In Vitro Assays Predictive of Telomerase Inhibitory Effect of G

Dec 12, 2013 - G-quadruplex-binding and telomerase-inhibiting capacities of G-quadruplex ligands were examined under a cell nuclei-mimicking condition...
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In Vitro Assays Predictive of Telomerase Inhibitory Effect of G‑Quadruplex Ligands in Cell Nuclei Hidenobu Yaku,†,‡,§ Takashi Murashima,‡,§ Daisuke Miyoshi,*,‡,§ and Naoki Sugimoto*,‡,§ †

Advanced Technology Research Laboratories, Panasonic Corporation, 3-4 Hikaridai, Seika-cho, Soraku-gun, Kyoto 619-0237, Japan FIRST (Faculty of Frontiers of Innovative Research in Science and Technology), Konan University, 7-1-20 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan § FIBER (Frontier Institute for Biomolecular Engineering Research), Konan University, 7-1-20 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan ‡

S Supporting Information *

ABSTRACT: G-quadruplex-binding and telomerase-inhibiting capacities of G-quadruplex ligands were examined under a cell nuclei-mimicking condition including excess double-stranded DNA (λ DNA) and molecular crowding cosolute (PEG 200). Under the cell nuclei-mimicking condition, a cationic porphyrin (TMPyP4) did not bind to the G-quadruplex despite the high affinity (Ka = 3.6 × 106 M−1) under a diluted condition without λ DNA and PEG 200. Correspondingly, TMPyP4 inhibited telomerase activity under the diluted condition (IC50 = 1.6 μM) but not under the cell nuclei-mimicking condition. In contrast, the Ka and IC50 values of an anionic copper phthalocyanine (Cu-APC) under the diluted (2.8 × 104 M−1 and 0.86 μM) and the cell nuclei-mimicking (2.8 × 104 M−1 and 2.1 μM) conditions were similar. In accordance with these results, 10 μM TMPyP4 did not affect the proliferation of HeLa cells, while Cu-APC efficiently inhibited the proliferation (IC50 = 1.4 μM). These results show that the cell nuclei-mimicking condition is effective to predict capacities of G-quadruplex ligands in the cell. In addition, the antiproliferative effect of Cu-APC on normal cells was smaller than that on HeLa cells, indicating that the cell nuclei-mimicking condition is also useful to predict side effects of ligands.

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INTRODUCTION Telomeric DNA can form a G-quadruplex, which is a tetraplex structure with π-planar G-quartets. Since the G-quadruplex inhibits telomerase,1 which plays a role in carcinogenesis by elongating telomeric DNA,2,3 G-quadruplex ligands are promising as anticancer drugs.4,5 Ligands containing a large π-planar core and cationic functional groups for π−π stacking and electrostatic interactions with the G-quadruplex, respectively, inhibit efficiently telomerase activity in vitro.5,6 However, many such ligands have relatively low anticancer effects in cellular assays.7−9 These low effects may result from intracellular environmental factors that inhibit the G-quadruplex ligands. Reportedly, abundant double-stranded DNA (dsDNA) prevents the conventional cationic G-quadruplex ligand, 5,10,15,20-tetra-(N-methyl-4-pyridyl)porphyrin (TMPyP4), from binding to the telomeric G-quadruplex and from © 2013 American Chemical Society

inhibiting telomerase activity because of electrostatic interactions between the dsDNA and TMPyP4.10 This finding indicates that TMPyP4 does not efficiently inhibit telomerase in cell nuclei because most genomic DNA forms dsDNA. In contrast, high selectivity of anionic ligands to the G-quadruplex over dsDNA has been reported, although the studies on anionic ligands have been only a few due to their lower affinity for Gquadruplexes than cationic ones.5,10−12 Because of this high selectivity, anionic G-quadruplex ligands inhibit telomerase very efficiently, even in the presence of excess dsDNA.5,10,12 Moreover, molecular crowding (MC), which mimics the high concentration of biomolecules in cells,13,14 critically affects properties and reactions of biomolecules, including dsDNA15,16 Received: October 29, 2013 Revised: December 9, 2013 Published: December 12, 2013 2605

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Figure 1. Ligands examined in this study.

