Recruitment of miR-8080 by luteolin inhibits androgen receptor splice variant 7 expression in castration-resistant prostate cancer.

A need exists for seeking effective treatments for castration-resistant prostate cancer (CRPC) in response to its emergence following androgen deprivation therapy as a major clinical problem. In the present study, we investigated the chemopreventive and chemotherapeutic potential of luteolin, a flavonoid with antioxidative properties, on prostate cancer, including CRPC. Luteolin inhibited the progression of rat prostate carcinogenesis by induction of apoptosis in a transgenic rat for adenocarcinoma of prostate (TRAP) model. Luteolin decreased cell proliferation in a dose-dependent manner and induced apoptosis with the activation of caspases 3 and 7 in both rat (PCai1, established from a TRAP prostate tumor) and human (22Rv1) CRPC cells. Dietary luteolin also suppressed tumor growth via an increase in apoptosis and inhibition of angiogenesis in PCai1 and 22Rv1 xenografts implanted in castrated nude mice. We also focused on androgen receptor splice variant 7 (AR-V7), which contributes to cell proliferation and therapeutic resistance in CRPC. Luteolin dramatically suppressed AR-V7 protein expression in 22Rv1 cells in vitro and ex vivo. Microarray analysis identified MiR-8080, which contains a possible target sequence for AR-V7 3'-UTR, as a gene upregulated by luteolin. MiR-8080 transfection decreased the AR-V7 expression level and the induction of apoptosis in 22Rv1 cells. Furthermore, miR-8080 knockdown canceled luteolin decreasing AR-V7 and the cell growth of 22Rv1. MiR-8080 induced by luteolin intake enhanced the therapeutic effect of enzalutamide on 22Rv1 xenografts under castration conditions. These results indicate luteolin inhibits CRPC by AR-V7 suppression through miR-8080, highlighting luteolin and miR-8080 as promising therapeutic agents for this disease.

Luteolin suppresses bladder cancer growth via regulation of mechanistic target of rapamycin pathway.

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 cell-cycle 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-3'-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-3'-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.

A novel model of non-alcoholic steatohepatitis with fibrosis and carcinogenesis in connexin 32 dominant-negative transgenic rats.

Non-alcoholic steatohepatitis (NASH) is a recognized risk factor for liver fibrosis and malignancies, and is associated with features of metabolic syndrome, such as obesity and insulin resistance (IR). We previously demonstrated that the disturbance of connexin 32 (Cx32), a gap junctional protein of hepatocytes, exacerbated NASH in Cx32 dominant-negative transgenic (Cx32ΔTg) rats fed methionine choline-deficient diet (MCDD). MCDD is well-established means of inducing NASH in rodents; however, the Cx32ΔTg-MCDD NASH model does not reproduce obesity and IR. In this study, we aimed to establish an improved NASH model. Eight-week-old male Cx32ΔTg and wild-type (Wt) rats received a high-fat diet (HFD) with dimethylnitrosamine (DMN) for 12 weeks. The HFD with DMN led to gains in body, liver, and visceral fat weights in both genotypes. IR was significantly greater in Cx32ΔTg than in Wt rats. Elevation of serum hepatic enzymes (AST, ALT), inflammatory cytokine expressions (Tnfα, Il-6, Tgf-ß1, Il-1ß, Timp2, and Col1a1), steatohepatitis, and fibrosis were significantly greater in Cx32ΔTg as compared with Wt rats. Regarding carcinogenesis, the number and area of glutathione S-transferase placental form (GST-P)-positive preneoplastic hepatic foci were significantly increased in Cx32ΔTg versus Wt rats. Moreover, activation of NF-κB and JNK contributed to the progression of NASH in Cx32ΔTg rats. These results suggest that Cx32 dysfunction promoted the progression of NASH, metabolic syndrome, and carcinogenesis. Therefore, the novel Cx32ΔTg-HFD-DMN NASH model may be a rapid and useful tool for evaluating the progression of NASH.

Pioglitazone, a Peroxisome Proliferator-Activated Receptor γ Agonist, Suppresses Rat Prostate Carcinogenesis.

