Lucia Picariello

Transforming growth factor beta signalling and matrix metalloproteinases in the mucosa overlying Crohn's disease strictures.

Background and Aims: In addition to its crucial role in dampening tissue-damaging immune responses in the gut, transforming growth factor β (TGFβ) is a potent profibrogenic agent inducing collagen synthesis and regulating the balance between matrix-degrading matrix metalloproteinases (MMPs) and their inhibitors (TIMPs). TGFβ signalling was investigated by analysis of Smad proteins and MMPs/TIMPs in the mucosa overlying strictures in patients with Crohn’s disease (CD). Methods: Specimens were collected from macroscopically normal mucosa overlying strictured and non-strictured gut of patients with fibrostenosing CD. Isolated myofibroblasts were cultured with anti-TGFβ blocking antibody or TGFβ1. TGFβ transcripts were analysed by quantitative reverse transcription-PCR (RT-PCR). Smad proteins and MMPs were determined by immunoblotting. MMP-12 activity was measured by a real-time MMP-12 activity assay. An in vitro wound-healing scratch assay was used to assess myofibroblast migration. Results: TGFβ transcripts, phosphorylated Smad2–Smad3 (pSmad2–3) and TIMP-1 proteins were higher in mucosa overlying strictures than in mucosa overlying non-strictured areas. In contrast, mucosa overlying strictured gut had lower expression of Smad7, MMP-12 and MMP-3. Myofibroblasts from mucosa overlying strictured gut showed higher TGFβ transcripts, a greater pSmad2–3 response to TGFβ, increased TIMP-1, lower Smad7, increased collagen production and reduced migration ability compared with myofibroblasts from mucosa overlying non-strictured gut. TGFβ blockade increased myofibroblast MMP-12 production and migration, more obviously in myofibroblasts isolated from mucosa overlying non-strictured compared with strictured gut. Conclusions: Changes in TGF-β signalling and MMP production were identified in the mucosa overlying strictures in CD which may give a window into the process of fibrosis.

Azidothymidine Induces Apoptosis and Inhibits Cell Growth and Telomerase Activity of Human Parathyroid Cancer Cells in Culture

Telomerase activity has been correlated to parathyroid carcinoma. Because its role in acquisition of a malignant phenotype by parathyroid cells is unclear, we treated telomerase‐positive cultured human parathyroid cancer cells with the telomerase inhibitor AZT, evaluating cell telomerase activity, cytotoxic effects, growth, and morphological changes. In vitro exposure of these cells to AZT correlated with inhibition of cell proliferation.

Estrogen synthesis in human colon cancer epithelial cells.

Epidemiological and experimental data suggest an involvement of estrogen in the development and progression of colorectal cancer. In order to determine whether local synthesis of estrogen occurred in human colonic cancer cells, two colorectal cancer cell lines, HCT8 and HCT116, were evaluated for gene expression and enzyme activity of cytochrome P450 aromatase. In addition, the effect on aromatase expression of charcoal-stripped fetal calf serum, of quercetin and genistein and of tamoxifen and raloxifene was investigated in both cell lines. RT-PCR analysis revealed that colorectal adenocarcinoma cell lines contain aromatase as a major component. The conversion of [(3)H]-androstenedione to estrone and labeled water was dose-dependently inhibited by 4-hydroxyandrostenedione and obeyed Michaelis-Menten kinetic with apparent Km values of approximately 20 nM and V(max) values of approx. 200 and 500 fmol/mg protein/h for HCT8 and HCT116 cells, respectively. After 24 h incubation, genistein (1 microM) significantly increased aromatase activity in HCT8 cells, with no effect on HCT116 cells. In accord with previous observation in reproductive tissues, quercetin (1 microM) significantly inhibited the enzyme activity in both cell lines. Also tamoxifen (100 nM) acted as inhibitor, while raloxifene (10 nM) decreased the enzyme activity only in HCT116 cells. The aromatase gene expression modulation by these effective agents was consistent with their effects on enzyme activity. These findings demonstrate for the first time that colorectal adenocarcinoma cell lines express aromatase. Interestingly, the enzyme activity was inhibited by quercetin, one major dietary flavonoid, by tamoxifen, a hormonal therapeutic agent for breast cancer, and by raloxifene, used in the prevention of postmenopausal osteoporosis.

Selective oestrogen receptor modulators in desmoid tumours

Selective oestrogen receptor modulators (SERMs) are compounds with a mixed agonist/antagonist activity on oestrogen receptors. An ideal SERM is a compound with an oestrogen antagonist effect on the breast and uterus but oestrogen agonist effect on bone. Beside tamoxifen, a group of well-investigated SERMs is represented by raloxifene, LY-353381 (SERM3), EM-800 and CP-336156. On an empirical basis, tamoxifen has been used to pharmacologically treat desmoid tumours. Recently, raloxifene, a second-generation SERM, has been used in the treatment of familial adenomatous polyposis patients affected by desmoid tumour. The mechanisms through which these molecules affect desmoid tumour growth appear to be due, in part, to the fact that SERMs may act independently of oestrogen receptors. The knowledge of the molecular basis of SERM action will make the development of novel synthetic compounds with engineered tissue selectivity possible.

