Solamargine induces apoptosis associated with p53 transcription-dependent and transcription-independent pathways in human osteosarcoma U2OS cells Xia Li a,b , Ying Zhao a , William K.K. Wu c , Shanshan Liu b , Min Cui b , Hongxiang Lou a, ⁎ a School of Pharmaceutical Sciences, Shandong University, Jinan 250012, PR China b School of Ocean, Shandong University, Weihai 264209, PR China c Institute of Digestive Diseases, LKS Institute of Health Sciences and Department of Medicine & Therapeutics, The Chinese University of Hong Kong, Hong Kong, PR China abstract article info Article history: Received 22 July 2010 Accepted 1 December 2010 Available online 14 December 2010 Keywords: Solamargine Apoptosis U2OS cells p53 Mitochondria Aims: Solamargine, a steroidal glycoalkaloid isolated from S. incanum, has been shown to induce apoptosis in several cancer cell lines. In this study, the involvement of p53 in the pro-apoptotic action of solamargine was investigated in human osteosarcoma U2OS cells. Main methods: The cytotoxicity of solamargine was evaluated by MTT assay. Solamargine-induced apoptosis was evidenced by chromatin condensation, formation of apoptotic bodies and exposure of phosphatidylserine on the extracellular surface as revealed by DAPI nuclear staining, transmission electron microscopy and flow cytometry, respectively. mRNA expressions of p53 and Bax were investigated using real time-PCR. Western blot was used to examine the changes in the expression levels of p53, Bax, Bcl-2, caspase-3, caspase-9 and cytochrome c. Subcellular localization of p53 was verified by immunofluorescence staining and cell fractionation. Key findings: Solamargine substantially reduced cell viability and induced apoptosis in osteosarcoma U2OS cells. In this connection, solamargine increased the mRNA and protein expressions of p53 and Bax (a pro- apoptotic protein downstream to p53). The expression of Bcl-2 (an anti-apoptotic protein) was also reduced. Furthermore, solamargine induced mitochondrial translocation of p53, loss of mitochondrial membrane potential, cytochrome c release and activation of caspase-9 and -3. p53-specific transcriptional inhibitor pifithrin-α or mitochondrial translocation inhibitor pifithrin-μ partially reversed solamargine-induced apoptosis. Significance: Solamargine activates the mitochondria-mediated apoptotic pathway in U2OS cells via both p53 transcription-dependent and -independent mechanisms. This compound may merit further investigation as a potential therapeutic agent for the treatment of cancer. © 2010 Elsevier Inc. All rights reserved. Introduction Osteosarcoma is the most common high-grade primary bone tumor and usually occurs in children and adolescents. In osteosarcoma, rapid proliferation of cancer cells with a high expression of anti-apoptotic genes is associated with poor prognosis. Resistance to apoptosis may also be induced as a secondary effect of conventional chemotherapy. Several anti- cancer agents, including methotrexate, doxorubicin, cisplatin, etoposide and cyclophosphamide, alone or in combination, are commonly employed to treat osteosarcoma (Wittig et al., 2002). Aside from the frequent acquisition of drug-resistant phenotypes, the occurrence of secondary malignancies is often associated with chemotherapy. The development of novel therapeutic agents therefore becomes greatly in need. This study was undertaken to investigate the effects and mechanisms that underlie the anti-cancer activity of solamargine (SM), a steroidal glycoalkaloid isolated from the Chinese herb Solanum incanum, in human osteosarcoma U2OS cells. Apoptosis is an evolutionarily conserved, orchestrated cell-death process characterized by membrane blebbing, shrinking of the cytoplasm, DNA fragmentation, and the formation of distinct apoptotic bodies that contain components of the dying cell. Activation of p53 is an important mechanism involved in apoptosis (Chipuk et al., 2004; Jung-Hynes and Ahmad, 2009; Smith et al., 2000). The striking pro-apoptotic and tumor suppressive potency of p53 lies in its pleiotropism, which includes transcription-dependent and -independent functions (Chipuk and Green, 2004; Schmitt et al., 2002; Schuler and Green, 2005). In this regard, p53 regulates the expression of multiple genes through its direct interaction with chromatin and other transcription regulators. However, there is also an emerging evidence supporting that p53 has an extranuclear role in the cytoplasm for the induction of apoptosis (Chipuk and Green, 2004). For instance, p53 has been shown to directly activate the pro-apoptotic protein Bax, a member of the Bcl-2 family of genes, resulting in mitochondrial membrane permeabilization and release of cytochrome c, Life Sciences 88 (2011) 314–321 ⁎ Corresponding author. School of Pharmaceutical Sciences, Shandong University, Wenhua West Road 44, 250012 Jinan, PR China. Tel.: + 86 531 88364778; fax: + 86 531 88382019. E-mail address: louhongxiang@sdu.edu.cn (H. Lou). 0024-3205/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2010.12.006 Contents lists available at ScienceDirect Life Sciences journal homepage: www.elsevier.com/locate/lifescie