Cell Biology International ISSN 1065-6995 doi: 10.1002/cbin.10308
RESEARCH ARTICLE
Induction of apoptosis by Fe(salen)Cl through caspase-dependent pathway specifically in tumor cells Nitika 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 2 3 4
Imgenex India, Infocity, Bhubaneswar, India School of Biological Sciences, National Institute of Science Education & Research, Bhubaneswar, India School of Chemical Sciences, National Institute of Science Education & Research, Bhubaneswar, India 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 their specificity towards cancer cells and the mechanism of action are hitherto less investigated. A Fe(salen)Cl derivative has been synthesized that remains active in monomer form. The efficacy of this compound as an anti-tumor agent has been investigated in 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 undergo apoptosis, but normal mouse spleen cells and human peripheral blood mononuclear cells (PBMC) remain largely unaffected by Fe(salen)Cl. Fe(salen)Cl treated tumor cells show significantly higher expression level of cytochrome c that might have triggered the cascade of reactions leading to apoptosis in cancer cells. A significant loss of mitochondrial membrane potential upon Fe(salen)Cl treatment suggests that Fe(salen)Cl induces apoptosis by disrupting mitochondrial membrane potential and homeostasis, leading to cytotoxity. We also established that apoptosis in the Fe(salen)Cl-treated tumor cells is mediated through caspase-dependent pathway. This is the first report demonstrating that Fe(salen)Cl can specifically target the tumor cells, leaving the primary cells least affected, indicating an excellent potential for this compound to emerge as a next-generation anti-tumor drug. Keywords: apoptosis; caspase; Fe(salen)Cl; leukemia cell line; mitochondria; tumor
Introduction Amongst the chemotherapeutic drugs for cancer, cisplatin is mostly being used, although it has serious side effects (Rafique et al., 2010). There is a need for suitable drugs that specifically target cancer cells while leaving normal cells unaffected or less affected. In this respect, transition metal complexes are of considerable research interest because of their structural diversity (Teyssot et al., 2009; To et al., 2009; Liu et al., 2011; Liu and Sadler, 2011). Amongst the metallodrugs, iron-based compounds have recently been explored for their anticancer properties in spite of having some side effects (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 ironbased nano-particles (Wu et al., 2011) have cytotoxic effects in vitro in cancer cells; but their mechanism of action remains largely undefined. FeIII(porphyrin) complexes are widely used to mimic the role of the cytochrome P-450 class of enzymes that are responsible for the oxidation of various organic substances (Meunier, 1992). In this context, we have synthesized Fe(salen)Cl complexes to mimic the activities of FeIII(porphyrin) complexes. A structural similarity exists between FeIII(porphyrin) and Fe(salen)Cl complexes where the iron remains in FeIII-state (Böttcher et al., 1996; Liou and Wang, 2000). Mandal and colleagues (Mandal et al., 1997) have synthesized a series of
Corresponding authors: e-mail: [email protected], [email protected], [email protected]
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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 normal primary cells in an effort to study its potential as an anticancer drug. From single crystal X-ray structural analysis, it is found that Fe(salen)Cl complexes could exist as monomeric or dimeric forms depending on the solvent used for crystallization. The single crystal data obtained from CH3CN medium suggest that the compound is indeed dimeric in nature in solid phase. The solution phase electrochemical studies indicate that the compound is monomer in solution. The interaction of Fe(salen)Cl with living 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 death in breast cancer cell line in vitro, suggesting that salen compounds may have anti-tumor properties, although the mechanism by which they induce cell death is unclear. Oxidative stress exerted by redox active metals like iron may be responsible for DNA/RNA damage in vitro, as has been suggested (Bhattacharya and Mandal, 1996; Czlapinski and Sheppard, 2001; Shao et al., 2006). We show here that Fe(salen)Cl specifically kills the cancerous cells but spares primary normal cells. In cancer cells it causes significant loss of mitochondrial membrane potential with a concomitant increase in cytochrome c level, which in turn induces apoptosis in cancer cells through a caspase-dependent pathway.
Fe(salen)Cl induces apoptosis in tumor cells
calomel reference electrode (SCE) were used in a 3-electrode configuration. Tetraethyl ammonium perchlorate (TEAP) was the supporting electrolyte (0.1 M) at a concentration of 103 M with respect to the complex. The half wave potential E 298 was set equal to 0.5(Epa þ Epc), where Epa and Epc are anodic and cathodic cyclic voltammetric peak potentials, respectively. The scan rate used was 100 mV s1.
Synthesis of salen ligand Salen ligand (H2L) was synthesized by following the procedure of Pfeiffer et al. (1933). A 2:1 molar ratio of salicylaldehyde and ethylenediamine were mixed together in 20 mL absolute methanol. The resulting mixture was kept for 30 min at 4 C. The precipitate was filtered and washed thoroughly with ice-cold methanol followed by ice-cold diethyl ether. The final product was recrystallized from CH3CN.
Synthesis of Fe(salen)Cl Fe(salen)Cl has been synthesized by the procedure of Matsushita et al. (1982). A 1:1 molar ratio of ferric chloride and salen ligand (H2L) were taken in 25 mL absolute CH3OH. The resulting mixture was heated to reflux under a dinitrogen atmosphere for 2.5 h. The concentrated solution was refrigerated overnight. The precipitate was filtered and washed thoroughly with ice-cold water followed by ice-cold diethyl ether. The product was recrystallized from acetone.
Materials and methods
Determination of crystal structure
General methods
Single crystals of complex were grown by slow evaporation of a solution of the complex in acetonitrile under atmospheric conditions. The crystal data of Fe(salen)Cl were collected on a Bruker Kappa APEX II CCD diffractometer at 293 K, which were corrected for Lorentz polarization and absorption effects. The program package SHELX-97 (ShelxTL) (Sheldrick, 1997) was used for structure solution and full matrix least squares refinement on F2. Hydrogen atoms were included in the refinement using the riding model. Contributions of H atoms for the water molecules were included but were not fixed.