Oxidative state and IL‐6 production in intestinal myofibroblasts of Crohn's disease patients

Background: Intestinal subepithelial myofibroblasts (ISEMFs) produce inflammatory cytokines in response to certain stimuli. In the intestine of patients with Crohns disease (CD), cytokine synthesis is modified and an increased number of myofibroblasts has been observed. The intracellular redox state influences cytokine production and oxidative stress is present in the intestinal mucosa of CD patients. Methods: This study was performed in ISEMFs isolated from the colon of patients with active CD and in a myofibroblast cell line derived from human colonic mucosa: 18Co cells. Cellular glutathione (GSH) levels were modulated by treatment with buthionine sulfoximine, an inhibitor of GSH synthesis, or N‐acetylcysteine, a GSH precursor. GSH and oxidized glutathione (GSSG) levels were measured by high‐performance liquid chromatography (HPLC) methods. Interleukin (IL)‐6 production was detected by enzyme‐linked immunosorbent assay (ELISA). Results: ISEMFs of CD patients exhibited an increased oxidative state due to a decrease in the GSH/GSSG ratio, which is related to an increase in basal IL‐6 production or is stimulated by tumor necrosis factor alpha (TNF&agr;) or bacterial products. This relationship was also confirmed in 18Co cells. Phosphorylation and activation of ERK1/2 and p38 MAPK, which are signaling factors involved in the IL‐6 synthesis, were also increased when there is oxidative stress in ISEMFs. Conclusions: This study shows for the first time in ISEMFs of CD patients an increased production of IL‐6 synthesis related to the decrease in the GSH/GSSH ratio, suggesting redox regulation with the involvement of specific kinase activation. The present data shed light on the pathogenesis of inflammatory chronic processes and relapses that occur in this pathology. (Inflamm Bowel Dis 2010;)

Apoptosis induced by sulindac sulfide in epithelial and mesenchymal cells from human abdominal neoplasms

We investigated whether the therapeutic action of sulindac, used for the treatment of familial adenomatous polyposis, desmoid tumors, and against colon cancer, could be mediated by its active metabolite, sulindac sulfide, in cell growth and apoptosis on cell lines derived from abdominal neoplasms. Sulindac sulfide actions on cell growth and apoptosis were evaluated in epithelial human colon tumor 8 (HCT8) cell line and mesenchymal cell lines (bovine bone endothelial (BBE) cell line, desmoid tumor-derived cells, human colorectal cancer-derived fibroblasts). Sulindac sulfide (0.1-60 microg/ml) induced a dose-dependent inhibition of cell proliferation of all cell lines tested. Apoptosis was induced at doses of 20 and 40 microg/ml, respectively, in BBE and HCT8 cells with no effect on desmoid tumor cells and colorectal cancer-derived fibroblasts. Since mesenchymal cells respond to clinically effective concentrations of the compound, its preferential action on the stromal compartment of intestinal polyps, desmoid tumors and colon cancer can be proposed, with consequent regression of the tumor.

Role of N-acetylcysteine and GSH redox system on total and active MMP-2 in intestinal myofibroblasts of Crohn's disease patients

PurposeIntestinal subepithelial myofibroblasts (ISEMFs)1 are the predominant source of matrix metalloproteinase-2 (MMP-2) in gut, and a decrease in glutathione/oxidized glutathione (GSH/GSSG) ratio, intracellular redox state index, occurs in the ISEMFs of patients with Crohn’s disease (CD). The aim of this study is to demonstrate a relationship between MMP-2 secretion and activation and changes of GSH/GSSG ratio in ISEMFs stimulated or not with tumor necrosis factor alpha (TNFα).MethodsISEMFs were isolated from ill and healthy colon mucosa of patients with active CD. Buthionine sulfoximine, GSH synthesis inhibitor, and N-acetylcysteine (NAC), precursor of GSH synthesis, were used to modulate GSH/GSSG ratio. GSH and GSSG were measured by HPLC and MMP-2 by ELISA Kit.ResultsIn cells, stimulated or not with TNFα, a significant increase in MMP-2 secretion and activation, related to increased oxidative stress, due to low GSH/GSSG ratio, was detected. NAC treatment, increasing this ratio, reduced MMP-2 secretion and exhibited a direct effect on the secreted MMP-2 activity. In NAC-treated and TNFα-stimulated ISEMFs of CD patients’ MMP-2 activity were restored to physiological value. The involvement of c-Jun N-terminal kinase pathway on redox regulation of MMP-2 secretion has been demonstrated.ConclusionFor the first time, in CD patient ISEMFs, a redox regulation of MMP-2 secretion and activation related to GSH/GSSG ratio and inflammatory state have been demonstrated. This study suggests that compounds able to maintain GSH/GSSG ratio to physiological values can be useful to restore normal MMP-2 levels reducing in CD patient intestine the dysfunction of epithelial barrier.

Redox regulation of MMP-3/TIMP-1 ratio in intestinal myofibroblasts: effect of N-acetylcysteine and curcumin.

