Antiangiogenic Activity of Mononuclear Copper(II) Polypyridyl

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Anti-angiogenic activity of mononuclear copper(II) polypyridyl complexes for the treatment of cancers penumaka nagababu, Ayan Kumar Barui, THULASIRAM BATHINI, Shobha Gampa, S. Satyanarayana, Chittaranjan Patra, and Bojja Sreedhar J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00651 • Publication Date (Web): 11 Jun 2015 Downloaded from http://pubs.acs.org on June 13, 2015

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Journal of Medicinal Chemistry

Anti-angiogenic activity of mononuclear copper(II) polypyridyl complexes for the treatment of cancers Penumaka Nagababu†*‡, Ayan K. BaruiΦ#‡, Bathini Thulasiram†‡, C. Shobha Devi♣‡, S. SatyanarayanaΩ, Chitta R. PatraΦ#*, Bojja Sreedhar†*#

†

Inorganic & Physical Chemistry Division, CSIR-Indian Institute of Chemical Technology, Uppal Road,

Tarnaka, Hyderabad - 500007, Telangana State, India. Φ

Biomaterials Group, CSIR-Indian Institute of Chemical Technology, Uppal Road, Tarnaka, Hyderabad

- 500007, Telangana State, India. ♣

Department of Chemistry, National Dong Hwa University, Hualien, Taiwan, ROC.

Ω

#

*

Department of Chemistry, Osmania University, Tarnaka, Hyderabad, Telangana State, India.

Academy of Scientific & Innovative Research (AcSIR), 2 Rafi Marg, New Delhi, India.

Corresponding authors: Inorganic & Physical Chemistry Division and Biomaterials Group, CSIR-

Indian Institute of Chemical Technology, Uppal road, Tarnaka, Hyderabad - 500007, Telangana, India.

ABSTRACT: A series of four new mononuclear copper(II) polypyridyl complexes (1-4) have been designed, developed and thoroughly characterized by several physico-chemical techniques. The CT-DNA binding properties of 1-4 have been investigated by absorption, emission spectroscopy and viscosity measurements. All the complexes especially 1 and 4 exhibit cytotoxicity towards several cancer cell lines suggesting their anti-cancer properties as observed by several in vitro assays. Additionally, the complexes show inhibition of endothelial cell (HUVECs) proliferation indicating their anti-angiogenic nature. In vivo chick embryo angiogenesis assay again confirms the anti-angiogenic properties of 1 and 4. The formation of excessive intracellular ROS (H2O2 and O2.-) and upregulation of BAX induced by copper(II) complexes may be the plausible mechanisms behind their anti-cancer activities. The present ACS Paragon Plus Environment


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study may offer a basis for the development of new transition metal complexes through suitable choice of ligands for cancer therapeutics by controlling tumor angiogenesis.

INTRODUCTION Studies of metal chelates, which bind to DNA strand as reactive models for protein-nucleic acid interaction, provide routes toward rational drug design as well as means to develop sensitive probes for DNA structure1 and to get information about drug design and tools of molecular biology.2,3 Binding of metal complexes with DNA has been studied extensively since DNA is the material of inherence and controls the structure and function of cells.4 Polypyridyl metal complexes can bind to DNA in a non-covalent interaction mode, such as groove binding for large ligands, electrostatic binding for cations,5 intercalative mode of binding for planar ligands and partial intercalative binding for incompletely planar ligands.6,7 According to the recent reports, cis-platin is one of the most widely used metal-based anti-tumor and anti-cancer drug targeting DNA8 through covalent bonding interaction.9 However, it has limited potential efficacy due to side-effects, drug resistance phenomena etc.10 Therefore, development of new alternative strategies for treatment of cancers is immediately required by finding out new materials based on different metals and ligands with an aim of reducing toxicity, enhancing specificity and thereby enhancing the therapeutic efficacy through non-covalent binding with DNA. It is well-known that the modes of non-covalent interaction between DNA and metal complexes include intercalation, groove binding and electrostatic binding. Among these non-covalent binding modes, intercalation has attracted special interests due to its various applications in cancer therapy and molecular biology.1 The complex [Cu([9]aneN3)Cl2] (Cu(II) 1,4,7, triazacyclononane chloride) is the first one to show an ability to cleave both protein bovine serum albumin (BSA) and phosphodiester bonds at near physiological pH.12 Chakravarty et al. reported several metal complexes that can show photo-induced protein and DNA cleavage activity for their potential application in photodynamic therapy.13 Sigman et

