Purpose: We have previously shown the reactivation of some methylation-silenced genes in cancer cells by (À)-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 h (RARb), p16INK4a , and O 6 -methylguanine methyltransferase (MGMT) in KYSE 510 esophageal squamous cell carcinoma cells treated with genistein alone or in combination with trichostatin, sulforaphane, or 2V -deoxy-5-aza-cytidine (5-aza-dCyd). Results: Genistein (2-20 Amol/L) reversed DNA hypermethylation and reactivated RARb, p16INK4a , and MGMT in KYSE 510 cells. Genistein also inhibited cell growth at these concentrations. Reversal of DNA hypermethylation and reactivation of RARb by genistein were also observed in KYSE 150 cells and prostate cancer LNCaP and PC3 cells. Genistein (20-50 Amol/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 RARb, 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.

The nervous systems affect immune functions by releasing neurohormones and neurotransmitters. A neurotransmitter dopamine signals via five different seven-transmembrane G protein-coupled receptors termed D1 to D5. The secondary lymphoid tissues are highly innervated by sympathetic nerve fibers that store dopamine at high contents. Lymphocytes also produce dopamine. In this study, we examined expression and function of dopamine receptors in lymphocytes. We found that D3 was the predominant subtype of dopamine receptors in the secondary lymphoid tissues and selectively expressed by naive CD8+ T cells of both humans and mice. Dopamine induced calcium flux and chemotaxis in mouse L1.2 cells stably expressing human D3. These responses were almost completely inhibited by pertussis toxin, indicating that D3 was coupled with the Gαi class of G proteins. Consistently, dopamine selectively induced chemotactic responses in naive CD8+ T cells of both humans and mice in a manner sensitive to pertussis toxin and D3 antagonists. Dopamine was highly synergistic with CCL19, CCL21, and CXCL12 in induction of chemotaxis in naive CD8+ T cells. Dopamine selectively induced adhesion of naive CD8+ T cells to fibronectin and ICAM-1 through activation of integrins. Intraperitoneal injection of mice with dopamine selectively attracted naive CD8+ T cells into the peritoneal cavity. Treatment of mice with a D3 antagonist U-99194A selectively reduced homing of naive CD8+ T cells into lymph nodes. Collectively, naive CD8+ T cells selectively express D3 in both humans and mice, and dopamine plays a significant role in migration and homing of naive CD8+ T cells via D3.

No abstract

The neoplastic environment is generally regarded as an immunosuppressive milieu. However, a group of cancers are characterized by the abundance of tumour-infiltrating lymphocytes (TILs). Here we examined the possible roles of chemokines in the formation of lymphoid stroma in lymphocyte-rich gastric carcinomas (GCs), including EBV(+) cases and conventional GCs. Regardless of EBV positivity, TILs in lymphocyte-rich GCs predominantly expressed CXCR3, while its ligand CXCL9 was abundantly expressed by stromal cells and a portion of cancer cells. CXCL9(+) stromal cells were judged to include dendritic cells, because they partly co-expressed fascin, DC-sign, CD83, DC-lamp or HLA-DR. T cells in close contact with CXCL9(+) cells showed frequent labelling of Ki-67 (approximately 10%), suggesting the immunostimulatory activity of CXCL9(+) stromal cells. The T-cell zone of the regional lymph nodes of lymphocyte-rich GCs also abounded with CXCR3(+) T cells and CXCL9(+) stromal cells. This indicated a close similarity between cancer stroma and regional lymph nodes of lymphocyte-rich GCs. Quantitative RT-PCR also confirmed the strong expression of CXCR3, CXCL9 and IFNgamma in lymphocyte-rich GCs. In contrast, conventional GCs contained less abundant CXCR3(+) T cells and few CXCL9(+) stromal cells. Collectively, the CXCL9-CXCR3 axis plays a pivotal role in the formation of lymphoid stroma in lymphocyte-rich GCs. Given similar findings in the regional lymph nodes, the lymphoid stroma of lymphocyte-rich GCs may represent a tertiary lymphoid tissue with predominantly Th1-shifted immune responses.

