Nicotinamide mononucleotide adenylyltransferase2 overexpression enhances colorectal cancer cell-kill by Tiazofurin.

Colorectal cancer cells exhibit limited cytotoxicity towards Tiazofurin, a pro-drug metabolized by cytosolic nicotinamide mononucleotide adenylyltransferase2 (NMNAT2) to thiazole-4-carboxamide adenine dinucleotide, a potent inhibitor of inosine 5'-monophosphate dehydrogenase required for cellular guanylate synthesis. We tested the hypothesis that colorectal cancer cells that exhibit low levels of NMNAT2 and are refractory to Tiazofurin can be rendered sensitive to Tiazofurin by overexpressing NMNAT2. Transfection of hNMNAT2 resulted in a six- and threefold cytoplasmic overexpression in Caco2 and HT29 cell lines correlating with Tiazofurin-induced enhanced cell-kill. Folate receptors expressed on the cell surface of 30-50% colorectal carcinomas were exploited for cellular targeting with Tiazofurin encapsulated in folate-tethered nanoparticles. Our results indicated that in wild-type colorectal cancer cells, free Tiazofurin-induced EC50 cell-kill was 1500-2000 µM, which was reduced to 66-156 µM in hNMNAT2-overexpressed cells treated with Tiazofurin encapsulated in non-targeted nanoparticles. This efficacy was improved threefold by encapsulating Tiazofurin in folate-tethered nanoparticles to obtain an EC(50) cell-kill of 22-59 µM, an equivalent of 100-300 mg m(-2) (one-tenth of the approved dose of Tiazofurin in humans), which will result in minimal toxicity leading to cancer cell-kill. This proof-of-principle study suggests that resistance of colorectal cancer cell-kill to Tiazofurin can be overcome by sequentially overexpressing hNMNAT2 and then facilitating the uptake of Tiazofurin by folate-tethered nanoparticles, which enter cells via folate receptors.

Cytotoxicity of a new IMP dehydrogenase inhibitor, benzamide riboside, to human myelogenous leukemia K562 cells.

COMPARE computer program suggested that benzamide riboside, BR, 3-(1-deoxy-beta-D-ribofuranosyl)benzamide, should have a similar mechanism of action as that of tiazofurin, an inhibitor of IMP dehydrogenase (IMPDH). This hypothesis was tested in K562 cells in culture. BR was cytotoxic to K562 cells with an IC50 of 2 microM. Incubation of K562 cells with BR resulted in a significant decrease in GMP and GTP levels with a concurrent increase in IMP pools, and with a significant inhibition of IMPDH activity. However, 290-fold higher BR concentration was needed to demonstrate in vitro inhibition of IMPDH activity, suggesting that the agent may require metabolism to exert its action. These results provide evidence that BR is a new inhibitor of IMPDH. This investigation should be helpful to design new analogues having activity against IMPDH.