and G-quadruplex DNA,17−19 by altering properties of the solution, including a decrease in the dielectric constant14 and water activity,16,17,20 and an increase in viscosity21 and excluded volume.22 Systematic studies demonstrated that MC reduces binding between dsDNA and a variety of dsDNA ligands including intercalators and groove binders.15 Furthermore, Tan’s group recently reported that MC also reduces binding between the telomeric G-quadruplex and cationic G-quadruplex ligands, including TMPyP4, and their inhibition of telomerase.19 This reduced affinity occurs because cationic ligands require hydration to bind to the G-quadruplex, which is unfavorable under decreased water activity conditions by MC.19 On the contrary, MC barely affects the telomerase inhibition mediated by anionic ligands because water molecules are not required for binding of these ligands to the G-quadruplex.23 These results strongly suggested that functions of the ligands in

cell nuclei should be significantly different those in a diluted test tube. The studies of effects of abundant dsDNA and MC on capacities of G-quadruplex ligands have shown striking findings, such as reduced binding affinity of cationic ligands by abundant dsDNA or MC, that we can never know from experiments under diluted test tube conditions. These studies should be essential to understand behaviors of G-quadruplex ligands in cell nuclei. However, these effects have been studied independently, although intracellular environmental multi factors, including abundant dsDNA and MC, simultaneously affect property and functions of molecules in living cells, and the effects should not be independent. Therefore, it is important to investigate systematically the effects of a condition with a combination of dsDNA and MC. Here, we systematically examined the DNA binding and telomerase inhibition activities of five G-quadruplex ligands: two anionic, copper(II) 2606

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Binding Assay. Absorbance for TMPyP4 (1.0 μM), CuAPC (2.5 μM), APC (2.5 μM), Cu-TMPyP4 (1.0 μM), or TTMAPP (1.0 μM) with various concentrations of Htelo-DNA was recorded at 25 °C; a UV-1700 spectrophotometer (Shimadzu) connected to a Shimadzu TMSPC-8 thermoprogrammer was used with a 1.0 cm path-length quartz cell. The measurements for these ligands were carried out in a buffer containing 50 mM MES-LiOH (pH 7.0) and 100 mM KCl in the absence or presence of 128 μg/mL λ DNA and 20 wt % PEG 200. Before measurement, each sample was heated to 80 °C for 2 min and gently cooled to 20 °C at 2 °C min−1. Evaluation of the Association Constant. The fractional degree (ν) of saturation of each ligand-binding site to DNA can be expressed by the following equation, which is based on a model that is based on the assumption of one binding site to estimate the equilibrium parameters:27

phthalocyanine 3,4′,4″,4‴-tetrasulfonic acid, tetrasodium salt (Cu-APC; Figure 1),5,10,12,23 phthalocyanine tetrasulfonate (APC; Figure 1); and three cationic, TMPyP4 (Figure 1),19,24 copper TMPyP4 (Cu-TMPyP4; Figure 1), and 5,10,15,20-Tetrakis(4-trimethylammoniophenyl) porphyrin (TTMAPP; Figure 1). Experimental conditions included λ DNA as excess and competitive dsDNA, and poly(ethylene glycol) with an average molecular weight of 200 (PEG 200) as MC cosolute to mimic conditions in cell nuclei. On the basis of the results obtained under the cell nuclei-mimicking condition, we further investigated the effects of each ligand on the proliferation of cancer and normal cells.

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EXPERIMENTAL SECTION Materials. High-performance liquid chromatography (HPLC), purification-grade DNA oligonucleotides were purchased from Tsukuba Oligo Service Co., Ltd. (Ibaraki, Japan). TMPyP4 was purchased from Dojindo Laboratories (Kumamoto, Japan), and Cu-APC, APC, and TTMAPP were purchased from Sigma-Aldrich Co. LLC (St. Louis, MO). These ligands were used without further purification. CuTMPyP4, a Cu2+ derivative of TMPyP4, was synthesized as follows. A 101.7 mg (0.15 mmol) sample of TMPyP4 was dissolved in 40 mL of freshly distilled water, and the solution was heated to reflux. To the resulting solution was added 6.65 g (50 mmol) of CuCl2, and the mixture was allowed to continue refluxing for 2 h with vigorous stirring. The reaction mixture was cooled below 4 °C, then the excess amount of sodium perchlorate was added to precipitate the Cu-TMPyP4. The resulting mixture was allowed to stand overnight below 0 °C. The precipitate was filtered and washed several times with dilute perchloric acid. PEG 200 was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) and was used without further purification. To generate stock solutions for ligands, each ligand was dissolved in distilled water. UV Melting Analysis. UV melting curves of G-quadruplex formed by a human telomeric oligo DNA (Htelo-DNA) in a buffer containing 50 mM MES-LiOH (pH 7.0) and 100 mM KCl were measured at 295 nm;25 a UV-1700 spectrometer (Shimadzu Co., Ltd., Kyoto, Japan) connected to a Shimadzu TMSPC-8 thermoprogrammer (Shimadzu) was used with a 0.1 cm path-length quartz cell. Before measurement, samples were heated at 90 °C for 1 min and gently cooled from 90 to 0 °C at 0.5 °C min−1. The DNA sample was then heated from 0 to 90 °C at 0.5 °C min−1. The temperature of the cell holder was controlled by a TMSPC-8 temperature controller, and water condensation on the cuvette exterior in the low-temperature range was avoided by flushing with a constant stream of dry N2 gas. Fluorescent Melting Analysis. Fluorescent melting analysis of G-quadruplex formed by Htelo-DNA modified with FITC and Dabcyl at the 5′ and 3′ ends,26 respectively, in a buffer containing 50 mM MES-LiOH (pH 7.0) and 100 mM KCl in the absence and presence of 128 μg/mL λ DNA and 20 wt % PEG 200 was carried out by measuring fluorescent intensity of FITC using a MyiQ2 real time PCR detection system (Bio-Rad Laboratories, Hercules, CA) with excitation and emission wavelengths of 485 and 530 nm, respectively. Before measurement, samples were heated at 90 °C for 1 min and gently cooled from 90 to 0 °C at 0.5 °C min−1. The DNA sample was then heated from 0 to 90 °C at 0.1 °C sec−1 with a dwell time of 55 s every increase of 0.5 °C.

