Pleiotropic effects of the sirtuin inhibitor sirtinol involves concentration-dependent modulation of multiple nuclear receptor-mediated pathways in androgen-responsive prostate cancer cell LNCaP.

Sirtinol is a purported specific inhibitor of the nicotinamide adenine dinucleotide (NAD)-dependent type III histone deacetylase (also known as sirtuin). Sirtinol has been used extensively to identify chemopreventive/chemotherapeutic agents that modulate the sirtuins. However, the molecular effect of sirtinol other than serving as sirtuin inhibitor in cells is less clear. The present study addressed this deficiency in the literature. Based on structural similarity with plant-derived cancer preventive/therapeutic compounds such as 3', 3'-diindolylmethane, resveratrol, and genistein, we hypothesized that sirtinol may act on pathways similar to that affected by these compounds in the human prostate cancer cell LNCaP. We found that treatment of LNCaP cells with sirtinol led to concentration-dependent effects on multiple pathways. Sirtinol inhibited LNCaP cell cycle and growth that was correlated with up-regulation of cyclin-dependent kinase inhibitor 1A mRNA and protein levels. This effect of sirtinol may due in part to modulation of androgen, estrogen, and insulin-like growth factor-1 mediated pathways as sirtinol treatment led to inhibition of mRNA and protein expression of marker genes involved in these pathways. We also found sirtinol activates aryl hydrocarbon-dependent pathways in LNCaP cells. The effects of sirtinol were observed at 25 µM, a concentration lower than Ki (38 µM) for sirtuin activity. Based on these results we reasoned that sirtinol exerts pleiotropic effects in cells and that biological effects of sirtinol may not be due solely to inhibition of sirtuin.

Cinnamon polyphenols regulate multiple metabolic pathways involved in insulin signaling and intestinal lipoprotein metabolism of small intestinal enterocytes.

OBJECTIVE: Increasing evidence suggests that dietary factors may affect the expression of multiple genes and signaling pathways, which regulate intestinal lipoprotein metabolism. The small intestine is actively involved in the regulation of dietary lipid absorption, intracellular transport, and metabolism and is closely linked to systemic lipid metabolism. Cinnamon polyphenols have been shown to improve glucose, insulin, and lipid metabolism and improve inflammation in cell culture, animal, and human studies. However, little is known of the effects of an aqueous cinnamon extract (CE) on the regulation of genes and signaling pathways related to intestinal metabolism. The aim of the study was to investigate the effects of a CE on the primary enterocytes of chow-fed rats. METHODS: Freshly isolated intestinal enterocytes were used to investigate apolipoprotein-B48 secretion by immunoprecipitation; gene expressions by quantitative reverse transcriptase-polymerase chain reaction and the protein and phosphorylation levels were evaluated by western blot and flow cytometric analyses. RESULTS: Ex vivo, the CE significantly decreased the amount of apolipoprotein-B48 secretion into the media, inhibited the mRNA expression of genes of the inflammatory cytokines, interleukin-1ß, interleukin-6, and tumor necrosis factor-α, and induced the expression of the anti-inflammatory gene, Zfp36. CE also increased the mRNA expression of genes leading to increased insulin sensitivity, including Ir, Irs1, Irs2, Pi3k, and Akt1, and decreased Pten expression. CE also inhibited genes associated with increased cholesterol, triacylglycerols, and apolipoprotein-B48 levels, including Abcg5, Npc1l1, Cd36, Mttp, and Srebp1c, and facilitated Abca1 expression. CE also stimulated the phospho-p38 mitogen-activated protein kinase, c-Jun N-terminal kinase, and extracellular-signal-regulated kinase expressions determined by flow cytometry, with no changes in protein levels. CONCLUSIONS: These results demonstrate that the CE regulates genes associated with insulin sensitivity, inflammation, and cholesterol/lipogenesis metabolism and the activity of the mitogen-activated protein kinase signal pathway in intestinal lipoprotein metabolism.

Selenium compounds activate ATM-dependent DNA damage response via the mismatch repair protein hMLH1 in colorectal cancer cells.

