Specific Conformational Change in Giant DNA Caused by

Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Suzuka, Mie 513-8670, Japan. ‡ Faculty of Pharmaceutical Sciences, Osaka ...
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Specific Conformational Change in Giant DNA Caused by Anticancer Tetrazolato-Bridged Dinuclear Platinum(II) Complexes: Middle-Length Alkyl Substituents Exhibit Minimum Effect Seiji Komeda,*,† Hiroki Yoneyama,‡ Masako Uemura,† Akira Muramatsu,§ Naoto Okamoto,† Hiroaki Konishi,∥ Hiroyuki Takahashi,⊥ Akimitsu Takagi,∥ Wakao Fukuda,# Tadayuki Imanaka,# Toshio Kanbe,¶ Shinya Harusawa,‡ Yuko Yoshikawa,§ and Kenichi Yoshikawa§ †

Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Suzuka, Mie 513-8670, Japan Faculty of Pharmaceutical Sciences, Osaka University of Pharmaceutical Sciences, Takatsuki, Osaka 569-1094, Japan § Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Kyoto 610-0394, Japan ∥ Yakult Central Institute, Yakult Honsha Co., Ltd., Kunitachi, Tokyo 186-8650, Japan ⊥ Pharmaceutical Research and Development Department, Yakult Honsha Co., Ltd., Chuo, Tokyo 104-0061, Japan # Department of Biotechnology, College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan ¶ Laboratory of Medical Mycology, Research Institute for Disease Mechanism and Control, School of Medicine, Nagoya University, Nagoya 464-0064, Japan ‡

S Supporting Information *

ABSTRACT: Derivatives of the highly antitumor-active compound [{cis-Pt(NH3)2}2(μ-OH)(μ-tetrazolato-N2,N3)]2+ (5-H-Y), which is a tetrazolato-bridged dinuclear platinum(II) complex, were prepared by substituting a linear alkyl chain moiety at C5 of the tetrazolate ring. The general formula for the derivatives is [{cisPt(NH3)2}2(μ-OH)(μ-5-R-tetrazolato-N2,N3)]2+, where R is (CH2)nCH3 and n = 0 to 8 (complexes 1−9). The cytotoxicity of complexes 1−4 in NCI-H460 human non-small-cell lung cancer cells decreased with increasing alkyl chain length, and those of complexes 5−9 increased with increasing alkyl chain length. That is, the in vitro cytotoxicity of complexes 1−9 was found to have a U-shaped association with alkyl chain length. This U-shaped association is attributable to the degree of intracellular accumulation. Although circular dichroism spectroscopic measurement indicated that complexes 1−9 induced comparable conformational changes in the secondary structure of DNA, the tetrazolatobridged complexes induced different degrees of DNA compaction as revealed by a single DNA measurement with fluorescence microsopy, which also had a U-shaped association with alkyl chain length that matched the association observed for cytotoxicity. Complexes 7−9, which had alkyl chains long enough to confer surfactant-like properties to the complex, induced DNA compaction 20 or 1000 times more efficiently than 5-H-Y or spermidine. A single DNA measurement with transmission electron microscopy revealed that complex 8 formed large spherical self-assembled structures that induced DNA compaction with extremely high efficiency. This result suggests that these structures may play a role in the DNA compaction that was induced by the complexes with the longer alkyl chains. The derivatization with a linear alkyl chain produced a series of complexes with unique cellular accumulation and DNA conformational change profiles and a potentially useful means of developing nextgeneration platinum-based anticancer drugs. In addition, the markedly high ability of these complexes to induce DNA compaction and their high intracellular accumulation emphasized the difference in mechanism of action from platinum-based anticancer drugs.

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reveal a novel pharmacophore. The clinical significance of transition-metal−DNA interactions was first demonstrated with the mononuclear platinum(II) complex cis-diamminedichloridoplatinum(II) (cisplatin),5 which has since become an essential

he secondary and higher-order structures of DNA can be altered via interactions with inorganic1 and organic cations.2 For example, in the presence of multivalent transitionmetal cations or cationic metal complexes such as Fe3+ and [Co(NH3)6]3+, high-molecular-weight DNA undergoes marked compaction.3,4 Examining the interactions involved in metalcomplex-mediated DNA compaction is important because it may © XXXX American Chemical Society

Received: September 16, 2016

A

DOI: 10.1021/acs.inorgchem.6b02239 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry

but also discover a new mechanism of action lying between these cationic tetrazolato-bridged dinuclear Pt(II) complexes and the DNA to which they are bound.

anticancer agent. Platinum-based anticancer agents form covalent platinum−DNA adducts,6 such as 1,2-intrastrand crosslinks7 and interstrand cross-links,8 which are thought to be how platinum-based anticancer drugs exert their anticancer effects.9 The particularly high therapeutic efficacy of platinum-based anticancer drugs compared with other anticancer agents has prompted the development of novel platinum-based anticancer drugs that are effective against chemotherapy-insensitive cancers3 and, possibly, cancers that have acquired resistance to the currently available platinum-based anticancer drugs.10 Through this research, a diverse range of covalent Pt(II)− DNA adducts with anticancer effects have been identified.11−15 We previously reported that a series of cationic tetrazolatobridged dinuclear Pt(II) complexes with the general formula [{cis-Pt(NH3)2}2(μ-OH)(μ-5-R-tetrazolato-N2,N3)]2+ were potent anticancer drug candidates that had remarkably high antitumor efficacy in a xenograft nude mouse model of pancreatic cancer.16,17 Substitution of two nucleobases for the μ-hydroxo group bridging the two Pt(II) ions resulted in the complexes forming covalent DNA adducts at a much slower rate than cisplatin.18 However, these complexes also formed DNA adducts via diffusion-controlled, noncovalent interactions, which induced dramatic conformational changes in the secondary and higherorder structures of the bound DNA, such as B- to C-form DNA transitioning and DNA compaction even at low concentrations,19−21 which has not been reported for the platinum-based drugs. These results suggest that there may be novel therapeutic applications for these complexes, especially in the treatment of cancers that are insensitive to the platinum-based anticancer drugs. In the present study, we prepared seven new derivatives of the anticancer drug candidate [{cis-Pt(NH3)}2(μ-OH)(μ-tetrazolato-N2,N3)]2+ (5-H-Y, Figure 1) by substituting linear alkyl

