Download Anti-HIV-1 activity of five anthraquinone derivatives* OUYANG Dong

Anti-HIV-1 activity of five anthraquinone derivatives*
OUYANG Dong-yun, LIU Chun-yong, ZENG Yao-ying△ , HE Xian-hui, ZENG Xiang-feng
(Institute of Tissue Transplantation and Immunology, College of Life Science and Technology, Jinan
University, Guangzhou 510632, China)
[ABSTRACT]
AIM: To investigate the anti-HIV-1 activity of five anthraquinone
derivatives (emodin, rhein, chrysophanol, physcion and aloe-emodin) in vitro. METHODS: Viral
replication was estimated by observation of cytopathogenesis and measurement of HIV-1 p24
antigen production in HIV-1IIIB acutely infected C8166 cells. The anti-HIV-1 activity was evaluated
by the 50% effective concentrations (EC50) and selective indexes (SI) of these derivatives.
RESULTS: These anthraquinone derivatives inhibited HIV-1IIIB replication on syncytia formation
induced by HIV-1IIIB infection with EC50 mean values of (11.44 ± 0.93) μmol/L (emodin), (51.28 ±
2.86) μmol/L (rhein), (90.58 ± 2.30) μmol/L (chrysophanol), (8.59 ± 0.38) μmol/L (physcion) and
(0.89 ± 0.08) μmol/L (aloe-emodin), respectively. The p24 antigen production with EC50 mean
values were (11.61 ± 0.56) μmol/L (emodin), (12.35 ± 4.73) μmol/L (rhein), (39.63 ± 2.87) μmol/L
(chrysophanol), >250 μmol/L (physcion) and (2.75 ± 0.20) μmol/L (aloe-emodin), respectively.
CONCLUSION: These structurally-related chemicals show different anti-HIV-1 activity in vitro.
Among them, aloe-emodin is the most potent inhibitor to HIV-1 replication.
[KEY WORDS]
HIV-1; Anthraquinone; Emodin; Rhein; Chrysophanol; Physcion;
Aloe-emodin
[CLC number]
R285.5
[Document code]
A
* [Foundation item] Supported by the National 973 Project of China (No. 2006CB504200); Natural Science
Fund of Guangdong Province (No. 8451063201000340)
△Corresponding author Tel: 020-85226219; E-mail: [email protected]
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Natural products are an important reservoir for developing new anti-HIV-1 drugs. Previous
reports indicate that anthraquinone derivatives possess anti-tumor, anti-bacterial and anti-fungal
activities. Some of the derivatives are also found to be effective against a broad spectrum of viruses,
such as human cytomegalovirus (HCMV)[1], poliovirus[2], vesicular stomatitis virus[3], herpes
simplex virus types 1 and 2 (HSV-1, -2)[4], parainfluenza virus[3], as well as hepatitis-B virus
(HBV)[5]. Radix et Rhizoma Rhei, which is usually processed from the roots of Rheum palmatum L.,
Rheum tanguticum Maxim. ex Balf. or Rheum officinale Baill, contains several anthraquinone
derivatives (including emodin, rhein, chrysophanol, physcion and aloe-emodin) and has multiple
activities such as laxation, hepatoprotection, prevention from obesity and atherosclerosis as well as
the development of cerebral ischemia-reperfusion injury on brain tissues[6-8]. In this study, we
examined the inhibitory effects of these five structure-related anthraquinones on HIV-1 replication
in vitro. Viral replication was estimated by observation of cytopathogenesis and measurement of
HIV-1 p24 antigen production. We also investigated whether emodin could inactivate HIV-1
particles directly, as it does on other enveloped viruses[9].
MATERIALS AND METHODS
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Reagents and chemicals
Five anthraquinone derivatives (emodin, rhein, chrysophanol, physcion and aloe-emodin) were
bought as reference materials from the National Institute for the Control of Pharmaceutical and
Biological Products (P. R. China). They were dissolved in dimethyl sulfoxide (DMSO; Sigma) at a
stock concentration of 0.1 mol/L and stored at -20 oC. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide (MTT) reagent was purchased from Sigma.