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al.14 have developed the first chemical nuclease, bis-(1,10-phenanthroline) copper(I) complex, that effectively minor groove cleavage of DNA in the presence of H2O2. Recently, Palaniandavar et al. reported mixed ligand copper(II) complexes containing phenolates and amino acids as coligands, which cleave and bind to DNA and also exhibit anti-cancer activity.15,16 Very recently, Rajendiran and coworker have reported that prominent anti-cancer activities of certain mixed ligand copper(II)-phenolate complexes17 are consistent with the ability to affect the double stand DNA cleavage. So, studies on interaction of copper(II) based complexes with DNA are useful in the rational design of small molecule anti-cancer therapeutics. With these considerations in mind, we in particular report the synthesis and characterization of four new copper(II) polypyridyl complexes (1-4). Interactions of these complexes with CT-DNA have been investigated by fluorescence and UV-visible spectroscopy as well as viscosity measurements. Recent reports demonstrate several biomedical applications of copper complexes showing their anti-diabetic, anti-bacterial and anti-fungal activities.18a-b However, copper based inorganic complexes have extensively been investigated for cancer therapy in recent years, assuming the less toxicity of endogeneous metal towards normal cells.19 However, copper may be toxic as it has redox property and it is prone to several binding sites that is supposed to be occupied by other metals. Though, there are recent reports of copper complexes as cytotoxic agents, the mechanism of their anti-proliferative nature has not been discussed much. In this context, the anti-cancer activity of newly synthesized copper(II) polypyridyl complexes (1-4) have been extensively studied by several in vitro assays. Angiogenesis is a critical process concerning formation of new blood vessels from the preexisting blood vessels.20 It has a noteworthy role in various physiological processes such as embryonic development, wound healing, menstrual cycle etc., as well as many pathological processes such as atherosclerosis, ischemic heart and ischemic limb disease, diabetic retinopathy, tumor growth and metastasis.21,22 Earlier reports demonstrate that copper is an essential cofactor during angiogenesis process where it can induce production of cytokines, endothelial cell proliferation and migration.19 However, 3 ACS Paragon Plus Environment


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recent study reveals that cuprous oxide nanoparticles exhibit anti-angiogenic properties through the inhibition of endothelial cell proliferation, migration and tube formation.23 To date the angiogenesis study of copper complexes has not been focused. In this circumstance, the angiogenesis study with the as synthesized new copper(II) polypyridyl complexes has been performed by in vitro as well as in vivo assays. In the present study, cell viability assay of the 1-4 in different cancer cell lines clearly indicates their anti-cancer activities. These complexes are also found to be cyto-toxic towards noncancerous COS-1 cells. Scratch wound healing assay in SKOV3 ovarian cancer cells indicates the ability of the complexes in inhibition of cancer cell migration while cell cycle assay in B16F10 melanoma cancer cells reveals the sub-G1 phase arrest leading to the cell apoptosis induced by these complexes. The generation of intracellular reactive oxygen species (H2O2 and O2.-) may be the plausible mechanism for the anti-proliferative activity. Further studies have manifested that the upregulation of pro-apoptotic protein BAX induced by the complexes may also play a vital role behind their anti-cancer property. The DNA binding affinity of 1-4 may put forward the idea that they could exhibit anti-tumor activity through DNA damage leading to the activation of cell apoptosis. Additionally, the in vitro cell viability study in HUVECs clearly shows the inhibition of endothelial cell proliferation (key step of angiogenesis) in presence of copper(II) polypyridyl complexes suggesting their anti-angiogenic nature. The in vivo chick embryo angiogenesis assay further confirms the anti-angiogenic potential of the complexes through damaging of blood vessels. The overall study reveals the anti-cancer and anti-angiogenic prospective of copper(II) polypyridyl complexes that may provide the sense for the fabrication of other metal based complexes for cancer therapy controlling the angiogenesis process in tumor cells.

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Scheme 1. Copper(II) polypyridyl complexes (1-4) and the imidazophenanthroline (IP) ligands.

