The Internalization of Bacillus Calmette-Guerin (BCG) in Bladder Cancer Cells May be Inhibited by Human beta-defensin 3

Lim, Song Won; Chang, In Ho; Kim, Tae Hyoung; Myung, Soon Chul; Kim, Kyung Do; Moon, Young Tae; Kim, Jin Wook; Chi, Byung Hoon

Abstract

PURPOSE
To investigate whether secretion of human beta-defensin 3 (HBD-3) is induced by bacillus Calmette-Guerin (BCG) and to determine whether HBD-3 affects BCG internalization in bladder cancer cells.
MATERIALS AND METHODS
RTPCR analysis was used to determine whether HBD-3 mRNA increases after incubation with BCG. HBD-3 proteins in 5637 and T24 human bladder cancer cell lines were assayed by ELISA. The internalization rate was evaluated by double immunofluorescence assay and confocal microscopy to test the optimal dose of HBD-3 for BCG internalization. We also investigated the difference in internalization rates and cell viability between recombinant HBD-3 protein, anti-HBD-3 antibody, and HBD-3 plus anti-HBD-3 antibody pretreatments.
RESULTS
BCG induced HBD-3 mRNA expression and HBD-3 production dose and time-dependently in bladder cancer cells and affected BCG internalization. Pretreatment with recombinant HBD-3 protein lowered internalization of BCG dose-dependently. Moreover, anti-HBD-3 antibody prevented the effect of HBD-3 on BCG internalization in bladder cancer cells. The internalization rate of BCG pretreated with anti-HBD-3 antibody was higher than that in the control. The BCG internalization rate in cells pretreated with anti-HBD-3 antibody plus recombinant HBD-3 protein was higher than that in the control. BCG decreased bladder cancer cell viability, and anti-HBD-3 antibody prevented the inhibitory role of HBD-3 on the anti-proliferative effects of M. bovis BCG in bladder cancer cells.
CONCLUSIONS
Bladder cancer cells produce HBD-3 when they are infected by BCG to defend themselves against BCG internalization, which plays an important role during the initiation and propagation of the immunotherapeutic response in bladder cancer cells.