Shuzo Moritani

P-glycoprotein as the drug efflux pump in primary cultured bovine brain capillary endothelial cells

The expression of a functional P-glycoprotein (P-gp) which pumps drugs out of brain capillary endothelial cells (BCEC) into blood was studied by evaluating the steady-state uptake and efflux of vincristine (VCR) by primary cultured bovine BCEC. The steady-state uptake of VCR was increased in the presence of metabolic inhibitors, and an anti-P-gp monoclonal antibody, MRK16, as well as verapamil and steroid hormones which are known to reverse multidrug resistance in tumor cells. Furthermore, efflux of VCR from BCEC was inhibited by verapamil. By immunohistochemistry, P-gp was localized at the luminal side of the capillary endothelial cells in both gray matter of bovine brain and primary cultured BCEC. These data suggest that P-gp functions as a drug efflux pump at the luminal side of BCEC and regulates the transfer of certain lipophilic drugs from the blood into the brain.

P-Glycoprotein-Dependent Disposition Kinetics of Tacrolimus: Studies in mdr la Knockout Mice

AbstractPurpose. This study was performed to evaluate the involvement of P-glycoprotein in disposition kinetics of tacrolimus (FK506), a substrate of P-glycoprotein, in the body. Methods. The blood and tissue concentrations of FK506 after i.v. or p.o. administration (2 mg/kg) to normal andmdrla knockout mice were measured by competitive enzyme immunoassay. Results. The blood concentrations in knockout mice were significantly higher than those in normal mice. The value of the total clearance (CLtot) for knockout mice (19.3 mL/min/kg) was about 1/3 of that for normal mice (55.8 mL/min/kg)(P < 0.001), although there was no significant difference in the distribution volume at the steady-state (Vdss) (about 4.6 L/kg) between both types of mice. FK506 rapidly penetrated the blood-brain barrier and the brain concentration reached a maximum, which was about 10 times higher in knockout mice than in normal mice, 1 hr after administration. The brain concentration in normal mice thereafter decreased slowly, whereas in knockout mice, an extremely high concentration was maintained for 24 hr. Conclusions. The pharmacokinetic behavior of FK506 in the tissue distribution is related with the function of P-glycoprotein encoded by themdr la gene. The brain distribution of FK506 is dominated by the P-glycoprotein-mediated drug efflux and presumably also by the binding to FK-binding proteins (immunophilins) in the brain.

Structure-activity relationship of flavonoids for inhibition of epidermal growth factor-induced transformation of JB6 Cl 41 cells.

We found that quercetin, myricetin, quercetagetin, fisetin, (−)‐epigallocatechin gallate (EGCG), and theaflavins, among 24 flavonoids examined, markedly inhibited epidermal growth factor (EGF)‐induced cell transformation of mouse epidermal JB6 Cl 41 cells. The six flavonoids suppressed the EGF‐induced activation of activator protein 1 (AP‐1). In addition, myricetin, quercetagetin, EGCG, and theaflavins directly inhibited EGF‐induced phosphatidylinositol 3‐kinase (PI3K) activation. The important structural features of flavonoids for cell transformation‐inhibitory activity are 3′‐ and 4′‐OH on the B‐ring, 3‐OH on the C‐ring, C2C3 double bond in the C‐ring, and the phenylchromone (C6C5C6) skeleton in the flavonols, and the galloyl group in EGCG and theaflavins. Our results provide new insight into possible mechanisms of the anti‐carcinogenic effects of flavonoids, and could help to provide a basis for the design of novel cancer chemopreventive agents.

Absorptive-Mediated Endocytosis of an Adrenocorticotropic Hormone (ACTH) Analogue, Ebiratide, into the Blood–Brain Barrier: Studies with Monolayers of Primary Cultured Bovine Brain Capillary Endothelial Cells

The internalization of a neuromodulatory adrenocorticotropic hormone (ACTH) analogue, [125I]ebiratide (H-Met(O2)-Glu[125I]His-Phe-D-Lys-Phe-NH(CH2)8NH2), was examined in cultured mono-layers of bovine brain capillary endothelial cells (BCEC). HPLC analysis of the incubation solution showed that [125I]ebiratide was not metabolized during the incubation with BCEC. The acid-resistant binding of [125I]ebiratide to BCEC increased with time for 120 min and showed a significant dependence on temperature and medium osmolarity. Pretreatment of BCEC with dansylcadaverine or phenylarsine oxide, endocytosis inhibitors, and 2,4-dinitrophenol, a metabolic inhibitor, decreased significantly the acid-resistant binding of [125I]ebiratide. The acid-resistant binding of [125I]ebiratide was saturable in the presence of unlabeled ebiratide (100 nM–1 mM). The maximal internalization capacity (Bmax) at 30 min was 7.96 ± 3.27 µmol/mg of protein with a half-saturation constant (Kd) of 15.9 ± 6.4 µM. The acid-resistant binding was inhibited by basic peptides such as poly-L-lysine, protamine, histone, and ACTH but was not inhibited by poly-L-glutamic acid, insulin, or transferrin. These results confirmed that ebiratide is transported through the blood-brain barrier via an absorptive-mediated endocytosis.

