In vitro activity: 3PO [full/chemical name of 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one] is an inhibitor of the PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase). 3PO inhibits the proliferation of cancer cells when combined with phenformin. Enhanced glycolysis in cancer cells presents a target for chemotherapy. 3PO reduces glycolytic flux and suppresses glucose uptake. PFKFB3 inhibitors can suppress glucose uptake, resulting in an increase in autophagy. The combination of selective autophagy inhibitors with 3PO may be useful for the treatment of cancer.

Kinase Assay: Glucose was assayed with 2-μl cell culture medium using the glucose oxidase and peroxidase reagent from Sigma-Aldrich. This is a colorimetric procedure in which the oxidation of glucose is coupled with glucose oxidase and peroxidase to the oxidation of dianisidine. Incubation at room temperature was performed for 30 min in a total volume of 0.3 ml, including 80 μl 1 M sodium phosphate (pH 6.0). The incubation was stopped by the addition of 0.2 ml 12 N sulfuric acid. Light absorbance at 540 nm was measured with 0.2 ml of the mixture using a plate reader and the glucose concentration was calculated by reference to a glucose standard solution.

Cell Assay: Cells and determination of growth. Human bladder cancer cell lines, namely 5637, HT1197, HT1376, RT4, SW780, T24, TCCSUP and UM-UC-3, and human colonic cancer cells (Caco-2, HCT116 and HT29) were obtained from the American Type Culture Collection (Rockville, MD, USA), and were incubated at 37°C with 5% carbon dioxide. After plating 5000 cells in 0.2 ml RPMI-1640 medium with 5% fetal calf serum for 24 hours, the medium was replaced with either control medium or medium containing drugs. Cell growth after a further 72 h was monitored by the increase in protein determined by staining with sulforhodamine B, essentially as described by Vichai and Kirtikara