ν = {Kd + [ligand] + [DNA] −

(Kd + [ligand] + [DNA])2 − 4[ligand][DNA] }

/2[ligand]

(1)

where Kd is the dissociation constant, [DNA] is the concentration of DNA, and [ligand] is the concentration of the ligand. Equation 1 can be transformed into eq 2. The association constant (Ka) for each DNA for each ligand was determined with eq 2: θ = a{K a[ligand] + K a[DNA] + 1 −

(K a[ligand] + K a[DNA] + 1)2 − 4K a 2[ligand][DNA] }

/2K a[ligand] + b

(2)

where θ is the absorbance value, Ka is the apparent association constant of DNA binding, a is a scale factor, and b is the initial θ value. Two-Step TRAP Assay. The two-step telomeric repeat amplification protocol (tsTRAP) assay was improved on the basis of the manufacturer’s protocol for the TRAPEZE telomerase detection kit manufactured by Millipore Corporation (Billerica, MA).3,10 In the tsTRAP assay, the telomerase reaction mixture containing G-quadruplex ligand was first diluted and then used as a template for the PCR step described below. Thus, diluted G-quadruplex ligand did not affect PCR efficiency. Each 10 μL telomerase reaction mixture contained telomerase, 1× TRAP reaction buffer, 1× dNTP mix, and 0.2 μL TS primer, and either lacked G-quadruplex ligand or contained 2 μL of a defined concentration of G-quadruplex ligand. In the first step, each reaction was incubated at 30 °C for 60 min; each mixture was then heated at 90 °C for 10 min. In the second step, telomerase reaction products were amplified in a 10 μL PCR reaction mixture containing 50-fold diluted telomerase mixture, 0.2 μL TS primer, 0.2 μL TRAP primer mix, 1× dNTP mix, 1× LA Taq polymerase buffer, and LA Taq polymerase manufactured by Takara Bio, Inc. (Shiga, Japan); the PCR was carried out over 30 cycles each comprising denaturation at 94 °C for 30 s, annealing at 59 °C for 30 s, and extension at 72 °C for 30 s. The TRAP assay products were resolved by nondenaturing electrophoresis at 400 V through a 10% nondenaturing polyacrylamide gel in Tris-borate-EDTA buffer (pH 8.5). The gels were stained with GelStar nucleic acid gel stain manufactured by Cambrex Corporation (East Rutherford, NJ) and imaged using FLA-5100 manufactured by Fujifilm Corporation (Tokyo, Japan). Telomerase activity 2607

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(TTAGGG)3-3′ (Htelo-DNA). In a diluted solution containing 100 mM KCl and 50 mM MES-LiOH (pH 7.0), Htelo-DNA showed a circular dichroism (CD) spectrum with a negative peak and a positive peak around 240 and 295 nm, respectively (Figure S1). This profile indicates that Htelo-DNA forms a (3 + 1) G-quadruplex, consistent with previous reports.29 Under the same condition, the melting temperature (Tm) of the Gquadruplex was estimated to be 69.0 °C based on the hypochromic UV melting profiles at 295 nm (Figure 2A),

was measured according to the protocol provided with the TRAPEZE telomerase detection kit. For the negative control reactions, lysis buffer was added in the place of telomerase. For the positive control reactions, telomerase extract was added to a reaction solution that lacked any G-quadruplex ligand. Relative activity (A) was calculated via the following equation: A = {(X − X 0)/C}/{(X p − X 0)/Cp}