Pioglitazone (PGZ), a peroxisome proliferator-activated receptor γ agonist, which is known as a type 2 diabetes drug, inhibits cell proliferation in various cancer cell lines, including prostate carcinomas. This study focused on the effect of PGZ on prostate carcinogenesis using a transgenic rat for an adenocarcinoma of prostate (TRAP) model. Adenocarcinoma lesions as a percentage of overall lesions in the ventral prostate were significantly reduced by PGZ treatment in a dose-dependent manner. The number of adenocarcinomas per given area in the ventral prostate was also significantly reduced by PGZ treatment. The Ki67 labeling index in the ventral prostate was also significantly reduced by PGZ. Decreased cyclin D1 expression in addition to the inactivation of both p38 mitogen-activated protein kinase (MAPK) and nuclear factor (NF)κB were detected in PGZ-treated TRAP rat groups. In LNCaP, a human androgen-dependent prostate cancer cell line, PGZ also inhibited cyclin D1 expression and the activation of both p38 MAPK and NFκB. The suppression of cultured cell growth was mainly regulated by the NFκB pathway as detected using specific inhibitors in both LNCaP and PC3, a human androgen-independent prostate cancer cell line. These data suggest that PGZ possesses a chemopreventive potential for prostate cancer.

Apolipoprotein A-I induces tubulin phosphorylation in association with cholesterol release in fetal rat astrocytes.

We previously identified cytosolic lipid-protein particles (CLPP) having size and density of HDL in rat astrocytes, to which apoA-I induces translocation of cholesterol, caveolin-1 and protein kinase Cα (PKCα) following its association with microtubules prior to cholesterol release/biogenesis of HDL (JBC 277: 7929, 2002; JLR 45: 2269, 2004). To further understand the physiological relevance of these findings, we investigated the CLPP/microtubule association and its role in intracellular cholesterol trafficking by using a technique of reconstituted microtubule-like filaments (rMT) in rat astrocyte cytosol. When the cells were pretreated with apoA-I, α-tubulin as a 52-kDa protein in rMT was found phosphorylated while α-tubulin in a soluble monomeric form was little phosphorylated. The phosphorylation took place coincidentally to apoA-I-induced association with rMT of CLPP, a complex containing PKCα, and was suppressed by a PKC inhibitor, Bis indolylmaleimide 1 (BIM). α-Tubulin dissociated from CLPP when phosphorylated, and it poorly bound to CLPP once dissociated. BIM did not influence association of PKCα with rMT but suppressed apoA-I-induced cholesterol translocation to the cytosol from the ER/Golgi apparatus and apoA-I-mediated cholesterol release. We thereby concluded that α-tubulin phosphorylation by PKCα on CLPP is involved in reversible CLPP association with the microtubules and intracellular cholesterol trafficking for apoA-I-dependent HDL biogenesis/cholesterol release in rat astrocytes.

Involvement of cdc42/Rho kinase in apoA-I-mediated cholesterol efflux through interaction between cytosolic lipid-protein particles and microtubules in rat astrocytes.

Activation of cdc42 reportedly enhances apoA-I-mediated cholesterol release through ATP-binding cassette transporter A1 (ABCA1). We examined the involvement of cdc42 and Rho kinase in intracellular cholesterol transport for release of cholesterol after the interaction between apoA-I and ABCA1 in astrocytes. Exogenously added apoA-I increased the GTP-bound form of cdc42 and enhanced Rho kinase activity in rat astrocytes. Suppression of ABCA1 expression by siRNA substantially repressed both cellular level of GTP-bound cdc42 and Rho kinase activity, indicating that these reactions require ABCA1. ApoA-I-mediated lipid release and Rho kinase activation were inhibited by not only Rho kinase inhibitor but also cdc42 siRNA. These findings suggest that cdc42 is activated by the interaction between apoA-I and ABCA1 and enhances cholesterol release through the activation of Rho kinase. ApoA-I increased the binding of Rock1, one of the Rho kinases, to reconstituted microtubule-like filaments (rMT). Y-27632 suppressed not only the association of rMT with the cytosolic lipid-protein particles (CLPP)-related proteins and lipids but also the intracellular transport of newly synthesized cholesterol to the plasma membrane in rat astrocytes treated with apoA-I without inhibiting cholesterol synthesis. Finally, cdc42 siRNA reduced apoA-I-induced interaction between rMT and major players in intracellular cholesterol trafficking, such as caveolin-1 and Rock1, suggesting a regulatory role of Rho family proteins in the apoA-I-mediated intracellular cholesterol transport. We conclude that ABCA1/cdc42/Rho kinase signaling is involved in apoA-I-induced intracellular cholesterol transport and apoA-I-mediated cholesterol release in rat astrocytes.

