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EVIDENCE-BASED COMPLIMENTARY AND ALTERNATIVE MEDICINE

eCAM Advance Access published online on May 5, 2010

eCAM, doi:10.1093/ecam/neq044

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Herbal Medicine and Hepatocellular Carcinoma: Applications and Challenges

Yan Li* and C. G. , II

Division of Surgical Oncology, Department of Surgery, University of Louisville

School of Medicine, Louisville, KY, USA

Abstract

Use of herbal medicine in the treatment of liver cancer has a long tradition.

The compounds derived from the herb and herbal composites are of considerable

interest among oncologists. In the past, certain herbal compounds and herbal

composite formulas have been studied through in vitro and in vivo as an

anti-hepatocellular carcinoma (HCC) agent, enhancing our knowledge about their

biologic functions and targets. However there is a significant distinction

between the herbal medicine and the herbal production even though both are the

plant-based remedies used in the practice. In this article, for the sake of

clarity, the effective herbal compounds and herbal composite formulas against

HCC are discussed, with emphasizing the basic conceptions of herbal medicine in

order to have a better understanding of the prevention and treatment of HCC by

herbal active compounds and herbal composite formulas.

Introduction

Herbal Medicine

Generally, the description of herbal medicine is the use of medicinal herbs,

preparation made from a plant or plants, to prevent and treat diseases and

ailments or to promote health and healing. However, it is important to

distinguish ‘herbal medicine’ and ‘herbal production’, which is often overlooked

(1,2). There is a significant distinction between the herbal medicine and the

herbal production, both are the plant-based remedies used in the practice.

Herbal production is the conventional medicine with definite ingredient(s) and

definite pharmacological effects when the ‘plant drug’ is for medical use.

Whereas, the use of herbs in herbal medicine divorced from the context of the

so-called ‘scientific information’ and thus not as strongly scientifically

validated is a specific discipline of herbal medicine that provides the

therapeutic understanding of the medicinal use of herbs (3,4).

A good example of herbal medicine is ‘Chinese herbal medicine’, one branch of

traditional Chinese medicine which focuses on naturalism and holistic health

that can be traced back as far as 2100 BC in ancient China (5). In Chinese

herbal medicine, the herbs are classified according to their properties. The use

of specific herb(s) to treat diseases depends on the sign and symptom of

patients. The herbalists believe that illness is an imbalance status of the

body, and the herbs, based on their various characteristics which are in the

accordance with the law of nature, can neutralize the sign and the symptom

thereby keep an overall balanced status in patient’s body (6). Similar to

Chinese herbal medicine, is the Ayurvedic alternative herbal medicine of India,

the balance between agni (representing strength, health and innovation) and ama

(representing weakness, disease and intoxication) is also emphasized. Therefore,

herbal medicine is a preparation made from a plant or plants and used for any of

such purposes, and the biological ingredients of herbal remedies are extracted

from natural substances such plants, animal parts, shells, insects and even

stones and minerals.

In general, the herbs used in herbal medicine often result from a combination of

herbs with multiple ingredients, called ‘herbal composite formula’, to ensure

effective actions on multiple targets simultaneously. Contrary to Western

medicine that prefers the analytical approach, most composite formulas in

Chinese herbal medicine are empirically based and the principles underlining the

composite formula are relatively simple; however, not as strongly defined and

can vary among herbalists. A ‘food model’ could be a good example to understand

the principles consisting of the composite formula. Foods are complex and

contain many different constituents. The furnished materials are used by body to

nourish, support and reproduce its vital activities, while the wastes and some

toxic materials are eliminated by the body itself. The rule of the composite

formula and the dosage for an individual with specific signs and symptoms is

also in the same way similar to the dosage of materials to treat marasmus and

diet materials to treat obesity. Although the choice of herbs or ‘composite

formula’ for some specific signs and symptoms may represent in part a trend

towards mysticism and thus highly variable, the fact is that some herbal drugs

contain ingredients that specifically treat diseases. These biological

ingredients extracted from natural substances result in multiple effective

actions on different biological molecular targets.

