Figure 5

Inhibition by chetomin of cytokine-stimulated lactate production, glucose utilization, and expression of SLC2A1 and HK2 by HT-29 cells. Serum-free cultures of HT-29 cells were pre-treated with chetomin or vehicle for 4 h, and TNFα (25 ng/ml) + IL-17 (50 ng/ml) was then added to some of the cultures for an additional 12 h. Medium samples were then collected for lactate and glucose assays, and protein extract samples of cell monolayers (30 μg of cellular protein/lane) were run in 7.5% gels. (A). Effect of increasing concentrations of chetomin on lactate production. Analysis of the data by linear regression showed a significant dose-dependent inhibition of lactate production by chetomin, P = 0.01. (B). Inhibition of lactate production (top) and glucose utilization (bottom) by 200 nM chetomin. Solid black and cross-hatched bars represent the means ± SE of assays performed with medium samples from quadruplicate cultures. Chetomin significantly inhibited lactate production and glucose utilization in cells stimulated by TNFα + IL-17, P < 0.001. (C). Western blots of cell extract proteins were probed for SLC2A1, HK2, or α-tubulin. (D). Results of Western blots shown in panel C were quantified by image analysis. Analysis of the data by linear regression showed significant dose-dependent inhibition by chetomin of SLC2A1 (P = 0.005) but not HK2 (P = 0.67, n.s.) expression. (E). Effect of chetomin on HT-29 cell numbers. Cultures were treated with vehicle or chetomin for 16 h, and cells were then harvested and counted. Each bar represents the mean ± SD of results obtained with six cultures. Bars with different letters were significantly different, P < 0.05.