(3)

where X is the signal intensity of the region of the gel lane corresponding to the TRAP product ladder bands, and C is the signal intensity of the region of the gel lane corresponding to the internal control product. Subscripts “p” and “0” indicate the positive and negative controls, respectively. Cell Culture. HeLa cells and normal human dermal fibroblast (NHDF) cells were purchased from Sigma-Aldrich Co. LLC (St. Louis, MO) and Toyobo Co., Ltd. (Tokyo, Japan), respectively. HeLa and NHDF cells were cultured in Dulbecco’s Modified Eagle’s Medium (Sigma-Aldrich Co. LLC, St. Louis, MO) and a special medium for NHDF (Toyobo Co., Ltd., Tokyo, Japan), respectively. All culture incubations were performed in a humidified 5% CO2 incubator at 37 °C. Cell Permeation Analysis. Subconfluent HeLa cells (100 μL containing 104 cells/mL) were subcultured in a 96-well microtiter plate in a humidified 5% CO2 incubator at 37 °C on day 0. After addition of 0 or 10 μM Cu-APC on day 0, the images of HeLa cells were taken using an inverted research microscope IX71 (Olympus Corporation, Tokyo, Japan) on days 1 and 7. Antiproliferation Assay. Subconfluent HeLa or NHDF cells (100 μL containing 104 cells/mL) were subcultured in the 96-well microtiter plate in the humidified 5% CO2 incubator at 37 °C on day 0. On day 1, 50 μL of each culture containing various concentrations of TMPyP4 or Cu-APC was added to cell cultures. On day 4, the cultured cells were twice washed with 150 μL of a new medium without any ligand, and the medium was completely removed from the plates. Then, cell counting was performed with Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan) as follows.28 Each reaction mixture, which comprised 100 μL of each culture and 10 μL of Cell Counting Kit-8 solution containing water-soluble tetrazolium salt, was added to the cultured cells, and the cultured cells in the reaction mixture were incubated in the humidified 5% CO2 incubator for 1 h at 37 °C. The reaction was terminated by adding 20 μL of 0.1 M HCl. Since the watersoluble tetrazolium salt is reduced by dehydrogenases in the cell and produces water-soluble formazan with a peak absorbance at 460 nm, the absorbance at 450 nm was measured in order to investigate the dehydrogenase activity correlating with the number of living cells. For the negative control reactions, no cell was cultured. For the positive control reactions, HeLa or NHDF cell was cultured without any G-quadruplex ligands. Relative dehydrogenase activity (A2) was calculated via the following equation: A 2 = (Y − Y0)/(Yp − Y0)

Figure 2. (A) Normalized absorbance melting curve recorded at 295 nm for Htelo-DNA in a buffer containing 50 mM MES-LiOH (pH 7.0) and 100 mM KCl. (B) Normalized fluorescence melting curve for Htelo-DNA modified with FITC and Dabcyl at the 5′ and 3′ ends, respectively, in a buffer containing 50 mM MES-LiOH (pH 7.0) and 100 mM KCl in the absence (solid line) and presence (dashed line) of 128 μg/mL λ DNA and 20 wt % of PEG 200. The fluorescence was monitored at 530 nm with an excitation wavelength of 485 nm.

which is a signature for the G-quadruplex structure.25 Moreover, fluorescence melting analyses of Htelo-DNA modified with FITC and Dabcyl at the 5′ and 3′ ends, respectively, were carried out under the same condition.26 The fluorescence intensity of FITC at 530 nm increased with higher temperature (Figure 2B). This is because FITC at the 5′ end and Dabcyl at the 3′ end are situated close to each other when the G-quadruplex is formed at lower temperature, and thermal denaturation of the G-quadruplex separates FITC and Dabcyl.26 The Tm value obtained from the fluorescence melting curve was 71.0 °C. Because Tm values evaluated from UV and fluorescence melting analyses were within the margin of error (2 °C), this confirmed that fluorescence melting analysis is useful for detecting G-quadruplex formation.26 Because of the excess of dsDNA (400 μM in base unit), it was not possible to confirm the G-quadruplex formation by CD spectroscopy and UV melting curve in a cell nuclei-mimicking solution that contained 100 mM KCl, 50 mM MES-LiOH (pH 7.0), 128 μg/ mL λ DNA (400 μM in base unit), and 20 wt % of PEG 200. By fluorescence analysis, a melting curve with a Tm value of 80.0 °C was observed in the cell nuclei-mimicking solution (Figure 2B). It has been reported that MC with PEG 200 stabilizes G-quadruplexes.17 Therefore, these results suggest that Htelo-DNA forms a G-quadruplex even under the condition that mimics cell nuclei. Binding of Cu-APC and TMPyP4 to G-Quadruplex under the Cell Nuclei-Mimicking Condition. Next, we studied the binding of Cu-APC to G-quadruplex under the cell nuclei-mimicking condition. In UV−vis absorbance spectra of Cu-APC with Htelo-DNA, a new peak around 690 nm increased as a function of Htelo-DNA concentration in a buffer of 100 mM KCl, 50 mM MES-LiOH (pH 7.0), 128 μg/ mL λ DNA, and 20 wt % of PEG 200 (Figure 3A). This increased peak was also observed in the absence of λ DNA and

(4)

where Y is the absorbance value. Subscripts “p” and “0” indicate the positive and negative controls, respectively.