Modern biomolecular science has contributed to the interpretation of these

multiple effective actions of herbs, and some important properties such as

anti-virus, anti-inflammatory and anticancer have been recognized. As more

information is becoming available, some ‘herbal drugs’ are identified for the

effects against hepatocellular carcinomas (HCC). This aim of this review article

is to present the current state of herbal medicine as a chemopreventitive and

chemotherapeutic agent in HCC.

HCC and Herbal Medicine

HCC is the fifth most common malignancy worldwide, and with a continuously

increasing incidence (7,8). Three curative methods are currently available:

orthotopic liver transplantation (OLT), surgical resection and local destruction

(LD). However, only few patients qualify for the ‘curative therapies’ because

these strategies depend largely on the extent and location of the tumor and the

underlying liver disease such as cirrhosis. Despite these curative options, the

recurrence rate may be as high as 50% at 2 years (9,10). Therefore, prevention

of recurrent HCC after (or before) successful curative therapeutic interventions

needs to be improved in order to make an impact on long-term survival of these

patients. Many adjuvant treatments such as trans-arterial chemo-embolization

(TACE), anti-viral treatments and immunotherapy have been used but never

confirmed if positive (11–14). Palliative treatments for HCC are indicated if

there is no curative treatment option, four palliative treatments are

transarterial chemo-embolization, systemic chemotherapy, interferon and

hormonotherapy (15). However, palliative therapy of patients with HCC remains

challenging as HCC is highly resistant to systemic therapies. Importantly, the

incidence still nearly equals the mortality rate and more than 80% of patients

present with advanced disease (15). The overall disappointing results of both

curative therapies and palliative treatments in advanced HCC patients support

the research for other more active and specific treatments to be administered

alone or in combination with the current therapy.

Currently, few medical interventions have been thoroughly tested in HCC, in

contrast with the many tested in other highly prevalent cancers, such as lung,

breast and colorectal cancer (16,17). There is an urgent need for new active and

well-tolerated treatments to improve survival among patients with advanced HCC

(palliative setting) and to increase long lasting remission after curative

treatments (adjuvant setting). Studies, especially in China, on the prevention

and treatment using herbal medicine against HCC have been accumulated during the

past decades. Herbal compounds could affect all phases of HCC, including

initiation, promotion and progression (18,19). The active development of

innovative therapeutic approaches and molecularly targeted agents using herbal

medicine could offer an opportunity to study the agents in HCC and gives new

hope for the future. Despite massive investigation and effort, no such herb-drug

for HCC treatment has been licensed to date. Actually, herbs are regulated in

the USA under Dietary Supplement Health and Education Act only as ‘dietary

supplements’, as opposed to the Chinese category ‘traditional Chinese medicine’.

Regarding the current use of herbs, many questions cannot be answered

definitively by the available scientific data. In some instances enough research

has not been performed, and in others the final end point of the research is

flawed.

Methods

In this article, for the sake of clarity, we divided the herbal medicine into

effective herbal compounds and herbal composite formula to discuss their

anticancer properties against HCC. Several reviews have provided major

contributions to the current knowledge on the herbal medicines for treatment of

liver fibrosis and cancer (20–23). The basic concepts of these ‘botanical drugs’

are emphasized in order to have a better understanding of the prevention and

treatment of HCC by herbal active compounds and herbal composite formula. The

reports from both Chinese and English language are reviewed to provide a full

picture of the progression of the use of herbal medicine against HCC. The

English literature searches were conducted through Medline, Embase, Science

Citation Index, Current Contents, PubMed databases, as well as relevant papers

from integrative and complementary medicine journals, such as Evidence Based

Complementary and Alternative Medicine, until January 2009. The outline of

articles reviewed is presented in a Quality of Reporting of Meta-Analysis

(QUORUM) flow chart showing the number of studies screened and included in the

meta-analysis (Fig. 1) (24). Search items were ‘herb’, ‘anticancer mechanism’,

‘traditional Chinese medicine’, ‘hepatoma’, ‘hepatocellular carcinoma’ and

‘hepatocellular adenocarcinoma’. Restrictions were placed on language of

publication, and only English was included. Studies lacking controls were

excluded. Case reports were excluded. The relevant Chinese literature searches

were carried out through Wanfang data until January 2009 using database of China

Medical Association Journals (CMAJ), which is a portal to medical research

materials published in China. The exclusion and inclusion for extracted data

from literatures were same as that in PubMed. These restrictions were placed in

order to have consistency among the reports reviewed. The aim of this review was

to evaluate the current literature for the efficacy and safety of current herbal

compounds in the treatment of hepatocellular cancer.

