Development of a histone deacetylase 6 inhibitorand its biological effectsJu-Hee Leea, Adaickapillai Mahendranb, Yuanshan Yaob, Lang Ngoa, Gisela Venta-Pereza, Megan L. Choya,Nathaniel Kimb, Won-Seok Hamb, Ronald Breslowb,1, and Paul A. Marksa,1

aDepartment of Cell Biology, Sloan–Kettering Institute, Memorial Sloan–Kettering Cancer Center, New York, NY 10065; and bDepartment of Chemistry,Columbia University, New York, NY 10027

Contributed by Paul A. Marks, August 1, 2013 (sent for review June 13, 2013)

Development of isoform-selective histone deacetylase (HDAC) inhib-itors is important in elucidating the function of individual HDACenzymes and their potential as therapeutic agents. Among theeleven zinc-dependent HDACs in humans, HDAC6 is structurally andfunctionally unique. Here, we show that a hydroxamic acid-basedsmall-molecule N-hydroxy-4-(2-[(2-hydroxyethyl)(phenyl)amino]-2-oxoethyl)benzamide (HPOB) selectively inhibits HDAC6 catalyticactivity in vivo and in vitro. HPOB causes growth inhibition of nor-mal and transformed cells but does not induce cell death. HPOBenhances the effectiveness of DNA-damaging anticancer drugs intransformed cells but not normal cells. HPOB does not block theubiquitin-binding activity of HDAC6. The HDAC6-selective inhibitorHPOB has therapeutic potential in combination therapy to enhancethe potency of anticancer drugs.

anticancer agents | epigenetics-based chemotherapy | drug discovery

Histone deacetylase 6 (HDAC6) is unique among the elevenzinc-dependent HDACs in humans. HDAC6 is located in the

cytoplasm, and it has two catalytic domains and an ubiquitin-binding domain at the C-terminal region (1–3). This study fo-cused on the development of a HDAC6-selective inhibitor and itsbiological effects. The substrates of HDAC6 include nonhistoneproteins such as α-tubulin, peroxiredoxin (PRX), cortactin, andheat shock protein 90 (Hsp90) but not histones (4–7). HDAC6plays a key role in the regulation ofmicrotubule dynamics includingcell migration and cell–cell interactions. The reversible acetylationof Hsp90, a substrate of HDAC6, modulates its chaperone activityand, accordingly, the stability of survival and antiapoptotic factors,including epidermal growth factor receptor (EGFR), protein ki-nase AKT, proto-oncogene C-RAF, survivin, and other factors.HDAC6, through its ubiquitin-binding activity and interactionwith other partner proteins, plays a role in the degradation ofmisfolded proteins by binding polyubiquitinated proteins anddelivering them to the dynein and motor proteins for transportinto aggresomes which are degraded by lysosomes (8–10). Thus,HDAC6 has multiple biological functions through deacetylase-dependent and -independent mechanisms modulating many cel-lular pathways relevant to normal and tumor cell growth, mi-gration, and death. HDAC6 is an attractive target for potentialcancer treatment.There are several previous reports on the development of

HDAC6-selective inhibitors (11–15). Themost extensively studiedis tubacin (16, 17). Tubacin has non–drug-like qualities, high lipo-philicity, and difficult synthesis and has proved to be more usefulas a research tool rather than as a potential drug (18). We andothers (12–15, 19) have developed HDAC6-selective inhibitorswhose pharmacokinetics, toxicity, and efficacy make them poten-tially more useful than tubacin as therapeutic agents. ACY-1215,2-(Diphenylamino)-N-(7-(hydroxyamino)-7-oxoheptyl)pyrimidine-5-carboxamide, a HDAC6-selective inhibitor, is currently beingevaluated in clinical trials (http://clinicaltrials.gov).HDAC inhibitors, such as suberoylanilide hydroxamic acid

(SAHA), consist of three structural domains: a metal-bindingdomain, a linker domain, and a surface domain (20). The catalytic

pocket of HDAC1 is deeper and narrower than the catalyticpocket of HDAC6 (14). To develop HDAC6-selective inhib-itors, we synthesized small molecules with bulkier and shorterlinker domains than the pan-HDAC inhibitor SAHA (20,21). A hydroxamic acid-based small-molecule N-hydroxy-4-(2-[(2-hydroxyethyl)(phenyl)amino]-2-oxoethyl)benzamide (HPOB)was synthesized that selectively inhibits HDAC6. We report theeffects of this HDAC6-selective inhibitor on normal and trans-formed cells. Further, we found that selective inhibition of HDAC6increases the effectiveness of anticancer agents, etoposide, doxo-rubicin, and SAHA in inducing cell death of transformed cells butnot normal cells.

