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NEOPLASIA
From the Institute of Human Virology, University of
Maryland Biotechnology Institute; Department of Microbiology and
Immunology, University of Maryland; and Department of Biology, Morgan
State University, Baltimore, MD.
Treatment of patients with human immunodeficiency virus (HIV)
protease inhibitors such as ritonavir can result in increases in
CD4+ T-cell counts that are independent of a reduction in
HIV-1 viral load. This lack of correlation between the 2 has led to the
identification of additional effects of ritonavir that potentially
alter HIV disease pathogenesis. Our previous studies indicated that
ritonavir directly affects immune cell activation, proliferation, and
susceptibility to apoptosis. We show here that ritonavir inhibited the
activation and proliferation of primary endothelial cells and decreased
the production of tumor necrosis factor Protease inhibitors (PIs) such as ritonavir have
been successfully used in the clinical treatment of human
immunodeficiency virus 1 (HIV-1) infection, with patients exhibiting a
marked decrease in HIV viral load and a subsequent increase in
CD4+ T-cell counts. The extent of the clinical benefits
achieved with HIV PI therapy thus far exceeds that of any other kind of
antiretroviral therapy.1-4 However, clinical studies in
some HIV patients treated with PIs have yielded unexpected results.
Increases in CD4+ T-cell counts have sometimes been noted
even though viral loads remained persistently high due to the presence
of PI-resistant viral strains.5,6 In past studies, we have
demonstrated that the HIV PI ritonavir improves immune cell viability
by decreasing cell susceptibility to apoptosis in HIV-free in vitro
systems.7-9 These observations provided some insight into
a possible explanation for the dissociation of CD4+ T-cell
counts from HIV viral loads in patients treated with ritonavir. Evidence of alternative effects by ritonavir on cellular proteases, such as the cysteine proteases cathepsin D and E, was presented in the
drug's original description.10 More recently, ritonavir has been shown to inhibit the chymotrypsinlike activity of the 20S
proteasome,11,12 although concentrations necessary to
demonstrate the in vitro effect exceeded the achievable therapeutic
drug levels.
Kaposi sarcoma (KS), a neoplasm of endothelial cell origin, is the most
common malignancy associated with HIV infection.21 Clinical treatments for the acquired immunodeficiency syndrome (AIDS)-related malignancy KS have generally revolved around aggressive chemotherapy and highly active antiretroviral therapies in the belief that decreasing the HIV viral load would eliminate a main contributory factor.2,13,14 The HIV PI ritonavir appeared to be particularly effective against KS.15-18 Similar to
the improvement sometimes seen in CD4+ T-cell counts in the
absence of reduction in viral loads, amelioration of KS lesions can
occur rapidly without a concomitant reduction in HIV-1
titers.16-18 With these findings in mind, we suspected that ritonavir might have direct inhibitory effects on KS development, independent of its effects on the HIV protease.
Inflammatory cytokines, chemokines, and angiogenic factors cooperate in
the induction of endothelial cell activation and are important for
tumor neovascularization and KS development.19-21 Specifically, interferon These observations suggest that cytokines and growth factors produced
by activated immune cells and endothelial cells predispose an
individual to KS development and progression by providing the initial
signals required for KS lesion formation via autocrine and paracrine
mechanisms. In addition, human herpesvirus 8 (HHV-8) has been shown to
be a causal factor for KS development and appears to contribute to the
inflammatory and angiogenic microenvironment.25,28,29 The
Tat protein of HIV-1, shown to be a contributing factor for KS growth
in vitro, also deregulates the expression of inflammatory and
angiogenic factors.30,31
In this study, we investigated whether ritonavir directly affects
angiogenesis-promoting factors and KS by assessing endothelial cell and
KS cell survival, activation, and proliferation. We also studied the
effects of ritonavir on tumor formation by KS cells in a mouse
xenotransplantation model. We evaluated how the HIV PI alters the
expression of proinflammatory cytokines, growth factors, and adhesion
molecules that may contribute to the development of KS. In addition,
because nuclear factor- Cell cultures
Monocytes were isolated from PBMCs using counterflow-centrifugal
