Membrane Localized Iridium(III) Complex Induces Endoplasmic

Article pubs.acs.org/jmc

Membrane Localized Iridium(III) Complex Induces Endoplasmic Reticulum Stress and Mitochondria-Mediated Apoptosis in Human Cancer Cells Rui Cao,†,‡ Junli Jia,† Xiaochuan Ma,†,‡ Ming Zhou,*,† and Hao Fei*,† †

Division of Nanobiomedicine, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, 398 Ruoshui Road, Suzhou Industrial Park, Suzhou, Jiangsu, 215123, P. R. China ‡ University of Chinese Academy of Sciences,19A Yuquan Road, Beijing, 100049, P. R. China S Supporting Information *

ABSTRACT: The cellular behavior and toxicity effect of organometallic complexes depend largely on their peripheral ligands. In this study, we have synthesized a series of novel luminescent cationic iridium(III) complexes by tuning the ancillary N∧N ligand based on a structure [Ir(ppy)2(N∧N)]+ (ppy = 1-phenyl-pyridine; N∧N = 2,2′-bipyridine (bpy, 1) or phenanthroline (phen, 2) or 4,7-diphenyl-1,10- phenanthroline (DIP, 3)). As the size of coordinated N∧N ligand increases, absorbance/emission efficiency, quantum yields, lipophilicity, and cell uptake rates of the complexes also increase, in a general order: 3 > 2 > 1. All three complexes display anticancer activity, with 3 exhibiting the highest cellular uptake efficiency and the greatest cytotoxic activities in several cancer cell lines with IC50s lower than that of cisplatin. Because of its strong hydrophobic nature, the death inducer 3 was found to accumulate favorably to endoplasmic reticulum (ER) and to cause ER stress in cells. The fast cytosolic release of calcium from stressed ER disturbed the morphology and function of mitochondria, initiating an intrinsic apoptotic pathway. Understanding of the cell death mechanism would help further structure−activity optimization on these novel Ir(III) complexes as emerging cancer therapeutics.

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INTRODUCTION Because of their coordination ability with peripheral ligands of different structures, transition metals can be regarded as versatile scaffolds for construction of compounds with desired functions.1−3 Such advantages would find best uses in the field of drug discovery. Studies thus far have shown that organometallic complexes exert different modes of anticancer activities depending on their intracellular targets, which includes nucleic acids, proteins, or cell organelles. The widely used anticancer drug cisplatin and its analogues target DNA in the cell nuclei and form platinum−DNA adducts that structurally distort the DNA, resulting in the occurrence of apoptosis.4 Some of the later developed ruthenium anticancer compounds were designed to interact with DNA duplexes in unique binding modes to induce structural distortions.5,6 More recently, a number of metal−arene complexes, including osmium-based complexes, were synthesized, also selecting nucleobases and DNA as the molecular targets.7 Taking advantages of threedimensional shapes and stabilities of metal complexes, Meggers group synthesized a number of ruthenium complexes-based protein kinase inhibitors using staurosporine-like structures as ligands, which exhibited anticancer activity.8−10 Additionally, several cytoplasmic mechanisms have been reported as the main pathways of metal complexes-induced cancer cell death. Mitochondrial cell death pathways caused by metal compounds, © 2013 American Chemical Society

such as ruthenium and gold complexes, have been documented.11−13 A different pathway of cell apoptotic death triggered by endoplasmic reticulum (ER) stress in cadmium compounds treated cells has been reported.14,15 Novel osmium(II) arene anticancer complexes were synthesized and exhibited their anticancer activity via interfering in the redox signaling pathways.16 Besides the classic apoptotic pathway, another mode of cell death called paraptosis in copper complexes treated human cancer cells has also been reported.17,18 In recent years, iridium complexes have been studied extensively as novel agents for biomedical applications in protein staining, cell imaging, peptide labeling, and cancer cell killing.19−24 As cytotoxic agents, several Ir(III) compounds with various peripheral ligands were proposed to exert their toxicity via DNA, enzyme, or cytoplasmic-based mechanisms.25−28 Accumulating evidence have suggested that the toxicity of iridium(III) complexes is largely determined by the coordinating ligands. The chemical nature, the size, and conjugation degree affect not only the photophysical and lipophilical properties but also the cytotoxic profiles of metal complexes significantly. Our previously studied histidine coordinated Received: January 31, 2013 Published: April 17, 2013 3636

dx.doi.org/10.1021/jm4001665 | J. Med. Chem. 2013, 56, 3636−3644

Journal of Medicinal Chemistry

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Ir(III) complex based on a simple model structure [Ir(ppy)2(N∧N)]+ (ppy = 1-phenyl-pyridine) exhibits little cytotoxicity.23 We also notice that earlier works have demonstrated metal compounds with several arene-based ligands show potent cytotoxicity toward cancer cells.11,12,24 In this study, we intend to investigate how a series of different N∧N arene ligands would influence the activity of compounds based on the structure [Ir(ppy)2(N∧N)]+. To this end, three N∧N ligands, i.e., 2,2′-bipyridine, 1,10phenanthroline, and 4,7-diphenyl-1,10-phenanthroline, were chosen to study the ligand effect. Their corresponding iridium(III) complexes (1−3) were synthesized (Chart 1).

