Characterization of cancer stem-like cells in the side population cells of human gastric cancer cell line MKN-45.

OBJECTIVE: Side population (SP) cells may play a crucial role in tumorigenesis and the recurrence of cancer. Many kinds of cell lines and tissues have demonstrated the presence of SP cells, including several gastric cancer cell lines. This study is aimed to identify the cancer stem-like cells in the SP of gastric cancer cell line MKN-45. METHODS: We used fluorescence activated cell sorting (FACS) to sort SP cells in the human gastric carcinoma cell line MKN-45 (cells labeled with Hoechst 33342) and then characterized the cancer stem-like properties of SP cells. RESULTS: This study found that the SP cells had higher clone formation efficiency than major population (MP) cells. Five stemness-related gene expression profiles, including OCT-4, SOX-2, NANOG, CD44, and adenosine triphosphate (ATP)-binding cassette transporters gene ABCG2, were tested in SP and MP cells using quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). Western blot was used to show the difference of protein expression between SP and MP cells. Both results show that there was significantly higher protein expression in SP cells than in MP cells. When inoculated into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, SP cells show higher tumorigenesis tendency than MP cells. CONCLUSIONS: These results indicate that SP cells possess cancer stem cell properties and prove that SP cells from MKN-45 are gastric cancer stem-like cells.

Tumor metastasis and the reciprocal regulation of heparanase gene expression by nuclear factor kappa B in human gastric carcinoma tissue.

AIM: To investigate whether NF-kappaB is activated in human gastric carcinoma tissues and, if so, to study whether there is any correlation between NF-kappaB activity and heparanase expression in gastric carcinoma. METHODS: NF-kappaB activation was assayed by immunohistochemical staining in formalin-fixed, paraffin-embedded specimens from 45 gastric carcinoma patients. Electrophoretic mobility shift assay (EMSA) method was used for nuclear protein from these fresh tissue specimens. Heparanase gene expression was quantified using quantitative RT-PCR. RESULTS: The nuclear translocation of RelA (marker of NF-kappaB activation) was significantly higher in tumor cells compared to adjacent and normal epithelial cells ((41.3+/-3.52)% vs (0.38+/-0.22) %, t = 10.993, P = 0.000<0.05; (41.3+/-3.52)% vs (0+/-0.31)%, t = 11.484, P = 0.000<0.05). NF-kappaB activation was correlated with tumor invasion-related clinicopathological features such as lymphatic invasion, pathological stage, and depth of invasion (Z = 2.148, P = 0.032<0.05; chi(2) = 8.758, P = 0.033<0.05; chi(2) = 18.531, P = 0.006<0.05). NF-kappaB activation was significantly correlated with expression of heparanase gene (r = 0.194, P = 0.046<0.05). CONCLUSION: NF-kappaB RelA (p65) activation was related with increased heparanase gene expression and correlated with poor clinicopathological characteristics in gastric cancers. This suggests NF-kappaB as a major controller of the metastatic phenotype through its reciprocal regulation of some metastasis-related genes.