Nuclear permeable ruthenium(ii) -carboline complexes induce autophagy to antagonize mitochondrial-mediated apoptosis

J. Med. Chem. 2010, 53, 7613–7624 7613 DOI: 10.1021/jm1009296 Nuclear Permeable Ruthenium(II) β-Carboline Complexes Induce AutophagyTo Antagonize Mitochondrial-Mediated Apoptosis Caiping Tan,† Sensen Lai,† Shouhai Wu,† Sheng Hu,§ Lingjun Zhou,† Yu Chen,† Minxu Wang,† Yiping Zhu,†Wu Lian,† Wenlie Peng,† Liangnian Ji,‡ and Anlong Xu*,† †State Key Laboratory of Biocontrol, Department of Biochemistry, College of Life Sciences, Sun Yat-sen University, Guangzhou,Guangdong 510006, P. R. China, ‡MOE Laboratory of Bioinorganic and Synthetic Chemistry,School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou 510275, P. R. China, and §Faculty of Chemical Engineering and Light Industry, Guangdong University of Technology, Guangzhou 510006, P. R. China Received July 22, 2010 The role of autophagy in cancer development and response to cancer therapy has been a subject of debate.
Here we demonstrate that a series of ruthenium(II) complexes containing a β-carboline alkaloid as ligandcan simultaneously induce autophagy and apoptosis in tumor cells. These Ru(II) complexes are nuclearpermeable and highly active against a panel of human cancer cell lines, with complex 3 displaying activitiesgreater than those of cisplatin. The antiproliferative potentialities of 1-3 are in accordance with theirrelative lipophilicities, cell membrane penetration abilities, and in vitro DNA binding affinities. Com-plexes 1-3 trigger release of reactive oxygen species (ROS) and attenuation of ROS by scavengers reducedthe sub-G1 population, suggesting ROS-dependent apoptosis. Inhibition of ROS generation also reducesautophagy, indicating that ROS triggers autophagy. Further studies show that suppression of autophagyusing pharmacological inhibitors (3-methyladenine and chloroquine) enhances apoptotic cell death.
mitochondrial pathway in otherwise highly chemoresistantmelanoma cells.11 Autophagy, or type II programmed cell death, has been Previously, a number of ruthenium compounds have been proposed as a third mode of cell death besides apoptosis and shown to display promising anticancer activity. Two Ru(III) necrosis.1,2 Autophagy is a double-edged sword in oncology, complexes have successfully entered clinical trials, namely, as it is involved in both cell survival and cell death. It is long known to provide a survival advantage to rapidly growing cells 4(DMSO)(Im)], where Im = imidazole and DMSO = dimethylsulfoxide)a and KP101913 under conditions of hypoxic or metabolic stresses, which thus contributes to normal and cancer cell survival. It also has a role 4(Ind)2], where Ind = indazole). Ru(II) complexes carrying labile ligands such as "half-sandwich" in the suppression of tumor growth, and autophagy defects are arene complexes exhibit both in vitro and in vivo activities.14,15 associated with increased tumorigenesis.3,4 The role of autop- Some of the R-[Ru(II)(azpy) hagy in cancer therapy is also a topic of intense debate, and 2Cl2] (azpy = 2-(phenylazo)- pyridine) type complexes show a cytotoxic potency similar autophagy can serve as a cell survival pathway by suppressing to or even better than that of cisplatin.16 It has also been apoptosis; thus, treating cancer cells by autophagy inhibition is reported that coordinatively saturated Ru(II) polypyridyl possible.5 It can also lead to death itself; thus, drugs can activate autophagy to kill cancer cells resistant to apoptosis.6 2(dppn)]Cl2 (dppn = 4,5,9-16-tetraazadi- benzo[a,c]naphthacene) exhibits cytotoxic activity against As autophagy is such a fundamental process, establishing how two cancer cell lines at low micromolar IC the functional status of autophagy influences the response to The β-carboline alkaloids are a class of synthetic and cancer treatment is important.4,7 naturally occurring compounds that possess a large spectrum The development of metal complexes with bioactive mole- cules as ligands offers possibilities for the discovery of novelanticancer drugs with enhanced and targeted activity.8 Sub- a Abbreviations: 1-Py-βC, 1-(2-pyridyl)-β-carboline; 3-MA, 3-methy- stitution of the β-phenyl ring of tamoxifens by ferrocenyl ladenine; AO, acridine orange; AVOs, acidic vesicular organelles; azpy, group affords ferrocifens, which exhibit a strong antiprolifera- 2-(phenylazo)pyridine; bpy, 2,20-bipyridine; CCCP, carbonyl cyanidem-chlorophenylhydrazone; CDKs, cyclin-dependent kinases; CQ, tive effect in hormone-independent breast cancer cells, where chloroquine; DIP, 4,7-diphenyl-1,10-phenanthroline; CT-DNA, calf tamoxifen and ferrocene are inactive.9 The combination of thymus DNA; DMSO, dimethylsulfoxide; dppn, 4,5,9-16-tetraazadi- Ru(II) arene complexes with staurosporine, a potent inhibi- benzo[a,c]naphthacene; dppz, dipyrido[3,2-a:20,30-c]phenazine; GF-AAS, graphite furnace atomic absorption spectrometry; H tor for various kinases, results in the discovery of nanomolar 20,70-dichlorofluorescein diacetate; Im, imidazole; Ind, indazole; JC-1, and even picomolar protein kinase inhibitors,10 and nota- bly, the GSK-3β inhibitor DW1/2 is highly cytotoxic dide; MLCT, metal-to-ligand charge transfer; MMP, mitochondrial in vitro and can induce p53-activated apoptosis via the membrane potential; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; NAC, N-acetylcysteine; PBS, phosphate bufferedsaline; phen, 1,10-phenanthroline; ROS, reactive oxygen species; TEM, *To whom correspondence should be addressed. Phone/Fax: 86-20- transmission electron microscopy; Tiron, 4,5-dihydroxy-1,3-benzenedi- 3933-2990. E-mail: [email protected]
sulfonic acid disodium salt.
r 2010 American Chemical Society Published on Web 10/19/2010


7614 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 ROS (reactive oxygen species) mediated mechanism. Autop-hagy caused by 1-3 is detected by TEM (transmissionelectron microscopy) and analysis of the localization of greenfluorescent protein-tagged autophagic marker microtubule-associated protein light chain 3 (GFP-LC3) in transfectedHeLa cells. Inhibition of autophagy by pretreatment with3-methyladenine (3-MA) or chloroquine (CQ) sensitizes thecells to apoptotic cell death, revealing that Ru(II)-inducedautophagy plays a cytoprotective role. Additionally, confocalmicroscopy studies show that 3 can penetrate the nuclearenvelope, making it possible that the primary target of thesecomplexes is genomic DNA, so the binding of 1-3 with DNAhas been studied by absorption and fluorescence spectro-scopic studies to further elucidate whether the mechanism of1-3 involves direct DNA damage.
