Targeting Mitochondrial Cell Death Pathway to Overcome Drug Resistance with a Newly Developed Iron Chelate

Targeting Mitochondrial Cell Death Pathway to Overcome Drug Resistance with a Newly Developed Iron Chelate
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  Targeting Mitochondrial Cell Death Pathway toOvercome Drug Resistance with a Newly Developed IronChelate Avishek Ganguly 1 , Soumya Basu 1 , Paramita Chakraborty 1 , Shilpak Chatterjee 1 , Avijit Sarkar 2 ,Mitali Chatterjee 2 , Soumitra Kumar Choudhuri 1 * 1 Department of   In Vitro  Carcinogenesis and Cellular Chemotherapy, Chittaranjan National Cancer Institute, Kolkata, India,  2 Department of Pharmacology, Institute of Post Graduate Medical Education and Research, Kolkata, India Abstract Background:   Multi drug resistance (MDR) or cross-resistance to multiple classes of chemotherapeutic agents is a majorobstacle to successful application of chemotherapy and a basic problem in cancer biology. The multidrug resistance gene,MDR1, and its gene product P-glycoprotein (P-gp) are an important determinant of MDR. Therefore, there is an urgent needfor development of novel compounds that are not substrates of P-glycoprotein and are effective against drug-resistantcancer. Methodology/Principal Findings:   In this present study, we have synthesized a novel, redox active Fe (II) complex (chelate),iron N- (2-hydroxy acetophenone) glycinate (FeNG). The structure of the complex has been determined by spectroscopicmeans. To evaluate the cytotoxic effect of FeNG we used doxorubicin resistant and/or sensitive T lymphoblastic leukemiacells and show that FeNG kills both the cell types irrespective of their MDR phenotype. Moreover, FeNG induces apoptosis indoxorubicin resistance T lymphoblastic leukemia cell through mitochondrial pathway via generation reactive oxygenspecies (ROS). This is substantiated by the fact that the antioxidant N-acetyle-cysteine (NAC) could completely block ROSgeneration and, subsequently, abrogated FeNG induced apoptosis. Therefore, FeNG induces the doxorubicin resistant Tlymphoblastic leukemia cells to undergo apoptosis and thus overcome MDR. Conclusion/Significance:   Our study provides evidence that FeNG, a redox active metal chelate may be a promising newtherapeutic agent against drug resistance cancers. Citation:  Ganguly A, Basu S, Chakraborty P, Chatterjee S, Sarkar A, et al. (2010) Targeting Mitochondrial Cell Death Pathway to Overcome Drug Resistance with aNewly Developed Iron Chelate. PLoS ONE 5(6): e11253. doi:10.1371/journal.pone.0011253 Editor:  Maxim Antopolsky, University of Helsinki, Finland Received  March 19, 2010;  Accepted  May 18, 2010;  Published  June 22, 2010 Copyright:    2010 Ganguly et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the srcinal author and source are credited. Funding:  This investigation received financial support from the Indian Council of Medical Research (ICMR), New Delhi, No. 3/2/2/200/2009/NCD-III and No. 5/13/18/2007/NCD-III. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests:  The authors have declared that no competing interests exist.* E-mail: Introduction Multidrug resistance (MDR), a phenotype of cross-resistance tomultiple drugs with diverse chemical structures is the majorimpediment of successful application of chemotherapy [1]. Although the underlying mechanisms are diverse, the role of  ATP-dependent drug efflux-proteins on the cell membrane isaccepted as a major cause behind MDR. Since the discovery of drug efflux proteins for last four decades, huge number of chemicals had been developed to inhibit these proteins and thusserve as resistance modifying agents (RMA). The development of non-toxic RMA capable of overcoming MDR clinically is stillelusive [2,3]. However, the basic target of chemotherapy is toinduce apoptosis to cancer cells irrespective of its phenotype. Is itpossible to induce apoptosis to cancer cells