Anti-HCV activities of selective polyunsaturated fatty acids
Guang-Zhou Leu,
aTiao-Yin Lin,
aand John T.A. Hsu
b,c,*aDepartment of Biological Science and Technology, National Chao Tung University, Hsinchu, Taiwan, ROC bDivision of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Taipei, Taiwan, ROC
cDepartment of Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan, ROC
Received 11 March 2004
Abstract
HCV infection can lead to chronic infectious hepatitis disease with serious sequelae. Interferon-a, or its PEGylated form, plus
ribavirin is the only treatment option to combat HCV. Alternative and more effective therapy is needed due to the severe side effects
and unsatisfactory curing rate of the current therapy. In this study, we found that several polyunsaturated fatty acids (PUFAs)
including arachidonic acid (AA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA) are able to exert anti-HCV
ac-tivities using an HCV subgenomic RNA replicon system. The EC
50(50% effective concentration to inhibit HCV replication) of AA
was 4 lM that falls in the range of physiologically relevant concentration. At 100 lM, a-linolenic acid, c-linolenic, and linoleic acid
only reduced HCV RNA levels slightly and saturated fatty acids including oleic acid, myristic acid, palmitic acid, and steric acid had
no inhibitory activities toward HCV replication. When AA was combined with IFN-a, strong synergistic anti-HCV effect was
observed as revealed by an isobologram analysis. It will be important to determine whether PUFAs can provide synergistic antiviral
effects when given as food supplements during IFN-based anti-HCV therapy. Further elucidation of the exact anti-HCV mechanism
caused by AA, DHA, and EPA may lead to the development of agents with potent activity against HCV or related viruses.
Ó 2004 Elsevier Inc. All rights reserved.
Keywords: HCV; Replicon; Polyunsaturated fatty acids including arachidonic acid; Eicosapentaenoic acid; Docosahexaenoic acid
Chronic hepatitis C virus (HCV) infection, previously
the most common causative agent of non-A, non-B
hepatitis, is a major health problem worldwide.
Ac-cording to the estimation made by the World Health
Organization, HCV affects 170 million individuals
worldwide [1]. Although the acute phase of infection is
usually associated with mild symptoms, approximate
80% of HCV infection results in chronic infection that
frequently leads to severe chronic liver disease; 20–30%
of infected individuals may develop cirrhosis and 1–3%
may develop liver cancer [2,3]. Neither a vaccine nor
specific antiviral agents are available for the treatment of
HCV infection. Currently, interferon-a (IFN-a), or
PEGylated IFN-a, combined with ribavirin, a guanosine
analogue, is the only recommended treatment [4].
Al-though the combination therapy represents a major
ad-vance in the treatment of HCV, this treatment is effective
only in around 50% of patients [5]. Given the high
prevalence and severe clinical sequels of HCV infection,
discovery, and development of molecular-based agents
for HCV therapy have become a focus of intensive
re-search. Equally important is to assess and better define
the risks and benefits of alternative and nontraditional
means for treating or stabilizing HCV infection [6].
HCV is a member of the family Flaviviridae. It
contains a single-stranded, positive-sense RNA genome
of approximate 9.5 kb encoding a unique polyprotein
of approximately 3000 amino acids [7,8]. The
poly-protein precursor is co- and post-translationally
pro-cessed by cellular and viral proteases to yield the
mature structural and nonstructural proteins arranged
in
the
sequence
of
NH
2–C–E1–E2–P7–NS2–NS3–
NS4A–NS4B–NS5A–NS5B–COOH. This polyprotein
is consisted of at least three structural (core, E1, and
E2) and seven nonstructural (NS) (p7, NS2, NS3,
HS4A/B, and NS5A/B) protein coding region. It has
been recently described that some HCV-encoded
pro-teins can be produced from alternate reading frames
through ribosomal frameshift [9]. The ORF is flanked
*
Corresponding author. Fax: +886-2-2789-0264. E-mail address:[email protected](J.T.A. Hsu).
0006-291X/$ - see front matterÓ 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.04.019
Biochemical and Biophysical Research Communications 318 (2004) 275–280
BBRC
at the 5
0end by a nontranslated region (NTR) that
functions in part as an internal ribosome entry site
(IRES) and at the 3
0end by a highly conserved
se-quence essential for replication of viral genome. In
recent years, many potent antiviral agents are being
developed based on viral enzymes that are essential for
HCV replication.