Synthesis and Characterization. The synthesis of the com- plexes [Ru(N-N)2(1-Py-βC)](PF6)2 (1-3) was carried out asfollows: (i) Synthesis of the ligand 1-Py-βC was achieved byreacting tryptamine and 2-pyridinecarboxaldehyde in dryanisole; (ii) [Ru(N-N)2(1-Py-βC)](PF6)2 (1-3) were syn-thesized by refluxing 1 equiv of 1-Py-βC and cis-[Ru-(N-N)2Cl2] 3 2H2O in 75% (v/v) aqueous ethanol for >3 h, followed by anion exchange with NH4PF6, purification bycolumn chromatography, and recrystallization. The com-plexes were obtained as racemic mixtures containing bothΔ- and Λ-isomers. The ligand and the complexes were charac- Figure 1. (A) Structures of the Ru(II) complexes. (B) ORTEP view terized by 1H NMR spectroscopy, ESI-MS (Supporting of 1 (30% probability ellipsoids). Hydrogen atoms, solvated molec- Information Figures S2-S9), and elemental analysis.
ules, and anions are omitted for clarity.
Complexes 1-3 show intense spin-allowed intraligand (1IL) absorption bands in the UV region at approximately of important pharmacological properties including sedative, 250-340 nm and less intense spin-allowed metal-to-ligand anxiolytic, hypnotic, anticonvulsant, antitumor, antiviral, charge transfer (1MLCT) absorption bands at approxi- antiparasitic, and antimicrobial activities.18 It has been re- mately 350-530 nm (Supporting Information Figure S10), ported that β-carboline alkaloids can exert antitumor activ- which are typical absorption properties of Ru(II) polypyr- ities through multiple mechanisms, such as intercalating into idine complexes. 1-3 display luminescence (550-700 nm) in DNA19 and inhibiting topoisomerases I and II,20 CDKs (cyclin- Tris-HCl buffer at 298 K upon excitation at 488 nm. The dependent kinases),21 and IκB kinases.22 Various synthetic emission maxima of all the complexes occur at approxi- derivatives with different substituents in positions 1, 3, and 9 mately 604-609 nm (Figure 9B). The emission is attributed of the β-carboline skeleton have been synthesized in order to to a 3MLCT (dπ(Ru) f π*(diimine)) excited state.25 The elucidate the structure-activity relationship of this class of DIP complex 3 displays more intense emission than 1 and 2 compounds.18 Al-Allaf and co-workers have reported the do, and the reason is that the π* orbitals of DIP are lying in synthesis and the preliminary biological results of some Pd(II) lower energy than bpy and phen, owing to the electron- and Pt(II) complexes based on monodentate β-carboline withdrawing phenyl substitutions.26 alkaloids.23 The Pd(II) complex trans-[Pd(DMSO)(harmine)- The X-ray crystal structures of 1-Py-βC and complex 1 Cl2] shows a potency better than those of cisplatin, fluorour- are depicted in Figure S1 (Supporting Information) and acil and carboplatin, while the mechanism of antitumor action Figure 1B, respectively. The crystallographic data and se- by these complexes still remains largely speculative.24 lected bond lengths and angles are listed in Tables S1 and S2 The objective of the present study is to investigate the (Supporting Information), respectively. The Ru(II) center of potential of ruthenium β-carboline complexes as anticancer complex 1 adopts a distorted octahedral geometry, and the drugs. Three luminescent Ru(II) complexes, [Ru(N-N)2- average Ru-N bond length is 2.059 A˚, which is comparable (1-Py-βC)](PF6)2 (N-N = 2,20-bipyridine (bpy, 1), 1,10- to those found for [Ru(bpy)3]2þ (2.056 A˚) and related com- phenanthroline (phen, 2), 4,7-diphenyl-1,10-phenanthroline pounds.27 The average bite angle of bpy (87.10(13)°) falls (DIP, 3); 1-Py-βC = 1-(2-pyridyl)-β-carboline) (Figure 1A), within the same range as those of related tris(diimine)- have been synthesized and characterized. [Ru(phen)2- (dppz)](PF6)2 (4, dppz = dipyrido[3,2-a:20,30-c]phenazine), Cellular Uptake. The cellular uptake properties of metal- which lacks a β-carboline moiety compared with 2, is used based anticancer drugs are important factors that can influ- as a reference in biological assays to investigate whether the ence their antiproliferative efficacies. For platinum com- biological activities of these complexes are associated with the plexes, increasing lipophilicities enhances the rate of cellular β-carboline moiety. We show for the first time that Ru(II) uptake and, consequently, the cytotoxic activities.29 The complexes can induce autophagy and apoptosis simulta- cellular uptake characteristics of Ru(II) complexes can neously in HeLa (human cervical cancer) cells through a be estimated by their lipophilicities, which are commonly


Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7615 referred to as the n-octan-1-ol/water partition coefficients flow cytometry was used to obtain semiquantitative data on (expressed as log Po/w).26,30 The log Po/w values of 1-3 were the uptake of Ru(II) complexes into HeLa cells (Figure 2A).
determined by reversed-phase HPLC (Supporting Infor- Cells not treated with Ru(II) exhibit neglectable background mation). The lipophilicities of the complexes are substan- luminescence. Incubation with 20 μM complexes for 2 h tially increased by incorporating more hydrophobic ligand causes changes in the luminescence profiles in the following DIP, and the log Po/w value of the DIP complex 3 (3.39) is order: 3 > 2 > 1. The luminescence intensity of the cell much larger than those of 1 (0.52) and 2 (1.43). The high population increases dramatically after cells are incubated lipophilicity of the DIP complex is anticipated to facilitate for 2 h with 20 μM 3. The time-dependent uptake profiles of the tissue and cellular uptake.
