Benzylglucosinolate Derived Isothiocyanate from Tropaeolum majus Reduces Gluconeogenic Gene and Protein Expression in Human Cells

Nasturtium (Tropaeolum majus L.) contains high concentrations of benzylglcosinolate. We found that a hydrolysis product of benzyl glucosinolate—the benzyl isothiocyanate (BITC)—modulates the intracellular localization of the transcription factor Forkhead box O 1 (FOXO1). FoxO transcription factors can antagonize insulin effects and trigger a variety of cellular processes involved in tumor suppression, longevity, development and metabolism. The current study evaluated the ability of BITC—extracted as intact glucosinolate from nasturtium and hydrolyzed with myrosinase—to modulate i) the insulin-signaling pathway, ii) the intracellular localization of FOXO1 and, iii) the expression of proteins involved in gluconeogenesis, antioxidant response and detoxification. Stably transfected human osteosarcoma cells (U-2 OS) with constitutive expression of FOXO1 protein labeled with GFP (green fluorescent protein) were used to evaluate the effect of BITC on FOXO1. Human hepatoma HepG2 cell cultures were selected to evaluate the effect on gluconeogenic, antioxidant and detoxification genes and protein expression. BITC reduced the phosphorylation of protein kinase B (AKT/PKB) and FOXO1; promoted FOXO1 translocation from cytoplasm into the nucleus antagonizing the insulin effect; was able to down-regulate the gene and protein expression of gluconeogenic enzymes; and induced the gene expression of antioxidant and detoxification enzymes. Knockdown analyses with specific siRNAs showed that the expression of gluconeogenic genes was dependent on nuclear factor (erythroid derived)-like2 (NRF2) and independent of FOXO1, AKT and NAD- dependent deacetylase sirtuin-1 (SIRT1). The current study provides evidence that BITC might have a role in type 2 diabetes T2D by reducing hepatic glucose production and increasing antioxidant resistance

The histone deacetylase inhibitor SAHA acts in synergism with fenretinide and doxorubicin to control growth of rhabdoid tumor cells

Background: Rhabdoid tumors are highly aggressive malignancies affecting infants and very young children. In many instances these tumors are resistant to conventional type chemotherapy necessitating alternative approaches. Methods: Proliferation assays (MTT), apoptosis (propidium iodide/annexin V) and cell cycle analysis (DAPI), RNA expression microarrays and western blots were used to identify synergism of the HDAC (histone deacetylase) inhibitor SAHA with fenretinide, tamoxifen and doxorubicin in rhabdoidtumor cell lines. Results: HDAC1 and HDAC2 are overexpressed in primary rhabdoid tumors and rhabdoid tumor cell lines. Targeting HDACs in rhabdoid tumors induces cell cycle arrest and apoptosis. On the other hand HDAC inhibition induces deregulated gene programs (MYCC-, RB program and the stem cell program) in rhabdoid tumors. These programs are in general associated with cell cycle progression. Targeting these activated pro-proliferative genes by combined approaches of HDAC-inhibitors plus fenretinide, which inhibits cyclinD1, exhibit strong synergistic effects on induction of apoptosis. Furthermore, HDAC inhibition sensitizes rhabdoid tumor cell lines to cell death induced by chemotherapy. Conclusion: Our data demonstrate that HDAC inhibitor treatment in combination with fenretinide or conventional chemotherapy is a promising tool for the treatment of chemoresistant rhabdoid tumors.<br

第1090回千葉医学会例会・第7回環境生命医学研究会

<p>HepG2 cells were incubated with BITC 30 μM, insulin 100 nM and insulin + BITC for 30 min. PathScan Signaling Array Kit was used for the detection of the phosphorylated AKT (Thr308). Data shown as mean of fold phosphorylation normalized to untreated control + SEM (n = 3) *p<0.05(Unpaired Student’s t test).</p

The histone deacetylase inhibitor SAHA acts in synergism with fenretinide and doxorubicin to control growth of rhabdoid tumor cells

BACKGROUND: Rhabdoid tumors are highly aggressive malignancies affecting infants and very young children. In many instances these tumors are resistant to conventional type chemotherapy necessitating alternative approaches. METHODS: Proliferation assays (MTT), apoptosis (propidium iodide/annexin V) and cell cycle analysis (DAPI), RNA expression microarrays and western blots were used to identify synergism of the HDAC (histone deacetylase) inhibitor SAHA with fenretinide, tamoxifen and doxorubicin in rhabdoidtumor cell lines. RESULTS: HDAC1 and HDAC2 are overexpressed in primary rhabdoid tumors and rhabdoid tumor cell lines. Targeting HDACs in rhabdoid tumors induces cell cycle arrest and apoptosis. On the other hand HDAC inhibition induces deregulated gene programs (MYCC-, RB program and the stem cell program) in rhabdoid tumors. These programs are in general associated with cell cycle progression. Targeting these activated pro-proliferative genes by combined approaches of HDAC-inhibitors plus fenretinide, which inhibits cyclinD1, exhibit strong synergistic effects on induction of apoptosis. Furthermore, HDAC inhibition sensitizes rhabdoid tumor cell lines to cell death induced by chemotherapy. CONCLUSION: Our data demonstrate that HDAC inhibitor treatment in combination with fenretinide or conventional chemotherapy is a promising tool for the treatment of chemoresistant rhabdoid tumors

