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A novel NF-κB inhibitor improves glucocorticoid sensitivity of canine neoplastic lymphoid cells by up-regulating expression of glucocorticoid receptors
Research in Veterinary Science 89 (2010) 378–382
Contents lists available at ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
A novel NF-jB inhibitor improves glucocorticoid sensitivity of canine neoplastic lymphoid cells by up-regulating expression of glucocorticoid receptors A. Matsuda a, A. Tanaka a, S. Muto b, K. Ohmori a, T. Furusaka c, K. Jung a, K. Karasawa a, N. Okamoto a, K. Oida a, A. Itai b, H. Matsuda a,* a Laboratory of Veterinary Molecular Pathology and Therapeutics, Division of Animal Life Science, Graduate School, Institute of Symbiotic Science and Technology, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan b The Institute of Medicinal Molecular Design Inc., Bunkyo-ku, Tokyo 113-0033, Japan c Department of Otorhinolaryngology-Head and Neck Surgery, Nihon University School of Medicine, Itabashi-ku, Tokyo 173-8610, Japan
a r t i c l e
i n f o
Article history: Accepted 8 March 2010
Keywords: Glucocorticoid receptor IMD-0354 Lymphoma/leukemia NF-jB
a b s t r a c t Lymphoid neoplasms including lymphoma and leukemia are one of the most life-threatening disorders in dogs. Many lymphoid malignancies are well-treated with glucocorticoid (GC); however, GC resistance sometimes develops and its mechanism remains uncertain. Since constitutive activation of nuclear factor-jB (NF-jB) has been reported to play roles in lymphoid malignancies, we examined whether inhibition of NF-jB activity with a synthetic inhibitor IMD-0354 affected GC sensitivity of canine neoplastic lymphoid cells, CL-1 and GL-1. Dexamethasone failed to inhibit proliferation of these cells, in which low expression of glucocorticoid receptors (GR) was identified. In the presence of IMD-0354, GR expressions in CL-1 and GL-1 were increased, consequently dexamethasone inhibited their proliferation. These results indicated that GR expression might be down-regulated by spontaneous activation of NF-jB, resulting in GC resistance. Taken together, interference of NF-jB activity may have the synergistic effect in combination chemotherapy with GC for treatment against lymphoid malignancies. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Lymphoid malignancies, including lymphoma and leukemia, are one of the most common neoplasms in dogs, and lymphoma is widely recognized as a model for human non-Hodgkin’s lymphoma (Khanna et al., 2006). Canine lymphoma can be distinguished on the basis of anatomic location, histologic criteria, and immunophenotypic characteristics. Different subtypes of canine lymphoma require different therapeutic intervention, and react quite different to therapy. Although many cases of this lymphoid malignancy can be controlled by aggressive chemotherapy in an initial stage of therapy, relapses occur at a high rate. Chemotherapy for dogs with relapsed lymphoma frequently fails and long term survival of the dogs remains poor (Vail and Young, 2007). Lots of attempts to develop therapies with few side effects have been continued. Nuclear factor-jB (NF-jB), a dimeric transcription factor of the Rel family, exists as an inactive form in the cytoplasm by binding to its endogenous inhibitor IjB family proteins (Baldwin, 2001; Ghosh et al., 1998). IjB kinase (IKK) a and b phosphorylate IjB, resulting in NF-jB release and translocation to the nucleus. NFjB moved into the nucleus binds to specific DNA sequences of tar* Corresponding author. Tel.: +81 423675784; fax: +81 423675916. E-mail address: [email protected] (H. Matsuda). 0034-5288/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2010.03.017
get genes and contributes to cell proliferation and functions (Baldwin, 2001; Ghosh et al., 1998; Joyce et al., 2001; Yamamoto and Gaynor, 2001). Constitutive activation of NF-jB plays a crucial role in the development and progression of various kinds of cancers, including lymphoma and leukemia. We have already demonstrated that a novel NF-jB inhibitor IMD-0354, which blocks ATP-binding site of IKKb, suppresses the proliferation of human neoplastic mast cells and breast cancer cells (Tanaka et al., 2005, 2006). Glucocorticoid (GC) induces apoptosis and/or cell cycle arrest in lymphoid malignancies (Distelhorst, 2002; Greenstein et al., 2002). However, some tumors have GC resistance a priori and others mostly develop GC resistance during therapy (Kaspers et al., 1994; Moalli and Rosen, 1994). Although these are common phenomena, the mechanisms of GC resistance are not well understood. Biological actions of GC are mediated by glucocorticoid receptors (GR), a ligand-activated transcription factor belonging to the nuclear receptor superfamily. Some studies found that a lower number of GR per cell contributed to poor treatment response in lymphoid malignancies (Costlow et al., 1982; Mastrangelo et al., 1980). Although much is known regarding suppressive effects of GC on NF-jB pathways (Neumann and Naumann, 2007), the exact role of NF-jB in GC resistance remains uncertain. In this study, we used GC-resistant GL-1 and CL-1 cells, and examined that a selective NF-jB inhibitor IMD-0354 suppressed
A. Matsuda et al. / Research in Veterinary Science 89 (2010) 378–382
neoplastic proliferation of these neoplastic lymphoid cells. Treatment of these cells with GC exhibited synergistic anti-tumor effect when IMD-0354 was applied at the concentration that has a little effect on cell proliferation. Expression of GR was promoted in GC-resistant lymphoid cells by the addition of IMD-0354. These results indicated that NF-jB had a serious role in development of GC resistance as well as neoplastic cell proliferation; therefore, the reagents that inhibit NF-jB signal pathway may be useful as an antitumor drug in the combination chemotherapy against lymphoid malignancies. 2. Materials and methods 2.1. Cell culture CL-1 cells derived from canine T cell lymphoma (Momoi et al., 1997) and GL-1 cells derived from canine B cell leukemia (Nakaichi et al., 1996) were kindly provided from Dr. Atsuhiko Hasegawa (The University of Tokyo). CL-1 and GL-1 cells were cultured in RPMI 1640 (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (Filtron, Brooklyn, Australia) and antibiotics. 2.2. Reagents Rabbit anti-phospho-IjBa antibody and rabbit anti-IjBa antibody were purchased from Cell Signaling Technology (Beverly, MA). Rabbit anti-NF-jB p65 antibody was obtained from Santa Cruz Biotechnology (Lake Placid, NY). Rabbit anti-GR antibody was purchased from Abcam (Cambridge, UK). IMD-0354, a novel NF-jB inhibitor, was kindly provided from the Institute of Medic-
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inal Molecular Design Inc. (Tokyo, Japan). Dexamethasone (Dex) was obtained from BIOMOL International (Plymouth Meeting, PA).
