Epstein-Barr virus (EBV) is a ubiquitous human γ herpes virus that establishes a latent infection more than 90% of adults worldwide.1 Immunocompromised individuals such as those with AIDS or transplant recipients are at increased risk for developing aggressive EBV-associated lymphoproliferative diseases. EBV is associated with malignant diseases, including Burkitt's lymphoma,1, 2 nasopharyngeal carcinoma3, 4 and immunoblastic B cell lymphoma of immunosuppressed individuals. Infection of primary B cells with EBV results in transformation with growth of the cells in tight clumps and immortalization of the cells. These immortalized B cells have an immunoblastic morphology and express each of the EBV-encoded small RNAs (EBERs), EBV nuclear antigen (EBNAs) and latent membrane proteins (LMPs).2, 5 EBERs have oncogenic potential through inhibition of PKR.6 EBNA-2 is a transactivator that up-regulates expression of cellular genes and LMPs. LMP-1 may mediate proliferative and survival effects not only in EBV-transformed B lymphocytes but also in these malignancies that occur long after primary infection. Many immunocompromised patients with EBV-associated immunoblastic lymphoma have tumors at extranodal sites such as the brain, lung, or gastrointestinal tract. The prognosis of EBV-associated lymphomas is very poor for patients with irreversible immunosuppression and treatment options are limited.
Despite the diversity in clinical manifestations of hematopoietic malignancies, strong and constitutive nuclear factor-kappaB (NF-κB) activation was reported to be a unique and common characteristic of malignant cells.7, 8 In resting cells, NF-κB is sequestered as an inactive precursor by association with inhibitory IκBs in the cytoplasm. On stimulation, IκBs are rapidly phosphorylated, ubiquitinated and degraded by a proteasome-dependent pathway allowing active NF-κB to translocate into the nucleus where it can activate the expression of a number of genes.9 LMP-1 is an oncoprotein that constitutively activates NF-κB to induce B cell proliferation.7 Lymphoblastoid cell lines (LCLs) express high level of the antiapoptotic proteins BCL-2, BCL-xL, c-IAP1, Bfl-1 and c-FLIP the targets of NF-κB.10, 11 NF-κB activation has been connected with multiple processes of oncogenesis including control of apoptosis, cell-cycle, differentiation and cell migration,9 and therefore, inhibition of NF-κB was suggested to be a useful strategy for cancer therapy.12–20 It has been also reported that inhibition of NF-κB in EBV-associated lymphomas results in induction of apoptosis.21 Therefore, targeting the NF-κB pathway and inhibition of NF-κB activity is a logical strategy for treating EBV-associated lymphomas.
Ritonavir, a human immunodeficiency virus type 1(HIV-1) protease inhibitor, has been successfully used in clinical treatments of HIV infection, with patients exhibiting a marked decrease in HIV viral load and a subsequent increase in CD4+ T-cell counts.22–25 Evidence of other effects by ritonavir on cellular proteases, such as the cysteine proteases cathepsin D and E, was presented in the drug's original description, albeit at concentrations >500-fold above the concentration required for inhibition of HIV protease.26 Protease inhibitors have also been shown to directly affect cell metabolism, interfere with host or fungal proteases and block T-cell activation and dendritic-cell function.27, 28 Ritonavir has been shown to inhibit the chymotrypsin-like activity of the 20S proteasome, and it activates the chymotrypsin-like activity of the 26S proteasome conversely.27, 29, 30 Ritonavir also has been reported to inhibit the transactivation of NF-κB induced by activators such as TNFα, HIV-1 Tat protein and the human herpesvirus 8 protein ORF74.31 It is possible that inhibition of NF-κB activation by ritonavir is linked to additional pathways other than inhibition of proteasome.31 Protease inhibitors also have been shown to have direct antiangiogenic and antitumor activity.31, 32 Recently, we reported that ritonavir inhibits growth and infiltration of ATL cells through targeting NF-κB.20
In the present, we demonstrate that inhibition of NF-κB activity by ritonavir results in marked increase of apoptosis and induce cell-cycle arrest in EBV-positive lymphoblastoid B cells. We found that ritonavir also suppresses the expression of genes involved in antiapoptosis and cell-cycle progression. In addition, we established preclinical models using newly developed NOD/SCID/γcnull (NOG) mouse,16 a unique type of animal, lacking T-, B- and NK-cells to evaluate the efficacy of antitumor and anti-NF-κB therapies. In the murine model, ritonavir at the clinically relevant dose potently inhibited the growth and infiltration of EBV-transformed LCL cells.