Table 1. Photophysical Data of 1−3 in CH3CN, PBS at Room Temperature complexes 1 λab/nm 254(41.08), (ε, 103 M−1 cm−1)a 310(18.10), 380(4.97) λab/nm 254(38.35), (ε, 103 M−1 cm−1)b 308(17.01), 373(4.49) λem/nma 586 λem/nmb 583 Φc 0.12 REId (CH3CN) 1 REId (PBS) 1 log Po/w 0.77

Chart 1. Chemical Structures of the Iridium(III) Complexes

2 266(48.56), 376 (6.17) 265(41.94), 371(5.85) 576 584 0.19 1.20 2.52 1.67

3 271(60.47), 333(19.46), 382(10.72) 289(32.57), 337(18.53), 390(11.65) 586 597 0.24 3.02 8.30 2.12

a The complexes were dissolved in CH3CN. bThe complexes were dissolved in PBS (final DMSO concentration, 0.1% v/v). cThe values are quantum yields or lifetimes measured in CH3CN saturated with argon gas. dREI = relative emission intensity, which was calculated relative to the emission intensity of 1.

than those of 1 (0.77) and 2 (1.67) in Table 1, which follows a similar trend reported in earlier works.29,30 Higher lipophilicity is anticipated to facilitate cellular uptake. Under the same experimental condition, luminescence images of cells incubated with these complexes were obtained. The luminescence intensity in the cytoplasm indicates that the ability of cell uptake of these compounds follows an order of 1 < 2 < 3 (Figure 2A). The stable luminescent property of these compounds in aqueous solution allows quantitative measurement of uptake rates by monitoring the residual luminescence in the medium after coincubation with cells. As shown in Figure 2B, coincubation of 1−3 with HeLa cells results in the weakening of the luminescence in medium over time. It is noteworthy that 3 entered the cells most rapidly, with the majority (>60%) of luminescence absorbed by cells in 30 min. These data indicate a correlation between compound lipophilicity and cell uptake efficiency. In Vitro Cytotoxicity. The cellular uptake properties of metal-based anticancer drugs can influence their antiproliferative efficacies, and increasing lipophilicities have often been linked to the enhanced rate of cellular uptake and, consequently, the cytotoxic activities.24,31 The antitumor potential of 1−3 on HeLa, A549, and MCF-7 cell lines were tested after 24 h incubation with varying concentrations, and cell viability was determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.32 The resulting IC50 values for the tested compounds are shown in Table 2. The results indicate that 1−3 show an increased cytotoxic potency with increasing size of the coordinated arene. Besides, 3 is much more potent than cisplatin against all the cancer cell lines screened (approximately 6-fold more potent than cisplatin in killing HeLa cells). It is noteworthy that the complex Ir(ppy)2−histamine shows very low cytotoxicity, with much higher IC50 values toward all cancer cell lines tested (Table 2). The results indicate that adjusting a single N∧N ancillary ligand on the same Ir(ppy)2 base structure can influence cytotoxicity very dramatically. Collectively, above characterizations demonstrate strong structure−activity relationship that could be applied to additional molecular designs in the future. In this study, the

Absorbance efficiency, emission intensity, photo/quantum yields, and lipophilicity of the complexes were compared as the size of coordinated N∧N ligand increases. All three complexes display anticancer activity, with 3 exhibiting the highest cellular uptake efficiency and the greatest cytotoxic activities in several cancer cell lines with IC50s lower than that of cisplatin. As the strongest death inducer, 3 was found to be a membrane-localized agent that induces endoplasmic reticulum stress and mitochondria-mediated apoptosis in human cancer cells.

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RESULTS Ancillary Ligand Tuning on Ir(ppy)2(N∧N) Enhances Functional Performance. Photophysical Characterization. The photophysical properties of 1−3 in CH3CN and PBS (final DMSO concentration, 0.1% v/v) were studied, and the summarized data are listed in Table 1. Figure 1 shows the UV−vis absorption and emission spectra recorded in CH3CN and PBS at room temperature. According to Table 1, these three complexes have a strong intraligand absorption band (π−π*) at around 250−300 nm (ε >15000 M−1cm−1) and a weaker metal-to-ligand charge-transfer (MLCT) transition band at around 350−500 nm. It should be noted that 3 with the largest conjugation ligand also have the strongest absorbance. Such effects have also been found in the relative emission intensity (REI) and quantum efficiency (Φ). As illustrated in Table 1, both REI and Φ increased along with the conjugation intensity of the ancillary ligand. Lipophilicity and Cell Uptake. The size of the ancillary ligand also influences the lipophilicity properties of these Ir(III) complexes. The lipophilicity was determined as a measure of the relative solubility in oil and water. Herein, the lipophilicity of 1−3 was measured by the partition coefficient in octanol/ water (as log Po/w values) through a classical method in accordance with the literature.29 The lipophilicity of the complexes substantially increased with increasing size of the coordinated arene, as the log Po/w value of 3 (2.12) is larger 3637