2.3. Cell proliferation assay For synchronization of cell cycle, cells were incubated in serumfree medium for 12 h before performing following assays. For a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, cells were incubated in serum-containing medium in the presence of various concentrations of IMD-0354 for 24, 48, and 72 h. A MTT assay was performed as described previously (Tanaka et al., 2005). After incubation with reagents for 20 h, a 5-bromo-20 deoxy-uridine (BrdU) incorporation assay was performed according to the manufacturer’s instructions of a cell proliferation ELISA, BrdU kit (Roche Molecular Biochemicals, Mannheim, Germany).
2.4. Western blot analysis After incubation with various concentrations of IMD-0354 for 24 h, whole cell lysates were prepared with a CelLytic-MT reagent (Sigma Chemicals, St. Louis, MO), and nuclear extractions and cytoplasmic extractions were prepared using NE-PER nuclear and cytoplasmic extraction reagents (Pierce, Rockford, IL). A Western blot analysis was performed as described previously (Tanaka et al., 2005). Relative intensities of each band in a Western blot analysis were calculated with the intensity of b-actin or Histon H1 as endogenous controls, and then standardized to a value without IMD-0354 as 1.
Fig. 1. Inhibitory effect of IMD-0354 on spontaneous activation of NF-jB in CL-1 and GL-1 cells. Inhibition of phosphorylation of IjB in CL-1 and GL-1 cells was detected after incubation with indicated concentrations of IMD-0354 for 24 h (A). Spontaneous translocation of NF-jB p65 into the nucleus was detected in extraction of the nuclear fraction of CL-1 and GL-1 cells (B). Incubation with IMD-0354 for 24-h suppressed translocation of NF-jB p65 into the nucleus. Histon H1 or b-actin was used for endogenous controls. Relative intensities of phospho-IjB and NFjB were standardized to a value without IMD-0354 as 1. Data represent mean ± SE of three individual experiments.
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2.5. Reverse transcription and polymerase chain reaction (RT-PCR)
2.7. Statistical analysis
Following incubation with various concentrations of IMD-0354 for 6 h, total RNA was extracted and reverse-transcribed into cDNA by using a PrimeScript 1st strand cDNA synthesis kit (Takara Bio, Shiga, Japan). PCR was performed with Platinum Taq DNA polymerase (Invitrogen, Tokyo, Japan) and specific primers as follows: forward (50 -TACGCAGGGTATGACAGCTC-30 ) and reverse (50 -AGCGTT GCCAGTTCTGGCTGG-30 ) for canine GR (NCBI reference number; XM_535225.2), and forward (50 -TGTGGCCATCCAGGCTGTGC-30 ) and reverse (50 -GTGGTCTCGTGGATACCGCA-30 ) for canine b-actin. PCR master mix was made according to a manufacturer’s manual of Platinum Taq DNA polymerase. The PCR amplification consisted of pre-denaturation (94 °C, 2 min), and 35 cycles of denaturation (94 °C, 30 s), annealing (55 °C, 30 s), and extension (72 °C, 1 min), followed by final extension (72 °C, 4 min). Relative intensities of each band in RT-RCR were calculated with the intensity of b-actin as an endogenous control, and then standardized to a value without IMD-0354 as 1.
The statistical significance was analyzed by one- or two-factor analysis of variance (ANOVA), followed by a Holm–Sidak test with the use of SigmaPlot 11 (Systat software, San Jose, CA). p values of <0.01 were considered statistically significant.
2.6. Collagen gel droplet embedded drug sensitivity test (CD-DST) Cells were cultured in collagen gel droplets with the use of Primaster (Nitta Gelatin, Osaka, Japan) according to the manufacturer’s instructions. Collagen gel droplets were covered with serum-free AIM V medium (Invitrogen, Carsbad, CA) with 1 lM IMD-0354 and/or 10 lM Dex, and then incubated for 7 days. Drug sensitivity was expressed as the T/C ratio (%), where T was the total volume of the treated group and C was the total volume of the control group.
Fig. 2. Inhibitory effect of IMD-0354 on proliferation of CL-1 and GL-1 cells. Cells were incubated with increasing concentrations of IMD-0354, and the inhibitory effect on proliferation was evaluated by a MTT assay (A) and a BrdU incorporation test (B). Cells were harvested at 20 h after incubation with IMD-0354 for a BrdU incorporation test. Data represent mean ± SE of three individual experiments. *p < 0.01, when compared with medium alone.
