Protein-bound polysaccharide from Phellinus linteus inhibits tumor growth, invasion, and angiogenesis and alters Wnt/ß-catenin in SW480 human colon cancer cells.

BACKGROUND: Polysaccharides extracted from the Phellinus linteus (PL) mushroom are known to possess anti-tumor effects. However, the molecular mechanisms responsible for the anti-tumor properties of PL remain to be explored. Experiments were carried out to unravel the anticancer effects of PL. METHODS: The anti-cancer effects of PL were examined in SW480 colon cancer cells by evaluating cell proliferation, invasion and matrix metallo-proteinase (MMP) activity. The anti-angiogenic effects of PL were examined by assessing human umbilical vein endothelial cell (HUVEC) proliferation and capillary tube formation. The in vivo effect of PL was evaluated in an athymic nude mouse SW480 tumor engraft model. RESULTS: PL (125-1000 µg/mL) significantly inhibited cell proliferation and decreased ß-catenin expression in SW480 cells. Expression of cyclin D1, one of the downstream-regulated genes of ß-catenin, and T-cell factor/lymphocyte enhancer binding factor (TCF/LEF) transcription activity were also significantly reduced by PL treatment. PL inhibited in vitro invasion and motility as well as the activity of MMP-9. In addition, PL treatment inhibited HUVEC proliferation and capillary tube formation. Tumor growth of SW480 cells implanted into nude mice was significantly decreased as a consequence of PL treatment, and tumor tissues from treated animals showed an increase in the apoptotic index and a decrease in ß-catenin expression. Moreover, the proliferation index and microvessel density were significantly decreased. CONCLUSIONS: These data suggest that PL suppresses tumor growth, invasion, and angiogenesis through the inhibition of Wnt/ß-catenin signaling in certain colon cancer cells.

Activity and expression of urokinase-type plasminogen activator and matrix metalloproteinases in human colorectal cancer.

BACKGROUND: Matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), and urokinase-type plasminogen activator (uPA) are involved in colorectal cancer invasion and metastasis. There is still debate whether the activity of MMP-2 and MMP-9 differs between tumors located in the colon and rectum. We designed this study to determine any differences in the expression of MMP-2, MMP-9 and uPA system between colon and rectal cancer tissues. METHODS: Cancer tissue samples were obtained from colon carcinoma (n = 12) and rectal carcinomas (n = 10). MMP-2 and MMP-9 levels were examined using gelatin zymography and Western blotting; their endogenous inhibitors, tissue inhibitor of metalloproteinase-2 (TIMP-2) and tissue inhibitor of metalloproteinase-1 (TIMP-1), were assessed by Western blotting. uPA, uPAR and PAI-1 were examined using enzyme-linked immunosorbent assay (ELISA). The activity of uPA was assessed by casein-plasminogen zymography. RESULTS: In both colon and rectal tumors, MMP-2, MMP-9 and TIMP-1 protein levels were higher than in corresponding paired normal mucosa, while TIMP-2 level in tumors was significantly lower than in normal mucosa. The enzyme activities or protein levels of MMP-2, MMP-9 and their endogenous inhibitors did not reach a statistically significant difference between colon and rectal cancer compared with their normal mucosa. In rectal tumors, there was an increased activity of uPA compared with the activity in colon tumors (P = 0.0266), however urokinase-type plasminogen activator receptor (uPAR) and plasminogen activator inhibitor-1 (PAI-1) showed no significant difference between colon and rectal cancer tissues. CONCLUSION: These findings suggest that uPA may be expressed differentially in colon and rectal cancers, however, the activities or protein levels of MMP-2, MMP-9, TIMP-1, TIMP-2, PAI-1 and uPAR are not affected by tumor location in the colon or the rectum.

Gabexate mesilate inhibits colon cancer growth, invasion, and metastasis by reducing matrix metalloproteinases and angiogenesis.

Gabexate mesilate (GM), a synthetic protease inhibitor, has an antiproteinase activity on various types of plasma serine proteases. However, its role on matrix metalloproteinases (MMPs) has not been identified. In this study, we investigated the effect of GM on MMPs and on the invasion and metastasis of human colon cancer cell lines and neoangiogenesis. The activities of MMPs secreted from these cells were significantly reduced by GM but unaffected by the serine protease inhibitor aprotinin. GM directly inhibited purified progelatinase A derived from T98G human glioblastoma cells. In vitro, GM significantly reduced the invasive ability of colon cancer cells but not cellular motility, whereas aprotinin affected neither. Liver metastatic ability and tumorigenic potential in nude mice were remarkably reduced on treatment with GM. Immunohistochemical analysis of GM-treated tumors in mice showed a marked increase in apoptosis and a significant reduction in tumor angiogenesis. Human umbilical vein endothelial cell proliferation, tube formation, and neoangiogenesis in the rabbit cornea and Matrigel implanted in mice were significantly inhibited by GM. These results suggest that GM is a novel inhibitor of MMPs and that it may inhibit the invasion and metastasis of human colon cancer cells by blocking MMPs and neoangiogenesis.

