Toxicology 234 (2007) 203–215 Apoptosis induced by uterine 24p3 protein in endometrial carcinoma cell line Hsiu Hsia Lin b , Wen-Wei Li b , Ying-Chu Lee a , Sin-Tak Chu a,b,∗ a Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan b Institute of Biochemical Science, College of Life Science, National Taiwan University, Taipei, Taiwan Received 10 January 2007; received in revised form 26 February 2007; accepted 26 February 2007 Available online 12 March 2007 Abstract The biological functions and reaction pathways of lipocalins in mammalian system were sought. Mouse uterine 24p3 protein is a secreted lipocalin from mouse uterus. To evaluate the effect of uterine 24p3 protein on the reproductive system, endometrial carcinoma cell line (RL95-2) was an experimental target for achieving the in vitro study. The cells were treated with 0.75 M dexamethasone (DEX) or under serum-free medium to mimic the stress environment for various time periods, then employing Western blot to measure the 24p3 protein secretion. It showed the time-dependent induction effect on 24p3 protein and suggested the level of protein secretion correlating to environmental stress. Furthermore, the supplementation of 24p3 protein to the medium accompanied the reduction of cell viability. It showed that the 24p3 protein may be a death factor under conditional media via PI and annexinV-FITC assay. Based on the autocrine hypothesis, we investigated the effect of 24p3 protein on cultured RL95-2 cells upon the 24p3 protein interaction. We have demonstrated significant increase in intracellular reactive oxygen species upon 24p3 protein interaction. While the changes of mitochondrial membrane potential and cytochrome c release from mitochondria occurred, the activities of caspase-8, -9 and -3 were found to have increased. The condensation of DNA was occurred suggesting that 24p3 protein induced irreparable DNA damage, which in turn triggered the process of apoptosis. It shows evidence for the direct effect of this protein on endometrial cells. These findings suggest that 24p3 protein creates an intracellular oxidative environment that induces apoptosis in RL95-2 cells. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Cell death; Endometrial cell; Lipocalin; Uterine secretory protein 1. Introduction Lipocalins are a large and ever expanding group of proteins exhibiting considerable structural and func- tional variation, both within and between species (Garibotti et al., 1997; Flower, 1994; Lopez-Boado et al., ∗ Corresponding author at: Institute of Biological Chemistry, Academia Sinica, P.O. Box 23-106, Taipei, Taiwan. Tel.: +886 2 23620261x4531; fax: +886 2 33664073. E-mail address: stc316@gate.sinica.edu.tw (S.-T. Chu). 1996). However, the functions of many lipocalins remain unclear to date. Human neutrophil gelatinase-associated lipocalin (hNGAL/24p3) is a 25 kDa glycoprotein, which has the alternative name of 24p3 protein like a lipocalin from mice (Akerstrom et al., 2000). It belongs to the lipocalin super-family with a hydrophobic bind- ing site (Flower, 1996), first found in the granules of the human neutrophil (Kjeldsen et al., 1994); homologous protein has been identified in mouse (24p3/uterocalin) (Akerstrom et al., 2000). NGAL/24p3 is an acute phase protein expressed in several tissues and organs, and iden- tified in human, mouse, and rat (SIP24) genomes as 0300-483X/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2007.02.017