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Experimental Therapeutics |
Cancer Immunology Program, The Peter MacCallum Cancer Institute, East Melbourne 3002, Victoria, Australia [M. J. P., K. M. T., A. A. R., K. A. S., N. J. W., J. A. T., R. W. J.]; Monash University Department of Medicine, Box Hill Hospital, Victoria, Australia [A. E. D.]; and Molecular Genetics of Cancer Division, The Walter and Eliza Hall Institute of Medical Research, Parkville 3050, Victoria, Australia [L. A. O.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chemotherapeutic drugs can induce cell death by activating either of two major apoptotic pathways (1) . The death receptor pathway involves ligation of receptors (i.e. Fas, tumor necrosis factor receptor, and TRAILR) by their cognate ligands, resulting in binding of adaptor proteins (i.e. FADD and TRADD) and recruitment and activation of membrane proximal caspases (i.e. caspase-8 and caspase-10). These enzymes subsequently activate effector caspases (i.e. caspase-3), resulting in the cleavage of a number of nuclear and cytoplasmic substrates to induce the morphological changes characteristic of apoptosis. The second pathway, known as the intrinsic pathway, is primarily activated by stress stimuli such as chemotherapeutic drugs, ionizing radiation, and growth factor withdrawal (2) . These stimuli induce the release of mitochondrial proteins such as cytochrome c, which, together with Apaf-1, induces caspase-9 activation, and HtrA2 and Smac/DIABLO, which neutralize the caspase-inhibitory actions of inhibitors of apoptosis proteins (IAP). Caspase-9 can then activate downstream effector caspases (i.e. caspase-3). The intrinsic pathway is primarily regulated by the Bcl-2 family of proteins, which includes the multidomain antiapoptotic (i.e. Bcl-2 and Bcl-XL) and proapoptotic (i.e. Bak and Bax) members, and BH3 domain-only proapoptotic members (i.e. Bid, Bad, NOXA, PUMA, and Hrk). Interestingly, death receptors can also activate the intrinsic pathway through caspase-8-mediated activation of Bid. In this way, Bid serves as a molecular link between the two pathways and can amplify the death receptor-stimulated response.
HDACIs3 are a novel class of chemotherapeutic agent initially identified by their ability to reverse the malignant phenotype of transformed cells. They have been shown to activate differentiation programs, inhibit the cell cycle, and induce apoptosis in a wide range of tumor-derived cell lines and to block angiogenesis and stimulate the immune system in vivo (3 , 4) . Whereas the mechanisms through which HDACIs exert these antitumor activities have not been fully delineated, induction of histone hyperacetylation and modulation of gene transcription through chromatin remodeling are thought to be primarily responsible, leading to the selective activation of genes associated with cell growth and survival. In addition, proteins other than histones, such as p53 and GATA-1, have been identified as targets for acetylation (5 , 6) , and it is possible that HDACIs may exert other effects independently of their direct effects on transcription through chromatin remodeling. Recently, there has been strong interest in HDACIs as anticancer agents due to their selective toxicity against tumor cells and synergistic activity with existing therapeutic agents, including retinoic acids (7) , vitamin D analogues (8) , and peroxisome proliferator-activated receptor ligands (9) . A number of HDACIs are currently in Phase I and II clinical trials, and encouraging results have been reported for the treatment of both hematological malignancies and solid tumors (3 , 4) .
Previously, we reported that the HDACI SAHA mediated apoptosis via a novel mechanism involving activation of the intrinsic death pathway by inducing the caspase-independent cleavage and activation of Bid (10) . The aim of this study was to determine whether the structurally diverse HDACIs oxamflatin and depsipeptide (officially called FR901228 or FK228) activate a similar apoptotic pathway to SAHA. Oxamflatin is a synthetic HDACI belonging to the same structural class (hydroxamic acid) as SAHA. Depsipeptide is a natural HDACI that was isolated from Chromobacterium violaceum and has a novel chemical structure (cyclic tetrapeptide). Both oxamflatin and depsipeptide exhibit potent cytotoxicity against various human tumor cell lines (11 , 12) and in vivo efficacy against human tumor xenografts and/or murine tumors (12) ; however, little is known about the molecular events underpinning cell death by these drugs.
We have identified key molecular events necessary for apoptosis induced by these drugs and found that all three agents induced death marked by alteration of the cell cycle profiles with a loss of cells in G1 and S phase and accumulation of cells with a 4n DNA content and the cleavage and activation of caspase-2 and the BH3-only Bcl-2 protein Bid. Inhibition of the death receptor pathway or addition of the polycaspase inhibitor zVAD-fmk, which can inhibit the activity of a number of key caspases (i.e. caspase-3, -7, -8, and -9) but is a poor inhibitor of caspase-2 (13) , had little or no effect on tumor cell death by these drugs. However, consistent with these agents mediating cell death after mitochondrial disruption, overexpression of Bcl-2 inhibited HDACI-induced mitochondrial membrane disruption and the cytotoxic effects of these drugs. Interestingly, initial processing of caspase-2 to its p33 intermediate form still occurred in Bcl-2-overexpressing cells, indicating that HDACIs can induce the initial cleavage of caspase-2 upstream of Bcl-2; however, full cleavage to the active p19 form of caspase-2 required mitochondrial membrane perturbation. This is consistent with a model for caspase activation requiring the release of mitochondrial Smac/DIABLO and/or HtrA2 to sequester IAPs and allow full caspase activation.
Inhibition of cell cycle progression at the G1-S boundary through overexpression of the cyclin-dependent kinase inhibitor p16INK4A abrogated cell death mediated by HDACIs. Moreover, inhibition of de novo protein synthesis inhibited the cleavage of Bid and subsequent death induced by SAHA, oxamflatin, and depsipeptide, indicating that their mechanism of action is mediated through a direct (through histone hyperacetylation) or indirect effect on gene transcription. Whereas all three drugs had similar effects on cell proliferation and survival, depsipeptide-induced Bid cleavage was perturbed by zVAD-fmk, indicating differences in the mechanisms of action of depsipeptide compared with SAHA and oxamflatin. In addition, we have determined that depsipeptide is a substrate for the drug efflux protein P-gp and is less effective against P-gp-expressing, MDR cells. Taken together, these data provide a mechanistic profile of a novel class of chemotherapeutic drugs and indicate that although these structurally diverse compounds activate a number of common molecular pathways, subtle yet significant differences in their mechanisms of action exist.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Culture.
