Homocysteine promotes intestinal fibrosis in rats with trinitrobenzene sulfonic acid-induced colitis.

BACKGROUND AND AIM: Previous studies have revealed significantly increased levels of plasma and mucosal homocysteine (Hcy) in patients with Crohn's disease (CD); however, whether Hcy is involved in intestinal fibrosis of CD remains unclear. This study aimed to investigate the effects of Hcy on intestinal fibrosis in TNBS/ethanol-induced colitis and to elucidate its potential mechanisms. METHODS: Sprague-Dawley rats were divided into 4 groups: normal control, normal + Hcy injection, TNBS model and TNBS model + Hcy injection. Hyperhomocysteinemia was induced by subcutaneous injection of Hcy. DAI, CMDI and HI were calculated to evaluate the severity of colitis. Masson trichrome staining was performed to assess the severity of fibrosis. The plasma and mucosal levels of Hcy were measured by HPLC-FD. The levels of IL-1ß, IL-6, TNF-α, TGF-ß1, CTGF, MMP-2,9 and collagen I, III in the colon were determined by ELISA, and the mRNA expressions of TGF-ß1, MMP-2,9 and TIMP-1 were detected by RT-PCR. RESULTS: Hcy was found to increase the scores of DAI, CMDI and HI; levels of IL-1ß, Il-6, TNF-α, TGF-ß1, CTGF, MMP-2,9 and collagen I, III; and mRNA expressions of TGF-ß1, MMP-2,9 and TIMP-1 in colonic tissue of rats with TNBS/ethanol-induced colitis. CONCLUSIONS: Hcy promotes intestinal fibrosis in rats with TNBS/ethanol-induced colitis, the underlying mechanisms of which may be attributed to its effects of increasing inflammatory damage, promoting the expression of profibrogenic cytokines and influencing MMPs/TIMPs balance.

Effect of homocysteine on intestinal permeability in rats with experimental colitis, and its mechanism.

OBJECTIVE: To investigate the effect of homocysteine (Hcy) on intestinal permeability in rats with TNBS/ethanol-induced colitis and elucidate its mechanism. METHODS: Sprague-Dawley rats were divided into four groups: normal, normal + Hcy injection, TNBS model, and TNBS model + Hcy injection. Experimental colitis was induced by trinitrobenzene sulfonic acid (TNBS) in 50% ethanol; rats were injected subcutaneously with Hcy from the first day after the induction of experimental colitis on 30 consecutive days. To determine the severity of colitis, the disease activity index (DAI) was evaluated; colon tissues were collected for the detection of the activity of myeloperoxidase (MPO) and the contents of MDA, IL-1ß, IL-6, TNF-α, MMP-2, and MMP-9. Intestinal epithelial permeability was assessed with Evans blue (EB) dye. The levels of Hcy in plasma and colon mucosa were measured by high-performance liquid chromatography-fluorescence detection (HPLC-FD). RESULTS: Compared with the normal group, the DAI scoring and MPO activity, contents of MDA, IL-1ß, IL-6, TNF-α, MMP-2, MMP-9 in the colon and EB in the small intestine were significantly increased in the TNBS group (P < 0.01). Compared with the TNBS model group, the DAI scoring, plasma and colonic mucosa Hcy levels, MPO activity and contents of MDA, IL-1ß, IL-6, TNF-α, MMP-2, MMP-9 in colon and EB in small intestine were significantly increased in the TNBS-induced colitis rats with simultaneous Hcy injection (P < 0.01). CONCLUSION: Hcy can increase intestinal permeability and aggravate inflammatory damage in rats with TNBS-induced colitis, the underlying mechanisms of which may be attributed to its effects of promoting the expression of MMP-2 and MMP-9, leading to injury of the intestinal barrier.

Retinoid receptors in gastric cancer: expression and influence on prognosis.

BACKGROUND: Gastric cancer is frequently lethal despite aggressive multimodal therapies, and new treatment approaches are therefore needed. Retinoids are potential candidate drugs: they prevent cell differentiation, proliferation and malignant transformation in gastric cancer cell lines. They interact with nuclear retinoid receptors (the retinoic acid receptors [RARs] and retinoid X receptors [RXRs]), which function as transcription factors, each with three subclasses, α, ß and γ. At present, little is known about retinoid expression and influence on prognosis in gastric cancers. PATIENTS AND METHODS: We retrospectively analyzed the expression of the subtypes RARα, RARß, RARγ, RXRα, RXRß, RXRγ by immunohistochemistry in 147 gastric cancers and 51 normal gastric epithelium tissues for whom clinical follow-up data were available and correlated the results with clinical characteristics. In addition, we quantified the expression of retinoid receptor mRNA using real- time PCR (RT-PCR) in another 6 gastric adenocarcinoma and 3 normal gastric tissues. From 2008 to 2010, 80 patients with gastric cancers were enrolled onto therapy with all-trans-retinoic acid (ATRA). RESULTS: RARα, RARß, RARγ and RXRγ positively correlated with each other (p<0.001) and demonstrated significantly lower levels in the carcinoma tissue sections (p<0.01), with lower RARß, RARγ and RXRα expression significantly related to advanced stages (p<=0.01). Tumors with poor histopathologic grade had lower levels of RARα and RARß in different histological types of gastric carcinoma (p<0.01). Patients whose tumors exhibited low levels of RARa expression had significantly lower overall survival compared with patients who had higher expression levels of this receptor (p<0.001, HR=0.42, 95.0% CI 0.24-0.73), and patients undergoing ATRA treatment had significantly longer median survival times (p=0.007, HR=0.41, 95.0% CI 0.21-0.80). CONCLUSIONS: Retinoic acid receptors are frequently expressed in epithelial gastric cancer with a decreased tendency of expression and RARa may be an indicator of a positive prognosis. This study provides a molecular basis for the therapeutic use of retinoids against gastric cancer.

