IntroductionChemotherapy using natural products extracted from various resources, including plants and microorganisms, has long been of interest in the study of anti-human immunodeficiency virus (HIV) drug development [1,2]. Many natural compounds have been widely tested, and some have been reported as possible candidates for inhibition of HIV replication or boosting of host immune response. They are mostly secondary metabolites, including terpenoids and polyphenols. Natural compounds can provide novel anti-AIDS chemotherapeutic leads that are structurally unique or have new mechanisms of action .The chemical constituents of some species of Euphorbia, which are succulent plants growing mostly in semi-desert areas, have been found to include chemotaxonomically important myrsinane diterpenoids acting as enzyme inhibitors against alpha-glycosidase, urease, HIV-1 reverse transcriptase, and prolyl endopeptidase . Ingenol, one of the active ingredients in an extract from Euphorbia peplus, was shown to have activity against human melanoma cells and the ability to induce the apoptosis-regulating tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) in preclinical study . While latex as well as total leaf extracts of Euphorbia plants exhibit Epstein–Barr virus (EBV) inducing activity , some ingenol derivatives were reported as potential HIV reverse transcriptase inhibitors .Ingenol derivatives are structurally close to phorbol 12-myristate 13-acetate (PMA) , but have been less studied than PMA because of their structural complexity. Prostratin , another nontumorigenic phorbol ester, was reported to have HIV-inhibitory activity related to PKC activation . PMA itself also showed inhibitory effects against HIV-1 . Other reports have indicated that treatment of ingenol derivatives such as ingenol triacetate and aurintricarboxylic acid in HIV-infected T cells inhibited HIV replication through downregulation of CD4 receptors [12,13] and CXCR4 receptors .We tested the anti-HIV activity of ingenol purified from Euphorbia ingens and subsequent viral and host event-influencing immune responses, which remain largely unknown despite some previous work on ingenol’s antiviral [12,14] and anti-inflammatory effects [15,16]. In addition to anti-HIV activity screening, it is essential to understand the mechanism of action related to HIV inhibition or immune response during ingenol action against infection, which might contribute to developing novel anti-HIV reagents or new therapeutic strategies for AIDS.
2.1 Cells culture and reagentsMT4 and H9 human T lymphocyte cell lines were obtained from the NIH AIDS Research and Reference Reagent Program (Bethesda, MD). Cells were maintained in RPMI-1640 medium (GIBCO, Carlsbad, NY) supplemented with 10% fetal bovine serum, penicillin 100 U/mL, streptomycin 100 μg/mL, and glutamine 2 mM.Ingenol (Figure 1) purified from E. ingens was obtained from Sigma-Aldrich (St. Louis MO). Compound was dissolved in dimethylsulfoxide (DMSO) and further diluted with RPMI-1640 medium. The final concentration of DMSO in stock solution was <1.5% (v/v).
2.2 Preparation of virus stock and titrationTo obtain HIV-1 virus stock we incubated HTLV-IIIB/H9 cells (CRL 8543; American Type Culture Collection, Manassas, VA) for 14 days and harvested cell culture supernatants twice/week. The virus was then harvested from cell culture supernatant by centrifugation at 4500 rpm for 20 minutes followed by filtration through 0.22-μm filter (Millipore, Bedford, MA). To quantify viable HIV-1 virions in culture supernatant we evaluated 50% tissue culture infectious dose (TCID50) using HIV-1 gag p24 antigen ELISA (BioMerieux, Durham, NC). Titration for TCID50 infectivity was measured by the Spearman-Karber statistical method. The titer of virus stock was determined in MT4 cells (6.56 × 105 TCID50/mL) then divided into aliquots of fixed volume. Prepared viruses were stored at –80°C until use.
2.3 Cytotoxicity and anti-HIV activity assayCytotoxicity of ingenol was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method (MTT assay; Roche, Basel, Switzerland) after 24-h incubation using MT4 and H9 cells. Anti-HIV infection activity was measured by cell viability of infected MT4 cells on day 6 postinfection of HIV-1 in the presence of serially diluted ingenol (0.1–250 μM). MT4 cells were infected with HIV-1 at various multiplicities of infection (MOI; 0.002–0.016 [25–200 TCID50]). The 50% effective dose (ED50) was calculated based on inhibition of virus-induced cell death after HIV infection, and 50% cytotoxic dose (CD50) was calculated based on reduction of viability in noninfected cells. Absorbance was measured at 550 nm wavelength with 690 nm reference wavelength using Bio-Tek microplate reader (EL800; Winooski, VT).