The acute T-cell leukemia cell line CEM-CCRF and its doxorubicin-selected and P-gp+ derivative CEM-P-gp and the colon carcinoma cell line LoVo and its Adriamycin-selected P-gp+ derivative Lovo-adr have been described previously (14
, 15)
. CEM cells expressing CrmA and p16INK4A have been described previously (16
, 17)
. CEM cells overexpressing human Bcl-2 were a gift from David Huang (Walter and Eliza Hall Institute, Melbourne, Victoria). All cells were grown in RPMI 1640 supplemented with 10% (v/v) FCS, 2 mm/liter glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Inc., Grand Island, NY). CEM-p16INK4A cells were additionally cultured in 250 ng/ml hygromycin B (Amersham, Castle Hill, Australia). The cell surface expression of P-gp was confirmed and monitored by fluorescence analysis using the anti-Pgp monoclonal antibody MRK 16 (Kamiya Biochemical Co., Thousand Oaks, CA).
Apoptosis Assays.
Cells were cultured for 0.5–48 h with 0–10 µM oxamflatin, depsipeptide, or SAHA or 0.5–100 ng/ml anti-Fas antibody clone CH-11 (Upstate Biotechnology, Lake Placid, NY). Cell death was assessed by 51Cr release assay as described previously (18)
. The spontaneous release of 51Cr was determined by incubating cells with medium alone. The maximum release was determined by adding HCl to a final concentration of 1N. The percentage of specific lysis was calculated as follows: 100 x [(experimental release - spontaneous release)/(maximum release - spontaneous release)]. Trypan blue exclusion assays were performed as described previously (19)
. In all assays, cells from at least four different fields of view were counted for each data point, and data were calculated as the mean ± SD and are representative of at least two separate experiments. The number of apoptotic or dead cells (blue cells) was expressed as a percentage of the total cell number. Translocation of the membrane phospholipid phosphatidylserine from the inner face of the plasma membrane to the outer leaflet was detected using Annexin-V-FLUOS according to the manufacturer’s protocol (Roche, Mannheim, Germany). Briefly, cells treated for 18 h with various apoptotic stimuli were washed in PBS, stained with Annexin-V-FLUOS for 10 min at room temperature, and then analyzed by flow cytometry using a FACScan flow cytometer (BD Biosciences, North Ryde, Australia). Colony assays were performed on cells treated for 24 h with various apoptotic stimuli as described previously (19)
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Cell Cycle Analysis.
Cells (2 x 105) treated with various apoptotic stimuli were assessed for DNA content by PI staining followed by flow cytometry as described previously (20)
. Briefly, cells were washed with PBS, fixed by incubation in 50% ice-cold ethanol/PBS for 30 min on ice, washed again with PBS, and resuspended and incubated in PI solution (69 mM PI, 388 nM sodium citrate, 100 µg/ml RNase A) for 15 min at 37°C.
DEVDase Assay.
Cells (2 x 106) treated with or without drug for 24 h were harvested, washed twice with PBS, and lysed in ice-cold NP40 lysis buffer without protease inhibitors. Lysate (45 µg) was incubated with 1.25 mM Ac-Asp-Glu-Val-Asp-pNA (Ac-DEVD-pNA) substrate (Bachem, Bubendorf, Switzerland) in BAADT buffer (pH 7.3) containing 0.1 M HEPES and 0.05 M CaCl2 at 37°C, and absorbance at (405 nm) was read every 15 min. Relative fold activity was determined by dividing absorbance readings of samples containing drug by absorbance of control untreated samples.
Permeabilized Cell Assay for 
m.
Experiments to examine m in the presence and absence of HDACIs were performed as described previously (21)
. CEM or CEM-Bcl-2 cells (2 x 105) incubated for 24 h at 37°C in the presence or absence of SAHA (2.5 µM), depsipeptide (0.5 µM), or oxamflatin (2.5 µM) were subsequently incubated in media containing TMRE (200 nM) for 20 min at 37°C. Cells were then analyzed by flow cytometry with TMRE fluorescence detected in FL2.
Anti-Bid mAb Production.
A mAb reactive against human Bid was produced as described previously (22)
. Briefly, BALB/c mice received i.p. injection with 50 µg of recombinant glutathione S-transferase-Bid fusion protein in Freund’s complete adjuvant and were then boosted twice at fortnightly intervals with 50 µg of glutathione S-transferase-Bid in Freund’s incomplete adjuvant. Ten days after fusion, hybridoma culture supernatants were tested for the secretion of antibody reactive with Bid by ELISA.
Western Blot Analysis.
Cells (6 x 105) were washed with PBS and lysed in ice-cold NP40 lysis buffer as described previously (19)
. Proteins (5–15 µg) were separated on 10% or 15% SDS-polyacrylamide gels electroblotted onto Immobilon-P nylon membranes (Millipore, Bedford, MA). Membranes were incubated with anti-acetylated histone H3 (Upstate Biotechnology), anti-acetylated histone H4 (Upstate Biotechnology), anti-caspase-2 (23)
, antihuman Bid mAb, antihuman PARP (Boehringer Mannheim), and antihuman 
-tubulin mAb (Sigma) overnight at 4°C, followed by subsequent incubation with horseradish peroxidase-conjugated secondary antibodies (DAKO, Glostrup, Denmark). Immunoreactive bands were visualized by enhanced chemiluminescence (Amersham).
To assess the release of cytochrome, cells (1 x 106) were harvested by centrifugation, washed once in PBS, and lysed in 100 µl of ice-cold plasma membrane permeabilization buffer (400 µg/ml digitonin in PBS containing 80 mM KCl and 250 mM sucrose) for 5 min (21) . Aliquots were analyzed by trypan blue to ensure that greater than 95% of the cells had permeabilized plasma membranes. The cells were centrifuged (800 x g for 5 min at 4°C), and the supernatant was analyzed by Western blotting using an anti-cytochrome c mAb (PharMingen).
Extraction of Histones.
Histones were isolated by acid extraction. Cells (5 x 106) treated with or without drug for varying times were harvested and washed with PBS. Cells were lysed in ice-cold lysis buffer [10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, and 1.5 mM phenylmethylsulfonyl fluoride], and 5 M H2SO4 was added to 0.4 N. After incubation on ice for 1 h, the suspension was centrifuged, and the supernatant was harvested, mixed with acetone at a ratio of 9:1, and incubated at -20°C overnight. After centrifugation, the pellet was washed with 70% ethanol and air dried, and the acid-soluble histone fraction was dissolved in H2O. A BCA protein assay was then used for quantitation (Pierce), and histones were analyzed by Western blot.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Death induced by oxamflatin (1 µM) and depsipeptide (0.5 µM) was compared with that induced by SAHA (2.5 µM). Significant death was observed after treatment with all three HDACIs by 12 h and steadily increased over the 24-h time period (Fig. 1A)
. Similar results were obtained using DNA fragmentation/PI staining, trypan blue exclusion, and Annexin V staining as markers of cell death (data not shown).
