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- W2003238218 abstract "The antitumor agent 11β (CAS 865070-37-7), consisting of a DNA-damaging aniline mustard linked to an androgen receptor (AR) ligand, is known to form covalent DNA adducts and to induce apoptosis potently in AR-positive prostate cancer cells in vitro; it also strongly prevents growth of LNCaP xenografts in mice. The present study describes the unexpectedly strong activity of 11β against the AR-negative HeLa cells, both in cell culture and tumor xenografts, and uncovers a new mechanism of action that likely explains this activity. Cellular fractionation experiments indicated that mitochondria are the major intracellular sink for 11β; flow cytometry studies showed that 11β exposure rapidly induced oxidative stress, mitochondria being an important source of reactive oxygen species (ROS). Additionally, 11β inhibited oxygen consumption both in intact HeLa cells and in isolated mitochondria. Specifically, 11β blocked uncoupled oxygen consumption when mitochondria were incubated with complex I substrates, but it had no effect on oxygen consumption driven by substrates acting downstream of complex I in the mitochondrial electron transport chain. Moreover, 11β enhanced ROS generation in isolated mitochondria, suggesting that complex I inhibition is responsible for ROS production. At the cellular level, the presence of antioxidants (N-acetylcysteine or vitamin E) significantly reduced the toxicity of 11β, implicating ROS production as an important contributor to cytotoxicity. Collectively, our findings establish complex I inhibition and ROS generation as a new mechanism of action for 11β, which supplements conventional DNA adduct formation to promote cancer cell death. The antitumor agent 11β (CAS 865070-37-7), consisting of a DNA-damaging aniline mustard linked to an androgen receptor (AR) ligand, is known to form covalent DNA adducts and to induce apoptosis potently in AR-positive prostate cancer cells in vitro; it also strongly prevents growth of LNCaP xenografts in mice. The present study describes the unexpectedly strong activity of 11β against the AR-negative HeLa cells, both in cell culture and tumor xenografts, and uncovers a new mechanism of action that likely explains this activity. Cellular fractionation experiments indicated that mitochondria are the major intracellular sink for 11β; flow cytometry studies showed that 11β exposure rapidly induced oxidative stress, mitochondria being an important source of reactive oxygen species (ROS). Additionally, 11β inhibited oxygen consumption both in intact HeLa cells and in isolated mitochondria. Specifically, 11β blocked uncoupled oxygen consumption when mitochondria were incubated with complex I substrates, but it had no effect on oxygen consumption driven by substrates acting downstream of complex I in the mitochondrial electron transport chain. Moreover, 11β enhanced ROS generation in isolated mitochondria, suggesting that complex I inhibition is responsible for ROS production. At the cellular level, the presence of antioxidants (N-acetylcysteine or vitamin E) significantly reduced the toxicity of 11β, implicating ROS production as an important contributor to cytotoxicity. Collectively, our findings establish complex I inhibition and ROS generation as a new mechanism of action for 11β, which supplements conventional DNA adduct formation to promote cancer cell death. Adaptive responses to hypoxia and oxidative stress allow tumor cells to exist and grow under adverse conditions and to acquire therapeutic resistance, contributing to the failure of chemotherapy for prostate and other cancers (1Ruan K. Song G. Ouyang G. J. Cell. Biochem. 