Differential effects of phorbol-13-monoesters on human immunodeficiency virus reactivation

Article abstract of DOI:10.1016/j.bcp.2007.12.004

The persistence of latent reservoirs of HIV-1 represents a major barrier to virus eradication in patients treated with antiretrovirals. Prostratin is a non-tumor promoting 12-deoxyphorbol monoester capable of up-regulating viral expression from latent provirus and therefore is potentially useful for HIV adjuvant therapy and similar properties might be elicited by related non-tumor promoting phorboids. We have therefore investigated a series of phorbol 13-monoesters for their capacity to reactivate HIV latency. Using a Jurkat T cell line containing latent HIV proviruses, we found that prostratin and phorbol-13-stearate effectively activate HIV-1 gene expression in these latently infected cells, with phorbol-13-stearate being at least 10-fold more potent than prostratin, and its activity rapidly decreasing with a shortening of the acyl side chain. We further demonstrated that phorbol-13-stearate and prostratin stimulate IKK-dependent phosphorylation and degradation of IκBα, leading to activation of NF-κB. Moreover, prostratin, phorbol-13-hexanoate and phorbol-13-stearate also activate the JNK and ERK pathways. Studies with isoform-specific PKC inhibitors suggest that the classical PKCs play a prominent role in the responses elicited by phorbol-13-stearate. Nevertheless, this compound induces a translocation pattern of the PKC isotypes α and δ to cellular compartments distinctly different from that elicited by prostratin and PMA.

Full text of DOI:10.1016/j.bcp.2007.12.004

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available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/biochempharm
Differential effects of phorbol-13-monoesters on human
immunodeficiency virus reactivation
a
a
a
a
´
´
´
´
Nieves Marquez , Marco A. Calzado , Gonzalo Sanchez-Duffhues , Moises Perez ,
Alberto Minassi b, Alberto Pagani b, Giovanni Appendino b, Laura Diaz c,
Maria Angeles Mun˜ oz-Fernandez , Eduardo Mun˜ oz
c
a,
´
´
*
a Departamento de Biolog´ıa Celular, Fisiolog´ıa e Inmunolog´ıa, Facultad de Medicina, Avda. de Menendez Pidal s/n,
14004 Universidad de Co´rdoba, Spain
b Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche, Universita` del Piemonte Orientale, Novara, Italy
c Laboratorio de Inmuno-Biolog´ıa Molecular, Hospital General Universitario Gregorio Maran˜o´n, Madrid, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
The persistence of latent reservoirs of HIV-1 represents a major barrier to virus eradication
in patients treated with antiretrovirals. Prostratin is a non-tumor promoting 12-deoxyphor-
bol monoester capable of up-regulating viral expression from latent provirus and therefore
is potentially useful for HIV adjuvant therapy and similar properties might be elicited by
related non-tumor promoting phorboids. We have therefore investigated a series of phorbol
13-monoesters for their capacity to reactivate HIV latency. Using a Jurkat T cell line
containing latent HIV proviruses, we found that prostratin and phorbol-13-stearate effec-
tively activate HIV-1 gene expression in these latently infected cells, with phorbol-13-
stearate being at least 10-fold more potent than prostratin, and its activity rapidly decreas-
ing with a shortening of the acyl side chain. We further demonstrated that phorbol-13-
stearate and prostratin stimulate IKK-dependent phosphorylation and degradation of IkBa,
leading to activation of NF-kB. Moreover, prostratin, phorbol-13-hexanoate and phorbol-13-
stearate also activate the JNK and ERK pathways. Studies with isoform-specific PKC inhi-
bitors suggest that the classical PKCs play a prominent role in the responses elicited by
phorbol-13-stearate. Nevertheless, this compound induces a translocation pattern of the
PKC isotypes a and d to cellular compartments distinctly different from that elicited by
prostratin and PMA.
Received 13 November 2007
Accepted 12 December 2007
Keywords:
HIV-1
Latency
Phorbol ester
Prostratin
PKC
NF-kB
# 2007 Elsevier Inc. All rights reserved.
1.
Introduction
voirs represents the major hurdle to virus eradication with
highly active anti-retroviral therapy (HAART), since latently
HIV infects several cell types during the course of infection
and progression to acquired immune deficiency syndrome
(AIDS). The persistence of latent HIV-infected cellular reser-
infected cells remain a permanent source of viral reactivation
[1]. As a result, a sudden rebound of the virus load after
interruption of HAART is generally observed [2–4]. The HIV-1
* Corresponding author. Tel.: +34 957 218267; fax: +34 957 218229.
E-mail address: fi1muble@uco.es (E. Munoz).
˜
Abbreviations: AP-1, activator protein-1; ERK, extracellular regulated kinase; HIV-LTR, HIV long terminal repeats; IkB, kB inhibitor; JNK,
c-Jun N-terminal kinase; PKC, protein kinase C; NF-kB, nuclear factor kappa B; PMA, phorbol 12-myristate 13-acetate; P-13S, phorbol-13-
stearate; TNFa, tumor necrosis factor-a.
0006-2952/$ – see front matter # 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.bcp.2007.12.004

b i o c h e m i c a l p h a r m a c o l o g y 7 5 ( 2 0 0 8 ) 1 3 7 0 – 1 3 8 0
1371
establishes a persistent infection in CD4+ T lymphocytes
creating a persistent reservoir consisting mainly of latently
infected resting memory CD4+ T cells carrying an integrated
provirus that is transcriptionally silent [5,6]. The extremely
long half-life of these cells, combined with a tight control of
HIV-1 expression, has been reported to make this reservoir
ideally suited to maintain hidden copies of the virus [6].