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RESULTS AND DISCUSSION Structural Analysis of a Telomeric DNA under a Cell Nuclei-Mimicking Condition. Initially, we examined the structure of a human telomeric oligo DNA, 5′-GGG2608

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the buffer that lacked λ DNA and PEG 200, 2.8 ± 0.1 × 104 M−1 (Figure 3B). Thus, Cu-APC binds to the G-quadruplex equally efficiently under the condition that mimics cell nuclei and under the test tube condition. Next, we studied TMPyP4 binding to the G-quadruplex under the diluted test tube condition and the cell nuclei-mimicking condition with λ DNA and PEG 200. In contrast to Cu-APC, the change in UV−vis spectra of TMPyP4 with Htelo-DNA was subtle in the presence of λ DNA and 20 wt % PEG 200 (Figure 3C), although a significant reduction at 424 nm was observed in the absence of λ DNA and PEG 200 (Figure S2B). These results indicate that TMPyP4 bound to the G-quadruplex only in the absence of λ DNA and PEG 200, as shown previously (Figure S2B).10,19 Previous studies using UV−vis and ITC showed stoichiometries of 1:1 (G-quadruplex:TMPyP4) and 1:2 for strong and weak binding modes, respectively.30 Based on the assumption that the dominant stoichiometry is 1:1, the Ka value was estimated to be 3.6 ± 1.8 × 106 M−1 in the absence of λ DNA and PEG 200 according to plots of absorbance at 424 nm vs Htelo-DNA concentration (Figure 3D). This Ka value is very similar to that for the stronger binding mode obtained by ITC experiments previously (4.0 ± 0.4 × 106 M−1).30 These results indicate that under the condition mimicking cell nuclei, CuAPC can bind to the G-quadruplex as efficiently as it does under dilute conditions, and that in contrast, TMPyP4 drastically lost binding affinity for the G-quadruplex under the condition mimicking cell nuclei. Effects of the Coordinating Metal and Functional Groups on the Ligand Binding. Because Cu-APC has a coordinating copper at its center, whereas TMPyP4 does not, the effect of the coordinating copper on the binding property of ligands was studied using an anionic ligand without coordinating metals, APC (Figure 1), and a cationic one, copper TMPyP4 (Cu-TMPyP4; Figure 1). The structures of APC and Cu-TMPyP4 are identical to Cu-APC and TMPyP4, respectively, except for the coordinating copper. Like Cu-APC, APC bound to Htelo-DNA with similar Ka values, 3.4 ± 0.2 × 104 and 5.6 ± 0.4 × 104 M−1, in the absence and presence of λ DNA and PEG 200, respectively, as shown in Figure 4A. In contrast, the binding property of Cu-TMPyP4 was repressed by the cell nuclei-mimicking condition (Figure 4B) as observed for TMPyP4 (Figure 3D). These results show that the coordinating metal has little impact on ligand binding to the G-quadruplex. In addition, in order to investigate the effects of functional groups on the binding of G-quadruplex ligands, the binding capacity of TTMAPP (Figure 1) was also studied in the absence or presence of λ DNA and PEG 200. TTMAP is also a cationic ligand with a porphyrin scaffold like TMPyP4, but its

Figure 3. UV−vis spectra of 2.5 μM Cu-APC with 0−250 μM HteloDNA (A) and 1 μM TMPyP4 with 0−10 μM Htelo-DNA (C) in a buffer containing 50 mM MES-LiOH (pH 7.0), 100 mM KCl, 128 μg/ mL λ DNA, and 20 wt % PEG 200 at 25 °C. Plots of absorbance at 690 nm of Cu-APC (B) and 424 nm of TMPyP4 (D) vs the concentration of Htelo-DNA in the absence (blue) and presence (red) of λ DNA and PEG 200.

PEG 200 (Figure S2A). These results indicate that Cu-APC bound to the G-quadruplex under both conditions in the presence and absence of λ DNA and PEG 200. The stoichiometry of the binding was studied by titration experiment with higher concentrations of Cu-APC and Htelo-DNA (100 μM Cu-APC and 0−500 μM Htelo-DNA) in a buffer containing 50 mM MES-LiOH (pH 7.0), 100 mM KCl, and 10 mM MgCl2 (Figure S3). We added MgCl2 in the buffer because Mg2+ reduces the electrostatic repulsion between Cu-APC and DNA, resulting in a higher affinity of Cu-APC with HteloDNA. A stoichiometric point around 100 μM Htelo-DNA was observed (Figure S3), suggesting a 1:1 binding of Cu-APC and Htelo-DNA. Based on the assumption that the 1:1 binding stoichiometry does not depend on the experimental conditions, the association constant (Ka) for Htelo-DNA in the presence of λ DNA and PEG 200 was estimated to be 2.8 ± 1.2 × 104 M−1 at 25 °C according to plots of absorbance at 690 nm versus Htelo-DNA concentration (Figure 3B) (see Experimental Section). Surprisingly, this Ka value was identical to that in

