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In vitro reactivation of latent HIV-1 by cytostatic bis(thiosemicarbazonate) gold(III) complexes.

Fonteh P, Meyer D - BMC Infect. Dis. (2014)

Bottom Line: Viral reactivation was absent for the complexes during co-stimulation with PMA indicating the lack of an additive effect between the chemicals as well as an absence of inhibition of PMA induced HIV reactivation but for HU inhibition of the stimulant's activity was observed (p = 0.01).The cytostatic effect of 1 and 2 and now HIV reactivation from a U1 latency model is consistent with that of the cytostatic agent, HU.These findings suggest that the complexes have a potential dual (cytostatic and reactivation) role in viral "activation/elimination".

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Faculty of Natural and Agricultural Sciences, University of Pretoria, Hatfield Campus, Pretoria 0002, South Africa. Debra.Meyer@up.ac.za.

ABSTRACT

Background: A number of cytostatic agents have been investigated for the ability to reactivate latent viral reservoirs, which is a major prerequisite for the eradication of HIV-1 infection. Two cytostatic bis(thiosemicarbazonate) gold(III) complexes (designated 1 and 2) were tested for this potential in the U1 latency model of HIV-1 infection.

Methods: Cell viability in the presence or absence of 1 and 2 was determined using a tetrazolium dye and evidence of reactivation was assessed by p24 antigen capture following exposure to a latency stimulant, phorbol myristate acetate (PMA) and or test compounds. The latency reactivation mechanism was explored by determining the effect of the complexes on protein kinase C (PKC), histone deacetylases (HDAC) and proinflammatory cytokine production.

Results: The CC50 of the complexes in U1 cells were 0.53 ± 0.12 μM for 1 and 1.0 ± 0.4 μM for 2. In the absence of PMA and at non toxic concentrations of 0.2 and 0.5 μM, 1 and 2 significantly (p ≤ 0.02) reactivated virus in U1 cells by 2.7 and 2.3 fold respectively. In comparison, a 2.6 fold increase (p = 0.03) in viral reactivation was observed for hydroxyurea (HU), which was used as a cytostatic and latent HIV reactivation control. Viral reactivation was absent for the complexes during co-stimulation with PMA indicating the lack of an additive effect between the chemicals as well as an absence of inhibition of PMA induced HIV reactivation but for HU inhibition of the stimulant's activity was observed (p = 0.01). Complex 1 and 2 activated PKC activity by up to 32% (p < 0.05) but no significant inhibition of HDAC was observed. Increases in TNF-α levels suggested that the reactivation of virus by the complexes may have been due to contributions from the latter and the activation of PKC.

Conclusion: The ethyl group structural difference between 1 and 2 seems to influence bioactivity with lower active concentrations of 1, suggesting that further structural modifications should improve specificity. The cytostatic effect of 1 and 2 and now HIV reactivation from a U1 latency model is consistent with that of the cytostatic agent, HU. These findings suggest that the complexes have a potential dual (cytostatic and reactivation) role in viral "activation/elimination".

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Effect of complex 1 and 2 on TNF-α production from U1 cells. Cell free supernatant from U1 cells treated with 1 and 2 was analysed using the CBA kit technology. TNF-α levels were increased by 9 and 3 fold for 1 and 2 respectively. The observed visual differences between treated and untreated cells was not statistically significant (p > 0.05) but could be contributing to the viral reactivation mechanism observed. PMA was used as a positive control and significantly stimulated TNF-α (p = 0.004). The data is plotted on a log scale, n = 3.
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Fig5: Effect of complex 1 and 2 on TNF-α production from U1 cells. Cell free supernatant from U1 cells treated with 1 and 2 was analysed using the CBA kit technology. TNF-α levels were increased by 9 and 3 fold for 1 and 2 respectively. The observed visual differences between treated and untreated cells was not statistically significant (p > 0.05) but could be contributing to the viral reactivation mechanism observed. PMA was used as a positive control and significantly stimulated TNF-α (p = 0.004). The data is plotted on a log scale, n = 3.

Mentions: Stimulants such as PMA are associated with stimulating endogenous production of proinflammatory cytokines such as TNF-α [16], which in turn stimulates the HIV-1 LTR leading to viral reactivation. In vitro stimulation with TNF-α result in viral reactivation [11], further supporting this. TNF-α has been implicated in the immune disregulation observed in HIV since it promotes systemic inflammation resulting in disease progression in vivo [42] making it an important molecule in HIV infection. To further probe the mechanism by which complexes 1 and 2 reactivated virus, determination of TNF-α production was performed using the BD BioSciences (California, USA) CBA kit. The cytokines quantified by the kit included IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ and IL-17A. For the purposes of this study, the main focus was on the endogenous production of TNF-α. TNF-α levels were shown to increase by 9 fold for complex 1 and 3 fold for complex 2 compared to the vehicle control (Figure 5) while for PMA treated cells, the increase was as a significant increase (p = 0.004) of up to 2353.86 ± 204 pg/mL (648 fold). Although increases were observed for 1, the findings were not statistically significant (p > 0.05) suggesting that TNF-α stimulation might only be playing a contributory role which together with the increase in kinase activity resulted in HIV reactivation. Complex 1’s effect on TNF-α was more elevated than 2 supporting the fact that reactivation of latent virus induced by 1 exceeded that of 2.Figure 5


In vitro reactivation of latent HIV-1 by cytostatic bis(thiosemicarbazonate) gold(III) complexes.

