Developing Anticancer Copper(II) Pro-drugs Based on the Nature of

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Developing anti-cancer copper(II) pro-drugs based on the nature of cancer cells and human serum albumin carrier IIA subdomain Yi Gou, Jinxu Qi, Joshua-Paul Ajayi, Yao Zhang, Zuping Zhou, Xiaoyang Wu, Feng Yang, and Hong Liang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00314 • Publication Date (Web): 09 Sep 2015 Downloaded from http://pubs.acs.org on September 14, 2015

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Molecular Pharmaceutics

Developing anti-cancer copper(II) pro-drugs based on the nature of cancer cells and human serum albumin carrier IIA subdomain Yi Gou1, Jinxu Qi1, Joshua-Paul Ajayi2, Yao Zhang1, Zuping Zhou3, Xiaoyang Wu2, Feng Yang1*, Hong Liang1* 1

State Key Laboratory Cultivation Base for the Chemistry and Molecular

Engineering of Medicinal Resources, Ministry of Science and Technology of China. Guangxi Normal University, Guilin, Guangxi, China. 2

Ben May Department for Cancer Research, University of Chicago,

Chicago, IL, USA. 3

Guangxi Universities Key Laboratory of Stem Cell and Pharmaceutical

Biotechnology, Guangxi Normal University, Guilin, Guangxi, China.

*To whom correspondence should be addressed: Feng Yang; Hong Liang E-mail address: [email protected] Postal address: 15 Yucai Road, Guilin, Guangxi, China. Zip code: 541004 Phone/Fax: 86-773-212-0958

1

ACS Paragon Plus Environment


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ABSTRACT To synergistically enhance the selectivity and efficiency of anti-cancer copper drugs, we proposed and built a model to develop anti-cancer copper pro-drugs based on the nature of human serum albumin (HSA) IIA subdomain and cancer cells. Three copper (II) compounds of a 2-hydroxy-1-naphthaldehyde benzoyl hydrazone Schiff-base ligand in the presence pyridine, imidazole, or indazole ligands were synthesized (C1–C3). The structures of three HSA complexes revealed that the Cu compounds bind to the hydrophobic cavity in HSA IIA subdomain. Among them, the pyridine and imidazole ligands of C1 and C2 are replaced by Lys199, and His242 directly coordinates with Cu(II). The indazole and Br ligands of C3 are replaced by Lys199 and His242, respectively. Compared with Cu(II) compounds alone, the HSA complexes enhance cytotoxicity in MCF-7 cells approximately 3–5 fold, but do not raise cytotoxicity levels in normal cells in vitro through selectively accumulating in cancer cells to some extent. We find HSA complex has a stronger capacity for cell cycle arrest in the G2/M phase of MCF-7 by targeting cyclin-dependent kinase 1 (CDK1), and down-regulating the expression of CDK1 and cyclin B1. Moreover, the HSA complex promotes MCF-7 cell apoptosis possibly through the intrinsic reactive oxygen species (ROS) mediated mitochondrial pathway, accompanied by the regulation of Bcl-2 family proteins. Keywords: Human serum albumin; Prodrug; Drug delivery systems; Anti-cancer activity; Anti-cancer mechanism.

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1 INTRODUCTION Although cisplatin is a promising anti-cancer agent, multifactorial resistance, serious side effects, and general toxicity have limited the use of cisplatin in cancer chemotherapy.1–3 Therefore, less toxic and more effective metal-based anti-cancer compounds have been extensively sought and developed,4–6 and drug delivery systems have been exploited to improve the selectivity of anti-cancer drugs and decrease their side effects.7 Human serum albumin (HSA) is a promising drug delivery system because HSA is a non-toxic, non-antigenic, biocompatible, and biodegradable endogenous protein.8 HSA is the most abundant protein in plasma and can bind a diverse group of endogenous and exogenous compounds.9–11 HSA contains three structurally similar α-helical domains (I–III). Each domain consists of sub-domains A and B, which contain six and four α-helices, respectively.12 Although HSA complex structures have revealed several binding sites for drugs in HSA, the majority of drugs bind to two main subdomains of HSA, namely, the IIA and IIIA subdomains.12–14 However, the current model of HSA binding built by Zsila has shown that the IB subdomain of HSA is the third major drug binding region.15 Owing to the presence of a free Cys34 of HSA, it has been possible to design Ru and Pt pro-drugs that will tether with HSA carriers by reaction at Cys34 of HSA. For instance, Kratz and co-workers 3



