Virginie Aires

Transport, stability, and biological activity of resveratrol

Numerous studies have reported interesting properties of trans‐resveratrol, a phytoalexin, as a preventive agent of several important pathologies: vascular diseases, cancers, viral infections, and neurodegenerative processes. These beneficial effects of resveratrol have been supported by observations at the cellular and molecular levels in both cellular and in vivo models, but the cellular fate of resveratrol remains unclear. We suggest here that resveratrol uptake, metabolism, and stability of the parent molecule could influence the biological effects of resveratrol. It appears that resveratrol stability involves redox reactions and biotransformation that influence its antioxidant properties. Resveratrols pharmacokinetics and metabolism represent other important issues, notably, the putative effects of its metabolites on pathology models. For example, some metabolites, mainly sulfate‐conjugated resveratrol, show biological effects in cellular models. The modifications of resveratrol stability, chemical structure, and metabolism could change its cellular and molecular targets and could be crucial for improving or decreasing its chemopreventive properties.

Docosahexaenoic Acid Induces Increases in [Ca2+]i via Inositol 1,4,5-Triphosphate Production and Activates Protein Kinase Cγ and -δ via Phosphatidylserine Binding Site: Implication in Apoptosis in U937 Cells

We investigated, in monocytic leukemia U937 cells, the effects of docosahexaenoic acid (DHA; 22:6 n-3) on calcium signaling and determined the implication of phospholipase C (PLC) and protein kinase C (PKC) in this pathway. DHA induced dose-dependent increases in [Ca2+]i, which were contributed by intracellular pool, via the production of inositol-1,4,5-triphosphate (IP3) and store-operated Ca2+ (SOC) influx, via opening of Ca2+ release-activated Ca2+ (CRAC) channels. Chemical inhibition of PLC, PKCγ, and PKCδ, but not of PKCβ I/II, PKCα, or PKCβI, significantly diminished DHA-induced increases in [Ca2+]i. In vitro PKC assays revealed that DHA induced a ∼2-fold increase in PKCγ and -δ activities, which were temporally correlated with the DHA-induced increases in [Ca2+]i. In cell-free assays, DHA, but not other structural analogs of fatty acids, activated these PKC isoforms. Competition experiments revealed that DHA-induced activation of both the PKCs was dose-dependently inhibited by phosphatidylserine (PS). Furthermore, DHA induced apoptosis via reactive oxygen species (ROS) production, followed by caspase-3 activation. Chemical inhibition of PKCγ/δ and of SOC/CRAC channels significantly attenuated both DHA-stimulated ROS production and caspase-3 activity. Our study suggests that DHA-induced activation of PLC/IP3 pathway and activation of PKCγ/δ, via its action on PS binding site, may be involved in apoptosis in U937 cells.

Thapsigargin-stimulated MAP kinase phosphorylation via CRAC channels and PLD activation: inhibitory action of docosahexaenoic acid.

This study was conducted on human Jurkat T‐cells to investigate the role of depletion of intracellular Ca2+ stores in the phosphorylation of two mitogen‐activated protein kinases (MAPKs), i.e. extracellular signal‐regulated kinase (ERK) 1 and ERK2, and their modulation by a polyunsaturated fatty acid, docosahexaenoic acid (DHA). We observed that thapsigargin (TG) stimulated MAPK activation by store‐operated calcium (SOC) influx via opening of calcium release‐activated calcium (CRAC) channels as tyrphostin‐A9, a CRAC channel blocker, and two SOC influx inhibitors, econazole and SKF‐96365, diminished the action of the former. TG‐stimulated ERK1/ERK2 phosphorylation was also diminished in buffer containing EGTA, a calcium chelator, further suggesting the implication of calcium influx in MAPK activation in these cells. Moreover, TG stimulated the production of diacylglycerol (DAG) by activating phospholipase D (PLD) as propranolol (PROP) (a PLD inhibitor), but not U73122 (a phospholipase C inhibitor), inhibited TG‐evoked DAG production in these cells. DAG production and protein kinase C (PKC) activation were involved upstream of MAPK activation as PROP and GF109203X, a PKC inhibitor, abolished the action of TG on ERK1/ERK2 phosphorylation. Furthermore, DHA seems to act by inhibiting PKC activation as this fatty acid diminished TG‐ and phorbol 12‐myristate 13‐acetate‐induced ERK1/ERK2 phosphorylation in these cells. Together these results suggest that Ca2+ influx via CRAC channels is implicated in PLD/PKC/MAPK activation which may be a target of physiological agents such as DHA.

Exploring new ways of regulation by resveratrol involving miRNAs, with emphasis on inflammation.