2009; 107: 1053-1062Crossref PubMed Scopus (336) Google Scholar). Strategies to overcome resistance include development of agents with multiple cytotoxic mechanisms; such strategies could include a primary toxicity mechanism (e.g. DNA damage) that is complemented by a secondary stressor, such as the intentional generation of oxidative stress or the inhibition of natural antioxidant enzymes (2Ozben T. J. Pharm. Sci. 2007; 96: 2181-2196Abstract Full Text Full Text PDF PubMed Scopus (556) Google Scholar). In fact, many clinically useful drugs have more than one mechanism of toxicity. Reactive oxygen species (ROS) 5The abbreviations used are: ROSreactive oxygen speciesARandrogen receptor11βcarbamic acid, [3-[4-[bis(2-chloroethyl)amino]phenyl]propyl]-,2-[[6-[(11β,17β)-17-hydroxy-3-oxoestra-4,9-dien-11-yl]hexyl]amino]ethyl ester (CAS 865070-37-7)11β-dimcarbamic acid, [3-[4-[bis(2-methoxyethyl)amino]phenyl]propyl]-,2-[[6-[(11β,17β)-17-hydroxy-3-oxoestra-4,9-dien-11-yl]hexyl]amino]ethyl ester (CAS 865070-39-9)CM-DCFoxidized form of CM-H2DCFDACM-DCFDA5-(and-6)-chloromethyl-2′,7′-fluorescein diacetateCM-H2DCFDA5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetateCTBCellTiter-BlueDMSOdimethyl sulfoxideETCelectron transport chainFCCP4-(trifluoromethoxy)phenylhydrazoneHBSSHanks' balanced salt solutionJC-15,5′,6,6′-tetrachloro-l,l′,3,3′-tetraethylbenzimidazol-carbocyanine iodideNACN-acetyl-l-cysteinePARPpoly(ADP-ribose) polymeraseTMPDN,N,N′,N′-tetramethyl-p-phenylenediamine. have been implicated to varying extents in the cytotoxic mechanisms of cisplatin (3Nowak G. J. Biol. Chem. 2002; 277: 43377-43388Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar), doxorubicin (4Ravid A. Rocker D. Machlenkin A. Rotem C. Hochman A. Kessler-Icekson G. Liberman U.A. Koren R. Cancer Res. 1999; 59: 862-867PubMed Google Scholar), bleomycin (5Wallach-Dayan S.B. Izbicki G. Cohen P.Y. Gerstl-Golan R. Fine A. Breuer R. Am. J. Physiol. Lung Cell Mol. Physiol. 2006; 290: L790-L796Crossref PubMed Scopus (183) Google Scholar), and etoposide (6Kapiszewska M. Cierniak A. Elas M. Lankoff A. Toxicol. in Vitro. 2007; 21: 1020-1030Crossref PubMed Scopus (29) Google Scholar). It is thought that ROS may be supplementing the primary mechanism of toxicity of each of these agents. reactive oxygen species androgen receptor carbamic acid, [3-[4-[bis(2-chloroethyl)amino]phenyl]propyl]-,2-[[6-[(11β,17β)-17-hydroxy-3-oxoestra-4,9-dien-11-yl]hexyl]amino]ethyl ester (CAS 865070-37-7) carbamic acid, [3-[4-[bis(2-methoxyethyl)amino]phenyl]propyl]-,2-[[6-[(11β,17β)-17-hydroxy-3-oxoestra-4,9-dien-11-yl]hexyl]amino]ethyl ester (CAS 865070-39-9) oxidized form of CM-H2DCFDA 5-(and-6)-chloromethyl-2′,7′-fluorescein diacetate 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate CellTiter-Blue dimethyl sulfoxide electron transport chain 4-(trifluoromethoxy)phenylhydrazone Hanks' balanced salt solution 5,5′,6,6′-tetrachloro-l,l′,3,3′-tetraethylbenzimidazol-carbocyanine iodide N-acetyl-l-cysteine poly(ADP-ribose) polymerase N,N,N′,N′-tetramethyl-p-phenylenediamine. The small molecule anticancer agent 11β (see Fig. 1A) was designed to kill androgen receptor (AR)-positive prostate cancer cells by targeting both DNA replication (via its aniline mustard moiety, which forms DNA adducts) and the expression of steroid-responsive genes (via its steroid ligand). The structure of 11β features the DNA-reactive p-N,N-bis-(2-chloroethly)aminophenyl group linked to an estradien-3-one ligand (see Fig. 1A), which has a high affinity for the AR (7Marquis J.C. Hillier S.M. Dinaut A.N. Rodrigues D. Mitra K. Essigmann J.M. Croy R.G. Chem. Biol. 2005; 12: 779-787Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). It was originally proposed that the DNA adducts formed by 11β could bind the AR and potentially disrupt tumor cell biology by two nonmutually exclusive mechanisms: (i) antagonism of AR-regulated gene expression and (ii) obstruction of the DNA repair process. Recent studies demonstrate that 11β induces apoptosis in AR+ prostate cancer cells, is stable in vivo, and effectively prevents the growth of AR+ LNCaP tumors as xenografts in mice (7Marquis J.C. Hillier S.M. Dinaut A.N. Rodrigues D. Mitra K. Essigmann J.M. Croy R.G. Chem. Biol. 2005; 12: 779-787Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 8Hillier S.M. Marquis J.C. Zayas B. Wishnok J.S. Liberman R.G. Skipper P.L. Tannenbaum S.R. Essigmann J.M. Croy R.G. Mol. Cancer Ther. 2006; 5: 977-984Crossref PubMed Scopus (15) Google Scholar). Although very effective against AR+ cells and tumors, 11β has surprisingly potent activity against AR− cells and tumors, leading to reconsideration of the role of the AR in the process leading to cell death. The present study is the result of the search for AR-independent mechanisms of 11β toxicity. Previous studies (7Marquis J.C. Hillier S.M. Dinaut A.N. Rodrigues D. Mitra K. Essigmann J.M. Croy R.G. Chem. Biol. 2005; 12: 779-787Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) revealed that 11β induces apoptosis at lower exposure levels and much more rapidly than DNA-damaging aniline mustard drugs such as chlorambucil (see Fig. 1A), which contains the same alkylating group. Within 6 h of treatment with >5 μm 11β, LNCaP cells undergo cytoplasmic contraction and detachment from the culture dish, whereas treatment with similar or even higher concentrations of chlorambucil has little effect on cell morphology and does not induce apoptosis (7Marquis J.C. Hillier S.M. Dinaut A.N. Rodrigues D. Mitra K. Essigmann J.M. Croy R.G. Chem. Biol. 2005; 12: 779-787Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). In addition, 11β-dim, a DNA-unreactive analog of 11β (see Fig. 1A), produces similar morphological changes but does not induce apoptosis in the same concentration range (7Marquis J.C. Hillier S.M. Dinaut A.N. Rodrigues D. Mitra K. Essigmann J.M. Croy R.G. Chem. Biol. 2005; 12: 779-787Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), further suggesting the possibility of additional mechanisms of cytotoxicity. The current study demonstrated that 11β potently induced apoptosis in AR-negative HeLa cells both in vitro and when grown as xenograft tumors in mice. Furthermore, both 11β and its analog, 11β-dim generated a burst of intracellular ROS, whereas in the same dose ranges, chlorambucil or the steroid moiety of 11β alone (estradien-3-one) did not. The functional role of ROS was evidenced by co-treatment with antioxidants, which reduced ROS formation and suppressed the cytotoxicity of both 11β and 11β-dim. Additional experiments indicated that mitochondria were the main intracellular sink for 11β and an important source of the ROS, which were produced due to the specific inhibition of complex I of the mitochondrial electron transport chain (ETC). Together, these findings established ROS production and complex I inhibition as new, DNA adduct-independent mechanisms of 11β that supplemented the ability of the compound to kill tumor cells by covalent DNA damage. The data also suggested that 11β could be active against a wide range of tumors, including those that do not express the AR and added support to the growing body of evidence that oxidative stress may synergize with conventional DNA adducts to promote cancer cell death. The compounds 11β and 11β-dim were synthesized as previously reported (7Marquis J.C. Hillier S.M. Dinaut A.N. Rodrigues D. Mitra K. Essigmann J.M. Croy R.G. Chem. Biol. 2005; 12: 779-787Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). [14C]11β was prepared as described in Ref. 8Hillier S.M. Marquis J.C. Zayas B. Wishnok J.S. Liberman R.G. Skipper P.L. Tannenbaum S.R. Essigmann J.M. Croy R.G. Mol. Cancer Ther. 2006; 5: 977-984Crossref PubMed Scopus (15) Google Scholar. Stock solutions of test compounds were prepared in DMSO and stored at −80 °C. All chemical reagents were purchased from Sigma-Aldrich unless indicated otherwise. 