostat lethality. Signaling by CD95 caused PERK activation that was responsible for both promoting caspase 8 association with CD95 and for increased eIF2α phosphorylation; suppression of eIF2α function abolished drug combination lethality. Cell killing was paralleled by PERK- and eIF2α-dependent lowering of c-FLIP-s protein levels and over-expression of c-FLIP-s maintained cell viability. In a CD95-, FADD- and PERK-dependent fashion, sorafenib and vorinostat increased expression of ATG5 that was responsible for enhanced autophagy. Expression of PDGFRβ and FLT3 were essential for high dose single agent sorafenib treatment to promote autophagy. Suppression of PERK function reduced sorafenib and vorinostat lethality whereas suppression of ATG5 levels elevated sorafenib and vorinostat lethality. Over-expression of c-FLIP-s blocked apoptosis and enhanced drug-induced autophagy. Thus sorafenib and vorinostat promote ceramide-dependent CD95 activation followed by induction of multiple downstream survival regulatory signals: ceramide-CD95-PERK-FADD-pro-caspase 8 (death); ceramide-CD95-PERK-eIF2α -↓c-FLIP-s (death); ceramide-CD95-PERK-ATG5-autophagy (survival).
PMCID: PMC2674577  PMID: 18787411
Vorinostat; Sorafenib; CD95; c-FLIP-s; PDGFRβ; FLT3; autophagy; ceramide; cell death; ASMase
2.  Sorafenib and HDAC inhibitors synergize to kill CNS tumor cells 
Cancer Biology & Therapy  2012;13(7):567-574.
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.
doi:10.4161/cbt.19771
PMCID: PMC3679096  PMID: 22406992
HDAC inhibitor; Sorafenib; apoptosis; glioma
3.  Sorafenib activates CD95 and promotes autophagy and cell death via Src family kinases in GI tumor cells 
Molecular cancer therapeutics  2010;9(8):2220-2231.
Sorafenib and vorinostat interact in a synergistic fashion to kill carcinoma cells by activating CD95, and the present studies have determined individually how sorafenib and vorinostat contribute to CD95 activation. Sorafenib (3-6 μM) promoted a dose-dependent increase in Src Y416, ERBB1 Y845 and CD95 Y232/Y291 phosphorylation, and Src Y527 dephosphorylation. Low levels of sorafenib (3 μM) –induced CD95 tyrosine phosphorylation did not promote surface localization whereas sorafenib (6 μM), or sorafenib (3 μM) and vorinostat (500 nM) treatment promoted higher levels of CD95 phosphorylation that correlated with DISC formation, receptor surface localization and autophagy. CD95 (Y232F, Y291F) was not tyrosine phosphorylated and was unable to plasma membrane localize or induce autophagy. Knock down / knock out of Src family kinases abolished sorafenib –induced: CD95 tyrosine phosphorylation; DISC formation; and the induction of cell death and autophagy. Knock down of PDGFRβ enhanced Src Y416 and CD95 tyrosine phosphorylation that correlated with elevated CD95 plasma membrane levels and autophagy, and with a reduced ability of sorafenib to promote CD95 membrane localization. Vorinostat increased ROS levels; and in a delayed NFκB-dependent fashion, those of FAS ligand and CD95. Neutralization of FAS-L did not alter the initial rapid drug-induced activation of CD95 however, neutralization of FAS-L reduced sorafenib + vorinostat toxicity by ~50%. Thus sorafenib contributes to CD95 activation by promoting receptor tyrosine phosphorylation whereas vorinostat contributes to CD95 activation via initial facilitation of ROS generation and subsequently of FAS-L expression.
doi:10.1158/1535-7163.MCT-10-0274
PMCID: PMC2933415  PMID: 20682655
Vorinostat; Sorafenib; CD95; c-FLIP-s; FAS-L; cell death; autophagy
4.  Vorinostat and sorafenib increase CD95 activation in gastrointestinal tumor cells through a Ca2+ - de novo ceramide - PP2A - ROS dependent signaling pathway 
Cancer research  2010;70(15):6313-6324.
The targeted therapeutics sorafenib and vorinostat interact in a synergistic fashion to kill carcinoma cells by activating CD95, and this drug combination is entering phase I evaluation. In this study we determined how CD95 is activated by treatment with this drug combination. Low doses of sorafenib and vorinostat but not the individual drugs rapidly increased ROS, Ca2+ and ceramide levels in GI tumor cells. The production of ROS was reduced in Rho zero cells. Quenching ROS blocked drug-induced CD95 surface localization and apoptosis. ROS generation, CD95 activation and cell killing was also blocked by quenching of induced Ca2+ levels or by inhibition of PP2A. Inhibition of acidic sphingomyelinase or de novo ceramide generation blocked the induction of ROS however combined inhibition of both acidic sphingomyelinase and de novo ceramide generation was required to block the induction of Ca2+. Quenching of ROS did not impact on drug-induced ceramide/dihydro-ceramide levels whereas quenching of Ca2+ reduced the ceramide increase. Sorafenib and vorinostat treatment radiosensitized liver and pancreatic cancer cells, an effect that was suppressed by quenching ROS or knock down of LASS6. Further, sorafenib and vorinostat treatment suppressed the growth of pancreatic tumors in vivo. Our findings demonstrate that induction of cytosolic Ca2+ by sorafenib and vorinostat is a primary event that elevates dihydroceramide levels, each essential steps in ROS generation that promotes CD95 activation.
doi:10.1158/0008-5472.CAN-10-0999
PMCID: PMC2918282  PMID: 20631069
5.  Regulation of autophagy by ceramide-CD95-PERK signaling 
Autophagy  2008;4(7):929-931.
