Rice bran, removed from whole grain rice for white rice milling, has demonstrated efficacy for the control and suppression of colitis and colon cancer in multiple animal models. Dietary rice bran intake was shown to modify human stool metabolites as a result of modifications to metabolism by gut microbiota. In this study, human stool microbiota from colorectal cancer (CRC) survivors that consumed rice bran daily was examined by fecal microbiota transplantation (FMT) for protection from azoxymethane and dextran sodium sulfate (AOM/DSS) induced colon carcinogenesis in germ-free mice. Mice transfaunated with rice bran-modified microbiota communities (RMC) harbored fewer neoplastic lesions in the colon and displayed distinct enrichment of Flavonifractor and Oscillibacter associated with colon health, and the depletion of Parabacteroides distasonis correlated with increased tumor burden. Two anti-cancer metabolites, myristoylcarnitine and palmitoylcarnitine were increased in the colon of RMC transplanted mice. Trimethylamine-N-oxide (TMAO) and tartarate that are implicated in CRC development were reduced in murine colon tissue after FMT with rice bran-modified human microbiota. Findings from this study show that rice bran modified gut microbiota from humans confers protection from colon carcinogenesis in mice and suggests integrated dietary-FMT intervention strategies should be tested for colorectal cancer control, treatment, and prevention.

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Epigenetic modifications of histones (e.g. hypoacetylation) and DNA (e.g. hypermethylation) cause reversible alterations in chromatin structure leading to aberrant changes in gene expression, which eventually impact cellular processes like cell cycle, apoptosis, and invasion during malignant transformation of cells. Several tumor suppressor genes like CDH1, RARB, RASSF1A, MGMT etc are silenced by these interlinked mechanisms in human lung cancer; the leading diagnosed and lethal malignancy world wide. Hence, the impetus has been on developing novel agents that target ‘epigenome’ for both cancer therapy and chemoprevention. Silibinin, a flavanolignan isolated from the seeds of milk thistle exerts strong anti-cancer efficacy against many epithelial cancers including lung. Recently, we showed that silibinin inhibits p53 null-H1299 cell growth through G1 arrest by modulating the expression and function of associated key cell-cycle regulators. Herein, we assessed the effect of silibinin on major chromatin-modification enzymes to delineate their possible involvement in the observed silibinin effects. A time response study for histone deacetylase (HDAC) activity in total cell extract showed that silibinin treatment causes a dose-dependent decrease in enzyme activity from 6h [(100 µM; p<0.05)]-72h [(75-100 µM; p<0.001)]. Concomitantly, under similar treatment conditions, a sustained accumulation of acetylated histones (H3 and H4) in total cellular chromatin was observed between 6-24h time points. We also addressed the question whether silibinin directly inhibits HDAC activity in a cell free system. In both total and nuclear cell extracts, a decreasing trend in enzyme activity was observed by silibinin addition in incubation reactions, but the concentration for 50% inhibition (IC50) was ∼ 400 µM, suggesting an indirect effect of silibinin in regulating HDAC activity in cell free system. Consistent with these results, silibinin (75-100 µM) also caused a decrease in the protein levels of HDAC1, HDAC2, HDAC3, DNMT1 and DNMT3A in nuclear and total extracts after 24, 48 and 72h of treatment. Next, we evaluated the expression profile of 84 key genes encoding chromatin-modification enzymes in H1299 cells after silibinin treatment (100 µM; 72h). Real time RT-PCR array analysis identified a subset of fifteen genes, primarily including DNMT3A, HDAC1, HDAC6, SET domain proteins (SETD1A, D4, D6) and lysine specific demethylases (KDM 5B, 5C, 4A) which were significantly down-regulated (∼2-4 fold) in silibinin treated samples compared to vehicle controls. Collectively, these results show that silibinin inhibits specific histone deacetylases, histone demethylases and DNA methyltransferases, which might cooperatively contribute to the anti-cancer efficacy of this non-toxic phytochemical. (Supported by CA113876) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3796.

Nuclear Speckles (NS) are phase-separated condensates of protein and RNA whose components dynamically coordinate RNA transcription, splicing, transport and DNA repair. NS, probed largely by imaging studies, remained historically well known as Interchromatin Granule Clusters, and biochemical properties, especially their association with Chromatin have been largely unexplored. In this study, we tested whether NS exhibit any stable association with chromatin and show that limited DNAse-1 nicking of chromatin leads to the collapse of NS into isotropic distribution or aggregates of constituent proteins without affecting other nuclear structures. Further biochemical probing revealed that NS proteins were tightly associated with chromatin, extractable only by high-salt treatment just like histone proteins. NS were also co-released with solubilised mono-dinucleosomal chromatin fraction following the MNase digestion of chromatin. We propose a model that NS-chromatin constitutes a “putative stable association” whose coupling might be subject to the combined regulation from both chromatin and NS changes. Abbreviations: NS: Nuclear speckles; DSB: double strand breaks; PTM: posttranslational modifications; DDR: DNA damage repair; RBP-RNA binding proteins; TAD: topologically associated domains; LCR: low complexity regions; IDR: intrinsically disordered regions.

Lung cancer is the leading cause of cancer-related mortalities, and the 5-year survival rate of this malignancy remains at 15%. This feature is attributed primarily to the early metastasis of the disease within the organ and to distant sites that involves the epithelial-to-mesenchymal transition (EMT) of lung cancer cells. Aggressive cancer cells with EMT are characterized by loss of cell adhesion, repression of E-cadherin expression and increased cell mobility. NSCLC cells widely differ in their basal level of E-cadherin expression, which is epigenetically silenced in most of them due to deacetylation by histone deactylases. This suggests that inhibitors of HDACs would be useful in NSCLC control. Several HDAC inhibitors (HDACi) have shown potential in this regard, and are in clinical trials; however, their toxicity and cancer cell resistance are the limiting factors. Silibinin (Sb), a non-toxic chemopreventive agent, has shown strong efficacy against epithelial cancers including NSCLC, and previously, we have demonstrated that Sb alters HDAC enzymatic activity and protein levels in a total cellular extract of H1299 cells. Herein, we sought to determine the putative role of Silibinin, either alone or in combination with other HDACi in modulating E-cadherin levels in NSCLC cell lines including H1299 cells (epigenetically silent E-cadherin levels). Treatment with HDAC inhibitors, Trichostatin A [TSA] (0.33-1 µM) or SAHA (0.5-5 µM) led to a dose-dependent induction of E-cadherin expression levels. Furthermore, the combinations of TSA (0.5 µM) with low doses of Sb (3.75-25 µM) in H1299 cells, significantly increased E-cadherin protein levels (24h), which remained sustained at later time points (48-72h) longer than with TSA alone treatments. Also, at these time points, Zeb1, a major transcriptional repressor of E-cadherin, was inversely down-modulated with the combination treatments. Substantial inhibition of invasion/migration of H1299 cells with similar treatment conditions further emphasized the biological significance of E-cadherin restoration. Unlike H1299 cells, treatment of H322 and H358 cells (with detectable E-cadherin level) with Sb (3.75-25 µM) alone was sufficient to consistently increase the basal levels of E-cadherin, with concomitant reduction of Zeb1 levels, which also led to inhibition of migratory potential of these cells. Overall these findings demonstrate that Sb alone or in combination with HDAC inhibitors upregulates E-cadherin expression levels in NSCLC cell lines, and this consequently leads to inhibition of invasion/migratory phenotype of these cells. 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 1608. doi:1538-7445.AM2012-1608