Wan-Mohaiza Dashwood

Sulforaphane Retards the Growth of Human PC-3 Xenografts and Inhibits HDAC Activity in Human Subjects

Sulforaphane (SFN) is an isothiocyanate found in cruciferous vegetables such as broccoli. This anticarcinogen was first identified as a potent inducer of Phase 2 enzymes, but evidence is mounting that SFN acts through other cancer chemopreventive mechanisms. We recently reported on a novel mechanism of chemoprotection by SFN in human colon cancer cells and prostate epithelial cells, namely the inhibition of histone deacetylase (HDAC). In the present investigation, we sought to test whether SFN also might inhibit HDAC activity in vivo. When consumed in the diet at an average daily dose of 7.5 μmol per animal for 21 days, SFN suppressed the growth of human PC-3 prostate cancer cells by 40% in male nude mice. There was a significant decrease in HDAC activity in the xenografts, as well as in the prostates and mononuclear blood cells (MBC), of mice treated with SFN, compared to controls. There also was a trend towards increased global histone acetylation in the xenografts, prostates, and MBC. In human subjects, a single dose of 68 g BroccoSprouts inhibited HDAC activity significantly in peripheral blood mononuclear cells (PBMC) 3 and 6 hrs following consumption. These findings provide evidence that one mechanism through which SFN acts as a cancer chemopreventive agent in vivo is through the inhibition of HDAC activity. Moreover, the data suggest that HDAC activity in PBMC may be used as a biomarker for assessing exposure to novel dietary HDAC inhibitors in human subjects.

HDAC turnover, CtIP acetylation and dysregulated DNA damage signaling in colon cancer cells treated with sulforaphane and related dietary isothiocyanates

Histone deacetylases (HDACs) and acetyltransferases have important roles in the regulation of protein acetylation, chromatin dynamics and the DNA damage response. Here, we show in human colon cancer cells that dietary isothiocyanates (ITCs) inhibit HDAC activity and increase HDAC protein turnover with the potency proportional to alkyl chain length, i.e., AITC < sulforaphane (SFN) < 6-SFN < 9-SFN. Molecular docking studies provided insights into the interactions of ITC metabolites with HDAC3, implicating the allosteric site between HDAC3 and its co-repressor. ITCs induced DNA double-strand breaks and enhanced the phosphorylation of histone H2AX, ataxia telangiectasia and Rad3-related protein (ATR) and checkpoint kinase-2 (CHK2). Depending on the ITC and treatment conditions, phenotypic outcomes included cell growth arrest, autophagy and apoptosis. Coincident with the loss of HDAC3 and HDAC6, as well as SIRT6, ITCs enhanced the acetylation and subsequent degradation of critical repair proteins, such as CtIP, and this was recapitulated in HDAC knockdown experiments. Importantly, colon cancer cells were far more susceptible than non-cancer cells to ITC-induced DNA damage, which persisted in the former case but was scarcely detectable in non-cancer colonic epithelial cells under the same conditions. Future studies will address the mechanistic basis for dietary ITCs preferentially exploiting HDAC turnover mechanisms and faulty DNA repair pathways in colon cancer cells vs. normal cells.

α-Keto acid metabolites of organoselenium compounds inhibit histone deacetylase activity in human colon cancer cells

Methylselenocysteine (MSC) and selenomethionine (SM) are two organoselenium compounds receiving interest for their potential anticancer properties. These compounds can be converted to beta-methylselenopyruvate (MSP) and alpha-keto-gamma-methylselenobutyrate (KMSB), alpha-keto acid metabolites that share structural features with the histone deacetylase (HDAC) inhibitor butyrate. We tested the organoselenium compounds in an in vitro assay with human HDAC1 and HDAC8; whereas SM and MSC had little or no activity up to 2 mM, MSP and KMSB caused dose-dependent inhibition of HDAC activity. Subsequent experiments identified MSP as a competitive inhibitor of HDAC8, and computational modeling supported a mechanism involving reversible interaction with the active site zinc atom. In human colon cancer cells, acetylated histone H3 levels were increased during the period 0.5-48 h after treatment with MSP and KMSB, and there was dose-dependent inhibition of HDAC activity. The proportion of cells occupying G(2)/M of the cell cycle was increased at 10-50 microM MSP and KMSB, and apoptosis was induced, as evidenced by morphological changes, Annexin V staining and increased cleaved caspase-3, -6, -7, -9 and poly(adenosine diphosphate-ribose)polymerase. P21WAF1, a well-established target gene of clinically used HDAC inhibitors, was increased in MSP- and KMSB-treated colon cancer cells at both the messenger RNA and protein level, and there was enhanced P21WAF1 promoter activity. These studies confirm that in addition to targeting redox-sensitive signaling molecules, alpha-keto acid metabolites of organoselenium compounds alter HDAC activity and histone acetylation status in colon cancer cells, as recently observed in human prostate cancer cells.

