Analysis of hair and plasma samples for methotrexate (MTX) and metabolite using high-performance liquid chromatography triple quadrupole mass spectrometry (LC-MS/MS) detection.

Methotrexate (MTX), a folate antagonist, is the anchor drug used to treat several diseases. Therapeutic effects are attributed to intracellular levels of various methotrexate conjugates that are present in the cell as polyglutamates (MTX-Glu). The present study was conducted to develop a new liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS)-based assay to separately quantitate the MTX-Glu in hair cells, red blood cells, and serum using internal standards. Sample preparation consisted of extraction with an organic solution followed by solid-phase extraction. The presented methodology was applied for the analysis of methotrexate and its polyglutamates in hair cells, red blood cells, and serum obtained from clinical patients. The developed LC-ESI-MS/MS method for the quantitative measurement of MTX-Glu was both sensitive and precise within the clinically relevant range. This method is possibly be superior with respect to sensitivity, selectivity, and speed than all previously described approaches and can be easily applied in routine clinical tests owing to the combination of a simple pretreatment process with robust LC-MS/MS.

Serum Exosomal miRNA-1915-3p Is Correlated With Disease Activity of Korean Rheumatoid Arthritis.

BACKGROUND/AIM: It has been found that microRNAs (miRNA) affect rheumatoid arthritis (RA) pathophysiology. This study aimed to identify novel serum exosomal miRNAs related to RA disease activity in patients with an inadequate treatment response. PATIENTS AND METHODS: The sample population comprised clinical remission (CR) and non-clinical remission (non-CR) groups of RA patients. To identify potent miRNA markers for RA disease activity, miRNA array and qPCR were performed after patient serum exosomes preparation. RESULTS: Has-miR-1915-3p and has-miR-6511b-5p were significantly higher in the serum exosomes of the CR group. The level of serum C-reactive protein (CRP) was negatively correlated with has-miR-1915-3p level in serum exosomes. CONCLUSION: Has-miR-1915-3p may be a potential marker for Korean RA disease activity.

Long-term Treatment with Anti-platelet Agents for Collagen-induced Arthritis Improves Radiological Findings.

OBJECTIVES: The objectives of this study were to evaluate the long-term effect of anti-platelet treatment on the radiological progression of collagen-induced arthritis in rats. METHODS: Female Lewis rats with collagen-induced arthritis were divided into three experimental groups: saline, aspirin monotherapy (n = 12), and aspirin-clopidogrel dual therapy (n = 12). Drugs were administered daily and continued up to 70 days after the induction of arthritis. The clinical arthritis index (weight, morphology score, and paw thickness) and radiological scores were evaluated. RESULTS: The clinical arthritis index peaked on day 20, while the radiological scores peaked on day 35. No intergroup difference was observed in the clinical arthritis index throughout the experiment. The aspirin-clopidogrel dual therapy group had a significantly higher mean radiological score than the other groups (p = 0.045) on day 35. Further treatments resulted in significantly improved radiological findings in the aspirin monotherapy and aspirin-clopidogrel dual therapy groups on day 70 but no significant improvement in the saline group. CONCLUSION: Anti-platelet agent treatment improved radiological findings on day 70. These observations emphasize the importance of a future long-term study of the effects of anti-platelet agent treatment on arthritis.

Paralog Specificity Determines Subcellular Distribution, Action Mechanism, and Anticancer Activity of TRAP1 Inhibitors.

Although Hsp90 inhibitors can inhibit multiple tumorigenic pathways in cancer cells, their anticancer activity has been disappointingly modest. However, by forcing Hsp90 inhibitors into the mitochondria with mitochondrial delivery vehicles, they were converted into potent drugs targeting the mitochondrial Hsp90 paralog TRAP1. Here, to improve mitochondrial drug accumulation without using the mitochondrial delivery vehicle, we increased freely available drug concentrations in the cytoplasm by reducing the binding of the drugs to the abundant cytoplasmic Hsp90. After analyzing X-ray cocrystal structures, the purine ring of the Hsp90 inhibitor 2 (BIIB021) was modified to pyrazolopyrimidine scaffolds. One pyrazolopyrimidine, 12b (DN401), bound better to TRAP1 than to Hsp90, inactivated the mitochondrial TRAP1 in vivo, and it exhibited potent anticancer activity. Therefore, the rationale and feasible guidelines for developing 12b can potentially be exploited to design a potent TRAP1 inhibitor.

