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Jun 12, 2014 - Previously, we have demonstrated that mitochondrial targeting of DNA damaging agents is a promising tool for evading a number of common...
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Structural Modifications of Mitochondria-Targeted Chlorambucil Alter Cell Death Mechanism but Preserve MDR Evasion Sae Rin Jean,† Mark P. Pereira,‡ and Shana O. Kelley*,†,‡,§ †

Department of Chemistry, Faculty of Arts and Science, ‡Department of Biochemistry, Faculty of Medicine, and §Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario Canada S Supporting Information *

ABSTRACT: Multidrug resistance (MDR) remains one of the major obstacles in chemotherapy, potentially rendering a multitude of drugs ineffective. Previously, we have demonstrated that mitochondrial targeting of DNA damaging agents is a promising tool for evading a number of common resistance factors that are present in the nucleus or cytosol. In particular, mitochondria-targeted chlorambucil (mt-Cbl) has increased potency and activity against resistant cancer cells compared to the parent compound chlorambucil (Cbl). However, it was found that, due to its high reactivity, mt-Cbl induces a necrotic type of cell death via rapid nonspecific alkylation of mitochondrial proteins. Here, we demonstrate that by tuning the alkylating activity of mt-Cbl via chemical modification, the rate of generation of protein adducts can be reduced, resulting in a shift of the cell death mechanism from necrosis to a more controlled apoptotic pathway. Moreover, we demonstrate that all of the modified mt-Cbl compounds effectively evade MDR resulting from cytosolic GST-μ upregulation by rapidly accumulating in mitochondria, inducing cell death directly from within. In this study, we systematically elucidated the advantages and limitations of targeting alkylating agents with varying reactivity to mitochondria. KEYWORDS: multidrug resistance (MDR), drug delivery, mitochondria, chlorambucil (Cbl), alkylating activity

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INTRODUCTION A major challenge in the area of cancer chemotherapy is combating multidrug resistance (MDR). MDR can be caused by a number of cellular changes including overexpression of cellular efflux pumps and xenobiotic modifying proteins, upregulation of cellular repair pathways, and disabled apoptotic triggering.1−3 This resistant phenotype may be intrinsic to the cancer cell, or may be acquired during the course of treatment necessitating increased dose concentration and frequency in order to maintain the effectiveness of the therapyoften leading to high levels of compound toxicity.4,5 In order to circumvent this problem, numerous strategies have been explored to overcome MDR including small molecule modulators for drug efflux pumps, nanoparticle drug delivery systems, stimulus-responsive drug delivery systems, or targeting of the expression of MDR proteins with RNAi.4,6−8 We recently developed a peptide-based drug delivery system that represents a new approach to combating MDR. The peptides control the intracellular localization of various bioactive cargo, specifically directing these molecules to mitochondria. These mitochondria-penetrating peptides (MPPs) are nontoxic, are stable in cells and animals, and can carry a wide variety of molecules into this organelle.9−11 Using this delivery system, it was determined that clinically used anticancer drugs such as chlorambucil (Cbl),9 doxorubicin,12 and cisplatin13 could be redirected from cytoplasmic and nuclear targets to specifically accumulate within the mitochondria of cells to act on alternate targets within this organelle.11 For each compound, it was © 2014 American Chemical Society

established that certain cellular resistance mechanisms specific to the particular drug, as well as those associated with MDR, could be overcome. This preservation of activityeven in the presence of resistance factorsis possible due to the sequestration of the compound in mitochondria thereby avoiding modifying or efflux proteins or interacting with alternate targets that are not affected by the overexpressed repair mechanisms.9,12,13 While it was determined that mitochondrial accumulation and sequestration can have a positive effect on the evasion of cellular resistance mechanisms, recent work has demonstrated that selective accumulation and sequestration of a small molecule drug within the mitochondrion can profoundly alter its mechanism of action.14 A very interesting effect was observed with the mitochondria-targeted version of Cbl (mt-Cbl(Cl/Cl)). While this compound displayed a large increase in potency compared to the parent compound Cbl and favorable in vivo efficacy, in vitro studies suggested that the major drug target and cell death mechanism had been altered.14 It was observed that redirection of Cbl, a highly reactive nitrogen mustard, to mitochondria triggered a rapid, necrotic cell death that appeared to be caused by the formation of nonspecific protein Special Issue: Drug Delivery and Reversal of MDR Received: Revised: Accepted: Published: 2675

February 3, 2014 June 2, 2014 June 12, 2014 June 12, 2014 dx.doi.org/10.1021/mp500104j | Mol. Pharmaceutics 2014, 11, 2675−2682