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Figure 1. UV−vis absorption (dashed) and emission (solid) spectra (λex = 350 nm) of 1 (black), 2 (red), and 3 (blue) in CH3CN (A) and PBS (B) at room temperature (40 μM for absorption spectra and 10 μM for emission spectra).

Figure 2. Uptake efficiency studies. (A) Bright-field images (upper panels) and luminescence images (lower panels) of HeLa cells incubated with complexes 1−3 (10 μM) for 30 min. Scale bar: 20 μm. (B) Measurement of the residual of luminescence in the culture media of HeLa cells incubated with 1−3 (10 μM) after 0, 10, 20, 30 min (Ex = 350 nm).

endoplasmic reticulum (ER). Co-staining with ER marker shows nearly complete overlap between 3 and ER-Tracker Red (Figure 3B), demonstrating that 3 can penetrate cell membrane and localize mainly to the ER. In previous work, cyclometalated Ir(III) compounds with different functional ligands such as [Ir(N∧C)2(phen-DPA)](PF6) and Ir(III)−octaethylporphyrin had also been reported to colocalize to the ER.33,34 The endoplasmic reticulum plays two fundamental roles in a cell. On one, ER is the place for protein synthesis, folding, and initial sorting in the cell. When under pressure, ER responds through coordinated signaling mechanism known as the unfolded protein response (UPR) to protect the cell from stress.35 On the other, ER is main storage for intracellular Ca2+, which is a key component in cellular signaling and survival. Calcium can be released into the cytoplasm in response to physiological changes.36 Several ER-stress pathways have been described in mammalian cells. The nuclear transcription factor C/EBP homologous protein (CHOP) operates as a downstream component of these pathways. Elevated expression level of this protein is considered a general marker of ER stress.37,38 To test if the accumulation of 3 in the ER further induces ER stress, protein extracts of 3 treated cells at different time points were analyzed for the expression level of CHOP using Western blot. The result shows that cells begin to show detectable expression level of CHOP after 12 h of treatment (Figure 4A), indicating elevated ER stress level.

Table 2. IC50 Values of Tested Compounds toward Different Cell Linesa IC50 (μM) complex

HeLa

A549

MCF-7

Ir(ppy)2−histamine 1 2 3 Cisplatin

>200 26.6 ± 1.4 21.5 ± 1.3 3.3 ± 0.2 20.2 ± 1.8

>200 22.2 ± 0.8 15.6 ± 0.5 2.0 ± 0.1 38.6 ± 0.9

107.6 ± 19.6 22.7 ± 3.2 17.4 ± 0.3 3.2 ± 0.2 31.4 ± 3.0

IC50 values are given in μM, and cisplatin is included for comparison. Data are presented as mean values ± standard deviations, and cell viability is assessed after 24 h of incubation.

a

promising cytotoxic activity of 3 warrants further investigation into its cell death mechanism. Ir(ppy)2DIP Induces Endoplasmic Reticulum Stress. By using a subcellular fractionation kit and following the luminescence of the iridium(III) complex, the intracellular distribution of 3 was determined in each fraction after 1 h coincubation with HeLa cells. Results in Figure 3A shows that the highest amount of 3 was in the cell membrane fraction, accounting for nearly 60% of 3 in the cell, which indicated that its cytotoxic effect may initiate from the membranous organelles in the cytoplasm. To directly observe its intracellular distribution, 3 μM of the compound was added to HeLa cells for 30 min. Under microscope, 3 displays extensive networklike staining in cytoplasm, resembling the typical structure of 3638



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Figure 3. Membrane localization of 3. (A) Luminescence intensity distribution in each subcellular fraction of 3 (10 μM) treated HeLa cells for 1 h. Results are the means of three independent experiments and are expressed as means ± SDs. (B) Confocal microscopy images of HeLa cells prestained with ER-Tracker Red (500 nM) for 30 min and then coincubated with 3 (3 μM) for 20 min at 37 °C. Images of ER-Tracker Red was collected at 570−620 nm (excitation: 561 nm), and 3 was collected at 570−620 nm (excitation: 403 nm), respectively. Scale bar: 20 μm.

