Gamma-tocotrienol modulation of senescence-associated gene expression prevents cellular aging in human diploid fibroblasts

OBJECTIVE: Human diploid fibroblasts undergo a limited number of cellular divisions in culture and progressively reach a state of irreversible growth arrest, a process termed cellular aging. The beneficial effects of vitamin E in aging have been established, but studies to determine the mechanisms of these effects are ongoing. This study determined the molecular mechanism of γ-tocotrienol, a vitamin E homolog, in the prevention of cellular aging in human diploid fibroblasts using the expression of senescence-associated genes. METHODS: Primary cultures of young, pre-senescent, and senescent fibroblast cells were incubated with γ-tocotrienol for 24 h. The expression levels of ELN, COL1A1, MMP1, CCND1, RB1, and IL6 genes were determined using the quantitative real-time polymerase chain reaction. Cell cycle profiles were determined using a FACSCalibur Flow Cytometer. RESULTS: The cell cycle was arrested in the G0/G1 phase, and the percentage of cells in S phase decreased with senescence. CCND1, RB1, MMP1, and IL6 were upregulated in senescent fibroblasts. A similar upregulation was not observed in young cells. Incubation with γ-tocotrienol decreased CCND1 and RB1 expression in senescent fibroblasts, decreased cell populations in the G0/G1 phase and increased cell populations in the G2/M phase. γ-Tocotrienol treatment also upregulated ELN and COL1A1 and downregulated MMP1 and IL6 expression in young and senescent fibroblasts. CONCLUSION: γ-Tocotrienol prevented cellular aging in human diploid fibroblasts, which was indicated by the modulation of the cell cycle profile and senescence-associated gene expression.

Effect of trolox addition to cryopreservation media on human sperm motility

Sperm parameters and motion kinetics are affected by cryopreservation. The main purpose of the current study was to determine the effect of different concentrations of Trolox as an antioxidant to freezing-thawing procedure on human sperm kinematic parameter. Semen was collected from 20 normal donors and divided into five aliquots prior to cryopreservation. The first aliquot was analyzed by computer-assisted sperm analysis [CASA]. Other aliquots were mixed with cryo-protective agent containing 0, 20, 40, and 80 micro mol Trolox and treated samples were cryopreserved in liquid nitrogen. After two weeks samples were thawed and sperm motion kinematics was measured by CASA. Percent motility [Mot], curvilinear velocity [VCL], straight-line velocity [VSL], average path velocity [VAP], linearity [LIN], and amplitude of lateral head displacement [ALH] were compared before and after freeze. Addition of 40 micro mol Trolox resulted in significantly higher [p<0.05] post thaw VCL, VSL and VAP compared to other groups. Therefore the percentage of post thaw motile spermatozoa were significantly higher [p<0.01]. The supplementation of Trolox significantly improved the post-thawed human semen quality, especially progressive motility and average path velocity

Duration and Magnitude of Extracellular Signal-Regulated Protein Kinase Phosphorylation Determine Adipogenesis or Osteogenesis in Human Bone Marrow-Derived Stem Cells

PURPOSE: Imbalances between osteogenic and adipogenic differentiation leads to diseases such as osteoporosis. The aim of our study was to demonstrate the differences in extracellular signal-regulated kinase (ERK) phosphorylation during both adipogenesis and osteogenesis of human bone marrow-derived stem cells (BMSCs). MATERIALS AND METHODS: Using troglitazone, GW9662 and U0126, we investigated their role in hBMSC differentiation to adipogenic and osteogenic fates. RESULTS: ERK1/2 inhibition by U0126 suppressed proliferator-activated receptor (PPAR)gamma expression and lipid accumulation, while it decreased the mRNA expression of adipogenic genes (lipoprotein lipase, PPARgamma, and adipocyte protein) and osteogenic genes (type I collagen and osteopontin). ERK phosphorylation was transient and decreased during adipogenesis, whereas it occurred steadily during osteogenesis. Troglitazone, a PPARgamma agonist, induced adipogenesis by inhibiting ERK phosphorylation even in an osteogenic medium, suggesting that ERK signaling needs to be shut off in order to proceed with adipose cell commitment. Cell proliferation was greatly increased in osteogenesis but was not changed during adipogenesis, indicating that ERK might play different roles in cellular proliferation and differentiation between the two committed cell types. CONCLUSION: The duration and magnitude of ERK activation might be a crucial factor for the balance between adipogenesis and osteogenesis in human bone marrow-derived stem cells.