The current therapies directed against viral proteins
(HAART) have been problematic because of long-term toxicity,
inhibitor resistance, and the inability to target persistent
reservoirs. Therefore, it has been suggested that reactivation
of the latent reservoirs could allow effective targeting and
possible eradication of the virus [7]. Immunoactivation
therapy to reduce the latent pool of HIV by treatment with
the anti-CD3 antibody OKT-3 alone or in combination with
interleukin-2, substantially failed to significantly decrease the
viral reservoir [8].
of localization of PKC, and therefore of access to substrates,
might underlie distinct patterns of biological response to a
given PKC ligand.
We report here the synthesis and biological evaluation of a
homologous series of phorbol 13-monoesters,
a type of
compounds that have been so far largely overlooked in terms
of bioactivity. The length of the fatty acid side chain of these
compounds is critical to induce HIV-1 reactivation in a cellular
model of HIV-1 latency.
2.
Materials and methods
2.1.
Cell lines and reagents
Jurkat T leukemia cells were grown at 37 8C and 5% CO2 in
supplemented RPMI 1640 medium (Cambrex Co., Barcelona,
Spain), containing 10% heat-inactivated fetal bovine serum,
2 mM glutamine, penicillin (50 U/ml) and streptomycin (50 mg/
ml). The CHO-K1 cells (CCL 61) were obtained from the
American Type Culture Collection (Manassas, VA) and
cultured in complete DMEM. The anti-IkBa mAb 10B was a
gift from Hay (St. Andrews, Scotland), the mAb anti-tubulin
and the anti-ERK 1+2 antibody were purchased from Sigma Co.
(St. Louis, MO, USA). The anti phospho-ERK 1 + 2 (sc-7383) and
the anti-IKKg (FL-419) were from Santa Cruz Biotechnology
(CA, USA). The antibody anti-phospho-JNK (9255S) was from
New England Biolabs (Hitchin, UK). The anti-p50 and anti-p65
Nevertheless, a host of small molecules including phorbol
esters [9], ingenols [10] and 1,2-diacylglycerol analogs [11], has
been suggested as agents to reactivate HIV and eradicate the
pool of latently HIV-infected CD4+ T cells. More recently, non-
tumor-promoting phorbol deoxyphorbol esters such as pros-
tratin and 12-deoxyphorbol 13-phenylacetate have been
directly evaluated for their ability to reactivate latent virus
both in latently infected cell lines and in primary memory T
cells from HIV infected patients [9,12]. Prostratin was isolated
for the first time from the poisonous New Zealand plant Pimela
prostrata [13] and was later identified as the anti-viral
constituent of the Samoan plant Homalanthus nutans [14].
Prostratin not only reactivates HIV-1 latency in ‘‘vitro’’ by
protein kinase C (PKC)-dependent NF-kB activation [14,15], but
also down-regulates the expression of the HIV-1 receptor CD4
and the co-receptor CXCR4, thus avoiding the new infection of
CD4+ cells [9,16].
antisera were a gift from Dr. Alain Israel (Institute Pasteur).
¨
The antibodies anti-PKCd and anti phospho- PKCd (Thr505)
were from Cell Signaling Technology (Danvers, MA). The mAb
anti-CD25 antibody (clone ACT-1, PE-labeled) was from Dako
(Glostrap, Denmark). The mAbs anti-CD69 (clone FN50, FITC-
labeled) and anti-CXCR4 (clone 12G5, PE-labeled) were from BD
Biosciences Pharmigen (San Diego, CA, USA). The mAb anti-
CD4 (clone 6D10, FITC-labeled) was from ImmunoTools
(Friesoythe, DE). Dual-Color Reagent Mouse IgG1/FITC + Mouse
IgG1/PE from DAKO (clone DAK-GO1 directed towards Asper-
gillus niger glucose oxidase) was used as negative control. The
inhibitors Rottlerin and PD 98059 were obtained from Alexis
Co. (Lausen, Switzerland) and the inhibitors Go6983, Go6976
The PKC family of serine/threonine kinases plays a central
role in mediating the signal transduction of extracellular
stimuli, which result in the production of the second
messenger 1,2-diacyl-sn-glycerol (DAG). PKC is also the
primary target of the phorbol ester tumor promoters and
consists of a family of 12 members that are classified into three
major subfamilies. The classical PKCs (a, bI, bII and g) are Ca2+
-
¨
¨
and DAG-dependent, whereas the novel PKCs (d, e, h and u) are
Ca2+-independent but DAG-responsive. The atypical PKCs (z
and l/i) lack the responses to both Ca2+ and DAG [17]. A highly
conserved cysteine-rich motif (C1 domain) in the regulatory
region of the PKCs acts as the specific receptor for DAG and
phorbol esters [18,19]. The C1 domain displays a hydrophobic
surface interrupted by a hydrophilic cleft. By sneaking into the
hydrophilic cleft, phorbol esters and DAG provide a hydro-
phobic cap on the hydrophilic cleft, masking its polarity and
facilitating the association of the C1 domain with the lipid
bilayer and other hydrophobic surfaces [20]. However, phorbol
derivatives with different lypophilicities exhibit different
biological activities and potencies [21].
and Go6850 were from Calbiochem (EMD Biosciences, Inc.
¨
Darmstadt, Germany). [g-32P]ATP (3000 Ci/mmol) was from MP
Biomedicals (Irvine, CA, USA). Prostratin was obtained from
Alexis Co. and all other reagents were from Sigma.
2.2.
Plasmids
The plasmid pEV731 (pHR0-tat2x.flag-IRES-eGFP) was obtained
from Dr. E. Verdin (Gladstone Institute of Virology, UCSF, CA,
USA) [23]. The pEGFP-N1 derived plasmids PKCa-GFP and
PKCd-GFP were obtained from Dr. P.M. Blumberg (NCI, MD,
USA). The GST-IkBa(1–54) plasmid has been described else-
where [24].
The translocation of PKCs from cytoplasm to plasma
membrane and other subcellular localizations is the hallmark
for PKC activation and several studies have shown that the
subcellular translocation of PKC is isoform-, cell type-, and
activator-specific, and, for phorbol 12, 13-diesters, is tightly
regulated by lipophilicity [21,22]. Therefore, distinct patterns
2.3.
