Pradhan N, Pratheek BM, Garai A, Kumar A, Meena VS, Ghosh S

Cell Biology International ISSN 1065-6995doi: 10.1002/cbin.10308

RESEARCH ARTICLE

Induction of apoptosis by Fe(salen)Cl through caspase-dependentpathway specifically in tumor cellsNitika Pradhan1, B.M. Pratheek2, Antara Garai3, Ashutosh Kumar2, Vikram S. Meena2,Shyamasree Ghosh2, Sujay Singh4, Shikha Kumari2, T.K. Chandrashekar3, Chandan Goswami2,Subhasis Chattopadhyay2*, Sanjib Kar3* and Prasanta K. Maiti1*

1 Imgenex India, Infocity, Bhubaneswar, India2 School of Biological Sciences, National Institute of Science Education & Research, Bhubaneswar, India3 School of Chemical Sciences, National Institute of Science Education & Research, Bhubaneswar, India4 Imgenex Corporation, San Diego, CA, USA

Abstract

Iron-based compounds possess the capability of inducing cell death due to their reactivity with oxidant molecules, but theirspecificity towards cancer cells and the mechanism of action are hitherto less investigated. A Fe(salen)Cl derivative has beensynthesized that remains active inmonomer form. The efficacy of this compound as an anti-tumor agent has been investigatedin mouse and human leukemia cell lines. Fe(salen)Cl induces cell death specifically in tumor cells and not in primary cells.Mouse and human T-cell leukemia cell lines, EL4 and Jurkat cells are found to be susceptible to Fe(salen)Cl and undergoapoptosis, but normal mouse spleen cells and human peripheral blood mononuclear cells (PBMC) remain largely unaffectedby Fe(salen)Cl. Fe(salen)Cl treated tumor cells show significantly higher expression level of cytochrome c that might havetriggered the cascade of reactions leading to apoptosis in cancer cells. A significant loss of mitochondrial membrane potentialupon Fe(salen)Cl treatment suggests that Fe(salen)Cl induces apoptosis by disrupting mitochondrial membrane potential andhomeostasis, leading to cytotoxity. We also established that apoptosis in the Fe(salen)Cl-treated tumor cells is mediatedthrough caspase-dependent pathway. This is the first report demonstrating that Fe(salen)Cl can specifically target the tumorcells, leaving the primary cells least affected, indicating an excellent potential for this compound to emerge as a next-generationanti-tumor drug.

Keywords: apoptosis; caspase; Fe(salen)Cl; leukemia cell line; mitochondria; tumor

Introduction

Amongst the chemotherapeutic drugs for cancer, cisplatin ismostly being used, although it has serious side effects(Rafique et al., 2010). There is a need for suitable drugs thatspecifically target cancer cells while leaving normal cellsunaffected or less affected. In this respect, transition metalcomplexes are of considerable research interest because oftheir structural diversity (Teyssot et al., 2009; To et al., 2009;Liu et al., 2011; Liu and Sadler, 2011). Amongst the metallo-drugs, iron-based compounds have recently been exploredfor their anticancer properties in spite of having some sideeffects (Wang and Pantopoulos, 2011; Huang et al., 2011;Dixon et al., 2012; Reed and Pellecchia, 2012; Yu et al., 2012).

This is because iron-based compounds such as FeIII(porphyrin)(Böttcher et al., 1996), Fe(salen) (Ansari et al., 2011), and iron-basednano-particles (Wuet al., 2011) have cytotoxic effects invitro in cancer cells; but their mechanism of action remainslargely undefined.FeIII(porphyrin) complexes are widely used to mimic the

role of the cytochrome P-450 class of enzymes that areresponsible for the oxidation of various organic substances(Meunier, 1992). In this context, we have synthesizedFe(salen)Cl complexes tomimic the activities of FeIII(porphyrin)complexes.A structural similarity existsbetweenFeIII(porphyrin)and Fe(salen)Cl complexes where the iron remains in FeIII-state(Böttcher et al., 1996; Liou and Wang, 2000). Mandal andcolleagues (Mandal et al., 1997) have synthesized a series of

�Corresponding authors: e-mail: [email protected], [email protected], [email protected]

1Cell Biol Int 9999 (2014) 1–14 © 2014 International Federation for Cell Biology

Fe(salen)Cl derivatives and studied their biochemical effects,mainly at the DNA level.We have recently synthesized a Fe(salen)Cl derivative and

studied its anti-cancer efficacy in tumor cells and normalprimary cells in an effort to study its potential as an anti-cancer drug. From single crystal X-ray structural analysis, itis found that Fe(salen)Cl complexes could exist asmonomeric or dimeric forms depending on the solventused for crystallization. The single crystal data obtainedfrom CH3CN medium suggest that the compound is indeeddimeric in nature in solid phase. The solution phaseelectrochemical studies indicate that the compound ismonomer in solution. The interaction of Fe(salen)Cl withliving cells is little known (Rokita et al., 2003). Mn(salen)(Mandal et al., 1997; Ansari et al., 2009a) and Fe(salen)(Ansari et al., 2009b) have been reported to induce cell deathin breast cancer cell line in vitro, suggesting that salencompounds may have anti-tumor properties, although themechanism by which they induce cell death is unclear.Oxidative stress exerted by redox active metals like iron maybe responsible for DNA/RNA damage in vitro, as has beensuggested (Bhattacharya and Mandal, 1996; Czlapinski andSheppard, 2001; Shao et al., 2006). We show here thatFe(salen)Cl specifically kills the cancerous cells but sparesprimary normal cells. In cancer cells it causes significantloss of mitochondrial membrane potential with a concomi-tant increase in cytochrome c level, which in turn inducesapoptosis in cancer cells through a caspase-dependentpathway.