Duration and Magnitude of Extracellular Signal-Regulated Protein Kinase Phosphorylation Determine Adipogenesis or Osteogenesis in Human Bone Marrow-Derived Stem Cells

PURPOSE: Imbalances between osteogenic and adipogenic differentiation leads to diseases such as osteoporosis. The aim of our study was to demonstrate the differences in extracellular signal-regulated kinase (ERK) phosphorylation during both adipogenesis and osteogenesis of human bone marrow-derived stem cells (BMSCs). MATERIALS AND METHODS: Using troglitazone, GW9662 and U0126, we investigated their role in hBMSC differentiation to adipogenic and osteogenic fates. RESULTS: ERK1/2 inhibition by U0126 suppressed proliferator-activated receptor (PPAR)gamma expression and lipid accumulation, while it decreased the mRNA expression of adipogenic genes (lipoprotein lipase, PPARgamma, and adipocyte protein) and osteogenic genes (type I collagen and osteopontin). ERK phosphorylation was transient and decreased during adipogenesis, whereas it occurred steadily during osteogenesis. Troglitazone, a PPARgamma agonist, induced adipogenesis by inhibiting ERK phosphorylation even in an osteogenic medium, suggesting that ERK signaling needs to be shut off in order to proceed with adipose cell commitment. Cell proliferation was greatly increased in osteogenesis but was not changed during adipogenesis, indicating that ERK might play different roles in cellular proliferation and differentiation between the two committed cell types. CONCLUSION: The duration and magnitude of ERK activation might be a crucial factor for the balance between adipogenesis and osteogenesis in human bone marrow-derived stem cells.

CD36 signaling inhibits the translation of heat shock protein 70 induced by oxidized low density lipoprotein through activation of peroxisome proliferators-activated receptor gamma

Oxidized LDL (OxLDL), a causal factor in atherosclerosis, induces the expression of heat shock proteins (Hsp) in a variety of cells. In this study, we investigated the role of CD36, an OxLDL receptor, and peroxisome proliferator-activated receptor gamma (PPAR gamma) in OxLDL-induced Hsp70 expression. Overexpression of dominant-negative forms of CD36 or knockdown of CD36 by siRNA transfection increased OxLDL-induced Hsp70 protein expression in human monocytic U937 cells, suggesting that CD36 signaling inhibits Hsp70 expression. Similar results were obtained by the inhibition of PPAR gamma activity or knockdown of PPAR gamma expression. In contrast, overexpression of CD36, which is induced by treatment of MCF-7 cells with troglitazone, decreased Hsp70 protein expression induced by OxLDL. Interestingly, activation of PPAR gamma through a synthetic ligand, ciglitazone or troglitazone, decreased the expression levels of Hsp70 protein in OxLDL-treated U937 cells. However, major changes in Hsp70 mRNA levels were not observed. Cycloheximide studies demonstrate that troglitazone attenuates Hsp70 translation but not Hsp70 protein stability. PPAR gamma siRNA transfection reversed the inhibitory effects of troglitazone on Hsp70 translation. These results suggest that CD36 signaling may inhibit stress- induced gene expression by suppressing translation via activation of PPAR gamma in monocytes. These findings reveal a new molecular basis for the anti-inflammatory effects of PPAR gamma.

15-Deoxy-delta12,14-PGJ2inhibits IL-6-induced Stat3 phosphorylation in lymphocytes

