The effects of short-chain fatty acids on human colon cancer cell phenotype are associated with histone hyperacetylation

The short-chain fatty acid (SCFA) butyrate is produced via anaerobic bacterial fermentation within the colon and is thought to be protective in regard to colon carcinogenesis. Although butyrate (C4) is considered the most potent of the SCFA, a variety of other SCFA also exist in the colonic lumen. Butyrate is thought to exert its cellular effects through the induction of histone hyperacetylation. We sought to determine the effects of a variety of the SCFA on colon carcinoma cell growth, differentiation and apoptosis. HT-29 or HCT-116 (wild-type and p21-deleted) cells were treated with physiologically relevant concentrations of various SCFA, and histone acetylation state was assayed by acid-urea-triton-X gel electrophoresis and immunoblotting. Growth and apoptotic effects were studied by flow cytometry, and differentiation effects were assessed using transient transfections and Northern blotting. Propionate (C3) and valerate (C5) caused growth arrest and differentiation in human colon carcinoma cells. The magnitude of their effects was associated with a lesser degree of histone hyperacetylation compared with butyrate. Acetate (C2) and caproate (C6), in contrast, did not cause histone hyperacetylation and also had no appreciable effects on cell growth or differentiation. SCFA-induced transactivation of the differentiation marker gene, intestinal alkaline phosphatase (IAP), was blocked by histone deacetylase (HDAC), further supporting the critical link between SCFA and histones. Butyrate also significantly increased apoptosis, whereas the other SCFA studied did not. The growth arrest induced by the SCFA was characterized by an increase in the expression of the p21 cell-cycle inhibitor and down-regulation of cyclin B1 (CB1). In p21-deleted HCT-116 colon cancer cells, the SCFA did not alter the rate of proliferation. These data suggest that the antiproliferative, apoptotic and differentiating properties of the various SCFA are linked to the degree of induced histone hyperacetylation. Furthermore, SCFA-mediated growth arrest in colon carcinoma cells requires the p21 gene.     

Effects of the in vivo supply of butyrate on histone acetylation of cecum in piglets

BACKGROUND: In vitro, butyrate inhibits histone deacetylase and down-regulates expression of cyclin D1. We hypothesized that an increased entry rate of butyrate into the cecal lumen would have similar effects in vivo. METHODS: We used frozen cecal tissue and data from previous studies, one showing that lactulose supplementation caused an increased rate of cecal synthesis of butyrate and decreased cecal cell proliferation and density of clostridia and the other showing that cecal cell proliferation was increased by an exogenous cecal butyrate infusion at a comparable rate. The ratio of acetylated to total histones (AH ratio) and cyclin D1 mRNA expression were measured in cecal tissue. RESULTS: Lactulose supplementation caused a 189% increase in the AH ratio (p = .004), which inversely correlated with cecal cell proliferation (r = -0.782; p = .008). With cecal butyrate infusion, we observed a significant decrease in histone acetylation (p = .02), which also inversely correlated with cecal cell proliferation (r = -0.797; p = .002). Cyclin D1 expression was increased 6.5-fold by lactulose feeding (p = .02) but decreased 50% with cecal butyrate infusion (p = .004). CONCLUSIONS: The effects on histone acetylation of increased "endogenous" butyrate production produced by lactulose feeding, but not exogenous cecal infusion of butyrate, mirror those in vitro. Thus, bacterial production and exogenous infusion of butyrate have opposite effects on histone acetylation and cyclin D1 expression, suggesting that the composition of bacterial flora may play a role in butyrate's in vivo effects on the cell cycle.     

组蛋白去乙酰化酶抑制剂联合顺铂对宫颈癌细胞凋亡作用的体外研究

目的:观察组蛋白去乙酰化酶抑制剂(SAHA)联合顺铂(DDP)对宫颈癌SiHa细胞生长抑制和促凋亡的效果。方法:以1μmol/L SAHA、2μmol/L SAHA、4μmol/L SAHA和1μg/ml、2μg/ml、4μg/ml DDP分别组成单药组和不同SA-HA+DDP联合用药组,分别应用MTT方法检测细胞增殖抑制率,应用流式细胞术检测SiHa细胞早期凋亡的情况。结果:联合用药组肿瘤细胞大量坏死明显,对细胞增殖的抑制率明显高于相应单药组(P<0.05);SAHA与中低剂量的DDP联合给药表现为协同作用,而与较高剂量的DDP联合用药表现为单纯相加作用;联合作用较二者单独用药细胞凋亡率明显增加(P<0.05)。结论:SAHA对宫颈癌细胞具有抑制增殖和促进凋亡的作用,SAHA联合DDP有协同增敏作用。     