This review presents recent evidence implicating microRNAs (miRNAs) in the beneficial effects of resveratrol (trihydroxystilbene), a nonflavonoid plant polyphenol, with emphasis on its anti‐inflammatory effects. Many diseases and pathologies have been linked, directly or indirectly, to inflammation. These include infections, injuries, atherosclerosis, diabetes mellitus, obesity, cancer, osteoarthritis, age‐related macular degeneration, demyelination, and neurodegenerative diseases. Resveratrol can both decrease the secretion of proinflammatory cytokines (e.g., IL‐6, IL‐8, and TNF‐α) and increase the production of anti‐inflammatory cytokines; it also decreases the expression of adhesion proteins (e.g., ICAM‐1) and leukocyte chemoattractants (e.g., MCP‐1). Resveratrols primary targets appear to be the transcription factors AP‐1 and NF‐κB, as well as the gene COX2. Although no mechanistic link between any particular miRNA and resveratrol has been identified, resveratrol effects depend at least in part upon the modification of the expression of a variety of miRNAs that can be anti‐inflammatory (e.g., miR‐663), proinflammatory (e.g., miR‐155), tumor suppressing (e.g., miR‐663), or oncogenic (e.g., miR‐21).

Absence of correlation between oxysterol accumulation in lipid raft microdomains, calcium increase, and apoptosis induction on 158N murine oligodendrocytes

There is some evidence that oxidized derivatives of cholesterol, 7-ketocholesterol (7KC) and 7β-hydroxycholesterol (7βOHC), are increased in the plasma of patients with neurodegenerative diseases associated with demyelinization of the central nervous system (CNS). It was therefore of interest to investigate the effects of these oxysterols on oligodendrocytes, the myelin-forming cells in the CNS. To this end, 158N murine oligodendrocytes were treated with 7KC or 7βOHC inducing an apoptotic mode of cell death characterized by condensation/fragmentation of the nuclei, dephosphorylation of Akt and GSK3, mitochondrial depolarization involving Mcl-1, and caspase-3 activation. In contrast, under treatment with 27-hydroxycholesterol (27OHC), no cell death was observed. When the cells were stained with Fura-2, no significant Ca(2+) rise was found with the different oxysterols, whereas strong signals were detected with ionomycin used as positive control. At concentrations which induced apoptosis, 7KC but not 7βOHC accumulated in lipid rafts. Although not cytotoxic, 27OHC was mainly detected in lipid rafts. It is noteworthy that α-tocopherol (but not ellagic acid and resveratrol) was able to counteract 7KC- and 7βOHC-induced apoptosis and to decrease the accumulation of 7KC and 27OHC in lipid rafts. Thus, in 158N cells, the ability of oxysterols to trigger a mode of cell death by apoptosis involving GSK-3 and caspase-3 activation is independent of the increase in the Ca(2+) level and of their accumulation in lipid raft microdomains.

Resveratrol initiates differentiation of mouse skeletal muscle-derived C2C12 myoblasts

Resveratrol is one of the most widely studied bio-active plant polyphenols. While its effect on endothelial blood vessel cells, cancer cells, inflammatory processes and neurodegenerative events is well documented, little is known about the implication of this phytophenol in differentiating processes, particularly in skeletal muscle cells. Here, we report the effects of resveratrol on mouse skeletal muscle-derived cells (C2C12) in either a nondifferentiated (myoblasts) or differentiated state (myotubes) by evaluating resveratrol uptake, cell proliferation, changes in cell shape, and the expression of genes encoding muscle-specific transcription factors or contractile proteins. Resveratrol: (1) rapidly accumulates within cells through passive and facilitated processes; (2) does not strongly affect cell viability, cell cycle and apoptosis; (3) behaves as a pro-differentiating agent as shown by the lengthening of cells, leading to a myotube phenotype; (4) upregulates muscular pro-differentiation markers and transcription factors (myogenin, Scrp3) starting after 12h of exposure and strongly increases heavy chain myosin content after 18h of exposure to resveratrol; (5) increases the Srf transcription factors transcript level, a target mRNA of the miRNA-133b, which is itself downregulated by this polyphenol. These results put forward new pro-differentiating regulatory properties of resveratrol on skeletal muscles at least partly via modulation of specific miRNAs.

Docosahexaenoic acid inhibits cancer cell growth via p27Kip1, CDK2, ERK1/ERK2, and retinoblastoma phosphorylation

Docosahexaenoic acid (DHA), a PUFA of the n-3 family, inhibited the growth of FM3A mouse mammary cancer cells by arresting their progression from the late-G1 to the S phase of the cell cycle. DHA upregulated p27Kip1 levels by inhibiting phosphorylation of mitogen-activated protein (MAP) kinases, i.e., ERK1/ERK2. Indeed, inhibition of ERK1/ERK2 phosphorylation by DHA, U0126 [chemical MAPK extracellularly signal-regulated kinase kinase (MEK) inhibitor], and MEKSA (cells expressing dominant negative constructs of MEK) resulted in the accumulation of p27Kip1. MAP kinase (MAPK) inhibition by DHA did not increase p27Kip1 mRNA levels. Rather, this fatty acid stabilized p27Kip1 contents and inhibited MAPK-dependent proteasomal degradation of this protein. DHA also diminished cyclin E phosphorylation, cyclin-dependent kinase-2 (CDK2) activity, and phosphorylation of retinoblastoma protein in these cells. Our study shows that DHA arrests cell growth by modulating the phosphorylation of cell cycle-related proteins.

