Histone Deacetylase Inhibitor Potentiates Anticancer Effect of ...

Histone Deacetylase Inhibitor Potentiates Anticancer Effect of Docetaxel via Modulation of Bcl-2 Family Proteins and Tubulin in Hormone Refractory Prostate Cancer Cells Jung Jin Hwang, Yong Sook Kim, Mi Joung Kim, Dong Eun Kim, In Gab Jeong and Choung-Soo Kim* From the Institute for Innovative Cancer Research (JJH, YSK, MJK, DEK) and Department of Urology (IGJ), University of Ulsan College of Medicine, Asan Medical Center (CSK), Seoul, Korea

Purpose: We evaluated the antitumor effects of docetaxel (Sigma®) and histone deacetylase inhibitors in hormone refractory prostate cancer cells, and analyzed the mechanism by which combination treatment induced cell death. Materials and Methods: We used LNCaP, DU145 and PC3 cells (ATCC®) to evaluate the in vitro apoptotic effects of histone deacetylase inhibitors and their combinations with docetaxel as well as the molecular mechanisms. The DU145 xenograft model was used to evaluate the in vivo efficacy of PXD101 combined with docetaxel. Results: Suberoylanilide hydroxamic acid or PXD101 inhibited the growth of hormone dependent LNCaP cells, and hormone independent DU145 and PC3 cells. It increased sub-G1 population and activated caspase-8, 9 and 3, indicating apoptosis induction. Pretreating DU145 cells with docetaxel followed by histone deacetylase inhibitors showed significant synergistic cytotoxicity compared with that of simultaneous co-treatment or reverse sequential treatment. Pretreatment with docetaxel followed by histone deacetylase inhibitors increased the apoptotic sub-G1 population, caspase activation and tubulin acetylation compared with that of docetaxel alone. Combination treatment decreased Mcl-1 and Bcl-xl, and increased t-Bid, Bik and Bim. Combined docetaxel and PXD101 reduced tumor size with efficacy equivalent to that of a double dose of docetaxel alone in the DU145 xenograft model. Conclusions: These preclinical results indicate that the sequential combination of docetaxel and histone deacetylase inhibitors led to a synergistic increase in the death of hormone refractory prostate cancer cells via intrinsic and extrinsic apoptotic pathways by modulating Bcl-2 family proteins and tubulin in vitro and in vivo. Results suggest that this combination may be a new therapeutic modality in patients with hormone refractory prostate cancer.

Abbreviations and Acronyms CI ⫽ combination index HDAC ⫽ histone deacetylase HDACI ⫽ HDAC inhibitor HRPC ⫽ hormone refractory prostate cancer MTS ⫽ 3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2(4-sulfophenyl)-2H-tetrazolium SAHA ⫽ suberoylanilide hydroxamic acid v/v ⫽ volume per volume Submitted for publication March 23, 2010. Study received institutional animal care and use committee approval. Supported by Grant A062254 from the Korea Health 21 R & D Project, Ministry of Health, Welfare and Family Affairs, and a grant (2009-450) from the Asan Institute for Life Sciences, Republic of Korea. * Correspondence: Department of Urology, Asan Medical Center, 388-1 Pungnap 2 dong, Songpa-gu, Seoul 138-736, Korea (telephone: 82-23010-3734; FAX: 82-2-477-8928; e-mail: cskim@ amc.seoul.kr).