Material and methods
Mice and cells
NOG mice were obtained from the Central Institute for Experimental Animals (Kawasaki, Japan). All mice were maintained under specific-pathogen-free conditions in the Animal Center of Tokyo Medical and Dental University (Tokyo, Japan). The Ethical Review Committee of the Institute approved the experimental protocol.
EBV-positive immortalized lymphoblatoid B-cell lines (LCL-Ya, LCL-Ao, LCL-Ka and LCL-Ku) were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (JRH Biosciences, Lenexa, KS), 100 U/ml penicillin, and 10 μg/ml streptomycin. Peripheral blood mononuclear cells (PBMCs) from 3 healthy volunteers were analyzed. Mononuclear cells were isolated by Ficoll-Paque density gradient centrifugation (GE Healthcare Biosciences, Uppsala, Sweden) and washed with PBS.
Cell viability assay
The effect of ritonavir on cell viability of LCLs and PBMCs from healthy donors was examined by the reagent, water-soluble tetrazolium (WST)-8 (Wako Chemicals, Osaka, Japan). Briefly, 2 × 105 cells were incubated in a 96-well microculture plate in the absence or presence of various concentrations of ritonavir. After 72 hr of culture, WST-8 (5 μl) was added for the last 4 hr of incubation and absorbance at 450 nm was measured using an automated microplate reader. Measurement of mitochondrial dehydrogenase cleavage of WST-8 to formazan dye provides an indication of the level of cell viability.
Cells were plated at a density of 3 × 105/ml in 60-mm tissue culture dishes. Twelve hours after plating, cells were exposed to 40 μM ritonavir for 24 h. Cell-cycle analysis was performed with the CycleTEST PLUS DNA reagent kit (Becton Dickinson, San Jose, CA). Briefly, cells were washed with a buffer solution containing sodium citrate, sucrose and dimethyl sulfoxide, suspended in a solution containing RNase A, and stained with 125 μg/ml propidium iodide (PI) for 10 min. Cell suspensions were analyzed on EPICS XL flow cytometer (Beckman Coulter, Fullerton, CA) using EXPO32 software. The cell population at each cell-cycle phase was determined with MultiCycle software (Beckman Coulter).
Assay for apoptosis
Cells were plated at a density of 3 × 105/ml in 60-mm tissue culture dishes. Twelve hours after plating, cells were exposed to ritonavir for 72 hr. Apoptosis was quantified by double staining with Annexin-V-Fluos (Roche Diagnostics, Mannheim, Germany) and PI (Backman Coulter) according to the instructions supplied by the manufacturer. Cells were analyzed on EPICS XL flow cytometer (Beckman Coulter) using EXPO32 software.
Western blot analysis
Treated cells were solubilized at 4°C in lysis buffer containing 62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 6% 2-mercaptoethanol and 0.01% bromophenol blue. Samples were subjected to electrophoresis on SDS-polyacrylamide gels followed by transfer to a polyvinylidine difluoride membrane and probing with the following specific antibodies: polyclonal antibodies against survivin, cyclin D2 (Santa Cruz Biotechnology, Santa Cruz, CA), Bcl-XL (BD Transduction Laboratories, San Jose, CA) and monoclonal antibodies against Bcl-2, p53, actin (NeoMarkers, Fremont, CA), PARP (BD Transduction Laboratories) and LMP-1 (DAKO, Kyoto, Japan). The protein bands recognized by the antibodies were visualized using the enhanced chemiluminescence system (Amersham, Piscataway, NJ).