17β-Hydroxy-estra-4(5),9(10)dien-3-one (estradien-3-one) was obtained from Brighton Co., Chang Sha, Hunan, China. CM-H2DCFDA, CM-DCFDA, MitoSOX, and JC-1 molecular dyes were obtained from Molecular Probes. All cell media, media supplements, PBS and HBSS were from Invitrogen. CellTiter-Blue (CTB) reagent was purchased from Promega. HeLa cells were obtained from American Type Culture Collection (Rockville, MD) and maintained in minimal essential medium (MEM) supplemented with 1 mm GlutaMAX, 1% nonessential amino acids, 1 mm pyruvate, and 10% fetal bovine serum (Hyclone, Logan, UT), in a humidified 5% CO2/air atmosphere at 37 °C. Clonogenic survival assays were performed by seeding 1 × 103 HeLa cells/well in 6-well plates, followed by growth for 24 h to allow cell attachment. Test compounds dissolved in DMSO were added to growth medium for 24 h, replaced with fresh medium, and growth was continued for 3–5 days until colonies were clearly visible. Colonies were fixed with water:methanol:acetic acid (4:5:1), stained with 0.5% crystal violet, and counted manually. The surviving fraction was calculated as the ratio of the number of colonies in treated wells to the number in untreated wells. Cell viability was estimated using the CTB assay as described in the manufacturer's instructions. Briefly, 2.5 × 103 cells/well were seeded in black/clear-bottom 96-well plates (Corning Inc., Corning, NY) and incubated for 24 h. Medium was then replaced with fresh drug-containing medium for the indicated times. CTB reagent was then added for 2–4 h after which fluorescence (ex/em: 555/585 nm) was measured. Cell viability was calculated as the ratio between the average background corrected signal in the treated wells and the untreated control wells. Whole cell extracts of HeLa cells were prepared by lysis in radioimmune precipitation assay solution (RIPA) according to the manufacturer's instructions (Santa Cruz Biotechnology, Santa Cruz, CA). Equal amounts of protein were separated by SDS-PAGE and transferred to Immobilon-P membranes (Millipore). Membranes were probed with primary antibodies anti-caspase-3 (9665), anti-caspase-9 (9502), anti-PARP (9542), and secondary antibody anti-rabbit IgG-HRP (7074). All antibodies were from Cell Signaling Technology (Danvers, MA). Detection of antibody complexes was achieved by PicoWest chemoluminescence reagent (Pierce). Intracellular ROS levels were measured with fluorometric dyes. 1 × 105 HeLa cells were seeded in 6-well plates and grown for 24 h. After the cells were exposed to test compounds for the indicated times, and the cells were washed with PBS and exposed to a molecular probe solution in HBSS at 37 °C. The molecular probes and their final concentrations were: CM-H2DCFDA (2.5 μm), CM-DCFDA (2.5 μm), and MitoSOX (2 μm). After incubation for 15 min at 37 °C, the cells were washed, trypsinized, resuspended in HBSS, and analyzed on the FL1 channel (CM-H2DCFDA, CM-DCFDA) or the FL2 channel (MitoSOX) of a Becton Dickinson FACSCanto II flow cytometer. The results were analyzed with FACSDiva 6.0 software (BD Biosciences). Mitochondrial inner membrane potential (ΔΨm) was estimated using the mitochondria-specific dye JC-1 according to a published flow cytometry protocol (9Cossarizza A. Baccarani-Contri M. Kalashnikova G. Franceschi C. Biochem. Biophys. Res. Commun. 