188 CALBINDIN-D28k IS INVOLVED IN THE APOPTOTIC PATHWAY AND PLAYS A ROLE AS AN ANTIAPOPTOTIC GENE IN HYDROGEN PEROXIDE-INDUCED CELL DEATH OF HUMAN ENDOMETRIAL ISHIKAWA CELLS

Abstract

Calbindin-D28k (CaBP-28k), a calcium-binding protein, buffers intracellular Ca²⁺ and eventually has antiapoptosis properties in neuron, osteoblast, and male germ cells. Although endometrial cancer is the most common invasive gynecological malignancy, CaBP-28k expression in apoptosis signalling is poorly understood. In this study, we investigated whether CaBP-28k expression is regulated by H₂O₂-induced apoptosis signalling in human endometrial Ishikawa cells. Ishikawa cells were treated with H₂O₂ in a dose-dependent manner (0, 0.25, 0.5, 1.0, 1.5, 2 mM) and a time-dependent manner (0, 15, 30, 60, 90, 120 min). The protein expressions of Bax, p53, and caspase 3 were determined by Western blot analysis. Treatment of Ishikawa cells with H₂O₂ induced an increase in Bax and p53 expression at the translational level at 1 mM for 1 h. Interestingly, overexpression of CaBP-28k caused a decrease in Bax, p53, and caspase 3 on H₂O₂-induced apoptosis in the Ishikawa cells. These results suggest that expression of CaBP-28k blocked up-regulation of apoptotic gene expression. In addition, knockdown of CaBP-28k expression using a small inhibitory RNA resulted in an elevation of H₂O₂-induced cell death and a increase in Bax, p53, and caspase 3 in H₂O₂-induced apoptosis, whereas cell survival was increased in overexpressing CaBP-28k cells, providing additional evidence that the induction of CaBP-28k expression may be associated with survival signalling during H₂O₂-mediated oxidative cell death. Taken together, these results imply that CaBP-28k expression is involved in the apoptotic pathway and may play a role as an antiapoptotic gene in the human endometrial Ishikawa cells.