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Recepteur d'Origine Nantais (RON) expression is known to induce oncogenic properties including tumor cell growth, survival, motility, angiogenesis and chemoresistance. In the present study, we evaluated whether RON affects chemosensitivity and oncogenic behavior of colorectal cancer cells and investigated its prognostic value in colorectal cancer. To evaluate the impact of RON on chemosensitivity and tumor cell behavior, we treated colorectal cancer cells with small interfering RNAs specific to RON. This was followed by flow cytometric analyses and migration, Matrigel invasion and endothelial tube formation assays. The expression of RON was investigated by immunohistochemistry in colorectal cancer tissues. TUNEL assay and immunohistochemical staining for CD34 and D2-40 were deployed to determine apoptosis, angiogenesis and lymphangiogenesis. RON knockdown enhanced 5-fluorouracil (FU)-induced apoptosis by upregulating the activities of caspases and expression of proapoptotic genes. Moreover, it enhanced 5-FU-induced cell cycle arrest by decreasing the expression of cyclins and cyclin‑dependent kinases and inducing that of p21. Furthermore, RON knockdown augmented the 5-FU-induced inhibition of invasion and migration of colorectal cancer cells. The β-catenin signaling cascade was blocked by RON knockdown upon 5-FU treatment. RON knockdown also decreased endothelial tube formation and expression of VEGF-A and HIF-1α and increased angiostatin expression. Furthermore, it inhibited lymphatic endothelial cell tube formation and the expression of VEGF-C and COX-2. RON expression was observed to be associated with age, tumor size, lymphovascular and perineural invasion, tumor stage, lymph node and distant metastasis, and poor survival rate. The mean microvessel density value of RON-positive tumors was significantly higher than that of RON-negative ones. These results indicate that RON is associated with tumor progression by inhibiting chemosensitivity and enhancing angiogenesis in colorectal cancer.

Early growth response-1 (Egr-1) is implicated in the regulation of cell growth, proliferation, differentiation and apoptosis. Egr-1 is considered tobe either a tumor-suppressor or tumor-promoter, depending on the cell type and environment. The aim of the present study was to evaluate the expression of Egr-1 in colorectal cancer and its correlation with tumor cell proliferation, apoptosis and clinicopathological features. The expression of Egr-1 in colorectal cancer tissues was investigated by reverse transcription-polymerase chain reaction (RT-PCR), western blotting and immunohistochemistry. Apoptosis was detected by terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL), and cellular proliferative activity was evaluated by immunohistochemical staining with the Ki-67 antibody. Egr-1 expression was significantly elevated in colorectal cancer tissues, when compared to that in the paired normal mucosa at the mRNA and protein levels. In addition, Egr-1 expression was significantly increased in the metastatic lymph node tissues, when compared to that in the non‑metastatic lymph node tissues at the protein level. The mean Ki-67 labeling index (KI) and apoptotic index (AI) values for 158 tumors were 53.6±15.4 and 9.0±1.0, respectively. Higher KI values were significantly associated with distant metastasis. Lower AI values were significantly associated with lymph node metastasis. However, KI or AI values were not associated with patient survival. The mean KI value of Egr-1-positive tumors was significantly higher than that of Egr-1-negative tumors. However, there was no significant difference between Egr-1 expression and AI value. Positive expression of Egr-1 was significantly associated with age, lymphovascular invasion, lymph node and distant metastasis, tumor stage and poor survival. These results indicate that Egr-1 may be associated with colorectal cancer progression via tumor cell proliferation.

Livin is one of the most important members of the inhibitor of apoptosis protein family. It is overexpressed in several types of tumors and may have prognostic significance. The present study investigated the biological role of Livin in the oncogenic behavior of gastric cancer cells, the expression of Livin in gastric cancer tissue and the relationship of its expression with various clinicopathological parameters and patient survival. Small interfering RNA blocked Livin gene expression in AGS and SNU638 human gastric cancer cell lines. The expression of Livin was investigated in gastric cancer tissues by RT-PCR, western blotting and immunohistochemistry. The associations with various clinicopathological parameters and survival were analyzed. Livin knockdown inhibited tumor cell migration, invasion and proliferation in AGS and SNU638 cells. Livin knockdown induced apoptosis by activating caspase-3, caspase-7 and PARP. Livin knockdown induced cell cycle arrest by a decrease in cyclin D1, cyclin-dependent kinase 4 and 6 and an increase in expression of p21 and p27. The ERK1/2 and JNK signaling pathways were inhibited by Livin knockdown. Livin expression was upregulated in gastric cancer tissues at the mRNA and protein levels. However, no significant correlation was found between Livin expression and various clinicopathological parameters including survival. In conclusion, Livin expression may be important in the alteration of invasive and oncogenic phenotypes of gastric cancer cells. The prognostic relevance of Livin remains unclear.