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Ethyl acetate (1.5 mL) was used to extract Cbl-NO2 from the reaction mixture. The organic phase was washed with saturated NaCl solution (3 × 2 mL) and dried using a centrifugal evaporator to yield Cbl-NO2 in 95% yield (0.529 g, 1.52 mmol). 1 H NMR (500 MHz, CDCl3): δ ppm 11.25 (s, 1H), 7.21 (d, J = 8.6 Hz, 1H), 7.19 (d, J = 2.8 Hz, 1H), 6.88 (dd, J = 8.6, 2.9 Hz, 1H), 3.77 (t, J = 7.0 Hz, 4H), 3.68−3.62 (m, 4H), 2.85−2.79 (m, 2H), 2.42 (t, J = 7.4 Hz, 2H), 1.99−1.92 (m, 2H) (Figure S2 in the Supporting Information). 13C NMR (126 MHz, CDCl3): δ ppm 179.61, 150.19, 145.19, 133.19, 124.70, 116.71, 107.58, 53.37, 40.18, 33.43, 31.18, 25.55 (Figure S3 in the Supporting Information). DART (positive ion mode) MS: m/z = 349.1. Synthesis and Characterization of mt-Cbl(Cl/Cl), mtCbl-NO2(Cl/Cl), and mt-Cbl(OH/Cl). Peptides were synthesized using a Prelude peptide synthesizer (Protein Technologies, Inc.) as described previously.14 FMOC-(Fxr)3 (50 μmol) on Rink amide resin was swelled in DMF (1 mL, N,N-dimethylformamide) for 20 min. The N-terminal FMOC was deprotected in piperidine (20% (v/v)) in DMF (2 × 1 mL, Protein Technologies, Inc.) for 20 min and washed with DMF (2 × 1 mL). A solution of Cbl or Cbl-NO2 (2 equiv), HBTU (4 equiv, O-(benzotriazol-l-yl)N,N,N′,N′-tetramethyluronium hexafluorophosphate, Protein Technologies, Inc.), and DIPEA (1.5 eq, N,N-diisopropylethylamine, Sigma-Aldrich) in DMF (1 mL) was added to the rinsed peptide on resin and was shaken at room temperature for 2 h. Peptides were then washed in DMF (1 mL), methanol (1 mL), and DCM (1 mL, dichloromethane) and dried under vacuum. A solution of TFA (trifluoroacetic acid, Sigma-Aldrich)/deionized water/TIPS (triisopropylsilane, Sigma-Aldrich) (95:2.5:2.5 (v/v)) were added to the dried resin and shaken at room temperature for 3 h. The cleaved peptide solution was drained into ice-cold diethyl ether (25 mL), and resulting precipitate was obtained as a solid by centrifugation at 2000 rpm for 5 min. Crude peptide conjugates were purified on a preparative RP-HPLC C18 column (mobile phase: acetonitrile (0.1% TFA) and deionized water (0.1% TFA)) and were dried using a rotary evaporator and a lyophilizer. To synthesize mt-Cbl(OH/Cl), mt-Cbl(Cl/Cl) was hydrolyzed in acetonitrile/deionized water (1:1) for 72 h and then purified on a preparative RP-HPLC C18 column. All peptide conjugates were characterized by electrospray ionization mass spectroscopy (ESI/MS) and quantified using a bincinchoninic acid (BCA) assay (Thermo Scientific). ESI/MS (positive ion mode): mt-Cbl(Cl/Cl), m/z = 1230.7; mt-Cbl-NO2(Cl/Cl), m/z = 1275.7; and mt-Cbl(OH/Cl), m/z = 1212.8. Synthesis and Characterization of TAMRA and Biotin (bt) Labeled Peptide Conjugates. FMOC-(Fxr)3K(Mtt) (50 μmol) on Rink amide resin was swelled in DMF (1 mL, N,N-dimethylformamide) for 20 min. Peptides were then washed in DMF (1 mL), methanol (1 mL), and DCM (1 mL, dichloromethane) and dried under vacuum. The Mtt protecting group on the C-terminal lysine was deprotected by adding 3% TFA in DCM (2 × 1 mL) to the peptide on resin and shaking for 30 min. A solution of 5(6)-TAMRA (2 equiv, carboxytetramethylrhodamine, AnaSpec Inc.), HBTU (4 equiv), and DIPEA (4 equiv) in DMF (1 mL) was added to the rinsed peptide on resin and was shaken at room temperature for 2 h or overnight. For bt-labeled peptides, Fmoc-Lys(Biotin)-OH (4 equiv, Anaspec, Inc.) was coupled to Rink amide resin in a solution of HBTU (4 equiv) and DIPEA (4 equiv) in DMF (1 mL) prior to synthesis of FMOC-(Fxr)3 on the peptide synthesizer. Cbl or Cbl-NO2 conjugation to the N-terminus of the labeled peptides was performed as described above for the unlabeled peptide conjugates. All TAMRA-labeled peptide conjugates were