3. Results 3.1. Spontaneous activation of NF-jB in CL-1 and GL-1 cells inhibited by IMD-0354 First, we examined spontaneous activity of NF-jB in CL-1 and GL-1 cells, as well as inhibitory effect of IMD-0354 by a Western blot analysis after incubation with IMD-0354 for 24 h. Phosphorylation of IjB was obviously identified in untreated cells, which was clearly suppressed by IMD-0354 treatment (Fig. 1A). NF-jB p65 was detected in a nuclear fraction (Fig. 1B), indicating that NF-jB automatically moved into the nucleus in CL-1 and GL-1 cells. Incubation with IMD-0354 reduced positive reactivity of p65 subunit of NF-jB in a nuclear fraction, while that of p65 in a cytoplasmic fraction was increased (Fig. 1B). These results indicated that IMD-0354 effectively suppressed NF-jB that was constitutively activated in CL-1 and GL-1 cells.
Fig. 3. GC resistance recovered by IMD-0354 treatment in CL-1 and GL-1 cells. Cells were incubated with increasing concentrations of Dex alone, and the inhibitory effect on proliferation was evaluated by a BrdU incorporation test (A). Cells were incubated for 20 h with increasing concentrations of Dex in the presence of 1 lM IMD-0354, and a BrdU incorporation test was performed (B). Synergistic effects of IMD-0354 and Dex on cell proliferation were also evaluated by CD-DST (C). Data represent mean ± SE of three individual experiments. *p < 0.01, compared with IMD-0354 alone.
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3.2. Effect of IMD-0354 on proliferation of CL-1 and GL-1 cells Next, effect of IMD-0354 on proliferation of CL-1 and GL-1 cells was evaluated by a MTT assay and a BrdU incorporation test. Proliferative activities of CL-1 and GL-1 cells were effectively suppressed by the addition of IMD-0354 to the culture medium in a dose-dependent manner (Fig. 2A). IMD-0354 also decreased DNA synthesis of CL-1 and GL-1 cells in a dose-dependent manner (Fig. 2B). As shown in Figs. 1 and 2, 1 lM IMD-0354 had a little effect on BrdU incorporation of CL-1 and Gl-1 cells while it suppressed NFjB activity; thus we used IMD-0354 at 1 lM in following experiments. 3.3. Low sensitivity to Dex treatment of CL-1 and GL-1 cells improved by IMD-0354 In the chemotherapy of canine lymphoma/leukemia, GC is fundamental and widely used. Thus we assessed the effect of Dex on the proliferation of CL-1 and GL-1 cells. Dex had no effect on DNA synthesis of CL-1 and GL-1 cells even at the dose of 10 lM (Fig. 3A). Next, we assessed the synergic effect of IMD-0354 and Dex on the proliferation of these cells to evaluate the possibility that inhibition of NF-jB may recover GC sensitivity. Combination of various concentrations of Dex with 1 lM IMD-0354 inhibited DNA synthesis of both CL-1 and GL-1 cells in a dose-dependent manner (Fig. 3B). The synergistic effect of IMD-0354 and Dex on the proliferation of CL-1 and GL-1 cells was also evaluated by a CD-DST. Although incubation with 10 lM Dex alone inhibited no or little cell proliferation, addition of 1 lM IMD-0354 to culture with Dex decreased colony volume in both CL-1 and GL-1 cells (Fig. 3C). 3.4. Modulation of GR expression by IMD-0354 To examine the possible involvement of NF-jB activation in GC resistance, we assessed expression levels of GR of CL-1 and GL-1 cells. Semi-quantitative RT-PCR revealed that incubation with IMD-0354 increased GR expression in a dose-dependent manner
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(Fig. 4A). Weak and/or little positive bands of GR protein were detected by Western blot analysis (Fig. 4B). After incubation with IMD-0354 for 24 h, the positive reaction for GR protein increased in a dose-dependent manner in CL-1 and GL-1 cells (Fig. 4B). 4. Discussion In this study, we showed that constitutive activation of NF-jB strongly correlated to the proliferation of canine neoplastic lymphoid cells. Although chemotherapy is a primary treatment in canine lymphoid malignancies, side effects of conventional anticancer drugs and drug resistance to chemotherapy are serious problems. Therefore, effective chemotherapeutic agents with few side effects are widely expected. Since auto-activation of NF-jB has been widely demonstrated to seriously contribute to neoplastic transformation of hematopoietic cells, NF-jB is one of the most important molecular targets in the development of effective chemotherapy for lymphoma, leukemia, and others (Davis et al., 2001; Keller et al., 2000; Mori et al., 2002; Tanaka et al., 2005, 2006). In CL-1 and GL-1 cells, NF-jB was spontaneously activated and inhibition of the activity by the NF-jB inhibitor IMD-0354 suppressed proliferation of these cells. Although there is no published study on side effects of IMD-0354 in dogs, daily injection with IMD-0354 for a month did not show any adverse effect in mice (Tanaka et al., 2006). Further studies must take place, but IMD-0354 has a possibility to be one of effective target drugs without adverse effects. In lymphoid malignancy, GC treatment is fundamental and used for many years (Distelhorst, 2002; Greenstein et al., 2002). However, the beneficial effects of GC treatment are limited due to development of GC resistance. It was reported that down-regulated expression of GR was associated with low sensitivity to GC treatment (Costlow et al., 1982; Mastrangelo et al., 1980). The present results also suggested that low expression of GR might induce hyporesponsiveness to GC in CL-1 and GL-1 cells. Moreover, we demonstrated that application of IMD-0354 recovered Dex sensitivity and synergistically suppressed neoplastic proliferation in CL-1 and GL-1 cells, which possessed a strong GC resistance. Expression of GR was elevated in the presence of IMD-0354, indicating that
Fig. 4. Expression of GR promoted by IMD-0354 treatment. RT-PCR revealed mRNA expression of GR in CL-1 and GL-1 cells after incubation with various concentrations of IMD-0354 for 6 h (A). Protein levels of GR in whole cell lysates of CL-1 and GL-1 cells were examined after incubation with various concentrations of IMD-0354 for 24 h (B). bactin was used for an endogenous control. Relative intensities were standardized to a value without IMD-0354 as 1. Data represent mean ± SE of three individual experiments. *p < 0.01, compared with medium alone.