Exosomal amyloid A and lymphatic vessel endothelial hyaluronic acid receptor-1 proteins are associated with disease activity in rheumatoid arthritis.

BACKGROUND: Exosomes are thought to play an important role in exchanging information between cells. The proteins and lipids in exosomes play roles in mediating inflammatory and autoimmune diseases. The aim of this study was to identify exosomal candidate proteins that are related to other inflammatory parameters in rheumatoid arthritis (RA). METHODS: The study population consisted of 60 patients with RA: 30 in the clinical remission (CR) group with a Disease Activity Score in 28 joints based on erythrocyte sedimentation rate (DAS28-ESR) ≤2.6 and 30 in the non-clinical remission (non-CR) group with a DAS28-ESR >2.6. Preparation of exosomes from patient serum samples was performed with the ExoQuick kit, and protein identification/quantification was performed using tandem mass tag labeling/mass spectrometry and an enzyme-linked immunosorbent assay. Comparisons between groups were made using Student's t test or the Mann-Whitney U test, as appropriate. Spearman's correlation coefficients (ρ) were calculated. RESULTS: We identified six candidate proteins. Exosomal levels of amyloid A (AA) and lymphatic vessel endothelial hyaluronic acid receptor-1 (LYVE-1) differed between the CR and non-CR groups. Both serum and exosomal AA levels were higher in the non-CR group than in the CR group (p = 0.001). Significant positive correlations were found between exosomal AA and C-reactive protein (CRP) as well as between serum AA and CRP (ρ = 0.614, p = 0.001, and ρ = 0.624, p = 0.001, respectively). Although serum levels of LYVE-1 did not differ between the non-CR and CR groups, exosomal levels of LYVE-1 were lower in the non-CR group than in the CR group (p = 0.01). We identified positive correlations between serum/exosomal LYVE-1 and CRP only in the non-CR group (serum ρ = 0.376, p = 0.04; exosome ρ = 0.545, p = 0.002). CONCLUSIONS: Exosomal LYVE-1 shows potential for use as an additional marker of disease activity in patients with RA, and exosomes may carry other useful markers for RA.

Relaxin Receptor RXFP1 and RXFP2 Expression in Ligament, Tendon, and Shoulder Joint Capsule of Rats.

Numerous musculoskeletal disorders are caused by thickened ligament, tendon stiffness, or fibrosis of joint capsule. Relaxin, a peptide hormone, can exert collagenolytic effect on ligamentous and fibrotic tissues. We hypothesized that local injection of relaxin could be used to treat entrapment neuropathy and adhesive capsulitis. Because hormonal effect depends on the receptor of the hormone on the target cell, it is important to confirm the presence of such hormonal receptor at the target tissue before the hormone therapy is initiated. The aim of this study was to determine whether there were relaxin receptors in the ligament, tendon, and joint capsular tissues of rats and to identify the distribution of relaxin receptors in these tissues. Transverse carpal ligaments (TCLs), inguinal ligaments, anterior cruciate ligaments (ACLs), Achilles tendons, and shoulder joint capsules were obtained from male Wistar rats. Western blot analysis was used to identify relaxin receptor isoforms RXFP1 and RXFP2. The distribution of relaxin receptors was determined by immunohistochemical staining. The RXFP1 isoform was found in all tissues examined. The RXFP2 isoform was present in all tissues but the TCLs. Its expression in ACLs tissues was relatively weak compared to that in other tissues. Our results revealed that RXFP1 and RXFP2 were distributed in distinctly different patterns according to the type of tissue (vascular endothelial cells, fibroblast-like cells) they were identified.

Rapid DNA extraction from dried blood spots on filter paper: potential applications in biobanking.