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EXPERIMENTAL SECTION

Materials. K2PtCl4 was obtained from Tanaka Kikinzoku Kogyo K.K. (Tokyo, Japan). The 5-alkyltetrazoles25 (5-propyl-1H-tetrazole, 5-butyl-1H-tetrazole, 5-pentyl-1H-tetrazole, 5-hexyl-1H-tetrazole, 5-heptyl-1H-tetrazole, 5-octyl-1H-tetrazole, 5-nonyl-1H-tetrazole) and the tetrazolate-bridged complexes of [{cis-Pt(NH3)2}2(μ-OH)(μ-5methyltetrazolato-N2,N3)](NO 3 ) 2 (1), 17 and [{cis-Pt(NH 3 ) 2 } 2 (μ-OH)(μ-5-ethyltetrazolato-N2,N3)](NO3)2(2),25 were prepared according to published methods. Preparation of [{cis-Pt(NH 3 ) 2 } 2 (μ-OH)(μ-5-n-propyltetrazolato-N2,N3)](NO3) 2 (3), [{cis-Pt(NH3)2}2(μ-OH)(μ-5-nbutyltetrazolato-N2,N3)](NO3)2 (4), [{cis-Pt(NH3)2}2(μ-OH)(μ-5-npentyltetrazolato-N2,N3)](NO3)2 (5), and [{cis-Pt(NH3)2}2(μ-OH) (μ-5-n-hexyltetrazolato-N2,N3)](NO3)2 (6). A solution of 1H-5-nalkyltetrazole (3.57 mmol) in 20 mL of MeOH was added to a solution of [cis-Pt(NH3)2(μ-OH)]2(NO3)2 (2.00 g, 3.25 mmol) in 80 mL of water. The solution was stirred at 40 °C for 48 h in the dark and lyophilized. The resulting white powder was collected on a glass filter, washed with 2-propanol and diethyl ether, and recrystallized from water. 3. Yield: 0.68 g (29%). Anal. Calcd for C4H20N10O7Pt2: C, 6.76; H, 2.84; N, 19.72. Found: C, 6.59; H, 2.93; N, 19.65. All values are given as percentages. 1H NMR (D2O): δ = 0.92 (3H, t), 1.74 (2H, sext), 2.86 (2H, t). 13C NMR (D2O): δ = 15.6, 23.9, 29.5, 168.6. 195Pt NMR: δ = −2173. MS [M − H]+: 585.1 (M = [{cis-Pt(NH3)2}2(μ-OH)(μ-5-npropyltetrazolato-N2,N3)]2+). 4. Yield: 0.52 g (22%). Anal. Calcd for C5H22N10O7Pt2: C, 8.29; H, 3.06; N, 19.33. Found: C, 8.13; H, 2.96; N, 18.56. All values are given as percentages. 1H NMR (D2O): δ = 0.91 (3H, t), 1.38 (2H, sext), 1.70 (2H, quint), 2.90 (2H, t). 13C NMR (D2O): δ = 15.8, 24.3, 27.2, 32.5, 168.8. 195Pt NMR: δ = −2173. MS [M − H]+: 599.1 (M = [{cisPt(NH3)2}2(μ-OH)(μ-5-n-butyltetrazolato-N2,N3)]2+). 5. Yield: 0.64 g (27%). Anal. Calcd for C6H24N10O7Pt2: C, 9.76; H, 3.28; N, 18.97. Found: C, 9.61; H, 3.39; N, 18.33. All values are given as percentages. 1H NMR (D2O): δ = 0.88 (3H, t), 1.26−1.37 (4H), 1.73 (2H, quint), 2.89 (2H, t). 13C NMR (D2O): δ = 16.1, 24.5, 27.5, 30.0, 33.3, 168.8. 195Pt NMR: δ = −2172. MS [M − H]+: 613.1 (M = [{cisPt(NH3)2}2(μ-OH)(μ-5-n-pentyltetrazolato-N2,N3)]2+). 6. Yield: 0.68 g (28%). Anal. Calcd for C7H28N10O7Pt2: C, 11.17; H, 3.48; N, 18.62. Found: C, 11.30; H, 3.70; N, 18.36. All values are given as percentages. 1H NMR (D2O): δ = 0.87 (3H, t), 1.2−1.4 (6H), 1.72 (2H, quint), 2.89 (2H, t). 13C NMR (D2O): δ = 16.2, 24.7, 27.5, 30.3, 30.7, 33.5, 168.9. 195Pt NMR: δ = −2174. MS [M − H]+: 627.1 (M = [{cisPt(NH3)2}2(μ-OH)(μ-5-n-hexyltetrazolato-N2,N3)]2+). Preparation of [{cis-Pt(NH 3 ) 2 } 2 (μ-OH)(μ-5-n-heptyltetrazolato-N2,N3)](NO3)2 (7), [{cis-Pt(NH3)2}2(μ-OH)(μ-5-n-octyltetrazolato-N2,N3)](NO3)2 (8), and [{cis-Pt(NH3)2}2(μ-OH)(μ-5-nnonyltetrazolato-N2,N3)](NO3)2 (9). A solution of 1H-5-alkyltetrazole (3.57 mmol) in 30 mL of MeOH was added to a solution of [cis-Pt(NH3)2(μ-OH)]2(NO3)2 (2.00 g, 3.25 mmol) in 60 mL of water. The solution was stirred vigorously at 50 °C for 48 h in the dark and then lyophilized. The resulting white powder was collected on a glass filter, washed with 2-propanol and diethyl ether, and recrystallized from water twice. 7. Yield: 0.72 g (29%). Anal. Calcd for C8H28N10O7Pt2: C, 12.54; H, 3.68; N, 18.27. Found: C, 13.33; H, 3.68; N, 18.12. All values are given as percentages. 1H NMR (CD3OD): δ = 0.90 (3H, t), 1.2−1.5 (8H), 1.74 (2H, quint), 2.86 (2H, t). 13C NMR (CD3OD): δ = 16.3, 25.5, 28.2, 31.1, 31.9, 32.0, 34.7, 168.6. 195Pt NMR (CD3OD): δ = −2178. MS [M − H]+: 641 (M = [{cis-Pt(NH3)2}2(μ-OH)(μ-5-n-hexyltetrazolato-N2,N3)]2+). 8. Yield: 0.82 g (32%). Anal. Calcd for C9H30N10O7Pt2: C, 13.85; H, 3.87; N, 17.94. Found: C, 14.46; H, 4.02; N, 17.94. All values are given as percentages. 1H NMR (CD3OD): δ = 0.90 (3H, t), 1.2−1.5 (10H), 1.73 (2H, quint), 2.86 (2H, t). 13C NMR (CD3OD): δ = 16.3, 25.6, 28.2, 31.1, 32.08, 32.18, 32.23, 34.9 168.6. 195Pt NMR (D2O): δ = −2175.

