U1 cell viability studies

A dose response curve from 0.2 – 25 μM of the viability of U1 cells was obtained for 1 and 2 and for the precursor ligands, HAuCl 4 .4H 2 O, and HU from 0.8-100 μM (Figure 2). The complexes were more toxic than the complementary ligands and gold starting material (p < 0.05) with 50% cytotoxic concentrations (CC 50 s) of 0.53 ± 0.12 μM and 1.00 ± 0.4 μM for 1 and 2 respectively. Ligands L1 and L2 as well as HU were non toxic with CC 50 of > 100 μM while that of HAuCl 4 .4H 2 O was > 50 μM. Considering that toxic concentrations could provide false information on the effect of the complexes in the other cell-based assays, only non toxic concentrations were subsequently used. For complex 1, concentrations of 0.2 μM and lower were used while for 2, concentrations of 0.5 μM and lower were used since these resulted in more than 85% cell viability.

Figure 2 Effect of complex 1 and 2 and the associated ligands on U1 cell viability. A dose response curve was obtained for complexes 1 and 2 from 0.2-25 μM with CC 50 values of 0.53 ± 0.12 and 1.00 ± 0.4 μM respectively. L1, L2 and HU were less toxic with CC 50 > 100 μM while that of the gold (synthesis) starting material, HAuCl 4 .4H 2 O was >50 μM. The numbers above the bar graphs are the CC 50 values. # Two fold serial dilutions from 100 down to 0.8 μM were performed. ****p < 0.0001, ***p = 0.0001, **p = 0.002. Full size image

Reactivation of latent HIV-1

For the activation studies, U1 cells were treated with complex only or co-stimulated with complex and PMA. Findings from these treatments are shown in Figure 3. To easily differentiate the effect of the complexes in each treatment type, the vehicle control and PMA treated cells were represented as 100% in Figure 3A and B respectively. For comparison of the differences between the vehicle control, complex treated and complex plus PMA, another representation of the data in Figure 3 is shown in Additional file 1: Figure S2 for complexes 1 and 2.

Figure 3 Complex 1 and 2 reactivate HIV replication in latently infected U1 cells. At non toxic concentrations of 0.2 and 0.5 μM viral p24 levels increased by 2.7 and 2.3 fold for complex 1 and 2 respectively (*p ≤ 0.03) while 200 μM HU, which was included in the study as a cytostatic positive control, reactivated latent virus by 2.6 fold (A). PMA (3 nM), which was used both as a positive control for virus activation and to monitor the effect of the complexes on activated virus, significantly activated viral production (p = 0.01). HU also significantly (p = 0.01) inhibited PMA mediated latency activation by 44% when compared to PMA treated cells only, an observation which was absent for 1 and 2 (B). The p24 levels for the vehicle control and PMA treated cells are represented as 100% so that differences resulting from complex effects could easily be differentiated. Full size image

In the absence of stimulant

The complexes were treated at three concentrations in a pre-screen (at CC 50 or below, Additional file 1: Figure S1); complex 1 significantly reactivated viral production from the U1 cells by 2.7 fold (p = 0.023) when tested at 0.2 μM while complex 2 (0.5 μM), reactivated HIV replication by 2.3 fold (p = 0.005) when compared to untreated or cells only control (Figure 3A). At 200 μM, HU, a cytostatic agent used as a positive control, reactivated virus by 2.6 fold (p = 0.03). Complexes 1 and 2 were previously reported as cytostatic [24] and now with the ability to reactivate virus, have another activity in common with HU although demonstrating this activity at much lower concentrations. Ligands L1 and L2 had no effect on viral reactivation supporting our findings and previous reports for metal-based drugs that the metal entity is important for bioactivity. Also as expected, the gold starting material, HAuCl4.4H 2 O did not reactivate virus, further supporting the role of ligand complexation in metal-based drugs, as being activity enhancing [34]. The HIV-1 reactivation findings for these gold-based complexes are also supported by those reported for auranofin, another gold-based drug which induced HIV-1 reactivation from primary monocyte-derived macrophages as reported in the Additional file one by Shytaj et al. [35]. PMA reactivated virus production from the U1 cells (7.8 fold increase, Figure 3A) when compared to the vehicle control, confirming previous reports [12],[17].

PMA co-stimulation

In the co-stimulation treatment, the expectation was that the complexes would either synergistically reactivate virus in concert with PMA or inhibit virus production effected by the phorbol ester. For 1 and 2, neither a synergistic reactivation nor inhibition of PMA mediated viral reactivation was observed at the tested concentrations since no significant increases or decreases in percentage reactivation was observed (Figure 3B and Additional file 1: Figure S2). Synergistic activation has been defined as the situation where the combination of two activators produces a level of activation that is greater than the sum of the effects produced with the individual activators [36]. This type of activation is important as it makes it possible to combine different latency activators to improve on the effectiveness of viral reactivation [18]. PMA’s activation of virus replication minimised the effects of all the treatments, except for that of HU which at 200 μM, inhibited p24 antigen production by 44% (p = 0.01, Figure 3B). This finding is not surprising since HU has been reported to inhibit HIV-1 LTR transactivation in the presence of both TNF-α and PMA as stimulants [19],[37]. According to Calzado and his colleagues [37], this inhibition of PMA mediated HIV-1 replication suggests that HU is capable of inhibiting HIV by another mechanism, in addition to RNR inhibition. HU inhibits activation mediated by PMA and TNF-α, which are both transcriptional activators but synergistically activates virus in the presence of the posttranslationally active cytokine, IL-6, by causing increases in Sp1 and Sp3 proteins which are involved in the expression of HIV-1 LTR [19],[38]. Considering that 1 and 2 did not inhibit viral production when co-stimulated with PMA, it is possible that the mechanism of activation is different from that of HU. Alternatively, it could be a concentration dependent issue and because higher concentrations of 1 and 2 were toxic to U1 cells, inhibition at higher concentrations would not be of value.