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have developed a series of maleimide-functionalized Pt(II) complexes that bind highly selectively to Cys34.16,17 The same strategy has been reported by Hartinger and co-workers to develop a series of Ru(arene) compounds in which the arene is functionalized with a maleimide moiety.18 In order to attempt to overcome the limitations of platinum(II) chemotherapeutics, Kowol and co-workers synthesized the first platinum(IV) complexes containing a maleimide moiety at the axial position that will simply and quickly couple with Cys34.19 Interestingly, Lay and co-workers developed a novel approach using ruthenium anti-cancer pro-drugs with potential leaving groups that are replaced by S-donor, N-donor, and carboxylate residues of albumin.20 Lippard and co-workers designed a series of Pt(IV) pro-drugs tethered to fatty acids that are delivered by HSA carriers having the fatty acids bound.21 The pro-drug strategy has been one of the most promising for improving the anti-cancer activity and selectivity of metal drugs.17–22 The current rule in design of HSA-based metal pro-drugs is that chemical coupling of metal drugs with special residues of albumin should be performed to generate HSA-drug conjugates. However, the shortcomings of these HSA-drug conjugates are unavoidable. For example, excess chemical modification linkers (succinyl HCl terephthalic hydrazine) used for tethering drugs to residues of HSA can result in precipitation of the protein.23,24 Tethering drugs to the Cys34 anchoring site is difficult 4


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because Cys34 is buried in a small cavity of HSA, and not freely accessible.12,25 Furthermore, two potential problems may exist when metal pro-drugs are delivered to cancer cells through the HSA carrier in vivo. First, metal pro-drugs tethered to HSA dangle outside HSA and are therefore exposed to other proteins in the plasma. Consequently, other proteins may compete with HSA for binding of metal pro-drugs. This allows pro-drugs to escape from the HSA carrier and bind to other proteins in plasma, leading to unexpected side effects. Second, metal pro-drugs are released from HSA prematurely because of weak binding, or not released at all from the HSA carrier in tumor cells owing to tight binding. Obviously, it is necessary to optimize the prodrug strategy to overcome the above problems. For example, the current studies demonstrated that the anti-cancer activity and selectivity of drugs can be improved through complexing with the HSA carrier.26,27 Interestingly, the current studies have revealed by X-ray crystallography that metal compounds can bind to HSA through coordinating bond. For example, Yang and co-workers discovered that the Ru compound ([RuCl5(ind)]2-) not only binds to IB subdomain through His146 of HSA by replacing its one Cl atom, but also IIA subdomain through Lys199 and His242 of HSA by replacing its two Cl atoms.28 However, Merlino and co-workers have revealed that cisplatin cannot bind to IIA subdomain; instead, it binds to 5



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other sites on HSA by reacting with certain residues of HSA.29 Obviously, the molecular structure and geometry of metal compounds have an important influence on their binding site and binding mode in HSA. Among the next generation of metal-based anti-cancer compounds, Cu anti-cancer compounds are especially promising because Cu is an essential element for humans because of its bioactivity and oxidative nature.30 In particular, copper-bound aroylhydrazone derivatives of pyridoxal isonicotinoyl hydrazone show great potential as effective antiproliferative agents.31,32 Taking into consideration the above factors, we proposed that we may develop appropriate Cu(II) anti-cancer pro-drugs with leaving groups that can initially bind in the HSA IIA subdomain where there is a large hydrophobic cavity. Once the prodrug is bound, the N-donor residues (Lys199 or/and His242) of the HSA IIA subdomain would displace the leaving groups of the Cu pro-drugs and coordinate to the Cu centre, forming stable HSA complexes. Subsequently, N-donor residues of HSA will be protonated because of the acidic environment of the cancer cell lysosomal compartment.33,34 This decreases their coordination ability with Cu ion, allowing Cu drugs to be released from the HSA carrier. To confirm our hypothesis on developing Cu anti-cancer pro-drugs based on nature of cancer cells and HSA IIA subdomain, the anti-cancer Cu compounds derived from aroylhydrazone Schiff-base and breast cancer (MCF-7) cells were used to build an in vitro 6


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model through conducting the following studies: (1) synthesizing the three aroylhydrazone Cu(II)-Schiff base anti-cancer compounds with one/two potential leaving groups (C1–C3) (Fig 1); (2) providing solid evidence on feasibility for developing Cu pro-drugs based on nature of HSA carrier IIA subdomain and cancer cells; (3) investigating the anti-cancer properties and possible anti-cancer mechanism of Cu prodrug/HSA complex; (4) determining the improvement of selectivity and efficiency of HSA complex relative to Cu prodrug alone.