Materials and methods

General methods

The precursor compound, ferric chloride was purchasedfrom Merck (Germany). The ligand, 2,20-((1E,10E)-(ethane-1,2 diylbis (azanylylidene)) bis(methanylylidene)) diphenol(H2L), was prepared according to Pfeiffer et al. (1933). Allother chemicals and solvents were of reagent grades. Forspectroscopy and electrochemical studies, HPLC gradesolvents were used. Commercial tetraethyl ammoniumbromide was converted to pure tetraethyl ammoniumperchlorate (TEAP) following the procedure of Sawyeret al. (1995).

Physical measurements

UV-Vis spectral work used a Perkin-Elmer LAMBDA-750spectrophotometer. FT-IR spectra were taken with samplesprepared as KBr pellets. Cyclic voltammetry measurementswere carried out using a CH instrument model CHI1120Aelectrochemistry system. A glassy-carbon working electrode,a platinum wire as an auxiliary electrode, and a saturated

calomel reference electrode (SCE) were used in a 3-electrodeconfiguration. Tetraethyl ammonium perchlorate (TEAP)was the supporting electrolyte (0.1M) at a concentrationof 10� 3M with respect to the complex. The half wavepotential E�298 was set equal to 0.5(EpaþEpc), where Epa andEpc are anodic and cathodic cyclic voltammetric peakpotentials, respectively. The scan rate used was 100mV s� 1.

Synthesis of salen ligand

Salen ligand (H2L) was synthesized by following theprocedure of Pfeiffer et al. (1933). A 2:1 molar ratio ofsalicylaldehyde and ethylenediamine were mixed together in20mL absolutemethanol. The resultingmixture was kept for30min at 4�C. The precipitate was filtered and washedthoroughly with ice-cold methanol followed by ice-colddiethyl ether. The final product was recrystallized fromCH3CN.

Synthesis of Fe(salen)Cl

Fe(salen)Cl has been synthesized by the procedure ofMatsushita et al. (1982). A 1:1 molar ratio of ferric chlorideand salen ligand (H2L) were taken in 25mL absoluteCH3OH. The resulting mixture was heated to reflux under adinitrogen atmosphere for 2.5 h. The concentrated solutionwas refrigerated overnight. The precipitate was filtered andwashed thoroughly with ice-cold water followed by ice-colddiethyl ether. The product was recrystallized from acetone.

Determination of crystal structure

Single crystals of complex were grown by slow evaporationof a solution of the complex in acetonitrile underatmospheric conditions. The crystal data of Fe(salen)Clwere collected on a Bruker Kappa APEX II CCDdiffractometer at 293 K, which were corrected for Lorentzpolarization and absorption effects. The program packageSHELX-97 (ShelxTL) (Sheldrick, 1997) was used forstructure solution and full matrix least squares refinementon F2. Hydrogen atoms were included in the refinementusing the riding model. Contributions of H atoms for thewater molecules were included but were not fixed.

Cell culture

Jurkat, Molt-4 (human acute lymphoblastic leukemia), Raji,Ramos, Daudi (Burkitt’s lymphoma), MCF-7 (human breastadenocarcinoma), THP-1 (human acute monocytic leukemia),A431 (human epithelial carcinoma), Hep G2 (humanhepatocellular carcinoma), HeLa (human cervix adenocarci-noma), U87 (human glioblastoma), A549 (human lungcarcinoma), SW1990 (human pancreatic carcinoma), F0

Fe(salen)Cl induces apoptosis in tumor cells N. Pradhan et al.

2 Cell Biol Int 9999 (2014) 1–14 © 2014 International Federation for Cell Biology

(mouse myeloma), B16 (mouse melanoma), and EL4 (mouseT cell leukemia) cells were procured from American TypeCulture Collection (ATCC). Cells were grown in T25 cultureflask (Nunc, Denmark) in RPMI (Rosewell Park MemorialInstitute, Invitrogen, USA) or IMDM (Iscove’s ModifiedDulbecco’s Medium, Invitrogen) supplemented with 10%heat inactivated FBS (Fetal Bovine Serum, Lonza, USA), 1%glutamine (HiMedia, India), and 1% penicillin and strepto-mycin (HiMedia). All cells were cultured in a CO2 incubatorat 37�C in air with 5% CO2 and 90% humidity.

Isolation of mouse splenocytes

All the experiments performed for the study were accordingto the Institutional guidelines for animal care and approvedby Institutional Animal Ethics Committee (IAEC) andCPCSEA (Committee for the Purpose of Control andSupervision of Experiments in Animals), Govt. of India.Male C57BL/6 mice (B6) aged 6–8 weeks were procuredfrom the breeding facility of Imgenex India Pvt. Ltd.,Bhubaneswar, India.Mouse splenocytes (from C57BL/6 mouse) were isolated

according to the procedure of Chattopadhyay et al. (2008).In brief, cells were isolated and centrifuged at 1500 rpm for10min and washed with PBS. Splenocytes were kept in 2mLof RBC lysis buffer (Imgenex, USA) for 5min to removeRBCs. After neutralizing and washing with PBS, cells wereresuspended in IMDM supplemented with 10% FBS, 1%glutamine, and 1% penicillin and streptomycin.