Herbal Compounds

There are a number of molecular compounds derived from the herbs that have been

proven to be effective against HCC. Modern research is confirming that many

compounds are active at some molecular targets which are being sought to find

out potential newer generation ‘targeted’ biological response modifier drugs

(25,26). These herbal compounds have been shown to engage various molecular

targets related HCC carcinogenesis and chemoprevention, which have been

identified by laboratory research findings and clinical observations. These

molecular compounds represent an enormous and almost untapped resource for HCC

treatment (Fig. 2). Some of the herbal compounds are discussed and summarized in

Table 1.

Curcumin

Curcumin (diferuloylmethane), a compound extracted from Curcuma aromatica widely

used as a spice and coloring agent in food, possesses potent antioxidant,

anti-inflammatory and anticarcinogenic properties. The anticarcinogenic property

has been widely studied in various cancers (27). Regarding HCC, three important

properties of curcumin have been studied: anti-HCC; anti-angiogenesis of HCC;

and anti-metastatic activity of HCC. Chuang et al. investigated the effect of

curcumin on a HCC mouse model induced by N-diethylnitrosamine (DEN). They found

that curcumin can inhibit effectively DEN-induced hepatocarcinogenesis in the

C3H/HeN mice. The hepatic tissue from the DEN-treated mice showed a remarkable

increase in the levels of p21(ras), expression of proliferating cell nuclear

antigen (PCNA) and CDC2 proteins, while curcumin reversed the levels of all

these biological markers. Another study performed by the same research group

showed that curcumin can also suppress effectively DEN-induced liver

inflammation and hyperplasia in a rat HCC model. Immunoblotting analysis showed

that curcumin inhibits DEN-mediated the increased expression of oncogenic

p21(ras), p53 proteins, PCNA, cyclin E, factor NF- and p34(cdc2), but not Cdk2,

c-Jun and c-Fos (28,29). Yoysungnoen et al. evaluated the effect of curcumin and

tetrahydrocurcumin on tumor angiogenesis of HCC mice. Human HCC cell line

(HepG2) inoculated onto a dorsal skin-fold chamber of male BALB/c nude mice, and

curcumin and tetrahydrocurcumin were fed oral daily at 300 and 3000 mg kg–1. The

tumor microvasculature was observed using fluorescence videomicroscopy and

capillary vascularity (CV) was measured. They found that treatment with curcumin

and tetrahydrocurcumin resulted in significant decrease in the CV. The

anti-angiogenic effects of curcumin and tetrahydrocurcumin were found to be

dose-dependent. They concluded that the anti-angiogenic properties of curcumin

and tetrahydrocurcumin represent a common potential mechanism for their

anti-cancer actions (30–32).

Curcumin has also been shown to have potent anti-metastatic activity. Ohashi et

al. analyzed the anti-metastatic mechanism using an orthotopic implantation HCC

model with CBO140C12 cells. They found that daily oral administration of

curcumin suppressed intrahepatic metastasis of orthotopic implanted HCC cells.

They further examined the effect of curcumin on the metastatic properties in

vitro, the results indicated that curcumin significantly inhibited adhesion and

haptotactic migration to fibronectin and laminin thereby inhibiting tumor cells

through Matrigel-coated filters (33). An in vitro study carried out by Lin et

al. also showed that curcumin could be a potential anti-metastatic agent against

HCC, they found that curcumin, at 10 µM, inhibited 17.4 and 70.6% of cellular

migration and invasion of SK-Hep-1 cells, a highly invasive SK-Hep-1 cell line

of human HCC. This anti-metastatic effect is associated with its inhibitory

action on MMP-9 secretion (34). There are also a number of studies for exploring

the mechanism of curcumin against HCC. Lv et al. reported that curcumin can

inhibit the level of histone deacetylase, enhance the expression of

P21(WAF1/CIP1) protein and mRNA in HepG2 cells. They concluded that inhibiting

histone deacetylase and increasing P21may be one of the possible mechanisms of

curcumin against HCC (35). Cao et al. demonstrated that HepG2 cells treated with

curcumin showed a transient elevation of the mitochondrial membrane potential,

followed by cytochrome c release into the cytosol and disruption of DeltaPsim.

