ABSTRACT
FK506, an immunosuppressive drug for T cells, reduces pain in patients with rheumatoid arthritis. However, the mechanism for pain reduction remains uncharacterized. In this study, we investigated the effect of FK506 on prostaglandin E(2) (PGE(2)) production from synovial cells in vitro. Human synovial cells were cultured with supernatant from peripheral blood mononuclear cells (PBMC) stimulated with anti-CD3 plus anti-CD28 antibodies. Cultured synovial cells with PBMC supernatant produced a high amount of PGE(2) and FK506 inhibited PGE(2) induction from synovial cells. Culture supernatant contained interleukin-1beta (IL-1beta) and TNFalpha, and FK506 suppressed both in PBMC supernatant. Anti-IL-1beta neutralizing antibody, but not anti-TNFalpha neutralizing antibody, completely inhibited PGE(2) induction by PBMC supernatant. These results suggest that FK506 suppresses inflammation by inhibiting PGE(2) production from synovial cells through suppression of IL-1beta production from leukocytes.
Subject(s)
Dinoprostone/biosynthesis , Immunosuppressive Agents/pharmacology , Leukocytes, Mononuclear/drug effects , Synovial Membrane/metabolism , Tacrolimus/pharmacology , Humans , Interleukin-1/biosynthesis , Synovial Membrane/cytology , Tumor Necrosis Factor-alpha/biosynthesisABSTRACT
FK506 (tacrolimus), an immunosuppressive drug, improves quality of life (QOL) for patients with rheumatoid arthritis (RA). However, the mechanism of FK506 behind the improvement in QOL is still uncharacterized. To explain the improvement of QOL by FK506, we investigated the effect of FK506 on spontaneous locomotor activity in rats with collagen-induced arthritis (CIA). CIA was induced in 7- to 8-week-old female Lewis rats by immunization with bovine type II collagen. After initiation of paw inflammation (paw swelling, histopathological analysis), CIA rats were therapeutically administered FK506 or methotrexate (MTX) from day 15. Therapeutic treatment with FK506 ameliorated spontaneous locomotor activity without suppressing paw inflammation in CIA rats from day 27. FK506 also improved hyperalgesia and grip strength from day 27. Therapeutic treatment with MTX did not improve spontaneous locomotor activity, and simultaneously did not recover hyperalgesia or grip strength in CIA rats. Our results indicate that spontaneous locomotor activity in CIA rats correlates mainly with hyperalgesia and muscle strength, but not paw inflammation, implying that therapeutic treatment with FK506 ameliorates spontaneous locomotor activity via improvement of hyperalgesia and muscle strength in CIA rats.
Subject(s)
Arthritis, Experimental/drug therapy , Inflammation/drug therapy , Motor Activity/drug effects , Tacrolimus/pharmacology , Animals , Arthritis, Experimental/pathology , Body Weight/drug effects , Female , Femur Head/drug effects , Femur Head/metabolism , Hyperalgesia/drug therapy , Immunosuppressive Agents/pharmacology , Inflammation/pathology , Methotrexate/pharmacology , Muscle, Skeletal/drug effects , Muscle, Skeletal/physiology , Pain/drug therapy , Proteoglycans/metabolism , Rats , Rats, Inbred LewABSTRACT
In this study, we detected genes sensitive to an histone deacetylase inhibitor, FK228 [(E)-(1S,4S,10S,21R)-7-[(Z)-ethylidene]-4,21-diisopropyl-2-oxa-12,13-dithia-5,8,20,23-tetraazabicyclo-[8,7,6]-tricos-16-ene-3,6,9,19,22-pentanone; FR901228, depsipeptide] in vitro and identified marker genes to predict sensitivity to FK228 in vivo using Affymetrix GeneChip. Three percent of genes (205/7070) were sensitive to FK228 in vitro, 105 and 100 genes, were up- and down-regulated, respectively, by FK228. Commonly up-regulated genes included p21(WAF1/Cip1), interleukin-8 (IL-8), histone family, JunB, caspase 9, mitogen-activated protein kinase phosphatase 1 (MKP-1) and mitogen-activated protein kinase (MAPK) family, and commonly down-regulated genes included cyclin A and MAPK family. One percent of genes (76/7070) showed native differences in patterns of expression, when FK228-sensitive (PC-3 prostate and SC-6-JCK (SC-6) stomach) and FK228-resistant (ACHN and A-498 renal) tumors implanted in BALB/c nu/nu mice were compared. Twenty-seven and forty nine of those genes were expressed at high or low levels, respectively, in FK228-sensitive tumors. Caspase 9 and MKP-1 genes showed distinct differences in patterns of expression between FK228-sensitive and resistant tumors and have been known to have roles in apoptosis and chromatin remodeling. The expression of caspase 9 gene was higher in FK228-sensitive tumors and the expression of MKP-1 gene was higher in FK228-resistant tumors. Caspase 9 and MKP-1 genes in the other FK228-sensitive tumors had the same patterns of expression as they did in PC-3 and SC-6 tumors. Our results present profiles of gene expression related to FK228 and marker genes to predict sensitivity to FK228, such as caspase 9 and MKP-1 genes.