ApoA-I enhances generation of HDL-like lipoproteins through interaction between ABCA1 and phospholipase Cγ in rat astrocytes.

In the previous paper, we reported that apolipoprotein (apo) A-I enhances generation of HDL-like lipoproteins in rat astrocytes to be accompanied with both increase in tyrosine phosphorylation of phospholipase Cγ (PL-Cγ) and PL-Cγ translocation to cytosolic lipid-protein particles (CLPP) fraction. In this paper, we studied the interaction between apoA-I and ATP-binding cassette transporter A1 (ABCA1) to relate with PL-Cγ function for generation of HDL-like lipoproteins in the apoA-I-stimulated astrocytes. ABCA1 co-migrated with exogenous apoA-I with apparent molecular weight over 260kDa on SDS-PAGE when rat astrocytes were treated with apoA-I and then with a cross-linker, BS3. The solubilized ABCA1 of rat astrocytes was associated with the apoA-I-immobilized Affi-Gel 15. An LXR agonist, To901317, increased the cellular level of ABCA1, association of apoA-I with ABCA1 and apoA-I-mediated lipid release in rat astrocytoma GA-1/Mock cells where ABCA1 expression at baseline is very low. PL-Cγ was co-isolated by apoA-I-immobilized Affi-Gel 15 and co-immunoprecipitated by anti-ABCA1 antibody along with ABCA1 from the solubilized membrane fraction of rat astrocytes. The SiRNA of ABCA1 suppressed not only the PL-Cγ binding to ABCA1 but also the tyrosine phosphorylation of PL-Cγ. A PL-C inhibitor, U73122, prevented generation of apoA-I-mediated HDL-like lipoproteins in rat astrocytes. To901317 increased the association of PL-Cγ with ABCA1 in GA-1/Mock cells dependently on the increase of cellular level of ABCA1 without changing that of PL-Cγ. These findings suggest that the exogenous apoA-I augments the interaction between PL-Cγ and ABCA1 to stimulate tyrosine phosphorylation and activation of PL-Cγ for generation of HDL-like lipoproteins in astrocytes.

PBK expression predicts favorable survival in colorectal cancer patients.

Colorectal cancer (CRC) is one of the most common gastrointestinal cancers worldwide with high morbidity and mortality rates. The discovery of small molecule anticancer reagents has significantly affected cancer therapy. However, the anticancer effects of these therapies are not sufficient to completely cure CRC. PDZ-binding kinase (PBK) was initially identified as a mitotic kinase for mitogen-activated protein kinase and is involved in cytokinesis and spermatogenesis. Aberrant expression of PBK has been reported to be closely associated with malignant phenotypes of many cancers and/or patient survival. However, the expression of PBK and its association to patient survival in CRC have not been fully elucidated. In the present study, 269 primary CRCs were evaluated immunohistochemically for PBK expression to assess its ability as a prognostic factor. CRC tumor cells variably expressed PBK (range, 0-100%; median, 32%) in the nucleus and cytoplasm. Univariate analyses identified a significant inverse correlation between PBK expression and pT stage (P<0.0001). Furthermore, patients carrying CRC with higher PBK expression showed significantly favorable survival (P=0.0094). Multivariate Cox proportional hazards regression analysis revealed high PBK expression (HR, 0.52; P=0.015) as one of the potential favorable factors for CRC patients. PBK expression showed significant correlation to Ki-67 labeling indices (ρ=0.488, P<0.0001). In vitro, the PBK inhibitor OTS514 suppressed cellular proliferation of CRC cells with PBK expression through downregulation of P-ERK and induction of apoptosis. These results suggest that PBK-targeting therapeutics may be useful for the treatment of PBK-expressing CRC patients.