1993; 197: 40-45Crossref PubMed Scopus (945) Google Scholar). Briefly, after treatment, cells were incubated at 37 °C with a solution of JC-1 in medium without phenol red for 30 min, then trypsinized, resuspended in PBS, and analyzed on a FACSCanto II flow cytometer. The mitochondrial membrane potential was estimated by the ratio between the red (FL2) and green (FL1) fluorescence. The results were analyzed with FACSDiva 6.0 software (BD Biosciences). Cellular levels of H2O2 were measured using the Quantichrom Peroxide Assay (Bioassay Systems, Hayward, CA) according to the manufacturer's instructions. Lipid peroxidation was monitored using the OxiSelect TBARS assay (Cell Biolabs, San Diego, CA) as described in the manufacturer's instructions. Cellular NADPH content was measured using the EnzyChrom NADP/NADPH assay (BioAssay Systems) according to the manufacturer's instructions. In all assays, the results were normalized for protein content, determined using the Bradford reagent (Bio-Rad). Cells were grown on polylysine-coated coverslips, in 6-well plates. The seeding and treatment with test compounds were performed identically to the viability assay. After treatment, the cells were stained for 10 min at 37 °C with 1 μm MitoSOX and 5 μm Hoechst 33342 in HBSS, washed three times in fresh HBSS, and imaged with a Nikon Eclipse fluorescence microscope. The protocol was adapted from Ref. 10Frezza C. Cipolat S. Scorrano L. Nat. Protoc. 2007; 2: 287-295Crossref PubMed Scopus (824) Google Scholar with modifications. All steps were performed at 4 °C. 3 × 107 HeLa cells were detached by scraping, washed in cold PBS, and resuspended in 3 ml of isolation buffer (buffer IB: 10 mm Tris-MOPS, 1 mm EGTA-Tris, 200 mm sucrose, pH 7.4). The cells were disrupted with a tight-fitting Dounce homogenizer, on ice (30 strokes). The homogenate was sedimented at 800 × g for 10 min to yield a nuclear pellet. The supernatant was further sedimented at 8000 × g for 10 min to yield the mitochondrial pellet. Finally, the postmitochondrial supernatant was centrifuged at 100,000 × g (Beckmann Ultracentrifuge) for 1 h to separate the microsomal fraction (pellet) from the cytosol (supernatant). For oxygen consumption studies, rat liver mitochondria were isolated using a similar protocol (10Frezza C. Cipolat S. Scorrano L. Nat. Protoc. 2007; 2: 287-295Crossref PubMed Scopus (824) Google Scholar). Mitochondria isolated from fresh rat liver were kept on ice and used the same day. Total mitochondrial protein was determined by hydrolyzing mitochondrial aliquots in 0.5 m NaOH and then using the Bradford method. To determine the cellular distribution of 11β, HeLa cells were treated with [14C]11β for 6 h, then subjected to fractionation. Aliquots of each fraction were suspended in scintillation fluid (EcoScint H), and radioactivity was measured using a Beckmann LS 6500 scintillation counter. Oxygen consumption was measured with a PreSens Fibox 3 (Precision Sensing, Regensburg, Germany) oxygen minisensor, according to the manufacturer's instructions. All measurements were done at room temperature. For cellular oxygen consumptions, HeLa cells were washed, trypsinized, and resuspended in cell culture medium at 5 × 106 cells/ml. Mitochondrial oxygen consumption was measured as described (10Frezza C. Cipolat S. Scorrano L. Nat. Protoc. 2007; 2: 287-295Crossref PubMed Scopus (824) Google Scholar) using the substrates for complex I (5 mm glutamate/2.5 mm malate) complex II (5 mm succinate) or cytochrome c/complex IV (6 mm ascorbate/300 μm TMPD). Mitochondria were suspended in experimental buffer (buffer EB: 12.5 mm KCl, 10 mm Tris-MOPS, 0.1 mm EGTA-Tris, 1 mm KH2PO4, pH 7.4), at a concentration of 2 mg/ml mitochondrial protein. To measure NADH-driven oxygen consumption, mitochondria were permeabilized to NADH using three freeze-thaw cycles. Rat liver mitochondria (0.5 mg/ml protein concentration) were incubated at 37 °C in EB, supplemented with 0.1% BSA, 