The inability to efficiently propagate HCV in cell
culture had impeded the development of antiviral agents
against this virus. This obstacle was partly overcome by
the development of a bicistronic subgenomic HCV
replicons in Huh-7 cells [10,11]. These subgenomic
rep-licon systems have greatly facilitated the studies of HCV
replication. With the aid of the HCV subgenomic RNA
replication system, we evaluated the effect of PUFAs on
HCV replication. In this report, we showed that AA,
DHA, and EPA are able to exert anti-HCV activity.
Detailed dose-dependent studies showed that AA was
effective at concentration that is achievable at normal
physiological conditions. Importantly, when combined
with IFN-a, AA was able to exert strong synergistic
anti-HCV activity.
Materials and methods
Reagents and cells. Dulbecco’s modified Eagle’s medium (DMEM) high glucose, Fetal calf serum (FCS), TRIZOL reagent, and G418 (geneticin) were purchased from Invitrogen (Carlsbad, CA). Arachi-donic acid (AA; 20:4, n6), docosahexaenoic acid (DHA; 22:6, n3), eicosapentaenoic acid (EPA; 20:5, n3), a-linolenic acid (18:3, n3), c-linolenic acid (18:3, n6), and linoleic acid (18:2, n6) were obtained from Cayman Chemical (Ann Arbor, MI). Oleic acid (18:1, n9), myristic acid (14:0), palmitic acid (16:0), and steric acid (18:0) were obtained from Sigma–Aldrich (St. Louis, MO). The [a-32P]dCTP was purchased
from Amersham Bioscience (Piscataway, NJ). Human hepatoma cells (Huh-7) was purchased from Japanese Collection of Research Biore-sources (JCRB, JCRB0403) and Huh-7 cell clone containing HCV replicon (Ava5) was provided by Apath (St. Louis, MO). Cells were maintained in DMEM supplemented with 10% heat-inactivated FCS in a humidified atmosphere containing 5% CO2. For Ava5, the culture
medium was additionally supplemented with 500 lg/ml G418. Cytotoxicity assay. Cell viability was determined by MTS assay that was essentially as described [12]. The principle of this convenient method is based on the conversion of tetrazolium salt (MTS) into a chro-matic, soluble formazan by a mitochondria enzyme, NAD-dependent
dehydrogenase, in live cells. MTS and PMS are purchased from Sigma (St. Louis, MO) or Promega (Madison, WI) as powder and prepared in DPBS (Dulbecco’s phosphate-buffered saline). For a measurement with a 96-well microtiter plate, 2 ml reagent containing both MTS and PMS in the ratio of 20:1 is mixed immediately with 8 ml serum-free DMEM before adding into drug-treated cells. Drug concentration is performed with four repeats.
Northern blotting. Total RNA was isolated from cells using TRI-ZOL reagent (one-step, guanidium thiocyanate phenol–chloroform total RNA isolation reagent) according to supplier’s instruction. RNA was isolated and concentration was determined by spectrophotometer. All reagents used for analysis are of ultra-pure grade. RNA samples, 10 lg each well, were loaded onto 1% TBE agarose gel and separated by electrophoresis at 10 V/cm for 1.5 h according to Kevil et al. [13]. The RNAs in the gel were then transferred to a positively charged nylon membrane, BrightStar-Plus (Ambion, Austin, TX), by a vacuum blotter (Vacu. GeneXL, Pharmacia, MI). After drying, RNA was then crosslinked to the membrane by UV irradiation (Stratagene, CA). The membrane was probed separately with the NS5B gene fragment of HCV and human glyceraldehydes-3-phosphate dehydrogenase (GADPH) fragment labeled with [a-32P]dCTP by rediprime II random prime
labeling system (Pharmacia, MI) in accordance with manufacturer’s instructions. Hybridization was carried out with denatured probes in Rapid-hyb hybridization buffer (Pharmacia) for 2 h at 65°C. After hybridization, membranes were washed once in 2 SSC–0.2% SDS for 20 min at 60°C and once in 1 SSC–0.2% SDS for 20 min at 60 °C and twice in 0.1 SSC–0.2% SDS for 15 min at 65 °C. The results of hybridization were visualized by autoradiography.
Synergistic statistics. Effect of combination of drugs was evaluated using an isobologram method [14,15]. Isobologram is a generalized method for analyzing the effects of multiple drugs and for determining their additivity, synergism, or antagonism. Different doses of AA and IFN-a were combined to generate dose–response curves of inhibition of HCV replication. Cells were treated with AA (0, 0.78, 3.13, 12.5, 50, and 200 lM) and IFN-a (0, 0.78, 3.13, 12.5, 50, and 200 IU/mL) that were combined in checkerboard manner. Antiviral response for inhibitory concentration for each combination was analyzed by sig-moid regression. The resulting data were used to generate isoeffect curves (isoboles) of 50% and 90% inhibition to determine drug–drug interactions.