Lactoferrin Prevents Hepatic Injury and Fibrosis via the Inhibition of NF-κB Signaling in a Rat Non-Alcoholic Steatohepatitis Model.

Non-alcoholic steatohepatitis (NASH) can cause liver cirrhosis and hepatocellular carcinoma (HCC), with cases increasing worldwide. To reduce the incidence of liver cirrhosis and HCC, NASH is targeted for the development of treatments, along with viral hepatitis and alcoholic hepatitis. Lactoferrin (LF) has antioxidant, anti-cancer, and anti-inflammatory activities. However, whether LF affects NASH and fibrosis remains unelucidated. We aimed to clarify the chemopreventive effect of LF on NASH progression. We used a NASH model with metabolic syndrome established using connexin 32 (Cx32) dominant negative transgenic (Cx32ΔTg) rats. Cx32ΔTg rats (7 weeks old) were fed a high-fat diet and intraperitoneally injected with dimethylnitrosamine (DMN). Rats were divided into three groups for LF treatment at 0, 100, or 500 mg/kg/day for 17 weeks. Lactoferrin significantly protected steatosis and lobular inflammation in Cx32ΔTg rat livers and attenuated bridging fibrosis or liver cirrhosis induced by DMN. By quantitative RT-PCR, LF significantly down-regulated inflammatory (Tnf-α, Il-6, Il-18, and Il-1ß) and fibrosis-related (Tgf-ß1, Timp2, and Col1a1) cytokine mRNAs. Phosphorylated nuclear factor (NF)-κB protein decreased in response to LF, while phosphorylated JNK protein was unaffected. These results indicate that LF might act as a chemopreventive agent to prevent hepatic injury, inflammation, and fibrosis in NASH via NF-κB inactivation.

NCL1, A Highly Selective Lysine-Specific Demethylase 1 Inhibitor, Suppresses Castration-Resistant Prostate Cancer Growth via Regulation of Apoptosis and Autophagy.

Recent studies have shown that epigenetic alterations lead to oncogenic activation, thus indicating that these are therapeutic targets. Herein, we analyzed the efficacy and therapeutic potential of our developed histone lysine demethylase 1 (LSD1) inhibitor, NCL1, in castration-resistant prostate cancer (CRPC). The CRPC cell lines 22Rv1, PC3, and PCai1CS were treated with NCL1, and LSD1 expression and cell viability were assessed. The epigenetic effects and mechanisms of NCL1 were also evaluated. CRPC cells showed strong LSD1 expression, and cell viability was decreased by NCL1 in a dose-dependent manner. Chromatin immunoprecipitation analysis indicated that NCL1 induced histone H3 lysine 9 dimethylation accumulation at promoters of P21. As shown by Western blot and flow cytometry analyses, NCL1 also dose-dependently induced caspase-dependent apoptosis. The stimulation of autophagy was observed in NCL1-treated 22Rv1 cells by transmission electron microscopy and LysoTracker analysis. Furthermore, WST-8 assay revealed that the anti-tumor effect of NCL1 was reinforced when autophagy was inhibited by chloroquine in 22Rv1 cells. Combination index analysis revealed that a concurrent use of these drugs had a synergistic effect. In ex vivo analysis, castrated nude mice were injected subcutaneously with PCai1 cells and intraperitoneally with NCL1. Tumor volume was found to be reduced with no adverse effects in NCL1-treated mice compared with controls. Finally, immunohistochemical analysis using consecutive human specimens in pre- and post-androgen deprivation therapy demonstrated that LSD1 expression levels in CRPC, including neuroendocrine differentiation cases, were very high, and identical to levels observed in previously examined prostate biopsy specimens. NCL1 effectively suppressed prostate cancer growth in vitro and ex vivo without adverse events via the regulation of apoptosis and autophagy, suggesting that NCL1 is a potential therapeutic agent for CRPC.

