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1.  Piper betle induces phase I & II genes through Nrf2/ARE signaling pathway in mouse embryonic fibroblasts derived from wild type and Nrf2 knockout cells 
Background
Nuclear factor-erythroid 2 p45 related factor 2 (Nrf2) is a primary transcription factor, protecting cells from oxidative stress by regulating a number of antioxidants and phase II detoxifying enzymes. Dietary components such as sulforaphane in broccoli and quercetin in onions have been shown to be inducers of Nrf2. Piper betle (PB) grows well in tropical climate and the leaves are used in a number of traditional remedies for the treatment of stomach ailments and infections among Asians. The aim of this study was to elucidate the effect of Piper betle (PB) leaves extract in Nrf2 signaling pathway by using 2 types of cells; mouse embryonic fibroblasts (MEFs) derived from wild-type (WT) and Nrf2 knockout (N0) mice.
Methods
WT and N0 cells were treated with 5 and 10 μg/ml of PB for 10 and 12-h for the determination of nuclear translocation of Nrf2 protein. Luciferase reporter gene activity was performed to evaluate the antioxidant response element (ARE)-induction by PB. Real-time PCR and Western blot were conducted on both WT and N0 cells after PB treatment for the determination of antioxidant enzymes [superoxide dismutase (SOD1) and heme-oxygenase (HO-1)], phase I oxidoreductase enzymes [NAD(P)H: quinone oxidoreductase (NQO1)] and phase II detoxifying enzyme [glutathione S-transferase (GST)].
Results
Nuclear translocation of Nrf2 by PB in WT cells was better after 10 h incubation compared to 12 h. Real time PCR and Western blot analysis showed increased expressions of Nrf2, NQO1 and GSTA1 genes with corresponding increases in glutathione, NQO1 and HO-1 proteins in WT cells. Reporter gene ARE was stimulated by PB as shown by ARE/luciferase assay. Interestingly, PB induced SOD1 gene and protein expressions in N0 cells but not in WT cells.
Conclusion
The results of this study confirmed that PB activated Nrf2-ARE signaling pathway which subsequently induced some phase I oxidoreductase, phase II detoxifying and antioxidant genes expression via ARE reporter gene involved in the Nrf2 pathway with the exception of SOD1 which may not be dependent on this pathway.
doi:10.1186/1472-6882-14-72
PMCID: PMC3936848  PMID: 24559113
Nrf2; PB; ARE; NQO1; HO-1; GST. SOD1
2.  Comparative Effects of Biodynes, Tocotrienol-Rich Fraction, and Tocopherol in Enhancing Collagen Synthesis and Inhibiting Collagen Degradation in Stress-Induced Premature Senescence Model of Human Diploid Fibroblasts 
Biodynes, tocotrienol-rich fraction (TRF), and tocopherol have shown antiaging properties. However, the combined effects of these compounds on skin aging are yet to be investigated. This study aimed to elucidate the skin aging effects of biodynes, TRF, and tocopherol on stress-induced premature senescence (SIPS) model of human diploid fibroblasts (HDFs) by determining the expression of collagen and MMPs at gene and protein levels. Primary HDFs were treated with biodynes, TRF, and tocopherol prior to hydrogen peroxide (H2O2) exposure. The expression of COL1A1, COL3A1, MMP1, MMP2, MMP3, and MMP9 genes was determined by qRT-PCR. Type I and type III procollagen proteins were measured by Western blotting while the activities of MMPs were quantified by fluorometric Sensolyte MMP Kit. Our results showed that biodynes, TRF, and tocopherol upregulated collagen genes and downregulated MMP genes (P < 0.05). Type I procollagen and type III procollagen protein levels were significantly increased in response to biodynes, TRF, and tocopherol treatment (P < 0.05) with reduction in MMP-1, MMP-2, MMP-3, and MMP-9 activities (P < 0.05). These findings indicated that biodynes, TRF, and tocopherol effectively enhanced collagen synthesis and inhibited collagen degradation and therefore may protect the skin from aging.
doi:10.1155/2013/298574
PMCID: PMC3874949  PMID: 24396567
3.  Gelam Honey Attenuates Carrageenan-Induced Rat Paw Inflammation via NF-κB Pathway 
PLoS ONE  2013;8(8):e72365.