Fonteh P, Meyer D - BMC Infect. Dis. (2014)

Effect of complex 1 and 2 on TNF-α production from U1 cells. Cell free supernatant from U1 cells treated with 1 and 2 was analysed using the CBA kit technology. TNF-α levels were increased by 9 and 3 fold for 1 and 2 respectively. The observed visual differences between treated and untreated cells was not statistically significant (p > 0.05) but could be contributing to the viral reactivation mechanism observed. PMA was used as a positive control and significantly stimulated TNF-α (p = 0.004). The data is plotted on a log scale, n = 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4265357&req=5

Fig5: Effect of complex 1 and 2 on TNF-α production from U1 cells. Cell free supernatant from U1 cells treated with 1 and 2 was analysed using the CBA kit technology. TNF-α levels were increased by 9 and 3 fold for 1 and 2 respectively. The observed visual differences between treated and untreated cells was not statistically significant (p > 0.05) but could be contributing to the viral reactivation mechanism observed. PMA was used as a positive control and significantly stimulated TNF-α (p = 0.004). The data is plotted on a log scale, n = 3.
Mentions: Stimulants such as PMA are associated with stimulating endogenous production of proinflammatory cytokines such as TNF-α [16], which in turn stimulates the HIV-1 LTR leading to viral reactivation. In vitro stimulation with TNF-α result in viral reactivation [11], further supporting this. TNF-α has been implicated in the immune disregulation observed in HIV since it promotes systemic inflammation resulting in disease progression in vivo [42] making it an important molecule in HIV infection. To further probe the mechanism by which complexes 1 and 2 reactivated virus, determination of TNF-α production was performed using the BD BioSciences (California, USA) CBA kit. The cytokines quantified by the kit included IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ and IL-17A. For the purposes of this study, the main focus was on the endogenous production of TNF-α. TNF-α levels were shown to increase by 9 fold for complex 1 and 3 fold for complex 2 compared to the vehicle control (Figure 5) while for PMA treated cells, the increase was as a significant increase (p = 0.004) of up to 2353.86 ± 204 pg/mL (648 fold). Although increases were observed for 1, the findings were not statistically significant (p > 0.05) suggesting that TNF-α stimulation might only be playing a contributory role which together with the increase in kinase activity resulted in HIV reactivation. Complex 1’s effect on TNF-α was more elevated than 2 supporting the fact that reactivation of latent virus induced by 1 exceeded that of 2.Figure 5

Bottom Line: Viral reactivation was absent for the complexes during co-stimulation with PMA indicating the lack of an additive effect between the chemicals as well as an absence of inhibition of PMA induced HIV reactivation but for HU inhibition of the stimulant's activity was observed (p = 0.01).The cytostatic effect of 1 and 2 and now HIV reactivation from a U1 latency model is consistent with that of the cytostatic agent, HU.These findings suggest that the complexes have a potential dual (cytostatic and reactivation) role in viral "activation/elimination".

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Faculty of Natural and Agricultural Sciences, University of Pretoria, Hatfield Campus, Pretoria 0002, South Africa. Debra.Meyer@up.ac.za.

ABSTRACT

Background: A number of cytostatic agents have been investigated for the ability to reactivate latent viral reservoirs, which is a major prerequisite for the eradication of HIV-1 infection. Two cytostatic bis(thiosemicarbazonate) gold(III) complexes (designated 1 and 2) were tested for this potential in the U1 latency model of HIV-1 infection.

Methods: Cell viability in the presence or absence of 1 and 2 was determined using a tetrazolium dye and evidence of reactivation was assessed by p24 antigen capture following exposure to a latency stimulant, phorbol myristate acetate (PMA) and or test compounds. The latency reactivation mechanism was explored by determining the effect of the complexes on protein kinase C (PKC), histone deacetylases (HDAC) and proinflammatory cytokine production.

Results: The CC50 of the complexes in U1 cells were 0.53 ± 0.12 μM for 1 and 1.0 ± 0.4 μM for 2. In the absence of PMA and at non toxic concentrations of 0.2 and 0.5 μM, 1 and 2 significantly (p ≤ 0.02) reactivated virus in U1 cells by 2.7 and 2.3 fold respectively. In comparison, a 2.6 fold increase (p = 0.03) in viral reactivation was observed for hydroxyurea (HU), which was used as a cytostatic and latent HIV reactivation control. Viral reactivation was absent for the complexes during co-stimulation with PMA indicating the lack of an additive effect between the chemicals as well as an absence of inhibition of PMA induced HIV reactivation but for HU inhibition of the stimulant's activity was observed (p = 0.01). Complex 1 and 2 activated PKC activity by up to 32% (p < 0.05) but no significant inhibition of HDAC was observed. Increases in TNF-α levels suggested that the reactivation of virus by the complexes may have been due to contributions from the latter and the activation of PKC.

Conclusion: The ethyl group structural difference between 1 and 2 seems to influence bioactivity with lower active concentrations of 1, suggesting that further structural modifications should improve specificity. The cytostatic effect of 1 and 2 and now HIV reactivation from a U1 latency model is consistent with that of the cytostatic agent, HU. These findings suggest that the complexes have a potential dual (cytostatic and reactivation) role in viral "activation/elimination".

Show MeSH
Related in: MedlinePlus