The flavones apigenin and luteolin induce FOXO1 translocation but inhibit gluconeogenic and lipogenic gene expression in human cells.

The flavones apigenin (4',5,7,-trihydroxyflavone) and luteolin (3',4',5,7,-tetrahydroxyflavone) are plant secondary metabolites with antioxidant, antiinflammatory, and anticancer activities. We evaluated their impact on cell signaling pathways related to insulin-resistance and type 2 diabetes. Apigenin and luteolin were identified in our U-2 OS (human osteosarcoma) cell screening assay for micronutrients triggering rapid intracellular translocation of the forkhead box transcription factor O1 (FOXO1), an important mediator of insulin signal transduction. Insulin reversed the translocation of FOXO1 as shown by live cell imaging. The impact on the expression of target genes was evaluated in HepG2 (human hepatoma) cells. The mRNA-expression of the gluconeogenic enzymes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pc), the lipogenic enzymes fatty-acid synthase (FASN) and acetyl-CoA-carboxylase (ACC) were down-regulated by both flavones with smaller effective dosages of apigenin than for luteolin. PKB/AKT-, PRAS40-, p70S6K-, and S6-phosphorylation was reduced by apigenin and luteolin but not that of the insulin-like growth factor receptor IGF-1R by apigenin indicating a direct inhibition of the PKB/AKT-signaling pathway distal to the IGF-1 receptor. N-acetyl-L-cysteine did not prevent FOXO1 nuclear translocation induced by apigenin and luteolin, suggesting that these flavones do not act via oxidative stress. The roles of FOXO1, FOXO3a, AKT, sirtuin1 (SIRT1), and nuclear factor (erythroid-derived2)-like2 (NRF2), investigated by siRNA knockdown, showed differential patterns of signal pathways involved and a role of NRF2 in the inhibition of gluconeogenic enzyme expression. We conclude that these flavones show an antidiabetic potential due to reduction of gluconeogenic and lipogenic capacity despite inhibition of the PKB/AKT pathway which justifies detailed investigation in vivo

CAT gene expression in HepG2 cells modulated by BITC.

<p>Dose-dependency is shown for the antioxidant enzyme CAT. Results are presented as fold mRNA expression, normalized to the housekeeping gene RPL32 and the control. Data shown as mean value ± SEM *(p<0.05), **(p<0.01) and ***(p<0.001). (Oneway ANOVA and posthoc Bonferroni).</p

CAT time dependency analyses in HepG2 cells modulated by BITC.

<p>Time dependent analyses for CAT. Results are presented as fold mRNA expression, normalized to the housekeeping gene RPL32 and the control. Data shown as mean value ± SEM *(p<0.05). (Oneway ANOVA and posthoc Bonferroni).</p

Primer sequences for qRT-PCR.

<p>Primer sequences for qRT-PCR.</p

U-2 OS-FOXO1-GFP cells treated with BITC at selected concentrations.

<p>Nuclear accumulation was estimated by measuring the ratio of FOXO1 nuclear/FOXO1 cytosolic (FOXO1 Nuc/Cyt) after 1 h treatment normalized to untreated control cells. Ratios higher than 1 showed a nuclear accumulation of FOXO1. Results are presented as mean values + standard error (SEM) (n = 4). Significant differences vs control (0 μM) are labelled for *(p<i><</i>0.05) and **(p<i><</i>0.01) (Oneway ANOVA and posthoc Bonferroni multiple comparisons).</p

FOXO1 translocation in stably transfected U-2 OS (human osteosarcoma cells).

<p>Fig 1A–1D show FOXO1-GFP expressing cells and nuclear DAPI-staining (red) changing to orange-yellow in overlays with GFP after incubation with BITC at concentrations of 0 (1A-A’), 1 (1B-B’), 10 (1C-C’) and 100 μM (1D-D’), respectively. Following 1 h of starvation, stimulation of cells was performed with BITC for 1 h. Cells were fixed and stained with DAPI for identification of nuclear areas by fluorescence microscopic detection, for segmentation of cells and calculation of GFP-intensity ratios in nuclei and cytoplasm (Nuc/Cyt).</p