Synthesis of 12-hydroxy-13 phorbol monoesters
The synthesis of 1b–h is outlined in Fig. 2. Aliphatic phorbol 13-
esters were prepared by treatment of phorbol (2) with an
excess (5–10 molar equivalents) of acylating agent (Anhy-

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drides for the synthesis of 1b, RCOOH/DCC/DMAP for the
synthesis of 1c–1g) in a suitable solvent (pyridine for Ac2O,
CH2Cl2 for all other cases). The resulting 13,20-diesters were
purified by gravity column chromatography on neutral
alumina to remove unreacted acids and next transesterified
by treatment with methanolic perchloric acid (pH 3). After
neutralization (NaOAc), filtration and evaporation, the crude
reaction mixture was purified by gravity column chromato-
graphy on silica gel (ca. 15 g/g of mixture) using ethyl acetate–
petroleum ether mixtures, to afford the 13-monoesters in
overall yield of 24–68% from phorbol. The 13-benzoate 1h
could not be prepared this way, since transesterification of the
allylic ester group was very sluggish, presumably because of
the decrease reactivity of the ester carbonyl toward nucleo-
philic addition. Compound 1h could be directly prepared from
phorbol by esterification with benzoic acid in the presence of
DCC (2 molar equivalents each). Without DMAP, acylation
occurred chemoselectively at the C-13 hydroxyl. The crude
product was purified from unreacted phorbol and benzoic acid
by gravity column chromatography on silica gel (petroleum
ether–ethyl acetate 1:1 as eluant) to afford 1h in 25% yield. All
final compounds were fully characterized spectroscopically
(1H and 13C NMR, MS), and showed purity >95% by HPLC.
buffer and three times in cold kinase buffer (20 mM Hepes/
KOH pH 7.4, 25 mM ß-glycerophosphate, 2 mM DTT, 20 mM
MgCl2). The kinase assay was performed in a final volume of
20 ml kinase buffer containing 40 mM ATP and 2 mg of the
purified substrate protein GST-IkBa(1–54). After incubation for
20 min at 30 8C, the reaction was stopped by the addition of 5ꢀ
SDS loading buffer. After separation by SDS-PAGE the proteins
were transferred to nitrocellulose membranes and IkBa
phosphorylation was detected by Western blot using the
antibody anti-phospho-IkBa 5A5 (Cell Signaling Technologies).
2.6.
Western blots
Jurkat-LAT-GFP cells (106 cells/ml) were stimulated with the
indicated compounds. Cells were then washed with PBS and
proteins extracted in 50 ml of lysis buffer (20 mM Hepes pH 8.0,
10 mM KCl, 0.15 mM EGTA, 0.15 mM EDTA, 0.5 mM Na3VO4,
5 mM NaF, 1 mM DTT, leupeptin 1 mg/ml, pepstatin 0.5 mg/ml,
aprotinin 0.5 mg/ml, and 1 mM PMSF) containing 0.5% NP-40.
Protein concentration was determined by the Bradford assay
(Bio-Rad, Richmond, CA, USA) and 30 mg of proteins were
boiled in Laemmli buffer and electrophoresed in 10% SDS/
polyacrylamide gels. Separated proteins were transferred to
nitrocellulose membranes (0.5 A at 100 V; 4 8C) for 1 h. Blots
were blocked in TBS solution containing 0.1% Tween 20 and 5%
non-fat dry milk overnight at 4 8C, and immunodetection of
specific proteins was carried out with primary antibodies
using an ECL system (GE Healthcare).
2.4.
Generation of Jurkat-LAT-GFP cells
For the production of viral particles containing an HIV-derived
vector, 5 ꢀ 105 293T cells were transfected with plasmids
pEV731 (10 mg), pCMV-R8.91 (6.5 mg) and pcDNA3-VSV (3.5 mg)
in10 cm dishes. After 16 h, medium was replaced, supernatants
containing viral particles were harvested 24 h later and viral
particles containing 150 ng of p24 were used to infect 106 Jurkat
cells. After 96 h, the efficiency of the infection process was
monitored by FACS analysis and the negative population was
sorted (FACSCvantage SE, BD Bioscience) and cultured again in
completed medium. Then the sorted cells were stimulated with
TNFa for 24 h and then the GFP+ population was analysed (Cell
Quest-Pro software), sorted and cloned by limit dilution in 96
well plates. After 3 weeks the clones were stimulated with PMA
(50 ng/ml) to induce the expression of the integrated LTR-GFP
vector for 24 h and 4 out of 72 clones were selected for
characterization. The percentage of GFP+ cells was analysed by
flow cytometry in an EPIC XL flow cytometer (Beckman-Coulter
Inc. CA, USA). Ten thousand gated events were collected per
sample. Finally, clon 8 was selected for further experiments and
renamed Jurkat-LAT-GFP cells.
2.7.
Isolation of nuclear extracts and mobility shift assays
Jurkat-LAT-GFP cells (106 /ml) were stimulated with the
agonists in complete medium as indicated. Cells were then
washed twice with cold PBS and proteins from nuclear extracts
isolated as previously described [24]. Protein concentration was
determined by the Bradford method (Bio-Rad). For the electro-
phoretic mobility shift assay (EMSA), the consensus oligonu-
cleotide probes NF-kB, 50-AGTTGAGGGGACTTTCCCAGG-30 and
the commercial AP-1 site (Promega Biotech Ibe´rica, ES) were
end-labelled with [g-32P]ATP. The binding reaction mixture
contained 3 mg of nuclear extract, 0.5 mg poly(dI-dC) (GE
Healthcare), 20 mM Hepes pH 7, 70 mM NaCl, 2 mM DTT,
0.01% NP-40, 100 mg/ml BSA, 4% Ficoll, and 100,000 cpm of end-
labelled DNA fragments in a total volume of 20 ml. When
indicated, 0.5 ml of rabbit anti-p50, anti-p65 or preimmune
serumwas added tothestandardreaction before the addition of
the radiolabelled probe. For cold competition, a 100-fold excess
of thedouble stranded oligonucleotidecompetitor was added to
the binding reaction. After 30 min incubation at 4 8C, the
mixture was electrophoresed through a native 6% polyacryla-
mide gel containing 89 mM Tris–borate, 89 mM boric acid and
1 mM EDTA. Gels were pre-electrophoresed for 30 min at 225 V
andthen for2 h afterloading thesamples. These gelsweredried
and exposed to X-ray film at ꢁ80 8C.
