Inhibition of STAT3 by RNA interference suppresses angiogenesis in colorectal carcinoma

In order to investigate signal transduction and activation of transcription 3 (STAT3) signaling on angiogenesis in colorectal carcinoma (CRC) after inhibiting STAT3 expression, we constructed the HT-29-shSTAT3 cell line by lentivirus-mediated RNAi. Cell growth was assessed with MTT and the cell cycle distribution by flow cytometry. CRC nude mouse models were established and tumor growth was monitored periodically. On day 30, all mice were killed and tumor tissues were removed. Microvessel density (MVD) was determined according to CD34-positive staining. The expression of vascular endothelial growth factor A (VEGFA), matrix metalloproteinase-2 (MMP2) and basic fibroblast growth factor (FGF2) was monitored by quantitative real-time PCR and Western blot analysis. Knockdown of STAT3 expression significantly inhibited cell growth in HT-29 cells, with a significantly higher proportion of cells at G0/G1 (P < 0.01). Consistently, in vivo data also demonstrated that tumor growth was significantly inhibited in mice injected with HT-29-shSTAT3 cells. MVD was 9.80 ± 3.02 in the HT-29-shSTAT3 group, significantly less than that of the control group (P < 0.01). mRNA and protein levels of VEGFA and MMP2 in the HT-29-shSTAT3 group were significantly lower than in the control group (P < 0.05), but no significant difference was observed in the mRNA or protein level of FGF2 (P > 0.05). Taken together, these results demonstrate that STAT3 signaling is important to the growth of CRC and promotes angiogenesis by regulating VEGFA and MMP2 expression.

Correlations of 3T DCE-MRI Quantitative Parameters with Microvessel Density in a Human-Colorectal-Cancer Xenograft Mouse Model

OBJECTIVE: To investigate the correlation between quantitative dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) parameters and microvascular density (MVD) in a human-colon-cancer xenograft mouse model using 3 Tesla MRI. MATERIALS AND METHODS: A human-colon-cancer xenograft model was produced by subcutaneously inoculating 1 x 106 DLD-1 human-colon-cancer cells into the right hind limbs of 10 mice. The tumors were allowed to grow for two weeks and then assessed using MRI. DCE-MRI was performed by tail vein injection of 0.3 mmol/kg of gadolinium. A region of interest (ROI) was drawn at the midpoints along the z-axes of the tumors, and a Tofts model analysis was performed. The quantitative parameters (Ktrans, Kep and Ve) from the whole transverse ROI and the hotspot ROI of the tumor were calculated. Immunohistochemical microvessel staining was performed and analyzed according to Weidner's criteria at the corresponding MRI sections. Additional Hematoxylin and Eosin staining was performed to evaluate tumor necrosis. The Mann-Whitney test and Spearman's rho correlation analysis were performed to prove the existence of a correlation between the quantitative parameters, necrosis, and MVD. RESULTS: Whole transverse ROI of the tumor showed no significant relationship between the MVD values and quantitative DCE-MRI parameters. In the hotspot ROI, there was a difference in MVD between low and high group of Ktrans and Kep that had marginally statistical significance (ps = 0.06 and 0.07, respectively). Also, Ktrans and Kep were found to have an inverse relationship with MVD (r = -0.61, p = 0.06 in Ktrans; r = -0.60, p = 0.07 in Kep). CONCLUSION: Quantitative analysis of T1-weighted DCE-MRI using hotspot ROI may provide a better histologic match than whole transverse section ROI. Within the hotspots, Ktrans and Kep tend to have a reverse correlation with MVD in this colon cancer mouse model.

Comparison of blood neoangiogenesis and lymphatic vascularization in colorectal adenomas from patients with and without concomitant colorectal cancer

Blood and lymphatic vessel proliferation is essential for tumor growth and progression. Most colorectal carcinomas develop from adenomas (adenoma-carcinoma sequence) in a process due to accumulation of molecular genetic alterations. About 5% of adenomatous polyps are expected to become malignant, but data on the differential angiogenic patterns of these lesions in patients with and without concomitant cancer are missing. The aim of the present study is to compare the angiogenic and lymphatic patterns of adenomatous polyps from patients with and without sporadic cancer. Thirty adenomatous polyps (15 from patients with another principal malignant lesion, and 15 from patients without cancer) were submitted to immunohistochemical staining for CD105 (marker for neoangiogenesis) and D2-40 (marker for lymphatic endothelium). Microvessel density and total vascular area were determined by computer image analysis to quantify the immunostained and total areas, and to assess the number of microvessels. Adenomas from patients with carcinoma showed significantly higher values of total vascular area determined by immunostaining for CD105 (cutoff value = 4386 µm²; P = 0.019) and of lymphatic microvessel density determined by immunostaining with D2-40 (cutoff value = 11.5; P = 0.041) when compared with those from patients without cancer. The present data indicate a significant increase in blood microvascular area and in lymphatic microvascular counts in adenomas removed from patients with cancer.

