Effects of CAPE-like compounds on HIV replication in vitro and modulation of cytokines in vivo

Chuan-Chen Ho1, Shih-Shen Lin1, Ming-Yung Chou1, Fang-Lung Chen1, Chao-Chin Hu2, Chung-Shih Chen3,4, Guan-Yu Lu4 and Chi-Chiang Yang5,*

1 Department of Dentistry, Chung Shan Medical University Hospital, Chung Shan Medical University, Taichung, Taiwan, R.O.C.; 2 Department of Applied Chemistry, Chung Shan Medical University, Taichung, Taiwan, R.O.C.; 3 Institute of Immunology, Chung Shan Medical University, Taichung, Taiwan, R.O.C.; 4 Department of Pharmacy, Kuang Tien General Hospital, Taichung, Taiwan, Taiwan, R.O.C.; 5 School of Medical Technology, Chung Shan Medical University, Taichung, Taiwan, R.O.C.


* Corresponding author. Tel: +886-4-24730022, ext. 11716; Fax: +886-4-23767469; E-mail: cyang{at}csmu.edu.tw

Received 11 March 2005; returned 16 May 2005; revised 31 May 2005; accepted 8 June 2005


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: Five CAPE-like compounds, namely caffeic acid phenethyl ester (CAPE), methyl caffeate (MC), ethyl 3-(3,4-dihydroxyphenyl)acrylate (EC), phenethyl dimethyl caffeate (PEDMC) and phenethyl 3-(4-bromophenyl)acrylic (BrCAPE) were tested for their anti-HIV replication in vitro and immune modulation effects in vivo.

Methods: Short-term cytotoxicity was assessed by Trypan Blue stain and MTT assay. For antiviral assays, M-tropic (strain JRCSF), T-tropic (strain NL-4-3) and dual tropic (strain 89.6) HIV isolates were used in peripheral blood mononuclear cell (PBMC) culture.

Results: None of these CAPE-like compounds showed significant cytotoxicity in the treatment of PBMCs. By P24 EIA tests, CAPE, MC and EC significantly inhibited HIV replication in PBMC cells, but PEDMC and BrCAPE showed only slightly inhibitory effects. The in vivo modulatory effects on six cytokines [interleukin (IL)-2, IL-4, IL-6, interferon (IFN)-{gamma}, granulocyte-macrophage colony-stimulating factor (GM-CSF) and soluble Fas] were analysed. BALB/c mice treated with different doses or not treated with these CAPE-like chemicals showed that cytokines were increased to different extents by the different treatments. However, the concentrations of IL-6 and GM-CSF were not significantly affected by administration of any of these compounds (P > 0.05).

Conclusions: The different effects of treatments on anti-HIV replication and cytokine modulation suggested that these compounds affect virological and immunological response via different mechanisms. The virological and immunological mechanisms and response to these treatments need to be elaborated in further studies in order to derive the structural features of more effective compounds. Since neither death nor pathological change in the mice were observed in this study, these CAPE-like compounds are worth studying further as potential chemotherapy agents for anti-HIV infection and cytokine modulation.

Keywords: cytotoxicity , integrase inhibitors , chemotherapy


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Caffeic acid phenethyl ester (CAPE) is an antioxidant component of the propolis. It is known that CAPE has anti-mitogenic, anti-carcinogenic, anti-inflammatory and immunomodulatory properties.13 Moreover, CAPE can selectively inhibit virus-transformed and oncogene-transformed rodent cells and human tumour cells, including colon adenocarcinoma (HT-29 and HCT116),4 glioblastoma multiforme (GBM-18),5 melanoma (HU-1, SK-MEL-28 and SK-MEL-MO),6,7 human oral cancer cells (GNM and TSCCa),8 human breast carcinoma (MCF-7) and Fischer rat embryo fibroblasts (CREF).6,9,10 CAPE can also stop the growth of human leukaemia HL-60 cells11 and inhibit the synthesis of DNA, RNA and protein in HeLa cells.12 The activation of NF-{kappa}B by tumour necrosis factor can be dose- and time-dependently blocked by CAPE.13 CAPE can also be used as a lipoxygenase inhibitor, performing anti-oxidation14,15 and anti-inflammatory activity.16

