|Ki: ||Kd:||Ic 50:||Ec50/Ic50:|
Novel mechanisms of apoptosis induced by histone deacetylase inhibitors.. Melissa J Peart; Kellie M Tainton; Astrid A Ruefli; Anthony E Dear; Karin A Sedelies; Lorraine A O'Reilly; Nigel J Waterhouse; Joseph A Trapani; Ricky W Johnstone (2003) Cancer research display abstract
Histone deacetylase inhibitors (HDACIs) are a new class of chemotherapeutic drugs able to induce tumor cell apoptosis and/or cell cycle arrest; however, the molecular mechanisms underpinning their anticancer effects are poorly understood. Herein, we assessed the apoptotic pathways activated by three HDACIs, suberoylanilide hydroxamic acid, oxamflatin, and depsipeptide. We determined that all three drugs induced the accumulation of cells with a 4n DNA content and apoptosis mediated by the intrinsic apoptotic pathway. HDACI-induced mitochondrial membrane damage and apoptosis were inhibited by overexpression of Bcl-2, but not by the polycaspase inhibitor N-tert-butoxy-carbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk). Moreover, induction of a G(1)-S checkpoint through overexpression of p16(INK4A) or suppression of de novo protein synthesis also inhibited HDACI-induced cell death. Proteolytic cleavage of caspase-2, which is poorly inhibited by zVAD-fmk, was concomitant with HDACI-induced death; however, full processing of caspase-2 to the p19 active form was blocked by Bcl-2. Whereas all three drugs induce the activation of the proapoptotic Bcl-2 protein Bid upstream of mitochondrial membrane disruption, Bid cleavage in response to depsipeptide was significantly attenuated by zVAD-fmk. Suberoylanilide hydroxamic acid and oxamflatin could kill both P-glycoprotein (P-gp)(+) MDR cells and their P-gp(-) counterparts, whereas depsipeptide was shown to be a substrate for P-gp and was less effective in killing P-gp(+) cells. These data provide insight into the functional profile of three HDACIs and are important for the development of more rational approaches to chemotherapy, where information regarding the genetic profile of the tumor is matched with the functional profile of a given chemotherapeutic drug to promote favorable clinical responses.
Histone deacetylase (HDAC) inhibitor activation of p21WAF1 involves changes in promoter-associated proteins, including HDAC1.. C-Y Gui; L Ngo; W S Xu; V M Richon; P A Marks (2004) Proceedings of the National Academy of Sciences of the United States of America display abstract
Histone deacetylase (HDAC) inhibitors (HDACi) cause cancer cell growth arrest and/or apoptosis in vivo and in vitro. The HDACi suberoylanilide hydroxamic acid (SAHA) is in phase I/II clinical trials showing significant anticancer activity. Despite wide distribution of HDACs in chromatin, SAHA alters the expression of few genes in transformed cells. p21(WAF1) is one of the most commonly induced. SAHA does not alter the expression of p27(KIPI), an actively transcribed gene, or globin, a silent gene, in ARP-1 cells. Here we studied SAHA-induced changes in the p21(WAF1) promoter of ARP-1 cells to better understand the mechanism of HDACi gene activation. Within 1 h, SAHA caused modifications in acetylation and methylation of core histones and increased DNase I sensitivity and restriction enzyme accessibility in the p21(WAF1) promoter. These changes did not occur in the p27(KIPI) or epsilon-globin gene-related histones. The HDACi caused a marked decrease in HDAC1 and Myc and an increase in RNA polymerase II in proteins bound to the p21(WAF1) promoter. Thus, this study identifies effects of SAHA on p21(WAF1)-associated proteins that explain, at least in part, the selective effect of HDACi in altering gene expression.
Sequence-specific potentiation of topoisomerase II inhibitors by the histone deacetylase inhibitor suberoylanilide hydroxamic acid.. Douglas C Marchion; Elona Bicaku; Adil I Daud; Victoria Richon; Daniel M Sullivan; Pamela N Munster (2004) Journal of cellular biochemistry display abstract
Acetylation of histones leads to conformational changes of DNA. We have previously shown that the histone deacetylase (HDAC) inhibitor, suberoylanilide hydroxamic acid (SAHA), induced cell cycle arrest, differentiation, and apoptosis. In addition to their antitumor effects as single agents, HDAC inhibitors may cause conformational changes in the chromatin, rendering the DNA more vulnerable to DNA damaging agents. We examined the effects of SAHA on cell death induced by topo II inhibitors in breast cancer cell lines. Topo II inhibitors stabilize the topo II-DNA complex, resulting in DNA damage. Treatment of cells with SAHA promoted chromatin decondensation associated with increased nuclear concentration and DNA binding of the topo II inhibitor and subsequent potentiation of DNA damage. While SAHA-induced histone hyperacetylation occurred as early as 4 h, chromatin decondensation was most profound at 48 h. SAHA-induced potentiation of topo II inhibitors was sequence-specific. Pre-exposure of cells to SAHA for 48 h was synergistic, whereas shorter pre-exposure periods abrogated synergy and exposure of cells to SAHA after the topo II inhibitor resulted in antagonistic effects. Synergy was not observed in cells with depleted topo II levels. These effects were not limited to specific types of topo II inhibitors. We propose that SAHA significantly potentiates the DNA damage induced by topo II inhibitors; however, synergy is dependent on the sequence of drug administration and the expression of the target. These findings may impact the clinical development of combining HDAC inhibitors with DNA damaging agents.
