Blue Nevus Associated With Acquired Dermal Melanocytosis on the Back.

Dermal melanocytosis includes various congenital and acquired pigmentary disorders characterized by dermal dendritic melanocytes. Blue nevi typically present as papulonodular lesions, whereas other dermal melanocytoses manifest as patches. This report describes a case of a blue nevus associated with acquired dermal melanocytosis on the back of a 46-year-old Japanese woman. The patient presented with a black nodule on a blue-greyish hyperpigmented area on the upper back. Histopathological analysis of the nodule confirmed a common blue nevus, whereas the adjacent hyperpigmented area showed features consistent with acquired dermal melanocytosis. Blue nevi and acquired dermal melanocytoses share a common pathophysiology involving ectopic melanocyte accumulation during embryogenesis. The coexistence of blue nevus and acquired dermal melanocytosis on the back is rare, highlighting the broad spectrum of dermal melanocytosis and the variability of its clinical manifestations. Recognition of such unusual presentations is critical for appropriate diagnosis and management.

Phase 1/2a clinical trial in ALS with ropinirole, a drug candidate identified by iPSC drug discovery.

iPSC-based drug discovery led to a phase 1/2a trial of ropinirole in ALS. 20 participants with sporadic ALS received ropinirole or placebo for 24 weeks in the double-blind period to evaluate safety, tolerability, and therapeutic effects. Adverse events were similar in both groups. During the double-blind period, muscle strength and daily activity were maintained, but a decline in the ALSFRS-R, which assesses the functional status of ALS patients, was not different from that in the placebo group. However, in the open-label extension period, the ropinirole group showed significant suppression of ALSFRS-R decline and an additional 27.9 weeks of disease-progression-free survival. iPSC-derived motor neurons from participants showed dopamine D2 receptor expression and a potential involvement of the SREBP2-cholesterol pathway in therapeutic effects. Lipid peroxide represents a clinical surrogate marker to assess disease progression and drug efficacy. Limitations include small sample sizes and high attrition rates in the open-label extension period, requiring further validation.

Protocadherin B9 Is Associated with Human Esophageal Squamous Cell Carcinoma Progression.

INTRODUCTION: Esophageal cancer is the sixth leading cause of cancer-related death worldwide. However, molecular targeted therapy and novel therapeutic targets are needed for esophageal squamous cell cancer (ESCC). In a previous study, we reported that protocadherin (PCDH) B9 plays an important role in several cancers. Therefore, in this study, we examined the clinical significance of PCDHB9 expression in ESCC. METHODS: PCDHB9 expression was examined using immunohistochemistry in 128 cases and using quantitative reverse transcription-polymerase chain reaction in 16 cases of ESCC. PCDHB9 function in ESCC cells was examined using RNA interference. RESULTS: High PCDHB9 expression was identified in 5 of 16 (31.3%). In total, 51 (40%) ESCC cases showed strong PCDHB9 expression, whereas nonneoplastic mucosa rarely showed its expression. High PCDHB9 expression was significantly associated with T classification, N grade, and stage in ESCC. In ESCC cell lines, PCDHB9 knockdown affected cell growth, migration, and adhesion. Further, the expression of integrin (ITG) A3, ITGA4, ITGA5, ITGB1, ITGB6, vimentin, snail family transcriptional repressor 1, and cadherin 2 (NCAD) was significantly reduced and cadherin 1 was significantly increased in PCDHB9 knockdown ESCC cells. CONCLUSION: These results suggest that PCDHB9 plays a tumor-promoting role and is a potential biomarker and therapeutic target in ESCC.

Overexpression of aldolase, fructose-bisphosphate C and its association with spheroid formation in colorectal cancer.

Colorectal cancer (CRC) is a leading cause of cancer-related death worldwide. The spheroid colony formation assay is a useful method to identify cancer stem cells (CSCs). Using the DLD-1 and WiDr CRC cell lines, we performed microarray analyses of spheroid body-forming and parental cells and demonstrated that aldolase, fructose-bisphosphate C (ALDOC) was overexpressed in the spheroid body-forming cells of both lines. Cells transfected with small interfering RNA against ALDOC demonstrated lower proliferation, migration, and invasion compared with negative control cells. Both the number and size of spheres produced by the CRC cells were significantly reduced by ALDOC knockdown. Additionally, inhibition of ALDOC reduced lactate production. Immunohistochemistry was used to analyze ALDOC protein expression in tissues from 135 CRC patients and revealed that 66 (49%) cases were positive for ALDOC. The ALDOC-positive cases were associated with higher T and M grades and, as determined by Kaplan-Meier analysis, a poorer prognosis. Univariate and multivariate analyses indicated that ALDOC expression was an independent prognostic factor for CRC patients. Furthermore, ALDOC expression was associated with CD44 expression. These results suggest that ALDOC contributes to CRC progression and plays an important role in CSCs derived from CRC.