去乙酰酶抑制剂在骨性关节炎中的应用研究

目的：对近年来在骨性关节炎方面的相关应用研究及新进展做了综合概述。方法：通过对动物的体内外造模、诱导或抑制的方法,探寻软骨降解的过程,并模拟机械应力对人软骨细胞作用,观察压力对软骨降解的影响。结果：去乙酰酶抑制剂不仅可以作用于组蛋白,对非组蛋白如转录因子也有影响,可以延缓软骨细胞凋亡,还可调节疼痛反应。结论：从体外实验的分子和细胞以及体外动物实验可以看出,去乙酰酶抑制剂的确对于OA有一定治疗作用,是潜在的OA临床治疗药物。     

The short chain fatty acid butyrate induces promoter demethylation and reactivation of RARbeta2 in colon cancer cells

It has been proposed that cancer prevention results from multiple dietary agents acting together as "action packages." Here we obtain evidence that butyrate, which is generated from dietary fiber, enhances the responsiveness of colon cancer cells to all-trans retinoic acid (ATRA). Evidence was obtained that this interaction depends on histone deactylase one (HDAC1) inhibition by butyrate and retinoic acid receptor alpha (RARalpha) activation by ATRA. The enhancement of RAR beta 2 (RARbeta2) activation was accompanied by a rapid demethylation of the RARbeta2 promoter. This demethylation could be achieved by butyrate alone, and it differed from that triggered by the DNA methyltransferase inhibitor 5-Aza-2' deoxycytidine in that it was 1) sporadic on the RARbeta2 promoter, 2) not genome wide, and 3) independent of extensive DNA replication. An analysis of inter-methylated sites assay indicated that only a few percent of loci analyzed showed reduced methylation. In colon cancer cells that were particularly resistant to RARbeta2 reactivation, the actions of butyrate could be further enhanced by the soy isoflavone genistein, which has also been reported to work through an epigenetic mechanism. These data suggest that dietary compounds that modulate epigenetic programming are likely to function best in the presence of retinoids and other cancer-preventing compounds that are sensitive to a cell's epigenetic state.     

Butyrate and propionate induced activated or non-activated neutrophil apoptosis via HDAC inhibitor activity but without activating GPR-41/GPR-43 pathways

ObjectiveDecreased neutrophil apoptosis is implicated in persistent inflammation resulting in systemic inflammatory response syndrome and multiple organ dysfunctions syndromes. Short-chain fatty acids (SCFAs) may be a candidate to control neutrophil apoptosis because SCFAs are normally produced in the gut and related products have been approved for human use. We investigated the effects of SCFAs on apoptosis of activated and non-activated neutrophils and their mechanisms.MethodsPurified neutrophils obtained from healthy volunteers were preincubated for 1 h with or without the G-protein receptor (GPR) inhibitor pertussis toxin (100 ng/mL) or U-73122 (50 ng/mL), extracellular signal-related protein kinase inhibitor PD98059 (10 μM), mitogen-activated protein kinase (MAPK) p38 inhibitor SB203580 (25 μM), Jun kinase inhibitor-I (2 μM), caspase-3 and -7 inhibitor Z-VAD-FMK (100 μM), caspase-8 inhibitor Z-IETD-FMK (50 μM), or caspase-9 inhibitor Z-LEHD-FMK (50 μM). The cells were then cultured with or without SCFAs or trichostatin A, a typical histone deacetylase inhibitor, in the presence or absence of lipopolysaccharide (1 μg/mL) or tumor necrosis factor-α (100 ng/mL). Neutrophil apoptosis was assessed by annexin V staining using flow cytometry. The GPR-41 and -43 and apoptosis-related proteins (bax, mcl-1, a1) mRNA were measured by quantitative real-time polymerase chain reaction and the expression of acetylated histone H3 was determined by western blot.ResultsThe caspase inhibitors inhibited butyrate- and propionate-induced neutrophil apoptosis treated or untreated with lipopolysaccharide or tumor necrosis factor-α, whereas GPR and MAPK inhibitors had no effect. The mRNA expressions of GPR-43 and a1 protein were reduced by butyrate and propionate. The expressions of acetylated histone H3 were induced by butyrate and propionate.ConclusionThese results suggest that butyrate and propionate increase apoptosis of neutrophils irrespective of their activation state, by factors other than GPRs and MAPKs, and their mechanisms likely relate to their histone deacetylase inhibition activity, which may control a1 mRNA expression.     