Reactivity of astrocytes to fibroblast growth factor-1 for biogenesis of apolipoprotein E-high density lipoprotein is down-regulated by long-time secondary culture.

We previously showed that astrocytes produce and release fibroblast growth factor-1 (FGF-1) upon 1-month primary and 1-week secondary culture (M/W cells) and stimulate themselves by an autocrine manner to produce apoE-high-density lipoproteins (HDL), closely associated with their generation of apoE-HDL in brain injury. Astrocytes prepared by 1-week primary and 1-month secondary culture (W/M cells), however, expressed FGF-1 as much as M/W cells but produce apoE-HDL much less. The W/M cells conditioned medium in fact contained FGF-1 activity to stimulate astrocytes prepared by 1-week primary and 1-week-secondary culture (W/W cells). FGF-1 did not stimulate W/M cells for apoE-HDL biogenesis while it stimulated W/W cells. Phosphorylation of Akt, ERK and MEK were induced by FGF-1 in W/W cells but not in W/M cells. Finally, fibroblast growth factor receptor-1 in the membrane decreased in W/M cells in comparison to W/W cells. Interestingly, the reactivity of astrocytes to FGF-1 was recovered when W/M cells were transferred to the tertiary culture of 1 week. We concluded that astrocytes decrease their reactivity to FGF-1 for apoE-HDL biogenesis in certain conditions. The findings indicate astrocyte FGF-1 enhances biogenesis of apoE-HDL also by a paracrine mechanism.

Orally administered nicotine effects on rat urinary bladder proliferation and carcinogenesis.

Tobacco smoking is a major risk factor for human cancers including urinary bladder carcinoma. Cigarette smoke inhalation in mice and orally administered nicotine in rats and mice increased urothelial cell proliferation. Nicotine, a major component of smoke, induced cell proliferation in multiple cell types in vitro. In the present study, the enhancing effects of nicotine on F344 rat bladder carcinogenesis induced by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) were examined. Nicotine administered in drinking water for 32 weeks following 4 weeks of BBN treatment significantly increased the incidence and number of urothelial carcinomas dose-dependently. Ki67 and pSTAT3 labeling indices and expression of nicotinic acetylcholine receptor alpha 7 (nAChRα7) in non-tumor bladder urothelial lesions were significantly increased by nicotine, but the TUNEL assay for apoptosis showed no increase. In a 4 week study, inhibitors of nicotinic acetylcholine receptor decreased nicotine-induced urothelial simple hyperplasia and Ki67 labeling index in the bladder and kidney pelvis at a single cytotoxic dose of nicotine (40 ppm). Urothelial cytotoxicity with regenerative proliferation was observed by light and scanning electron microscopy. In vitro, nicotine was not cytotoxic to rat or human immortalized urothelial cells (do not express nicotine receptors) below millimolar concentrations, nor in human RT4, T24 or UMUC3 urothelial carcinoma cells (express nicotine receptors). However, nicotine slightly, but statistically significantly, increased cell proliferation at micromolar concentrations in human urothelial carcinoma cells. These data suggest that nicotine enhances urinary bladder carcinogenesis by inducing cytotoxicity with regenerative proliferation. The possible role of direct mitogenesis, involving nAChR and STAT3 signaling and of nicotine receptors requires further investigation at non-cytotoxic doses of nicotine.

Cyclosporin A inhibits apolipoprotein A-I-induced early events in cellular cholesterol homeostasis in rat astrocytes.