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, treatment with oxamflatin, depsipeptide, or SAHA led to a marked increase in both acetylated histones H3 and H4 within 30 min. The accumulation of acetylated histones remained elevated to 24 h. Thus, similarly to SAHA, oxamflatin and depsipeptide induce apoptosis of CEM cells in a dose- and time-dependent manner, preceded by the accumulation of acetylated histones H3 and H4.
Apoptosis Induced by HDACIs Requires Cell Cycle Progression.
Because HDACIs can affect both cell proliferation and cell survival, we assessed the effect of oxamflatin, depsipeptide, and SAHA on cell cycle progression. CEM cells were treated with the different HDACIs for 24 h and stained with PI. Flow cytometric analysis revealed that, in addition to inducing apoptosis as denoted by the increase in the percentage of the sub-G1 population of cells, treatment with oxamflatin, depsipeptide, or SAHA caused an accumulation of cells with 4n DNA content, consistent with a G2-M cell cycle arrest (Fig. 2A)
. A time course analysis revealed that the percentage of cells with 4n DNA content decreased over time, concomitant with an increase in the percentage of apoptotic cells (Table 1)
. These data indicate that the movement of cells into and through the G2-M phases of the cell cycle may precede apoptosis induced by these HDACIs.
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, p16INK4A-expressing CEM cells were almost completely arrested in G1 and were significantly less sensitive to death induced by the three HDACIs compared with cycling CEM cells. Treatment of CEM-p16INK4A (-dox) cells with oxamflatin, depsipeptide, or SAHA led to an accumulation of cells with a 4n DNA content and induction of apoptosis after 24 h. In contrast, there was no increase in cells within G2-M, and there was a marked reduction (>50%) in the sub-G1 population of the HDACI-treated p16INK4A-expressing cells. Remarkably, these cells remained largely protected from HDACI-induced death over 48 h (data not shown). Comparable results were also obtained by trypan blue exclusion assays, and similar effects were seen with an independent dox-inducible, p16INK4A-expressing clone (data not shown). Thus, it appears that transition through the cell cycle is necessary for HDACI-mediated apoptosis and, taken together with Table 1
, suggests that these drugs induce apoptosis during or immediately after movement of cells through G2-M.
Oxamflatin, but not Depsipeptide, Induces Death of P-gp+ and P-gp- Cells.
MDR is a major obstacle for anticancer therapy, and one common mechanism of MDR is overexpression of the multidrug transporter P-gp. P-gp can protect cells from drug-induced apoptosis through its combined ability to efflux drugs and inhibit caspase activation (19)
. However, not all drugs are inhibited by P-gp because we have demonstrated that both hexamethylene bisacetamide and SAHA, which are not P-gp substrates and function in a caspase-independent manner, can equivalently kill P-gp+ and P-gp- tumor cells (10
, 20) . To determine whether oxamflatin and depsipeptide were able to kill P-gp-expressing cells, we treated two paired P-gp+ and P-gp- cell lines with various doses of oxamflatin or depsipeptide for 24 h and measured cell death. The expression of P-gp on these cell lines was assessed before and during the course of the assays by immunofluorescence analysis using the anti-P-gp mAb MRK 16. As shown in Fig. 3A
, P-gp was highly expressed on CEM-P-gp and Lovo-adr cells, weakly expressed on Lovo cells, and not expressed at all on CEM cells. Treatment of the cells with oxamflatin or depsipeptide did not alter the expression levels of P-gp over the 24-h time period (data not shown). As seen in Fig. 3B
, oxamflatin induced equivalent death of P-gp+ and P-gp- cells in a dose-dependent manner. In contrast, death induced by depsipeptide was significantly inhibited in P-gp+ cells. The protection by P-gp against depsipeptide-induced death but not oxamflatin-induced death was confirmed in sensitive long-term clonogenic assays (data not shown). Verapamil, a pharmacological inhibitor of P-gp, reversed the resistance of the P-gp+ cells to depsipeptide (Fig. 3C)
, supporting the notion that depsipeptide is a substrate for P-gp (27)
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. Because depsipeptide is a substrate for P-gp, we expected a loss of caspase-3 activity in depsipeptide-treated CEM-P-gp cells. However, CEM-P-gp cells treated with SAHA or oxamflatin also show a strong decrease in caspase-3 activity, even though these cells are as sensitive to the drugs as the parental CEM cells. These data support our previous findings that P-gp can inhibit the activation of caspases by drugs that are not substrates for P-gp (29)
and suggest that caspase-3 activation is not required for death induced by SAHA, depsipeptide, or oxamflatin.
Oxamflatin and Depsipeptide Do Not Require an Intact Death Receptor Pathway or zVAD-fmk-inhibitable Caspases to Induce Cell Death.
A number of chemotherapeutic drugs, including the HDACIs apicidin and CBHA, have been reported to induce cell death via activation of the death receptor pathway (30
, 31)
, whereas others, such as SAHA and butyrate, show no requirement for this pathway (10
, 24) . To determine whether oxamflatin or depsipeptide utilize this pathway to induce cell death, we used CEM cells expressing the cowpox virus protein CrmA, which inhibits caspase-8 and -10, effectively short circuiting the death receptor pathway. As shown in Fig. 4A
, expression of CrmA had no effect on apoptosis induced by either oxamflatin or depsipeptide, indicating that they do not require a functional death receptor pathway to induce apoptosis. This result was confirmed in long-term clonogenic assays (Fig. 4B)
. As expected, cell death mediated by anti-Fas antibody was significantly inhibited in the CrmA-expressing cells in both short- and long-term cell death assays, whereas CrmA had no effect on SAHA-mediated death (Fig. 4A
; Ref. 10
). Thus, similarly to SAHA, oxamflatin and depsipeptide do not require activation of the death receptor pathway for their cytotoxic action.