Figure 4. Plots of absorbance at 710 nm of APC (A), at 424 nm of Cu-TMPyP4 (B), and at 424 nm of TTMAPP (C), vs the concentration of Htelo-DNA in a buffer containing 50 mM MES-LiOH (pH 7.0) and 100 mM KCl in the absence (blue) and presence (red) of 128 μg/mL λ DNA and 20 wt % PEG 200 at 25 °C. 2609

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Figure 5. Results of the tsTRAP assay. Various concentrations of Cu-APC (A) or TMPyP4 (C) were used in the absence (left) or presence (right) of 128 μg/mL λ DNA and 20 wt % PEG 200. I.C. is the internal control. The relative activity of telomerase vs the concentration of Cu-APC (B) and TMPyP4 (D) in the absence (blue) and presence (red) of λ DNA and PEG 200.

required to rationalize how cosolutes affect the functions of the G-quadruplex ligands, it is considered that reduced water activity by PEG 200 has a significant impact on ligand binding to the G-quadruplex in the presence of λ DNA and PEG 200. In addition, it was demonstrated that twisted intercalating nucleic acids (TINA)-modified telomeric oligonucleotides contributed more to stabilization of the G-quadruplex and the subsequent telomerase inhibition in the presence of PEG 200 than in the absence of PEG 200.34 That is attributed to the fact that TINA insertions stabilize the Hoogsteen bonds of the Gquadruplex, the formation of which releases water molecules. This study also supports that reduced water activity by PEG 200 is a key factor affecting G-quadruplex-targeting drugs. Telomerase Inhibition by CuAPC and TMPyP4 under the Cell Nuclei-Mimicking Condition. The robust binding properties of Cu-APC should allow for efficient inhibition of telomerase activity under the condition mimicking cell nuclei. We utilized a two-step telomeric repeat amplification protocol (tsTRAP) assay to study the inhibitory effect of Cu-APC (Figure 5A,B).3,10 In the tsTRAP assay, PCR was carried out with 50-fold diluted products of the telomerase reaction to avoid the false-negative problem caused by PCR inhibition by ligands and experimental conditions (see details in the Experimental Section). The tsTRAP products were reduced as a function of the concentration of Cu-APC under the diluted condition, as reported previously (left panel of Figure 5A).10 Importantly, even in the presence of 128 μg/mL λ DNA and 20 wt % PEG 200, Cu-APC inhibited the telomerase activity (right panel of Figure 5A). The IC50 (half maximal inhibitory concentration) of Cu-APC in the presence of λ DNA and PEG 200 was 2.1 μM, which was similar to the IC50 value, 0.86 μM, in the absence of both λ DNA and PEG 200 (Figure 5B).

cationic functional group differs from that of TMPyP4. TTMAPP exhibited a similar binding property as TMPyP4 and Cu-TMPyP4 (Figure 4C) both in the absence and presence of λ DNA and PEG 200. These systematic results of the cationic and anionic ligands may generalize that cationic groups do not contribute to ligand binding to the G-quadruplex under the cell nuclei-mimicking condition. This lesser contribution of cationic groups should be partially caused by electrostatic binding to λ DNA based on the previous studies on TMPyP4 binding to dsDNA.10,31 On the other hand, as pointed out by the recent reports,32 the effects of PEGs on biomolecular properties are difficult to rationalize because the cosolutes such as PEGs not only alter a variety of properties of the solution, but they also directly bind to biomolecules. Furthermore, Chaires and co-workers recently demonstrated by molecular dynamics simulations that PEG 200 interacts with the planar region of the G-tetrad faces of the propeller Gquadruplex,33 which may inhibit ligand binding to the Gquadruplex. In this study, we found that the binding affinity of Cu-APC in the presence and absence of λ DNA and PEG 200 was identical, while TMPyP4 reduced the binding affinity in the presence of λ DNA and PEG 200. Thus, it is possible to conclude that the direct binding of PEG 200 to the G-tetrad may not largely affect ligand binding to the G-quadruplex in our experimental condition. One way to assess which effect of PEGs plays a major role is to compare the effects of different cosolutes, such as PEGs with different molecular weight. Our previous studies for ligand binding to the G-quadruplex in the presence of etylene glycol, PEG 200, and PEG 8000 indicated that reduced water activity by these cosolutes decreased the affinity of TMPyP4 to the G-quadruplex.23 Although further studies are 2610

