Valuable Insight into the Anticancer Activity of the Platinum-Histone

May 16, 2012 - Celine J. Marmion,. ‡ and Jana Kasparkova*. ,†. †. Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Kr...
0 downloads 0 Views 764KB Size
Download PDF
Article pubs.acs.org/molecularpharmaceutics

Valuable Insight into the Anticancer Activity of the Platinum-Histone Deacetylase Inhibitor Conjugate, cis-[Pt(NH3)2malSAHA−2H)] Viktor Brabec,† Darren M. Griffith,‡ Anna Kisova,† Hana Kostrhunova,† Lenka Zerzankova,† Celine J. Marmion,‡ and Jana Kasparkova*,† †

Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Kralovopolska 135, 61265 Brno, Czech Republic Centre for Synthesis and Chemical Biology, Department of Pharmaceutical & Medicinal Chemistry, Royal College of Surgeons in Ireland, 123 St. Stephen’s Green, Dublin 2, Ireland

‡

S Supporting Information *

ABSTRACT: cis-[PtII(NH3)2(malSAHA−2H)], a cisplatin adduct conjugated to a potent histone deacetylase inhibitor (HDACi), suberoylanilide hydroxamic acid (SAHA), was previously developed as a potential anticancer agent. This Pt− HDACi conjugate was demonstrated to have comparable cytotoxicity to cisplatin against A2780 ovarian cancer cells but significantly reduced cytotoxicity against a representative normal cell line, NHDF. Thus, with a view to (i) understanding more deeply the effects that may play an important role in the biological (pharmacological) properties of this new conjugate against cancer cells and (ii) developing the next generation of Pt−HDACi conjugates, the cytotoxicity, DNA binding, cellular accumulation and HDAC inhibitory activity of cis-[PtII(NH3)2(malSAHA−2H)] were investigated and are reported herein. cis[PtII(NH3)2(malSAHA−2H)] was found to have marginally lower cytotoxicity against a panel of cancer cell lines as compared to cisplatin and SAHA. cis-[PtII(NH3)2(malSAHA−2H)] was also found to accumulate better in cancer cells but bind DNA less readily as compared to cisplatin. DNA binding experiments indicated that cis-[PtII(NH3)2(malSAHA−2H)] bound DNA more effectively in cellulo as compared to in cell-free media. Activation of the Pt−HDACi conjugate was therefore investigated. The binding of cis-[PtII(NH3)2(malSAHA−2H)] to DNA was found to be enhanced by the presence of thiol-containing molecules such as glutathione and thiourea, and activation occurred in cytosolic but not nuclear extract of human cancer cells. The activity of cis[Pt(NH3)2(malSAHA−2H)] as a HDAC inhibitor was also examined; the conjugate exhibited no inhibition of HDAC activity in CH1 cells. In light of these results, novel Pt−HDACi conjugates are currently being developed, with particular emphasis, through subtle structural modifications, on enhancing the rate of DNA binding and enhancing HDAC inhibitory activity. KEYWORDS: cisplatin, SAHA, histone deacetylase inhibitor, cytotoxicity, cellular accumulation, DNA binding

■

INTRODUCTION Since the discovery of the antitumor properties of cisplatin and its introduction into the clinical arena in 1978, platinum (Pt) complexes have been vigorously investigated as potential cancer chemotherapeutics.1 Surprisingly to date though, only two additional structurally related Pt drugs have been approved for worldwide clinical use, namely, carboplatin and oxaliplatin, and three others, heptaplatin, lobaplatin and nedaplatin, are in limited use.2−4 These PtII-based drugs are among the most effective chemotherapeutics in medical oncology today. They are widely used to treat a variety of tumors including ovarian, colorectal, non-small-cell lung, and genitourinary cancers and are believed to operate by a similar mechanism.5 The activity of cisplatin and its analogues in killing tumor cells is based on their ability to enter the cell, to bind DNA and to induce cellular responses.6,7 Upon entering the cell, the Pt drugs are activated by aquation and bind nuclear DNA, forming various types of DNA covalent adducts.8 Formation of DNA lesions is an important determinant of the anticancer activity of Pt-based drugs, leading to arrest of key cellular functions, such as replication or transcription, and triggering apoptosis or necrosis.5 © 2012 American Chemical Society

Unfortunately, the clinical use of cisplatin and its direct analogues is associated with several drawbacks, such as limited activity against a range of sensitive human tumors, severe side effects and acquired resistance.9 These limitations have undoubtedly been the impetus behind the search for novel Pt drugs with improved pharmacological properties and a broader range of antitumor activity. One strategy to overcome these drawbacks is to develop novel Pt drug candidates that can not only bind DNA but also interact with other molecular targets, thus enhancing their therapeutic potential.10 In one such search for new, therapeutically more effective Pt drugs, cisplatin was combined with a derivatized form of another anticancer drug, suberoylanilide hydroxamic acid (SAHA, vorinostat).11 SAHA is a potent inhibitor of histone deacetylases (HDACs), a class of zinc metalloenzymes that play a key role in the epigenetic regulation of gene expression. Histones are the main protein component of chromatin around which DNA coils. HDACs Received: Revised: Accepted: Published: 1990

January 20, 2012 May 10, 2012 May 16, 2012 May 16, 2012 dx.doi.org/10.1021/mp300038f | Mol. Pharmaceutics 2012, 9, 1990−1999

Molecular Pharmaceutics

Article

confer selectivity to the drug candidate thereby reducing the nonselective toxicity of classical Pt drugs. In their preliminary study, cis-[Pt(NH3)2(malSAHA−2H)] was found to possess dual DNA binding and HDAC inhibitory activity and selectivity for cancer cells over normal cells.11 Thus, with a view to (i) understanding more deeply the effects that may play an important role in the biological (pharmacological) properties of this new Pt−HDACi conjugate (Figure 1) against cancer cells and (ii) developing the next generation of Pt−HDACi conjugates, the cytotoxicity, DNA binding, cellular accumulation and HDAC inhibitory activity of cis[PtII(NH3)2(malSAHA−2H)] were thoroughly investigated and are reported herein.

mediate the deacetylation of core histone lysine residues, resulting in a condensed chromatin structure and ultimately transcriptional repression.12 Inhibition of HDAC function can thus affect chromatin structure and function. SAHA has been shown to cause growth arrest, differentiation and/or apoptosis of many tumor types in vitro and in vivo.13−16 It binds directly to the catalytic site of the enzyme, blocking substrate access. It inhibits class I, II and IV HDACs at nanomolar concentrations and was the first FDA-approved pan-HDAC inhibitor (HDACi) to enter the clinic as a treatment for cutaneous Tcell lymphoma.17 SAHA is well tolerated in patients, particularly at doses which exhibit a potent anticancer effect.18,19 More recently inhibitors of HDACs have been shown to target non-histone proteins such as those involved in cellular proliferation, migration, death, DNA repair, angiogenesis, inflammation and the immune response.20 A range of structurally diverse inhibitors of HDAC have been shown to be effective anticancer agents via multiple mechanisms, including inducing cell-cycle arrest, intrinsic and extrinsic apoptotic mechanisms, mitotic cell death, autophagic cell death, generation of reactive oxygen species, inhibiting angiogenesis and improving NK cell-mediated tumor immunity.12,20 Several of these inhibitors are now undergoing clinical trials.21 Marmion et al. devised a strategy to derivatize SAHA in such a way as to facilitate its binding to Pt, while minimizing any impact on its HDAC inhibitory activity. A molecular modeling study (data not shown) indicated that the introduction of malonate, as a linker, onto the phenyl ring of SAHA would not limit its HDAC inhibitory activity. In turn they developed a novel bifunctional anticancer Pt drug candidate, cis-[Pt(NH3)2(malSAHA−2H)], Figure 1. In doing so they anticipated

