Soy isoflavones genistein and daidzein exert anti-apoptotic actions via a selective ER-mediated mechanism in neurons following HIV-1 Tat(1-86) exposure

Abstract

Background: HIV-1 viral protein Tat partially mediates the neural dysfunction and neuronal cell death associated with HIV-1 induced neurodegeneration and neurocognitive disorders. Soy isoflavones provide protection against various neurotoxic insults to maintain neuronal function and thus help preserve neurocognitive capacity.

Methodology/principal findings: We demonstrate in primary cortical cell cultures that 17β-estradiol or isoflavones (genistein or daidzein) attenuate Tat(1-86)-induced expression of apoptotic proteins and subsequent cell death. Exposure of cultured neurons to the estrogen receptor antagonist ICI 182,780 abolished the anti-apoptotic actions of isoflavones. Use of ERα or ERβ specific antagonists determined the involvement of both ER isoforms in genistein and daidzein inhibition of caspase activity; ERβ selectively mediated downregulation of mitochondrial pro-apoptotic protein Bax. The findings suggest soy isoflavones effectively diminished HIV-1 Tat-induced apoptotic signaling.

Conclusions/significance: Collectively, our results suggest that soy isoflavones represent an adjunctive therapeutic option with combination anti-retroviral therapy (cART) to preserve neuronal functioning and sustain neurocognitive abilities of HIV-1 infected persons.

Figure 1. Soy isoflavones genistein and daidzein protect primary cortical cultures from Tat neurotoxicity.

Primary cortical neurons were exposed to estrogen (0.1, 2.0 and 10 nM), or isoflavones (0.05, 0.2 and 1 µM) 24 hr prior to the start of Tat_1–86 B (50 nM) treatment. Cell viability was assessed by Live/Dead assay. Live/Dead ratios were determined after 48 hr (A-C) or 5 days (D-F) of the continuous exposure to Tat or equal volume of vehicle in cell culture groups that were treated or not treated with estrogen, GEN, or DAI. Data represents mean values ± SEM, n of cultures analyzed  = 7–12 per each group. *- indicates significant (p≤0.05) protective effects of the selected compounds against Tat neurotoxicity (cell viability decrease) in cortical cell cultures. Repeated (2–3) trials using cell culture preparations from different litters were carried out to ensure the reproducibility of the results.

Figure 2. Genistein and daidzein attenuate Tat-induced caspase activation in primary cortical cultures.

Cortical cultures were treated with 1 µM GEN or DAI 24 hr prior to Tat exposure. Expression of activated apoptotic proteins A. Caspase 9 (4 hr of Tat exposure) and B. caspase 3 (4 hr Tat exposure) was assessed by ELISA experiments. Data represents mean values ± SEM, with experiments performed in triplicate, *p≤0.05 as compared to Tat-treated cultures.

Figure 3. Isoflavones prevent Tat-induced expression of Bcl-2 and Bax protein levels.

Cortical cultures were treated with 1 µM GEN or DAI 24 hr prior to Tat exposure. Expression of apoptotic proteins A. Bcl-2 (16 hr of Tat exposure) and B. Bax (4 hr Tat exposure) were assessed by ELISA experiments. Data represents mean values ± SEM, with experiments performed in triplicate, *p≤0.05 as compared to Tat-treated cultures.

Figure 4. Estrogen receptors mediate isoflavone effects on Caspase 3 and Bax expression following Tat exposure.

A. GEN or DAI effects on caspase 3 expression were blocked in the presence of ER antagonist, ICI 182,780. B. GEN or DAI effects on Tat-induced expression of Bax were reversed by ICI 182,780, suggesting that estrogenic actions on caspase 3 and Bax are ER mediated. Data represents mean values ± SEM, *p≤0.05 vs. GEN/DAI+T treated cultures.

Figure 5. ER subtype specific effects against caspase activity and Bax expression.

Similar to 17β-estradiol (A), GEN (B) and DAI (C) effects against Tat-induced caspase 3 activity were maintained in the presence of specific antagonists for ERα (MPP) and ERβ (PHTPP). ER subtype antagonists reveal that ERβ signaling was preferential for GEN (E) effects on Bax. DAI (F) effects on Bax were blocked in the presence of both ER subtype antagonists. Data represents mean values ± SEM, *p≤0.05 as compared to GEN/DAI+Tat treated cultures.