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inhibition of NF-jB activity might restore GC sensitivity, at least in part, through up-regulation of GR expression and post-transcriptional mechanisms in both CL-1 and GL-1 cells. Some reagents were reported to up-regulate the expression of GR in human hematopoietic cells (Oerlemans et al., 2007; Goecke et al., 2007), thus further studies on the mechanism of regulation of GR expression should be done in the future. Taken together, activated NF-jB contributed to proliferation of canine neoplastic lymphoid cells, and inhibition of its activity effectively suppressed malignant cell proliferation. Interestingly, auto-activation of NF-jB may associate with GC resistance by decreasing GR expression; thereby, treatment with a lower concentration of IMD-0354 that hardly induce cell death increased GR expression and recovered GC sensitivity of canine neoplastic lymphoid cells. Therefore, we conclude that a NF-jB inhibitor may have therapeutic advantages in treatment for GC-resistant lymphoid malignancies. Acknowledgements This work was supported by the grant from Grant-in-Aid for Scientific Research on Priority Areas A and on Grant-in-Aid for Scientific Research Priority Areas B provided by Japan Society for the Promotion of Science, Japan. We wish to thank Dr. A. Hasegawa (The University of Tokyo) for providing CL-1 and GL-1 cells. References Baldwin, A.S., 2001. Control of oncogenesis and cancer therapy resistance by the transcription factor NF-jB. J. Clin. Invest. 107, 241–246. Costlow, M.E., Pui, C.H., Dahl, G.V., 1982. Glucocorticoid receptors in childhood acute lymphocytic leukemia. Cancer Res. 42, 4801–4806. Davis, R.E., Brown, K.D., Siebenlist, U., Staudt, L.M., 2001. Constitutive nuclear factor-jB activity is required for survival of activated B cell-like diffuse large B cell lymphoma cells. J. Exp. Med. 194, 1861–1874. Distelhorst, C.W., 2002. Recent insights into the mechanism of glucocorticosteroidinduced apoptosis. Cell Death Differ. 9, 6–19. Ghosh, S., May, M.J., Kopp, E.B., 1998. NF-jB and Rel proteins: evolutionarily conserved mediators of immune responses. Annu. Rev. Immunol. 16, 225–260. Goecke, I.A., Alvarez, C., Henríquez, J., Salas, K., Molina, M.L., Ferreira, A., Gatica, H., 2007. Methotrexate regulates the expression of glucocorticoid receptor alpha
and beta isoforms in normal human peripheral mononuclear cells and human lymphocyte cell lines in vitro. Mol. Immunol. 44, 2115–2123. Greenstein, S., Ghias, K., Krett, N.L., Rosen, S.T., 2002. Mechanisms of glucocorticoidmediated apoptosis in hematological malignancies. Clin. Cancer Res. 8, 1681– 1694. Joyce, D., Albanese, C., Steer, J., Fu, M., Bouzahzah, B., Pestell, R.G., 2001. NF-jB and cell-cycle regulation: the cyclin connection. Cytokine Growth Factor Rev. 12, 73–90. Kaspers, G.J., Pieters, R., Klumper, E., DeWaal, F.C., Veerman, A.J., 1994. Glucocorticoid resistance in childhood leukemia. Leuk. Lymphoma 13, 187–201. Keller, S.A., Schattner, E.J., Cesarman, E., 2000. Inhibition of NF-jB induces apoptosis of KSHV-infected primary effusion lymphoma cells. Blood 96, 2537–2542. Khanna, C., Lindblad-Toh, K., Vail, D., London, C., Bergman, P., 2006. The dog as a cancer model. Nat. Biotechnol. 24, 1065–1066. Mastrangelo, R., Malandrino, R., Riccardi, R., Longo, P., Ranelletti, F.O., Iacobelli, S., 1980. Clinical implications of glucocorticoid receptor studies in childhood acute lymphoblastic leukemia. Blood 56, 1036–1040. Moalli, P.A., Rosen, S.T., 1994. Glucocorticoid receptors and resistance to glucocorticoids in hematologic malignancies. Leuk. Lymphoma 15, 363–374. Momoi, Y., Okai, Y., Watari, T., Goitsuka, R., Tsujimoto, H., Hasegawa, A., 1997. Establishment and characterization of a canine T-lymphoblastoid cell line derived from malignant lymphoma. Vet. Immunol. Immunopathol. 59, 11–20. Mori, N., Yamada, Y., Ikeda, S., Yamasaki, Y., Tsukasaki, K., Tanaka, Y., Tomonaga, M., Yamamoto, N., Fujii, M., 2002. Bay 11-7082 inhibits transcription factor NF-jB and induces apoptosis of HTLV-I-infected T-cell lines and primary adult T-cell leukemia cells. Blood 100, 1828–1834. Nakaichi, M., Taura, Y., Kanki, M., Mamba, K., Momoi, Y., Tsujimoto, H., Nakama, S., 1996. Establishment and characterization of a new canine B-cell leukemia cell line. J. Vet. Med. Sci. 58, 469–471. Neumann, M., Naumann, M., 2007. Beyond IjBs: alternative regulation of NF-jB activity. FASEB J. 21, 2642–2654. Oerlemans, R., Vink, J., Dijkmans, B.A., Assaraf, Y.G., van Miltenburg, M., van der Heijden, J., Ifergan, I., Lems, W.F., Scheper, R.J., Kaspers, G.J., Cloos, J., Jansen, G., 2007. Sulfasalazine sensitises human monocytic/macrophage cells for glucocorticoids by upregulation of