2.5.
IKK kinase assay
Cells were lysed in NP-40 lysis buffer (20 mM Tris/HCl pH 7.5,
150 mM NaCl, 1 mM PMSF, 10 mM NaF, 0.5 mM sodium
vanadate, leupeptine (10 mg/ml), aprotinin (10 mg/ml), 1% (v/
v) NP-40 and 10% (v/v) glycerol) for 15 min at 4 8C, and after
centrifugation for 10 min at 13000 rpm, the supernatant was
incubated with non-specific IgG and 25 ml protein A/G
sepharose (preclearing) and incubated overnight on a spinning
wheel. After centrifugation, the supernatants were incubated
with 2 mg of anti-IKK-g antibody and 25 ml protein A/G
sepharose and incubated for 2–4 h on a spinning wheel at
4 8C. The precipitate was washed three times in cold lysis
2.8.
Expression of PKCs-GFP in cultured cells and
visualization by confocal microscopy
CHO-K1 cells were grown on 12 mm round coverslips to 50–
75% confluence. Transient transfection was conducted using

b i o c h e m i c a l p h a r m a c o l o g y 7 5 ( 2 0 0 8 ) 1 3 7 0 – 1 3 8 0
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Jet Pei reagent (Polyplus transfection, France), following the
manufacturer’s instructions. The fluorescence became detect-
able 24 h after transfection, and all experiments were
performed 3 days after transfection. Prior to observation,
transiently transfected CHO-K1 cells were washed twice with
PBS containing 1% FCS prewarmed to 37 8C. All PKC activators
were diluted to specified concentrations in the same medium,
and the final concentration of solvent (DMSO) was always less
than 1%. After 10 min of treatment the cells were fixed and
analysed with a confocal microscopy (Espectral TCS-SP2-
AOBS, Leica).
the control of the HIV-1 LTR promoter was used [23]. Jurkat
cells were infected with viral particles containing this vector,
and 96 h later the GFPꢁ cells were selected by fluorescence-
activated cell sorting (FACS). This population seemingly
contained both uninfected cells and cells with transcription-
ally inactive proviruses. To activate HIV-1 expression, this
population was stimulated with TNFa (10 ng/ml) for 24 h and
the GFP+ cells were sorted again and cloned by limiting
dilution. Four out of 72 clones were selected and characterized
for their responsiveness to OKT3, PMA and TNFa (Fig. 1A).
Clone 8 (Jurkat-LAT-GFP cells) was selected for further studies,
and, after treatment with PMA, OKT3 or TNFa, GFP expression
was induced to a different extent, with PMA being the stronger
activator (Fig. 1B). Re-analysis 14 and 28 days after Jurkat-LAT-
GFP cells selection showed that more than 95% of the cells had
no GFP expression, indicating transcriptional silencing in the
absence of stimuli. As expected, the HIV-1 Tat protein was
detectable only in stimulated Jurkat-LAT-GFP cells (data not
shown).
2.9.
Isolation of peripheral human T cells and
cytofluorimetric analyses of cell surface antigen
Human PBMC, from healthy adult volunteer donors, were
isolated by centrifugation of venous blood on Ficoll-hypaque1
density gradients (GE Healthcare). Macrophages were removed
by incubating the PBMC on 100 mm plastic Petri plates at 37 8C
for 60 min, and the remaining cells were passed twice through a
nylon wool column to deplete residual B cells and macrophages
[25]. Nylon Wool T cells were usually 95% CD3+ and less than 5%
CD25+. Cell surface expression of CD4, CXCR4, CD25 and CD69
antigens was measured by direct fluorescence using specific
mAbs and analyzed by flow cytometry in an EPIC XL flow
cytometer (Beckman-Coulter Inc. CA, USA).
3.2.
Effects of phorbol-13-monoesters in HIV-1
reactivation
12-Deoxyphorbol-13-acetate (prostratin, 1a) (Fig. 2), has been
shown to reactivate HIV-1 latency in a cellular model similar to
the Jurkat-LAT-GFP cells [26]. To study the effect of hydro-
xylation at C-12, phorbol 13-acetate (1b) was synthesized from
phorbol (2) (Fig. 2), and Jurkat-LAT-GFP cells were stimulated
with increasing concentrations of both 1b and prostratin (1a)
for 6 h. As expected, prostratin (1a) activated HIV-1 latency
with an EC50 of 0.41 mM, while 1b was totally inactive for HIV-1
reactivation (Fig. 3). Thus, the presence of a 12b-hydroxyl is
detrimental for the activity of prostratin (1a). Having estab-
lished that the substitution pattern of C-12 is critical for
3.
Results
3.1.
Generation of latent HIV-1 infected Jurkat cells
To establish cell lines with latent integrated HIV-1, an HIV-
based retroviral vector containing the Tat and GFP genes under
Fig. 1 – Characterization of the Jurkat-LAT-GPF cell line. (A) Cellular clones from latently infected Jurkat cells were stimulated
with the agonists PMA (50 ng/ml), TNFa (10 ng/ml) and plastic coated OKT3 (2 mg/ml) for 24 h, and analyzed for GFP
expression by flow cytometry. Results are represented as the percentage of GFP positive cells W S.D. of three different
experiments. (B) Clone 8 (Jurkat LAT-GFP) was selected for the further experiments, and the histograms represent the
typical response of Jurkat-LAT-GFP cells to different stimuli. One out of three independent experiments is shown.