A phase I/IIa double blind single institute trial of low dose sirolimus for Pendred syndrome/DFNB4.

INTRODUCTION: Pendred syndrome (PDS)/DFNB 4 is a disorder with fluctuating and progressive hearing loss, vertigo, and thyroid goiter. We identified pathophysiology of a neurodegenerative disorder in PDS patient derived cochlear cells that were induced via induced pluripotent stem cells and found sirolimus, an mTOR inhibitor, as an inhibitor of cell death with the minimum effective concentration less than 1/10 of the approved dose for other diseases. Given that there is no rational standard therapy for PDS, we planned a study to examine effects of low dose oral administration of sirolimus for the fluctuating and progressive hearing loss, and the balance disorder of PDS by daily monitor of their audio-vestibular symptoms. METHODS AND ANALYSIS: This is a phase I/IIa double blind parallel-group single institute trial in patient with PDS/DFNB4. Sixteen of outpatients with fluctuating hearing diagnosed as PDS in SLC26A4 genetic testing aged in between 7 and 50 years old at the time of consent are given either placebo or sirolimus tablet (NPC-12T). In NPC-12T placebo arm, placebo will be given for 36 weeks; in active substance arm, placebo will be given for 12 weeks and the NPC-12T for 24 weeks. Primary endpoints are safety and tolerability. The number of occurrences and types of adverse events and of side effects will be sorted by clinical symptoms and by abnormal change of clinical test results. A 2-sided 95% confidence interval of the incidence rate by respective dosing arms will be calculated using the Clopper-Pearson method. Clinical effects on audio-vestibular tests performed daily and precise physiological test at each visit will also be examined as secondary and expiratory endpoints. TRIAL REGISTRATION NUMBER: JMA-IIA00361; Pre-results.

Regulation of insulin-like growth factor binding protein-1 and lipoprotein lipase by the aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (Ahr), a ligand-activated transcriptional factor, mediates the transcriptional activation of a battery of genes encoding drug metabolism enzymes. In the present study, we investigated the hepatic mRNA expression profile in Ahr-null (Ahr KO) mice compared to wild-type mice by microarray analysis to find new Ahr target genes. Pooled total RNA samples of liver extracted from 7- and 60-week-old Ahr KO or wild-type mice were studied by DNA microarray representing 19,867 genes. It was demonstrated that 23 genes were up-regulated and 20 genes were down-regulated over 2 fold in Ahr KO mice compared with wild-type mice commonly within the different age groups. We focused on insulin-like growth factor binding protein-1 (Igfbp-1) and lipoprotein lipase (Lpl) that were up-regulated in Ahr KO mice. The higher expression in Ahr KO mice compared to wild-type mice were confirmed by real-time RT-PCR analysis. In the wild-type mice but not in the Ahr KO mice, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treatment increased the Igfbp-1 and Lpl mRNA levels. The expression profile of Igfbp-1 protein was consistent with that of Igfbp-1 mRNA. Since Lpl is the primary enzyme responsible for hydrolysis of lipids in lipoproteins, the serum triglyceride levels were determined. Indeed, the serum triglyceride levels in Ahr KO mice was lower than that in wild-type mice in accordance with the Lpl mRNA levels. Contrary to our expectation, TCDD treatment significantly increased the serum triglyceride levels in wild-type, but did not in Ahr KO mice. These results suggest that serum triglyceride levels are not correlated with hepatic Lpl expression levels. In the present study, we found that Ahr paradoxically regulates Igfbp-1 and Lpl expressions in the liver.

Genetic polymorphisms of CYP2B6 affect the pharmacokinetics/pharmacodynamics of cyclophosphamide in Japanese cancer patients.