Isolation of human PBMC

All experiments for human PBMCs were performedaccording to the guidelines set by the Institutional HumanEthics Committee of National Institute of Science Educationand Research (Bhubaneswar). PBMC from humanblood (hPBMC) was separated following the protocolpublished elsewhere (Chattopadhyay and Chakraborty,2005; Chattopadhyay et al., 2006) and were cultured inIMDM supplemented with 10% FBS, 1% glutamine, and 1%penicillin and streptomycin. Adherent hPBMC weregenerated by using HiSepTM LSM 1077 (HiMedia) densitygradient-cut of human peripheral blood of healthy donor(s).Monocyte/macrophage rich cells were isolated after 1 h ofadherence from hPBMC in 6 well cell culture plates(BD Biosciences, USA). The adherent cells were culturedin IMDM supplemented with 10% FBS supplementedwith L-arginine (0.55mM), L-asparagine (0.24mM), andL-glutamine (1.5 nM) (Invitrogen) with 50U/mL penicillin,and 50mg/mL streptomycin; henceforth described ascomplete medium. The non-adherent and loosely adherentcells were separated by vigorous washing in microbiologi-cally sterile PBS and the adherent cells were kept in culture in

6-well cell culture plates for 1–2 weeks in complete mediumfor downstream experiments.

Cytotoxicity assay

Cytotoxicity of Fe(salen)Cl was measured by Trypan blueexclusion (Patel et al., 2009). For suspension cells, 1� 106

cells/mL in RPMI media, and for adherent cells, 0.25� 106

cells/mL in IMDM media, were seeded in 24-well cultureplates (Nunc). Fe(salen)Cl was dissolved in DMSO (Sigma)and diluted with in culture media before being added totriplicate wells at 0 (control), 0.625, 1.25, 2.5, 5, and 10mM,and in some experiments 20mM. Control wells were treatedwith an equivalent amount of diluted DMSO. After 24 h ofincubation, cells were collected and assayed by the trypanblue exclusion method. IC50 value for the Fe(salen)Clmetallo-complex was determined by counting the percent-age of live and dead cells under an inverted microscope(Olympus, Japan).

Flow cytometry

For a better understanding of the pathway by whichFe(salen)Cl induced cell death in cancer cells, apoptosiswas measured by staining the cells with IANBD [N-((2-(iodoacetoxy) ethyl)-N-Methyl) amino-7-Nitrobenz-2-Oxa-1,3-Diazole] conjugated p-SIVATM (polarity sensitiveindicator of viability and apoptosis, Imgenex, USA). pSIVAis an Annexin XII based, polarity sensitive probe for thespatiotemporal analysis of apoptosis to detect phosphati-dylserine (PS) exposure. PS exposure is a hallmarkphenomenon, occurs early during apoptosis and persiststhroughout the cell death process (Bevers and Williamson,2010; Kim et al., 2010a, 2010b; Yamazaki and Piette, 1990).In brief, cells were washed twice with 1mL cold PBS andwere added with 5mL pSIVA-IANBD to each sample andwere incubated for 10min at room temperature (25�C) inthe dark. After the incubation, the cells were analyzed bya flow cytometer (FACS Calibur, BD Biosciences). Thedata were analyzed with CellQuestProTM software (BDBiosciences).Expression of apoptotic proteins, Apaf-1 (Apoptotic

protease activating factor 1), BAD (Bcl2-associated antago-nist of cell death), active caspases (cleaved caspase-3 andcaspase-9) and the anti-apoptotic protein, Bcl2, weredetected by flow cytometry. Jurkat cells (1� 106 cells/mL)were seeded in a 24-well culture plate (Nunc) with Fe(salen)Cl as described earlier. After 24 h of incubation, thecells were harvested and analyzed by flow cytometry.The cell pellet was fixed with fixation buffer (Imgenex) for30min at room temperature followed by the addition ofpermeabilization buffer (Imgenex). Cells were incubatedseparately with anti-Apaf-1, anti-Bcl2, anti-BAD (cleaved),

N. Pradhan et al. Fe(salen)Cl induces apoptosis in tumor cells


anti-caspase-3 (cleaved), and anti-caspase-9 (cleaved) anti-bodies (Imgenex) appropriately diluted with staining buffer(Imgenex) for 30min, followed by washing twice withpermeabilization buffer and finally resuspended in fluores-cent labeled goat anti-rabbit secondary antibody (Jackson,USA) for 30min in the dark. The cells were washed withstaining buffer, resuspended in 400mL of staining buffer andanalyzed by flow cytometry.

Western blot analyses

Cytochrome c is a trigger of the cascade of reactions leadingto the induction of apoptotic pathway. The enhancedapoptosis in Fe(salen)Cl treated EL4 and Jurkat cellsencouraged us to investigate the expression status ofcytochrome c in the apoptotic cells. Western blot analyseswere done on Fe(salen)Cl treated Jurkat cell lysate. In brief,Jurkat and EL4 cells were cultured in presence (2.5mM) orabsence of Fe(salen)Cl for 24 h. After the incubation, cellswere harvested, washed in PBS, and resuspended in lysisbuffer containing protease inhibitors (Sigma). The cell lysatewas stored in � 80�C until further processing. Lysate proteinswere separated by SDS-PAGE and transferred on PVDFmembrane (Millipore, USA), which had been blocked with5% non-fat milk in PBS. The membrane was probed withanti-cytochrome c (Imgenex) andwasdetectedwith goat anti-rabbit antibody conjugated to peroxidase (Jackson). Super-SignalTMWest Pico Chemiluminescent kit (Pierce, USA) wasused for chemiluminescent signal and the signal was exposedto anX-ray film (Kodak, Japan). GAPDH (Clone IMG13H12,Imgenex) antibody was used as loading control.