1-3 have also been measured (Supporting Information Because of their stability in aqueous solution and lumines- Figure S11). The complexes have accumulated in HeLa cells cence (the luminescence intensity is increased upon binding in 30 min. In addition, either lowering the incubation tem- with DNA, Figure 9B), the cellular uptake properties of perature or depleting the cellular ATP with CCCP (carbonyl Ru(II) polypyridyl complexes can be studied using flow cyto- cyanide m-chlorophenylhydrazone) shows little influence metry and confocal microscopy conveniently.26,30,31 First, on uptake (Supporting Information Figure S12), which leadsus to conclude that uptake might occur via an energy-independent process, i.e., passive diffusion, as proposed for[Ru(DIP)2(dppz)]Cl2.31 Flow cytometry cannot discriminate among membrane- associated, cytoplasmic, and nuclear localization, while thecellular localization characteristics of anticancer drugs arefundamental to their efficacy,32,33 thus the cellular distribu-tion Ru(II) has been studied by confocal microscopy. Inter-estingly, unlike [Ru(DIP)2(dppz)]Cl2, of which almost nonuclear staining can be observed,26 complex 3 (5 μM) graduallypenetrates into the interior of the nucleus and shows diffusecytoplasmic and nuclear fluorescence after 2 h of incubationwith HeLa cells (Figure 2B). The impact of the incorporationof 1-Py-βC on cellular distribution of Ru(II) complexes hasbeen further investigated by using 4 as a reference, whichlacks the β-carboline moiety. After incubation at 40 μM for24 h, 2 produces a diffuse cytoplasmic and nuclear fluorescencein HeLa cells. In contrast, 4 accumulates in the cytoplasmand is predominantly excluded from the nucleus. Notably,cells treated with 2 show marked morphological changes(e.g., cell shrinkage and membrane blebbing).
The cellular ruthenium content of the samples was also determined by GF-AAS (graphite furnace atomic absorptionspectrometry) quantitatively using the literature method.17 Figure 2. (A) Flow cytometric results of HeLa cells incubated with The protein content was determined by the Bradford meth- blank medium and complexes 1 (20 μM), 2 (20 μM), and 3 (20 μM) at37 °C for 2 h. 10 000 events were collected in the FL2 channel od, and results were reported as ng of ruthenium per mg of (excitation, 488 nm; emission, 585 ( 21 nm). (B) Confocal images of cellular protein. The uptake levels are relatively low for HeLa cells treated with 3. Cells were incubated with 5 μM complex 3 complexes 1 (148.1 ( 20.5) and 2 (173.9 ( 15.6). An incuba- for different time intervals at 37 °C and observed by confocal tion concentration of 500 μM has to be used for 1 and 2 in microscopy (excitation, 488 nm; emission, 600-630 nm). (C) Com- order to reach detectable cellular Ru level, while 3 (103.1 ( parison of the cellular localization of 2 and 4 taken by confocal 5.8) shows a comparable Ru level after exposure to a much microscopy (excitation, 488 nm; emission, 600-630 nm). Note themorphological changes (cell shrinkage and membrane blebbing) lower concentration (10 μM). The cellular uptake data ob- caused by treatment of 2 (indicated by arrows).
tained for complexes 1-3 show a clear correlation between Table 1. IC50 Values of Tested Compounds Towards Different Cell Linesa a IC50 values are given in μM, and those of 1-Py-βC, the Ru(II) precursors, 4, NAMI-A, and cisplatin are included for comparison. Data are presented as mean values ( standard deviations, and cell viability is assessed after 48 h of incubation.


7616 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 Figure 3. Quantitative cell cycle distribution data for HeLa cells after treatment with 1-3. Data shown are mean values ( standard deviationsof three samples for each treatment.
the lipophilicity and the amount of ruthenium associated and 2 cause an increase of the sub-G1 fraction (0.8 ( 0.1%, with the cells.
12.9 ( 0.2%, and 20.7 ( 2.8% for control, 1, and 2, In Vitro Cytotoxicity. To explore the antitumor potential respectively), an index of apoptotic DNA fragmentation.
of the Ru(II) complexes, HepG2 (human hepatocellular Notably, 3 (2.5 μM) causes a significant and time-dependent liver carcinoma), HeLa, MCF-7 (human breast adeno- increase in the percentage of cells in sub-G1 phase. After 48 h carcinoma), and MCF-10 (immortalized human breast of incubation, most of the cells treated with 3 (63.2 ( 4.9%) epithelial) cells were treated with varying concentrations have undergone apoptosis.
of Ru(II) for 48 h, and cell viability was determined by the To observe the morphologic characteristics of apoptotic nuclei, HeLa cells were stained with Hoechst 33342 after lium bromide) assay. 1-Py-βC, three Ru(II) synthetic pre- exposure to serial concentrations of 1-4 and cisplatin for cursors, complex 4, NAMI-A, and cisplatin were included 24 h and detected by fluorescence microscopy. Representa- as controls. The resulting IC50 values for the tested com- tive images of the cells treated with vehicle (1% DMSO) and pounds are shown in Table 1. The result indicates that, in 3 are shown in Figure 4A. Control cells exhibit homogeneous general, on the basis of calculated IC50 values, the following nuclear staining, and apoptotic cells increase gradually in a order of in vitro antiproliferative activity of the compounds dose-dependent manner and display typical apoptotic changes can be considered: 3 > cisplatin > 2 > 1 > 4 > NAMI-A.