1374
b i o c h e m i c a l p h a r m a c o l o g y 7 5 ( 2 0 0 8 ) 1 3 7 0 – 1 3 8 0
Fig. 2 – Chemical structures of prostratin (1a), 12-hydroxyprostratin (1b) and the other 12-hydroxyphorbol-13 monoesters
used in the study (1c–h) and synthesis of 1b–h.
activity, a series of analogues having a longer acyl residue at C-
13 (C4, C6, C10, C14 and C18) was prepared (1c–h) (Fig. 2).
Remarkably, recovery of activity was observed starting from
the C6 ester, with the C18 analogue even outperforming
prostratin by an order or potency (EC50 around 30 nM) for the
capacity to induce HIV-1 reactivation from latency (Fig. 3).
(1a) and phorbol-13 monoesters in the activation of PKC-
inducible transcription factors, Jurkat-LAT-GFP cells were
stimulated with compounds 1b–1h (10 mM) for 60 min, and
nuclear extracts were obtained and analysed by electrophore-
tic mobility shift assay (EMSA) using NF-kB and AP-1-specific
32P-labelled oligonucleotides. As shown in Fig. 4A, PMA
stimulation strongly increased two NF-kB bands (Fig. 4A,
compare lane 2 to lane 1) and one AP-1 band. The specificity of
NF-kB bands was confirmed by cold oligonucleotide competi-
tion: wildtype kB oligonucleotides competed away-induced
bands (Fig. 4B, lane 6), whereas AP-1 oligonucleotides did not
diminish the intensity of the induced bands (Fig. 4B, lane 7).
The addition of p65 (Fig. 4B, lane 4) and p50 (Fig. 4B, lane 3)
3.3.
Effects of prostratin and 12-hydroxyphorbol-13-
monoesters on NF-kB, JNK and ERK signaling pathways
Several groups have independently demonstrated that pros-
tratin reactivates HIV-1 latency by activating a PKC-dependent
NF-kB pathway [15,26]. In order to study the role of prostratin
Fig. 3 – 12-Hydroxyphorbol-13 monoesters and prostratin antagonize HIV-1 latency. Jurkat LAT-GFP cells were stimulated
with the 12-hydroxyphorbol-13 monoesters and prostratin at the indicated concentrations for 6 h, and next analyzed by
flow cytometry. Results are represented as the percentage of GFP positive cells W S.D. of four different experiments.

b i o c h e m i c a l p h a r m a c o l o g y 7 5 ( 2 0 0 8 ) 1 3 7 0 – 1 3 8 0
1375
Fig. 4 – Prostratin and phorbol-13 stearate (P-13S) activate the NF-kB and the MAPK pathways. (A) Effect of phorbol 13
monoesters and prostratin on NF-kB and AP-1-DNA binding activity. Jurkat LAT-GFP cells were incubated with compounds
1a–1h (10 mM) and PMA (50 ng/ml) for 60 min. Nuclear cell extracts were then assayed by EMSA using g-32P-labelled NF-kB
and AP-1 oligonucleotides (left panel). (B) The specificity of the NF-kB binding to DNA was demonstrated by supershift and
cold competition assays. Supershift analysis with the indicated antibodies is shown in lanes 3, 4 and control with pre-
immune serum (PIS) in lane 5. (C) Effect of phorbols 1a, 1d, 1e, 1f and 1g on IkBa degradation and JNK activation. Jurkat LAT-
GFP cells were incubated with the selected phorbols (1 and 10 mM) during 15 min and IkBa degradation, JNK 1 + 2 activation,
and the steady state levels of a-tubulin were analyzed using specific antibodies by Western blots. (D) Prostratin (1a) and
phorbol-13-stearate (P-13S, 1g) activated IKK kinase activity. Cells were treated with the indicated compounds (10 mM) for
5 min, and half of the cell lysates were subjected to Western blot for the detection of endogenous IkBa phosphorylation
(input). The other half of the lysates was used for IKKg immunoprecipitation and the kinase activity was monitored using
recombinant GST-IkBa (1–54) as substrate (IP KA). IKKg was also detected by immunoblots as a control of the
immunoprecipitated fraction.
antibodies indicated that the upper and lower bands con-
tained p65 homodimers and p65-p50 heterodimers, respec-
tively. The addition of pre-immune serum (PIS, lane 5) did not
modify NF-kB EMSA complexes. PMA, prostratin (1a), phorbol
13-hexanoate (1d), phorbol 13-decanoate (1e), phorbol 13-
miristate (1f) and phorbol 13-stearate (1g) (P-13S) induced a
strong NF-kB and AP-1 binding to DNA (Fig. 4A, compare lanes
2, 3, 7–10 to lane 1). In contrast, phorbol 13-acetate (1b),
phorbol 13-butyrate (1c) and phorbol 13-benzoate (1h) did not
induce DNA binding to these transcription factors (Fig. 4A,
compare lanes 4–6 to lane 1). Therefore, a close correlation
between the NF-kB and AP-1 binding activity and HIV-1
latency reactivation was observed for phorbol derivatives in
Jurkat-LAT-GFP cells.
degradation and JNK1 + 2 activation (Fig. 4C, compare lanes 4
and 6 to lane 1). In contrast, those biochemical pathways
were clearly activated when the cells were treated with 1 mM
concentration of the compounds 1e, 1f and 1g (Fig. 4C,
compare lanes 7, 9 and 11 to lane 1). Additionally, we found
that phorbol-12-hydroxy-13-acetate (1b) and phorbol 13-
butyrate (1c) were ineffective to induce the activation of the
signalling pathways investigated (data not shown). The
degradation of IkB proteins occurs after signal-induced
phosphorylation of IkB proteins at specific serine residues,
a reaction catalysed by IKKs present in the IkB kinase
complex (IKC) [27]. We therefore investigated if the induction
of IkBa degradation by prostratin (1a) and P-13S (1g) is also
due to the activation of kinase activity of the IKC. Endogenous
IKC was isolated by immunoprecipitation with an anti-IKKg
Ab, and its activity was analyzed by immune complex kinase
assays using recombinant IkBa protein as substrate. Fig. 4D
shows that stimulation of Jurkat-LAT-GFP cells with pros-
tratin (1a) and P-13S (1g) induced both the activation of IKC
and the phosphorylation of IkBa (Fig. 4D, compare lanes 2, 3 to
lane 1).