Purpose: Aberrant arachidonic acid (AA) metabolism, especially through the cyclooxygenase (Cox) and 5-lipoxygenase (5-Lox) pathways, has been suggested to play an important role in the development of esophageal adenocarcinoma (EAC). The purpose of this study was to investigate the expression of 5-Lox in EAC of a rat model and in human samples as well as the chemopreventive effects of zileuton (a specific 5-Lox inhibitor) and celecoxib (a specific Cox2 inhibitor) in the rat EAC model.Experimental Design: 5-Lox expression in EAC of a rat esophagogastroduodenal anastomosis model and of humans was examined with immunohistochemistry. A chemoprevention study was designed to test whether zileuton and celecoxib could suppress aberrant AA metabolism and esophageal adenocarcinogenesis.Results: With immunohistochemistry, we found that 5-Lox was overexpressed during esophageal adenocarcinogenesis in our rat model and in humans. In the chemoprevention study, EAC incidence was reduced in a dose-dependent manner from 68.8% (11 of 16) to 44.4% (8 of 18; P > 0.05) and 31.3% (5 of 16; P < 0.05) by 500 and 1,000 ppm zileuton, respectively, and to 33.3% (7 of 21; P < 0.05) and 20% (3 of 15; P < 0.05) by 500 and 1,000 ppm celecoxib, respectively. With isobolographic analysis, zileuton and celecoxib, both at a dose of 500 ppm, had an additive effect by reducing the tumor incidence to 16.7% (3 of 18, P < 0.01). Leukotriene B 4 and prostaglandin E 2 levels in the esophageal tissues were also significantly reduced by zileuton and celecoxib.Conclusions: This study clearly demonstrated that 5-Lox and Cox2 play important roles in the development of EAC. Both zileuton and celecoxib had inhibitory effects on esophageal adenocarcinogenesis through inhibition on their respective enzymes of AA metabolism.

No abstract

Impaired phosphatase activity contributes to the persistent activation of STAT3 in tumors. Given that STAT family members with various or even opposite functions are often phosphorylated or dephosphorylated by the same enzymes, the mechanism for STAT3-specific dephosphorylation in cells remains largely unknown. Here, we report that GdX (UBL4A) promotes STAT3 dephosphorylation via mediating the interaction between TC45 (the nuclear isoform of TC-PTP) and STAT3 specifically. GdX stabilizes the TC45-STAT3 complex to bestow upon STAT3 an efficient dephosphorylation by TC45. Inasmuch, GdX suppresses tumorigenesis and tumor development by reducing the level of phospho-STAT3 (p-STAT3), whereas deletion of GdX results in a high level of p-STAT3 and accelerated colorectal tumorigenesis induced by AOM/DSS. Thus, GdX converts TC45, a nonspecific phosphatase, into a STAT3-specific phosphatase by bridging an association between TC45 and STAT3.

In the B cell lineage, CCR10 is known to be selectively expressed by plasma cells, especially those secreting IgA. In this study, we examined the regulation of CCR10 expression in terminally differentiating human B cells. As reported previously, IL-21 efficiently induced the differentiation of activated human CD19+ B cells into IgD−CD38+ plasma cells in vitro. A minor proportion of the resulting CD19+IgD−CD38+ cells expressed CCR10 at low levels. 1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3), the active metabolite of vitamine D3, dramatically increased the proportion of CD19+IgD−CD38+ cells expressing high levels of CCR10. The 1,25-(OH)2D3 also increased the number of CCR10+ cells expressing surface IgA, although the majority of CCR10+ cells remained negative for surface IgA. Thus, 1,25-(OH)2D3 alone may not be sufficient for the induction of IgA expression in terminally differentiating human B cells. To further determine whether 1,25-(OH)2D3 directly induces CCR10 expression in terminally differentiating B cells, we next performed the analysis on the human CCR10 promoter. We identified a proximal Ets-1 site and an upstream potential vitamin D response element to be critical for the inducible expression of CCR10 by 1,25-(OH)2D3. We confirmed the specific binding of Ets-1 and 1,25-(OH)2D3-activated vitamin D receptor to the respective sites. In conclusion, 1,25-(OH)2D3 efficiently induces CCR10 expression in terminally differentiating human B cells in vitro. Furthermore, the human CCR10 promoter is cooperatively activated by Ets-1 and vitamin D receptor in the presence of 1,25-(OH)2D3.