ResultsSynthesis of the HDAC6-Selective Inhibitor. HPOB was synthesizedfrom commercially available materials in five steps with an overallyield of 36% (Fig. 1A). (i) Reaction of aniline with glycolaldehydein dichloroethane yielded an imine intermediate, which was sub-sequently reduced with sodium triacetoxyborohydride to give 2-(phenylamino)ethanol, compound 2. (ii) The reactive hydrophilichydroxyl group of compound 2 was protected with tert-butyl-dimethylsilyl-chloride (TBDMS-Cl) to give N-(2-[(tert-butyl-dimethylsilyl)oxy]ethyl)aniline, compound 3. (iii) Compound8 was obtained from oxidation of commercially available 4-(Hydroxymethyl)phenylacetic acid with calcium hypochloritein the presence of methanol in acetonitrile, using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide to yield methyl 4-(2-[(2-[(tert-butyldimethylsilyl) oxy]ethyl)(phenyl)amino]-2-oxoethyl)benzoate. (iv) Compound 3 was then coupled with 2-[4-(methox-ycarbonyl) phenyl]acetic acid, compound 8. (v) Further, the hydro-xamic acid functional group was introduced to compound 4 byreacting with aqueous hydroxylamine and a catalytic amount of po-tassium cyanide to yield 4-(2-[(2-[(tert-butyldimethylsilyl)oxy]ethyl)(phenyl)amino]-2-oxoethyl)-N-hydroxybenzamide, compound 5.

Significance

Wediscovered ahydroxamic acid-based small-moleculeN-hydroxy-4-(2-[(2-hydroxyethyl)(phenyl)amino]-2-oxoethyl)benzamide selec-tively inhibits histone deacetylase 6 catalytic activity in vivo andin vitro.

Author contributions: J.-H.L., A.M., R.B., and P.A.M. designed research; A.M., Y.Y., N.K.,W.-S.H., and R.B. designed the small molecule; A.M and N.K. synthesized the smallmolecule; J.-H.L., A.M., Y.Y., L.N., G.V.-P., and M.L.C. performed research; J.-H.L., R.B.,and P.A.M. analyzed data; and J.-H.L., A.M., R.B., and P.A.M. wrote the paper.

Conflict of interest statement: Memorial Sloan–Kettering Cancer Center and ColumbiaUniversity hold patents on suberoylanilide hydroxamic acid (SAHA, vorinostat) and re-lated compounds that were exclusively licensed in 2001 to ATON Pharma, a biotechnologystart-up that was wholly acquired by Merck, Inc., in April 2004.

Freely available online through the PNAS open access option.1To whom correspondence may be addressed. E-mail: marksp@mskcc.org or rb33@columbia.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1313893110/-/DCSupplemental.

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(vi) Finally, removal of the TBDMS group from compound 5 using2% (vol/vol) HCl in ethanol resulted in compound 6, HPOB.

HPOB Is a Selective Inhibitor of HDAC6. To determine if HPOB isa selective inhibitor of HDAC6, it was assayed for inhibition ofrecombinant HDAC1 compared with HDAC6. HPOB has anIC50 inhibitory activity for HDAC6 of 0.056 μM compared withHDAC1 of 2.9 μM (Fig. 1B). HPOB inhibitory activity againstthe 11 zinc-dependent HDACs ranged from 0.056 μM for HDAC6to ≥1.7 μM for the other enzymes (Fig. 1C). By comparison,SAHA is a potent inhibitor of Class I HDACs and HDAC6 (Fig.

1C). Tubacin, a HDAC6-selective inhibitor discovered by Schreiberand coworkers (16) has an IC50 inhibitory activity for HDAC6 of0.045 μM and for HDAC1 of 0.193 μM (Fig. 1B). HPOB is a morepotent inhibitor of HDAC6 than tubacin.

