Mingzhu Fang

Reversal of Hypermethylation and Reactivation of p16INK4a, RARβ, and MGMT Genes by Genistein and Other Isoflavones from Soy

Purpose: We have previously shown the reactivation of some methylation-silenced genes in cancer cells by (−)-epigallocatechin-3-gallate, the major polyphenol from green tea. To determine whether other polyphenolic compounds have similar activities, we studied the effects of soy isoflavones on DNA methylation. Experimental Design: Enzyme assay was used to determine the inhibitory effect of genistein on DNA methyltransferase activity in nuclear extracts and purified recombinant enzyme. Methylation-specific PCR and quantitative real-time PCR were employed to examine the DNA methylation and gene expression status of retinoic acid receptor β (RARβ), p16INK4a, and O6-methylguanine methyltransferase (MGMT) in KYSE 510 esophageal squamous cell carcinoma cells treated with genistein alone or in combination with trichostatin, sulforaphane, or 2′-deoxy-5-aza-cytidine (5-aza-dCyd). Results: Genistein (2-20 μmol/L) reversed DNA hypermethylation and reactivated RARβ, p16INK4a, and MGMT in KYSE 510 cells. Genistein also inhibited cell growth at these concentrations. Reversal of DNA hypermethylation and reactivation of RARβ by genistein were also observed in KYSE 150 cells and prostate cancer LNCaP and PC3 cells. Genistein (20-50 μmol/L) dose-dependently inhibited DNA methyltransferase activity, showing substrate- and methyl donor–dependent inhibition. Biochanin A and daidzein were less effective in inhibiting DNA methyltransferase activity, in reactivating RARβ, and in inhibiting cancer cell growth. In combination with trichostatin, sulforaphane, or 5-aza-dCyd, genistein enhanced reactivation of these genes and inhibition of cell growth. Conclusions: These results indicate that genistein and related soy isoflavones reactivate methylation-silenced genes, partially through a direct inhibition of DNA methyltransferase, which may contribute to the chemopreventive activity of dietary isoflavones.

Overexpression of 5-Lipoxygenase and Cyclooxygenase 2 in Hamster and Human Oral Cancer and Chemopreventive Effects of Zileuton and Celecoxib

Purpose: Previous studies have suggested an important role of aberrant arachidonic acid metabolism, especially the cyclooxygenase (Cox) pathway, in oral carcinogenesis. However, it is unknown whether the 5-lipoxygenase (5-Lox) pathway contributes to oral carcinogenesis, and whether combination of inhibitors of both pathways may have synergistic or additive effects of chemoprevention. Experimental Design: 5-Lox expression was examined in 7,12-dimethylbenz[a]anthracene (DMBA)–induced hamster and human oral cancer tissues by immunohistochemistry, and Cox2 expression was investigated in hamster oral tissues using in situ hybridization. Zileuton (a specific 5-Lox inhibitor) and celecoxib (a specific Cox2 inhibitor), either alone or in combination, were investigated for their chemopreventive effects on the DMBA-induced hamster model at the post-initiation stage through topical application. Results: 5-Lox was overexpressed during oral carcinogenesis in hamsters and humans, as well as Cox2 in the hamster tissues. In a chemoprevention study using the post-initiation DMBA model, incidence of hamster oral squamous cell carcinoma was reduced from 76.9% (20 of 26) to 45.8% (11 of 24, P < 0.05) and 32.1% (9 of 28, P < 0.01) by 3% and 6% topical zileuton, respectively; and to 57.6% (15 of 26, P > 0.05) and 50% (12 of 24, P < 0.05) by 3% and 6% topical celecoxib, respectively. When used in combination, celecoxib and zileuton (3% of each) had an additive inhibitory effect on the incidence of squamous cell carcinoma (36%, 9 of 25, P < 0.01). Other pathologic variables and the levels of leukotriene B4 and prostaglandin E2 of the hamster tissues were reduced as well. Conclusions: The results clearly showed that both 5-Lox and Cox2 played important roles in oral carcinogenesis. Zileuton and celecoxib prevented oral carcinogenesis at the post-initiation stage through their inhibitory effects on arachidonic acid metabolism.