It is well known that human disorders in patients with acquired immune deficiency syndrome (AIDS) are caused by human immunodeficiency virus (HIV). Current therapy for AIDS patients involves the use of combined therapies to inhibit replication of HIV via targeting viral reverse transcriptase and protease. However, the development of HIV resistance to known antiviral agents remains a threat to the effectiveness of these regimens.1721 The combination of antiviral agents is necessary to achieve effective treatment or prophylaxis of AIDS or related disorders and inhibition of viral replication. An improved understanding of HIV pathogenesis has demonstrated the need for aggressive antiretroviral therapy in most patients with HIV infection. Combinations of at least three antiviral drugs are effective to fully suppress viral replication.22 Suppression of viral replication prevents the development of drug resistance and allows immune reconstitution to occur. The correlates of immune reconstitution include resolution of existing opportunistic infections and a markedly reduced risk of new infections. In certain settings, immune reconstitution may allow the discontinuation of opportunistic infection prophylaxis or treatments. The net result has been a substantial improvement in the quality and quantity of life of HIV-infected patients. This study investigated the effects of five CAPE-like compounds, synthesized from commercially available caffeic acid, on HIV replication in vitro and the modulation of cytokines in vivo.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals

Phenethyl 3-(3,4-dihydroxyphenyl)acrylate (CAPE), phenethyl 3-(3,4-dimethoxyphenyl)acrylate (PEDMC), methyl 3-(3,4-dihydroxyphenyl)-acrylate (MC), ethyl 3-(3,4-dihydroxyphenyl)acrylate (EC) and phenethyl 3-(4-bromophenyl)acrylic (BrCAPE) (Figure 1) were prepared and provided by Dr Yean-Jang Lee, Department of Chemistry, National Changhua University of Education and Chao-Chin Hu, Department of Applied Chemistry, Chung Shan Medical University according to previously described procedures.8,23



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Figure 1. Structural formula of CAPE, MC, EC, PEDMC, and BrCAPE.

 
Virus

M-tropic (strain JR-CSF),24 T-tropic (strain NL4-3)24 and dual tropic (strain 89.6)25,26 HIV isolate titred virus stocks were kindly provided by Dr David D. Ho and Dr Linqi Zhang of the Aaron Diamond AIDS Research Center at Rockefeller University in New York. Phytohaemagglutinin (PHA)-activated peripheral blood mononuclear cells (PBMCs) from healthy donors were separated by means of Ficoll-Hypaque gradients and cultured in human recombinant interleukin (IL)-2 (10 U/mL) conditioned R-20 medium.

Trypan Blue dye exclusion method

The Trypan Blue dye exclusion method was performed as described by Phillips.27 For determination of the growth inhibitory effect of MC, CAPE, EC, PEDMC and BrCAPE, 2 x 106 cells were placed in 24-well dishes with several different sub-cytotoxic concentrations of each of these CAPE-like agents for 48 h. In each well, 100 µL of DMSO or one of the different concentrations of CAPE-like agents in DMSO adjusted to be less than 0.5% (v/v) was added. After treatment, cells from quadruplicate dishes were washed once with Hank's balanced salt solution (HBSS) and then trypsinized. Then the cells were stained with 0.4% Trypan Blue and counted. The dead cells were separated from the live monolayer cells. The total number of viable cells in the control group was considered as 100% viability and the agent-treated cells were compared with the control group for the determination of percentage viability. The percentage viability of agent-treated cells was calculated from the total number of viable cells in the control group and in the agent-treated group.

MTT assay

The cytotoxicities of CAPE-like agents on PBMCs were assessed with MTT assays according to the method of Alley et al.28 as reported previously.8,29

In vitro analysis of antiviral efficacy

In order to evaluate the antiviral efficacy, 2 x 106 PBMCs were treated with various concentrations (0, 0.1, 0.5, 1, 5, 10, 25, 50, 100, 200 and 400 µM) of CAPE, MC, EC, PEDMC or BrCAPE and simultaneously infected with macrophage-tropic (JRCSF), T cell-tropic (NL-43) or macrophage and T cell dual tropic (89.6) HIV-1 isolates, respectively at 100 TCID50/106 cells in duplicate. A fully inhibitory dose of 1 µM zidovudine was used as a positive control. After 24 h, the added compounds were removed by washing methods or maintained at the same concentration. Samples were divided into two groups and the supernatants of the culture medium were collected at 7 days after infection. The P24 EIA reagent kit was used to compare the concentrations of viral P24 antigen with or without the compounds tested. For quantification of HIV-1, P24 was measured by a quantitative EIA (Abbott Laboratories, North Chicago, IL, USA) according to the manufacturer's instructions. Unknown HIV P24 antigen values were determined from the standard curve, which was prepared from calibrated positive controls and expressed as pg/mL.