Epidemiological and animal studies indicate that selenium supplementation suppresses risk of colorectal and other cancers. The majority of colorectal cancers are characterized by a defective DNA mismatch repair (MMR). Here, we have employed the MMR-deficient HCT 116 colorectal cancer cells and the MMR-proficient HCT 116 cells with hMLH1 complementation to investigate the role of hMLH1 in selenium-induced DNA damage response, a tumorigenesis barrier. The ATM (ataxia telangiectasia mutated) protein responds to clastogens and initiates DNA damage response. We show that hMLH1 complementation sensitizes HCT 116 cells to methylseleninic acid, methylselenocysteine, and sodium selenite via reactive oxygen species and facilitates the selenium-induced oxidative 8-oxoguanine damage, DNA breaks, G(2)/M checkpoint response, and ATM pathway activation. Pretreatment of the hMLH1-complemented HCT 116 cells with the antioxidant N-acetylcysteine or 2,2,6,6-tetramethylpiperidine-1-oxyl or the ATM kinase inhibitor KU55933 suppresses hMLH1-dependent DNA damage response to selenium exposure. Selenium treatment stimulates the association between hMLH1 and hPMS2 proteins, a heterodimer critical for functional MMR, in a manner dependent on ATM and reactive oxygen species. Taken together, the results suggest a new role of selenium in mitigating tumorigenesis by targeting the MMR pathway, whereby the lack of hMLH1 renders the HCT 116 colorectal cancer cells resistant to selenium-induced DNA damage response.

Enhanced Gadd45 expression and delayed G2/M progression are p53-dependent in zinc-supplemented human bronchial epithelial cells.

Zinc is an essential nutrient for humans; however, this study demonstrated for the first time that an elevated zinc status, created by culturing cells at optimal plasma zinc concentration attainable by oral zinc supplementation, is cytotoxic for normal human bronchial epithelial (NHBE) cells. p53 plays a central role in the modulation of cell signal transduction in response to the stress from DNA damage, hypoxia and oncogene activation. The present study was designed to determine whether the previously reported increased Gadd45 expression and delayed G2/M cell cycle progression in zinc-supplemented NHBE cells is p53-dependent, and to decipher the mechanisms responsible for the regulation of Gadd45 expressions by p53, and elucidate the Gadd45 functions in impaired cell growth and cell cycle progression in NHBE cells. Cells were cultured for one passage in different concentrations of zinc: <0.4 micromol/L (ZD) as severe zinc-deficient; 4 micromol/L (ZN) as normal zinc level in culture medium; 16 micromol/L (ZA) as normal human plasma zinc level; and 32 micromol/L (ZS) as the high end of plasma zinc attainable by oral supplementation. Inhibition of cell growth and upregulation of p53 mRNA and protein expression were observed in ZS cells. Most importantly, ZS treatment also enhanced Gadd45 nuclear protein level and promoter activity, decreased CDK1-Cyclin B1 level and delayed G2/M cell cycle progression. These changes were normalized to those observed in ZN by treating ZS cells with Pifitherin, an inhibitor of p53 transactivation activity. Thus, our findings support the p53 dependency of the Gadd45-CDK1/Cyclin B1-G2/M cell cycle progression pathway in ZS NHBE cells.

Influence of zinc deficiency on Akt-Mdm2-p53 and Akt-p21 signaling axes in normal and malignant human prostate cells.

Phosphorylated Akt (p-Akt), a phosphoinositide-3-OH-kinase-activated protein kinase, is highly expressed in prostate tumors. p-Akt can indirectly hinder p53-dependent growth suppression and apoptosis by phosphorylating Mdm2. Alternatively, p-Akt can directly phosphorylate p21 and restrict it to the cytoplasm for degradation. Because the prostate is the highest zinc-accumulating tissue before the onset of cancer, the effects of physiological levels of zinc on Akt-Mdm2-p53 and Akt-p21 signaling axes in human normal prostate epithelial cells (PrEC) and malignant prostate LNCaP cells were examined in the present study. Cells were cultured for 6 days in low-zinc growth medium supplemented with 0 [zinc-deficient (ZD)], 4 [zinc-normal (ZN)], 16 [zinc-adequate (ZA)], or 32 [zinc-supplemented (ZS)] microM zinc. Zinc status of both cell types was altered in a dose-dependent manner, with LNCaP cells reaching a plateau at >16 microM zinc. For both cell types, p-Akt was higher in the ZD than in the ZN cells and was normalized to that of the ZN cells by treatment with a PI3K inhibitor, LY-294002. PTEN, an endogenous phosphatase targeting Akt dephosphorylation, was hyperphosphorylated (p-PTEN, inactive form) in ZD PrEC. Nuclear p-Mdm2 was raised, whereas nuclear p53 was depressed, by zinc deficiency in PrEC. Nuclear p21 and p53 were lowered by zinc deficiency in LNCaP cells. Higher percentages of ZD, ZA, and ZS than ZN LNCaP cells were found at the G(0)/G(1) phase of the cell cycle, with proportionally lower precentages at the S and G(2)/M phases. Hence, the increased p-PTEN in ZD PrEC would result in hyperphosphorylation of p-Akt and p-Mdm2, as well as reduction of nuclear p53 accumulation. For ZD LNCaP cells, Akt hyperphosphorylation was probably mediated through p21 phosphorylation and degradation, thus restricting p21 nuclear entry to induce cell cycle arrest. Thus zinc deficiency differentially modulated the Akt-Mdm2-p53 signaling axis in normal prostate cells vs. the Akt-p21 signaling axis in malignant prostate cells.