Modulation of radiation response by histone deacetylase inhibition.. Prakash Chinnaiyan; Geetha Vallabhaneni; Eric Armstrong; Shyh-Min Huang; Paul M Harari (2005) International journal of radiation oncology, biology, physics display abstract
PURPOSE: Histone deacetylase (HDAC) inhibitors, which modulate chromatin structure and gene expression, represent a class of anticancer agents that hold particular potential as radiation sensitizers. In this study, we examine the capacity of the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) to modulate radiation response in human tumor cell lines and explore potential mechanisms underlying these interactions. METHODS AND MATERIALS: Cell proliferation: Exponentially growing tumor cells were incubated in medium containing 0-10 microM of SAHA for 72 h. Cells were fixed/stained with crystal violet to estimate cell viability. Apoptosis: Caspase activity was analyzed by fluorescence spectroscopy using a fluorescein labeled pan-caspase inhibitor. Cells were harvested after 48 h of exposure to SAHA (1.0 microM), radiation (6 Gy), or the combination. Whole cell lysates were evaluated for poly(ADP-ribose) polymerase (PARP) cleavage by western blot analysis. Radiation survival: Cells were exposed to varying doses of radiation +/- 3 days pretreatment with SAHA (0.75-1.0 microM). After incubation intervals of 14-21 days, colonies were stained with crystal violet and manually counted. Immunocytochemistry: Cells were grown and treated in chamber slides. At specified times after treatment with SAHA, cells were fixed in paraformaldehyde, permeabilized in methanol, and probed with primary and secondary antibody solutions. Slides were analyzed using an epifluorescent microscope. RESULTS: SAHA induced a dose-dependent inhibition of proliferation in human prostate (DU145) and glioma (U373vIII) cancer cell lines. Exposure to SAHA enhanced radiation-induced apoptosis as measured by caspase activity (p < 0.05) and PARP cleavage. The impact of SAHA on radiation response was further characterized using clonogenic survival analysis, which demonstrated that treatment with SAHA reduced tumor survival after radiation exposure. We identified several oncoproteins and DNA damage repair proteins (epidermal growth factor receptor, AKT, DNA-PK, and Rad51) that show differential expression after exposure to SAHA. These proteins may contribute to mechanistic synergy between HDAC inhibition and radiation response. CONCLUSION: These preclinical results suggest that treatment with the HDAC inhibitor SAHA can enhance radiation-induced cytotoxicity in human prostate and glioma cells. We are examining the capacity of HDAC inhibitors to modulate radiation response and tumor control in animal xenograft model systems to strengthen the rationale for future clinical trial exploration.
Apoptosis signal-regulating kinase 1 is a direct target of E2F1 and contributes to histone deacetylase inhibitor-induced apoptosis through positive feedback regulation of E2F1 apoptotic activity.. Jing Tan; Li Zhuang; Xia Jiang; Kevin K Yang; Krishina M Karuturi; Qiang Yu (2006) The Journal of biological chemistry display abstract
The oncogenic retinoblastoma protein (Rb)/E2F pathway links cellular proliferation control to apoptosis as a fail-safe mechanism to protect aberrant oncogenic transformation. We have previously shown that histone deacetylase inhibitors (HDACIs) activate the E2F1-Bim apoptotic pathway, leading to efficient cell killing in cancer cells with deregulated E2F1 activity. To identify additional gene cassettes that might contribute HDACI-induced apoptosis upon E2F1 activation, we investigated the apoptotic transcriptional network affected by HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) in cancer cells with inducible E2F1. Data analysis focusing on 220 apoptosis-related genes identified apoptosis signal-regulating kinase 1 (ASK1) as one of a few genes in addition to Bim that are substantially up-regulated by SAHA upon E2F1 activation. We show that ASK1 is directly regulated by E2F1 and that prevention of ASK1 induction by RNA interference decreases SAHA-induced apoptosis. We further show that the role of ASK1 in the SAHA apoptotic response is not associated with its downstream effectors p38 or JNK. Instead, ASK1 knockdown results in reduced E2F1 transcriptional activity, leading to decreased Bim induction by SAHA. Moreover, ASK1 expression reverses the negative effect of Rb on E2F1 activity. These results indicate that ASK1 induction by E2F1 provides positive feedback regulation of E2F1 activity via Rb inhibition, which allows an efficient E2F1-Bim activation. Thus, the concomitant induction of E2F1 targets ASK1 and Bim by HDACIs warrants an effective activation of E2F1-dependent apoptosis in response to SAHA.