(e.g., staining bright, condensed chromatin, and fragmented The Ru(II) precursors are far more cytotoxic than the nuclei).34 The percentages of cells showing abnormal nuclei corresponding Ru(II) β-carboline complexes against all are shown in Figure 4B, and Ru(II) (1, 100 μM; 2, 50 μM; 3, the cell lines screened. 1-3 also show an increased cytotoxic 3.75 μM; 4, 50 μM) treatment increases the percentage of potency if compared with the ligand 1-Py-βC alone, which abnormal nuclei (1, 35.0 ( 2.0%; 2, 55.3 ( 3.1%; 3, 86.3 ( is inactive against all the cell lines tested (IC50 > 200 μM).
3.5%; 4, 14.3 ( 1.5%) compared with the vehicle-treated Notably, complex 3 is more potent than cisplatin against all cells (2.3 ( 1.2%).
the cancer cell lines screened (approximately 9-fold more The activation of cysteine proteases (caspases) is the best potent than cisplatin in killing HeLa cells), while its cyto- recognized biochemical hallmark of both early and late toxicity against the normal-like breast epithelial cell line, stages of apoptosis.35 Thus, we determined the effect of Ru(II) MCF-10 (IC50 ≈ 40.6 μM), is only slightly higher than treatment on caspase-3/7 activity. HeLa cells were treated that of cisplatin (IC50 ≈ 56.2 μM). NAMI-A shows a very with complexes 1-4 and cisplatin at serial concentrations for low cytotoxic potency, with IC50 values ranging between 6 h, after which caspase-3/7 activity was determined using the 533.5 and 647.3 μM, which is consistent with the literature Caspase-Glo assay. As shown in Figure 4C, treatment of HeLa cells with 1-4 and cisplatin results in a concentration- Cell Cycle Arrest and Induction of Apoptosis. The effect of dependent increase in caspase-3/7 activity.
1-3 on cell cycle was investigated by flow cytometry in PI Compared with 4, more apoptotic nuclei, consistent with (propidium iodide) stained cells after Ru(II) treatment for 3, higher level of caspase-3/7 activation, are observed when 12, 24, 36, and 48 h (Figure 3 and Supporting Information HeLa cells are incubated with 2, implying that the β-carbo- Figure S13). 1 (50 μM) and 2 (25 μM) cause pronounced G0/ line moiety is involved in the cytotoxicity induced by 1-3. It G1 arrest after 24 h of incubation (83.4 ( 4.3% and 80.0 ( is evident from these studies that 3 displays a significantly 3.4% for 1 and 2, respectively) compared with the vehicle- higher potency in apoptosis induction than cisplatin does treated cells (53.7 ( 3.4%). After treatment for 48 h, both 1 under similar conditions.



Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7617 Figure 5. Representative TEM images showing the ultrastructureof HeLa cells treated with vehicle (1% DMSO) (A) or 2.5 μM 3 (B-H)for 12 h. Nuclei are labeled N. Autophagic structures including auto-lysosomes (/) and autophagic vacuoles (#) are detected in Ru-treatedcells. Parts C, D, F, and G are pictures with higher magnificationshowing detailed autophagic structures.
has been examined to determine whether LC3 is concen-trated in HeLa cells after Ru(II) treatment. In cells treatedwith vehicle (1% DMSO), GFP-LC3 protein is diffuselydistributed throughout the cytoplasm, whereas cells treatedwith 1-4 and cisplatin show an increase in number andfrequency of GFP-LC3 dots (g5 dots per cell, Figure 6A).
The percentage of autophagy quantified by counting thenumber of cells showing the punctate pattern of GFP-LC3 in300 GFP-positive cells is shown in Figure 6B, and 65.4 (5.3% of HeLa cells treated with 3 (2.5 μM, 12 h) show GFP-LC3 dots, whereas these autophagic features are detected inonly 2.3 ( 1.3% of cells treated with vehicle.
Autophagy is the process of sequestering cytoplasmic proteins into lytic components and is characterized by theformation and promotion of AVOs (acidic vesicular organel-les), which can be accessed by AO (acridine orange) stain- Figure 4. (A) Hochest stained HeLa cells after treatment of 3 at ing.38,39 As shown in Figure 6C, after Ru(II) treatment for indicated concentrations after 24 h of incubation. (B) Histogramsshowing the number of cells with abnormal (condensed or fragmented) 12 h, AO staining reveals an increase in the number of cyto- nuclei. (C) Caspase-3/7 activity after drug treatment for 6 h at plasmic AVOs, characteristic of autophagy. To quantify the indicated concentrations. Data shown are mean values ( standard accumulation of the acidic components, flow cytometry was deviations from three independent experiments: (/) P < 0.01, (//) applied to analyze AO-stained cells using the FL3 channel to P < 0.005, compared with the vehicle-treated cells.
evaluate the bright red fluorescence and the FL1 channel toevaluate the green fluorescence. As shown in Figure 6D, Autophagy Induced by Ruthenium Complexes. TEM is the 1 (100 μM), 2 (50 μM), and 3 (2.5 μM) treatment increases the most convincing method for the analysis of autophagy.36 strength of red fluorescence from 2.2% to 47.2%, 53.3%, Very few autophagical vacuoles are observed, and microvilli and 79.0%, respectively. Both 3 and cisplatin significantly are preserved around the cytoplasm in control cells (Figure 5A).
develop AVOs in HeLa cells, and the AVO-inducing ability Typical autophagic structures, such as autolysosomes and of 3 is higher than that of cisplatin (62.3%). The formation of autophagic vacuoles containing cellular material, can be observed red fluorescent AVOs caused by 2 is more pronounced than in 3-treated cells (Figure 5B-G). Notably, some of the cells that caused by 4 (17.7%).
containing autophagic vacuoles simultaneously show mor- Phosphoinositide 3-kinase (PI3K) is an essential compo- phologies of apoptosis (apoptotic nuclear condensation and nent of core machinery involved in autophagic vesicle for- fragmentation, Figure 5H).
mation. 3-MA, the specific class III PI3K inhibitor, can One of the hallmarks of autophagy is the conversion of the inhibit autophagy at an early stage by preventing the forma- soluble form of LC3 (LC3-I) to the lipidated and autophago- tion of autophagosomes.40 CQ, a lysosomotropic agent, some-associated form (LC3-II);37 thus, the presence of a inhibits autophagy by raising the lysosomal pH, which leads punctate pattern of GFP-LC3 expression (GFP-LC3 dots) to inhibition of lysosome-autophagosome fusion and lysosomal


7618 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 Figure 6. (A) Fluorescence staining of GFP-LC3 in response to drug treatment. (B) Autophagosome formation was quantified after 12 htreatment. Data are presented as percentage of GFP-LC3-transfected cells with punctate fluorescence: (/) P < 0.01, (//) P < 0.005, comparedwith vehicle-treated control cells. (C) HeLa cells were stained with AO and examined by fluorescence microscopy after 12 h of treatment.