HPOB Inhibits Growth, However, Not Viability, of Normal or TransformedCells. We next determined the effect of HPOB on cell growthand viability of normal human foreskin fibroblast (HFS) andtransformed (LNCaP, human prostate adenocarcinoma; A549, lungadenocarcinoma; and U87, glioblastoma) cells cultured with 8, 16,or 32 μM HPOB for up to 72 h. HPOB inhibited cell growth of

Fig. 1. HPOB is a HDAC6-selective inhibitor. (A)Synthesis of a HDAC6-selective inhibitor, HPOB. (B)IC50 values of HDAC6-selective inhibitors. IC50 valuesof HPOB and tubacin. (C) IC50 values of HPOB andSAHA for the 11 zinc-dependent HDAC enzymes.HPOB selectively inhibits HDAC6 in vitro comparedwith pan-HDAC inhibitors. The table shows the av-erage values of two runs for each case, with differ-ences between duplicate runs within less than 10%.P < 0.001. P value was derived from the Student’sone-tail t test.

Fig. 2. Effects of HPOB on cell growth and viabilityand acetylated patterns of proteins and histones innormal and transformed cells in culture. Normal (HFS)and transformed (LNCaP, A549, and U87) cells werecultured with indicated doses of HPOB for 72 h. Fivemicromolars SAHA is a positive control. (A) Cellgrowth. (B) Cell viability. Inhibition of cell growth ofnormal and transformed cells is concentration-de-pendent. Viable cells were evaluated by trypan bluestaining. Data are represented as mean ± SD, P <0.001. P value was derived from the two-way ANOVA.(C) HPOB causes accumulation of acetylated α-tubulinbut not acetylated histone H3 in normal (HFS) andtransformed (LNCaP, U87, and A549) cells. Cells wereculturedwith 5 μMSAHA, 4 μM tubacin, or 4, 8, and 16μM compound HPOB for 24 h as indicated. SAHA, thepan-HDAC inhibitor, and tubacin, the HDAC6 relativeinhibitor, were controls. Cell lysates were prepared forimmunoblot analysis of acetylated α-tubulin (Acet-Tub), acetylated peroxiredoxin (Acet-PRX), and acety-lated histone H3 (Acet-H3). GAPDH and total H3 areloading controls. (D) SAHA and tubacin induce accu-mulation of γH2AX, an early marker of DNA damagein LNCaP cells. HPOB did not induce accumulation ofγH2AX in LNCaP cells. Cells were cultured with 5 μMSAHA, 4 μM tubacin, or 8 or 16 μM HPOB for 24 h.Immunoblots are of phosphorylated H2AX (γH2AX).Total H3 is the loading control.

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normal and transformed cells in a concentration-dependent manner(Fig. 2A) but did not induce cell death of normal or transformedcells (Fig. 2B).

HPOB Induces Acetylation of α-Tubulin, However, Not Histones, inNormal and Transformed Cells. In normal (HFS) and transformed(LNCAP, U87, and A549) cells, HPOB causes accumulation ofacetylated α-tubulin and acetylated peroxiredoxin, substrates ofHDAC6 (4, 5), but not of acetylated histones (Fig. 2C). As pre-viously reported (20, 22, 23), SAHA induced the accumulation ofacetylated α-tubulin and histone H3, and tubacin induced accu-mulation of acetylated α-tubulin but not of histone H3 (Fig. 2C).

HPOB Does Not Induce Accumulation of γH2AX, an Early Indicator ofDNA Double-Strand Breaks. We previously found that the HDAC6-selective inhibitor, tubacin, causes accumulation of phosphorylatedhistone H2AX (γH2AX), an early indicator of DNA double-strandbreaks (DSB), in transformed cells (23). Tubacin and SAHA in-duced the accumulation of γH2AX in LNCaP cells. HPOB did notinduce detectable accumulation of γH2AX in LNCaP cells (Fig.2D). These data suggest that HPOB, unlike tubacin and SAHA,does not cause DNA damage in transformed cells.

HPOB Does Not Inhibit Trehalose-Induced Autophagy. We next de-termined if HPOB inhibited HDAC6 ubiquitin-binding complexformation induced by the complex carbohydrate, trehalose. Ithas been shown that HDAC6 has a role in ubiquitin-binding-complex–forming aggresomes in the autophagic pathway of celldeath (8, 9, 24, 25). Trehalose induces aggresome formation andautophagy (26). Trehalose-induced inhibition of cell growth wasnot blocked by HPOB in normal and transformed cells (Fig. 3A).Consistent with these findings, HPOB was found not to altertrehalose-induced LC3-II (microtubule-associated protein 1A/1B-light chain 3-II) accumulation, an indication of autophago-some formation which is responsible for degradation of poly-ubiquitinated complexes (Fig. 3B).To further evaluate the effect of HPOB on formation of the