RESULTS AND DISCUSSION Synthesis and structures of complexes. All the (aqua)bis(imidazophenanthroline) copper(II) complexes (1-4) have been prepared from Cu(ClO4)2.6H2O with imidazophenanthroline (IP) ligands (Scheme 1) in aqueous methanol. Synthesized complexes were characterized from analytical and spectral data (A detail description of synthesis and characterization of 1-4 are given in supporting information Figure S1S12). The formulae of the complexes were determined by elemental analysis (Table S1). Mass spectra of 1-4 exhibit [M+-H2O] peak at respective m/z values, indicating the dissociation of aqua ligand in solution. The infrared spectra of complexes show a broad band around 3200-3400 cm-1 that can be attributed to the coordinated water molecule. Complexes (1-4) show weight loss (TGA studies) corresponding to one water molecule at 120 -200 °C suggesting their presence in the coordination sphere around the metal center24 (Figure S12). All the complexes exhibit a broad band in the visible region (700-710 nm) with very low absorptivities (Table S2, Figure S6) corresponds to d-d* transition revealing a distorted trigonal–bipyramidal geometry25,26 that is consistent with the obtained EPR spectral data of the complexes. 5 ACS Paragon Plus Environment


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Electron paramagnetic resonance (EPR) spectroscopy. EPR spectroscopy is the most useful technique for studying chemical compounds with unpaired electrons. This technique is useful benchmarks for verifying the magnetic properties of 1–4. The sample temperature has been maintained at 77 K by using a liquid nitrogen finger Dewar. The EPR spectra of complexes show isotropic signals at parallel and perpendicular regions as shown in Figure 1. EPR spectra of 1-4 showed g|| in the range 2.25 to 2.27 and a pronounced peak at 2.043 ± 0.002 corresponds to g⊥. Further, EPR spectra of complexes are axial with g||> g⊥> 2.0023 indicating a dx2−y2 ground state27 and the factor g||/A|| (range 150-160 × 10-4) reflects the distortion of coordination geometry. Higher the value of this factor, greater is the distortion. This is because when the geometry is no longer planar, the interaction of dx2-y2 ground state orbital with ligand orbitals is lowered and thus repulsion between unpaired electron in dx2-y2 orbital and ligand electrons decreases.27 So, the observed values of g||/A|| for 1−4 indicate significant distortion of coordination geometry.

Figure 1. EPR spectra of the complex (1) in CH3CN solution. DNA binding studies. The binding character of the complexes towards calf thymus DNA (CT-DNA) has been investigated by different spectroscopic techniques. Electronic spectra. The electronic spectra of the four copper(II) polypyridyl complexes are measured 6 ACS Paragon Plus Environment

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from 200 nm to 800 nm in buffer at pH 7.1 [2% CH3OH 5 mM TrisHCl, 50 mM NaCl] and diluting suitably with corresponding buffer to required concentrations for all the experiments. Absorption spectral titration experiments have been performed by maintaining a constant concentration of the complex and varying the DNA concentration. This has been achieved by dissolving an appropriate amount of the copper complex and DNA stock solutions while maintaining the total volume constant (3 mL). The solution has been mixed thoroughly and allowed to stand for 5 min before measurement. The observed peaks are characteristic of π–π* for ligand transitions in the UV region and metal-to-ligand charge transfer (MLCT) transition in the visible region (Table S2). In order to further elucidate the binding strength of 1-4, the intrinsic constants Kb have been determined by monitoring the changes of absorbance with increasing concentration of CT-DNA using the following equation.28 [DNA]/(εa-εf) = [DNA]/(εb-εf)+1/(Kb (εb-εf)) whereεa is the extinction coefficient observed for the MLCT absorption band at a given DNA concentration, εf is the extinction coefficient of DNA and εb is the extinction coefficient of the complex fully bound to DNA. In plots of [DNA]/(εb-εf) versus [DNA], Kb is given by the ratio of slope to intercept. Intrinsic binding constants (Kb) of complexes 1–4 are3.1 × 103 (1), 5.2 × 105 (2), 6.3 × 103(3) and 2.2 × 104 M-1 (4) respectively. As shown in Table 1 the obtained Kb values are good agreement with previous reports on Cu(II) complexes.29,30 These data confirm that 1–4 bind to DNA and also show that as the ancillary ligand PYIP binding is stronger than other due to the more planarity. When compared to TfPIP and CN-PIP ligands, CF3 group possess more electronegativity then -CN. The ligand Tf-PIP shows least binding with DNA due to repulsion between DNA base pairs and Tf. The affinity of complexes binding to DNA follows the order 2>4>3>1 in agreement with the conclusion that these complexes bind with the base pairs of DNA. Figure 2a and2b represents the absorption spectra of the 3 and 4 with and without DNA respectively while the absorption spectra for 1 and 2 have been given in supporting information (Figure S6).



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Figure 2. Absorption spectra of 3(a) and 4(b) in the absence (top) and presence (lower) of DNA. Arrow shows the absorbance changes upon increasing DNA concentrations. Inset plots of [DNA]/[εa-εf ] versus [DNA] for the titration of DNA with complexes; (•) experimental data points; solid lines, linear fitting of the data.