■

MATERIALS AND METHODS Material and Reagents. cis-[PtII(NH3)2(malSAHA−2H)], cis-[Pt(NH3)2(mal−2H)], and malSAHA were prepared as described previously.11 Cisplatin and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich sro (Prague, Czech Republic). Calf thymus (CT) DNA (42% G + C, mean molecular mass ca. 20 000 kDa) was prepared and characterized as described previously.23,24 MTT [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide] was from Calbiochem (Darmstadt, Germany). RPMI 1640 medium, fetal bovine serum (FBS), trypsin/EDTA and DMEM medium were from PAA (Pasching, Austria). Antibodies: Ac-histone H3 (Lys 24):sc-34262 was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA); monoclonal anti-β-actin clone AC-15 was purchased from Sigma (St. Louis, MO, USA). Secondary antibodies: for H3 antibody, sc-2004 (goat-antirabbit Igb-HRP) was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA); for actin, peroxidase conjugated goatanti-mouse Igb (H+L) was from Thermo Scientific Pierce (Rockford, IL, USA). Nitrocellulose membranes (0.45 μm) were from Bio-Rad Laboratories (Hercules, CA, USA). Dithiothreitol (DTT) was from Merck KgaA (Darmstadt, Germany). Sodium dodecyl sulfate (SDS) and gentamycin were from Serva (Heidelberg, Germany).The Bradford reagent and phenylmethanesulfonyl fluoride (PMSF) were from Sigma (Prague, Czech Republic). The BSA standard was from Thermo Scientific Pierce (Rockford, IL, USA). The DNAzol kit was from Invitrogen Dynal AS (Oslo, Norway). Cell Lines. The A2780, CH1 human ovarian carcinoma, human colon adenocarcinoma SW480, human pulmonary adenocarcinoma A549, and human brest carcinoma SK-BR-3 cell lines were kindly supplied by Prof. B. Keppler from University of Vienna. A2780 cells were grown in RPMI 1640 medium (PAA) supplemented with gentamycin (50 μg mL−1; Serva) and heat inactivated fetal bovine serum (10%, PAA). SW480, SK-BR-3, A549 and CH1 cells were grown in Dulbecco’s modified Eagle’s medium (PAA) supplemented with gentamycin and 10% heat-inactivated fetal bovine serum. Cytotoxicity Assay. The cells were seeded in 96-well tissue culture plates at a density of 10 000 cells per well. After overnight incubation, the cells were treated with the compounds tested. After 72 h of incubation 10 μL of MTT (2.5 mg mL−1; CALBIOCHEM) was added to each well and incubated for 4 h in culture conditions. At the end of the incubation period, the medium was removed and the formazan product was dissolved in DMSO (100 μL). The cell viability was evaluated by measurement of the absorbance at 570 nm, using an absorbance reader SUNRISE TECAN SCHOELLER. IC50 values (compound concentration that produces 50% of

Figure 1. Structures of compounds used in the present work.

that the novel Pt−HDACi conjugate would be activated inside cells releasing a Pt moiety and a derivatized HDACi. The Pt moiety could subsequently bind DNA in much the same way as classical Pt drugs, but because of the release of the derivatized HDACi, the conjugate might, via additive or synergistic effects, be active against a broader spectrum of human cancer cells and/or cells that have acquired resistance to Pt-based regimes. It has also been previously proposed that the observed synergism between SAHA (or other HDACis) and cytotoxic agents is due to the relaxed nature of the chromatin structure following HDAC inhibition providing greater access for the cytotoxic agent to its target DNA.22 In addition as Pt drugs react indiscriminately in the body giving rise to many of their drawbacks, Marmion et al. speculated that the presence of the inhibitor, with its known affinity for tumor cells, might also 1991

dx.doi.org/10.1021/mp300038f | Mol. Pharmaceutics 2012, 9, 1990−1999

Molecular Pharmaceutics

Article

scraped into tubes and centrifuged for 10 min at 0 °C (1000g). The pellets were resuspended in the lysis buffer [Tris·HCl (10 mM, pH 8.0), KCl (60 mM), EDTA (1.2 mM), DTT (1 mM), PMSF (0.1 mM), NP-40 (0.05%)] for 10 min on ice and centrifuged for 4 min at 0 °C (800g). The pellets were rinsed with the above lysis buffer without PMSF and NP-40 and incubated in nuclear extraction buffer [Tris·HCl (20 mM, (pH 8.0), NaCl (420 mM), MgCl2 (0.7 mM), EDTA (0.25 mM), glycerol (25%)] for 30 min at 4 °C, and then centrifuged for 15 min at 4 °C (15000g). Protein concentration of the nuclear extracts was determined by the Bradford assay, and stored at −80 °C until used. Histone Deacetylase Assay. The CH1 nuclear extracts were tested for their HDAC activity following the protocol recommended by the manufacturer of the Colorimetric HDAC Activity Assay Kit (BioVision Research Products, Mountain View, CA, USA). The standard curve was constructed using the known amount of the deacetylated standard included in the kit. The absolute amount of deacetylated product generated in the samples was calculated from the standard curve. The values in the bar graph are expressed as the concentration (μM) of deacetylated product in the sample minus the concentration of deacetylated product in the control. 50 μg of nuclear protein was added to each sample.25 Acid Extraction of Histones. Acid extraction of histones was performed as described previously26 with modifications. Cells were grown on 100 mm Petri dishes for 24 h. The compounds were added to isotoxic concentrations (IC50), and after another 24 h the cells were harvested. The dishes were washed three times with ice-cold PBS and the cells scraped into tubes and centrifuged for 10 min at 0 °C (1000g). Cells were then suspended with five volumes of lysis buffer [HEPES (10 mM, pH 7.9), MgCl2 (1.5 mM), KCl (10 mM), DTT (0.5 mM), and PMSF (1.5 mM)] and hydrochloride acid at a final concentration of 0.2 M and subsequently lysed on ice for 30 min. After centrifugation at 11000g for 10 min at 4 °C, the supernatant fraction containing the acid-soluble proteins was retained. Supernatant was dialyzed against acetic acid (0.1 M) twice for 1−2 h each and then dialyzed against H2O for 1 h, 3 h and overnight. Dialysis was performed using Spectra/Pore 3 Dialysis Membranes 3500 MWCO. The protein concentration of the extracts was determined by Bradford assay using bovine serum albumin as a standard. Western Blot Analysis. The extracts containing 10 μg of protein were subjected to SDS−PAGE (15% PAA gel) in loading buffer [Tris·HCl (50 mM, pH 8.0), SDS (2%), bromphenol blue (0.1%), glycerol (10%)] and blotted onto nitrocellulose membrane in a transfer buffer containing TrisHCl (25 mM, pH 8.0), glycine (192 mM), methanol (20%). Detection of acetylated histones H3 was carried out by overnight incubation of the membrane with primary antibody (Ac-histone H3 (Lys 24):sc-34262; dilution 1:200). Then the membrane was washed with TBS·Tween and incubated with secondary horseradish perixodase conjugated antibody (sc2004, goat-anti-rabbit Igb-HRP, dilution 1:5000) for 2 h. Equal loading of transferred proteins was verified by detection of βactin using monoclonal anti-β-actin clone AC-15 primary antibody, dilution 1:5000 followed by incubation of the membrane with secondary peroxidase conjugated goat-antimouse Igb (H+L) (dilution 1:500). The proteins were visualized using Super Signal West Pico Chemiluminiscent Substrate Kits (Pierce) with luminiscent image analyzer (LAS3000, FUJIFILM). Quantification of visualized bands was

