Elsevier

Food and Chemical Toxicology

Volume 45, Issue 11, November 2007, Pages 2155-2164
Food and Chemical Toxicology

Solamargine induces apoptosis and sensitizes breast cancer cells to cisplatin

https://doi.org/10.1016/j.fct.2007.05.009Get rights and content

Abstract

Solamargine (SM), a major steroidal alkaloid glycoside, was purified from Solanum incanum plant. SM exhibited the most cytotoxic effect comparing with that of cisplatin (cDDP), methotrexate (MTX), 5-fluorouracil (5-FU), epirubicin (EPI) and cyclophosphamide (CP) against human breast cancer cells. In this study, SM induces apoptosis of the breast cancer cells and the mechanism was characterized. SM up-regulated the expressions of external death receptors, such as tumor necrosis factor receptor I (TNFR-I), Fas receptor (Fas), TNFR-I-associated death domain (TRADD), and Fas-associated death domain (FADD). SM also enhanced the intrinsic ratio of Bax to Bcl-2 by up-regulating Bax and down-regulating Bcl-2 and Bcl-xL expressions. These effects resulted in the release of mitochondrial cytochrome c and activation of caspase-8, -9 and -3 in the cells, indicating that SM triggered extrinsic and intrinsic apoptotic pathways of breast cancer cells. Similar to function way of SM, cDDP causes cancer cell apoptosis though caspase-8/caspase-3 and Bax/cytochrome c pathways, but the resistance to cDDP is correlated with Bcl-2 and Bcl-xL overexpression. However, the overexpression of Bcl-2 and Bcl-xL can be broken through by SM. The combined treatment of SM and cDDP significantly reduced Bcl-2 and Bcl-xL expressions, and enhanced Bax, cytochrome c, caspase-9 and -3 expressions in breast cancer cells. Thus, the combined use of SM and cDDP may be effective in cDDP-resistant breast cancer.

Introduction

Apoptosis has a key role in the regulation of growth of normal and neoplastic tissues (Ghobrial et al., 2005). Extrinsic and intrinsic-mediated pathways lead to apoptosis. The extrinsic-mediated pathway is also called the receptor-mediated pathway. It is characterized by the activation of cell surface ligand-gated death receptors, including those of the superfamily of tumor necrosis factor receptors, such as TNFR, the Fas and TRAIL receptors (Ashkenazi and Dixit, 1998). When specific ligands bind to the extracellular domain of death receptors, receptor trimerization will be triggered. The TRADD and the FADD adaptor molecules then bind to the cytoplasmic domain of the receptor. Initiator caspase-8 then causes activation and further cleaves and activates effector caspase-3, inducing irreversible cell death (Thorburn, 2004). The intrinsic or mitochondria-mediated pathway can be activated directly without being triggered by a death receptor. In this process, mitochondria will be disrupted by cell stress, releasing of cytochrome c into the cytoplasm. This is a known means of converting procaspase-9 into its activated form, caspase-9, which can active caspase-3, and trigger the irreversible apoptotic program (Li et al., 1997, Thomas et al., 2003). The Bcl-2 protein family is the most important regulator of the intrinsic and apoptotic processes, and this family includes a pro-apoptotic member, such as Bax and such anti-apoptotic members as Bcl-2 and Bcl-xL. Bcl-2 and Bcl-xL act as repressors of apoptosis by blocking the release of cytochrome c and also are important in resistance to chemotherapy and radiotherapy (Tortora et al., 2003, Tsujimoto, 2003). However, Bax has been demonstrated to play a key role in initiating mitochondrial dysfunction (Wei et al., 2001). Mitochondria dysfunction leads to the release of cytochrome c, which activates downstream effectors and develops a DNA damage-initiated apoptotic pathway (Plas and Thompson, 2002). In fact, the apoptotic process is regulated by the ratio between Bax and Bcl-2, and the Bax:Bcl-2 ratio is enhanced by anti-cancer drugs, before apoptosis (Del Bello et al., 2001).

Many chemotherapeutic drugs cause the cytotoxicity of cancer cells by apoptosis. cDDP [cis-diamminedichloroplatinum(II)] is commonly used as a chemotherapeutic agent in the treatment of human breast cancers. The cellular responses to cDDP include the suppression of DNA replication, cell cycle arrest, transcriptional inhibition, and apoptosis. Two of the signaling pathways that are involved in cDDP-induced DNA damage to irreversible program cell death are the caspase-8/caspase-3 pathway and the Bax/cytochrome c pathway (Siddik, 2003). However, the resistance of Bcl-2 and Bcl-xL has also been reported in breast cancer chemotherapy (Del Bufalo et al., 2002, Mercatante et al., 2002). Bcl-2 and Bcl-xL molecules can prevent permeability transition and stabilize the outer-membrane of mitochondria (Shimizu et al., 1998). A recent study has shown that the Bcl-2-mediated cDDP resistance in breast cancer cells depends on the up-regulation of glutathione production (Rudin et al., 2003).

SM is a major glycoalkaloid that is found in at least 100 Solanum species (Lorey et al., 1996). Structurally, SM has a steroidal aglycone and the trisaccharide (bis-α-l-rhamnopyranosyl-β-glucopyranose) chain attaches to the 3-hydroxy group of steroidal aglycone (Alzerreca and Hart, 1982). Our previous studies demonstrated that SM-caused apoptosis of human hepatoma and lung cancer cells though extrinsic pathway (Kuo et al., 2000, Liang et al., 2004, Liu et al., 2004). This study determined the extrinsic and intrinsic apoptosis pathways of SM on human breast cancer cells. Besides, SM and cDDP synergistically up-regulated the Bax, cytochrome c, caspase-9 and -3 expressions, and down regulated the Bcl-2 and Bcl-xL expressions. That may increase the susceptibility of breast cancer cells to cDDP.

Section snippets

Drugs

Drugs were provided as pure substances. SM was purified from Solanum incanum according to the previous procedures (Lin et al., 1990). SM (CAS number: 20311-51-7) and 5-FU (CAS number: 51-21-8; Sigma Chemical Co., St Louis, MO) were diluted in dimethyl sulfoxide (DMSO). cDDP (CAS number: 15663-27-1; Bristol-Myers Squibb, Woerden, The Netherlands), CP (CAS number: 6055-19-2; Sigma Chemical Co., St Louis, MO), MTX (CAS number: 59-05-2; Lederle Arzneimittel GMBH & Co.) and EPI (CAS number:

The cytotoxic effect of SM is stronger than that of chemotherapeutic agents against human breast cancer cells

The cytotoxicities of SM, cDDP, MTX, 5-FU, EPI and CP in human breast cancer cells (HBL-100, SK-BR-3 and ZR-75-1) were compared by the MTS assay. SM exhibited greatest cytotoxicity among the chemotherapeutic drugs against breast cancer cells, and the action was dose-dependent (Fig. 1a). The concentrations (IC50) of the SM that caused 50% cell death were approximately 2.07, 3.00 and 2.15 μM for HBL-100, SK-BR-3 and ZR-75-1 cells, while those of most of the chemotherapeutic drugs exceeded 250 μM

Discussion

Apoptosis is an essential physiological process for the normal development and maintenance of tissue homeostasis. During apoptosis, morphological changes can be observed, including cytoplasm shrinking, chromatin condensation, plasma membrane blebs, DNA fragmentation and apoptotic body formation. In our present study, SM-caused cell death more rapidly than conventional chemotherapeutic agents and the maximum of SM-caused cytotoxicity was at 3 h (Fig. 1b). The apoptotic features can be visualized

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