5 mm glutamate, 2.5 mm malate, 0.05 units/ml horseradish peroxidase (HRP), and 10 μm Amplex Red reagent, in the presence of DMSO (control) or test compound, with or without 2000 units/ml bovine liver catalase (Roche Applied Science). After 20 min in the dark, the fluorescence of the resorufin generated by Amplex Red was monitored for 20 min in a SpectraMax M3 spectrophotometer (Molecular Devices). The amount of H2O2 generated was calculated from a H2O2 standard curve obtained in similar conditions. Complex I activity was measured by using the synthetic electron acceptor decylubiquinone (Sigma) and monitoring the rate of NADH disappearance (11Estornell E. Fato R. Pallotti F. Lenaz G. FEBS Lett. 1993; 332: 127-131Crossref PubMed Scopus (191) Google Scholar). Rat liver mitochondria were permeabilized to NADH with three freeze-thaw cycles, diluted to 0.25 mg/ml (total mitochondrial protein) in potassium phosphate buffer (25 mm, pH 7.4) and supplemented with 0.35% BSA, 1 μm antimycin, 1 mm KCN, and 70 μm decylubiquinone. NADH (0.1 mm) and the test compound (or DMSO control) were added last. After a brief and thorough mixing, the drop in the absorbance difference A340–A380 was monitored in a quartz cuvette on a Beckmann Coulter DU730 spectrophotometer at room temperature. Complex I activity was then calculated using ϵNADH for A340–A380 as 5.5 mm−1 cm−1 (11Estornell E. Fato R. Pallotti F. Lenaz G. FEBS Lett. 1993; 332: 127-131Crossref PubMed Scopus (191) Google Scholar). HeLa cells (6 × 106) suspended in 50% PBS/50% Matrigel (BD Biosciences) were injected subcutaneously in the right flank of 6-week-old NIH Swiss nu/nu female mice. Mice developing subcutaneous tumors within 2 weeks were randomized to treatment and control groups. Administration of test compound dissolved in cremophore EL, saline, ethanol (43:30:27) was begun when established tumors reached ∼7-mm diameter. Tumor measurements were obtained with vernier calipers and tumor volumes estimated using the formula (width2 × length × 3.14)/6. Experiments were carried out under the guidelines of the MIT Animal Care Committee. All results are expressed as mean ± S.D. (error bars). The significance of the difference between two populations was calculated using Student's two-tailed t test for unpaired data sets with unequal variances. p values <0.05 were considered significant. The LC50 and EC50 values were calculated from the dose-response curves by using a four variable logistic curve fitting function in Prism 3.0 for Windows (GraphPad Software Inc., San Diego, CA). Our investigations sought to uncover mechanisms of toxicity that were unrelated to the affinity of 11β for the AR. HeLa cells, which do not express the AR (12Nelson-Rees W.A. Hunter L. Darlington G.J. O'Brien S.J. Cytogenet. Cell Genet. 1980; 27: 216-231Crossref PubMed Scopus (25) Google Scholar), were chosen to assess the respective contributions of the steroid and alkylating portions of the compound (Fig. 1). Chlorambucil was used as a model of the reactive alkylating portion of 11β because both molecules share the p-N, N-bis-(2-chloroethyl)aminophenyl group that can form a reactive aziridine capable of covalent modification of DNA and other cellular molecules. To investigate the noncovalent interactions of 11β, an analog, 11β-dim (Fig. 1A) was prepared. This compound was unreactive toward the model nucleophile 4-(4-nitro-benzyl)-pyridine (supplemental data and Fig. S1), indicating that it lacked the ability to modify DNA covalently. It was thus expected that any observed 11β-dim toxicity would arise only from noncovalent interactions with cellular targets. Cell viability analysis, performed using the CTB assay (Fig. 1B), revealed a steep dose response to concentrations of 11β > 5 μm, and a similar steep response was