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Cells and virus
Cell lines of C8166, H9 and H9/HIV-1IIIB were maintained in RPMI-1640 (Gibco)
supplemented with 10% heat-inactivated fetal calf serum and antibiotics (complete medium). The
cells used in all experiments were in log-phase growth. HIV-1IIIB was obtained from the culture
supernatant of H9/HIV-1IIIB cells. The 50% HIV-1 tissue culture infectious dose (TCID50) in C8166
cells was determined and calculated by the Reed and Muench method. Virus stocks were stored in
aliquots at −70 C. The titer of the virus stocks was 2.3×109 TCID50/L.
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Cytotoxicity assay
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Cells were seeded in triplicate in complete medium for 72 h in the presence of varying
concentrations of anthraquinone derivatives or vehicle (DMSO) alone. Cytotoxicity was measured
by MTT assay under the manufacturer’s instruction. The percentage of viable cells was quantified at
570 nm with a reference wave length of 630 nm in a microplate reader (Bio-Rad, model 680). To
reduce the photoabsorption interference of the anthraquinone rings at 570 nm, the cell culture
medium was removed before adding haemolyzing solution. The cytotoxic concentration that caused
50% reduction of viable cells (CC50) was determined from the dose-response curve.
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Syncytium reduction assay
In the presence of 100 μL anthraquinone derivatives at varying concentrations, 50 μL of C8166
cells (5×105/mL) were infected with HIV-1IIIB at a multiplicity of infection (MOI) of 0.018. The
final volume per well was 200 μL. After cultured for 72 h, syncytia in the culture wells were
observed and counted under an inverted microscope to evaluate the cytopathic effects (CPE).
Inhibition rates of syncytial cell formation were measured by comparing the syncytium numbers in
drug-treated culture wells with that in control (vehicle alone) wells. Fifty percent effective
concentration (EC50) was calculated from the dose-response curve.
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ELISA for HIV-1 p24 antigen
C8166 cells (6×108 cells/L, 50 μL/well) were inoculated with HIV-1IIIB (MOI=0.015) in the
presence of different concentrations of anthraquinone derivatives in triplicate and cultured at 37 C
in a humidified chamber with 5% CO2 for 72 h. The cells in six wells treated with vehicle alone
(control) were used as positive controls (HIV-1IIIB infected) and negative controls (HIV-1IIIB absent).
The levels of HIV-1 p24 antigen in the culture supernatant were determined by a sandwich ELISA
as described previously[10].
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Co-cultivation assay
HIV-1IIIB chronically infected H9 cells (H9/HIVIIIB, 3×104 cells/well) were pretreated for 1 h
with different concentrations of anthraquinones or vehicle alone (control) prior to mixing with
3×104 C8166 cells in a 96-well plate. After co-cultured for 4 h at 37 C in a humidified chamber
with 5% CO2, the syncytium formation was scored as described above.
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Direct drug-virus incubation assay
Serial diluted emodin solutions were incubated with an equal volume of HIV-1 stock
supernatant at room temperature for 40 min. H2O2 or complete medium was used as positive and
negative controls, respectively. The mixtures (5 μL) were then diluted with 995 μL of complete
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medium to stop further inactivation of HIV-1 particles. The remaining infectivities of drug-treated
HIV-1 particles in 150 μL of the diluted solutions were indicated by cytopathic effects of C8166
cells (2×104 cells/well) in a 96-well microtiter plate. After 60 h incubation at 37 C in a humidified
chamber with 5% CO2, the syncytia were scored as described above. The HIV-1 particle
infectivities were estimated by comparing the syncytium numbers in drug-treated culture wells with
that in negative control (vehicle alone) wells. Each experiment was done in triplicate.
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Statistical analysis
Data were collected from three independent experiments performed in triplicates. Values were
analyzed by Prism software 4.0 (GraphPad San Diego, CA). One-way ANOVA followed by
Newman-Keuls post test was used to compare the results between treatment groups. Results were
presented asx ± sE and a P-value <0.05 was considered significant.
RESULTS
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Cytotoxicity of anthraquinone derivatives
Cell viability of C8166 cells incubated with anthraquinone derivatives was evaluated by MTT
assay. Emodin, rhein and aloe-emodin suppressed the cell proliferation in a dose-dependent manner.