HDAC6 ubiquitin-binding complex, we assayed polyubiquitincomplex accumulation in normal and transformed cells culturedwith SAHA, tubacin, or HPOB (Fig. 3C). There was no detectabledifference in the accumulation of the polyubiquitin complex innormal or transformed cells cultured with SAHA, tubacin, or

HPOB. These results indicate that HPOBmay not inhibit HDAC6activity which is important in proteasome inhibition.To further evaluate whether ubiquitin-binding activity of HDAC6

is inhibited by HPOB, we performed an immunoprecipitation assaywith HDAC6 antibody in LNCaP cells. HPOB causes an increasein the accumulation of acetylated α-tubulin, but does not blockHDAC6 binding to the ubiquitin complex (Fig. 3D, Right). Immu-noprecipitation assay with HDAC6 antibody showed that HDAC6binds to mono- and di-ubiquitin complex (<75 kDa) rather thanpolyubiquitin complex. Immunoprecipitation assay with ubiquitinantibody showed that ubiquitin complex binds to HDAC6 protein.The binding of ubiquitin complex with acetylated tubulin did notchange in LNCaP cells cultured with HDAC inhibitors comparedwith the control. These data indicate that HPOB inhibits thedeacetylase activity of HDAC6 but not its ubiquitin-binding activity.

HPOB Enhances Transformed Cell Death Induced by the AnticancerDrugs Etoposide, Doxorubicin, or SAHA. We previously reportedthat inhibition of HDAC6 by either si-RNA or tubacin potentiatesthe cytotoxicity of anticancer drugs in transformed but not innormal cells (23). To assesswhether selective inhibition ofHDAC6by HPOB enhances cell death of normal and transformed cells inculture with anticancer agents, cells were cultured with HPOB andthe topoisomerase II inhibitors etoposide or doxorubicin or thepan-HDAC inhibitor SAHA for 72 h. InHFS cells, HPOBalone orin combination with doxorubicin, etoposide, or SAHA inhibitedcell growth but did not induce loss of cell viability (Fig. 4A).LNCaP cells cultured with 50 μM etoposide and 8 μM HPOB

demonstrated inhibition in cell growth and loss of cell viability toa greater extent than LNCaP cells cultured with etoposide alone(Fig. 4B). LNCaP cells cultured with 400 nM doxorubicin and8 μM HPOB had increased cell death compared with cultureswith 400 nM doxorubicin alone. LNCaP cell death was enhancedin cultures with HPOB and 2.5 μM SAHA compared with cul-tures with SAHA alone (Fig. 4B).In U87 cells, combination treatment with HPOB and 400 nM

doxorubicin resulted in an increase in cell death compared withcultures with doxorubicin alone (Fig. 4C). There was no enhancedcell death in U87 cells cultured with 8 μM HPOB and 50 μMetoposide or SAHA. In A549 cells, HPOB in combination with50 μM etoposide or 400 nM doxorubicin showed enhanced celldeath compared with cultures with either drug alone (Fig. 4D).

Fig. 3. HPOB does not block trehalose-inducedautophagy and cell death in LNCaP. (A) Trehalose, aninducer of aggresome autophagy, inhibits cell growthin HFS and LNCaP cells and induces cell death inLNCaP. HPOB does not block trehalose-induced LNCaPcell death. Cells were cultured with 16 μM HPOB, 200mM trehalose, or combination for 72 h. Cell growthand viability were determined by trypan blue stain-ing. Data are represented as mean ± SD, P < 0.001. Pvalue was derived from the two-way ANOVA. (B)Trehalose-induced accumulation of LC3 is not inhibi-ted by HPOB in HFS and LNCAP cells. Cells were cul-tured with 8 or 16 μM HPOB, 200 mM trehalose, orcombination for 24 h and were prepared for immu-noblot analysis. Immunoblot is of LC3. GAPDH is theloading control. (C) HPOB, SAHA, or tubacin, inhib-itors of HDAC6 catalytic activity, do not block poly-ubiquitin complex formation in HFS and LNCaP cells.Cells were cultured with 5 μM SAHA, 4 μM tubacin, or8 or 16 μM compound HPOB for 24 h. Immunoblotsare of polyubiquitin complex. GAPDH is the loadingcontrol. (D) Immunoprecipitation of HDAC6 bringsdown ubiquitin complex, which is not blocked bySAHA or HPOB. LNCaP cells were cultured with 5 μMSAHA or 16 μM HPOB for 24 h. Cell lysates were immunoprecipitated with HDAC6 or ubiquitin antibody and prepared for immunoblot analysis. Immunoblots areof HDAC6, polyubiquitin complex, and acetylated α-tubulin (Acet-Tub).