Figure 1. Structures of 5-H-Y and the series of 5-alkyl-tetrazolatobridged dinuclear Pt(II) complexes with the general formula [{cis-Pt(NH3)2}2(μ-OH)(μ-5-R-tetrazolato-N2,N3)]2+, where R = (CH2)nCH3.

chains of various lengths at C5 of the tetrazolate ring; substitution at this position has been suggested to affect the intracellular accumulation and DNA conformational changes induced by these complexes.22 At present, lung cancers are the most common cause of cancer death,23 with 80% classified as chemoresistant non-small-cell lung cancers (NSCLC).24 It has also been reported that tetrazolato-bridged complexes exhibited the highest cytotoxicity against NSCLC cells in a panel of 39 human cancer cell lines (JFCR39).17 Therefore, here we used a human NSCLC cell line to determine the cytotoxicity and intracellular accumulation of our study complexes. We also determined the effects of these complexes on the secondary and higher-order structures of DNA by means of circular dichroism spectroscopy, fluorescence microscopy, and electron microscopy techniques. The results of these investigations enabled us to not only deduce structure−activity relationships for these complexes B

DOI: 10.1021/acs.inorgchem.6b02239 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry MS [M − H]+: 655 (M = [{cis-Pt(NH3)2}2(μ-OH)(μ-5-n-octyltetrazolato-N2,N3)]2+). 9. Yield: 0.42 g (16%). Anal. Calcd for C10H32N10O7Pt2: C, 15.12; H, 4.06; N, 17.63. Found: C, 15.26; H, 4.05; N, 17.49. All values are given as percentages. 1H NMR (CD3OD): δ = 0.90 (3H, t), 1.2−1.5 (12H), 1.74 (2H, quint), 2.86 (2H, t). 13C NMR (CD3OD): δ = 16.3, 25.6, 28.2, 31.1, 32.1, 32.26, 32.29, 32.5, 34.9, 168.6. 195Pt NMR (CD3OD): δ = −2179. MS [M − H]+: 669 (M = [{cis-Pt(NH3)2}2(μ-OH)(μ-5-nnonyltetrazolato-N2,N3)]2+). NMR Measurement. The 1H, 13C, and 195Pt NMR spectra of 3−9 were recorded on a Varian NMR System (600 MHz, Agilent, Santa Clara, CA, US) at 20 °C and are shown in Figures S2, S3, and S4, respectively. All 1H and 13C NMR spectra were referenced to TSP [sodium 3-trimethylsilylpropionate-2,2,3,3-d(4), δ = 0], and 195Pt chemical shifts to K2PtCl4 (δ = −1614). MS Measurement. For complexes 3−6, high-resolution MS were performed using a micrOTOF-Q quadrupole−time-of-flight mass spectrometer (Bruker, Billerica, MA, US) in the positive ion mode. For 7−9, FAB-MS were performed using a JMS-700(2) mass spectrometer (JEOL Ltd., Tokyo, Japan) in the positive ion mode. Cytotoxicity Study. NCI-H460 human non-small-cell lung cancer cells were maintained in RPMI 1640 medium containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin (Invitrogen [Life Technologies], Carlsbad, CA, USA) in a humidified atmosphere of 5% CO2 at 37 °C. On the day before exposure to one of the complexes, 50 μL/well of the cell suspension (2500 cells/well) was seeded into 96-well microplates. For each complex, an aqueous solution was prepared by dissolving a sample of the complex in saline to a concentration of 50 to 100 μmol/L. The aqueous solutions were then further diluted to the required concentrations before adding 50 μL of the dilution to the cell suspensions. After incubation of the microplates for 48 h at 37 °C, 20 μL of Cell Counting Kit-8 solution (Dojindo, Kumamoto, Japan) was added to each well, and the incubation was continued for an additional 60 min at 37 °C. After incubation, the absorbance of each well at a wavelength of 450 nm was measured with a Benchmark microplate reader (Molecular Devices, Sunnyvale, CA, USA). Each experiment was performed independently three times, with three replicate wells for each complex concentration. The IC50 value was calculated as the concentration of complex that provided 50% formazan production relative to that of the control (no complex added). Cellular Accumulation Study. Three milliliters of NCI-H460 cell suspension (2 × 105 cells/mL) was seeded on a 60 mm culture dish. Dimethyl sulfoxide or water solution was prepared for cisplatin or each tetrazolato-bridged complex by dissolving the complex to a concentration of 100 μM. Thirty microliters of solution was added to each dish, and the dishes were incubated at 37 °C to expose the cells to the complex. The exposure times for NCI-H460 were 0, 3, and 6 h. After incubation and cell counting, the cell suspension was collected in centrifuge tubes and centrifuged at 6000g for 5 min. The supernatant was discarded by decantation. To remove extracellular Pt complexes, 2 mL of phosphate-buffered saline was added to the pellet, and the cells were resuspended by pipeting. The suspension was centrifuged at 6000g for 5 min, and the supernatant discarded by decantation a total of three times. The pellet was then resuspended in 200 μL of ultrapure water. The suspension was transferred to a glass container, and 800 μL of concentrated nitric acid was added. The mixture was heated and evaporated to dryness at 120 °C on a hot plate. The residue was then dissolved in 2 mL of 2%(v/v) HNO3, and the amount of Pt was quantified by using a 7500CX inductively coupled plasma mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). Data were acquired and processed by using an Agilent 7500 series inductively coupled plasma mass spectrometer MassHunter workstation (G7200A). The instrument settings were optimized for platinum (m/z 195), and the internal standard used was thallium (m/z 205). A blank sample was prepared by adding 30 μL of 10% dimethyl sulfoxide to 3 mL of cell suspension followed by mineralization, as described above. Each experiment was performed independently three times. Quantitative values were calculated and are shown in the figures in units of nanomoles of Pt complex per 107 cells (Figure 2) because cisplatin