Key Words: prostate, prostatic neoplasms, histone deacetylases, docetaxel, apoptosis PROSTATE cancer is the most common nonskin cancer and the second most common cause of cancer death in men in the United States with an estimated 186,320 newly diagnosed patients and 27,360 prostate cancer deaths in 2008.1

Although docetaxel based regimens have palliative and survival benefits, men with metastatic HRPC have only 16 to 18-month median survival.2,3 Thus, new therapeutic modalities aimed to improve man-

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Vol. 184, 2557-2564, December 2010 Printed in U.S.A. DOI:10.1016/j.juro.2010.07.035

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HISTONE DEACETYLASE INHIBITOR POTENTIATES DOCETAXEL IN PROSTATE CANCER

agement for advanced HRPC include docetaxel plus other agents.4 The importance of epigenetic changes in cancer cells led to the development of agents that modify histones via acetylation, methylation, phosphorylation, ubiquitination or adenosine triphosphate ribosylation, or that methylate DNA. Of these modifications the acetylation of lysine residues at the histone N-terminal tail decreases the affinity of histone for DNA, resulting in the expression of genes related to tumor suppression and/or differentiation.5 Thus, HDACIs show potent activity against many cancers.6 Five HDACI classes have been characterized, including the hydroxamic acids SAHA, PXD101, LAQ-824, LBH 589 and trichostatin A, the shortchain fatty acids 4-phenylbutyrate, pivaloyloxymethyl butyrate and valproic acid, the cyclic tetrapeptides trapoxin, apicidin and depsipeptide (FK 228), and benzamide (MS-275).7–10 Many reports show the usefulness of various HDACIs for prostate cancer.11 Cell death mechanisms of HDACIs include induction of the cyclin dependent kinase inhibitors p21 and p27, apoptotic Bcl-2 family proteins, death receptors, death ligands and retinoic acid receptor.8,12 HDACI also acetylates many nonhistone proteins, including tubulin, heat shock proteins and Ku70, disrupting protein function and resulting in cell death.13–15 Although it was reported that SAHA, valproic acid, MS-275, PXD101 and FR235222 decreased prostate specific antigen, and increased caspase activation, and p21 and annexin A1 expression in a prostate cancer cell line,11,16 –19 the precise mechanism of HDACIs must be further elucidated. The merit of HDACIs is that cytotoxic effects are specific to cancer cells and not to normal cells or tissues. HDACIs are well tolerated with a good toxicity profile compared to that of other anticancer agents.7 Many clinical trials have been done or are under way using HDACIs combined with other chemotherapy, for example the taxane paclitaxel or docetaxel combined with the HDACI pivaloyloxymethyl butyrate, LBH589, SAHA or PXD101 for solid tumors, including prostate, breast and lung cancer.9,20 –22 However, the exact mechanism of action is not fully understood. We determined whether HDACIs would enhance the effects of docetaxel in advanced prostate cancer cells. We also assessed the mechanism of cell death. Thus, we evaluated the growth inhibitory effects of HDACI alone in hormone dependent and independent prostate cancer cells. Finally, we determined the antitumor effect of docetaxel and HDACI in HRPC DU145 cells in vitro and in vivo, and analyzed the mechanism by which combination treatment induced cell death.

MATERIALS AND METHODS Cell Culture and Drug Treatment We cultured the human prostate cancer cell lines LNCaP, DU145 and PC3 in RPMI 1640 medium containing 100 U/ml penicillin, 100 ␮g/ml streptomycin and 10% (v/v) fetal bovine serum (Invitrogen™). The HDACIs SAHA and PXD101 were synthesized elsewhere. We treated cells with SAHA, PXD101 or docetaxel in 5% (v/v) RPMI 1640 medium containing fetal bovine serum. For sequential combination treatment with HDACI and docetaxel cells were exposed to the former drug for 24 hours and then to the next drug for another 48 hours.