Results and discussion
Effects of PUFAs on HCV RNA replication
To study the effects of PUFAs on HCV replication,
the HCV sub-genomic recplicon cells (Ava5) containing
HCV subgenomic RNA were employed [11]. Cells were
Fig. 1. Effect of fatty acids on HCV RNA replication in replicon cells containing HCV subgenomic RNA (Ava5 cells). Ava5 cells were treated with AA (lane 2), DHA (lane 3), EPA (lane 4), a-linolenic acid (lane 5), c-linolenic acid (lane 6), linoleic acid (lane 7), oleic acid (lane 8), myristic acid (lane 9), palmitic acid (lane 10), and steric acid (lane 11) at 100 lM for 24 h. Lane 1 was mock treatment containing only solvent used for preparation of stock solutions. Cellular RNAs were extracted and analyzed by Northern blotting as described in Materials and methods. The percentage of HCV RNA remained upon each treatment is shown in the bottom of the figure.
treated with fatty acids, including polyunsaturated,
monounsaturated, and saturated fatty acids, for 24 h. As
shown in Fig. 1 (lanes 2–4), several PUFAs, including
arachidonic acid (AA), docosahexaenoic acid (DHA),
and eicosapentaenoic acid (EPA), were able to exert
potent anti-HCV activities at 100 lM. At the same
concentration, a-linolenic acid, c-linolenic, and linoleic
acid only reduced HCV RNA levels slightly (Fig. 1,
Fig. 2. Effect of AA on inhibition of HCV RNA replication and cell viability. (A) Inhibition of HCV-RNA replication by AA in a dose-dependent manner. Ava5 cells were treated with various concentrations of AA for 24 h. Total cellular RNA was extracted and analyzed for the HCV sub-genomic RNA and GAPDH mRNA levels by Northern blotting. (B) As a reference standard, IFN-a was used to treat Ava5 cells and cellular RNAs were analyzed for HCV subgenomic RNA and GAPDH mRNA. (C) Viability of cells as measured by MTS assay. Cells were treated with different concentrations of AA for 24 and 72 h. Each data point was derived from six identical repeats.
lanes 5–7). In contrast, saturated fatty acids including
oleic acid, myristic acid, palmitic acid, and steric acid
slightly enhanced HCV RNA levels (Fig. 1, lanes 8–11).
The RNA levels of GADPH, a house-keeping gene,
were not affected by drug treatments.
Arachidonic acid reduced HCV RNA level
dose-depen-dently
To further confirm the results, we chose to analyze
the effect of AA for its anti-HCV activity in more
details. Ava5 cells were treated with AA at various
concentrations for 24 h. In Fig. 2A, it is clear that AA
was able to suppress HCV RNA levels in a
dose-dependent manner. When cells were treated with
100 lM AA, there was only 7.5% of HCV RNA left
compared to untreated cells while treatment with
500 IU/mL IFN-a reduced HCV RNA level to 13.4% of
the control (Fig. 2B). The EC
50(effective concentration
required to inhibit 50% of HCV RNA level) of AA is
approximate 4 lM which is physiologically relevant.
The plasma concentration for AA varied from 5.8 to
49.3 lM [16]. As a comparison, the anti-HCV activity of
IFN-a was measured in parallel and the EC
50of IFN-a
was around 3.1 IU/ml (Fig. 2B).
There was no cellular toxicity for cells treated with
AA at this range as revealed by MTS assay (Fig. 2C)
and the IC
50(concentration required to inhibit 50% of
cell viability) was measured to be around 350 and
380 lM after 24 and 72 h, respectively, of drug
treat-ment. The cellular morphology did not change after
treatment with AA for 24 h at 100 lM (data not shown).
Thus, AA might exert its anti-HCV effects through a
specific pathway but not because of its cellular toxicity.