Subject(s)
Depsipeptides/pharmacology , Enzyme Inhibitors/pharmacology , Histone Deacetylase Inhibitors , Animals , Caspase 9 , Caspases/genetics , Cell Cycle Proteins/genetics , Cell Line, Tumor , Dual Specificity Phosphatase 1 , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/drug effects , Humans , Immediate-Early Proteins/genetics , Male , Mice , Mice, Nude , Oligonucleotide Array Sequence Analysis , Phosphoprotein Phosphatases/genetics , Protein Phosphatase 1 , Protein Tyrosine Phosphatases/genetics , Time FactorsABSTRACT
FK506 suppresses activation of T cells; however, it down-regulates E-selectin, ICAM-1 and VCAM-1 expression in inflamed tissues. In this study, we investigated the effect of FK506 on expression of those adhesion molecules on human vascular endothelial cells (HMVEC). Culture supernatant from peripheral blood mononuclear cells (PBMC) stimulated with anti-CD3 plus anti-CD2 antibodies effectively induced the expression of E-selectin, ICAM-1 and VCAM-1 on HMVEC, and treatment with FK506 down-regulated their expression. Culture supernatant contained tumor necrosis factor (TNF) alpha and interleukin (IL)-1beta, which effectively induced adhesion molecules, and FK506 suppressed both cytokine secretions. TNFalpha content in culture supernatant was parallel to the induction of adhesion molecules by the culture supernatant. IL-1beta content was not enough to induce those adhesion molecules. Anti-TNFalpha antibody completely inhibited those expressions. FK506 did not inhibit either TNFalpha- or IL-1beta-induced expression of adhesion molecules, or viability of HMVEC. These results indicate that FK506 suppresses migration of inflammatory cells through the inhibition of TNFalpha secretion from leukocytes.
Subject(s)
E-Selectin/drug effects , Endothelium, Vascular/drug effects , Intercellular Adhesion Molecule-1/drug effects , Monocytes/drug effects , Tacrolimus/pharmacology , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Vascular Cell Adhesion Molecule-1/drug effects , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , Enzyme-Linked Immunosorbent Assay , Humans , Interleukin-1/metabolism , Interleukin-1/physiology , Monocytes/metabolism , Tumor Necrosis Factor-alpha/metabolismABSTRACT
BACKGROUND: FK506 ointment (tacrolimus ointment, protopic) is a new drug therapeutically effective for patients with atopic dermatitis (AD). However, the mechanism of action of FK506 ointment on AD is not fully understood. METHODS: We examined the effect of FK506 ointment on mite antigen-induced dermatitis in NC/Nga mice. Clinical symptoms and ear thickness were recorded, and histopathological studies and in vitro analyses were performed. RESULTS: Topical application of FK506 ointment (0.03-0.3%) suppressed the development of dermatitis. In the lesional skin, both interleukin (IL)-4 and interferon (IFN)-gamma were detected, even though the IL-4+/IFN-gamma- T helper 2 (Th2) population was predominant in the regional lymph nodes (LNs). Topical application of FK506 treatment reduced the elevated level of both IL-4 and IFN-gamma in the skin, but did not decrease the expansion of the Th2 population in the LNs. CONCLUSIONS: Topical application of FK506 ointment suppresses dermatitis by inhibiting the activation of inflammatory cells locally, without systemic immune suppression, in this AD model.
