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2.
Toxicology ; 218(2-3): 172-85, 2006 Feb 01.
Article in English | MEDLINE | ID: mdl-16337070

ABSTRACT

Human liver microsomes, supersomes from baculovirus-transformed insect cells expressing different human CYP450 isoforms, and control and CYP3A4 cDNA-transfected V79 Chinese hamster cells were tested for their ability to metabolize territrem B (TRB) and territrem C (TRC). Two TRB metabolites, designated MB(2) and MB(4), and one TRC metabolite, designated MC, were formed by all of these preparations. Of the nine supersomes from baculovirus-transformed insect cells expressing different human CYP450 isoforms (1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, and 3A5), only those expressing CYP3A4 or CYP3A5 metabolized TRB and TRC. MB(2), MB(4), and MC were formed by CYP3A4 cDNA-transfected V79MZ Chinese hamster cells, but not by non-transfected cells. In order to investigate which CYP450 isoforms were responsible for MB(2), MB(4) and MC formation in human liver microsomal preparations, six isoform-specific chemical inhibitors (furafylline, sulfaphenazole, omeprazole, quinidine, ketoconazole, and diethyldithiocarbamate) and antibodies against CYP3A4 were used. MB(2), MB(4), and MC formation was markedly inhibited by ketoconazole, but less affected by quinidine and sulfaphenazole. Anti-CYP3A4 antibody markedly inhibited MB(2), MB(4), and MC formation and also 6 beta-hydroxytestosterone formation from testosterone. The CYP3A-dependent reaction of testosterone 6 beta-hydroxylation showed a high correlation with 4 beta-C hydroxylation of TRB (r(2)=0.97, P<0.0001), O-demethylation of TRB (r(2)=0.95, P<0.0001), and 4 beta-C hydroxylation of TRC (r(2)=0.99, P<0.0001). Immunoblotting and RT-PCR showed that CYP3A4 and CYP3A5 were expressed in all four human liver microsomal preparations tested (HLM1-HLM4). The amount of MB(2), MB(4), and MC formed using different HLM preparations was related to the 6 beta-testosterone hydroxylase activity of the preparations. However, the extent of MB(2), MB(4), and MC formation was not related to the age or gender of the person from whom the microsomal sample was prepared. It was therefore suggest that CYP3A4 and CYP3A5 are the major enzymes responsible for TRB and TRC metabolism by human liver microsomes.


Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Pyrans/metabolism , Steroid Hydroxylases/metabolism , Adult , Aged , Animals , Baculoviridae/genetics , Cell Line , Cells, Cultured , Cricetinae , Cricetulus , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme System/genetics , DNA, Complementary/genetics , Female , Humans , Insecta/genetics , Male , Microsomes, Liver/metabolism , Middle Aged , Transfection
3.
Mol Pharmacol ; 68(4): 1061-73, 2005 Oct.
Article in English | MEDLINE | ID: mdl-15998873

ABSTRACT

Diosgenin, extracted from the root of wild yam (Dioscorea villosa), has been reported to demonstrate an opportunity for medical application. Vascular endothelial growth factor-A (VEGF-A) plays an important role in bone-related angiogenesis, a critical process occurring during bone formation and fracture healing. In this study, we examine whether diosgenin is able to induce VEGF-A expression and to promote angiogenesis in osteoblasts. For murine MC3T3-E1 preosteoblast-like cells, VEGF-A mRNA and protein expression seemed to be significantly elevated in response to diosgenin in a concentration-dependent fashion. Conditioned media prepared from cells treated with diosgenin induced strong angiogenic activity in either in vitro or ex vivo angiogenesis assay. Furthermore, diosgenin treatment increased the stability and activity of HIF-1alpha protein. Inhibition of HIF-1alpha activity by transfection with DN-HIF-1alpha significantly diminished diosgenin-mediated VEGF-A up-regulation. The use of pharmacological inhibitors or genetic inhibition revealed that both the phosphatidylinositol 3-kinase (PI3K)/Akt and p38 signaling pathways were potentially required for diosgenin-induced HIF-1 activation and subsequent VEGF-A up-regulation. It is noteworthy that an estrogen receptor binding assay revealed that diosgenin has the strong ability to replace [(3)H]estradiol bound to estrogen receptor (IC(50), 10 nM). In addition, the specific estrogen receptor antagonists ICI 182,780 (faslodex) and tamoxifen were noted to be able to strongly inhibit diosgenin-induced, src kinase-dependent Akt and p38 MAPK activation. Taken together, such results provide evidence that diosgenin up-regulates VEGF-A and promotes angiogenesis in preosteoblast-like cells by a hypoxia-inducible factor-1alpha-dependent mechanism involving the activation of src kinase, p38 MAPK, and Akt signaling pathways via estrogen receptor.


