We examined the effect of δ-tocotrienol on melanin content in mouse melanoma B16 cells. Melanin content was significantly reduced in cells treated with 50 and 100 μM δ-tocotrienol, but not 10 μM δ-tocotrienol. The activity and amount of tyrosinase also significantly decreased in cells treated with 10, 50, and 100 μM δ-tocotrienol. Furthermore, the mRNA level of tyrosinase as measured using realtime PCR was significantly decreased compared to controls in cells treated with 100 μM δ-tocotrienol, but not 10 or 50 μM δ-tocotrienol. These results indicated that at first δ-tocotrienol caused tyrosinase degradation, and then caused a further decrease in the tyrosinase protein level via both tyrosinase degradation and a decrease in the mRNA level of tyrosinase. We conclude that the decrease of melanin content in the cells by δ-tocotrienol was the result of the decrease of the protein level of tyrosinase (tyrosinase degradation is more important than the decrease of mRNA).

We previously reported that chloroquine disrupted lysosomes, but not the shift to low-density lysosomes. In the present study, the effects of primaquine on lysosomal integrity in cultured rat hepatocytes were studied by measuring lysosomal enzyme β-glucuronidase (β-G) or lysosomal-associated membrane glycoprotein (lamp-1) in the cytosolic fraction obtained from cells permeabilized by digitonin, and in the cytosolic fraction obtained by conventional cell fractionation or in Percoll density gradient fractions. The percentage disruption of lysosomes in living cells by 50 μM or 100 μM of primaquine was 1% or 4%, respectively, and lysosomes disrupted by homogenization or centrifugation during cell fractionation by 50 μM or 100 μM of primaquine were 2% or 7%, respectively. The decrease of β-G and lamp-1 in lysosome fractions (fractions 16 to 18) on a Percoll density gradient (1 to 18 fractions) in 50 μM or 100 μM primaquine-treated cells was 9% or 19% for β-G, and 16% or 24% for lamp-1, respectively. The decrease of β-G and lamp-1 in the lysosome fraction was higher than the disruption of lysosomes in living cells or by homogenization or centrifugation during cell fractionation by 50 μM or 100 μM of primaquine. Also, the peak fraction numbers of the subcellular distribution of β-G and lamp-1 on a Percoll density gradient by 50 μM primaquine-treated cells were fraction 17 (high density) and 4 (low density), while those by 100 μM primaquine-treated cells were fraction 6 (low density) or 4 (low density). From these data, we infer that the main effect of primaquine is to cause a shift of lysosomal protein to low density, and then to cause differences in the proportion of membrane and luminal proteins of lysosomes in low-density fractions, although the main effect of chloroquine was the disruption of lysosomes.

Recently, it has been questioned whether mevalonate pyrophosphate decarboxylase (MPD) is predominantly located in the peroxisomes or cytosol. We previously reported that a small amount of MPD in the liver of rats fed a CP diet (5% cholestyramine and 0.1 % pravastatin) existed in the peroxisomes, although MPD is predominantly located in the cytosol in the liver of rats fed normal chow and a CP diet for 12 days. In the present study, we examined the subcellular distribution of MPD in mouse melanoma cells (1316 and B16F10) treated with or without lovastatin, using digitonin permeabilization and immunoblotting. In permeabilized B16 by digitonin after treatment with or without lovastatin, 95% and 5%, or 98 % and 2 % of MPD existed in the cytosol and membrane/organelle (M/O) fraction, respectively. Using B16F10 under the same conditions, 80% and 20%, or 91 % and 9 % of MPD existed in the cytosol and M/O fraction, respectively. These results indicated that MPD was predominantly located in the cytosol in both mouse melanoma cells treated with or without lovastatin.

Recently, it has been questioned whether mevalonate pyrophosphate decarboxylase (MPD) is predominantly located in the peroxisomes or cytosol. We previously reported that MPD was predominantly present in the cytosol of rat hepatocytes, normal rat kidney cells, or mouse melanoma cells. In the present study, we examined whether MPD was predominantly present in the cytosol of HepG2 (human hepatoma) cells and Cos7 (monkey kidney) cells using digitonin permeabilization. In HepG2 cells permeabilized with digitonin, 90 % and 10 % of MPD existed in the cytosol and membrane/organelle (M/O) fraction, respectively, while in Cos7 cells permeabilized with digitonin, 20 % and 80 % of MPD existed in the cytosol and M/O fraction, respectively. These data suggest that the difference in subcellular distribution of MPD is due to the cell type.

