Figure 1.
Copper chelation selectively affects proliferation, survival and migration of colon cancer HT-29 cells bearing BRAFV600E mutation compared to HCT-116 cells carrying BRAFwt. (a) Copper depletion by treatment with the chelating agent tetrathiomolybdate (TM) affects short-term proliferation and survival of colon cancer cells bearing BRAFV600E mutation. Colon cancer cell lines BRAFwt (HCT-116) and BRAFV600E (HT-29) were treated with TM (dose range 1-100 μM). After 24 hours the WST-1 cell proliferation assay was performed. Results are reported as means ± S.D. of three independent experiments. (b) TM treatment affects clonogenic ability of BRAFV600E colon cancer cells. Long-term response to TM treatment was evaluated by clonogenic assay. BRAFwt and BRAFV600E colon cancer cells were seeded at a density of 1.5x103/60-mm tissue culture dish and treated with TM, CuSO4 or both. Media were changed every 3–4 days. After 10 days of culture colonies were stained with crystal violet and then observed under an inverted microscope. Representative images are shown. The experiment was performed two times in duplicate. (c) TM treatment reduces bioluminescent emission from BRAFV600E compared to BRAFwt colon cancer cells expressing luciferase. Non-invasive bioluminescent analysis using D-luciferin as substrate performed on BRAFwt and BRAFV600E colon cancer cells engineered to express luciferase exposed to TM for 1 week. (d) TM treatment inhibits cells migration ability of BRAFV600E colon cancer cells. Scratch assay was performed on colon cancer HCT-116 (upper panel) and HT-29 cells (lower panel). A 1-mm wide scratch was performed on cells monolayer, then cells were treated with 1 μM TM. Cells migration and scratch closure was assessed every 24 hours. Left panels show representative images taken at different time points. The scratched area was determined using the ImageJ software, and the quantitative data are shown in the right panels as mean ± S.D. of three independent experiments. In all panels * denotes p < 0.05.