Chemical genetics analysis of an aniline mustard anticancer agent reveals complex I of the electron transport chain as a target

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.

FIGURE 1.

Structures of compounds and their effects on HeLa cell viability and apoptosis. A, the antitumor agent 11β is an aniline mustard linked to the steroid ligand estradiene-3-one (EDO). Similarly to chlorambucil (CMB), 11β contains the p-N,N-bis-(2-chloroethly)aminophenyl moiety capable of forming reactive aziridinium ions that produce covalent DNA adducts (DNA damage). The unreactive analog 11β-dim substitutes methoxy groups for chlorine atoms, which prevent formation of aziridinium ions, and hence abolishes the ability to interact covalently with DNA. B, cell viability was estimated with the CTB assay following 24-h treatment with 11β, 11β-dim, CMB, or EDO. C, cell viability was assayed using the CTB assay at 24 h following treatments with the indicated concentrations of 11β for 3, 6, or 24 h. The viable cell fraction was calculated relative to vehicle-treated controls. Data represent average ± S.D. (error bars), n = 3. D, Western blot analysis investigating the cleavage of PARP, caspase-9, and caspase-3 following treatment with 11β for 6 h. β-Actin is included as a loading control. Staurosporine (STS) is shown as a positive control.

FIGURE 2.

Cellular uptake of 11β and intracellular localization. A, 11β distribution between the medium and HeLa cells after 6-h exposure to 5 μm 11β. B, relative intracellular localization of 11β in HeLa cells exposed for 6 h to 5 μm 11β. Subcellular fractionation was performed as described under “Experimental Procedures.” C, concentration of 11β (in pmol/μg protein) in the subcellular fractions from B. Data represent mean ± S.D. (error bars), n = 3.

FIGURE 3.

Effects of 11β compounds on ROS levels and markers of oxidative stress in HeLa cells. A, representative histograms of flow cytometry experiments demonstrating increased fluorescence intensity of CM-DCF following treatment with 3 μm or 5 μm 11β for 6 h. B, quantitative estimates of changes in mean fluorescence intensity of CM-DCF following 6-h treatments with 5 μm 11β, 10 μm 11β-dim, 10 μm estradien-3-one (EDO) or 10 μm chlorambucil (CMB) as measured by flow cytometry. Antimycin A (AMA), used at 1 μm, is shown as a positive control. C–F, time course of the levels of ROS and markers of oxidative stress after exposure to 5 μm 11β. C, time-dependent changes in mean fluorescence intensity of CM-DCF as measured by flow cytometry. D, H₂O₂ concentration. E, malonic dialdehyde (MDA) concentration. F, total cellular NADPH concentration. Assays were performed as described under “Experimental Procedures.” Data represent mean ± S.D. (error bars), n = 3. *, p < 0.05; **, p < 0.01.

FIGURE 4.

Effect of antioxidants on the toxicity of 11β compounds in HeLa cells. A, HeLa cells were treated with 5 μm 11β for 6 h in the presence of NAC (10 mm), vitamin E (100 μm), or vehicle (DMSO). Cells were then loaded with CM-H₂DCFDA and the mean fluorescence intensity determined by flow cytometry. B, cell viability following 24-h treatment with 11β alone or in the presence of NAC (10 mm) or vitamin E (100 μm) was assessed with the CTB assay. C, cell viability following 24-h treatment with 11β-dim alone or in the presence of NAC (10 mm) or vitamin E (100 μm) was assessed with the CTB assay. Data represent average ± S.D. (error bars), n = 3. *, p < 0.05; **, p < 0.01.

FIGURE 5.

Effects of 11β on superoxide levels and ΔΨ_m in HeLa cells. A, cells were treated with DMSO (left) or 5 μm 11β for 6 h (right) then loaded with the mitochondrial O₂^˙̄ indicator MitoSOX Red and Hoechst 33342 and imaged by fluorescence microscopy. Top panels display enhanced mitochondrial O₂^˙̄ production in 11β-treated cells. Middle panels show nuclear staining for reference. Bottom panels show merged images. Magnification is ×100. B, quantitative estimates of dose-dependent changes in mean fluorescence intensity of MitoSOX Red following 6-h treatments with 11β as measured by flow cytometry. C, 11β-induced changes in ΔΨ_m. Cells were treated with the indicated concentrations of 11β for 6 h in the absence or presence of NAC (10 mm) or vitamin E (100 μm). The cells were then loaded with JC-1 dye and the fluorescence measured by flow cytometry. Data represent mean ± S.D. (error bars), n = 3. *, p < 0.05; **, p < 0.01.

FIGURE 6.

Effects of 11β on cellular and mitochondrial respiration. A, cellular oxygen consumption was measured as described under “Experimental Procedures.” HeLa cells were treated with 5 μm 11β or DMSO for 2 h, then oxygen consumption was measured in the presence of FCCP (1 nm), a mitochondrial ETC uncoupler. B, oxygen consumption in isolated rat liver mitochondria uncoupled with FCCP (1 nm) was measured. To test the electron flow through ETC via complexes I-III-IV, a glutamate/malate mix was used as a substrate. Rotenone (ROT) (1 μm) was used as a control. To test the electron flow via complexes II-III-IV, succinate and rotenone (1 μm final) were used. In each case, oxygen consumption was determined in the presence of DMSO control, 11β, or 11β-dim at the indicated concentrations. C, oxygen consumption was determined in isolated rat liver mitochondria, using NADH as a substrate. The mitochondria were permeabilized to NADH using three freeze-thaw cycles. Rotenone (1 μm) was used as a control. D, complex I activity was measured in permeabilized mitochondria, by spectrophotometrically monitoring the disappearance of the NADH substrate. Decylubiquinone was used as the final electron acceptor. Rotenone (1 μm) was used as a positive control. Data represent average ± S.D. (error bars), n = 3. E, production of H₂O₂ in isolated rat liver mitochondria was measured following exposure to the 11β compounds. Mitochondria, charged with glutamate/malate, were exposed for 20 min at 37 °C to the indicated amounts of 11β, 11β-dim, or rotenone control (1 μm), in the presence or absence of 2000 units/ml bovine liver catalase. The rate of H₂O₂ produced was then determined using the Amplex Red method. Data represent average ± S.D., n = 4. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 7.

Clonogenic survival of HeLa cells treated with 11β, 11β-dim, chlorambucil, or 11β-dim/chlorambucil (CMB) equimolar mixture. Surviving fraction was determined by staining and counting colonies after 7 days. Data represent mean ± S.D., n = 3.

FIGURE 8.

Effect of 11β treatment on HeLa tumor xenografts. Mice bearing established HeLa tumors were dosed by daily injection with 30 mg/kg 11β or vehicle only. Points, mean tumor volume; bars, S.D.; horizontal bars, periods of dosing.

FIGURE 9.

Proposed mechanisms of toxicity. The substantial antitumor activity of 11β is proposed to be due to its ability to utilize two distinct mechanisms of toxicity: (i) DNA adduct formation and (ii) mitochondrial ROS production. Similarly to other nitrogen mustards, such as chlorambucil (top) 11β forms covalent DNA adducts (DNA damage). However, 11β is also a potent mitochondrial respiration inhibitor and oxidative stress inducer. The second mechanism of toxicity is recapitulated by 11β-dim (bottom), the dimethoxy derivative of 11β that cannot form DNA adducts, suggesting that the two mechanisms are distinct. The concurrent ability of 11β to damage DNA and induce mitochondrial ROS appears to be a useful paradigm for the design of broad specificity antitumor agents.