Electrophoresis mobility shift assay (EMSA)
Cells were placed in culture at 1 × 106 cells/ml and examined for inhibition of NF-κB 24 hr after exposure to ritonavir. Nuclear proteins were extracted, and NF-κB binding activities to κB element were examined by EMSA as described previously.8 In brief, 5 μg of nuclear extracts were preincubated in a binding buffer containing 1 μg of poly (dI:dC) (Amersham Biosciences), followed by addition of 32P-labeled oligonucleotide probe containing NF-κB element (5 × 104 c.p.m.). These mixtures were incubated for 15 min at room temperature. The DNA-protein complexes were separated on a 4% polyacrylamide gel and visualized by autoradiography. To examine the specificity of the NF-κB element probe, unlabeled competitor oligonucleotides were preincubated with nuclear extracts for 15 min before incubation with probes. The probe or competitors used were prepared by annealing the sense and antisense synthetic oligonucleotides as follows: a typical NF-κB element from the IL-2Rα gene, 5′-gatcCGGCAGGGGAATCTCCCTCTC-3′; and AP-1 element of the IL-8 gene, 5′-gactGTGATGACTCAGGTT-3′. Underlined sequences represent the NF-κB or AP-1 binding site. To identify NF-κB protein in the DNA protein complex revealed by EMSA, we used antibodies specific for various NF-κB proteins, including p50, p65, c-Rel, RelB and p52 (Santa Cruz Biotechnology), to elicit a supershift DNA protein complex formation. These antibodies were incubated with the nuclear extracts for 45 min at room temperature before incubation with radiolabeled probes.
Inoculation of EBV-positive immortalized LCLs and collection of samples
LCL Cells [LCL-Ya, LCL-Ao, LCL-Ka and LCL-Ku] were washed twice with serum-free RPMI-1640 medium and resuspended in same medium. Mice were anaesthetized with ether and cells were inoculated subcutaneously (sc) in the postauricular region of NOG mice at a dose of 1 × 107 cells per mouse. All mice were sacrificed 3 weeks after inoculation with lymphoma cells. We measured tumor size 3 weeks after inoculation. Tissues and various organs of mice were collected and fixed with Streck Tissue Fixative, then processed to paraffin wax-embedded sections for staining with hematoxylin and eosin (HE) and immunostaining.
PCR primer and conditions
Detection of the BamHI W repeat region of the EBV genome was performed using 100 ng of genomic DNA extracted from LCLs as follows. LCLs were lysed with genomic DNA extraction buffer (100 mM Tris-HCl pH8.0, 5 mM EDTA, 0.2% SDS, 200 mM NaCl and 200 μg/mL proteinase K) and the lysate was incubated at 50°C for 3 hr. After phenol-chloroform extraction, genomic DNA was purified by ethanol precipitation procedure. A 121-bp fragment of the EBV W repeat region was amplified by the forward primer 5′-CGCATAATGGCGGACCTAG-3′ and reverse primer 5′-CAAACAAGCCCACTCCCC-3′ in a 25 μl reaction mixture comprising 1 × AmpliTaq Gold buffer, 3.5 mM MgCl2, 200 μM dNTP, 300 nM primers, 200 nM probe and 0.025 U/μl AmpliTaq Gold. The PCR cycle conditions were as follows: a DNA denaturation and polymerase activation step of 10 min at 95°C and then 40 cycles of amplification (95°C for 15 sec, 60°C for 1 min). PCR products were separated by electrophoresis on agarose gels, stained with ethidium bromide and visualized by UV-light.
Treatment of tumor-bearing mice with ritonavir
Ritonavir was obtained from Abbott Labs, North Chicago, IL. LCL-Ku cells (1 × 107) were inoculated s.c. in the post-auricular region of NOG mice. The drug was administered s.c. into the tumor cells inoculated site of mice at doses of 30 mg/kg/day, beginning on day 0 for 3 weeks. The control mice received RPMI-1640 (200 μl) simultaneously. In other experiments, ritonavir or RPMI-1640 was also administered intraperitoneally into mice as the same doses stated above, beginning on day 4 for 18 days.