Apoptosis was detected after curcumin treatment but the expression of Bcl-2

remained unchanged. They conclude that mitochondrial hyperpolarization is a

prerequisite for curcumin-induced apoptosis in HepG2 cells (36,37). Chan et al.

investigated the effect of curcumin on methylglyoxal-induced apoptotic events in

HepG2 cells. In contrary, they report that curcumin significantly attenuates

methylglyoxal-induced reactive oxygen species (ROS) formation thereby prevented

cell apoptosis and apoptotic biochemical changes such as mitochondrial release

of cytochrome c, caspase-3 activation, and cleavage of PARP (poly [ADP-ribose]

polymerase) (38).

Although numerous in vitro and animal studies have shown that curcumin exhibits

significant chemopreventitive effects and thus reported anti-HCC effect, the

exact mechanism is largely unknown. The current reason for this lack of

understanding comes from the fact that Curcumin is a complex herb made up of

many potential active agents, Curcuma aromatica, Curcuma longa and curcumin oil

to name a few, all of which have not been defined as to where the most active

agent(s) are effective in chemoprevention. In addition, poor systemic absorption

of curcumin is still a major obstacle for its application (39,40).

Resveratrol

Resveratrol, a polyphenol found in grape skins, peanuts, berries and red wine,

has been shown to possess potent growth inhibitory effects against various human

cancer cells including HCC. Resveratrol can be absorbed rapidly and accumulate

in the liver. Lancon et al. studied the absorption and the efflux of resveratrol

in the HepG2 cells. They found that resveratrol was rapidly conjugated and it

entirely metabolized at 8 h to form two main resveratrol metabolites:

monosulfate and disulfate (41). The effect of resveratrol on HCC has been also

extensively studied. Bishayee et al. evaluated the inhibitory effect of

resveratrol against hepatocarcinogenesis using a two-stage HCC rat model. The

HCC model was reproduced by a single intraperitoneal injection of

diethylnitrosamine (DENA), followed by promotion with phenobarbital in drinking

water. They found that resveratrol exerts a significant chemopreventive effect

on DENA-initiated hepatocarcinogenesis through inhibition of cell proliferation

and induction of apoptosis. They concluded the possible mechanism could be that

the resveratrol-induced apoptogenic signal is mediated through the

downregulation of Bcl-2 and upregulation of Bax expression (42). An in vitro

study carried out by Stervbo et al. also showed that the inhibitory effects of

resveratrol on cell proliferation and apoptosis in the HepG2 cells. They found

that resveratrol inhibited DNA synthesis and increased nuclear size and

granularity at G1 and S phases of HepG2 cells. Apoptosis was also stimulated by

resveratrol in a concentration-dependent manner in HepG2 cells. They concluded

that resveratrol inhibits cell proliferation by interfering with different

stages of the cell cycle, and causes stimulation of apoptosis (43). Notas et al.

also used the HepG2 cells to address the action of resveratrol on cell growth

and to examine some possible mechanisms. Their results indicate that the

stilbene resveratrol inhibits cell proliferation, reduces the production of ROS

and induces apoptosis, through cell-cycle arrest in G1 and G2/M phases. They

also found that stilbene resveratrol modulates the NO/NOS system, by increasing

iNOS and eNOS expression, NOS activity and NO production (44). Yan et al.

investigated the effects of resveratrol on proliferation and gap-junctional

intercellular communication (GJIC) in HepG2 cells. They found that resveratrol

arrests HepG2 cell growth in S phase, inhibits DNA synthesis and induces cell

apoptosis. The levels of GJIC increased sharply after resveratrol treatment

implied that the increased GJIC level could play a role on the effect of

resveratrol in the cancer chemopreventive activity (45). The study carried out

by Ma et al. agreed with the observations above. They further investigated the

effects of resveratrol on mitochondrial membrane potential and cell morphology

of HepG2 cells. Their results showed that resveratrol at high concentrations can

obviously cause sharp increment of the mitochondria membrane potential. They

concluded that the capacity of resveratrol for inhibiting proliferation and

inducing cell apoptosis could be resulted in depolarizing mitochondrial membrane

potential (46).

