Potential anti-cancer activity of 7-O-pentyl quercetin: Efficient, membrane-targeted kinase inhibition and pro-oxidant effect.

Quercetin is a redox-active plant-derived flavonoid with potential anticancer effects, stemming largely from its interaction with a number of proteins, and in particular from inhibition of pro-life kinases. To improve efficacy, we reasoned that a local increase in concentration of the compound at the level of cell membranes would result in a more efficient interaction with membrane-associated signaling kinases. We report here the synthesis of all five isomeric quercetin derivatives in which an n-pentyl group was linked via an ether bond to each hydroxyl of the flavonoid kernel. This strategy proved effective in directing quercetin to cellular membranes, and revealed a remarkable dependence of the derivatives' bioactivity on the specific site of functionalization. The isomer bearing the pentyl group in position 7, Q-7P, turned out to be the most effective and promising derivative, selectively inducing apoptosis in tumoral and fast-growing cells, while sparing slow-growing, non-tumoral ones. Cytotoxicity for tumoral cells was strongly enhanced compared to quercetin itself. Q-7P induced massive ROS production, which however accounted only partially for cell death. Alterations in the levels of various signaling phospho-proteins were observed in a proteomics screen. An important contribution seems to come from inhibition of the PI3K/Akt pathway. This work opens new perspectives in developing membrane-associating, polyphenol-based anticancer agents.

Cytotoxicity of mitochondria-targeted resveratrol derivatives: interactions with respiratory chain complexes and ATP synthase.

We recently reported that mitochondria-targeted derivatives of resveratrol are cytotoxic in vitro, selectively inducing mostly necrotic death of fast-growing and tumoral cells when supplied in the low µM range (N. Sassi et al., Curr. Pharm. Des. 2014). Cytotoxicity is due to H2O2 produced upon accumulation of the compounds into mitochondria. We investigate here the mechanisms underlying ROS generation and mitochondrial depolarization caused by these agents. We find that they interact with the respiratory chain, especially complexes I and III, causing superoxide production. "Capping" free hydroxyls with acetyl or methyl groups increases their effectiveness as respiratory chain inhibitors, promoters of ROS generation and cytotoxic agents. Exposure to the compounds also induces an increase in the occurrence of short transient [Ca(2+)] "spikes" in the cells. This increase is unrelated to ROS production, and it is not the cause of cell death. These molecules furthermore inhibit the F0F1 ATPase. When added to oligomycin-treated cells, the acetylated/methylated ones cause a recovery of the cellular oxygen consumption rates depressed by oligomycin. Since a protonophoric futile cycle which might account for the uncoupling effect is impossible, we speculate that the compounds may cause the transformation of the ATP synthase and/or respiratory chain complex(es) into a conduit for uncoupled proton translocation. Only in the presence of excess oligomycin the most effective derivatives appear to induce the mitochondrial permeability transition (MPT) within the cells. This may be considered to provide circumstantial support for the idea that the ATP synthase is the molecular substrate for the MPT pore.

Cytotoxicity of a mitochondriotropic quercetin derivative: mechanisms.

The mitochondriotropic compound 7-O-(4-triphenylphosphoniumbutyl)quercetin iodide (Q-7BTPI) in the µM concentration range caused necrotic death of cultured cells by acting as a prooxidant, with generation of superoxide anion in the mitochondria. Externally added membrane-permeating superoxide dismutase or catalase largely prevented death. Rescue by permeant catalase indicates that the toxicant is H(2)O(2), or reactive species derived from it. Rescue by permeant dismutase suggests the possibility of a chain mechanism of H(2)O(2) production, in which dismutation of superoxide constitutes a termination step. Oxidative stress was due to the presence of free phenolic hydroxyls and to accumulation in mitochondria, since the analogous mitochondriotropic per-O-methylated compound -3,3',4',5-tetra-O-methyl,7-O-(4-triphenylphosphoniumbutyl) quercetin iodide (QTM-7BTPI)-or Quercetin itself induced no or little superoxide production and cell death. Q-7BTPI did not cause a significant perturbation of the mitochondrial transmembrane potential or of respiration in cells. On the other hand its presence led to inhibition of glutathione peroxidase, an effect expected to accentuate oxidative stress by interfering with the elimination of H(2)O(2). An exogenous permeable glutathione precursor determined a strong increase of cellular glutathione levels but did not rescue the cells. Death induction was selective for fast-growing C-26 tumoral cells and mouse embryonic fibroblasts (MEFs) while sparing slow-growing MEFs. This suggests a possible use of Q-7BTPI as a chemotherapeutic agent.