The precursor compound, ferric chloride was purchased from Merck (Germany). The ligand, 2,20 -((1E,10 E)-(ethane1,2 diylbis (azanylylidene)) bis(methanylylidene)) diphenol (H2L), was prepared according to Pfeiffer et al. (1933). All other chemicals and solvents were of reagent grades. For spectroscopy and electrochemical studies, HPLC grade solvents were used. Commercial tetraethyl ammonium bromide was converted to pure tetraethyl ammonium perchlorate (TEAP) following the procedure of Sawyer et al. (1995).
Cell culture Physical measurements UV-Vis spectral work used a Perkin-Elmer LAMBDA-750 spectrophotometer. FT-IR spectra were taken with samples prepared as KBr pellets. Cyclic voltammetry measurements were carried out using a CH instrument model CHI1120A electrochemistry system. A glassy-carbon working electrode, a platinum wire as an auxiliary electrode, and a saturated
Jurkat, Molt-4 (human acute lymphoblastic leukemia), Raji, Ramos, Daudi (Burkitt’s lymphoma), MCF-7 (human breast adenocarcinoma), THP-1 (human acute monocytic leukemia), A431 (human epithelial carcinoma), Hep G2 (human hepatocellular carcinoma), HeLa (human cervix adenocarcinoma), U87 (human glioblastoma), A549 (human lung carcinoma), SW1990 (human pancreatic carcinoma), F0
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(mouse myeloma), B16 (mouse melanoma), and EL4 (mouse T cell leukemia) cells were procured from American Type Culture Collection (ATCC). Cells were grown in T25 culture flask (Nunc, Denmark) in RPMI (Rosewell Park Memorial Institute, Invitrogen, USA) or IMDM (Iscove’s Modified Dulbecco’s Medium, Invitrogen) supplemented with 10% heat inactivated FBS (Fetal Bovine Serum, Lonza, USA), 1% glutamine (HiMedia, India), and 1% penicillin and streptomycin (HiMedia). All cells were cultured in a CO2 incubator at 37 C in air with 5% CO2 and 90% humidity.
Isolation of mouse splenocytes All the experiments performed for the study were according to the Institutional guidelines for animal care and approved by Institutional Animal Ethics Committee (IAEC) and CPCSEA (Committee for the Purpose of Control and Supervision of Experiments in Animals), Govt. of India. Male C57BL/6 mice (B6) aged 6–8 weeks were procured from 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 for 10 min and washed with PBS. Splenocytes were kept in 2 mL of RBC lysis buffer (Imgenex, USA) for 5 min to remove RBCs. After neutralizing and washing with PBS, cells were resuspended in IMDM supplemented with 10% FBS, 1% glutamine, and 1% penicillin and streptomycin.
Isolation of human PBMC All experiments for human PBMCs were performed according to the guidelines set by the Institutional Human Ethics Committee of National Institute of Science Education and Research (Bhubaneswar). PBMC from human blood (hPBMC) was separated following the protocol published elsewhere (Chattopadhyay and Chakraborty, 2005; Chattopadhyay et al., 2006) and were cultured in IMDM supplemented with 10% FBS, 1% glutamine, and 1% penicillin and streptomycin. Adherent hPBMC were generated by using HiSepTM LSM 1077 (HiMedia) density gradient-cut of human peripheral blood of healthy donor(s). Monocyte/macrophage rich cells were isolated after 1 h of adherence from hPBMC in 6 well cell culture plates (BD Biosciences, USA). The adherent cells were cultured in IMDM supplemented with 10% FBS supplemented with L-arginine (0.55 mM), L-asparagine (0.24 mM), and L-glutamine (1.5 nM) (Invitrogen) with 50 U/mL penicillin, and 50 mg/mL streptomycin; henceforth described as complete medium. The non-adherent and loosely adherent cells were separated by vigorous washing in microbiologically sterile PBS and the adherent cells were kept in culture in 1120
6-well cell culture plates for 1–2 weeks in complete medium for downstream experiments.
Cytotoxicity assay Cytotoxicity of Fe(salen)Cl was measured by Trypan blue exclusion (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 culture plates (Nunc). Fe(salen)Cl was dissolved in DMSO (Sigma) and diluted with in culture media before being added to triplicate wells at 0 (control), 0.625, 1.25, 2.5, 5, and 10 mM, and in some experiments 20 mM. Control wells were treated with an equivalent amount of diluted DMSO. After 24 h of incubation, cells were collected and assayed by the trypan blue exclusion method. IC50 value for the Fe(salen)Cl metallo-complex was determined by counting the percentage of live and dead cells under an inverted microscope (Olympus, Japan).
Flow cytometry For a better understanding of the pathway by which Fe(salen)Cl induced cell death in cancer cells, apoptosis was measured by staining the cells with IANBD [N-((2(iodoacetoxy) ethyl)-N-Methyl) amino-7-Nitrobenz-2-Oxa1,3-Diazole] conjugated p-SIVATM (polarity sensitive indicator of viability and apoptosis, Imgenex, USA). pSIVA is an Annexin XII based, polarity sensitive probe for the spatiotemporal analysis of apoptosis to detect phosphatidylserine (PS) exposure. PS exposure is a hallmark phenomenon, occurs early during apoptosis and persists throughout the cell death process (Bevers and Williamson, 2010; Kim et al., 2010a, 2010b; Yamazaki and Piette, 1990). In brief, cells were washed twice with 1 mL cold PBS and were added with 5 mL pSIVA-IANBD to each sample and were incubated for 10 min at room temperature (25 C) in the dark. After the incubation, the cells were analyzed by a flow cytometer (FACS Calibur, BD Biosciences). The data were analyzed with CellQuestProTM software (BD Biosciences). Expression of apoptotic proteins, Apaf-1 (Apoptotic protease activating factor 1), BAD (Bcl2-associated antagonist of cell death), active caspases (cleaved caspase-3 and caspase-9) and the anti-apoptotic protein, Bcl2, were detected 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, the cells were harvested and analyzed by flow cytometry. The cell pellet was fixed with fixation buffer (Imgenex) for 30 min at room temperature followed by the addition of permeabilization buffer (Imgenex). Cells were incubated separately with anti-Apaf-1, anti-Bcl2, anti-BAD (cleaved),
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anti-caspase-3 (cleaved), and anti-caspase-9 (cleaved) antibodies (Imgenex) appropriately diluted with staining buffer (Imgenex) for 30 min, followed by washing twice with permeabilization buffer and finally resuspended in fluorescent labeled goat anti-rabbit secondary antibody (Jackson, USA) for 30 min in the dark. The cells were washed with staining buffer, resuspended in 400 mL of staining buffer and analyzed by flow cytometry.