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indicate that caspase-3 activity is not necessary for SAHA- or oxamflatin-induced death. CEM cells were pretreated with zVAD-fmk or the cathepsin B inhibitor zFA-fmk for 1 h before the addition of oxamflatin, depsipeptide, SAHA, or anti-Fas antibody as a control, and cell death was then assessed by Annexin V staining (Fig. 5A)
. Inhibition of caspase activity by zVAD-fmk had no effect on the ability of oxamflatin, depsipeptide, or SAHA to induce apoptosis, and the effectiveness of zVAD-fmk in inhibiting caspase activation in our assay systems was confirmed because Fas-mediated apoptosis was significantly inhibited in the presence of this inhibitor. Similar results were also obtained by trypan blue exclusion (data not shown) and clonogenic assays (Fig. 5B)
. Taken together with the results seen in Figs. 3
and 4
and previous published data (10
, 29)
, these data indicate that although oxamflatin, depsipeptide, or SAHA do induce caspase activation, the activity of caspases that are blocked by zVAD-fmk are not necessary for the cytotoxic activities of these drugs.
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m and release of cytochrome c from the mitochondria into the cytosol. The overexpression of Bcl-2 in CEM-Bcl-2 cells was confirmed by intracellular fluorescence-activated cell sorting (data not shown). As shown in Fig. 6A
, treatment of cells with oxamflatin, depsipeptide, or SAHA induced a loss of TMRE staining indicative of mitochondrial membrane disruption and a loss of mitochondrial transmembrane potential. The effect of these HDACIs on m was completely inhibited by overexpression of Bcl-2. The activation of the intrinsic apoptotic pathway by oxamflatin, depsipeptide, and SAHA was confirmed by Western blotting to assess the release of cytochrome c from the mitochondria into the cytosol. As expected, all three HDACIs induced the release of mitochondrial cytochrome c that was effectively blocked by overexpression of Bcl-2 (Fig. 6B)
.
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. By contrast, the release of cytochrome c mediated by cross-linking of Fas was inhibited by zVAD-fmk (Fig. 6C)
. Taken together, these data indicate that oxamflatin, depsipeptide, and SAHA can perturb the mitochondrial membrane that can be blocked by overexpression of Bcl-2 but not by the inhibition of caspase activation using zVAD-fmk.
Overexpression of Bcl-2 Inhibits Oxamflatin- and Depsipeptide-induced Cell Death but not Cell Cycle Arrest.
To determine the role of mitochondrial membrane perturbation in death induced by HDACIs, CEM-Bcl-2 and parental CEM cells were treated with SAHA, oxamflatin, or depsipeptide for 24 h, and cell death was measured by 51Cr release. Overexpression of Bcl-2 significantly inhibited death mediated by oxamflatin and depsipeptide (Fig. 7A)
over various doses of drug, indicating that mitochondrial membrane perturbation is required for death induced by these drugs. As seen previously (10)
, overexpression of Bcl-2 also inhibited SAHA-induced cell death. Interestingly, although Bcl-2 increased survival of drug-treated cells in short-term assays, the clonogenic potential of the CEM-Bcl-2 cells was diminished (Fig. 7B)
. PI staining of the HDACI-treated CEM-Bcl-2 cells revealed that although the cells were less sensitive to DNA damage induced by HDACIs the effects on the cell cycle, marked by accumulation of cells with a 4n DNA content, were not affected (Fig. 7C)
, suggesting that the cytostatic effects of these drugs cannot be blocked by Bcl-2. Because clonogenic assays are a measure of both cell survival and proliferation, it is likely that the alteration in cell cycle progression is responsible for the loss of clonogenicity of the CEM-Bcl-2 cells.
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, oxamflatin (Fig. 8B)
, or depesipeptide (Fig. 8C)
resulted in the processing of pro-caspase-2 (p48) to produce the intermediate (p33) and active (p19) proteins. Interestingly, the production of the p33 form of caspase-2 still occurred after treatment of CEM-Bcl-2 cells with SAHA, oxamflatin, or depesipeptide; however, further cleavage to produce p19 caspase-2 was inhibited by Bcl-2. These data demonstrate that the initiation of caspase-2 activation after treatment with SAHA, oxamflatin, or depsipeptide can occur upstream of the mitochondria, indicating that caspase-2 processing may be an important step for the induction of cell death after treatment with these drugs.
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, all three HDACIs induced the cleavage of Bid to its characteristic Mr 16,000 activated tBid form. The molecular weight of tBid produced in response to the HDACIs was indistinguishable from that produced after addition of anti-Fas antibody. Cleavage of Bid by SAHA and oxamflatin was not affected by zVAD-fmk (Fig. 9A)
; however, depsipeptide-induced Bid cleavage was significantly attenuated after the addition of zVAD-fmk, to an extent similar to that seen following the cross-linking of Fas in the presence of zVAD-fmk. This suggests that depsipeptide may be more reliant on caspases to cleave Bid than are oxamflatin and SAHA. As expected, Fas-induced Bid cleavage was inhibited by zVAD-fmk. The effectiveness of zVAD-fmk in inhibiting caspase activity at this time point and concentration was demonstrated by Western blotting for cleavage of the prototypic caspase substrate PARP. PARP was cleaved after treatment of cells with SAHA, oxamflatin, depsipeptide, and Fas, both alone and in the presence of zFA-fmk; however, cleavage was significantly inhibited after treatment with all HDACIs or with anti-Fas antibodies when zVAD-fmk was present (Fig. 9A)
.
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. Treatment with increasing doses of all three HDACIs induced cleavage of Bid in a dose-dependent manner in both the CEM and CEM-Bcl-2 cells, as shown by loss of the Mr 22,000 pro-form and an increase in the Mr 16,000 tBid cleavage product. Thus, it appears that Bid is cleaved upstream of mitochondria membrane disruption by these drugs. As expected, there was less processing of Bid in the CEM-Bcl-2, compared with CEM cells, because overexpression of Bcl-2 would suppress the release of caspase-activating molecules such as cytochrome c, HtrA2, and Smac/DIABLO from the mitochondria, thereby inhibiting Bid cleavage after secondary caspase activation.
De Novo Protein Synthesis Is Required for Oxamflatin- and Depsipeptide-induced Apoptosis.
HDACIs induce histone acetylation, resulting in chromatin remodeling and new gene transcription. We have shown previously that de novo protein synthesis is necessary for SAHA-induced cell death, and we therefore determined whether alteration of gene expression is necessary for the antitumor activities of oxamflatin and depsipeptide. Pretreatment of cells with 5, 50, or 500 ng/ml CHX had no effect on cell cycle progression of CEM cells over the 24-h time course of this experiment (data not shown). However, treatment of cells with CHX for 1 h before the addition of oxamflatin, depsipeptide, or SAHA significantly inhibited the ability of the drugs to affect the cell cycle (data not shown) and induce apoptosis (Fig. 10A)
, suggesting that oxamflatin and depsipeptide require new protein synthesis to exert their antitumor effects. The effectiveness of CHX in inhibiting protein synthesis was demonstrated by Western blotting for proteins with short half-lives (i.e. p27; data not shown).