observed at higher concentrations (>12 μm) with 11β-dim. By contrast, neither chlorambucil nor estradiene-3-one at 20 μm decreased the CTB signal by >25% (Fig. 1B). Both 11β and 11β-dim exhibited a threshold effect above which cell viability rapidly decreased; such an effect was not observed with either chlorambucil or estradiene-3-one. Given the striking decrease in viability that was observed after a 24-h treatment with 11β, it was of interest to determine the effect of shorter exposure times on cell viability. HeLa cells were exposed to increasing concentrations of 11β for 3, 6, or 24 h, and cell viability was measured at 24 h. Comparable toxicities were achieved for 6- or 24-h exposures to 11β (6 h, EC50 = 5.3 ± 0.3 μm; 24 h, EC50 = 4.9 ± 0.3 μm), whereas a 3-h exposure period resulted in lower levels of toxicity (Fig. 1C). Together, these data suggested that a 6-h exposure was sufficient for achieving maximum toxicity of 11β for most doses. Therefore, subsequent mechanistic experiments focused on cellular changes that occur during the initial 6 h. Activation of apoptotic pathways by 11β was observed previously in LNCaP cells (7Marquis J.C. Hillier S.M. Dinaut A.N. Rodrigues D. Mitra K. Essigmann J.M. Croy R.G. Chem. Biol. 2005; 12: 779-787Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). To determine whether apoptosis was also the mechanism of cell death in HeLa cells, the proteolytic cleavage of caspase-9, caspase-3, and PARP were investigated. Western blot analysis with corresponding antibodies revealed that a 6-h treatment with 7.5 μm or 10 μm 11β induced cleavage of caspase-9, caspase-3, and PARP (Fig. 1D). To understand better the putative targets of 11β, an intracellular localization study was performed. HeLa cells were treated with 5 μm 14C-labeled 11β for 6 h, isolated, and subjected to subcellular fractionation (see “Experimental Procedures”). By measuring the radioactivity associated with the medium and the cell pellet, it was found that a significant portion of the total compound (>30%) becomes associated with the cells in 6 h (Fig. 2A). Subcellular fractionation revealed that 50 ± 2% of the radioactive compound is found in the mitochondrial fraction, whereas the rest is distributed among the nucleus, microsomes, and cytosol (Fig. 2B). The radioactivity measurements also allowed the estimation of the local concentration of 11β (Fig. 2C), the compound being most concentrated in the mitochondria (9.9 ± 0.4 pmol/μg protein) and the microsomes (8.5 ± 0.5 pmol/μg protein). By assuming the volume of a HeLa cell of 4.2 pl (measured average diameter = 20 μm), the molar concentration of 11β in the cell after 6 h was estimated to be ∼300 μm, a 60-fold increase from the 5 μm concentration in the medium. Moreover, using the published value for the mitochondrial volume of 1.6 μl/mg mitochondrial protein (13Petronilli V. Pietrobon D. Zoratti M. Azzone G.F. Eur. J. Biochem. 1986; 155: 423-431Crossref PubMed Scopus (19) Google Scholar), the molar concentration of 11β in the mitochondria was estimated to be ∼6 mm, which is more than 1000-fold increase from the medium concentration of 11β. These findings suggest that mitochondria might be one of the targets of the compound. The observation that the DNA-unreactive 11β-dim is nevertheless toxic (Fig. 1B) indicated that 11β may be employing several mechanisms of toxicity. Given the rapid activation of the apoptotic pathway (Fig. 1C) and the mitochondrial localization of 11β (Fig. 2B), we hypothesized that one such mechanism may involve direct mitochondrial toxicity. One of the hallmarks of mitochondrial dysregulation is the generation of ROS and oxidative stress (14Ott M. Gogvadze V. Orrenius S. Zhivotovsky B. Apoptosis. 2007; 12: 913-922Crossref PubMed Scopus (1477) Google Scholar, 15Hengartner M.O. Nature. 