The Expression of Matrix Metalloproteinases (MMPs), Tissue Inhibitor of Metalloproteinases (TIMPs) and Angiogenesis in Relation to the Depth of Tumor Invasion and Lymph Node Metastasis in Submucosally Invasive Colorectal Carcinoma / 대한소화기학회지

BACKGROUND/AIMS: Lymph node (LN) metastasis occurs in approximately 10% of patients with submucosally invasive colorectal carcinoma. This study was performed to determine the role of matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs) production and microvessel formation on the LN metastasis in submucosally invasive colorectal carcinoma. METHODS: A total of forty-one subjects with surgically resected submucosally invasive colorectal carcinoma were included in this study. Immunohistochemical staining of MMP-2, MMP-9, TIMP-1, TIMP-2, and urokinase-type plasminogen activator were performed. Angiogenesis was evaluated by counting the number of microvessels in each pathologic specimen as identified by CD34 immunohistochemical staining. RESULTS: The depth of submucosal invasion was not significantly correlated with the expression of MMP-2, MMP-9, TIMP-1, TIMP-2, or urokinase-type plasminogen activator, but the microvessel count was significantly correlated with the absolute depth of invasion (r=0.312, p<0.05). Upregulation of TIMP-2 was positively correlated with adjacent lymphatic invasion (p<0.05) and increased TIMP-2 expression was correlated with LN metastasis in submucosally invasive colorectal carcinoma (p=0.088). CONCLUSIONS: These results suggest that the expression of TIMP-2 and the microvessel count may be useful parameters for considering additional surgery after endoscopic treatment of submucosally invasive colorectal carcinoma.

Determination of carcinoembryonic antigen levels in peripheral and draining venous blood in patients with colorectal carcinoma

RACIONAL: O problema da relação entre os níveis de CEA no sangue e o conteúdo de CEA tissular no carcinoma colorretal e os mecanismos de liberação do CEA das células neoplásicas nos tecidos vizinhos à neoplasia e sua conseqüente entrada dentro do sangue periférico são importantes para o entendimento da biologia do carcinoma colorretal. Ainda não foi convenientemente elucidado se o CEA no sangue é drenado principalmente pelo sangue do sistema portal ou pelos linfáticos para o ducto torácico ou, ainda, por ambos os sistemas. OBJETIVO: Estudar o comportamento dos níveis do CEA no sangue periférico (CEA-p) e no sangue do efluente venoso (CEA-d) de doentes com tumores colorretais operados curativamente. MÉTODO: foram estudados 28 doentes, sendo 12 (42,9%) homens e 16 (57,1%) mulheres. A média de idade foi de 66,1 anos (43 a 84 anos). Imediatamente após a laparotomia, o sangue venoso periférico foi extraído por punção venosa antecubital e o sangue do efluente venoso coletado da veia principal de drenagem das lesões. Os valores de CEA-p, CEA-d e do gradiente entre o CEA-d e CEA-p abaixo de 5,0 ng/mL foram considerados normais. RESULTADOS: Oito (28,6%) doentes foram classificados no estádio A de Dukes, 9 (32,1%) no estádio B e 11 (39,3%) no estádio C. A neoplasia estava localizada no reto em 14 (50,0%), no cólon transverso em 5 (17,9%), no cólon sigmóide em 4 (14,3%), no ceco e/ou cólon ascendente em 3 (10,7%), e no cólon descendente em 2 (7,1%) enfermos. O exame histopatológico revelou adenocarcinoma bem diferenciado em todos os enfermos. Em apenas um (3,6%) doente a neoplasia, estadiada como Dukes C, exibia invasão venosa. O gradiente entre os níveis de CEA-p e de CEA-d estava normal em 25 (89,3%) doentes e elevado em 3 (10,7%). O valor médio do CEA-p foi de 3,8 ± 4,1 ng/mL (0,1 a 21,1 ng/mL) e do CEA-d foi de 4,5 ± 4,3 ng/mL (0,3 a 20,2 ng/mL), sem diferença significativa entre esses valores. Houve diferença significativa entre a média dos valores dos níveis do CEA-p e do CEA-d maiores que 5 ng/mL. CONCLUSAO: Os níveis de CEA-p e do CEA-d nos doentes com carcinoma colorretal não se mostraram diferentes. Os resultados deste estudo sugerem que, nas neoplasias colorretais sem invasão venosa, o CEA não é drenado expressivamente pelo sangue do efluente venoso portal do tumor.

Correlation between expression of matrix metalloproteinase-2 (MMP-2), and matrix metalloproteinase-9 (MMP-9) and angiogenesis in colorectal adenocarcinoma

Matrix metalloproteinases-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9), which degrade extracellular matrix, are believed to play a crucial role in tumor invasion and metastasis. Angiogenesis is also perceived as an important step in tumor growth and metastasis. To investigate the expression of MMPs and the correlation between the expression of MMPs and angiogenesis in colorectal adenocarcinoma, we studied 72 cases of colorectal adenocarcinoma in Inha University Hospital from 1996 to 1997. We evaluated the expression of MMPs by immunohistochemistry and angiogenesis by counting the microvessels. The expression of MMP-2 was increased according to the Astler-Coller stage (p< 0.05). Angiogenesis in the metastatic group was higher than that of the localized one (p<0.05). The expression of MMP-2 positively correlated with angiogenesis (p<0.05), and marked expression of MMP-9 positively correlated with angiogenesis (p<0.05). The present results suggest that the expression of MMP-2 provides clues for tumor progression and angiogenesis provides significant information to predict whether metastasis is present in colorectal adenocarcinoma.