Bcl-2 overexpression in PhIP-induced colon tumors: cloning of the rat Bcl-2 promoter and characterization of a pathway involving β -catenin, c-Myc and E2F1

β-Catenin/T-cell factor (Tcf) signaling is constitutively active in the majority of human colorectal cancers, and there are accompanying changes in Bcl-2 expression. Similarly, 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP)-induced colon tumors in the rat have increased β-catenin and elevated Bcl-2. To examine the possible direct transcriptional regulation of rat Bcl-2 by β-catenin/Tcf, we cloned and characterized the corresponding promoter region and found 70.1% similarity with its human counterpart, BCL2. Bcl-2 promoter activity was increased in response to LiCl and exogenous β-catenin, including oncogenic mutants of β-catenin found in PhIP-induced colon tumors. Protein/DNA arrays identified E2F1, but not β-catenin/Tcf, as interacting most strongly with the rat Bcl-2 promoter. Exogenous E2F1 increased the promoter activity of rat Bcl-2, except in mutants lacking the E2F1 sites. As expected, β-catenin induced its downstream target c-Myc, as well as E2F1 and Bcl-2, and this was blocked by siRNA to c-Myc or E2F1. These findings suggest an indirect pathway for Bcl-2 over-expression in PhIP-induced colon tumors involving β-catenin, c-Myc and E2F1.

NADPH oxidase overexpression in human colon cancers and rat colon tumors induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)

NADPH oxidase/dual‐oxidase (Nox/Duox) family members have been implicated in nuclear factor kappa‐B (NFκB)‐mediated inflammation and inflammation‐associated pathologies. We sought to examine, for the first time, the role of Nox/Duox and NFκB in rats treated with the cooked meat heterocyclic amine carcinogen 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP). In the PhIP‐induced colon tumors obtained after 1 year, Nox1, Nox4, NFκB‐p50 and NFκB‐p65 were all highly overexpressed compared with their levels in adjacent normal‐looking colonic mucosa. Nox1 and Nox4 mRNA and protein levels also were markedly elevated in a panel of primary human colon cancers, compared with their matched controls. In HT29 human colon cancer cells, Nox1 knockdown induced G1 cell cycle arrest, whereas in Caco‐2 cells there was a strong apoptotic response, with increased levels of cleaved caspase‐3, ‐6, ‐7 and poly(ADP‐ribose)polymerase. Nox1 knockdown blocked lipopolysaccharide‐induced phosphorylation of IκB kinase, inhibited the nuclear translocation of NFκB (p50 and p65) proteins, and attenuated NFκB DNA binding activity. There was a corresponding reduction in the expression of downstream NFκB targets, such as MYC, CCND1 and IL1β. The results provide the first evidence for a role of Nox1, Nox4 and NFκB in PhIP‐induced colon carcinogenesis, including during the early stages before tumor onset. Collectively, the findings from this investigation and others suggest that further work is warranted on the role of Nox/Duox family members and NFκB in colon cancer development.

Competitive inhibition of carcinogen‐activating CYP1A1 and CYP1B1 enzymes by a standardized complex mixture of PAH extracted from coal tar