irrespective of itsphenotype? To address this question, we had earlier showed thatreactive oxygen species (ROS) plays important role in inducing apoptosis to MDR cells through generation of host protectivecytokines by a copper chelate (CuNG) formed with copper salt andN-(2hydroxyacetophenone) glycinate (NHAG), synthesized by us[4,5,6]. In the present work we tried to understand the role of ROS generation and consequent induction of apoptosis toovercome MDR by the iron chelate (FeNG) formed with thesame ligand (NHAG) [7] and iron salt. As iron is less toxic thancopper and is required in high amount in normal humanphysiology [8], we tried to harness the ROS generating effect of ferrous ion to induce apoptosis to MDR cells.Redox regulation has been shown to be an importantcomponent of malignant cell survival. Tipping the cellular redoxbalance through pharmacologic regulation in favor of increasing intracellular ROS and/or depleting protective reducing metabo-lites (such as glutathione (GSH) and nicotinamide adeninedinucleotide phosphate) may lead to oxidative stress and resulting in induction of apoptosis for the treatment of cancer [9]. It hasbeen postulated that the intrinsic ROS stress associated withoncogenic transformation makes the cells highly dependent ontheir antioxidant systems to counteract the damaging effect of ROS and to maintain redox balance in a dynamic state (increased PLoS ONE | 1 June 2010 | Volume 5 | Issue 6 | e11253  ROS generation and active ROS scavenging). This situationrenders cancer cell highly vulnerable to further oxidative insults byexogenous agents [10]. Cancer cells, especially those in advanceddisease stages, become highly adapted to intrinsic oxidative stresswith up regulated antioxidant capacity. This redox adaptationgenerally enables the cancer cells to survive under increased ROSstress and also provides a mechanism of resistance to manyanticancer agents. Owing to the presence of redox adaptationmechanisms, the use of ROS-generating agents alone may not besufficient to kill cancer cells that have an up-regulated antioxidantcapacity (mostly cellular GSH). Agents those disable such adaptivemechanisms becomes more effective against these cancer cells.Combinations of ROS-generating agents with compounds capableof abrogating cellular antioxidant systems are likely to have anadditive or synergistic effect [11].Inthepresentwork,wehavesynthesizedandcharacterizedanovel,nontoxic iron complex (FeNG) and harnessed its anti proliferativeproperties against drug resistant T lymphobastic leukemia  in vitro . Wehave investigated the underlying molecular mechanisms of apoptosisupon treatment with FeNG in CEM/ADR5000 cells. The pro-apoptotic activity of iron chelate involves mitochondrial apoptoticpathway through generation of ROS and hints at a possibility of utilizing redox active metal chelates in combating cancer. Results UV_Vis spectral study UV spectrum for the ligand  l  max (water) was observed at: 211,253 and 325 nm.UV-VIS spectrum for the complex  l  max (water) was observedat: 218, 254, 340 and 475 nm.The electronic absorption spectrum of the complex shows fourbands in aqueous solution at 218 nm, 254 nm, 340 nm and475 nm. The first three absorption bands are also observed inalmost the same  l  value in free ligand. These bands are perhapsdue to intra-ligand transition.In the metal complex these three bands appear with higher  l  values, as there may decrease electron donating ability aftercomplexation of ligand. The 475 nm band in the complex may beassigned as d-d transition band, due to the spin forbidden transitionsfrom 6  A 1g  to 4 T 1g  andfrom 6  A 1g  to 4 T 2g  [4].Thisbandpositionofthecomplex suggests an octahedral geometry of the Fe(II) complex. Infra Red spectral study Important infrared (i.r.) bands for the ligand appear at:3410 2 3360, 1689, 1619, 1524, 1466, 1421, 1395, 1318, 1269,1205, 1163, 969, 931, 752 and 730 cm 