Microscopic detection of mitochondrial potential change

Tomonitor the change in mitochondrial oxidative potential,MCF7 cells and human PBMC were treated with Mito-tracker-green-FM dye (MTG, Invitrogen; Zhu et al., 2010).MCF7 cells were maintained in IMDM (Invitrogen)supplemented with 10% fetal bovine serum (Lonza) in thepresence of antibiotics and glutamine (Invitrogen) at 37�Cincubator in air with 5% CO2. Human PBMCs weremaintained as mentioned before. All cells were grown on22mm glass coverslips and loaded with MTG (1mM) for20min before imaging. After loading with MTG, the cellswere washed gently twice with PBS buffer and taken into alive cell imaging chamber where they were imaged for someexperiments as a time series images acquired at regularintervals. In some experiments, Fe(salen)Cl was added(1.25mM and 5.0mM) after taking a few initial images andwas imaged again to monitor the changes in mitochondrialmembrane potential/oxidative potential. Cells loaded withMTG were excited by wavelength set at 490–543 (by usingArgon laser, 1.8% power).

JC-1 cationic dye (Sigma) was used to follow the mito-chondrial potential (c) in Jurkat cells and humanPBMC. Thisdye shows potential-dependent accumulation inside themitochondria and also has fluorescence emission propertiesthat shifts fromgreen (525 nm) to red (590nm) and reflects theapoptotic state of cells (Kimet al., 2010a, 2010b). Briefly, Jurkatcells (1� 106 cells/mL) were grown in 35mm dishes andFe(salen)Cl drug (5mM) was added for 6 h. JC-1 (1mM) dyewas added to the cells for 20min prior to imaging. Cells weremaintained at 37�C in the presence of air plus 5% CO2 in ahumid condition. As Jurkat cells are non-adherent in nature,cells were pelleted at 2000 rpm for 4min and resuspended inIMDM. The cells were placed onto the live cell chamber forimaging with a confocal microscope (LSM780, Zeiss,Germany) using a 63� or 20� objective. All images weretaken under the same conditions for comparative purposes,and were analyzed and processed using LSM Image Examinersoftware(Zeiss).Therelativefluorescence intensity is represen-ted in pseudo-color, where red indicates highest and blueindicates lowest intensity of the respective fluorophores.

Caspase inhibition

zVAD-fmk (Benzyloxycarbonyl-Val-Ala-Asp fluoromethyl-ketone) is a broad spectrum caspase inhibitor that bindswith active caspases and effectively blocks their biologicalactivity (Gregoli and Bondurant, 1999; Slee et al., 1996). Weexamined whether inhibition of caspases rescues the cellsfrom Fe(salen)Cl-induced apoptosis. Jurkat cells wereincubated with Fe(salen)Cl in the absence and presence ofzVAD-fmk (Invitrogen) for 24 h. Cells were harvested andwere stained with IANBD conjugated p-SIVA as describedearlier and were incubated at room temperature for 10min.The samples were analyzed by flow cytometry andCellQuestProTM software (BD Biosciences).

Results

Synthesis and characterization of Fe(salen)Cl

Fe(salen)Cl has been characterized by using variousspectroscopic techniques, like UV-Vis spectroscopy, IRspectroscopy, CHN analysis, and also from single crystalX-ray analysis. Purity and identity of the Fe(salen)Cl isdemonstrated by its satisfactory elemental analyses. Thehexa-coordinated dimeric nature of Fe(salen)Cl had beenestablished by its single crystal X-ray structure. Singlecrystals of Fe(salen)Cl were grown by slow evaporation oftheir acetonitrile solution. The molecule crystallized in thespace group of P21/c and the corresponding cellparameters are a¼ 11.33 A

�, b¼ 6.8738 A

�, c¼ 19.1689 A

�,

and b¼ 91.446�. It matches well with the correspondingdimeric structure of Fe(salen)Cl.



Redox properties of the Fe(salen)Cl

The redox properties of Fe(salen)Cl were studied inacetonitrile solvent by cyclic voltammetric techniques(Figure 1). The reduction processes at the negative side ofSCE were recorded by using a platinum working electrode.The complex exhibited one reversible reductive couple:E�298, V (DEp, mV): � 0.590 (90) versus SCE. The observedresponse was assigned as electron-transfer process involvingthe metal center, FeIII/FeII.