Cell Survival and CI Analysis Cells were cultured in 96-well plates and treated with various drug concentrations for the indicated times. We measured viability with the CellTiter 96® Aqueous One Solution Cell proliferation assay (MTS assay). MTS reagent was added to each well according to manufacturer instructions. After 2-hour incubation we determined cell viability by measuring absorbance at 490 nm. We calculated the CI with CalcuSyn (Biosoft®), which is based on the Chou and Talalay median effect principle. The isobologram is a graphic representation of the interaction between 2 drugs that is formed by plotting the individual drug doses required to achieve a single agent effect on the respective x and y-axes. A line connecting the 2 points is drawn and the concentration of the 2 drugs used in combination to achieve the same effect is plotted on the isobologram. Combination data points that fall on the line represent an additive interaction while points above and below represent antagonism and synergy, respectively. Similar to the isobologram, CI analysis provides qualitative information on the drug interaction. A numerical CI value is calculated based on the equation, CI ⫽ (D)1/(Dx)1 ⫹ (D)2/(Dx)2 ⫹ (D)1(D)2/(Dx)1(Dx)2, where (D)1 and (D)2 are the doses of drugs 1 and 2 with a certain percent effect when used in combination, and (Dx)1 and (Dx)2 are the doses of drugs 1 and 2, respectively, with the same certain percent effect when used alone. CI greater than 1 indicates antagonism, 1 indicates an additive effect and less than 1 indicates synergy.

Cell Cycle Analysis Cells were treated with drugs, fixed with 70% (v/v) ethanol and stained with 60 ␮g/ml propidium iodide (Sigma) containing 10 U/ml ribonuclease A for 30 minutes. We measured the percent of 10,000 cells in the different cell cycle phases using the FACSCalibur™ flow cytometer built-in ModFit LT™ 3.0 software.

Western Blot Equal amounts of protein were electrophoresed in sodium dodecyl sulfate-polyacrylamide gel and transferred to Millipore polyvinylidene difluoride membranes (Chemicon®). After blocking with 3% (weight per volume) nonfat dry milk the membranes were incubated with various primary antibodies (1:1,000) in blocking solution overnight at 4C. Appropriate secondary antibodies (1:5,000) conjugated to horseradish peroxidase (Pierce, Rockford, Illinois) were incubated for 1 hour at room temperature. Immobilon™ Western ECL solution and Image Station 4000MM


(Kodak, Rochester, New York) were used to visualize immunoreactive bands. We used antibodies to acetylated histone 3, histone 3, caspase-3, 8 and 9, Bid, Bik, tubulin, p21 (Cell Signaling Technology®), actin, acetylated tubulin (Sigma), Bim, Mcl-1 and Bcl-xl (Santa Cruz Biotechnology, Santa Cruz, California).

Xenograft Animal Model Four-week-old male BALB/C nude mice (OrientBio, Seoul, Korea) were subcutaneously inoculated with 5 ⫻ 106 DU145 cells. Mice bearing tumors with a volume of about 100 mm3 intraperitoneally received docetaxel (10 or 5 mg/kg for 1 day per week at 9:00 a.m.) and PXD101 (30 mg/kg for 5 days per week at 6:00 p.m.) for 3 weeks. Tumor volume was measured twice weekly and calculated using the formula, tumor volume in mm3 ⫽ 1/2 (␻1 x ␻22), where ␻1 and ␻2 represent the larger and smaller tumor diameters, respectively.

Statistical Analysis All data are shown as the mean ⫾ SD. Statistical significance was considered at p ⬍0.05 and determined by 1-way ANOVA.

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RESULTS HDAC Inhibitors Prostate cancer cell growth inhibition. Incubating hormone dependent LNCaP prostate cancer cells, and DU145 and PC3 HRPC cells with 0.04, 0.2, 1, 5, 25 or 100 ␮M SAHA or PXD101 for 48 hours decreased cell viability in a dose dependent manner (fig. 1, A). PXD101 was more potent than SAHA in all preparations. DU145 cells were as sensitive as LNCaP and more sensitive than PC3 cells (fig. 1, A). The half maximum inhibitory concentration of SAHA and PXD101 was 2.80 and 0.75 ␮M in LNCaP cells, 2.50 and 0.70 ␮M in DU145 cells, and 6.60 and 1.20 ␮M in PC3 cells, respectively. These results indicate that hormone dependent and independent prostate cancer cells are sensitive to HDACIs. To assess the effects of these 2 HDACIs on intracellular HDAC activity we analyzed histone 3 and tubulin acetylation by Western blot in the 3 cell lines

Figure 1. Cytotoxicity and activity of HDACIs SAHA and PXD101 in LNCaP, DU145 and PC3 cell lines. A, percent viability of 4 preparations of each cell type exposed to 0.04, 0.2, 1, 5, 25 and 100 ␮M SAHA or PXD101 for 48 hours. B, histone 3 acetylation (Ac-H3) was increased by HDACIs in cells exposed to 1 or 10 ␮M SAHA or PXD101. h, hours. C, HDACIs increased acetylated tubulin (Ac-tubulin) in cells on 15% gel. Conc, concentration.