Western blot analyses Cytochrome c is a trigger of the cascade of reactions leading to the induction of apoptotic pathway. The enhanced apoptosis in Fe(salen)Cl treated EL4 and Jurkat cells encouraged us to investigate the expression status of cytochrome c in the apoptotic cells. Western blot analyses were done on Fe(salen)Cl treated Jurkat cell lysate. In brief, Jurkat and EL4 cells were cultured in presence (2.5 mM) or absence of Fe(salen)Cl for 24 h. After the incubation, cells were harvested, washed in PBS, and resuspended in lysis buffer containing protease inhibitors (Sigma). The cell lysate was stored in 80 C until further processing. Lysate proteins were separated by SDS-PAGE and transferred on PVDF membrane (Millipore, USA), which had been blocked with 5% non-fat milk in PBS. The membrane was probed with anti-cytochrome c (Imgenex) and was detected with goat antirabbit antibody conjugated to peroxidase (Jackson). SuperSignalTM West Pico Chemiluminescent kit (Pierce, USA) was used for chemiluminescent signal and the signal was exposed to an X-ray film (Kodak, Japan). GAPDH (Clone IMG13H12, Imgenex) antibody was used as loading control.
Microscopic detection of mitochondrial potential change To monitor the change in mitochondrial oxidative potential, MCF7 cells and human PBMC were treated with Mitotracker-green-FM dye (MTG, Invitrogen; Zhu et al., 2010). MCF7 cells were maintained in IMDM (Invitrogen) supplemented with 10% fetal bovine serum (Lonza) in the presence of antibiotics and glutamine (Invitrogen) at 37 C incubator in air with 5% CO2. Human PBMCs were maintained as mentioned before. All cells were grown on 22 mm glass coverslips and loaded with MTG (1 mM) for 20 min before imaging. After loading with MTG, the cells were washed gently twice with PBS buffer and taken into a live cell imaging chamber where they were imaged for some experiments as a time series images acquired at regular intervals. In some experiments, Fe(salen)Cl was added (1.25 mM and 5.0 mM) after taking a few initial images and was imaged again to monitor the changes in mitochondrial membrane potential/oxidative potential. Cells loaded with MTG were excited by wavelength set at 490–543 (by using Argon laser, 1.8% power).
Fe(salen)Cl induces apoptosis in tumor cells
JC-1 cationic dye (Sigma) was used to follow the mitochondrial potential (c) in Jurkat cells and human PBMC. This dye shows potential-dependent accumulation inside the mitochondria and also has fluorescence emission properties that shifts from green (525 nm) to red (590 nm) and reflects the apoptotic state of cells (Kim et al., 2010a, 2010b). Briefly, Jurkat cells (1 106 cells/mL) were grown in 35 mm dishes and Fe(salen)Cl drug (5 mM) was added for 6 h. JC-1 (1 mM) dye was added to the cells for 20 min prior to imaging. Cells were maintained at 37 C in the presence of air plus 5% CO2 in a humid condition. As Jurkat cells are non-adherent in nature, cells were pelleted at 2000 rpm for 4 min and resuspended in IMDM. The cells were placed onto the live cell chamber for imaging with a confocal microscope (LSM780, Zeiss, Germany) using a 63 or 20 objective. All images were taken under the same conditions for comparative purposes, and were analyzed and processed using LSM Image Examiner software (Zeiss). The relative fluorescence intensity is represented in pseudo-color, where red indicates highest and blue indicates lowest intensity of the respective fluorophores.
Caspase inhibition zVAD-fmk (Benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone) is a broad spectrum caspase inhibitor that binds with active caspases and effectively blocks their biological activity (Gregoli and Bondurant, 1999; Slee et al., 1996). We examined whether inhibition of caspases rescues the cells from Fe(salen)Cl-induced apoptosis. Jurkat cells were incubated with Fe(salen)Cl in the absence and presence of zVAD-fmk (Invitrogen) for 24 h. Cells were harvested and were stained with IANBD conjugated p-SIVA as described earlier and were incubated at room temperature for 10 min. The samples were analyzed by flow cytometry and CellQuestProTM software (BD Biosciences). Results
Synthesis and characterization of Fe(salen)Cl Fe(salen)Cl has been characterized by using various spectroscopic techniques, like UV-Vis spectroscopy, IR spectroscopy, CHN analysis, and also from single crystal X-ray analysis. Purity and identity of the Fe(salen)Cl is demonstrated by its satisfactory elemental analyses. The hexa-coordinated dimeric nature of Fe(salen)Cl had been established by its single crystal X-ray structure. Single crystals of Fe(salen)Cl were grown by slow evaporation of their acetonitrile solution. The molecule crystallized in the space group of P21/c and the corresponding cell parameters are a ¼ 11.33 A, b ¼ 6.8738 A, c ¼ 19.1689 A, and b ¼ 91.446 . It matches well with the corresponding dimeric structure of Fe(salen)Cl.
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Redox properties of the Fe(salen)Cl The redox properties of Fe(salen)Cl were studied in acetonitrile solvent by cyclic voltammetric techniques (Figure 1). The reduction processes at the negative side of SCE 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 observed response was assigned as electron-transfer process involving the metal center, FeIII/FeII.