In situ hybridization
EBERs were detected by in situ hybridization using fluorescein isothiocyanate (FITC)-conjugated EBER PNA (peptide nucleic acid)-probe (DAKO). Briefly, formalin-fixed, paraffin-embedded tissue sections of tumor and various organs were deparaffinized and hydrated in xylenes and graded alcohol series, then rinsed for 5 min in PBS. Deparaffinized samples were incubated with 10 ng/μl of proteinase K for 20 min at 37°C followed by washing, and then incubated with 0.3% methanol for 30 min at room temperature. After washing in PBS, the sections were hybridized with FITC-conjugated EBER-PNA probe in the hybridization solution for 90 min at 56°C. The slides were washed twice in 0.2 × SSC for 20 min at 56°C, and incubated with anti-FITC monoclonal antibody (DAKO) for 45 min at 37°C. Followed by washing, the slides were incubated with horse-radish peroxidase-conjugated polymer reagent (Envision, DAKO) for 30 min at room temperature. Positive staining was visualized after incubation of these samples with a mixture of 0.05% 3,3′-diaminobenzidine tetrahydrochloride in 50 mM Tris-HCl buffer pH7.6 and 0.01% hydrogen peroxide for 5 min. The samples were counterstained with hematoxlin for 2 min, hydrated completely, cleaned in xylene and then mounted. The samples were visualized and photographed under light microscopy (BX41 and DP70; Olympus, Tokyo, Japan).
Ritonavir induces cell-cycle arrest and apoptosis of LCLs
Ritonavir was examined for its effect on cell-cycle distribution of EBV-immortalized LCLs (Fig. 1a). Ritonavir effectively inhibited cell-cycle progression, as evidenced by increased proportion of the cells in G1 phase of LCL-Ao, LCL-Ka, LCL-Ku and LCL-Ya (LCL-Ao: from 60.1% to 67.1%; LCL-Ka: from 61.2% to 69.9%; LCL-Ku: from 52.6% to 57.3%; and LCL-Ya: from 67.4% to 72.1%). These results indicated that ritonavir induced cell-cycle arrest at G1-phase. The weak accumulation of cells in G1 phase by ritonavir suggests that it might rather be an apoptosis inducer than a cell growth inhibitor.
Furthermore, we evaluated the effect of ritonavir on the cell viability of LCLs and PBMCs from healthy individuals (Fig. 1d). Ritonavir effectively reduced the survival of LCLs (LCL-Ao, LCL-Ka and LCL-Ku) as measured by WST-8 on the third day of culture in a dose-dependent manner. In contrast, ritonavir hardly affected the survival of PBMCs from healthy volunteers.
The effect of ritonavir on apoptosis was examined by the Annexin-V and PI method. Annexin-V binds to the cells that express phosphatidylserine on the outer layer of the cell membrane, a characteristic feature of cells entering apoptosis. Early apoptotic cells were stained with Annexin V but not with PI. Late apoptotic and necrotic cells were stained with both fluorescent. Ritonavir induced increased proportion of cells positive for Annexin-V and negative for PI in all cell lines (LCL-Ao: from 14.8% to 62.3%; LCL-Ka: from 13.8% to 37.8%; LCL-Ku: from 9.9% to 37.9% and LCL-Ya: from 10.1% to 66.9%) (Fig. 1b). Ritonavir also induced dose-dependent increasing of Annexin-V positive and PI negative cells in LCL-Ku cells (Fig. 1c), indicating increasing apoptosis of ritonavir-treated cells.
Ritonavir down-regulates the expression of the cell-cycle- and apoptosis-associated genes
The antiproliferative and proapoptotic effects of ritonavir were explored by examining the levels of intracellular regulators of cell-cycle and apoptosis after exposure to ritonavir (Fig. 2). Ritonavir down-regulated the levels of survivin and cyclin D2 in EBV-immortalized B-cell lines. We also observed increased cleavage of PARP in these cells. However, ritonavir did not modulate the other regulators of cell-cycle and apoptosis such as Bcl-XL, Bcl-2 and p53. Ritonavir had no effect on the expression of viral proteins such as LMP-1, suggesting that ritonavir may induce cell-cycle arrest and apoptosis by down-regulating the levels of survivin and cyclin D2 without reducing the virus levels in the cells.
Ritonavir suppresses constitutive NF-κB expressed by EBV-transformed LCLs
To examine the effect of ritonavir on NF-κB DNA binding, EMSA was performed. EBV-immortalized B-cell lines were incubated with or without 40 μM ritonavir for 24 h, and nuclear extracts were prepared and examined for NF-κB by EMSA. Down-regulation of NF-κB occurred in all cell lines (Fig. 3a, upper panels). Inhibition appeared specific to NF-κB, because no significant change in binding activity of AP-1 was observed after treatment of cells with ritonavir (Fig. 3a, lower panels). Also, the observed protein/DNA binding was specific for NF-κB, because the binding was effectively competed and abrogated by excess unlabeled NF-κB oligonucleotide but not by mutant NF-κB or AP-1 oligonucleotide (Fig. 3b). LCL-Ku cell extract without ritonavir treatment contained p50, p65 and Rel B proteins in the NF-κB complex (Fig. 3b, upper panel), and ritonavir did not affect components of the NF-κB complex (Fig. 3b, lower panel).