cell growth inhibition) were calculated from curves constructed by plotting cell survival (%) versus drug concentration (μM). All experiments were made in triplicate. The reading values were converted to the percentage of control (% cell survival). Cytotoxic effects were expressed as IC50, which is the concentration inducing 50% inhibition of metabolic activity of the cells. Interaction of Platinum Compounds with Cells Monitored by Real-Time Cell Electronic Sensing. Background of the E-plates was determined in 100 μL of medium (RPMI), and subsequently, 50 μL of the A2780 cell suspension was added (104 cells/well). E-plates were immediately placed into the real-time cell analyzer (RTCA) station (xCELLigence RTCA SP Instrument, ROCHE). Cells were grown for 24 h (in a humidified incubator at 37 °C in a 5% CO2). Subsequently, the cells were treated with 50 μL of medium alone (control) or with 50 μL of medium containing varying inhibitory concentrations of cis-[Pt(NH3) 2(malSAHA−2H)], cis-[Pt(NH3)2(mal−2H)], malSAHA, SAHA or cisplatin, and impedance was monitored for the first 6 h every 15 min and for the rest of the test period every 30 min. The electronic readout, cell-sensor impedance, is displayed as an arbitrary unit called cell index (CI). CI at each time point is defined as (Rt − Rb)/15 where Rt is defined as the cell-electrode impedance of the well with the cells at different time points, and Rb is defined as the background impedance of the well with the medium alone. Normalized CI is calculated by dividing the cell index at particular time points by the CI at the time of interest. Each treatment was performed in triplicate. Treatment of Cells Used for Determination of Cellular Accumulation. The A2780, SW480, A549, SK-BR-3 and CH1 cells were seeded in 100 mm tissue culture dishes (30 000 per cm2). After overnight incubation, the cells were treated with the compounds (10 μM) for 24 h or SW480 cells also for 0.5, 1, 2, 4 and 8 h. The attached cells were washed twice with PBS (4 °C) and the pellet stored at −80 °C. Pellet was digested by high pressure microwave mineralization. The metal content in the samples was determined by flameless atomic absorption spectrometry (FAAS) or inductively coupled plasma mass spectroscopy (ICP-MS). Determination of Metal Binding to DNA in Culture Cells. Cells were washed, pelleted and resuspended in lysis buffer. Afterward, cell suspension was treated with proteinase K and subsequently with RNase A. DNA was extracted with phenol−chloroform−isoamyl alcohol, precipitated and washed with ethanol, dried and resuspended in water. The DNA content in each sample was measured by UV spectrophotometry, and metal bound to DNA was determined by ICP-MS. Preparation of Cytosolic Extracts. HeLa cells were grown to confluence on 100 mm Petri dishes, washed three times with ice-cold PBS, scraped into tubes and centrifuged for 10 min at 0 °C (1000g). The pellet was resuspended in the lysis buffer [Tris·HCl (10 mM, pH 8.0), KCl (60 mM), EDTA (1.2 mM), DTT (1 mM), PMSF (0.1 mM), NP-40 (0.05%)] for 10 min on ice and centrifuged for 10 min at 0 °C (1200g). Protein concentration of the cytosolic extract was determined by the Bradford assay, and stored at −80 °C until used. Preparation of Nuclear Extracts. Nuclear extracts for the determination of HDAC activity were prepared as follows: Cells were grown on 100 mm Petri dishes for 24 h. The compounds were added to isotoxic concentrations (IC50), and after another 24 h the cells were harvested. Briefly, the dishes were washed three times with ice-cold PBS and the cells 1992

dx.doi.org/10.1021/mp300038f | Mol. Pharmaceutics 2012, 9, 1990−1999

Molecular Pharmaceutics

Article

Table 1. Inhibition of Growth of Cancer Cells by Single Agents Tested in the Present Work and Comparison with Cisplatina,b,c cis-[Pt(NH3)2(malSAHA−2H)] cisplatin cis-[Pt(NH3)2(mal−2H)] SAHA malSAHA a

SKBR

SW480

A549

A2780

9.9 ± 1.8 7.5 ± 1.2 14.6 ± 2.5 1.7 ± 0.8 >200

13.4 ± 3.9 8.0 ± 0.3 23.2 ± 1.5 5.3 ± 1.8 >200

12.7 ± 1.8 6.3 ± 0.1 10.0 ± 1.9 7.2 ± 1.7 >200

13.7 ± 2.8 2.9 ± 0.3 6.5 ± 1.8 11.9 ± 1.9 >200

CH1 3.8 ± 1.0 ± 2.5 ± 1.2 ± >200

0.5 0.5 0.5 0.2

NHDFd 83 ± 7.6 10 ± 1.8 48 ± 2.2 4.5 ± 0.3 335 ± 3.1

IC50 mean values ± standard deviations (μM). bDrug-treatment period was 72 h. cThe experiments were made in triplicate. dTaken from ref 11.

compounds.28 Figure 2 shows the TCRPs of A2780 cells that have been treated with cis-[Pt(NH3)2(malSAHA−2H)], cis[Pt(NH3)2(mal−2H)], malSAHA, SAHA or cisplatin.

performed by densitometry using AIDA software (Advanced Image Data Analyzer). Other Physical Methods. Absorption spectra of DNA were measured with a Beckmann DU-7400 spectrophotometer. The analysis with the aid of ICP-MS was perfomed using Agilent 7500 (Agilent, Japan). The FAAS measurements were carried out on a Varian AA240Z Zeeman atomic absorption spectrometer equipped with a GTA 120 graphite tube atomizer.

■

RESULTS AND DISCUSSION Cytotoxicity of Single Agents. The effect on cell-growth inhibition of cis-[PtII(NH3)2(malSAHA−2H)] and the related cisplatin, cis-[Pt(NH3)2(mal−2H)] and SAHA was screened against a panel of cisplatin-sensitive and -resistant human cancer cell lines using the MTT test, which measures mitochondria dehydrogenase activity as a marker of cell viability.27 The corresponding IC50 values (concentrations of agents inhibiting cell growth by 50%) are reported in Table 1. cis-[PtII(NH3)2(malSAHA−2H)] exhibited significant toxicity in all tumor cells tested in this work; its cytotoxicity was comparable to or slightly less than that of cisplatin or its cis[Pt(NH3)2(mal−2H)] congener as well as SAHA alone. The results, at first glance, might indicate that combining the DNAbinding Pt with the HDACi SAHA in one molecule represents no significant advantage with respect to its toxic potency in cancer cell lines when compared with cisplatin, cis-[Pt(NH3)2(mal−2H)] or SAHA. However, cis[PtII(NH3)2(malSAHA−2H)] has been shown from a previous study11 to be significantly less toxic to normal human dermal fibroblast (NHDF) cells as compared to cisplatin, cis-[Pt(NH3)2(mal−2H)] or SAHA indicating that the presence of the SAHA ligand might be conferring selectivity for tumor cells over a representative normal cell line. In an effort to optimize the therapeutic profile of cis[PtII(NH3)2(malSAHA−2H)], it was deemed important to elucidate the biochemical and pharmacological factors that may be affecting its cytotoxic properties. Hence, our primary objective in the present study was to understand more fully molecular and cellular mechanisms underlying cytostatic effects of the combined agent cis-[PtII(NH3)2(malSAHA−2H)] with a view to elucidating why cis-[PtII(NH3)2(malSAHA−2H)] failed to exhibit significantly improved toxicity in a number of cancer cell lines as compared with cisplatin and SAHA. Impedance-Based Monitoring in Real Time of the Effects on Cell Growth. We have used impedance-based time-dependent cell response profiling (TCRP) to measure and characterize cellular responses to cis-[Pt(NH3)2(malSAHA−2H)], cis-[Pt(NH3)2(mal−2H)], malSAHA, SAHA and cisplatin. It has been demonstrated that impedance-based monitoring of cellular responses to biologically active small molecule compounds produces timedependent cellular response profiles (TCRPs), which can be predictive of mechanism of action of small molecule

Figure 2. Interactions of cis-[Pt(NH3)2(malSAHA−2H)], cis-[Pt(NH3)2(mal−2H)], malSAHA, SAHA and cisplatin with A2780 cells monitored by a real-time cell analyzer (RTCA). The vertical line indicates the start of treatment after allowing the cells to grow and adhere to the microelectrodes for 24 h. Cell indices were normalized to account for differences in cell counts that exist across the wells prior to treatment. Incubations were performed in triplicate with 104 cells/ well using inhibitory drug concentrations determined for 72 h of incubation in a MTT assay (Table 1).

