Paeoniae Radix, a Chinese herbal extract, inhibit hepatoma cells growth by inducing apoptosis in a p53 independent pathway
Introduction
Paeoniae Radix (PR) is the roots of Paeonia lactiflora Pallas and it is a crude drug used in many traditional prescriptions in China and Japan. It is commonly used in nourishing blood, activating circulation, alleviating pain, regulating menstruation, treating liver disease and cancer. The extract from Paeoniae Radix (PRE) can improve blood flow through its endothelium-dependent vasodilator action on aorta [1] and inhibitory effects on thrombosis and platelet aggregation [2]. PRE causes endothelium-dependent relaxation and antioxidant enzyme activity in rats administered high-fat diet [3]. Paeoniflorin, a glycoside purified from the PR is capable of reversing guanethidine-induced hypotension via activation of central adenosine A1 receptors in Wistar rats [4]. Besides, PR is a potential anti-aging and anti-carcinogenesis agent as it has been reported to inhibit oxidative DNA cleavage induced by various oxidative DNA damage chemicals [5], [6]. The role of PR as a potential anticancer agent in clinical applications has been reported. 110 premenopausal patients consume Kuei-chih-fu-ling-wan (Keishi-bukuryo-gan; KBG), a traditional Chinese herbal remedy, which consists of PR as one of the components, achieved shrinking of uterine myomas in roughly 60% of the cases. The underlying mechanism of PR as an anticancer agent has not been defined. Several polysaccharides isolated from PR have been found to have immunological activities [7], [8]. Thus, the inhibitory effect of PR on tumor growth may be due to activation of immunological action against the tumor. The study of the anticancer effects in vivo and in vitro and identification of drug targets would provide a rationale for clinical development of this agent as a therapeutic drug in cancer therapy. Our results showed that PRE exhibited a marked direct cytotoxic effect to both HepG2 and Hep3B human hepatoma cells. The cytotoxicity of PRE to the cells is through activation of the cell death program, apoptosis, evidenced by induction of internucleosomal DNA fragmentation and chromatin condensation appearance, and accumulation of sub-G1 phase of cell cycle profile. The activation of apoptosis by PRE is independent of the p53 pathway and probably through altering the expression level of genes encoding BNIP3, ZK1, RAD23 and HSPD1 proteins identified by cDNA microarray and RT-PCR analysis.
Section snippets
Preparation of PRE
100 g dried roots were cut into approximately 0.5 cm long pieces before boiling with 500 ml distilled water under reflux. Then the filtrate was collected after filtration and then lyophilized. The powder obtained was dissolved in 500 ml phosphate-buffered saline (PBS) for storage in –20 °C freezer and used in all subsequent experiments. The final storage concentration was 200 mg/ml and the concentration used in the experiment was based on the dry weight of the extract (mg/ml).
Cell culture
Human hepatoma
Inhibition of cell growth by PRE
Initially, the cytotoxicity of the water extract of PR was assessed by a cell growth inhibition assay using the HepG2 and Hep3B human hepatoma cell lines. The growth of HepG2 and Hep3B cells in the presence of various concentration of PRE was examined. Under the experimental conditions used (2 day continuous exposure), PRE exhibited a marked growth inhibitory effect to both HepG2 and Hep3B human hepatoma cells and the IC50 for HepG2 and Hep3B are approximately 4.6 and 4.4 mg/ml, respectively
Discussion
Paeoniae Radix (PR) is a commonly used traditional Chinese medicine to treat vascular problems as well as liver diseases such as hepatitis virus infection, liver fibrosis and liver cancer in Asia. Moreover, it is used in combination with other herbs as a remedy. PR can inhibit liver fibrosis and damage induced by CCL4 and D-galactosamine in rat; thus PR has a hepatocyte protective role [10], [11]. Animal models and clinical studies have revealed that PR included in some herbal remedies are
Acknowledgements
This study was supported by earmarked grants from the Research Grant Council, Hong Kong (Ref. No.: CUHK 418/95M and 205/96M), and grants from the Industrial Department, the government of the Hong Kong SAR (Ref. No.: AF/47/98 and AF/9/97). S.M.Y. Lee is supported by a post-doctoral fellowship from the Chinese University of Hong Kong.
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