Vorinostat, a histone deacetylase inhibitor, enhances the response of human tumor cells to ionizing radiation through prolongation of gamma-H2AX foci.. Anupama Munshi; Toshimitsu Tanaka; Marvette L Hobbs; Susan L Tucker; Victoria M Richon; Raymond E Meyn (2006) Molecular cancer therapeutics display abstract
Vorinostat (suberoylanilide hydroxamic acid) is the prototype of a family of hybrid polar compounds that can induce growth arrest in transformed cells and shows promise for the treatment of cancer. Vorinostat specifically binds to and inhibits the activity of histone deacetylases resulting in acetylation of nucleosomal histones and an activation of gene transcription. Because histone deacetylases modulate chromatin structure and gene expression, both of which can influence radioresponse, this study was designed to examine the capacity of Vorinostat to influence radiation response in human tumor cells and investigate the mechanism underlying these interactions. Vorinostat induced hyperacetylation of histone H4 in a dose-dependent manner. We tested its ability to radiosensitize three human tumor cell lines (A375, MeWo, and A549) using clonogenic cell survival assays. Clonogenic cell survival assay showed that Vorinostat significantly radiosensitized all three tumor cell lines, substantially reducing the surviving fraction at 2 Gy. We examined potential mechanisms that may contribute to the enhanced radiation response induced by Vorinostat. Vorinostat and radiation alone did not induce apoptosis in the melanoma cell line. However, enhanced apoptosis was observed when cells were exposed to both Vorinostat and radiation, suggesting that Vorinostat renders tumor cells more susceptible to radiation-induced apoptosis. Results from DNA damage repair analysis in cultured A375 cells showed that Vorinostat had a strong inhibitory effect on the nonhomologous end joining pathway after radiation. A detailed examination of the involvement of the DNA repair pathway following Vorinostat treatment showed that Vorinostat reduced the expression of the repair-related genes Ku70, Ku80, and Rad50 in A375 cells as detected by Western blot analysis. We also examined gamma-H2AX phosphorylation as a predictive marker of radiotherapy response to Vorinostat and observed that the combination of Vorinostat and radiation caused a prolongation of expression of DNA repair proteins such as gamma-H2AX. Overall, we conclude that Vorinostat enhances tumor radioresponse by multiple mechanisms that may involve antiproliferative growth inhibition and effects on DNA repair after exposure to radiation.
Histone deacetylase inhibitors induce cell death in supratentorial primitive neuroectodermal tumor cells.. K Saravana Kumar; Jürgen Sonnemann; James F Beck (2006) Oncology reports display abstract
Histone deacetylase inhibitors (HDIs) are a promising new class of antineoplastic agents with the capacity to induce differentiation and/or apoptosis of cancer cells. The objective of this study was to evaluate the activity of HDIs against supratentorial primitive neuroectodermal tumor (sPNET) cells. We show that the HDIs, suberoylanilide hydroxamic acid, sodium butyrate, and trichostatin A, induced cell death, and activated caspase-3 and -9 in a sPNET cell line, PFSK. The poly-caspase inhibitor z-VAD-fmk partially prevented the action of HDIs, as judged by determining the mitochondrial membrane potential and by quantifying internucleosomal DNA fragmentation. In conclusion, the HDIs explored possess potent activity against sPNET cells, suggesting that HDIs may be effective in the treatment of sPNET.
Intrinsic apoptotic and thioredoxin pathways in human prostate cancer cell response to histone deacetylase inhibitor.. Weisheng Xu; Lang Ngo; Gisela Perez; Milos Dokmanovic; Paul A Marks (2006) Proceedings of the National Academy of Sciences of the United States of America display abstract
There is a great need to develop better mechanism-based therapies for prostate cancer. In this investigation, we studied four human prostate cancer cell lines, LNCaP, DU145, LAPC4, and PC3, which differ in response to the histone deacetylase inhibitor, suberoylanilide hydroxamic acid (vorinostat), a new anticancer drug. Examining the role of intrinsic mitochondrial caspase-dependent apoptosis and caspase-independent, reactive oxygen species (ROS) facilitated cell death, has provided an understanding of mechanisms that may determine the varied response to the histone deacetylase inhibitor. We found striking differences among these cancer cells in constitutive expression and response to suberoylanilide hydroxamic acid in levels of antiapoptotic and proapoptotic proteins, mitochondria membrane integrity, activation of caspases, ROS accumulation, and expression of thioredoxin, the major scavenger of ROS. Identifying these differences can have predictive value in assessing therapeutic response and identifying targets to enhance therapeutic efficacy.
