Functional analysis of dog multidrug resistance-associated protein 2 (Mrp2) in comparison with rat Mrp2.

We investigated whether the species difference in the biliary excretion activity of some Mrp2 substrates was attributable to the intrinsic transport potential or the expression level of Mrp2, especially in rat and dog. Dog Mrp2 cDNA was isolated from beagle dog liver, and a vesicle transport study was performed using recombinant rat and dog Mrp2 expressed in insect Sf9 cells. The ATP-dependent transport of 17beta-estradiol 17-(beta-D-glucuronide) ([3H]E(2)17betaG) and leukotriene C4 ([3H]LTC4), normalized by the absolute protein expression level, was similar in both Mrp2s. The Mrp2 protein expression in dog liver was only 10% of that in rat liver and was comparable with the reported difference in the biliary excretion clearance of temocaprilat as Mrp2 substrate. In contrast to LTC4, unique transport kinetics for E(2)17betaG were evident in dog Mrp2. In addition to the high-affinity site with a K(m) value of 3.25 +/- 0.10 microM, which is similar to that in rat Mrp2 (4.81 +/- 1.21 microM), dog Mrp2 has an additional low-affinity site (>75 microM), which makes a major contribution to the transport of E(2)17betaG (65% of the total transport capacity at tracer concentration). In summary, the difference in the biliary excretion activity of Mrp2 substrates between rat and dog depends on the Mrp2 protein expression level rather than the intrinsic transport activity of the transporter molecules. The unique transport properties of glucuronide conjugates by dog Mrp2 may lead to the species difference involving the drug-drug interaction or drug-induced hyperbilirubinemia on the bile canalicular membrane.     

Non-ionic detergent Tween 80 modulates VP-16 resistance in classical multidrug resistant K562 cells via enhancement of VP-16 influx

The non-ionic detergent Tween 80, which is used as a solvent for lipophilic drugs such as VP-16 and Taxotere, was found to reverse VP-16 resistance of the P-glycoprotein-associated multidrug resistance phenotype via increasing VP-16 influx. In adriamycin-resistant human chronic myelogenous leukemia K562 cells (K562/ADM), which overexpress mdr1 mRNA, the accumulation of VP-16 was only about 10% that in wild-type K562 cells. Tween 80 enhanced VP-16 accumulation in K562/ADM cells but did not influence VP-16 accumulation in parental K562 cells. VP-16 efflux was rapid and similar in both sensitive and resistant cell lines and was not blocked by Tween 80 or verapamil. Under glucose-free conditions, VP-16 accumulation in K562/ADM cells was only half of that in K562 cells. Tween 80 increased VP-16 accumulation in K562/ADM cells in glucose-free medium. In growth inhibition assay, Tween 80 reversed K562/ADM sensitivity to VP-16 without cell damage. Taken together, Tween 80 reverses VP-16 sensitivity in multidrug-resistant K562 cells by increasing influx, which is considered to be the primary mechanism of VP-16 resistance in K562/ADM cells.     

Genetic aberration on chromosome 10 in human oral squamous cell carcinoma

Recently a tumor suppressor gene, a deleted in malignant brain tumor gene (DMBT1), was detected on chromosome 10. In some types of tumors, the frequent deletion of DMBT1 locus have been reported as well as loss of heterozygosity (LOH) on chromosome 10. However, little is known relating to human oral squamous cell carcinoma (OSCC). To study the genetic aberrations on chromosome 10 in OSCC, we performed polymerase chain reaction (PCR) analysis of microsatellite polymorphisms corresponding to 16 loci, containing 2 DMBT1 loci. We examined 38 oral primary squamous cell carcinoma (SCC) tissues and corresponding normal tissues. Microsatellite instability (MI) was detected at least on 1 of the 16 loci in 15 (39.5%) of 38 cases, and loss of heterozygosity (LOH) at least 1 of the 16 loci was also observed in 28 (73.7%) of 38 cases. LOH was accumulated at D10S202 (34.6%) and D10S217 (28.6%), suggesting the presence of two putative tumor suppressor genes associated with OSCC. The 2 DMBT1 loci, D10S209 and D10S587, had comparatively high frequent LOH (20.0 and 22.7%, respectively), maybe indicating the important role of DMBT1 in OSCC. No significant correlation between histological differentiation and LOH was found. These results suggest that genetic aberrations on chromosome 10 play important roles in the oncogenesis of OSCC.     

