|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Blood, 15 January 2006, Vol. 107, No. 2, pp. 716-724. Prepublished online as a Blood First Edition Paper on September 20, 2005; DOI 10.1182/blood-2005-02-0735.
NEOPLASIA Efficient intervention of growth and infiltration of primary adult T-cell leukemia cells by an HIV protease inhibitor, ritonavirFrom the Department of Molecular Virology, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan; the AIDS Research Center, National Institute of Infectious Diseases, Tokyo, Japan; the Division of Molecular Virology and Oncology, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan; the Department of Pathology, National Institute of Infectious Diseases, Tokyo, Japan; the Central Institute for Experimental Animals, Kanagawa, Japan; the Department of Bioorganic Medical Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan; the Department of Epidemiology and Preventive Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan; the Department of Hematology, Molecular Medicine Unit, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan; the Second Department of Internal Medicine, Miyazaki Medical College, University of Miyazaki, Miyazaki, Japan; the Department of Laboratory Medicine, Miyazaki Medical College, University of Miyazaki, Miyazaki, Japan; and the Division of Clinical Trials and Research, Breast Cancer Research and Treatment Program, Tokyo Metropolitan Komagome Hospital, Tokyo Medical Center for Cancer and Infectious Disease, Tokyo, Japan.
Adult T-cell leukemia (ATL), an aggressive malignancy of CD4+ T cells associated with human T-cell leukemia virus type I (HTLV-I) infection, carries a very poor prognosis because of the resistance of leukemic cells to any conventional regimen, including chemotherapy. We examined the effect of ritonavir, an HIV protease inhibitor, on HTLV-I-infected T-cell lines and primary ATL cells and found that it induced apoptosis and inhibited transcriptional activation of NF-  B in these cells. Furthermore, ritonavir inhibited expression of Bcl-xL, survivin, c-Myc, and cyclin D2, the targets of NF-B. In nonobese diabetic/severe combined immunodeficient (NOD/SCID)/ cnull (NOG) mice, ritonavir very efficiently prevented tumor growth and leukemic infiltration in various organs of NOG mice at the same dose used for treatment of patients with AIDS. Our data indicate that ritonavir has potent anti-NF-B and antitumor effects and might be clinically applicable for treatment of ATL. These results would provide a new concept and novel platform for new drug development of leukemia and solid cancer as well. (Blood. 2006;107:716-724)
Human T-cell leukemia virus type I (HTLV-I) is the causative agent of an aggressive form of CD4+ T-cell leukemia designated adult T-cell leukemia (ATL).1-3 ATL was first identified in Japan in 1977.4,5 Common findings for patients with ATL include enlargement of peripheral lymph nodes, hepatomegaly, splenomegaly, hypercalcemia, and skin lesions. At present, there is no accepted curative therapy for ATL, and patients progress to death with a median survival duration of 13 months in aggressive ATL.6 ATL has a poor prognosis mainly because of its resistance to conventional as well as high-dose chemotherapy.
ATL develops after a long period of latent infection. This long latency suggests that multiple genetic events, which accumulate in HTLV-I-infected cells, are involved in the development of ATL. However, the precise molecular mechanism of leukemogenesis and the development of ATL after HTLV-I infection are not fully elucidated. A unique viral gene Tax is considered to play a central role in HTLV-I-induced transformation, which is responsible for transactivation of the HTLV-I long terminal repeat,7,8 as well as numerous cellular genes involved in T-cell activation and growth, such as those encoding IL-2,9 and the
HTLV-I-infected cell lines derived from a leukemic cell clone and primary ATL cells failed to express significant amounts of Tax and other viral proteins, suggesting that the expression of viral proteins is not always necessary for leukemic proliferation at the late stage of the disease. However, HTLV-I-infected cell lines and leukemic cells from patients with ATL display constitutive NF-
Ritonavir, a human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PI), 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.25-28 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 greater than 500-fold above the concentration required for inhibition of HIV protease.29 PIs have also been shown to directly affect cell metabolism, interfere with host or fungal proteases, and block T-cell activation and dendritic cell function.30,31 Recently, 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.30,32,33 Ritonavir also has been reported to inhibit the transactivation of NF-
In this study, we investigated the antitumor effects of ritonavir on HTLV-I-infected cell lines and primary ATL cells. We found that ritonavir decreases NF-