adducts rather than DNA adducts. Interestingly, these changes were not observed for mitochondria-targeted cisplatin analogue (mt-Pt), a compound that reacts with DNA with slower kinetics.13 In this study, we sought to investigate the impact of modulation of mt-Cbl(Cl/Cl) reaction kinetics on activity and to reestablish an apoptotic mechanism of cell deaththereby limiting potential inflammation from necrosis of dying cells within a tumor.15 It has previously been established that the reactivity of Cbl is controlled by the chemical structure of the appended nitrogen mustard moiety.16 Taking this into consideration, we have synthesized a mt-Cbl(Cl/Cl) derivative modified with a strong electron withdrawing nitro group (mt-Cbl-NO2(Cl/Cl)) and a monoalkylating conjugate (mt-Cbl(OH/Cl)) where one of the alkylating groups was inactivated via partial hydrolysis (Figure 1). It was observed that these chemical modifications

Figure 1. Structures of mt-Cbl(Cl/Cl), mt-Cbl-NO2(Cl/Cl), and mt-Cbl(OH/Cl). Cbl and Cbl-NO2 were conjugated to the N-terminus of a mitochondria-penetrating peptide (MPP) composed of repeating units of L-cyclohexylalanine (Fx) and D-arginine (r) to permit their entry into mitochondria.

resulted in a marked decrease in the reactivity of mt-Cbl(Cl/Cl), but with potencies that remained in a clinically relevant range. Indeed, with attenuation of mt-Cbl(Cl/Cl) alkylation rates, the mechanism of cell death reverted back to a primarily apoptotic mechanism and fewer protein adducts were observed. Moreover, these novel compounds retained their ability to circumvent a MDR phenotype with only a marginal loss in potency compared to the parent mitochondria-targeted compound mt-Cbl(Cl/Cl).

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MATERIALS AND METHODS Synthesis of Cbl-NO2. Cbl (0.5 g, 1.6 mmol, Oakwood Products, Inc.) was dissolved in concentrated sulfuric acid (350 μL, 6.2 mmol) with stirring at room temperature. Concentrated nitric acid (100 μL, 1.8 mmol) and concentrated sulfuric acid (115 μL, 1.8 mmol) were added while the reaction mixture was stirred at 0 °C for 15 min and then for 1 h at room temperature. 2676