Efficient engraftment and infiltration of EBV-transformed LCLs in NOG mice
EBV-immortalized LCLs (LCL-Ya, LCL-Ao, LCL-Ka and LCL-Ku) were inoculated s.c. in the post-auricular region of NOG mice (Fig. 4 and Table I). Mice inoculated with LCL cells (LCL-Ya, LCL-Ao, LCL-Ka and LCL-Ku) produced a visible tumor within 3 weeks in all NOG mice. LCL-Ku cell was very efficient in the formation of a large tumor (Fig. 4a), as well as development of clinical signs of near-death, such as pilorection, weight loss and cachexia in mice at the time point of sacrifice. The average tumor size (LCL-Ya, LCL-Ao, LCL-Ka and LCL-Ku) in NOG mice inoculated s.c. with lymphoma cells was shown in Figure 4a. To test whether tumors maintain original histomorphology and expression patterns of tumor markers in NOG, we performed HE and in situ hybridization for EBER of normal mice spleen not receiving tumor cells and tumor tissues obtained from mice inoculated with LCLs. Histological analysis revealed that morphologically immunoblastic cells with large nucleus, clear nuclear membrane and broad cytoplasm expressed EBER, whereas EBER was not detected in spleen tissue collected from mice not receiving tumor cells, suggesting that in vivo tumor cells preserved well morphology as well as expressed viral gene EBER (Fig. 4b). Tumor cells from mice inoculated with EBV-immortalized B-cell lines were positive for DNA of EBV by PCR (Fig. 4c). These results showed that EBV-immortalized B-cell lines inoculated s.c. into the postauricular region of NOG mice were able to produce a visible tumor very efficiently. To assess the tissue distribution of lymphoma cells, we carried out histological examinations of the different organs of NOG mice after inoculation of the cells. Proliferation and infiltration of tumor cells were found not only in primary tumor tissues but also in spleen and to a lesser extent in liver and lung of NOG mice inoculated with tumor cells (Table I). HE and in situ hybridization staining for EBER showed a degree of infiltration of tumor cells at the site of inoculation and various organs with lymphoma cells (Fig. 4d). Interestingly, LCL-Ku cells appeared to infiltrate in various organs of mice more aggressively and massively than other cells. This extremely rapid tumor formation and infiltration in all mice is one of the hallmarks of our clinically relevant animal model without change of histomorphology or tumor marker expression.
Table I. In Vivo Charateristics of EBV-Positive Lymphoblastoid B Cells in NOG Mice
Mice were inoculated with 1 × 107 cells per mouse.
Number of animals in which tumor developed.
Organ-infiltration was examined by histological analysis.−, no infiltration; +, slight infiltration; ++, marked infiltration; +++, massive infiltration.
Ritonavir suppresses the LCLs growth and infiltration in vivo
To determine the effect of ritonavir on tumor growth and infiltration, we injected LCL-Ku cells (1 × 107) s.c. into the postauricular region of NOG mice. Mice were treated with either RPMI-1640 (as control) or ritonavir (30 mg/kg/day), beginning on either day 0 or day 4. A significant decrease in the size of tumors in mice treated with ritonavir was demonstrated when compared with controls 3 weeks after the injection of tumor cells (Fig. 5a). Gross appearance of the mice treated by ritonavir showed apparent reduction of the tumor mass at 3 weeks after inoculation of tumor cells (Fig. 5b). Ritonavir also inhibited the size and growth of established tumors (Fig. 5c and 5d). Ritonavir at this treatment dosage (30 mg/kg/day for 3 weeks) is well tolerated without adverse findings such as standing of hair, weight loss and cachexia of treated mice, all of which are signs of near death. Clinical evaluation of organ invasion 3 weeks after injection of tumor cells showed that ritonavir treatment inhibited their infiltration into spleen (Fig. 5e). In contrast, all control mice showed infiltration with tumor cells into spleen. Organ infiltration of lymphoma cells were analyzed and evaluated by HE and in situ hybridization of EBER. Together, these data indicate that ritonavir significantly inhibits lymphoma cell growth and infiltration in various organs of NOG mice (Fig. 5).These results suggest that ritonavir contributes to the reduction of the tumor growth and inhibits the organ infiltration in the mice through targeting the constitutive NF-κB activity.