elutriation using a Beckman JE-5.0 rotor and a type A chamber (Beckman
Instruments, Palo Alto, CA). Purity of the separated monocyte fraction
was approximately 95% as determined by cytomorphology in Pappenheim
stain and approximately 96% as determined by the expression of CD14
antigen (LeuM3; Becton Dickinson, Mountain View, CA) and measured by
flow cytometry. Elutriated monocytes were cultured in medium (Cellgrow
40-101-LV, Mediatech, Herndon, VA) at a density of
3 × 106 cells/mL. Stimulation with lipopolysaccharide
(LPS; Sigma Chemicals, St Louis, MO) was used in some experiments for
inducing NF- The PBMCs were cultured in RPMI-1640, containing 10% FCS, antibiotics,
and IL-2 (Life Technologies) at an initial concentration of
6 × 105 cells/mL. Inhibition by ritonavir of PBMC
activation as induced by anti-CD3 monoclonal antibodies (no. 30110D,
0.5 µg/mL, Pharmingen, San Diego, CA) was measured over time as
indicated. Recombinant TNF- Caspase assays
To determine the direct effects of ritonavir on activated caspases-1, -3, and -8, PBMCs were stimulated with anti-CD3 monoclonal antibody (mAb) and subsequently primed to apoptosis using CD95-agonistic mAbs (clone CH-11; 0.5 mg/L; Kamyia, San Francisco, CA). Cell lysates were prepared as the source for activated caspases. Specific inhibitors of caspases (zVAD-FMK for caspase-1, DEVD-FMK for caspase-3, and IETD-FMK for caspase-8, Enzyme System Products, Livermore, CA) and respective specific substrates (WEHD-AMC, DEVD-AMC, and IETD-AMC; Enzyme System Products) were used to evaluate the effect of ritonavir on the enzymatic reactions. Adhesion assays A shear force assay, as previously described,40 was adapted to measure adhesion of leukocytes to endothelial cells. HUVECs (passage 5) were grown to form a monolayer in 96-well plates. Wells were treated with indicated drugs for 2 hours and subsequently activated with TNF- (5 ng/mL) for 4 hours. Jurkat, HL60, and U937
cells (American Type Culture Collection, Rockville, MD) were labeled
with Calcein AM (0.1 µM; Molecular Probes, Eugene, OR) for 30 minutes
in separate reactions, and then allowed to adhere to HUVECs for 2 hours. Nonadherent cells were removed by minimal shear force in 0.85%
NaCl solution. Fluorescence, which is proportional to cell number, was
measured using a Victor-2 fluorometer (Wallac). Standard
curves with known numbers of cells were generated to ensure a linear
relation between fluorescence and cell number. SDs were generated from
3 separate wells in 4 experiments performed with Jurkat cells and from
8 separate wells for each treatment condition in experiments with HL60
and U937 cells.
Flow cytometry analysis For quantitative and qualitative determination of cell surface adhesion molecules, HUVEC cultures (passage 5) were treated with TNF (10 ng/mL) in presence or absence of 20 µmol/L ritonavir for 4 hours. Cells were detached with cell dissociation solution, containing 5 mmol/L EDTA (Life Technologies). Aliquots of 0.5 × 106 cells were stained with phycoerythrin (PE)-conjugated antibodies for VCAM-1, ICAM-1, and E-selectin, as well as with appropriate isotype control antibodies (Pharmingen) and then fixed in 0.5 mL 2% paraformaldehyde. Samples were analyzed using a Becton Dickinson FACSCalibur flow cytometer and Cell Quest Analysis software.Tumor studies in mice Male immunodeficient beige-nude-xid (BNX) mice (obtained from Jackson Laboratory, Bar Harbor, ME) at the age of 6 to 10 weeks (with no more than a 2-week spread in a single experiment) were used for tumor formation and inhibition experiments with in vivo-selected KSIMM cells. Animals were housed in microisolator cages under sterile conditions and observed for at least 1 week to ensure proper health before study initiation. Lighting, temperature, and humidity were controlled centrally and recorded daily. Animals were injected with 7 × 106 KS cells (suspended in 300 µL phosphate-buffered saline [PBS]) subcutaneously into the posterior flanks. Treatment was started approximately 6 days after cell injections, when the tumors became visible and palpable. Randomized groups of mice (n = 10) were treated with ritonavir (30 mg/kg) or PBS by intraperitoneal injections daily for 15 days. Tumor growth and progression were monitored by biweekly measurements of tumors with calipers.Assays for detection of NF- Transfections and luciferase reporter gene assays.
To assess drug effects on NF- Electrophoretic mobility shift assays.
Electrophoretic mobility shift assays (EMSAs) were performed with
nuclear extracts from elutriated human monocytes to measure effects of
ritonavir on NF- Immunoblot analysis.