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quantified using a molar extinction coefficient of 92 000 M−1 cm−1 at 547 nm in methanol, and bt-labeled peptide conjugates were quantified using a BCA assay. ESI/MS (positive ion mode): mtCbl(Cl/Cl)-TAMRA, m/z = 1771.0; mt-Cbl-NO2(Cl/Cl)TAMRA, m/z = 1816.0; mt-Cbl(OH/Cl)-TAMRA, m/z = 1753.0; mt-Cbl(Cl/Cl)-bt, m/z = 1585.0; mt-Cbl-NO2(Cl/Cl)bt, m/z = 1630.0; and mt-Cbl(OH/Cl)-bt, m/z = 1567.0. Colorimetic Alkylation Assay. A modified method of the 4-(4-nitrobenzyl)pyridine (NBP) assay described previously was used to measure the rate of alkylation of mt-Cbl derivatives.17 An appropriate amount of each compound was dissolved in deionized water (4 × 50 μL, 1 sample per time point) and added to 0.7% (w/v) 4-NBP in acetone/2% triethanolamine in water (1:1, 180 μL). Each mixture was incubated at 37 °C for a specific time point (30, 60, 90, 120 min) in triplicate. Immediately after each time point, the reaction was terminated by freezing the solution in a dry ice/ethanol bath. Each solution was heated to room temperature and mixed vigorously with 25 μL of 5 M NaOH and 100 μL of ethyl acetate. The absorbance of the organic acetate phase was measured at 540 nm. Cell Culture. HeLa cells were cultured in Minimum Essential Medium alpha (MEMα, Invitrogen) supplemented with 10% (v/v) fetal bovine serum (FBS, Invitrogen). A2780 wild-type (WT) and Cbl-resistant (CblR) cell lines were cultured in RPMI 1640 (Invitrogen) supplemented with 10% (v/v) FBS. Resistance was maintained by incubating A2780 CblR cells with 100 μM Cbl for 1 h on a weekly basis. All cell lines were incubated at 37 °C with 5% CO2. Live Cell Imaging. 5000 HeLa cells per well were seeded in 8-well μ-slides (iBidi) 1 day prior to treatment. Cells were washed once with serum-free MEMα and then treated with TAMRA-labeled peptide conjugates by serial dilution and incubated at 37 °C with 5% CO2 for 30 min. MitoTracker Deep Red (100 nM, Invitrogen) was added during the last 10 min of the incubation. Cells were then washed three times with MEMα and then imaged on an inverted Zeiss Observer.Z1 fluorescence microscope. Cell Viability Assay. 4000 cells per well were seeded in 96-well flat-bottom tissue culture plates (Sarstedt) 1 day prior to treatment. Cells were washed with appropriate media once before treatment with various concentrations of each compound by serial dilution. The treated cells were incubated for 90 min, 24 h, or 72 h at 37 °C with 5% CO2. Following each time point, cells were washed once with appropriate serum-free media, and then 10 μL of CCK-8 viability dye (Dojindo) and 90 μL of media were added to each well. Absorbance at 450 nm was then measured upon color development. Detection of Modified Mitochondrial Proteins. HeLa cells were plated to 80% confluency in T75 cm2 flask with vented caps. Cells were incubated with 2 μM bt-labeled peptide conjugates for 30 min at 37 °C with 5% CO2. Cells were then harvested using 0.25% Trypsin-EDTA (Invitrogen), and their mitochondria were isolated using Mitochondrial Isolation Kit for Cultured Cells (Thermo Scientific) as per the manufacturer’s instructions. The mitochondrial pellets were resuspended in RIPA buffer plus protease inhibitors (Cell Signaling Technology) and placed on ice for 30 min with intermittent vortexing. The total mitochondrial protein concentration was quantified using a 660 nm protein assay (Thermo Scientific). Mitochondrial protein lysates (15 μg) were loaded onto 4−15% polyacrylamide gel (Biorad), and standard Western blotting procedure was followed. (1:1000) VDAC1/Porin rabbit antibody (Abcam) and (1:2000) Biotin mouse antibody (Jackson-ImmunoResearch)

primary antibodies were diluted in 2.5% BSA-TBST and incubated with the membrane for 1 h at room temperature or overnight at 4 °C. (1:200) goat anti-rabbit IgG-HRP antibody (Abcam) and (1:1000) donkey anti-mouse IgG-HRP antibody (Cell Signaling Technology) secondary antibodies were diluted in 2.5% BSA-TBST and incubated with the membrane for 1 h at room temperature. The chemiluminescence signal was detected on a VersaDoc Imager following exposure of the membrane to ECL Western blotting substrate (Thermo Scientific). Detection of Cytochrome c Release. HeLa cells were plated to 80% confluency in 4 T75 cm2 flask with vented caps. Each flask was incubated with 500 μM Cbl, 2 μM mt-Cbl(Cl/Cl), 2 μM mt-Cbl-NO2(Cl/Cl), or 4 μM mt-Cbl(OH/Cl) for 6 h at 37 °C with 5% CO2. Cells were then harvested using 0.25% Trypsin-EDTA (Invitrogen), and nuclear, cytoplasmic, and mitochondrial fractions were obtained using Mitochondrial Isolation Kit for Cultured Cells (Thermo Scientific) as per the manufacturer’s instructions. The cytosolic protein concentration was quantified using a 660 nm protein assay (Thermo Scientific). Cytosolic protein lysates (40 μg) were loaded onto 4−15% polyacrylamide gel (Biorad), and standard Western blotting procedure was followed. (1:500) cytochrome c rabbit antibody (Cell Signaling Technology) and (1:1000) GAPDH rabbit antibody (Cell Signaling Technology) primary antibodies were diluted in 2.5% milk-TBST and incubated with the membrane for 1 h at room temperature or overnight at 4 °C. (1:1000) anti-rabbit IgG-HRP antibody (Cell Signaling Technology) secondary antibody was diluted in TBST and incubated with the membrane for 1 h at room temperature. The chemiluminescence signal was detected on a VersaDoc Imager following exposure of the membrane to ECL Western blotting substrate (Thermo Scientific). Annexin V Assay. 20000 HeLa cells per well were seeded in a 24-well flat-bottom tissue culture plate (BD Falcon) 1 day prior to treatment. Cells were washed with MEMα once before treatment with various concentrations of each compound by serial dilution. The treated cells were incubated for 90 min, 6 h, or 24 h at 37 °C with 5% CO2. The steps outlined in the manufacturer’s instructions for annexin V-FITC (Invitrogen) were followed using a SYTOX Red dead cell stain (Invitrogen). Cells were incubated with annexin V-FITC in annexin binding buffer (100 μL, 5% (v/v), Invitrogen) and incubated for 10 min at room temperature. Subsequently, SYTOX Red in annexin binding buffer (400 μL, final concentration of 5 nM) was added and incubated for 10 additional min at room temperature. Immediately following incubation, a minimum of 10000 cells per sample were analyzed by flow cytometry using FACSCanto flow cytometer (BD Biosciences). Assessment of Caspase 3/7 Activity. 4000 HeLa cells per well were seeded in white opaque flat-bottom 96-well plates (Greiner Bio-one) 1 day prior to treatment. Cells were washed once with MEMα before treatment with various concentrations of each compound by serial dilution. Cells were incubated for 6 h at 37 °C with 5% CO2, and then caspase 3/7 activity was measured using Caspase-Glo 3/7 Assay (Promega) as per the manufacturer’s instructions. TUNEL Assay. 200000 cells per well were seeded in a 12-well flat-bottom tissue culture plate (BD Falcon) 1 day prior to treatment. Cells were washed with appropriate media once before treatment with various concentrations of each compound by serial dilution. The treated cells were incubated for 6 h at 37 °C with 5% CO2. The manufacturer’s instructions in APO-DIRECT Flow Cytometry Kit for Apoptosis (Milipore) or In situ Direct DNA Fragmentation (TUNEL) Assay Kit (Abcam) were followed. Immediately following incubation, a 2677