Cytotoxic effects of Fe(salen)Cl on tumor and normal cells The effectiveness of an ideal anti-cancer candidate should selectively target the cancer cells and not the normal cells. Thus the ability of Fe(salen)Cl to induce cell death was investigated in cancer cell lines compared with its effect on normal cells. Initially Fe(salen)Cl was tested on a series of cancer cell lines: Jurkat, Molt4 (both are human acute T cell leukemia), Raji, Ramos, Daudi (all human B cell leukemia), MCF7 (human breast carcinoma), THP-1 (human acute monocytic leukemia), A431 (human epithelial carcinoma), Hep G2 (human hepatocellular carcinoma), HeLa (human cervix adenocarcinoma), U87 (human glioblastoma), A549 (human lung carcinoma), SW1990 (human pancreatic carcinoma), EL4 (mouse T cell lymphoma), F0 (mouse myeloma), and B16 (mouse melanoma) cells. Figure 2 shows that most cell lines are susceptible to this compound and the cell 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, we chose EL4 and Jurkat cells and studied to investigate the efficacy of Fe(salen)Cl in inducing cell death. Mouse
splenocytes and normal human peripheral blood mononuclear cells (PBMCs) were used as primary cells to monitor the effects of Fe(salen)Cl on normal cells. Cells were cultured in the presence and absence of Fe(salen)Cl and were incubated at different concentrations of Fe(salen)Cl. After 24 h of incubation, live and dead cells were counted by Trypan blue dye exclusion method. Fe(salen)Cl induced cell death in EL4 in a dose-dependent manner and the LC50 for EL4 was about 1.25 mM (Figure 3). Normal mouse splenocytes were largely unaffected by Fe(salen)Cl compared to its cancerous counterparts and 95% EL4 cells were dead. Similarly, the result showed that Jurkat cells were susceptible to Fe(salen)Cl even below 1.25 mM and >20% of the cells were dead. The LC50 of Jurkat cells was ~2.0 mM. Interestingly, normal human PBMCs were significantly insensitive to Fe(salen)Cl in comparison to Jurkat cells (Figure 3), with only 3–4% cells dead at 10 mM, whereas at this dose, >95% Jurkat cells were found to be dead. Thus, it is evident that Fe(salen)Cl induces cell death specifically in cancer cells both in mouse and human, whereas it has little effect on normal cells. In addition, a time-kinetic study reveals 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 in susceptible cells is a salient feature of most anti-cancer agents. Thus, the effect of Fe(salen)Cl in tumor cells was investigated in detail to find out whether death was due to apoptosis (Figure 4). IANBD conjugated p-SIVA was used to detect apoptosis induced by Fe(salen)Cl in EL4 and Jurkat cells. The cells were incubated with different doses of Fe(salen)Cl for 24 h and were stained with p-SIVA-IANBD. Both EL4 and Jurkat cells were apoptotic after treatment with Fe(salen)Cl in a dose-dependent manner. On the other hand, mouse splenocyte and human PBMC did not undergo apoptosis 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 in Fe(salen)Cl treated cells compared to untreated cells (Figure 5), suggesting that Fe(salen)Cl induces the expression of cytochrome c level that in turn initiates the apoptotic pathway.
Fe(salen)Cl lowers the mitochondrial oxidative potential Figure 1 Cyclic voltammograms (——) of Fe(salen)Cl in CH3CN/0.1 M TEAP at 298 K. The potentials are versus SCE.
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To understand the molecular mechanism of how Fe(salen)Cl causes cell death, especially in cancer cells, we tested whether
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Fe(salen)Cl induces apoptosis in tumor cells
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 different doses (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 are mean 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. Normal mouse splenocytes, human PBMC, EL4 and Jurkat cells were cultured in a 24-well plate in the presence of different concentrations of Fe(salen)Cl as indicated and were cultured for 24 h (A and B). For time kinetics, cells were incubated with 2.5 mM 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 splenocytes and normal human PBMCs. The values are mean standard error of means. The data are representative of three independent experiments.
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Figure 4 Fe(salen)Cl induces apoptosis in EL4 and Jurkat cells. Flow cytometric analysis of apoptosis in mouse splenocytes and EL4 (left panles); and human 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, and were stained with IANBD-p-SIVA. Data were analyzed by flow cytometry. Numbers in the lower right 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 cells increases. Data are representative of three independent experiments.
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Figure 5 Fe(salen)Cl upregulates cytochrome c expression in EL4 and Jurkat cells but not in mouse splenocytes and human PBMCs. Cells were cultured for 24 h in presence of Fe(salen)Cl (2.5 mM) and the cell lysates were subjected to Western blot analysis using anti-cytochrome c antibody (lower panel). Anti-GAPDH antibody was used as loading control (upper panel).
it has any effect on the mitochondrial properties, especially on mitochondrial membrane potential. For that purpose we did two independent experiments using Jurkat cells and MCF-7 as the cancerous cells and human PMBC as noncancerous cells. To confirm that Fe(salen)Cl alters mitochondrial properties, we measured mitochondrial potential of Jurkat cells by using fluorescent dye, 5,50 ,6,60 -tetrachloro- 1,10 ,3,30 -tetraethylbenzimidazolcarbocyanine iodide (JC-1), which is reliable and very sensitive. The green fluorescence (at 525 nm, i.e., low mitochondrial membrane potential) indicates the uptake of JC1 monomer and the red fluorescence (590 nm) indicates the presence of high J-aggregates (higher mitochondrial membrane potential) of JC-1 (Pfeiffer et al., 1933). Under control conditions, the majority of cells had high fluorescence in the 590 nm region, while Fe(salen)Cl-treated cells had much less signal in this region. In contrast, emission at 525 nm was higher in Fe(salen)-treated cells. These fluorescence results suggest that the mitochondrial membrane potential is significantly low in Fe(salen)Cl-treated Jurkat cells with respect to untreated control cells (Figure 6a). Some of the Fe(salen)Cltreated cells even showed both red and green fluorescence, indicating the co-existence of mitochondria at both high and low potential, a status which indicates an early phase of cell death. In experiments with human PBMC, similar changes in mitochondrial membrane potential in Fe(salen)Cl-treated cells were not observed, being similar to the untreated controls (Figure 6b). The results strongly indicate that Fe(salen)Cl-treatment reduces the mitochondrial potentiality in cancerous cells, but not in non-cancerous cells. The MCF7 is also susceptible to Fe(salen)Cl and undergoes apoptosis. The adherent nature of MCF-7 allowed us to use live cell fluorescence microscopy and measure changes in the mitochondrial oxidative potential. In MCF-7 cells treated with Fe(salen)Cl (1.25 mM) for 24 h and then labeled with Mitotracker Green FM dye (MTG), the treated cells had very low fluorescence compared with untreated cells (Figure 7), which indicates that the oxidative potential of MCF-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 1.25 mM caused an immediate (