Dual inhibitors of inosine monophosphate dehydrogenase and histone deacetylases for cancer treatment.. Liqiang Chen; Daniel Wilson; Hiremagalur N Jayaram; Krzysztof W Pankiewicz (2007) Journal of medicinal chemistry display abstract
Mycophenolic acid (MPA), an inhibitor of IMP-dehydrogenase (IMPDH), is used worldwide in transplantation. Recently, numerous studies showed its importance in cancer treatment. Consequently, MPA entered clinical trials in advanced multiple myeloma patients. Suberoylanilide hydroxamic acid (SAHA), a potent differentiation agent acting through inhibition of histone deacetylases (HDACs), was recently approved for treatment of cutaneous T cell lymphoma. We report herein the synthesis of dual inhibitors of IMPDH and HDACs. We found that mycophenolic hydroxamic acid (9, MAHA) inhibits both IMPDH (Ki=30 nM) and HDAC (IC50=5.0 microM). A modification of SAHA with groups known to interact with IMPDH afforded a SAHA analogue 14, which inhibits IMPDH (Ki=1.7 microM) and HDAC (IC50=0.06 microM). Both MAHA (IC50=4.8 microM) and SAHA analogue 14 (IC50=7.7 microM) were more potent than parent compounds as antiproliferation agents. They were also significantly more potent as differentiation inducers.
SAHA induces caspase-independent, autophagic cell death of endometrial stromal sarcoma cells by influencing the mTOR pathway.. A Hrzenjak; M-L Kremser; B Strohmeier; F Moinfar; K Zatloukal; H Denk (2008) The Journal of pathology display abstract
Endometrial stromal sarcomas are rare and molecular mechanisms involved in their pathogenesis are poorly understood. Covalent modifications of histone proteins, in particular de/acetylation of lysine residues, play an important role in the regulation of gene transcription in normal and neoplastic cells, but there are only limited data about these processes in solid mesenchymal tumours. We treated endometrial stromal sarcoma cells (ESS-1) and non-malignant human endometrial stromal cells (HESCs) with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor. SAHA was able to mediate the cell cycle and expression of genes related to the malignant phenotype of endometrial stromal tumours, eg p21(WAF1) and HDAC7. SAHA led to dose-dependent differentiation and death of ESS-1 cells but not of HESCs. Exposure of HESCs to SAHA resulted only in slightly decreased cell proliferation. SAHA also increased the p21(WAF1) expression and caused significant changes in the cell cycle by inhibiting the G1/S transition in ESS-1 cells. Recovery experiments indicated that these changes became irreversible when the tumour cells were treated with SAHA for longer than 24 h. In our experimental system, not apoptotic but autophagic processes were responsible for the cell death. Monodansyl cadaverine accumulation in treated ESS-1 cells and decreased expression of the mTOR and phospho-S6 ribosomal protein (S6rp) additionally supported this observation. Taken together, our study indicates that HDACs might be considered as potential drug targets in the therapy of stromal sarcomas and that SAHA might be a promising therapeutic agent for endometrial stromal sarcoma.
A class of hybrid polar inducers of transformed cell differentiation inhibits histone deacetylases.. V M Richon; S Emiliani; E Verdin; Y Webb; R Breslow; R A Rifkind; P A Marks (1998) Proceedings of the National Academy of Sciences of the United States of America display abstract
Hybrid polar compounds (HPCs) have been synthesized that induce terminal differentiation and/or apoptosis in various transformed cells. We have previously reported on the development of the second-generation HPCs suberoylanilide hydroxamic acid (SAHA) and m-carboxycinnamic acid bishydroxamide (CBHA) that are 2,000-fold more potent inducers on a molar basis than the prototype HPC hexamethylene bisacetamide (HMBA). Herein we report that CBHA and SAHA inhibit histone deacetylase 1 (HDAC1) and histone deacetylase 3 (HDAC3) activity in vitro. Treatment of cells in culture with SAHA results in a marked hyperacetylation of histone H4, but culture with HMBA does not. Murine erythroleukemia cells developed for resistance to SAHA are cross-resistant to trichostatin A, a known deacetylase inhibitor and differentiation inducer, but are not cross-resistant to HMBA. These studies show that the second-generation HPCs, unlike HMBA, are potent inhibitors of HDAC activity. In this sense, HMBA and the second-generation HPCs appear to induce differentiation by different pathways.