Next, we investigated the biochemical pathways involved
in NF-kB and AP-1 activation. Jurkat-LAT-GFP cells were
stimulated with compounds 1a, 1d, 1e, 1f and 1g (1 and
10 mM) for 15 min, and the phosphorylation of both the NF-kB
inhibitor IkBa, and the JNK were investigated by Western
blots using specific mAbs. Prostratin (1a) and compound 1d
only at the 10 mM concentration were able to induce IkBa

1376
b i o c h e m i c a l p h a r m a c o l o g y 7 5 ( 2 0 0 8 ) 1 3 7 0 – 1 3 8 0
3.4.
Activation of latent HIV-1 by prostratin (1a), phorbol
lanes 3–5 to lane 2). Rottlerin did not affect PMA-induced JNK
activation, but clearly prevented the activation of this MAPK
induced by prostratin (1a) and P-13S (1g) (Fig. 5B, compare lane
7 to lane 2). These results indicate that different phorbol-based
compounds activate distinct signal pathways to antagonise
HIV-1 latency, and that the contribution of the novel PKCd to
this activity is negligible.
13-hexanoate (1d) and P-13S (1g) requires activity of classical
PKC isoforms
Previous studies on prostratin-induced HIV-1 activation have
suggested that both classical [15] and novel PKCs [26] are
involved in this response. To further assess the ERK and PKC
dependence of prostratin (1a) and phorbol-13 monoesters (1d–
1g)-mediated antagonism of HIV latency, we pretreated
Jurkat-LAT-GFP cells with medium or the chemical inhibitors
It has been shown that the ERK pathway is activated by the
PKC pathway, and is involved in HIV-1 activation [28]. Thus,
we studied this pathway in Jurkat-LAT-GFP cells stimulated
with compounds 1a, 1d and 1g. Cells were preincubated with
the MEK inhibitor PD 98059, and then stimulated with the
various phorboids. PD 98059 partially inhibited GFP expression
induced by these compounds (1a, 1d, 1g), further implicating
an ERK-dependent signaling pathway in this response
(Fig. 5A). Accordingly, PD 98059 also prevented prostratin
(1a) and P-13S (1g)-induced ERK activation (Fig. 5B, compare
lane 6 to lane 2). Altogether, these results suggest that HIV-1
reactivation in response to non-tumor promoter phorbol
esters can be mediated by both the NF-kB and the ERK
pathways.
Go6976 (classical PKCs inhibitor), Go6850 (classical and novel
¨
¨
PKCs inhibitor), Go6983 (pan-PKC inhibitor), rottlerin (PKCd
¨
inhibitor) and PD 98059 (MEK inhibitor) at the indicated
concentrations. Go6976, Go6850 and Go6983 strongly inhibited
¨
¨
¨
GFP expression induced by Prostratin and the phorbol-13
monoesters 1d and 1g, further implicating a PKC-dependent
signaling step in this response (Fig. 5A). TNFa induction of the
latent HIV provirus was unaffected by any of these inhibitors,
thus excluding non-specific toxic effects of these drugs (data
not shown). In contrast, the PKCd inhibitor rottlerin did not
affect phorbol-induced GFP expression, ruling out the involve-
ment of this PKC in HIV-1 reactivation, at least in Jurkat-LAT-
GFP cells. Given the effects of both classical and novel PKCs
inhibitors on activation of latent HIV-1 by prostratin (1a),
phorbol 13-hexanoate (1d) and P-13S (1g), we next explored
the biochemical pathways involved in NF-kB and AP-1
activation by Western blots. As shown in Fig. 5B, Prostratin
(1a) and P-13S (1g)-induced IkBa degradation and JNK
phosphorylation were completely inhibited by the presence
of PKC inhibitors that target classical PKCs (Fig. 5B, compare
3.5.
Effect of PKC activators on the translocation and
activation of PKCa-GFP and PKCd-GFP
The translocation of PKC from the cytoplasm to subcellular
localizations is the hallmark for PKC activation [29], and their
isozyme-specific functions may result in part from differences
in subcellular localization [30,31]. There is a general agreement
that only phorbol esters inducing a sustained PKC transloca-
Fig. 5 – Prostratin, phorbol 13-hexanoate, and phorbol 13-stearate antagonize HIV-1 latency through a classical PKC-
dependent pathway. (A) Jurkat LAT-GFP cells were pretreated with the indicated inhibitors for 30 min at the indicated dose,
and then stimulated with the agonists (10 mM) for 6 h. The percentage of GFP+ cells was measured by flow cytometry.
Results are represented as percentage of activation W S.D. compared to cells treated with agonists in the absence of the
chemical inhibitors (100% activation). (B) The cells were pretreated with the inhibitors as indicated and then stimulated
with the agonists for 15 min. Total cell extracts were analyzed by Western blot with specific antibodies.