glucocorticoid receptor alpha and glucocorticoid induced apoptosis. Ann. Rheum. Dis. 66, 1289–1295. Tanaka, A., Konno, M., Muto, S., Kambe, N., Morii, E., Nakahata, T., Itai, A., Matsuda, H., 2005. A novel NF-jB inhibitor, IMD-0354, suppresses neoplastic proliferation of human mast cells with constitutively activated c-kit receptors. Blood 105, 2324–2331. Tanaka, A., Muto, S., Konno, M., Itai, A., Matsuda, H., 2006. A new IjB inhibitor prevents human breast cancer progression through negative regulation of cell cycle transition. Cancer Res. 66, 419–426. Vail, D.M., Young, K.M., 2007. Hematopoietic tumors. In: Withrow, S.J., Vail, D.M. (Eds.), Small Animal Clinical Oncology, fourth ed. W.B. Saunders, Philadelphia, USA, pp. 699–784. Yamamoto, Y., Gaynor, R.B., 2001. Therapeutic potential of inhibition of the NF-jB pathway in the treatment of inflammation and cancer. J. Clin. Invest. 107, 135– 142.
Contents lists available at ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
A novel NF-jB inhibitor improves glucocorticoid sensitivity of canine neoplastic lymphoid cells by up-regulating expression of glucocorticoid receptors A. Matsuda a, A. Tanaka a, S. Muto b, K. Ohmori a, T. Furusaka c, K. Jung a, K. Karasawa a, N. Okamoto a, K. Oida a, A. Itai b, H. Matsuda a,* a Laboratory of Veterinary Molecular Pathology and Therapeutics, Division of Animal Life Science, Graduate School, Institute of Symbiotic Science and Technology, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan b The Institute of Medicinal Molecular Design Inc., Bunkyo-ku, Tokyo 113-0033, Japan c Department of Otorhinolaryngology-Head and Neck Surgery, Nihon University School of Medicine, Itabashi-ku, Tokyo 173-8610, Japan
a r t i c l e
i n f o
Article history: Accepted 8 March 2010
Keywords: Glucocorticoid receptor IMD-0354 Lymphoma/leukemia NF-jB
a b s t r a c t Lymphoid neoplasms including lymphoma and leukemia are one of the most life-threatening disorders in dogs. Many lymphoid malignancies are well-treated with glucocorticoid (GC); however, GC resistance sometimes develops and its mechanism remains uncertain. Since constitutive activation of nuclear factor-jB (NF-jB) has been reported to play roles in lymphoid malignancies, we examined whether inhibition of NF-jB activity with a synthetic inhibitor IMD-0354 affected GC sensitivity of canine neoplastic lymphoid cells, CL-1 and GL-1. Dexamethasone failed to inhibit proliferation of these cells, in which low expression of glucocorticoid receptors (GR) was identified. In the presence of IMD-0354, GR expressions in CL-1 and GL-1 were increased, consequently dexamethasone inhibited their proliferation. These results indicated that GR expression might be down-regulated by spontaneous activation of NF-jB, resulting in GC resistance. Taken together, interference of NF-jB activity may have the synergistic effect in combination chemotherapy with GC for treatment against lymphoid malignancies. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Lymphoid malignancies, including lymphoma and leukemia, are one of the most common neoplasms in dogs, and lymphoma is widely recognized as a model for human non-Hodgkin’s lymphoma (Khanna et al., 2006). Canine lymphoma can be distinguished on the basis of anatomic location, histologic criteria, and immunophenotypic characteristics. Different subtypes of canine lymphoma require different therapeutic intervention, and react quite different to therapy. Although many cases of this lymphoid malignancy can be controlled by aggressive chemotherapy in an initial stage of therapy, relapses occur at a high rate. Chemotherapy for dogs with relapsed lymphoma frequently fails and long term survival of the dogs remains poor (Vail and Young, 2007). Lots of attempts to develop therapies with few side effects have been continued. Nuclear factor-jB (NF-jB), a dimeric transcription factor of the Rel family, exists as an inactive form in the cytoplasm by binding to its endogenous inhibitor IjB family proteins (Baldwin, 2001; Ghosh et al., 1998). IjB kinase (IKK) a and b phosphorylate IjB, resulting in NF-jB release and translocation to the nucleus. NFjB moved into the nucleus binds to specific DNA sequences of tar* Corresponding author. Tel.: +81 423675784; fax: +81 423675916. E-mail address: [email protected] (H. Matsuda). 0034-5288/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2010.03.017
