The effects of combinations of a green tea extract and an active ingredient thereof, with standard antiretroviral drugs on SC-1 cells infected with the LP-BM5 virus

Abstract

The introduction of highly active antiretroviral therapy (HAART) has resulted in a significant decrease in the mortality and morbidity associated with the acquired immunodeficiency syndrome (AIDS). Several problems are associated with HAART and include high costs of treatments, poor availability of drugs in low-income countries, poor compliance, severe adverse effects and drug resistance. Therefore, the focus of current research is the development of new antiretroviral drugs, improved treatment strategies and the discovery of new drugs derived from plants. Green tea (GT) and its active constituent epigallocatechin gallate (EGCg) have been found to be protective against cancer, cardiovascular and neurodegenerative diseases and were found also to have antimicrobial, antimalarial and more importantly antiviral activity. EGCg, in vitro has been shown to inhibit the human immunodeficiency virus (HIV) viral enzymes reverse transcriptase and protease, destroy viral particles and interfere with the attachment of gp120 to cellular receptor CD4. The aims of this study were firstly to investigate the in vitro antiretroviral activity of GT and EGCg on the LP-BM5 defective murine leukemia virus (MuLV) that induces a disease in C57BL/6 mice similar to AIDS in humans and secondly to investigate the effects of GT and EGCg on the in vitro cytotoxicity and antiretroviral activity of current antiretroviral drugs zidovudine (AZT), indinavir (IDV), hydroxyurea (HU) and chloroquine (CQ). To achieve the above aims an in vitro model that represents cell-to-cell spreading of the LP-BM5 MuLV was developed. Firstly the presence of the LP-BM5-defective virus in the BM5 cell line was confirmed using transmission electron microscopy (TEM) to identify viral particles, PCR and RT-PCR were used to determine the presence of viral DNA and RNA respectively and viral infectivity was confirmed in C57BL/10 mice. The cytotoxicity of each drug and combination was evaluated with the MTT assay in the SC-1 cell line, the predominant cell type in the in vitro cell culture model. GT was the least cytotoxic, followed by AZT, IDV, EGCg, HU and CQ. Co-cultures (BM5:SC-1, 1:10000) that represented cell-to-cell transmission of the virus were established. Real time PCR for proviral DNA revealed that IDV, AZT and HU completely suppressed, CQ dose dependently reduced while GT and EGCg had no effect on viral transmission. Findings using AZT and IDV thus validated the use of this in vitro co-culture model for first line screening of new drugs and plant extracts. The effect of GT or EGCg in combination with AZT, IDV, HU or CQ was also evaluated as GT or EGCg could enhance the antiretroviral effects or decrease cellular toxicity of these drugs. For GT with AZT a mix of synergism and antagonism on cell toxicity was observed with little to no effect on the antiretroviral activity of AZT. Antagonism on cell toxicity was observed for GT with IDV, with no effect on the antiretroviral activity of IDV. In contrast EGCg significantly reduced the antiretroviral activity of IDV. A strong antagonistic effect was observed for GT with HU, with GT reducing the antiretroviral effect of HU. For combinations of AZT with EGCg and HU with EGCg a similar effect was observed as for AZT and HU respectively combined with GT. Synergism in cytotoxicity was observed between GT and CQ associated with a significant decrease in viral loads while EGCg combined with CQ had an opposite effect at higher concentrations. In conclusion, the in vitro co-culture model of BM5 and SC-1 cells was successfully used to evaluate combinations of GT and EGCg with AZT, IDV, HU and CQ. Interesting and often contradicting effects were observed, such as seen for IDV in combination with GT and EGCg as well as CQ in combination with GT and EGCg. These effects may be of clinical relevance and further investigation is warranted.