To measure the effect of ritonavir on activation-induced degradation of
I
Ritonavir reduces the proliferation of primary endothelial cells and induces apoptosis in KS cells in vitro Because we had previously observed that ritonavir inhibited the activation, proliferation, and apoptotic cell death of primary human immune cells, but induced cell death in immortalized cell lines,35 we compared effects of ritonavir treatment on the KS cell line KSIMM36 and on primary HUVECs. Cells were treated for 24 hours with different concentrations of ritonavir (range, 0-50 µmol/L) and viability was assessed by a standardized WST-1 metabolic assay and correlated to trypan blue exclusion staining. The number of viable primary endothelial cells (HUVECs) counted was approximately 25% lower at the highest concentration (50 µmol/L) after 24 hours of treatment when compared with untreated cultures (Figure 1A). Because no significant cell death was detectable in these cultures, this decrease suggests that ritonavir inhibits HUVEC proliferation. In contrast, the number of viable KSIMM cells fell by about 60%, most likely a due to both an inhibition of cell proliferation and increased cell death.
To determine if the cell death induced by ritonavir was due to increased apoptosis, we analyzed caspase-3 activation. This cysteine protease, which is activated during the early stages of apoptosis,37 cleaves and activates other caspases in a cascade leading to apoptosis. In a concentration window between 6 and 25 µmol/L, caspase-3 activity was increased in KSIMM but not in the nonproliferating HUVEC cultures (Figure 1B). These studies suggest that tumorigenic KS cells are more susceptible to cell death mediated by ritonavir than are their primary cell counterparts. Because previous studies showed that rapidly proliferating cells are
more susceptible to apoptosis than are slowly proliferating, quiescent,
or differentiated cells,38 we determined whether we could
alter the susceptibility of HUVECs to apoptosis by inducing activation
with TNF- In previous experiments, we reported that treatment of PBMC cultures
with ritonavir resulted in decreased caspase-1 expression and lower
caspase-3 activity.8,9 In the context of differential effects on endothelial cells, we were interested to know if ritonavir alters the enzymatic activity of caspases in a direct manner. Activation of caspases was induced in PBMC cultures by stimulation with
an anti-CD3 mAb and a CD95 agonistic mAb CH11. Cell lysates of these
cultures served as the source for activated caspase-1, -3, and -8, and
caspase-specific fluorogenic substrates and inhibitors were used to
measure the effect of ritonavir in enzymatic reactions. As depicted in
Figure 2, ritonavir has no direct
inhibitory effects on the cleavage of the tetrapeptide substrates that
are specific for each respective caspase-1, -3, and -8. Thus, the drug
alters cellular events prior to the induction of caspases, when
affecting cell activation and apoptosis pathways.
Ritonavir inhibits the production of KS-promoting inflammatory cytokines and growth factors To investigate how the inhibition of cell activation, proliferation, and the induction of apoptosis by the drug affected the expression of different inflammatory cytokines, we treated HUVEC cultures with ritonavir and measured the released cytokines by ELISA. After 48 hours, the level of IL-8 decreased in a dose-dependent manner from 172 ± 17 pg/mL (untreated) to 48 ± 10 pg/mL (20 µmol/L) in cultures that were kept at 70% to 90% cell confluence (data not shown). Decreases in TNF-, IL-6 (38%), and VEGF (30%) in HUVEC
culture supernatants after 24 hours of treatment (20 µmol/L) were
only detectable by ELISA when cells were grown at the highest density
possible without inducing significant cell death. We were not able to
determine changes in other cytokines and growth factors such as
IL-1, IL-1 , IL-12, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), and
INF- levels in these cultures (data not shown). As mentioned previously, KS lesions are composed of a mixed cellularity that includes the predominant KS spindle cells and a large immune cell infiltrate. PBMCs interact closely with KS spindle cells and
endothelial cells within KS lesions. Because PBMCs are believed to be
important sources of inflammatory factors that promote KS pathogenesis, we tested whether ritonavir could inhibit the induction of cytokine expression following activation through the T-cell receptor by anti-CD3
antibodies. The production of INF-, TNF-, IL-1, and IL-6 was
decreased in a dose- and time-dependent manner (Figure 3) without significant toxicity to cells,
because the inhibition was reversible after removal of the drug from
cultures (data not shown).
Ritonavir inhibits cell adhesion and expression of cell adhesion molecules Leukocyte adhesion to endothelial cells is an early step in inflammation and is believed to play an important role in the promotion of KS lesions. The adhesion of leukocytes to endothelial cells is mediated by endothelial cell adhesion molecules (ECAMs) such as ICAM-1, VCAM-1, and E-selectin. Expression of these molecules is highly dependent on NF- B and is induced by proinflammatory stimuli such as
TNF-.39 We investigated by flow cytometry the effects
of ritonavir treatment on the expression of these ECAMs in HUVECs,
stimulated with a low dose of TNF- (5 ng/mL). After 4 hours of treatment with ritonavir (20 µmol/L) the expression of
VCAM-1, ICAM-1, and E-selectin induced by TNF- was inhibited (Figure
4). This effect of ritonavir was
reversible, because 24 hours after the initial dose, the differences in
ECAM expression between treated and untreated cells declined markedly
(data not shown).
