Western blot analyses of primary CRC cell lines treated with IL-6.

<p>To investigate EMT and STAT3 activation of human CRC cells, cytokine IL-6 was treated on a gradient level of 0, 1, 5 and 10 u/ml. A: HT-29 was treated with a gradient of IL-6, then STAT3 phosphorylation and EMT phenotype was observed. Another important stem cell marker Oct-4 expression was also monitored. B: DLD-1 cells were treated with IL-6 and subjected to the western analyses.</p

Western analyses of normal colon cell FHC, primary and metastatic colon cancer cell lines of SW480, SW620, HT-29 and T84.

<p>Immunoblotting was performed for the VEGF specifically upregulated in metastatic colon cancer and TIMP-1, amphiregulin, endostatin, angiogenin and GM-CSF. Protein expression levels were measured and normalized by the normal colon cell FHC expression.</p

Identification of Novel Biomarkers for Metastatic Colorectal Cancer Using Angiogenesis-Antibody Array and Intracellular Signaling Array

<div><p>Colorectal cancer (CRC) is one of the three leading causes for cancer mortality. CRC kills over 600,000 people annually worldwide. The most common cause of death from CRC is the metastasis to distant organs. However, biomarkers for CRC metastasis remain ill-defined. We compared primary and metastatic CRC cell lines for their angiogenesis-protein profiles and intracellular signaling profiles to identify novel biomarkers for CRC metastasis. To this end, we used primary and metastatic CRC cell lines as a model system and normal human colon cell line as a control. The angiogenesis profiles two isogenic CRC cell lines, SW480 and SW620, and HT-29 and T84 revealed that VEGF was upregulated in both SW620 and T84 whereas coagulation factor III, IGFBP-3, DPP IV, PDGF AA/AB, endothelin I and CXCL16 were downregulated specifically in metastatic cell lines. Furthermore, we found that TIMP-1, amphiregulin, endostatin, angiogenin were upregulated in SW620 whereas downregulated in T84. Angiogenin was downregulated in T84 and GM-CSF was also downregulated in SW620. To induce CRC cell metastasis, we treated cells with pro-inflammatory cytokine IL-6. Upon IL-6 treatment, epithelial-mesenchymal transition was induced in CRC cells. When DLD-1 and HT-29 cells were treated with IL-6; Akt, STAT3, AMPKα and Bad phosphorylation levels were increased. Interestingly, SW620 showed the same signal activation pattern with IL-6 treatment of HT-29 and DLD-1. Our data suggest that Akt, STAT3, AMPKα and Bad activation can be biomarkers for metastatic colorectal cancer. IL-6 treatment specifically reduced phosphorylation levels of EGFR, HER2 receptor, Insulin R and IGF-1R in receptor tyrosine kinase array study with HT-29. Taken together, we have identified novel biomarkers for metastatic CRC through the angiogenesis-antibody array and intracellular signaling array studies. Present study suggests that those novel biomarkers can be used as CRC prognosis biomarkers, and as potential targets for the metastatic CRC therapy.</p></div

Human phosphor-receptor tyrosine kinase array of SW480, SW620 and T84.

<p>We investigated the receptor tyrosine kinase phosphorylation with IL-6 treatment in SW480. Untreated control SW480 and IL-6 treated SW480 arrays showed no RTK activation. Moreover, metastatic cancer cell line SW620 and T84 did not show the RTK activation, either.</p

Human phosphor-receptor tyrosine kinase array of HT-29.

<p>We investigated the receptor tyrosine kinase phosphorylation with IL-6 treatment in HT-29. A: Untreated control HT-29 array showed the phosphorylated EGFR, HER2, Insulin R and IGF-1R receptors. B: When we treated HT-29 with IL-6, the phosphorylation of EGFR, HER2, Insulin R and IGF-1R was abolished.</p

The cellular functions of angiogenesis-related proteins specifically downregulated in the metastatic colorectal cancer cells.

<p>The cellular functions of angiogenesis-related proteins specifically downregulated in the metastatic colorectal cancer cells.</p

Intracellular signaling arrays of human CRC cell lines.

<p>To monitor the intracellular signaling, important signaling component activation was observed using antibody arrays. A: Two isogenic CRC cell lines, SW480 and SW620, were analyzed for the signaling activation. Phosphorylation levels of STAT3, Akt, AMPKα and BAD proteins were elevated in SW620 compared to SW480 cells. B: HT-29 was treated with IL-6 and subjected to intracellular signaling array study. Interestingly, the same protein set of STAT3, Akt, AMPKα and BAD phosphorylation levels were all increased upon IL-6 treatment. C: DLD-1 cells were treated with IL-6 and observed for the signaling component activation. DLD-1 also showed the STAT3, Akt, AMPKα and BAD phosphorylation enhancement upon IL-6 stimulation.</p

Western blot analyses of normal colon cell line FHC, primary cell line SW480 and HT-29 and metastatic SW620 and T84 for angiogenesis protein expression profiles.

<p>Western analyses were performed with the normal cell line and primary and metastatic colorectal cancer cell lines. Angiogenesis proteins identified from the array study, coagulation factor III, IGFBP-3, DPP IV, PDGF-A, endothelin I and CXCL16 were run on the gel and analyzed. Protein bands were measured for intensity and quantified using normal colon cell FHC as a reference.</p