Elsevier

Journal of Ethnopharmacology

Volume 114, Issue 2, 1 November 2007, Pages 227-233
Journal of Ethnopharmacology

Cimicifuga foetida extract inhibits proliferation of hepatocellular cells via induction of cell cycle arrest and apoptosis

https://doi.org/10.1016/j.jep.2007.08.008Get rights and content

Abstract

The purpose of this study is to determine whether the ethyl acetate fraction (EAF) from the aerial part of Cimicifuga foetida Linnaeus possesses the anti-tumor action on hepatoma, and therefore, provide evidence for the traditional use of the plant as a detoxification agent. EAF was extracted and its cytotoxicity was evaluated on a panel of Hepatocytes by MTT assay. The IC50 values of EAF on HepG2, R-HepG2 and primary cultured normal mouse hepatocytes were 21, 43 and 80 μg/mL, respectively. Morphology observation, Annexin V-FITC/PI staining, cell cycle analysis and western blot were used to further elucidate the cytotoxic mechanism of EAF. EAF induced G0/G1cell cycle arrest at lower concentration (25 μg/mL), and triggered G2/M arrest and apoptosis at higher concentrations (50 and 100 μg/mL, respectively). An increase in the ratio of Bax/Bcl-2, activation of downstream effector Caspase 3, and cleavage of poly-ADP-ribose polymerase (PARP) were implicated in EAF-induced apoptosis. In addition, EAF inhibited the growth of the implanted mouse H22 tumor in a dose-dependent manner with the growth inhibitory rate of 63.32% at 200 mg/kg. In conclusion, EAF may potentially find use as a new therapy for the treatment of hepatoma.

Introduction

Tumorigenesis is the manifestation of a delicate balancing act gone awry (Mathew and White, 2006). Deregulated cell growth and suppressed cell death together provide the underlying platform for neoplastic progression (Evan and Vousden, 2001). Cell cycle progression and apoptosis are two pivotal signaling mechanisms used to maintain homeostasis in healthy tissue. Cell proliferation is governed by cell cycle, which is an order of events that is tightly regulated by a number of cyclin-dependent kinases (CDK) and cyclins (Buecher et al., 2003). A shift in favor of proliferative signals, for example, over-activation of cyclin/CDK, often leads to uncontrolled cell division and malignancy. Necrosis and apoptosis are two distinct mechanisms of cell death. Unlike necrosis, apoptosis, also known as programmed cell death, is an expedient way of eliminating superfluous abnormal cells by phagocytosis without an inflammatory response. Multiple lines of evidence suggest that the loss of control of cell cycle or apoptosis is responsible for cancer initiation and progression (Tu et al., 1996, Vitale-Cross et al., 2004). In turn, one essential strategy for cancer therapy is focused on target proteins that suppress cell cycle progression and apoptosis in the tumor cells. It has been found that many anti-cancer agents arrest the cell cycle at the G1, S or G2/M phase and then induce apoptotic cell death (Fujimoto et al., 1999, Cheng et al., 2005, Qi et al., 2005, Yang et al., 2005, Wang et al., 2007). In the search for new cancer therapeutics, the herbs used in traditional medicines for cancer treatment are promising candidates.

The genus Cimicifuga (Ranunculaceae) consists of more than 18 species and many rhizomes of these plants have been widely used in traditional medicine worldwide. Rhizomes of Cimicifuga foetida Linnaeus and Cimicifuga dahurica, two of the most common Asiatic species, have been employed as cooling and detoxification agents by Chinese people since ancient times. As we know, tumor is a kind of toxin in the theory of Chinese Medicine, so it is of interest to investigate the antitumor activity of Cimicifuga plants. Cimicifuga racemosa, a famous European species has been shown remarkable antitumor activities in recent studies. Several kinds of extracts from rhizomes of Cimicifuga racemosa were demonstrated to kill estrogen receptor-positive (MCF-7), estrogen receptor negative (MDA-MB231 and MDA-MB-453) human breast carcinoma and androgen-sensitive LNCaP human prostate cancer-derived cell lines. The cytotoxic mechanisms of these extracts were related to the induction of cell cycle arrest, apoptosis and degradation of cytokeratin (CK) 18 (Einbond et al., 2004, Hostanska et al., 2004, Hostanska et al., 2005, Jarry et al., 2005, Seidlova-Wuttke et al., 2006). The standard Cimicifuga racemosa extract BNO 1055 also inhibits the formation and/or proliferation of tumors induced by subcutaneous inoculation of LNCaP cells in immunodeficient nu/nu mice (Seidlova-Wuttke et al., 2006). Recently, two cycloartane triterpenoid glycosides isolated from the rhizomes and aerial part of Cimicifuga foetida Linnaeus have demonstrated the cytotoxicity (Tian et al., 2006a, Sun et al., 2007). However, up till now, the aerial part of Cimicifuga foetida has been discarded and few reports been conducted studying its pharmacological effects.

In the present study, we reported the antitumor activity and mechanisms of action of EAF from the aerial part of Cimicifuga foetida Linnaeus to provide evidence for its traditional use as a detoxification agent.

Section snippets

Extraction and concentration measurement

The plant material was collected in Ankang, Shannxi Province, China, in August 1998 and was identified by Professor Ruile Pan of the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College. A voucher specimen has been deposited in the Herbarium of the same Institute (No. 19980816-1). The powdered aerial part of the plant was extracted exhaustively with 80% ethanol under refluxing. After combination, the solvent was evaporated under vacuum

Effects of EAF on the growth of hepatoma cells and normal hepatocytes

The effects of EAF and betulinic acid on cellular proliferation of HepG2 cells, its drug-resistant subline R-HepG2 and normal mouse hepatocyte were evaluated with MTT assay. A 48 h exposure to EAF and betulinic acid decreased the proliferation of all cells in a concentration-dependent manner (Fig. 1). The IC50 values of EAF on HepG2, R-HepG2, and mouse hepatocyte were 21, 43, and 80 μg/mL while the values of betulinic acid in each cell were 12, 12 and >100 μM, respectively. It is of interest that

Discussion

The present studies provide the first detailed examination on antitumor activity of EAF in order to help to understand the detoxification effect of Cimicifuga foetida Linnaeus in traditional use. The antiproliferative activity of EAF was evaluated on hepatoma cells and normal mouse hepatocytes. It is well known that primary cultured cells more closely resemble to normal cells in vivo. Hence, we used mouse hepatocytes instead of normal human liver cells, which is difficult to obtain. Our results

Acknowledgements

This work is funded by National Natural Science Foundation of China (30470195). Thanks for Dr. Vishal Saxena (Brigham and Women's Hospital) and Annie Park Moseman (Children's Hospital Boston) for thoughtful reading of the manuscript. Thanks for Dr. Isaac Kohane (Children's Hospital Boston) for his support.

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