The activation of nuclear factor kappa B (NF-κB) plays a major role in the pathogenesis of a number of inflammatory diseases. In this study, we investigated the anti-inflammatory mechanism of Gelam honey in inflammation induced rats via NF-κB signalling pathway. Rats paw edema was induced by subplantar injection of 1% carrageenan into the right hind paw. Rats were pre-treated with Gelam honey at different doses (1 or 2 g/kg, p.o.) and NSAID Indomethacin (10 mg/kg, p.o.), in two time points (1 and 7 days). Our results showed that Gelam honey at both concentrations suppressed the gene expressions of NF-κB (p65 & p50) and IκBα in inflamed rats paw tissues. In addition, Gelam honey inhibited the nuclear translocation and activation of NF-κB and decreased the cytosolic degradation of IκBα dose dependently in inflamed rats paw tissues. The immunohistochemical expressions of pro-inflammatory mediators COX-2 and TNF-α were also decreased in inflamed rats paw tissues when treated with Gelam honey. The results of our findings suggest that Gelam honey exhibits its inhibitory effects by attenuating NF-κB translocation to the nucleus and inhibiting IκBα degradation, with subsequent decrease of inflammatory mediators COX-2 and TNF-α.
doi:10.1371/journal.pone.0072365
PMCID: PMC3756081  PMID: 24015236
4.  Comparative effect of Piper betle, Chlorella vulgaris and tocotrienol-rich fraction on antioxidant enzymes activity in cellular ageing of human diploid fibroblasts 
Background
Human diploid fibroblasts (HDFs) undergo a limited number of cellular divisions in culture and progressively reach a state of irreversible growth arrest, a process termed cellular ageing. Even though beneficial effects of Piper betle, Chlorella vulgaris and tocotrienol-rich fraction (TRF) have been reported, ongoing studies in relation to ageing is of interest to determine possible protective effects that may reverse the effect of ageing. The aim of this study was to evaluate the effect of P. betle, C. vulgaris and TRF in preventing cellular ageing of HDFs by determining the activity of antioxidant enzymes viz.; catalase, superoxide dismutase (SOD) and glutathione peroxidase.
Methods
Different passages of HDFs were treated with P. betle, C. vulgaris and TRF for 24 h prior to enzymes activity determination. Senescence-associated beta-galactosidase (SA β-gal) expression was assayed to validate cellular ageing.
Results
In cellular ageing of HDFs, catalase and glutathione peroxidase activities were reduced, but SOD activity was heightened during pre-senescence. P. betle exhibited the strongest antioxidant activity by reducing SA β-gal expression, catalase activities in all age groups, and SOD activity. TRF exhibited a strong antioxidant activity by reducing SA β-gal expression, and SOD activity in senescent HDFs. C. vulgaris extract managed to reduce SOD activity in senescent HDFs.
Conclusion
P. betle, C. vulgaris, and TRF have the potential as anti-ageing entities which compensated the role of antioxidant enzymes in cellular ageing of HDFs.
doi:10.1186/1472-6882-13-210
PMCID: PMC3847057  PMID: 23948056
Antioxidant; Piper betle; Chlorella vulgaris; Tocotrienol-rich fraction (TRF); Cellular ageing; Fibroblasts
5.  Gamma-Tocotrienol Modulated Gene Expression in Senescent Human Diploid Fibroblasts as Revealed by Microarray Analysis 
The effect of γ-tocotrienol, a vitamin E isomer, in modulating gene expression in cellular aging of human diploid fibroblasts was studied. Senescent cells at passage 30 were incubated with 70 μM of γ-tocotrienol for 24 h. Gene expression patterns were evaluated using Sentrix HumanRef-8 Expression BeadChip from Illumina, analysed using GeneSpring GX10 software, and validated using quantitative RT-PCR. A total of 100 genes were differentially expressed (P < 0.001) by at least 1.5 fold in response to γ-tocotrienol treatment. Amongst the genes were IRAK3, SelS, HSPA5, HERPUD1, DNAJB9, SEPR1, C18orf55, ARF4, RINT1, NXT1, CADPS2, COG6, and GLRX5. Significant gene list was further analysed by Gene Set Enrichment Analysis (GSEA), and the Normalized Enrichment Score (NES) showed that biological processes such as inflammation, protein transport, apoptosis, and cell redox homeostasis were modulated in senescent fibroblasts treated with γ-tocotrienol. These findings revealed that γ-tocotrienol may prevent cellular aging of human diploid fibroblasts by modulating gene expression.