Acetal derivatives as prodrugs of resveratrol.

The pharmacological exploitation of resveratrol is hindered by rapid phase-II conjugative metabolism in enterocytes and hepatocytes. One approach to the solution of this problem relies on prodrugs. We report the synthesis and characterization as well as the assessment of in vivo absorption and metabolism of a set of prodrugs of resveratrol in which the OH groups are engaged in the formal (-OCH2OR) or the more labile acetal (-OCH(CH3)OR) linkages. As carrier group (R) of the prodrug, we have used short ethyleneglycol oligomers (OEG) capped by a terminal methoxy group: -O-(CH2CH2O)n-CH3 (n = 0, 1, 2, 3, 4, 6). These moieties are expected to exhibit, to a degree, the favorable properties of longer polyethyleneglycol (PEG) chains, while their relatively small size makes for a more favorable drug loading capacity. After administration of formal-based prodrugs to rats by oral gavage, significant concentrations of derivatives were measured in blood samples over several hours, in all cases except for n = 0. Absorption was maximal for n = 4. Complete deprotection to give resveratrol and its metabolites was however too slow to be of practical use. Administration of the acetal prodrug carrying tetrameric OEG chains resulted instead in the protracted presence of resveratrol metabolites in blood, consistent with a progressive regeneration of the parent molecule from the prodrug after its absorption. The results suggest that prodrugs of polyphenols based on the acetal bond and short ethyleneglycol oligomers of homogeneous size may be a convenient tool for the systemic delivery of the unconjugated parent compound.

Role of Kv1.3 mitochondrial potassium channel in apoptotic signalling in lymphocytes.

Mitochondria have been shown to play a pivotal role in apoptotic signalling in various cell types. We have recently reported that in lymphocytes the voltage-gated potassium channel Kv1.3, known to reside in the plasma membrane, is active also in the inner mitochondrial membrane. Upon induction of apoptosis, outer-membrane inserted Bax binds to and inhibits Kv1.3 resulting in hyperpolarization, an increase in reactive oxygen species production and cytochrome c release. In cells lacking Kv1.3 these events do not take place. Here, we present new data which further corroborates an important role of this channel in the sequence of events leading to Bax-induced cytochrome c release. Recombinant Kv1.3, when pre-incubated with Bax, prevents the actions of Bax at the level of mitochondria. Furthermore, we report the presence of Kv1.3 protein in mitochondria from PC3 and MCF-7 cancer cells, suggesting that this channel might play a role in the apoptotic signalling not only in lymphocytes but also in other cells.

An investigation of the occurrence and properties of the mitochondrial intermediate-conductance Ca2+-activated K+ channel mtKCa3.1.

The mitochondrial intermediate-conductance Ca2+-activated K+ channel mtKCa3.1 has recently been discovered in the HCT116 colon tumor-derived cell line, which expresses relatively high levels of this protein also in the plasma membrane. Electrophysiological recordings revealed that the channel can exhibit different conductance states and kinetic modes, which we tentatively ascribe to post-translational modifications. To verify whether the localization of this channel in mitochondria might be a peculiarity of these cells or a more widespread feature we have checked for the presence of mtKCa3.1 in a few other cell lines using biochemical and electrophysiological approaches. It turned out to be present at least in some of the cells investigated. Functional assays explored the possibility that mtKCa3.1 might be involved in cell proliferation or play a role similar to that of the Shaker-type KV1.3 channel in lymphocytes, which interacts with outer mitochondrial membrane-inserted Bax thereby promoting apoptosis (Szabò, I. et al., Proc. Natl. Acad Sci. USA 105 (2008) 14861-14866). A specific KCa3.1 inhibitor however did not have any detectable effect on cell proliferation or death.

Impact of mitochondriotropic quercetin derivatives on mitochondria.