Differential regulation of vimentin mRNA by 12-O-tetradecanoylphorbol 13-acetate and all-trans-retinoic acid correlates with motility of Hep 3B human hepatocellular carcinoma cells.

Vimentin is a growth-related gene and often expressed when epithelial cells are stimulated to proliferate by growth factors. In cancer, vimentin expression is associated with a dedifferentiated malignant phenotype, increased motility, invasive ability and poor prognosis. We studied the regulation of vimentin mRNA and multistep invasion processes following treatment of 12-O-tetradecanoylphorbol 13-acetate (TPA) and all-trans-retinoic acid (RA) in Hep 3B hepatocellular carcinoma cells. TPA showed marked induction of vimentin mRNA, while RA decreased the mRNA level. TPA or RA did not affect cell proliferation, cell-matrix protein adhesion, and matrix metalloproteinases and urokinase plasminogen activator activities. In vitro invasion ability was significantly increased or decreased with TPA or RA treatment, paralleled to the in vitro motile activity, respectively. These findings suggest that TPA and RA could modulate the invasive potential of Hep 3B cells by altering cellular motility related to differential regulation of vimentin mRNA.

Protein-bound polysaccharide from Phellinus linteus induces G2/M phase arrest and apoptosis in SW480 human colon cancer cells.

The cytotoxic mechanism of protein-bound polysaccharide isolated from Phellinus linteus (PL, Mesima) has been investigated. PL inhibited the proliferation and colony formation of SW480 human colon cancer cells. Flow cytometry analysis showed that PL increased the populations of both apoptotic sub-G1 and G2/M phase. The result obtained from TUNEL assay corroborated apoptosis which was shown in flow cytometry. Western blot analysis suggested that PL-induced apoptosis and growth inhibition were associated with decrease in Bcl-2, increase of the release of cytochrome c, and reduced expression of cyclin B1. These results suggest that PL has a direct antitumor effect through apoptosis and cell cycle blockade in certain cancer cells.

Radiation-induced thymidine phosphorylase upregulation in rectal cancer is mediated by tumor-associated macrophages by monocyte chemoattractant protein-1 from cancer cells.

PURPOSE: The mechanisms of thymidine phosphorylase (TP) regulation induced by radiation therapy (XRT) in various tumors are poorly understood. We investigated the effect and mechanisms of preoperative XRT on TP expression in rectal cancer tissues. METHODS AND MATERIALS: TP expression and CD68 and monocyte chemoattractant protein-1 (MCP-1) levels in rectal cancer tissues and cancer cell lines were evaluated before and after XRT in Western blotting, immunohistochemistry, enzyme-linked immunoassay, and reverse transcription-polymerase chain reaction studies. Isolated peripheral blood monocytes were used in the study of chemotaxis under the influence of MCP-1 released by irradiated colon cancer cells. RESULTS: Expression of TP was significantly elevated by 9 Gy of XRT in most rectal cancer tissues but not by higher doses of XRT. In keeping with the close correlation of the increase in both TP expression and the number of tumor-associated macrophages (TAMs), anti-TP immunoreactivity was found in the CD68-positive TAMs and not the neoplastic cells. Expression of MCP-1 was increased in most cases after XRT, and this increase was strongly correlated with TP expression. However, this increase in MCP-1 expression occurred in tumor cells and not stromal cells. The XRT upregulated MCP-1 mRNA and also triggered the release of MCP-1 protein from cultured colon cancer cells. The supernatant of irradiated colon cancer cells showed strong chemotactic activity for monocyte migration, but this activity was completely abolished by neutralizing antibody. CONCLUSIONS: Use of XRT induces MCP-1 expression in cancer cells, which causes circulating monocytes to be recruited into TAMs, which then upregulate TP expression in rectal cancer tissues.

The tumorigenic, invasive and metastatic potential of epithelial and round subpopulations of the SW480 human colon cancer cell line.

It has been reported that the SW480 human colon cancer cell line consists of E-type and R-type cells. The long-term tumorigenic potential, invasive and metastatic properties of these subclones have not been characterized. E-type and R-type cells were subcloned using limiting dilution methods from parental SW480 cells. The cell growth rate was determined by MTT colorimetric assay, and colony forming efficiency was analyzed using Matrigel-coated plates. The activity of matrix metalloproteinase (MMP) and of urokinase plasminogen activator (uPA) was assessed by zymography. Invasive and locomotive ability was analyzed using transwell chambers. In situ apoptosis detection of these subclones was also performed. In vivo long-term tumorigenicity and nodal metastasis were evaluated using nude mice. E-type cells produced spontaneously regressive tumors in spite of invasion and lymph node metastasis. In contrast, R-type cells revealed progressively growing tumors without invasion or metastasis. E-type cells exhibited increased apoptosis and invasive and motile ability, as well as strong MMP-9 and -2 activity. Although phorbol 12-myristate 13-acetate treatment induced MMP-9 activity in E-type cells, it had no effect on R-type cells. These findings suggest that E- and R-type cells may have different biological properties in terms of colon cancer progression, regression, invasion and nodal metastasis, and might serve as a useful model for these studies.