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Figure 2. Subcellular localization of TAMRA labeled mt-Cbl(Cl/Cl), mt-Cbl-NO2(Cl/Cl), and mt-Cbl(OH/Cl). Cells were stained with 2 μM of each compound. A characteristic mitochondrial staining was observed with each of the TAMRA labeled compounds (green) consistent with the commercially available mitochondrial stain Mitotracker Deep Red (red).

minimum of 10000 cells per sample were analyzed by flow cytometry using FACSCanto flow cytometer (BD Biosciences).

for structures) and the compounds were incubated with a mitochondria-specific dye, MitoTracker Deep Red, in live HeLa cells. Each compound displayed a characteristic mitochondrial staining pattern consistent with the MitoTracker dye, confirming that the MPP vector can effectively drive the accumulation of the modified compounds into mitochondria (Figure 2). To determine if the modifications would have an effect on the alkylating activity, we first studied the rate of alkylation using a chemical model of 7-alkylguanine, 4-(4-nitrobenzyl)pyridine (NBP).17 The greatest reduction in the rate of alkylation was observed with mt-Cbl(OH/Cl), and a more moderate reduction for mt-Cbl-NO2(Cl/Cl) was observed using this assay compared to the original mt-Cbl(Cl/Cl) (Figure 3A). This data is consistent with the expected impact on the chemical reactivity of the derivatives. Next, we investigated the effect of reducing the alkylating activity on the rate of induction of cell death. It is established that necrosis occurs on a much more rapid time frame in comparison to apoptosis, which involves a cascade of biological events that systematically eliminates the cell over a longer period of time.23−25 By testing the cytotoxicity of each mt-Cbl conjugate at different time points (90 min, 24 h, and 72 h), a decline in LC50 values over time would then provide evidence that an apoptotic type of cell death was operative, whereas LC50 values that did not decrease would suggest a necrotic type of cell death. Interestingly, LC50 values declined from 90 min to 72 h for mt-Cbl-NO2(Cl/Cl) (3.7 μM to 2.1 μM) and mt-Cbl(OH/Cl) (12 μM to 5 μM), suggesting a slow, apoptotic type of cell death (Figure 3B). In the case of mt-Cbl(Cl/Cl), the LC50 did not decrease with time, consistent with a necrotic mechanism (Figure 3B). Previous studies have demonstrated that when Cbl is redirected to mitochondria, its main cellular target shifts from DNA to protein.14 We sought to investigate if the decrease in LC50 values over time is linked to the reduction of mitochondrial protein alkylation. To test this, a biotin (bt) labeled conjugate of mt-Cbl and its derivatives were synthesized (see Figure S1 in the