b i o c h e m i c a l p h a r m a c o l o g y 7 5 ( 2 0 0 8 ) 1 3 7 0 – 1 3 8 0
1377
Fig. 6 – Effects of PMA and P-13S on PKCa-GFP and PKCd-GFP expressed in CHO-K1 cells (A). Fluorescent images of CHO-K1
cells expressing PKCa-GFP and PKCd-GFP proteins treated at 37 8C for 10 min with either PMA (1 mM), prostratin (1 and
10 mM) or P13-S (1 and 10 mM). Three additional experiments gave similar results. (B) PMA and phorbol-13 stearate
phosphorylate PKCd. Jurkat LAT-GFP cells were incubated with either PMA or P-13S (10 mM) for the indicated period of time.
Total PKCd and phosphorylated PKCd (Thr505) were analyzed using specific antibodies by Western blots.
tion to the cell membrane are endowed with tumor promoter
activity. Thus, while the tumor promoter 12-deoxyphorbol 13-
tetradecanoate closely resembled PMA in its translocation
pattern, its antihyperplastic and antipromoting 12-deoxy-
phorbol 13-phenylacetate analog caused punctuate intracel-
lular accumulation of PKCd on the nuclear membrane [31]. In
order to explore the specificity of PKC a- and d-GFP transloca-
tion in response to prostratin (1a), P-13S (1g) and PMA, we used
CHO-K1 cells transiently transfected with constructs contain-
ing the full length open reading frame of either PKCa or PKC d
fused to the GFP gene. In the absence of stimuli, PKCa and d
were scattered in the cytoplasm. When a single dose of the
tumor promoter PMA was added, both PKC isotypes were
mainly translocated to the plasma membrane. Prostratin
induced a transient translocation of PKCa to the plasma
membrane and a perinuclear translocation of PKCd. Con-
3.6.
P-13S downregulates HIV receptors in peripheral
blood lymphocytes
The anti-HIV activity of phorbol esters has been linked to
downregulation of HIV-1 receptors thus interfering with the
viral entry step [9,16]. To analyze the effects of P-13S on
cellular surface in primary T cell surface antigens, CD3+ T cells
were isolated from healthy volunteer donors, treated with
either prostratin (10 mM) or P-13S (1 mM) and incubated for
24 h. The cells were then analyzed by FACS for CD4, CXCR4,
CD69 and CD25 expression. Fig. 7 demonstrates several-fold
CD4 and CXCR4 down-regulation in response to prostratin and
P-13S. Therefore P13-S is also at least ten fold more potent
than prostratin to downregulate HIV-1 receptors. Similar
results were found when the activation antigens CD69 and
CD25 were measured in peripheral T cells stimulated with
either prostratin or P-13S (Fig. 7). However, none of these
compounds showed mitogenic activity in human peripheral T
cells (data not shown).
versely, P-13S induced only
a little plasma membrane
translocation of PKCa-GFP, without apparently affecting the
cellular distribution of PKCd (Fig. 6A). Similar results were
found after 20 min of exposure to either PMA or P-13S (data not
shown). In general PKCs are also regulated by phosphorylation
and upon activation PKCd exhibits phosphorylation of the
activation loop (Thr-505) [32], which has been suggested to be
required for the activity of PKCd [33]. Thus, we studied by using
a specific antibody anti-phospho PKCd (Thr505) the effects of
P-13S on PKCd activation and phosphorylation, and we found
that both PMA and P-13S activated PKCd with a similar kinetic
(Fig. 6B, compare lanes 2–7 to lane 1). Altogether our results
strongly suggest that PMA, prostratin and P-13S differ
substantially in their biological activities (activation vs.
subcellular translocation).
4.
Discussion
The identification of potent natural or synthetic PKC agonists
lacking tumor-promoter and cellular proliferative activities
has opened new research avenues for the treatment of HIV-1.
HIV-1 latency represents a major hurdle to the complete
eradication of the HIV from patients under HAART regimens
[5,6]. One solution would be the reactivation of the latent
reservoirs in presence of HAART to prevent spreading of the
infection by the newly synthesized viruses [7]. In this sense, a

1378
b i o c h e m i c a l p h a r m a c o l o g y 7 5 ( 2 0 0 8 ) 1 3 7 0 – 1 3 8 0
Fig. 7 – Effects of Prostratin and P-13S on cell surface antigen expression in primary T cells. Purified T cells were treated with
either prostratin (10 mM) or P-13S (1 mM) for 24 h and the expression of CD4, CXCR4, CD69 and CD25 detected by flow
cytometry as described in Materials and Methods. The results are representative of three experiments.
recent study shows that the therapy with HIV protease
inhibitors (PIs) should be compatible with an inductive
therapy for latent reservoir reduction/elimination in associa-
tion with HAART regimens [34].
phorbol-13 monoesters was prepared and investigated for
their capacity to antagonize HIV-1 latency. As a cellular model
of HIV-1 latency we have used the Jurkat-LAT-GFP cells, which
contain
a full-length latent HIV provirus. This provirus
In this study we have synthesized novel 12-hydroxyphor-
bol-13 monoesters and explored the mechanism by which
these compounds and the non-tumor promoter PKC activa-
tors, prostratin and bryostatin, antagonized HIV-1 latency.
The capacity of prostratin to behave as an in vivo agent to purge
latent HIV-1 proviruses [35] has raised considerable interest,
owing to a potential clinical application in combination with
HAART to eradicate HIV-1 infection. However, relatively high
concentrations of prostratin are required to reactivate HIV-1
latency and it has been suggested that prostratin may have
negative side effects and therefore it is unlikely that high-
doses or long-term treatment would be well tolerated [26].
Since the PKC-dependent activation of the NF-kB and AP-1
pathways are well known mechanisms to reactivate HIV-1
latency, the identification of novel PKC activators lacking
tumor-promoter activity such as synthetic diacylglycerol
lactones [11] and 12-deoxyphorbol 13-phenylacetate [12] are
of special interest for the clinical development of drugs that
antagonize HIV-1 latency.
contains GFP instead of Nef and transcriptional activation of
the latent provirus can be easily detected by flow cytometry.
