Ruth Freeman

α-Tocopheryl succinate induces apoptosis by targeting ubiquinone-binding sites in mitochondrial respiratory complex II

α-Tocopheryl succinate (α-TOS) is a selective inducer of apoptosis in cancer cells, which involves the accumulation of reactive oxygen species (ROS). The molecular target of α-TOS has not been identified. Here, we show that α-TOS inhibits succinate dehydrogenase (SDH) activity of complex II (CII) by interacting with the proximal and distal ubiquinone (UbQ)-binding site (QP and QD, respectively). This is based on biochemical analyses and molecular modelling, revealing similar or stronger interaction energy of α-TOS compared to that of UbQ for the QP and QD sites, respectively. CybL-mutant cells with dysfunctional CII failed to accumulate ROS and underwent apoptosis in the presence of α-TOS. Similar resistance was observed when CybL was knocked down with siRNA. Reconstitution of functional CII rendered CybL-mutant cells susceptible to α-TOS. We propose that α-TOS displaces UbQ in CII causing electrons generated by SDH to recombine with molecular oxygen to yield ROS. Our data highlight CII, a known tumour suppressor, as a novel target for cancer therapy.

Suppression of Tumor Growth In vivo by the Mitocan α-tocopheryl Succinate Requires Respiratory Complex II

Purpose: Vitamin E analogues are potent novel anticancer drugs. The purpose of this study was to elucidate the cellular target by which these agents, represented by α-tocopoheryl succinate (α-TOS), suppress tumors in vivo, with the focus on the mitochondrial complex II (CII). Experimental Design: Chinese hamster lung fibroblasts with functional, dysfunctional, and reconstituted CII were transformed using H-Ras. The cells were then used to form xenografts in immunocompromized mice, and response of the cells and the tumors to α-TOS was studied. Results: The CII-functional and CII-reconstituted cells, unlike their CII-dysfunctional counterparts, responded to α-TOS by reactive oxygen species generation and apoptosis execution. Tumors derived from these cell lines reciprocated their responses to α-TOS. Thus, growth of CII-functional and CII-reconstituted tumors was strongly suppressed by the agent, and this was accompanied by high level of apoptosis induction in the tumor cells. On the other hand, α-TOS did not inhibit the CII-dysfuntional tumors. Conclusions: We document in this report a novel paradigm, according to which the mitochondrial CII, which rarely mutates in human neoplasias, is a plausible target for anticancer drugs from the group of vitamin E analogues, providing support for their testing in clinical trials.

MicroRNAs regulate tumor angiogenesis modulated by endothelial progenitor cells

Bone marrow-derived endothelial progenitor cells (EPC) contribute to the angiogenesis-dependent growth of tumors in mice and humans. EPCs regulate the angiogenic switch via paracrine secretion of proangiogenic growth factors and by direct luminal incorporation into sprouting nascent vessels. miRNAs have emerged as key regulators of several cellular processes including angiogenesis; however, whether miRNAs contribute to bone marrow-mediated angiogenesis has remained unknown. Here, we show that genetic ablation of miRNA-processing enzyme Dicer, specifically in the bone marrow, decreased the number of circulating EPCs, resulting in angiogenesis suppression and impaired tumor growth. Furthermore, genome-wide deep sequencing of small RNAs revealed tumor EPC-intrinsic miRNAs including miR-10b and miR-196b, which have been previously identified as key regulators of HOX signaling and adult stem cell differentiation. Notably, we found that both miR-10b and miR-196b are responsive to vascular endothelial growth factor stimulation and show elevated expression in human high-grade breast tumor vasculature. Strikingly, targeting miR-10b and miR-196b led to significant defects in angiogenesis-mediated tumor growth in mice. Targeting these miRNAs may constitute a novel strategy for inhibiting tumor angiogenesis.

Mitochondrial targeting of α-tocopheryl succinate enhances its pro-apoptotic efficacy: A new paradigm for effective cancer therapy

Mitochondria are emerging as intriguing targets for anti-cancer agents. We tested here a novel approach, whereby the mitochondrially targeted delivery of anti-cancer drugs is enhanced by the addition of a triphenylphosphonium group (TPP(+)). A mitochondrially targeted analog of vitamin E succinate (MitoVES), modified by tagging the parental compound with TPP(+), induced considerably more robust apoptosis in cancer cells with a 1-2 log gain in anti-cancer activity compared to the unmodified counterpart, while maintaining selectivity for malignant cells. This is because MitoVES associates with mitochondria and causes fast generation of reactive oxygen species that then trigger mitochondria-dependent apoptosis, involving transcriptional modulation of the Bcl-2 family proteins. MitoVES proved superior in suppression of experimental tumors compared to the untargeted analog. We propose that mitochondrially targeted delivery of anti-cancer agents offers a new paradigm for increasing the efficacy of compounds with anti-cancer activity.