The 50% cytotoxic concentrations (CC50) of emodin and rhein for C8166 cells were (144.19 ± 2.43)
μmol/L and (174.70 ± 4.15) μmol/L, respectively, while the other three derivatives were much less
cytotoxic to C8166 cells. The CC50 value of aloe-emodin was approximately 750 μmol/L, and the
CC50 values of chrysophanol and physcion were greater than 750 μmol/L (Table 1, Figure 2A).
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Anti-HIV-1 activity of anthraquinone derivatives
HIV-1 replication in C8166 cells was evaluated by two methods: (1) CPE method to count the
syncytium numbers induced by HIV-1 infection and (2) ELISA method to detect the HIV-1 p24
levels in cell culture supernatant. Results from both methods clearly demonstrated that the five
anthraquinone derivatives suppressed HIV-1 replication in C8166 cells in a dose-dependent manner
(Figure 2B and Figure 2C). Table 1 summarizes their 50% effective concentrations (EC50) and
selective indexes (SI=CC50/EC50). Emodin, rhein and aloe-emodin, when their concentrations were
more than 10 μmol/L, showed strong inhibitory effects on HIV-1 replication; the selective indexes
were 12.6 ± 1.2, 3.4 ± 0.3 and approximately 838, respectively (by method 1), or 12.4 ± 0.7, 14.2 ±
4.9 and approximately 273, respectively (by method 2). When used at the same concentration,
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chrysophanol was less effective against virus as compared to emodin, rhein and aloe-emodin,
though it was less cytotoxic to host cells than the other three derivatives. Although physcion
inhibited the cytopathogenesis in HIV-1IIIB infected C8166 cells [with an EC50 value of (8.59 ± 0.38)
μmol/L and a SI value >87], but it was less effective against HIV-1 p24 production (EC50 >250
μmol/L).
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Effect of anthraquinone derivatives on the syncytium formation
The syncytia are induced by budding virus particles on cell surface binding to receptors on an
adjacent cell. This cytopathic effect can be easily observed in a short time (usually within 4 h) after
co-culture of uninfected C8166 cells with HIV-1IIIB chronically infected H9 cells (H9/HIVIIIB). This
system is often used to evaluate whether a chemical can prevent (or impede) HIV-1 from entering
host cells at the fusion stage of the viral life cycle. The co-culture assay indicated that the
anthraquinone derivatives (except chrysophanol) had little influence on the syncytium formation
even when their concentrations were >10 μmol/L (Figure 2D).
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Direct incubation of HIV-1IIIB particles with emodin
We also tested the ability of emodin in inactivated HIV-1 particles by pre-incubating HIV-1IIIB
virus with emodin before infection of C8166 cells. No significant difference in the syncytium
formation was observed between drug-treated groups and the negative control one (virus treated
with vehicle only), even though the concentration of emodin reached as high as 1.25 mmol/L. As a
positive control, 0.12% H2O2 totally inactivated HIV-1IIIB and RPMI-1640 dilution of the
inactivated HIV-1IIIB could not reactivate the viral particles (Figure 3).
DISCUSSION
Combination therapy of anti-HIV drugs (ART) has significantly increased the length and
quality of life for HIV-1 infected individuals. Unfortunately, due to the side effects and drug
resistance of mutated HIV-1, as well as the high cost to patients, new anti-HIV-1 compounds need
to be developed.
Chinese medical herbs have a long history of treating various diseases. Many natural
compounds purified from these herbs have demonstrated inhibitory effects on HIV-1 replication,
targeting nearly every stage of the HIV-1 life cycle. These compounds vary in chemical
composition and include alkaloids, sulphated polysaccharides, polyphenolics, flavonoids,
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coumarines, phenolics, tannins, triterpenes, lectins, phloroglucinols, lactones, iridoids, depsidones,
O-caffeoyl derivatives, lignans, ribosome inactivating proteins, saponins, xanthones,
naphthodianthrones, phospholipids, quinines and peptides.
As mentioned above, previous studies indicate that anthraquinone derivatives can inactivate a
wide-spectrum of viruses. Of note is that emodin shows effectiveness against HBV[5], which like
HIV-1, is a retrovirus.