RESEARCH ARTICLE
Induction of apoptosis by Fe(salen)Cl through caspase-dependent pathway specifically in tumor cells Nitika 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 2 3 4
Imgenex India, Infocity, Bhubaneswar, India School of Biological Sciences, National Institute of Science Education & Research, Bhubaneswar, India School of Chemical Sciences, National Institute of Science Education & Research, Bhubaneswar, India 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 their specificity towards cancer cells and the mechanism of action are hitherto less investigated. A Fe(salen)Cl derivative has been synthesized that remains active in monomer form. The efficacy of this compound as an anti-tumor agent has been investigated in 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 undergo apoptosis, but normal mouse spleen cells and human peripheral blood mononuclear cells (PBMC) remain largely unaffected by Fe(salen)Cl. Fe(salen)Cl treated tumor cells show significantly higher expression level of cytochrome c that might have triggered the cascade of reactions leading to apoptosis in cancer cells. A significant loss of mitochondrial membrane potential upon Fe(salen)Cl treatment suggests that Fe(salen)Cl induces apoptosis by disrupting mitochondrial membrane potential and homeostasis, leading to cytotoxity. We also established that apoptosis in the Fe(salen)Cl-treated tumor cells is mediated through caspase-dependent pathway. This is the first report demonstrating that Fe(salen)Cl can specifically target the tumor cells, leaving the primary cells least affected, indicating an excellent potential for this compound to emerge as a next-generation anti-tumor drug. Keywords: apoptosis; caspase; Fe(salen)Cl; leukemia cell line; mitochondria; tumor
Introduction Amongst the chemotherapeutic drugs for cancer, cisplatin is mostly being used, although it has serious side effects (Rafique et al., 2010). There is a need for suitable drugs that specifically target cancer cells while leaving normal cells unaffected or less affected. In this respect, transition metal complexes are of considerable research interest because of their structural diversity (Teyssot et al., 2009; To et al., 2009; Liu et al., 2011; Liu and Sadler, 2011). Amongst the metallodrugs, iron-based compounds have recently been explored for their anticancer properties in spite of having some side effects (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 ironbased nano-particles (Wu et al., 2011) have cytotoxic effects in vitro in cancer cells; but their mechanism of action remains largely undefined. FeIII(porphyrin) complexes are widely used to mimic the role of the cytochrome P-450 class of enzymes that are responsible for the oxidation of various organic substances (Meunier, 1992). In this context, we have synthesized Fe(salen)Cl complexes to mimic the activities of FeIII(porphyrin) complexes. A structural similarity exists between FeIII(porphyrin) and Fe(salen)Cl complexes where the iron remains in FeIII-state (Böttcher et al., 1996; Liou and Wang, 2000). Mandal and colleagues (Mandal et al., 1997) have synthesized a series of
Corresponding authors: e-mail: [email protected], [email protected], [email protected]
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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 normal primary cells in an effort to study its potential as an anticancer drug. From single crystal X-ray structural analysis, it is found that Fe(salen)Cl complexes could exist as monomeric or dimeric forms depending on the solvent used for crystallization. The single crystal data obtained from CH3CN medium suggest that the compound is indeed dimeric in nature in solid phase. The solution phase electrochemical studies indicate that the compound is monomer in solution. The interaction of Fe(salen)Cl with living 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 death in breast cancer cell line in vitro, suggesting that salen compounds may have anti-tumor properties, although the mechanism by which they induce cell death is unclear. Oxidative stress exerted by redox active metals like iron may be responsible for DNA/RNA damage in vitro, as has been suggested (Bhattacharya and Mandal, 1996; Czlapinski and Sheppard, 2001; Shao et al., 2006). We show here that Fe(salen)Cl specifically kills the cancerous cells but spares primary normal cells. In cancer cells it causes significant loss of mitochondrial membrane potential with a concomitant increase in cytochrome c level, which in turn induces apoptosis in cancer cells through a caspase-dependent pathway.
Fe(salen)Cl induces apoptosis in tumor cells
calomel reference electrode (SCE) were used in a 3-electrode configuration. Tetraethyl ammonium perchlorate (TEAP) was the supporting electrolyte (0.1 M) at a concentration of 103 M with respect to the complex. The half wave potential E 298 was set equal to 0.5(Epa þ Epc), where Epa and Epc are anodic and cathodic cyclic voltammetric peak potentials, respectively. The scan rate used was 100 mV s1.
Synthesis of salen ligand Salen ligand (H2L) was synthesized by following the procedure of Pfeiffer et al. (1933). A 2:1 molar ratio of salicylaldehyde and ethylenediamine were mixed together in 20 mL absolute methanol. The resulting mixture was kept for 30 min at 4 C. The precipitate was filtered and washed thoroughly with ice-cold methanol followed by ice-cold diethyl ether. The final product was recrystallized from CH3CN.
Synthesis of Fe(salen)Cl Fe(salen)Cl has been synthesized by the procedure of Matsushita et al. (1982). A 1:1 molar ratio of ferric chloride and salen ligand (H2L) were taken in 25 mL absolute CH3OH. The resulting mixture was heated to reflux under a dinitrogen atmosphere for 2.5 h. The concentrated solution was refrigerated overnight. The precipitate was filtered and washed thoroughly with ice-cold water followed by ice-cold diethyl ether. The product was recrystallized from acetone.
Materials and methods
Determination of crystal structure
General methods
Single crystals of complex were grown by slow evaporation of a solution of the complex in acetonitrile under atmospheric conditions. The crystal data of Fe(salen)Cl were collected on a Bruker Kappa APEX II CCD diffractometer at 293 K, which were corrected for Lorentz polarization and absorption effects. The program package SHELX-97 (ShelxTL) (Sheldrick, 1997) was used for structure solution and full matrix least squares refinement on F2. Hydrogen atoms were included in the refinement using the riding model. Contributions of H atoms for the water molecules were included but were not fixed.
The precursor compound, ferric chloride was purchased from Merck (Germany). The ligand, 2,20 -((1E,10 E)-(ethane1,2 diylbis (azanylylidene)) bis(methanylylidene)) diphenol (H2L), was prepared according to Pfeiffer et al. (1933). All other chemicals and solvents were of reagent grades. For spectroscopy and electrochemical studies, HPLC grade solvents were used. Commercial tetraethyl ammonium bromide was converted to pure tetraethyl ammonium perchlorate (TEAP) following the procedure of Sawyer et al. (1995).