The TCRPs of all compounds are characterized by an initial negligible change in cell index in comparison with the control followed by a decrease in cell index below control levels in the case of the cellular responses to cis-[Pt(NH3)2(malSAHA−2H)], cis-[Pt(NH3)2(mal−2H)], SAHA and cisplatin, reflecting cytotoxic response. Thus, the TCRP of the latter compounds coclustered with the TCRPs of compounds interfering with DNA synthesis and replication, transcription and translation,28 which are also known to induce cell-cycle arrest followed by induction of cell death. Nevertheless, cis-[Pt(NH3)2(malSAHA−2H)] and cis-[Pt(NH3)2(mal−2H)] produced TCRP different from that produced by cisplatin and SAHA. Most strikingly, while cisplatin and SAHA cause moderate or even complete killing, respectively, of adherent cells at the longest times of cell growth, cis-[Pt(NH3)2(malSAHA−2H)] and cis-[Pt(NH3)2(mal−2H)] killed adherent cells much less pronouncedly even at the longest times of their growth. Moreover, the TCRP for SAHA and malSAHA is characterized by an initial and sustained increase in cell index above that of the control. This increase observed for SAHA was followed by a decrease in cell index below control levels, reflecting ultimate cytotoxic response. In contrast, this increase observed for malSAHA was followed by only a very small decrease in cell index below control levels and only at the longest times of cell growth, suggesting that efficiency of malSAHA to kill adherent cells is markedly lowered compared to SAHA. The results of impedance-based real-time monitoring of the e ff e c t s o f ci s - [ P t ( NH 3 ) 2 ( m a l S A H A − 2 H ) ] , ci s - [ P t 1993

dx.doi.org/10.1021/mp300038f | Mol. Pharmaceutics 2012, 9, 1990−1999

Molecular Pharmaceutics

Article

relation to its cytotoxicity in cellulo as compared to that of cisplatin. DNA Binding in a Cell-Free Medium. Nuclear DNA is an important molecular target for Pt anticancer drugs, which bind purine bases.31,32 The resulting DNA damage triggers downstream effects including inhibition of replication and transcription and cell cycle arrest.1,6 The bifunctional Pt−HDACi conjugate cis-[Pt(NH3)2(malSAHA−2H)] was designed with the intention to endow the conjugate with HDAC inhibitory activity while retaining its DNA binding properties.11 In the first instance therefore, the ability of cis-[Pt(NH3)2(malSAHA−2H)] to bind DNA in cell-free media was investigated and compared with that of cisplatin and cis[Pt(NH3)2(mal−2H)]. The efficiency of binding to CT DNA was determined at an ri (molar ratio of free Pt complex to nucleotide phosphate) of 0.09 in NaClO4 (10 mM) at 37 °C in the dark. The Pt complexes were incubated with the CT DNA, and aliquots withdrawn at indicated time intervals were quickly cooled in an ice bath and then exhaustively dialyzed against NaClO4 (10 mM) at 4 °C to remove free (unbound) Pt compounds. The content of Pt in these DNA samples was then determined by FAAS (Table 3).

(NH3)2(mal−2H)], malSAHA, SAHA and cisplatin on cell growth indicate that there are differences in extent and dynamics of cell cycle perturbations induced by these compounds. In contrast to standard methods for cell viability or proliferation, such as MTT, representing end-point analysis of whole cell population, this method makes it possible to register very small and rapid changes in cell count, cell adhesion, and cell morphology due to drug toxicity. The results of these experiments suggest that the reduced cell viability determined in the colorimetric assay translates into cell death. Cellular Accumulation. The early phases by which Pt compounds exert their anticancer activity are cellular entry, activation, and DNA binding.6 Before the active form of a Pt drug reaches its major pharmacological target in the cell nucleus, the Pt drug has to first accumulate in cells. It has been demonstrated that higher accumulation of Pt drugs generally correlates with enhanced cytotoxicity.29,30 To examine the cellular accumulation of cis-[Pt(NH3)2(malSAHA−2H)] and its parent Pt complexes cisplatin and cis-[Pt(NH3)2(mal−2H)], the cellular levels of Pt were first measured after a 24 h exposure of human cancer cells A2780, SW480, A549, SK-BR-3 and CH1 to equimolar concentrations of these Pt agents (10 μM). The uptake of the complexes in different cells is listed in Table 2. The intracellular

Table 3. Binding of Platinum Complexes (%a) Tested in This Work to Calf Thymus DNA in a Cell-Free Mediumb,c

Table 2. Uptake (pmol of Pt/2 × 106 cells) of Pt Complexes into Cellsa,b cis[Pt(NH3)2(malSAHA−2H)] SKBR SW480 A549 A2780 CH1

6568 5422 6419 5535 1595

± ± ± ± ±

106 122 119 165 58

cisplatin 876 392 615 612 425

± ± ± ± ±

30 12 25 38 22

cis[Pt(NH3)2(mal−2H)] 488 127 357 221 283

± ± ± ± ±

18 7 17 6 7

time (h)

cis[Pt(NH3)2(malSAHA−2H)]

cisplatin

cis[Pt(NH3)2(mal−2H)]

24 48 72

3.9 ± 0.5 5.4 ± 0.9 7.7 ± 0.9

98 ± 1 100 ± 2 99 ± 2

6.3 ± 0.4 11.3 ± 0.6 14.8 ± 0.9

a

Pt bound after incubation of DNA with cisplatin for 48 h was taken as 100%. bNaClO4 (10 mM), 37 °C after incubation lasting 24−72 h. c Each value represents the mean ± standard deviation for three independent experiments.

a Table shows the uptake of Pt complexes at their 10 μM concentrations after 24 h of cell treatment. Each value shown in the table (mean ± standard deviation) is in pmol of Pt/2 × 106 cells. bThe experiments were performed in triplicate.

Cisplatin bound quantitatively as expected, whereas only ca. 4 or 6% of cis-[Pt(NH 3 ) 2 (malSAHA −2H )] or cis-[Pt(NH3)2(mal−2H)], respectively, bound after 24 h or ca. 8 or 15% after 72 h. The DNA binding experiments carried out in the present work indicated that modification reactions resulting in the irreversible coordination of cis-[Pt(NH3)2(malSAHA−2H)] or cis-[Pt(NH3)2(mal−2H)] were very slow, such that only a small portion of the Pt present in the bulk of solution bound to DNA even after a relatively long time of incubation (72 h). Bidentate ligands bound to Pt are much more stable than chlorido ligands as leaving ligands. While the half-life for displacement of the chlorido ligands in an aquous solution of cisplatin is approximately 2 h at 37 °C,33 the half-life for the displacement of the malonato ligand in ethylenediamine platinum(II) malonate approaches 5 days.34 Similar stabilities were reported for other bidentate ligands, such as oxalate, ethylmalonate and 1,1-cyclobutane dicarboxylate.35,36 Hence, given their stability in aqueous solutions, Pt compounds containing bidentate ligands have been shown to react slowly with DNA in vitro.37,38 In fact, most available evidence suggests that the displacement of the bidentate ligand in Pt complexes to form the monoaquated species is rate-limiting for their reaction with DNA. Thus, it is not surprising that cis-[Pt(NH3)2(malSAHA−2H)] or cis-[Pt(NH3)2(mal−2H)] bound to DNA only very slowly in comparison to cisplatin. It is therefore reasonable to suggest that the reduced potency of cis[Pt(NH3)2(malSAHA−2H)] and cis-[Pt(NH3)2(mal−2H)] in

concentration of cis-[Pt(NH3)2(mal−2H)] was lower than that of cisplatin (1.5−3.1-fold), consistent with the lower toxicity of cis-[Pt(NH3)2(mal−2H)] relative to cisplatin (1.6−2.9-fold). In contrast, the accumulation of cis-[Pt(NH3)2(malSAHA−2H)] was markedly higher than that of cisplatin (∼4−14-fold). Thus, the trend was cis-[Pt(NH3)2(malSAHA-2H)] ≫ cis-[Pt(NH3)2(mal-2H)] > cisplatin. The same trend in the cellular accumulation was observed if SW480 cells were treated for 24 h with the platinum compounds at equitoxic concentrations corresponding to their IC50 values (Table S1 in the Supporting Information) or at the equimolar concentration (10 μM) for 0.5, 1, 2, 4 and 8 h (Table S2 in the Supporting Information). Thus, the results revealed that the presence of SAHA in cis[Pt(NH3)2(malSAHA−2H)] significantly facilitated transport of this conjugate across cell membranes. This observation is consistent with the lipophilic character of the suberoyl chain in cis-[Pt(NH3)2(malSAHA−2H)]. Interestingly, this markedly enhanced accumulation of cis-[Pt(NH3)2(malSAHA−2H)] was not accompanied by a corresponding enhanced cytotoxicity in cancer cells; in fact cis-[Pt(NH3)2(malSAHA−2H)] had similar or slightly less potency as compared to cisplatin (1.3−4.7-fold, Table 1). Hence, the cellular accumulation of cis-[Pt(NH3)2(malSAHA−2H)] appears not to be a critical factor in 1994

