Allyl sulfides are characteristic flavor components obtained from garlic. These sulfides are thought to be responsible for their epidemiologically proven anticancer effect on garlic eaters. This study was aimed at clarifying the molecular basis of this anticancer effect of garlic by using human colon cancer cell lines HCT-15 and DLD-1. The growth of the cells was significantly suppressed by diallyl trisulfide (DATS, HCT-15 IC 50 ‫؍‬ 11.5 M, DLD-1 IC 50 ‫؍‬ 13.3 M); however, neither diallyl monosulfide nor diallyl disulfide showed such an effect. The proportion of HCT-15 and that of DLD-1 cells residing at the G 1 and S phases were decreased by DATS, and their populations at the G 2 /M phase were markedly increased for up to 12 h. The cells with a sub-G 1 DNA content were increased thereafter. Caspase-3 activity was also dramatically increased by DATS. Fluorescence-activated cell sorter analysis performed on the cells arrested at the G 1 /S boundary revealed cell cycle-dependent induction of apoptosis through the transition of the G 2 /M phase to the G 1 phase by DATS. DATS inhibited tubulin polymerization in an in vitro cell-free system. DATS disrupted microtubule network formation of the cells, and microtubule fragments could be seen at the interphase. Peptide mass mapping by liquid chromatography-tandem mass spectrometry analysis for DATS-treated tubulin demonstrated that there was a specific oxidative modification of cysteine residues Cys-12␤ and Cys-354␤ to form S-allylmercaptocysteine with a peptide mass increase of 72.1 Da. The potent antitumor activity of DATS was also demonstrated in nude mice bearing HCT-15 xenografts. This is the first paper describing intracellular target molecules directly modified by garlic components.Allyl sulfides, e.g. diallyl monosulfide (DAS), 4 diallyl disulfide (DADS), and diallyl trisulfide (DATS), are characteristic flavor components of the essential oil prepared from garlic (Allium sativum L.). Garlic is widely served around the world, and it has been reported that allyl sulfides inhibit both the initiation and promotion stages of tumorigenesis in experimental carcinogenesis models for various types of cancer (1-5). Recently, several lines of investigation have shown that allyl sulfides suppress cell growth and induce apoptosis in multiple cancer cell lines (6 -12). We previously reported that the sulfur-containing volatile oils prepared from garlic and onion inhibit proliferation and induce differentiation of the human promyelocytic leukemia cell line HL-60 (13). However, the molecular mechanisms underlying the antitumorigenesis of allyl sulfides are still not fully understood.Microtubules are ubiquitous proteins present in eukaryotes as components of the cytoskeleton and play pivotal roles in a variety of cellular processes involving cell division, motility, and intracellular trafficking (14). The microtubules are dynamic polymers composed of ␣␤-tubulin heterodimers, and they form the mitotic spindles, which are known to introduce the replicated DNA molecules to the res...

