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Mechanisms of [gamma]-linolenic acid induction of apoptosis in T cells chronically infected with HIV-1

University of British Columbia

BACKGROUND: The polyunsaturated fatty acid (PUFA) gamma-linolenic acid (GLA) kills HIV-1- infected cells at concentrations that are relatively harmless to uninfected cells. The mechanism of this effect is unclear but evidence implicates oxidative stress. The aim of this study was to define mechanisms of GLA cytotoxicity in HIV-infected T cells. METHODS: The test model comprised uninfected A3.01 and HIV-1-infected 8E5 T cells. Cell proliferation was measured using WST-1 and Trypan blue dye-exclusion assays. Colorimetric analysis for lipid peroxidation, and supplementation with antioxidants and lipoxygenase (LOX) or cyclooxygenase (COX)-mediated PUFA metabolism inhibitors probed for involvement of oxidative stress. Apoptosis was assayed flow cytometrically using FITC-conjugated Annexin V and bromodeoxyuridine. We examined cells for specific apoptosis signaling using caspase inhibitors, and by ribonuclease protection and immunoblot assays. Propidium iodide staining helped to investigate association of apoptosis and cell cycling. As validation, GLA effects were assessed in CD4⁺ cells infected demuowith isolates of HIV-1, or freshly isolated from HIV⁺ patients. RESULTS: GLA was more cytotoxic to 8E5 cells (IC₅₀ = 14.5 μg/ml) than A3:01 cells (IC₅₀ = 43.0 μg/ml). GLA-induced cytotoxicity in 8E5 was associated with a four-fold increase in concentration of lipid peroxidation metabolites. The thiol antioxidant, 2-mercaptoethanol, and glutathione peroxidasernimic, ebselen, inhibited GLA actions. Concentration-specific inhibition was also achieved with LOX and COX inhibitors. Flow cytometric analysis with Annexin V-FITC and BrdU-FITC, and inhibition by caspase inhibitor BD.fmk confirmed occurrence of apoptosis in GLA-treated cells. The majority of apoptotic cells were found in the G₀/G₁ cell cycle-phase. Immunoblot analysis demonstrated cleavage of caspase-3 and poly (ADP-ribose) polymerase (PARP). Differential GLA cytotoxicity was also observed in cells acutely infected with HIV-l[sub IIIB] but not in cells infected with other isolates. There was a negative correlation between GLA IC₅₀ in CD4⁺ cells from HIV⁺ patients and plasma viral load. CONCLUSION: GLA induces apoptosis in 8E5 cells through a mechanism that involves lipid peroxidation and cleavage of caspase-3 and PARP. GLA cytotoxicity against CD4⁺ cells of HIV⁺ patients increases with viral load. Therefore, GLA and its homologues deserve further study in the search for compounds that target HIV-infected cells for selective killing.

Thesis/Dissertation

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2009-10-08

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http://hdl.handle.net/2429/13785

Experimental Medicine

University of British Columbia

UBCV

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