OBJECTIVES: Dried blood spot (DBS) technology is a microsampling alternative to traditional plasma or serum sampling for pharmaco- or toxicokinetic evaluation. DBS technology has been applied to diagnostic screening in drug discovery, nonclinical, and clinical settings. We have developed an improved elution protocol involving boiling of blood spots dried on Whatman filter paper. METHODS: The purpose of this study was to compare the quality, purity, and quantity of DNA isolated from frozen blood samples and DBSs. We optimized a method for extraction and estimation of DNA from blood spots dried on filter paper (3-mm FTA card). A single DBS containing 40 µL blood was used. RESULTS: DNA was efficiently extracted in phosphate-buffered saline (PBS) or Tris-EDTA (TE) buffer by incubation at 37°C overnight. DNA was stable in DBSs that were stored at room temperature or frozen. The housekeeping genes GAPDH and beta-actin were used as positive standards for polymerase chain reaction (PCR) validation of general diagnostic screening. CONCLUSION: Our simple and convenient DBS storage and extraction methods are suitable for diagnostic screening by using very small volumes of blood collected on filter paper, and can be used in biobanks for blood sample storage.

Profiling and semiquantitative analysis of the cell surface proteome in human mesenchymal stem cells.

Mulitpotent mesenchymal stem cells (MSCs) derived from human bone marrow are promising candidates for the development of cell therapeutic strategies. MSC surface protein profiles provide novel biological knowledge concerning the proliferation and differentiation of these cells, including the potential for identifying therapeutic targets. Basic fibroblast growth factor (bFGF) affects cell surface proteins, which are associated with increased growth rate, differentiation potential, as well as morphological changes of MSCs in vitro. Cell surface proteins were isolated using a biotinylation-mediated method and identified using a combination of one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry. The resulting gel lines were cut into 20 bands and digested with trypsin. Each tryptic fragment was analyzed by liquid chromatography-electrospray ionization tandem mass spectrometry. Proteins were identified using the Mascot search program and the International Protein Index human database. Noble MSC surface proteins (n = 1,001) were identified from cells cultured either with (n = 857) or without (n = 667) bFGF-containing medium in three independent experiments. The proteins were classified using FatiGO to elucidate their function. We also confirmed the proteomics results using Western blotting and immunofluorescence microscopic analysis. The nature of the proteins identified makes it clear that MSCs express a wide variety of signaling molecules, including those related to cell differentiation. Among the latter proteins, four Ras-related Rab proteins, laminin-R, and three 14-3-3 proteins that were fractionated from MSCs cultured on bFGF-containing medium are implicated in bFGF-induced signal transduction of MSCs. Consequently, these finding provide insight into the understanding of the surface proteome of human MSCs.

Proteome analysis of human cerebrospinal fluid as a diagnostic biomarker in patients with meningioma.

BACKGROUND: To identify meningioma-specific proteins, cerebrospinal fluid (CSF) from 4 patients with a meningioma and 4 patients with a non-brain tumorous lesion were analyzed. MATERIAL/METHODS: Two-dimensional electrophoresis and electrospray quadrupole time-of-flight tandem mass spectrometry analyses revealed 10 unique spots, containing 11 independent proteins (spot #2 and #4 each contained 2 proteins and spot #3 was not identified) were evident in CSF associated with human meningioma: serum albumin precursor (3 different isoforms), Apolipoprotein E (Apo E), Apolipoprotein J precursor (Apo J), Transthyretin precursor (TTR), Prostaglandin D2 synthase 21 kDa (PTGDS), proapolipoprotein, Chain D hemoglobin Ypsilanti, alpha-1-antitrypsin (AAT), and beta-2-microglobulin precursor (ß2M). RESULTS: The contents of Apo E, Apo J and AAT were increased, while PTGDS, TTR and ß2M were decreased. CONCLUSIONS: The results observed by 2-dimensional electrophoresis were verified by Western blot analysis. The unique proteins may represent possible candidate biomarkers of meningioma.

Gene Expression Profile of T-cell Receptors in the Synovium, Peripheral Blood, and Thymus during the Initial Phase of Collagen-induced Arthritis.