Subject(s)
Dermatitis, Atopic/drug therapy , Immunosuppressive Agents/administration & dosage , Tacrolimus/administration & dosage , Administration, Topical , Animals , Antigens, Dermatophagoides/immunology , CD4 Antigens/metabolism , Dermatitis, Atopic/immunology , Dermatitis, Atopic/pathology , Disease Models, Animal , Female , Flow Cytometry , Immunohistochemistry , Intercellular Adhesion Molecule-1/metabolism , Interferon-gamma/metabolism , Interleukin-1/metabolism , Interleukin-4/metabolism , Lymph Nodes/immunology , Lymph Nodes/metabolism , Mice , Mice, Inbred Strains , Specific Pathogen-Free Organisms , Tumor Necrosis Factor-alpha/metabolism , Vascular Cell Adhesion Molecule-1/metabolismABSTRACT
UNLABELLED: It has been recently demonstrated that histone deacetylase inhibitors inhibit angiogenesis, but their mechanism of action has not been characterized well. In this study, we examined the in vitro and in vivo effects of FK228 [(E)-(1S,4S,10S,21R)-7-[(Z)-ethylidene]-4,21-diisopropyl-2-oxa-12,13-dithia-5,8,20,23-tetraazabicyclo-[8,7,6]-tricos-16-ene-3,6,9,19,22-pentanone; FR901228, depsipeptide], an HDAC inhibitor, on the expression of angiogenesis factors in FK228-sensitive PC-3 prostate and FK228-resistant ACHN renal cancer cells. FK228 suppressed the expression of VEGF mRNA in PC-3 cells, but not in ACHN cells. FK228 also suppressed the expression of basic fibroblast growth factor (bFGF) mRNA in both PC-3 and ACHN cells. Under conditions of hypoxia, FK228 suppressed the expression of VEGF mRNA without modulating the expression of hypoxia-inducible factor-1 alpha mRNA in PC-3 cells. FK228 induced the highest acetylation of histone H3 and H4 in the P2 region of the VEGF promoter, which includes the hypoxia-inducible factor-1 alpha binding site that plays an important role in regulating the expression of VEGF gene. Moreover, FK228 reduced the amount of VEGF and bFGF protein, and their mRNA levels in PC-3 xenograft implanted in nude mice, but did not reduce them in ACHN xenograft. IN CONCLUSION: (i) FK228 showed a suppressive effect on the expression of angiogenesis factors, such as VEGF and bFGF, in PC-3 xenograft but not in ACHN xenograft, which suggests that the effect on the expression of angiogenesis factors is important for the antitumor efficacy of FK228; (ii) FK228 caused histone acetylation of the VEGF promoter regions, which may contribute to the suppression of VEGF gene expression.
