Matrix metalloproteinases in the restorative proctocolectomy pouch of pediatric ulcerative colitis

AIM:To investigate matrix metalloproteinases(MMPs) and their tissue inhibitors(TIMPs) in pouch mucosa of pediatric onset ulcerative colitis(UC).METHODS:In this cross-sectional study,28 patients with pediatric onset UC underwent ileal pouch biopsy 13 years(median) after proctocolectomy.Expression of MMPs-3,-7,-8,-9,-12 and-26 and TIMPs-1,-2 and-3 in samples was examined using immunohistochemichal methods,and another biopsy was used to evaluate the grade of histological inflammation.Two investigators independently graded the immunohistochemical specimens in a semiquantitative fashion,using a scale marking staining intensity as follows:0 = less than 20 positive cells;1 = 20-50 positive cells;2 = 50-200 positive cells;3 = over 20 positive cells.Fecal calprotectin and blood inflammatory markers [serum C-reactive protein(CRP) and erythrocyte sedimentation rate] were determined during a follow-up visit to examine correlations between these markers and the expression of MMPs and TIMPs.RESULTS:Of the 28 patients with pediatric onset UC,nine had not experienced pouchitis,whereas thirteen reported a single episode,and six had recurrent pouchitis(≥ 4 episodes).At the time of the study,six patients required metronidazole.In all of the others,the most recent episode of pouchitis had occurred over one month earlier,and none were on antibiotics.Only four samples depicted no sign of inflammation,and these were all from patients who had not had pouchitis.Two samples were too small to determine the grade of inflammation,but both had suffered pouchitis,the other recurrent.No sample depicted signs of colonic metaplasia.Most pouch samples showed expression of epithelial(e) and stromal(s) MMP-3(e,n = 22;s,n = 20),MMP-7(e,n = 28;s,n = 27),MMP-12(e,n = 20;s,n =24),TIMP-2(e,n = 23;s,n = 23) and MMP-3(e,n = 23;s,n = 28) but MMP-8(e,n = 0;s,n = 1),MMP-9(e,n = 0;s,n = 9) and MMP-26(e,n = 0;s,n = 3) and TIMP-1(n = 0,both) were lacking.In samples with low grade of inflammatory activity,the epithelial MMP-3 and MMP-7 expression was increased(r =-0.614 and r =-0.472,respectively,P < 0.05 in both).MMPs and TIMPs did not correlate with the markers of inflammation,fecal calprotectin,erythrocyte sedimentation rate,or CRP,with the exception of patients with low fecal calprotectin(< 100 μg/g) in whom a higher expression of epithelial MMP-7 was found no differences in MMPor TIMP-profiles were seen in patients with a history of pouchitis compared to ones with no such episodes.Anastomosis with either straight ileoanal anastomosis or ileoanal anastomosis with J-pouch did depict differences in MMP-or TIMP-expression.CONCLUSION:The expression of MMPs pediatric UC pouch in the long-term shares characteristics with inflammatory bowel disease,but inflammation cannot be classified as a reactivation of the disease.

Arsenic trioxide up-regulates Fas expression in human osteosarcoma cells

Background Osteosarcoma is a common primary malignant tumor of bone with a poor prognosis due to its propensity for metastasis. The prognosis of patients is highly dependent on the presence or absence of lung metastasis and on the effectiveness of treatment against it. It has been reported that low level expression of Fas protein in human osteosarcoma cell is closely associated with lung metastasis. A large number of studies have shown that arsenic trioxide （ATO） can inhibit proliferation and induce apoptosis of many cancer cell lines; however, its effects on human osteosarcoma cells （Saos-2 cell line） remains unknown. The aim of this study was to investigate the effects of ATO on Saos-2 cells and to characterize its mechanism of Fas-expressing. Methods A group of Saos-2 cells was treated with or without 0.5, 1, 2, 4 and 8 pmol/L ATO for three successive days, and the cytotoxicity of ATO was determined by an 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide （MTT） assay. Morphological changes in cells were studied by acridine orange/ethidium bromide （AO/EB） double staining. Flow cytometry （FCM） was used to assay cell DNA distribution. Another group of cells was pretreated with 10 nmol/L matrix metalloproteinase 7 （MMP-7） for 3 hours. They were then incubated with or without 2 pmol/L ATO for 24, 48 and 72 hours. Cytotoxicity, Fas protein and mRNA levels were systematically studied using MTT, Western blotting and real-time PCR, respectively. Cell proliferation, cell cycle progression and apoptosis were examined in this study. Results Proliferation of Saos-2 cells was inhibited by ATO in both a dose- and time-dependent manner. The IC50 values at 24, 48 and 72 hours were 9.30, 5.54 and 3.49 pmol/L, respectively. The survival rate of Saos-2 cells in the MMP-7 and ATO co-treated group was significantly higher than the ATO group, but it was lower than the control group. ATO induced G1 phase arrest of the cell cycle and very efficiently stimulated apoptosis in Saos-2 cells, as evidenced by flow cytometric detection of sub-G1 DNA content and AO/EB staining. Western blotting results indicated that Fas （FasL） protein expression in osteosarcoma cultures markedly increases in a time dependent manner after exposure to ATO. Compared with control, treatment with ATO 2 IJmol/L and 4 pmol/L for 48 hours, resulted in increase of Fas gene expression to 28.31% and 56.74%, respectively. Our results indicated that ATO induced-apoptosis of Saos-2 cells may be mediated through the Fas pathway. Conclusions ATO suppressed cell proliferation of Saos-2 cell in a dose- and time-dependent manner and increased Fas protein expression. However, Fas-mediated apoptosis was incompletely interrupted by MMP-7, which suggested that other molecular mechanisms may mediate this process.