BACKGROUND: Current management strategies attempt to diagnose rheumatoid arthritis (RA) at an early stage. Transcription profiling is applied in the search for biomarkers for detecting early-stage disease. Even though gene profiling has been reported using several animal models of RA, most studies were performed after the development of active arthritis, and conducted only on the peripheral blood and joint. Therefore, we investigated gene expression during the initial phase of collagen-induced arthritis (CIA) before the arthritic features developed in the thymus in addition to the peripheral blood and synovium. METHODS: For gene expression analysis using cDNA microarray technology, samples of thymus, blood, and synovium were collected from CIA, rats immunized only with type II collagen (Cll), rats immunized only with adjuvant, and unimmunized rats on days 4 and 9 after the first immunization. Arrays were scanned with an Illumina bead array. RESULTS: Of the 21,910 genes in the array, 1,243 genes were differentially expressed at least 2-fold change in various organs of CIA compared to controls. Among the 1,243 genes, 8 encode T-cell receptors (TCRs), including CD3ζ, CD3δ, CD3ε, CD8α, and CD8ß genes, which were down-regulated in CIA. The synovium was the organ in which the genes were differentially expressed between CIA and control group, and no difference were found in the thymus and blood. Further, we determined that the differential expression was affected by adjuvant more than Cll. The differential expression of genes as revealed by real-time RT-PCR, was in agreement with the microarray data. CONCLUSION: This study provides evidence that the genes encoding TCRs including CD3ζ, CD3δ, CD3ε, CD8α, and CD8ß genes were down-regulated during the initial phase of CIA in the synovium of CIA. In addition, adjuvant played a greater role in the down-regulation of the CD3 complex compared to CII. Therefore, the down-regulation of TCR gene expression occurred dominantly by adjuvant could be involved in the pathogenesis of the early stage at CIA.

Associations of vitamin d binding protein gene polymorphisms with the development of peripheral arthritis and uveitis in ankylosing spondylitis.

OBJECTIVE: Genetic factors account for more than 90% of overall susceptibility to ankylosing spondylitis (AS), and recent studies have focused on non-major histocompatibility complex genes. Vitamin D binding protein (DBP) is a highly polymorphic protein that transports vitamin D and its metabolites. In addition to its sterol binding capacity, DBP has many other roles in the inflammatory and immune systems, and has been reported to be associated with autoimmune diseases. We investigated the association between DBP polymorphisms and susceptibility to AS. METHODS: This case-control study was conducted in 223 patients with AS and 239 ethnically matched controls who were genotyped for 8 single-nucleotide polymorphisms (SNP) in the DBP and its promoter. Genomic DNA was isolated from peripheral blood leukocytes using the standard phenolchloroform method, and the GoldenGate assay was used for genotyping. RESULTS: No significant association was found between the susceptibility to AS and DBP polymorphisms. In a subgroup analysis of patients with AS, G alleles at rs222016 and rs222020 (OR 0.63, 95% CI 0.42-0.95, p = 0.03; OR 0.63, 95% CI 0.42-0.95, p = 0.03, respectively) and A allele at rs3733359 (OR 0.59, 95% CI 0.39-0.90, p = 0.01) showed the decreased risk of peripheral arthritis. G allele at rs4752 showed increased risk of uveitis (OR 2.04, 95% CI 1.12-3.72, p = 0.02). On the haplotype analyses, haplotype 2 (AGGA) protected against the development of peripheral arthritis (p = 0.01) and haplotype 3 (GAAG) was associated with an increased likelihood of uveitis (p = 0.02). CONCLUSION: DBP gene polymorphisms are associated with the development of peripheral arthritis and uveitis in Korean patients with AS. Given the influence of different DBP variants on the immune system, larger-scale studies are warranted to elucidate the role of DBP in the pathogenesis of AS.

Identification of trimethylation at C-terminal lysine of pilin in the cyanobacterium Synechocystis PCC 6803.