The manuscripts by Park et al.1 and Zhang et al.2 were initially planned as studies to understand the regulation of cell survival in transformed cells treated with sorafenib and vorinostat, and in primary hepatocytes treated with a bile acid+MEK1/2 inhibitor. In both cell systems we discovered that the toxicity of sorafenib and vorinostat or bile acid+MEK1/2 inhibitor exposure depended on the generation of ceramide and the ligand-independent activation of the CD95 death receptor, with subsequent activation of pro-caspase 8. We noted, however, in these systems that, in parallel with death receptor–induced activation of the extrinsic pathway, CD95 signaling also promoted increased phosphorylation of PKR-like endoplasmic reticulum kinase (PERK) and eIF2α, increased expression of ATG5, and increased processing of LC3 and vesicularization of a GFP-LC3 construct. The knockdown of ATG5 expression blocked GFP-LC3 vesicularization and enhanced cell killing. Thus ceramide-CD95 signaling promoted cell death via activation of pro-caspase 8 and cell survival via autophagy. PERK was shown to signal in a switch-hitting fashion; PERK promoted CD95-DISC formation and an eIF2α-dependent reduction in c-FLIP-s levels that were essential for cell killing to proceed, but in parallel it also promoted autophagy that was protective. The death receptor-induced apoptosis and autophagy occur proximal to the receptor rather than the mitochondrion, and the relative flow of death receptor signaling into either pathway may determine cell fate. Finally, death receptor induced apoptosis and autophagy could be potential targets for therapeutic intervention.
PMCID: PMC3292039  PMID: 18719356
Vorinostat; Sorafenib; bile acid; CD95; autophagy; ceramide; cell death; ASMase
6.  Combined anticancer effects of sphingosine kinase inhibitors and sorafenib 
Investigational new drugs  2010;29(6):1132-1142.
Summary
The pro-apoptotic lipid sphingosine is phosphorylated by sphingosine kinases 1 and 2 (SK1 and SK2) to generate the mitogenic lipid sphingosine-1-phosphate (S1P). We previously reported that inhibition of SK activity delays tumor growth in a mouse mammary adenocarcinoma model. Because SK inhibitors and the multikinase inhibitor sorafenib both suppress the MAP kinase pathway, we hypothesized that their combination may provide enhanced inhibition of tumor growth. Therefore, we evaluated the effects of two SK inhibitors, ABC294640 (a SK2-specific inhibitor) and ABC294735 (a dual SK1/SK2 inhibitor), alone and in combination with sorafenib on human pancreatic adenocarcinoma (Bxpc-3) and kidney carcinoma (A-498) cells in vitro and in vivo. Exposure of either Bxpc-3 or A-498 cells to combinations of ABC294640 and sorafenib or ABC294735 and sorafenib resulted in synergistic cytotoxicity, associated with activation of caspases 3/ 7 and DNA fragmentation. Additionally, strong decreases in ERK phosphorylation were observed in Bxpc-3 and A-498 cells exposed to either the sorafenib/ABC294640 or the sorafenib/ABC294735 combination. Oral administration of either ABC294640 or ABC294735 to mice led to a delay in tumor growth in both xenograft models without overt toxicity to the animals. Tumor growth delay was potentiated by co-administration of sorafenib. These studies show that combination of an SK inhibitor with sorafenib causes synergistic inhibition of cell growth in vitro, and potentiates antitumor activity in vivo. Thus, a foundation is established for clinical trials evaluating the efficacy of combining these signaling inhibitors.
doi:10.1007/s10637-010-9452-0
PMCID: PMC3089696  PMID: 20473784
Targeted therapy; Sphingosine kinase; Sorafenib; Apoptosis; MAPK pathway
7.  Histone deacetylase inhibitor vorinostat suppresses the growth of uterine sarcomas in vitro and in vivo 
Molecular Cancer  2010;9:49.
Background
Uterine sarcomas are very rare malignancies with no approved chemotherapy protocols. Histone deacetylase (HDAC) inhibitors belong to the most promising groups of compounds for molecular targeting therapy. Here, we described the antitumor effects of suberoylanilide hydroxamic acid (SAHA; vorinostat) on MES-SA uterine sarcoma cells in vitro and in vivo. We investigated effects of vorinostat on growth and colony forming ability by using uterine sarcoma MES-SA cells. We analyzed the influence of vorinostat on expression of different HDACs, p21WAF1 and activation of apoptosis. Finally, we examined the antitumor effects of vorinostat on uterine sarcoma in vivo.
Results
Vorinostat efficiently suppressed MES-SA cell growth at a low dosage (3 μM) already after 24 hours treatment. Decrease of cell survival was even more pronounced after prolonged treatment and reached 9% and 2% after 48 and 72 hours of treatment, respectively. Colony forming capability of MES-SA cells treated with 3 μM vorinostat for 24 and 48 hours was significantly diminished and blocked after 72 hours. HDACs class I (HDAC2 and 3) as well as class II (HDAC7) were preferentially affected by this treatment. Vorinostat significantly increased p21WAF1 expression and apoptosis. Nude mice injected with 5 × 106 MES-SA cells were treated for 21 days with vorinostat (50 mg/kg/day) and, in comparison to placebo group, a tumor growth reduction of more than 50% was observed. Results obtained by light- and electron-microscopy suggested pronounced activation of apoptosis in tumors isolated from vorinostat-treated mice.
Conclusions
Our data strongly indicate the high therapeutic potential of vorinostat in uterine sarcomas.
doi:10.1186/1476-4598-9-49
PMCID: PMC2843655  PMID: 20202195
8.  Sorafenib inhibits STAT3 activation to enhance TRAIL-mediated apoptosis in human pancreatic cancer cells 
Molecular Cancer Therapeutics  2010;9(3):742-750.