[Expressions of 6 microRNAs in prostate cancer].

OBJECTIVE: Experimental evidence shows that microRNAs play an important role in the initiation and progression of human malignancies. The present study aimed to investigate the expressions of 6 microRNAs in prostate cancer (PCa) and their clinical significance. METHODS: We investigated the expression profiles of 6 microRNAs (let-7g, let-7d, miR-98, miR-96, miR-182 and miR-183) using the method of locked nucleic acid (LNA)-modified oligonucleotide in situ hybridization (ISH) and the technology of tissue microarray (TMA) with the formalin-fixed paraffin-embedded (FFPE) specimens from 52 patients with PCa and 38 with benign prostatic hyperplasia (BPH). Then we analyzed the correlation among the expressions of the 6 microRNAs in PCa and their correlation with the Gleason score and clinical stages of PCa. RESULTS: Compared with BPH, the PCa patients showed decreased expressions of miR-98, let-7d and let-7g, and decreased expressions of miR-96, miR-182 and miR-183, with statistically significant differences between the two groups (P < 0.05). The positive rate of the 6 microRNAs was significantly correlated with the Gleason grades of PCa (P < 0.05), but not with the age and serum PSA concentration of the patients (P > 0.05). The expressions of miR-96 and miR-182 were correlated with the clinical stages of the tumor (P < 0.05). There was a positive correlation among the expressions of miR-96, miR-182 and miR-183 (P = 0.00, r = 0.41), as well as between the expressions of let-7d and let-7g (P = 0.00, r = 0.46) in the PCa tissues. And the expression of miR-98 was positively correlated with those of let-7d and let-7g (P = 0.00, r = 0.46). CONCLUSION: The expression profiles of the microRNAs let-7d, let-7g, miR-98, miR-96, miR-182 and miR-183 reflect the biological behavior of PCa to some extent, and might be important biomarkers for the early detection and prognostic assessment of prostate cancer.

Blocking effects of siRNA on VEGF expression in human colorectal cancer cells.

AIM: To investigate the expression of vascular endothelial cell growth factor (VEGF) and its receptors Fms-like tyrosine kinase 1 (FLT-1) and fetal liver kinase 1 (FLK-1) in colorectal carcinoma (CRC), and the blocking effects of small interfering RNAs (siRNAs) on VEGF expression in human colorectal cancer HCT116 cells. METHODS: Immunohistochemical staining for VEGF, FLT-1 and FLK-1 proteins was performed in 82 cases of CRC and 14 normal colorectal mucosae. A siRNA targeting VEGF was synthesized and transfected into HCT116 cells using lipofectamine 2000. Immunocytochemical staining and Western blotting analyses were performed to detect the expression of VEGF protein. The suppressive effect of the siRNA on cell proliferation was detected using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltertrazolium bromide (MTT) assay. Cellular apoptosis was detected using flow cytometry (FCM). RESULTS: The expression of VEGF, FLT-1 and FLK-1 in tumor tissues was significantly higher than that in normal tissues (P = 0.008, P = 0.000, P = 0.000). The expression of VEGF was positively correlated with both lymph node metastasis and clinical stage (P = 0.009 and P = 0.025, respectively). Immunocytochemistry showed that the expression of VEGF was weakly positive and Western blotting indicated a significant reduction in VEGF-siRNA cell protein levels. VEGF-siRNA cell growth inhibition was assessed by the MTT assay, and the tumor cell proliferation rate was significantly different at 24, 48, and 72 h after transfection. FCM results showed that the VEGF-siRNA group had an apparent aneuploid peak. CONCLUSION: VEGF, FLT-1 and FLK-1 are associated with colorectal carcinogenesis. siRNA silencing of the VEGF gene suppresses proliferation, and induces apoptosis in HCT116 cells. The results suggest that VEGF may be a new gene therapy target for colorectal cancer.

Expression and clinical significance of S100A2 and p63 in esophageal carcinoma.

AIM: To investigate the expression and clinical significance of S100A2 mRNA and protein, p63 protein in esophageal squamous cell carcinoma (ESCC) and their roles in carcinogenesis and progression of esophageal carcinoma (EC). METHODS: Immunohistochemical staining (S-P method) for S100A2 and p63 protein were performed in 40 samples of ESCC and 40 samples of normal esophageal mucosa. In situ hybridization (ISH) was used to detect the expression of S100A2 mRNA. RESULTS: Expression of S100A2 mRNA in ESCC was positive in 77.5% of samples, which was lower than that in normal mucosa (100%) by ISH (P = 0.002). The expression level of S100A2 mRNA was closely related to differentiation and and node-metastasis (P = 0.012, P = 0.008). Expression of S100A2 protein was positive in 72.5% of ESCC samples and expression of p63 protein was positive in 37.5% of ESCC samples, and was lower than that in normal mucosa (100%) (P = 0.000). The expression of S100A2 protein was correlated with the differentiation and node-metastasis (P = 0.007, P = 0.001), but no relationship was observed between the expression of p63 protein and clinical pathological manifestations. S100A2 protein was positively correlated with the expression of S100A2 mRNA, and negatively associated with the expression of p63 protein (P = 0.000, P = 0.002). CONCLUSION: S100A2 and p63 protein both play important roles in the carcinogenesis of ESCC. An investigation into the combined expression of S100A2 and p63 may be helpful in early diagnosis and in evaluating the prognosis of ESCC.