2 MATERIALS AND METHODS Fatty acid free HSA (catalogue number A3782) was purchased from Sigma Chemical Company and used without further purification. All other chemicals and solvents used were of high purity and available from commercial sources. Water used in the reactions was distilled prior to use. Elemental analyses (C, N, and H) were carried out on a Perkin-Elmer 2400 analyser. Infrared (IR) spectra were recorded using KBr pellets (4000–400 cm-1) on a Nexus 870 FT-IR spectrophotometer. 2.1 Synthesis and structure determination of three Cu(II) compounds 2-Hydroxy-1-naphthaldehyde benzoyl hydrazone (HL) was synthesized according to literature procedures.35,36 Synthesis of [Cu(L)(pyridine)] (C1) and [Cu(L)(imidazole)] (C2). The C1 and C2 were prepared according to the previously reported method.36,37 In brief, HL (0.58 g, 2 mmol) and the heterocyclic nitrogen 7



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ligands (pyridine: 0.16 g, 2 mmol for C1 and imidazole: 0.14 g, 2 mmol for C2, ) were dissolved in an aqueous methanol solution (15 mL) and stirred for 1 h to give an orange solution, which was added to a methanol solution (15 mL) of copper(II) salts (Cu(NO3)2·3H2O: 0.48 g, 2 mmol for C1 and Cu(CH3COO)2·H2O: 0.40 g, 2 mmol for C2). The mixture was stirred for another 30 min at room temperature to give a translucent solution and then filtered. The filtrate was kept in air for a week, forming blue black crystals. The crystals were isolated, washed three times with distilled water and dried in a vacuum desiccator containing anhydrous CaCl2. C1: Yield: 681 mg (79%). Anal. Calcd for C23H17CuN3O2 (430.94): C, 64.10; H, 3.98 and N, 9.75. Found: C, 64.07; H, 3.95 and N, 9.78. IR (KBr, cm-1): 1621 ν(C=N); 544, 518, 458, 430 ν(Cu–N/Cu–O). C2: Yield: 655 mg (78%). Anal. Calcd for C21H16CuN4O2 (419.92): C, 60.06; H, 3.84 and N, 13.34. Found: C, 60.07; H, 3.83 and N, 13.35. IR (KBr, cm-1): 1620 ν(C=N); 545, 514, 455, 432 ν(Cu–N/Cu–O). Synthesis of [Cu(Br)(L)(indazole)] (C3). Benzoylhydrazine (0.27 g, 2 mmol), 2-hydroxy-1-naphthaldehyde (0.34 g, 2 mmol) and indazole (0.23 g, 2 mmol) were dissolved in an aqueous methanol solution (15 mL) and stirred for 1 h to give an orange solution, which was added to a methanol solution (15 mL) of CuBr2 (0.44 g, 2 mmol). The mixture was stirred for another 30 min at room temperature to give a translucent solution and then filtered. The filtrate was kept in air for a week, forming blue black 8


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crystals. The crystals were isolated, washed three times with distilled water and dried in a vacuum desiccator containing anhydrous CaCl2. Yield: 837 mg (76%). Anal. Calcd for C25H19BrCuN4O2 (550.89): C, 54.51; H, 3.47 and N, 10.17. Found: C, 54.53; H, 3.44 and N, 10.20. IR (KBr, cm-1): 1617 ν(C=N); 547, 504, 457, 436 ν(Cu–N/Cu–O). X-ray crystallographic data were collected on a Bruker SMART Apex II CCD diffractometer using graphite-monochromated Mo-Kα (λ = 0.71073 Å) radiation. Empirical adsorption corrections were applied to all data using SADABS. The structures were solved by direct methods and refined against F2 by full-matrix least-squares methods using the SHELXTL version 5.1.38 All of the non-hydrogen atoms were refined anisotropically. All other hydrogen atoms were placed in geometrically ideal positions and constrained to ride on their parent atoms. The crystallographic data for compound C1–C3 are summarized in Table S1. Selected bond lengths and angles are given in Table S2. Crystallographic data for the structural analyses have been deposited at the Cambridge Crystallographic Data Centre, reference numbers 1046819 for C2 and 1046818 for C3. The crystallographic data can be obtained free of charge from

the

Cambridge

Crystallographic

Data

Centre

via

http://www.ccdc.cam.ac.uk/data_request/cif. 2.2 Studies on copper pro-drugs bound to HSA Fluorescence spectrum. HSA solution (2 µM) was titrated by 9