15-deoxy-delta12,14-PGJ2(15d-PGJ2) is a natural ligand that activates the peroxisome proliferators-activated receptor (PPAR) gamma, a member of nuclear receptor family implicated in regulation of lipid metabolism and adipocyte differentiation. Recent studies have shown that 15d-PGJ2 is the potent anti-inflammatory agent functioning via PPARgamma-dependent and -independent mechanisms. Most postulated mechanisms for anti-inflammatory action of PPARgamma agonists are involved in inhibiting NF-kappaB signaling pathway. We examined the possibility that IL-6 signaling via the Jak-Stat pathway is modulated by 15d-PGJ2 in lymphocytes and also examined whether the inhibition of IL-6 signaling is dependent of PPARgamma. 15d-PGJ2 blocked IL-6 induced Stat1 and Stat3 activation in primary human lymphocytes, Jurkat cells and immortalized rheumatoid arthritis B cells. Inhibition of IL-6 signaling was induced rapidly within 15 min after treatment of 15d-PGJ2. Other PPARgamma-agonists, such as troglitazone and ciglitazone, did not inhibit IL-6 signaling, indicating that 15d-PGJ2 affect the IL-6-induced Jak-Stat signaling pathway via PPARgamma-independent mechanism. Although cycloheximide reversed 15d-PGJ2-mediated inhibition of Stat3 activation, actinomycin D had no effect on 15d-PGJ2-mediated inhibition of IL-6 signaling, indicating that inhibition of IL-6 signaling occur independent of de novo gene expression. These results show that 15d-PGJ2 specifically inhibit Jak-Stat signaling pathway in lymphocytes, and suggest that 15d-PGJ2 may regulate inflammatory reactions through the modulation of different signaling pathway other than NF-kappaB in lymphocytes.

Ox-LDL suppresses PMA-induced MMP-9 expression and activity through CD36-mediated activation of PPAR-gamma

During chronic inflammatory response, mono- cytes/macrophages produce 92-kDa matrix metalloproteinase-9 (MMP-9), which may contribute to their extravasation, migration and tissue remodeling. Activation of peroxisome proliferator- activated factor receptor-gamma (PPAR-gamma) has been shown to inhibit MMP-9 activity. To evaluate whether ox-LDL, a PPAR-gamma activator, inhibits PMA-induced MMP-9 expression and activity, and if so, whether CD36 and PPAR-gamma are involved in this process, we investigated the effect of ox-LDL on MMP-9 expression and activity in PMA-activated human monocytic cell line U937. PMA-induced MMP-9 expression and activity were suppressed by the treatment with ox-LDL (50 micrigram/ml) or PPAR-gamma activators such as troglitazone (5 micrometer), ciglitazone (5 micrometer), and 15d- PGJ2 (1 micrometer) for 24 h. This ox-LDL or PPAR-gamma activator-mediated inhibition of micrometer P-9 activity was diminished by the pre-treatment of cells with a blocking antibody to CD36, or PGF2a (0.3 micrometer), which is a PPAR-gamma inhibitor, as well as overexpression of a dominant-negative form of CD36. Taken together, these results suggest that ox-LDL suppresses PMA-induced MMP-9 expression and activity through CD36-mediated activation of PPAR-gamma.

Effect of a water soluble derivative of alpha-tocopherol on radiation response of Saccharomyces cerevisiae.

The radioprotection conferred by a highly water soluble glucose derivative of alpha-tocopherol, namely, 2-(alpha-D-glucopyranosyl) methyl-2,5,7,8-tetramethylchroman-6-ol (TMG) in Saccharomyces cerevisiae was studied. Cells grown in standard YEPD-agar medium and irradiated in the presence of TMG showed a concentration dependent higher survival up to 10 mM of TMG in comparison to cells irradiated in distilled water. Treatment of TMG to cells given either before or immediately after irradiation but not during irradiation, had no effect on their radiation response. S. cerevisiae strain LP1383 (rad52) which is defective in recombination repair showed enhanced radioresistance only when subjected to irradiation in presence of TMG. Cells of rad52 strain grown in the medium containing TMG showed a radiation response similar to that of cells grown in the medium without TMG. The nature of TMG dependent enhanced radioresistance was studied by scoring the mutations in the strain D-7, which behaved like wild type strain in complete medium, at trp and ilv loci. Our study indicated that TMG confers radioresistance in S. cerevisiae possibly by two mechanisms viz. (i), by eliminating radiation induced reactive free radicals when the irradiation is carried out in the presence of TMG and (ii), by activating an error prone repair process involving RAD52 gene, when the cells are grown in the medium containing TMG.