Modulation of intracellular calcium concentrations and T cell activation by prickly pear polyphenols

Opuntia ficus indica(prickly pear) polyphenolic compounds (OFPC) triggered an increase in [Ca2+]i in human Jurkat T-cell lines. Furthermore, OFPC-induced rise in [Ca2+]i was significantly curtailed in calcium-free buffer (0% Ca2+) as compared to that in 100% Ca2+ medium. Preincubation of cells with tyrphostin A9, an inhibitor of Ca2+ release-activated Ca2+(CRAC) channels, significantly diminished the OFPC-induced sustained response on the increases in [Ca2+]i. Lanthanum and nifedipine, the respective inhibitors of voltage-dependent and L-type calcium channels, failed to curtail significantly the OFPC-induced calcium response. As OFPC still stimulated increases in [Ca2+]i in 0% Ca2+ medium, the role of intracellular calcium was investigated. Hence, addition of thapsigargin (TG), an inhibitor of Ca2+-ATPase of the endoplasmic reticulum (ER), during the OFPC-induced peak response exerted an additive effect, indicating that the mechanism of action of these two agents are different. Furthermore, U73122, an inhibitor of IP3 production, completely abolished increases in [Ca2+]i, induced by OFPC, suggesting that these polyphenols induce the production of IP3 that recruits calcium from ER pool. Polyphenolic compounds do act extracellularly as addition of fatty acid-free bovine serum albumin (BSA) significantly diminished the rise in [Ca2+]i evoked by the formers. OFPC also induced plasma membrane hyperpolarisation which was reversed by addition of BSA. OFPC were found to curtail the expression of IL-2 mRNA and T-cell blastogenesis. Together these results suggest that OFPC induce increases in [Ca2+]i via ER pool and opening of CRAC channels, and exert immunosuppressive effects in Jurkat T-cells.

Importance of lipid microdomains, rafts, in absorption, delivery, and biological effects of resveratrol.

The preventive effects of the phytoalexin trans‐resveratrol toward cancer have been largely described at the cellular and molecular levels in both in vivo and in vitro models; however, its primary targets are still poorly identified. In this review, we show the crucial role of cell membrane microdomains, that is, lipid rafts, not solely in the initiation of the early biochemical events triggered by resveratrol leading to cancer cell death, but also in resveratrol absorption and distribution. Resveratrol accumulates in lipid rafts and is then taken up by cells through raft‐dependent endocytosis. These events allow early activation of kinase pathways and redistribution of cell death receptors within lipid microdomains, events ultimately leading to apoptotic cell death.

Docosahexaenoic acid and other fatty acids induce a decrease in pHi in Jurkat T-cells

Docosahexaenoic acid (DHA) induced rapid (t1/2=33 s) and dose‐dependent decreases in pHi in BCECF‐loaded human (Jurkat) T‐cells. Addition of 5‐(N,N‐dimethyl)‐amiloride, an inhibitor of Na+/H+ exchanger, prolonged DHA‐induced acidification as a function of time, indicating that the exchanger is implicated in pHi recovery. Other fatty acids like oleic acid, arachidonic acid, eicosapentaenoic acid, but not palmitic acid, also induced a fall in pHi in these cells. To assess the role of calcium in the DHA‐induced acidification, we conducted experiments in Ca2+‐free (0% Ca2+) and Ca2+‐containing (100% Ca2+) buffer. We observed that there was no difference in the degree of DHA‐induced transient acidification in both the experimental conditions, though pHi recovery was faster in 0% Ca2+ medium than that in 100% Ca2+ medium. In the presence of BAPTA, a calcium chelator, a rapid recovery of DHA‐induced acidosis was observed. Furthermore, addition of CaCl2 into 0% Ca2+ medium curtailed DHA‐evoked rapid pHi recovery. In 0% Ca2+ medium, containing BAPTA, DHA did not evoke increases in [Ca2+]i, though this fatty acid still induced a rapid acidification in these cells. These observations suggest that calcium is implicated in the long‐lasting DHA‐induced acidosis. DHA‐induced rapid acidification may be due to its deprotonation in the plasma membrane (flip‐flop model), as suggested by the following observations: (1) DHA with a –COOH group induced intracellular acidification, but this fatty acid with a –COOCH3 group failed to do so, and (2) DHA, but not propionic acid, ‐induced acidification was completely reversed by addition of fatty acid‐free bovine serum albumin in these cells. These results suggest that DHA induces acidosis via deprotonation and Ca2+ mobilization in human T‐cells.