Figure 4. Induction of endoplasmic reticulum stress by 3. (A) ER stress as indicated by increased expression of CHOP using Western blot. HeLa cells were incubated with 3 (10 μM) for 3, 5, and 12 h. β-Tubulin was used as loading control. (B) In the time-lapse confocal images, the fluorescence intensity of Fluo 4-AM gradually increased after HeLa cells treated with 3. Scale bar: 100 μm. (C) Quantitative analysis of changes in fluorescence emission intensity of Fluo 4-AM in 3 treated cells using confocal microscope software (F0: basal fluorescence emission intensity obtained at the start of the experiment; Ft: the fluorescence intensity at different time points).

mitochondria-localized aldehyde dehydrogenase fused with GFP41) was used as a live imaging probe for the labeling of mitochondrial morphology in cells. HeLa cells transiently expressing ALDH-GFP shows typical fibrous structure as shown in Figure 5A and Figure S2 in Supporting Information. Adding 3 to these HeLa cells causes obvious fragmentation and swelling of mitochondria (Figure5A,B). Time-lapse confocal imaging recorded the continuous morphological changes of the mitochondria that began to appear after 4 min and continued to evolve until 40−60 min, in a lagged time frame than the previous calcium changes, suggesting a downstream effect of ER stress and calcium release. The fragmentation and swelling of mitochondria strongly indicate the onset of intrinsic pathway of apoptosis. Classic biochemical markers in the mitochondria-mediated pathway, including cytoplasmic cytochrome c level and caspase-3 activity, were next examined.42 Western blot result shows that after HeLa cells were incubated with 3, cytochrome c appeared in the cytosol fractions (Figure 5C). Using Caspase-Glo assay, the activity of caspase-3 was detected, with highest activity occurring after 6 h of treatment. For comparison, cisplatin can also induce caspase-3 activity in HeLa cells but with a significant lag in time, suggesting a different mechanism in apoptosis induction (Figure 5D). Apoptosis can also be

Change in nuclear protein expression level is a relatively slow procedure, whereas the release of calcium from ER is a far more sensitive indicator of ER function. Using a calcium fluorescence probe Fluo 4-AM, the time dependent changes of cytoplasmic calcium ion levels can be recorded (Figure 4B). Quantitative analysis of the change in fluorescence intensity shows there is a sharp increase during the time period of 4−6 min before reaching a plateau beyond 6 min, indicating a large pulse of calcium influx into the cytoplasm (Figure 4C). Thus, the accumulation of 3 in the ER produces a rapid functional interference to the organelle, leading to stress and release of ER stored calcium. ER Stress Leads to Mitochondrial Fragmentation and Apoptosis. ER and mitochondria physically associate and functionally correlate in the cell. A Ca2+ rise in cytoplasm allows the rapid accumulation of the cation in the matrix of mitochondria, which would trigger the bioenergetics failure of the organelle through the change of membrane permeability, causing morphological changes such as swelling and fragmentation, leading to the onset of mitochondria mediated apoptosis.39,40 To investigate the influence of 3 induced ER stress toward mitochondria, a mitochondria-targeted green fluorescent protein (ALDH-GFP, the N-terminal targeting sequence of 3639



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Figure 5. Mitochondrial fragmentation and onset of apoptosis. (A) In the time-lapse confocal images of 3 treated HeLa cells, ALDH-GFP labeled mitochondria became fragmented, resulting in small, rounded, and numerous organelles in 40 min. (B) Representative image of ALDH-GFP labeled mitochondria (green) and membrane localized 3 (red). (C) Western blot of cytochrome c in cytosol (s) verses membrane (p) fractions extracted from HeLa cells after exposure to 3 for indicated periods of time. β-Tubulin was used as loading control: supernatant (s); precipitates (p). (D) Caspase-Glo assay showing increased caspase-3 activity after cell exposure to 3 at indicated times. Cisplatin (50 μM) was included for comparison of time dependent effect. Data shown are mean values (standard deviations from three independent experiments). (E) Nuclear condensation in 3 (10 μM) treated HeLa cells for indicated times (Hoechest staining). (F) Bright-field image showing plasma membrane blebbing of 3 treated HeLa cells in 2 h. All scale bars: 20 μm.

characterized by cytoplasmic shrinkage, nuclear condensation, and blebbing of the plasma membrane.43 In Figure 5E,F, apparent membrane blebbing and nuclear condensation can be observed in 3 treated cells. In summary, 3 can induce apoptosis in cancer cells within a short period of time, and the death employs intrinsic apoptotic pathway associated with mitochondria.