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MTT assay.28 Dehydrogenase activity within HeLa cells decreased as a function of Cu-APC concentration (Figure 7A). The IC50 value was evaluated to be 1.4 ± 0.3 μM. It has been known that the copper ion itself is toxic to cells.37 However, the coordinating copper should not dissociate from phthalocyanines because of its tight-binding to phthalocyanines.38 In addition, the IC50 value of the copper ion for cell proliferation is usually more than several tens of micromolar concentration,37 which is much larger than that of Cu-APC for HeLa cells (Figure 7A). Therefore, the decrease in the dehydrogenase activity shown in Figure 7A indicates that CuAPC inhibits HeLa cell proliferation via the telomerase inhibition as expected from the results obtained under the cell nuclei-mimicking condition. The inhibitory effect of CuAPC was comparable with that of other superior G-quadruplex ligands such as BRACO-19 (IC50 = 13, 10, and 10 μM for SKOV-3, CHI, and A2780, respectively),8 12459 (IC50 = 1.2 μM for A549),39 PIPER (IC50 = 9 μM for HT29),9 and MST312 (IC50 = 1.7 μM for U937).40 Furthermore, our previous study using Cu-APC, APC, and anionic nickel phthalocyanine (Ni-APC) showed that the capacities of anionic phthalocyanines to bind to the G-quadruplex and to inhibit the telomerase activity depended little on the coordinating metal in the diluted condition.10 Thus, it is expected that APC and Ni-APC show the similar cell antiproliferative effects with that of Cu-APC, although only Cu-APC was tested as a representative example here. Moreover, the main purposes of this study are the following: (1) to show that the cell nuclei-mimicking condition with λ DNA and PEG 200 is useful to evaluate ligand functions in cells and (2) to demonstrate that anionic molecules can be promising for targeting telomeric DNA and telomerase. Although the role of metal on the telomerase inhibition should be studied in detail, it is out of the scope of this study. On the contrary, TMPyP4 did not affect HeLa cell proliferation at all even at high concentration (10 μM) (Figure 7A). This difference of antiproliferation effects between Cu-APC and TMPyP4 is in good agreement with the results of titration experiments and telomerase inhibitory assays under the cell nuclei-mimicking condition but not under the diluted condition. These results suggest that conventional test tube conditions totally differ from intracellular conditions, and that the cell nuclei-mimicking condition in this study is effective to predict capacities of G-quadruplex ligands in cell. In addition, the antiproliferation effect of Cu-APC on normal human dermal fibroblast (NHDF) cells was also examined to investigate specificity of Cu-APC. Interestingly, NHDF cell proliferation was less affected by Cu-APC than was HeLa cell proliferation (Figure S4). The IC50 value with NHDF cells was 18 ± 13 μM, which was 13 times larger than that with HeLa cells (Figure 7B), indicating that the effects of Cu-APC on cell proliferation are reasonably specific for HeLa cells. Although further mechanisms of the effects should be studied as we will discuss in the next paragraph, these results support that specific binding of Cu-APC to G-quadruplex can inhibit telomerase function in living cells. The selectivity of the antiproliferative property of Cu-APC was a little lower than that of telomestatin, a nonionic G-quadruplex ligand; 3 days of treatment with 5 μM telomestatin kills about 60% of all HeLa cells but less than 10% of all normal cells;41 it was found here that about 60% of HeLa cells and 20% of NHDF cells were killed by 5 μM Cu-APC (Figures 7A and S4). The selective antiproliferative effect of Cu-APC on cancer cells may lead to fewer side effects for normal cells.

The efficient Cu-APC-mediated inhibition of telomerase even under the condition mimicking cell nuclei was attributed to the robust binding of Cu-APC to the telomeric G-quadruplex, as indicated in the UV−vis titration assay. TMPyP4 also inhibited the telomerase activity with an IC50 value of 1.6 μM under the diluted condition (left panel in Figure 5C and blue line in Figure 5D). However, under the condition mimicking cell nuclei, TMPyP4-mediated inhibition of the telomerase activity was completely abrogated (right panel in Figure 5C and red line in Figure 5D); this finding is consistent with the decreased binding of TMPyP4 to the G-quadruplex under the condition. In the tsTRAP assay, λ DNA and PEG 200 should affect the telomerase activity. For example, we previously reported that PEG 200 itself inhibited slightly the telomerase activity, which is attributable to the stabilization of the G-quadruplex of the telomeric DNA, and the destabilization of hybridization between the telomerase RNA and the telomeric DNA.35 Therefore, to study the telomerase inhibitory effect of each ligand in Figure 5B,D, telomerase activities in the presence of various concentrations of ligands were evaluated as relative activities that were normalized with the telomerase activity without ligands in the presence of λ DNA and PEG 200. Taken together, the results of the binding and tsTRAP assays imply that in cell nuclei, where dsDNA and highly concentrated biomolecules are very abundant, Cu-APC will inhibit telomerase activity more efficiently than will TMPyP4. Cell-Based Assay with CuAPC and TMPyP4. On the basis of the results of the binding assays and telomerase inhibition assays, we further assessed the inhibitory effects of Cu-APC on the proliferation of HeLa cells as a model of cancer cells. First, we examined a permeation behavior of Cu-APC into living HeLa cells. It was found that HeLa cells were stained gradually by Cu-APC during culture of HeLa cells in a medium containing Cu-APC (Figure 6), indicating that Cu-APC