Subject(s)
DNA-Binding Proteins/physiology , Diosgenin/pharmacology , Neovascularization, Physiologic/drug effects , Nuclear Proteins/physiology , Osteoblasts/drug effects , Phosphatidylinositol 3-Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Receptors, Estrogen/physiology , Transcription Factors/physiology , p38 Mitogen-Activated Protein Kinases/metabolism , 3T3 Cells , Animals , Base Sequence , Culture Media, Conditioned , DNA Primers , Hypoxia-Inducible Factor 1 , Hypoxia-Inducible Factor 1, alpha Subunit , Mice , Mice, Inbred BALB C , Osteoblasts/enzymology , Osteoblasts/metabolism , Proto-Oncogene Proteins c-akt , Reverse Transcriptase Polymerase Chain Reaction , Vascular Endothelial Growth Factor A/biosynthesis
4.
Cancer Res ; 65(11): 4827-35, 2005 Jun 01.
Article in English | MEDLINE | ID: mdl-15930303

ABSTRACT

Experimental and clinical findings support the essential role of interleukin (IL)-6 in the pathogenesis of various human cancers and provide a rationale for targeted therapeutic investigations. A novel peptide, S7, which selectively binds to IL-6 receptor (IL-6R) alpha chain (gp80) and broadly inhibits IL-6-mediated events, was identified using phage display library screening. The synthetic S7 peptide specifically bound to soluble IL-6R as well as cognate human IL-6R alpha, resulting in a dose-dependent blockade of the interaction between IL-6 and IL-6R alpha. S7 peptide prevents IL-6-mediated survival signaling and sensitizes cervical cancer cells to chemotherapeutic compounds in vitro. The in vitro analysis of antiangiogenic activity showed that S7 peptide substantially inhibits IL-6-induced vascular endothelial growth factor-A expression and angiogenesis in different cancer cell lines. Furthermore, S7 peptide was bioavailable in vivo, leading to a significant suppression of IL-6-induced vascular endothelial growth factor-mediated cervical tumor growth in severe combined immunodeficient mice. These observations show the feasibility of targeting IL-6/IL-6R interaction using the small peptide and highlight its potential in the clinical applications.


Subject(s)
Angiogenesis Inhibitors/metabolism , Angiogenesis Inhibitors/pharmacology , Neoplasms/blood supply , Neoplasms/drug therapy , Oligopeptides/metabolism , Oligopeptides/pharmacology , Receptors, Interleukin-6/metabolism , Animals , Cell Line, Tumor , Female , HT29 Cells , HeLa Cells , Humans , Interleukin-6/antagonists & inhibitors , Interleukin-6/pharmacology , Mice , Mice, SCID , Neoplasms/metabolism , Neoplasms/pathology , Neovascularization, Pathologic/drug therapy , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/pathology , Peptide Library , Protein Binding , Receptors, Interleukin-6/antagonists & inhibitors , Substrate Specificity , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Vascular Endothelial Growth Factor A/biosynthesis , Xenograft Model Antitumor Assays
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