Effect of resveratrol and zinc on intracellular zinc status in normal human prostate epithelial cells.

To evaluate the influence of resveratrol on cellular zinc status, normal human prostate epithelial (NHPrE) cells were treated with resveratrol (0, 0.5, 1, 2.5, 5, and 10 microM) and zinc [0, 4, 16, and 32 microM, representing zinc-deficient (ZD), zinc-normal (ZN), zinc-adequate (ZA), and zinc-supplemented (ZS) conditions, respectively]. A progressive reduction in cell growth was observed in cells treated with increasing amounts of resveratrol (2.5-10 microM). Resveratrol at 5 and 10 microM resulted in a dramatic increase in cellular total zinc concentration, especially in ZS cells. Flow cytometry indicated that 10 microM resveratrol induced arrest of the cell cycle at the G(2)/M phase in association with the observed cell growth inhibition. Data from an in vitro experiment using zinquin as an indicator of intracellular free Zn(II) status demonstrated complex interactions between resveratrol and Zn(II). Fluorescence spectrofluorometry and fluorescence microscopic analyses revealed that intracellular free labile zinc was progressively elevated from nearly twofold in ZS cells with no resveratrol to multifold in ZA and ZS cells with 10 microM resveratrol compared with the corresponding ZN cells. Furthermore, increases in cellular zinc status were associated with elevated levels of reactive oxygen species and senescence, as evidenced by morphological and histochemical changes in cells treated with 2.5 or 10 microM resveratrol, especially in ZA and ZS cells. Taken together, the interaction between resveratrol and zinc in NHPrE cells increases total cellular zinc and intracellular free labile zinc status and, subsequently, reactive oxygen species production and senescence.

A polyphenol mixture from cinnamon targets p38 MAP kinase-regulated signaling pathways to produce G2/M arrest.

We recently demonstrated that treatment of three leukemic cell lines with an aqueous extract of cinnamon (CE) for 24 h produced dose-dependent arrests in the G2/M phase of the cell cycle. To accomplish the goal of understanding underlying mechanisms, we selected the cell line most responsive to the CE treatment to study the effects of the extract on signaling molecules regulating cell cycle progression. Cell cycle analyses were conducted on treated versus nontreated cells from 0-6 h. The percentages of cells in G2/M in CE-treated cells increased significantly from 11.0+/-1.0 to 23.6+/-1.4 after 6 h, while the percentage for nontreated cells remained unchanged (12.3+/-0.8). Multiparametric flow cytometric analyses were used to associate activation of p38 mitogen-activated protein kinase (MAPK) with cells arrested in G2/M, the size of these cells, and the presence or absence of cyclin B1. After 4 h, there was a 26% increase in the activated phosphorylated form of p38 MAPK in CE-treated cells compared with the nontreated control cells, with larger cells showing the greater increases. Although the proportion of CE-treated cells in G2/M was higher than controls, this population was shown to be less positive for cyclin B1 than the control G2/M population. Our results demonstrate that CE significantly modulated two signaling proteins, p38 MAPK and cyclin B, that regulate progression through G2/M. Overall, the data provide evidence that CE affects proliferation in a leukemic cell line by disrupting critical phosphorylating/dephosphorylating signaling events that propel cells through the G2/M phase.

Zinc-induced G2/M blockage is p53 and p21 dependent in normal human bronchial epithelial cells.

