Andrographolide induces cell cycle arrest at G2/M phase and cell death in HepG2 cells via alteration of reactive oxygen species Jieliang Li a , Hon-Yeung Cheung a,b, ⁎ , Zhiqiang Zhang a , Gallant K.L. Chan b , Wang-Fun Fong a a Research Group for Bioactive Products, Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Ave., Kowloon, Hong Kong SAR, China b Pharmaceutical & Chemical Technology Center Ltd., City University of Hong Kong, 83 Tat Chee Ave., Kowloon, Hong Kong SAR, China Received 17 August 2006; received in revised form 27 March 2007; accepted 1 April 2007 Available online 22 April 2007 Abstract The cytotoxicity of andrographolide to HepG2 human hepatoma cells was investigated in the present study. Growth of HepG2 cells was affected in the presence of andrographolide with an IC 50 of 40.2 μM after 48 h treatment. Flow cytometric analysis and DNA fragmentation assay revealed that andrographolide induced cell cycle arrest at G2/M phase and a late apoptosis of the cells. The occurrence of cell cycle arrest was accompanied by the collapse of mitochondrial membrane potential (MMP) and an intracellular increase of hydrogen peroxide (H 2 O 2 ) but a decrease of superoxide radicals (O 2 U - ) and reduced glutathione. In the treated cells, expression of Bax as well as the transcriptional controller of this pro-apoptotic gene, p53, was upregulated but not other apoptotic proteins such as Bad, Bcl-2 and Bcl-X L . Although the activity of caspase-3, which has direct effect on apoptosis, was also enhanced by the presence of andrographolide, cell death of HepG2 could neither be prevented by a specific inhibitor of capsase-3 nor the pan-caspase inhibitor-zVAD (Val-Ala-Asp), indicating that it was a caspase-independent cell death. Since the overall percentage of apoptotic cells was relatively small throughout the experimental studies, we conclude that the cytotoxic effect of andrographolide on HepG2 cells is primary attributed to the induction of cell cycle arrest via the alteration of cellular redox status. © 2007 Published by Elsevier B.V. Keywords: Andrographolide; Cell cycle arrest; Cell death; Mitochondrial membrane potential; Reactive oxygen species; HepG2 cell 1. Introduction Andrographis paniculata (Burm. f) Nees (Acathaceae), also known as “King of Bitters” is an important herbal medicine widely used in China, India and other Southeastern Asian countries. It is a prominent component of many traditional Indian and Chinese formulae for treatment of liver disorders. There are reports indicating that A. paniculata has protective effects against damage of the liver produced in mice by giving them carbon tetrachloride, alcohol or other chemicals (Visen et al., 1993; Kapil et al., 1993). Andrographolide is the major bioactive constituent found in A. paniculata, and it contributes to a broad range of effects including antibacterial, anti-inflammatory and immunos- timulant effects (Thisoda et al., 2006; Tsai et al., 2004; Kumar et al., 2004; Singha et al., 2003). Andrographolide is colorless, crystalline in appearance and has a very bitter taste. A γ-lactone ring is connected to a decalin ring system via an unsaturated C 2 moiety (Fig. 1A). In our previous study (Cheung et al., 2005), we reported that andrographolide in comparison to other two diterpenoids, deoxyandrographolide and neoandrographolide, had more potent anti-cancer activity against human leukemia HL-60 cells and other cancer cells. As demonstrated by chromosomal DNA fragmentation, accumulation of HL-60 cells in the sub-G1 cell cycle stage, disappearance of mitochondrial cytochrome c and an increased expression of Bax and down- regulation of Bcl-2 in the inhibited cells, it induced apoptosis of HL-60 cells. Apoptosis, or programmed cell death, is an active form of cell death that plays a crucial role in the normal development and differentiation of multicellular organisms (Lawen, 2003). It is characterized by a highly stereotypical series of morpholog- ical and biological changes, such as cytoplasmic shrinkage, chromatin condensation and DNA degradation (Kerr et al., European Journal of Pharmacology 568 (2007) 31 – 44 www.elsevier.com/locate/ejphar ⁎ Corresponding author. Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong SAR, China. Tel.: +86 852 2788 7746; fax: +86 852 2788 7406. E-mail address: bhhonyun@cityu.edu.hk (H.-Y. Cheung). 0014-2999/$ - see front matter © 2007 Published by Elsevier B.V. doi:10.1016/j.ejphar.2007.04.027