To determine if this decrease in adhesion molecule expression had
functional consequences, we carried out static binding assays in which
leukocyte cell adhesion to activated endothelial cell monolayers was
measured. Human T-lymphoid Jurkat cells, and monocytoid cell lines U937
and HL60 were fluorescently labeled with Calcein-AM and allowed to
adhere to endothelial cells. Nonadherent cells were removed by limited
shear force.40 Fluorescence of remaining cells was
measured and the cell number determined using a standard curve of
labeled cells. As controls, cyclosporin A (calcineurin inhibitor),
ketokonazole (cytochrome P450 inhibitor), and lactacystin (proteasome
inhibitor) were included at comparable concentrations. The inhibition
of leukocyte adhesion to endothelial cells by ritonavir was dose
dependent (Figure 5), with less than 60%
of adherent cells remaining at concentrations higher than 30 µmol/L.
Ritonavir inhibits KS tumor growth in a mouse model To determine if the in vitro effects on cells and tumor-promoting factors would translate into in vivo effects, a KS tumor xenotransplantation mouse model was used. The KSIMM cell line was adapted by in vivo selection and repeated passages to form tumors in immunodeficient BNX mice. Mice (n = 10/group) were inoculated with KSIMM cells subcutaneously (7 × 106 cells/mouse). After visible tumor formation (5-7 days), mice were given intraperitoneal injections of ritonavir, 30 mg/kg per day, for 15 days. The control group received PBS. The growth was monitored by measuring the tumor size. Treatment of mice with ritonavir inhibited KSIMM tumor development and growth significantly (Figure 6A). Figure 6B contains photographs that show an example of a treated and untreated tumor in BNX mice. No adverse effect of the treatment was noted on mice in a control group without tumors during a follow-up period of 3 months.
Ritonavir inhibits NF- B activity
as induced by the inflammatory cytokine TNF- and by the viral
proteins HIV-1 Tat and HHV-8 ORF74, which are all believed to
contribute to the pathogenesis of KS.41-45 NF-B
transcriptional activity was determined by luciferase-reporter
transient transfection assays, using a promoter construct with
consensus NF-B binding sites. Ritonavir inhibited the induction of
NF-B transcriptional activity by Tat, ORF74, and TNF- in a
dose-dependent manner at concentrations ranging from 0.3 to 30 µmol/L
(Figure 7A-C). To determine whether the
effect of ritonavir was specific for NF-B activity, we tested 2 control luciferase expression vectors that lack responsive elements,
but have constitutive expression of luciferase from a CMV and SV40
promoter. We found no differences in luciferase readings (data not
shown) between the treated and untreated samples. EMSAs were used to
confirm the validity of reporter expression assays. NF-B
translocation, measured in nuclear extracts from activated primary
human monocyte cultures, indicated a dose-dependent inhibition by
ritonavir of TNF-induced activation (Figure 7D).
To further understand the mechanism of the observed NF-
Clinical use of HIV PIs such as ritonavir in the prevention and treatment of HIV-associated KS has led to a drastic decline of this malignancy in the patient population with access to these drugs. Past studies have described the direct effects of ritonavir on immune cells, effects that are independent of HIV protease inhibition.9,35,75 Inhibition of cell activation correlated with a lower susceptibility to apoptosis in primary cells.8,9 In our present work, we demonstrate that ritonavir treatment directly inhibits KS-promoting factors and the growth of tumors from KS-derived cells xenotransplanted into immunodeficient mice. This is independent of any effects of ritonavir on HIV-1 or HHV-8, because KSIMM cells are not infected with either virus. Although this model of KS may not accurately represent KS lesions in patients, these mouse tumors are composed of KS cells that behave similarly and are dependent on the same cytokines and growth factors as KS spindle cells within lesions. To substantiate this unexpected finding, possible reasons for the antitumor effects were investigated and confirmed at the cellular and molecular biologic levels. Ritonavir decreased the production of the NF- Ritonavir treatment of endothelial cells inhibited ICAM-1, VCAM-1, and
E-selectin expression-suppressed leukocyte adhesion to these cells.