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HPOB Induces Increased Apoptotic Cell Death of Transformed CellsCultured with Anticancer Drugs. To investigate the pathway of celldeath in transformed cells cultured with the combination of HPOBwith etoposide, we determined the levels of poly(ADP ribose)polymerase (PARP) and its cleavage fragments. In LNCaP cellscultured with HPOB and etoposide, there was an increase incleaved PARP, a marker of apoptosis (27) (Fig. 4E). There was noincrease in accumulation of cleaved PARP in HFS cells culturedwith HPOB alone and in combination with etoposide. Thesefindings are consistent with HPOB enhancing etoposide-inducedtransformed cell death via the apoptotic pathway.We next examined whether selective inhibition of HDAC6 with

HPOB activates a DNA damage response in combination withanticancer drugs. Combination of HPOBwith etoposide increasedthe accumulation of DNAdamage compared with etoposide aloneas evidenced by accumulation of γH2AX in LNCaP cells (Fig. 4F).

HPOB is Well-Tolerated in Mice and Enhances Cytotoxicity of anAnticancer Drug. We next determined the toxicity of HPOB.HPOB was intraperitoneally injected daily for 5 d with 100, 200,or 300 mg/kg HPOB. There was no weight loss in these mice,

suggesting that HPOB is well-tolerated in these animals (Fig. 5A).The effect of HPOB on the acetylation of α-tubulin and histonesin the spleen isolated from mice treated with HPOB was analyzedat three time points after the administration of the drug. At 1.5 hafter injection of HPOB, an increased accumulation of acetylatedtubulin was found in the spleen (Fig. 5B). By 5 h after injection ofHPOB, the accumulation of acetylated tubulin was reduced to thelevel seen in vehicle-treated controls. There was no detectableaccumulation of acetylated histones in the spleen from the micereceiving HPOB. One hundred milligrams/kilogram SAHA in-creased the accumulation of acetylation of both α-tubulin andhistones which persisted up to 3 h, but the increased acetylatedlevels were reduced to the level seen in vehicle-treated controls by5 h. These data are consistent with previous reports (11).Next, we examined the effects of HPOB in combination with

an anticancer drug, SAHA, in nude mice with the androgen-dependent CWR22 human prostate cancer xenograft, which wasgrown s.c. Daily administration of either 300 mg/kg HPOB or50 mg/kg SAHA alone for 18 d caused no significant suppressionof the growth of established CWR22 tumors and no weight loss

Fig. 4. HPOB enhances etoposide-, doxorubicin-, and SAHA-induced transformed cell death but not normal cell death. Cell growth and viability of (A) HFS,(B) LNCaP, (C) U87, and (D) A549 cells cultured with HPOB with 50 μM etoposide, 400 nM doxorubicin, or 5 μM SAHA alone and in combination with HPOB for72 h. Cell growth and viability were determined as previously described. Data are represented as mean ± SD, P < 0.001. P value was derived from the two-wayANOVA. (E) LNCaP and HFS cells were cultured with HPOB in combination with etoposide for 24 h. Immunoblots show PARP degradation and acetylatedα-tubulin. GAPDH is a loading control. (F) Immunoblots for phosphorylated H2AX (γH2AX) and acetylated histone H3 (Acet-H3). Total H3 is a loading control.

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(Fig. 5C). Daily administration of HPOB and SAHA causedsuppression of the growth of established CWR22 tumors, suchthat doses of 300 mg/kg/d HPOB in combination with SAHA50 mg/kg/d caused reductions of 50% in the mean final tumorvolume compared with vehicle-treated control animals. Tumors,spleen, and brain were removed from the animals, and histonesand proteins were extracted for the detection of acetylatedlysine patterns. There was increased accumulation of acetylatedα-tubulin in CWR22 tumors and spleen from mice treated withHPOB, SAHA, or combination of HPOB and SAHA. In thebrains of mice treated with HPOB, there was increased accu-mulation of acetylation of PRX1, a substrate of HDAC6 (Fig.5D). Increased levels of accumulation of histones were found intumors of mice injected with SAHA or a combination of SAHAand HPOB, but not with HPOB alone. These data indicate thatHPOB is a selective inhibitor against HDAC6 in vivo, and HPOBcan enhance the antitumor effect of chemotherapeutic agents.