Figure 2. Intracellular content (nmol Pt complex [107 cells]−1) of cisplatin, 5-H-Y, and 1−9 in NCI-H460 human NSCLC cells after 1, 3, or 6 h exposure to 1 μM of the complexes, as determined by means of inductively coupled plasma mass spectrometry (n = 3: mean ± SE). contains one Pt(II) center, whereas the tetrazolato-bridged complexes contained two. Determination of Critical Micelle Concentration (CMC). CMC was determined by using the plot of conductivity versus complex concentration obtained for 6−9. Solutions of the complexes at different concentrations were dissolved and diluted in Millipore water, and conductivities were measured by using an ES-51 conductivity meter (Horiba, Kyoto, Japan) with a cell constant of 100 m−1, calibrated with 10 mM KCl. During each measurement, the solution temperature was maintained at 25 °C by using a water bath with an accuracy of ±0.2 °C. Circular Dichroism (CD) Spectroscopy. Each complex (2−9) was mixed with calf thymus (CT)-DNA in 0.3 mM citrate buffer (pH 7.4) at various molar ratios (r) at room temperature (r = the concentration of complex (μM)/the phosphate concentration of nucleotide (30 μM) = 0, 0.033, 0.067, 0.17, 0.33, 0.67, 1.0). Spectra were determined by using a circular dichroism spectrophotometer (J-805; JASCO, Tokyo, Japan) in the wavelength range of 220 to 340 nm immediately after the addition of the complex to the buffered CT-DNA solution at 25 °C. The path length of the cell was 0.5 cm, and the scan rate was 50 nm/min. Fluorescence Microscopy Observation of the Higher-Order Conformational Changes in T4 DNA Induced by the Complexes. T4 phage DNA (166 kbp) was dissolved in 10 mM Tris-HCl buffer and 4%(v/v) 2-mercaptoethanol at pH 7.6 in the presence of various concentrations (0.2−200 μM) of Pt(II) complexes. Measurements were conducted at a DNA concentration low enough that multimolecular aggregation was not induced (0.1 μM in nucleotide units). To visualize individual DNA molecules under a fluorescence microscope, the cyanine dye YOYO-1 was added to the DNA solution after incubation with the complexes. YOYO-1 binds to double-stranded DNA through base-pair intercalation, which markedly increases its fluorescence. To minimize the effects of YOYO-1 on DNA−complex interactions, a low concentration of YOYO-1 (0.05 μM) was added to the samples immediately prior to the single-DNA observation. Fluorescent DNA images were obtained by using an inverted microscope (Axiovert 200; Carl Zeiss, Oberkochen, Germany) equipped with a 100× oil-immersion objective lens and a highly sensitive electron-bombarded charge-coupleddevice camera (Hamamatsu Photonics, Shizuoka, Japan), which made it possible to record images onto DVD. The video images were analyzed by using ImageJ, which is Java-based image-processing software developed by the National Institutes of Health (Bethesda, MD, USA). C

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Inorganic Chemistry Transmission Electron Microscopy. Samples used for transmission electron microscopy analysis were mounted on carbon-coated copper grids, negatively stained with 2% ammonium molybdate, and observed under a transmission electron microscope (JEM-1400EX; JEOL, Tokyo, Japan) at 100 kV. The DNA concentration was 0.1 μM.