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. These data are consistent with our previous studies demonstrating the importance of Bid cleavage in SAHA-induced apoptosis (10)
. Although depsipeptide-induced Bid cleavage was partially inhibited by zVAD-fmk, whereas the poly-caspase inhibitor did not affect cleavage of Bid by SAHA and oxamflatin, all three drugs required new protein synthesis to mediate Bid cleavage and cell death. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We found that all three HDACIs were comparable in their ability to alter the cell cycle, resulting in an accumulation of cells with a 4n DNA content, and all induced apoptosis via activation of the intrinsic apoptotic pathway, which was accompanied by the cleavage and activation of caspase-2 and Bid. However, differences emerged with relation to the protease(s) responsible for Bid cleavage induced by these drugs because zVAD-fmk inhibited Bid cleavage induced by depsipeptide, but not that induced by oxamflatin or SAHA. Furthermore, oxamflatin and SAHA, but not depsipeptide, were able to kill P-gp-expressing MDR cells.
Treatment of cells with oxamflatin, depsipeptide, or SAHA induced two cellular responses: (a) accumulation of cells with a 4n DNA content at what has been termed a G2 cell cycle checkpoint (34) and (b) induction of apoptosis. With time, those cells that initially accumulate with a 4n DNA content also undergo apoptosis. A number of studies have demonstrated that the cell cycle inhibitor p21WAF1/CIP1 is commonly induced in response to HDACIs (35 , 36) and plays an important role in the G1 cell cycle arrest of many cancer cells, although it is clear from studies with p21-/- cells that it is not the sole determinant responsible for this arrest (37 , 38) . The molecular events leading to the effect of HDACIs on cells during or after DNA replication have not yet been defined. It is possible that histone modification during DNA replication and cell division may be important for HDACI function. Histone acetylation in heterochromatin is tightly regulated during S phase, and disruption of this process triggers cell cycle arrest within G2-M (39) . The HDACI-associated accumulation of cells with a 4n DNA content may be related to hyperacetylation of the centromere, allowing release of heterochromatin proteins resulting in abnormal chromosomal segregation (40) . In cancer cells, a proposed HDACI-associated G2 checkpoint may be lost, leading to aberrant mitoses and apoptosis (34) . Hence, it is formally possible that dysregulated histone acetylation during replicative S phase and/or mitosis may play an important role in the induction of HDACI-mediated cell death.
Progression through the G1-S checkpoint was necessary for the induction of apoptosis by SAHA, oxamflatin, and depsipeptide because CEM cells overexpressing p16INK4A and arrested in G1 were resistant to HDACI-induced death. A previous study using these p16INK4A-expressing CEM cells similarly reported that butyrate-induced cell death was abrogated by forced G1 arrest (24) . In contrast to these studies, Burgess et al. (41) demonstrated that melanoma cells arrested at G1 using a similar inducible p16INK4A system remained sensitive to the HDACI azelaic bishydroxamic acid. However, we have shown that when p16INK4A-expressing CEM cells were treated with azelaic bishydroxamic acid, death was inhibited to a similar extent to that seen with the HDACIs investigated in this study,4 suggesting that these differences may relate to cell type. Interestingly, other chemotherapeutic drugs, including vincristine, staurosporine, and etoposide, were also ineffective in killing these p16INK4A-expressing CEM cells, whereas anti-Fas antibody could kill the cells to the same extent as cycling cells (data not shown), demonstrating that these cells are not resistant to all death stimuli and that diverse chemotherapeutic drugs rely on the ability of cells to cycle to exert their effects.
Apoptosis mediated by oxamflatin, depsipeptide, and SAHA did not require activation of the death receptor pathway. Likewise, another HDACI, sodium butyrate, has also been shown to induce apoptosis independently of this pathway in CEM cells (24) . Interestingly, butyrate did not alter the expression of either the CD95 receptor or ligand (CD95L) in these cells but enhanced sensitivity to CD95-mediated apoptosis (42) . These results are in contrast to studies with the HDACIs CBHA and apicidin, which have been reported to induce the expression of CD95/CD95L in neuroblastoma and promyelocytic leukemia cells, respectively, thereby activating the death receptor pathway (30 , 31) . Moreover, apicidin-induced apoptosis was abrogated when CD95 signaling was inhibited (30) . Thus, the molecular pathway(s) initiated in response to HDACIs may be related to intrinsic characteristics of the particular cell type. Alternatively, it is possible that structurally similar HDACIs may preferentially target different HDACs and therefore initiate diverse molecular pathways to induce cell death.
With its broad substrate specificity, P-gp can efflux a wide variety of compounds, thereby conferring resistance to structurally diverse and functionally unrelated chemotherapeutic agents (43) . In addition to lowering intracellular drug concentration, P-gp can protect cells against caspase-dependent forms of apoptosis by inhibiting caspase activation after chemotherapeutic drug treatment (29) . Consistent with this, we showed here that HDACI-induced caspase-3 activation was inhibited in P-gp-expressing tumor cells. The poly-caspase inhibitor zVAD-fmk did not attenuate killing induced by oxamflatin, depsipeptide, or SAHA, demonstrating the ability of these drugs to kill in the absence of the activation of certain caspases. Therefore, all three HDACIs had the potential to kill P-gp-expressing MDR cells. However, we found that whereas oxamflatin and SAHA induced equivalent death of P-gp-expressing and non-P-gp-expressing cells, depsipeptide was a substrate for P-gp and was therefore less effective against these cells. Our data and those of others (12 , 27) indicate that the anticancer activities of depsipeptide may be somewhat diminished against P-gp-expressing MDR tumors. A recent report demonstrating that depsipeptide is a prodrug and is activated by glutathione-mediated reduction after cellular uptake indicates that this agent may be effective in killing tumors that have become resistant to agents such as cisplatin that are inhibited by high glutathione levels (44) . Whether depsipeptide is a substrate for the drug efflux pump MRP1, which extrudes glutathione-conjugated xenotoxins from tumor cells, remains to be determined.