2 1 [7].Important i.r. bands for the complex appear at: 3219–3385,1598, 1538, 1434, 1390, 1331, 1309, 1231, 1162, 1134, 1083,1024, 963, 865, 745, 605, 525, and 433 cm 2 1 .The  n  CN characteristic stretching band in the ligand appears at1619 cm 2 1 and shifts in the lower frequency region in the complexat 1598 cm 2 1 ; such shifting towards lower frequency regionsuggests the coordination between nitrogen atom of the ligand andthe Fe-metal. In the ligand, one strong band appears at1689 cm 2 1 due to asymmetric stretching vibration of –COO  –  and another strong band appears at 1395 cm 2 1 due to symmetricstretching vibration of – COO  –  group [7]. In the metal ligandsystem, 1689 cm 2 1 band is not observed and the band at1395 cm 2 1 band shifted to 1434 cm 2 1 ; so there is strong indication that the COO  —  group coordinates through deprotona-tion. The  n  C-O ligand band (1269 cm 2 1  ) shifted towards higherfrequency side in the complex at 1309 cm 2 1 . This high frequencyshift of   n  C-O phenolic band confirms the formation of covalentbond between oxygen atom of phenolic –OH and metal ionthrough deprotonation [12]. The –OH group participation incoordination is also indicated by the shift of 3394 cm 2 1 band(  2 OH group) towards 3219–3385 cm 2 1 through deprotonationand formation of a metal-oxygen bond.Metal ligand vibrations are generally located in the region 600– 250 cm 2 1 . The skeletal vibrations of the ligand appearing in thisregion complicate the scope of interpretation. However, thecomparison of the complex and ligand spectra allowed theassignment of metal sensitive bands. In the present case, we haveassigned the band at 605 cm 2 1 to  n  M-O in the complex [4]. Proton NMR Study Proton nmr peak of the ligand in D 2 O appears at  d  7.38–7.51(S, 5H) and  d  6.59–6.76 (S, 3H) for aromatic protons. -CH 2 protons appear at  d  4.09 (1H, M). -CH 3  protons appear at  d  2.26– 2.29 (4H, M).Proton nmr peak of the complex FeNG in D 2 O appear at  d 6.88–7.8 (5H, S) for aromatic protons. -CH 2 - protons appear at  d 3.49. CH 3  protons appear at  d  2.55.The characteristic proton signals due to aryl group in the ligand(  d  6.59–7.5) are almost unaffected in the complex and appear at  d 6.88–7.8. Complexation causes drastic changes of proton signals of -CH 2  and -CH 3  groups in the ligand. The signal in the ligand dueto -CH 2  group (  d  4.09) shifts to higher field  d  3.49 in the complex;This is an indication of considerable drift of electrons from twoneighboring groups viz.,  2 N=C and –COOH to the metalmoiety. The signal for CH 3  shifts to lower field in the complex (inthe complex at  d  2.55 and in the ligand at  d  2.26–2.29) due todeshielding of protons and indicates participation of CH 3 -C=N-group to coordination with iron atom [4]. Mass Spectral Study The formation of molecular ion peaks indicates that thestructure of the iron complex in Fig. 1A and Mass spectral datais presented in Fig. 1B. Antiproliferative effects of FeNG In an initial approach, to determine the antiproliferative effect of FeNGonTlymphoblasticleukemiacellsweperformedMTT(3-[4,5-dimethylthiazol- 2-yl]-2, 5-diphenyltetrazolium bromide) assay em-ploying CEM/ADR5000 in comparison to CCRF-CEM or humanPBMC (peripheral blood mononuclear cells). FeNG induced growthinhibitory effect occurred in time as well as dose dependent manner inCEM/ADR5000 (fig. 2A) and CCRF-CEM (fig. 2B) cell line withIC 50  values (at 72 h treatment) 0.75 6 10 2 3 M and 0.79 6 10 2 3 Mrespectively (Table 1). However under the same condition FeNGdidn’t display cytotoxic effect on normal human PBMC (fig. 2C) atgiven experimental concentration range. The results presented in theTable 1 would suggest that CEM/ADR5000 and CCRF-CEM cellswere equally sensitive to FeNG as the difference in IC 50  valuesbetween two different cell lines were statistically not significant. Inaddition, data obtained for FeNG displayed