In vivo cytokine analysis by ELISA

The design of this study was in accordance with the Institutional Guidelines of Animal Experiments from the Animal Center, Chung Shan Medical University for the care and use of laboratory animals. Female BALB/c mice, purchased from National Cheng Kung University (Tainan, Taiwan) at 4 weeks of age and weighing between 14 and 16 g, were used in this study. The mice were randomly divided into six groups. The diets of mice in five groups included different CAPE-like compounds for 2 weeks. Four different doses, i.e. 50, 100, 200 and 400 µmol/kg per day, were used in each group. The diet of another group of mice contained a DMSO solution (DMSO final concentration <0.5% v/v) without a CAPE-like compound and these animals served as the normal control. A minimum of two mice were killed at the end of each week for 2 weeks after the regimen had commenced. Sera were then separated from the blood samples by centrifugation at 3500g for 15 min, and aliquotted and stored at –70°C until required for cytokine assay. The brain, heart, liver, spleen and kidneys were sectioned for haematoxylin–eosin (H&E) staining. The cytokine [IL-2, IL-4, IL-6, interferon (IFN)-{gamma}, granulocyte-macrophage colony-stimulating factor (GM-CSF) and soluble Fas (sFas)] assays were performed using commercial ELISA kits purchased from Pharmingen, San Diego, CA, USA (or, in the case of sFas, from R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturers' instructions as reported previously.3032 For every test, a standard curve was also derived for IL-4, IL-6, IFN-{gamma}, GM-CSF or sFas. The range of the standard curve was 7.8–1000 pg for IL-2, 1.95–1000 pg for IL-4, 4.7–600 pg for IL-6, 15.6–8000 pg for IFN-{gamma}, 4.7–600 pg for GM-CSF, and 31.3–15 000 pg for sFas.

Haematoxylin–eosin (H&E) staining

All specimens were examined by routine fixation with H&E staining. All brains, hearts, livers, spleens and kidneys were fixed immediately in 10% formalin solution overnight at 4°C and embedded in paraffin. Serial sagittal sections 5 µm thick were cut, and stained with H&E for histological study.

Statistical analyses

The Kruskal–Wallis test with SPSS Version 10.0 software was used to evaluate the significance of different cytotoxicities and antiviral activities observed. The Wilcoxon rank sum test was used to test the differences in cytokine ratio between each of the groups and P values <0.05 were taken as significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Short-term cytotoxicity assays

In 24-well plates, PBMCs were exposed to various concentrations (0.1, 0.5, 1, 5, 10, 25, 50, 100, 200 and 400 µM) of CAPE, MC, EC, PEDMC or BrCAPE for 48 h. Viability of the cells was assayed by the Trypan Blue dye exclusion method and microculture tetrazolium test. Viable cells are impermeable to Trypan Blue dye. When PBMCs were exposed to various concentrations of these chemicals, the number of dead cells in a count of 400 cells ranged from 7 to 12 (the control number of dead cells was 9). The viabilities of PBMCs under high-dose treatments of CAPE, MC, EC, PEDMC or BrCAPE (391.8 ± 1.6 cells), as high as 400 µM, were not significantly different from those under low-dose treatments (390.9 ± 1.7 cells) (e.g. 0.1–10 µM) and controls (391.1 ± 0.9 cells) (P > 0.05).

The microculture tetrazolium test method is based on the principle that live cells can reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (tetrazolium salt) to formazan crystals through dehydrogenase metabolism in mitochondria. In this study, the crystals were dissolved in propanol and OD values were determined. The OD values were used to indicate the numbers of live cells. The percentage of dehydrogenase activity at each concentration, compared with that of the control, was calculated from the absorbance values. As shown in Table 1, no significant cytotoxic effects were found among PBMCs treated with CAPE, MC, EC, PEDMC and BrCAPE (P > 0.05). These results indicate that the compounds tested do not cause cytotoxicity to cells even under a high dose up to 400 µM.


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Table 1. Cytotoxicities of CAPE, MC, EC, PEDMC and BrCAPE to PBMCs assayed by MTT

 
In vitro analysis of antiviral efficacy

P24 antigen values were quantified to assess the replication of HIV. As shown in Figure 2, treatment with CAPE, MC and EC significantly inhibited viral replication. However, PEDMC and BrCAPE appeared to only slightly inhibit viral replication. CAPE and MC at a concentration of 100 µM or greater and EC at a concentration of 200 µM or greater, inhibited 100% of viral replication of various HIV isolates. The inhibitory effects of PEDMC and BrCAPE were less prominent than that of EC, and HIV could not be 100% inhibited even at a concentration of 400 µM. After 24 h of treatment with these CAPE-like compounds, differences were observed between the compound-removed and compound-maintained wells. Some of these CAPE-like compounds, i.e. CAPE, MC and EC, significantly inhibited HIV replication regardless of whether they were removed after 24 h of treatment. Moreover, the overall P24 antigen curves of these CAPE-like compounds indicated that when their concentration gradually decreases, the inhibition of viral replication gradually decreases. Furthermore, the antiviral activities were relatively consistent among the various HIV isolates (NL-43, JRCSF or 89.6) tested.