get genes and contributes to cell proliferation and functions (Baldwin, 2001; Ghosh et al., 1998; Joyce et al., 2001; Yamamoto and Gaynor, 2001). Constitutive activation of NF-jB plays a crucial role in the development and progression of various kinds of cancers, including lymphoma and leukemia. We have already demonstrated that a novel NF-jB inhibitor IMD-0354, which blocks ATP-binding site of IKKb, suppresses the proliferation of human neoplastic mast cells and breast cancer cells (Tanaka et al., 2005, 2006). Glucocorticoid (GC) induces apoptosis and/or cell cycle arrest in lymphoid malignancies (Distelhorst, 2002; Greenstein et al., 2002). However, some tumors have GC resistance a priori and others mostly develop GC resistance during therapy (Kaspers et al., 1994; Moalli and Rosen, 1994). Although these are common phenomena, the mechanisms of GC resistance are not well understood. Biological actions of GC are mediated by glucocorticoid receptors (GR), a ligand-activated transcription factor belonging to the nuclear receptor superfamily. Some studies found that a lower number of GR per cell contributed to poor treatment response in lymphoid malignancies (Costlow et al., 1982; Mastrangelo et al., 1980). Although much is known regarding suppressive effects of GC on NF-jB pathways (Neumann and Naumann, 2007), the exact role of NF-jB in GC resistance remains uncertain. In this study, we used GC-resistant GL-1 and CL-1 cells, and examined that a selective NF-jB inhibitor IMD-0354 suppressed
A. Matsuda et al. / Research in Veterinary Science 89 (2010) 378–382
neoplastic proliferation of these neoplastic lymphoid cells. Treatment of these cells with GC exhibited synergistic anti-tumor effect when IMD-0354 was applied at the concentration that has a little effect on cell proliferation. Expression of GR was promoted in GC-resistant lymphoid cells by the addition of IMD-0354. These results indicated that NF-jB had a serious role in development of GC resistance as well as neoplastic cell proliferation; therefore, the reagents that inhibit NF-jB signal pathway may be useful as an antitumor drug in the combination chemotherapy against lymphoid malignancies. 2. Materials and methods 2.1. Cell culture CL-1 cells derived from canine T cell lymphoma (Momoi et al., 1997) and GL-1 cells derived from canine B cell leukemia (Nakaichi et al., 1996) were kindly provided from Dr. Atsuhiko Hasegawa (The University of Tokyo). CL-1 and GL-1 cells were cultured in RPMI 1640 (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (Filtron, Brooklyn, Australia) and antibiotics. 2.2. Reagents Rabbit anti-phospho-IjBa antibody and rabbit anti-IjBa antibody were purchased from Cell Signaling Technology (Beverly, MA). Rabbit anti-NF-jB p65 antibody was obtained from Santa Cruz Biotechnology (Lake Placid, NY). Rabbit anti-GR antibody was purchased from Abcam (Cambridge, UK). IMD-0354, a novel NF-jB inhibitor, was kindly provided from the Institute of Medic-
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inal Molecular Design Inc. (Tokyo, Japan). Dexamethasone (Dex) was obtained from BIOMOL International (Plymouth Meeting, PA).
2.3. Cell proliferation assay For synchronization of cell cycle, cells were incubated in serumfree medium for 12 h before performing following assays. For a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, cells were incubated in serum-containing medium in the presence of various concentrations of IMD-0354 for 24, 48, and 72 h. A MTT assay was performed as described previously (Tanaka et al., 2005). After incubation with reagents for 20 h, a 5-bromo-20 deoxy-uridine (BrdU) incorporation assay was performed according to the manufacturer’s instructions of a cell proliferation ELISA, BrdU kit (Roche Molecular Biochemicals, Mannheim, Germany).
2.4. Western blot analysis After incubation with various concentrations of IMD-0354 for 24 h, whole cell lysates were prepared with a CelLytic-MT reagent (Sigma Chemicals, St. Louis, MO), and nuclear extractions and cytoplasmic extractions were prepared using NE-PER nuclear and cytoplasmic extraction reagents (Pierce, Rockford, IL). A Western blot analysis was performed as described previously (Tanaka et al., 2005). Relative intensities of each band in a Western blot analysis were calculated with the intensity of b-actin or Histon H1 as endogenous controls, and then standardized to a value without IMD-0354 as 1.
Fig. 1. Inhibitory effect of IMD-0354 on spontaneous activation of NF-jB in CL-1 and GL-1 cells. Inhibition of phosphorylation of IjB in CL-1 and GL-1 cells was detected after incubation with indicated concentrations of IMD-0354 for 24 h (A). Spontaneous translocation of NF-jB p65 into the nucleus was detected in extraction of the nuclear fraction of CL-1 and GL-1 cells (B). Incubation with IMD-0354 for 24-h suppressed translocation of NF-jB p65 into the nucleus. Histon H1 or b-actin was used for endogenous controls. Relative intensities of phospho-IjB and NFjB were standardized to a value without IMD-0354 as 1. Data represent mean ± SE of three individual experiments.