The reactivation for the complexes is shown here for the U1 promonocytic cell line model of latency as proof of concept that the cytostatic compounds also reactivate virus like HU. To have a better in vivo representation of the potential of these complexes as latency reactivators, a test on reactivation on T cells will be important since HIV predominantly resides in resting memory CD4+ T cells [39]. Unfortunately, another gold-based drug, auranofin was unable to activate latent HIV from primary CD4+ T cells [40]. The possibility that this could be applicable for complexes 1 and 2 which are also gold-based complexes exists. That notwithstanding, the complexes could play a role in reactivating virus from a subset of the immune system cells and further confirmation in a monocyte-derived macrophage cell line will be important in further exploring this potential.

Viral reactivation mechanism

Complex effect on HDAC and PKC activity

Because HDAC plays a role in maintaining latency, inhibitors of these enzymes reactivate latent virus. To determine whether complex 1 and 2’s ability to reactivate virus was as a result of inhibiting HDAC, the activity of this enzyme in the presence of non-toxic concentrations of the complexes was evaluated using a fluorometric assay. Complexes 1 and 2 did not appreciably inhibit HDAC activity when a cut-off of 50% inhibition was considered. For 2, a 27.4% inhibition was not significant (p > 0.05). TSA which was used as the positive control significantly (p < 0.0005) inhibited HDAC by 103.7% (Figure 4A).

Figure 4 The effects of the complexes on HDAC and PKC activity. At a cut-off of ≥50% inhibition as inhibitory, complex 1 and 2 did not appreciably inhibit HDAC. The positive control, TSA, inhibited the enzyme by 103.7% with a p value of <0.0001 (A). The enzyme control is represented as 100% inhibition. In the PKC assay, significant increases in PKC activity of 31.5 (p = 0.033) and 32.7% (p = 0.036) were observed for 1 and 2 respectively compared to the untreated vehicle control (B) suggesting that reactivation was as a result of an effect on NF-κB. Prostratin, a PKC activator caused significant increases in PKC of 80.3% (p = 0.02). The vehicle control sample is represented as 100% activity. Full size image

Another mechanism by which viral latency activators function is by activating PKC. PKC activation by 1 and 2 significantly (p < 0.05) increased by 31.5 and 32.6% at 0.2 and 0.5 μM respectively compared to the vehicle only which was the 100% reference (Figure 4B). Kinase activity increased significantly by 80.3% for cells treated with 1 μg/mL of the positive control, prostratin.

These results indicate that PKC activation contributed to the HIV-1 reactivation observed for complexes 1 and 2 from the latently infected U1 cells. While complexes 1 and 2, which are gold(III) thiosemicabazonate-based are structurally different from the known PKC activators, it is interesting to note that reactivation of HIV-1 is associated to the activation of this enzyme. Interestingly, in the co-stimulation studies with another PKC activator, PMA, no significant differences in viral reactivation was observed at the tested concentrations (Figure 3B and Additional file 1: Figure S2). A co-stimulation study with or without a PKC inhibitor should provide additional mechanistic information.

For the HDAC assay, the observed 27.4% inhibition for 2, was not significant. The latter assay was not cell-based and higher concentrations of the complexes did not inhibit the enzyme (data not shown). Future studies to confirm the absence of HDAC activity will include doing the reactivation studies in the presence or absence of a histone acetyltransferases (HAT) inhibitor. Considering that the balance between protein acetylation and deacetylation controls several physiological and pathological cellular processes, the inhibition of HAT and thus alteration of the HAT/HDAC enzymes which maintain this balance [41] should provide additional mechanistic information. As part of continual investigations on the functionality of these complexes in HIV reactivation, these types of analysis will be considered.

Endogenous TNF-α production as viral reactivation mechanism

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 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. Full size image

At 200 μM, HU had no major effect on the production of TNF-α and because this compound also had no effect on PKC and HDAC, the mechanism by which HU reactivates virus probably differs from that of the complexes. In 1997, Navarra and his colleagues reported HU as capable of stimulating TNF-α production in vivo [43], and the lack thereof in this in vitro set up means other necessary factors required for an effect by HU on this cytokine, were absent. The cytostatic compound actinomycin D reactivates virus by modulating the cytokines IL-6 and TNF-β [20]. IL-6 levels were not affected by either the complexes or HU in our case. Complexes 1 and 2 also increased TNF-α levels in peripheral blood mononuclear cells from HIV negative donors (Additional file 1: Figure S3). This indicates that the in vitro upregulation of TNF-α occurs not only in the promonocytic U1 cell line but also ex vivo in primary immune system cells.

Upregulation of proinflammatory cytokine production by 1 and 2 (although not statistically significant) could imply that these complexes might be reactivating virus through a non specific mechanism, which is a concern with most latency activating agents [18]. Such a nonspecific mechanism might present these complexes as non selective meaning structural modifications for improved efficacy is needed. These modifications should also address the toxicity issues associated with the complexes with better targeting to latent reservoirs such as the use of nanotechnology [44].

Cytostatic agents arrest the cell cycle but according to Oguariri et al. [19], HU did not arrest U1 cells in the S and G2/M phases of the cell cycle as expected. This could be the reason why viral reactivation and hence replication occurred since retrovirus replication depends on cell cycling. Cell cycle analysis was performed for 1 and 2 and similarly to HU, cell cycle arrest of U1 cells was absent in the S and G2/M phases (data not shown).