dx.doi.org/10.1021/mp300038f | Mol. Pharmaceutics 2012, 9, 1990−1999

Molecular Pharmaceutics

Article

Table 4. DNA-Bound Platinum in SW 480 Cells Exposed to 5 or 10 μM Platinum Complexes for 24 ha,b concn (μM)

cis-[Pt(NH3)2(malSAHA−2H)]

cisplatin

cis-[Pt(NH3)2(mal−2H)]

untreated cells

5 10

(37 ± 4) × 10−15 (77 ± 7) × 10−15

(58 ± 3) × 10−15 (140 ± 5) × 10−15

(50 ± 5) × 10−15 (114 ± 9) × 10−15

1.5 × 10−15 1.5 × 10−15

a Table shows the Pt contents on DNA after 24 h of the cell treatment with Pt complexes at their 5 μM and 10 μM concentrations. Each value shown in the table (means ± standard deviations) is in mol of Pt per μg of DNA. bThe experiments were made in triplicate.

were withdrawn after 48 h and exhaustively dialyzed against NaClO4 (10 mM). DNA concentrations were determined by absorption spectrophotometry and Pt concentrations by FAAS. The results (Figure 3A) showed that although DNA binding of

comparison with cisplatin (Table 1) might be at least partially associated with the reduced ability of the former two complexes to bind to DNA, very likely due to the relatively high complex stability caused by the presence of the bidentate malonato ligands. DNA Binding in Cells Exposed to Pt Complexes. In contrast to cell-free experiments, the situation in vivo may be somewhat more complex since, during transportation of metallodrugs into cells and inside cells, they interact with various biomolecules. These interactions may inhibit but also activate drugs with respect to their interaction with their pharmacological target. Although the rate of DNA binding of malonate-containing complexes (cis-[Pt(NH3)2(malSAHA−2H)] and cis-[Pt(NH3)2(mal−2H)]) in cell-free media was markedly lower than that of the analogous compound with chlorido leaving ligands (i.e., cisplatin) (15−25-fold after 24 h, 7−14fold after 72 h, Table 3), Pt compounds containing malonato ligands were only ca. 2−4 times less effective than cisplatin against tumor cells in culture (Table 1). Thus, the cytotoxic effects of cis-[Pt(NH 3 ) 2 (malSAHA −2H )] and cis-[Pt(NH3)2(mal−2H)] do not correlate with their ability to bind to DNA in cell-free media. In order to compare our results observed in cell-free media (Table 3) relative to living tumor cells, DNA-bound Pt was examined in SW480 cells after exposure to cis-[Pt(NH3)2(malSAHA−2H)], cis-[Pt(NH3)2(mal−2H)], or cisplatin for 24 h. While the results of these measurements (Table 4) revealed that exposure of SW480 cells to cis-[Pt(NH3)2(malSAHA−2H)] or cis-[Pt(NH3)2(mal−2H)] resulted in cellular DNA-bound Pt lower than that found after exposure of the cells to cisplatin (1.6−1.8-fold in the case of cis[Pt(NH3)2(malSAHAH−2)] or 1.2-fold in the case of cis[Pt(NH3)2(malH−2)]), these differences were pronouncedly lower than differences in the amounts of DNA-bound Pt found when malonate complexes were incubated with DNA in cellfree medium (for instance, after 24 h incubation, 25-fold in the case of cis-[Pt(NH3)2(malSAHA−2H)] or 16-fold in the case of cis-[Pt(NH3)2(mal−2H)], Table 3). This comparison suggests that there might be an alternative mechanism that facilitates DNA binding of malonate-containing Pt complexes inside living cells. It has been suggested39,40 that monodentate sulfurcontaining ligands may play a role in the antitumor mechanism of action of carboplatin due to its nucleophilic-facilitated binding to DNA. It seems reasonable therefore to assume that similar effects could be applicable also in the case of Pt complexes containing other bidentate ligands, such as malonate. To prove this hypothesis, we tested the effect of sulfurcontaining species, such as glutathione, L-methionine and thiourea on DNA binding of cis-[Pt(NH3)2(malSAHA−2H)] and cis-[Pt(NH3)2(mal−2H)] in a cell-free medium. CT DNA at a concentration of 1 × 10−4 M (related to the phosphorus content) was incubated with cis-[Pt(NH3)2(malSAHA−2H)], cis[Pt(NH3)2(mal−2H)] or cisplatin at ri = 0.05 in NaClO4 (10 mM), in the presence or absence of glutathione (1 mM), thiourea or L-methionine at 37 °C in the dark. The aliquots

Figure 3. Effects of sulfur-containing compounds (A) and cellular extracts (B) on DNA binding of cis-[Pt(NH3)2(malSAHA−2H)], cis[Pt(NH3)2(mal−2H)], and cisplatin. The results are means of three independent experiments, and standard deviations are shown by error bars.

cisplatin was reduced by the presence of glutathione in the reaction mixture, the binding of cis-[Pt(NH3)2(malSAHA−2H)] or cis-[Pt(NH3)2(mal−2H)] was enhanced by a factor of 5.0 and 3.4, respectively (Figure 3A). Similar results were obtained also for other sulfur-containing nucleophiles (thiourea and Lmethionine, Figure 3A). This increased DNA-binding activity of cis-[Pt(NH3)2(malSAHA−2H)] and cis-[Pt(NH3)2(mal−2H)] in the presence of sulfur-containing species (which quench the binding of cisplatin) indicates the difference in mechanisms of action of these malonate complexes on the one hand and cisplatin on the other. It has been proposed40 that in the case of carboplatin sulfur-containing compounds such as glutathione or thiourea, with their easily polarizable sulfur atoms, can replace the first arm of the cyclobutandicarboxylato ligand. The coordination of the Pt with the remaining second arm could then be labilized via a ligand−cis-effect41 facilitating DNA binding either through direct attack by a nucleobase or through aquation. This explanation can also be applicable to the malonato ligand. Thus, because of the slow dissociation of the malonato ligand in cis-[Pt(NH3)2(malSAHA−2H)], the activation of the prodrug may not primarily involve aquation but rather the reaction with a sulfur-containing bionucleophile. To investigate the relevance of this proposed activation mechanism to (i) the situation in cellulo and (ii) its cellular localization, we also performed experiments in a cell-free 1995

dx.doi.org/10.1021/mp300038f | Mol. Pharmaceutics 2012, 9, 1990−1999

Molecular Pharmaceutics

Article

was significantly reduced as manifested by the lower level of histone H3 acetylation. The presence of the malonate moiety in malSAHA therefore appears to affect its inhibitory activity. In a previously reported study undertaken by Marmion et al., the HDAC inhibitory activity of SAHA and malSAHA was investigated using a commercial colorimetric cell-free assay kit and recombinant HDAC1.11 In the said study the IC50 value for malSAHA (142 ± 29 nM), though less, compared favorably with that found for SAHA (83 ± 23 nM), which suggests that the data from the commercial assay, which used recombinant HDAC1, does not correlate closely with the current study under cellular conditions. The presence of the Pt moiety in cis-[Pt(NH3)2(malSAHA−2H)] and cis-[Pt(NH3)2(mal−2H)] further reduced the inhibitory effect of SAHA. The level of histone H3 acetylation in cells treated with cis-[Pt(NH3)2(malSAHA−2H)] was lower than the basal level of acetylated histone H3 in the untreated cells. The treatment of CH1 cells with cisplatin resulted in a decrease of the level of histone H3 acetylation slightly below the basal level of acetylated histone H3 in the untreated CH1 cells, consistent with previously reported results.42,43 Qualitatively identical results were also obtained if the levels of acetylation of histone H4 were examined in CH1 cells treated with the agents tested in the present work (not shown). The results obtained by Western blotting were complemented by the direct analysis of HDAC activity by using a commercial colorimetric HDAC Activity Assay Kit. The results confirmed that the inhibitory effect of malSAHA was markedly reduced compared with that of SAHA (Table 5 and Figure 5).