In the biogenesis of HDL by exogenous apolipoprotein (apo) A-I in rat astrocytes, apoA-I induced translocation of phospholipase Cgamma (PLCgamma) and PKCalpha to cytosolic lipid protein particle (CLPP) [Ito et al., 2004. J. Lipid Res. 45, 2269] and caused tyrosine-phosphorylation of PLCgamma in CLPP in the initial 5 min. It also induced translocation of caveolin-1 and newly synthesized cholesterol and phospholipid to CLPP, and increased cholesterol biosynthesis prior to the HDL biogenesis [Ito et al., 2002a. J. Biol. Chem. 277, 7929]. Cyclosporin A (CsA), an indirect inhibitor of protein phosphatase 2B (PP2B) and a potential inhibitor of ABC transporter A1 (ABCA1), suppressed all of these apoA-I-induced cellular events. CsA, however, did not affect the basal lipid release by the production of HDL with endogenous apoE, except for moderate decrease of its cholesterol content. Direct inhibitors of PP2B, inhibited only the release of lipids by apoA-I and had no effect on other apoA-I-induced events. CsA thus interferes with cellular cholesterol homeostasis independently of PP2B inhibition, perhaps by direct inhibition of ABCA1 reactivity to exogenous apoA-I, although PP2B may be involved in the lipid release step. CsA could therefore cause some neurological side effects by interfering with cellular cholesterol homeostasis in the brain.

Preventive Effects of Fermented Brown Rice and Rice Bran against Prostate Carcinogenesis in TRAP Rats.

Fermented brown rice and rice bran with Aspergillus oryzae (FBRA) is considered to have the potential to prevent chemically-induced carcinogenesis in multiple organs of rodents. In the present study, we evaluated the possible chemopreventive effects of FBRA against prostate tumorigenesis. Six-week-old male rats of the transgenic rat for adenocarcinoma of prostate (TRAP) strain were fed diets containing 5% or 10% FBRA for 15 weeks. Animals were sacrificed at 21 weeks of age, and the ventral and lateral prostate were removed for histopathological evaluation and immunoblot analyses. FBRA decreased the incidence of adenocarcinoma in the lateral prostate and suppressed the progression of prostate carcinogenesis. Treatment with FBRA induced apoptosis and inhibited cell proliferation in histologically high-grade prostatic intraepithelial neoplasias. Phospho-AMP-activated kinase α (Thr172) was up-regulated in the prostate of rats fed the diet supplemented with FBRA. These results indicate that FBRA controls tumor growth by activating pathways responsive to energy deprivation and suggest that FBRA has translational potential for the prevention of human prostate cancer.

An acidic fibroblast growth factor-like factor secreted into the brain cell culture medium upregulates apoE synthesis, HDL secretion and cholesterol metabolism in rat astrocytes.

Production and release of apolipoprotein (apo) E and cholesterol were highly upregulated in the astrocytes prepared by 1-week secondary culture after 1-month primary culture of rat fetal brain cells (M/W cells) in comparison to the cells prepared by a conventional method of 1-week primary and 1-week secondary culture (W/W cells). Both cell preparations were mostly composed of astrocytes with small population of other glial cells, except that type-2 astrocyte-like cells accounted for 5-15% of M/W cells indicating more activated and/or matured status. The conditioned medium of the 1-month primary culture stimulated W/W cells to increase the release of apoE and cholesterol into the medium. The treatment of W/W cells by acidic fibroblast growth factor (aFGF) similarly upregulated biosyntheses and release of apoE and cholesterol. The effect of the conditioned medium was completely inhibited by pretreatment with an anti-aFGF antibody. The increase of the aFGF message was demonstrated in the brain cells after 1-month primary culture. The findings suggested that an aFGF-like trophic factor upregulates biosynthesis and secretion of apoE-high density lipoprotein (HDL) in astrocytes probably by autocrine stimulation in this culture system. Since this cytokine is highly expressed in the development or post-injury period of the brain, it putatively activates intercellular cholesterol transport to support construction or recovery of the brain.

Biochemical properties in membrane of rat astrocytes under oxidative stress.