Prostaglandin E2‐Mediated Anabolic Effect of a Novel Inhibitor of Phosphodiesterase 4, XT‐611, in the In Vitro Bone Marrow Culture

The mechanism of osteoblast formation by a novel PDE4 inhibitor, XT‐611, was studied in the in vitro bone marrow culture system. The compound potentiated the osteoblast differentiation through accumulation of cyclic AMP after autocrine stimulation of EP4 receptor by PGE2 in pro‐osteoblastic cells.

Inhibition of epidermal growth factor-induced cell transformation and Akt activation by caffeine

We found that caffeine significantly inhibited epidermal growth factor (EGF)‐ and 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA)‐induced cell transformation in the JB6 mouse epidermal cell line. The tumor promoter‐induced cell transformation was also blocked by treatment with an adenosine A1 receptor antagonist, 8‐phenyltheophylline (8‐PTH). Caffeine slightly attenuated activation of EGF‐induced activator protein 1 (AP‐1) activation, which play important roles in cell transformation, but only at the highest concentration examined (1 mM). Interestingly, pretreatment with caffeine suppressed EGF‐induced phosphorylation and activation of Akt and ribosomal p70 S6 protein kinase (p70 S6K), a target of Akt, without inhibiting phosphatidylinositol 3‐kinase (PI3K) activation. The inhibition of Akt activation of caffeine was not a result of its adenosine receptor antagonism. Because Akt plays a key role in signal transduction pathways leading to cell proliferation and apoptosis, our results provide novel insight into possible mechanisms of the chemotherapeutic effect of caffeine.

Inhibition of osteoclastogenesis by a phosphodiesterase 4 inhibitor XT-611 through synergistic action with endogenous prostaglandin E2

We examined the effect of a phosphodiesterase 4 (PDE4) inhibitor, 3,4-dipropyl-4,5,7,8-tetrahydro-3H-imidazo[1,2-i]-purin-5-one (XT-611) on osteoclast formation in three different mouse bone-marrow cell (BMC) culture systems. We confirmed that selective inhibitors of PDE4, including XT-611, among several PDE inhibitors decreased osteoclast formation in the BMC culture system. XT-611 also inhibited osteoclast formation in co-culture of mouse bone-marrow stromal cell line ST2 and adherent cell-depleted (ACD)-BMCs. However, it did not inhibit osteoclastogenesis in culture of ACD-BMCs alone in the presence of macrophage-colony stimulating factor (M-CSF) and soluble receptor activator of NF-kappaB ligand (sRANKL). XT-611 significantly increased prostaglandin E(2) (PGE(2)) production from ST2 cells and, in combination with PGE(2), synergistically increased cAMP concentration in osteoclast progenitors. In the ST2 co-culture system, XT-611 did not influence the expression of RANKL, osteoprotegerin and RANK mRNAs. By combined treatment with XT-611 and PGE(2) of ACD-BMCs, osteoclast multinucleation was clearly inhibited with decrease in the expression of calcitonin receptor mRNA, while the expression of RANK and c-fms (an M-CSF receptor) mRNAs was unchanged. These results indicate that the PDE4 inhibitor inhibits osteoclastogenesis by acting on osteoclast progenitors synergistically with PGE(2) secreted from stromal cells, but not by influencing the cell-to-cell interaction between stromal cells and osteoclast progenitors.

In vivo cisplatin resistance of rat ascites hepatoma AH66

The rat ascites hepatoma AH66 cell line had higher resistance to cisplatin than a variant AH66F cell line in in vivo experiments, though in vitro sensitivities to cisplatin of both cell lines were similar in a medium containing 5% fetal calf serum (FCS). When AH cells were cultured in the medium containing 5% ascites fluid (ASF), the sensitivity of AH66 cells to cisplatin was significantly lower than that in the medium containing 5% FCS, but the sensitivity of AH66F cells was almost same in the case of 5% FCS. The metallothionein (MT) contents in AH66 cells, but not in AH66F cells, increased with the incubation time in the medium containing ASF. MT in AH66 cells was also induced by treatment with zinc ion, but the induction in AH66F cells was very low. These results indicate that AH66 cells have a high ability to induce MT and thereby may acquire in vivo resistance to cisplatin.

Inhibition by protein kinase C inhibitor of expression of leukocyte function-associated antigen-1 molecules in rat hepatoma AH66F cells

To examine the mechanism of inhibition by protein kinase C (PKC) inhibitors of the adhesion of highly malignant hepatoma AH66F cells to the mesentery‐derived mesothelial cell (M‐cell) layer through leukocyte function‐associated antigen‐1 (LFA‐1)/intercellular adhesion molecule‐1, the effects of a PKC inhibitor, NA‐382, on the expression of LFA‐1 molecules in AH66F cells were examined and compared with those in thymocytes from normal rats. NA‐382 inhibited the adhesion of AH66F cells to the M‐cell layer and the expression of LFA‐1 on the membrane of the hepatoma cells after treatment for more than 24 h. It was confirmed that AH66F cells express similar mRNAs for LFA‐1 subunits to those of thymocytes, and their levels were also decreased after treatment with NA‐382. On the other hand, the LFA‐1‐mediated adhesion and the expression of both protein and mRNA for LFA‐1 subunits in thymocytes were not changed by the PKC inhibitor. These results suggest that the expression of LFA‐1 molecules in AH66F cells may be regulated by PKC via quite different mechanisms from those in normal lymphocytes