Cytotoxic effects of Fe(salen)Cl on tumor and normal cells

The effectiveness of an ideal anti-cancer candidate shouldselectively target the cancer cells and not the normal cells.Thus the ability of Fe(salen)Cl to induce cell death wasinvestigated in cancer cell lines compared with its effect onnormal cells. Initially Fe(salen)Cl was tested on a series ofcancer cell lines: Jurkat, Molt4 (both are human acute T cellleukemia), Raji, Ramos, Daudi (all human B cell leukemia),MCF7 (human breast carcinoma), THP-1 (human acutemonocytic leukemia), A431 (human epithelial carcinoma),Hep G2 (human hepatocellular carcinoma), HeLa (humancervix adenocarcinoma), U87 (human glioblastoma), A549(human lung carcinoma), SW1990 (human pancreaticcarcinoma), EL4 (mouse T cell lymphoma), F0 (mousemyeloma), and B16 (mouse melanoma) cells. Figure 2 showsthat most cell lines are susceptible to this compound and thecell death is dose-dependent. Amongst the cell lines used,B16 cells were less susceptible than the others.Since both mouse (EL4) and human (Jurkat) T cell

leukemia cells responded significantly to Fe(salen)Cl, wechose EL4 and Jurkat cells and studied to investigate theefficacy of Fe(salen)Cl in inducing cell death. Mouse

splenocytes and normal human peripheral blood mononu-clear cells (PBMCs) were used as primary cells to monitorthe effects of Fe(salen)Cl on normal cells. Cells were culturedin the presence and absence of Fe(salen)Cl and wereincubated at different concentrations of Fe(salen)Cl. After24 h of incubation, live and dead cells were counted byTrypan blue dye exclusion method. Fe(salen)Cl induced celldeath in EL4 in a dose-dependent manner and the LC50 forEL4 was about 1.25mM (Figure 3). Normal mousesplenocytes were largely unaffected by Fe(salen)Cl com-pared to its cancerous counterparts and <10% cells weredead at a dose as high as 10mM, at which >95% EL4 cellswere dead. Similarly, the result showed that Jurkat cells weresusceptible to Fe(salen)Cl even below 1.25mM and>20% ofthe cells were dead. The LC50 of Jurkat cells was ~2.0mM.Interestingly, normal human PBMCs were significantlyinsensitive to Fe(salen)Cl in comparison to Jurkat cells(Figure 3), with only 3–4% cells dead at 10mM, whereas atthis dose,>95% Jurkat cells were found to be dead. Thus, it isevident that Fe(salen)Cl induces cell death specifically incancer cells both in mouse and human, whereas it has littleeffect on normal cells. In addition, a time-kinetic studyreveals cell death only in tumor cells (Figure 3).

Fe(salen)Cl induces apoptosis in tumor cells but not in

normal cells

Induction of ‘programmed cell death’ or apoptosis insusceptible cells is a salient feature of most anti-canceragents. Thus, the effect of Fe(salen)Cl in tumor cells wasinvestigated in detail to find out whether death was due toapoptosis (Figure 4). IANBD conjugated p-SIVAwas used todetect apoptosis induced by Fe(salen)Cl in EL4 and Jurkatcells. The cells were incubated with different doses ofFe(salen)Cl for 24 h and were stained with p-SIVA-IANBD.Both EL4 and Jurkat cells were apoptotic after treatmentwith Fe(salen)Cl in a dose-dependent manner. On the otherhand, mouse splenocyte and human PBMC did not undergoapoptosis when treated with Fe(salen)Cl (Figure 4).

Fe(salen)Cl induces enhanced expression of cytochrome c

in tumor cells

Endogenous levels of cytochrome c were much higher inFe(salen)Cl treated cells compared to untreated cells(Figure 5), suggesting that Fe(salen)Cl induces the expres-sion of cytochrome c level that in turn initiates the apoptoticpathway.

Fe(salen)Cl lowers the mitochondrial oxidative potential

To understand themolecularmechanism of how Fe(salen)Clcauses cell death, especially in cancer cells, we tested whether

Figure 1 Cyclic voltammograms (——) of Fe(salen)Cl in CH3CN/0.1M TEAP at 298 K. The potentials are versus SCE.



Figure 2 Fe(salen)Cl induces cell death in different cancer cell lines. Cells were cultured for 24 h in the absence (control) and presence of differentdoses (as indicated) of Fe(salen)Cl and were subjected to live and dead assay. Most cell lines were found to be susceptible to Fe (salen)Cl. The values aremean� standard error of means. The data are representative of three independent experiments.

Figure 3 Fe(salen)Cl induces cell death in EL4 and Jurkat cells but not in mouse splenocytes and human PBMCs. Dose (upper panels A and B)and time kinetics (lower panels, C and D) of Fe(salen)Cl on cell survival in mouse splenocyte and EL4 cells and in human PBMC and Jurkat cells. Normalmouse splenocytes, human PBMC, EL4 and Jurkat cells were cultured in a 24-well plate in the presence of different concentrations of Fe(salen)Cl asindicated and were cultured for 24 h (A and B). For time kinetics, cells were incubated with 2.5mM of Fe(salen)Cl for 0, 2, 4, 6, 12, 18, and 24 h,respectively (C and D). Fe(salen)Cl efficiently induced cell death in EL4 (A and C) and Jurkat cells (B and D) but did not affect normal mouse splenocytesand normal human PBMCs. The values are mean� standard error of means. The data are representative of three independent experiments.