Methylselenocysteine Resets the Rhythmic Expression of Circadian and Growth Regulatory Genes Disrupted by Nitrosomethylurea in vivo

Epidemiologic and animal studies indicate that disruption of circadian rhythm increases breast cancer risk. Previously, we showed that methylselenocysteine reduced the incidence of N-nitroso-N-methylurea (NMU)–induced mammary carcinomas in Fischer 344 rats by 63%. Methylselenocysteine also increased the expression of Period 2 (Per2) and D-binding protein (DBP), providing evidence for a link between circadian rhythm and chemoprevention. Here, we report that NMU disrupted the expression of core circadian genes (Per1, Per2, Cry1, Cry2, and RevErbAα) and circadian-controlled genes, including melatonin receptor 1α (MTNR1A), estrogen receptors (ERα and ERβ), and growth-regulatory genes (Trp53, p21, Gadd45α, and c-Myc) in mammary glands of Fischer 344 rats. By contrast, dietary methylselenocysteine (3 ppm selenium) given for 30 days, significantly enhanced the circadian expression of these genes (except for Cry1 and Cry2). The largest effect was on the levels of the Per2, MTNR1A, and ERβ mRNAs, which showed 16.5-fold, 4.7-fold, and 9.5-fold increases in their rhythm-adjusted means, respectively, and 44.5-fold, 6.5-fold, and 9.7-fold increases in amplitude as compared with the control diet, respectively. Methylselenocysteine also shifted the peak expression times of these genes to Zeitgeber time 12 (ZT12; lights off). Methylselenocysteine also induced rhythmic expression of Trp53, p21, and Gadd45α mRNAs with peak levels at ZT12, when c-Myc expression was at its lowest level. However, methylselenocysteine had no significant effect on the circadian expression of these genes in liver. These results suggest that dietary methylselenocysteine counteracted the disruptive effect of NMU on circadian expression of genes essential to normal mammary cell growth and differentiation. Cancer Prev Res; 3(5); 640–52. ©2010 AACR.

Sleep interruption associated with house staff work schedules alters circadian gene expression

BACKGROUND Epidemiological studies indicate that disruption of circadian rhythm by shift work increases the risk of breast and prostate cancer. Our studies demonstrated that carcinogens disrupt the circadian expression of circadian genes (CGs) and circadian-controlled genes (CCGs) during the early stages of rat mammary carcinogenesis. A chemopreventive regimen of methylselenocysteine (MSC) restored the circadian expression of CGs and CCGs, including PERIOD 2 (PER2) and estrogen receptor β (ERS2), to normal. The present study evaluated whether changes in CG and CCG expression in whole blood can serve as indicators of circadian disruption in shift workers. METHODS Fifteen shift workers were recruited to a crossover study. Blood samples were drawn before (6 PM) and after (8 AM) completing a night shift after at least seven days on floating night-shift rotation, and before (8 AM), during (1 PM), and after (6 PM) completing seven days on day shift. The plasma melatonin level and messenger RNA (mRNA) expression of PER2, nuclear receptor subfamily 1, group d, member 1 (NR1D1), and ERS2 were measured, and the changes in levels of melatonin and gene expression were evaluated with statistical analyses. RESULTS The mRNA expression of PER2 was affected by shift (p = 0.0079); the levels were higher in the evening for the night shift, but higher in the morning for the day shift. Increased PER2 expression (p = 0.034) was observed in the evening on the night versus day shifts. The melatonin level was higher in the morning for both day shifts (p = 0.013) and night shifts (p <0.0001). CONCLUSION Changes in the level of PER2 gene expression can serve as a biomarker of disrupted circadian rhythm in blood cells. Therefore, they can be a useful intermediate indicator of efficacy in future MSC-mediated chemoprevention studies.

Enhancement of NAD + -dependent SIRT1 deacetylase activity by methylselenocysteine resets the circadian clock in carcinogen-treated mammary epithelial cells

We previously reported that dietary methylselenocysteine (MSC) inhibits N-methyl-N-nitrosourea (NMU)-induced mammary tumorigenesis by resetting circadian gene expression disrupted by the carcinogen at the early stage of tumorigenesis. To investigate the underlying mechanism, we developed a circadian reporter system comprised of human mammary epithelial cells with a luciferase reporter driven by the promoter of human PERIOD 2 (PER2), a core circadian gene. In this in vitro model, NMU disrupted cellular circadian rhythm in a pattern similar to that observed with SIRT1-specific inhibitors; in contrast, MSC restored the circadian rhythms disrupted by NMU and protected against SIRT1 inhibitors. Moreover, NMU inhibited intracellular NAD+/NADH ratio and reduced NAD+-dependent SIRT1 activity in a dose-dependent manner, while MSC restored NAD+/NADH and SIRT1 activity in the NMU-treated cells, indicating that the NAD+-SIRT1 pathway was targeted by NMU and MSC. In rat mammary tissue, a carcinogenic dose of NMU also disrupted NAD+/NADH oscillations and decreased SIRT1 activity; dietary MSC restored NAD+/NADH oscillations and increased SIRT1 activity in the mammary glands of NMU-treated rats. MSC-induced SIRT1 activity was correlated with decreased acetylation of BMAL1 and increased acetylation of histone 3 lysine 9 at the Per2 promoter E-Box in mammary tissue. Changes in SIRT1 activity were temporally correlated with loss or restoration of rhythmic Per2 mRNA expression in NMU-treated or MSC-rescued rat mammary glands, respectively. Together with our previous findings, these results suggest that enhancement of NAD+-dependent SIRT1 activity contributes to the chemopreventive efficacy of MSC by restoring epigenetic regulation of circadian gene expression at early stages of mammary tumorigenesis.