In this study, we investigated whether emodin and related anthraquinone derivatives (natural
products from Radix et Rhizoma Rhei) could suppress HIV-1 replication in vitro. The results
demonstrated that 4 of the 5 anthraquinone derivatives tested (emodin, rhein, chrysophanol and
aloe-emodin) had anti-HIV-1 activity with EC50 values lower than 100 μmol/L. Among them,
aloe-emodin was the most potent inhibitor, with its EC50 values of (0.89 ± 0.08) μmol/L (CPE
method) and (2.75 ± 0.20) μmol/L (p24 ELISA method). The SI values of aloe-emodin were higher
(≈838 by CPE method and SI≈273 by p24 ELISA method) than that of other derivatives (Table 1).
These results suggest that some of the derivatives have the potential to be developed as anti-HIV-1
lead compounds.
The mechanism of the anthraquinones’ anti-HIV-1 activity is still exclusive. Other studies
reported that emodin, aloe-emodin and other derivatives were effective against a broad spectrum of
enveloped viruses, the likely mechanism may beassociated with the disruption of virus envelope[3].
Showing affinity to phospholipid membranes, emodin weakened the hydrophobic interactions
between hydrocarbon chains in phospholipid bilayers. However, membrane disruption by emodin
was only specific to phospholipid bilayers containing phosphatidylethanolamine and
phosphatidylglycerol, the two major phospholipids present in the membranes of bacteria. In
disagreement with this report, our study demonstrated that pre-incubation with emodin did not
significantly reduce the infectivity of HIV-1 particles (Figure 3), suggesting that direct inactivation
is not sufficient to explain why emodin suppressed HIV-1 replication in C8166 cells. One possible
explanation is that the components of HIV-1 envelope may be different from that of other viruses.
Furthermore, emodin did not influence HIV-1 binding or fusion with host cell membranes.
When C8166 cells were co-cultured with the HIV-1IIIB chronically infected H9 cell line,
cytopathogenesis was not significantly reduced in the presence of emodin, rhein, physcion or
aloe-emodin. The only exception existed in the syncytium reduction in response to chrysophanol
when its concentration was >10 μmol/L (Figure 2D), suggesting that high concentrations of
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chrysophanol could delay the virus-receptor fusion. Of note was that chrysophanol was only one of
the derivatives to inactivate non-enveloped viruses (poliovirus types 2 and 3)[2].
Several compounds with anthraquinone pharmacophore (i.e., hypericin, quinalizarin, purpurin
and alizarin) were found to be inhibitors of HIV-1 integrase or the subviral preintegration complex
(PIC). However, emodin displayed no inhibition of purified integrase or PIC[11].
Anthraquinones, at least for emodin, were likely to inhibit HIV-1 replication at a stage/stages
after the virus enters the host cells, acting either directly on the viral life cycle per se or indirectly
through host cell components. Emodin has demonstrated pro-apoptotic activity in several cancer
cell lines via several different mechanisms. Emodin has shown the ability to decrease the
mitochondrial transmembrane potential, decrease the expression of anti-apoptotic proteins bcl-2,
c-myc, and increase the expression of pro-apoptotic protein caspase-3, or by inducing oxidative
stress. Emodin has also demonstrated as an inhibitor of tyrosine protein kinases, protein kinase CK2,
tumor necrosis factor-induced NF-κB activation (IκB degradation) and other signaling proteins
involved in proliferation.
In conclusion, emodin and other structurally related derivatives have shown their anti-HIV-1
activity in vitro. The active chemicals of the traditional Chinese medicine Radix et Rhizoma Rhei
have the potential to become lead compounds for counteracting HIV-1 infection. However, the
mechanism(s) for inhibition of HIV-1 replication by these anthraquinones needs to be further
studied, focusing on the stages after virus fusion with host cells.
ACKNOWLEDGEMENT
We thank Professor ZHENG Yong-tang for providing us with cell lines for HIV-1 production
and antibodies for HIV-1 p24 ELISA assay.
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Alves DS, Pérez-Fons L, Estepa A, et al. Membrane-related effects underlying the biological
activity of the anthraquinones emodin and barbaloin [J]. Biochem Pharmacol, 2004,
68(3):549-561.
[10] Ouyang DY, Chan H, Wang YY, et al. An inhibitor of c-Jun N-terminal kinases (CEP-11004)
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1996, 93(18):9742-9747.
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Figure 1.
Structures and molecular weights of the five anthraquinone derivatives.
