Galectin-3 Regulates Mitochondrial Stability and Antiapoptotic Function in Response to Anticancer Drug in Prostate Cancer Tomoharu Fukumori, 1 Natsuo Oka, 1 Yukinori Takenaka, 3 Pratima Nangia-Makker, 4 Essam Elsamman, 1 Toshinori Kasai, 1 Masayuki Shono, 2 Hiro-omi Kanayama, 1 Julie Ellerhorst, 5 Reuben Lotan, 6 and Avraham Raz 4 1 Department of Urology and 2 Support Center for Advanced Medical Sciences, The University of Tokushima Graduate School, Institute of Health Biosciences, Tokushima, Japan; 3 Department of Otolaryngology and Sensory Organ Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan; 4 Tumor Progression and Metastasis Program, Karmanos Cancer Institute, Wayne State University, Detroit, Michigan; and Departments of 5 Experimental Therapeutics and 6 Thoracic/Head and Neck Medical Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas Abstract Prostate cancer is one of the malignant tumors which exhibit resistance to anticancer drugs, at least in part due to enhanced antiapoptotic mechanisms. Therefore, the under- standing of such mechanisms should improve the design of chemotherapy against prostate cancer. Galectin-3 (Gal-3), a multifunctional oncogenic protein involved in the regulation of tumor proliferation, angiogenesis, and apoptosis has shown antiapoptotic effects in certain cell types. Here, we show that the expression of exogenous Gal-3 in human prostate cancer LNCaP cells, which do not express Gal-3 constitutively, inhibits anticancer drug–induced apoptosis by stabilizing the mito- chondria. Thus, Gal-3-negative cells showed 66.31% apoptosis after treatment with 50 Mmol/L cis -diammine-dichloroplati- num for 48 hours, whereas two clones of Gal-3-expressing cells show only 2.92% and 1.42% apoptotic cells. Similarly, Gal-3- negative cells showed 43.8% apoptosis after treatment with 300 Mmol/L etoposide for 48 hours, whereas only 15.38% and 14.51% of Gal-3-expressing LNCaP cells were apoptotic. The expression of Gal-3 stimulated the phosphorylation of Ser 112 of Bcl-2-associated death (Bad) protein and down-regulated Bad expression after treatment with cis -diammine-dichlor- oplatinum. Gal-3 also inhibited mitochondrial depolarization and damage after translocation from the nuclei to the cytoplasm, resulting in inhibition of cytochrome c release and caspase-3 activation. These findings indicate that Gal-3 inhibits anticancer drug–induced apoptosis through regula- tion of Bad protein and suppression of the mitochondrial apoptosis pathway. Therefore, targeting Gal-3 could improve the efficacy of anticancer drug chemotherapy in prostate cancer. (Cancer Res 2006; 66(6): 3114-9) Introduction Prostate cancer is the most common cancer in men, with f230,110 new cases and 29,900 deaths annually in the U.S. in 2004 (1). About 10% to 20% of men with prostate cancer present with metastatic disease. Initially, primary androgen ablation therapy leads to a reduction in serum levels of prostate-specific antigen in patients with metastasis of prostate cancer, but in almost every patient, the disease eventually becomes hormone-refractory and apoptosis-resistant (2). The prognosis for these patients is poor because conventional chemotherapeutic drugs have little effect on hormone-refractory prostate cancer and do not provide a marked survival advantage (3). Therefore, it is necessary to clarify the mechanism of chemotherapeutic drug resistance of prostate cancer and develop a novel strategy for the treatment of prostate cancer. The galectins comprise a family of 14 members of h-galactoside- binding proteins, characterized by their affinity for h-galactosides and by a conserved sequence of the carbohydrate recognition domain that bind to the carbohydrate portion of cell surface gly- coproteins or glycolipids. Galectin-3 (Gal-3), a 31-kDa chimeric gene product, is a multifunctional oncogenic protein which regu- lates cell growth, cell adhesion, cell proliferation, angiogenesis, and apoptosis (4–12). We and others have shown that endogenous Gal-3, which contains the NWGR anti-death motif of the Bcl-2 family, inhibits epithelial cell apoptosis induced by staurosporine, chemotherapeutic agents such as cisplatin, genistein, tumor necrosis factor, and nitric oxide (13–16). We also reported that nuclear export of phosphorylated Gal-3 regulates its antiapoptotic activity in response to chemotherapeutic drugs (17). Phospho- rylated wild-type Gal-3 was exported from the nucleus to the cytoplasm and protected cancer cells from drug-induced apoptosis, whereas nonphosphorylated Ser 6 mutant Gal-3 was neither exported from the nucleus nor protected cancer cells from drug- induced apoptosis (17). Other investigators also reported that under certain conditions, Gal-3 was found in the cytoplasm and perinuclear mitochondrial membranes (18, 19), where it was involved in the control of apoptosis, possibly through an inter- action with the bcl-2 protein (20). Expression and cellular localization of Gal-3 are important for the prognosis of a variety of cancers. Sanjuan et al. reported on the down-regulation of Gal-3 in colorectal cancer with increased cytoplasmic expression of Gal-3 at more advanced stages (21). Down-regulation of Gal-3 was also observed in prostate cancer, and both nuclear exclusion and cytoplasmic localization of Gal-3 are correlated with cancer progression (19, 22). Furthermore, Califice et al. reported that cytoplasmic Gal-3 expression in LNCaP cells induced tumor growth, invasion, angiogenesis, and decreased inducible apoptosis (23). Here, we investigated the effects of introducing Gal-3 into non-expressing human prostate cancer cells (LNCaP) on their response to proapoptotic chemotherapeutic agents and explored the mechanisms involved in the apoptotic function of Gal-3. We show that the expression of Gal-3 in LNCaP cells inhibits cis -diammine-dichloroplatinum (CDDP)– and etoposide-induced Requests for reprints: Avraham Raz, Tumor Progression and Metastasis Program, Karmanos Cancer Institute, Wayne State University, 110 East Warren Avenue, Detroit, MI 48201. Phone: 313-833-0960; Fax: 313-831-7518; E-mail: raza@karmanos.org. I2006 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-05-3750 Cancer Res 2006; 66: (6). March 15, 2006 3114 www.aacrjournals.org Research Article Research. on September 5, 2015. © 2006 American Association for Cancer cancerres.aacrjournals.org Downloaded from