doi:10.1155/2013/454328
PMCID: PMC3619550  PMID: 23634235
6.  Modulation of Cell Cycle Profile by Chlorella vulgaris Prevents Replicative Senescence of Human Diploid Fibroblasts 
In this study, the effects of Chlorella vulgaris (CV) on replicative senescence of human diploid fibroblasts (HDFs) were investigated. Hot water extract of CV was used to treat HDFs at passages 6, 15, and 30 which represent young, presenescence, and senescence ages, respectively. The level of DNA damage was determined by comet assay while apoptosis and cell cycle profile were determined using FACSCalibur flow cytometer. Our results showed direct correlation between increased levels of damaged DNA and apoptosis with senescence in untreated HDFs (P < 0.05). Cell cycle profile showed increased population of untreated senescent cells that enter G0/G1 phase while the cell population in S phase decreased significantly (P < 0.05). Treatment with CV however caused a significant reduction in the level of damaged DNA and apoptosis in all age groups of HDFs (P < 0.05). Cell cycle analysis showed that treatment with CV increased significantly the percentage of senescent HDFs in S phase and G2/M phases but decreased the population of cells in G0/G1 phase (P < 0.05). In conclusion, hot water extract of Chlorella vulgaris effectively decreased the biomarkers of ageing, indicating its potential as an antiageing compound.
doi:10.1155/2013/780504
PMCID: PMC3610352  PMID: 23573154
7.  Antioxidant enzyme activity and malondialdehyde levels can be modulated by Piper betle, tocotrienol rich fraction and Chlorella vulgaris in aging C57BL/6 mice 
Clinics  2012;67(12):1447-1454.
OBJECTIVE:
The aim of this study was to determine the erythrocyte antioxidant enzyme activity and the superoxide dismutase, catalase, glutathione peroxidase, and plasma malondialdehyde levels in aging mice and to evaluate how these measures are modulated by potential antioxidants, including the tocotrienol-rich fraction, Piper betle, and Chlorella vulgaris.
METHOD:
One hundred and twenty male C57BL/6 inbred mice were divided into three age groups: young (6 months old), middle-aged (12 months old), and old (18 months old). Each age group consisted of two control groups (distilled water and olive oil) and three treatment groups: Piper betle (50 mg/kg body weight), tocotrienol-rich fraction (30 mg/kg), and Chlorella vulgaris (50 mg/kg). The duration of treatment for all three age groups was two months. Blood was withdrawn from the orbital sinus to determine the antioxidant enzyme activity and the malondialdehyde level.
RESULTS:
Piper betle increased the activities of catalase, glutathione peroxidase, and superoxide dismutase in the young, middle, and old age groups, respectively, when compared to control. The tocotrienol-rich fraction decreased the superoxide dismutase activity in the middle and the old age groups but had no effect on catalase or glutathione peroxidase activity for all age groups. Chlorella vulgaris had no effect on superoxide dismutase activity for all age groups but increased glutathione peroxidase and decreased catalase activity in the middle and the young age groups, respectively. Chlorella vulgaris reduced lipid peroxidation (malondialdehyde levels) in all age groups, but no significant changes were observed with the tocotrienol-rich fraction and the Piper betle treatments.