medium analogous to those described in the preceding paragraph in the presence of glutathione, thiourea or Lmethionine (Figure 3A), but these sulfur-containing compounds were replaced by cellular extracts (cytoplasmic and nuclear). CT DNA at a concentration of 1 × 10−4 M was incubated with cisplatin, cis-[Pt(NH3)2(mal−2H)] or cis-[Pt(NH3)2(malSAHA−2H)] at ri = 0.05, in NaClO4 (10 mM), in the presence or absence of cytosolic or nuclear extract of HeLa cells (0.37 mg of protein mL−1) at 37 °C, in the dark. After 48 h of incubation, the samples were extracted by phenol and chloroform to remove proteins and subsequently exhaustively dialyzed against NaClO4 (10 mM). DNA concentrations were determined by absorption spectrophotometry and Pt concentrations by FAAS. The results showed (Figure 3B) that cytosolic, but not nuclear, extract of human cancer HeLa cells was able to activate the malonate-containing Pt complexes to facilitate their DNA binding. This result can be interpreted to mean that the natural nucleophiles which are able to activate cis-[Pt(NH3)2(mal−2H)] or cis-[Pt(NH3)2(malSAHA−2H)] are present preferably in the cytosol. Inhibition of Histone Deacetylase Activity. cis-[Pt(NH3)2(malSAHA−2H)] was designed such that it would bind DNA in much the same way as cisplatin or its analogues and because of its additional functionality (HDAC inhibitory activity) be active against a broader spectrum of human cancer cells as well as being endowed with selectivity for tumor cells over normal cells.10,11 Thus, further studies were aimed at determining the effect of the treatment of CH1 cells with cis[Pt(NH3)2(malSAHA−2H)] on HDAC activity. Western blot analysis (Figure 4) indicated that the levels of histone H3 acetylation in CH1 cells were increased compared to the basal (control) level, if CH1 cells were treated with SAHA. This observation is in good agreement with the wellknown HDAC inhibitory effect of SAHA. Interestingly, the inhibitory effect of SAHA conjugated to malonate (malSAHA)

Table 5. Effect of the Single Agents Tested in the Present Work on HDAC Activity in CH1 Cells and Comparison with Cisplatina,b C (μM)c control, untreated cells cis-[Pt(NH3)2(malSAHA−2H)] cisplatin cis-[Pt(NH3)2(mal−2H)] SAHA malSAHA

77 114 86 107 55 72

± ± ± ± ± ±

6 6 3 5 4 2

a

The cells were treated for 24 h with the isotoxic concentrations of the agents corresponding to the IC50 values (Table 1). bThe experiments were made in triplicate. cConcentration of the deacetylated product. The results are means ± standard deviations of three independent experiments.

The HDAC activity in cells treated with cis-[Pt(NH3)2(malSAHA−2H)], cis-[Pt(NH3)2(mal−2H)] or cisplatin was increased, which is in agreement with the results obtained by Western blot analysis (Figure 4). Thus, both methods used to assay the HDAC inhibitory effects showed that cis[Pt(NH3)2(malSAHA−2H)] exhibited no inhibition of HDAC activity in CH1 tumor cells although it should be noted that cells were harvested after only 24 h exposure to the test compounds. Cytotoxicity of the Mixtures (1:1) of the Two Agents. CH1 cells were treated with a 1:1 mixture of cisplatin + SAHA, cis-[Pt(NH3)2(mal−2H)] + SAHA or cisplatin + malSAHA (Table 6). Cotreatment of CH1 cells with SAHA and cisplatin increased the killing efficiency of the Pt drug (Table 6). This observation is in agreement with the results of previously

Figure 4. Western blot analysis for acetylated histone H3 in CH1 cells treated with the agents tested in the present work. The cells were treated for 24 h with the isotoxic concentrations of the agents corresponding to the IC50 values (Table 1). (A) The proteins were transferred onto nitrocellulose membranes and stained with antiacetylated histone H3 antibody. β-Actin is shown as equal loading control. Lanes: 1, control, untreated cells; 2, cells treated with malSAHA; 3, cells treated with SAHA; 4, cells treated with cis[Pt(NH3)2(malSAHA−2H)] (PtmalSAHA); 5, cells treated with cis[Pt(NH3)2(mal−2H)] (Ptmal); 6, cells treated with cisplatin (cisPt). (B) Quantification of the acetylated histone H3 (H3ac). The levels of H3ac were computed by subtracting the H3ac level measured for the control, the untreated cells, from the value measured for the treated cells. The results are means of three independent experiments, and standard deviations are shown by error bars. 1996

dx.doi.org/10.1021/mp300038f | Mol. Pharmaceutics 2012, 9, 1990−1999

Molecular Pharmaceutics

Article

displacement of malonate to give a cis-diammine Pt-DNA binding moiety and release of a HDACi derivative and that each entity might work independently of the other. The current study indicates that sulfur-containing molecules may have a role to play in the in vitro activation of this conjugate. In contrast with a molecular modeling study in which the binding constants for SAHA and malSAHA were comparable (data not shown), it is clear from the current study that the presence of the malonate, if bound to SAHA, reduces its HDAC inhibitory activity (Figures 4 and 5 and Table 5). Requirements for effective HDAC inhibitors, such as SAHA, include not only the hydroxamate moiety of SAHA to bind to the ZnII ion at the HDAC active site but also interactions of their hydrophobic motifs with the periphery of the HDAC binding pocket.44,45 Hence, a plausible explanation for the observation that the HDAC inhibitory effect of malSAHA was markedly reduced compared with that of SAHA is that the malonate in malSAHA impedes important interactions between SAHA and the HDAC enzyme, necessary for its HDAC inhibitory activity. In addition, while cellular accumulation of cis-[Pt(NH3)2(malSAHA−2H)] was significantly higher than that of cis-[Pt(NH3)2(mal−2H)] or cisplatin, the rate of DNA platination of cis-[Pt(NH3)2(malSAHA−2H)] was significantly less than that of cisplatin. In aggregate, the presence of the malonate moiety either as the linker substituent of the HDAC inhibitor SAHA or as the leaving ligand in the DNA binding PtII complex considerably reduces one of the dual functionalities anticipated to be effective after the novel cisplatin−SAHA conjugate reaches the nucleus. This is consistent with and supported by the results of the experiments in which CH1 cells were treated with the 1:1 mixtures of cis-[Pt(NH3)2(mal−2H)] + SAHA or cisplatin + malSAHA (Table 6). No synergic or additive effects were observed when CH1 cells were treated with the 1:1 mixture of cisplatin + malSAHA (Table 6). This study has undoubtedly offered a valuable insight into facets of the activity of cis-[PtII(NH3)2(malSAHA−2H)] against cancer cells and will aid in the design of the next generation of Pt−HDACi conjugates. These conjugates should incorporate linker groups which do not negatively impact on the HDAC inhibitory activity of the HDACi in cellulo. While the lipophilic character associated with nonpolar groups in the HDACi for example offers a significant advantage in terms of cellular accumulation of the conjugate, the rate of loss of the HDACi and subsequent release of the Pt moiety free to bind DNA should be carefully considered. The conjugate should also be tested against a range of normal cell lines to further validate its observed lower tocixity against the representative NHDF cell line as compared to cisplatin. The preferential selectivity of HDACis such as SAHA for cancer cells over normal cells12,46,47 and their potential to synergistically enhance the anticancer activity of many chemotherapeutics48−50 will continue to provide the motivation behind our approach to combine both entities into one drug molecule. Ultimately an in vivo study will be necessary to more fully understand the anticancer properties of this new class of potential anticancer agents.

