Conjugate of Pt(IV)–Histone Deacetylase Inhibitor as a Prodrug for

Sep 6, 2012 - The cytosolic VAAP was intracellular reduced to Pt(II) and released VA eliciting a HDAC inhibitory effect and subsequently induced cell ...
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Conjugate of Pt(IV)−Histone Deacetylase Inhibitor as a Prodrug for Cancer Chemotherapy Jun Yang, Xuanrong Sun, Weiwei Mao, Meihua Sui, Jianbin Tang,* and Youqing Shen* Center for Bionanoengineering and the State Key Laboratory of Chemical Engineering, Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China S Supporting Information *

ABSTRACT: Platinum(IV) prodrug diaminedichlorodihydroxyplatinum (ACHP) conjugated with a histone deacetylase (HDAC) inhibitor valproic acid (VA), VAAP, exhibited strong synergistic cytotoxicity, about 50−100 times more cytotoxic than ACHP or its simple mixture with VA, against various human carcinoma cell lines. VAAP could be quickly absorbed in the cell membrane and diffused into the cytosol. VAAP loaded in polyethylene glycol−polycaprolactone micelles (PEG-PCL) was taken up via endocytosis. The cytosolic VAAP was intracellular reduced to Pt(II) and released VA eliciting a HDAC inhibitory effect and subsequently induced cell cycle arrest at the S phase in 24 h and cell apoptosis in a timedependent manner. The in vivo antitumor experiment on A549-xenograft tumor model showed that VAAP dispersed in Tween 80 or loaded in PEG-PCL nanoparticles had long blood circulation times and thereby high accumulation in tumors and exerted a significant in vivo inhibitory effect on tumor growth with low systemic toxicity. Therefore, this novel conjugate is very promising for cancer chemotherapy. KEYWORDS: histone deacetylase inhibitor, valproic acid, platinum(IV) prodrug, synergistic cytotoxicity, cell cycle arrest and apoptosis

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As a first-line chemotherapeutic drug against a variety of cancer types,26,27 cisplatin has severe toxic side effects to normal tissues, particularly acute nephrotoxicity and chronic neurotoxicity.28−30 Its prodrugs, octahedrally coordinated platinum(IV) compounds, have low toxicity but can be intracellularly reduced to platinum(II) to regain their cytotoxicity. 31,32 The high kinetic inertness of Pt(IV) complexes enables them to overcome many problems associated with cisplatin and its analogues;33 thus, they are advantageous in delivering platinum-based drugs to target tumor cells. Furthermore, various carboxylic ligands can be introduced to the octahedral Pt(IV) center to further tailor prodrugs' lipophilicity, stability, reduction behavior, and biological activity.34 Currently, satraplatin composed of two acetate ligands in the axial positions is in phase III clinical trials for hormone refractory prostate cancer.35 Inspired by the structure of satraplatin, we proposed that using VA as ligands of Pt(IV) to form a satraplatin-like Pt(IV)− VA prodrug would combine the advantages of VA and Pt(IV); VA would serve as not only a ligand but also an HDAC inhibitor once the Pt(IV)−VA prodrug was intracelluarly reduced to Pt(II) and VA, and they might elicit synergistic antitumor activity. Moreover, the Pt(IV)-based prodrug should

INTRODUCTION Histone deacetylase (HDAC) inhibitors are prospective anticancer agents that can inhibit proliferation and stimulate differentiation and apoptosis of cancer cells.1,2 They have been tested alone3,4 or in combination with other conventional chemotherapeutic agents5−7 as well as to resensitize resistant cancer cells.8,9 For instance, FR901228 (depsipeptide, FK228), showing its antiproliferative effects on non small cell lung cancer and small cell lung cancer, was found to effectively arrest cell growth at the G2/M phase and induce subsequent apoptosis.10 Currently, several HDAC inhibitors are in phase I and phase II clinical trials.11,12 Valproic acid (VA), a clinically used antiepileptic and anticonvulsant drug,13−15 has recently been demonstrated as a short-chain fatty acid type HDAC inhibitor.16−18 Like other HDAC inhibitors, VA shows antitumor effects by modulating cell cycle arrest, apoptosis, angiogenesis, metastasis, differentiation, and senescence.19,20 VA can potentiate chemotherapy by enhancing tumor growth suppression and apoptosis.21,22 The reported mechanism is that VA binds to the catalytic center and blocks substrate access, causing hyperacetylation of the N-terminal tails of core histones H3 and H4 that influence the expression of target genes and subsequently inhibit HDAC activity.23,24 VA exhibited synergistic cytotoxicity with cisplatin to ovarian carcinoma cells.25 For example, Lin et al. demonstrated that VA (at near-nontoxic dose, 0.6 mM) could upregulate the cisplatin-mediated DNA damage and thus enhanced cytotoxicity to the cisplatin-resistant ovarian cancer.25 © 2012 American Chemical Society