CONCLUSION:
We found equivocal age-related changes in erythrocyte antioxidant enzyme activity when mice were treated with Piper betle, the tocotrienol-rich fraction, and Chlorella vulgaris. However, Piper betle treatment showed increased antioxidant enzymes activity during aging.
doi:10.6061/clinics/2012(12)16
PMCID: PMC3521809  PMID: 23295600
Aging; Antioxidant Enzymes; Piper Betle; Tocotrienol-Rich Fraction; Chlorella Vulgaris
8.  Effects of Palm Vitamin E on Bone-Formation-Related Gene Expression in Nicotine-Treated Rats 
The study determines the effects of palm vitamin E on the gene expression of bone-formation-related genes in nicotine-treated rats. Male rats were divided into three groups: normal saline olive oil (NSO), nicotine olive oil (NO), and nicotine palm vitamin E (NE). The treatment was carried out in 2 phases. During the first 2 months, the NSO group received normal saline while the NO and NE groups received nicotine 7 mg/kg, 6 days a week, intraperitoneally. The following 2 months, normal saline and nicotine administration was stopped and was replaced with oral supplementation of olive oil for the NSO and NO groups and oral supplementation of palm vitamin E (60 mg/kg) for the NE group. Both femurs were harvested to determine the gene expression of bone morphogenetic protein-2 (BMP-2), Osterix (OSX), and Runt-related transcription factor 2 (RUNX2). Nicotine significantly downregulated the gene expression. This effect was reversed by palm vitamin E treatment. In conclusion, palm vitamin E may play a role in osteoblast differentiation and can be considered as an anabolic agent to treat nicotine-induced osteoporosis.
doi:10.1155/2012/656025
PMCID: PMC3434599  PMID: 23049610
9.  Inhibition of Mitochondrial Cytochrome c Release and Suppression of Caspases by Gamma-Tocotrienol Prevent Apoptosis and Delay Aging in Stress-Induced Premature Senescence of Skin Fibroblasts 
In this study, we determined the molecular mechanism of γ-tocotrienol (GTT) in preventing cellular aging by focusing on its anti-apoptotic effect in stress-induced premature senescence (SIPS) model of human diploid fibroblasts (HDFs). Results obtained showed that SIPS exhibited senescent-phenotypic characteristic, increased expression of senescence-associated β-galactosidase (SA β-gal) and promoted G0/G1 cell cycle arrest accompanied by shortening of telomere length with decreased telomerase activity. Both SIPS and senescent HDFs shared similar apoptotic changes such as increased Annexin V-FITC positive cells, increased cytochrome c release and increased activation of caspase-9 and caspase-3 (P < 0.05). GTT treatment resulted in a significant reduction of Annexin V-FITC positive cells, inhibited cytochrome c release and decreased activation of caspase-9 and caspase-3 (P < 0.05). Gene expression analysis showed that GTT treatment down regulated BAX mRNA, up-regulated BCL2A1 mRNA and decreased the ratio of Bax/Bcl-2 protein expression (P < 0.05) in SIPS. These findings suggested that GTT inhibits apoptosis by modulating the upstream apoptosis cascade, causing the inhibition of cytochrome c release from the mitochondria with concomitant suppression of caspase-9 and caspase-3 activation. In conclusion, GTT delays cellular senescence of human diploid fibroblasts through the inhibition of intrinsic mitochondria-mediated pathway which involved the regulation of pro- and anti-apoptotic genes and proteins.
doi:10.1155/2012/785743
PMCID: PMC3418699  PMID: 22919441
10.  Gelam Honey Inhibits the Production of Proinflammatory, Mediators NO, PGE2, TNF-α, and IL-6 in Carrageenan-Induced Acute Paw Edema in Rats 
Natural honey is well known for its therapeutic value and has been used in traditional medicine of different cultures throughout the world. The aim of this study was to investigate the anti-inflammatory effect of Malaysian Gelam honey in inflammation-induced rats. Paw edema was induced by a subplantar injection of 1% carrageenan into the rat right hind paw. Rats were treated with the nonsteroidal anti-inflammatory drug (NSAID) Indomethacin (10 mg/kg, p.o.) or Gelam honey at different doses (1 or 2 g/kg, p.o.). The increase in footpad thickness was considered to be edema, which was measured using a dial caliper. Plasma and paw tissue were collected to analyze the production of inflammatory mediators, such as NO, PGE2, TNF-α, and IL-6, as well as iNOS and COX-2. The results showed that Gelam honey could reduce edema in a dose-dependent fashion in inflamed rat paws, decrease the production of NO, PGE2, TNF-α, and IL-6 in plasma, and suppress the expression of iNOS, COX-2, TNF-α, and IL-6 in paw tissue. Oral pretreatment of Gelam honey at 2 g/kg of body weight at two time points (1 and 7 days) showed a significantly decreased production of proinflammatory cytokines, which was similar to the effect of the anti-inflammatory drug Indomethacin (NSAID), both in plasma and tissue. Thus, our results suggest that Gelam honey has anti-inflammatory effects by reducing the rat paw edema size and inhibiting the production of proinflammatory mediators. Gelam honey is potentially useful for treating inflammatory conditions.