A prospective analysis of concentration of 25-OHD between northern and southern district in Japan in year-round study

Although osteoporotic patients have already been recognized as having a low-volume vitamin D status, the concentration of active vitamin D precursor has not been studied in detail. This trial aimed to clarify the concentration of 25-hydroxyvitamin D (25-OHD), which is a natural type of vitamin D and compare between 2 separate areas in Japan. To compare and clarify the concentration of 25-OHD between 2 separate areas, Japanese patients who were diagnosed as having osteoporosis based on bone mineral density were studied. We analyzed 2 different hospitals’ patients whose residence is separated into a northern district (Akita city: north latitude 39” 43’) and a southern district (Shizuoka city: north latitude 34” 58’). Both of them have completely different daylight hours. Three-hundred sixty eight patients (174 in Akita, 194 in Shizuoka) were enrolled in this trial to compare the differences of concentration of 25-OHD by Welch’s t t-test. There were significant differences in the concentration of 25-OHD and age between them. Akita patients were significantly higher than that of Shizuoka patients despite Shizuoka having much daylight hours of Akita. In conclusion, there might be no relationship between the concentration of 25 OHD and exposure to sunlight.     

The Impact of Antiviral Therapy for Hepatitis C Virus on the Survival of Patients after Hepatocellular Carcinoma Treatment

Objective Owing to advances in direct-acting antiviral (DAA) therapy, a considerable number of patients with hepatitis C virus (HCV)-positive hepatocellular carcinoma (HCC) are now able to achieve a sustained viral response (SVR) after curative treatment of HCC. However, the beneficial effect of a DAA-SVR on the survival remains unclear. Methods A total of 205 patients with HCC who were HCV-positive with Child-Pugh A at the onset from 2008 to 2018 were categorized into 2 groups: 140 patients untreated for HCV throughout the entire course after HCC development (untreated group) and 65 patients treated for HCV with DAAs following HCC treatment who achieved an SVR (SVR group). After propensity score matching, 63 patients from each group were selected. Using these patients, the survival and maintenance of Child-Pugh A after HCC treatment were compared between the untreated group and SVR group. Results There was a significant difference in the overall survival (p<0.001) and the rate of maintaining Child-Pugh A (p<0.001) between the groups. The 5-year survival rates were 96% (SVR group) and 60% (untreated group), and the proportions of patients with Child-Pugh A at 5 years after HCC treatment were 96% (SVR group) and 38% (untreated group). Conclusion In patients with HCV-positive HCC, achieving a DAA-SVR after HCC treatment significantly improved the overall survival rate compared with HCV-untreated patients. The contribution of DAA-SVR during the course of HCC treatment to a longer survival is mainly due to the prevention of the progression of Child-Pugh A to B/C. Further research is needed to determine whether aggressive antiviral therapy is also effective for HCC patients with Child-Pugh B/C.     

Quercetin-induced PC12 cell death accompanied by caspase-mediated DNA fragmentation

Flavonoids have been reported to be potent antioxidants and beneficial in oxidative stress related diseases. Quercetin, a major flavonoid in food, deserves much attention because of its antioxidative activity. However, the actions of flavonoids including quercetin are complex and paradoxical. Quercetin caused apoptosis and/or cell death in various cells including cancer cells and normal cells. In this study, we investigated the effects of quercetin with or without hydrogen peroxide (H2O2) on cell death of PC12 cells, a neuronal cell line. We showed that quercetin at 10-30 microM alone caused cell death accompanied by caspase-mediated DNA fragmentation in undifferentiated PC12 cells. Quercetin did not inhibit and rather enhanced 0.1 mM H2O2-induced cell death. The toxic effect of quercetin was not inhibited by antioxidants such as N-acetylcysteine and GSH, although H2O2-induced cell death was inhibited by the antioxidants. Quercetin-induced cell death was reduced by 2 h treatment with nerve growth factor and serum. In addition, quercetin caused cell death in differentiated PC12 cells that were cultured with nerve growth factor for 6 d. Genistein, a soy isoflavone that has the pro-apoptotic activity, also caused cell death with DNA fragmentation. Further evaluation of the potential of dietary flavonoids as neuroprotective reagents is needed.