a considerable lowerresistance factor than doxorubicin [13] suggested that the complexwas not a potential MDR1 substrate (Table 2). Selective cellular and nuclear morphological changes inCEM-ADR5000 cells after FeNG treatment The Hoechst 33342 staining is sensitive to DNA and was used toassess changes in nuclear morphology. A concentration of 0.75 6 10 2 3 M is high enough to inhibit cell growth (fig. 2A).CEM/ADR5000 cell were treated with FeNG for 24 h, 48 h, and72 h and the iron complex induced nuclear condensation as well Iron Complex Induces ApoptosisPLoS ONE | 2 June 2010 | Volume 5 | Issue 6 | e11253  as nuclear fragmentation (a typical apoptosis associated markers)was determined by fluorescence microscopy (fig. 3A). As shown infig. 3B, percentage of apoptotic cells was increased in timedependent manner when cells were exposed to FeNG. FeNG induces apoptosis in CEM/ADR5000 cell lines intime dependent fashion Early cellular changes in apoptosis are characterized by thetranslocation of phosphatidylserine (PS) to the external surface of theplasma membrane where it can be detected by binding to annnexinV- FITC. As cell membrane is further compromised and cell deathoccurs, cellular DNA becomes accessible for staining with PI [14,15].The flow cytometric analysis showed that (fig. 4A), the CEM/ ADR5000 cells that had been incubated with FeNG for 24 h, 48 h,and 72 h and dual stained with annexin V-FITC and PI, there was aprogressive increase in the annnexin V-FITC positive population of cells (39.17%, 46.65%, 60.70% for 24 h, 48 h, 72 h respectively) in atemporal manner as compared to untreated control (0.09%). Figure 1. Structure and Mass spectral study of Iron Complex.  (A)Chemical Structure of iron complex, iron (II) N-(2-hydroxyacetophenone)glycinate (FeNG). (B) Mass fragments of FeNG.doi:10.1371/journal.pone.0011253.g001Iron Complex Induces ApoptosisPLoS ONE | 3 June 2010 | Volume 5 | Issue 6 | e11253  In addition, FeNG induced apoptosis was also determined bycell cycle analysis of PI stained CEM/ADR5000 cell line byflowcytometry after FeNG treatment. It was found that (fig. 4B),the increase in the counts of sub diploidal (sub G1/G0) cells in atime dependent way as compared to untreated control. FeNG induced apoptosis involves mitochondriamediated pathway in CEM/ADR5000 cell line Since apoptotic cell death may be actuated through the extrinsic(transmembrane death receptor mediated) or the intrinsic(mitochondria mediated) pathway, we enquired which pathwaywas involved in FeNG induced cell death. To look into thisquestion, we treated CEM/ADR5000 cells with FeNG for 24 h,48 h, and 72 h and FasR expression on cell were ascertained byflowcytometry. Fig. 5A showed that FeNG was not able to induceFasR expression on CEM/ADR5000 cells. On the contrary Figure 2. Comparison of the cytotoxic effect of iron complex on different cell types.  Dose response curves for iron complex (FeNG) using(A) CEM/ADR5000 (B) CCRF-CEM, and (C) Human PBMC cells, as assessed by MTT assay. Cells were seeded into 96-well plates (4 6 10 4 cells/well) andallowed to overnight incubation at 37 u C in 5% CO 2  incubator. Next day, cells were treated with increasing concentrations of FeNG for 24 h, 48 h, and72 h incubation. Results are expressed as percentage viability of solvent-treated control cells. Value represents the mean 6 SD of three independentexperiments with four replicates in each.doi:10.1371/journal.pone.0011253.g002 Table 1.  IC 50  Values of FeNG for CEM/ADR5000, CCRF-CEMand Human PBMC. IC 50  values (mM ± SD)Compound CEM/ADR5000 CCRF-CEM Human PBMC FeNG 0.75 6 0.06 0.79 6 0.11 Not determinedAnti-proliferative activity of FeNG was determined using CEM/ADR5000, CCRF-CEM, and Human PBMC following 72 h continuous incubation. All the data arerepresentative of three similar experiments. Values represent mean  6 S.D.doi:10.1371/journal.pone.0011253.t001 Table 2.  