Effects and mechanisms of emodin on cell death in human lung squamous cell carcinoma.

Emodin (1,3,8-trihydroxy-6-methylanthraquinone) is an active component from the root and rhizome of Rheum palmatum that has been reported to exhibit antitumour effects, but the mechanism is not known. The study investigated the effects and mechanisms of emodin-induced cell death in human lung squamous carcinoma cell line CH27. Emodin (50 μM)-induced CH27 cell apoptosis was confirmed by cell morphological change, sub-G1 formation in flow cytometry analysis, viability assay and degradation of focal adhesion kinase in this study. Emodin-induced apoptosis of CH27 cells does not involve modulation of endogenous Bcl-XL protein expression, but appears to be associated with the increased expression of cellular Bak and Bax proteins. This study also demonstrated the translocation of Bak and Bax from cytosolic to particulate fractions. This study has shown that emodin-treated CH27 cells revealed the increases in the relative abundance of cytochrome c for the indicated time intervals in cytosolic fraction. This study demonstrates that the activation of caspase-3, caspase-9 and caspase-8 is an important determinant of apoptotic death induced by emodin. These results suggested that emodin induces CH27 cell death by Bax death pathway and Fas pathway. Keywords: Emodin, human lung squamous carcinoma cell line CH27, apoptosis, Bcl-XL, Bak, Bax, cytochrome c, caspase Introduction Emodin, an active component contained in the root and rhizome of Rheum palmatum l. (Polygonaceae) (Tsai & Chen, 1992; Liang et al., 1993), was found to have antitumour, antibacterial, diuretic and vasorelaxant effects (Koyama et al., 1988; Zhou & Chen, 1988; Huang et al., 1991). Emodin has also been reported to sensitize HER-2/neu-overexpressing lung cancer cells to chemotherapeutic drugs (Zhang & Hung, 1996) and repress transformation and metastasis-associated properties of HER-2/neu overexpression breast cancer cells (Zhang et al., 1995; 1998). However, the reason why the molecular mechanisms of emodin produced its biological effects remained unknown. Apoptosis is a major form of cell death characterized by a series of stereotypic molecular features, such as expression and translocation of Bcl-2 family proteins, release of cytochrome c, and activation of caspases. Cytochrome c, which is usually present in the mitochondrial intermembrane space, is released into the cytosol following the induction of apoptosis by many different stimuli including Fas, tumour necrosis factor (TNF) and chemotherapeutic agents (Liu et al., 1996; Kluck et al., 1997; Reed, 1997). Caspases, a family of cysteine proteases, play a critical role during apoptosis. There are at least two major mechanisms by which a caspase cascade resulting in the activation of effector caspases (caspase-3, -6 and -7) may be initiated by the most apical caspase, one involving caspase-8 and the other involving caspase-9 (Srinivasula et al., 1996; Zou et al., 1997; Srinivasula et al., 1998). Therefore, two typical apoptosis pathways, receptor (Fas)-mediated (involving caspase-8) and chemical-induced (involving caspase-9) apoptosis, have been suggested (Deveraux et al., 1999; Sun et al., 1999). The Bcl-2 family proteins, such as Bcl-2, Bcl-XL, Bax and Bak, are the best-characterized regulators of apoptosis (Adams & Cory, 1998; Evan & Littlewood, 1998; Nomura et al., 1999). Many reports have indicated that the release of mitochondrial cytochrome c and activation of caspase-3 is blocked by anti-apoptosis members of the Bcl-2 family, such as Bcl-2 and Bcl-XL, and promoted by proapoptotic members, such as Bax and Bak (Kluck et al., 1997; Yang et al., 1997; Jurgensmeier et al., 1998). The major purpose of this study was to investigate whether emodin induced cancer cell death. Since the anticancer mechanisms of emodin were unclear, this study also investigated the mechanisms of emodin-induced cell death in the human lung squamous carcinoma cell line CH27. The results of the present study suggest that emodin induces CH27 cell death by the Bax death pathway and the Fas pathway. In other words, the pathway of emodin-induced CH27 cell apoptosis involved receptor-mediated and chemical-induced apoptosis.