Effects of 7,8-Dihydroxyflavone on Lipid Isoprenoid and Rho Protein Levels in Brains of Aged C57BL/6 Mice.

Synaptic impairment may be the main cause of cognitive dysfunction in brain aging that is probably due to a reduction in synaptic contact between the axonal buttons and dendritic spines. Rho proteins including the small GTPase Rac1 have become key regulators of neuronal morphogenesis that supports synaptic plasticity. Small Rho- and Ras-GTPases are post-translationally modified by the isoprenoids geranylgeranyl pyrophosphate (GGPP) and farnesyl pyrophosphate (FPP), respectively. For all GTPases, anchoring in the plasma membrane is essential for their activation by guanine nucleotide exchange factors (GEFs). Rac1-specific GEFs include the protein T lymphoma invasion and metastasis 1 (Tiam1). Tiam1 interacts with the TrkB receptor to mediate the brain-derived neurotrophic factor (BDNF)-induced activation of Rac1, resulting in cytoskeletal rearrangement and changes in cellular morphology. The flavonoid 7,8-dihydroxyflavone (7,8-DHF) acts as a highly affine-selective TrkB receptor agonist and causes the dimerization and autophosphorylation of the TrkB receptor and thus the activation of downstream signaling pathways. In the current study, we investigated the effects of 7,8-DHF on cerebral lipid isoprenoid and Rho protein levels in male C57BL/6 mice aged 3 and 23 months. Aged mice were daily treated with 100 mg/kg b.w. 7,8-DHF by oral gavage for 21 days. FPP, GGPP, and cholesterol levels were determined in brain tissue. In the same tissue, the protein content of Tiam1 and TrkB in was measured. The cellular localization of the small Rho-GTPase Rac1 and small Rab-GTPase Rab3A was studied in total brain homogenates and membrane preparations. We report the novel finding that 7,8-DHF restored levels of the Rho proteins Rac1 and Rab3A in membrane preparations isolated from brains of treated aged mice. The selective TrkB agonist 7,8-DHF did not affect BDNF and TrkB levels, but restored Tiam1 levels that were found to be reduced in brains of aged mice. FPP, GGPP, and cholesterol levels were significantly elevated in brains of aged mice but not changed by 7,8-DHF treatment. Hence, 7,8-DHF may be useful as pharmacological tool to treat age-related cognitive dysfunction although the underlying mechanisms need to be elucidated in detail.

Gallic acid inhibits migration and invasion in human osteosarcoma U-2 OS cells through suppressing the matrix metalloproteinase-2/-9, protein kinase B (PKB) and PKC signaling pathways.

Advanced cancer is a multifactorial disease which complicates treatment if the cancer cells have metastasized calling for the targeting of multiple cellular pathways. Gallic acid (GA) is known to possess multiple pharmacological activity including antitumor effects. This study investigated the mechanisms for the anticancer properties of GA on migration and invasion of human osteosarcoma U-2 OS cells. The migration and invasion in U-2 OS cells were determined by a Boyden chamber transwell assay. The expression levels and activities of MMP-2 and MMP-9 were measured by Western blotting, real-time PCR and gelatin zymography assays. All examined proteins levels from Western blotting indicated that GA decreased the protein levels of GRB2, PI3K, AKT/PKB, PKC, p38, ERK1/2, JNK, NF-κB p65 in U-2 OS cells. GA also inhibited the activities of AKT, IKK and PKC by in vitro kinase assay. GA suppressed the migration and invasive ability of U-2 OS cells, and it decreased MMP-2 and MMP-9 protein and mRNA levels and secreted enzyme activities in vitro. These results suggest that potential signaling pathways of GA-inhibited migration and invasion in U-2 OS cells may be due to down-regulation of PKC, inhibition of mitogen-activated protein kinase (MAPK) and PI3K/AKT, resulting in inhibition of MMP-2 and MMP-9 expressions.

Nucleotides released from Aß1₋42 -treated microglial cells increase cell migration and Aß1₋42 uptake through P2Y2 receptor activation.