(D) Quantification of AVOs with AO using flow cytometry. HeLa cells were treated with the complexes for 12 h. For inhibitory experiments, cellswere treated 3-MA (5 mM) or CQ (2.5 μM) for 1 h and further incubated with or without Ru(II) for 12 h. FL1-H indicates green color intensity,while FL3-H shows red color intensity. Top of the grid is considered as AVOs. Data shown are representative of three independent experiments.
protein degradation.41 Preincubation of HeLa cells with these causes a red to green color shift, indicating loss of MMP, in inhibitors of autophagy, 3-MA (5 mM) and CQ (2.5 μM), most of the treated cells. Representative JC-1 red/green ratio before drug treatment markedly suppresses the induction of signals recorded by flow cytometry in vehicle-treated (1% AVOs in HeLa cells (Figure 6D).
DMSO) cells and Ru(II)-treated cells are shown in Figure 7B.
Induction of Mitochondrial Dysfunction. Mitochondrial After 6 h of incubation, 1 (100 μM), 2 (50 μM), and 3 (25 μM) dysfunction is involved in both apoptotic and autophagic treatment decreases the JC-1 red/green ratio signal from 14.5 ( cell death. Mitochondria play important roles in apoptosis 1.1 to 1.2 ( 0.2, 1.0 ( 0.1, and 0.6 ( 0.1, respectively.
through the release of proapoptotic factors such as cyto- ROS Accumulation. It is well-known that apoptosis can be chrome c and apoptosis-inducing factor.2,42 As a mechanism triggered by increased intracellular ROS levels,45 and there is to maintain genomic integrity in the face of metabolic stress, strong evidence that ROS are also involved in the induction drug treatment, or radiation, autophagy can selectively remove of autophagy.46 Therefore, we investigated whether Ru(II) damaged mitochondria, which are major sites of genotoxic treatment could increase the ROS level in HeLa cells. ROS ROS production.43 Thus, mitochondrial dysfunction was accumulation was quantified by the 20,70-dichlorofluorescein determined by measuring changes in the mitochondrial diacetate (H2DCF-DA) assay. Confocal microscopic anal- membrane potential (MMP, ΔΨm) using confocal micro- ysis of DCF-stained Ru(II)-treated cells shows significant scopy and flow cytometry after staining live cells with the concentration-dependent increase in intensity of DCF stain- cationic dye JC-1 (5,50,6,60-tetrachloro-1,10,3,30-tetraethyl- ing compared with the vehicle-treated cells (Figure 8A, left).
benzimidazolylcarbocyanine iodide). JC-1 exhibits poten- This result was further confirmed by flow cytometry (Figure 8A, tial-dependent accumulation in mitochondria, indicated by right) and microplate analyzer with DCF staining (Figure 8B).
a fluorescence emission shift from red (∼590 nm) to green 3 (10 μM) treatment results in a 7.5-fold increase of fluorescence (∼525 nm).44 Representative images taken by confocal mi- signal compared with the vehicle-treated control, at 6 h post- croscope are shown in Figure 7A. 3 (25 μM, 6 h) treatment treatment (Figure 8B).


Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7619 Figure 7. (A) Fluorescence imaging of JC-1 labeled cells taken by confocal microscope. (B) Effects of 1-3 on MMP analyzed by flowcytometry. Representative histograms of two independent experiments done in triplicate are shown: (unfilled curve) cultures treated withvehicle (1% DMSO); (filled curve) cultures treated with Ru(II).
Having determined that Ru(II) induced generation of Ru(II) treatment alone. These data therefore further suggest ROS, we proceeded to examine whether ROS played a role that Ru(II) complexes stimulate autophagy as a cytoprotec- in Ru(II)-elicited cell death, autophagy, and apoptosis. We tive mechanism.
used 4,5-dihydroxy-1,3-benzenedisulfonic acid disodium salt DNA Binding Studies. It has been reported that DNA- (Tiron, 5 mM) and N-acetylcysteine (NAC, 10 mM), two damaging agents can activate the intrinsic pathway of apop- ROS scavengers, and examined their effect on cell viability tosis involving the release of cytochrome c and other mito- after Ru(II) treatment. Both Tiron and NAC substantially chondrial apoptogenic factors47 and trigger autophagy suppress the ROS accumulation (Figure 8B) and reduce the simultaneously as a self-defense mechanism.48,49 Previously, cytotoxicity of 3 in HeLa cells (Figure 8C). To investigate the confocal microscopy studies show that 1-3 can readily pass involvement of ROS in Ru(II)-induced apoptosis, we mon- the cell membrane and penetrate into the nucleus (Figure 2), itored drug-induced sub-G1 population in the absence and so genomic DNA may serve as the target of these complexes.
presence of Tiron and NAC using flow cytometry. There is a DNA binding studies have been performed to determine decline in the drug-induced sub-G1 population when ROS is whether Ru(II)-induced apoptosis and autophagy are corre- inhibited by the antioxidants, suggesting that ROS play a lated with their abilities to cause DNA damage.
crucial role in induction of apoptosis (Figure 8D). Attenua- The absorption spectra of 1, 2, and 3 in the absence tion of ROS levels by Tiron and NAC significantly decreases and presence of CT-DNA (calf thymus DNA) are shown in the number of AVOs upon Ru(II) treatment (Figure 8E), Figure 10A. With increasing concentration of CT-DNA, suggesting ROS also mediate induction of autophagy in absorption bands of the complexes display clear hypochro- HeLa cells. Taking all these results together, we conclude mism. The hypochromism (H% = 100  (Afree - Abound)/ that ROS have an important role in Ru(II)-induced autop- Afree) of the 1MLCT bands at ∼470 nm of 1, 2, and 3 are hagy and apoptosis.