Figure 5. HDAC activity in CH1 cells treated with the single agents tested in the present work. Other details were the same as indicated in Table 5. The ΔC values were computed by subtracting the concentration of deacetylated product in the control, the untreated cells, from the concentration of deacetylated product in the treated cells. The standard deviations are shown by error bars.

Table 6. Inhibition of Growth of CH1 Cancer Cells by the Mixtures (1:1) of Two Agents Tested in the Present Work and Comparisons with cis-[Pt(NH3)2(malSAHAH−2)], Cisplatin and SAHA Used as Single Agentsa,b IC50 (μM)d cis-[Pt(NH3)2(malSAHA−2H)] cisplatin SAHA cis-[Pt(NH3)2(mal−2H)] malSAHA cis-[Pt(NH3)2(mal−2H)] + SAHAc cisplatin + malSAHAc cisplatin + SAHAc

3.8 ± 0.5e 1.0 ± 0.5e 1.2 ± 0.2e 2.5 ± 0.5e >200e 1.13 ± 0.04 0.9 ± 0.1 0.21 ± 0.02

a

Drug-treatment period was 72 h. bThe experiments were made in triplicate. cBoth agents were at the same molar concentrations. dThe IC50 value (means ± standard deviations) corresponds to the concentration of one agent. eThe data taken from Table 1.

published studies22 carried out with several human tumor cell lines. The results of these studies were interpreted to mean that loosening up the chromatin structure by histone acetylation can increase the efficiency of anticancer drugs including cisplatin to target DNA. In contrast, no increase of the killing efficiency of cisplatin was observed if CH1 cells were cotreated with this metallodrug and malSAHA. Similarly, no increase of the killing efficiency of cis-[Pt(NH3)2(mal−2H)] was observed if CH1 cells were cotreated with this analogue of cisplatin containing malonate and SAHA (Table 6). Conclusions. cis-[Pt(NH3)2(malSAHA−2H)], in which cisplatin and SAHA are combined in one molecule, exhibited lower cytotoxicity in tumor cells as compared to cisplatin or SAHA if applied alone and less cytotoxicity than might be anticipated if they are used as single agents in a 1:1 mixture (Table 6). It seems reasonable to assume that the lower cytotoxicity associated with cis-[Pt(NH3)2(malSAHA−2H)] is due to the reduced rate of DNA platination in tumor cells treated with cis-[Pt(NH3)2(malSAHA−2H)] (Table 4) and also with the fact that this complex exhibited no HDAC inhibitory activity in this current study (Table 5, Figures 4 and 5)]. It would appear that the presence of the malonate linker in the conjugated complex cis-[Pt(NH3)2(malSAHA−2H)] is responsible for these observations. It was expected11 that the Pt−HDACi conjugate, upon reaching the nucleus, would undergo hydrolysis via a classical ring-opening process followed by hydration and consequent

■

ASSOCIATED CONTENT

S Supporting Information *

Tables S1 and S2 showing uptake of Pt complexes into cells. This material is available free of charge via the Internet at http://pubs.acs.org. 1997

dx.doi.org/10.1021/mp300038f | Mol. Pharmaceutics 2012, 9, 1990−1999

Molecular Pharmaceutics

■

Article

(17) Marks, P. A. Discovery and development of SAHA as an anticancer agent. Oncogene 2007, 26, 1351−1356. (18) Kelly, W. K.; Marks, P. A. Drug insight: histone deacetylase inhibitors - development of the new targeted anticancer agent suberoylanilide hydroxamic acid. Nat. Clin. Pract. Oncol. 2005, 2, 150−157. (19) Duvic, M.; Vu, J. Vorinostat: a new oral histone deacetylase inhibitor approved for cutaneous T-cell lymphoma. Expert Opin. Invest. Drugs 2007, 16, 1111−1120. (20) Shabason, J. E.; Tofilon, P. J.; Camphausen, K. HDAC inhibitors in cancer care. Oncol.-N.Y. 2010, 24, 180−185. (21) Cang, S.; Ma, Y.; Liu, D. New clinical developments in histone deacetylase inhibitors for epigenetic therapy of cancer. J. Hematol. Oncol. 2009, 2, 22−32. (22) Kim, M. S.; Blake, M.; Baek, J. H.; Kohlhagen, G.; Pommier, Y.; Carrier, F. Inhibition of histone deacetylase increases cytotoxicity to anticancer drugs targeting DNA. Cancer Res. 2003, 63, 7291−7300. (23) Brabec, V.; Palecek, E. The influence of salts and pH on polarographic currents produced by denatured DNA. Biophysik 1970, 6, 290−300. (24) Brabec, V.; Palecek, E. Interaction of nucleic acids with electrically charged surfaces. II. Conformational changes in doublehelical polynucleotides. Biophys. Chem. 1976, 4, 76−92. (25) Chavez-Blanco, A.; Segura-Pacheco, B.; Perez-Cardenas, E.; Taja-Chayeb, L.; Cetina, L.; Candelaria, M.; Cantu, D.; GonzalezFierro, A.; Garcia-Lopez, P.; Zambrano, P.; Perez-Plasencia, C.; Cabrera, G.; Trejo-Becerril, C.; Angeles, E.; Duenas-Gonzalez, A. Histone acetylation and histone deacetylase activity of magnesium valproate in tumor and peripheral blood of patients with cervical cancer. A phase I study. Mol. Cancer 2005, 4, 3−11. (26) Zhu, W.-G.; Lakshmanan, R. R.; Beal, M. D.; Otterson, G. A. DNA methyltransferase inhibition enhances apoptosis induced by histone deacetylase inhibitors. Cancer Res. 2001, 61, 1327−1333. (27) Alley, M. C.; Scudiero, D. A.; Monks, A.; Hursey, M. L.; Czerwinski, M. J.; Fine, D. L.; Abbott, B. J.; Mayo, J. G.; Shoemaker, R. H.; Boyd, M. R. Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay. Cancer Res. 1988, 48, 589−601. (28) Abassi, Y. A.; Xi, B.; Zhang, W. F.; Ye, P. F.; Kirstein, S. L.; Gaylord, M. R.; Feinstein, S. C.; Wang, X. B.; Xu, X. Kinetic cell-based morphological screening: Prediction of mechanism of compound action and off-target effects. Chem. Biol. 2009, 16, 712−723. (29) Harris, A. L.; Yang, X.; Hegmans, A.; Povirk, L.; Ryan, J. J.; Kelland, L.; Farrell, N. P. Synthesis, characterization, and cytotoxicity of a novel highly charged trinuclear platinum compound. Enhancement of cellular uptake with charge. Inorg. Chem. 2005, 44, 9598− 9600. (30) Kasparkova, J.; Novakova, O.; Vrana, O.; Intini, F.; Natile, G.; Brabec, V. Molecular aspects of antitumor effects of a new platinum(IV) drug. Mol. Pharmacol. 2006, 70, 1708−1719. (31) Johnson, N. P.; Butour, J.-L.; Villani, G.; Wimmer, F. L.; Defais, M.; Pierson, V.; Brabec, V. Metal antitumor compounds: The mechanism of action of platinum complexes. Prog. Clin. Biochem. Med. 1989, 10, 1−24. (32) Jamieson, E. R.; Lippard, S. J. Structure, recognition, and processing of cisplatin-DNA adducts. Chem. Rev. 1999, 99, 2467− 2498. (33) Bancroft, D. P.; Lepre, C. A.; Lippard, S. J. 195Pt NMR kinetic and mechanistic studies of cis-diamminedichloroplatinum and transdiamminedichloroplatinum(II) binding to DNA. J. Am. Chem. Soc. 1990, 112, 6860−6871. (34) Cutbush, S. D.; Kuroda, R.; Neidle, S.; Robins, A. B. The antitumor complex ethylenediamine platinum(II) malonate: X-ray structure analysis and studies of its stability in solution. J. Inorg. Biochem. 1983, 18, 213−220. (35) Cleare, M. J.; Hydes, P. C.; Malerbi, B. W.; Watkins, D. M. Antitumor platinum complexes: relationship between chemical properties and activity. Biochimie 1978, 60, 835−850.

