Lubomir Prochazka

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.

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.

Hippo/Mst1 Stimulates Transcription of the Proapoptotic Mediator NOXA in a FoxO1-Dependent Manner

The proapoptotic protein Noxa, a member of the BH3-only Bcl-2 protein family, can effectively induce apoptosis in cancer cells, although the relevant regulatory pathways have been obscure. Previous studies of the cytotoxic effects of α-tocopheryl succinate (α-TOS) on cancer cells identified a mechanism whereby α-TOS caused apoptosis requiring the Noxa-Bak axis. In the present study, ab initio analysis revealed a conserved FoxO-binding site (DBE; DAF-16 binding element) in the NOXA promoter, and specific affinity of FoxO proteins to this DBE was confirmed by fluorescence anisotropy. FoxO1 and FoxO3a proteins accumulated in the nucleus of α-TOS-treated cells, and the drug-induced specific FoxO1 association with the NOXA promoter and its activation were validated by chromatin immunoprecipitation. Using siRNA knockdown, a specific role for the FoxO1 protein in activating NOXA transcription in cancer cells was identified. Furthermore, the proapoptotic kinase Hippo/Mst1 was found to be strongly activated by α-TOS, and inhibiting Hippo/Mst1 by specific siRNA prevented phosphorylation of FoxO1 and its nuclear translocation, thereby reducing levels of NOXA transcription and apoptosis in cancer cells exposed to α-TOS. Thus, we have demonstrated that anticancer drugs, exemplified by α-TOS, induce apoptosis by a mechanism involving the Hippo/Mst1-FoxO1-Noxa pathway. We propose that activation of this pathway provides a new paradigm for developing targeted cancer treatments.

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.

Regulation of alternative splicing of CD44 in cancer

CD44 is a hyaluronan binding cell surface signal transducing receptor that influences motility, cell survival and proliferation as well as the formation of tumor microenvironment. CD44 contains two variable regions encoded by variable exons. Alternative splicing, which is often deregulated in cancer, can produce various isoforms of CD44 with properties that may have different tissue specific effects and therefore even diverse effects on cancer progression. This review summarizes and puts together all major regulators of alternative splicing of CD44 in cancer that have been documented so far and that have an experimentally proved effect on CD44 isoform switching. It is important to better understand the mechanisms of alternative splicing of CD44, where all the variability of CD44 originates, to be able to explain the isoform switching and occurrence of variant isoforms of CD44 (CD44v) in cancer.

Liposomal formulation of α-tocopheryl maleamide: In vitro and in vivo toxicological profile and anticancer effect against spontaneous breast carcinomas in mice

The vitamin E analogue alpha-tocopheryl succinate (alpha-TOS) is an efficient anti-cancer drug. Improved efficacy was achieved through the synthesis of alpha-tocopheryl maleamide (alpha-TAM), an esterase-resistant analogue of alpha-tocopheryl maleate. In vitro tests demonstrated significantly higher cytotoxicity of alpha-TAM towards cancer cells (MCF-7, B16F10) compared to alpha-TOS and other analogues prone to esterase-catalyzed hydrolysis. However, in vitro models demonstrated that alpha-TAM was cytotoxic to non-malignant cells (e.g. lymphocytes and bone marrow progenitors). Thus we developed lyophilized liposomal formulations of both alpha-TOS and alpha-TAM to solve the problem with cytotoxicity of free alpha-TAM (neurotoxicity and anaphylaxis), as well as the low solubility of both drugs. Remarkably, neither acute toxicity nor immunotoxicity implicated by in vitro tests was detected in vivo after application of liposomal alpha-TAM, which significantly reduced the growth of cancer cells in hollow fiber implants. Moreover, liposomal formulation of alpha-TAM and alpha-TOS each prevented the growth of tumours in transgenic FVB/N c-neu mice bearing spontaneous breast carcinomas. Liposomal formulation of alpha-TAM demonstrated anti-cancer activity at levels 10-fold lower than those of alpha-TOS. Thus, the liposomal formulation of alpha-TAM preserved its strong anti-cancer efficacy while eliminating the in vivo toxicity found of the free drug applied in DMSO. Liposome-based targeted delivery systems for analogues of vitamin E are of interest for further development of efficient and safe drug formulations for clinical trials.