bridged polynuclear Pt(II) complexes was much higher than that of cisplatin.29 In the present study, the intracellular accumulation of the complexes, which all had a +2 charge, was dependent on the length of the alkyl chain. The intracellular accumulation of 5-H-Y and 1−3 decreased as the length of the alkyl chain increased, whereas that of 4−9 increased as the length of the alkyl chain increased. The intracellular content of 9 was 40 times (at 1-h exposure) and 7 times (at 6 h exposure) higher than that of 5-H-Y at the respective times, suggesting that 5-H-Y and the complexes with short alkyl chains (1−3) were taken up into the cells via a different mechanism than were the complexes with a long alkyl chain (7−9). Complexes with an intermediate-length alkyl chain (4−6) may be taken up by both mechanisms but less efficiently. Determination of Critical Micelle Concentration. The complexes examined in the present study were all water-soluble cationic complexes with a +2 charge. However, the alkyl chain substituted at C5 of the tetrazole ring is a hydrophobic moiety, so the complexes with an alkyl chain lipophilic enough to induce micelle formation (complexes 7−9) were expected to have surfactant-like properties. Table 2 shows the CMC determined for 7−9 by means of an electrical conductance method (Table 2.)

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RESULTS Cytotoxicity of the Complexes. First, we determined the cytotoxicity of cisplatin, 5-H-Y, and 1−9 in the NCI-H460 human NSCLC cell line (Table 1). Compared with cisplatin, Table 1. Cytotoxicity of Cisplatin, 5-H-Y, and 1−9 in NCIH460 Human Non-Small-Cell Lung Cancer (NSCLC) Cells complex

IC50/μM

cisplatin 5-H-Y 1 2 3 4 5 6 7 8 9

4.5 1.5 5.3 9.8 52.3 95.1 >100 62.7 34.0 28.4 44.7

Table 2. Critical Micelle Concentration (CMC) of 6−9 at 25°C

5-H-Y, 1, and 2 had comparable cytotoxicity, whereas 3−9 mostly had moderate cytotoxicity. Overall, the cytotoxicity of the series resembled a U-shaped curve, with 5 having the lowest cytotoxicity (an IC50 value higher than 100 μM). For 5-H-Y and 1−4, cytotoxicity decreased with increasing alkyl chain length, whereas for 5−9 cytotoxicity increased with increasing alkyl chain length, with the exception that 9 was found to be less cytotoxic than 7 and 8. Thus, the complexes that possessed an alkyl chain of intermediate length (4 and 5) were less cytotoxic than were those with shorter or longer alkyl chains. Intracellular Accumulation of the Complexes. Next, we examined the intracellular accumulation of cisplatin, 5-H-Y, and 1−9 in NCI-H460 human NSCLC cells by using inductively coupled plasma mass spectrometry.26 Note that in Figure 2 the intracellular content of the Pt(II) complex is expressed in units of nanomoles of Pt complex per 107 cells, not in units of nanograms of Pt per 107 cells, so that the results can be compared because cisplatin contains only one Pt(II) center, whereas the tetrazolatobridged complexes contained two. The intracellular platinum atom content can be calculated by multiplying the Pt(II) complex content by 195 (cisplatin) or 390 (tetrazolato-bridged complexes). At 3 and 6 h after exposure to the complexes (final concentration, 1 μM), the intracellular accumulation for all of the complexes was greater than that of cisplatin (Figure 2). Furthermore, when the units of nmol Pt complex (107 cells)−1 were converted to ng Pt (107 cells)−1, that is, a comparison was made based on intracellular Pt atom content, a major difference was observed between the intracellular Pt accumulation of cisplatin and the tetrazolato-bridged complexes. It has been suggested that cisplatin enters cells via both passive diffusion and active transport.27,28 The greater intracellular accumulation of 5-H-Y compared with that of cisplatin adds weight to our recent results in the cisplatin-resistant L1210 murine leukemia cell line, where more 5-H-Y than cisplatin accumulated within the cells.22 The present results are also consistent with those of Farrell’s group, which demonstrated that the intracellular accumulation of a series of cationic polyamine-

a

complex

CMC (mM)

6 7 8 9

N.D.a 7.03 6.77 0.883

No determinable CMC below saturating concentration.

For comparison, the CMC of the structurally similar surfactant− cobalt(III) complexes cis-[Co(2,2′-bipyridyl)2{NH2(CH2)10CH3}Cl]2+ and cis-[Co(1,10-phenanthroline)2{NH2(CH2)10CH3}Cl]2+ are 0.895 and 0.787 mM, respectively, at 25 °C.30 Circular Dichroism Spectroscopy Analysis of the Conformational Changes Induced by the Complexes in the Secondary Structure of Calf Thymus DNA. We previously reported that 5-H-Y and 1 induced conformational changes in the secondary structure of CT-DNA (30 μM effective phosphate concentration), as determined by CD spectroscopy.20,21 In the present study, we expanded these results by examining the conformational changes in the secondary structure of CT-DNA induced by 2−9. CD measurements were performed immediately after the addition of 2−9 at various molar ratios (r), which we defined as the ratio of the amount of Pt(II) complex added to the total concentration of phosphate in the sample of CT-DNA. The concentration-dependent CD spectral changes for 120 and 2−9 are shown in Figure 3A. At r = 0, the secondary structure of the CT-DNA was in the B-form, as demonstrated by the positive ellipticity around 278 nm and the negative ellipticity of a similar magnitude around 248 nm. However, when 5-H-Y,19 1,20 or 2−9 was added, the positive ellipticity decreased, indicating a B- to C-form transition in the structure of the CT-DNA, which we hypothesized to be a result of electrostatic attractions between the phosphate groups of the CT-DNA and the positively charged Pt(II) complexes.20,21 To test this hypothesis, we measured the CD spectra for CT-DNA in the presence of 2−9 (r = 0.067) with the addition of a high concentration of NaCl (200 mM) to weaken electrostatic attractions between the Pt(II) complex and DNA. The CD spectra D