Apolipoprotein E (apoE) is a major apolipoprotein in the brain. The ⑀4 allele of apoE is a major risk factor for Alzheimer disease, and apoE deficiency in mice leads to blood-brain barrier (BBB) leakage. However, the effect of apoE isoforms on BBB properties are as yet unknown. Here, using an in vitro BBB model consisting of brain endothelial cells and pericytes prepared from wild-type (WT) mice, and primary astrocytes prepared from human apoE3-and apoE4-knock-in mice, we show that the barrier function of tight junctions (TJs) was impaired when the BBB was reconstituted with primary astrocytes from apoE4-knock-in mice (apoE4-BBB model). The phosphorylation of occludin at Thr residues and the activation of protein kinase C (PKC) in mBECs were attenuated in the apoE4-BBB model compared with those in the apoE3-BBB model. The differential effects of apoE isoforms on the activation of PKC, the phosphorylation of occludin at Thr residues, and TJ integrity were abolished following the treatment with an anti-low density lipoprotein receptor-related protein 1 (LRP1) antibody or a LRP1 antagonist receptor-associated protein. Consistent with the results of in vitro studies, BBB permeability was higher in apoE4-knock-in mice than in apoE3-knock-in mice. Our studies provide evidence that TJ integrity in BBB is regulated by apoE in an isoform-dependent manner. Apolipoprotein E (apoE)2 is a polymorphic glycoprotein with a molecular mass of 34 kDa. Its three isoforms, apoE2, apoE3, and apoE4, are all products of the same gene, which exists as three alleles (⑀2, ⑀3, and ⑀4) at a single locus (1). Among these three isoforms, apoE4 is a major risk factor for Alzheimer disease (AD) (2, 3). ApoE is expressed in several organs, with the liver showing the highest expression level, followed by the brain. In the brain, apoE is a major apolipoprotein and plays a major role in the transportation of lipids as a lipid acceptor (1). ApoE-containing lipoprotein particles are mainly produced by astrocytes and deliver cholesterol and other essential lipids to neurons through low density lipoprotein (LDL) receptor family members (4 -6). A number of studies revealed that astrocytes are involved in the control of endothelium blood-brain barrier (BBB) properties (7,8) and that apoE deficiency leads to BBB leakage (9 -11).BBB is formed by brain endothelial cells and is essential for the protection of the central nervous system from harmful blood molecules and cells (12). Brain endothelial cells form tight junctions (TJs), which are the fundamental characteristics of BBB (13,14). The assembly of TJs requires at least three types of transmembrane protein, namely, occludin, claudin, and junctional adhesion molecule (15). Protein kinases are localized at TJs or interact directly with TJ proteins (16 -18). Among protein kinases, PKC has been shown to regulate the phosphorylation of occludin at its Thr residues and play a crucial role in the assembly and/or maintenance of TJs (19). Cells surrounding brain capillaries, such as astrocytes and pericytes, con...

Adipose tissue fibrosis is a hallmark of malfunction that is linked to insulin resistance and type 2 diabetes; however, what regulates this process remains unclear. Here we show that the PRDM16 transcriptional complex, a dominant activator of brown/beige adipocyte development, potently represses adipose tissue fibrosis in an uncoupling protein 1 (UCP1)-independent manner. By purifying the PRDM16 complex, we identified GTF2IRD1, a member of the TFII-I family of DNA-binding proteins, as a cold-inducible transcription factor that mediates the repressive action of the PRDM16 complex on fibrosis. Adipocyte-selective expression of GTF2IRD1 represses adipose tissue fibrosis and improves systemic glucose homeostasis independent of body-weight loss, while deleting GTF2IRD1 promotes fibrosis in a cell-autonomous manner. GTF2IRD1 represses the transcription of transforming growth factor β-dependent pro-fibrosis genes by recruiting PRDM16 and EHMT1 onto their promoter/enhancer regions. These results suggest a mechanism by which repression of obesity-associated adipose tissue fibrosis through the PRDM16 complex leads to an improvement in systemic glucose homeostasis.

SUMMARY Catecholamines promote lipolysis both in brown and white adipocytes, whereas the same stimuli preferentially activate thermogenesis in brown adipocytes. Molecular mechanisms for the adipose-selective activation of thermogenesis remain poorly understood. Here, we employed quantitative phosphoproteomics to map global and temporal phosphorylation profiles in brown, beige, and white adipocytes under β3-adrenenoceptor activation and identified kinases responsible for the adipose-selective phosphorylation profiles. We found that casein kinase2 (CK2) activity is preferentially higher in white adipocytes than brown/beige adipocytes. Genetic or pharmacological blockade of CK2 in white adipocytes activates the thermogenic program in response to cAMP stimuli. Such activation is largely through reduced CK2-mediated phosphorylation of class I HDACs. Notably, inhibition of CK2 promotes beige adipocyte biogenesis and leads to an increase in whole-body energy expenditure and ameliorates diet-induced obesity and insulin resistance. These results indicate that CK2 is a plausible target to rewire the β3-adreneno-ceptor signaling cascade that promotes thermogenesis in adipocytes.

Background: Apolipoprotein E allele ε4 (apoE4) is a strong risk factor for developing Alzheimer's disease (AD). Secreted apoE has a critical function in redistributing lipids among central nervous system cells to maintain normal lipid homeostasis. In addition, previous reports have shown that apoE4 is cleaved by a protease in neurons to generate apoE4(1-272) fragment, which is associated with neurofibrillary tanglelike structures and mitochondria, causing mitochondrial dysfunction. However, it still remains unclear how the apoE fragment associates with mitochondria and induces mitochondrial dysfunction.