In-vitro growth characteristics were tested by a colony formation assay.25 Briefly, after 48 hr of siRNA transfection, cells were harvested, and 500 trypan blue-negative viable cells were replated in each well of six-well plates. The cells were cultured in RPMI 1640 supplemented with 5% serum, and surviving colonies were counted 10 days later after staining with methylene blue. Cell migration was measured in a modified Boyden selleck screening library
chamber.27 Briefly, polycarbonate filters with 8 ��m pores were coated with 100 ��g/ml of collagen in 0.5 M acetic acid for 16 hr. The coated filter was then placed on a 12-blind-well chemotaxis chamber, and 105 cells in 100 ��l per well were loaded into the upper wells. The cells were incubated for 15 min before loading. After incubation at 37��C in 5% CO2 for 4 hr, the filter was disassembled. The upper side of the filter was then scraped free of cells. The cells on the lower side of the filter were fixed with methanol and stained with a Diff-Quick staining kit (International Reagent, Kobe, Japan). The number of cells that migrated to the lower side of the filter was counted. The number of migrated cells in each experiment was adjusted by the cell viability assay (trypan-blue assay) to correct for anti-proliferation effects of the IL-8 neutralizing antibody (corrected migrated cell number = counted migrated cell number/percentage this website
of viable cells).28 Statistical analyses were performed using version 5.0 of GraphPad Prism for Windows (GraphPad Software, San Diego, CA). p < 0.05 was considered significant, and p �� 0.05 but p < 0.1 was considered borderline significant. To assess the association between IL-8 expression and KRAS mutations in lung cancer, we first examined IL-8 mRNA expression in a panel of lung cancer cell lines by quantitative RT-PCR analysis. IL-8 mRNA levels were significantly higher in NSCLCs harboring KRAS mutation or EGFR mutations than those with wild-type KRAS/EGFR (Fig. 1a). In contrast, IL-8 expression was low or undetectable in SCLCs, in agreement with a previous study.29 We next examined IL-8 protein levels in cultured NSCLC cell lines. IL-8 protein levels were significantly higher in KRAS mutants or EGFR mutants than those with wild-type Ibrutinib
KRAS/EGFR (Fig. 1b). Quantitatively, differences in IL-8 levels were more prominent in KRAS mutants than EGFR mutants compared to NSCLCs with wild-type KRAS/EGFR. Notably, we were able to confirm the IL-8 overexpression in NCI-H1792 cells with a baseline protein level of 5,662 pg/ml, and microarray analysis revealed transcriptional downregulation of IL-8 expression (fold-change was ?17.4) by shRNA-mediated KRAS knockdown.21 We also examined the expression of IL-8 receptors, CXCR1 and CXCR2, to determine whether these receptors are also differentially expressed in NSCLC cell lines according to the mutation status of EGFR and KRAS.