OBJECTIVE: To evaluate the effects of genetic polymorphisms of drug metabolizing enzymes on the pharmacokinetics of cyclophosphamide and its active metabolite, 4-hydroxycyclophosphamide, and on the pharmacodynamics. EXPERIMENTAL DESIGN: One hundred and three Japanese patients with malignant lymphoma or breast cancer treated with cyclophosphamide (500-750 mg/m) participated in this study. The plasma concentrations of cyclophosphamide and 4-hydroxycyclophosphamide were determined by high-performance liquid chromatography, and pharmacokinetic parameters were calculated. The genotypes of CYP2B6, CYP2C19, CYP3A4, CYP3A5, ALDH1A1, GST genes were determined by allele-specific polymerase chain reaction or polymerase chain reaction-restriction-fragment length polymorphism. RESULTS: A large interindividual difference (54-fold) was observed in the area under the curve ratio of 4-hydroxycyclophosphamide/cyclophosphamide calculated as the metabolic index. We first proved that leukocytopenia and neutropenia were significantly (P<0.01) related to the area under the curve of 4-hydroxycyclophosphamide. We found that the homozygotes of CYP2B6*6 (Q172H and K262R) showed significantly (P<0.05) higher clearance and shorter half-life of cyclophosphamide than heterozygotes and homozygotes of CYP2B6*1. The small sample size, however, limited the impact. On the other hand, it was clearly demonstrated that the patients possessing the single nucleotide polymorphisms of the CYP2B6 gene, g.-2320T>C, g.-750T>C (5'-flanking region), g.15582C>T (intron 3), or g.18492T>C (intron 5), had significantly lower area under the curve ratios of 4-hydroxycyclophosphamide/cyclophosphamide, indicating a decreased cyclophosphamide 4-hydroxylation. Of particular importance was the finding that leukocytopenia was significantly related to the single nucleotide polymorphisms g.-2320T>C, g.-750T>C, and g.18492T>C in CYP2B6 gene, which are highly linked. No relationship was observed between the pharmacokinetics of cyclophosphamide or 4-hydroxycyclophosphamide and genetic polymorphisms of the other enzymes. CONCLUSIONS: We clarified that the single nucleotide polymorphisms in the promoter region or introns in the CYP2B6 affect the potency of cyclophosphamide activation to 4-hydroxycyclophosphamide. This information would be valuable for predicting adverse reactions and the clinical efficacy of cyclophosphamide.

Pharmacokinetics of paclitaxel in ovarian cancer patients and genetic polymorphisms of CYP2C8, CYP3A4, and MDR1.

Interindividual differences in the pharmacokinetics of paclitaxel and its metabolites in Japanese ovarian cancer patients were investigated in relation to genetic polymorphisms of the CYP2C8, CYP3A4, and MDR1 genes. The area under the concentration-time curve (AUC) ratios of paclitaxel/6alpha-hydroxypaclitaxel and paclitaxel/3 -p-hydroxypaclitaxel calculated as the metabolic index of CYP2C8 and CYP3A4 showed 13- and 12-fold interindividual variations, respectively. No patient had any CYP2C8 variants, while 2 patients were heterozygotes of CYP3A4*16. For the MDR1 gene, the frequencies of -129C, 1236C, 2677T, 2677A, and 3435T alleles were 2.2%, 8.7%, 56.5%, 4.4%, and 52.2%, respectively. Subjects possessing the 3435T allele had a significantly (P < .05) higher AUC of 3'- p-hydroxypaclitaxel compared to those possessing the 3435C allele. Leukocytopenia was significantly (P < .05) related to the AUC of paclitaxel. Genotyping of the CYP2C8, CYP3A4, and MDR1 genes might not be essential to predict adverse effects of paclitaxel in Japanese patients, although an allelic variant of MDR1 may functionally affect the pharmacokinetics of its metabolite.

A novel polymorphism of human CYP2A6 gene CYP2A6*17 has an amino acid substitution (V365M) that decreases enzymatic activity in vitro and in vivo.

Cytochrome P450 (CYP) 2A6 is a major CYP responsible for the metabolism of nicotine and coumarin in humans. We identified a novel allele, designated CYP2A6*17 , which contains A51G (exon 1), C209T (intron 1), G1779A (exon 3), C4489T (intron 6), G5065A (V365M, exon 7), G5163A (intron 7), C5717T (exon 8), and A5825G (intron 8). We developed a genotyping method by polymerase chain reaction-restriction fragment length polymorphism for the CYP2A6*17 allele, targeting the G5065A mutation. The allele frequency in black subjects (n = 96) was 9.4% (95% confidence interval [CI], 5.3%-13.5%). The allele was not found in white subjects (95% CI, 0%-0.9%; n = 163), Japanese subjects (95% CI, 0%-1.6%; n = 92), and Korean subjects (95% CI, 0%-0.7%; n = 209). To examine the effects of the amino acid change in the CYP2A6*17 allele on the enzymatic activity, we expressed a wild-type or variant (V365M) CYP2A6 together with NADPH-CYP reductase in Escherichia coli . For coumarin 7-hydroxylation, the apparent Michaelis-Menten constant value of variant CYP2A6 (1.06 +/- 0.11 micromol/L) was significantly (P < .005) higher than that of wild type (0.60 +/- 0.05 micromol/L). The maximum velocity values of the wild-type and variant CYP2A6 were 0.61 +/- 0.06 and 0.64 +/- 0.07 pmol . min -1 . pmol -1 CYP, respectively. For nicotine C -oxidation, the apparent Michaelis-Menten constant values of the wild-type or variant CYP2A6 were 31.6 +/- 2.9 micromol/L and 31.3 +/- 3.1 micromol/L, respectively. The maximum velocity value of variant CYP2A6 (0.72 +/- 0.21 pmol . min -1 . pmol -1 CYP) was significantly (P < .05) lower than that of the wild type (1.80 +/- 0.42 pmol . min -1 . pmol -1 CYP). Thus the intrinsic clearance values for coumarin 7-hydroxylation and nicotine C -oxidation by the variant were both significantly (P < .05) decreased to 40% to 60% compared with the wild type. Furthermore, cotinine/nicotine ratios after 1 piece of nicotine gum was chewed, used as an index of in vivo nicotine metabolism, were significantly (P < .05) decreased in heterozygotes of the CYP2A6*17 allele (5.4 +/- 2.7, n = 12) compared with homozygotes of the wild type (11.5 +/- 10.5, n = 37). A subject with CYP2A6*17 / CYP2A6*17 revealed the lowest cotinine/nicotine ratio (1.8). We found a novel allele in black subjects that affects the nicotine metabolism in vitro and in vivo.