DiscussionWe report the discovery of a HDAC6-selective inhibitor, HPOB,and its biological effects in normal and transformed cells. HPOBinhibits HDAC6 in vitro with ∼50-fold selectivity against HDAC6over HDAC1 enzyme. Concentrations as high as 16 μMof HPOBinduce accumulation of acetylated α-tubulin and acetylated PRX,substrates of HDAC6, but not of acetylated histones, not a sub-strate of HDAC6, in both normal and transformed cells. HPOB inconcentrations≤16 μMdoes not induce normal cell death. HPOBenhances etoposide, doxorubicin, or SAHA-induced transformedcell death. These findings (12, 25, 28) provide evidence that se-lective inhibition of HDAC6 in combination with anticancerdrugs may be an important avenue to enhance the therapeuticefficacy of such drugs in treating human cancers.

HPOB selectively inhibits the catalytic activity of HDAC6 butdoes not block HDAC6 binding to form a polyubiquitinatedprotein complex. The levels of LC3-II, a marker of autophago-some formation, do not change in cells cultured with HPOB.Combination of HPOB and trehalose, an inducer of autophagy,causes cell growth inhibition but not cell death of normal cells.HPOB does not induce cell death in normal or transformed

cells. Culture with HPOB in transformed cells enhances the cy-totoxicity of DNA-damaging anticancer drugs through increasedinduction of apoptosis and accumulation of DNA damage.HPOB is well-tolerated in animals. HPOB in combination with

SAHA significantly enhances the antitumor effect of SAHA againstthe androgen-dependent CWR22 human prostate cancer xenograftin nude mice.In summary, we have discovered a HDAC6-selective inhibitor,

HPOB, that has the potential to enhance anticancer drug efficacy incombination therapy of human cancers, suggesting the promise ofdrugs targetingHDAC6 to improve therapeutic strategies in cancers.

Experimental ProceduresThe section discussing materials and methods is included in SI ExperimentalProcedures. This section describes preparation of cells, reagents, proteins,and histone extracts used in this study. Assay procedures for determi-nation of in vitro enzymatic assay for histone deacetylases and animalexperiments are also detailed in SI Experimental Procedures. Animalstudies were carried out under protocol 12-02-003, approved by theMemorial Sloan–Kettering Cancer Center Institutional Animal Care andUse Committee. Institutional guidelines for the proper, humane use ofanimals in research were followed.

ACKNOWLEDGMENTS. We thank Joann Perrone for her assistance in thepreparation of this manuscript. These studies were supported, in part, byNational Institute of Cancer Grant P30CA08748-44, The David Koch Foun-dation, and a grant from Servier.

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Fig. 5. HPOB enhances anticancer effects of SAHAin mice bearing human prostate cancer CWR22xenograft. (A) HPOB is well-tolerated in animals.Mice were injected with indicated doses of HPOB andSAHA intraperitoneally daily for 5 d. Data are repre-sented as mean ± SD, P < 0.001. P value was derivedfrom the two-way ANOVA. (B) HDAC6 selectivity ofHPOB in spleen isolated from immune-deficient mice.Spleens were isolated from mice injected with in-dicated drugs at 1.5, 3, and 5 h after the last injectionon day 5. Immunoblots for acetylated α-tubulin (Acet-Tubulin) and acetylated histone H3 (Acet-H3). Hsp90and total H3 are loading controls. (C) Mice injectedwith HPOB in combination with SAHA showed sig-nificant shrinkage of CWR22 tumors. There was noweight loss in the animals. Data are represented asmean ± SD, *P < 0.05, ***P < 0.001. P value was de-rived from the two-wayANOVA. (D) HDAC6 selectivityof HPOB in spleen, brain, and tumors isolated frommice bearing human prostate cancer CWR22 xeno-graft. Mice were injected with SAHA, HPOB, or com-bination of SAHA and HPOB intraperitoneally dailyfor 18 d. Tissues were isolated on day 25 and preparedfor immunoblot analysis. Immunoblots are shown foracetylated α-tubulin (Acet-Tubulin), acetylated per-oxiredoxin (Acet-PRX), and acetylated histone H3(Acet-H3). Hsp90 and total H3 are loading controls.

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