More advantageous than many reported metal complexes, such as platinum, copper, or other metal-based compounds, these iridium complexes exhibit tunable luminescent property that allows more direct and precise optical observations of their intracellular destinations. Similar to several previously reported iridium complexes, these compounds mostly localized to the cytoplasm, with little or no presence in the cell nucleus. Thus, although nuclear changes such as morphological condensation can be observed in a longer time scale in our study, this is most likely a downstream effect of the cell death mechanism initiated from the cytoplasm. In fact, our data shows by far the most dramatic morphological changes such as mitochondria fragmentation occurred in the cytoplasm of Ir(ppy)2DIP treated cells very rapidly. Besides optical properties, tuning of the ancillary ligand of these iridium(III) complexes also improves lipophilicity/ hydrophobicity along with cytotoxicity. Increased lipophilicity facilitates the partition of the compound into lipid, thus disturbing the hydrophobic environment required by membrane/lipid embedded proteins (such as many transmembrane proteins or integral membrane proteins). In fact, in the earlier years, bathophenanthroline (i.e., DIP) conjugated metal complexes had been shown to inhibit ATPase activity in vitro.44,45 Detailed studies had later suggested this effect may

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DISCUSSION AND CONCLUSIONS In this paper, we report a series of three novel organometalated Ir(III) complexes based on a simple common structure [Ir(ppy)2(N∧N)]+, where three ancillary N∧N ligands with increasing conjugation intensity, namely bipyridine, phenanthroline, and diphenylphenanthroline, were synthesized. Comparative studies show that emission intensity, quantum efficiency, as well as cell physiology relevant parameters including compound lipophilicity, cell uptake efficiency, and cytotoxicity all enhanced significantly along with the ligand size. Detailed studies on the most potent cytotoxic complex, Ir(ppy)2DIP, suggest that it localizes mainly in the ER membranes, causing ER stress and calcium release into cytoplasm, which further triggered mitochondria dysfunction and the onset of apoptotic cell death mechanism. 3640



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secondary antibody to mouse IgG(H + L) (A0216, Beyotime Institute of Biotechnology), BeyoECL Plus (P0018, Beyotime Institute of Biotechnology), Caspase-Glo 3/7 assay (G 8091, Promega, Madison, WI, USA), and Lipofectamine 2000 reagent (11668−027, Invitrogen) were used according to reference guide. Fetal bovine serum, penicillin−streptomycin solution (SV30010) and the culture media were obtained from Hyclone. Cisplatin was dissolved at a concentration of 1 mM in 0.154 M NaCl and frozen at −20 °C. Stock solutions (10 mM) of 1−3 were dissolved in dimethylsulfoxide (DMSO) at room temperature. Synthetic Protocols and Characterization. A dichloromethane/ methanol (V:V = 3:1) mixture solution of [Ir(μ-Cl)(ppy)2]2 (0.5 mmol) and corresponding N∧N bidentate coordinating ligands (1.2 mmol) were refluxed for 24 h. After cooling to room temperature, the solvents were removed by rotoevaporation. The residue were chromatographed on a silica gel column with CH2Cl2/CH3OH (various ratios based on the complexes properties) eluent to give pure product. Ultrahigh pressure liquid chromatography (UHPLC) was used to determine the purity of three complexes using two mobile phases of methanol and 0.1% (v/v) trifluoroacetic acid (Waters ACQUITY UPLC). The complexes 1−3 were dissolved in CH3CN or PBS (final DMSO concentration, 0.1% v/v). Time of flight mass spectra (TOFMS) were obtained by preparing the samples in CH3OH and infusing into the mass spectrometer (Agilent Technologies TOF 1200/6220). The mass spectra were recorded with a scan range of m/z 100−1000 for positive ions. 1H NMR spectra were recorded with a Varian spectrometer at 400 MHz. Luminescence quantum yields were measured in CH3CN saturated by argon gas with an aqueous solution of [Ir(ppy)3]Cl2 (Φem = 0.4) as the standard solution. UV−visible spectra of the complexes were recorded on Perkin-Elmer Lambda 25 spectrophotometer at the concentration of 40 μM, and emission spectra were obtained on Hitachi F4600 fluorescence spectrophotometer at the concentration of 10 μM (Ex = 350 nm). [Ir(ppy)2bpy]Cl. (1). Complex 1 was isolated as yellow crystals. Yield: 0.588 g (85%). Purity: 99.81%. TOF-MS: calcd for [Ir(ppy)2(bpy)]+ m/z 655.2, found 655.2. 1H NMR (400 MHz, CDCl3) δ ppm: 9.59 (d, J = 8 Hz, 2 H), 8.27 (t, J = 8 Hz, 2 H), 7.91 (m, 4 H), 7.76 (m, 2 H), 7.68 (dd, J = 0.8, 8 Hz, 2 H), 7.48 (d, J = 5.2 Hz, 2 H), 7.40 (m, 2 H), 7.03 (m, 4 H), 6.91 (td, J = 0.16, 7.4 Hz, 2 H), 6.3 (dd, J = 0.8, 7.6 Hz, 2 H). [Ir(ppy)2phen]Cl. (2). Complex 2 was isolated as yellow crystals. Yield: 0.526 g (76%). Purity: 99.76%. TOF-MS: calcd for [Ir(ppy)2(phen)]+ m/z 679.2, found 679.2. 1H NMR (400 MHz, CDCl3) δ ppm: 8.98 (m, 2 H), 8.49 (m, 2 H), 8.26 (dd, J = 0.16, 5.2 Hz, 2 H), 7.92 (m, 4 H), 7.73 (m, 4 H), 7.32 (m, 2 H), 7.10 (td, J = 0.12, 7.2 Hz, 2 H), 6.98 (td, J = 0.12, 7.2 Hz, 2 H), 6.90 (m, 2 H), 6.41 (dd, J = 0.8, 7.6 Hz, 2 H). [Ir(ppy)2DIP]Cl. (3). Complex 3 was isolated as orange crystals. Yield: 0.642 g (74%). Purity: 98.71%. TOF-MS: calcd for [Ir(ppy)2(DIP)]+ m/z 831.2, found 831.2. 1H NMR (400 MHz, CDCl3) δ ppm: 8.33 (d, J = 5.2 Hz, 2 H), 8.16 (s, 2 H), 7.95 (d, J = 8 Hz, 2 H), 7.83 (td, J = 0.12, 7.2 Hz, 2 H), 7.74 (m, 4 H), 7.57 (m, 12 H), 7.18 (m, 2 H), 7.07 (td, J = 0.12, 7.2 Hz, 2 H), 6.97 (td, J = 0.12, 7.6 Hz, 2 H), 6.42 (dd, J = 0.8, 7.2 Hz, 2 H). Cell Lines and Culture. The cell human cancer cell lines HeLa, MCF-7, and A549 were provided by the Institute of Biochemistry and Cell Biology, SIBS, CAS (China). HeLa cells were grown in DMEM supplemented with 10% fetal bovine serum and 1% penicillin/ streptomycin. MCF-7 and A549 cell lines were grown in RPMI 1640 supplemented with 10% fetal bovine serum and 1% penicillin/ streptomycin. Cells were incubated at 37 °C under a 5% CO2 atmosphere. Cell Viability Assay. We used MTT assay to examine the cytotoxic effect of the compounds toward cell lines. Cells were seeded in a complete growth medium in 96-well plates (Costar Corning, NY), at a density of 1 × 104 cells/well, and grown for 24 h before treatment. The growth medium was then substituted with fresh medium containing the compounds to be tested at appropriate concentrations around IC50s. After incubation for 24 h, 20 μL of aqueous MTT