doi:10.1155/2012/109636
PMCID: PMC3418690  PMID: 22919407
11.  Gamma-tocotrienol modulation of senescence-associated gene expression prevents cellular aging in human diploid fibroblasts 
Clinics  2012;67(2):135-143.
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.
doi:10.6061/clinics/2012(02)08
PMCID: PMC3275122  PMID: 22358238
Vitamin E; Molecular mechanism; Cellular aging; Senescence-associated genes; Human diploid fibroblasts
12.  5-Azacytidine Is Insufficient For Cardiogenesis In Human Adipose-Derived Stem Cells 
Background
Adipose tissue is a source of multipotent adult stem cells and it has the ability to differentiate into several types of cell lineages such as neuron cells, osteogenic cells and adipogenic cells. Several reports have shown adipose-derived stem cells (ASCs) have the ability to undergo cardiomyogenesis. Studies have shown 5-azacytidine can successfully drive stem cells such as bone marrow derived stem cells to differentiate into cardiomyogenic cells. Therefore, in this study, we investigated the effect 5-azacytidine on the cardiogenic ability of ASCs.
Methods
The cardiogenic potential of ASCs was analysed by studying the morphological changes after induction, the changes in the cardiogenic genes expression i.e. GATA4, MLC-2v, MLC-2a, NKX2.5, β-MHC, α-MHC, Atrial natriuretic peptide (ANP), Connexin 43, Cardiac Troponin C, Cardiac Troponin I and myocyte enhancer factor (MEF2C) and the changes of embryonic stem cells genes expression at P5 and P10 using quantitative PCR.
Results
Our results showed that the induced ASCs did not show significant morphological difference compared to the non-induced ASCs. While quantitative PCR data indicated that most cardiogenic genes and stemness genes expression level decreased after induction at P5 and P10.
Conclusion
5-azacytidine is insufficient for the cardiogenic induction of the ASCs.
doi:10.1186/1477-5751-11-3
PMCID: PMC3274438  PMID: 22221649
5-Azacytidine; Cardiogenic; Ischemia; Adipose; Stem Cells; Differentiation; Heart
13.  Modulation of collagen synthesis and its gene expression in human skin fibroblasts by tocotrienol-rich fraction 
Introduction
Skin aging may occur as a result of increased free radicals in the body. Vitamin E, the major chain-breaking antioxidant, prevents propagation of oxidative stress, especially in biological membranes. In this study, the molecular mechanism of tocotrienol-rich fraction (TRF) in preventing oxidative stress-induced skin aging was evaluated by determining the rate of total collagen synthesis and its gene expression in human skin fibroblasts.
Material and methods
Primary culture of human skin fibroblasts was derived from circumcision foreskin of 9 to 12 year-old boys. Fibroblast cells were divided into 5 different treatment groups: untreated control, hydrogen peroxide (H2O2)-induced oxidative stress (20 µM H2O2 exposure for 2 weeks), TRF treatment, and pre- and post-treatment of TRF to H2O2-induced oxidative stress.
Results
Our results showed that H2O2-induced oxidative stress decreased the rate of total collagen synthesis and down-regulated COL I and COL III in skin fibroblasts. Pre-treatment of TRF protected against H2O2-induced oxidative stress as shown by increase in total collagen synthesis and up-regulation of COL I and COL III (p<0.05) genes. However, similar protective effects against H2O2-induced oxidative stress were not observed in the post-treated fibroblasts.