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exposed to 1 or 10 ␮M SAHA or PXD101 for the indicated times (fig. 1, B and C). SAHA and PXD101 increased histone 3 and tubulin acetylation in a time and dose dependent manner. The histone 3 or tubulin level was not changed by HDACIs. Effect on cell cycle and HRPC cell apoptotic signaling. To assess the effects of these HDACIs on the cell cycle we analyzed DNA contents by flow cytometry. We observed an increase in the hypodiploid population (sub-G1) of LNCaP, DU145 and PC3 cells treated with 10 ␮M SAHA or PXD101 for 48 hours (fig. 2, A and B). Exposure of these cells to 5 ␮M SAHA or PXD101 for 72 hours resulted in activation of caspase-8, an enzyme with a role in the extrinsic apoptotic pathway (fig. 2, C), and the activation of caspase-9 and 3, which mediate intrinsic apoptosis. Induction of DU145 cell apoptosis with docetaxel. Since HDACI had an antiproliferative effect on HRPC cells, we tested the effect of HDACIs on docetaxel toxicity in DU145 cells, which are sensitive to HDACIs. Although treatment with 1.5 nM docetaxel, 1.5 ␮M SAHA or 0.5 ␮M PXD101 alone resulted in modest toxicity (mean ⫾ SD 69.3% ⫾ 3.5%, 65.6% ⫾ 7.7% and 73% ⫾ 2.7% viability, respectively), pretreatment with 1.5 nM docetaxel followed by 1.5 ␮M SAHA or 0.5 ␮M PXD101 increased toxicity significantly (35.8% ⫾ 8.0% and 35.0% ⫾ 3.6% viability, respectively, fig. 3, A). However, administering HDACIs followed by docetaxel (HDACIs ¡docetaxel) had no effect on cell death while simultaneous treatment with HDACIs and docetaxel was less effective than docetaxel followed by HDACIs (fig. 3, B and C). To establish whether the combined effects of docetaxel, and the HDACIs SAHA and PXD101 were synergistic we exposed DU145 cells to the drugs while keeping a constant ratio of each drug to the other. Growth inhibition was then measured by MTS assay (fig. 3, D and G). Cell growth was mark-

edly inhibited by docetaxel and HDACI applied in combination compared with that of each drug alone. Analysis of the dose effect relationship and isobolograms of these results revealed that sequential treatment with a high concentration of docetaxel and SAHA (docetaxel ¡SAHA) was synergistic (fig. 3, E and F). The docetaxel and PXD101 combination (docetaxel ¡PXD101) similarly showed synergy except for the highest concentration of the combination on isobologram (fig. 3, H and I). To investigate apoptosis induced by combination treatment we performed flow cytometry of DU145 cells sequentially exposed to docetaxel and HDACIs. Histograms showed that combination treatment increased the sub-G1 population compared with cells exposed to a single agent, that is docetaxel, SAHA or PXD101 (fig. 3, J and K). Combination Treatment Apoptosis inducing mechanisms. Since pan-HDACIs and docetaxel increase in tubulin acetylation, we examined the accumulation of tubulin acetylation in DU145 cells using combination treatment (fig. 4, A). Although 1.5 nM docetaxel, 1.5 ␮M SAHA or 0.5 ␮M PXD101 slightly increased tubulin acetylation, combined docetaxel/HDACIs increased it strongly. On the other hand, adding docetaxel had no effect on the level of acetylation on histone 3 due to HDACIs. Since combination treatment also significantly increased activated caspase-8, 9 and 3, we analyzed the level of proteins that regulate cell survival and death (fig. 4, B and C). Combination treatment decreased levels of the anti-apoptotic Bcl-2 family proteins (Mcl-1 and Bcl-xl) and increased levels of pro-apoptotic Bcl-2 family proteins (t-Bid, Bik and Bim). This indicates that combined docetaxel and HDACI treatment (docetaxel¡HDACIs) increased apoptosis by modulating Bcl-2 family protein expression. The cell cycle inhibitor p21 protein was accumulated by combination treatment, as reported previously.8