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successive additions of the Cu(II) compound using micropipettes for all of the experiments. The fluorescence emission spectra were scanned from 300 to 420 nm after excitation at 280 nm (the maximum emission was obtained at 347). The binding constant of HSA for compound can be analyzed according to the Scatchard equation:39 log[(F0 - F)/F] = logK + n × log(Q) where F and F0 are the fluorescence intensities of protein in the presence and absence of the quencher, respectively; n is the number of binding sites; K is the binding constant and [Q] is quencher concentration. From the plot of log[(F0 - F)/F] versus log [Q], the number of binding sites (n) and the binding constant (K) was calculated. UV–visible spectrum. UV-visible absorption spectra were measured on a Cary 1E UV-Visible spectrophotometer in the 200–800 nm range, connected to a Haake F3 water bath, which maintained the temperature of each sample at 37 °C. Measurements were performed using 25 µM HSA and 25 µM solutions of each sample in 1 mL volumes. Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF-MS) Analyses. HSA protein solution (1.5 mM) was added to solutions of compounds C1–C3 in order to achieve a 4:1 metal/protein ratio and shaken for 24 h at room temperature. The samples were prepared using the dried droplet method with freshly prepared sinapinic acid [10 mg/mL in CH3CN/H2O/trifluoroacetic acid 10


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(70:29.9:0.1)] as the matrix solution. The protein sample solution (0.1 mL, series of 1:10 dilutions) was mixed on the target with the matrix solution (0.1 mL) and allowed to air-dry. The MS spectra were recorded in the m/z 30000−200000 range in a positive linear mode. X-ray crystallography. Palmitic acid (PA) was dissolved in alcohol, and diluted to 2.5 mM with 20 mM potassium phosphate (pH 7.5). Fatty acid (FA) free HSA was purified by removing HSA dimers and multimers as published.40 The complexes of Cu compounds and HSA were prepared by mixing 100 µL HSA (100 mg/mL), 380 µL PA (2.5 mM) and 90 µL Cu compounds (5 mM) overnight, then the mixture was concentrated to 100 mg/ml with a Millipore spin filter (10,000 dalton cutoff). Crystallization was carried out by sitting drop vapor diffusion at room temperature. An equal volume of the HSA complex was mixed with the reservoir solution, consisting of 28–32% (w/v) polyethylene glycol 3350, 50 mM potassium phosphate (pH 7.5), 5% glycerol, and 5% DMSO. Crystals were directly selected from the drop solution and then frozen in liquid nitrogen. X-ray diffraction data were collected under cryo-conditions (100 K) using the Shanghai Synchrotron Radiation Facility. The data were integrated and scaled with HKL2000. The three data sets obtained from the HSA complexes were processed in space group P1. The structure of the HSA complexes were solved by molecular replacement with AMORE program using the HSA-MYR structure (PDB code 1BJ5) stripped of its 11



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ligands as the model. The model was initially refined using a rigid body protocol in CNS and then subjected to cycles of positional and B-factor refinement before the calculation of the initial Fo-Fc and 2Fo-Fc maps. These maps were used to guide the position of the fatty acid and ligands and to make manual adjustments to the protein prior to further cycles of refinement. Figures depicting the structure were prepared by PyMOL.41 Data collection details and unit cell parameters are given in Table 1. 2.3 Anti-cancer properties of HSA complex in vitro Culture medium DMEM (with L-glutamin), Foetal bovine serum (FBS),

PBS

(phosphate

buffered

saline)

(pH

=

7.2)

and

Antibiotic-Antimycotic were from E.U. Gibco BRL. Human breast cancer cell lines MCF-7 and human liver cell lines HL-7702 (purchased from the American Type Culture Collection and the German Collection of Microorganisms and Cell Cultures) were maintained in DMEM supplemented with 10% FBS, 50 U/mL penicillin, 50 mg/mL streptomycin at 37 °C and 5% CO2. Cytotoxicity assay (MTT). To gain deeper insight into the effect of HSA interactions on the activity of the compounds, two sets of experiments were performed: (1) all compounds were dissolved in PBS with 0.5% DMSO and incubated with HSA for 24 h at room temperature, after which the HSA-complexes incubated with above cell lines for 48 h, and (2) all compounds were dissolved in PBS with 0.5% DMSO and then 12