Angiogenesis and lymphangiogenesis are involved in the dissemination of tumor cells from solid tumors to regional lymph nodes and various distant sites. KAI1 COOH-terminal interacting tetraspanin (KITENIN) contributes to tumor progression and poor clinical outcomes in various cancers including colorectal cancer. The aim of the present study was to evaluate whether KITENIN affects tumor angiogenesis and lymphangiogenesis in colorectal cancer. A KITENIN small interfering RNA vector was used to silence KITENIN expression in colorectal cancer cell lines including DLD1 and SW480 cells. To evaluate the ability of KITENIN to induce angiogenesis and lymphangiogenesis in human umbilical vein endothelial cells (HUVECs) and lymphatic endothelial cells (HLECs), we performed Matrigel invasion and tube formation assays. Immunohistochemistry was used to determine the expression of KITENIN in colorectal cancer tissues. Angiogenesis and lymphangiogenesis were evaluated by immunostaining with CD34 and D2-40 antibodies. KITENIN silencing inhibited both HUVEC invasion and tube formation in the DLD1 and SW480 cells. KITENIN silencing led to decreased expression of the angiogenic inducers vascular endothelial growth factor (VEGF)-A and hypoxia-inducible factor-1α and increased expression of the angiogenic inhibitor angiostatin. KITENIN silencing did not inhibit either HLEC invasion or tube formation in all tested cells, but it resulted in decreased expression of the lymphangiogenic inducer VEGF-C. KITENIN expression was significantly associated with tumor stage, depth of invasion, lymph node and distant metastases and poor survival. The mean microvessel density was significantly higher in the KITENIN-positive tumors than that in the KITENIN-negative tumors. However, the mean lymphatic vessel density of KITENIN-positive tumors was not significantly higher than that of the KITENIN-negative tumors. These results suggest that KITENIN promotes tumor progression by enhancing angiogenesis in colorectal cancer.

Epithelial-mesenchymal transition (EMT) is a critical process that occurs during cancer progression, and cancer stem cells have been shown to acquire the EMT phenotype. Myeloid cell leukemia-1 (Mcl-1) has been implicated in cancer progression and is overexpressed in a variety of human cancers. However, the interaction between Mcl-1 and EMT in human gastric cancer (GC) is unclear. We investigated the impact of Mcl-1 expression levels on EMT and the underlying signaling pathways in human GC cells. We used the human GC cell lines, AGS and SNU638, and small interfering RNAs (siRNAs) to evaluate the effects of Mcl-1 knockdown on cell adhesion, migration and invasion. Expression of Mcl-1 and other target genes was determined using reverse transcription-polymerase chain reaction assays and western blotting. The results revealed that expression levels of Mcl-1 mRNA and protein in the AGS and SNU638 cells were reduced following transfection with Mcl-1 siRNAs. Knockdown of Mcl-1 led to increased cellular adhesion to fibronectin and collagen. Expression levels of vimentin, MMP-2, MMP-9 and Snail protein were decreased following knockdown of Mcl-1. However, expression of E-cadherin was increased in the AGS cells following knockdown of Mcl-1. The expression of cancer stemness markers, such as CD44 and CD133, was not altered by knockdown of Mcl-1. Knockdown of Mcl-1 suppressed tumor cell migration and invasion in both human GC cell lines. Signaling cascades, including the β-catenin, MEK1/2, ERK1/2 and p38 pathways, were significantly blocked by knockdown of Mcl-1. Our results indicate that Mcl-1 expression induces EMT via β-catenin, MEK1/2 and MAPK signaling pathways, which subsequently stimulates the invasive and migratory capacity of human GC cells.

Leptin, which is encoded by the obese gene, is a multifunctional neuroendocrine peptide that regulates appetite, bone formation, reproductive function and angiogenesis. The aims of the present study were to investigate the expression of leptin in 80 patients with colorectal cancer (CRC) and to determine the effects of leptin on the malignant properties of CRC cells. We evaluated the expression of leptin in tissues of 80 patients with CRC. Suspension cultures were used to isolate CRC stem cells following pretreatment with leptin. We analyzed the effects of leptin on the adhesion and invasive capacities of CRC cell lines. The effects of leptin on JAK and ERK activation were examined using western blotting. Leptin expression was associated with CRC progression and increased the number and size of spheroid formation by CRC cell lines. Leptin enhanced cell invasion and adhesion and activated JAK and ERK signaling in the CRC cell lines. The present study demonstrated that leptin influences the growth and survival of CRC stem cells and regulates adhesion and invasion of colorectal carcinoma through activation of the JAK and ERK signaling pathways.

The recepteur d'origine nantais (RON) receptor tyrosine kinase is highly expressed in various cancers including human hepatocellular carcinoma (HCC) and involved in tumor progression. The aims of the current study were to evaluate whether RON affects tumor cell behavior and oncogenic signaling cascades in HCC cells. We investigated the biologic role of RON on tumor cell behavior and oncogenic signaling cascades including Akt, c-Raf and extracellular signal-regulated kinase (ERK) by using the small interfering RNA (siRNA) in HCC cell lines, chang, HepG2 and Huh7. Knockdown of RON suppressed tumor cell migration and invasion in all tested HCC cell lines. The proportion of apoptotic cells induced by knockdown of RON was greater than that induced by transfection of the scramble siRNA in all tested HCC cell lines. Knockdown of RON resulted in cell cycle arrest in the G2/M phase of chang and Huh7 cells, and sub G1 phase of HepG2 cells. Knockdown of RON activated cleaved caspase-3 and PARP, and down-regulated the expression of Bcl-2, Bcl-xL and survivin, leading to induction of apoptosis in all tested cell lines. Knockdown of RON negatively regulates the progression of the cell cycle by decreasing cyclin D1 and D3, and increasing p21 and p27 in all tested cell lines. The phosphorylation of Akt, c-Raf and ERK1/2 signal proteins was significantly blocked by knockdown of RON in all tested cell lines. These results suggest that RON is associated with invasive and oncogenic phenotypes such as tumor cell migration, invasion, resistance to apoptosis and cell cycle arrest through the modulation of Akt, c-Raf and ERK signaling cascades in HCC cells.