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Figure 2. The P24 concentration PBMCs infected with (a) T cell-tropic (NL-43), (b) macrophage-tropic (JRCSF) or (c) dual tropic (89.6) viruses, after treatment with CAPE, MC, EC, PEDMC or BrCAPE at different concentrations. The horizontal axis represents the concentrations (µM) of the compounds tested. The vertical axis represents the concentration (units) of the viral P24 antigen. Filled diamonds indicate maintaining the original concentration of the compound added after washing at the end of the infection. Filled squares indicate removing the compound after washing. The error bars represent standard deviations.

 
Pathological examination and in vivo cytokine analysis

Mice were fed diets containing 0, 50, 100, 200 and 400 µmol of different CAPE-like compounds per kg per day for 2 weeks. None of the mice in any of these treatment groups died during the experimental period. Moreover, no pathological change was found in the brain, heart, liver, spleen and kidneys of the mice given these CAPE-like compounds (histological data not shown).

The modulatory effects of these CAPE-like compounds on six cytokines (IL-2, IL-4, IL-6, IFN-{gamma}, GM-CSF and sFas) were analysed. The mean cytokine values of the control group were 4.35 ± 0.43 pg/mL for IL-2, 0.33 ± 0.08 pg/mL for IL-4, 2.25 ± 0.56 pg/mL for IL-6, 1.94 ± 0.10 pg/mL for IFN-{gamma} 2.10 ± 0.43 pg/mL for GM-CSF and 864.38 ± 146.62 pg/mL for sFas. Comparing the cytokine values of treated groups with the control (Table 2), the results demonstrated that the ratios of cytokines, i.e. IL-2, IL-4, IL-6, IFN-{gamma}, GM-CSF and sFas, were different for different treatment amounts of the CAPE-like compounds. Among the tested cytokines, however, the ratios of IL-6 and GM-CSF were not significantly changed by differences in these treatment amounts (P > 0.05). The most obvious increase in IL-2 occurred with BrCAPE treatment for 2 weeks; for IL-4, an obvious increase occurred with all five compounds (treatment for 1 and 2 weeks) except for MC treatment for which IL-4 declined to control levels after another week; for IFN-{gamma}, similar results were demonstrated among all five compounds; and for sFas, the most marked increase occurred with MC treatment for 1 week, however, this increase declined to control levels after another week.


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Table 2. Ratio of mouse sera cytokines analysis of different doses of CAPE, MC, EC, PEDMC, or BrCAPE treated for 1 or 2 weeks compared with controls

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Recently, it has been found that the integrase essential for viral replication may be a possible target for another class of antiviral agents.33,34 It appears to have the greatest potential for use in developing anti-HIV inhibitors. In HIV and other retroviruses, the integration of a DNA copy obtained from the RNA genome into the host cells chromosome is essential for effective viral replication.35,36 Moreover, no integrase has been found in human cells, suggesting that integrase inhibitors may be useful for treating retroviral infections.

Many compounds including DNA-binding agents,3739 topoisomerase inhibitors37 and aurintricarboxylic acid40 have been reported to inhibit HIV integrases in biochemical assays. However, most of these compounds possess little or no activities in tissue cultures and have no selectivity in their action mechanisms. These results indicate that these compounds do not have selectivity to eliminate the activation of HIV integrases or that the compounds inhibiting HIV integrase do not enter the cells. In particular, although most compounds exhibit an inhibitory activity on integrases in in vitro enzymic assays, it has not yet been demonstrated that such compounds have an ex vitro anti-HIV activity.41 For instance, both actinomycin D and baicalein have in vitro inhibitory activity on integrases,42 but it has not been reported if they have anti-HIV activity.