Cell lines The T-cell leukemia cell line Jurkat, HTLV-I-infected T-cell lines MT-2,36 MT-4,37 C5/MJ,38 SLB-1,39 HUT-102,2 MT-1,40 and ED-40515(-),41 and bcr-abl+ leukemic cell line K562 were cultured in RPMI 1640 medium supplemented with 2% heat-inactivated fetal bovine serum (JRH Biosciences, Lenexa, KS), 100 U/mL penicillin, and 10 µg/mL streptomycin. MT-2, MT-4, C5/MJ, and SLB-1 are HTLV-I-transformed T-cell lines. MT-1 and ED-40515(-) are T-cell lines of leukemic cell origin established from patients with ATL. The clonal origin of HUT-102 is unclear. Human specimens Leukemic cells from 38 patients (8 patient samples for in vitro studies, 20 for establishment of ATL model, 10 for in vivo ritonavir studies) diagnosed as either acute type or chronic type were used in this study. The diagnosis of ATL was based on clinical features, hematologic findings, and the presence of anti-HTLV-I antibodies in the sera. Baseline characteristics for the patients who entered the study are shown in Table 1. Monoclonal HTLV-I provirus integration into the DNA of leukemia cells was confirmed by Southern blot hybridization in all cases (data not shown). All samples were collected after obtaining informed consent from patients. Peripheral blood mononuclear cells (PBMCs) from healthy volunteers and patients with ATL were purified by Ficoll-Hypaque gradient centrifugation (Amersham Biosciences, Uppsala, Sweden) and washed with RPMI 1640.
Growth inhibition assay The effect of ritonavir on cell growth was assayed by the WST-8 method as described previously.42 The WST-8 Cell Counting Kit was obtained from Wako Chemicals (Osaka, Japan). Briefly, 2 x 105 cells were incubated in a 96-well microculture plate in the absence or presence of various concentrations of ritonavir. After 72 hours of culture, 10 µL WST-8 solution was added, and the cells were incubated for another 2 hours. The number of surviving cells was measured with a microplate reader at a reference wavelength of 655 nm and test wavelength of 450 nm. Cell viability was determined as the percentage of the control (ie, absence of ritonavir). Assay for apoptosis Quantification of apoptosis was performed by immunostaining cells with Apo2.7, which specifically detects the 38-kDa mitochondrial membrane antigen 7A6 present only on the mitochondrial membrane of apoptotic cells, and so can be used as an early apoptotic marker in cells.43,44 Cells cultured for 72 hours in the absence or presence of various concentrations of ritonavir were labeled with the Apo2.7-phycoerythrin-conjugated monoclonal antibody (Beckman-Coulter/Immunotech, Miami, Florida) or mouse IgG1 isotype control (Beckman-Coulter/Immunotech) and subsequently analyzed by flow cytometry. EMSA
Cells were placed in culture at 1 x 106 cells/mL (cell line) or 5 x 106 cells/mL (PBMCs) and examined for inhibition of NF- 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 polyvinylidene difluoride membrane and probing with the following specific antibodies: polyclonal antibodies against I Plasmids and transfection
Reporter plasmid Inoculation of ATL cells and collection of samples 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 National Institute of Infectious Diseases (Tokyo, Japan). The Ethical Review Committee of the Institute approved the experimental protocol. Mice were anesthetized with ether, and cells were inoculated either intraperitoneally in abdominal region or subcutaneously in the postauricular region of NOG mice without injection of human recombinant IL-2 at a dose of 1 to 2 x 107 cells per mouse. All mice were killed 30 or 60 days after inoculation with primary ATL cells. Blood was collected from the tail to make a smear, as well as from the heart with heparinized syringes. PBMCs and splenocytes were isolated by density gradient concentration with Ficoll-Hypaque. Blood smear slides were fixed in methyl alcohol for May-Grunwald and Giemsa staining. PBMCs and splenocytes were stored at -80°C for further experiments. 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. Treatment of ATL mice with ritonavir Ritonavir was obtained from Abbott Labs, North Chicago, IL. Primary ATL cells (2 x 107) from 10 patients were inoculated subcutaneously in the postauricular region of NOG mice. One day after inoculation of ATL cells, mice were treated with either RPMI 1640 (control mice) or drug (ritonavir 30 mg/kg/d) intraperitoneally daily for 30 days followed by observation for another 30 days without treatment. ATL cell growth and progression were monitored by observation of physical condition of mice during a 2-month follow-up period. Immunohistochemistry Paraffinized cryosections of various organs were deparaffinized and hydrated in xylenes or clearing agents and graded alcohol series, then rinsed for 5 minutes in water. Deparaffinized samples were incubated with 0.025% trypsin/PBS for 30 minutes followed by washing, and then incubated with 0.3% methanol for 30 minutes at room temperature and washed 2 times with PBS. Immunostaining was done as described previously46 using Vector MOM immunodetection kit (Vector Labs, Burlingame CA) for ATL cells with a 1:500 dilution of primary mouse monoclonal antibody specific for human CD4 and CD25 (Dako, Caterpillar, CA). This was followed by washing in PBS and incubation with a secondary antibody MOM biotinylated anti-mouse IgG, after which cells were again washed in PBS and incubated with VECTASTAIN Elite ABC for 20 minutes 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 and 0.01% hydrogen peroxide for 5 minutes. The samples were counterstained with hematoxylin for 2 minutes, hydrated completely, cleaned in xylene, and then mounted. Photographs were taken by light microscopy (BX41, Olympus, Tokyo, Japan) using UplanF1 lenses (DP70, Olympus; magnification x40).