Received: Revised: Accepted: Published: 2793

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Scheme 1. Synthesis of VAAP

with less nephrotoxicity than cisplatin, and can be intracellularly reduced to platinum(II) for cytotoxicity.42 VA shows inhibition of HDAC at a millimolar concentration. Enlightened by satraplatin, we synthesized a conjugate of ACHP and VA, VAAP, to simultaneously deliver these two antitumor agents into cancer cells for synergistic antitumor activity. ACHP could react with an excess VAn in DMSO to produce VAAP (Scheme 1). The excess VAn and produced VA could be simply removed by repeated reprecipitation. The VAAP structure was verified by the element analysis and NMR spectra (Figures S1 and S2 in the Supporting Information). VAAP is water-insoluble. For in vitro tests, it was first dissolved in DMSO and then diluted with medium (DMSO content was less than 5‰). For in vivo administration, it was either formulated with 10% Tween 80 (3.52 mg/mL VAAP) or loaded into the PEG-PCL micelles with a loading content of 4.2 wt %. The hydrodynamic diameter of the PEG-PCL/VAAP was 106 nm (Figures S3 and S4 in the Supporting Information). This size is suitable for tumor targeting via passive accumulation through the enhanced permeability and retention (EPR) effect.43 The nanoparticles released VAAP slowly (Figure S5 in the Supporting Information), only 30% in pH 7.4 buffer in 24 h. HDAC Activity Assay and in Vitro Cytotoxic Activity. The HDAC inhibitory activities of VA and VAAP were determined using HDAC activity assay, and the results are summarized in Figure 1. VA was previously reported to have an inhibitory effect on HDAC1, 2, 3, 4, and 8 in vitro.23 In the present study, we found that 5 mM VA could inhibit 66.0% of

also have low side effects. Herein, we report the synthesis of a Pt(IV)−VA complex, cis,cis,trans-diaminedichlorobisvalproatoplatinum(IV) (VAAP) (Scheme 1) and its in vitro and in vivo anticancer activity. VAAP was also dispersed in Tween 80 or loaded in polyethylene glycol−polycaprolactone (PEG-PCL) nanoparticles to prolong its blood circulation time and improve its anticancer activity in vivo.

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MATERIALS AND METHODS Materials. The sources of the used chemicals are included in the Supporting Information. cis,cis,trans-Diaminedichlorodihydroxy-platinum(IV) [Pt(NH3)2(OH)2Cl2, ACHP] was synthesized according to the literature.36−38 PEG-PCL was prepared as reported (Mn of PEG = 1900, Mn of PCL = 5800, calculated from 1H NMR spectrum, data not shown).39,40 Synthesis of VAAP [Pt(NH3)2Cl2(COOCH(CH2CH2CH3)2)] (Scheme 1). ACHP (201.0 mg, 0.6 mmol) and valproic anhydride (VAn, 652.0 mg, 2.4 mmol) were stirred in 0.8 mL of dried DMSO at 70 °C in the dark. After 24 h, the yellow solution was poured in ether. The precipitates were isolated and washed with ether. The yield was 82.4%. 1H NMR (DMSO-d6, 400 MHz, δ ppm, Figure S2 in the Supporting Information): 0.8 (m, −CH3, 12H), 1.2 (m, −CH2−, 12H), 1.4 (m, −CH2−, 4H), 2.2 (m, −CH−, 2H), 6.5 (b, −NH3, 6H). Anal. calcd for C16H36Cl2N2O4Pt: C, 32.77; H, 6.19; N, 4.78. Found: C, 33.54; H, 6.56; N, 4.89. The platinum content determined by brilliantgreen method41 was 35.82% (calcd, 33.26%). VAAP Encapsulation into Nanoparticles (PEG-PCL/ VAAP). VAAP (10 mg) and PEG-PCL (100 mg) (weight ratio = 1:10) were dissolved in a 200 μL of DMSO/800 μL of THF mixture with stirring. Distilled water (3 mL) was added dropwise into the organic solution slowly. The solution was loaded in a dialysis bag (Spectra/Pro; molecular weight cutoff, 3500; Spectrum, United States) and dialyzed against water. PEG-PCL nanoparticles loaded with VAAP (PEG-PCL/VAAP) had an average size of 106 nm with PDI of 0.319 as measured by a dynamic laser scattering spectrometer (DLS, nano series ZEN4002, Malvern Instruments Ltd., United Kingdom) (Figure S4 in the Supporting Information). The platinum content of the particles determined by brilliant-green method was 2.1 mg/mL in terms of VAAP. Preparation of Rhodamine B Isothiocyanate (RITC)Labeled Nanoparticles (PEG-PCL-RITC/VAAP) for Cell Uptake Study. The PEG-PCL-RITC was prepared by stirring PEG-PCL with RITC at room temperature in THF in the dark for 24 h. Then, the solution was dialyzed by cutoff 3500 dialysis bag against water before it was lyophilized. The RITC-labeled VAAP-loaded nanoparticles were prepared from PEG-PCLRITC with the same procedure as PEG-PCL/VAAP preparation.

Figure 1. HDAC inhibition activity of VAAP. The nuclear extract of HeLa cells was used as the negative HDAC inhibition control and trichostatin A as the positive control. The HDAC inhibitory activities of the nuclear extract treated with 5 mM VA-equivalent dose of VA, VAAP, or VAAP pretreated with 5 mM AA (VAAP + AA) at 37 °C for 10 h were determined using a Colorimetric HDAC Activity Assay Kit based on the OD values of the treated/untreated samples (at 405 nm). The corresponding HDAC inhibition efficiencies were calculated accordingly. Data are shown as the mean ± SD of three independent experiments.

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RESULTS AND DISCUSSION Synthesis of VAAP and Formulations. Dihydroxyl platinum(IV) complex ACHP is an oral antitumor prodrug 2794

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Figure 2. IC50 values of cisplatin, ACHP, the mixture of ACHP with VA (ACHP + VA), VAAP, and PEG-PCL/VAAP against A549, BCap-37, SKOV-3, and HepG2 cells (A). Cells were treated with the compounds for 48 h and post-treated for an additional 24 h. Data are shown as means ± SDs of three independent experiments. Pt concentrations in the cytosol or cell membrane determined using ICP-MS after the BCap-37 cells were treated with 1 μM compounds (equivalent to 1 μM ACHP) for 4 and 24 h (B). Their dose-responsive curves of cell viability are shown in Figure S7 in the Supporting Information.