Amyloid ß-protein (Aß) deposits in brains of Alzheimer's disease patients generate proinflammatory cytokines and chemokines that recruit microglial cells to phagocytose Aß. Nucleotides released from apoptotic cells activate P2Y(2) receptors (P2Y(2) Rs) in macrophages to promote clearance of dead cells. In this study, we investigated the role of P2Y(2) Rs in the phagocytosis and clearance of Aß. Treatment of mouse primary microglial cells with fibrillar (fAß(1-42) ) and oligomeric (oAß(1-42) ) Aß(1-42) aggregation solutions caused a rapid release of ATP (maximum after 10 min). Furthermore, fAß(1-42) and oAß(1-42) treatment for 24 h caused an increase in P2Y(2) R gene expression. Treatment with fAß(1-42) and oAß(1-42) aggregation solutions increased the motility of neighboring microglial cells, a response inhibited by pre-treatment with apyrase, an enzyme that hydrolyzes nucleotides. The P2Y(2) R agonists ATP and UTP caused significant uptake of Aß(1-42) by microglial cells within 30 min, which reached a maximum within 1 h, but did not increase Aß(1-42) uptake by primary microglial cells isolated from P2Y(2) R(-/-) mice. Inhibitors of α(v) integrins, Src and Rac decreased UTP-induced Aß(1-42) uptake, suggesting that these previously identified components of the P2Y(2) R signaling pathway play a role in Aß phagocytosis by microglial cells. Finally, we found that UTP treatment enhances Aß(1-42) degradation by microglial cells, but not in cells isolated from P2Y(2) R(-/-) mice. Taken together, our findings suggest that P2Y(2) Rs can activate microglial cells to enhance Aß clearance and highlight the P2Y(2) R as a therapeutic target in Alzheimer's disease.

Rutin inhibits human leukemia tumor growth in a murine xenograft model in vivo.

Numerous studies have shown that rutin has anticancer effects. We have previously reported that rutin induced cell cycle arrest and apoptosis in murine leukemia WEHI-3 cells in vitro and in vivo. However, there are no data showing that rutin inhibits human leukemia HL-60 cells in vivo in a murine xenograft animal model. Human leukemia HL-60 cells were implanted into mice and treated with vehicle (1% DMSO), rutin (120 mg/kg of body weight) or vinblastine (120 µg/kg of body weight). Compounds and agents were injected once every four days intraperitoneally (i.p.) for 36 days. Treatment with 120 mg/kg of rutin or with 120 µg/kg of vinblastine resulted in a reduction of tumor weight and volume when compared with the control groups. Tumor size in xenograft mice treated with 120 mg/kg of rutin was significantly smaller than that in the untreated-control group. These novel findings indicate that rutin inhibits tumor growth in a xenograft animal model. Rutin may be useful in treating leukemia but certainly much more research is needed. © 2011 Wiley Periodicals, Inc. Environ Toxicol, 2012.

The quinolone derivative CHM-1 inhibits murine WEHI-3 leukemia in BALB/c mice in vivo.

CHM-1 [2-(2-fluorophenyl)-6,7-methylenedioxyquinolin-4-one] is a quinolone derivative that has been reported to induce apoptosis and inhibit invasion of cancer cells. However, there is no available information to address the effects of CHM-1 on leukemia cells in vivo. Therefore, the present study examined the effects of CHM-1 using a mouse model of leukemia. We established leukemia in mice by injecting WEHI-3 cells into BALB/c mice. Mice were then treated with or without CHM-1 (5 and 10 mg/kg). CHM-1 promoted the total survival rate of leukemic mice and these effects were dose-dependent. CHM-1 increased body weight and decreased spleen weight, but did not affect liver weight. The levels of cell markers Mac-3 and CD11b were reduced by CHM-1, indicating that the differentiation of macrophage precursor cells was inhibited. Levels of CD3 and CD19 were induced by CHM-1, suggesting that the differentiation of precursors of T and B cells was promoted in PBMC. Results of the present study indicate that CHM-1 has an inhibitory effect on leukemia induced in mice in vivo and warrants further study as to the mechanisms and effects in other types of cancer.

Enlarging the scope of cell-penetrating prenylated peptides to include farnesylated 'CAAX' box sequences and diverse cell types.

Protein prenylation is a posttranslational modification that is present in a large number of proteins; it has been proposed to be responsible for membrane association and protein-protein interactions, which contribute to its role in signal transduction pathways. Research has been aimed at inhibiting prenylation with farnesyltransferase inhibitors based on the finding that the farnesylated protein Ras is implicated in 30% of human cancers. Despite numerous studies on the enzymology of prenylation in vitro, many questions remain about the process of prenylation as it occurs in living cells. Here we describe the preparation of a series of farnesylated peptides that contain sequences recognized by protein farnesyltransferase. Using a combination of flow cytometry and confocal microscopy, we show that these peptides enter a variety of different cell types. A related peptide where the farnesyl group has been replaced by a disulfide-linked decyl group is also shown to be able to efficiently enter cells. These results highlight the applicability of these peptides as a platform for further study of protein prenylation and subsequent processing in live cells.

Mitochondrial-dependent caspase activation pathway is involved in baicalein-induced apoptosis in human hepatoma J5 cells.