have been due to the hydrophobic interference of the compound with this membrane embedded protein.46 The observation of hydrophobicity-based interaction with biomolecules is not limited to membranes. In another model system, the metalloinsertion of organo-ligated ruthenium complexes into the base-stacking of double-stranded DNA has also been studied extensively.47−49 From such perspectives, hydrophobicity-based interaction between organometallic complexes and biomolecules or cell structures may have broad implication in the development of novel therapeutics or diagnostics. In recent years, the molecular link between ER stress and cell death has been firmly established, and several important signaling pathways initiated from ER have been well documented.35,50 For example, the occurrence of ER stress can induce the activation of transmembrane stress sensors in the ER, including double-stranded RNA-dependent protein kinase, (PKR)-like ER kinase (PERK), inositol-requiring protein-1 (IRE-1), and activating transcription factor 6 (ATF6).51 All these signaling converge to the increased expression of CHOP, which then promotes the expression of death related proteins required for mitochondria-mediated apoptosis.38,52 Additionally, calcium plays a messenger role between the ER and mitochondria. Stress can lead to the release of calcium from its ER storage. The accumulation of calcium in the cytoplasm and uptake by the mitochondria causes mitochondria fragmentation, triggering cytochrome c release and thus initiating apoptotic pathway.39,53 Our results demonstrated that Ir(ppy)2DIP can induce the release of calcium and the increased expression of CHOP, pointing to an elevated stress level in the ER. Further morphological changes of mitochondria, accompanied by the release of cytochrome c and activation of caspase-3, strongly suggest the pathway of ER-induced and mitochondria-mediated apoptosis in these cells. In conclusion, our study explored the structure−activity relationship of a series of novel iridium(III) complexes via systematic tuning of the ancillary ligand groups. One compound, Ir(ppy)2DIP, with optimized optical property and potent cytotoxicity was obtained. The hydrophobic nature of this compound contributes to its cell uptake rate and membrane distribution in the cell. An accumulation in the endoplasmic reticulum appears to be a main factor for triggering the rapid downstream events of mitochondriamediated apoptosis in human cancer cells. On the basis of these findings, future optimization with variations of DIP ligand on both main and ancillary positions will likely yield compounds with even more enhanced optical and cytotoxic properties for cancer diagnostic and therapeutic applications.