calculated to be about 18.7%, 20.7% and 28.3%, respec- Inhibition of Apoptosis and Autophagy. To gain better tively. In order to compare quantitatively the binding affinity insight into the mechanism of Ru(II) cell-killing action, of 1-3, the intrinsic binding constants K of 1-3 to DNA autophagy inhibitors (3-MA and CQ) and caspase inhibitor were determined by monitoring the changes in absorbance at (z-VAD-FMK) were used to analyze the interconnection 470 nm (shown in the insets of Figure 10A) using the equation between Ru(II)-induced cell death, autophagy, and apoptosis.
previously described in the literature.50 The intrinsic binding First, we determined whether autophagy inhibitors pro- constants derived for 1, 2, and 3 are (1.08 ( 0.21)  106 M-1, moted Ru(II)-induced apoptosis using PI staining and flow (1.93 ( 0.35)  106 M-1, and (3.22 ( 0.38)  106 M-1, cytometry. Pretreatment with 3-MA (5 mM) promotes Ru- respectively, which are in the same range as those reported for (II)-induced apoptosis, which is demonstrated by the in- Δ-[Ru(phen)2(dppz)]2þ (1.7  106 M-1) and Λ-[Ru(phen)2- creased percentage of sub-G1 phase cells. Similar results (dppz)]2þ (3.2  106 M-1).51 The changes in emission spectra of are obtained by pretreatment with CQ (2.5 μM) (Figure 9A 1-3 in aqueous solution with increasing DNA concentrations and Supporting Information Figure S14). These observa- are shown in Figure 10B. As DNA is successively added, an tions suggest that Ru(II)-mediated autophagy has a cyto- obvious increase in emission intensity can be observed. The protective role in nature.
emission intensities of 1, 2, and 3 increase to about 2.49, 2.97, Effect of autophagic and caspase inhibitors on Ru(II)- and 7.48 times of the original intensities, respectively.
induced cell death is shown in Figure 9B. Cells pretreated Both the electronic absorption titration and fluorescence with z-VAD-FMK (50 μM) show a marked improvement in spectroscopic studies indicate that 1-3 have a strong inter- cell viability relative to cells treated with Ru(II) complexes action with DNA and the order of DNA binding affinity is alone, suggesting that Ru(II)-induced cell death is caspase- 3 > 2 > 1. The DNA binding affinities of 1-3 are consistent dependent, and autophagy may not be an apoptosis-alter- with their cytotoxicities and potential to induce apoptosis native pathway to induce cell death. In all cases, the CQ/ and autophagy, implying that DNA may be their primary Ru(II) and 3-MA/Ru(II) combinations result in reductions target, and genotoxic stress caused by DNA damage triggers in cell viability when compared with those observed for mitochondria-mediated apoptosis and autophagy.


7620 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 Figure 8. Analysis of ROS production after HeLa cells were treated with Ru(II) for 6 h. (A) The intracellular ROS level was detected byconfocal microscope (excitation at 488 nm and emission at 530 nm) and flow cytometry (FL-1 channel; excitation at 488 nm and emission at525 nm). (B) ROS level is expressed as relative fluorescence intensity measured in a microplate reader after 6 h of Ru(II) treatment with orwithout antioxidants at the indicated concentrations. Data are presented as mean values ( standard deviations and are obtained in threeindependent experiments. (C) Effects of Tiron (5 mM) and NAC (10 mM) on 3-induced cytotoxicity. HeLa cells were exposed to different dosesof 3 with or without antioxidants for 48 h. Cell viability was assessed by MTT assay. (D) Attenuating ROS levels by Tiron (5 mM) and NAC(10 mM) reduces the sub-G1 population upon 3 (2.5 μM, 24 h) treatment. (E) Effects of Tiron (5 mM) and NAC (10 mM) on 3-inducedautophagy formation. HeLa cells were exposed to 2.5 μM 3 with or without antioxidants for 12 h. The percentage of autophagic cells wasquantified by AO using flow cytometry as described in Figure 7D: (/) P < 0.01, (//) P < 0.005, compared with the cells treated with 3 alone.
Discussion and Conclusions space.54 For example, organometallic kinase inhibitors derived from the class of indolocarbazole alkaloids (e.g., staurosporine) -Carboline alkaloids are originally isolated from the plant Peganum harmala. Powdered seeds of Peganum harmala have can match the size of the active site of targeting kinases and long been used in herbal formulas of traditional Chinese discriminate between otherwise closely related binding sites.55 medicine to cure digestive tract tumors.52 Because of their In the present study, we use the chemically inert and easily affinity with benzodiazepine, imidazoline, serotonin receptors synthesized Ru(II) polypyridyl complexes to complement the of the central nervous system, β-carbolines display considerable molecular diversity of β-carboline derivatives in the quest for acute neurotoxicity, which hinders their clinical applications as the discovery of compounds with superior biological activities anticancer drugs.18,52 It has long been recognized that, com- (e.g., improved solubility and selectivity, diminished neurotoxi- pared with platinum-based anticancer agents, ruthenium com- city). We present here the first synthesis of a series of ruthenium- plexes have particularly low general toxicity, allowing admin- β-carboline complexes that display markedly enhanced cyto- istration of larger doses.8,53 On the other hand, bulky and rigid toxicities, compared with the original alkaloid.
octahedral ruthenium complexes can serve as structural scaf- It has been reported that Ru(II) dipyridophenazine com- folds to organize the organic ligands in three-dimensional plexes can readily accumulate in the cytoplasm of live cells but Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7621 are mostly excluded from the nucleus and mainly localized inthe cytoplasm.26,31 For anticancer agents targeting genomicDNA, nuclear accumulation is highly desirable.32,33 Thesubcellular localization of polypyridylruthenium complexescan be greatly influenced by the functional groups (e.g., cell-penetrating peptide,56,57 fluorescein,57 phenanthridine,58 car-boxylic groups,59 and estradiol30) attached to the ligandsystems. It has been reported that β-carboline analogues canpass through the cell membrane after 1 min of drug exposureand enter the nuclear envelope gradually,60 and we speculatethat may partly explain the nuclear penetrating properties ofruthenium β-carboline complexes. A detailed comparison ofcomplexes 2 and 4 indicates that the coordination of the β-carboline ligand 1-Py-βC to polypyridyl-Ru(II) centersinfluences their cellular localization and results in morecytotoxic complexes. 2 can readily pass through the nuclearenvelope and shows higher ROS, apoptosis, and autophagy-inducing capabilities than 4 does.