Baicalein has been reported to induce growth-inhibitory activity in vitro in human cancer cells; however, the molecular mechanism of action is not completely understood. A pharmacological dose (10-100 microM) of baicalein exerted a cytotoxic effect on human hepatoma J5 cells resulting in G2/M arrest and apoptosis. In addition to cytotoxicity in J5 cells, several apoptotic events including mitochondrial cytochrome c release, activation of caspase-9 and -3 occurred. Baicalein induced AIF and Endo G release from mitochondria indicating that baicalein stimulates apoptosis through the caspase-independent pathway, while undergoing apoptosis, there was a remarkable accumulation of G2/M cells. Also, the ratio of Bax/Bcl-2 was increased leading to changes in mitochondria membrane potential (DeltaPsim) and release of cytochrome c, whereas the baicalein-induced apoptosis was partially abrogated by pretreatment with the pan-caspase inhibitor z-VAD-fmk, the accumulation of G2/M cells remained. These results demonstrate that the cytotoxicity of baicalein in J5 cells is attributable to apoptosis mainly involving G2/M-arrest in an ER-dependent manner, via a mitochondria-dependent caspase pathway and as well as contributions of AIF and Endo G pathways.

DNA damage and endoplasmic reticulum stress mediated curcumin-induced cell cycle arrest and apoptosis in human lung carcinoma A-549 cells through the activation caspases cascade- and mitochondrial-dependent pathway.

Curcumin, a major component of the Curcuma species, is known to have antioxidant, anti-inflammatory properties and induce apoptosis of cancer cells, however, the precise molecular mechanisms of apoptosis in vitro are unclear. In this study, we showed that curcumin, a plant product containing the phenolic phytochemical, caused DNA damage and endoplasmic reticulum (ER) stress and mitochondrial-dependent-induced apoptosis through the activation of caspase-3 at a treatment concentration of 30 microM in human lung cancer A-549 cells. In contrast, treatment with 5-10 microM of curcumin did not induce significant apoptosis, but rather induced G2/M-phase arrest in A-549 cells. Flow cytometric analysis indicated that curcumin directly increased intracellular oxidative stress based on the cell permeable dye, 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) acting as an indicator of reactive oxygen species (ROS) generation. GADD153 and GRP78 were increased by curcumin which was indicative of ER stress. Curcumin increased Ca(2+) levels and the mitochondrial membrane potential (DeltaPsi(m)), was decreased in A-549 cells. Overall, our results demonstrated that curcumin treatment causes cell death by activating pathways inducing G2/M-phase arrest and apoptosis.

Bcl-2 upregulation and neuroprotection in guinea pig brain following chronic simvastatin treatment.

The present study determined if chronic simvastatin administration in vivo would provide neuroprotection in brain cells isolated from guinea pigs after challenge with the Bcl-2 inhibitor HA 14-1 or the NO donor sodium nitroprusside (SNP). Bcl-2 levels were significantly increased in brains of simvastatin-treated guinea pigs while levels of the pro-apoptotic protein Bax were significantly reduced. The ratio of Bax/Bcl-2, being a critical factor of the apoptotic state of cells, was significantly reduced in simvastatin-treated animals. Cholesterol levels in the brain remained unchanged in the simvastatin group. Brain cells isolated from simvastatin-treated guinea pigs were significantly less vulnerable to mitochondrial dysfunction and caspase-activation. These results provide new insight into potential mechanisms for the protective actions of statins within the CNS where programmed cell death has been implicated.

Amyloid beta-protein interactions with membranes and cholesterol: causes or casualties of Alzheimer's disease.

Amyloid beta-protein (Abeta) is thought to be one of the primary factors causing neurodegeneration in Alzheimer's disease (AD). This protein is an amphipathic molecule that perturbs membranes, binds lipids and alters cell function. Several studies have reported that Abeta alters membrane fluidity but the direction of this effect has not been consistently observed and explanations for this lack of consistency are proposed. Cholesterol is a key component of membranes and cholesterol interacts with Abeta in a reciprocal manner. Abeta impacts on cholesterol homeostasis and modification of cholesterol levels alters Abeta expression. In addition, certain cholesterol lowering drugs (statins) appear to reduce the risk of AD in human subjects. However, the role of changes in the total amount of brain cholesterol in AD and the mechanisms of action of statins in lowering the risk of AD are unclear. Here we discuss data on membranes, cholesterol, Abeta and AD, and propose that modification of the transbilayer distribution of cholesterol in contrast to a change in the total amount of cholesterol provides a cooperative environment for Abeta synthesis and accumulation in membranes leading to cell dysfunction including disruption in cholesterol homeostasis.

Ethanol and lipid metabolic signaling.

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Shivendra D. Shukla and Grace Y. Sun. The presentations were (1) Metabolic turnover of ethanol into cellular lipids and platelet activating factor, by Shivendra D. Shukla; (2) Ethanol action on the phospholipase A2 signaling pathways in astrocytes, by Grace Y. Sun; (3) Mechanisms of ethanol-induced perturbation of lipoprotein cholesterol transport, by W. Gibson Wood; (4) Transfer of an abnormal ethanol-induced phospholipid, phosphatidylethanol, between lipoproteins, by Markku J. Savolainen; (5) Phospholipase-d-mediated formation of phosphatidylethanol, by Christer Alling; and (6) Changes in phosphoinositide signaling after chronic ethanol treatment, by Jan B. Hoek.