Purpose
STAT3 is constitutively active in human pancreatic cancer cells and can promote cell growth and apoptosis resistance that contribute to tumorigenesis. We determined if sorafenib, a multikinase inhibitor, can induce apoptosis by targeting STAT3 signaling to enhance apoptosis induction by TRAIL.
Experimental Design
Human pancreatic cancer cell lines (PANC-1 and BxPC-3) were pre-incubated with sorafenib (Nexavar®) alone or followed by TRAIL. Apoptosis was determined by Annexin V labeling, caspase cleavage and Bax/Bak activation. Protein expression was analyzed by immunoblotting. Knockdown of STAT3, Mcl-1 and Bim were achieved by lentiviral shRNA. Adenoviral dominant negative (DN) or retroviral constitutively active (CA) STAT3 were also utilized.
Results
Sorafenib inhibited constitutive STAT3 phosphorylation (Tyr705) and suppressed Mcl-1 and Bcl-xL proteins in a dose- and time-dependent manner. CA-STAT3 overexpression was shown to attenuate caspase-3 cleavage and suppression of Mcl-1 by sorafenib. STAT3 knockdown or a DN STAT3 was shown to down-regulate Mcl-1 and Bcl-xL and to sensitize cells to TRAIL-mediated apoptosis. Treatment with sorafenib enhanced TRAIL-induced Annexin V staining and release of mitochondrial cytochrome c and AIF. Since the BH3-only Bim protein is a potent inducer of mitochondrial apoptosis, Bim knockdown was shown to attenuate caspase-3,-9 cleavage and Bax/Bak activation by sorafenib plus TRAIL.
Conclusions
Suppression of STAT3 by genetic means or using sorafenib was shown to down-regulate Mcl-1 and Bcl-xL and to sensitize cells to TRAIL-mediated apoptosis. These data indicate that targeting STAT3 may enhance treatment efficacy against pancreatic cancer.
doi:10.1158/1535-7163.MCT-09-1004
PMCID: PMC3281304  PMID: 20197401
sorafenib; STAT3; Mcl-1; TRAIL; pancreatic cancer
9.  Vorinostat Eliminates Multicellular Resistance of Mesothelioma 3D Spheroids via Restoration of Noxa Expression 
PLoS ONE  2012;7(12):e52753.
When grown in 3D cultures as spheroids, mesothelioma cells acquire a multicellular resistance to apoptosis that resembles that of solid tumors. We have previously found that resistance to the proteasome inhibitor bortezomib in 3D can be explained by a lack of upregulation of Noxa, the pro-apoptotic BH3 sensitizer that acts via displacement of the Bak/Bax-activator BH3-only protein, Bim. We hypothesized that the histone deacetylase inhibitor vorinostat might reverse this block to Noxa upregulation in 3D. Indeed, we found that vorinostat effectively restored upregulation of Noxa protein and message and abolished multicellular resistance to bortezomib in the 3D spheroids. The ability of vorinostat to reverse resistance was ablated by knockdown of Noxa or Bim, confirming the essential role of the Noxa/Bim axis in the response to vorinostat. Addition of vorinostat similarly increased the apoptotic response to bortezomib in another 3D model, the tumor fragment spheroid, which is grown from human mesothelioma ex vivo. In addition to its benefit when used with bortezomib, vorinostat also enhanced the response to cisplatin plus pemetrexed, as shown in both 3D models. Our results using clinically relevant 3D models show that the manipulation of the core apoptotic repertoire may improve the chemosensitivity of mesothelioma. Whereas neither vorinostat nor bortezomib alone has been clinically effective in mesothelioma, vorinostat may undermine chemoresistance to bortezomib and to other therapies thereby providing a rationale for combinatorial strategies.
doi:10.1371/journal.pone.0052753
PMCID: PMC3530471  PMID: 23300762
10.  Sorafenib inhibits ERK1/2 and MCL-1L phosphorylation levels resulting in caspase-independent cell death in malignant pleural mesothelioma 
Cancer biology & therapy  2009;8(24):2406-2416.
Malignant pleural mesothelioma (MPM) is an aggressive, rapidly progressive malignancy without effective therapy. We evaluate sorafenib efficacy and impact on the cellular pro-survival machinery in vitro, efficacy of sorafenib as monotherapy and in combination with the naturally occurring death receptor agonist, TRAIL using human MPM cell lines, MSTO-211H, M30, REN, H28, H2052 and H2452. In vitro studies of the six MPM lines demonstrated single agent sensitivity to the multikinase inhibitor sorafenib and resistance to TRAIL. H28 and H2452 demonstrated augmented apoptosis with the addition of TRAIL to sorafenib in vitro. Treated cell lines demonstrated sorafenib-induced rapid dephosphorylation of AKT followed shortly by near complete dephosphorylation of the constitutively phosphorylated ERK1/2. Sorafenib therapy also decreased phosphorylation of B-Raf and mTOR in several cell lines. Within 3 h of sorafenib treatment, a number of known pro-survival molecules were dephosphorylated and/or downregulated in expression including MCL-1L, c-FLIPL, survivin and cIAP 1. These changes and eventual cell death did not elicit significant caspase-3 activation or PARP cleavage and pretreatment with the pan-caspase inhibitor, Z-VAD-FMK, did not block sorafenib efficacy but did block the effect of TRAIL monotherapy. Pre-treatment with Z-VAD-FMK did not block the synergistic effect of TRAIL and sorafenib in H28. In summary, single agent treatment with sorafenib results in widespread inhibition of the pro-survival machinery in vitro leading to cell death via a primarily caspase-independent mechanism. Combining sorafenib therapy with TRAIL, may be useful in order to provide a more efficient death signal and this synergistic effect appears to be caspase-independent. Pilot in vivo data demonstrates promising evidence of therapeutic efficacy in human tumor bearing xenograft nu/nu mice. We document single agent activity of sorafenib against MPM, unravel novel effects of sorafenib on anti-apoptotic signaling mediators, and suggest the combination of sorafenib plus TRAIL as possible therapy for clinical testing in MPM.
