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Polyphenolic antioxidant (–)-epigallocatechin-3-gallate from green tea as a candidate anti-HIV agent

Fassina, Gianfrancoa,b; Buffa, Annac; Benelli, Robertoa,c; Varnier, Oliviero E.d; Noonan, Douglas M.a; Albini, Adrianaa

Research Letters

aIstituto Nazionale per la Ricerca sul Cancro, Genoa, Italy; bCentro di Studio per la Neurofisiologia Cerebrale, Consiglio Nazionale delle Ricerche, Genoa, Italy; cCentro per le Biotecnologie Avanzate, Genoa, Italy; and dInstitute of Microbiology, School of Medicine, University of Genoa, Italy.

Sponsorship: This work was partly supported by ISS grant 3rd Programma Nazionale di Ricerca sull'AIDS.

Received: 10 August 2001;

revised: 30 October 2001; accepted: 14 November 2001.

Epigallocatechin-3-gallate (EGCG), one of the components of green tea, has been suggested to have antiviral activity. To determine the effects of EGCG on HIV infection, peripheral blood lymphocytes were incubated with either LAI/IIIB or Bal HIV strains and increasing concentrations of EGCG. EGCG strongly inhibited the replication of both virus strains as determined by reverse transcriptase and p24 assays on the cell supernatants.

Green tea extracts obtained from dried leaves of the tea plant (Camelia sinensis) contain polyphenols, such as flavonols, commonly known as catechins. Among these, (–)-epigallocatechin-3-gallate (EGCG), a strong antioxidant, accounts for over 50% of the catechins present in green tea. The consumption of green tea has been associated with a variety of effects, including delays in cancer insurgence, the suppression of angiogenesis and leukocyte infiltration, and the inhibition of several proteases.

It has also been reported that some plant extracts have an inhibitory effect on the activity of Rauscher murine leukemia virus and HIV-reverse transcriptases (RT). EGCG inhibits the biochemical activity of HIV-RT measured as cellular protein or enzyme activity in in-vitro assays [1,2], but not on the viral packaging. HIV-associated RT is considered to be one of the best targets for chemoterapeutic approaches to AIDS, but most RT inhibitors are toxic or not selective enough. For this reason, natural inhibitors of RT are of great interest. Here we report that EGCG acts as a strong inhibitor of HIV (LAI/IIIB and Bal strains) replication in cultured peripheral blood cells of healthy donors.

Peripheral blood mononuclear cells from seronegative healthy donors were separated on a ficoll gradient, suspended in DMEM supplemented with 10% heat-inactivated fetal calf serum (FCS) at 8 × 106 cells/ml and allowed to adhere to plastic. Non-adherent cells, subjected to a second round of adhesion to eliminate contaminating monocytes, were collected by centrifugation, resuspended in RPMI 1600 medium plus 10% heat-inactivated FCS, and stimulated with 5 μg/ml phytohaemoagglutinin for 3 days; 106 cells/ml, resuspended in RPMI plus 10% FCS, were added with 20 U/ml IL-2 and infected with either HIV-1 LAI/IIIB or HIV-1Bal strains at a multiplicity of infection of 0.1, in 96-well flat-bottom multiwell plates. EGCG (0.1–50 μM) was added to peripheral blood mononuclear cells 30 min before HIV infection. Stromal derived factor-1α (CXCL12) (1 μM) or regulated upon activation: normal T cell expressed/secreted (CCL5) (0.04 μM), the natural ligands of CXCR4 and CCR5 respectively, used as positive infection inhibitor controls, were also added 30 min before infection.

The RT assay was performed as previously described [3] on culture supernatants, collected every 72 h, which were added to a 32P dTTP (2′-deoxythymidine triphosphate) and 0.5 M dithiothreitol reaction cocktail in 96-V bottom multiwell plates. Plates were incubated for 4 h at 37°C and then blotted on chromatography paper. Radioactivity is directly proportional to the protein amount, which in turn is directly proportional to the amount of HIV-1 virus in the cells.

The results were confirmed using the HIV-1 p24 core profile enzyme-linked immunosorbent assay. An aliquot of 200 μl of cell-free supernatants were added to 0.5% Triton-100 in order to release HIV-1 p24 antigen. The antigen was captured by a highly specific mouse monoclonal antibody to p24 antigen, complexed with 100 μl of biotinylated polyclonal antibodies for 60 min at 37°C, followed by probing with a streptavidin-horseradish peroxidase conjugate for 30 min at room temperature. The complex was detected by incubation with orthophenylendiamine- hydrochloride and quantified by Quantikin Detection System software (RILAB s.r.l., Genoa, Italy) using a combination of kinetic and endpoint readings of the optical densities [4].

EGCG dose-dependently inhibited the replication of both viral strains as assessed by the RT assay, with doses of 25 and 50 μM giving nearly 100% inhibition of viral replication (Fig. 1a,b). To confirm that the effect on HIV replication was caused by the inhibition of viral infection and not direct inhibition of RT, we also analysed p24, a constitutive HIV protein whose release corresponds to an effective packaging of viral particles. We found a dose-dependent, significant reduction of p24 concentration in the supernatants of HIV LAI/IIIB-infected T lymphoblasts (Fig. 1c), demonstrating that infection itself was hampered. No apoptosis or necrosis was observed in cells treated with EGCG (data not shown), thus excluding the possibility that the assays were affected by cell death.

Fig. 1.

Fig. 1.

This report is the first demonstration of EGCG efficacy in the inhibition of HIV infection and replication, as previous results have been obtained only by using biochemical assays on purified enzymes. The mechanism by which EGCG interferes with viral infection is not yet clear. The antioxidant N-acetylcysteine has been reported to inhibit HIV replication and glutathione depletion in HIV-infected individuals [5–7]. Recent studies [8,9] have shown that one enzyme affected by EGCG is casein kinase II, responsible for the phosphorylation and activation of HIV-RT. EGCG is also able to modulate the cell cycle, downregulating natural factor kappa B [10] and cyclin D1, cdk4 and cdk6, while inducing p21, p27, p16, p18 [11]. Further studies are required to elucidate the exact mechanism by which EGCG inhibits HIV replication. Green tea extracts or EGCG represent potential low-cost inhibitors of HIV infection that could be associated with current anti-HIV therapy.

Gianfranco Fassinaa,b

Anna Buffac

Roberto Benellia,c

Oliviero E. Varnierd

Douglas M. Noonana

Adriana Albinia

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