Histone-deacetylase inhibition and butyrate formation: Fecal slurry incubations with apple pectin and apple juice extracts

OBJECTIVE: Butyrate plays a major role among the short-chained fatty acids formed by the microbial flora of the colon. It is considered to be an important nutrient of the colon mucosa and has been shown to trigger differentiation and apoptosis of colon-derived cells in culture. Inhibition of histone deacetylase (HDAC) seems to play a central role in these effects. Butyrate was thus suggested to act as a chemopreventive metabolite that can prevent the occurrence of colorectal cancer, one of the most abundant types of cancer in Western industrialized countries. Some polymeric carbohydrates such as pectin, resistant to digestion in the small intestine, have been shown to serve as substrates for butyrate formation by the microflora of the colon. METHODS: In this study we investigated fermentation supernatants (FSs) from incubations of human fecal slurry with apple pectin and with polyphenol-rich apple juice extracts (AJEs). RESULTS: We found that FSs from fermentations with pectin were rich in butyrate and very active in HDAC inhibition in nuclear extracts prepared from the colon tumor cell lines HT-29 and Caco-2 and in intact HeLa Mad 38 cells bearing a reporter gene driven by HDAC inhibition. The butyrate levels explained most of the HDAC-inhibitory potency in FSs from pectin-rich fermentations. FSs from fermentations with AJEs showed lower butyrate yields but comparable HDAC inhibition. Combined incubations of pectin with AJEs led to effects similar to those with FSs from incubations with pectin as the only substrate added. These effects could not be explained by a direct HDAC-inhibitory potency of AJEs. Furthermore, the FSs were not cytotoxic at the HDAC-inhibitory concentrations. CONCLUSION: These findings suggest that butyrate is the most relevant HDAC inhibitor formed in fermentations of human fecal slurry with apple pectin, whereas addition of AJEs leads to the formation of butyrate and other, yet unknown, HDAC inhibitors.     

The Short Chain Fatty Acid Butyrate Induces Promoter Demethylation and Reactivation of RARβ2 in Colon Cancer Cells

It has been proposed that cancer prevention results from multiple dietary agents acting together as “action packages.” Here we obtain evidence that butyrate, which is generated from dietary fiber, enhances the responsiveness of colon cancer cells to all-trans retinoic acid (ATRA). Evidence was obtained that this interaction depends on histone deactylase one (HDAC1) inhibition by butyrate and retinoic acid receptor alpha (RARα) activation by ATRA. The enhancement of RAR beta 2 (RARβ2) activation was accompanied by a rapid demethylation of the RARβ2 promoter. This demethylation could be achieved by butyrate alone, and it differed from that triggered by the DNA methyltransferase inhibitor 5-Aza-2′ deoxycytidine in that it was 1) sporadic on the RARβ2 promoter, 2) not genome wide, and 3) independent of extensive DNA replication. An analysis of inter-methylated sites assay indicated that only a few percent of loci analyzed showed reduced methylation. In colon cancer cells that were particularly resistant to RARβ2 reactivation, the actions of butyrate could be further enhanced by the soy isoflavone genistein, which has also been reported to work through an epigenetic mechanism. These data suggest that dietary compounds that modulate epigenetic programming are likely to function best in the presence of retinoids and other cancer-preventing compounds that are sensitive to a cell's epigenetic state.     