Various post-translational modifications (PTMs) of pilin in Synechocystis sp. PCC 6803 have been proposed. In this study, we investigated previously unidentified PTMs of pilin by mass spectrometry (MS). MALDI-TOF MS and TOF/TOF MS showed that the molecular mass of the C-terminal lysine of pilin was increased by 42Da, which could represent acetylation (ΔM=42.0470) or trimethylation (ΔM=42.0106). To discriminate between these isobaric modifications, the molecular mass of the C-terminal tryptic peptide was measured using 15T Fourier transform ion cyclotron resonance (FT-ICR) MS. The high magnetic field FT-ICR provided sub-ppm mass accuracy, revealing that the C-terminal lysine was modified by trimethylation. We could also detect the existence of mono- and di-methylation of the C-terminal lysine. Cells expressing a pilin point mutant with glutamine replacing the C-terminal lysine showed dramatically reduced motility and short pili. These findings suggest that trimethylation of pilin at the C-terminal lysine may be essential for the biogenesis of functional pili.

Effect of calcium phosphate cements on growth and odontoblastic differentiation in human dental pulp cells.

OBJECTIVE: Calcium phosphate cements (CPCs) are an interesting class of bone substitute materials. However, the biological effects of CPCs have not been well studied in human dental pulp cells (HDPCs). The purpose of this study was to investigate the effects of CPCs on the mechanical properties, growth, and odontoblastic differentiation in HDPCs compared with Portland cement (PC) and mineral trioxide aggregate (MTA). METHODS: Experimental CPCs either containing chitosan (Ch-CPC) or without chitosan (CPC) were composed from the alpha-tricalcium phosphate powder. Setting time, compressive strength measurements, cell growth, alkaline phosphatase (ALP) activity, the levels of messenger RNA for differentiation-related genes, and mineralization of the HDPCs on various cements were assessed. RESULTS: The setting time for CPC-Ch was 7.5 minutes, which was significantly less than the 8.6 minutes for the CPC. On the seventh day of immersion, the compressive strength of CPC-CH reached 13.1 MPa, which was higher than 10.8 MPa of CPC. CPC and Ch-CPC-treated cells showed decreased cell proliferation but increased the levels of ALP activity, enhanced mineralized nodule formation, and upregulated odontoblastic markers messenger RNA including osteonectin, osteopontin, bone sialoprotein, dentin matrix protein-1, matrix extracellular phosphoglycoprotein, and dentin sialophosphoprotein (DSPP), compared with untreated control. The response of CPC and CP-CPC were similar to that of PC and MTA. However, the adhesion, growth, and differentiation in Ch-CPC-treated cells were similar to that in the CPC. CONCLUSION: CPC may be useful for pulp-capping applications based on its abilities to promote HDPC differentiation.

Differential expression of cell surface proteins in human bone marrow mesenchymal stem cells cultured with or without basic fibroblast growth factor containing medium.

Mesenchymal stem cells (MSCs) are multipotent cells, which have the capability to differentiate into various mesenchymal tissues such as bone, cartilage, fat, tendon, muscle, and marrow stroma. However, they lose the capability of multi-lineage differentiation after several passages. It is known that basic fibroblast growth factor (bFGF) increases growth rate, differentiation potential, and morphological changes of MSCs in vitro. In this report, we have used 2-DE coupled to MS to identify differentially expressed proteins at the cell membrane level in MSCs growing in bFGF containing medium. The cell surface proteins isolated by the biotin-avidin affinity column were separated by 2-DE in triplicate experiments. A total of 15 differentially expressed proteins were identified by quadrupole-time of flight tandem MS. Nine of the proteins were upregulated and six proteins were downregulated in the MSCs cultured with bFGF containing medium. The expression level of three actin-related proteins, F-actin-capping protein subunit alpha-1, actin-related protein 2/3 complex subunit 2, and myosin regulatory light chain 2, was confirmed by Western blot analysis. The results indicate that the expression levels of F-actin-capping protein subunit alpha-1, actin-related protein 2/3 complex subunit 2, and myosin regulatory light chain 2 are important in bFGF-induced morphological change of MSCs.

Island clustering analysis for the comparison of the membrane and the soluble protein fractions of human brain proteome.