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tested. The 100 µL of cell suspensions at a density of 5 × 104 cells/mL was seeded in triplicates in 96-well plates and incubated for 24 h at 37 °C in 5% CO2. Then the medium was replaced with the respective medium with 10% FBS containing the compounds at various concentrations and incubated at 37 °C under conditions of 5% CO2 for 48 h. The final DMSO concentration in all experiments was 20

HL-HSA

9.68 ± 0.73

>20

C1

0.73 ± 0.06

1.41 ± 0.09

HSA-C1

0.26 ± 0.12

1.52 ± 0.06

C2

1.45 ± 0.36

1.47 ± 0.21

HSA-C2

0.27 ± 0.11

1.51 ± 0.18

C3

1.28 ± 0.11

1.37 ± 0.15

HSA-C3

0.26 ± 0.14

1.52 ± 0.13

Cisplatin

18.9 ± 1.65

9.39 ± 0.95

>20

>20

HSA-Cisplatin a

Antitumor activity IC50 (µM)

IC50 values are presented as the mean ± SD from three separated experiments.

45



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Graphic Abstract 131x101mm (300 x 300 DPI)


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Figure 1 Chemical structure of three copper pro-drugs (C1–C3). 55x17mm (600 x 600 DPI)



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Figure 2 The experimental sigmaA weighted 2Fo-Fc electron density map of Cu pro-drugs in HSA. (A) C1 (B) C2 (C) C3. 50x14mm (300 x 300 DPI)


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Figure 3 (A) The overall structure of HSA complex. (B) The structural binding environment of Cu prodrug in HSA. 74x32mm (300 x 300 DPI)



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Figure 4 Induction of apoptosis by C1 and HSA-C in MCF-7 cells. (A) Representative dot plots of PI and annexin V double staining on the MCF-7 cells in the presence of the indicated concentrations of C1 and HSAC for 12 h. (B) Representative images of AO/EB double stained MCF-7 cells after treatment with C1 and HSA-C at the indicated concentrations for 12 h. 123x88mm (300 x 300 DPI)


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Figure 5 (A) Cell cycle contributions resulting from treatment with C1 and HSA-C (0.25 µM) for 24 h. (B) Representative Western blots of the effects of C1 and HSA-C on the protein expression levels of CDK1 and cyclin B1. β-Actin was assessed as a loading control. 73x69mm (300 x 300 DPI)



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Figure 6 Analysis of ROS levels by flow cytometry after MCF-7 cells were treated with vehicle, C1 (A) or HSA-C (B) at indicated concentrations for 12 h and stained with H2DCFDA. (C) Quantification of the flow cytometric results in (A) and (B) showing the percentage of cells with increased intracellular DCF oxidation compared to control cells. Results are the mean ± SD (n = 5): (**) p < 0.01. 192x210mm (300 x 300 DPI)


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Figure 7 Effects of C1 and HSA-C on mitochondrial membrane potential analyzed by JC-1 staining and flow cytometry. MCF-7 cells were treated with vehicle, C1 or HSA-C complex at the indicated concentrations indicated for 12 h. 91x96mm (600 x 600 DPI)



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Figure 8 (A) Western blot analysis of cleaved Caspase-3, cleaved Caspase-9, Bcl-2, Bcl-xl, Bad, Bax and cytochrome c in MCF-7 cells treated with different concentrations of C1 and HSA-C complex for 48 h. (B) Percentage expression levels of Bcl-2, Bcl-xl, Bad, and Bax. (C) Percentage expression levels of cleaved Caspase-3, cleaved Caspase-9 and Cytochrome c. The percentage values are those relative to the control. Results are the mean ± SD (n = 3): (*) p < 0.05, (**) p < 0.01, (***) p < 0.001. 147x295mm (300 x 300 DPI)


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Figure 9 (A) Cu content (%) in cells. (B) Distribution of HSA-C complex in MCF-7 cells. (C) The profiles of Cu release from HSA at different pH. Data plotted are mean ± SD of 3 separate experiments, (**) p < 0.01. 159x343mm (600 x 600 DPI)


Developing Anticancer Copper(II) Pro-drugs Based on the Nature of

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