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2.5. Reverse transcription and polymerase chain reaction (RT-PCR)
2.7. Statistical analysis
Following incubation with various concentrations of IMD-0354 for 6 h, total RNA was extracted and reverse-transcribed into cDNA by using a PrimeScript 1st strand cDNA synthesis kit (Takara Bio, Shiga, Japan). PCR was performed with Platinum Taq DNA polymerase (Invitrogen, Tokyo, Japan) and specific primers as follows: forward (50 -TACGCAGGGTATGACAGCTC-30 ) and reverse (50 -AGCGTT GCCAGTTCTGGCTGG-30 ) for canine GR (NCBI reference number; XM_535225.2), and forward (50 -TGTGGCCATCCAGGCTGTGC-30 ) and reverse (50 -GTGGTCTCGTGGATACCGCA-30 ) for canine b-actin. PCR master mix was made according to a manufacturer’s manual of Platinum Taq DNA polymerase. The PCR amplification consisted of pre-denaturation (94 °C, 2 min), and 35 cycles of denaturation (94 °C, 30 s), annealing (55 °C, 30 s), and extension (72 °C, 1 min), followed by final extension (72 °C, 4 min). Relative intensities of each band in RT-RCR were calculated with the intensity of b-actin as an endogenous control, and then standardized to a value without IMD-0354 as 1.
The statistical significance was analyzed by one- or two-factor analysis of variance (ANOVA), followed by a Holm–Sidak test with the use of SigmaPlot 11 (Systat software, San Jose, CA). p values of <0.01 were considered statistically significant.
2.6. Collagen gel droplet embedded drug sensitivity test (CD-DST) Cells were cultured in collagen gel droplets with the use of Primaster (Nitta Gelatin, Osaka, Japan) according to the manufacturer’s instructions. Collagen gel droplets were covered with serum-free AIM V medium (Invitrogen, Carsbad, CA) with 1 lM IMD-0354 and/or 10 lM Dex, and then incubated for 7 days. Drug sensitivity was expressed as the T/C ratio (%), where T was the total volume of the treated group and C was the total volume of the control group.
Fig. 2. Inhibitory effect of IMD-0354 on proliferation of CL-1 and GL-1 cells. Cells were incubated with increasing concentrations of IMD-0354, and the inhibitory effect on proliferation was evaluated by a MTT assay (A) and a BrdU incorporation test (B). Cells were harvested at 20 h after incubation with IMD-0354 for a BrdU incorporation test. Data represent mean ± SE of three individual experiments. *p < 0.01, when compared with medium alone.
3. Results 3.1. Spontaneous activation of NF-jB in CL-1 and GL-1 cells inhibited by IMD-0354 First, we examined spontaneous activity of NF-jB in CL-1 and GL-1 cells, as well as inhibitory effect of IMD-0354 by a Western blot analysis after incubation with IMD-0354 for 24 h. Phosphorylation of IjB was obviously identified in untreated cells, which was clearly suppressed by IMD-0354 treatment (Fig. 1A). NF-jB p65 was detected in a nuclear fraction (Fig. 1B), indicating that NF-jB automatically moved into the nucleus in CL-1 and GL-1 cells. Incubation with IMD-0354 reduced positive reactivity of p65 subunit of NF-jB in a nuclear fraction, while that of p65 in a cytoplasmic fraction was increased (Fig. 1B). These results indicated that IMD-0354 effectively suppressed NF-jB that was constitutively activated in CL-1 and GL-1 cells.
Fig. 3. GC resistance recovered by IMD-0354 treatment in CL-1 and GL-1 cells. Cells were incubated with increasing concentrations of Dex alone, and the inhibitory effect on proliferation was evaluated by a BrdU incorporation test (A). Cells were incubated for 20 h with increasing concentrations of Dex in the presence of 1 lM IMD-0354, and a BrdU incorporation test was performed (B). Synergistic effects of IMD-0354 and Dex on cell proliferation were also evaluated by CD-DST (C). Data represent mean ± SE of three individual experiments. *p < 0.01, compared with IMD-0354 alone.
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3.2. Effect of IMD-0354 on proliferation of CL-1 and GL-1 cells Next, effect of IMD-0354 on proliferation of CL-1 and GL-1 cells was evaluated by a MTT assay and a BrdU incorporation test. Proliferative activities of CL-1 and GL-1 cells were effectively suppressed by the addition of IMD-0354 to the culture medium in a dose-dependent manner (Fig. 2A). IMD-0354 also decreased DNA synthesis of CL-1 and GL-1 cells in a dose-dependent manner (Fig. 2B). As shown in Figs. 1 and 2, 1 lM IMD-0354 had a little effect on BrdU incorporation of CL-1 and Gl-1 cells while it suppressed NFjB activity; thus we used IMD-0354 at 1 lM in following experiments. 3.3. Low sensitivity to Dex treatment of CL-1 and GL-1 cells improved by IMD-0354 In the chemotherapy of canine lymphoma/leukemia, GC is fundamental and widely used. Thus we assessed the effect of Dex on the proliferation of CL-1 and GL-1 cells. Dex had no effect on DNA synthesis of CL-1 and GL-1 cells even at the dose of 10 lM (Fig. 3A). Next, we assessed the synergic effect of IMD-0354 and Dex on the proliferation of these cells to evaluate the possibility that inhibition of NF-jB may recover GC sensitivity. Combination of various concentrations of Dex with 1 lM IMD-0354 inhibited DNA synthesis of both CL-1 and GL-1 cells in a dose-dependent manner (Fig. 3B). The synergistic effect of IMD-0354 and Dex on the proliferation of CL-1 and GL-1 cells was also evaluated by a CD-DST. Although incubation with 10 lM Dex alone inhibited no or little cell proliferation, addition of 1 lM IMD-0354 to culture with Dex decreased colony volume in both CL-1 and GL-1 cells (Fig. 3C). 3.4. Modulation of GR expression by IMD-0354 To examine the possible involvement of NF-jB activation in GC resistance, we assessed expression levels of GR of CL-1 and GL-1 cells. Semi-quantitative RT-PCR revealed that incubation with IMD-0354 increased GR expression in a dose-dependent manner