A protein identified in multiple separate bands of a 1-D gel reflects variation in the molecular weight caused by alternative splicing, endoproteolytic cleavage, or PTMs, such as glycosylation or ubiquitination. To characterize such a protein distribution over the bands, we defined an entity called an 'island' as the band region including the bands of the same protein identified sequentially. We quantified the island distribution using a new variable called an Iscore. Previously, as described in Park et al.. (Proteomics 2006, 6, 4978-4986.), we analyzed human brain tissue using a multidimensional MS/MS separation method. Here, the new method of island analysis was applied to the previous proteome data. The soluble and membrane protein fractions of human brain tissue were reanalyzed using the island distribution. The proteome of the soluble fraction exhibited more variation in island positions than that of the membrane fraction. Through the island analysis, we identified protein modifications and protein complexes over the 1-D gel bands.

The tumorigenic, invasive and metastatic potential of epithelial and round subpopulations of the SW480 human colon cancer cell line.

It has been reported that the SW480 human colon cancer cell line consists of E-type and R-type cells. The long-term tumorigenic potential, invasive and metastatic properties of these subclones have not been characterized. E-type and R-type cells were subcloned using limiting dilution methods from parental SW480 cells. The cell growth rate was determined by MTT colorimetric assay, and colony forming efficiency was analyzed using Matrigel-coated plates. The activity of matrix metalloproteinase (MMP) and of urokinase plasminogen activator (uPA) was assessed by zymography. Invasive and locomotive ability was analyzed using transwell chambers. In situ apoptosis detection of these subclones was also performed. In vivo long-term tumorigenicity and nodal metastasis were evaluated using nude mice. E-type cells produced spontaneously regressive tumors in spite of invasion and lymph node metastasis. In contrast, R-type cells revealed progressively growing tumors without invasion or metastasis. E-type cells exhibited increased apoptosis and invasive and motile ability, as well as strong MMP-9 and -2 activity. Although phorbol 12-myristate 13-acetate treatment induced MMP-9 activity in E-type cells, it had no effect on R-type cells. These findings suggest that E- and R-type cells may have different biological properties in terms of colon cancer progression, regression, invasion and nodal metastasis, and might serve as a useful model for these studies.

Profiling human brain proteome by multi-dimensional separations coupled with MS.

In our initial attempt to analyze the human brain proteome, we applied multi-dimensional protein separation and identification techniques using a combination of sample fractionation, 1-D SDS-PAGE, and MS analysis. The complexity of human brain proteome requires multiple fractionation strategies to extend the range and total number of proteins identified. According to the method of Klose (Methods Mol. Biol. 1999, 112, 67), proteins of the temporal lobe of human brain were fractionated into (i) cytoplasmic and nucleoplasmic, (ii) membrane and other structural, and (iii) DNA-binding proteins. Each fraction was then separated by SDS-PAGE, and the resulting gel line was cut into approximately 50 bands. After trypsin digestion, the resulting peptides from each band were analyzed by RP-LC/ESI-MS/MS using an LTQ spectrometer. The SEQUEST search program, which searched against the IPI database, was used for peptide sequence identification, and peptide sequences were validated by reversed sequence database search and filtered by the Protein Hit Score. Ultimately, 1533 proteins could be detected from the human brain. We classified the identified proteins according to their distribution on cellular components. Among these proteins, 24% were membrane proteins. Our results show that the multiple separation strategy is effective for high-throughput characterization of proteins from complex proteomic mixtures.

Modulation of human cytochrome P450 1B1 expression by 2,4,3',5'-tetramethoxystilbene.

We have previously shown that 2,4,3',5'-tetramethoxystilbene (TMS), a synthetic trans-stilbene analog, is one of the most potently selective inhibitors of recombinant human cytochrome P450 1B1 (CYP1B1) in vitro. In the present studies, the effects of TMS on CYP1B1 expression were investigated in human cancer cells. TMS significantly inhibited CYP1-mediated 7-ethoxyresorufin O-deethylation activity in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced MCF-7 cells or lung microsomes of Sprague-Dawley rats treated with 7,12-dimethylbenz[a]anthracene. TCDD-stimulated CYP1B1 protein and mRNA expression was significantly suppressed by TMS in a concentration-dependent manner in MCF-7, MCF-10A, and HL-60 cells. Whereas TMS down-regulated TCDD-induced CYP1B1 gene expression, the levels of aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator mRNA expression were not changed by TMS treatment. In human cancer cells, TMS induced apoptotic cell death, and the cytotoxic effects of TMS were significant when the cells were incubated with TCDD. CYP1B1 was able to convert TMS to a metabolite(s) when incubated with NADPH. Metabolic activation of TMS by CYP1B1 induced by TCDD may mediate cellular toxicity of TMS in human cancer cells because the sensitivity to TMS in MCF-7 cells treated with TCDD was more significant than in HL-60 cells treated with TCDD. Taken together, our results indicate that TMS acts as a strong modulator of CYP1B1 gene expression as well as a potent selective inhibitor in vitro. The ability of TMS to induce apoptotic cell death in tumor cells, as well as CYP1B1 inhibition, may contribute to its usefulness for cancer chemoprevention.