Subject(s)
Antibiotics, Antineoplastic/pharmacology , Depsipeptides , Endothelial Growth Factors/metabolism , Fibroblast Growth Factor 2/metabolism , Histone Deacetylase Inhibitors , Intercellular Signaling Peptides and Proteins/metabolism , Lymphokines/metabolism , Peptides, Cyclic/pharmacology , Transcription Factors , Acetylation/drug effects , Angiogenesis Inducing Agents , Animals , Antibiotics, Antineoplastic/therapeutic use , Chromatin/metabolism , DNA-Binding Proteins/metabolism , Disease Models, Animal , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , Gene Expression/drug effects , Hypoxia-Inducible Factor 1 , Hypoxia-Inducible Factor 1, alpha Subunit , Male , Mice , Mice, Inbred BALB C , Neoplasm Transplantation , Nuclear Proteins/metabolism , Peptides, Cyclic/therapeutic use , Promoter Regions, Genetic , RNA, Messenger/drug effects , RNA, Messenger/metabolism , Tumor Cells, Cultured , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors , Xenograft Model Antitumor AssaysABSTRACT
In this study, we examined the effects of FK228 (FR901228, depsipeptide) on tumor growth and expression of p21 and c-myc genes in vivo. FK228 induced the expression of p21 mRNA and decreased c-myc mRNA in tumor xenograft sensitive to FK228. However, FK228 did not sufficiently modulate the expression of p21 mRNA and increased the expression of c-myc in tumor xenograft less sensitive to FK228. The modulation of p21 and/or c-myc genes may be critical for the marked antitumor activity of FK228 in vivo.
Subject(s)
Cyclins/biosynthesis , Depsipeptides , Enzyme Inhibitors/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Histone Deacetylase Inhibitors , Neoplasm Proteins/antagonists & inhibitors , Peptides, Cyclic/pharmacology , Protein Processing, Post-Translational/drug effects , Proto-Oncogene Proteins c-myc/biosynthesis , Acetylation/drug effects , Adenocarcinoma/drug therapy , Adenocarcinoma/metabolism , Adenocarcinoma/pathology , Adrenal Gland Neoplasms/metabolism , Adrenal Gland Neoplasms/pathology , Animals , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Carcinoma, Renal Cell/metabolism , Carcinoma, Renal Cell/pathology , Carcinoma, Small Cell/metabolism , Carcinoma, Small Cell/pathology , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/genetics , Enzyme Inhibitors/therapeutic use , Genes, myc , Humans , Kidney Neoplasms/metabolism , Kidney Neoplasms/pathology , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/genetics , Peptides, Cyclic/therapeutic use , Pheochromocytoma/metabolism , Pheochromocytoma/pathology , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , RNA, Messenger/biosynthesis , RNA, Neoplasm/biosynthesis , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/metabolism , Tumor Cells, Cultured/transplantation , Xenograft Model Antitumor AssaysABSTRACT
FK228 [(E)-(1S,4S,10S,21R)-7-[(Z)-ethylidene]-4,21-diisopropyl-2-oxa-12,13-dithia-5,8,20,23-tetraazabicyclo-[8,7,6]-tricos-16-ene-3,6,9,19,22-pentanone; FR901228, depsipeptide] is a novel histone deacetylase inhibitor that shows therapeutic efficacy in Phase I trials of patients with malignant lymphoma. However, its mechanism of action has not been characterized. In this study, we examined the in vitro and in vivo effects of FK228 on human lymphoma U-937 cells. FK228 very strongly inhibited the growth of U-937 cells with an IC(50) value of 5.92 nM. In a scid mouse lymphoma model, mice treated with FK228 once or twice a week survived longer than control mice, with median survival times of 30.5 (0.56 mg/kg) and 33 days (0.32 mg/kg), respectively (vs. 20 days in control mice). Remarkably, 2 out of 12 mice treated with FK228 (0.56 mg/kg once or twice a week) survived past the observation period of 60 days. The apoptotic population of U-937 cells time-dependently increased to 37.7% after 48 hr of treatment with FK228. In addition, FK228 induced G1 and G2/M arrest and the differentiation of U-937 cells to the CD11b(+)/CD14(+) phenotype. Expression of p21(WAF1/Cip1) and gelsolin mRNA increased up to 654- and 152-fold, respectively, after 24hr of treatment with FK228. FK228 caused histone acetylation in p21(WAF1/Cip1) promoter regions, including the Sp1-binding sites. In conclusion, (i) FK228 prolonged the survival time of scid mice in a lymphoma model, and (ii) the beneficial effects of FK228 on human lymphoma may be exerted through the induction of apoptosis, cell cycle arrest, and differentiation via the modulation of gene expression by histone acetylation.