Bmi1は胃腺峡部に存在する幹細胞をマークする

京都大学0048新制・課程博士博士(医学)甲第21994号医博第4508号新制||医||1037(附属図書館)京都大学大学院医学研究科医学専攻(主査)教授 羽賀 博典, 教授 武藤 学, 教授 山下 潤学位規則第4条第1項該当Doctor of Medical ScienceKyoto UniversityDFA

Epithelial EP4 plays an essential role in maintaining homeostasis in colon

Colonic epithelial cells comprise the mucosal barrier, and their dysfunction promotes microbial invasion from the gut lumen and induces the development of intestinal inflammation. The EP4 receptor is known to mediate the protective effect of prostaglandin (PG) E2 in the gastrointestinal tract; however, the exact role of epithelial EP4 in intestinal pathophysiology remains unknown. In the present study, we aimed to investigate the role of epithelial EP4 in maintaining colonic homeostasis by characterizing the intestinal epithelial cell-specific EP4 knockout (EP4 cKO) mice. Mice harboring the epithelial EP4 deletion showed significantly lower colonic crypt depth and lower numbers of secretory cell lineages, as well as impaired epithelial cells in the colon. Interestingly, EP4-deficient colon epithelia showed a higher number of apoptotic cells. Consistent with the defect in mucosal barrier function of colonic epithelia and secretory cell lineages, EP4 cKO colon stroma showed enhanced immune cell infiltration, which was accompanied by increased production of inflammatory cytokines. Furthermore, EP4-deficient colons were susceptible to dextran sulfate sodium (DSS)-induced colitis. Our study is the first to demonstrate that epithelial EP4 loss resulted in potential "inflammatory" status under physiological conditions. These findings provided insights into the crucial role of epithelial PGE2/EP4 axis in maintaining intestinal homeostasis

Sulfasalazine, an inhibitor of the cystine-glutamate antiporter, reduces DNA damage repair and enhances radiosensitivity in murine B16F10 melanoma

<div><p>The sodium-independent cystine-glutamate antiporter plays an important role in extracellular cystine uptake. It comprises the transmembrane protein, xCT and its chaperone, CD98. Because glutathione is only weakly cell membrane permeable, cellular uptake of its precursor, cystine, is known to be a key step in glutathione synthesis. Moreover, it has been reported that xCT expression affects the progression of tumors and their resistance to therapy. Sulfasalazine is an inhibitor of xCT that is known to increase cellular oxidative stress, giving it anti-tumor potential. Here, we describe a radio-sensitizing effect of sulfasalazine using a B16F10 melanoma model. Sulfasalazine decreased glutathione concentrations and resistance to H₂O₂ in B16F10 melanoma cells, but not in mouse embryonic fibroblasts. It synergistically enhanced the cyto-killing effect of X-irradiation in B16F10 cells. It inhibited cellular DNA damage repair and prolonged cell cycle arrest after X-irradiation. Furthermore, in an <i>in vivo</i> transplanted melanoma model, sulfasalazine decreased intratumoral glutathione content, leading to enhanced susceptibility to radiation therapy. These results suggest the possibility of using SAS to augment the treatment of radio-resistant cancers.</p></div

Inhibitory role of Gas6 in intestinal tumorigenesis.

Growth arrest-specific gene (Gas) 6 is a γ-carboxyglutamic acid domain-containing protein, which shares 43% amino acid identity with protein S. Gas6 has been shown to enhance cancer cell proliferation in vitro. On the other hand, recent studies have demonstrated that Gas6 inhibits toll-like receptor-mediated immune reactions. Immune reactions are known to affect intestinal tumorigenesis. In this study, we investigated how Gas6 contributes to tumorigenesis in the intestine. Administration of recombinant Gas6 weakly, but significantly, enhanced proliferation of intestinal cancer cells (SW480 and HT29), whereas it suppressed the inflammatory responses of Lipopolysaccharide (LPS)-stimulated monocytes (THP-1). Compared with Gas6(+/+) mice, Gas6(-/-) mice exhibited enhanced azoxymethane/dextran sulfate sodium (DSS)-induced tumorigenesis and had a shorter survival. Gas6(-/-) mice also exhibited more severe DSS-induced colitis. DSS-treated Gas6(-/-) mice showed attenuated Socs1/3 messenger RNA expression and enhanced nuclear factor-kappaB activation in the colonic stroma, suggesting that the target of Gas6 is stromal cells. Bone marrow transplantation experiments indicated that both epithelial cells and bone marrow-derived cells are Gas6 sources. Furthermore, the number of intestinal tumors in Apc(Min) Gas6(-/-) mice was higher than that in Apc(Min) Gas6(+/+) mice, resulting in shorter survival. In a group of 62 patients with advanced colorectal cancer, Gas6 immunoreactivity in cancer tissues was positively correlated with prognosis. Thus, we revealed a unique in vivo inhibitory role of Gas6 during the progression of intestinal tumors associated with suppression of stromal immune reactions. These results suggest a novel therapeutic approach for colorectal cancer patients by regulation of stromal immune responses

SAS sensitizes transplanted B16F10 tumors to irradiation.

<p>The radio-sensitizing effect of SAS was evaluated on tumors formed by B16F10 cells inoculated into the hind legs of C57BL/6N mice. (A) Images of xCT immunostaining in a representative tumor. The transplanted B16F10 tumor was excised and immunostained for xCT. White T, tumor region; scale bar = 200 μm. (B) Intratumoral GSH concentration with and without SAS treatment. After 12 days, transplanted B16F10 tumors were treated with SAS (24 h, 250 mg/kg intraperitoneally). Error bars = SD, *p<0.05. (C) Representative images of γH2AX staining at 3 h after 1 Gy X-irradiation with or without pretreatment with SAS. IR, irradiation, scale bar = 200 μm. (D) Experimental radiotherapy to validate the effect of SAS. B16F10 tumor-bearing mice were treated with SAS for 3 days at 24 h intervals, and then X-irradiated at a dose of 4 Gy (n = 4). Tumor volumes were measured as described in the Materials and Methods section. IR, irradiation, error bars = SD, *p<0.05.</p

Knockdown of xCT decreases cellular resistance to H₂O₂.

<p>The cellular viability, GSH levels, ROS levels, and sensitivity to H₂O₂ were evaluated in B16F10 cells after siRNA treatment. (A) xCT expression after siSLC7A11 treatment. (B) Cellular ROS measurement after siRNA (72 h). ROS levels were analyzed by DCFDA staining. (C) Quantitative analysis of intracellular GSH concentration after siRNA. (D) The effect of SAS on H₂O₂ cytotoxicity. Cells were treated with siRNA (72 h) followed by treatment with H₂O₂ (24 h). Error bars = SD, *p<0.05.</p

Murine B16F10 melanoma cells highly express xCT.

<p>B16F10 cells and MEF were subjected to RT-qPCR and western blot analyses of xCT expression. (A) RT-qPCR analysis for <i>xCT</i> mRNA expression in both cell types. (B) Image of a representative western blot for xCT in both cell types. (C) Western blot images were analyzed and the relative xCT expression was calculated for both cell types. Error bars = SD, *p<0.05.</p