AUTHOR INFORMATION

Corresponding Author

*Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Kralovopolska 135, 61265 Brno, Czech Republic. E-mail: [email protected]. Tel: +420-541517148. Fax: +420-541240499. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by the Czech Science Foundation (Grants P301/10/0598 and P303/11/P047). This material is also based upon works supported by the Science Foundation Ireland under Grants 07/RFP/CHEF570 and 08/RFP/ CHE1675. We also gratefully acknowledge the Programme for Research in Third Level Institutions (PRTLI), administered by the HEA for funding. We acknowledge COST D39 for being a platform to progress fruitful collaborations.

■

REFERENCES

(1) Kelland, L. The resurgence of platinum-based cancer chemotherapy. Nat. Rev. Cancer 2007, 7, 573−584. (2) Lokich, J. What is the “best” platinum: Cisplatin, carboplatin, or oxaliplatin? Cancer Invest. 2001, 19, 756−760. (3) Alderden, R. A.; Hall, M. D.; Hambley, T. W. The discovery and development of cisplatin. J. Chem. Educ. 2006, 83, 728−734. (4) 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. (5) Cepeda, V.; Fuertes, M.; Castilla, J.; Alonso, C.; Quevedo, C.; Perez, J. M. Biochemical mechanisms of cisplatin cytotoxicity. AntiCancer Agents Med. Chem. 2007, 7, 3−18. (6) Wang, D.; Lippard, S. J. Cellular processing of platinum anticancer drugs. Nat. Rev. Drug Discovery 2005, 4, 307−320. (7) Jung, Y.; Lippard, S. J. Direct cellular responses to platinuminduced DNA damage. Chem. Rev. 2007, 107, 1387−1407. (8) Brabec, V. DNA modifications by antitumor platinum and ruthenium compounds: their recognition and repair. Prog. Nucleic Acid Res. Mol. Biol. 2002, 71, 1−68. (9) Wong, E.; Giandomenico, C. M. Current status of platinumbased antitumor drugs. Chem. Rev. 1999, 99, 2451−2466. (10) Griffith, D.; Parker, J. P.; Marmion, C. J. Enzyme inhibition as a key target for the development of novel metal-based anti-cancer therapeutics. Anti-Cancer Agents Med. Chem. 2010, 10, 354−370. (11) Griffith, D.; Morgan, M. P.; Marmion, C. J. A novel anti-cancer bifunctional platinum drug candidate with dual DNA binding and histone deacetylase inhibitory activity. Chem. Commun. 2009, 6735− 6737. (12) Botrugno, O. A.; Santoro, F.; Minucci, S. Histone deacetylase inhibitors as a new weapon in the arsenal of differentiation therapies of cancer. Cancer Lett. 2009, 280, 134−144. (13) Butler, L. M.; Zhou, X.; Xu, W.-S.; Scher, H. I.; Rifkind, R. A.; Marks, P. A.; Richon, V. M. The histone deacetylase inhibitor SAHA arrests cancer cell growth, up-regulates thioredoxin-binding protein-2, and down-regulates thioredoxin. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 11700−11705. (14) Xu, W. S.; Parmigiani, R. B.; Marks, P. A. Histone deacetylase inhibitors: molecular mechanisms of action. Oncogene 2007, 26, 5541− 5552. (15) Tan, J.; Cang, S.; Ma, Y.; Petrillo, R.; Liu, D. Novel histone deacetylase inhibitors in clinical trials as anti-cancer agents. J. Hematol. Oncol. 2010, 3, 5−17. (16) Lane, A. A.; Chabner, B. A. Histone deacetylase inhibitors in cancer therapy. J. Clin. Oncol. 2009, 27, 5459−5468. 1998

dx.doi.org/10.1021/mp300038f | Mol. Pharmaceutics 2012, 9, 1990−1999

Molecular Pharmaceutics

Article

(36) Cleare, M. J.; Hoeschele, J. D. Antitumor activity of group VIII transition metal complexes. 1. Platinum(ll) complexes. Bioinorg. Chem. 1973, 2, 187−210. (37) Macquet, J. P.; Butour, J. L. Modifications of the DNA secondary structure upon platinum binding: a proposed model. Biochimie 1978, 60, 901−914. (38) Macquet, J. P.; Butour, J. L. Platinum - amine compounds: Importance of the labile and inert ligands for their pharmacological activities toward L1210 leukemia cells. J. Natl. Cancer Inst. 1983, 70, 899−905. (39) Barnham, K. J.; Djuran, M. I.; Murdoch, P. d. S.; Ranford, J. D.; Sadler, P. J. Ring-opened adducts of the anticancer drug carboplatin with sulfur amino acids. Inorg. Chem. 1996, 35, 1065−1072. (40) Natarajan, G.; Malathi, R.; Holler, E. Increased DNA binding activity of cis-1,1-cyclobutanedicarboxylatodiammineplatinum(II) (Carboplatin) in the presence of nucleophiles and human breast cancer MCF-7 cell cytoplasmic extracts: Activation theory revisited. Biochem. Pharmacol. 1999, 58, 1625−1629. (41) Howe-Grant, M.; Lippard, S. J. Binding of platinum(II) intercalation reagents to DNA. Dependence on base pair composition, nature of the intercalator, and ionic strength. Biochemistry 1979, 18, 5762−5769. (42) Tikoo, K.; Ali, I. Y.; Gupta, J.; Gupta, C. 5-Azacytidine prevents cisplatin induced nephrotoxicity and potentiates anticancer activity of cisplatin by involving inhibition of metallothionein, pAKT and DNMT1 expression in chemical induced cancer rats. Toxicol. Lett. 2009, 191, 158−166. (43) Koprinarova, M.; Markovska, P.; Iliev, I.; Anachkova, B.; Russev, G. Sodium butyrate enhances the cytotoxic effect of cisplatin by abrogating the cisplatin imposed cell cycle arrest. BMC Mol. Biol. 2010, 11, 49−57. (44) Mai, A. The therapeutic uses of chromatin-modifying agents. Expert Opin. Ther. Targets 2007, 11, 835−851. (45) Codd, R.; Braich, N.; Liu, J.; Soe, C. Z.; Pakchung, A. A. H. Zn(II)-dependent histone deacetylase inhibitors: Suberoylanilide hydroxamic acid and trichostatin A. Int. J. Biochem. Cell Biol. 2009, 41, 736−739. (46) Dokmanovic, M.; Perez, G.; Xu, W. S.; Ngo, L.; Clarke, C.; Parmigiani, R. B.; Marks, P. A. Histone deacetylase inhibitors selectively suppress expression of HDAC7. Mol. Cancer Ther. 2007, 6, 2525−2534. (47) Lee, J. H.; Choy, M. L.; Ngo, L.; Foster, S. S.; Marks, P. A. Histone deacetylase inhibitor induces DNA damage, which normal but not transformed cells can repair. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 14639−14644. (48) Ozaki, K.; Kishikawa, F.; Tanaka, M.; Sakamoto, T.; Tanimura, S.; Kohno, M. Histone deacetylase inhibitors enhance the chemosensitivity of tumor cells with cross-resistance to a wide range of DNAdamaging drugs. Cancer Sci. 2008, 99, 376−384. (49) Frew, A. J.; Johnstone, R. W.; Bolden, J. E. Enhancing the apoptotic and therapeutic effects of HDAC inhibitors. Cancer Lett. 2009, 280, 125−133. (50) Rasheed, W.; Bishton, M.; Johnstone, R. W.; Prince, H. M. Histone deacetylase inhibitors in lymphoma and solid malignancies. Expert Rev. Anticancer Ther. 2008, 8, 413−432.

1999

dx.doi.org/10.1021/mp300038f | Mol. Pharmaceutics 2012, 9, 1990−1999