PMCID: PMC3052759  PMID: 20038816
mesothelioma; cancer therapy; cell death; sorafenib; MCL-1; imaging; TRAIL; caspase-independent
11.  Bortezomib-Induced Sensitization of Malignant Human Glioma Cells to Vorinostat-Induced Apoptosis Depends on Reactive Oxygen Species Production, Mitochondrial Dysfunction, Noxa Upregulation, Mcl-1 Cleavage, and DNA Damage 
Molecular carcinogenesis  2011;52(2):118-133.
Glioblastomas are invasive tumors with poor prognosis despite current therapies. Histone deacetylase inhibitors (HDACIs) represent a class of agents that can modulate gene expression to reduce tumor growth, and we and others have noted some antiglioma activity from HDACIs, such as vorinostat, although insufficient to warrant use as mono-therapy. We have recently demonstrated that proteasome inhibitors, such as bortezomib, dramatically sensitized highly resistant glioma cells to apoptosis induction, suggesting that proteasomal inhibition may be a promising combination strategy for glioma therapeutics. In this study, we examined whether bortezomib could enhance response to HDAC inhibition in glioma cells. Although primary cells from glioblastoma multiforme (GBM) patients and established glioma cell lines did not show significant induction of apoptosis with vorinostat treatment alone, the combination of vorino-stat plus bortezomib significantly enhanced apoptosis. The enhanced efficacy was due to proapoptotic mitochondrial injury and increased generation of reactive oxygen species. Our results also revealed that combination of bortezomib with vorinostat enhanced apoptosis by increasing Mcl-1 cleavage, Noxa upregulation, Bak and Bax activation, and cytochrome c release. Further downregulation of Mcl-1 using shRNA enhanced cell killing by the bortezomib/vorinostat combination. Vorinostat induced a rapid and sustained phosphorylation of histone H2AX in primary GBM and T98G cells, and this effect was significantly enhanced by co-administration of bortezomib. Vorinostat/bortezomib combination also induced Rad51 downregulation, which plays an important role in the synergistic enhancement of DNA damage and apoptosis. The significantly enhanced antitumor activity that results from the combination of bortezomib and HDACIs offers promise as a novel treatment for glioma patients.
doi:10.1002/mc.21835
PMCID: PMC4068609  PMID: 22086447
bortezomib; vorinostat; glioma; synergy; apoptosis
12.  Synergistic Anticancer Effects of Vorinostat and Epigallocatechin-3-Gallate against HuCC-T1 Human Cholangiocarcinoma Cells 
The aim of this study was to investigate the effect of the combination of vorinostat and epigallocatechin-3-gallate against HuCC-T1 human cholangiocarcinoma cells. A novel chemotherapy strategy is required as cholangiocarcinomas rarely respond to conventional chemotherapeutic agents. Both vorinostat and EGCG induce apoptosis and suppress invasion, migration, and angiogenesis of tumor cells. The combination of vorinostat and EGCG showed synergistic growth inhibitory effects and induced apoptosis in tumor cells. The Bax/Bcl-2 expression ratio and caspase-3 and -7 activity increased, but poly (ADP-ribose) polymerase expression decreased when compared to treatment with each agent alone. Furthermore, invasion, matrix metalloproteinase (MMP) expression, and migration of tumor cells decreased following treatment with the vorinostat and EGCG combination compared to those of vorinostat or EGCG alone. Tube length and junction number of human umbilical vein endothelial cells (HUVECs) decreased as well as vascular endothelial growth factor expression following vorinostat and EGCG combined treatment. These results indicate that the combination of vorinostat and EGCG had a synergistic effect on inhibiting tumor cell angiogenesis potential. We suggest that the combination of vorinostat and EGCG is a novel option for cholangiocarcinoma chemotherapy.
doi:10.1155/2013/185158
PMCID: PMC3706064  PMID: 23864881
13.  Inhibition of autophagy significantly enhances combination therapy with sorafenib and HDAC inhibitors for human hepatoma cells 
AIM: To clarify whether histone deacetylase inhibitors histone deacetylase inhibitors (HDACIs) can sensitize hepatocellular carcinoma (HCC) cells to sorafenib treatment.
METHODS: Bax, Bcl-2, ATG5-ATG12, p21, and p27 protein levels in Hep3B, HepG2, and PLC/PRF/5 cells were examined by Western blot. CCK8 and a fluorometric caspase-3 assay were used to examine cellular viability and apoptosis levels. The effect of Beclin-1 on sensitization of HCC cells to sorafenib was examined by transfecting Beclin-1 siRNA into Hep3B, HepG2, and PLC/PRF/5 cells.
RESULTS: Autophagy inhibition enhances the inhibitory effects of vorinostat and sorafenib alone or in combination on HCC cell growth. Vorinostat and sorafenib synergistically induced apoptosis and cell cycle alterations. Western blot data indicated that HDACIs and Beclin-1 knockdown increased the p53 acetylation level. The knockdown of Beclin-1 enhanced the synergistic effect of the combination of vorinostat with sorafenib.
