Mitochondrial targeting of vitamin E succinate enhances its pro-apoptotic and anti-cancer activity via mitochondrial complex II

Abstract

Mitochondrial complex II (CII) has been recently identified as a novel target for anti-cancer drugs. Mitochondrially targeted vitamin E succinate (MitoVES) is modified so that it is preferentially localized to mitochondria, greatly enhancing its pro-apoptotic and anti-cancer activity. Using genetically manipulated cells, MitoVES caused apoptosis and generation of reactive oxygen species (ROS) in CII-proficient malignant cells but not their CII-dysfunctional counterparts. MitoVES inhibited the succinate dehydrogenase (SDH) activity of CII with IC(50) of 80 μM, whereas the electron transfer from CII to CIII was inhibited with IC(50) of 1.5 μM. The agent had no effect either on the enzymatic activity of CI or on electron transfer from CI to CIII. Over 24 h, MitoVES caused stabilization of the oxygen-dependent destruction domain of HIF1α fused to GFP, indicating promotion of the state of pseudohypoxia. Molecular modeling predicted the succinyl group anchored into the proximal CII ubiquinone (UbQ)-binding site and successively reduced interaction energies for serially shorter phytyl chain homologs of MitoVES correlated with their lower effects on apoptosis induction, ROS generation, and SDH activity. Mutation of the UbQ-binding Ser(68) within the proximal site of the CII SDHC subunit (S68A or S68L) suppressed both ROS generation and apoptosis induction by MitoVES. In vivo studies indicated that MitoVES also acts by causing pseudohypoxia in the context of tumor suppression. We propose that mitochondrial targeting of VES with an 11-carbon chain localizes the agent into an ideal position across the interface of the mitochondrial inner membrane and matrix, optimizing its biological effects as an anti-cancer drug.

FIGURE 1.

Structures of the compounds used in this study.

FIGURE 2.

MitoVE₁₁S is superior to α-TOS, requires high ΔΨ_m,i, and acts by targeting the mitochondrial respiratory CII. A, Jurkat cells were exposed to MitoVE₁₁S or α-TOS at the concentrations shown (μm) for 10 or 20 h and assessed for apoptosis by flow cytometry. B, Jurkat cells were exposed to 4 μm MitoVE₁₁S (MVES) for 4 h or 100 μm α-TOS (TOS) for 6 h in the presence or absence of 5 μm FCCP (FC) and assessed for apoptosis. The inset shows that exposure of Jurkat cells to increasing doses (μm) of FCCP for 6 h results in dissipation of ΔΨ_m,i, as assessed using the fluorescent probe tetramethylrhodamine methyl ester and flow cytometry (mean fluorescence intensity (MFI)). B1_Ras, B9_Ras, and B9_Ras-SDHC cells were exposed to 5 μm MitoVE₁₁S for the times shown (C and D) or for 2 h (E and F) and assessed for apoptosis induction (C) and ROS accumulation by flow cytometry (D) and EPR spectroscopy (E, EPR spectra; F, double integration evaluation of the signal; AU, arbitrary units). G, Jurkat cells were exposed to 5 μm MitoVE₁₁S (MV) for 6 h, their mitochondria were lysed, and the lysates were fractionated using size exclusion chromatography. Individual fractions were probed by Western blotting for the presence of CI–CIII using specific antibodies. H, Jurkat cells were exposed to 5 μm MitoVE₁₁S for 6 h, and mRNA levels of the mtDNA genes coding subunits of mitochondrial complexes were assessed using RT-PCR (left lanes, control; right lanes, MitoVE₁₁S; M, markers). The data shown are mean ± S.D. (error bars) (n = 3); the images are representative of three independent experiments. * (A), statistically significant differences between corresponding treatments with MitoVE₁₁S and α-TOS; * (B), statistically significant differences between treatments in the absence and presence of FCCP; * and ** (C–F), significant differences between B1_Ras or B9_Ras-SDHC cells and B9_Ras cells treated with MitoVE₁₁S for 3 and 6 h, respectively (p < 0.05).

FIGURE 3.

MitoVE₁₁S efficiently inhibits the oxidoreductase activity of CII. A, SMPs were assessed for the transfer of electrons from CI to CIII and from CII to CIII (left) and the activity of CI (NDH) and CII (SDH) (right) in the presence of MitoVE₁₁S. HCT116_ODD-GFP cells were assessed for stabilization of GFP by Western blotting after exposure to MitoVE₁₁S at the levels shown (μm) for 24 h by Western blotting (B) or by fluorescence microscopy following 24-h exposure to 5 μm MitoVE₁₁S (C). The data in A are average values from two independent experiments; the images are representative of three independent experiments.