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RESULTS AND DISCUSSION Reducing the Alkylating Activity of MitochondriaTargeted Cbl Conjugates Alters the Kinetics of Cell Death. The driving hypothesis of this study was that, through a carefully tuned reduction of alkylating activity of mt-Cbl(Cl/Cl), the potency and the MDR-evading properties of the drug could be retained, but with activity that would not cause the rapid necrotic cell death. It was postulated that slower reaction kinetics would limit the rapid nonspecific reactions of mt-Cbl with mitochondrial proteins and permit a more controlled apoptotic mechanism of cell death to dominate. The electron density within the aromatic ring of Cbl has a key role in modulating the rate-limiting formation of the highly reactive aziridinium ring.16,18,19 Therefore, one possibility to decrease the reactivity is to add a strong electron-withdrawing substituent in the ortho position of the nitrogen mustard moiety on the aromatic ring to further reduce the nucleophilicity of the nitrogen.16,20 An alternate compound with decreased alkylating activity was designed that featured partial hydrolysis of the nitrogen mustard moiety to inactivate one of the chloroethyl groups to identify the importance of cross-linking activity.21,22 In order to test whether these changes would trigger the desired changes in the alkylating activity, a nitro derivative (mt-Cbl-NO2(Cl/Cl)) and one featuring an inactivated chloroethyl group (mt-Cbl(OH/Cl)) were synthesized and studied to explore this hypothesis. In previous studies of mt-Cbl(Cl/Cl), we demonstrated the compound’s ability to localize within mitochondria using confocal microscopy.9 We first sought to verify the mitochondrial localization of the newly synthesized mt-Cbl derivatives in order to determine if the new alkylating agents maintained their localization within mitochondria. To study this, a carboxytetramethylrhodamine (TAMRA) fluorophore was conjugated to a C-terminal lysine of mt-Cbl(Cl/Cl), mt-Cbl-NO2(Cl/Cl), and mt-Cbl(OH/Cl) (see Figure S1 in the Supporting Information 2678

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Figure 3. Modifications of mt-Cbl alter chemical reactivity and biological activity. (A) Alkylating activity is reduced with mt-Cbl-NO2(Cl/Cl) and mtCbl(OH/Cl) compared to the original mt-Cbl(Cl/Cl). Student’s t test, versus mt-Cbl(Cl/Cl), *p < 0.01. (B) LC50 values for compounds in HeLa cells at various time points (90 min, 24 h, and 72 h). A decline in LC50 values is observed with mt-Cbl-NO2(Cl/Cl) and mt-Cbl(OH/Cl) over time. LC50 values of mt-Cbl-(Cl/Cl) remain unchanged. Student’s t test, versus 90 min, *p < 0.05; versus 24 h, °p < 0.05. (C) Degree of protein alkylation correlates to the rate of alkylation of mitochondria-targeted compounds. HeLa cells were treated with 2 μM of each biotin (bt)-labeled compound, and then mitochondrial protein lysates were immunoblotted. Reduction in overall mitochondrial protein alkylation is observed in mt-Cbl-NO2(Cl/Cl) and mtCbl(OH/Cl) compared to the original mt-Cbl(Cl/Cl). Lane 1: Untreated. Lane 2: Treated with mt-Cbl(Cl/Cl)-bt. Lane 3: Treated with mt-CblNO2(Cl/Cl)-bt. Lane 4: Treated with mt-Cbl(OH/Cl)-bt. The VDAC protein is shown as a loading control. Mean values are plotted for all experiments, and error bars represent SEM.

not show this pattern evolving as a function of time, and the rapid appearance of A-FITC+/SR+ cells is consistent with necrosis being the dominant cell death pathway (Figure 4A). However, Cbl, mt-Cbl-NO2(Cl/Cl), and mt-Cbl(OH/Cl) all showed an apoptotic population of cells that was much more pronounced at later time points (6 and 24 h), consistent with this being the true mechanism of cell death. Additionally, we tested for cytochrome c release as a marker for apoptosis (Figure S4 in the Supporting Information). Interestingly, we observed cytochrome c in the cytosol for all of the mt-Cbl conjugates as well as the parent compound Cbl. The release of cytochrome c from the intermembrane space of mitochondria to cytosol is the result of mitochondrial membrane permeabilization that occurs in both necrosis and apoptosis; thus other events further downstream were subsequently studied to characterize the mechanism of cell death of mt-Cbl conjugates.29,30 We next tested if the mechanism of apoptosis induced by mt-Cbl-NO2(Cl/Cl) and mt-Cbl(OH/Cl) is a classical caspase-dependent type of apoptosis or caspase-independent.14,31,32 In contrast to the parent compound Cbl, the mechanism of apoptosis appeared to be caspase-independent (Figure 4B). To confirm that the mt-Cbl derivatives are triggering apoptosis, we looked for the presence of nuclear DNA (nDNA) degradation using a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay.27,28,33 This particular apoptotic marker is present in both caspase-dependent and independent types of apoptosis.32,34 Flow cytometry analysis of TUNEL stained cells (positive for nDNA degradation) was used to measure the degree of apoptosis triggering. Treatment of HeLa cells with mt-CblNO2(Cl/Cl) and mt-Cbl(OH/Cl) for 6 h resulted in a dosedependent increase in TUNEL positive cells similar to the Cbl positive control, whereas no increase in TUNEL staining was observed for mt-Cbl(Cl/Cl) compared to the untreated negative