α-Tocopheryl succinate causes mitochondrial permeabilization by preferential formation of Bak channels

Mitocans are drugs selectively killing cancer cells by destabilizing mitochondria and many induce apoptosis via generation of reactive oxygen species (ROS). However, the molecular events by which ROS production leads to apoptosis has not been clearly defined. In this study with the mitocan α-tocopheryl succinate (α-TOS) the role of the Bcl-2 family proteins in the mechanism of malignant cell apoptosis has been determined. Exposure of several different cancer cell lines to α-TOS increased expression of the Noxa protein, but none of the other proteins of the Bcl-2 family, an event that was independent of the cellular p53 status. α-TOS caused a profound conformational change in the pro-apoptotic protein, Bak, involving oligomerization in all cell types, and this also applied to the Bax protein, but only in non-small cell lung cancer cells. Immunoprecipitation studies indicated that α-TOS activates the two BH1-3 proteins, Bak or Bax, to form high molecular weight complexes in the mitochondria. RNAi knockdown revealed that Noxa and Bak are required for α-TOS-induced apoptosis, and the role of Bak was confirmed using Bak- and/or Bax-deficient cells. We conclude that the major events induced by α-TOS in cancer cells downstream of ROS production leading to mitochondrial apoptosis involve the Noxa-Bak axis. It is proposed that this represents a common mechanism for mitochondrial destabilization activated by a variety of mitocans that induce accumulation of ROS in the early phases of apoptosis.

Role of thioredoxin‐1 in apoptosis induction by α‐tocopheryl succinate and TNF‐related apoptosis‐inducing ligand in mesothelioma cells

Malignant mesothelioma (MM) is a fatal type of cancer. We studied the role of the redox‐active protein thioredoxin‐1 (Trx‐1) in apoptosis induced in MM cells and their non‐malignant counterparts (Met‐5A) by α‐tocopheryl succinate (α‐TOS) and TNF‐related apoptosis‐inducing ligand (TRAIL). MM cells were susceptible to α‐TOS and less to TRAIL, while Met‐5A cells were susceptible to TRAIL and resistant to α‐TOS. MM cells expressed very low level of the Trx‐1 protein, which was high in Met‐5A cells, while the level of Trx‐1 mRNA was similar in all cell lines. Downregulation of Trx‐1 further sensitised Met‐5A cells to TRAIL but not to α‐TOS. Our data suggest that the role of Trx‐1 in apoptosis modulation is unrelated to its anti‐oxidant properties.

Cancer cell CCL5 mediates bone marrow independent angiogenesis in breast cancer

It has recently been suggested that the chemokine receptor (CCR5) is required for bone marrow (BM) derived endothelial progenitor cell (EPC) mediated angiogenesis. Here we show that suppression of either cancer cell produced CCL5, or host CCR5 leads to distinctive vascular and tumor growth defects in breast cancer. Surprisingly, CCR5 restoration in the BM alone was not sufficient to rescue the wild type phenotype, suggesting that impaired tumor growth associated with inhibiting CCL5/CCR5 is not due to defects in EPC biology. Instead, to promote angiogenesis cancer cell CCL5 may signal directly to endothelium in the tumor-stroma. In support of this hypothesis, we have also shown: (i) that endothelial cell CCR5 levels increases in response to tumor-conditioned media; (ii) that the amount of CCR5+ tumor vasculature correlates with invasive grade; and (iii) that inhibition of CCL5/CCR5 signaling impairs endothelial cell migration, associated with a decrease in activation of mTOR/AKT pathway members. Finally, we show that treatment with CCR5 antagonist results in less vasculature, impaired tumor growth, reduced metastases and improved survival. Taken as a whole, this work demonstrates that directly inhibiting CCR5 expressing vasculature constitutes a novel strategy for inhibiting angiogenesis and blocking metastatic progression in breast cancer.

Corrigendum to: “Mitochondrial targeting of α-tocopheryl succinate enhances its pro-apoptotic efficacy: A new paradigm for effective cancer therapy” [Free Radic Biol Med. 50 (2011) 1546–1555]

Lan-Feng Dong , Victoria J.A. Jameson , David Tilly , Lubomir Prochazka , Jakub Rohlena , Karel Valis , Jaroslav Truksa , Renata Zobalova , Elahe Mahdavian , Katarina Kluckova , Marina Stantic , Jan Stursa , Ruth Freeman , Paul K. Witting , Erik Norberg , Jacob Goodwin , Brian A. Salvatore , Jana Novotna , Jaroslav Turanek , Miroslav Ledvina , Pavel Hozak , Boris Zhivotovsky , Mark J. Coster , Stephen J. Ralph , Robin A.J. Smith , Jiri Neuzil a,e,n