Cell culture Physical measurements UV-Vis spectral work used a Perkin-Elmer LAMBDA-750 spectrophotometer. FT-IR spectra were taken with samples prepared as KBr pellets. Cyclic voltammetry measurements were carried out using a CH instrument model CHI1120A electrochemistry system. A glassy-carbon working electrode, a platinum wire as an auxiliary electrode, and a saturated
Jurkat, Molt-4 (human acute lymphoblastic leukemia), Raji, Ramos, Daudi (Burkitt’s lymphoma), MCF-7 (human breast adenocarcinoma), THP-1 (human acute monocytic leukemia), A431 (human epithelial carcinoma), Hep G2 (human hepatocellular carcinoma), HeLa (human cervix adenocarcinoma), U87 (human glioblastoma), A549 (human lung carcinoma), SW1990 (human pancreatic carcinoma), F0
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Fe(salen)Cl induces apoptosis in tumor cells
(mouse myeloma), B16 (mouse melanoma), and EL4 (mouse T cell leukemia) cells were procured from American Type Culture Collection (ATCC). Cells were grown in T25 culture flask (Nunc, Denmark) in RPMI (Rosewell Park Memorial Institute, Invitrogen, USA) or IMDM (Iscove’s Modified Dulbecco’s Medium, Invitrogen) supplemented with 10% heat inactivated FBS (Fetal Bovine Serum, Lonza, USA), 1% glutamine (HiMedia, India), and 1% penicillin and streptomycin (HiMedia). All cells were cultured in a CO2 incubator at 37 C in air with 5% CO2 and 90% humidity.
Isolation of mouse splenocytes All the experiments performed for the study were according to the Institutional guidelines for animal care and approved by Institutional Animal Ethics Committee (IAEC) and CPCSEA (Committee for the Purpose of Control and Supervision of Experiments in Animals), Govt. of India. Male C57BL/6 mice (B6) aged 6–8 weeks were procured from 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 for 10 min and washed with PBS. Splenocytes were kept in 2 mL of RBC lysis buffer (Imgenex, USA) for 5 min to remove RBCs. After neutralizing and washing with PBS, cells were resuspended in IMDM supplemented with 10% FBS, 1% glutamine, and 1% penicillin and streptomycin.
Isolation of human PBMC All experiments for human PBMCs were performed according to the guidelines set by the Institutional Human Ethics Committee of National Institute of Science Education and Research (Bhubaneswar). PBMC from human blood (hPBMC) was separated following the protocol published elsewhere (Chattopadhyay and Chakraborty, 2005; Chattopadhyay et al., 2006) and were cultured in IMDM supplemented with 10% FBS, 1% glutamine, and 1% penicillin and streptomycin. Adherent hPBMC were generated by using HiSepTM LSM 1077 (HiMedia) density gradient-cut of human peripheral blood of healthy donor(s). Monocyte/macrophage rich cells were isolated after 1 h of adherence from hPBMC in 6 well cell culture plates (BD Biosciences, USA). The adherent cells were cultured in IMDM supplemented with 10% FBS supplemented with L-arginine (0.55 mM), L-asparagine (0.24 mM), and L-glutamine (1.5 nM) (Invitrogen) with 50 U/mL penicillin, and 50 mg/mL streptomycin; henceforth described as complete medium. The non-adherent and loosely adherent cells were separated by vigorous washing in microbiologically sterile PBS and the adherent cells were kept in culture in 1120
6-well cell culture plates for 1–2 weeks in complete medium for downstream experiments.
Cytotoxicity assay Cytotoxicity of Fe(salen)Cl was measured by Trypan blue exclusion (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 culture plates (Nunc). Fe(salen)Cl was dissolved in DMSO (Sigma) and diluted with in culture media before being added to triplicate wells at 0 (control), 0.625, 1.25, 2.5, 5, and 10 mM, and in some experiments 20 mM. Control wells were treated with an equivalent amount of diluted DMSO. After 24 h of incubation, cells were collected and assayed by the trypan blue exclusion method. IC50 value for the Fe(salen)Cl metallo-complex was determined by counting the percentage of live and dead cells under an inverted microscope (Olympus, Japan).
Flow cytometry For a better understanding of the pathway by which Fe(salen)Cl induced cell death in cancer cells, apoptosis was measured by staining the cells with IANBD [N-((2(iodoacetoxy) ethyl)-N-Methyl) amino-7-Nitrobenz-2-Oxa1,3-Diazole] conjugated p-SIVATM (polarity sensitive indicator of viability and apoptosis, Imgenex, USA). pSIVA is an Annexin XII based, polarity sensitive probe for the spatiotemporal analysis of apoptosis to detect phosphatidylserine (PS) exposure. PS exposure is a hallmark phenomenon, occurs early during apoptosis and persists throughout the cell death process (Bevers and Williamson, 2010; Kim et al., 2010a, 2010b; Yamazaki and Piette, 1990). In brief, cells were washed twice with 1 mL cold PBS and were added with 5 mL pSIVA-IANBD to each sample and were incubated for 10 min at room temperature (25 C) in the dark. After the incubation, the cells were analyzed by a flow cytometer (FACS Calibur, BD Biosciences). The data were analyzed with CellQuestProTM software (BD Biosciences). Expression of apoptotic proteins, Apaf-1 (Apoptotic protease activating factor 1), BAD (Bcl2-associated antagonist of cell death), active caspases (cleaved caspase-3 and caspase-9) and the anti-apoptotic protein, Bcl2, were detected 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, the cells were harvested and analyzed by flow cytometry. The cell pellet was fixed with fixation buffer (Imgenex) for 30 min at room temperature followed by the addition of permeabilization buffer (Imgenex). Cells were incubated separately with anti-Apaf-1, anti-Bcl2, anti-BAD (cleaved),
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anti-caspase-3 (cleaved), and anti-caspase-9 (cleaved) antibodies (Imgenex) appropriately diluted with staining buffer (Imgenex) for 30 min, followed by washing twice with permeabilization buffer and finally resuspended in fluorescent labeled goat anti-rabbit secondary antibody (Jackson, USA) for 30 min in the dark. The cells were washed with staining buffer, resuspended in 400 mL of staining buffer and analyzed by flow cytometry.