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This phenomenon was more prominent in the presence of the complexes with longer alkyl chains (7−9). It has also been reported that different concentrations of the cationic surfactant n-cetyltrimethylammonium bromide (CTAB) was attributed to the compaction and decompaction of single CT-DNA molecules.31 Thus, CT-DNA molecules are likely compacted by 7−9 at low molar ratios (up to r = 0.333) but decompacted at high molar ratios (up to r = 1). This assumption is consistent with the findings that 7−9 had surfactant-like properties and a markedly high potential to induce DNA compaction. Fluorescence Microscopy Observation of the HigherOrder Conformational Changes in Giant DNA Induced by the Complexes. We next used fluorescence microscopy to investigate how efficiently 2−9 induced higher-order conformational changes in T4 DNA (166 kbp), which is ca. 1000 times larger than the persistence length of usual double-stranded DNA and therefore is large enough to be used as a model of the giant genomic DNA found in living cells. The conditions used to examine 2−9 were identical to those used previously to examine 5-H-Y and 1.20,21 Figure 4 shows representative fluorescence

Figure 3. (A) Circular dichroism (CD) spectra of calf thymus DNA in the presence of different concentrations of 120 and 2−9. The phosphate concentration of the calf thymus DNA was 30 μM. Molar ratio (r) was calculated as the amount of platinum complex added divided by the total phosphate concentration. (B, C) Plots of Δθ at 278 nm versus molar ratio.

Figure 4. Representative fluorescence microscopy images of individual T4 DNA molecules moving freely in solution, and the corresponding quasi-three-dimensional profiles of fluorescence intensity. (A) Control: Snapshot of a fluorescence image of a single T4 DNA molecule exhibiting Brownian motion in a buffer solution. (B−D) Upon addition of 2, 5, or 9, concentration-dependent conformational changes in the structure of the T4 DNA were observed. EC, elongated coil; C, coil; PG, partial globule; G, compact globule.

were comparable with those determined at r = 0 (see Figure S1), indicating that the noncovalent Pt···DNA interactions inducing the B- to C-form transition were electrostatic attractions. The difference in ellipticity at 278 nm (Δθ) was calculated by subtracting the ellipticity observed in the absence of a tetrazolatobridged complex (θ0) and that obtained at a specific molar ratio of a tetrazolato-bridged complex (θr) as follows:

microscopy images of individual DNA molecules mixed with various concentrations of 2, 5, or 9 in aqueous solution, together with the corresponding quasi-three-dimensional profiles of fluorescence intensity. In the absence of a sufficient amount of cationic species to neutralize the DNA backbone, the higherorder structure of T4 DNA existed predominantly as an elongated coil (Figure 4A, control). The addition of a multivalent cation, such as transition metal ions or polyamines, induces a conformational change from the elongated coil to a compact globule accompanied by a large density difference on the order of 103 to 105.2,32 The addition of 5-H-Y or 1 also induced a conformational change to a compact globule; however, this change proceeded via an intermediate partial globule state in which different parts of the same DNA were elongated or condensed.19,20 The potency of 5-H-Y and 1 to induce DNA compaction was much greater than that of a polyamine.19,20 Similarly, upon addition of 2−9 to the DNA

Δθ = (θ0 − θr) Plots of r versus Δθ are shown in Figure 3B (1−6) and C (7−9). As the molar ratio increased, Δθ also increased, peaking at r = 0.333 for 1−6. Further addition of complex caused a slight decrease in Δθ (i.e., an increase in θ), which is consistent with previous reports for other series of tetrazolato-bridged complexes.20,21 A similar change in Δθ was observed for 7−9 (Figure 3C). By increasing the molar ratio further (up to r = 1), Δθ was further decreased, indicating that that positive band around 278 nm increased in intensity until it was comparable with that for the DNA that was predominantly in the B-form. E

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Inorganic Chemistry solution, the individual DNA molecules underwent a concentration-dependent conformational change from the coil state to the compact globule state via a partial globule state. By using atomic force microscopy, Farrell’s group monitored DNA after the addition of derivatives of polyamine-bridged polynuclear Pt(II) complexes and argued that DNA condensation and aggregation occurred via noncovalent interactions,33,34 such as hydrogen bonding and electrostatic attractions.35,36 Figure 5 shows the conformational states of T4 DNA after the addition of various concentrations of 5-H-Y and 1−9; cells

Figure 5. Conformational states of T4 DNA in the presence of various concentrations of 5-H-Y,19 1,20 or 2−9. C, coil; PG, partial globule; G, compact globule. Cells marked with C, PG, or G represent the results of our observation of 50−100 DNA molecules.