Mitochondria-targeted polyphenols are being developed with the intent to intervene on the levels of reactive oxygen species (ROS) in mitochondria. Polyphenols being more than just anti-oxidants, the interaction of these derivatives with the organelles needs to be characterised. We have studied the effects of two quercetin derivatives, 3-(4-O-triphenylphosphoniumbutyl)quercetin iodide (Q3BTPI) and its tetracetylated analogue (QTA3BTPI), on the inner membrane aspecific permeability, transmembrane voltage difference and respiration of isolated rat liver mitochondria. While the effects of low concentrations were too small to be reliably defined, when used in the 5-20 microM range these compounds acted as inducers of the mitochondrial permeability transition (MPT), an effect due to pro-oxidant activity. Furthermore, Q3BTPI behaved as an uncoupler of isolated mitochondria, causing depolarisation and stimulating oxygen consumption. When applied to tetramethylrhodamine methyl ester (TMRM)-loaded HepG2 or Jurkat cells uptake of the compounds was predictably associated with a loss of TMRM fluorescence, but there was no indication of MPT induction. A production of superoxide could be detected in some cells upon prolonged incubation of MitoSOX-loaded cells with QTA3BTPI. The overall effects of these model mitochondriotropic polyphenols may thus differ considerably depending on whether their hydroxyls are protected or not and on the experimental system. In vivo assays will be needed for a definitive assessment of their bioactivities.

A mitochondriotropic derivative of quercetin: a strategy to increase the effectiveness of polyphenols.

Mitochondria-targeted compounds are needed to act on a variety of processes that take place in these subcellular organelles and that have great pathophysiological relevance. In particular, redox-active molecules that are capable of homing in on mitochondria provide a tool to intervene on a major cellular source of reactive oxygen species and on the processes they induce, notably the mitochondrial permeability transition and cell death. We have linked the 3-OH of quercetin (3,3',4',5,7-pentahydroxy flavone), a model polyphenol, and the triphenylphosphonium moiety, a membrane-permeant cationic group, to produce proof-of-principle mitochondriotropic quercetin derivatives. The remaining hydroxyls were sometimes acetylated to hinder metabolism and improve solubility. The new compounds accumulate in mitochondria in a transmembrane potential-driven process and are only slowly metabolised by cultured human colon cells. They inhibit mitochondrial ATPase activity much as quercetin does, and are toxic for fast-growing cells.

Development of mitochondria-targeted derivatives of resveratrol.

To target natural polyphenols to the subcellular site where their redox properties might be exploited at best, that is, mitochondria, we have synthesised new proof-of-principle derivatives by linking resveratrol (3,4',5-trihydroxy-trans-stilbene) to the membrane-permeable lipophilic triphenylphosphonium cation. The new compounds, (4-triphenylphosphoniumbutyl)-4'-O-resveratrol iodide and its bis-acetylated derivative, the latter intended to provide transient protection against metabolic conjugation, accumulate into energized mitochondria as expected and are cytotoxic for fast-growing but not for slower-growing cells. They provide a powerful potential tool to intervene on mitochondrial and cellular redox processes of pathophysiological relevance.

Resveratrol derivatives as a pharmacological tool.

Prodrugs of resveratrol are under development. Among the long-term goals, still largely elusive, are (1) modulating physical properties (e.g., water-soluble derivatives bearing polyethylene glycol chains), (2) changing distribution in the body (e.g., galactosyl derivatives restricted to the intestinal lumen), (3) increasing absorption from the gastrointestinal tract (e.g., derivatives imitating the natural substrates of endogenous transporters), and (4) hindering phase II metabolism (e.g., temporarily blocking the hydroxyls), all contributing to (5) increasing bioavailability. The chemical bonds that have been tested for functionalization include carboxyester, acetal, and carbamate groups. A second approach, which can be combined with the first, seeks to reinforce or modify the biochemical activities of resveratrol by concentrating the compound at specific subcellular sites. An example is provided by mitochondria-targeted derivatives. These proved to be pro-oxidant and cytotoxic in vitro, selectively killing fast-growing and tumor cells when supplied in the low micromolar range. This suggests the possibility of anticancer applications.

Mitochondria-targeted resveratrol derivatives act as cytotoxic pro-oxidants.

Resveratrol derivatives bearing an O-linked mitochondria-targeting 4-triphenylphosphoniumbutyl group at either position 3 or position 4' are prooxidant and cytotoxic for cultured cells, selectively killing fast-growing cells when supplied in the low µM range. Resveratrol is essentially without effect under these experimental conditions, while the cytotoxicity of the mitochondriotropic derivatives increases if they are methylated on the remaining hydroxyls. Experiments with Bax(-/-)/Bak(-/-) cells and a pan-caspase inhibitor show that cell death is mostly of the necrotic type. Cytotoxicity is due to ROS produced upon accumulation of the compounds into mitochondria, and specifically to H2O2, since externally added membrane-permeant catalase largely prevents cell death while superoxide dismutase potentiates toxicity. The mitochondriotropic compounds cause ROS-independent depolarization of in situ mitochondria. Effectiveness is increased if resveratrol hydroxyls are acetylated or methylated; this excludes the involvement of autooxidation of the polyphenolic nucleus and a protonophoric cycle as the causes of ROS generation and of depolarization, respectively. Resveratrol-triphenylphosphonium conjugates may thus represent a new class of chemotherapeutic agents, redox-active "mitocans", whose mechanisms of action and in vivo activity are worthy of further investigation.