The involvement of p53 and p21 signal pathway in the G2/M cell cycle progression of zinc-supplemented normal human bronchial epithelial (NHBE) cells was examined using the small interferring RNA (siRNA) approach. Cells were cultured for one passage in a different concentration of zinc: <0.4 microM (ZD) as zinc deficient; 4 microM as normal zinc level (ZN) in culture medium; 16 microM (ZA) as normal human plasma zinc level; and 32 microM (ZS) as the high end of plasma zinc attainable by oral supplementation. Nuclear p21 protein and mRNA levels as well as promoter activity in ZS cells, but not in ZD cells, were markedly elevated to almost twofold compared with ZN control cells. G2/M blockage in ZS cells was coupled with the observation of elevated p21 gene expression. In ZS cells, the abrogation of p21 protein induction by the transfection of p21 siRNA was shown to alleviate the G2/M blockage, demonstrating the positive linkage of p21 elevation and G2/M blockage. Abolishment of the increase in p53 protein in ZS cells with transfection of p53 siRNA normalized the elevated p21 protein to a similar level as in ZN control cells, which demonstrated that the p21 induction is p53 dependent. Furthermore, the normalization of p53 protein by siRNA treatment in ZS cells alleviated cell growth depression and G2/M blockage, which demonstrated that p53 was involved in the high zinc status-induced G2/M blockage and growth depression. Thus high zinc status in NHBE cells upregulates p53 expression which in turn elevates p21 that eventually induces G2/M blockage.

Suppression of Gadd45 alleviates the G2/M blockage and the enhanced phosphorylation of p53 and p38 in zinc supplemented normal human bronchial epithelial cells.

An adequate zinc status is essential for optimal cellular functions and growth. Yet, excessive zinc supplementation can be cytotoxic and can impair cell growth. Gadd45 plays a vital role as cellular stress sensor in the modulation of cell signal transduction in response to stress. The present study was designed to determine the influence of zinc status on Gadd45 expression and cell cycle progression in zinc deficient and supplemented normal human bronchial epithelial (NHBE) cells, and to decipher the molecular mechanism(s) exerted by the suppression of Gadd45 expression on cell growth and cell cycle progression in this cell type. Cells were cultured for one passage in different concentration of zinc: <0.4 muM (ZD) as severe zinc deficient; 4 muM as normal zinc level in culture medium; 16 microM (ZA) as normal human plasma zinc level; and 32 muM (ZS) as the high end of plasma zinc attainable by oral supplementation. Inhibition of cell growth, upregulation of Gadd45 mRNA and protein expression, and blockage of G2/M cell cycle progression were observed in ZS cells. In contrast, little or no changes in these parameters were seen in ZD cells. The siRNA-mediated knocking down of Gadd45 was found to relieve G2/M blockage in ZS cells, which indicated that the blockage was Gadd45 dependent. Moreover, the enhanced phosphorylation of p38 and p53 (ser15) in ZS cells was normalized after suppression of Gadd45 by siRNA, implicating that the enhanced phosphorylation of these proteins was Gadd45 dependent. Thus, we demonstrated for the first time that an elevated zinc status modulated signal transduction to produce a delay at G2/M during cell cycle progression in NHBE cells.

Zinc deficiency depresses p21 gene expression: inhibition of cell cycle progression is independent of the decrease in p21 protein level in HepG2 cells.

The influence of zinc status on p21 gene expression was examined in human hepatoblastoma (HepG2) cells. Cells were cultured for one passage in a basal medium depleted of zinc to induce severely zinc-deficient (ZD) cells or in basal medium supplemented with 0.4, 4.0, 16, or 32 microM zinc to represent mild zinc deficiency (ZD0.4), the amount of zinc in most normal media (ZN), the normal human plasma zinc level (zinc-adequate; ZA), or the high end of plasma zinc attainable by oral supplementation (ZS), respectively. In ZD and ZD0.4 cells, the nuclear p21 protein level, mRNA abundance, and promoter activity were reduced to 40, 70, and 65%, respectively, of ZN cells. However, p21 protein and mRNA levels, as well as p21 promoter activity, were not altered in ZA and ZS cells compared with ZN cells. Moreover, the amounts of acetylated histone-4 associated with the proximal and distal p21 promoter regions, as a measure of p21 promoter accessibility, were decreased in ZD (73 and 64%, respectively) and ZD0.4 (82 and 77%, respectively) cells compared with ZN cells (100 and 100%, respectively). Thus multiple lines of evidence indicate that the transcriptional process of p21 is downregulated by depressed zinc status in HepG2 cells. Furthermore, the transfection of 5 microg of plasmid cytomegalovirus-p21 plasmid, which constitutively expressed p21, was able to normalize the reduction in p21 protein level and cyclin D1-cdk4 complex activity but not the inhibition of cell growth and G1/S cell cycle progression in ZD cells.