Calculation of Resistance factor for FeNG. IC 50  values (mM ± SD)Compound CEM/ADR5000 CCRF-CEM Resistance factor FeNG 0.75 6 0.06 0.79 6 0.11 0.95Doxorubicin 0.00025 6 0.0001 0.1 6 0.009 400 * Anti-proliferative activity and resistance factor used to confirm multi-drugresistance phenotype and demonstrating whether iron complex (FeNG) was asubstrate for P-glycoprotein. The resistance factor was calculated by division of the IC 50  for the drug resistance CEM-ADR 5000 cell line by the IC 50  for the drugsensitive CCRF-CEM cell line. Results presented are representative of threeindependent experiments.*Reference[13].doi:10.1371/journal.pone.0011253.t002 Iron Complex Induces ApoptosisPLoS ONE | 4 June 2010 | Volume 5 | Issue 6 | e11253  increased expression of FasR was detected on CEM/ADR5000cells after 72 h treatment of 100 pg/ml of human recombinantIFN- c . This data indicated that the extrinsic pathways might notbe involved in FeNG mediated apoptosis.Cell death through the mitochondrion involves an increase inmitochondrial permeability transition that results in the release of cytochrome  c   and downstream activation of effector caspases.The increase in mitochondrial permeability transition is accom-panied by a collapse in mitochondrial membrane potential (  DY m  )[16,17] that can be measured by JC-1 dye staining. In healthynonapoptotic cells, JC-1 is accumulated in mitochondria inproportion to inner membrane potential and form a ‘‘Jaggregates’’ that fluoresce red; however, with the loss of mitochondrial membrane potential, the dye remains in thecytoplasm where JC-1 exist as monomer that fluoresce green.The ratio of red to green fluorescence provides a measure of  DY m . After exposure to FeNG for 2 h to 6 h there was a timedependent increase in  DY m , indicating that mitochondria werehyperpolarized followed by a decline in  DY m  which was detectedat around 12 h (fig. 5B). Our data indicating that exposure toFeNG results in a biphasic change in  DY m  with an early hyperpolarization, followed by a later depolarization and  DY m collapse.Mitochondrial swelling induced by permeability transition isknown to cause the outer membrane rupture and followed byrelease of cytochrome c from mitochondria to cytosol [18]. Toanalyse the involvement of mitochondria in the apoptosis inducedby FeNG, a cytochrome c release assay was performed. Asillustrated in the fig. 5C, FeNG treatment induced the release of cytochrome c to cytosol as detected by western blot analysis of cytosolic fraction. The intensity of immunoreactive band wasfound to increase in a time dependent fashion after FeNGtreatment (fig. 5D). Reactive oxygen species is critical for FeNG inducedapoptosis in CEM/ADR5000 cells The intrinsic pathway of apoptosis can be triggered by manystimuli including ROS. Mitochondria are the major site for ROS Figure 3. Changes in nuclear morphology of CEM/ADR5000 cells after FeNG treatment.  (A) Morphological changes of CEM/ADR5000 cellstreated with 0.75 6 10 2 3 M FeNG alone or in combination with 5 mM NAC (one hour prior to FeNG treatment). CEM/ADR5000 cells after treatmentswith drugs were fixed with 1% paraformaldehyde and stained with Hoechst 33258. The cells were observed under a fluorescence microscope.Apoptotic cells showed condensed or fragmented chromatin in the nucleus (arrowhead). (B) Represents the temporal kinetics of apoptoticpercentage of CEM/ADR5000 cells. Cells were treated with FeNG alone or in combination with 5 mM NAC for the indicated times. After treatment,cells were harvested and stained with Hoechst 33258. Apoptotic cells were examined by counting the cells with condensed and fragmented nuclei.Each point represents an average of three independent experiments, and standard deviation bars are shown.doi:10.1371/journal.pone.0011253.g003Iron Complex Induces ApoptosisPLoS ONE | 5 June 2010 | Volume 5 | Issue 6 | e11253
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