Midori Hirai

MDR1 genotype-related pharmacokinetics of digoxin after single oral administration in healthy Japanese subjects.

AbstractPurpose. To evaluate the MDR1 genotype frequency in the Japanese population and to study the relationship between the MDR1 genotype and the pharmacokinetics of digoxin after single oral administration in healthy subjects. Methods. The MDR1 genotype at exon 26 was determined in 114 healthy volunteers by polymerase chain reaction-restriction fragment length polymorphism. The serum concentration-time profile of digoxin was examined after single oral administration at a dose of 0.25 mg. Results. It was found that 35.1 % (40/114) of subjects were homozygous for the wild-type allele (C/C), 52.6 % (60/114) were compound heterozygotes with a mutant T-allele (C3435T) (C/T), and 12.3 % (14/114) were homozygous for the mutant allele (T/T). There was no effect of gender or age on the distribution. The serum concentration of digoxin after a single oral administration increased rapidly, attaining a steady state in all subjects; however, it was lower in the subjects harboring the T-allele. AUC0-4 h values (±SD) were 4.11 ± 0.57, 3.20 ± 0.49, and 3.27± 0.58 ng h/ml, respectively, with a significant difference between C/C and C/T or T/T. Conclusions. The serum concentration of digoxin after single oral administration was lower in the subjects harboring a mutant allele (C3435T) at exon 26 of the MDR1 gene.

Chitosan Induces Apoptosis via Caspase‐3 Activation in Bladder Tumor Cells

Recently, because of its low toxicity and biological effects, chitosan has been widely used in the medical and pharmaceutical fields, e.g., for nasal or oral delivery of peptide or polar drug delivery. Here, we report a growth‐inhibitory effect of chitosan on tumor cells. The growth inhibition was examined by WST‐1 colorimetric assay and cell counting. We also observed DNA fragmentation, which is characteristic of apoptosis, and elevated caspase‐3‐like activity in chitosan‐treated cancer cells. The findings suggest that chitosan may have potential value in cancer therapy.

MDR1 up-regulated by apoptotic stimuli suppresses apoptotic signaling.

AbstractPurpose. Recently, MDR1 (P-glycoprotein) and related transporters have been suggested to play a fundamental role in regulating apo- ptosis, but little information is available concerning the role of MDR1. Here, the effect of apoptotic stimuli on the MDR1 mRNA and apoptotic signaling was examined in MDR1-overexpressing cells. Methods. The expression levels of mRNA for MDR1, MRP1, MRP2, p53, p21, Bax, and Bcl-2 were measured by real time quantitative polymerase chain reaction in HeLa and its MDR1-overexpressing sublines. The effects of apoptotic stimuli by cisplatin (CDDP) on their levels were also assessed as well as on caspase 3, 8, and 9 activities. Results. MDR1 was rapidly upregulated when the cells were exposed to apoptotic stimuli by CDDP. The increase in Bax mRNA to Bcl-2 mRNA ratio after treatment with CDDP was suppressed in MDR1-overexpressing cells. The increases in caspase 3 and 9 activities after treatment with CDDP were suppressed in MDR1-overexpression cells. Conclusion. MDR1 is upregulated by apoptotic stimuli suppressed apoptotic signaling presumably via the mitochondrial pathway.

Multidrug Resistance‐associated Protein‐mediated Multidrug Resistance Modulated by Cyclosporin A in a Human Bladder Cancer Cell Line

A doxorubicin‐resistant subline (5637/DR5.5) from human bladder cancer cells (5637) was induced by stepwise increase in the doxorubicin concentration. 5637/DR5.5 cells were cross‐resistant to vinblastine and etoposide but not to mitomycin C and cisplatin. We analyzed the mdr1, MRP (multidrug resistance‐associated protein), and DNA topoisomerase II gene expression using the reverse transcription polymerase chain reaction assay (RT‐PCR) and investigated possible differences in the accumulation and efflux of radiolabeled daunorubicin. 5637/DR5.5 cells do not express the mdr1 gene, but the expression levels of MRP are markedly higher than in drug‐sensitive 5637 cells. The intracellular accumulation of radiolabeled daunorubicin was markedly decreased in the 56377 DR5.5 cells in comparison with the parent cells. This reduced drug accumulation was associated with an enhanced drug efflux, but was reversed when cells were incubated with cyclosporin A. Cyclosporin A at the concentration of 5 (M caused 3.4‐fold enhancement of daunorubicin‐sensitivity in the 56377 DR5.5 cells. On the other hand, there was no difference in DNA‐topoisomerase II activity between the parent and resistant cells. The resistance of the 5637/DR5.5 cells is therefore associated with an enhanced drug efflux mediated by the MRP gene overexpression, as distinct from P‐glycoprotein, and is modulated by cyclosporin A.