The poly-caspase inhibitor zVAD-fmk can inhibit the activity of most caspases; however, it is a poor inhibitor of caspase-2 (13) . In addition, caspase-2 has recently been demonstrated to be an important initiator caspase, capable of activating the intrinsic apoptotic pathway in response to DNA damage (32) , and recombinant caspase-2 can cleave and activate Bid in vitro (45) . It is therefore possible that caspase-2 can mediate the cleavage of Bid induced by SAHA or oxamflatin and/or may be responsible for the induction of cell death induced by all three HDACIs in the presence of zVAD-fmk. SAHA, oxamflatin, and depsipeptide activated caspase-2 consistent with a putative role of this caspase in HDACI-mediated cell death. However, full processing and activation of caspase-2 were inhibited in CEM-Bcl-2 cells, whereas the cleavage of Bid still occurs in these cells, although to a lesser degree. Thus, active caspase-2, produced after treatment of cells with HDACIs, may cleave Bid; however, it appears that fully processed caspase-2 is not necessary for Bid activation. Whether the p33 intermediate form of caspase-2 is capable of cleaving Bid needs to determined following a thorough biochemical analysis. In addition, the molecular processes necessary for the activation and cleavage of caspase-2 in response to HDACI treatment also remain to be elucidated.
A number of studies have demonstrated that overexpression of Bcl-2 protects cells against diverse apoptotic stimuli that induce death via the intrinsic apoptotic pathway (1)
. Bcl-2 overexpression inhibited the reduction of m, release of mitochondrial cytochrome c, and subsequent apoptosis induced by oxamflatin, depsipeptide, and SAHA, suggesting that mitochondrial membrane disruption is important for HDACI-induced apoptosis of CEM cells. The proapoptotic BH3-only Bcl-2 family member Bid can activate the intrinsic apoptotic pathway and is involved in apoptosis mediated by SAHA (10)
. We found that Bid was also cleaved after treatment of cells with oxamflatin or depsipeptide, even in cells overexpressing Bcl-2, placing HDACI-induced Bid cleavage upstream of mitochondrial membrane perturbation. Interestingly, however, zVAD-fmk inhibited Bid cleavage in response to depsipeptide, but not oxamflatin or SAHA, suggesting that caspases may be primarily responsible for mediating Bid cleavage by depsipeptide, whereas an as yet unidentified protease may be activated and/or induced by oxamflatin and SAHA to cleave Bid. Identification of the protease responsible for the processing of Bid by each of these drugs should aid in dissecting the upstream signaling pathways leading to mitochondrial membrane disruption triggered by these drugs. Interestingly, the accumulation of cells with a 4n DNA content by HDACIs was not affected by Bcl-2 overexpression, suggesting that HDACI-induced cell cycle alterations occur independently or upstream of mitochondrial membrane disruption. In addition to its role as an antiapoptotic protein, Bcl-2 has a separate function as a cell cycle inhibitor (46)
. Overexpression of Bcl-2 has been reported to delay transition from the M to G1 phase (47)
and prolong the G0-G1 phase (48)
. Movement through the cell cycle is required for HDACI-induced apoptosis; thus, in addition to its ability to inhibit the release of proapoptotic proteins from the mitochondria, the antiproliferative property of Bcl-2 could contribute to its protective effect against HDACI-induced apoptosis.
Our study demonstrated that oxamflatin, depsipeptide, and SAHA required de novo protein synthesis to induce their antitumor activities, supporting the notion that HDACIs mediate their effects through regulation of new gene expression. Similar results have been reported with other HDACIs, including butyrate, trichostatin A, CBHA, apicidin, and depudecin (30 , 31 , 49 , 50) . The induction of new gene expression by HDACIs is presumed to be mediated primarily through the inhibition of histone deacetylases, and whereas histones may be their primary targets, non-histone proteins (i.e. p53) can be acetylated and are therefore potential targets for HDACIs (3 , 4) . Thus it is unclear whether the cytostatic and cytotoxic effects of HDACIs are mediated through histone acetylation, thereby directly affecting gene expression, or by acetylation of other proteins that also require new gene expression to mediate their effects. Interestingly, we found depsipeptide to be a significantly more potent agent against P-gp- cells compared with oxamflatin and SAHA. In addition, Bid cleavage after depsipeptide treatment was inhibited by zVAD-fmk, whereas oxamflatin- and SAHA-mediated Bid cleavage was unaffected by the poly-caspase inhibitor. These data highlight functional differences between these structurally dissimilar compounds that are thought to have the same intracellular targets.
These and our other recent studies (10 , 29) identify novel mechanisms of apoptosis induced by HDACIs. Our findings demonstrate that oxamflatin, depsipeptide, and SAHA require activation of a number of common molecular pathways to induce both growth arrest and apoptosis. However, these HDACIs differed in their ability to kill P-gp-expressing cells, which is related to structural characteristics of the drugs, and in the molecular events triggered upstream of Bid cleavage and mitochondrial membrane disruption. This illustrates the value of profiling the mechanism of action of individual agents, information that will be important for the improvement of existing therapies. Ongoing studies in our laboratory are using microarray analysis to examine the gene expression changes induced in response to these drugs to identify key pathways and components linking histone deacetylase inhibition to cell cycle arrest and apoptosis induction to further elucidate the underlying mechanisms of action of HDACIs.
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FOOTNOTES |
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1 Supported by grants from the Wellcome Trust, the National Health and Medical Research Council of Australia (NHMRC), and the Anti-Cancer Council of Victoria. R. W. J. is a Wellcome Trust Senior Research Fellow. J. A. T. is a Principal Research Fellow of the NHMRC, and N. J. W. is a Peter Doherty Fellow of the NHMRC. L. A. O. is supported by a grant from the Dr. Josef Steiner Cancer Research Foundation (Bern, Switzerland). 
2 To whom requests for reprints should be addressed, at The Peter MacCallum Cancer Institute, Gene Regulation Laboratory, Locked Bag 1, Melbourne, Victoria 8006, Australia. Phone: 61-3-9656-3727; Fax: 61-3-9656-1411; E-mail: r.johnstone{at}pmci.unimelb.edu.au
3 The abbreviations used are: HDACI, histone deacetylase inhibitor; P-gp, P-glycoprotein; PI, propidium iodide; mAb, monoclonal antibody; PARP, poly(ADP-ribose) polymerase; MDR, multidrug resistance; CHX, cycloheximide; SAHA, suberoylanilide hydroxamic acid; dox, doxycycline; CBHA, m-carboxycinnamic acid bishydroxamide; zVAD-fmk, N-tert-butoxy-carbonyl-Val-Ala-Asp-fluoromethylketone; TRAILR, TNF related apoptosis inducing ligand receptor; FADD, Fas-associated death domain; TRADD, TNF receptor-associated death domain; PUMA, p53 upregulated modulator of apoptosis; ZFA-fmk, N-tert-butoxy-carbonyl-Phe-Ala-fluoromethylketone; TMRE, tetra-methyl rhodamine ethyl ester.
