Figure 2

Moderate increase in the concentration of D -(+)-glucose down-regulated, but deficiency of D -(+)-glucose, L -leucine, L -methionine, L -cysteine or combination of L -methionine and L -cysteine up-regulated the relative luciferase activity of p27-5'UTR in human MDA-MB-231 breast cancer cells in vitro. (a) Schematic drawing of the luciferase reporter plasmid containing a proximal 5'-upstream region (-575) of the p27 gene (p27-5'UTR). (b) Moderate increase in the concentration of D-(+)-glucose down-regulated the relative luciferase activity of p27-5'UTR in MDA-MB-231 cells. This panel also shows that rotenone (inhibitor of NADH dehydrogenase (Complex 1) of the mitochondrial respiratory oxidation-phosphorylation chain) and AICA riboside (inhibitor of AMPK (5'-AMP-activated protein kinase)) up-regulated the relative luciferase activity of p27-5'UTR in MDA-MB-231 cells. In contrast, compound C (activator of AMPK) down-regulated the relative luciferase activity of p27-5'UTR in these cells. Metformin (the most widely prescribed anti-diabetic drug that activates AMPK in the cells by mechanisms that are dependent on its upstream kinase, the tumor suppressor LKB1) did not either up or down-regulate the relative luciferase activity of p27-5UTR probably because MDA-MB-231 cells lack LKB1. (c) This panel shows that deficiency of D-(+)-glucose, L-leucine, L-methionine, L-cysteine, or combination of L-methionine and L-cysteine up-regulated the relative luciferase activity of p27-5'UTR in MDA-MB-231 cells. All assays were performed in triplicates and repeated three times.