Figure 2. Apoptosis induction by SAHA or PXD101 in LNCaP, DU145 and PC3 cells. A, flow cytometry profiles show that HDACI increased sub-G1 apoptotic population of cells treated with 10 ␮M SAHA or PXD101 for 48 hours. B, sub-G1 population of 3 cell lines in 3 preparations each. PXD, PXD101. Asterisk indicates p ⬍0.05 vs control. C, Western blots of caspase-8, 9 and 3 in cells treated with 5 ␮M SAHA or PXD101 for 72 hours on 15% gel. CTL, control.


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Figure 3. Cytotoxicity of combined HDACI and docetaxel treatment in DU145 HRPC cells. A, 6 preparations each of cells were incubated with 1.5 nM docetaxel (DTX) for 24 hours and then with or without 1.5 ␮M SAHA, or 0.5 ␮M PXD101 for 48 hours (DTX ¡ HDACI). HDACI posttreatment significantly potentiated docetaxel induced cell death. B, 6 preparations each of cells were incubated with 1.5 ␮M SAHA or 0.5 ␮M PXD101 for 24 hours and then with or without 1.5 nM docetaxel for 48 hours (HDACI ¡ DTX). C, 6 preparations each of cells were exposed to HDACI, that is 1.5 ␮M SAHA or 0.5 ␮M PXD101, and 1.5 nM docetaxel simultaneously for 48 hours. D, viability curves in 3 cell preparations each of single agent docetaxel and SAHA, and sequential combinations in cells treated with docetaxel and SAHA at 1:1,000 ratio. E, CI plot of combined docetaxel and SAHA (D). F, isobologram shows combined docetaxel and SAHA (D). G, viability curves of single agent docetaxel and PXD101, and sequential combinations in 3 preparations each of cells treated with docetaxel and PXD101 at 1:333 ratio. H, CI plot of the combined docetaxel and PXD101 (G). I, isobologram shows combined docetaxel and PXD101 (G). J, flow cytometry profiles reveal that sub-G1 apoptotic population was greater after sequential treatment with 1.5 nM docetaxel followed by 1.5 ␮M SAHA or 0.5 ␮M PXD101. CTL, control. K, bars represent sub-G1 population of sequentially (DTX ¡ HDACI) treated cells in 3 preparations each.

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Figure 4. Western blot shows effect of sequential combination treatment with docetaxel followed by HDACI on histone acetylation, caspase activation and Bcl-2 family protein expression in DU145 cells. A, acetylated histone 3 (Ac-H3) and acetylated tubulin (Ac-Tubulin) from cells incubated with 1.5 nM docetaxel for 24 hour and then next with or without 1.5 ␮M SAHA or 0.5 ␮M PXD101 for 48 hours on 15% gel. B, for caspase-8, 9 and 3 from cells treated with sequential combinations on 15% gel. C, Bcl-2 family proteins from cells treated with sequential combination on 13% gel. Minuses indicate negative. Plus signs indicate positive.