4 M. J. Peart, A. A. Ruefli, and R. W. Johnstone, unpublished observations.
Received 1/15/03. Accepted 5/19/03.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
,25 dihydroxyvitamin D3 and its 19-nor-hexafluoride analogs in combination with either sodium butyrate or trichostatin A. Oncogene, 20: 1860-1872, 2001.[Medline]
and inhibitors of histone deacetylase in adenocarcinoma of the lung. Clin. Cancer Res., 8: 1206-1212, 2002.This article has been cited by other articles:
|
|
 |
|
  A. A. Lane and B. A. Chabner Histone Deacetylase Inhibitors in Cancer Therapy J. Clin. Oncol., November 10, 2009; 27(32): 5459 - 5468. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. W. Schaefer, A. Loaiza-Bonilla, M. Juckett, J. F. DiPersio, V. Roy, J. Slack, W. Wu, K. Laumann, I. Espinoza-Delgado, S. D. Gore, et al. A phase 2 study of vorinostat in acute myeloid leukemia Haematologica, October 1, 2009; 94(10): 1375 - 1382. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. HORIUCHI, A. MORINOBU, T. CHIN, Y. SAKAI, M. KUROSAKA, and S. KUMAGAI Expression and Function of Histone Deacetylases in Rheumatoid Arthritis Synovial Fibroblasts J Rheumatol, August 1, 2009; 36(8): 1580 - 1589. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Ellis, M. Bots, R. K. Lindemann, J. E. Bolden, A. Newbold, L. A. Cluse, C. L. Scott, A. Strasser, P. Atadja, S. W. Lowe, et al. The histone deacetylase inhibitors LAQ824 and LBH589 do not require death receptor signaling or a functional apoptosome to mediate tumor cell death or therapeutic efficacy Blood, July 9, 2009; 114(2): 380 - 393. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. M. Prince, M. J. Bishton, and S. J. Harrison Clinical Studies of Histone Deacetylase Inhibitors Clin. Cancer Res., June 15, 2009; 15(12): 3958 - 3969. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. F. Whitecross, A. E. Alsop, L. A. Cluse, A. Wiegmans, K.-M. Banks, C. Coomans, M. J. Peart, A. Newbold, R. K. Lindemann, and R. W. Johnstone Defining the target specificity of ABT-737 and synergistic antitumor activities in combination with histone deacetylase inhibitors Blood, February 26, 2009; 113(9): 1982 - 1991. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. S. de Bono, R. Kristeleit, A. Tolcher, P. Fong, S. Pacey, V. Karavasilis, M. Mita, H. Shaw, P. Workman, S. Kaye, et al. Phase I Pharmacokinetic and Pharmacodynamic Study of LAQ824, a Hydroxamate Histone Deacetylase Inhibitor with a Heat Shock Protein-90 Inhibitory Profile, in Patients with Advanced Solid Tumors Clin. Cancer Res., October 15, 2008; 14(20): 6663 - 6673. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Sami, N. Hoti, H.-M. Xu, Z. Shen, and X. Huang Valproic Acid Inhibits the Growth of Cervical Cancer both In Vitro and In Vivo J. Biochem., September 1, 2008; 144(3): 357 - 362. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L.-P. Wu, X. Wang, L. Li, Y. Zhao, S. Lu, Y. Yu, W. Zhou, X. Liu, J. Yang, Z. Zheng, et al. Histone Deacetylase Inhibitor Depsipeptide Activates Silenced Genes through Decreasing both CpG and H3K9 Methylation on the Promoter Mol. Cell. Biol., May 15, 2008; 28(10): 3219 - 3235. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Newbold, R. K. Lindemann, L. A. Cluse, K. F. Whitecross, A. E. Dear, and R. W. Johnstone Characterisation of the novel apoptotic and therapeutic activities of the histone deacetylase inhibitor romidepsin Mol. Cancer Ther., May 1, 2008; 7(5): 1066 - 1079. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Herrmann, L. Grosse-Hovest, T. Otz, P. H. Krammer, H.-G. Rammensee, and G. Jung Construction of Optimized Bispecific Antibodies for Selective Activation of the Death Receptor CD95 Cancer Res., February 15, 2008; 68(4): 1221 - 1227. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. R. Fantin and V. M. Richon Mechanisms of Resistance to Histone Deacetylase Inhibitors and Their Therapeutic Implications Clin. Cancer Res., December 15, 2007; 13(24): 7237 - 7242. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Sonnemann, B. Bumbul, and J. F. Beck Synergistic activity of the histone deacetylase inhibitor suberoylanilide hydroxamic acid and the bisphosphonate zoledronic acid against prostate cancer cells in vitro Mol. Cancer Ther., November 1, 2007; 6(11): 2976 - 2984. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. K. Lindemann, A. Newbold, K. F. Whitecross, L. A. Cluse, A. J. Frew, L. Ellis, S. Williams, A. P. Wiegmans, A. E. Dear, C. L. Scott, et al. Analysis of the apoptotic and therapeutic activities of histone deacetylase inhibitors by using a mouse model of B cell lymphoma PNAS, May 8, 2007; 104(19): 8071 - 8076. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Baradari, A. Huether, M. Hopfner, D. Schuppan, and H. Scherubl Antiproliferative and proapoptotic effects of histone deacetylase inhibitors on gastrointestinal neuroendocrine tumor cells Endocr. Relat. Cancer, December 1, 2006; 13(4): 1237 - 1250. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.-L. Chen, J. Sung, M. Cohen, W. H. Chowdhury, M. D. Sachs, Y. Li, Y. Lakshmanan, B. Y. M. Yung, S. E. Lupold, and R. Rodriguez Valproic Acid Inhibits Invasiveness in Bladder Cancer but Not in Prostate Cancer Cells J. Pharmacol. Exp. Ther., November 1, 2006; 319(2): 533 - 542. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Xu, L. Ngo, G. Perez, M. Dokmanovic, and P. A. Marks Intrinsic apoptotic and thioredoxin pathways in human prostate cancer cell response to histone deacetylase inhibitor PNAS, October 17, 2006; 103(42): 15540 - 15545. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. V. Abramova, T. V. Pospelova, F. P. Nikulenkov, C. M. Hollander, A. J. Fornace Jr., and V. A. Pospelov G1/S Arrest Induced by Histone Deacetylase Inhibitor Sodium Butyrate in E1A + Ras-transformed Cells Is Mediated through Down-regulation of E2F Activity and Stabilization of beta-Catenin J. Biol. Chem., July 28, 2006; 281(30): 21040 - 21051. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q. Xia, J. Sung, W. Chowdhury, C.-l. Chen, N. Hoti, S. Shabbeer, M. Carducci, and R. Rodriguez Chronic Administration of Valproic Acid Inhibits Prostate Cancer Cell Growth In vitro and In vivo. Cancer Res., July 15, 2006; 66(14): 7237 - 7244. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. A. Cao, K. E. Bass, S. Balasubramanian, L. Liu, B. Schultz, E. Verner, Y. Dai, R. A. Molina, J. R. Davis, S. Misialek, et al. CRA-026440: a potent, broad-spectrum, hydroxamic histone deacetylase inhibitor with antiproliferative and antiangiogenic activity in vitro and in vivo. Mol. Cancer Ther., July 1, 2006; 5(7): 1693 - 1701. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. L. Piekarz, A. R. Frye, J. J. Wright, S. M. Steinberg, D. J. Liewehr, D. R. Rosing, V. Sachdev, T. Fojo, and S. E. Bates Cardiac Studies in Patients Treated with Depsipeptide, FK228, in a Phase II Trial for T-Cell Lymphoma. Clin. Cancer Res., June 15, 2006; 12(12): 3762 - 3773. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Maiso, X. Carvajal-Vergara, E. M. Ocio, R. Lopez-Perez, G. Mateo, N. Gutierrez, P. Atadja, A. Pandiella, and J. F. San Miguel The Histone Deacetylase Inhibitor LBH589 Is a Potent Antimyeloma Agent that Overcomes Drug Resistance Cancer Res., June 1, 2006; 66(11): 5781 - 5789. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. W. Johnstone The Intrinsic and Extrinsic Apoptotic Pathways in HDAC Inhibitor Lethality Am. Assoc. Cancer Res. Educ. Book, April 1, 2006; 2006(1): 313 - 317. [Full Text] [PDF]
|
|
|
|
|
|
R. W. Robey, Z. Zhan, R. L. Piekarz, G. L. Kayastha, T. Fojo, and S. E. Bates Increased MDR1 Expression in Normal and Malignant Peripheral Blood Mononuclear Cells Obtained from Patients Receiving Depsipeptide (FR901228, FK228, NSC630176) Clin. Cancer Res., March 1, 2006; 12(5): 1547 - 1555. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O. W. Rokhlin, R. B. Glover, N. V. Guseva, A. F. Taghiyev, K. G. Kohlgraf, and M. B. Cohen Mechanisms of Cell Death Induced by Histone Deacetylase Inhibitors in Androgen Receptor-Positive Prostate Cancer Cells Mol. Cancer Res., February 1, 2006; 4(2): 113 - 123. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Gao, M. Rahmani, X. Shi, P. Dent, and S. Grant Synergistic antileukemic interactions between 2-medroxyestradiol (2-ME) and histone deacetylase inhibitors involve Akt down-regulation and oxidative stress Blood, January 1, 2006; 107(1): 241 - 249. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. R. Acharya, A. Sparreboom, J. Venitz, and W. D. Figg Rational Development of Histone Deacetylase Inhibitors as Anticancer Agents: A Review Mol. Pharmacol., October 1, 2005; 68(4): 917 - 932. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W.-S. Xu, G. Perez, L. Ngo, C.-Y. Gui, and P. A. Marks Induction of Polyploidy by Histone Deacetylase Inhibitor: A Pathway for Antitumor Effects Cancer Res., September 1, 2005; 65(17): 7832 - 7839. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. N. Bhalla Epigenetic and Chromatin Modifiers As Targeted Therapy of Hematologic Malignancies J. Clin. Oncol., June 10, 2005; 23(17): 3971 - 3993. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. A. Strait, C. T. Warnick, C. D. Ford, B. Dabbas, E. H. Hammond, and S. J. Ilstrup Histone deacetylase inhibitors induce G2-checkpoint arrest and apoptosis in cisplatinum-resistant ovarian cancer cells associated with overexpression of the Bcl-2-related protein Bad Mol. Cancer Ther., April 1, 2005; 4(4): 603 - 611. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. Peart, G. K. Smyth, R. K. van Laar, D. D. Bowtell, V. M. Richon, P. A. Marks, A. J. Holloway, and R. W. Johnstone Identification and functional significance of genes regulated by structurally different histone deacetylase inhibitors PNAS, March 8, 2005; 102(10): 3697 - 3702. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. S. Ungerstedt, Y. Sowa, W.-S. Xu, Y. Shao, M. Dokmanovic, G. Perez, L. Ngo, A. Holmgren, X. Jiang, and P. A. Marks Role of thioredoxin in the response of normal and transformed cells to histone deacetylase inhibitors PNAS, January 18, 2005; 102(3): 673 - 678. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Doi, H. Soda, M. Oka, J. Tsurutani, T. Kitazaki, Y. Nakamura, M. Fukuda, Y. Yamada, S. Kamihira, and S. Kohno The histone deacetylase inhibitor FR901228 induces caspase-dependent apoptosis via the mitochondrial pathway in small cell lung cancer cells Mol. Cancer Ther., November 1, 2004; 3(11): 1397 - 1402. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Bahar, D. J. Simpson, S. J. Cutty, J. E. Bicknell, P. R. Hoban, S. Holley, M. Mourtada-Maarabouni, G. T. Williams, R. N. Clayton, and W. E. Farrell Isolation and Characterization of a Novel Pituitary Tumor Apoptosis Gene Mol. Endocrinol., July 1, 2004; 18(7): 1827 - 1839. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Geller, I. Petak, K. S. Szucs, K. Nagy, D. M. Tillman, and J. A. Houghton Interferon-{gamma}-Induced Sensitization of Colon Carcinomas to ZD9331 Targets Caspases, Downstream of Fas, Independent of Mitochondrial Signaling and the Inhibitor of Apoptosis Survivin Clin. Cancer Res., December 15, 2003; 9(17): 6504 - 6515. [Abstract] [Full Text] [PDF]
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