Fluorescence spectra. In general, when small molecules, often referred to as metal complexes bind to DNA, show spectral changes in fluorescence spectra relative to what is observed for solutions without 8 ACS Paragon Plus Environment

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DNA. The luminescence of complexes at room temperature exhibits λmax at around 550 nm to 700 nm and its interaction with CT-DNA has been monitored with luminescence.

Figure 3. Fluorescence emission spectra of 1 (a) and 2 (b) in aqueous buffer (tris 5 mM, NaCl 50 mM, pH 7.0) in the presence of CTDNA, where [copper] = 20 µM, [DNA]/Cu = 0, 5 and 10. The arrows indicate the intensity changes upon increasing concentration.

The results of emission titrations for 1 and 2 with CT-DNA are illustrated in Figure 3a & 3b respectively and for 3 and 4 have been given in supporting information (Figure S7). For complexes the emission intensity increases two-fold over that of free DNA. This implies that complexes strongly interact with DNA and can be efficiently protected by DNA as the hydrophobic environment inside the DNA helix 9 ACS Paragon Plus Environment


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reduces the accessibility of solvent water, leading to decrease in vibrational mode of relaxation and hence increase in fluorescence intensity. The flattening of fluorescence intensity indicates that the complex saturates all binding sites on DNA. Fluorescence emission enhancement is based on comparison of emission intensity of complexes in the absence and presence of CT-DNA. Complexes exhibit fluorescence at ambient temperature with λmax at 559 (1), 558 (2), 601 (3) and 598 nm (4) respectively. As can be seen in Figure 3a and3b upon addition of CT-DNA the fluorescence emission intensities of 1-4 increases indicating their strong binding with DNA and the consequent restriction of their mobility at the binding site. Viscosity studies. The mode of binding of 1-4 to DNA has been explained by viscosity measurements. Optical and photophysical probes are necessary but it does not give sufficient clues to support the binding mode. Hydrodynamic measurements that are sensitive to length change are regarded as the least ambiguous. A classical intercalative mode demands that the DNA helix must lengthen as base pairs are separated to accommodate the binding ligand, leading to an increase in DNA viscosity.31 The viscosity of DNA increases with increasing concentration of the complex that is similar to proven intercalator ethidium bromide (EtBr) (Figure 4) suggesting that 1-4 bind to DNA through intercalative mode.

Figure 4. Effect of increasing amount of 1-4 on the relative viscosities of CT-DNA at room temperature.


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Cytotoxicity assay. Cytotoxicity assay is a well-known method to elucidate the cell viability of new drugs/biomaterials. Therefore, we have investigated the cytotoxicity profile of as synthesized complexes

Figure 5. Cell viability assay in different cancer cells [(a): B16F10, (b): SKOV3, (c): A549 and (d): MDA-MB-231] using MTT reagent. Results reveal that all the complexes, especially 1 and 4, significantly inhibit the proliferation of cancer cells (anti-cancer activity) in a dose dependant manner (0.1–20 µM; 48 h). The precursor material of these complexes, Cu(ClO4)2 is found to be biocompatible in all cancer cell lines at same doses (0.1–20 µM). DMSO and doxorubicin (DOX: 4 µM) have been used as vehicle control and positive control, respectively. Values are mean ± SD of three independent experiments; *p ≤0.05; **p ≤0.005 compared to control.



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1-4 in different type of cancer cells e.g. mouse malignant melanoma cells: B16F10 (Figure 5a), human ovarian carcinoma cells: SKOV3 (Figure 5b), human lung cancer cells: A549 (Figure 5c) and human breast cancer cells: MDA-MB-231 (Figure 5d). Table 1. Comparison of IC50 values of 1-4 with recently reported copper complexes.

It has been found that all the complexes, especially 1 and 4 exhibit significant cytotoxicity in a dose de

IC50 (µM) Complexes

Kb(M-1)