Like curcumin, potent anti-metastatic activity of resveratrol has been also

investigated. Yu et al. investigated the effects of resveratrol on invasion

ability of human HCC cells and tumor necrosis factor-alpha (TNF-)-mediated MMP-9

expression. They found that resveratrol significantly inhibited TNF--mediated

MMP-9 expression, NF-kappa B expression and invasion in HepG2 cells. They

concluded that the inhibition of TNF--mediated MMP-9 expression and the

potential invasion by resveratrol are partly associated with the downregulation

of the NF-kappa B signaling pathway (47). Studies carried out by Miura et al.

demonstrated that dietary resveratrol can inhibit metastasis of hepatoma in

Donryu rats subcutaneously implanted with an ascites hepatoma cell line of

AH109A. They found that ROS accelerated the invasive capacity of a rat ascites

hepatoma cells, and resveratrol suppressed the ROS-potentiated invasion of the

hepatoma cells (48,49). Despite the encouraging achievement regarding the

anti-HCC effects of resveratrol, the low bioavailability and the potential

toxicity by the modulation of liver genes at the high dose are also observed

(50,51). Again, as with Curcumin the specific active agents in resveratrol have

not been accurately defined, and clinical trial researchers have been plagued by

the lack of systemic absorption that can be achieved through oral

administration.