marked with a “G” represent the concentration of complex required to convert more than 80% of the DNA molecules in the sample to the compact globule state. The overall trend in DNA compaction efficiency was similar to the trend observed for cytotoxicity; that is, for 5-H-Y and 1−4, DNA compaction efficiency decreased as the length of the alkyl chain increased. For 5−7, DNA compaction efficiency increased as the length of the alkyl chain increased. DNA compaction efficiency plateaued with 7−9, probably due to the surfactant-like properties of these complexes and their ability to form micelles. DNA compaction induced by polycationic species or cationic surfactants is accompanied by almost complete charge neutralization.37 At a concentration of 20 μM, 5-H-Y induced DNA compaction 50 times more efficiently than spermidine,19 which is often used as a positive control in DNA compaction experiments. At a concentration of 1 μM, complexes 7−9 induced DNA compaction 20 or 1000 times more efficiently than 5-H-Y or spermidine, respectively. The highly efficient induction of DNA compaction by the complexes with a long alkyl chain may be a result of their surfactant-like properties, especially given that cationic surfactants such as CTAB also efficiently induce DNA compaction.38 Transmission Electron Microscopy. Finally, we examined the higher-order DNA conformational changes induced by the complexes by using transmission electron microscopy. It has been reported that DNA compacted by the present series of tetrazolato-bridged complexes are irregularly packed,19,20 whereas those compacted by transition-metal ions or cationic polyamines are in a toroidal form. Transmission electron microscopy images revealed that T4 DNA (0.1 μM) compacted by 2 (20 μM, Figure 6A,B) had a morphology similar to T4 DNA compacted by 5-H-Y. Interestingly, a 1 μM solution of 8 in 10 mM Tris-HCl buffer (pH 7.6) was found to self-assemble into large spherical structures ranging from 10 to 200 nm in diameter

Figure 6. Representative transmission electron microscopy images of compacted T4 DNA particles (0.1 μM) in the presence of 20 μM 2 (A, B); the spherical self-assembled structures formed by 8 (1 μM) (C); and the rod-like compacted T4 DNA molecules on the surface of the spherical self-assembled structures formed by 8 (1 μM) (D−F).

(Figure 6C); these structures were too large to be micelles and were maintained for at least an hour. When 8 was mixed with T4 DNA (0.1 μM), DNA molecules on the surface of the spherical self-assembled structures were compacted into a rod-like morphology, possibly due to the cationic charges on the surface of the spherical structures (Figure 6D,E,F). This result suggests that these structures may play a role in the DNA compaction that was induced by the complexes with the longer alkyl chains. For example, it is known that the CMC of surfactants is lowered in the presence of certain electrolytes, which reduce the forces of repulsion between the charged head groups at the micelle surface. Furthermore, DNA compaction can be induced by cationic micelles of CTAB, and the micelle formation has been observed at concentrations far below the CMC in the presence of DNA molecules.39

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DISCUSSION In the present study, we first examined the cytotoxicity of a series of tetrazolato-bridged Pt(II) complexes with a linear alkyl chain substituted at C5 of the tetrazolate ring. We found a U-shaped association between alkyl chain length and cytotoxicity, where cytotoxicity decreased with increasing alkyl chain length up to an alkyl chain length of four or five, which agrees with the previous F

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Inorganic Chemistry finding that this class of Pt(II) complexes with a bulky substituent at C5 are generally less cytotoxic than those with a small substituent.20,40 However, after an alkyl chain length of five, cytotoxicity tended to increase with increasing alkyl chain length. Complexes 7−9, which have surfactant-like properties, exhibited a higher cytotoxicity compared with complexes 4−6, which had a medium-length alkyl chain. Our previous studies have suggested that there is a relationship between the cytotoxicity of tetrazolato-bridged complexes and their degree of intracellular accumulation22 or DNA compaction efficiency,20 which are critical processes in the cytotoxicity of these complexes. Therefore, we next examined the intracellular accumulation of the complexes in a human NSCLC cell line and the DNA conformational changes these complexes induce in T4 DNA. A summary of our findings is shown in Figure 7. The process by which the long-chain complexes were taken up by the cancer cells is likely different from the process by which 5-H-Y and the short-chain complexes were taken up because the inflection point in the trend in intracellular accumulation was at complex 3 or 4. The present results suggest that 5-H-Y and the complexes with a short alkyl chain were taken up by the cancer cells via an unidentified active transporter because the addition of a small number of alkyl groups to 5-H-Y significantly decreased its cellular accumulation. This is similar to the suggestion that the copper transporter CTR1 partially regulates cisplatin uptake.41 In contrast, the complexes with a long alkyl chain appeared to enter the cancer cells via passive diffusion as a result of the lipophilicity of the long alkyl chain, although dramatically higher accumulation and lack of time dependency imply that the total intracellular Pt concentration contained some membrane-bound Pt. Surfactants are often used to solubilize cell membrane bilayers because they can enter the inner membrane monolayer.42 The alkyl chains of 7−9 were hydrophobic enough to confer surfactant properties to the complexes, which likely enhanced their plasma membrane permeability. Indeed, the intracellular accumulation of 7−9 was found to be markedly higher than that of 3−6, which was similar to the trend in cytotoxicity. However, it should be noted that despite this high intracellular accumulation, the cytotoxicity of these complexes was only moderate, compared to that of 5-H-Y and the short-chain complexes. It has been reported that cationic surfactants such as benzalkonium chloride induce apoptosis in cancer cells, whereas anionic or amphoteric surfactants do not.43 Some surfactant− cobalt(III) complexes have also been reported to induce apoptosis or necrosis in human cancer cell lines.44 The cytotoxicity of cationic surfactants might be a result of their interactions with DNA, which is a negatively charged biomolecule. If so, this implies that the same could be true for 7−9. Although the complexes examined in the present study all induced conformational changes in the secondary structure of CT-DNA with similar efficiencies, they had different DNA compaction profiles. Electrostatic interactions between the complexes and the phosphate backbone of the DNA molecules appeared to cause the conformational changes observed in the secondary structure of the DNA, since these conformational changes were prevented by the addition of a high concentration of an inorganic salt. It was previously reported that DNA secondary structural changes are not necessarily linked to higher-order changes and that the noncoordinative interaction can be divided into two distinct interactions.20 Accordingly, the conformational changes in the secondary structure of DNA induced by these electrostatic interactions may not be related to the cytotoxicity of the