2.4 HIV-1 p24 antigen ELISA assay and luminex assay for cytokine measurementMT4 cells were infected with HIV-1 at 0.004 MOI (50 TCID50) after treatment of 5 μM ingenol. Gag p24 antigen level in culture supernatant was measured by Vironostika HIV-1 Antigen MicroELISA kit (BioMerieux, Durham, NC). Absorbance was read at 450 nm wavelength using EL800 Bio-Tek microplate reader.Cytokine and chemokine levels in culture supernatants were measured at the same day of p24 antigen measurement. Procarta™ human cytokine 5-plex kit and Procarta™ human chemokine 5-plex kit were purchased from Parnomics (Fremont, CA); assay was performed following the manufacturer’s protocol. Results were read using Bio-Plex 200 Suspension array system (Bio-Rad, Hercules, CA) and calculated with Bio-Plex Manager software 4.1 (Bio-Rad).
2.5 Flow cytometric assayCells were incubated with Golgi-Plug™ (BD Biosciences, San Jose, CA) for 4 hours and permeabilized with PermFix/PermWash (BD Biosciences). Then, cells were stained using Annexin V-Mitotracker Vybrant® Apoptosis assay kit (Molecular Probes, Eugene, OR) and APO-BrdUTM TUNEL assay kit (Invitrogen, Carlsbad, CA) following the manufacturers’ protocols. Stained cells were analyzed by FC500 flow cytometer (Beckman Coulter, Fullerton, CA).
2.6 Western blot analysisCellular lysate was prepared using ice-cold RIPA buffer (50 mM Tris pH 7.5, 0.5% deoxycholate, 0.5% NP-40, 0.5% SDS, and 100 mM NaCl) supplemented with freshly added protease inhibitor cocktail. Protein concentration in cell lysate was determined by BCA protein assay kit (Pierce, Rockford, IL); proteins were separated by SDS-PAGE. After transfer of separated proteins to Immobilon™–P PVDF membrane (Millipore, Danvers, MA), primary antibodies were applied for hybridization: rabbit anti-human inducible nitric oxide synthase (iNOS) antibody (Abcam, Cambridge, MA) and rabbit anti-human zeta chain-associated protein kinase 70 (ZAP-70) antibody (Cell Signaling, Danvers, MA). Hybridized bands were developed with HRP-conjugated anti-rabbit secondary antibody. β-Actin antibody was used as a loading control, TMB chemiluminescence substrate for visualization.
3.1 Ingenol exerts low cytotoxicity and dose-dependent anti-HIV infection activityIngenol revealed no toxic effects in MT4 cells and comparatively low cytotoxicity in H9 cells at the range 0.1–100 μM (Figure 2A). Survival of MT4 cells was more stable up to high concentration than that of H9 cells (Figure 2A).Cell viability after HIV infection in MT4 cells preincubated with ingenol at concentrations 1.95–250 μM was observed as a dose- and MOI-dependent pattern around day 6 postinfection (Figure 2B). ED50 for 100 and 200 TCID50 of HIV-1 was 5.06 and 16.87 μM, respectively. This dose-dependent anti-HIV infection activity of ingenol was similar to that of azidothymidine (AZT), which is widely used for AIDS treatment (data not shown).
3.2 Ingenol reduced HIV-1 gag p24 antigen production by inducing cytokines and chemokinesGag p24 antigen production in HIV-infected MT-4 cells when 5 μM ingenol was treated into cell culture media was reduced by approximately 25% on day 6 postinfection (Figure 3A).Th1 cytokines such as TNF-α and IL-6 were highly induced by ingenol treatment in HIV-infected MT4 cells, whereas Th2 cytokines were not detected (Figure 3B). TNF-α was considerably increased by 14 times and IL-6 was also induced by ingenol treatment.Chemokines such as RANTES and MIP-1α were remarkably induced by ingenol treatment in HIV-infected cells on day 6 postinfection of HIV (Figure 3C). MIP-1β, MCP-1, and eotaxin were moderately increased or recovered by ingenol treatment compared with HIV-infected cells without ingenol (Figure 3C).