Conclusions
Tocotrienol-rich fraction protects against H2O2-induced oxidative stress in human skin fibroblast culture by modulating the expression of COL I and COL III genes with concomitant increase in the rate of total collagen synthesis. These findings may indicate TRF protection against oxidative stress-induced skin aging.
doi:10.5114/aoms.2011.25567
PMCID: PMC3258810  PMID: 22291837
tocotrienol-rich fraction; skin aging; collagen synthesis; gene expression
14.  Tocotrienol rich fraction supplementation improved lipid profile and oxidative status in healthy older adults: A randomized controlled study 
Background
Vitamin E supplements containing tocotrienols are now being recommended for optimum health but its effects are scarcely known. The objective was to determine the effects of Tocotrienol Rich Fraction (TRF) supplementation on lipid profile and oxidative status in healthy older individuals at a dose of 160 mg/day for 6 months.
Methods
Sixty-two subjects were recruited from two age groups: 35-49 years (n = 31) and above 50 years (n = 31), and randomly assigned to receive either TRF or placebo capsules for six months. Blood samples were obtained at 0, 3rd and 6th months.
Results
HDL-cholesterol in the TRF-supplemented group was elevated after 6 months (p < 0.01). Protein carbonyl contents were markedly decreased (p < 0.001), whereas AGE levels were lowered in the > 50 year-old group (p < 0.05). Plasma levels of total vitamin E particularly tocopherols were significantly increased in the TRF-supplemented group after 3 months (p < 0.01). Plasma total tocotrienols were only increased in the > 50 year-old group after receiving 6 months of TRF supplementation. Changes in enzyme activities were only observed in the > 50 year-old group. SOD activity was decreased after 3 (p < 0.05) and 6 (p < 0.05) months of TRF supplementation whereas CAT activity was decreased after 3 (p < 0.01) and 6 (p < 0.05) months in the placebo group. GPx activity was increased at 6 months for both treatment and placebo groups (p < 0.05).
Conclusion
The observed improvement of plasma cholesterol, AGE and antioxidant vitamin levels as well as the reduced protein damage may indicate a restoration of redox balance after TRF supplementation, particularly in individuals over 50 years of age.
doi:10.1186/1743-7075-8-42
PMCID: PMC3148956  PMID: 21702918
15.  Tocotrienol-Rich Fraction Prevents Cell Cycle Arrest and Elongates Telomere Length in Senescent Human Diploid Fibroblasts 
This study determined the molecular mechanisms of tocotrienol-rich fraction (TRF) in preventing cellular senescence of human diploid fibroblasts (HDFs). Primary culture of HDFs at various passages were incubated with 0.5 mg/mL TRF for 24 h. Telomere shortening with decreased telomerase activity was observed in senescent HDFs while the levels of damaged DNA and number of cells in G0/G1 phase were increased and S phase cells were decreased. Incubation with TRF reversed the morphology of senescent HDFs to resemble that of young cells with decreased activity of SA-β-gal, damaged DNA, and cells in G0/G1 phase while cells in the S phase were increased. Elongated telomere length and restoration of telomerase activity were observed in TRF-treated senescent HDFs. These findings confirmed the ability of tocotrienol-rich fraction in preventing HDFs cellular ageing by restoring telomere length and telomerase activity, reducing damaged DNA, and reversing cell cycle arrest associated with senescence.
doi:10.1155/2011/506171
PMCID: PMC3085479  PMID: 21541185
16.  Hot water extract of Chlorella vulgaris induced DNA damage and apoptosis 
Clinics  2010;65(12):1371-1377.
OBJECTIVES:
The aim of this study was to determine the antiproliferative and apoptotic effects of hot water extracts of Chlorella vulgaris on hepatoma cell line HepG2.
INTRODUCTION:
The search for food and spices that can induce apoptosis in cancer cells has been a major study interest in the last decade. Chlorella vulgaris, a unicellular green algae, has been reported to have antioxidant and anti‐cancer properties. However, its chemopreventive effects in inhibiting the growth of cancer cells have not been studied in great detail.
METHODS:
HepG2 liver cancer cells and WRL68 normal liver cells were treated with various concentrations (0‐4 mg/ml) of hot water extract of C. vulgaris after 24 hours incubation. Apoptosis rate was evaluated by TUNEL assay while DNA damage was assessed by Comet assay. Apoptosis proteins were evaluated by Western blot analysis.
