OBJECTIVETo investigate the reactivity of colon cancer cell line SW480 and CD133(+) SW480 subsets to hypoxia in vitro and the changes in the expressions of anti-apoptosis and angiogenesis genes.

METHODSSW480 cells was subjected to CoCl(2) exposure at varying concentrations and for different time lengths to induce hypoxia, and the protein expression of hypoxia induced factor 1α (HIF-1α) was detected by Western blotting. The CD133(+) SW480 cells were sorted by magnetic activated cell sorting (MACS) and their proportion was assayed by flow cytometry (FCM). The CD133(+) SW480 subsets were exposed to CoCl(2) at the optimal concentration with exposure time selected in terms of HIF-1α level, and their tumor stem cell sphere formation ability was evaluated. Real-time PCR was used to compare the mRNA expression levels of the surface markers of colon cancer stem cells (CD133 and PROM1), survivin, and vascular endothelial growth factor (VEGF).

RESULTSExposure to 200 µmol/L CoCl(2) for 8 h resulted in the highest HIF-1α expression in SW480 cells, but the same exposure failed to induce HIF-1α expression in CD133(+) SW480 subsets. The CD133(+) SW480 subsets, after CoCl(2)-induced hypoxia, showed significantly enhanced ability of cell sphere formation. Hypoxia of SW480 cells caused significant increases in CD133, survivin and VEGF mRNA levels by 1.607∓0.103, 2.745∓0.370 and 3.798∓0.091 folds, respectively (P<0.05).

CONCLUSIONCoCl(2) can simulate hypoxia in colon cancer cells in vitro to induce stable HIF-1α expression, which is concentration- and time-dependent. The hypoxia-stimulated tumor stem sells show an enhanced sphere formation and anti-apoptotic and anti-angiogenic abilities.

Apoptosis ; physiology ; Cell Hypoxia ; Cell Line, Tumor ; Colonic Neoplasms ; pathology ; Computer Simulation ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Neoplastic Stem Cells ; pathology ; Neovascularization, Pathologic ; physiopathology

BACKGROUND/AIMS: Deoxycholic acid (DCA), a secondary bile acid, has been implicated to promote colon cancer growth and progression. However, its molecular mechanisms are largely unknown. In this study, we investigated the effects of DCA on proliferation, migration, and invasiveness of colon cancer cells (HT-29). METHODS: HT-29 cells were incubated with either medium (control) only or DCA for 24-48 hours. Time courses of RT-PCR for vascular endothelial growth factor (VEGF) and hypoxia-inducible factor (HIF)-1alpha mRNA expression, Western blotting for VEGF and matrix metalloproteinase (MMP)-9, zymography for MMP-9 activation, and wound-migration assay were determined after various concentrations of DCA (0-80mum) treatment. Moreover, these experiments were reassessed after pretreatments (2-6 hours) with specific inhibitors of various signal pathways. RESULTS: DCA enhanced HIF-1alpha mRNA expression, VEGF mRNA and VEGF protein expression, MMP-9 protein expression/activation, and cell migration ability in a dose-related manner. DCA-induced VEGF protein expression was inhibited by pretreatment with NS-398 (COX-2 inhibitor), PDTC (NF-kappaB inhibitor), or tauroursodeoxycholic acid (TUDC). DCA-induced cell migration ability was inhibited by pretreatment of GF109203X, a protein kinase C inhibitor. DCA-induced MMP-9 protein expression/activation was inhibited by pretreatment with SB203580, U0126, or PDTC. CONCLUSIONS: DCA significantly upregulates invasive and angiogenic potentials of human colon cancer cells through multiple signal transduction pathways.
Cell Movement/drug effects ; Colonic Neoplasms/metabolism/pathology/*physiopathology ; Deoxycholic Acid/*pharmacology ; HT29 Cells ; Humans ; Hypoxia-Inducible Factor 1/metabolism ; Matrix Metalloproteinase 9/metabolism ; Neoplasm Invasiveness ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction/drug effects ; Vascular Endothelial Growth Factor A/metabolism