B16F10

SKOV3

A549

MDAMB-231

HeLa

H460

1

3.1×103

2.1±0.2

2.1±0.9

1.5±0.04

0.6±0.03

-

-

2

5.2×105

25.5±0.3

23.4±0.2

38.2±1.1

26.5±1.5

-

-

3

6.3×103

36.6±0.4

70.8±4.8

71.8±4.7

40.0±4.6

-

-

4

2.2×104

0.5±0.02

1.1±0.1

0.6±0.05

0.1±0.00

-

-

-

5 29

4.3×105

-

-

-

-

3.7

29

5.6×105

-

-

-

-

6.1

30

4.0×103

-

-

-

-

-

3400

30

6.5×103

-

-

-

-

-

900

30

9.8×103

-

-

-

-

-

2400

[Cu(Fc-tpy)(dppz)](ClO4)2 [Cu(Ph-tpy)(dppz)](ClO4)2 [Cu(L-tyr)(phen)]+ [Cu(L-tyr)(5,6-dmp)]+ [Cu(L-tyr)(dpq)]+

pendent manner towards all the cancer cell lines suggesting their anti-cancer activity. The IC50 values of all the complexes (including Kb value) have been compared with other recently reported copper complexes29,30 in different cancer cells and represented in Table 1. This comparison reveal that complexes 1 and 4 exhibit better cytotoxic responses than other reported copper complexes where IC50 values does not depend on Kb values of the complexes as shown by Maity et al. and Ramakrishnan et al.29,30 Additionally, the IC50 values of lead complexes 1 & 4 and their corresponding ligands L1 & L4 have been 12 ACS Paragon Plus Environment

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presented in Table S3 which shows the better cytotoxic responses of the complexes compared to their corresponding ligands irrespective of cancer cells. Further, the ligands are insoluble in water

Figure 6. Cell viability assay in non-cancerous (a) COS-1 and (b) HUVEC cell lines using MTT reagent. All the complexes (1-4; 0.1-20 µM; 24 h) exhibit cytotoxicity towards COS-1 cells. Additionally, the complexes (0.1-20 µM; 24 h) show inhibition of endothelial cell (HUVECs) proliferation revealing their anti-angiogenic nature. The precursor material of the complexes, Cu(ClO4)2 is found to be biocompatible in both the non-cancerous cell lines at same doses (0.1–20 µM). DMSO and doxorubicin (DOX: 4 µM) have been used as vehicle control and positive control, respectively. Values are mean ± SD of three independent experiments; *p ≤0.05; **p ≤0.005 compared to control. 13 ACS Paragon Plus Environment


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while corresponding copper complexes are soluble in water. These facts suggest the advantages of copper complexes over their respective ligands in practical application as anti-cancer agent. The complexes 1-4 are also found to be cytotoxic towards non-cancerous fibroblast like cell line COS-1 (Figure 6a). Moreover, Figure 6b shows that these complexes inhibit the proliferation of endothelial HUVECs in a dose dependent fashion indicating their anti-angiogenic properties as endothelial cell proliferation is known as one of the key/vital steps of angiogenesis process.21 The IC50 values of the complexes 1-4 in non-cancerous cells have also been presented in Table S4. To compare the relative anti-proliferative effects of 1-4 on cancerous versus normal cells, their therapeutic index (TI) has been calculated as the ratio of the IC50 values of complexes in normal cells to that of cancer cells and presented in Table S5. It is well-known that FDA approved anticancer drugs are toxic towards both cancerous and noncancerous cells.32a-b In present study, the lead complexes 1 and 4 show comparable cytotoxicity towards both cancerous (Figure 5) as well as non-cancerous (Figure 6) cells. In order to compare the cytotoxicity results with FDA approved drug, doxorubicin has been used as a positive control experiment that also exhibits cytotoxicity towards both cancerous (Figure 5) as well as non-cancerous cell lines (Figure 6). Therefore, we believe that though the complexes are cytotoxic towards both cancerous and normal cell lines, the possibility of using the complexes as therapeutic agent cannot be fully disregarded. However, for practical applications of 1 and 4 as potent anti-cancer therapeutic agents, targeted delivery of these complexes through nanoparticles/liposome/bio-molecules would be preferred that will inhibit the proliferation of cancer cells without affecting the normal cells which is beyond the scope of the present study. Analysis of cell morphology. Figure 7 depicts the effect of the treatment of 1-4 on the morphology of B16F10 cells. The untreated control cells are of spindle shaped whereas the cells treated with complexes especially 1 and 4, have been found to be mostly spherically shaped demonstrating the damaged cell bodies.23 This result suggests the evident change in cell morphology induced by the cytotoxic complexes. 14 ACS Paragon Plus Environment

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Cellular uptake study: In order to understand whether the internalization of the complexes plays a vital role on their cytotoxicity, cellular uptake study32c has been carried out in B16F10 cells. It has been found that after 6 h of incubation of the complexes (1-4: 20 µM) in B16F10 cells, the intracellular uptake of 4 (1.5 picogram/cell) is maximum compared to other complexes (Table S6). This study reveals that the cellular internalization of complexes follow the order 3

Antiangiogenic Activity of Mononuclear Copper(II) Polypyridyl

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