Expression of PAR4 and TFF2 in colorectal cancer tissues.

<p>(A) The matched normal (normal) and cancerous (cancer) tissues from each patients selected randomly were analyzed by RT-PCR using PAR4- and GAPDH- specific primers (n = 4). After the samples were normalized to GAPDH levels, mRNA levels of PAR4 and TFF2 were significantly increased in cancer compared to the normal tissues; (B) Western blot of tissue lysates from four cases of colorectal cancer (cancer) and relevant adjacent non-neoplastic mucosa (normal). The expression of actin was served as a control. The significant up-regulation expression of PAR4 was observed in the cancerous tissues in contrast to their matched normal tissues (n = 4).</p

5-Aza-dC induced PAR4 expression in colorectal cancer cell, LoVo.

<p>LoVo, a non- PAR4 expressing cells, were incubated with 5-Aza-dC (10 μM) or DMSO for 72 hours, and the cells were used to extract mRNA and protein. (A) The expression level of PAR4 mRNA was examined by RT-PCR; (B) The expression level of PAR4 protein was examined by western blot. The colorectal cancer cell line HT-29 expressed PAR4 was used as a positive control.</p

Genomic bisulfite sequencing of CpG methylation sites of PAR4 promoter in colorectal cancer tissues and cell lines.

<p>(A) PAR4 promoter methylation was analyzed in DNA from three colorectal cancer and their matched non-neoplastic tissues. (B) PAR4 promoter methylation in DNA from colorectal cancer cells lines, LoVo and HT-29. Average methylation at each analyzed CpG site in the PAR4 promoter is indicated based on bisulfite sequencing of 19 individual clones.</p

Representative photomicrographs of immunohistochemical staining of PAR4 and TFF2 in paraffin sections of colorectal tissues.

<p>(A) immunohistochemical staining for PAR4. (a) normal colorectal mucosa; (b) colorectal cancer tissue of number one patient. (c) colorectal cancer tissue of number two patient; (B) immunohistochemical staining for TFF2. (d) normal colorectal mucosa; (e) colorectal cancer tissue of number one patient; (f) colorectal cancer tissue of number two patient. Bar, 100 μm for each panel, and 50 μm for insets.</p

Association between the protein levels of PAR4 and TFF2 with clinic-pathological data of colorectal cancer.

<p>Association between the protein levels of PAR4 and TFF2 with clinic-pathological data of colorectal cancer.</p

Effect of recombinant TFF2 on the invasion of LoVo-mock and LoVo-PAR4 cells.

<p>(A) Cell invasion stimulated by TFF2 was tested using an InnoCyte cell invasion assay. Invasion of LoVo-mock and LoVo-PAR4 cells stimulated by 200nM TFF2 was tested, with BSA and 10nM EGF as controls. The results showed significant difference of 200nM TFF2 on LoVo-mock and LoVo-PAR4 cells invasion activity. Data were presented as means ± SD of three independent experiments. *p<0.05; (B) Invasion of LoVo-mock and LoVo-PAR4 cells stimulated by 200nM TFF2 were investigated by Confnocal laser fluorescence microscopy.</p

Cadherin-11 in Renal Cell Carcinoma Bone Metastasis

<div><p>Bone is one of the common sites of metastases from renal cell carcinoma (RCC), however the mechanism by which RCC preferentially metastasize to bone is poorly understood. Homing/retention of RCC cells to bone and subsequent proliferation are necessary steps for RCC cells to colonize bone. To explore possible mechanisms by which these processes occur, we used an <i>in vivo</i> metastasis model in which 786-O RCC cells were injected into SCID mice intracardially, and organotropic cell lines from bone, liver, and lymph node were selected. The expression of molecules affecting cell adhesion, angiogenesis, and osteolysis were then examined in these selected cells. Cadherin-11, a mesenchymal cadherin mainly expressed in osteoblasts, was significantly increased on the cell surface in bone metastasis-derived 786-O cells (Bo-786-O) compared to parental, liver, or lymph node-derived cells. In contrast, the homing receptor CXCR4 was equivalently expressed in cells derived from all organs. No significant difference was observed in the expression of angiogenic factors, including HIF-1α, VEGF, angiopoeitin-1, Tie2, c-MET, and osteolytic factors, including PTHrP, IL-6 and RANKL. While the parental and Bo-786-O cells have similar proliferation rates, Bo-786-O cells showed an increase in migration compared to the parental 786-O cells. Knockdown of Cadherin-11 using shRNA reduced the rate of migration in Bo-786-O cells, suggesting that Cadherin-11 contributes to the increased migration observed in bone-derived cells. Immunohistochemical analysis of cadherin-11 expression in a human renal carcinoma tissue array showed that the number of human specimens with positive cadherin-11 activity was significantly higher in tumors that metastasized to bone than that in primary tumors. Together, these results suggest that Cadherin-11 may play a role in RCC bone metastasis.</p></div

Expression of Cad11 in 786-O cell lines derived from metastases to various organs.

<p>(A) Quantitative PCR for the message levels of <i>Cad11</i> in the four 786-O cell lines. (B) Western blotting for the protein levels of Cad11 in four 786-O cell lines. Upper panel: A representative image of Western blot. Lower panel: Quantification of band density using Image J software. Data were expressed as folds of parental 786-O cells and the values were the Mean ± S.E. n = 5. *: <i>p</i><0.05; **: <i>p</i><0.01 as compared to parental 786-O cells. (C) FACS for surface expression of Cad11 in the four 786-O cell lines. Data were expressed as percentage of gated cells. (D) Immunofluorescence staining of cells with anti-Cad11 antibody. P, Parental 786-O; Liv, Liv-786-O; LN, LN-786-O, and Bo, Bo-786-O RCC cells.</p

Effect of Cad11 on cell proliferation and migration of Bo-786-O cells.

<p>(<b>A</b>) Proliferation and migration of parental and bone-derived 786-O cells. Left: Western blot of Cad11 protein. Middle: Cell proliferation. Right: Cell migration. A representative image of cell migration and the quantification of cells that migrated to the other side of migration inserts were shown. Values for migration were expressed as the average of migrated cells per microscope field (X100). (B) Effect of Cad11 knockdown on the proliferation and migration of Bo/shCont and Bo/shCad11 cells.</p

Cad11 expression in human primary RCC and bone metastatic RCC specimens.

<p>Staining of human RCC samples for cadherin-11.</p><p>*: chi-square analysis.</p