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EXPERIMENTAL SECTION

Materials and General Methods. All solvents were of analytical grade. All buffer components were of biological grade and used as received. The dichloro-bridged dimer [Ir(μ-Cl)(ppy)2]2 was received from SunaTech Inc. N∧N bidentate coordinating ligands, i.e., 2phenylpyridine (bpy), 1,10-phenanthroline (phen), and 4,7-diphenyl1,10- phenanthroline (DIP), were purchased from Aladdin Inc. Cisplatin (Alfa Aesar or Sigma), MTT (Sigma), and SRB(Sigma) were used without purification. Subcellular protein fractionation kit (78840, Pierce Biotechnology), ER tracker (E34250, Invitrogen), Fluo 4-AM Special Packaging (F312, Dojindo Molecular Technologies, Inc.), Hoechest 33342 (C1022, Beyotime Institute of Biotechnology), a mouse monoclonal anticytochrome c antibody (AC909, Beyotime Institute of Biotechnology), a mouse monoclonal β-Tublin antibody (T5201, Sigma), a mouse monoclonal anti-CHOP antibody (AC532, Beyotime Institute of Biotechnology), a HRP-labeled goat monoclonal 3641



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times, then 500 μL of 2.5 μM Fluo 4-AM solution diluted in HBSS was added (HBSS, no calcium, no magnesium, Gibco cat. no. 14170−112), the cells were incubated for 30 min at 37 °C. Then the staining solution was replaced, rinsed with HBSS for three times again, and 500 μL of HBSS was added and the cells were incubated for another 30 min at 37 °C. At the end, the HBSS solution was removed, 500 μL fresh medium containing 10 μM of 3 was added, and the cells were viewed using a confocal microscope immediately. Emission was collected at 500−560 nm (excitation light: 488 nm) for the cells incubated with Fluo 4-AM. Mitochondrial Fragmentation. HeLa cells were grown on Fluorodish at 37 °C under a 5% CO2 atmosphere for 24 h and transfected with a mixture consisting of 1.6 μg plasmid ALDH -GFP encoding an mitochondrial-targeted GFP-fusion construct and 3 μL of Lipofectamine 2000 reagent. After 3 h, the transfection mixture was replaced with 1 mL of culture medium without penicillin− streptomycin. Cell were used 24 h after transient transfection. In the time-lapse confocal microscope, continuous morphological changes of mitochondria after cells exposure to 10 μM of 3 were viewed for 2 h. Emission was collected at 500−560 nm (excitation light: 488 nm) for mitochondria-targeted green fluorescent protein ALDH-GFP. Emission was collected at 570−620 nm (excitation light: 403 nm) for the cells incubated with 3. Western Blot. HeLa cells were plated at a density of 8 × 105 cells/ 100 mm tissue culture dish in 8 mL of culture medium for 24 h. For cytochrome c, the cells were treated with 8 mL of medium containing 10 μM of 3 for 2 and 4 h, respectively. Then the drug-containing medium was removed, and the cells were washed, trypsinized, centrifuged, and resuspended with 200 μL of PBS. The supernatant contained the cytochrome c, and the cell precipitate was obtained using cytoplasmic extraction buffer of Subcellular Protein Fractionation Kit centrifuged at 3000 g for 5 min at 4 °C. For CHOP, the cells were treated with 8 mL medium containing 10 μM of 3 for 3, 5, and 12 h, respectively. Then the drug-containing medium was removed, and the cells were washed, trypsinized, centrifuged, and resuspended with 200 μL of PBS and the samples were boiled in loading buffer for 10 min. All of the samples were subjected to electrophoresis (15% SDSPAGE for cytochrome c, 10% SDS-PAGE for CHOP), followed by transfer to a PVDF membrane. Then the blots were blocked with 10% nonfat dry milk for 1 h at room temperature and further incubated with primary cytochrome c antibody (1:100) at 4 °C overnight. After incubation, the PVDF membranes were washed with TBST plus 0.5% Tween-20 for 30 min, followed by incubation for 2 h at room temperature with a HRP-labeled goat monoclonal secondary antibody to mouse IgG(H + L) antibody. Then the blots were again washed with TBST plus 0.5% Tween-20 for 30 min at 5 min interval of time. The cytochrome c bands were revealed by BeyoECL Plus system using a Luminescent image analyzer (LAS 4000 EPUV mini, FuJiFilm) Statistical Analysis. All biological experiments were performed in three repeats or at least twice with triplicates in each experiment. Data were analyzed using Origin 8.0 and SPSS 16.0 and presented as mean values ± standard deviations