CONCLUSION: HDACIs can sensitize HCC cells to sorafenib treatment by regulating the acetylation level of Beclin-1.
doi:10.3748/wjg.v20.i17.4953
PMCID: PMC4009527  PMID: 24833845
Hepatocellular carcinoma; Histone deacetylase inhibitors; Autophagy; Sorafenib; Chemoresistance
14.  PLK1 inhibitors synergistically potentiate HDAC inhibitor lethality in IM -sensitive or – resistant BCR/ABL+ leukemia cells in vitro and in vivo 
Purpose
To determine whether PLK1 inhibitors (e.g. BI2536) and HDAC inhibitor (e.g. vorinostat) interact synergistically in CML cells sensitive or resistant to imatinib mesylate (IM) in vitro and in vivo.
Experimental Design
K562 and LAMA84 cells sensitive or resistant to IM and primary CML cells were exposed to BI2536 and vorinostat. Effects on cell viability and signaling pathways were determined using flow cytometry, western blotting, and gene transfection. K562 and BV173/E255K animal models were used to test in vivo efficacy.
Results
Co-treatment with BI2536 and vorinostat synergistically induced cell death in parental or IM-resistant BCR/ABL+ cells and and primary CD34+ bone marrow cells but was minimally toxic to normal cells. BI2536/vorinostat co-treatment triggered pronounced mitochondrial dysfunction, inhibition of p-BCR/ABL, caspase activation, PARP cleavage, ROS generation, and DNA damage (manifest by increased expression of γH2A.X, p-ATM, p-ATR), events attenuated by the anti-oxidant TBAP. PLK1 shRNA knockdown significantly increased HDACI lethality, whereas or HDAC 1–3 shRNA knockdown reciprocally increased BI2536-induced apoptosis. Genetic interruption of the DNA damage linker H1.2 partially but significantly reduced PLK1/HDAC inhibitor-mediated cell death, suggesting a functional role for DNA damage in lethality. Finally, BI2536/vorinostat co-treatment dramatically reduced tumor growth in both subcutaneous and systemic BCR/ABL+ leukemia xenograft models and significantly enhanced animal survival.
Conclusions
These findings suggest that concomitant PLK1 and HDAC inhibition is active against IM-sensitive or refractory CML cells both in vitro and in vivo, and that this strategy warrants further evaluation in the setting of BCR/ABL+ leukemias.
doi:10.1158/1078-0432.CCR-12-2799
PMCID: PMC3548959  PMID: 23204129
BI2536; vorinostat; CML; PLK-1; BCR/ABL+
15.  Enhancing sorafenib-mediated sensitization to gemcitabine in experimental pancreatic cancer through EMAP II 
Background
Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive human malignancies and tends to be relatively resistant to conventional therapies. Activated Ras oncogene mutations are found in up to 90% of PDAC, leading to activation of the Ras/Raf/MEK/ERK signaling pathway. Sorafenib is a multikinase inhibitor of the Ras/Raf/MEK/ERK pathway and of tumor angiogenesis. Endothelial monocyte activating polypeptide II (EMAP) enhances gemcitabine effects in PDAC. Antitumor activity of sorafenib was evaluated in combination with gemcitabine (Gem) and the antiangiogenic agent EMAP in experimental PDAC.
Methods
Cell proliferation and protein expression were analyzed by WST-1 assay and Western blotting. Animal survival studies were performed in murine PDAC xenografts.
Results
Sorafenib decreased phospho-MEK, phospho-ERK1/2, phospho-p70S6K and phospho-4EBP-1 expression in PDAC cells. Sorafenib inhibited in vitro proliferation of all four PDAC cell lines tested. Additive effects on cell proliferation inhibition were observed in the gemcitabine-sorafenib combination in PDAC cells, and in combinations of sorafenib or EMAP with gemcitabine in endothelial (HUVEC) and fibroblast (WI-38) cells. Sorafenib, alone or in combination with gemcitabine and EMAP, induced apoptosis in HUVECs and WI-38 cells as observed via increased expression of cleaved poly (ADP-ribose) polymerase-1 (PARP-1) and caspase-3 proteins. Compared to controls (median survival: 22 days), animal survival increased after Gem therapy (29 days) but not in sorafenib (23 days) or EMAP therapy alone (25 days). Further increases in survival occurred in combination therapy groups Gem+sorafenib (30 days, p=0.004), Gem+EMAP (33 days, p=0.002), and Gem+sorafenib+EMAP (36 days, p=0.004), but not after the sorafenib+EMAP combination (24 days).
Conclusions
These findings demonstrate that the addition of a polymechanistic antiangiogenic agent such as EMAP can enhance the combination treatment effects of sorafenib and cytotoxic PDAC therapy.
doi:10.1186/1756-9966-32-12
PMCID: PMC3618297  PMID: 23497499
16.  Inhibiting oncogenic signaling by sorafenib activates PUMA via GSK3β and NF-κB to suppress tumor cell growth 
Oncogene  2012;31(46):4848-4858.
Aberrant Ras/Raf/MEK/ERK signaling is one of the most prevalent oncogenic alterations and confers survival advantage to tumor cells. Inhibition of this pathway can effectively suppress tumor cell growth. For example, sorafenib, a multi-kinase inhibitor targeting c-Raf and other oncogenic kinases, has been used clinically for treating advanced liver and kidney tumors, and also has shown efficacy against other malignancies. However, how inhibition of oncogenic signaling by sorafenib and other drugs suppresses tumor cell growth remains unclear. In this study, we found that sorafenib kills cancer cells by activating PUMA, a p53 target and a BH3-only Bcl-2 family protein. Sorafenib treatment induces PUMA in a variety of cancer cells irrespective of their p53 status. Surprisingly, the induction of PUMA by sorafenib is mediated by IκB-independent activation of NF-κB, which directly binds to the PUMA promoter to activate its transcription. NF-κB activation by sorafenib requires GSK3β activation, subsequent to ERK inhibition. Deficiency in PUMA abrogates sorafenib-induced apoptosis and caspase activation, and renders sorafenib resistance in colony formation and xenograft tumor assays. Furthermore, the chemosensitization effect of sorafenib is dependent on PUMA, and involves concurrent PUMA induction through different pathways. BH3 mimetics potentiate the anticancer effects of sorafenib, and restore sorafenib sensitivity in resistant cells. Together, these results demonstrate a key role of PUMA-dependent apoptosis in therapeutic inhibition of Ras/Raf/MEK/ERK signaling. They provide a rationale for manipulating the apoptotic machinery to improve sensitivity and overcome resistance to the therapies that target oncogenic kinase signaling.