In this study, some of these CAPE-like compounds, i.e. CAPE, MC and EC, significantly inhibited HIV replication regardless of whether they were removed after 24 h of treatment. The mechanism for their inhibition of viral replication is unclear. If the inhibition was due to the blockage of the attachment by the small molecules of these CAPE-like compounds, then the viral antigen amounts which represent the viral replication should rebound as in the mock-treated control when the added compounds were removed. However, the results did not support this assumption. This reasoning suggests that the occurrence of inhibition was not achieved in the attachment stage only but rather after the subsequent viral penetration stage as well. Moreover, a previous study43 reported that the associated integrase IC50 value was 7 µM for CAPE, and >100 µM for MC, EC and PEDMC. The in vitro virology results of this study support previous findings indicating that CAPE-like compounds exhibit activities against HIV in cell culture. Notably, the new compound synthesized in our laboratory, BrCAPE, and PEDMC demonstrated less prominent inhibitory activities than the other CAPE-like compounds, i.e. CAPE, MC and EC. For the less prominent inhibitory activity of BrCAPE, the substitution of hydroxyl group with bromine44 does not result in better inhibitory activity on HIV replication as in the oral cancer cells.8 For that of PEDMC, the results indicated the hydroxyl group(s) on at least one aryl ring45 is not essential but is important for viral inhibitory potency. Recently, Nagaoka et al.46 suggested that the synthetic analogues of CAPE, 3-phenylpropyl caffeate (IC50 7.34 µM) and 4-phenylbutyl caffeate (IC50 6.77 µM), possessed stronger NO inhibitory activity than CAPE. They also reported that elongation of the alkyl side chain of alcoholic parts of caffeic acid esters enhanced the NO inhibitory activity and that CAPE analogues having a longer carbon chain (>C5) in the alcoholic parts showed toxic effects toward J774.1 cells. Taken together, these results may provide more information about the structural features important to design better viral inhibitory or anti-tumour compounds.

The success of long-term anti-HIV therapy depends not only on suppression of viral replication, but also on at least partial restoration of immune function.47 Analysis of the cytokine modulatory effects of CAPE-like compounds has shown that different compounds exhibit different abilities to up-regulate different cytokines. Recently, Radtke et al.48 reported that caffeic acid showed no cytotoxicity against RAW cells (IC50 >600 to >2200 nM; >400 µg/mL) and that different degrees of modulatory effects were found on IL-6, interferon and tumour necrosis factor-{alpha}. IL-2 and IFN-{gamma} are key Th1 cytokines that activate cytotoxic T cells. IL-4 is generally described as an anti-inflammatory and a Th2 cytokine that affects IgG antibody production. These cytokines are all important for immunity. In this study, although BrCAPE did not significantly inhibit HIV replication in comparison with other compounds, IL-2, IL-4 and IFN-{gamma} were significantly increased by this treatment in vivo. Treatment with CAPE, which showed the best results of HIV inhibition in enzymology43 and in vitro virology assays, also increased sFas levels after 1 week, followed by a decrease after another week. As sFas is capable of inhibiting FasL-induced apoptosis,49,50 these results indicate that these compounds may induce an immediate response to release sFas for anti-apoptosis. Moreover, CAPE exhibited a prominent anti-apoptotic effect suggesting that CAPE treatment may significantly enhance immune response. It is known that immune competence prevents the onset of HIV-related opportunistic diseases, and also may synergize with antiviral agents to control viral replication. Chun et al.51 found that patients who received IL-2 in addition to HAART had a significantly lower frequency of latently infected CD4+ T cells harbouring replication-competent virus compared with patients who received HAART alone. However, although an effect on murine cytokines can be observed following treatment of mice with these compounds, further research is necessary to clarify the significance and mechanism of cytokine modulation by these CAPE-like compounds in primates.

In summary, this study showed that CAPE-like compounds can inhibit replication of HIV isolates with various tropisms in vitro and that the modulation of cytokines in vivo was different when different amounts of CAPE-like compounds were used in the treatment. The different effects of treatments on HIV replication and cytokine modulation suggested that these compounds affect virological and immunological response via different mechanisms. Both the virological and immunological mechanisms and response to these treatments need to be elaborated in further studies in order to derive the structural features of more effective compounds. In this study, neither death nor pathological change occurred following these treatments in mice. This suggests that it is likely that the CAPE-like compounds are safe in vivo, at least in the short-term. The CAPE-like compounds are worth studying further as potential new chemotherapy agents for HIV infection and cytokine modulation.


    Acknowledgements
 
We thank Drs David D. Ho and Linqi Zhang of the Aaron Diamond AIDS Research Center at Rockefeller University in New York for providing virus strains and reagents and useful discussions. Dr Chi-Chiang Yang is appreciative of and indebted to the Aaron Diamond AIDS Research Center for the practice of in vitro analysis of antiviral efficacy during his sabbatical at the Center. This study was partly funded by a grant (DOH89-DC-1001) from the Center for Disease Control, Department of Health, Taiwan, R.O.C.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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