A complex mixture of polycyclic aromatic hydrocarbons (PAH) extracted from coal tar, the Standard Reference Material (SRM) 1597, was recently shown to decrease the levels of DNA binding of the 2 strong carcinogens benzo[a]pyrene (BP) and dibenzo[a,l]pyrene (DBP) in the human mammary carcinoma‐derived cell line MCF‐7 (Mahadevan et al., Chem Res Toxicol 2005;18:224–231). The present study was designed to further elucidate the biochemical mechanisms involved in this inhibition process. We examined the effects of SRM 1597 on the metabolic activation of BP and DBP toward DNA‐binding derivatives in Chinese hamster cells expressing either human cytochrome P450 (CYP) 1A1 or CYP1B1. SRM 1597 inhibited BP‐DNA adduct formation through the entire exposure time in cells expressing human CYP1A1, while it significantly inhibited adduct formation only up to 48 hr when co‐treated with DBP. Conversely, human CYP1B1‐expressing cells were unable to catalyze PAH‐DNA adduct formation on treatment with SRM 1597 alone, and on co‐treatment with BP or DBP. The data obtained from biochemical experiments revealed that SRM 1597 competitively inhibited the activity of both human enzymes as analyzed by 7‐ethoxyresorufin O‐deethylation assays. While the Michaelis‐Menten constant (KM) was <0.4 μM in the absence of SRM 1597, this value increased up to 1.12 (CYP1A1) or 4.45 μM (CYP1B1) in the presence of 0.1 μg/ml SRM 1597. Hence the inhibitory effects of the complex mixture on human CYP1B1 were much stronger when compared to human CYP1A1. Taken together, the decreases in PAH‐DNA adduct formation on co‐treatment with SRM 1597 revealed inhibitory effects on the CYP enzymes that convert carcinogenic PAH into DNA‐binding metabolites. The implications for the tumorigenicity of complex environmental PAH mixtures are discussed.

Abstract LB-184: Differential effects of sulforaphane and related isothiocyanates on HDAC turnover and the DNA damage response in colon cancer cells

Protein acetylation is mediated by histone deacetylases (HDACs) and acetyltransferases, which influence chromatin dynamics, protein turnover, and the DNA damage response. HDACs overexpressed in cancer cells have been implicated in protecting against genotoxic insults, whereas HDAC inhibitors circumvent this protection (Rajendran et al. Clin Epigenetics 2011,3:4). Here, we show in human colon cancer cells that sulforaphane and related isothiocyanates (ITCs) inhibited HDAC activity and increased HDAC protein turnover, with the potency directly proportional to alkyl chain length. Under these conditions, DNA damage signaling was triggered by ATR kinases, leading to increased double-strand breaks and histone (H2AX) phosphorylation. Activation of checkpoint kinase-2 was followed by growth arrest and cell death. HDAC inhibition by ITCs enhanced the acetylation of repair proteins, like CtIP, leading to their degradation. Notably, cancer cells were more susceptible than normal cells, the latter exhibiting efficient double-strand-break processing and repair. Thus, dietary ITCs preferentially exploit the HDAC turnover pathways, faulty DNA repair mechanisms, and genomic instability in cancer cells. Supported by NIH grants CA090890, CA65525, CA122906, CA122959, CA80176, and ES007060. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-184. doi:1538-7445.AM2012-LB-184

Abstract B84: NADPH oxidase 1 (NOX1) and related NOX isoforms as key mediators of NFκB signaling in colon cancer

B84 NADPH oxidase/dual-oxidase (Nox/Duox) family members have been implicated in nuclear factor kappa-B (NFκB)-mediated inflammation and inflammation-associated pathologies. In rats given 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) alternating with a high-fat diet, Nox1, Nox4 and Duo2, but not Nox2, were over-expressed in colon tumors at 1 year, as shown by quantitative RT-PCR and immunoblotting. Over-expression of Nox/duox isoforms coincided with increased nuclear NFκB protein, and elevated levels of downstream targets IL-1β, IL6, TNF-α and TNFR1. Over-expression of Nox1 and activation of NFκB also occurred at early stages of colon carcinogenesis, prior to tumor development. Nox/Duox isoforms were over-expressed in primary human colon cancers and in several human colorectal cancer cell lines. In HT29 cells, silencing of Nox1 inhibited nuclear translocation and DNA binding activity of NFκB, attenuated downstream targets IL1β, c-myc, and cyclin D1, and blocked lipopolysaccharide-induced phosphorylation of IκB kinaseα/β, resulting in G1 arrest. We conclude that the Nox1-NFκB pathway is important in colon tumorigenesis, and that Nox1 may provide a target for novel therapeutic strategies against colon cancer development. Citation Information: Cancer Prev Res 2008;1(7 Suppl):B84.