Chromatin-regulating proteins as targets for cancer therapy

Chromatin-regulating proteins represent a large class of novel targets for cancer therapy. In the context of radiotherapy, acetylation and deacetylation of histones by histone acetyltransferases (HATs) and histone deacetylases (HDACs) play important roles in the repair of DNA double-strand breaks generated by ionizing irradiation, and are therefore attractive targets for radiosensitization. Small-molecule inhibitors of HATs (garcinol, anacardic acid and curcumin) and HDACs (vorinostat, sodium butyrate and valproic acid) have been shown to sensitize cancer cells to ionizing irradiation in preclinical models, and some of these molecules are being tested in clinical trials, either alone or in combination with radiotherapy. Meanwhile, recent large-scale genome analyses have identified frequent mutations in genes encoding chromatin-regulating proteins, especially in those encoding subunits of the SWI/SNF chromatin-remodeling complex, in various human cancers. These observations have driven researchers toward development of targeted therapies against cancers carrying these mutations. DOT1L inhibition in MLL-rearranged leukemia, EZH2 inhibition in EZH2-mutant or MLL-rearranged hematologic malignancies and SNF5-deficient tumors, BRD4 inhibition in various hematologic malignancies, and BRM inhibition in BRG1-deficient tumors have demonstrated promising anti-tumor effects in preclinical models, and these strategies are currently awaiting clinical application. Overall, the data collected so far suggest that targeting chromatin-regulating proteins is a promising strategy for tomorrow''s cancer therapy, including radiotherapy and molecularly targeted chemotherapy.     

Histone deacetylase inhibitor prodrugs in nanoparticle vector enhanced gene expression in human cancer cells

We developed histone deacetylase inhibitor (HDACI) prodrugs to enhance the expression of the external genes transfected into human cells with cationic nanoparticles (NPs). We synthesized five kinds of lipid-linked HDACI prodrugs in which n-dodecanoic acid or cholesterol is linked with a potent HDACI, K-182, by an ester bond or a disulfide carbonate linker. The prodrugs were able to admix as a component of NPs, although the intact K-182 was not incorporated into NPs. Namely, NPs composed of cholesteryl-3β-carboxyamidoethylene-N-hydroxyethylamine and Tween 80 with the 10 mol% K-182 prodrug were prepared as a DNA vector to transfect plasmid DNAs into human prostate cancer cells, PC-3, or human breast cancer cells, Sk-Br-3. The NPs containing K-182 prodrugs with n-dodecanoic acid exhibited two to four times higher the gene expression than the original NPs. The enhancement of the gene expression will be due to the hyperacetylation of core histones caused by intact K-182 degraded from the prodrug in the vector incorporated into the cells.     

Butyrate and trichostatin A attenuate nuclear factor κB activation and tumor necrosis factor α secretion and increase prostaglandin E2 secretion in human peripheral blood mononuclear cells

The effects of short-chain fatty acids (butyrate, propionate, and acetate) and trichostatin A (TSA), a typical histone deacetylase inhibitor, on tumor necrosis factor (TNF)-α secretion and nuclear factor κB (NF-κB) activation in peripheral blood mononuclear cells induced with lipopolysaccharide were evaluated in relation to prostaglandin E₂ (PGE₂) secretion. Treatment of cells with butyrate; tributyrin, a prodrug of butyrate; propionate; acetate; and TSA down-regulated TNF-α secretion but all up-regulated PGE₂ secretion. Butyrate, propionate, and TSA inhibited NF-κB activation. The effects of the cyclooxygenase-nonspecific inhibitor, indomethacin; the cyclooxygenase-2 selective inhibitor, N-[2-(cyclohexyloxy)-4-nitro-phenyl] methanesulfonamide; and the general lipoxygenase inhibitor, nordihydroguaiaretic acid, varied in cells treated with each short-chain fatty acids. N-[2-(cyclohexyloxy)-4-nitro-phenyl] methanesulfonamide inhibited the effect of propionate on TNF-α secretion, and nordihydroguaiaretic acid inhibited that of acetate. The results showed that butyrate, propionate, and TSA inhibited TNF-α production via PGE₂ secretion and down-regulated NF-κB activation by lipopolysaccharide. These data suggest that the mechanism of butyrate and propionate action is through histone deacetylation and acetate through lipoxygenase activation in the regulation of proinflammatory responses in cells.     