Supporting Information for structures). Following treatment of HeLa cells with equimolar concentration of each bt-labeled conjugate, mitochondrial lysate was obtained and immunoblotted to observe and compare levels of protein adducts. Indeed, there were fewer protein adducts formed with mt-Cbl-NO2(Cl/Cl) and mt-Cbl(OH/Cl) compared to mt-Cbl(Cl/Cl) (Figure 3C). By reducing the alkylating activity of mt-Cbl(Cl/Cl), we were able to control the degree of covalent modification of its major target, possibly limiting the rapid, nonspecific alkylation of nearby proteins. Induction of Apoptosis Is Observed with Mitochondria-Targeted Cbl Conjugates with Reduced Alkylating Activity. In general, drugs that induce apoptosis are preferred over ones that induce necrosis. The main reason for this is that necrosis causes a debilitating systemic or localized inflammatory response in vivo, which ultimately limits the efficacy of the drug.26 We hypothesized that by reducing the reactivity of mt-Cbl(Cl/Cl) conjugates we can generate alkylated adducts at a slower rate without overwhelming the cell and revert the dominant mechanism of action from necrosis to apoptosis. In order to determine if an apoptotic cell death mechanism was reestablished for the mt-Cbl derivatives with reduced alkylating activity, we tested for various apoptotic markers using Cbl as the positive control. One of the universal apoptotic markers that is commonly assessed is the externalization of phosphotidylserine residues on the plasma membrane that occurs during the early stages of apoptosis.27,28 Here, we used fluorescein labeled annexin V (A-FITC) and a counter dye SYTOX Red (SR) that detects membrane-comprised cells to be able to distinguish the various stages of apoptosis. Typically for an apoptotic mechanism of cell death, migration of cells from live (A-FITC−/SR−) to early apoptosis (A-FITC+/SR−) to late apoptosis (A-FITC+/SR+) is observed over time.25 Treatment of cells with mt-Cbl(Cl/Cl) did 2679

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Figure 4. Reducing the reactivity of mitochondria-targeted Cbl shifts cell death mechanism from necrosis to apoptosis in HeLa cells. (A) Analysis of annexin V-FITC and SYTOX Red staining. Slow migration of cell population from live to early apoptosis to late apoptosis is apparent for Cbl, mt-CblNO2(Cl/Cl), and mt-Cbl(OH/Cl) over time (90 min, 6 h, and 24 h), whereas mt-Cbl(Cl/Cl) induces rapid necrosis which results in no migration after 90 min. (B) Assessment of caspase dependence of cell death. Treatment of HeLa cells for 6 h with mitochondria-targeted compounds did not result in an increase in caspase 3/7 activity compared to the untreated cells. The parent compound Cbl follows a dose-dependent increase in caspase 3/7 activity. Student’s t test, versus untreated, *p < 0.01. (C) Flow cytometry analysis of TUNEL staining. Following a 6 h treatment of HeLa cells, mt-Cbl(Cl/Cl) shows no increase in TUNEL positive cells compared to the untreated cells. Dose dependent increase in TUNEL positive cells induced by Cbl, mt-CblNO2(Cl/Cl), and mt-Cbl(OH/Cl), consistent with an apoptotic cell death mechanism. Student’s t test, versus untreated, *p < 0.05. Mean values are plotted for all experiments, and error bars represent SEM.

control (Figure 4C). This finding provides evidence that the mtCbl derivatives with reduced alkylating activity rely on a primarily apoptotic pathway to initiate cell death. It is also interesting to note that the lack of cross-linking activity seen with the monoalkylating mt-Cbl(OH/Cl) has a profound effect on moderating the cell death profile rather than simply having the same activity of mtCbl(Cl/Cl) at half the concentration. The bifunctional alkylating activity of mt-Cbl(Cl/Cl) appears to be important in inducing a necrotic type of cell death, possibly through intramolecular and intermolecular cross-linking of proteins and also of proteins to mtDNA, resulting in aggregation and loss of function.35