Effect on DU145 xenografts. To confirm the effects of docetaxel and HDACI on HRPC cells in vivo we subcutaneously injected nude mice with 5 ⫻ 106 DU145 cells, maintained them until the tumor size reached 100 mm3 and then injected them intraperitoneally with docetaxel (10 mg/kg per week) and/or PXD101 (30 mg/kg per day) for 3 weeks. Combined treatment reduced tumor volume compared with that in control mice or mice treated with docetaxel or PXD101 alone but combined treatment had no effect on body weight (fig. 5, A and B). To determine whether PXD101 decreased the required dose of docetaxel we treated tumor bearing mice with 5 mg/kg per week docetaxel and/or 30 mg/kg per day PXD101 for 3 weeks and then calculated tumor size (fig. 5, C). Injecting 5 mg/kg per week docetaxel plus 30 mg/kg per day PXD101 re-

sulted in a mean tumor volume of 64% ⫾ 0.2% that in saline injected controls, similar to the 64% ⫾ 0.3% tumor volume in mice treated with 10 mg/kg per week docetaxel. These results suggest that when combined, PXD101 may allow a dose reduction of docetaxel, thus decreasing its side effects.

DISCUSSION The finding that HDAC activity is increased in tumors, including prostate cancer, led to the development of HDACIs as antitumor agents.11,23 Many HDACIs are in phase I/II clinical trials of treatment for hematopoietic cancer and solid tumors6 but to date only SAHA has been approved by the Food and Drug Administration in the United States as treatment for cutaneous T-cell lymphoma only. We eval-

Figure 5. Growth inhibitory effect of combined HDACI and docetaxel (DTX) in DU145 xenografts in mice. A, tumor volume changes after 3-week treatment with 10 mg/kg per week docetaxel and/or 30 mg/kg per day PXD101 (PXD) for 5 days per week. Asterisk indicates p ⬍0.05 vs control, PXD101 alone and docetaxel alone. B, total body weight in 3 treatment groups (A). C, tumor volume percent reduction after treatment with 5 or 10 mg/kg per week docetaxel combined with 30 mg/kg per day PXD101 for 3 weeks. Minuses indicate negative. Plus signs indicate positive. Asterisk indicates p ⬍0.05.


uated the antitumor effects of HDACIs on hormone dependent (LNCaP) and hormone independent (DU145 and PC3) prostate cancer cells. The panHDACIs SAHA and PXD101 inhibited the growth of hormone dependent and hormone independent cells in a dose dependent manner (fig. 1, A). Cell cycle analysis and Western blotting for caspase revealed that HDACIs induced prostate cancer cell apoptosis (fig. 2). These results suggest that pan-HDACIs may be used for hormone dependent and independent prostate cancer. Since HDACIs are thought to act by potentiating the antitumor effects of chemotherapeutic agents and/or radiation,20,24 –26 and they showed cytotoxicity as monotreatment in HRPC cells in our study, we tested whether SAHA and PXD101 could potentiate the activity of docetaxel, an agent widely used for HRPC. Using DU145 cells, the cell line most sensitive to HDACIs of the 3 lines tested, we found that sequential treatment with docetaxel followed by HDACI (docetaxel ¡SAHA or PXD101) resulted in the greatest growth inhibition (fig. 3, A). Although HDACI cytotoxicity was lower in PC3 than in DU145 cells, docetaxel ¡HDACI combination treatment had a potent antiproliferative effect in PC3 cells (data not shown). Also, median effect analysis and isobolograms indicated that the docetaxel and HDACI combination had synergy (fig. 3, D to I). These results suggest that sequential treatment with docetaxel followed by SAHA or PXD101 may be a useful therapy for HRPC. However, further study is needed to elucidate the influence of the combination schedule on the response. Docetaxel stabilizes polymerized tubulin and increased tubulin acetylation is associated with such stabilization.27 Also, HDACIs enhance tubulin acetylation by inhibiting HDAC 6, an enzyme that acetylates cytosolic nonhistone proteins.7 In accordance with these reports we found that the docetaxel and HDACI combination increased the acetylated tubulin level compared with that using either reagent alone (fig. 4, A). To elucidate other mechanisms of