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Figure 2.
Cytotoxicity and anti-HIV-1 activity of the anthraquinone derivatives. A:
cytotoxicity on C8166 cells was measured by MTT assay; B: inhibition of HIV-1 p24 antigen
production in cell-culture supernatant was performed by ELISA; C: inhibition of the syncytium
formation induced by HIV-1 infection was quantified under inverted microscope; D: inhibition of
the syncytium formation between co-cultured normal C8166 and HIV-1IIIB chronically infected H9
cells was measured under inverted microscope. x ± sE. n=3.
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Figure 3.
Influence on HIV-1 infectivity of the anthraquinone derivatives by direct
incubation with virus. Virus infectivity after drug treatment was measured by syncytium formation
in C8166 cell culture. Serial diluted H2O2 solutions were used as positive control to inactivate
HIV-1 particles. x ± sE. n=3. ** P<0.01 vs emodin.
Table 1.
Anti-HIV-1 activity of five anthraquinone derivatives (x ± sE)
CPE method
Chemicals
ELISA method
CC50 (μmol/L)
EC50 (μmol/L)
SI
EC50 (μmol/L)
SI
Emodin
144.19 ± 2.43
11.44 ± 0.93a
12.6 ± 1.2
11.61 ± 0.56f
12.4 ± 0.7
Rhein
174.70 ± 4.15
51.28 ± 2.86b
3.4 ± 0.3
12.35 ± 4.73g
14.2 ± 4.9
Chrysophanol
>750
90.58 ± 2.30c
>8
39.63 ± 2.87h
>18
Physcion
>750
8.59 ± 0.38d
>87
>250m
-
Aloe-emodin
~750
0.89 ± 0.08e
~838
2.75±0.20n
~273
Anti-HIV-1 activity was determined by HIV-1 p24 ELISA and the cytopathic effect (CPE)
method. Their 50% cytotoxic concentrations (CC50), 50% effective concentrations (EC50) and
selective indexes (SI) were shown. Difference of EC50 values among a, b, c, d, e groups, P<0.01
except for a vs d (P>0.05) and e vs a, d (P<0.01); among f, g, h, m, n groups, P<0.01 except for f vs
g, n (P>0.05) and g vs n (P>0.05).
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大黄素等五种蒽醌衍生物的体外抗 HIV-1 活性
欧阳东云，刘春永，曾耀英，何贤辉，曾祥凤
（暨南大学生命科学技术学院组织移植与免疫实验中心，广东 广州，510632）
[摘 要] 目的：检测大黄素、大黄酸、大黄酚、大黄素甲醚和芦荟大黄素等蒽醌衍生物
的体外抗HIV-1活性。方法：在96孔培养板中加入不同浓度的衍生物，在倒置显微镜下观察
HIV-1IIIB急性感染的C8166细胞形成合胞体的数量，并用ELISA的方法检测培养上清中HIV-1
p24的抗原水平，得到相应的50%抗HIV-1有效浓度（EC50）。结果：大黄素等蒽醌衍生物有
较好的体外抗HIV-1活性，表现为：(1) 抑制HIV-1IIIB感染诱导的合胞体形成，EC50均值分别
为11.44±0.93 μmol/L (大黄素), 51.28±2.86 μmol/L (大黄酸), 90.58±2.30 μmol/L (大黄酚),
8.59±0.38 μmol/L (大黄素甲醚) 和 0.89±0.08 μmol/L (芦荟大黄素)；（2）抑制HIV-1IIIB急性感
染的C8166细胞p24抗原产生，EC50均值分别为(11.61 ± 0.56) μmol/L（大黄素）、(12.35 ± 4.73)
μmol/L（大黄酸）、(39.63 ± 2.87) μmol/L（大黄酚）、>250 μmol/L（大黄素甲醚）和(2.75 ± 0.20)
μmol/L（芦荟大黄素）。 结论：大黄素等五种蒽醌衍生物具有不同水平的体外抗HIV-1活性，
其中以芦荟大黄素的抑制活性最强。
[关键词] 人类免疫缺陷病毒-1；蒽醌；大黄素；大黄酸；大黄酚；大黄素甲醚；芦荟大
黄素
[中图分类号] R285.5
[文献标识码] A
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