DNA methylome and transcriptome alterations and cancer prevention by curcumin in colitis-accelerated colon cancer in mice

Inflammation is highly associated with colon carcinogenesis. Epigenetic mechanisms could play an important role in the initiation and progression of colon cancer. Curcumin, a dietary phytochemical, shows promising effects in suppressing colitis-associated colon cancer in azoxymethane-dextran sulfate sodium (AOM-DSS) mice. However, the potential epigenetic mechanisms of curcumin in colon cancer remain unknown. In this study, the anticancer effect of curcumin in suppressing colon cancer in an 18-week AOM-DSS colon cancer mouse model was confirmed. We identified lists of differentially expressed and differentially methylated genes in pairwise comparisons and several pathways involved in the potential anticancer effect of curcumin. These pathways include LPS/IL-1-mediated inhibition of RXR function, Nrf2-mediated oxidative stress response, production of NO and ROS in macrophages and IL-6 signaling. Among these genes, Tnf stood out with decreased DNA CpG methylation of Tnf in the AOM-DSS group and reversal of the AOM-DSS induced Tnf demethylation by curcumin. These observations in Tnf methylation correlated with increased and decreased Tnf expression in RNA-seq. The functional role of DNA methylation of Tnf was further confirmed by in vitro luciferase transcriptional activity assay. In addition, the DNA methylation level in a group of inflammatory genes was decreased in the AOM+DSS group but restored by curcumin and was validated by pyrosequencing. This study shows for the first time epigenomic changes in DNA CpG methylation in the inflammatory response from colitis-associated colon cancer and the reversal of their CpG methylation changes by curcumin. Future clinical epigenetic studies with curcumin in inflammation-associated colon cancer would be warranted.

Uncoupling genotoxic stress responses from circadian control increases susceptibility to mammary carcinogenesis

We previously demonstrated that chemopreventive methylselenocysteine (MSC) prevents N-Nitroso-N-methylurea (NMU)-induced mammary carcinogenesis in the susceptible Fischer 344 (F344) rats by enhancing NAD+-dependent SIRT1 activity, restoring circadian expression of Period 2 (Per2) and circadian controlled genes. Here, we show that compared to the genetically resistant Copenhagen (COP) rat strain, mammary glands of the F344 rats have a 4-hour phase delay in circadian expression of Per2. Consequently, F344 rats failed to increase SIRT1 activity and circadian expression of Per2 and DDRR genes after exposure to NMU. Exposure of COP rats to NMU had the opposite effect, enhancing SIRT1 activity, increasing circadian expression of Per2 and DDRR genes. Significantly, SIRT1 activity and circadian expression of Per2 and DDRR genes in NMU-treated F344 rats on a chemopreventive regimen of MSC approximated those in NMU-treated COP rats. These results indicated that COP rats have an increased capacity to maintain NAD+-dependent SIRT1 activity under genotoxic stress. This contention was supported by increased stability of the period and phase of circadian locomotor activity in COP vs F344 rats exposed to changing light conditions. The increased sensitivity and rapid response of COP to changing light were correlated with the enhanced circadian response of this strain to carcinogen. Disturbance of circadian rhythm by jet lag also disrupted circadian expression of Per2 and DDRR genes, and accelerated mammary tumorigenesis in rodent models. These results suggested that uncoupling of DDRR responses from circadian control by environmental stresses and endogenous factors increases susceptibility to mammary carcinogenesis, possibly by inducing a promutagenic state.