Western blot analyses Cytochrome c is a trigger of the cascade of reactions leading to the induction of apoptotic pathway. The enhanced apoptosis in Fe(salen)Cl treated EL4 and Jurkat cells encouraged us to investigate the expression status of cytochrome c in the apoptotic cells. Western blot analyses were done on Fe(salen)Cl treated Jurkat cell lysate. In brief, Jurkat and EL4 cells were cultured in presence (2.5 mM) or absence of Fe(salen)Cl for 24 h. After the incubation, cells were harvested, washed in PBS, and resuspended in lysis buffer containing protease inhibitors (Sigma). The cell lysate was stored in 80 C until further processing. Lysate proteins were separated by SDS-PAGE and transferred on PVDF membrane (Millipore, USA), which had been blocked with 5% non-fat milk in PBS. The membrane was probed with anti-cytochrome c (Imgenex) and was detected with goat antirabbit antibody conjugated to peroxidase (Jackson). SuperSignalTM West Pico Chemiluminescent kit (Pierce, USA) was used for chemiluminescent signal and the signal was exposed to an X-ray film (Kodak, Japan). GAPDH (Clone IMG13H12, Imgenex) antibody was used as loading control.
Microscopic detection of mitochondrial potential change To monitor the change in mitochondrial oxidative potential, MCF7 cells and human PBMC were treated with Mitotracker-green-FM dye (MTG, Invitrogen; Zhu et al., 2010). MCF7 cells were maintained in IMDM (Invitrogen) supplemented with 10% fetal bovine serum (Lonza) in the presence of antibiotics and glutamine (Invitrogen) at 37 C incubator in air with 5% CO2. Human PBMCs were maintained as mentioned before. All cells were grown on 22 mm glass coverslips and loaded with MTG (1 mM) for 20 min before imaging. After loading with MTG, the cells were washed gently twice with PBS buffer and taken into a live cell imaging chamber where they were imaged for some experiments as a time series images acquired at regular intervals. In some experiments, Fe(salen)Cl was added (1.25 mM and 5.0 mM) after taking a few initial images and was imaged again to monitor the changes in mitochondrial membrane potential/oxidative potential. Cells loaded with MTG were excited by wavelength set at 490–543 (by using Argon laser, 1.8% power).
Fe(salen)Cl induces apoptosis in tumor cells
JC-1 cationic dye (Sigma) was used to follow the mitochondrial potential (c) in Jurkat cells and human PBMC. This dye shows potential-dependent accumulation inside the mitochondria and also has fluorescence emission properties that shifts from green (525 nm) to red (590 nm) and reflects the apoptotic state of cells (Kim et al., 2010a, 2010b). Briefly, Jurkat cells (1 106 cells/mL) were grown in 35 mm dishes and Fe(salen)Cl drug (5 mM) was added for 6 h. JC-1 (1 mM) dye was added to the cells for 20 min prior to imaging. Cells were maintained at 37 C in the presence of air plus 5% CO2 in a humid condition. As Jurkat cells are non-adherent in nature, cells were pelleted at 2000 rpm for 4 min and resuspended in IMDM. The cells were placed onto the live cell chamber for imaging with a confocal microscope (LSM780, Zeiss, Germany) using a 63 or 20 objective. All images were taken under the same conditions for comparative purposes, and were analyzed and processed using LSM Image Examiner software (Zeiss). The relative fluorescence intensity is represented in pseudo-color, where red indicates highest and blue indicates lowest intensity of the respective fluorophores.
Caspase inhibition zVAD-fmk (Benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone) is a broad spectrum caspase inhibitor that binds with active caspases and effectively blocks their biological activity (Gregoli and Bondurant, 1999; Slee et al., 1996). We examined whether inhibition of caspases rescues the cells from Fe(salen)Cl-induced apoptosis. Jurkat cells were incubated with Fe(salen)Cl in the absence and presence of zVAD-fmk (Invitrogen) for 24 h. Cells were harvested and were stained with IANBD conjugated p-SIVA as described earlier and were incubated at room temperature for 10 min. The samples were analyzed by flow cytometry and CellQuestProTM software (BD Biosciences). Results
Synthesis and characterization of Fe(salen)Cl Fe(salen)Cl has been characterized by using various spectroscopic techniques, like UV-Vis spectroscopy, IR spectroscopy, CHN analysis, and also from single crystal X-ray analysis. Purity and identity of the Fe(salen)Cl is demonstrated by its satisfactory elemental analyses. The hexa-coordinated dimeric nature of Fe(salen)Cl had been established by its single crystal X-ray structure. Single crystals of Fe(salen)Cl were grown by slow evaporation of their acetonitrile solution. The molecule crystallized in the space group of P21/c and the corresponding cell parameters are a ¼ 11.33 A, b ¼ 6.8738 A, c ¼ 19.1689 A, and b ¼ 91.446 . It matches well with the corresponding dimeric structure of Fe(salen)Cl.
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Redox properties of the Fe(salen)Cl The redox properties of Fe(salen)Cl were studied in acetonitrile solvent by cyclic voltammetric techniques (Figure 1). The reduction processes at the negative side of SCE 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 observed response was assigned as electron-transfer process involving the metal center, FeIII/FeII.
