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Intercellular cholesterol transport in the brain is carried by high density lipoprotein (HDL) generated in situ by cellular interaction with the apolipoprotein apoE, which is mainly synthesized by astrocytes, and with apoA-I secreted by cells such as endothelial cells. Rat astrocytes in fact generate HDL with extracellular apoA-I in addition to releasing HDL with endogenously synthesized apoE, seemingly by the same mechanism as the HDL assembly for systemic circulation. Relating to this reaction, apoA-I induced translocation of newly synthesized cholesterol and phospholipid to the cytosol prior to extracellular assembly of HDL, accompanied by an increase of caveolin-1 in the cytosol, activation of sterol regulatory element-binding protein, and enhancement of cholesterol synthesis. The lipid translocated into the cytosol was recovered in the fraction with a density of 1.09 -1.16 g/ml as well as caveolin-1 and cyclophilin A. Cyclosporin A inhibited these apoA-I-mediated reactions and suppressed apoA-I-mediated cholesterol release. The findings suggest that such translocation of cholesterol and phospholipid into the cytosol is related to the apo A-I-mediated HDL assembly in astrocytes through functional association with caveolin-1 and a cyclosporin A-sensitive cyclophilin protein(s). The central nervous system (CNS)1 is sheltered from interaction with the lipoproteins of the systemic circulation by the blood-brain barrier. Therefore, extracellular cholesterol transport in the CNS is mediated by its own lipoprotein system, consisting mainly of the particles equivalent to plasma HDL (1). The main apolipoproteins are apoE produced by astrocytes (2), microglias (3), and apoA-I from an unknown source but reportedly secreted by brain endothelial cells (4). The astrocytes were shown to generate HDL, not only with endogenously synthesized apoE but also with exogenous apoE and apoA-I (5).The apolipoprotein-cell interaction that generates HDL is common for many somatic cells from various origins and is distinct from the diffusion-mediated cholesterol efflux from the cell surface (6). The reaction is a main source of plasma lipoprotein (7) and is also one of the major pathways for cholesterol release from the cells (8). Generation of HDL with cellular phospholipid seems to require a cellular interaction site with apolipoprotein. An intracellular cholesterol trafficking system linked to such interaction is responsible for incorporation of cholesterol into the HDL (9, 10). Mutations in the ABCA1 transporter protein were identified in patients with plasma HDL deficiency who lack the ability to generate HDL by this reaction (11-13). Thus, the reaction depends on the cellular system to export materials. A specific intracellular cholesterol transport system is important to make the HDL cholesterolrich. In macrophages and smooth muscle cells, protein kinase C was shown to be involved in this trafficking (9, 10). It is not known whether the mechanism for generation of HDL in somatic cells is different from that for the HDL assembly i...

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Luteolin is a natural flavonoid with strong anti-oxidative properties that is reported to have an anti-cancer effect in several malignancies other than bladder cancer. In this study, we describe the effect of luteolin on a human bladder cancer cell line, T24, in the context of the regulation of p21, thioredoxin-1 (TRX1) and the mechanistic target of rapamycin (mTOR) pathway. Luteolin inhibited cell survival and induced G2/M cellcycle arrest, p21 upregulation and downregulation of phospho(p)-S6, which is downstream of mTOR signaling. Luteolin also upregulated TRX1 and reduced intracellular reactive oxygen species production. In a subcutaneous xenograft mouse model using the rat bladder cancer cell line, BC31, tumor volumes were significantly decreased in mice orally administered luteolin compared to control. Immunohistochemical analysis revealed that increased p21 and decreased p-S6 expression were induced in the luteolin treatment group. Moreover, in another in vivo N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced rat bladder cancer model, the oral administration of luteolin led to a trend of decreased bladder tumor dimension and significantly decreased the Ki67-labeling index and p-S6 expression. Furthermore, the major findings on the metabolism of luteolin suggest that both plasma and urine luteolin-3ʹ-O-glucuronide concentrations are strongly associated with the inhibition of cell proliferation and mTOR signaling. Moreover, a significant decrease in the squamous differentiation of bladder cancer is attributed to plasma luteolin-3ʹ-glucuronide concentration. In conclusion, luteolin, and in particular its metabolized product, may represent another natural product-derived therapeutic agent that acts against bladder cancer by upregulating p21 and inhibiting mTOR signaling. K E Y W O R D Sanimal model, bladder cancer, chemoprevention, mTOR, oxidative stress

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