doi:10.1038/onc.2011.644
PMCID: PMC3342476  PMID: 22286758
sorafenib; PUMA; apoptosis; NF-κB; GSK3β; colon cancer
17.  Antioxidants Impair Anti-Tumoral Effects of Vorinostat, but Not Anti-Neoplastic Effects of Vorinostat and Caspase-8 Downregulation 
PLoS ONE  2014;9(3):e92764.
We have recently demonstrated that histone deacetylase inhibitor, Vorinostat, applied as a single therapy or in combination with caspase-8 downregulation exhibits high anti-tumoral activity on endometrial carcinoma cell lines. In the present study, we have assessed the signalling processes underlying anti-tumoral effects of Vorinostat. Increasing evidence suggests that reactive oxygen species are responsible for histone deacetylase inhibitor-induced cell killing. We have found that Vorinostat induces formation of reactive oxygen species and DNA damage. To investigate the role of oxidative stress as anti-neoplastic mechanism, we have evaluated the effects of different antioxidants (Bha, Nac and Tiron) on endometrial carcinoma cell line Ishikawa treated with Vorinostat. We show that Bha, Nac and Tiron markedly inhibited the cytotoxic effects of Vorinostat, increasing cell viability in vitro. We found that all three antioxidants did not inhibited accumulation of acetyl Histone H4, so that antioxidants did not inhibit Vorinostat activity. Finally, we have evaluated the effects of antioxidants on anti-tumoral activity of Vorinostat as monotherapy or in combination with caspase-8 downregulation in vivo. Interestingly, antioxidants blocked the reduction of tumour growth caused by Vorinostat, but they were unable to inhibit anti-tumoral activity of Vorinostat plus caspase-8 inhibition.
doi:10.1371/journal.pone.0092764
PMCID: PMC3961419  PMID: 24651472
18.  Biomarker Modulation Following Short Term Vorinostat in Women with Newly-Diagnosed Primary Breast Cancer 
Purpose
Agents that target the epigenome demonstrate activity in breast cancer models. In preclinical studies, the histone deacetylase inhibitor vorinostat induces cell cycle arrest, apoptosis and differentiation. We evaluated biomarker modulation in breast cancer tissues obtained from women with newly-diagnosed invasive disease who received vorinostat and those who did not.
Experimental Design
Tumor specimens were collected from 25 women who received up to 6 doses of oral vorinostat 300 mg twice daily and from 25 untreated controls in a non-randomized study. Candidate gene expression was analyzed by RT-PCR using the Oncotype DX® 21-gene assay, and by immunohistochemistry for Ki-67 and cleaved caspase-3. Matched samples from treated women were analyzed for gene methylation by QM-MSP. Wilcoxon non-parametric tests were used to compare changes in quantitative gene expression levels pre- and post-vorinostat with changes in expression in untreated controls, and changes in gene methylation between pre- and post-vorinostat samples.
Results
Vorinostat was well-tolerated and there were no study-related delays in treatment. Compared to untreated controls, there were statistically significant decreases in the expression of proliferation-associated genes Ki-67 (p=0.003), STK15 (p=0.005), and Cyclin B1 (p=0.03) following vorinostat, but not in other genes by the Oncotype DX® assay, or in expression of Ki-67 or cleaved caspase-3 by immunohistochemistry. Changes in methylation were not observed.
Conclusions
Short term vorinostat administration is associated with a significant decrease in expression of proliferation-associated genes in untreated breast cancers. This demonstration of biological activity supports investigation of vorinostat in combination with other agents for the management of breast cancer.
doi:10.1158/1078-0432.CCR-13-0033
PMCID: PMC3718062  PMID: 23719261
Breast cancer; epigenetics; histone deacetylase inhibitors; vorinostat; predictive biomarkers
19.  The Mechanism of Action of the Histone Deacetylase Inhibitor Vorinostat Involves Interaction with the Insulin-Like Growth Factor Signaling Pathway 
PLoS ONE  2011;6(9):e24468.
A correlation between components of the insulin-like growth factor (IGF) system and endometrial cancer risk has been shown in recent studies. The antitumor action of vorinostat, a histone deacetylase inhibitor, involves changes in the expression of specific genes via acetylation of histones and transcription factors. The aim of this study was to establish whether vorinostat can modify the expression of specific genes related to the IGF-I receptor (IGF-IR) signaling pathway and revert the transformed phenotype. Human endometrioid (Type I, Ishikawa) and uterine serous papillary (Type II, USPC-2) endometrial cancer cell lines were treated with vorinostat in the presence or absence of IGF-I. Vorinostat increased IGF-IR phosphorylation, produced acetylation of histone H3, up-regulated pTEN and p21 expression, and reduced p53 and cyclin D1 levels in Ishikawa cells. Vorinostat up-regulated IGF-IR and p21 expression, produced acetylation of histone H3, and down-regulated the expression of total AKT, pTEN and cyclin D1 in USPC-2 cells. Of interest, IGF-IR activation was associated with a major elevation in IGF-IR promoter activity. In addition, vorinostat treatment induced apoptosis in both cell lines and abolished the anti-apoptotic activity of IGF-I both in the absence or presence of a humanized monoclonal IGF-IR antibody, MK-0646. Finally, vorinostat treatment led to a significant decrease in proliferation and colony forming capability in both cell lines. In summary, our studies demonstrate that vorinostat exhibits a potent apoptotic and anti-proliferative effect in both Type I and II endometrial cancer cells, thus suggesting that endometrial cancer may be therapeutically targeted by vorinostat.