Adherence of HIV-infected or HHV-8-infected monocytes to activated
endothelial cells is thought to be an important contributor to KS
lesion development.27 KS lesions are a complex mixture of
different cell types, including the characteristic spindle cells, but
also fibroblasts, microvascular endothelial cells, dendritic cells, and
a prominent infiltrate of extravasated erythrocytes and
lymphocytes.25,29 During tissue inflammation, endothelial cells become adhesive for circulating blood cells and support their
transmigration into inflamed tissue. This process is influenced by
NF- Primary endothelial cells responded differently than KS cell lines to
ritonavir treatment in vitro at a concentration range comparable to
that achieved clinically. Ritonavir has been directly linked to
inhibition of the cell cycle at the G1/S
checkpoint.10,12 Possibly, the drug causes apoptosis in
rapidly dividing cells due to conflicting signals, similar to the
induction of apoptosis by c-myc in serum-deprived
fibroblasts.52,53 For example, a decrease in NF- Ritonavir inhibited NF- The exact role of NF- Recent in vitro studies have shown ritonavir to be an inhibitor of the
chymotrypsinlike activity of the 20S proteasome.11 It is
tempting to base a mechanistic explanation for the action of ritonavir
on KS on these findings, because the proteasome represents the cell's
major nonlysosomal tool to rapidly degrade and process proteins by
adenosine triphosphate/ubiquitin-dependent proteolysis. Proteins
involved in cell cycle progression and survival, such as p53, cyclins,
the cyclin-dependent kinase inhibitors p21 and p27, bax, and
bcl-2,65-69 are substrates of the proteasome. One of the
best known pathways regulated by the proteasome is that of NF- Deregulation of the cellular protease network has been shown to be responsible for aggressive clinical behavior in several human malignancies.73,74 One key group of proteases involved in invasion and metastases includes urokinase plasminogen activator, cathepsins B, D, and L, and matrix metalloproteinases.74 These proteases catalyze the degradation of the interstitial matrix and basement membrane, and enhanced activity can be used as prognostic marker for rapid progression in various cancers. It is currently not known whether ritonavir affects any of these proteases or whether the drug targets unknown cellular aspartyl proteases. In this paper, we demonstrate antineoplastic features of the HIV PI,
ritonavir, effective against KS both in vitro and in vivo. These
findings may help to explain why ritonavir therapy can result in the
clinical improvement of AIDS-KS without a concomitant decrease in
HIV viral load. It would be interesting to evaluate the drug's effect
in patients with non-AIDS-related KS. Our results justify further
studies to evaluate the utility of ritonavir for other tumors in
addition to chemotherapy and radiation, which are often hindered in
their effectiveness by resistance due to deregulated NF-
Submitted August 9, 2001; accepted January 9, 2002.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Frank F. Weichold, Morgan State University, 1700 E Coldspring La, Spencer Hall, Rm G12, Baltimore, MD 21251; e-mail: fweichol{at}morgan.edu.
1.
Collier AC, Coombs RW, Schoenfeld DA, et al.
Treatment of human immunodeficiency virus infection with saquinavir, zidovudine, and zalcitabine. AIDS Clinical Trials Group.
N Engl J Med.
1996;334:1011-1017 2. Collier AC. Efficacy of combination antiretroviral therapy. Adv Exp Med Biol. 1996;394:355-372[Medline] [Order article via Infotrieve]. 3. Collier AC, Coombs RW, Schoenfeld DA, Bassett R, Baruch A, Corey L. Combination therapy with zidovudine, didanosine and saquinavir. Antiviral Res. 1996;29:99 |
Top
Abstract
Introduction
80°C.
Oligonucleotide probes containing NF-
), compared to HUVECs (
), correlated
with increased caspase-3 activity. Ritonavir-treated HUVECs differed in
cell death from KSIMM (P = .015). Wilcoxon rank-sum tests
were performed for all comparisons of the means. (B) Cultures were
treated with ritonavir at indicated concentrations for 8 hours and
enzymatic activity of caspase-3 was determined in protein extracts from
cell lysates using a specific fluorogenic assay. Values represent
means ± SD of triplicate determinations in relative fluorescence
units (RFU). Caspase-3 values for treated HUVECs (
) and compared with HUVECs that were not activated by
TNF-
) for the respective
caspases as controls. Uninhibited reactions are depicted as open
circles (
). Values shown represent means of triplicate
determinations ± SD. For caspase-1, the ritonavir-treated group
did not differ significantly from the control group, but differed from
the inhibitor group (P = .0019). P values for
differences between inhibitor and ritonavir-treated groups for
caspase-3 and caspase-8 were .011 and 019, respectively. The
ritonavir-treated group did not differ significantly from the control
group for these as well. Wilcoxon rank-sum tests were performed for all
comparisons of the means. Results from one experiment are shown of 3 performed using cells from 3 different donors, with similar
results.
], 10 µmol/L
[
12%.