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(Fig. 4A). Weak and/or little positive bands of GR protein were detected by Western blot analysis (Fig. 4B). After incubation with IMD-0354 for 24 h, the positive reaction for GR protein increased in a dose-dependent manner in CL-1 and GL-1 cells (Fig. 4B). 4. Discussion In this study, we showed that constitutive activation of NF-jB strongly correlated to the proliferation of canine neoplastic lymphoid cells. Although chemotherapy is a primary treatment in canine lymphoid malignancies, side effects of conventional anticancer drugs and drug resistance to chemotherapy are serious problems. Therefore, effective chemotherapeutic agents with few side effects are widely expected. Since auto-activation of NF-jB has been widely demonstrated to seriously contribute to neoplastic transformation of hematopoietic cells, NF-jB is one of the most important molecular targets in the development of effective chemotherapy for lymphoma, leukemia, and others (Davis et al., 2001; Keller et al., 2000; Mori et al., 2002; Tanaka et al., 2005, 2006). In CL-1 and GL-1 cells, NF-jB was spontaneously activated and inhibition of the activity by the NF-jB inhibitor IMD-0354 suppressed proliferation of these cells. Although there is no published study on side effects of IMD-0354 in dogs, daily injection with IMD-0354 for a month did not show any adverse effect in mice (Tanaka et al., 2006). Further studies must take place, but IMD-0354 has a possibility to be one of effective target drugs without adverse effects. In lymphoid malignancy, GC treatment is fundamental and used for many years (Distelhorst, 2002; Greenstein et al., 2002). However, the beneficial effects of GC treatment are limited due to development of GC resistance. It was reported that down-regulated expression of GR was associated with low sensitivity to GC treatment (Costlow et al., 1982; Mastrangelo et al., 1980). The present results also suggested that low expression of GR might induce hyporesponsiveness to GC in CL-1 and GL-1 cells. Moreover, we demonstrated that application of IMD-0354 recovered Dex sensitivity and synergistically suppressed neoplastic proliferation in CL-1 and GL-1 cells, which possessed a strong GC resistance. Expression of GR was elevated in the presence of IMD-0354, indicating that
Fig. 4. Expression of GR promoted by IMD-0354 treatment. RT-PCR revealed mRNA expression of GR in CL-1 and GL-1 cells after incubation with various concentrations of IMD-0354 for 6 h (A). Protein levels of GR in whole cell lysates of CL-1 and GL-1 cells were examined after incubation with various concentrations of IMD-0354 for 24 h (B). bactin was used for an endogenous control. Relative intensities were standardized to a value without IMD-0354 as 1. Data represent mean ± SE of three individual experiments. *p < 0.01, compared with medium alone.
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inhibition of NF-jB activity might restore GC sensitivity, at least in part, through up-regulation of GR expression and post-transcriptional mechanisms in both CL-1 and GL-1 cells. Some reagents were reported to up-regulate the expression of GR in human hematopoietic cells (Oerlemans et al., 2007; Goecke et al., 2007), thus further studies on the mechanism of regulation of GR expression should be done in the future. Taken together, activated NF-jB contributed to proliferation of canine neoplastic lymphoid cells, and inhibition of its activity effectively suppressed malignant cell proliferation. Interestingly, auto-activation of NF-jB may associate with GC resistance by decreasing GR expression; thereby, treatment with a lower concentration of IMD-0354 that hardly induce cell death increased GR expression and recovered GC sensitivity of canine neoplastic lymphoid cells. Therefore, we conclude that a NF-jB inhibitor may have therapeutic advantages in treatment for GC-resistant lymphoid malignancies. Acknowledgements This work was supported by the grant from Grant-in-Aid for Scientific Research on Priority Areas A and on Grant-in-Aid for Scientific Research Priority Areas B provided by Japan Society for the Promotion of Science, Japan. We wish to thank Dr. A. Hasegawa (The University of Tokyo) for providing CL-1 and GL-1 cells. References Baldwin, A.S., 2001. Control of oncogenesis and cancer therapy resistance by the transcription factor NF-jB. J. Clin. Invest. 107, 241–246. Costlow, M.E., Pui, C.H., Dahl, G.V., 1982. Glucocorticoid receptors in childhood acute lymphocytic leukemia. Cancer Res. 42, 4801–4806. Davis, R.E., Brown, K.D., Siebenlist, U., Staudt, L.M., 2001. Constitutive nuclear factor-jB activity is required for survival of activated B cell-like diffuse large B cell lymphoma cells. J. Exp. Med. 194, 1861–1874. Distelhorst, C.W., 2002. Recent insights into the mechanism of glucocorticosteroidinduced apoptosis. Cell Death Differ. 9, 6–19. Ghosh, S., May, M.J., Kopp, E.B., 1998. NF-jB and Rel proteins: evolutionarily conserved mediators of immune responses. Annu. Rev. Immunol. 16, 225–260. Goecke, I.A., Alvarez, C., Henríquez, J., Salas, K., Molina, M.L., Ferreira, A., Gatica, H., 2007. Methotrexate regulates the expression of glucocorticoid receptor alpha
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