2000; 407: 770-776Crossref PubMed Scopus (6170) Google Scholar). Generation of ROS in HeLa cells was monitored using the molecular probe CM-H2DCFDA, which can detect most ROS at a cellular level (16Jakubowski W. Bartosz G. Cell Biol. Int. 2000; 24: 757-760Crossref PubMed Scopus (236) Google Scholar). Once inside a cell, this probe is deacetylated by cellular esterases, and, when oxidized, it becomes the fluorescent compound CM-DCF, which can be detected in the FL1 channel of a flow cytometer. Fig. 3A is a fluorescence histogram showing that treatment of HeLa cells for 6 h with 3 or 5 μm 11β resulted in a notable increase in CM-DCF fluorescence intensity (right shift) compared with DMSO (vehicle)-treated cells. The possibility that increased fluorescence was due to increased uptake of the probe was ruled out by use of a related molecular probe, CM-DCFDA, which does not require oxidation to become fluorescent. When using CM-DCFDA, no significant increase in CM-DCF fluorescence was detected in the treated cells compared with controls, indicating that cellular uptake of the dye was unaffected by 11β. These findings together established that 11β treatment triggered a significant production of ROS. To determine whether the production of ROS required the ability of 11β to modify cellular molecules covalently, ROS levels were compared between cells treated for 6 h with 11β (5 μm), 11β-dim (10 μm), chlorambucil (10 μm), or estradien-3-one (10 μm) (Fig. 3B); antimycin A, a known ROS inducer, was included as a comparative control. The data revealed that both 11β compounds were potent inducers of ROS, whereas chlorambucil and estradien-3-one were not. Several other doses of each compound were investigated, revealing a dose-response dependence for 11β and 11β-dim (supplemental data and Fig. S2). Thus, neither the ability to form covalent adducts (characteristic of 11β and chlorambucil) nor the presence of the steroid group (11β, 11β-dim, and estradien-3-one) correlates with the ability to induce ROS. The operative structural feature responsible for the ROS induction appears to be the entire molecular framework of 11β or 11β-dim. To investigate the kinetics of intracellular ROS generation, a time course of ROS levels was measured in HeLa cells treated with 5 μm 11β. Increased levels of ROS were detected as early as 30 min after the addition of 11β to culture media, and then the levels continued to increase in the first 6 h of exposure (Fig. 3C), suggesting that 11β caused both rapid-onset and sustained ROS generation. Several markers of oxidative stress in 11β-treated cells were also increased, including levels of hydrogen peroxide (Fig. 3D) and the secondary oxidation product, malondialdehyde (Fig. 3E). Additionally, the cellular levels of NADPH, the principal cellular reducing co-factor, decreased rapidly during the same time interval (Fig. 3F). These findings are consistent with the rapid formation of ROS in HeLa cells exposed to 11β, suggesting that ROS and generalized oxidative stress may contribute to 11β cytotoxicity. To establish a link between ROS production and the toxicity of 11β, HeLa cells were treated with 11β in the presence of antioxidants. A significantly lower level of ROS (p < 0.01) was observed when HeLa cells were exposed to 5 μm 11β for 6 h in the presence of antioxidants N-acetyl-" @default.
- W2003238218 created "2016-06-24" @default.
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- W2003238218 date "2011-09-01" @default.
- W2003238218 modified "2023-10-06" @default.
- W2003238218 title "Chemical Genetics Analysis of an Aniline Mustard Anticancer Agent Reveals Complex I of the Electron Transport Chain as a Target" @default.
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