RESULTS:
Chlorella vulgaris decreased the number of viable HepG2 cells in a dose dependent manner (p < 0.05), with an IC50 of 1.6 mg/ml. DNA damage as measured by Comet assay was increased in HepG2 cells at all concentrations of Chlorella vulgaris tested. Evaluation of apoptosis by TUNEL assay showed that Chlorella vulgaris induced a higher apoptotic rate (70%) in HepG2 cells compared to normal liver cells, WRL68 (15%). Western blot analysis showed increased expression of pro‐ apoptotic proteins P53, Bax and caspase‐3 in the HepG2 cells compared to normal liver cells WRL68, and decreased expression of the anti‐apoptotic protein Bcl‐2.
CONCLUSIONS:
Chlorella vulgaris may have anti‐cancer effects by inducing apoptosis signaling cascades via an increased expression of P53, Bax and caspase‐3 proteins and through a reduction of Bcl‐2 protein, which subsequently lead to increased DNA damage and apoptosis.
doi:10.1590/S1807-59322010001200023
PMCID: PMC3020351  PMID: 21340229
HepG2; Chlorella vulgaris; DNA damage; chemopreventive; apoptosis
17.  Effect of experimental treatment on GAPDH mRNA expression as a housekeeping gene in human diploid fibroblasts 
BMC Molecular Biology  2010;11:59.
Background
Several genes have been used as housekeeping genes and choosing an appropriate reference gene is important for accurate quantitative RNA expression in real time RT-PCR technique. The expression levels of reference genes should remain constant between the cells of different tissues and under different experimental conditions. The purpose of this study was to determine the effect of different experimental treatments on the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA so that the reliability of GAPDH as reference gene for quantitative real time RT-PCR in human diploid fibroblasts (HDFs) can be validated. HDFs in 4 different treatment groups viz; young (passage 4), senescent (passage 30), H2O2-induced oxidative stress and γ-tocotrienol (GTT)-treated groups were harvested for total RNA extraction. Total RNA concentration and purity were determined prior to GAPDH mRNA quantification. Standard curve of GAPDH expression in serial diluted total RNA, melting curve analysis and agarose gel electrophoresis were used to determine the reliability of GAPDH as reference gene.
Results
HDFs with different experimental treatments exhibited diverse cell morphology with different expression of senescence-associated β-galactosidase (SA β-gal) activity. However the expression level of GAPDH was consistent in all treatment groups.
Conclusion
The study demonstrated that GAPDH is reliable as reference gene for quantitative gene expression analysis in HDFs. Therefore it can be used as housekeeping gene for quantitative real time RT-PCR technique in human diploid fibroblasts particularly in studying cellular senescence.
doi:10.1186/1471-2199-11-59
PMCID: PMC2930638  PMID: 20707929
18.  Chlorella Vulgaris Modulates Hydrogen Peroxide-Induced DNA Damage and Telomere Shortening of Human Fibroblasts Derived from Different Aged Individuals 
The objective of this study was to investigate the modulatory effect of Chlorella vulgaris on cultured fibroblast cells derived from young and old aged individuals focusing on DNA damage, telomere length and telomerase activity. Dose-response test of the algal extract on cells in both age groups revealed that optimum viability was observed at a concentration of 50 µg/ml. Results obtained showed that Chlorella vulgaris exhibited protective effects against H2O2-induced oxidative stress as shown by the reduction in damaged DNA caused by H2O2 treatment (p<0.05) in Chlorella vulgaris pre- and post-treated groups (p<0.05). Pre-treatment of Chlorella vulgaris resulted in a significant decrease in DNA damage suggesting a bioprotective effect against free radical attacks. A decline in DNA damage was observed in post-treated cells which proves Chlorella vulgaris to present bioremediative properties. In cells induced with oxidative stress, telomere length decreased significantly coupled with a concomitant decline of telomerase activity (p<0.05). However, these reductions were prevented with prior and post treatment of Chlorella vulgaris. Therefore, we concluded that Chlorella vulgaris exhibited bioprotective effects especially in cells obtained from young donor but were more bioremediative for cells obtained from old donor as indicated by DNA damage, telomere shortening and reduction in telomerase activity.