Figure 3. Cellular uptake and intracellular localization of PEG-PCL-RITC/VAAP of SKOV-3 cells after incubation for 1.5 h. CLSM images were taken from the RITC channel (A, red), the LysoTracker channel (B, green), and the overlap of the images (C).

The dihydroxyl Pt(IV) compound ACHP was mildly cytotoxic to the cancer cells with IC50 values of about 5−10 μM. The presence of free VA (ACHP + VA mixture) did not affect its cytotoxicity. This is agreeable with the low cytotoxicity of VA itself. Very surprisingly, the conjugate of ACHP and VA, VAAP, showed significantly enhanced cytotoxicity to all of the four tumor cell lines. For instance, the IC50 value of VAAP to A549 cells was 0.15 μM, while the value of ACHP was 13.99 μM, a 90-fold increase in cytotoxicity. Similarly, the IC50 values of VAAP versus ACHP were 0.20 versus 10.25 μM against BCap37, 0.17 versus 5.75 μM against SKOV-3, and 0.14 versus 10.24 μM against HepG2 cells, respectively. Furthermore, the cytotoxicity of VAAP was even significantly higher than cisplatin. For instance, VAAP was 12-fold more cytotoxic than cisplatin to A549 cells. In addition, PEG-PCL/VAAP showed the same cytotoxicity against cancer cells as the free VAAP. Cellular Pt Accumulation and Distribution. To probe the mechanism of the enhanced cytotoxicity of VAAP, cellular Pt levels after the BCap-37 cells were treated with the compounds at 1 μM ACHP-equivalent dose for 4 and 24 h were determined and are shown in Figure 2B. The accumulation of ACHP in the cytosol and cell membrane (cell debris) increased with time. In the presence of VA, the

the HDAC activity of the negative control. VAAP at 5 mM VAequivalent dose showed 24.3% inhibition on the HDAC activity. When VAAP was first incubated with a reducing agent ascorbic acid (AA, 5 mM) for 10 h, its HDAC inhibitory activity dramatically increased to 70.2%. This indicates that once VAAP is reduced to platinum(II) and thus release free VA, the VA will effectively elicit HDAC inhibition activity, and the produced Pt(II) ion does not interfere with VA's HDAC inhibition. The in vitro antitumor activities of cisplatin, ACHP, VA, the mixture of ACHP with VA (ACHP + VA), VAAP, PEG-PCL, and PEG-PCL/VAAP to human cancer cell lines from different origins were determined using the MTT assay. PEG-PCL had negligible cytotoxicity to the four cell lines at doses ranging from 0.05 to 200 μM (Figure S6 in the Supporting Information). VA itself did not suppress cell growth at the same dose range (Figure S6 in the Supporting Information). This is agreeable with the above HDAC inhibition result and the reports in literature that VA needs millimolar doses to elicit its HDAC inhibition and cytotoxicity.44,45 The dose-responsive curves of cell viability of VAAP and its controls are shown in Figure S7 in the Supporting Information. Their IC50 values (the concentration inhibiting cell growth to 50% of control) are shown in Figure 2A. 2795

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Figure 4. Flow cytometric analysis of cell cycle distribution and apoptosis (A). A549 cancer cells were incubated with the indicated compounds for 24, 72, and 96 h. Peaks are corresponding to the fraction of the cells in G1/G0, G2/M, and S phases or apoptosis. The percentages of the cells arrested in the different phases of cell cycle as well as apoptosis cells (AP) after administration of indicated drugs for 24 (B) and 96 h (C) are calculated accordingly.

accumulation of ACHP in cytosol for 4 h was 28.6 ng/2 × 107 cells but surprisingly decreased to 9.4 ng/2 × 107 cells after 24 h of incubation. It seems that free VA could help ACHP to enter the cells quickly, but afterward, the cytosolic ACHP was cleared up rapidly. When the cells were treated with VAAP, VAAP accumulated in the cell debris (mainly cell membrane) at 80.6 ng/2 × 107 cells in 4 h culture and 252.2 ng/2 × 107 cells in 24 h presumably due to the hydrophobic nature of VAAP. It then slowly further diffused into the cytosol, reaching 6.2 ng/2 × 107 cells at 4 h and 42.8 ng/2 × 107 cells in 24 h.

These data show that VAAP could attach a cell membrane efficiently due to its hydrophobicity and further entered cytosol to have high cytosolic Pt concentrations, resulting in enhanced cytotoxicity as compared to ACHP. However, it seems that the high cytotoxicity of VAAP was not simply due to the increased cytosolic Pt concentration. The cytosolic Pt concentration of the cells treated with VAAP for 24 h was just about twice that of the cells treated with ACHP, while its IC50 was about 50− 100 times lower than that of ACHP. Thus, the combination of the high cytosolic Pt concentration and the synergy of the 2796