Potent inhibition of recombinant human cytochrome p-450 1A1 by pentamethoxystilbene.

Previously it was reported that various hydroxystilbene compounds strongly inhibit human cytochrome P-450 1 enzymes and were postulated as candidate chemopreventive agents. In this study, the inhibitory potential of P-450 1 enzyme activities by 3,5,3,4,5-pentamethoxystilbene (PMS), a synthetic stilbene compound, was evaluated with the Escherichia coli (E. coil) membranes of recombinant human cytochrome P-450 1A1, 1A2, or 1B1 coexpressed with human NADPH-P-450 reductase. PMS produced a significant inhibition of ethoxyresorufin O-deethylation (EROD) activities with IC50 values of 0.14, 934, and 3.2 M for 1A1, 1A2, and 1B1, respectively. PMS did not significantly inhibit EROD activities in human liver microsomes. To elucidate the mechanism of inhibition by PMS, kinetic studies were performed. Analysis of the mode of inhibition indicated a mixed-type inhibition of P-450 1A1. The inhibition of P-450 1A1-mediated EROD activity by PMS was not irreversible-mechanism based. The loss of EROD activity of P-450 1A1 with PMS was blocked by trapping agents such as glutathione, N-acetylcysteine, or dithiothreitol. Moreover, PMS significantly suppressed P-450 1A1-mediated EROD activity and P-450 1A1 gene expression in HepG2 cells induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Taken together, the results suggested that PMS is a potent and selective inhibitor of human P-450 1A1 and may be considered for use as a cancer chemopreventive agent in humans.

Potent inhibition of human cytochrome P450 1 enzymes by dimethoxyphenylvinyl thiophene.

Cytochrome P450 (P450) 1 enzymes such as P450 1A1, 1A2, and 1B1 are known to be involved in the oxidative metabolism of various procarcinogens and are regarded as important target enzymes for cancer chemoprevention. Previously, several hydroxystilbene compounds were reported to inhibit P450 1 enzymes and were rated as candidate chemopreventive agents. In this study, we investigated the inhibitory effect of 2-[2-(3,5-dimethoxyphenyl)vinyl]-thiophene (DMPVT), produced from the chemical modification of oxyresveratrol, on the activities of P450 1 enzymes. The inhibitory potential by DMPVT on the P450 1 enzyme activity was evaluated with the Escherichia coli membranes of the recombinant human cytochrome P450 1A1, 1A2, or 1B1 coexpressed with human NADPH-P450 reductase. DMPVT significantly inhibited ethoxyresorufin O-deethylation (EROD) activities with IC50 values of 61, 11, and 2 nM for 1A1, 1A2, and 1B1, respectively. The EROD activity in DMBA-treated rat lung microsomes was also significantly inhibited by DMPVT in a dose-dependent manner. The modes of inhibition by DMPVT were non-competitive for all three P450 enzymes. The inhibition of P450 1B1-mediated EROD activity by DMPVT did not show the irreversible mechanism-based effect. The loss of EROD activity in P450 1B1 with DMPVT incubation was not blocked by treatment with the trapping agents such as glutathione, N-acetylcysteine, or dithiothreitol. Taken together, the results suggested DMPVT to be a strong noncompetitive inhibitor of human P450 1 enzymes that should be considered as a good candidate for a cancer chemopreventive agent in humans.