Transglutaminase 2 (TGase2) is a calcium-dependent, cross-linking enzyme that catalyzes iso-peptide bond formation between peptide-bound lysine and glutamine residues. TGase 2 can activate NF-kappa B through the polymerization-mediated depletion of I-kappa B alpha without IKK activation. This NF-kappa B activation mechanism is associated with drug resistance in cancer cells. However, the polymers cannot be detected in cells, while TGase 2 over-expression depletes free I-kappa B alpha, which raises the question of how the polymerized I-kappa B alpha can be metabolized in cells. Among proteasome, lysosome and calpain systems, calpain inhibition was found to effectively increase the accumulation of I-kappa B alpha polymers in MCF7 cells transfected with TGase 2, and induced high levels of polymers as well in MDA-MB-231 breast cancer cells that naturally express a high level of TGase 2. Inhibition of calpain also boosted the level of I-kappa B alpha polymers in HEK-293 cells in case of TGase 2 transfection either with I-kappa B alpha or I-kappa B alpha mutant (S32A, S36A). Interestingly, the combined inhibition of calpain and the proteasome resulted in an increased accumulation of both I-kappa B alpha polymers and I-kappa B alpha, concurrent with an inhibition of NF-kappa B activity in MDA-MB-231 cells. This suggests that mu-calpain proteasome-dependent I-kappa B alpha polymer degradation may contribute to cancer progression through constitutive NF-kappa B activation. (C) 2010 Elsevier Inc. All rights reserved.