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resulting Pt(II) and VA may account for VAAP's high cytotoxicity. HDAC inhibitor VA potentiated the acetylation of the nuclear histones, which loosened the histone−DNA interactions and facilitated the binding of Pt(II) to DNA.46 The cells treated with PEG-PCL/VAAP had a Pt concentration in their membranes similar to those treated with VAAP at both 4 and 24 h time points, but their cytosolic VAAP concentrations were higher, particularly at 24 h incubation. The cellular uptake of nanoparticles was observed by confocal laser scanning microscopy. As shown in Figure 3, PEG-PCL-RITC/VAAP nanoparticles were quickly taken up and localized into the tumor cells after 1.5 h of incubation at 37 °C. The overlap of the images taken from RITC, LysoTracker, and transmittance channels showed that PEG-PCL/VAAP nanoparticles were mainly localized in lysosomes. These results indicated that the micelles were internalized into tumor cells mainly by endocytosis, resulting in a higher cytosolic Pt concentration. Cell Cycle and Apoptosis Analysis. The cell cycle distribution and induction of apoptosis after treatment for 24, 72, or 96 h were evaluated by flow cytometry using A549 cells. The dose of each compound was chosen to induce 80% cell viability in the MTT assay. Figure 4A shows the flow cytometric profiles, and the results are summarized in Figure 4B,C and Figure S8 in the Supporting Information. The data show that tumor cells mainly accumulated at the S phase after 24 h of incubation with ACHP (100 μM), VAAP (1 μM), or PEG-PCL/VAAP nanoparticles (1 μM VAAP-equivalent dose). Furthermore, while 100 μM ACHP or 200 μM VA alone could not induce cell apoptosis, their mixture (ACHP + VA) led to about 15.9% cell apoptosis in 24 h and 79.9% in 96 h, indicating that free VA at high doses could promote ACHP to induce apoptosis. VAAP arrested the cells mainly in the S phase and induced no apoptosis in 24 h and 31.6% apoptosis in 96 h culture. The fraction of the cells under apoptosis treated with PEG-PCL/VAAP increased from 26.0 to 66.2% as the incubation time extended from 72 to 96 h. Interestingly, the presence of reducing agent AA did not alter ACHP- or VAAP-induced cell cycle arrest profile but influenced the cell apoptosis. The presence of AA significantly prevented ACHP-induced cell apoptosis but greatly promoted the VAAPinduced apoptosis. This is because that while AA can reduce Pt(IV) to Pt(II), it is known to have a cell-protection effect32,47 and thus reduced ACHP's cytotoxicity. In the case of VAAP, AA not only reduced Pt(IV) to Pt(II) but also released VA to prompt cell apoptosis. Thus, the flow cytometric analysis indicates that conjugation of the HDAC inhibitor VA to the platinum(IV) prodrug did not regulate the cell cycle pattern but enhanced the cell apoptosis. Blood Clearance. The blood platinum concentration levels as a function of time after single intravenous administration of ACHP, VAAP, or PEG-PCL/VAAP nanoparticles (at a dose equivalent to 12 mg/kg ACHP) are shown in Figure 5. ACHP was dissolved in 0.9% saline. VAAP is hydrophobic and could not be dissolved in water and thus was dispersed with 10% Tween 80. PEG-PCL/VAAP was well-dispersible in 10 mM PBS (pH 7.4). ACHP was cleared out very quickly, but VAAP dispersed in Tween 80 or loaded in PEG-PCL nanoparticles had extended blood circulation times. After 24 h of treatment, the blood of the mice administered with PEG-PCL/VAAP nanoparticles still maintained 4.7% of the injected dose, and that of the mice with VAAP in Tween 80 was about 2.2% of the injected dose. The AUC (area under the concentration−time

Figure 5. Blood platinum concentration as a function of time after single iv administration of ACHP, VAAP in Tween 80, or PEG-PCL nanoparticles at an ACHP-equivalent dose of 12 mg/kg. Data are expressed as means ± SDs (n = 3). *, p < 0.05; **, p < 0.01; and ***, p < 0.001. The corresponding AUC was calculated based on the trapezoidal rule up to 24 h.

curve)29,48 of PEG-PCL/VAAP for 24 h was twice of that of VAAP and seven times of that of ACHP. These data show that the administration of VAAP using PEG-PCL nanoparticles or Tween 80 surfactant led to a slow blood clearance, which is needed for passive accumulation in tumor via EPR effect. In Vivo Antitumor Studies. The in vivo antitumor activities of the compounds were compared using A549 tumor xenograft model. Mice bearing the tumors were iv treated with PBS, ACHP, VAAP/Tween 80, or PEG-PCL/ VAAP at an ACHP-equivalent dose of 10 mg/kg (Figure 6). As shown in Figure 6A,B, all of the treatments led to significant inhibition of A549 tumor growth as compared to the control group. The inhibition rates of tumor growth (IRT) of ACHP, VAAP, and PEG-PCL/VAAP on A549 tumors were 65.0, 87.3, and 85.2%, respectively (Figure 6D). The statistical analysis showed that VAAP and PEG-PCL/VAAP induced significantly high IRTs as compared to ACHP (both p < 0.05). The body weights of all of the mice gradually increased throughout the experiments (Figure 6C), indicating that the treatments did not cause severe side effects. To further investigate the drug efficacy, the Pt levels of the tumor tissues were measured after the treatment (Figure 6E). PEG-PCL/VAAP exhibited significantly higher Pt accumulation than ACHP (five times, p < 0.01) and VAAP (two times, p < 0.05), respectively. This is agreeable with their blood clearance data shown in Figure 5. A longer blood circulation time of the drug accumulated more concentration in the tumor due to the EPR effect. The removed A549 xenograft tumors and normal organs were fixed and prepared for histological analysis. In the control group, the tumor tissue sections were composed of tightly packed tumor cells interspersed with various amounts of stroma, while apoptotic tumor cells were rarely observed (Figure 6F). After the treatment of ACHP, VAAP/Tween 80, or PEG-PCL/VAAP, the histological features of tumors exhibited significant difference from the control group, particularly in tumors treated with VAAP/Tween 80 and PEG-PCL/VAAP. For example, many tumor cells after the treatment with ACHP became much larger, and the tumor cellularity,49,50 as evaluated by average tumor cell numbers of each microscopic field, reduced significantly when compared with the control group. In addition, many tumor cells exhibited vacuolization and typical apoptotic characteristics; that is, they 2797