Vasorelaxant effects of the potassium channel opener SR 47063 on the isolated human saphenous vein and rat aorta

The vasorelaxant effects of SR 47063 (4-(2-cyanimino-1,2-dihydropyrid-1-yl)-2,2-dimethyl-6-nitrochromene), a new K+-channel opener structurally related to levcromakalim, were examined in isolated human saphenous vein (HSV) and rat aorta (RA). HSV or RA rings were precontracted with either KCl or noradrenaline and cumulative relaxant concentration-response curves were obtained for SR 47063 (0.1 nM to 1 µM) in the presence or absence of 3 µM glibenclamide. SR 47063 potently relaxed HSV and RA precontracted with 20 mM (but not 60 mM) KCl or 10 µM noradrenaline in a concentration-dependent manner, showing slightly greater activity in the aorta. The potency of the effect of SR 47063 on HSV and RA was 12- and 58-fold greater, respectively, than that reported for the structurally related K+-channel opener levcromakalim. The vasorelaxant action of SR 47063 in both blood vessels was strongly inhibited by 3 µM glibenclamide, consistent with a mechanism of action involving ATP-dependent K+-channels

Anti-oxidative neuroprotection by estrogens in mouse cortical cultures

Estrogen replacement therapy in postmenopausal women may reduce the risk of Alzheimer's disease, possibly by ameliorating neuronal degeneration. In the present study, we examined the neuroprotective spectrum of estrogen against excitotoxicity, oxidative stress, and serum-deprivation-induced apoptosis of neurons in mouse cortical cultures. 17beta-estradiol as well as 17alpha-estradiol and estrone attenuated oxidative neuronal death induced by 24 hr exposure to 100 microM FeCl2, excitotoxic neuronal death induced by 24 hr of exposure to 30 microM N-methyl-D-aspartate (NMDA) and serum-deprivation induced neuronal apoptosis. Furthermore, estradiol attenuated neuronal death induced by Abeta25-35. However, all these neuroprotective effects were mediated by the anti-oxidative action of estrogens. When oxidative stress was blocked by an antioxidant trolox, estrogens did not show any additional protection. Addition of a specific estrogen receptor antagonist ICI182,780 did not reverse the protection offered by estrogens. These findings suggest that high concentrations of estrogen protect against various neuronal injuries mainly by its anti-oxidative effects as previously shown by Behl et al. Our results do not support the view that classical estrogen receptors mediate neuroprotection.

Anti-oxidative neuroprotection by estrogens in mouse cortical cultures

Estrogen replacement therapy in postmenopausal women may reduce the risk of Alzheimer's disease, possibly by ameliorating neuronal degeneration. In the present study, we examined the neuroprotective spectrum of estrogen against excitotoxicity, oxidative stress, and serum-deprivation-induced apoptosis of neurons in mouse cortical cultures. 17beta-estradiol as well as 17alpha-estradiol and estrone attenuated oxidative neuronal death induced by 24 hr exposure to 100 microM FeCl2, excitotoxic neuronal death induced by 24 hr of exposure to 30 microM N-methyl-D-aspartate (NMDA) and serum-deprivation induced neuronal apoptosis. Furthermore, estradiol attenuated neuronal death induced by Abeta25-35. However, all these neuroprotective effects were mediated by the anti-oxidative action of estrogens. When oxidative stress was blocked by an antioxidant trolox, estrogens did not show any additional protection. Addition of a specific estrogen receptor antagonist ICI182,780 did not reverse the protection offered by estrogens. These findings suggest that high concentrations of estrogen protect against various neuronal injuries mainly by its anti-oxidative effects as previously shown by Behl et al. Our results do not support the view that classical estrogen receptors mediate neuroprotection.

The reaction between ABTS radical cation and antioxidants and its use to evaluate the antioxidant status of serum samples

The 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical cation can be generated by incubation of ABTS and 2,2'-azo-bis(2-amidinopropane) at 45 degrees Celsius. The ABTS radical cation is stable for several minutes at room temperature and reacts quantitatively and instantaneously with several antioxidants, such as Trolox, ascorbic acid, uric acid, cysteine, glutathione and bilirubin. In contrast, the ABTS radical cation reacts slowly with albumin. When serum is added to a solution of the ABTS radical cation, the bleaching of the radical follows biphasic kinetics, with a fast decay followed by a slow decay that takes place within several minutes. The fast decay is primarily due to uric acid, while the slow decay is related to the protein content of the sample. We propose that this procedure can provide an independent and simultaneous evaluation of the low molecular weight and protein antioxidants present in biological samples such as serum.