The main exposure pathway of benzo[a]pyrene (Bap) for humans is considered to be via the daily diet. The purpose of this study was to investigate the effect of BaP on the intestinal transport of chemicals mediated by P-glycoprotein (P-gp). The intestinal epithelial membrane transport of rhodamine-123 (Rho-123), a substrate of P-gp, was examined using a monolayer of the human Caco-2 cell line grown in transwells. In the monolayer exposed to Bap for 72 h before transport experiments, the ratio of the apparent permeability coefficients (P(app)) of Rho-123 efflux increased compared to that of the control. The permeability of rhodamine-B (Rho-B), not a substrate of P-gp, showed no difference between the monolayers. Treatment with quinidine or cyclosporine A, which are P-gp inhibitors, decreased the P(app) of Rho-123 to the same degree in both monolayers. The transport of Rho-123 was not influenced by the presence of Bap. Thus, Bap seemed not to act directly on the efflux activity of P-gp and be a binding site competitor of Rho-123. In the Caco-2 cells that enhanced the efflux of Rho-123 by the treatment with Bap, an increase in mRNA expression of MDR 1 (P-gp) was confirmed compared to that of control by RT-PCR. Furthermore, Western blot analysis using a monoclonal antibody, C219, demonstrated the increase of P-gp in Caco-2 cells exposed to Bap, compared with controls. It was inferred that Bap exposure induced the expression of P-gp, which led to the observed increase in efflux transport of Rho-123. The possibility was suggested that Bap might affect the disposition of medicines by increasing P-gp expression.

We examined the change in the subcellular distribution of a lysosomal enzyme, beta-glucuronidase (beta-G), caused by decreased cholesterol levels in mouse melanoma cells using an HMG-CoA reductase inhibitor, lovastatin and lipoprotein-deficient serum (LDS). There was a decrease in the cholesterol content of the cells and increased secretion of the mature form of beta-G located in lysosomes, as documented by Percoll density gradient fractionation, digitonin permeabilization and immunoprecipitation. Furthermore, another lysosomal enzyme, cathepsin H, was found to be released in the medium from cells treated with lovastatin. Both the precursor and mature forms of cathepsin H were detected in the medium of treated cells. Next, when cells were treated with LDS without lovastatin, concomitantly with the decrease in the levels of cholesterol and beta-G activity in the cells, beta-G activity in the medium increased. Also, the ratio of beta-G (3.2-fold) released in the medium from cells treated with Dulbecco's modified Eagle medium (D-MEM) containing lovastatin and LDS was higher than that (2.3-fold) on treatment with D-MEM containing LDS without lovastatin. From these results, it was suggested that the exocytosis of mature enzymes from lysosomes into the medium or mis-sorting of the lysosomal precursor forms to the medium was caused by the lovastatin- and/or LDS-induced decrease in the cholesterol content of the cells, although the mechanism of secretion by lysosomal enzymes differed somewhat.

Lysosomal membrane glycoprotein termed LGP85 or LIMP II extends a COOH-terminal cytoplasmic tail of R459GQGSMDEGTADERAPLIRT478, in which an L475 I476 sequence lies as a di-leucine-based motif for lysosomal targeting. In the present study, we explored the role of the I476 residue in the localization of LGP85 to the endocytic organelles using two substitution mutants called I476A and I476L in which alanine and leucine are replaced at I476, respectively, and I476R477T478-deleted LGP85 called Δ 476–478. Immunofluorescence analyses showed that I476A and I476L are largely colocalized in intracellular organelles with an endogenous late endosomal and lysosomal marker, LAMP-1, but there were some granules in which staining for the LGP85 mutants was prominent, while Δ 476–478 is detected in LAMP-1-positive and LAMP-1-negative intracellular organelles, and on the cell surface. The subcellular fractionation studies revealed that I476A, I476L, and Δ 476–478 are different from wild-type LGP85 in the distribution of early endosomes, late endosomes, and lysosomes. I476A and I476L are present more in late endosomes than in the densest lysosomes, whereas wild-type LGP85 is mainly lysosomal. Substitution of I476 for A and L differentially modified the ratios of late endosomal to lysosomal LGP85. A major portion of Δ 476–478 resided in the light buoyant density fraction containing plasma membrane and early endosomes. Taken together, these results indicate that the existence of the 476th amino acid residue is essential for localization of LGP85 to late endocytic compartments. The fact that isoleucine but not leucine is in the 476th position is especially of importance in the proper distribution of LGP85 in late endosomes and lysosomes.