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Figure 6. Antitumor activities of ACHP, VAAP/Tween 80, and PEG-PCL/VAAP against A549 xenograft tumors. Nude mice bearing A549 tumors were treated with ACHP, VAAP/Tween 80, or PEG-PCL/VAAP at an ACHP-equivalent dose of 10 mg/kg (q4d × 3). The tumor volume, body weight, and tumor weight were measured and calculated as described in the Supporting Information and Materials and Methods. The results are summarized as tumor volumes of mice bearing A549 tumors exposed to various treatments (bars, SD; *, differs from control) (A), the image of tumors (B), the body weight changes (C), the tumor weights of each group of the mice at the end of the experiment (D), and the averaged platinum concentration in the tumor in each group after the final treatment (E); the results were expressed as means ± SDs (n = 4 for A−D; n = 3 for E). IRT, inhibition rates of tumor growth. *, p < 0.05; and **, p < 0.01. Representative histological features of A549 tumors (upper panel) and kidneys (lower panel) from the treated mice are shown in panel F. Scale bar = 50 and 100 μm, for upper and bottom panels, respectively.

renal tissue, such as alterations of renal tissue architecture, decreased cellularity of glomeruli, pyknotic and karyorhectic debris of tubular cells, and tubular cell detachment (Figure 6F). Importantly, the renal tissues treated with VAAP/Tween 80 and PEG-PCL/VAAP showed quite similar structures and histological features with the normal renal tissue of the untreated mice. These data indicated that VAAP/Tween 80 and PEG-PCL/VAAP were much less nephrotoxic than ACHP.

were composed of membrane-bound, small nuclear fragments surrounded by a rim of cytoplasm. In tumors treated with VAAP/Tween 80 and PEG-PCL/VAAP, the tumor cellularity decreased much more significantly, and the normal structure of tumor tissue was lost, which was consistent with the higher IRT induced by VAAP/Tween 80 and PEG-PCL/VAAP than ACHP. Moreover, we found that VAAP/Tween 80 and PEG-PCL/ VAAP showed less cytotoxicity to normal tissues. For instance, clinical application of platinum(II) drugs, such as cisplatin, was significantly limited by their side effects, particularly nephrotoxicity.28,30 Platinum(IV) prodrugs are expected to have minimized side effects to normal tissues. However, the treatment of ACHP still caused significant toxicity to normal

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CONCLUSION A conjugate of platinum(IV) prodrug, ACHP, with HDAC inhibitor VA, was synthesized and loaded in PEG-PCL nanoparticles. The conjugate VAAP showed similar inhibition on the HDAC activity to VA once intracellularly reduced. VA 2798