While phorbol 13-acetate, a compound differing from pros-
tratin only for the presence of a 12-hydroxyl, was totally
inactive, activity could be induced by increasing the length of
the acyl moiety, with the stearoyl analogue (P-13S) even
outperforming the potency of prostratin as an HIV-1 latency
antagonist by more than 10-fold. Activity was not due to a non-
specific buffering of the increased polarity associated to the
12-hydroxyl, since the benzoate analogue of prostratin was
inactive.
Since prostratin and phorbol 13-monoesters possess the
critical O-4 and O-20 hydroxyls essential for binding the C1
domain of PKC, it does not seem unreasonable to assume
these compounds can interact with PKC in the mainstream
way of phorboid ligands. However, the presence of a polar 12b-
hydroxyl in the upper moiety of the molecule might interfere
with the translocation of PKCs to specific cell compartment,
with an overall detrimental effect on the functional activity of
these enzymes, since a hydrophobic complex is required for
translocation to cell compartment other than plasma mem-
brane and the activation of PKC substrates [12]. In accordance
with this view, 12-deoxyphorbol derivatives bearing large
lypophilic chains at the position 13, like 12-deoxyphorbol-13-
tetradecanoate have been reported to be potent tumor
promoters that translocate PKCs to plasma membrane [31].
On the other hand, the corresponding compound from the
phorbol series (phorbol 13-myristate) (data not shown) as well
In addition to the above limitation of prostratin, its
development as a drug has been hampered by its very limited
availability by isolation. Prostratin is a derivative of 12-
deoxyphorbol. While this polyol is not more available than
prostratin itself, phorbol can be easily obtained in syntheti-
cally useful yields from a commercial source (Croton oil). We
have therefore started an investigation aimed at discovering if
prostratin-like properties could be induced in non-tumor
promoting phorbol derivatives. To this purpose, a series of

b i o c h e m i c a l p h a r m a c o l o g y 7 5 ( 2 0 0 8 ) 1 3 7 0 – 1 3 8 0
1379
as P-13S, though potent antagonists of HIV-1 latency through a
classical PKC-dependent pathway, failed to translocate PKCa
and d to the plasma membrane, and therefore may be devoid
of tumor-promoting activity. This observation is in full
accordance with the in vivo data reported in the pioneering
investigations by Hecker in this area [36], and clearly indicate
that the presence of a 12-hydroxyl, though compatible with
interaction with PKCs, is detrimental for the transfer of the
resulting complex to plasma membranes.
and activate Ras-GRP-1 through a PKC-independent pathway,
a hypothesis we are currently pursuing.
In summary, we have provided strong evidence that
phorbol 13-monoesters containing medium or long aliphatic
fatty acid chains are potent antagonists of HIV-1 latency,
acting by activation classical PKCs and downstream triggering
of the NF-kB pathway. These compounds do not translocate
PKC to the plasma membrane, seemingly lack tumor promoter
activity, downregulate the expression of the HIV-1 receptors
CD4 and CXCR4 and therefore represent new lead compounds
for the treatment of HIV-1 latency in combination with
HAART.
The discovery of a very promising HIV-1 purging activity of
P-13S prompted us to investigate the mechanistic details by
which this compound, in comparison to prostratin, reactivates
HIV-1 latency. In our cell model both prostratin and P-13S
could antagonize HIV-1 latency through a classical PKCs-
dependent pathway and irrespective of the activation of the
Acknowledgements
isoenzyme PKCd. Moreover, the classical PKC inhibitor Go6976
¨
also inhibited IkBa phosphorylation and degradation and JNK
activation induced by either prostratin or P-13S. Rottlerin
(which is more selective for PKCd) at the concentration tested
did not affect P-13S-induced HIV-1 reactivation and NF-kB
activation. Our results are in sharp contrast with those
reported by Williams et al. using a similar model of HIV-1
latency and the same chemical PKC inhibitors. These authors
The authors wish to thank Dr. P.M. Blumberg (NCI, Besthesda,
MD, USA) for the PKCa-GFP and PKCd-GFP plasmids. We thank
Ms. Carmen Cabrero-Doncel for her assistance with the
manuscript.
This work was supported by the Junta de Andalucı´a Grant
P06-CTS-01353 (E.M.), the FIS grant PI040526 (E.M.) and by the
ISCIII-RETIC RD06/006.
showed that Go6976 could not inhibit prostratin-induced HIV-
¨
1 reactivation, and similar results were obtained with the
specific PKCu inhibitor TER14867 suggesting that novel PKCs
other than PKCu are involved in prostratin signaling [26]. This
discrepancy might be due to the parental Jurkat cells used to
generate HIV-1 latently infected clones. In this sense, a critical
role for PKCa in the HIV-1 antagonizing effects of prostratin
has also been reported in Jurkat cells, suggesting that a
coordinate action between PKCa and PKCu is required for NF-
kB activation and HIV-1 reactivation by this compound [15].
Jurkat-LAT-GFP express very low levels of PKCe (data not
shown) and therefore our results suggest that in this cell line
activated with either prostratin or P-13S, the novel PKCs are
not critical for NF-kB activation and HIV-1 reactivation, but
they are absolutely required for JNK activation (at least PKCd).
Although PKCd signaling and JNK activation are not
required for P-13S-induced HIV-1 reactivation, we obtained
evidence that activation of the ERK pathway may also be
involved in prostratin signaling. In other latency models, it has
been shown that the ERK pathway is involved in PMA- and
cytokine activation of HIV-1 latency, while ERK cooperate with
NF-kB to fully reactivate HIV-1 latency [28]. We found that, in
addition to IKK activation, prostratin and P-13S also activated
the ERK 1 + 2, while the MEK inhibitor PD 98059 partially
prevented the HIV-1 antagonizing effects of these compounds.
These results might be related to the ability of P-13S to
relocalize RasGRP1 to the nuclear membrane and other
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