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cell death we analyzed caspase activation and Bcl-2 family protein levels. Western blotting for caspase showed that combination treatment activated extrinsic and intrinsic apoptosis pathways (fig. 4, B). Moreover, combination treatment increased the proapoptotic proteins t-Bid, Bik and Bim, and decreased the anti-apoptotic proteins Mcl-1 and Bcl-xl (fig. 4, C). Depsipeptide (FK228) potentiated docetaxel toxicity in HRPC cells, as shown by p21 expression assays.28,29 We have extended these findings by noting that SAHA or PXD101 potentiated docetaxel toxicity, and using tubulin and Bcl-2 family proteins as biomarkers in prostate cancer cell lines. Since all of our in vitro experiments indicated that combination treatment with docetaxel and HDACIs has an antitumor effect on HRPC cells by inducing apoptosis, we tested the effects of combining docetaxel and PXD101 on tumor growth in nude mice injected with DU145 cells. Because it was reported that combining SAHA plus docetaxel is poorly tolerated in HRPC cases30 and PXD101 is not a substrate of the multidrug resistance gene, we chose the combination of PXD101 plus docetaxel. Our results show that this combination was also effective in vivo against HRPC cells (fig. 5, A).

CONCLUSIONS Results indicate that HDACI can be used to treat patients with hormone independent and hormone dependent prostate cancer. Moreover, a sequential combination of docetaxel and HDACIs resulted in a synergistic increase in the antiproliferative effects of either drug given alone in DU145 HRPC cells in vitro and in vivo. These preclinical findings support the clinical evaluation of HDACIs combined with docetaxel for HRPC.

ACKNOWLEDGMENTS SAHA and PXD101 were synthesized at Crystal Genomics, Seoul, Korea.

REFERENCES 1. Jemal A, Siegel R, Ward E et al: Cancer statistics, 2009. CA Cancer J Clin 2009; 59: 225. 2. Petrylak DP, Tangen CM, Hussain MH et al: Docetaxel and estramustine compared with mitoxantrone and prednisone for advanced refractory prostate cancer. N Engl J Med 2004; 351: 1513. 3. Tannock IF, de Wit R, Berry WR et al: Docetaxel plus prednisone or mitoxantrone plus prednisone for advanced prostate cancer. N Engl J Med 2004; 351: 1502.

4. Galsky MD and Vogelzang NJ: Docetaxel-based combination therapy for castration-resistant prostate cancer. Ann Oncol 2010; Epub ahead of print. 5. Johnstone RW and Licht JD: Histone deacetylase inhibitors in cancer therapy: is transcription the primary target? Cancer Cell 2003; 4: 13. 6. Acharya MR, Sparreboom A, Venitz J et al: Rational development of histone deacetylase inhibitors as anticancer agents: a review. Mol Pharmacol 2005; 68: 917.

7. Minucci S and Pelicci PG: Histone deacetylase inhibitors and the promise of epigenetic (and more) treatments for cancer. Nat Rev Cancer 2006; 6: 38. 8. Xu WS, Parmigiani RB and Marks PA: Histone deacetylase inhibitors: molecular mechanisms of action. Oncogene 2007; 26: 5541. 9. Glaser KB: HDAC inhibitors: clinical update and mechanism-based potential. Biochem Pharmacol 2007; 74: 659.

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10. Mai A, Massa S, Rotili D et al: Histone deacetylation in epigenetics: an attractive target for anticancer therapy. Med Res Rev 2005; 25: 261.

17. Qian DZ, Wei YF, Wang X et al: Antitumor activity of the histone deacetylase inhibitor MS-275 in prostate cancer models. Prostate 2007; 67: 1182.

11. Abbas A and Gupta S: The role of histone deacetylases in prostate cancer. Epigenetics 2008; 3: 300.

18. Qian X, Ara G, Mills E et al: Activity of the histone deacetylase inhibitor belinostat (PXD101) in preclinical models of prostate cancer. Int J Cancer 2008; 122: 1400.