Figure 7. Plots of complex versus the logarithm of the reciprocal of the IC50 (top) and the logarithm of the intracellular concentration of the Pt(II) complex (middle) in NCI-H460 NSCLC cells. Plot of complex versus the logarithm of the reciprocal of the Pt(II) complex concentration required to convert 80% of the DNA to the compact globule form (bottom).

complexes, but instead could be an important step that facilitates other noncovalent DNA interactions such as minor-groove binding (Komeda, unpublished data), which can also result in DNA compaction. The complexes examined in the present study were different from other DNA compaction agents in that they induced higher-order conformational changes not directly but via an intermediate partial globule state. The intermediate form may suggest the formation of covalent Pt−DNA adducts, such as interhelix DNA cross-links. The generation of the irregularly shrunken structure as in Figure 6A is also attributable to the G

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CONCLUSION The introduction of a linear alkyl chain moiety at C5 of the tetrazolate ring of tetrazolato-bridged complexes produced a series of complexes with unique cellular accumulation and DNA conformational change profiles. The derivatization of tetrazolatobridged complexes is a potentially useful means of developing next-generation platinum-based anticancer drugs. Although the precise identity of the spherical self-assembled structures formed by the complexes with a long alkyl chain remains unknown, the markedly high ability of these complexes to induce DNA compaction and their high intracellular accumulation emphasize the difference in mechanism of action from currently available platinum-based anticancer drugs.

Pt−DNA adducts with markedly different morphology from the tightly packed structure induced by multivalent cationic species.4,32 The trends in DNA compaction efficiency and cytotoxicity in NCI-H460 human NSCLC cells were similar; however, it should be noted that the cytotoxicity of 7−9 was low relative to the DNA compaction efficiency, which was much higher than that of 5-H-Y. Transmission electron microscopy revealed that the DNA compaction induced by the short and long alkyl chain complexes was different and that the DNA compaction induced by the long alkyl chain complexes was mediated via spherical self-assembled structures. These structures may act as multications that provoke DNA compaction through ion correlation effects. The efficiency of DNA compaction agents depends on the distance between charges in multivalent cations, which is maximal when there is a perfect geometrical fit between the DNA and the charged multications. It has been reported that the DNA compaction induced by long-tail cationic surfactants of alkyl trimethylammonium salts such as CTAB, which are used in DNA extraction, occurs below the CMC of the free surfactant.39 Similarly, the formation of the spherical self-assembled structures by 8 was observed at a concentration (1 μM) that was far below its CMC (6.77 mM) (Figure 6C). Attempts have been made to develop transfection reagents that increase the transfection efficiency of RNA or plasmid DNA into cell cultures in vitro by harnessing the DNA neutralization ability of cationic surfactants.32,45,46 Although it remains unclear precisely what the spherical self-assembled structures formed by 8 were, it is possible that tetrazolato-bridged complexes with surfactant properties may be able to chaperone nucleic acids inside target cells via these structures. Given that the cytotoxicity of 7−9 is low in normal, noncancerous cells, the potential of these complexes to enter cells and neutralize DNA could be harnessed to produce novel transfection agents, although further structural modification of the complexes would be necessary. Platinum-based anticancer drugs, such as cisplatin, are electronically neutral, and marked noncovalent associations with DNA have not been reported. Therefore, their anticancer effects are likely a result of their covalent binding to DNA via 1,2-intrastrand cross-links, which produces severe distortions in the DNA structure. The prototype azolato-bridged dinuclear Pt(II) complexes [{cis-Pt(NH3)2}2(μ-OH)(μ-pyrazolato]2+ and [{cis-Pt(NH3)2}2(μ-OH)(μ-1,2,3-triazolato-N1,N2]2+, as well as the tetrazolato-bridged complexes, were predicted to form 1,2-intrastrand cross-links but with minimal kinking of the DNA structure.47−50 The formation of unique covalent DNA adducts appears to be essential for their high cytotoxicity against cisplatin-resistant cancer cells18,40,51 and is an important part of their unique cytotoxicity profiles.17 Tetrazolato-bridged complexes, as well as the prototype complexes, bind to DNA via relatively tight noncovalent associations such as minor-groove binding, which is then followed by relatively slow covalent interactions.18,21,52 In addition to the multimodal DNA binding, their unique cellular accumulation mechanisms and highly efficient DNA compaction abilities may also be the underlying reason for their high efficacy against cisplatin-resistant cancer cell lines. Moreover, it was reported recently that 5-H-Y induces not only compaction of bare DNA strings but also more efficient chromatin folding compared with Mg2+.52 In addition to DNA, the existence of unknown target molecules should not be excluded at present.53

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.6b02239.

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Additional information (PDF)

AUTHOR INFORMATION

Corresponding Author

*Tel (S. Komeda): 059-340-0581. Fax: 059-368-1274. E-mail: [email protected]. ORCID

Seiji Komeda: 0000-0001-5532-3251 Kenichi Yoshikawa: 0000-0002-2751-7136 Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by JSPS KAKENHI Grant Number 15K07905 (S.K. and M.U.). We are grateful to Dr. T. Matsuzaki and Dr. S. Ishii at Yakult Honsha Co., Ltd., for their fruitful discussions.

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REFERENCES

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