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(5) Sonnemann, J.; Gänge, J.; Pilz, S.; Stötzer, C.; Ohlinger, R.; Belau, A.; Lorenz, G.; Beck, J. F. Comparative Evaluation of the Treatment Efficacy of Suberoylanilide Hydroxamic Acid (SAHA) and Paclitaxel in Ovarian Cancer Cell Lines and Primary Ovarian Cancer Cells from Patients. BMC Cancer 2006, 6, 183−191. (6) Tatebe, H.; Shimizu, M.; Shirakami, Y.; Sakai, H.; Yasuda, Y.; Tsurumi, H.; Moriwaki, H. Acyclic Retinoid Synergises with Valproic Acid to Inhibit Growth in Human Hepatocellular Carcinoma Cells. Cancer Lett. 2009, 285, 210−217. (7) Jin, K. L.; Park, J. Y.; Noh, E. J.; Hoe, K. L.; Lee, J. H.; Kim, J. H.; Nam, J. H. The Effect of Combined Treatment with Cisplatin and Histone Deacetylase Inhibitors on HeLa cells. J. Gynecol. Oncol. 2010, 21 (4), 262−268. (8) Hirokawa, Y.; Arnold, M.; Nakajima, H.; Zalcberg, J.; Maruta, H. Signal Therapy of Breast Cancers by the HDAC Inhibitor FK228 That Blocks the Activation of PAK1 and Abrogates the Tamoxifenresistance. Cancer Biol. Ther. 2005, 4 (9), 956−960. (9) Maiso, P.; Carvajal-Vergara, X.; Ocio, E. M.; López-Pérez, R.; Mateo, G.; Gutiérrez, N.; Atadja, P.; Pandiella, A.; Miguel, J. F. S. The Histone Deacetylase Inhibitor LBH589 is a Potent Antimyeloma Agent That Overcomes Drug Resistance. Cancer Res. 2006, 66 (11), 5781−5789. (10) Tsurutani, J.; Soda, H.; Oka, M.; Suenaga, M.; Dor, S.; Nakamura, Y.; Nakatomi, K.; Shiozawa, K.; Yamada, Y.; Kamihira, S.; Kohno, S. Antiproliferative Effects of the Histone Deacetylase Inhibitor FR901228 on Small-cell Lung Cancer Lines and Drugresistant Sublines. Int. J. Cancer 2003, 104, 238−242. (11) Pathil, A.; Armeanu, S.; Venturelli, S.; Mascagni, P.; Weiss, T. S.; Gregor, M.; Lauer, U. M.; Bitzer, M. HDAC Inhibitor Treatment of Hepatoma Cells Induces Both TRAIL-independent Apoptosis and Restoration of Sensitivity to TRAIL. Hepatology 2006, 43 (3), 425− 434. (12) Bovenzi, V.; Momparler, R. L. Antineoplastic Action of 5-aza-2′Deoxycytidine and Histone Deacetylase Inhibitor and Their effect on The expression of Retinoic Acid Receptor β and Estrogen Receptor α Genes in Breast Carcinoma Cells. Cancer Chemother. Pharmacol. 2001, 48, 71−76. (13) Blabeta, R. A.; Micbaelis, M.; Driever, P. H.; Cinatl, J. J. Evolving anticancer drug valproic acid: Insight into the mechanism and clinical studies. Med. Res. Rev. 2005, 25 (4), 383−397. (14) Löscher, W. Basic Pharmacology of Valproate: A Review after 35 Years of Clinical Use for The Treatment of Epilepsy. CNS Drugs 2002, 16, 669−694. (15) Brodie, M. J.; Dichter, M. A. Antiepileptic Drugs. N. Engl. J. Med. 1996, 334, 168−175. (16) Kuendgen, A.; Schmid, M.; Schlenk, R.; Knipp, S.; Hildebrandt, B.; Steidl, C.; Germing, U.; Haas, R.; Dohner, H.; Gattermann, N. The Histone Deacetylase (HDAC) Inhibitor Valproic Acid As Monotherapy or In Combination With All-trans Retinoic Acid in Patients with Acute Myeloid Leukemia. Cancer 2006, 106, 112−119. (17) Chavez-Blanco, A.; Perez-Plasencia, C.; Perez-Cardenas, E.; Carrasco-Legleu, C.; Rangel-Lopez, E.; Segura-Pacheco, B.; TajaChayeb, L.; Trejo-Becerril, C.; Gonzalez-Fierro, A.; Candelaria, M.; Cabrera, G.; Duenas-Gonzalez, A. Antineoplastic Effects of the DNA Methylation Inhibitor Hydralazine and the Histone Deacetylase Inhibitor Valproic Acid in Cancer Cell Lines. Cancer Cell Int. 2006, 6, 2−10. (18) Drummond, D. C.; Noble, C. O.; Kirpotin, D. B.; Guo, Z.; Scott, G. K.; Benz, C. C. Clinical Development of Histone Deacetylase Inhibitors As Anticancer Agents. Annu. Rev. Pharmacol. Toxicol. 2005, 45, 495−528. (19) Duenas-Gonzalez, A.; Candelaria, M.; Perez-Plascencia, C.; Perez-Cardenas, E.; Cruz-Hernandez, E. d. l.; Herrera, L. A. Valproic Acid As Epigenetic Cancer Drug: Preclinical, Clinical and Transcriptional Effects on Solid Tumors. Cancer Treat. Rev. 2008, 34, 206−222. (20) Venkataramani, V.; Rossner, C.; Iffland, L.; Schweyer, S.; Tamboli, I. Y.; Walter, J.; Wirths, O.; Bayer, T. A. Histone Deacetylase Inhibitor Valproic Acid Inhibits Cancer Cell Proliferation via Down-

simply mixed with ACHP had no effect on the ACHP cytotoxicity, while the VAAP exerted a much enhanced cytotoxicity toward human cancer cell lines as compared to ACHP and even cisplatin. The flow cytometric analysis indicates that the introduction of the HDAC inhibitor VA to the Pt(IV) prodrug did not alter its cell cycle pattern but promoted apoptosis with the effective concentration significantly lower than ACHP (1/100 dose). In vivo, VAAP dispersed in Tween 80 or loaded in PEG-PCL nanoparticles had prolonged blood circulation times and led to 3−5 times increase in drug levels in tumor as compared to ACHP. They showed significantly increased in vivo antitumor activity but with decreased nephrotoxicity in comparison with ACHP. Therefore, this conjugate provides a new strategy to build potential anticancer agents for future chemotherapy.

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ASSOCIATED CONTENT

S Supporting Information *

Materials and methods including in vitro HDAC activity assay, determination of intracellular Pt accumulation, cellular uptake, flow cytometric analysis, blood clearance, in vivo antitumor activity, histological analysis, and statistical analysis as well as figures for the characterizations of VAAP and its loading in PEG-PCL micelles, dose-responsive curves of in vitro cytotoxicity, and the cell cycle analysis. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Address: Center for Bionanoengineering, Zhejiang University, Hangzhou 310027, China. Tel/Fax: +86 571 87953993. E-mail: [email protected] (J.T.) or [email protected] (Y.S.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was funded by the National Fund for Distinguished Young Scholars (50888001), National Science Foundation of China (21090352 and 20904046), Program for Changjiang Scholars and Innovative Research Team in University of China, Fundamental Research Funds for Central Universities (2010QNA4022), and the Public Welfare Program (2011C21055) and Qianjiang Talent Program of Zhejiang Province (2010R10050).