Improving the efficacy of plant polyphenols.

Plant polyphenols exhibit potentially useful effects in a wide variety of pathophysiological settings. They interact with proteins such as signalling kinases, transcription factors and ion channels, and modulate redox processes, such as those taking place in mitochondria. Biomedical applications of these natural compounds are however severely hindered by their low bioavailability, rapid metabolism, and often by unfavourable physico-chemical properties, e.g. a generally low water solubility. Derivatives are under development with the aim of improving their bioavailability and/or bioefficacy. Various strategies can be adopted. An increase in circulating blood levels of non-metabolized natural compound may be attainable through prodrugs. In the ideal prodrug, phenolic hydroxyls are protected by capping groups which a) help or at least do not hinder permeation of epithelia; b) prevent conjugative modifications during absorption and first-pass through the liver; c) are eliminated with opportune kinetics to regenerate the parent compound. Moreover, prodrugs may be designed with the goals of modulating physical properties of the parent compound, and/or changing its distribution in the body. A more specific action may be achieved by concentrating the compounds at specific sites of action. An example of the second approach is represented by mitochondria-targeted redox-active polyphenol derivatives, designed to intervene on radical processes in these organelles and as a tool either to protect cells from oxidative insults or to precipitate their death. Mitochondrial targeting can be achieved through conjugation with a triphenylphosphonium lipophilic cation. Quercetin and resveratrol were chosen as model polyphenols for these proof-of-concept studies. Data available at the moment show that both quercetin and resveratrol mitochondria-targeted derivatives are pro-oxidant and cytotoxic in vitro, selectively killing fast-growing and tumoural cells when supplied in the low µM range; the mechanism of ROS generation appears to differ between the two classes of compounds. These approaches are emerging as promising strategies to obtain new efficient chemopreventive and/or chemotherapeutic drugs based on polyphenols derivatives.

Inhibitors of mitochondrial Kv1.3 channels induce Bax/Bak-independent death of cancer cells.

Overcoming the resistance of tumours to chemotherapy, often due to downregulation of Bax and Bak, represents a significant clinical challenge. It is therefore important to identify novel apoptosis inducers that bypass Bax and Bak. Potassium channels are emerging as oncological targets and a crucial role of mitochondrial Kv1.3 in apoptosis has been demonstrated. Here we report for the first time that Psora-4, PAP-1 and clofazimine, three distinct membrane-permeant inhibitors of Kv1.3, induce death by directly targeting the mitochondrial channel in multiple human and mouse cancer cell lines. Importantly, these drugs activated the intrinsic apoptotic pathway also in the absence of Bax and Bak, a result in agreement with the current mechanistic model for mitochondrial Kv1.3 action. Genetic deficiency or short interfering RNA (siRNA)-mediated downregulation of Kv1.3 abrogated the effects of the drugs. Intraperitoneal injection of clofazimine reduced tumour size by 90% in an orthotopic melanoma B16F10 mouse model in vivo, while no adverse effects were observed in several healthy tissues. The study indicates that inhibition of mitochondrial Kv1.3 might be a novel therapeutic option for the induction of cancer cell death independent of Bax and Bak.

Intermediate conductance Ca2+-activated potassium channel (KCa3.1) in the inner mitochondrial membrane of human colon cancer cells.

Patch-clamping mitoplasts isolated from human colon carcinoma 116 cells has allowed the identification and characterization of the intermediate conductance Ca(2+)-activated K(+)-selective channel K(Ca)3.1, previously studied only in the plasma membrane of various cell types. Its identity has been established by its biophysical and pharmacological properties. Its localisation in the inner membrane of mitochondria is indicated by Western blots of subcellular fractions, by recording of its activity in mitochondria made fluorescent by a mitochondria-targeted fluorescent protein and by the co-presence of channels considered to be markers of the inner membrane. Moderate increases of mitochondrial matrix [Ca(2+)] will cause mtK(Ca)3.1 opening, thus linking inner membrane K(+) permeability and transmembrane potential to Ca(2+) signalling.