Oxidative stress induced by the treatment with 100 µM hydrogen peroxide (H2O2) for 10 min enhances release of cytosolic proteins along with fibroblast growth factor 1 (FGF-1) from rat astrocytes without inducing apoptosis. FGF-1 promotes the generation of apolipoprotein E-containing high-density lipoprotein-like particles (apoE/HDL) in astrocytes, which contributes to cholesterol homeostasis in the brain. In this work, we studied various effects of oxidative stress on rat astrocyte׳s membrane to understand the mechanism underlying release of cytosolic proteins and FGF-1. The oxidative stress using 100 µM H2O2 enhanced lipid release from rat astrocytes in addition to suppression of lipid synthesis. The lipid synthesis, however, was hardly suppressed by H2O2 in the cell lines such as bovine endothelial cells and HepG2 cells from which the release of cytosolic proteins is not increased by H2O2 unlike rat astrocytes. The treatment of rat astrocytes with H2O2 changed the distribution of lipids and proteins in the caveolin-1-rich domain of membrane to the non-raft domain, which was canceled by the pretreatment of cells with low-density lipoproteins (LDL). These findings suggest that oxidative stress induced by H2O2 changes lipid level of the plasma membrane to make the membrane structure fragile in rat astrocytes. The direct treatment with H2O2 of membrane fraction prepared from rat astrocytes did not enhance lipid release from the membrane. The lipid release, however, was enhanced from the isolated membrane fraction, after the cells were treated with H2O2 and incubated in H2O2-free DPBS. Hydrogen peroxide enhanced phosphorylation of protein kinases such as Akt, MEK, and ERK in intact astrocytes without injury and stress. A MEK inhibitor, U0126, suppressed not only the H2O2-induced ERK phosphorylation but also cytosolic protein release from rat astrocytes. These findings suggest that the H2O2-induced release of cytosolic proteins depends on imbalance of lipid level in the membrane through suppressing lipid synthesis and increasing lipid release induced by the intracellular biochemical reaction such as signal transduction generated in intact rat astrocytes treated with H2O2.

Astrocyte׳s endogenous apoE generates HDL-like lipoproteins using previously synthesized cholesterol through interaction with ABCA1.

Apolipoprotein E (apoE) in the brain is predominantly synthesized in and secreted from astrocytes to generate apoE-containing high-density lipoprotein-like particles (apoE/HDL). However, the mechanism underlying generation of apoE/HDL has not been completely understood. The newly synthesized cholesterol, which is synthesized in rat astrocytes within 24 h using [(3)H]-acetate as a cholesterol precursor, was assembled as lipoproteins with densities of 1.12-1.17 g/mL (higher density HDL), although apoE was secreted as lipoproteins with lower densities of 1.08-1.12 g/mL from the cells. This finding suggests that the newly synthesized cholesterol is released without the association with apoE, which is like that from apoE-deficient mouse (apoE-KO) astrocytes. The cholesterol released from rat astrocytes at 3 days after the onset of its synthesis (previously synthesized cholesterol) was assembled as apoE/HDL with the densities of 1.08-1.12 g/mL (lower density HDL). These findings indicate that the endogenous apoE participates in the release of previously synthesized cholesterol but not newly synthesized one. Whereas, exogenously added human apoE induced release of both newly synthesized and previously synthesized cholesterols to generate apoE/HDL with lower density, suggesting that the cellular pool of cholesterol released by endogenous and exogenous apoE is different. The endogenous apoE was distributed in the caveolin-1-rich domain along with ATP-binding cassette transporter A1 (ABCA1) in the membrane fraction and immuno-precipitated using an anti-ABCA1 antibody. However, this is not the case for ABCA1-KO astrocytes. These findings suggest that endogenous apoE generates lower density HDL to produce more lipidated HDL using previously synthesized cholesterol through the interaction with ABCA1 in caveolin-1-rich domain of astrocytes.

Increasing cellular level of phosphatidic acid enhances FGF-1 production in long term-cultured rat astrocytes.