Silibinin

Silibinin, a polyphenolic flavonoid, is the major biologically active compound

of milk thistle. It is well known that milk thistle is safe and well-tolerated,

and it protects the liver from drug or alcohol-related injury (52,53). Silibinin

and its crude form, silymarin, are used clinically and as dietary supplements

against liver toxicity. A randomized controlled multicenter trial has shown that

daily administration of silymarin for several years results in a significant

reduction in the mortality of patients suffering from alcoholic liver cirrhosis

(54). Studies have demonstrated the inhibitory effects of silibinin on multiple

cancer cell lines including HCC (55,56). Varghese et al. investigated the

effects of silibinin on cell growth, cytotoxicity, apoptosis and cell cycle in

two different HCC cell lines, HepG2 (hepatitis B virus negative; p53 intact) and

Hep3B (hepatitis B virus positive; p53 mutated). They found that silibinin

strongly inhibited growth of both HepG2 and Hep3B cells. Silibinin also caused

G1 arrest in HepG2, and G1 and G2-M arrests in Hep3B cells. Further studies

showed that silibinin induces Kip1/p27 but decreases cyclin D1, cyclin D3,

cyclin E, cyclin-dependent kinase (CDK)-2, and CDK4 levels in these two cell

lines. In Hep3B cells, silibinin also reduced the protein levels of G2-M

regulators. CDK2, CDK4, and CDC2 kinase activity were strongly inhibited in

these HCC cells by silibinin (57). Lah et al. investigated the effect of

silibinin on HCC cell growth in four human HCC cell lines: HuH7, HepG2,

PLC/PRF/5 and Hep3B cells. After treated with different doses of silibinin,

proliferation, apoptosis, cell-cycle progression, histone acetylation and other

related signal transductions were examined. They demonstrated that silibinin

significantly inhibited the growth of HuH7, HepG2, Hep3B and PLC/PRF/5 human

hepatoma cells. In addition, they also found downregulated levels of

metalloproteinase-2 (MMP2) and CD34 in the HCC cells, which could be a possible

anti-angiogenic mechanism of silibinin. They also demonstrated that silibinin

increased acetylation of histone H3 and H4 (AC-H3 and AC-H4), indicating a

possible role of altered histone acetylation in chemoprevention of silibinin

against HCC cells (58). Momeny et al. evaluated the effect of silibinin on

HepG-2 cells regarding the biomarkers of cell proliferation, cytotoxicity,

metastatic potential, nitric oxide (NO) production, ERK 1/2 phosphorylation and

activation in HepG-2 cells. They found that silibinin inhibited cell

proliferation, matrix MMP-2 enzymatic activity, NO production and ERK 1/2

phosphorylation without exerting any cytotoxicity effect. The possible mechanism

of silibinin against HCC could be inhibiting cell proliferation and invasive

potential of HepG-2 cells through inhibition of ERK 1/2 cascade (59).

Currently, silibinin has not been evaluated for human effects or toxicity in

human clinical trials and thus only holds potential promise as an active

chemopreventitive agent.

Tanshinone II-A

Tanshinone IIA, one of the most abundant diterpenes isolated from Salvia

miltiorrhiza Bunge (Danshen in Chinese). Tanshinone IIA has been shown to

possess pharmacological activities including antioxidant (60), protecting and/or

preventing angina pectoris and myocardial infarction (61). Report has shown that

inhibition of proliferation and cytotoxic effects on cell lines derived from

various human carcinomas (62,63). Yuan et al. evaluated the effects of

tanshinone II-A on growth inhibition and apoptosis of human HCC cells (cell line

SMMC-7721). The growth and colony-forming of SMMC-7721 cells were obviously

suppressed after tanshinone II-A treatment. The apoptosis index was

significantly increased and the cells were arrested in G(0)/G(1) phase. In

addition, expressions of apoptosis-related genes bcl-2 and c-myc were

downregulated, while fas, bax, p53 upregulated (64). Zhong et al. investigated

the effect of tanshinone IIA on the growth and apoptosis in HepG2 cells. They

found that tanshinone IIA not only inhibited the cell growth, but also induced

apoptosis in HepG2 cells (65). Tang et al. studies the effect of tanshinone IIA

on growth and apoptosis in human HCC cell line BEL-7402. Growth suppression and

induced cell apoptosis were found as BEL-7402 cells treated with tanshinone IIA

(66). Wang et al. evaluated the proliferation of human HCC cell line (SMMC-7721)

treated with tanshinone by Brdu labeling and PCNA immunohistochemical staining.

They found decreased indexes of Brdu labeling and PCNA detection after

tanshinone treatment. The inhibitory effect of tanshinone on cancer cell growth

might associate with inhibiting DNA synthesis (67,68). Li et al. performed in

vitro and in vivo studies using polylactic acid nanoparticles containing

tanshinone IIA (TS-PLA-NPs) against HCC. They found that tanshinone IIA in

TS-PLA-NPs were effective in destroying the human liver cancer cells. Tanshinone

IIA in TS-PLA-NPs also prevented tumor growth and increased survival rate of

mice with hepatoma (69).

Other Reported Compounds

Beside these widely investigated compounds, some other potential compounds for

the chemopreventive effect against HCC have been also evaluated. Salvianolic

acid B is a major water-soluble polyphenolic acid extracted from Radix Salviae

miltiorrhizae (Sm). Studies have shown that Salvianolic acid B can improve acute

and chronic liver, decrease the serum alanine aminotransferase (ALT) and

aspartate aminotransferase (AST) levels and enhance the total prostaglandin

content in liver mesenchymal cells in injured rats (70). Baicalein is a

flavonoid from baikal skullcap root. Matsuzaki et al. investigated the anti- HCC

effect of baicalein. They found that treatment with baicalein strongly inhibited

the activity of topoisomerase II, induced apoptosis and suppressed the

proliferation the HCC cell lines (71). Pheophorbide a (Pa) is an active compound

from Scutellaria barbata. Tang et al. used a multi-drug resistant (MDR) HCC cell

line (R-HepG2) to evaluate the anti-proliferative effect of Pa. They found that

Pa can significantly inhibit the growth of R-HepG2 cells (72). It has been

showed that Pa can enhance the efficacy of photodynamic therapy (PDT) for HCC,

very likely due to its antiproliferative and pro-apoptotic effect (73–75).