CEA producing primary hepatic carcinoid

Imaging studies of a hepatic tumor in a 53-year-old woman with elevated serum levels of neuron-specific enolase (NSE), carcinoembryonic antigen (CEA) and 5-hydroxyindole acetic acid (5HIAA) revealed a hypervascular tumor in the right lobe. Grossly, the brownish tumor was measured 13.5x12 cm with four daughter nodules. Microscopically, the majority of these columnar and round tumor cells had ribbon-or rosette-like patterns with the expression of neuroendocrine marker proteins, such as Grimelius, NSE, chromogranin A, and synaptophysin, and moderate expression of CEA but without the expression of cytokeratin nos 7,8,14,18,19 and OV-6; the minority had glandular patterns with a strong expression of CEA but without the expression of cytokeratin nos 7,8,14,18,19 and OV-6. Ultrastructurally, most tumor cells contained populations of electron-dense core granules ranging between 100 and 200 nm in diameter. After hepatectomy, serum CEA, NSE, and 5HIAA reverted to normal ranges and persisted for 19 months. These findings suggested that the diagnosis of primary hepatic carcinoid was tenable and that the tumor might derive from hepatic stem cells which acquired the additional nature of producing CEA without cytokeratins characteristic of hepatocytes or bile duct cells. Some molecular based approaches have attributed unique biological behavior and histogenesis to this carcinoid tumor.

Eosionophilic pseudotumor of the liver due to Ascaris suum infection

A case of eosinophilic pseudotumor of the liver due to Ascaris (A) suum is described in a 34-year-old-man with a high serum level of immunoglobulin E and hypereosinophilia ascribed to a history of atopic dermatitis since childhood. Multiple hepatic hypoechoic nodules detected by ultrasound were confirmed as low-density nodules on computed tomography (CT), and as low and high signal intensity lesions on T1-and T2-weighted magnetic resonance imaging (MRI), respectively. CT during arteriography (CTA) and arterial portography revealed multiple nodules with ring-shaped enhancement and perfusion defect, respectively. Biopsied liver tissue specimens did not contain tumor cells or atypical cells; instead, they showed marked infiltration of eosinophils with necrosis and Charcot-Leyden crystals in the portal tracts and hepatic sinusoides, suggesting parasitic infection, although neither larvae nor eggs were detected. The diagnosis of visceral larva migrans (VLM) due to A. suum was based on immunoserological tests. The patient was a habitual consumer of raw bovine liver, which may explain the A. suum infection. After drug therapy with albendazole, the hypoechoic nodules disappeared. Differential diagnoses and the possible transfection route of A. suum are discussed.

Phospholipase A2 products retain a neuron specific γ isoform of PKC on the plasma membrane through the C1 domain—a molecular mechanism for sustained enzyme activity

To clarify molecular mechanism for sustained activation of gamma protein kinase C (gammaPKC), a neuron-specific subtype, we investigated the involvement of phospholipase A2 (PLA2) products in the membrane association of gammaPKC upon activation of G protein coupled purinoceptors in CHO-K1 and NG 108-15 cells. In addition, the functional domain responsible for PLA2-product mediated retention of gammaPKC on the plasma membrane was determined by simultaneously monitoring two different fluorescence-tagged gammaPKCs and mutants in the same living CHO-K1 cells. Purinoceptor activation by UTP induced a transient translocation of gammaPKC from the cytoplasm to the plasma membrane. Interestingly, PLA2 inhibitors, bromoenol lactone (BEL) and arachidonyl-trifluoromethyl ketone (AACOF3), shortened the retention time of gammaPKC on the plasma membrane in cells treated with UTP, while a DAG kinase inhibitor did not affect it. The C1 domain deficient mutant (DeltaC1-gammaPKC) also showed short membrane association compared with wild type gammaPKC, when cells are treated with UTP or arachidonic acid (AA) plus a Ca(2+) ionophore. However, deletion of C1A or C1B subdomains (DeltaC1A-gammaPKC or DeltaC1B-gammaPKC) did not alter the retention time on the plasma membrane, whereas PLA2 inhibitor shortened the retention times of both mutants. These results indicate that PLA2 products prolong the retention of gammaPKC on the plasma membrane through the C1A and/or C1B subdomain in purinoceptor-stimulated CHO-K1 cells. The importance of PLA2 product and C1 domain for the retention of gammaPKC on the membrane was also confirmed using neuronal cell line, suggesting that these are part of molecular machinery for sustaining enzyme activity in neurons.

Development of multicentric hepatocellular carcinoma after completion of interferon therapy

6 international units (IU) of IFNα, 3 days a week for a total of 24 weeks. After the IFN therapy, the patient demonstrated a normal serum ALT level, and was continuously negative for HCV-RNA, and histology improved from chronic active hepatitis to chronic persistent hepatitis. Follow-up studies with ultrasonography (US) every 3 months and computed tomography (CT) every 6 months revealed no space-occupying lesion (SOL) for 3 years after IFN treatment.US-guided biopsies of two 15-mm hypoechoic SOLs in segments eight (S8) and seven (S7) 34 and 74 months, respectively, after IFN treatment showed well-differentiated hepatocellular carcinoma (HCC). Clinical data, imaging studies, and histologic examinations showed that both tumors were multicentric HCC. Further studies may provide insights into the possible role of HCV in hepatocarcinogenesis in patients demonstrating HCV eradication by IFN treatment.

Apoptotic pathway related to oval cell proliferation

Background and Aim:  Oval cells, liver stem cell‐derived cells, are generated from the liver periportal region and spread into the parenchyma by an autocrine signaling pathway. The mechanism behind how oval cells take their place among packed silent hepatocytes, however, is not well understood. We hypothesized that apoptosis involves a decrease in hepatocytes surrounding oval cells.