Ritonavir reduces cell growth and induces apoptosis of HTLV-I-infected cell lines and primary ATL cells Ritonavir was examined for its effect on proliferation of HTLV-I-infected cell lines (Figure 1A). Ritonavir effectively inhibited the proliferation of HTLV-I-infected cell lines as measured by WST-8 on the third day of culture in a dose-dependent manner, but not that of K562 cells. Further experiments using Apo2.7 showed that ritonavir caused apoptosis of HTLV-I-infected cell lines in a dose-dependent manner, but not that of K562 cells (Figure 1B-C). In addition, we explored the anti-ATL effect of ritonavir on freshly isolated ATL cells from patients. In all ATL cases, ritonavir reduced the survival of ATL cells in a dose-dependent manner (Figure 1D). Ritonavir also caused apoptosis of ATL cells (Figure 1E). In contrast, ritonavir hardly affected the survival of peripheral blood mononuclear cells (PBMCs) from 3 healthy volunteers as measured by WST-8 (Figure 1D).
Ritonavir suppresses constitutive NF- B expressed by HTLV-I-infected cell lines and primary ATL cells
To examine the effect of ritonavir on NF-
Ritonavir represses Tax-induced and constitutive transcriptional activity of NF- B
We examined whether ritonavir inhibits the transcriptional activity of NF-
Ritonavir down-regulates the expression of NF- B-regulated gene products
The antiproliferative and proapoptotic effects of ritonavir were explored by examining the level of intracellular regulators of cell cycle and apoptosis after exposure to ritonavir (Figure 3C). Ritonavir down-regulated levels of Bcl-XL, survivin, cIAP2, c-Myc, cyclin D2, regulated by NF-
Establishment of a novel ATL model
PBMCs from patients with ATL were inoculated either intraperitoneally into the abdominal region or subcutaneously in the postauricular region of unconditional NOD/SCID/
Ritonavir inhibits ATL cell growth and infiltration in NOG mice To study the effect of ritonavir on ATL, we injected primary ATL cells (2 x 107) from 10 patients subcutaneously into the postauricular region of NOG mice. One day after inoculation, mice were treated with either RPMI-1640 (as control) or ritonavir (30 mg/kg/d) intraperitoneally daily for 30 days followed by observation for another 30 days without any treatment. ATL cell inoculation promoted the development of piloerection, weight loss, and cachexia, all of which are signs of near-death, in addition to the enlargement of lymph nodes, spleen, lungs, and liver in all control mice 2 months after inoculation (Figure 5A). In contrast, ritonavir-treated mice appeared to be healthy and had almost no enlargement of these organs (Figure 5A). Clinical evaluation of organ invasion 2 months after injection of primary ATL cells showed that ritonavir treatment inhibited their infiltration into lymph nodes, spleen, lungs, and liver (Figure 5B-D). Samples from 7 patients of 10 injected in mice treated with ritonavir presented substantial inhibition of organ invasion, and 2 (patients 5 and 7) showed partial inhibition, whereas one sample (patient 6) failed to do so (Table 2). In contrast, all control mice showed formation of new lymph nodes and infiltration with ATL cells into various organs (Figure 5B-D; Table 2). Organ infiltration of primary ATL cells was analyzed and evaluated by HE staining and immunostaining of CD4 and CD25. Together, these data indicate that ritonavir significantly inhibits ATL cell growth and infiltration in various organs of NOG mice (Figure 5).