FIGURE 4.

Molecular modeling predicts binding of MitoVE₁₁S at the Q_P site. The CII model was refined by the addition of POPE molecules simulating the MIM. The position of the heme group is indicated in green, and the predicted position of MitoVE₁₁S is shown in red. The bottom panel shows the detail of the interaction of MitoVE₁₁S with CII.

FIGURE 5.

Shortening the aliphatic chain of MitoVE₁₁S successively reduces its cancer cell cytotoxic activity. A, Jurkat cells were exposed to homologs of MitoVES at 5 μm and evaluated for the level of apoptosis. HCT116_ODD-GFP cells were exposed to MitoVES homologs at 5 μm for 3 h and assessed for SDH activity (B) and ROS accumulation (C) or exposed for 12 h and assessed for the level of apoptosis (D) and expression of GFP (E). The data in A–D are mean ± S.D. (error bars) (n = 3), and data in E are representative of three independent experiments. *, significant difference (p < 0.05) between control cells and cells treated with different MitoVES homologs.

FIGURE 6.

Apoptogenic efficacy of MitoVE₁₁S depends on its chirality. A, molecular modeling documents the predicted interaction of R- (blue) and S-MitoV₁₁ES (brown) with the UbQ-interacting Ser⁶⁸ in the Q_P site of SDHC. B, Jurkat cells were exposed to rac-, R-, or S-MitoVE₁₁S at 2.5 μm for the times shown and assessed for apoptosis induction. The data are mean ± S.D. (error bars) (n = 3). *, significant difference (p < 0.05) between cells treated with S- and R-MitoVE₁₁S.

FIGURE 7.

The UbQ-binding Ser⁶⁸ in the Q_P site of CII is important for the effect of MitoVE₁₁S. B9_Ras-SDHC (WT), B9_Ras-SDHCS68A (S68A), or B9_Ras-SDHCS68L cells (S68L) were exposed to MitoVE₁₁S at 2 μm for the times shown and assessed for ROS accumulation (A) and apoptosis levels (B). The data are mean ± S.D. (error bars) (n = 3). *, significant difference (p < 0.05) between MitoVE₁₁S-treated B9_Ras cells stably transfected with WT SDHC or with S68A or C68L mutant SDHC.

FIGURE 8.

MitoVE₁₁S suppresses tumor progression and causes the state of pseudohypoxia. A, BALB/c nu/nu mice with xenografts derived from HCT116_ODD-GFP cells were treated by intraperitoneal injection of 1–2 μmol of MitoVE₁₁S or 15 μmol of α-TOS per mouse every 3–4 days, and tumors were visualized and their volume was quantified using USI on days 11, 18, and 28. B, tumors were paraffin-embedded, sectioned, and stained with H&E and photographed in a light microscope (left images) or mounted in DAPI-containing Vectashield and imaged using a confocal microscope (right images). The data are mean ± S.D. (error bars) (n = 6–7). *, significant difference between corresponding control and MitoVE₁₁S-treated animals (p < 0.05).

FIGURE 9.

Model for the molecular mechanism of action of MitoVE₁₁S on CII. A, in unstimulated cells, electrons generated from the conversion of succinate to fumarate at SDHA move via a series of Fe-S clusters down SDHB to the Q_P and, possibly, Q_D made up by amino acid residues of SDHC and SDHD. UbQ undergoes a two-electron reduction, when its affinity for the Q site(s) of CII is low, and it directs the two electrons to CIII, where it reoxidizes and shuttles back to CII. B, in the presence of MitoVE₁₁S, the SDH activity of CII is only mildly reduced (IC₅₀ ∼80 μm). Therefore, electrons are formed by conversion of succinate to fumarate, moving to the Q_P (and possibly Q_D) within the MIM. However, MitoVE₁₁S, which is positioned so that its TPP⁺ group lies at the matrix face of the MIM and the tocopheryl succinyl group, interacts with the Q_P, displacing access to UbQ and very efficiently suppresses transfer of electrons from Q_P of CII to CIII (IC₅₀ ∼1.5 μm). Consequently, this situation is highly unstable and gives rise to generation of superoxide as a by-product.