EBV-positive malignancies in immunocompromised patients are associated with high mortality and reduce overall survival period. The various chemotherapies so far developed have not increased significantly the survival of patients with EBV-associated malignancies in immunocompromised patients. Given disappointing results using conventional chemotherapy, new treatment strategies that specially target EBV-transformed cells are need.1, 2 LMP-1 is an oncogene that constitutively activates NF-κB to induce B cell proliferation.7 It has been previously reported that suppression of high NF-κB activity inhibited cell growth and induced apoptosis of cancer cells as well as EBV-transformed cells both in vitro and in vivo.12–20, 33 Ritonavir is cytotoxic for different types of malignant cells in vitro through affecting proteasomal proteolysis, although concentrations necessary to show the invitro effect exceed the achievable therapeutic drugs level.27, 29, 30 It may affect the stabilization of p21, p27 and p53 proteins. Recently, ritonavir has been shown to inhibit NF-κB activity and induce the apoptosis of ATL cells.20 This led us to investigate whether this drug exhibits anti-tumor effects against EBV-transformed cells in vitro and in our preclinical murine model. In the present study, we established a unique murine model that presents aggressive features concerning cell growth and infiltration in SCID mice within 3 weeks. Thus, it represents a novel model to evaluate tissue toxicity and the efficacy of therapeutic agents directed toward the treatment of EBV-associated lymphoproliferative diseases.
The blood-plasma ritonavir concentrations obtained in the therapy of HIV-infection are between 5 to 15 μM,34 but much higher maximal concentrations (up to 46 μM) have been demonstrated in individual patients.35 In the present study, we used the concentration of ritonavir for doing in vitro experiments from 0 to 40 μM and in vivo 30 mg/kg/day used for treatment of AIDS patients. Constitutive and strong NF-κB activation was reported to be a characteristic of LCL and important for LCL growth and survival.7 Our results indicate that inhibition of NF-κB activity by ritonavir reduced cell growth and induced apoptosis of these cells. This is consistent with down-modulation of NF-κB regulated genes such as antiapoptotic and cell-cycle related genes. Our murine model clearly indicate that 30 mg/kg/day of ritonavir (the same dose used clinically for treating HIV/AIDS patients) significantly inhibits EBV-transformed cell growth and infiltration into various organs of NOG mice. The plasma exposure produced by this dose in mice is only approximately one-half of the plasma exposure observed with the licensed dose of ritonavir in human (600 mg BID). In our murine model, ritonavir at this treatment dosage is well tolerated without severe adverse effects observed in the mice during the treatment period. These data strongly suggest that the HIV protease inhibitor, ritonavir, is a promising antitumor agent against EBV-transformed cells and could be used clinically for treatment of EBV-associated malignancies. These results suggest that anti-tumor activity of ritonavir correlates with suppression of NF-κB activity.
In summary, we have established a novel NOG EBV-associated lymphoma model that presents features similar to patients with EBV-infection in immunocompromised patients. These results also indicate that the HIV protease inhibitor, ritonavir, showed antitumor and anti-NF-κB activity against EBV-transformed cells. Finally, our results strongly suggest that NF-κB serves as a potential molecular target to treat EBV-associated malignancies, and that ritonavir might be used clinically as a single compound or in combination with the reducing dose of chemotherapeutic agents for treatment of patients with life-threatening EBV-associated lymphoproliferative diseases and AIDS-associated lymphomas.
We thank D. Kempf and T. Yamada of Abbott Laboratories, S. Ichinose of Instrumental Analysis Research Center and S. Endo of Animal Research Center, Tokyo Medical and Dental University for their advice and assistance with the experiments. We also thank Y. Sato of the National Institute of Infectious Diseases for her excellent technical assistance.