Synergistic antiviral activity of AA combined with IFN-a
Whether AA and IFN-a combination exerts
syner-gistic, additive, or antagonistic effects was assessed by
an isobologram method [14,15]. In general,
represen-tation of an isobologram to measure drug–drug
inter-action is shown in Fig. 3A. It was proposed that
synergy, additivity, and antagonism would be
repre-sented by concave, linear, and convex isoeffective curves
(isoboles), respectively. The anti-HCV effects of AA
and IFN-a in combination were evaluated. Ava5 cells
were treated with these two drugs in combination in a
checkerboard titration manner. HCV subgenomic RNA
levels in cells were then measured. Dose–response
inhibition of HCV RNA replication was evaluated for
varying AA concentrations (0, 0.78, 3.13, 12.5, 50,
and 200 lM) in the presence of various doses of IFN-a
(0, 0.78, 3.13, 12.5, 50, and 200 IU/mL) (Table 1). The
data in Table 1 were used to generate isoboles of 50%
and 90% inhibition of HCV replication (Fig. 3B). AA
and IFN-a exerted strong synergistic anti-HCV
activi-ties as revealed by the curvy concave plots of 50% and
90% isoboles.
Current IFN-based therapy for treating HCV
infec-tion is not satisfactory and development of more
effec-tive drugs has not been very fruitful over the past few
years. In fact, many HCV patients often seek other
complement and alternative medicine (CAM) and some
may even avoid or abandon standard IFN-based
ther-apy and seek other types of therther-apy [17,18]. Many forms
of CAM have shown scientific evidence as
cytoprotec-tive agents. However, few were known to possess specific
antiviral activity. PUFAs such as AA, DHA, and EPA
were all recognized as essential nutrients in human diet.
The metabolites of PUFAs also play numerous
impor-tant roles in normal physiological conditions and
pro-gression of diseases [19]. In this study, AA was found to
be able to inhibit HCV replication at physiologically
relevant concentration. Further research is needed to
evaluate the therapeutic role of PUFAs in the clinical
Fig. 3. Effect on HCV RNA levels by combination of AA and IFN-a. (A) In general, representation of an isobologram for measuring in-teraction between two drugs is shown in (A). The synergy, additivity, and antagonism would be represented by concave, linear, and convex isoeffective curves (isoboles) as shown. (B) Isoboles of 50% (EC50) and
90% (EC90) inhibition of HCV replication. Each data point in the
management for HCV-infected patients. It may be
possible that these fatty acids can be both as adjunctive
or complementary treatment to benefit HCV patients
through dietary control. In this study, we also found
that antiviral activity of IFN-a can be accentuated by
AA and probably also by other PUFAs. Thus, further
studies are warranted if management of AA or other
PUFAs through dietary control could increase the
ef-fectiveness of current IFN-based treatment as antiviral
therapy.
Polyunsaturated fatty acids (PUFAs) are important
for many physiologic functions [20–22]. AA, the
pre-cursor of eicosanoids, can be catalyzed by at least three
types of enzymes in cells, cyclooxygenases (COXs),
lip-oxygenases (LOXs), and P450 epoxygenase (CYPs), to
generate numerous metabolites that can mediate diverse
physiological and pathological responses such as blood
pressure, inflammation, phagocyte activation, pain, and
fever [22–25]. The mechanism of action of PUFAs in
inhibition of HCV replication is not clear. Nevertheless,
this study provides a potentially favorable observation
of drug–food interactions. A human trial is mandatory
to understand the clinical value of PUFAs in HCV
therapy. It is also important to elucidate of the exact
anti-HCV mechanism caused by the PUFAs identified
this study. Such understanding may lead to the
devel-opment of agents with potent activity against HCV or
related viruses.
Acknowledgments
We thank Dr. Charles Rice for providing the Ava5 cells. We also thank Drs. Yu-Sheng Yeh and Chau-Ting Chao for their helpful dis-cussion. This work was supported by the NHRI in Taiwan to John T.-A. Hsu.
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IFN-a concentration (IU/mL)
Arachidonic acid concentration (lM)
0 0.78 3.13 12.5 50 200 0 100 9.3 88.0 8.3 65.8 5.1 38.0 1.1 17.5 3.0 11.6 0.9 0.78 98.6 5.6 71.6 7.3 63.1 4.0 41.6 4.5 20.4 0.7 10.4 0.3 3.13 56.9 4.2 47.3 3.3 23.6 2.6 19.0 3.0 15.7 1.5 9.8 0.5 12.5 51.0 3.2 38.0 8.3 24.5 5.0 14.5 0.8 7.0 1.0 7.0 0.4 50 16.9 0.6 18.5 1.1 12.3 2.0 6.8 0.3 4.2 0.3 2.0 0.08 200 9.8 0.6 8.4 0.4 6.7 0.4 2.8 0.4 4.1 0.2 2.8 0.1 a
Relative levels of remaining HCV RNA in cells (% of control). Results are expressed as means SD of three experiments.
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