Figure 4 Fe(salen)Cl induces apoptosis in EL4 and Jurkat cells. Flow cytometric analysis of apoptosis in mouse splenocytes and EL4 (left panles); andhuman PBMC and Jurkat cells (right panels) treated with Fe(salen)Cl. Cells were cultured for 24 h in the presence of different concentrations of Fe(salen)Cl as indicated. Cells were harvested, washed in PBS, andwere stainedwith IANBD-p-SIVA. Data were analyzed by flow cytometry. Numbers in the lowerright quadrants are percentage of p-SIVA positive cells and show that with the increasing dose of Fe(salen)Cl the number of p-SIVA positive cellsincreases. Data are representative of three independent experiments.



it has any effect on the mitochondrial properties, especiallyon mitochondrial membrane potential. For that purpose wedid two independent experiments using Jurkat cells andMCF-7 as the cancerous cells and human PMBC as non-cancerous cells.To confirm that Fe(salen)Cl alters mitochondrial proper-

ties, we measured mitochondrial potential of Jurkat cells byusing fluorescent dye, 5,50,6,60-tetrachloro- 1,10,3,30-tetra-ethylbenzimidazolcarbocyanine iodide (JC-1), which isreliable and very sensitive. The green fluorescence (at525 nm, i.e., low mitochondrial membrane potential)indicates the uptake of JC1 monomer and the redfluorescence (590 nm) indicates the presence of highJ-aggregates (higher mitochondrial membrane potential)of JC-1 (Pfeiffer et al., 1933). Under control conditions, themajority of cells had high fluorescence in the 590 nm region,while Fe(salen)Cl-treated cells had much less signal in thisregion. In contrast, emission at 525 nm was higher inFe(salen)-treated cells. These fluorescence results suggestthat the mitochondrial membrane potential is significantlylow in Fe(salen)Cl-treated Jurkat cells with respect tountreated control cells (Figure 6a). Some of the Fe(salen)Cl-treated cells even showed both red and green fluorescence,indicating the co-existence of mitochondria at both high andlow potential, a status which indicates an early phase of celldeath. In experiments with human PBMC, similar changesinmitochondrial membrane potential in Fe(salen)Cl-treatedcells were not observed, being similar to the untreatedcontrols (Figure 6b). The results strongly indicate thatFe(salen)Cl-treatment reduces the mitochondrial potential-ity in cancerous cells, but not in non-cancerous cells.The MCF7 is also susceptible to Fe(salen)Cl and under-

goes apoptosis. The adherent nature of MCF-7 allowed us touse live cell fluorescence microscopy and measure changesin the mitochondrial oxidative potential. In MCF-7 cellstreated with Fe(salen)Cl (1.25mM) for 24 h and then labeledwithMitotracker Green FM dye (MTG), the treated cells hadvery low fluorescence compared with untreated cells(Figure 7), which indicates that the oxidative potential ofMCF-7 cells decreases under Fe(salen)Cl.To see whether the Fe(salen)Cl-mediated drop in

mitochondrial potential takes longer or occurs instantly,we loaded MCF-7 cells with MTG dye and took live cell

images. Fe(salen)Cl at 1.25mM caused an immediate(<5min) sharp drop in fluorescence intensity (Figure 7). Incontrast, humanPBMCshowednodrop inMTGfluorescenceeven after a long time. For example, 30min after addition of5mMFe(salen)Cl there was no decrease in fluorescence in themajority of the PBMC cells. These results strongly suggest thatFe(salen)Cl affects mitochondrial oxidative potential specifi-cally in cancer cells and not normal cells.

Fe(salen)Cl upregulates Apaf-1, BAD and downregulates

Bcl2 in tumor cells

The effects of Fe(salen)Cl on Apoptotic protease activatingfactor-1 (Apaf-1) were explored since this factor mediatescytochrome c-dependent activation of caspase-9, triggeringthe activation of caspase-3 and apoptosis (Saleh et al., 1999;Zhan et al., 1999; Ogawa et al., 2003). Activated caspase-3 inturn activates BAD (Bcl2-associated agonist of cell death, aBcl2 family of protein) which is a potent inducer of apoptosis(Condorelli et al., 2001). Flow cytometry analysis showed anincreased expression of Apaf-1 correlating with enhancedactivated BAD (cleaved BAD) in Fe(salen)Cl-treated Jurkatcell. This was accompanied by a concomitant decrease in thelevel of Bcl2 protein (Figure 8), thus consolidating theinvolvement of the mitochondrial pathway of apoptosis inFe(salen)Cl treated tumor cells.

Fe(salen)Cl induces expression of activated caspase-9 and

caspase-3

Apaf-1 binds to procaspase-9 to activate the later byproteolytic cleavage. This in turn recruits procaspase-3 toactivate it, leading to triggering of apoptosis. We thereforeinvestigated the levels of cleaved caspase-9 and cleavedcaspase-3 by flow cytometry. The data suggest that bothcaspase-9 and caspase-3 were activated and cleaved, i.e.upregulated, after Fe(salen)Cl treatment (Figure 9).

zVAD-fmk rescues the Fe(salen)Cl-treated cells from

apoptosis

We also inhibited caspase activities with a pan caspaseinhibitor, zVAD-fmk, which binds irreversibly to the activesite of activated caspases (Slee et al., 1996; Zhan et al., 1999).

Figure 5 Fe(salen)Cl upregulates cytochrome c expression in EL4 and Jurkat cells but not inmouse splenocytes andhumanPBMCs. Cells werecultured for 24 h in presence of Fe(salen)Cl (2.5mM) and the cell lysates were subjected toWestern blot analysis using anti-cytochrome c antibody (lowerpanel). Anti-GAPDH antibody was used as loading control (upper panel).