Liposomal delivery systems for anti-cancer analogues of vitamin E

Pro-apoptotic analogues of vitamin E (VE) exert selective anti-cancer effect on various animal cancer models. Neither suitable formulation of α-tocopheryl succinate (α-TOS), representative semi-synthetic VE analogue ester, nor suitable formulations of the other VE analogues for clinical application have been reported yet. The major factor limiting the use of VE analogues is their low solubility in aqueous solvents. Due to the hydrophobic character of VE analogues, liposomes are predetermined as suitable delivery system. Liposomal formulation prevents undesirable side effects of the drug, enhances the drug biocompatibility, and improves the drug therapeutic index. Liposomal formulations of VE analogues especially of α-TOS and α-tocopheryl ether linked acetic acid (α-TEA) have been developed. The anti-cancer effect of these liposomal VE analogues has been successfully demonstrated in pre-clinical models in vivo. Present achievements in: (i) preparation of liposomal formulations of VE analogues, (ii) physico-chemical characterization of these developed systems and (iii) testing of their biological activity such as induction of apoptosis and evaluation of anti-cancer effect are discussed in this review.

Multi-layered nanofibrous mucoadhesive films for buccal and sublingual administration of drug-delivery and vaccination nanoparticles - important step towards effective mucosal vaccines

&NA; Nanofibre‐based mucoadhesive films were invented for oromucosal administration of nanocarriers used for delivery of drugs and vaccines. The mucoadhesive film consists of an electrospun nanofibrous reservoir layer, a mucoadhesive film layer and a protective backing layer. The mucoadhesive layer is responsible for tight adhesion of the whole system to the oral mucosa after application. The electrospun nanofibrous reservoir layer is intended to act as a reservoir for polymeric and lipid‐based nanoparticles, liposomes, virosomes, virus‐like particles, dendrimers and the like, plus macromolecular drugs, antigens and/or allergens. The extremely large surface area of nanofibrous reservoir layers allows high levels of nanoparticle loading. Nanoparticles can either be reversibly adsorbed to the surface of nanofibres or they can be deposited in the pores between the nanofibres. After mucosal application, nanofibrous reservoir layers are intended to promote prolonged release of nanoparticles into the submucosal tissue. Reversible adsorption of model nanoparticles as well as sufficient mucoadhesive properties were demonstrated. This novel system appears appropriate for the use in oral mucosa, especially for sublingual and buccal tissues. To prove this concept, trans‐/intramucosal and lymph‐node delivery of PLGA‐PEG nanoparticles was demonstrated in a porcine model. This system can mainly be used for sublingual immunization and the development of “printed vaccine technology”. Graphical abstract Figure. No caption available.

Mitochondrial targeting overcomes ABCA1-dependent resistance of lung carcinoma to α-tocopheryl succinate

Abstractα-Tocopheryl succinate (α-TOS) is a promising anti-cancer agent due to its selectivity for cancer cells. It is important to understand whether long-term exposure of tumour cells to the agent will render them resistant to the treatment. Exposure of the non-small cell lung carcinoma H1299 cells to escalating doses of α-TOS made them resistant to the agent due to the upregulation of the ABCA1 protein, which caused its efflux. Full susceptibility of the cells to α-TOS was restored by knocking down the ABCA1 protein. Similar resistance including ABCA1 gene upregulation was observed in the A549 lung cancer cells exposed to α-TOS. The resistance of the cells to α-TOS was overcome by its mitochondrially targeted analogue, MitoVES, that is taken up on the basis of the membrane potential, bypassing the enhanced expression of the ABCA1 protein. The in vitro results were replicated in mouse models of tumours derived from parental and resistant H1299 cells. We conclude that long-term exposure of cancer cells to α-TOS causes their resistance to the drug, which can be overcome by its mitochondrially targeted counterpart. This finding should be taken into consideration when planning clinical trials with vitamin E analogues.

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