Lunasin: a cancer-preventive soy peptide

Lunasin is a novel, cancer-preventive peptide whose efficacy against chemical carcinogens and oncogenes has been demonstrated in mammalian cells and in a skin cancer mouse model. Isolated and characterized in soy, lunasin peptide is also documented in barley. Lunasin is found in all of the genotypes analyzed from the US soy germ plasm collection and in commercially available soy proteins. Pilot studies show that lunasin is bioavailable in mice and rats when orally ingested, opening the way for dietary administration in cancer prevention studies. Lunasin internalizes into mammalian cells within minutes of exogenous application, and localizes in the nucleus after 18 hours. It inhibits acetylation of core histones in mammalian cells. In spite of its cancer-preventive properties, lunasin does not affect the growth rate of normal and established cancer cell lines. An epigenetic mechanism of action is proposed whereby lunasin selectively kills cells being transformed or newly transformed by binding to deacetylated core histones exposed by the transformation event, disrupting the dynamics of histone acetylation-deacetylation and leading to cell death.     

蛋白质乙酰化与精子DNA稳定性和精子活力

表观遗传学参与调节精子发生和精子形成,其中组蛋白及非组蛋白乙酰化对生精细胞染色质结构重建以及精子活力起关键作用。环磷酸腺苷（cAMP）反应元件连接蛋白（CREB）的结合蛋白（CREB-binding protein,CBP）和p300是两个乙酰化酶,能使核心组蛋白发生乙酰化,其乙酰化作用能被乙酰化酶抑制剂（INHAT）所抑制。睾丸特异的布罗莫结构域（BRDT）蛋白为保守的核蛋白,能够识别乙酰化和非乙酰化的组蛋白。在精子发生早期,BRDT调节基因转录;在精子形成阶段,BRDT识别高度乙酰化组蛋白,接着组蛋白被鱼精蛋白替代,从而使精子形成致密的染色质。非组蛋白α微管蛋白（α-Tubulin）去乙酰化则降低精子活力,在少弱精患者精子中乙酰化α微管蛋白（acetylated α-tubulin,Ac-α-Tu）明显下降。     

Dietary compounds that induce cancer preventive phase 2 enzymes activate apoptosis at comparable doses in HT29 colon carcinoma cells.

Dietary agents that induce glutathione S-transferases and related detoxification systems (Phase 2 enzyme inducers) are thought to prevent cancer by enhancing elimination of chemical carcinogens. The present study shows that compounds of this group (benzyl isothiocyanate, allyl sulfide, dimethyl fumarate, butylated hydroxyanisole) activated apoptosis in human colon carcinoma (HT29) cells in culture over the same concentration ranges that elicited increases in enzyme activity (5-25, 25-100, 10-100, 15-60 micromol/L, respectively). Pretreatment of cells with sodium butyrate, an agent that induces HT29 cell differentiation, resulted in parallel increases in Phase 2 enzyme activities and induction of apoptosis in response to the inducers. Cell death characteristics included apoptotic morphological changes, appearance of cells at sub-G1 phase on flow cytometry, caspase activation, DNA fragmentation and TUNEL-positive staining. The results suggest that dietary Phase 2 inducers may protect against cancer by a mechanism distinct from and in addition to that associated with enhanced elimination of carcinogens. If this occurs in vivo, diets high in such compounds could eliminate precancerous cells by apoptosis at time points well after initial exposure to chemical mutagens and carcinogens.     

Dietary factors and epigenetic regulation for prostate cancer prevention

The role of epigenetic alterations in various human chronic diseases has gained increasing attention and has resulted in a paradigm shift in our understanding of disease susceptibility. In the field of cancer research, e.g., genetic abnormalities/mutations historically were viewed as primary underlying causes; however, epigenetic mechanisms that alter gene expression without affecting DNA sequence are now recognized as being of equal or greater importance for oncogenesis. Methylation of DNA, modification of histones, and interfering microRNA (miRNA) collectively represent a cadre of epigenetic elements dysregulated in cancer. Targeting the epigenome with compounds that modulate DNA methylation, histone marks, and miRNA profiles represents an evolving strategy for cancer chemoprevention, and these approaches are starting to show promise in human clinical trials. Essential micronutrients such as folate, vitamin B-12, selenium, and zinc as well as the dietary phytochemicals sulforaphane, tea polyphenols, curcumin, and allyl sulfur compounds are among a growing list of agents that affect epigenetic events as novel mechanisms of chemoprevention. To illustrate these concepts, the current review highlights the interactions among nutrients, epigenetics, and prostate cancer susceptibility. In particular, we focus on epigenetic dysregulation and the impact of specific nutrients and food components on DNA methylation and histone modifications that can alter gene expression and influence prostate cancer progression.