Most cancer chemotherapy drugs act through induction of apoptosis.61 The relationship between autophagy and apop-tosis is complex and depends on particular cell type, stimulus,and environment.62 Evidence has been accumulated to con-firm the importance of autophagy in determining the responseof tumor cells to chemotherapy.63 Under certain circum-stances, autophagy can manifest a cytoprotective role in drugtreatments, such as temozolomide,39 timosaponin A-III,64sulforaphane,65 tamoxifen,66 cisplatin,67 ionizing radiation,68and G-quadruplex ligand.69 Inhibition of autophagy may betherapeutically beneficial in these contexts, as it can sensitize Figure 9. (A) Apoptosis (formation of sub-G1 peak) was deter- cancer cells to chemotherapies. Autophagy can also play a mined after cells were treated with Ru(II) for 48 h at the indicated destructive role in promoting cell death, as in arsenic concentrations in the absence or presence of the inhibitors: (/) P < trioxide-,70,71 resveratrol-,72,73 cannabinoid-,74 and ceramide- 0.01, (//) P < 0.005, compared with the percentage of sub-G1 cells induced75 autophagy. In these cases, the induction of autop- for Ru(II) treatment alone. (B) Cell viability was determined by hagy may be used as a potential therapy for some apoptosis- MTT assay after cells were treated with 1-3 for 48 h at the indicated resistant cancers (i.e., breast and pancreatic cancers). For the concentrations in the absence or presence of 3-MA, CQ, or z-VAD-FMK: (/) P < 0.01, (//) P < 0.005, compared with the cell viability first time, we find that ruthenium complexes can induce both of Ru(II) treatment alone.
autophagy and apoptosis in cancer cells. Such dual properties Figure 10. Absorption spectra of 1-3 in the absence or presence of increasing amounts of CT-DNA. [Ru] = 10 μM, [DNA] = 0-200 μM fromtop to bottom. Arrows indicate the change in absorbance upon increasing the DNA concentration. Inset: plots of (εa - εf)/(εb - εf) vs [DNA]and the nonlinear fit for the titration of the complexes with DNA. Also shown are changes in the emission spectra (λem = 488 nm) of 1-3(10 μM) with increasing concentrations of CT-DNA (0-200 μM).
7622 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 have never been reported for ruthenium complexes or (Corning). After overnight incubation, the cells were treated β-carboline derivatives as far as we know. Inhibition of Ru- with 1 (500 μM), 2 (500 μM), and 3 (10 μM) for 2 h (final DMSO (II)-induced autophagy by 3-MA or CQ enhances the apop- concentration, 1% v/v). The cells were trypsinized and washed tosis-inducing effect with concomitant increased cell death, twice with cold PBS, and ruthenium content determined by GF- suggesting that Ru(II)-induced autophagy is a protective AAS was presented as ng of ruthenium per mg of cellular proteinas described in the literature.17 All experiments were performed response to the treatment.
in triplicate.
In conclusion, our study shows that the combination of Autophagy Detection. Transmission Electron Microscopy.
ruthenium complexes with β-carboline alkaloids has a great HeLa cells (5  105) were treated with Ru(II) at 37 °C for 12 h.
potential for developing new anticancer agents. The most Cells were washed twice and fixed with 2% glutaraldehyde at active drug, complex 3, exhibits higher cytotoxic potency than 4 °C for 1 h and postfixed with 2% osmium tetroxide. Cells were the widely used clinical chemotherapeutic agent cisplatin. The then dehydrated with sequential washes in ethanol and then antiproliferative effects on tumor cells exerted by 1-3 are embedded in Spurr's resin. The ultrathin sections obtained were consistent with their intracellular uptake properties, which are mounted in copper grids, counterstained with uranyl acetate significantly enhanced by the increase of lipophilicity with and lead citrate, and visualized in an electron microscope (JEM extended π-systems. Further study shows that Ru(II) β-carbo- 100 CX, JEOL, Tokyo, Japan). Images were photographed andscanned by using the Eversmart Jazz program (Scitex).
line complexes exhibit effective cell growth inhibition by Plasmid and Transfection. Transfection of HeLa cells with triggering G0/G1 phase arrest and inducing apoptosis through GFP-tagged LC3 was performed using jetPEI transfection a ROS-mediated mitochondrial dysfunction pathway. In vitro reagent (Q-Biogen, France) according to the manufacturer's DNA binding studies show that genomic DNA may serve as instructions. Cells were transfected with 1 μg of GFP-LC3 the primary target of these nuclear permeable complexes.
expressing plasmid in each well of 24-well plates. After 12 h, Notably, these newly synthesized Ru(II) β-carboline com- cells were treated with 1-3 for 12 h. The cells were then fixed plexes are dual autophagy- and apoptosis-inducing agents, with 0.4% paraformalclehyde for 30 min and washed twice with and they activate autophagy as a cytoprotective response in PBS. The fluorescence of GFP-LC3 was viewed, and the rate of HeLa cells, and both the apoptosis- and autophagy-inducing GFP-LC3 vacuoles was counted under a fluorescent microscope activities are at least partially related to ROS accumulation.
(Axio Observer Z1, Carl Zeiss, Germany). A minimum of 300GFP-LC3-transfected cells were counted. Cells treated withvehicle solution (1% DMSO) were included as controls.
