Abstract

In our previous studies, Rat-1 cells treated with Vibrio parahaemolyticus thermostable direct hemolysin (TDH) developed morphological changes including shrinkage and rounding of the cells. Such alterations suggest that TDH stimulates reorganization of the cellular cytoskeleton. In the current study, the effects of TDH on actin cytoskeleton, chromatin, and caspase-3 were evaluated. Rat-1 cells treated with TDH 5 µg/ml showed redistribution of actin with loss of stress fibers, a floccular staining pattern, cellular membrane blebbing, and cell rounding as assessed by fluorescent actin-staining. This actin redistribution was time dependent. Actin was rapidly disappeared within 10 min after TDH exposure, and the maximal effect was detected by 30 min. TDH-treated cells also showed chromatin condensation which is time dependent. The changes were also detected by as early as 10 min, with the maximal effect by 20 min. Moreover, caspase-3 was activated in TDH-treated Rat-1 cells by time-dependent manner. Pretreatment with a caspase inhibitor (ZVAD-FMK) partially reduced the activation of caspase-3 in the TDH-treated cells. All of the changes found in this study appear to be linked, and the findings, especially activation of caspase-3 in TDH-treated Rat-1 cells, provide a new insight into TDH-mediated cytotoxicity.

Key words: Thermostable direct hemolysin (TDH), Vibrio parahaemolyticus, Rat-1 cells, actin disruption, caspase-3 activation, chromatin condensation.