Role of the aryl hydrocarbon receptor and Cyp1b1 in the antiestrogenic activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The role of aryl hydrocarbon receptor (AhR) and cytochrome P450 (Cyp) 1 family in the antiestrogenic activity of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) was investigated in vivo. Immature (21 days old) AhR, Cyp1a2, or Cyp1b1 knockout (-/-) mice were treated intraperitoneally with estradiol (E2, 20 ng/mouse per day, for 14 consecutive days) and/or TCDD (200 ng/mouse per day, on days 7, 9, 11, and 13). Uterine wet weight and uterine peroxidase activity (UPA) were measured as markers of estrogen responsiveness. UPA was a better marker of estrogen responsiveness than the uterine wet weight. In AhR wild-type (+/+) mice, UPA (208.1+/-81.6 units/g tissue) was increased by the administration of E2 (to 297.2+/-178.7 units/g). The administration of TCDD significantly ( p<0.01) decreased the UPA (10.5+/-3.4 units/g) compared with that in the control mice. Co-administration of TCDD with E2 also significantly ( p<0.05) decreased the UPA (18.8+/-19.9 units/g) compared with that in E2-treated mice. In AhR(-/-) mice, UPA (162.9+/-146.7 units/g) was significantly ( p<0.01) increased by the administration of E2 (486.8+/-108.2 units/g). In contrast to the results in AhR(+/+) mice, UPA was not affected by the administration of TCDD (51.8+/-70.6 units/g) compared with control, and co-administration of TCDD with E2 (545.8+/-189.4 units/g) compared with that in E2-treated mice. In Cyp1a2/1b1(+/+) mice, UPA was significantly ( p<0.05) increased by the administration of E2 (70.0+/-36.4 units/g). Co-administration of TCDD with E2 significantly ( p<0.05) decreased the UPA (29.6+/-22.2 units/g) compared with that in E2-treated mice. In Cyp1a2(-/-) mice, co-administration of TCDD with E2 significantly ( p<0.01) decreased the UPA (6.8+/-5.1 units/g) compared with that in E2-treated mice. In Cyp1b1(-/-) mice, UPA (5.5+/-8.1 units/g) was significantly ( p<0.05) increased by the administration of E2 (56.6+/-34.1 units/g). In contrast to the results in Cyp1a2/1b1(+/+) mice or Cyp1a2(-/-) mice, UPA was not affected by the co-administration of TCDD and E2 (52.6+/-30.1 units/g) compared with that in E2-treated mice. This is the first demonstration that Cyp1b1 as well as AhR is involved in the antiestrogenic effects of TCDD.

Genetic polymorphisms of CYP2C8 in Japanese population.

CYP2C8 plays important roles in metabolizing therapeutic drugs and endogenous compounds. Although genetic polymorphisms of CYP2C8 were reported, there is little information on CYP2C8 polymorphisms in the Japanese population. In the present study, we screened for previously described polymorphisms in the coding region of this gene using polymerase chain reaction (PCR)-restriction fragment length polymorphism or allele specific-PCR analyses. Eleven polymorphisms of CYP2C8*2 (I269F), CYP2C8*3 (R139K, K399R), CYP2C8*4 (I264M), CYP2C8*5 (frameshift), T130N, E154D, N193K, K249R, L390S, P404A, and H411L have been comprehensively investigated in at least 200 Japanese individuals. A single subject was heterozygous for CYP2C8*5, and the allele frequency was calculated as 0.0025. The other single nucleotide polymorphisms (SNPs) were not found in the Japanese subjects in the present study. Thus, it appears that the frequencies of these alleles in Japanese are extremely low. In addition, concerning the SNPs of T130N, E154D, N193K, K249R, and H411L, it remains clear that these alleles exist as polymorphisms or represent sequence errors or cloning artifacts. Although several SNPs such as CYP2C8*2, CYP2C8*3, CYP2C8*4, and P404A have been reported to reduce the enzymatic activity, pharmacokinetic abnormalities of drugs metabolized by polymorphic CYP2C8 might be rare in Japanese.