Mitochondrial Targeting Evades Resistance Resulting from Overexpression of Cytosolic GST-μ. Cbl, like most chemotherapeutics, is susceptible to drug inactivation or efflux.36,37 One of the main resistance mechanisms that diminishes the efficacy of Cbl in particular is the upregulation of cytoplasmic glutathione S-transferase (GST)-μ, which conjugates glutathione to Cbl and facilitates the export of Cbl out of the cells via efflux pumps present in the plasma membrane.38,39 We hypothesized that this common resistance mechanism could be overcome by sequestrating the Cbl derivatives in mitochondria, away from cytosolic GST-μ and 2680

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promising method to effectively evade cytosolic drug inactivation and efflux.

efflux pumps. To test this hypothesis, TUNEL staining of wildtype (A2780 WT) and GST-μ overexpressing Cbl-resistant (A2780 CblR) ovarian cancer cell lines was analyzed to evaluate if the mt-Cbl conjugates are able to retain their ability to trigger apoptosis in a Cbl-resistant cell line (Figure 5). In this study, the

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CONCLUSIONS In summary, we have demonstrated that by tuning the alkylating activity of the mitochondria-targeted anticancer agent Cbl (mt-Cbl)(Cl/Cl), we are able to modulate its mechanism of cell death. Moreover, mitochondrial targeting can effectively evade cytosolic drug inactivation via mitochondrial sequestration as displayed by the potent cytotoxic activity of mt-Cbl(Cl/Cl) and its derivatives in both wild-type and Cbl-resistant cell lines. Taken together, these findings can be used to determine appropriate drug candidates that can be combined with mitochondrial targeting to invoke a desirable activity profile.

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ASSOCIATED CONTENT

S Supporting Information *

Additional characterization data. This material is available free of charge via the Internet at http://pubs.acs.org.

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Figure 5. Mitochondrial sequestration overcomes GST-μ upregulation. A2780 WT and CblR cell lines were treated with each compound for 6 h. TUNEL staining revealed that the parent compound Cbl only induces apoptosis in A2780 WT cells. mt-Cbl-NO2(Cl/Cl) and mt-Cbl(OH/ Cl) are capable of inducing apoptosis in both A2780 WT and CblR cell lines. Student’s t test, versus untreated, *p < 0.01. Mean values are plotted (n = 3), and error bars represent SEM.

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Work in the Kelley Laboratories is supported by the Canadian Institutes of Health Research (CIHR).

parent compound showed a 9-fold reduction in apoptosis triggering in the A2780 CblR cell line compared to A2780 WT. In contrast, mt-Cbl-NO2(Cl/Cl) and mt-Cbl(OH/Cl) were able to induce apoptosis in a dose-dependent manner in both cell lines to a similar degree. This finding was significant since it indicates that rapid accumulation of mitochondria-targeted compounds permits apoptosis triggering from directly within mitochondria in both wild-type and Cbl-resistant cell lines. Additionally, 24 h LC50 values of each compound in A2780 WT and CblR cells were compared (Table 1). In accordance with the

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LC50 (μM) A2780 WT

A2780 CblR

RF

117 ± 7 2.3 ± 0.4 1.5 ± 0.4 7.3 ± 1.2

>1000 2.4 ± 0.3 2.2 ± 0.1 13.7 ± 0.3

>8.5 1.0 1.5 2.0

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Table 1. Summary of LC50 Values and Corresponding Resistance Factors in Wild-Type (A2780 WT) and CblResistant (A2780 CblR) Ovarian Cancer Cell Linesa,b

Cbl mt-Cbl(Cl/Cl) mt-Cbl-NO2(Cl/Cl) mt-Cbl(OH/Cl)

AUTHOR INFORMATION

Corresponding Author

a

Mean values are reported (n = 3), and the errors are SEM. bRF (resistance factor) = A2780 CblR LC50/A2780 WT LC50.

9-fold reduction of TUNEL staining observed in A2780 CblR cells compared to A2780 WT cells, the parent compound Cbl had a calculated resistance factor (RF) value of >8.5, suggesting that the cytotoxic activity of this compound is severely attenuated by the overexpression of GST-μ. However, for all of the mitochondria-targeted compounds, little to no attenuation was observed with RF values of ≤2. Taken together, these findings indicate that, irrespective of the type of the cell death that is induced, mitochondrial targeting of chemotherapeutics is a 2681

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