World Trade Center (WTC) dust exposure in mice is associated with inflammation, oxidative stress and epigenetic changes in the lung

Exposure to World Trade Center (WTC) dust has been linked to respiratory disease in humans. In the present studies we developed a rodent model of WTC dust exposure to analyze lung oxidative stress and inflammation, with the goal of elucidating potential epigenetic mechanisms underlying these responses. Exposure of mice to WTC dust (20μg, i.t.) was associated with upregulation of heme oxygenase-1 and cyclooxygenase-2 within 3days, a response which persisted for at least 21days. Whereas matrix metalloproteinase was upregulated 7days post-WTC dust exposure, IL-6RA1 was increased at 21days; conversely, expression of mannose receptor, a scavenger receptor important in particle clearance, decreased. After WTC dust exposure, increases in methylation of histone H3 lysine K4 at 3days, lysine K27 at 7days and lysine K36, were observed in the lung, along with hypermethylation of Line-1 element at 21days. Alterations in pulmonary mechanics were also observed following WTC dust exposure. Thus, 3days post-exposure, lung resistance and tissue damping were decreased. In contrast at 21days, lung resistance, central airway resistance, tissue damping and tissue elastance were increased. These data demonstrate that WTC dust-induced inflammation and oxidative stress are associated with epigenetic modifications in the lung and altered pulmonary mechanics. These changes may contribute to the development of WTC dust pathologies.

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells

The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to sleep behavior. In vertebrates, circadian rhythm is controlled by a molecular oscillator that functions in both the suprachiasmatic nucleus (SCN; central pacemaker) and individual cells comprising most peripheral tissues. More importantly, disruption of circadian rhythm by exposure to light-at-night, environmental stressors and/or toxicants is associated with increased risk of chronic diseases and aging. The ability to identify agents that can disrupt central and/or peripheral biological clocks, and agents that can prevent or mitigate the effects of circadian disruption, has significant implications for prevention of chronic diseases. Although rodent models can be used to identify exposures and agents that induce or prevent/mitigate circadian disruption, these experiments require large numbers of animals. In vivo studies also require significant resources and infrastructure, and require researchers to work all night. Thus, there is an urgent need for a cell-type appropriate in vitro system to screen for environmental circadian disruptors and enhancers in cell types from different organs and disease states. We constructed a vector that drives transcription of the destabilized luciferase in eukaryotic cells under the control of the human PERIOD 2 gene promoter. This circadian reporter construct was stably transfected into human mammary epithelial cells, and circadian responsive reporter cells were selected to develop the in vitro bioluminescence assay. Here, we present a detailed protocol to establish and validate the assay. We further provide details for proof of concept experiments demonstrating the ability of our in vitro assay to recapitulate the in vivo effects of various chemicals on the cellular biological clock. The results indicate that the assay can be adapted to a variety of cell types to screen for both environmental disruptors and chemopreventive enhancers of circadian clocks.

Abstract A20: Decreased nuclear expression of the FRY protein identifies a subset of breast cancers with poor clinical outcomes

The goal of the present study was to determine if decreased nuclear expression of the human FRY protein can serve as a biomaker of breast cancer progression and outcomes. We previously identified the rat Fry gene as a putative mammary carcinoma susceptibility (Mcs) gene. We further demonstrated that FRY expression was decreased in several human breast cancer cell lines. Moreover, ectopic expression of wildtype Fry suppressed tumorigenicity of the triple negative MDA-MD-231 breast cancer cells in vitro and in vivo by promoting epithelial cell differentiation. To evaluate the contribution of altered FRY expression to the clinical progression of human breast cancer, we first compared FRY mRNA expression in >4,800 clinically annotated human breast cancers using DNA microarray data from 19 distinct cohorts available from the Oncomine 3.0 Cancer Profiling Database. The analysis indicated that decreased FRY mRNA was significantly correlated with poorly differentiated tumor histopathology, increased Elston tumor grade, and triple negative status (loss of estrogen, progesterone and Her2 receptors). Using commercially available breast cancer tissue microarrays, we used semi-quantitative immunohistochemistry and quantitative image analysis to compare FRY protein expression in normal mammary tissue and tumors. The results indicated that loss of FRY expression was a frequent event during progression of mammary carcinomas. We therefore analyzed >1200 clinically annotated breast cancers represented on tissue microarrays from commercial suppliers and the National Cancer Institute (NCI) Cancer Diagnosis Program, including the Breast Cancer Diagnosis and Progression panels. FRY protein expression was evaluated by immunohistochemical staining and was graded on a scale of 0 to 4 (0, +, ++, +++, ++++). The analysis indicated that decreased nuclear expression of the FRY protein was significantly correlated with poorly differentiated histopathologies, increasing Elston grade and loss of the estrogen receptor. Tumors with decreased FRY expression were also more likely to metastasize and were associated with decreased tumor-free survival. These findings indicated that loss of nuclear FRY expression identifies a subset of breast cancer with poor clinical outcomes, suggesting that FRY may serve as an early biomarker for breast cancer progression and prognosis. Citation Format: Helmut Zarbl, Jessica C. Graham, Norio Takizawa, Mingzhu Fang, Zhihong Gong, Brian Estrella, Xuefeng Ren. Decreased nuclear expression of the FRY protein identifies a subset of breast cancers with poor clinical outcomes. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A20.