doi:10.1371/journal.pone.0024468
PMCID: PMC3169604  PMID: 21931726
20.  The Kinase Inhibitor Sorafenib Induces Cell Death through a Process Involving Induction of Endoplasmic Reticulum Stress▿ †  
Molecular and Cellular Biology  2007;27(15):5499-5513.
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 2α (eIF2α) 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 IRE1α markedly reduced sorafenib-mediated apoptosis, whereas knockdown of IRE1α or XBP1, disruption of PERK activity, or inhibition of eIF2α 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.
doi:10.1128/MCB.01080-06
PMCID: PMC1952105  PMID: 17548474
21.  Vorinostat synergises with capecitabine through upregulation of thymidine phosphorylase 
British Journal of Cancer  2010;103(11):1680-1691.
Background:
Potentiation of anticancer activity of capecitabine is required to improve its therapeutic index. In colorectal cancer (CRC) cells, we evaluated whether the histone deacetylase-inhibitor vorinostat may induce synergistic antitumour effects in combination with capecitabine by modulating the expression of thymidine phosphorylase (TP), a key enzyme in the conversion of capecitabine to 5-florouracil (5-FU), and thymidylate synthase (TS), the target of 5-FU.
Methods:
Expression of TP and TS was measured by real-time PCR, western blotting and immunohistochemistry. Knockdown of TP was performed by specific small interfering RNA. Antitumour activity of vorinostat was assessed in vitro in combination with the capecitabine active metabolite deoxy-5-fluorouridine (5′-DFUR) according to the Chou and Talay method and by evaluating apoptosis as well as in xenografts-bearing nude mice in combination with capecitabine.
Results:
Vorinostat induced both in vitro and in vivo upregulation of TP as well as downregulation of TS in cancer cells, but not in ex vivo treated peripheral blood lymphocytes. Combined treatment with vorinostat and 5′-DFUR resulted in a synergistic antiproliferative effect and increased apoptotic cell death in vitro. This latter effect was impaired in cells where TP was knocked. In vivo, vorinostat plus capecitabine potently inhibited tumour growth, increased apoptosis and prolonged survival compared with control or single-agent treatments.
Conclusions:
Overall, this study suggests that the combination of vorinostat and capecitabine is an innovative antitumour strategy and warrants further clinical evaluation for the treatment of CRC.
doi:10.1038/sj.bjc.6605969
PMCID: PMC2994231  PMID: 21045833
HDAC inhibitor; vorinostat; thymidine phosphorylase; thymidylate synthase; colon cancer; capecitabine
22.  Activity of histone deacetylase inhibitors and an Aurora kinase inhibitor in BCR-ABL-expressing leukemia cells: Combination of HDAC and Aurora inhibitors in BCR-ABL-expressing cells 
Background
The use of imatinib, an ABL tyrosine kinase inhibitor, has led to a dramatic change in the management of BCR-ABL-positive leukemia patients. However, resistance to imatinib mediated by mutations in the BCR-ABL domain has become a major problem in the treatment of these patients.
Methods
In the present study, we examined the activity of histone deacetylase (HDAC) inhibitors in combination with an Aurora kinase inhibitor in BCR-ABL-expressing cells.
Results
We found the HDAC inhibitors vorinostat and/or pracinostat (SB939) induced apoptosis in BCR-ABL-expressing cells. Additionally, HDAC inhibitors reduced levels of Aurora A and B protein. An Aurora kinase inhibitor, tozasertib (VX-680), inhibited growth, promoted pro-apoptotic activity, reduced the phosphorylation of BCR-ABL and Crk-L, and activated caspase-3 and poly (ADP-ribose) polymerase (PARP) in BCR-ABL-positive cells. Moreover, after treatment with tozasertib, HDAC protein expression was decreased. Combination of vorinostat or pracinostat with tozasertib had a synergistic inhibitory effect on the proliferation of T315I cells. Phosphorylation of Crk-L decreased, and PARP activation increased after treatment with vorinostat or pracinostat and tozasertib. Moreover, combination of vorinostat or pracinostat and tozasertib significantly increased the extent of apoptosis in primary chronic myeloid leukemia cells.
Conclusions
This study demonstrated that combination of HDAC and Aurora inhibitors was highly effective against BCR-ABL-expressing cells.
doi:10.1186/1475-2867-13-32
PMCID: PMC3635933  PMID: 23556431
HDAC inhibitor; Aurora kinase inhibitor; T315I mutation
23.  Co-treatment with vorinostat enhances activity of MK-0457 (VX-680) against acute and chronic myelogenous leukemia cells 
Purpose
We determined the effects of vorinostat [suberoylanalide hydroxamic acid (SAHA)] and/or MK-0457 (VX-680), an Aurora kinase inhibitor on the cultured human (HL-60, OCI-AML3 and K562) and primary acute (AML) and chronic myelogenous leukemia (CML), as well as on the murine pro-B BaF3 cells with ectopic expression of the unmutated and mutant forms of Bcr-Abl.
Experimental Design
Following exposure to MK-047 and/or vorinostat, apoptosis, loss of viability, as well as activity and levels of Aurora kinase and Bcr-Abl proteins were determined.