12. Insinga A, Monestiroli S, Ronzoni S et al: Inhibitors of histone deacetylases induce tumor-selective apoptosis through activation of the death receptor pathway. Nat Med 2005; 11: 71.

19. D’Acunto CW, Fontanella B, Rodriquez M et al: Histone deacetylase inhibitor FR235222 sensitizes human prostate adenocarcinoma cells to apoptosis through up-regulation of Annexin A1. Cancer Lett 2010; 295: 85.

13. Haggarty SJ, Koeller KM, Wong JC et al: Domain-selective small-molecule inhibitor of histone deacetylase 6 (HDAC6)-mediated tubulin deacetylation. Proc Natl Acad Sci U S A 2003; 100: 4389. 14. Bali P, Pranpat M, Bradner J et al: Inhibition of histone deacetylase 6 acetylates and disrupts the chaperone function of heat shock protein 90: a novel basis for antileukemia activity of histone deacetylase inhibitors. J Biol Chem 2005; 280: 26729. 15. Subramanian C, Opipari AW Jr, Bian X et al: Ku70 acetylation mediates neuroblastoma cell death induced by histone deacetylase inhibitors. Proc Natl Acad Sci U S A 2005; 102: 4842. 16. Thelen P, Schweyer S, Hemmerlein B et al: Expressional changes after histone deacetylase inhibition by valproic acid in LNCaP human prostate cancer cells. Int J Oncol 2004; 24: 25.

20. Rathkopf D, Wong BY, Ross RW et al: A phase I study of oral panobinostat alone and in combination with docetaxel in patients with castrationresistant prostate cancer. Cancer Chemother Pharmacol 2010; 66: 181. 21. Daugaard G, Fizazi K, Huebner G et al: An openlabel randomized phase II trial of belinostat (PXD101) in combination with carboplatin and paclitaxel (BelCaP) compared to carboplatin and paclitaxel in patients with previously untreated carcinoma of unknown primary. J Clin Oncol, suppl., 2010; 28: 15s, abstract TPS185. 22. Reid T, Thorne S, Hedjran F et al: Impact of the histone deacetylase inhibitors on taxotere resistance due to P-gp (MDR) and tubulin acetylation. Proc Amer Assoc Cancer Res 2005; 46. 23. Di Croce L, Buschbeck M, Gutierrez A et al: Altered epigenetic signals in human disease. Cancer Biol Ther 2004; 3: 831.

24. Di Lorenzo G, Figg WD, Fossa SD et al: Combination of bevacizumab and docetaxel in docetaxel-pretreated hormone-refractory prostate cancer: a phase 2 study. Eur Urol 2008; 54: 1089. 25. Kim MS, Blake M, Baek JH et al: Inhibition of histone deacetylase increases cytotoxicity to anticancer drugs targeting DNA. Cancer Res 2003; 63: 7291. 26. Chen Y, Sawyers CL and Scher HI: Targeting the androgen receptor pathway in prostate cancer. Curr Opin Pharmacol 2008; 8: 440. 27. Piperno G, LeDizet M and Chang XJ: Microtubules containing acetylated alpha-tubulin in mammalian cells in culture. J Cell Biol 1987; 104: 289. 28. Zhang Z, Stanfield J, Frenkel E et al: Enhanced therapeutic effect on androgen-independent prostate cancer by depsipeptide (FK228), a histone deacetylase inhibitor, in combination with docetaxel. Urology 2007; 70: 396. 29. Kanzaki M, Kakinuma H, Kumazawa T et al: Low concentrations of the histone deacetylase inhibitor, depsipeptide, enhance the effects of gemcitabine and docetaxel in hormone refractory prostate cancer cells. Oncol Rep 2007; 17: 761. 30. Schneider BJ, Bradley D, Smith DC et al: Phase I study of vorinostat plus docetaxel in patients with solid tumor malignancies. J Clin Oncol, suppl., 2009; 27: 15s, abstract 2528.