Matrix metalloproteinases (MMPs) play a critical role in inflammation and ulcerations in gut of Crohn׳s disease (CD) patients. Intestinal subepithelial myofibroblasts (ISEMFs) secrete MMPs in response to inflammatory stimuli. Previous data showed in CD-ISEMFs increased oxidative status. The aim of this study was to investigate the role of ISEMFs in modulating the production of MMP-3 and TIMP-1, an inhibitor of MMPs activity. A relationship among oxidative stress, activity of antioxidants and MMP-3/TIMP-1 was also studied. ISEMFs isolated from CD patient colon and human colonic cell line of myofibroblasts (18Co) were used. Oxidative state was modulated by buthionine sulfoximine, an inhibitor of glutathione (GSH) synthesis, and N-acetylcysteine (NAC), GSH precursor. An up-regulation of MMP-3 due to increased oxidative state was found in CD-ISEMFs. Stimulation by tumor necrosis factor (TNF)α increased further MMP-3 levels. On the contrary, no change in TIMP-1 production was determined. NAC treatment decreased MMP-3 production in CD-ISMEFs and removed the enhancement due to TNFα. Similar effects were observed in 18Co cells treated with curcumin, antioxidant with anti-inflammatory properties. The involvement of MAPKs on MMP-3 redox regulation was also shown. This study demonstrates the involvement of ISEMFs and high oxidative state in the increased MMP-3 production found in intestinal mucosa of CD patients. NAC and curcumin normalize MMP-3 levels mainly in TNFα stimulated cells. A modulation of MMP-3 production by NAC and curcumin due to their direct action on transcriptional factors has been also suggested. Therefore, they could have a therapeutic use for the prevention and treatment of fistulaes in CD.

Transcriptional Subtyping and CD8 Immunohistochemistry Identifies Patients With Stage II and III Colorectal Cancer With Poor Prognosis Who Benefit From Adjuvant Chemotherapy

Purpose Transcriptomic profiling of colorectal cancer (CRC) has led to the identification of four consensus molecular subtypes (CMS1 to 4) that have prognostic value in stage II and III disease. More recently, the Colorectal Cancer Intrinsic Subtypes (CRIS) classification system has helped to define the biology specific to the epithelial component of colorectal tumors; however, the clinical value of these classification systems in the prediction of response to standard-of-care adjuvant chemotherapy remains unknown. Patients and Methods Using samples from four European sites, we assembled a novel cohort of patients with stage II and III CRC (n = 156 samples) and performed transcriptomic profiling and targeted sequencing and generated a tissue microarray to enable integrated multiomics analyses. We also accessed data from two published cohorts of patients with stage II and III CRC: GSE39582 and GSE14333 (n = 479 and n = 185 samples, respectively). Results The epithelial-rich CMS2 subtype of CRC benefitted significantly from treatment with adjuvant chemotherapy in both stage II and III disease (P = .02 and P < .001, respectively), whereas the CMS3 subtype significantly benefitted in stage III only (P = .001). After CRIS substratification of CMS2, we observed that only the CRIS-C subtype significantly benefitted from treatment with adjuvant chemotherapy in stage II and III disease (P = .0081 and P < .001, respectively), whereas the CRIS-D subtype significantly benefitted in stage III only (P = .0034). We also observed that CRIS-C patients with low levels of CD8+ tumor-infiltrating lymphocytes were most at risk for relapse in both stage II and III disease (log-rank P = .0031; hazard ratio, 12.18 [95% CI, 1.51 to 98.58]). Conclusion Patient stratification using a combination of transcriptional subtyping and CD8 immunohistochemistry analyses is capable of identifying patients with poor prognostic stage II and III disease who benefit from adjuvant standard-of-care chemotherapy. These findings are particularly relevant for patients with stage II disease, where the overall benefit of adjuvant chemotherapy is marginal.

Desmoid Tumours in Familial Adenomatous Polyposis

The term desmoid was first used by Muller [1] to describe the tendon-like aspect and the hard consistency of this type of proliferation (desmos in Greek means band). Desmoid tumours (DTs) are classified as extraor intra-abdominal. The extra-abdominal DTs arise from fascial or musculoaponeurotic structures predominantly of the abdominal wall (Fig. 1a, b, c) and occasionally of the shoulder girdle, chest wall, inguinal region and extremities. They present as a firm, smooth, painless, progressively growing mass. Imaging investigations are useful in better defining the extent of the tumour which displays an iceberg growth with only a small proportion being clinically manifest. Interestingly, multiple extra-abdominal DTs are frequently discovered in very young patients under the age of 3 [2]. The intra-abdominal DTs develop in the folds of the mesentery or the mesocolon (Fig. 2a, b) even reaching the retroperitoneal tissue or they may grow exclusively in this region. These proliferations are usually single, round or oval in shape and up to 60 cm in size. Rather than a mass, a thickening of the mesentery that appears to be covered with hard white spots and causes retraction of the peritoneal folds is frequently reported in familial Desmoid Tumours in Familial Adenomatous Polyposis