Nuclear accumulations of p53 and Mdm2 are accompanied by reductions in c-Abl and p300 in zinc-depleted human hepatoblastoma cells.

The influence of zinc status on the expression of proteins known to be involved in the stability of p53, the human tumor suppressor gene product, was examined in hepatoblastoma (HepG2) cells. Cells were cultured in zinc-deficient (ZD0.2, ZD0.4), zinc normal (ZN), zinc adequate (ZA), or zinc-supplemented (ZS) medium, which contained 0.2, 0.4, 4, 16, or 32 microM zinc, respectively. Nuclear p53 levels were almost 100% and 40% higher in ZD0.2 and ZD0.4 cells, respectively, than in ZN cells. Mdm2 protein, which mediates p53 degradation, was 174% and 148% higher in the nucleus of ZD0.2 and ZD0.4 cells, respectively, than in ZN cells. In addition, the observed reductions of nuclear c-Abl in ZD0.2 and ZD0.4 cells to 50% and 60% of ZN cells, respectively, may be a cellular response attempting to normalize nuclear p53 accumulation because nuclear c-Abl is known to down-regulate ubiquitination and nuclear export of p53. Moreover, no changes in total cellular p53, Mdm2, and c-Abl or nuclear Mdmx were observed among the treatment groups. Furthermore, in ZD0.2 and ZD0.4 cells, the reduction in total and nuclear p300, which is known to complex with CREB-binding protein and Mdm2 in the nucleus for the generation of degradable polyubiquitinated form of p53, may have depressed the degradation pathway for p53 and Mdm2, and contributed to the nuclear accumulation of these proteins in ZD cells.

Water-soluble polymeric polyphenols from cinnamon inhibit proliferation and alter cell cycle distribution patterns of hematologic tumor cell lines.

To explore possible anti-cancer properties of water-soluble, polymeric polyphenols from cinnamon, three myeloid cell lines (Jurkat, Wurzburg, and U937) were exposed to increasing concentrations of an aqueous extract prepared from cinnamon (CE) for 24 h. Cell growth and cell cycle distribution patterns responded in a dose-dependent manner to CE. That is, an increase in the percentage of cells distributed in G2/M was observed in all three cell lines as the amount of CE increased. At the highest dose of CE, the percentage of Wurzburg cells in G2/M was 1.5- and 2.0-fold higher than those observed for Jurkat and U937 cells, respectively. Wurzburg cells lack the CD45 phosphatase and may be more sensitive to imbalances in signaling through kinase/phosphatase networks that promote growth. The results suggest the potential of CE to interact with phosphorylation/dephosphorylation signaling activities to reduce cellular proliferation in tandem with a block at the G2/M phase of the cell cycle.

Zinc depletion reduced Egr-1 and HNF-3beta expression and apolipoprotein A-I promoter activity in Hep G2 cells.

We examined the influence of zinc status on expression of certain transcription factors involved in regulation of apolipoprotein A-I (apoAI) expression in human hepatoblastoma Hep G2 cells. A low zinc basal medium (zinc deficient, ZD) consisting of DMEM and 10% Chelex100-treated fetal bovine serum was used to deplete cellular zinc over one passage. Cells were also cultured for one passage in medium supplemented with 0.4 (ZD0.4), 4.0 (zinc normal, ZN), 16.0 (zinc adequate, ZA), or 32.0 microM zinc (zinc supplemented, ZS). Compared with ZN cells, cellular zinc levels were 43 and 31% lower in ZD and ZD0.4 cells but 70 and 146% higher in ZA and ZS cells, respectively. Supplementation of 0.4 microM zinc significantly increased DNA contents per plate, from 65% in ZD cells to 83% in ZD0.4 cells compared with ZN cells. Addition of >4 microM zinc in medium did not further increase DNA contents. The proportion of cells in G(1)/S and S phase was about fourfold higher and threefold lower, respectively, in ZD cells compared with ZN and other groups. Nuclear Egr-1 protein was markedly decreased in ZD and ZD0.4 cells. Moreover, hepatocyte nuclear factor (HNF)-3beta was severely degraded in ZD and ZD0.4 cells. In contrast, HNF-4alpha remained stable in all groups and was not significantly lower in ZD and ZD0.4 cells. Furthermore, downregulation of trans-acting factor Egr-1 and cleavage of HNF-3beta were associated with reduction of apoAI promoter activity in zinc-deficient Hep G2 cells. Thus zinc is critical in transcriptional regulation of apoAI gene expression in hepatocytes.