ATL is a malignancy of CD4+ T-lymphocytes etiologically linked to a retrovirus, HTLV-I.1-3 The malignant cells associated with all phases of ATL express very high levels of IL-2R  (CD25).12-14 The median survival duration of all patients with aggressive ATL was 13 months, and overall survival at 2 years was estimated to be 31.3%.6 The various chemotherapies so far developed have not increased significantly the survival of patients with ATL. Given disappointing results using conventional chemotherapy, new approaches for the treatment of ATL are required. Previous reports have shown that primary ATL cells proliferate and infiltrate into various organs of SCID mice.54-56 Our ATL model was consistent with others, but it represented more aggressive features about cell growth and infiltration in SCID mice. The tumor cells massively infiltrated into various organs in a manner similar to a leukemia-expressing T-cell marker CD4 and an activating marker CD25, especially into the spleen, lymph nodes, liver, and lungs of NOG mice. Our NOG ATL model presents many features 6 to 8 weeks after inoculation of ATL cells such as the clinical signs observed in patients with ATL. Two clinical types, acute and chronic, carry very different prognosis. However, no difference of cell growth, surface phenotype, and NF-B activity is observed in primary leukemic cells from patients with acute- and chronic-type ATL. Therefore, the same characteristics of freshly isolated ATL cells with acute- and chronic-type were observed in NOG mice. Thus, it represents a novel model to evaluate tissue toxicity and the efficacy of therapeutic agents directed toward the treatment of ATL.
Constitutive NF-
In the therapy of HIV infection, the blood plasma ritonavir concentrations are between 5 and 15 µM,58 but much higher maximal concentrations (up to 46 µM) have been determined in individual patients.59 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/d used for treatment of patients with AIDS. Our murine ATL model clearly indicates that 30 mg/kg/d of ritonavir significantly inhibits ATL 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 humans (600 mg twice daily). In our NOG ATL 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 PI, ritonavir, is a promising antitumor agent against ATL and could be used clinically for ATL regimens. Ritonavir exhibited anti-ATL activity against leukemic cells from patients with acute- and chronic-type ATL in vitro and in vivo. The expression of CD25 and NF-
In summary, using a large number of patient samples we have established a novel NOG ATL model that presents features similar to patients with ATL. These results also indicate that the HIV PI, ritonavir, showed antitumor and anti-NF-
We thank D. Kempf and T. Yamada of Abbott Laboratories, N. Yamamoto and S. Takeda of Molecular Virology, S. Ichinose of the Instrumental Analysis Research Center, S. Endo of the Animal Research Center, Tokyo Medical and Dental University, and P.J. Richard of Cardiff University for their advice and assistance with the experiments, and Y. Sato of the National Institute of Infectious Diseases for her excellent technical assistance. We thank J. Fujisawa for providing a luciferase reporter construct, B-LUC, K. Matsumoto for providing the Tax expression plasmid, M. Maeda for providing ED-40 515(-), and the Fujisaki Cell Center, Hayashibara Biomedical Laboratories, for providing the MT-1, HUT-102, and C5/MJ cell lines.
Submitted February 23, 2005; accepted September 1, 2005.
Prepublished online as Blood First Edition Paper, September 20, 2005; DOI 10.1182/blood-2005-02-0735.
Supported by grants from the Ministry of Education, Science, and Culture; the Ministry of Health, Labor, and Welfare; and Human Health Science of Japan.
M.Z.D. and J.U. contributed equally to the study.
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: Naoki Yamamoto, Department of Molecular Virology, Graduate School of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan; e-mail: yamamoto.mmb{at}tmd.ac.jp; and Naoki Mori, Division of Molecular Virology and Oncology, Graduate School of Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa 903-0215, Japan; e-mail: n-mori{at}med.u-ryukyu.ac.jp.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Top
Abstract
Introduction
) and ritonavir-treated (
) cells. (C) Dose-response effect of ritonavir on induction of apoptosis of MT-4 and HUT-102 cells. (D) Dose-response effect of ritonavir on the cell viability of freshly isolated ATL cells. Cells (106/mL) were cultured for 72 hours in the presence of various concentrations (2.5-40 µM) of ritonavir. (E) Dose-response effect of ritonavir on induction of apoptosis of ATL cells.