Cytotoxic effects of Fe(salen)Cl on tumor and normal cells The effectiveness of an ideal anti-cancer candidate should selectively target the cancer cells and not the normal cells. Thus the ability of Fe(salen)Cl to induce cell death was investigated in cancer cell lines compared with its effect on normal cells. Initially Fe(salen)Cl was tested on a series of cancer cell lines: Jurkat, Molt4 (both are human acute T cell leukemia), Raji, Ramos, Daudi (all human B cell leukemia), MCF7 (human breast carcinoma), THP-1 (human acute monocytic leukemia), A431 (human epithelial carcinoma), Hep G2 (human hepatocellular carcinoma), HeLa (human cervix adenocarcinoma), U87 (human glioblastoma), A549 (human lung carcinoma), SW1990 (human pancreatic carcinoma), EL4 (mouse T cell lymphoma), F0 (mouse myeloma), and B16 (mouse melanoma) cells. Figure 2 shows that most cell lines are susceptible to this compound and the cell 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, we chose EL4 and Jurkat cells and studied to investigate the efficacy of Fe(salen)Cl in inducing cell death. Mouse
splenocytes and normal human peripheral blood mononuclear cells (PBMCs) were used as primary cells to monitor the effects of Fe(salen)Cl on normal cells. Cells were cultured in the presence and absence of Fe(salen)Cl and were incubated at different concentrations of Fe(salen)Cl. After 24 h of incubation, live and dead cells were counted by Trypan blue dye exclusion method. Fe(salen)Cl induced cell death in EL4 in a dose-dependent manner and the LC50 for EL4 was about 1.25 mM (Figure 3). Normal mouse splenocytes were largely unaffected by Fe(salen)Cl compared to its cancerous counterparts and 95% EL4 cells were dead. Similarly, the result showed that Jurkat cells were susceptible to Fe(salen)Cl even below 1.25 mM and >20% of the cells were dead. The LC50 of Jurkat cells was ~2.0 mM. Interestingly, normal human PBMCs were significantly insensitive to Fe(salen)Cl in comparison to Jurkat cells (Figure 3), with only 3–4% cells dead at 10 mM, whereas at this dose, >95% Jurkat cells were found to be dead. Thus, it is evident that Fe(salen)Cl induces cell death specifically in cancer cells both in mouse and human, whereas it has little effect on normal cells. In addition, a time-kinetic study reveals 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 in susceptible cells is a salient feature of most anti-cancer agents. Thus, the effect of Fe(salen)Cl in tumor cells was investigated in detail to find out whether death was due to apoptosis (Figure 4). IANBD conjugated p-SIVA was used to detect apoptosis induced by Fe(salen)Cl in EL4 and Jurkat cells. The cells were incubated with different doses of Fe(salen)Cl for 24 h and were stained with p-SIVA-IANBD. Both EL4 and Jurkat cells were apoptotic after treatment with Fe(salen)Cl in a dose-dependent manner. On the other hand, mouse splenocyte and human PBMC did not undergo apoptosis 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 in Fe(salen)Cl treated cells compared to untreated cells (Figure 5), suggesting that Fe(salen)Cl induces the expression of cytochrome c level that in turn initiates the apoptotic pathway.
Fe(salen)Cl lowers the mitochondrial oxidative potential Figure 1 Cyclic voltammograms (——) of Fe(salen)Cl in CH3CN/0.1 M TEAP at 298 K. The potentials are versus SCE.
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To understand the molecular mechanism of how Fe(salen)Cl causes cell death, especially in cancer cells, we tested whether
Cell Biol Int 38 (2014) 1118–1131 © 2014 International Federation for Cell Biology
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Fe(salen)Cl induces apoptosis in tumor cells
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 different doses (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 are mean 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. Normal mouse splenocytes, human PBMC, EL4 and Jurkat cells were cultured in a 24-well plate in the presence of different concentrations of Fe(salen)Cl as indicated and were cultured for 24 h (A and B). For time kinetics, cells were incubated with 2.5 mM 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 splenocytes and normal human PBMCs. The values are mean standard error of means. The data are representative of three independent experiments.
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Figure 4 Fe(salen)Cl induces apoptosis in EL4 and Jurkat cells. Flow cytometric analysis of apoptosis in mouse splenocytes and EL4 (left panles); and human 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, and were stained with IANBD-p-SIVA. Data were analyzed by flow cytometry. Numbers in the lower right 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 cells increases. Data are representative of three independent experiments.
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Figure 5 Fe(salen)Cl upregulates cytochrome c expression in EL4 and Jurkat cells but not in mouse splenocytes and human PBMCs. Cells were cultured for 24 h in presence of Fe(salen)Cl (2.5 mM) and the cell lysates were subjected to Western blot analysis using anti-cytochrome c antibody (lower panel). Anti-GAPDH antibody was used as loading control (upper panel).
it has any effect on the mitochondrial properties, especially on mitochondrial membrane potential. For that purpose we did two independent experiments using Jurkat cells and MCF-7 as the cancerous cells and human PMBC as noncancerous cells. To confirm that Fe(salen)Cl alters mitochondrial properties, we measured mitochondrial potential of Jurkat cells by using fluorescent dye, 5,50 ,6,60 -tetrachloro- 1,10 ,3,30 -tetraethylbenzimidazolcarbocyanine iodide (JC-1), which is reliable and very sensitive. The green fluorescence (at 525 nm, i.e., low mitochondrial membrane potential) indicates the uptake of JC1 monomer and the red fluorescence (590 nm) indicates the presence of high J-aggregates (higher mitochondrial membrane potential) of JC-1 (Pfeiffer et al., 1933). Under control conditions, the majority of cells had high fluorescence in the 590 nm region, while Fe(salen)Cl-treated cells had much less signal in this region. In contrast, emission at 525 nm was higher in Fe(salen)-treated cells. These fluorescence results suggest that the mitochondrial membrane potential is significantly low in Fe(salen)Cl-treated Jurkat cells with respect to untreated control cells (Figure 6a). Some of the Fe(salen)Cltreated cells even showed both red and green fluorescence, indicating the co-existence of mitochondria at both high and low potential, a status which indicates an early phase of cell death. In experiments with human PBMC, similar changes in mitochondrial membrane potential in Fe(salen)Cl-treated cells were not observed, being similar to the untreated controls (Figure 6b). The results strongly indicate that Fe(salen)Cl-treatment reduces the mitochondrial potentiality in cancerous cells, but not in non-cancerous cells. The MCF7 is also susceptible to Fe(salen)Cl and undergoes apoptosis. The adherent nature of MCF-7 allowed us to use live cell fluorescence microscopy and measure changes in the mitochondrial oxidative potential. In MCF-7 cells treated with Fe(salen)Cl (1.25 mM) for 24 h and then labeled with Mitotracker Green FM dye (MTG), the treated cells had very low fluorescence compared with untreated cells (Figure 7), which indicates that the oxidative potential of MCF-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 1.25 mM caused an immediate (