3.3 Apoptosis more advanced to late phase by ingenol in HIV-infected cellsPercent apoptotic events MT4 cells was measured by flow cytometric assay using annexin V as an early phase marker and dUTP as a late phase marker at 24 h postinfection. Annexin-V expression was slightly reduced by 4% in ingenol-treated MT4 cells compared with nontreated cells; however, the change was not statistically significant (Figure 4A). The dUTP level in HIV-infected MT4 cells was further increased by 25% with ingenol treatment (Figure 4B).
3.4 iNOS and ZAP-70 recovered by ingenol treatment in HIV-infected cellsWestern blot results of iNOS and ZAP-70 expression are shown in Figure 5. Total cell lysate was analyzed at 24 h postinfection; both iNOs and ZAP-70 were recovered by ingenol treatment compared with the remarkably reduced levels of HIV infection. Especially, iNOS was increased to a higher level than that in uninfected cells, whereas ZAP-70 recovered to almost the same level of uninfected cells.
DiscussionIngenol and its derivatives are known to have several biological activities. In particular, they have a degree of potential to activate protein kinase C (PKC) . In previous studies, some ingenol derivatives were demonstrated to be potent inhibitors of acutely HIV-infected cells , whereas others were potent activators of HIV-1 transcription in chronically infected cells . We focused on the anti–HIV-1 activity of ingenol, so as further to elucidate the mechanism of inhibition, and to identify cellular factors related to inhibition of HIV-1 replication in infected cells.Ingenol-treated MT4 cells showed reduced p24 level compared with PBS-treated HIV-infected cells on day 6 postinfection. Gag p24 antigen of ingenol-treated cells was reduced by approximately 25% compared with mock-infected cells. Cell survival of ingenol-treated MT4 cells on day 6 postinfection was reduced to approximately 20% lower than AZT control (data not shown). Although our results showed lower inhibition of HIV infectivity compared with AZT, we nonetheless observed significant anti-HIV activity of ingenol.HIV-1 induces apoptotic and anti-apoptotic signaling in infected and uninfected T lymphocytes, and thereby counteracts the antiviral response and immune effector cell functions . Excessive induction of apoptosis may induce downregulation of the cellular immune response and favor pathogen immune evasion .Annexin V binding of ingenol-treated cells was reduced by approximately 4% compared with nontreated cells. On the other hand, dUTP of ingenol-treated MT4 cells was increased by approximately 9% compared with nontreated cells. Inhibition of apoptosis in HIV-1-infected T cells enhances virus production and facilitates persistent infection . HIV-1–infected and ingenol-treated MT4 cells suppressed virus production and early apoptosis arising compared with uninfected and AZT-treated cells. A reduced level of annexin V may suggest apoptosis of uninfected and ingenol-susceptible cells. Otherwise, DNA strand breakages of HIV-1-infected and ingenol-treated MT4 cells were not protected from later apoptosis. However, increased level of dUTP may have been induced by apoptosis of HIV-1 completely infected cells.Although little is known of the ability of TNF to induce apoptosis in HIV-infected cells, it may contribute to apoptosis induced by gp120-mediated cross-linking of CD4 .TNF-α and p24 antigen production were positively correlated at 6 days postinfection. In addition, IL-6 was very slightly increased in HIV-1-infected and ingenol-treated cells compared with days 1 and 3, suggesting that elevated TNF-α and IL-6 expression may induce high levels of HIV-1 infectivity. Indeed, this result is supported by elevated serum TNF levels seen in symptomatic HIV-infected patients [22,23], but not in asymptomatic patients [24,25]. Although the mechanisms to control the apoptotic pathway by ingenol has not been elucidated in detail, there is evidence that TNF-α may be involved in regulation of apoptosis when HIV-infected cells were treated with ingenol derivatives .The mechanism of anti-HIV-1 activity of ingenol requires further study. In future, we will focus on elucidating the steps of HIV-1-mediated immune-related mechanisms, so as to gain insight into new applications for anti-HIV treatment.