Figure 6. Images of HeLa cells cultured in a medium containing 0 (upper panels) or 10 μM (lower panels) Cu-APC in a humidified 5% CO2 incubator at 37 °C.

permeated HeLa cells. This cell permeation of Cu-APC is consistent with the previous study showing that zinc anionic phthalocyanine was taken up in tumor cells.36 Next, after 3 days in culture of HeLa cells in the presence of Cu-APC, the viability of the cells was evaluated via an assay based on dehydrogenase activity within cells; this assay correlates with the conventional 2611

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Figure 7. (A) Relative activity of dehydrogenase within HeLa cells as a function of Cu-APC (red) or TMPyP4 (blue) concentration. Relative activity value of 1 corresponds to the positive control, which was without ligand. (B) Comparison of IC 50 values of Cu-APC for HeLa cells and NHDF cells.

Possible Mechanism of Anticancer Effects of Cu-APC. Conventional telomerase inhibitors require long-term treatment to inhibit cell proliferation.41 However, Cu-APC exhibited anticancer effects after only 3 days of treatment (Figure 7A); this finding indicates that Cu-APC may inhibit HeLa cell proliferation not only by telomerase inhibition but also via other mechanisms. For example, some G-quadruplex ligands regulate the transcription of oncogenes by binding to Gquadruplexes found in the promoter regions.42 Also, the binding of telomestatin to telomeric G-quadruplex leads to disruption of the capping function of telomeric DNA, and disrupting this function induces rapid apoptosis of cancer cells.41 On the basis of these results, we postulate that Cu-APC may have anticancer effects that result from binding to telomeric G-quadruplex and from binding to G-quadruplexes within oncogene promoters. Although further studies are required, the specific binding of Cu-APC to G-quadruplex and the persistence of this binding under the condition that mimics cell nuclei as demonstrated here are likely critical to its anticancer effects. Furthermore, phthalocyanines preferentially accumulate in cancer cells,43 and anionic compounds are expected to exhibit long blood retention properties and high transfection efficiency because they do not participate in electrostatic interactions with lipoproteins or seric proteins.44 These properties of Cu-APC may facilitate its development as an anticancer drug that has very few side effects.

of TMPyP4. Furthermore, in accordance with the results of binding affinity and telomerase inhibitory function under the cell nuclei-mimicking condition, Cu-APC showed a greater antiproliferative effect on HeLa cell than TMPyP4 did. These results show that the cell nuclei-mimicking condition in this study is effective to predict capacities of G-quadruplex ligands in cell. In addition, the antiproliferative effect of Cu-APC on NHDF cell was smaller than that on HeLa cell, indicating that the cell nuclei-mimicking condition is also useful to predict side effects of ligands because it can evaluate specificity of ligands for G-quadruplex. Furthermore, Cu-APC is promising as an anticancer drug, although the antiproliferative mechanism still remains to be fully elucidated.

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ASSOCIATED CONTENT

S Supporting Information *

CD spectrum of Htelo-DNA in a diluted solution (Figure S1), UV−vis spectra of Cu-APC or TMPyP4 with Htelo-DNA in a diluted solution (Figure S2), stoichiometric titration of CuAPC with HteloDNA (Figure S3), and relative activity of dehydrogenase within NHDF cultured with Cu-APC (Figure S4). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

CONCLUSIONS We systematically examined the telomeric G-quadruplexbinding and telomerase-inhibiting capacities of five Gquadruplex ligands under the diluted condition and the cell nuclei-mimicking condition where λ DNA and PEG 200 cosolute coexist. Although cationic ligands such as TMPyP4, Cu-TMPyP4, and TTMAPP bound to the telomeric Gquadruplex under the diluted condition without λ DNA and PEG 200, the binding affinity was drastically reduced under the cell nuclei-mimicking condition. On the contrary, the cell nuclei-mimicking condition did not affect the binding capacities of anionic ligands such as Cu-APC and APC. These results indicate that electrostatic attraction was reduced under the cell nuclei-mimicking condition, and the coordinating metal, copper, made little contribution on the robust binding capacity of anionic phthalocyanines. Correspondingly, the telomerase inhibitory effect of Cu-APC under the diluted condition and the cell nuclei-mimicking condition was similar, although the addition of λ DNA and PEG 200 drastically reduced the effect

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We appreciate Dr. T. Endoh for education of cell-based assay and Chieko Hijiriyama for conducting the binding assays. This work is supported by Grants-in-Aid for Scientific Research, the Scientific Research on Innovative Areas “Nanomedicine Molecular Science”, the “Strategic Research Foundation at Private Universities” (2009−2014) from the Ministry of Education, Culture, Sports, Science and Technology, Japan, Kurata Grants from the Kurata Memorial Hitachi Science and Technology Foundation, and the grant for Research on Chemical and Biological Materials from the Japan Biochemistry Association. 2612

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