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REFERENCES

(1) Shao, Y.; Gao, Z.; Marks, P. A.; Jiang, X. Apoptotic and Autophagic Cell Death Induced by Histone Deacetylase Inhibitors. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 18030−18035. (2) Marks, P. A.; Richon, V. M.; Breslow, R.; Rifkind, R. A. Histone Deacetylase Inhibitors as New Cancer Drugs. Curr. Opin. Oncol. 2001, 13 (6), 477−483. (3) Long, J.; Zhao, J.; Yan, Z.; Liu, Z.; Wang, N. Antitumor Effects of a Novel Sulfur-containing Hydroxamate Histone Deacetylase Inhibitor H40. Int. J. Cancer 2009, 124, 1235−1244. (4) Thaler, F.; Colombo, A.; Mai, A.; Amici, R.; Bigogno, C.; Boggio, R.; Cappa, A.; Carrara, S.; Cataudella, T.; Fusar, F.; Gianti, E.; Ventimiglia, S. J. d.; Moroni, M.; Munari, D.; Pain, G.; Regalia, N.; Sartori, L.; Vultaggio, S.; Dondio, G.; Gagliardi, S.; Minucci, S.; Mercurio, C.; Varasi, M. Synthesis and Biological Evaluation of NHydroxyphenylacrylamides and N-Hydroxypyridin-2-ylacrylamides as Novel Histone Deacetylase Inhibitors. J. Med. Chem. 2010, 53, 822− 839. 2799

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Molecular Pharmaceutics

Article

Regulation of the Alzheimer Amyloid Precursor Protein. J. Biol. Chem. 2010, 285 (14), 10678−10689. (21) Catalano, M. G.; Poli, R.; Pugliese, M.; Fortunati, N.; Boccuzzi, G. Valproic Acid Enhances Tubulin Acetylation and Apoptotic Activity of Paclitaxel on Anaplastic Thyroid Cancer Cell Lines. Endocr. Relat. Cancer 2007, 14, 839−845. (22) Ziauddin, M. F.; Yeow, W. S.; Maxhimer, J. B.; Baras, A.; Chua, A.; Reddy, R. M.; Tsai, W., Jr.; Cole, G. W.; Schrump, D. S.; Nguyen, D. M. Valproic Acid, An Antiepileptic Drug with Histone Deacetylase Inhibitory Activity, Potentiates The Cytotoxic Effect of Apo2L/TRAIL on Cultured Thoracic Cancer Cells through Mitochondria-dependent Caspase Activation. Neoplasia 2006, 8, 446−457. (23) Göttlicher, M.; Minucci, S.; Zhu, P.; Krämer, O. H.; Schimpf, A.; Giavara, S.; Sleeman, J. P.; Coco, F. L.; Nervi, C.; Pelicci, P. G.; Heinzel, T. Valproic Acid Defines A Novel Class of HDAC Inhibitors Inducing Differentiation of Transformed Cells. EMBO J. 2001, 20 (24), 6969−6978. (24) Gurvich, N.; Tsygankova, O. M.; Meinkoth, J. L.; Klein, P. S. Histone Deacetylase Is A Target of Valproic Acid-mediated Cellular Differentiation. Cancer Res. 2004, 64, 1079−1086. (25) Lin, C. T.; Lai, H. C.; Lee, H. Y.; Lin, W. H.; Chang, C. C.; Chu, T. Y.; Lin, Y. W.; Lee, K. D.; Yu, M. H. Valproic Acid Resensitizes Cisplatin-resistant Ovarian Cancer Cells. Cancer Sci. 2008, 99, 1218− 1226. (26) Boulikas, T.; Vougiouka, M. Cisplatin and Platinum Drugs At the Molecular Level. Oncol. Rep. 2003, 10, 1663−1682. (27) Jamieson, E. R.; Lippard, S. J. Structure, Recognition, and Processing of Cisplatin-DNA Adducts. Chem. Rev. 1999, 99, 2467− 2498. (28) Wheate, N. J.; Walker, S.; Craig, G. E.; Oun, R. The Status of Platinum Anticancer Drugs in the Clinic and in Clinical Trials. Dalton Trans. 2010, 39, 8113−8127. (29) Uchino, H.; Matsumura, Y.; Negishi, T.; Koizumi, F.; Hayashi, T.; Honda, T.; Nishiyama, N.; Kataoka, K.; Naito, S.; Kakizoe, T. Cisplatin-incorporating Polymeric Micelles (NC-6004) Can Reduce Nephrotoxicity and Neurotoxicity of Cisplatin In Rats. Br. J. Cancer 2005, 93, 678−687. (30) Pinzani, V.; Bressolle, F.; Haug, I. J.; Galtier, M.; Blayac, J. P.; Balmès, P. Cispatin-induced Renal Toxicity and Toxicity-modulating Strategies: A Review. Cancer Chemother. Pharmacol. 1994, 35, 1−9. (31) Hall, M. D.; Hambley, T. W. Platinum (IV) Antitumor Compounds: Their Bioinorganic Chemistry. Coord. Chem. Rev. 2002, 232, 49−67. (32) Nemirovski, A.; Kasherman, Y.; Tzaraf, Y.; Gibson, D. Reduction of cis,trans,cis-[PtCl2(OCOCH3)2(NH3)2] by Aqueous Extracts of Cancer Cells. J. Med. Chem. 2007, 50, 5554−5556. (33) Bruijnincx, P. C.; Sadler, P. J. New Trends for Metal Complexes with Anticancer Activity. Curr. Opin. Chem. Biol. 2008, 12, 197−206. (34) Galanski, M.; Jakupec, M. A.; Keppler, B. K. Update of The Preclinical Situation of Anticancer Platinum Complexes: Novel Design Strategies and Innovative Analytical Approaches. Curr. Med. Chem. 2005, 12, 2075−2094. (35) Choy, H.; Park, C.; Yao, M. Current Status and Future Prospects for Satraplatin, An Oral Platinum Analogue. Clin. Cancer Res. 2008, 14, 1633−1638. (36) Brandon, R. J.; Dabrowiak, J. C. Synthesis, Characterization, and Properties of A Group of Platinum (IV) Complexes. J. Med. Chem. 1984, 27, 861−865. (37) Ang, W. H.; Pilet, S.; Scopelliti, R.; Bussy, F.; Juillerat-Jeanneret, L.; Dyson, P. J. Synthesis and Characterization of Platinum (IV) Anticancer Drugs with Functionalized Aromatic Carboxylate Ligands: Influence of The Ligands on Drug Efficacies and Uptake. J. Med. Chem. 2005, 48, 8060−8069. (38) Rieter, W. J.; Pott, K. M.; Taylor, K. M. L.; Lin, W. Nanoscale Coordination Polymers for Platinum-based Anticancer Drug Delivery. J. Am. Chem. Soc. 2008, 130, 11584−11585. (39) Zhou, S.; Deng, X.; Yang, H. Biodegradable Poly(εcaprolactone)-poly(ethylene glycol) Block Copolymers: Charateriza-

