Inhibition of eukaryotic elongation factor-2 confers to tumor suppression by a herbal formulation Huanglian-Jiedu decoction in human hepatocellular carcinoma

Abstract

Ethnopharmacological Relevance

An oriental medicinal formulation, Huanglian Jiedu Decoction (HLJDD), has been well documented in few Traditional Chinese Medicine Classics 1300 years ago for treatment of heat and dampness-related diseases. Its effect is well accepted in Asian community, including China, Japan and Korea. Recent studies have postulated HLJDD as a regimen for cancer treatment, especially liver cancer, but the underlying mechanism is unknown. The aim of this study was to examine the suppressive effect of HLJDD on the growth of hepatocellular carcinoma (HCC) and its possible underlying mechanism.

Methods

Chemical composition of HLJDD was analyzed by high performance liquid chromatography. The tumor suppressive effect of HLJDD was determined on both HCC cells and xenograft model. Nascent protein synthesis was detected with Click-IT protein labeling technology; protein expression was determined by immunoblotting and imunnohistochemical analysis.

Results

Quality analysis revealed that HLJDD of different batches is consistent in both chemical composition and bioactivities. HLJDD inhibited HCC cell proliferation at its non-toxic doses, and suppressed growth and angiogenesis in xenografted murine model. HLJDD suppressed the synthesis of nascent protein via inactivation of eEF2 without deregulating the translation initiation factors. The major components in HLJDD, geniposide, berberine and baicalin, additively act on eEF2, and contributed to the responsible activity. HLJDD-activated eEF2 kinase (eEF2K) led to eEF2 inactivation, and activation of AMPK signaling may be responsible for the eEF2K induction. Blocked AMPK activity in HLJDD-treated HCC cells attenuated eEF2K activation as well as the inhibitory effect of the formula. In nutrient deprived HCC cells with inactivated eEF2, the inhibitory effect of HLJDD in tumor cell expansion was interfered.

Conclusion

Our results indicate that HLJDD has potential in blocking HCC progression with involvement of eEF2 inhibition.

Introduction

Hepatocellular carcinoma (HCC) is one of the world leading causes of death and the fifth ranked common human malignant tumor (Ng et al., 2010). HCC is more threatening in south-eastern Asia, with 33 out of 100,000 deaths per year was reported in male inhabitants. In Hong Kong, HCC is the second major cause of cancer deaths (Yip et al., 2009). For HCC patients, orthotropic liver transplantation is the best option amongst variety therapeutic strategies. However, only a small proportion of HCC patients is suitable for liver surgery (Liu et al., 2011). Different treatments have been developed in fighting against the unresectable HCC; nevertheless, the outcome remains poor due to the rapid growth of HCC cells.

Herbal medicine has been shown to be effective in treating HCC in clinical practice (Feng et al., 2008, Feng et al., 2011, Lin et al., 2005, Wang et al., 2013). However, the evidence-based research of herbal medicine remains challenging due to the concerns on the reproducibility of its pharmacological actions. Recent study on PHY906, a herbal product derived from ancient formula Huangqin Decoction has postulated its beneficial effect in reducing chemotherapy-induced gastrointestinal toxicity (Lam et al., 2010). Furthermore, another study on the Realgar-Indigo naturalis, suggested the synergy of the compounds rendering pharmacological actions of the whole formula in treatment of promyelocytic leukemia (Wang et al., 2008). These impacted and pioneered studies indicate that reproducibility of the pharmacological effects of herbal medicine could be achieved with stringent quality control of bioactive components. The concept of cancer appeared in modern medical theories; however, it is not very difficult to find some description of cancer-like symptoms in ancient medical records of China. It was recorded in an ancient Chinese medical monograph “Zhong-Zang-Jing” that “Yong, Yang, Chuang and Zhong” (which described the cancer-like symptoms) are caused by retention of various pathogens including heat and dampness. Huanglian Jiedu decoction (HLJDD, Oren-gedoku-to in Japanese and Hwangryun-Hae-Dok-tang in Korean) is an ancient herbal formula and its therapeutic effects have been well-documented in many Traditional Chinese Medicine Classics such as Medical Secretes of an Official (wai tai mi yao in Chinese) and Prescriptions for Emerent Reference (zhou hou fang in Chinese) (Ge. et al., 1996, Wang, 1955). It was used to treat internal heat-related mania, delirium, insomnia, irritability, dry mouth and throat, heat-induced blood omitting, skin spots, and sore furuncle (Chuang and Yang in Chinese) according to Medical Secretes of an Official. The formula was also used to treat heat pathogen-induced pyrostagnant rhinorrhagia, jaundice, and carbuncle (Yong in Chinese) as summarized by Prescriptions for Emerent Reference. It is composed of four herbal species: the rhizome of Coptis chinensis Franch. (Huang lian; Rhizoma Coptidis), the bark of Phellodendron amurense Rupr. (Huang bo; Amur cork tree), the fruit of Gardenia jasminoides J. Ellis (Zhi zi; Fructus Gardenia or cape jasmine), and the root of Scutellaria baicalensis Georgi (Huang qin; Chinese Skullcap). Modern studies have revealed the various pharmacological effects of HLJDD such as anti-ulcer, anti-trypanosomal, anti-inflammation and anti-oxidation (Mizukawa et al., 1993, Stefek and Benes, 1994, Takase et al., 1989, Yabu et al., 1998). It is also found to reduce neutrophil infiltration (Hwang et al., 2002) and suppress pre-adipocyte differentiation (Ikarashi et al., 2012). These actions rendered possibility of the decoction in treating various diseases including acute gastric mucosal lesions, liver injury, colitis, hypercholesterolemia, transient cerebral ischemia, allergic dermatitis and Alzheimer׳s disease (Gao et al., 2005, Ohta et al., 1999, Ohta et al., 1997, Sekiya et al., 2005, Xu et al., 2000, Yu et al., 2010, Zhou and Mineshita, 1999). Our clinical observation and review studies also showed that HJLDD has liver protection and anticancer effects and therefore possesses high potential in treatment of liver diseases and cancer (Feng et al., 2008, Tang et al., 2009). A recent study showed that HLJDD inhibited human liver cancer in vitro (Hsu et al., 2008), however, the underlying mechanism regarding its anti-tumor effect has not been fully understood.

In this study, we reported eukaryotic elongation factor-2 (eEF2) as a new target of HLJDD in inhibiting HCC progression. We found that HLJDD inhibited proliferation of HCC cells in vitro, and suppressed the tumor growth in vivo. This inhibitory effect of HLJDD may be due to the suppression of nascent protein synthesis, which supports proliferation of the cancer cells (Shin et al., 2014). HLJDD controlled nascent protein synthesis by suppressing eEF2 activity, the protein essential for nascent peptide elongation (Kaul et al., 2011). The effect may be contributed by the activation of eEF2 kinase (eEF2K), and the regulation of AMPK/mTOR activity by HLJDD. In addition, we found that the combinatory effect of major active compounds of HLJDD inhibited eEF2 activity. Berberine is the major active component in inhibiting eEF2 while other compounds such as baicalin, geniposide and palmatine have additive actions. Our study reveals a novel mechanism involved in anti-cancer effect of HLJDD and may indicate the translational potential of the decoction as a complementary agent in HCC treatment.