Results
Treatment with MK-0457 decreased phosphorylation of Aurora kinase substrates including serine (S)10 on histone H3 and survivin, as well as led to aberrant mitosis, DNA endoreduplication and apoptosis of the cultured human acute leukemia HL-60, OCI-AML3 and K562 cells. Combined treatment with vorinostat and MK-0457 resulted in greater attenuation of Aurora and Bcr-Abl (in K562) kinase activity and levels as well as synergistically induced apoptosis of OCI-AML3, HL-60 and K562 cells. MK-0457 plus vorinostat also induced synergistic apoptosis of BaF3 cells with ectopic overexpression of wild-type or mutant Bcr-Abl. Finally, co-treatment with MK-0457 and vorinostat induced more loss of viability of primary AML and imatinib-refractory CML than treatment with either agent alone, but exhibited minimal toxicity to normal CD34+ progenitor cells.
Conclusions
Combined in vitro treatment with MK-0457 and vorinostat is highly active against cultured and primary leukemia cells. These findings merit in vivo testing of the combination against human AML and CML cells, especially against imatinib mesylate-resistant Bcr-AblT315I expressing CML cells.
doi:10.1158/1078-0432.CCR-08-0721
PMCID: PMC2665710  PMID: 18829489
Aurora kinase; MK-0457; vorinostat
24.  The novel Chk1 inhibitor MK-8776 sensitizes human leukemia cells to HDAC inhibitors by targeting the intra-S checkpoint and DNA replication and repair 
Molecular cancer therapeutics  2013;12(6):878-889.
Interactions between the novel Chk1 inhibitor MK-8776 and the HDAC inhibitor (HDACI) vorinostat were examined in human leukemia cells harboring wild-type (wt) or deficient p53. MK-8776 synergistically potentiated vorinostat-mediated apoptosis in various p53-wild type (wt) or -deficient leukemia cell lines, while p53 knock-down by shRNA sensitized p53-wt cells to lethality of this regimen. Leukemia cell lines carrying FLT3-ITD were also sensitive to the MK-8776/vorinostat regimen. Synergistic interactions were associated with inhibition of Chk1 activity, interference with the intra-S phase checkpoint, disruption of DNA replication, and down-regulation of proteins involved in DNA replication (e.g.,CDT1) and repair (e.g., CtIP and BRCA1), resulting in sharp increases in DNA damage, reflected by enhanced γH2A.X formation, and apoptosis. Moreover, leukemia cells expressing kinase-dead Chk1 (D130A) or Chk1 shRNA were significantly more sensitive to HDACIs compared to their wild-type counterparts, and displayed down-regulation of CtIP and BRCA1 phosphorylation following HDACI exposure. Finally, the MK-8776/vorinostat regimen was active in primary AML blasts, particularly against the CD34+/CD38-/CD123+ population enriched for leukemia-initiating cells. In contrast, identical regimens were relatively sparing toward normal cord blood CD34+ cells. Together, these findings indicate that the novel Chk1 inhibitor MK-8776 markedly potentiates HDACI lethality in leukemia cells displaying various genetic backgrounds through mechanisms involving disruption of the intra-S checkpoint, DNA replication, and DNA repair. They also argue that leukemic cells, including those bearing oncogenic mutations associated with poor prognosis e.g., p53 deletion/mutation or FLT3-ITD, may also be susceptible to this strategy.
doi:10.1158/1535-7163.MCT-12-0902
PMCID: PMC3681875  PMID: 23536721
Chk1 inhibitor; HDAC inhibitor; S phase; DNA damage/repair; leukemia
25.  Vorinostat increases carboplatin and paclitaxel activity in non-small cell lung cancer cells 
We observed a 53% response rate in non-small cell lung cancer (NSCLC) patients treated with vorinostat plus paclitaxel/carboplatin in a Phase I trial. Studies were undertaken to investigate the mechanism (s) underlying this activity. Growth inhibition was assessed in NSCLC cells by MTT assay after 72 h of continuous drug exposure. Vorinostat (1 µM) inhibited growth by: 17±7% in A549, 28±6% in 128-88T, 39±8% in Calu1, and 41±7% in 201T cells. Vorinostat addition to carboplatin or paclitaxel led to significantly greater growth inhibition than chemotherapy alone in all 4 cell lines. Vorinostat (1 µM) synergistically increased the growth inhibitory effects of carboplatin/paclitaxel in 128-88T cells. When colony formation was measured after drug withdrawal, vorinostat significantly increased the effects of carboplatin but not paclitaxel. The % colony formation was: control 100%; 1 µM vorinostat 83% ± 10%; 5 µM carboplatin, 41% ± 11%; carboplatin/vorinostat, 8% ± 4%; 2 nM paclitaxel, 53% ± 11%; paclitaxel/vorinostat 46% ± 21%. In A549 and 128-88T, vorinostat potentiated carboplatin induction of gamma-H2AX (a DNA damage marker) and increased α-tubulin acetylation (a marker for stabilized mictrotubules). In A549, combination of vorinostat with paclitaxel resulted in a synergistic increase in α-tubulin acetylation, which reversed upon drug wash-out. We conclude that vorinostat interacts favorably with carboplatin and paclitaxel in NSCLC cells, which may explain the provocative response observed in our clinical trial. This likely involves a vorinostat-mediated irreversible increase in DNA damage in the case of carboplatin and a reversible increase in microtubule stability in the case of paclitaxel.
doi:10.1002/ijc.24759
PMCID: PMC2795066  PMID: 19621389
Vorinostat; paclitaxel; carboplatin; histone deacetylase; non-small cell lung cancer

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