Experimental Section Quantification of AVOs with AO. HeLa cells were cultured Materials and General Methods. All solvents were of analy- with medium containing vehicle (1% DMSO) or the complexes tical grade. All buffer components were of biological grade and for 12 h. For microscopy studies, the cells grown on slides were used as received. Ruthenium(III) chloride hydrate (Alfa Aesar), washed twice with PBS, stained with medium containing 1 μg/ bpy (Alfa Aesar), phen (Alfa Aesar), DIP (Alfa Aesar), cisplatin mL AO for 15 min, and examined immediately by fluorescence (Acros), MTT (Sigma), 3-MA (Sigma), CQ (Sigma), PI (Sigma), microscopy (Axio Imager Z1, Carl Zeiss, Germany) using 490 nm band-pass blue excitation filters and a 515 nm long-pass 4PF6 (Alfa Aesar) were used without purification.
barrier filter according to a published protocol.70 For flow cyto- 2Cl2] 3 2H2O,76,77complex 4,78 and NAMI-A79 were prepared according to reported methods. The purity of metry studies, cells were removed from 60 mm dishes (Corning) synthesized compounds was analyzed via reversed-phase HPLC with trypsin-EDTA and washed twice with PBS. After being and was found to be g95% pure. Stock solutions of cisplatin stained with AO for 15 min, the cells were collected in phenol (5 mM in PBS) and NAMI-A (10 mM in PBS) were freshly red-free growth medium. Green (510-530 nm) and red (650 nm) prepared for every experiment. Stock solutions (20 mM) of 1-4 fluorescence emission from 10 000 cells illuminated with blue were prepared in DMSO, which were proved to be stable for at (488 nm) excitation light was analyzed on a flow cytometer least 48 h at room temperature as monitored by UV-visible (Becton Dickinson, Franklin Lakes, NJ) using CellQuest Cellular Uptake. Flow Cytometry. HeLa cells in growth Intracellular ROS Measurement. After treatment with Ru(II) medium were seeded in 35 mm tissue culture dishes (Corning) complexes for 6 h, the cells were incubated with 10 μM H2DCF- and incubated at 37 °C under a 5% CO DA (Sigma-Aldrich) for 20 min at 37 °C. The fluorescence 2 atmosphere until 70% confluent. The culture medium was removed and replaced with intensity of cells was measured by flow cytometry (Becton medium (final DMSO concentration, 1% v/v) containing the Dickinson, Franklin Lakes, NJ), confocal microscope (Leica Ru(II) complexes at 20 μM. After incubation for 2 h, the Microsystems, Wetzlar, Germany), and microplate analyzer cell layer was trypsinized and washed twice with cold PBS (Infinite M200, TECAN, Switzerland) with excitation set at (phosphate buffered saline). The samples were raised in 500 μL 488 nm and emission at 530 nm. When necessary, Tiron (5 mM) of cold PBS and analyzed by a FACSCalibur flow cytometer and NAC (10 mM) were applied 1 h before Ru(II) treatment and (Becton Dickinson & Co., Franklin Lakes, NJ) immediately.
kept in the medium during Ru(II) treatment until the cells were The samples were collected in FL2 channel (excitation at 488 nm and emission at 585 ( 21 nm), and the number of cells analyzed Inhibition of Apoptosis and Autophagy. HeLa cells were pre- for each sample was 10 000.30 incubated with 25 μM CQ, 5 mM 3-MA, or 50 μM z-VAD- Live Cell Confocal Microscopy. HeLa cells were grown on FMK for 1 h before the complexes were added. Percentage of chamber slides to 70% confluence. Complex 3 (5 μM) was added apoptosis was analyzed by flow cytometry, and cell viability was to the culture medium (final DMSO concentration, 0.1% v/v) determined by MTT as described in Supporting Information.
and incubated for varying amounts of time at 37 °C. The cells Statistical Analysis. All biological experiments were per- were then washed with PBS (2  200 μL) and photographed with formed at least twice with triplicates in each experiment. Re- a Leica TCS SP5 confocal microscope (Leica Microsystems, presentative results were depicted in this report. Data were Wetzlar, Germany) using a planapochromate 63/NA 1.4 oil presented as mean values ( standard deviations, and compar- immersion objective. The confocal microscope was equipped isons were made using Student's t test (two-tailed). A probabil- with an ArKr laser which was used to excite Ru(II) (488 nm ity of 0.01 or less was considered statistically significant.
excitation, 600-620 nm emission).
GF-AAS (Graphite Furnace Atomic Absorption Spectro- Acknowledgment. We thank Prof. Z. P. Chen for providing metry). HeLa cells were seeded in 60 mm tissue culture dishes the GFP-LC3 expression vector. This work is supported by Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7623 National Basic Research Program (973 Program, Grant (17) Schatzschneider, U.; Niesel, J.; Ott, I.; Gust, R.; Alborzinia, H.; 2007CB815800), International S&T Cooperation Program Wolfl, S. Cellular uptake, cytotoxicity, and metabolic profilingof human cancer cells treated with ruthenium(II) polypyridyl (Grant 2007DFA30840), State High-Tech Development Pro- complexes [Ru(bpy)2(N-N)]Cl2 with N-N = bpy, phen, dpq, dppz, gram (863 Program, Grants 2006AA090504, 2007AA091401, and dppn. ChemMedChem 2008, 3, 1104–1109.
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LIECHTENSTEINER VATERLAND DONNERSTAG, 3. MAI 2012 29 Ein aktiver Wanderer kommt zurück erwartetNew York. – Eines der berühmtes- Sie sind zurück – die pelzigen ten Gemälde der modernen Kunst und fleissigen Nager. Am Mitt- kam in der zurückliegenden Nacht wochabend führte Holger Frick,

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Curriculum Vitae JACK MERRIT GWALTNEY, JR. December 24, 1930, Norfolk, Virginia B.A. University of Virginia 1948-1952 M.D. University of Virginia 1952-1956 Summary of Career: University Hospitals of Cleveland, Cleveland, Ohio Residency, Internal Medicine University Hospitals of Cleveland, Cleveland, Ohio Chief Resident, Internal University of Virginia Hospital