Figure 6 Fe(salen)Cl lowers mitochondrial potential in cancerous cells. Shown are the confocal images of Jurkat (a) and human PBMC (b) cellslabeledwith JC-1 dye. Two different excitation and emission regions (Ex 488/Em 535 and Ex 535/Em 590) were used to image cell population. a. Majorityof the Jurkat cells reveal red fluorescence in Ex 535/Em 590 region in control conditions (upper panel) while most of the cells reveal decreased redfluorescence after 6 hour of Fe(salen)Cl (5 mM) treatment (lower panel). B. Human PBMC cells do not reveal significant changes in red fluorescence afterthe treatment of Fe(salen)Cl (5 mM) (lower panel) for 6 hour in contrast to control (upper panel). Scale bar 20 mM.

Figure 7 Fe(salen)Cl causes quick drop in mitochondrial oxidative potential specifically in cancer cells. Shown are the live confocal images ofMCF-7 cells in (upper panel) or human PBMC (lower panel) loaded with Mitotracker green dye and treated with Fe(salen)Cl. All MCF-7 cells revealimmediate drop of fluorescence of Mitotracker green dye upon application of Fe(salen)Cl (1.25 mM). In contrast majority of the Human PBMCs retainfluorescence ofMitotracker green dye for a long time even after addition of Fe(salen)Cl at a higher dose (5mM). The fluorescence intensity ofMitotrackergreen dye is represented in a pseudo color (Red indicates highest and blue indicates lowest fluorescence intensity). Scale bar 50 mm.



Jurkat cells were incubated with Fe(salen)Cl in the absenceor presence of zVAD-fmk before flow cytometry analysis tomeasure apoptosis. Apoptosis was analyzed by a moresensitive method, namely staining the cells with IANBDconjugated p-SIVA which binds only with apoptotic cells(Kim et al., 2010a; 2010b). Figure 10 shows that Jurkat cellsundergo apoptosis when incubated with Fe(salen)Cl alone,and that zVAD-fmk rescued the cells from Fe(salen)Cl-induced apoptosis, confirming that Fe(salen)Cl indeedinduces apoptosis in tumor cells through the caspase-dependent pathway.

Discussion

One of the major limitations in modern cancer treatment iseffective and economic chemotherapy (Denny, 1988). Themajority of the drugs used for the treatment of cancer todayare cytotoxic in nature and interfere with essential cellularprocesses and often cause DNA damage (Bakshi et al., 2010).In cancer therapy, the challenge is to design new drugs withextreme selectivity for cancer cells but with few side effects.

With the advancement in the field of inorganic chemistry,the role of transition metal complexes as therapeuticcompounds is becoming increasingly important and thesecomplexes may provide a realistic approach for cancertreatment (Lum et al., 2010). These metal complexes offer agreat diversity in their action; as these compounds not onlyhave anti-cancer properties but also have anti-inflammatory(Chohan et al., 2002), anti-infective (Ahmed et al., 2010),and anti-diabetic (Yasumatsu et al., 2007) effects.Metallo-salen compounds may have the potential to

induce cell death in cancer cells. Mn(salen) (Ansari et al.,2009) and Ru(salen) (Khan et al., 2009) kill the cells bydamaging DNA. Our results show that Fe(salen)Cl is indeedable to induce cell death in a dose-dependentmanner in bothmouse and human T cell leukemia cell lines (EL4 and Jurkat,respectively). The LC50 of Fe(salen)Cl in EL4 cells wasaround 1.25mM, whereas in Jurkat cells it was around 2mM.At a dose of 10mM of Fe(salen)Cl, the cell death percentagereached above 95% in both EL4 and Jurkat cells. Notably,Fe(salen)Cl merely affects the cell viability in normal mousesplenocytes and human PBMC primary cultures, even at a

Figure 8 Fe(salen)Cl upregulates expression ofApaf-1 (A) and BAD (B) anddownregulates Bcl2 (C) in Jurkat cells. Jurkat cells were cultured for24 h in presence of different concentrations of Fe(salen)Cl as indicated andwere subjected to flow cytometric analysis. Apaf-1 (A) and BAD (B) expressionin Jurkat cells were upregulated when cells were incubated with Fe(salen)Cl at 1.25 mM and 2.5 mM. Bcl2 (C) expression was downregulated in a dosedependent manner with the increasing doses of Fe(salen)Cl. Data were representative of three independent experiments.



dose of 10mM. These comparative results strongly justifyFe(salen)Cl as an ideal anti-tumor drug which selectivelytargets the tumor cells, but not the normal cells.Although the Fe(salen)Cl complexes induce cell death in

vitro, it is not well-defined how this compound works. Wehave now shown that Fe(salen)Cl induced programmed celldeath in EL4 and Jurkat cells, making it a strong candidate asan anti-cancer drug. Ansari et al. (2011) reported thatFe(salen)Cl compound is cytotoxic to breast (MCF7) andcolon (CCL228) cancer cell lines. Notably, MCF10, a non-malignant breast epithelial cell line, had shown greaterresistance to Fe(salen)Cl than MCF7 (Ansari et al., 2009).The pro-apoptotic nature of Fe(salen)Cl and its selectivity

against cancer cells led us to investigate the apoptoticpathway in details. Studies on this metallo drug demonstratethat it triggers the intrinsic apoptotic pathway by permeabi-