We found in a previous study that both mRNA expression and release of fibroblast growth factor 1 (FGF-1) are greater in rat astrocytes that are long term-cultured for one month (W/M cells) than in the cells cultured for one week (W/W cells). However, FGF-1 does not enhance phosphorylation of Akt, MEK, and ERK in W/M cells, while it does in W/W cells. In this work we studied the mechanism to cause these differences between W/W and W/M cells in culture. As it is known that long term culture generates oxidative stress, we characterized the stresses which W/M cells undergo in comparison with W/W cells. The levels of superoxide dismutase 1 (SOD1) and mitochondrial Bax were higher in W/M cells than in W/W cells. W/M cells recovered their ability to respond to FGF-1 to enhance phosphorylation of Akt, MEK, and ERK in the presence of antioxidants. Oxidative stress induced by hydrogen peroxide (H2O2) had no effect on mRNA expression of FGF-1 in W/W cells, although H2O2 enhances release of FGF-1 from W/W cells without inducing apoptosis. The influence of cell density was studied on mRNA expression of FGF-1 and cellular response to FGF-1, as an increasing cell density is observed in W/M cells. The increasing cell density enhanced mRNA expression of FGF-1 in W/W cells without suppression of responses to FGF-1. The decrease in cell density lowered the FGF-1 mRNA expression in W/M cells without recovery of the response to FGF-1 to enhance phosphorylation of Akt, MEK, and ERK. These findings suggest that oxidative stress attenuate sensitivity to FGF-1 and higher cell density may enhance FGF-1 expression in W/M cells. In addition, we found that the cellular level of phosphatidic acid (PA) increased in H2O2-treated W/W and W/M cells and decreased by the treatment with antioxidants, and that PA enhances the mRNA expression of FGF-1 in the W/W cells. These findings suggest that the increasing PA production may enhance FGF-1 expression to protect astrocytes against oxidative stress induced by long-term culture.

Enhancement of FGF-1 release along with cytosolic proteins from rat astrocytes by hydrogen peroxide.

We previously observed that the production and release of fibroblast growth factor (FGF-1) are increased in rat astrocytes during in vitro long-term culture, that FGF-1 enhances the generation of apoE-containing high density lipoproteins (apoE/HDL), and that the wound healing of brain cryoinjury delays in apoE-deficient mouse. The detail mechanism underlying these phenomena remains unknown. In this study, we examined effects of oxidative stress on release of FGF-1 from cultured rat astrocytes. The treatment of rat astrocytes with 100µM hydrogen peroxide (H2O2) for 10min enhanced FGF-1 release without inducing apoptosis. The conditioned medium prepared from the cells cultured in a fresh medium after the treatment with H2O2 had the FGF-1-like activities, which enhanced cholesterol synthesis, signalings to phosphorylate Akt and ERK, and apoE secretion. The oxidative stress induced by H2O2 enhanced the release of cytosolic proteins such as HSP70 and HSP90 in addition to FGF-1. Antioxidants such as ascorbic acid and ebselen suppressed the release of cytosolic proteins induced by H2O2 treatment. The addition of lipoproteins such as low density lipoproteins (LDL), furthermore, canceled H2O2-induced release of FGF-1 and cytosolic proteins. Proteolysis of cytosolic proteins in the H2O2-treated rat astrocytes was enhanced in the presence of exogenous trypsin, which was attenuated by the pretreatment with LDL, suggesting that H2O2 increases the permeability of the membrane of cells, which was prevented by the addition of lipoproteins. These findings suggest that oxidative stress is one of the candidates which triggers FGF-1 release from astrocytes in the brain, and that the lipid homeostasis in the cell membrane may regulate H2O2-induced release of FGF-1.

FGF-1-induced reactions for biogenesis of apoE-HDL are mediated by src in rat astrocytes.

Fibroblast growth factor-1 (FGF-1) is released from astrocytes in stress and stimulates MEK/ERK and PI3K/Akt pathways in autocrine fashion to increase synthesis of cholesterol and 25-OH-cholesterol, and to induce transport and secretion of apoE, respectively. FGF-1-induced phosphorylation of Src, and phosphorylation of MEK, ERK and Ark was inhibited by Src inhibitors in rat astrocytes. Src inhibitors also suppressed FGF-1-induced increase of biosynthesis and release of cholesterol and increase of apolipoprotein E (apoE) secretion. The results were reproduced in rat astrocytoma cells transfected by rat apoE and in 3T3-L1 cells. Down-regulation of Src expression reduced FGF-1-induced phosphorylation of the signalling protein and subsequent reactions. Increase by FGF-1 of messages of apoE and HMG-CoA reductase was not influenced by Src inhibitors or by its down-regulation. We conclude that FGF-1 activates Src for activation of MEK/ERK and PI3K/Akt pathways, while Src may not be involved in enhancement of transcription of the cholesterol-related genes.