solution (5 mg/mL) was added to each cell, and the cells were incubated continually for another 4 h, then the medium and MTT mixtures were removed. Furthermore, 150 μL of DMSO was added to each well and incubated at 37 °C for 10 min. The absorbance of each sample at 490 nm were measured using a microplate reader (PerkinElmer, Victor X4). As the study later found that the complexes could disturb mitochondria function, the IC50 values toward HeLa cells were also determined using the sulforhodamine B (SRB) assay for comparison.54 Cell culture and treatment were consistent with MTT assay, and the absorbance of each sample at 510 nm was measured using a microplate reader (Perkin-Elmer, Victor X4), the data were summarized in Supporting Information Table S1. Uptake Efficiency, Fluorescence Microscope. HeLa cells were seeded in a complete growth medium in 12-well plates (Costar Corning, NY), at a density of 1 × 105 cells/well, and grown for 24 h before treatment. The cells were incubated with fresh medium containing 1−3 at the concentration of 10 μM for 20 min at 37 °C, respectively. Then the medium was removed and cells were washed three times with PBS, the luminescence images were obtained using fluorescence microscope (Eclipse Ti-E, Nikon). Uptake Efficiency, Fluorescence Spectrophotometer. HeLa cells were seeded in a complete growth medium in 6-well plates (Costar Corning, NY), at a density of 2 × 105 cells/well, and grown for 24 h before treatment. Then the culture medium was removed, and cells were washed three times with PBS, incubated with 1× PBS containing 1−3 at the concentration of 10 μM at room temperature. The fluorescence intensity of the complexes at the indicated time were tested on Hitachi F4600 Fluorescence spectrophotometer (Ex = 350 nm). Membrane Localization of 3. HeLa cells were plated at a density of 8 × 105 cells/100 mm tissue culture dish (Costar Corning, NY) in 8 mL of culture medium (one dish was prepared for 3, and one untreated control dish, in three independent experiments). After incubation for 24 h, cells were exposed to 3, stock solutions of the compound was prepared fresh in DMSO and diluted in the culture medium (final DMSO concentration, 0.1% v/v) to a final concentration of 10 μM. After 1 h of drug exposure at 37 °C under a 5% CO2 atmosphere, the drug-containing medium was removed, and the cells were washed, trypsinized, and counted using a hematocytometer. Then the cells were centrifuged, washed with PBS, and used for cytosol, nucleus, membrane/particulate, and cytoskeleton fractionation, using a Subcellular Protein Fractionation Kit from Pierce Biotechnology. All of the supernatant were diluted 6-fold in the MilliQ water, and each component fluorescence intensity of 3 was tested on Hitachi F4600 Fluorescence spectrophotometer (Ex = 350 nm). Colocalized with Endoplasmic Reticulum. 3 colocalized with endoplasmic reticulum marker was shown using the Nikon A1R confocal laser scanning microscope and a 60 oil-immersion objective lens. HeLa cells were seeded at a density of 1 × 104 cells/35 mm Fluorodish (FD35−100, World Precision Instruments Inc.) and incubated at 37 °C under a 5% CO2 atmosphere for 24 h. For the cells, the medium was removed from the culture dish, rinsed with HBSS (HBSS/Ca/Mg, Gibco cat. no. 14025−092), and prewarmed staining solution was added at the concentration of 1 μM. The cells were incubated for 30 min at 37 °C. Staining solution was replaced with fresh medium containing 3 μM of 3 and the cells were incubated for another 20 min, the drug solution was replaced with fresh medium, and the cells were viewed using a confocal microscope. Emission was collected at 570−620 nm (excitation light: 561 nm) for cells incubated with ER-Tracker red. Emission was collected at 570−620 nm (excitation light: 403 nm) for the cells incubated with 3. ER Stress. The Release of Calcium. The release of ER stored calcium was indicated by rapid increase of Fluo 4-AM fluorescence intensity using Nikon A1R confocal laser scanning microscope. HeLa cells were seeded in 24-well plates (Costar Corning, NY) at the density of 5 × 104 cells/well in 500 μL of culture medium and incubated at 37 °C under a 5% CO2 atmosphere for 24 h. For the cells, the medium was removed from the wells, rinsed with HBSS (HBSS, no calcium, no magnesium, Gibco cat. no. 14170−112) for three

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ASSOCIATED CONTENT

S Supporting Information *

Details of methods; tables of IC50 values measured using the sulforhodamine B (SRB) assay and the UHPLC results of 1−3 complexes; UHPLC diagram; TOF-MS and 1H NMR sprectra of 1−3 complexes; Colocalized ALDH-GFP with Mito-Tracker Red. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*For M.Z.: phone, (+86)512 62872558; fax, (+86) 512 62872181; E-mail, [email protected]. For H.F.: 3642



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phone, (+86) 512 62872717; fax, (+86) 512 62872181; E-mail, [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Natural Science Foundation of China grants 31170777 and 30900341. We are grateful to all group members for invaluable discussions and technical support.

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ABBREVIATIONS USED ATF-6, activating transcription factor 6; bpy, 2,2′-bipyridine; CHOP, C/EBP homologous protein; DIP, 4,7-diphenyl-1,10phenanthroline; ER, endoplasmic reticulum; IRE-1, inositolrequiring protein-1; MLCT, metal-to-ligand charge transfer; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; UPR, unfolded protein response; PERK, RNAdependent protein kinase (PKR)-like ER kinase; phen, phenanthroline; ppy, 1-phenyl-pyridine; REI, relative emission intensity; SRB, sulforhodamine B

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Membrane Localized Iridium(III) Complex Induces Endoplasmic

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