lization of the outer mitochondrial membrane and thus maycause release of proteins from the mitochondrial intermem-brane space (Woldemariam and Mandal, 2008). Amongthese released factors, cytochrome c is important as it caninitiate caspase activation. The enhanced level of cyto-chrome c in Fe(salen)Cl treated EL4 and Jurkat cells matcheswell with the typical hallmark of apoptosis and thus suggestsactivation of intrinsic apoptotic pathway.Different iron-compounds, such as iron core-gold shell

nano-particles and iron-chelators, can be effectively used fortreating cancer as they selectively target cancer cells (Wuet al., 2011; Yu et al., 2012). These recent reports, in general,indicate that iron compounds can induce apoptosisspecifically in cancer cells, a phenomenon termed “Ferrop-tosis” (Dixon et al., 2012; Reed and Pellecchia, 2012). Weconclude that Fe(salen)Cl causes selective death of cancerous

Figure 10 zVAD rescues Jurkat cells from Fe(salen)-inducedapoptosis. Jurkat cells were cultured in a 24-well plate in absence orin presence of zVAD along with different concentration of Fe(salen)Cl asindicated and were cultured for 24 h. Cells were harvested and werestained with p-SIVA-IANBD for flow cytometric analysis of apoptosis. Thevalues indicate the percentage of apoptotic cells. The data arerepresentative of three independent experiments.

Figure 9 Fe(salen)Cl activates caspase 9 and caspase 3 in Jurkatcells. Jurkat cells were cultured in presence of indicated doses ofFe(salen) in 24-well culture plates for 24 h. Cells were harvested andweresubjected to flow cytometric analysis for cleaved caspase 9 and cleavedcaspase 3. Expression of activated form of caspase 9 was induced uponincubation with Fe(salen)Cl in a dose dependent manner and that ofcaspase 3 was induced at higher two doses, i.e., 1.25 mM and 2.5 mMconcentration. The data are representative of three independentexperiments.



cells, though the exact molecular mechanism by which itinduces apoptosis is still not entirely clear. However,involvement of iron signaling in the regulation ofmitochondrial function and dynamics by altering oxidativestress is possible (Zhu et al., 2010; Huang et al., 2011; Myerset al., 2011; Wang and Pantopoulos, 2011). Our results alsoagree with the other previously established observations, andthat Fe(salen)Cl causes a significant loss of mitochondrialmembrane potential in Jurkat cells, but not in humanPBMCs. A collapse in the mitochondrial membranepotential caused by Fe(salen)Cl might lead to mitochondrialdamage and an efflux of mitochondrial proteins, includingcytochrome c, which in turn results in the activation ofcascade of reactions leading to cell death.A decline in the mitochondrial membrane potential

concomitant with the increase in the level of cytochrome copens up a series of novel questions that need to be answered.We have investigated the expression of different pro- andanti-apoptotic molecules upon Fe(salen)Cl treatment, andhave shown that Fe(salen)Cl downregulates the anti-apopto-tic protein Bcl2 expression while at the same time inducingthe expression of pro-apoptotic factors, BAD and Apaf-1.These changes can be the consequences of mitochondrialmembrane disintegration and/or release of cytochrome c. Thecaspases can be sparked off by mitochondrial membranedamage caused here by Fe(salen)Cl as our results showinvolvement of caspase-3 and caspase-9.Therefore, it is evident that the Fe(salen) metallo

compound indeed has cytotoxic effects in cancer cells viainduction of apoptosis and our data suggested that the salencompound may induce apoptosis via disruption of mito-chondrial membrane potential, preceding the efflux ofcytochrome c into the cytoplasm. The cytochrome c binds toApaf-1 in the presence of dATP forming heptamericapoptosome complex. The apoptosome induces the activa-tion of caspase-9. Activated caspase-9 further mediates theactivation of effector caspases, such as procaspase-3(Guerrero et al., 2008; Yuan et al., 2011). These datastrongly indicated that caspase-mediated apoptotic pathwaymight be involved that led to the induction of apoptosis. Toconfirm this hypothesis, the caspase activity was blocked byzVAD in an attempt to rescue the cells from undergoingapoptosis in the presence of Fe(salen)Cl. Interestingly, Jurkatcells indeed remained unaffected to Fe(salen)Cl whencaspase pathway was inhibited. This finding confirmedthat Fe(salen)Cl-induced apoptosis was caspase dependent.In conclusion, we have demonstrated that treatment of

EL4 and Jurkat cells with Fe(salen)Cl is capable of inducingapoptosis via a cytochrome c-mediated caspase-dependentpathway suggesting that this compound is effective inarresting tumor cell growth in vitro. We also demonstratethat Fe(salen)Cl specifically targets the tumor cells and notthe normal cells. The concept proposed here not only

elucidates the possiblemechanism of action of Fe(salen)Cl ininducing apoptosis specifically in cancer cell but alsoprovides us with the information for consideration ofFe(salen)Cl as a next generation anti-cancer agent whichoffer almost no adverse effect on normal cells.

Acknowledgements and funding

Financial support received from the Department of AtomicEnergy, (India) is gratefully acknowledged. The work hasbeen supported by DBT (Grant No BT/PR13684/BRB/10/787/2010 to S.K., P.K.M. and S.C.), India.We also extend ourthanks to Lisa Stein, Imgenex Corporation, USA fortechnical support.

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Received 4 October 2013; accepted 14 April 2014.



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