PMCID: PMC2816465  PMID: 20606778
Chlorella vulgaris; DNA damage; telomere; telomerase; ageing
19.  Ginger Extract (Zingiber Officinale) has Anti-Cancer and Anti-Inflammatory Effects on Ethionine-Induced Hepatoma Rats 
Clinics (Sao Paulo, Brazil)  2008;63(6):807-813.
OBJECTIVE
To evaluate the effect of ginger extract on the expression of NFκB and TNF-α in liver cancer-induced rats.
METHODS
Male Wistar rats were randomly divided into 5 groups based on diet: i) control (given normal rat chow), ii) olive oil, iii) ginger extract (100mg/kg body weight), iv) choline-deficient diet + 0.1% ethionine to induce liver cancer and v) choline-deficient diet + ginger extract (100mg/kg body weight). Tissue samples obtained at eight weeks were fixed with formalin and embedded in paraffin wax, followed by immunohistochemistry staining for NFκB and TNF-α.
RESULTS
The expression of NFκB was detected in the choline-deficient diet group, with 88.3 ± 1.83% of samples showing positive staining, while in the choline-deficient diet supplemented with ginger group, the expression of NFκB was significantly reduced, to 32.35 ± 1.34% (p<0.05). In the choline-deficient diet group, 83.3 ± 4.52% of samples showed positive staining of TNF-α, which was significantly reduced to 7.94 ± 1.32% (p<0.05) when treated with ginger. There was a significant correlation demonstrated between NFκB and TNF-α in the choline-deficient diet group but not in the choline-deficient diet treated with ginger extract group.
CONCLUSION
In conclusion, ginger extract significantly reduced the elevated expression of NFκB and TNF-α in rats with liver cancer. Ginger may act as an anti-cancer and anti-inflammatory agent by inactivating NFκB through the suppression of the pro-inflammatory TNF-α.
doi:10.1590/S1807-59322008000600017
PMCID: PMC2664283  PMID: 19061005
Inflammatory markers; TNF-α; NFκB; Chemopreventive; Ginger
20.  γ-Tocotrienol prevents oxidative stress-induced telomere shortening in human fibroblasts derived from different aged individuals 
The effects of palm γ-tocotrienol (GGT) on oxidative stress-induced cellular ageing was investigated in normal human skin fibroblast cell lines derived from different age groups; young (21-year-old, YF), middle (40-year-old, MF) and old (68-year-old, OF). Fibroblast cells were treated with γ-tocotrienol for 24 hours before or after incubation with IC50 dose of H2O2 for 2 hours. Changes in cell viability, telomere length and telomerase activity were assessed using the MTS assay (Promega, USA), Southern blot analysis and telomere repeat amplification protocol respectively. Results showed that treatment with different concentrations of γ-tocotrienol increased fibroblasts viability with optimum dose of 80 µM for YF and 40 µM for both MF and OF. At higher concentrations, γ-tocotrienol treatment caused marked decrease in cell viability with IC50 value of 200 µM (YF), 300 µM (MF) and 100 µM (OF). Exposure to H2O2 decreased cell viability in dose dependent manner, shortened telomere length and reduced telomerase activity in all age groups. The IC50 of H2O2 was found to be; YF (700 µM), MF (400 µM) and OF (100 µM). Results showed that viability increased significantly (p < 0.05) when cells were treated with 80 µM and 40 µM γ-tocotrienol prior or after H2O2-induced oxidative stress in all age groups. In YF and OF, pretreatment with γ-tocotrienol prevented shortening of telomere length and reduction in telomerase activity. In MF, telomerase activity increased while no changes in telomere length was observed. However, post-treatment of γ-tocotrienol did not exert any significant effects on telomere length and telomerase activity. Thus, these data suggest that γ-tocotrienol protects against oxidative stress-induced cellular ageing by modulating the telomere length possibly via telomerase.
PMCID: PMC2835887  PMID: 20716926
γ-tocotrienol; telomere; telomerase; fibroblasts; oxidative stress; aging

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