tion and Their Use As Drug Carriers for A Controlled Delivery System. Biomaterials 2003, 24, 3563−3570. (40) Bogdanov, B.; Vidts, A.; Bulcke, A. V. D.; Verbeeck, R.; Schacht, E. Synthesis and Thermal Properties of Poly(ethylene glycol)-poly(εcaprolactone) Copolymers. Polymer 1998, 39, 1631−1636. (41) Chu, Q.; Zhang, J.; Tian, F. Determination of Platinum (IV) in Platinum (IV)-tin (II) Chloride-Brilliant Green System by Sepectrophotometry. Hydrometal. China 2006, 25, 54−56. (42) Olszewski, U.; Ach, F.; Ulsperger, E.; Baumgartner, G.; Zeillinger, R.; Bednarski, P.; Hamilton, G. In Vitro Evaluation of Oxoplatin: An Oral Platinum (IV) Anticancer Agent. Met. Based Drugs 2009, 2009, 1−11. (43) Yokoyama, M.; Miyauchi, M.; Yamada, N.; Okano, T.; Sakurai, Y.; Kataoka, K.; Inoue, S. Characterization and Anticancer Activity of the Micelle-forming Polymeric Anticancer Drug Adriamy-cinconjugated Poly(ethylene glycol)-poly(aspartic acid) Block Copolymer. Cancer Res. 1990, 50, 1693−1700. (44) Schuchumann, M.; Schulze-Bergkamen, H.; Fleischer, B.; Schattenberg, J. M.; Siebler, J.; Weinmann, A.; Teufel, A.; Wörns, M.; Fischer, T.; Strand, S.; Lohse, A. W.; Galle, P. R. Histone Deacetylase Inhibition by Valproic Acid Down-regulates c-FLIP/ CASH and Sensitizes Hepatoma Cells towards CD95- and TRAIL Receptor-mediated Apoptosis and Chemotherapy. Oncol. Rep. 2006, 15, 227−230. (45) Takai, N.; Kawamata, N.; Gui, D.; Said, J. W.; Miyakawa, I.; Koeffler, H. P. Human Ovarian Carcinoma Cells: Histone Deacetylase Inhibitors Exhibits Antiproliferative Activity and Potently Induce Apoptosis. Cancer 2004, 101, 2760−2770. (46) Lee, D. Y.; Hayes, J. J.; Pruss, D.; Wolffe, A. P. A Positive Role for Histone Acetylation In Transcription Factor Access To Nucleosomal DNA. Cell 1993, 72, 73−84. (47) Ellis, L. T.; Er, H. M.; Hambley, T. W. The Influence of The Axial Ligands of A Series of Platinum (IV) Anti-cancer Complexes on Their Reduction to Platinum (II) and Reaction With DNA. Aust. J. Chem. 1995, 48, 793−806. (48) Nishiyama, N.; Okazaki, S.; Cabral, H.; Miyamoto, M.; Kato, Y.; Sugiyama, Y.; Nishio, K.; Matsumura, Y.; Kataoka, K. Novel Cisplatinincorporated Polymeric Micelles Can Eradicate Solid Tumors In Mice. Cancer Res. 2003, 63, 8977−8983. (49) Sağol, O.; Culhaci, N.; Yörükoğlu, K.; Ozen, E.; Canda, T. Quantification of Tumor Cellularity and Mitotic Index In Invasive Ductal Carcinoma of The Breast. Anal. Quant. Cytol. Histol. 2001, 23, 21−26. (50) Rajan, R.; Poniecka, A.; Smith, T. L.; Yang, Y.; Frye, D.; Pusztai, L.; Fiterman, D. J.; Gal-gombos, E.; Whitman, G.; Rouzier, R.; Green, M.; Kuerer, H.; Buzdar, A. U.; Hortobagyi, G. N.; Symmans, W. F. Change In Tumor Cellularity of Breast Carcinoma After Neoadjuvant Chemotherapy As A Variable In The Pathologic Assessment of Response. Cancer 2004, 100, 1365−1373.

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