Barbara Malewicz

Potent Antiandrogen and Androgen Receptor Activities of an Angelica gigas–Containing Herbal Formulation: Identification of Decursin as a Novel and Active Compound with Implications for Prevention and Treatment of Prostate Cancer

Androgen and androgen receptor (AR)-mediated signaling are crucial for the development of prostate cancer. Identification of novel and naturally occurring phytochemicals that target androgen and AR signaling from Oriental medicinal herbs holds exciting promises for the chemoprevention of this disease. In this article, we report the discovery of strong and long-lasting antiandrogen and AR activities of the ethanol extract of a herbal formula (termed KMKKT) containing Korean Angelica gigas Nakai (AGN) root and nine other Oriental herbs in the androgen-dependent LNCaP human prostate cancer cell model. The functional biomarkers evaluated included a suppression of the expression of prostate-specific antigen (PSA) mRNA and protein (IC50, approximately 7 microg/mL, 48-hour exposure) and an inhibition of androgen-induced cell proliferation through G1 arrest and of the ability of androgen to suppress neuroendocrine differentiation at exposure concentrations that did not cause apoptosis. Through activity-guided fractionation, we identified decursin from AGN as a novel antiandrogen and AR compound with an IC50 of approximately 0.4 microg/mL (1.3 micromol/L, 48-hour exposure) for suppressing PSA expression. Decursin also recapitulated the neuroendocrine differentiation induction and G1 arrest actions of the AGN and KMKKT extracts. Mechanistically, decursin in its neat form or as a component of AGN or KMKKT extracts inhibited androgen-stimulated AR translocation to the nucleus and down-regulated AR protein abundance without affecting the AR mRNA level. The novel antiandrogen and AR activities of decursin and decursin-containing herbal extracts have significant implications for the chemoprevention and treatment of prostate cancer and other androgen-dependent diseases.

Lipids in gap junction assembly and function

Gap junctions (GJ) are important regulators of cellular function. They provide channels for the direct movement of small molecules between cells and thus control cell-to-cell transfer of metabolites and the transmission of various stimuli. Gap junctions have been shown to be involved in a multitude of cellular processes ranging from cell synchronization and neuronal function to cell differentiation and carcinogenesis. Much knowledge has been gained in recent years concerning the structure and molecular organization of GJ proteins; yet, the mechanisms that control and modulate gap junction assembly and function are still not well understood. Although it is quite apparent that the GJ proteins assemble in the lipid milieu of the plasma membrane, and that the cluster of proteins assembled in the junction do function in a lipid environment, there is a general paucity of information on the role of lipids in the gap junction assembly process and in the function of gap junctions.The present review is a comprehensive account of current knowledge on gap junction lipids. We also discuss what is known to date on the involvement of lipids in gap junction formation. Special emphasis is being placed on the potential role of membrane cholesterol in gap junction assembly and function.

A novel class of pyranocoumarin anti–androgen receptor signaling compounds

Androgen and the androgen receptor (AR)–mediated signaling are crucial for prostate cancer development. Novel agents that can inhibit AR signaling in ligand-dependent and ligand-independent manners are desirable for the chemoprevention of prostate carcinogenesis and for the treatment of advanced prostate cancer. We have shown recently that the pyranocoumarin compound decursin from the herb Angelica gigas possesses potent anti-AR activities distinct from the anti–androgen bicalutamide. Here, we compared the anti-AR activities and the cell cycle arrest and apoptotic effects of decursin and two natural analogues in the androgen-dependent LNCaP human prostate cancer cell culture model to identify structure-activity relationships and mechanisms. Decursin and its isomer decursinol angelate decreased prostate-specific antigen expression with IC50 of ∼1 μmol/L. Both inhibited the androgen-stimulated AR nuclear translocation and transactivation, decreased AR protein abundance through proteasomal degradation, and induced G0/1 arrest and morphologic differentiation. They also induced caspase-mediated apoptosis and reactive oxygen species at higher concentrations. Furthermore, they lacked the agonist activity of bicalutamide in the absence of androgen and were more potent than bicalutamide for suppressing androgen-stimulated cell growth. Decursinol, which does not contain a side chain, lacked the reactive oxygen species induction and apoptotic activities and exerted paradoxically an inhibitory and a stimulatory effect on AR signaling and cell growth. In conclusion, decursin and decursinol angelate are members of a novel class of nonsteroidal compounds that exert a long-lasting inhibition of both ligand-dependent and ligand-independent AR signaling. The side chain is critical for sustaining the anti-AR activities and the growth arrest and apoptotic effects. [Mol Cancer Ther 2007;6(3):907–17]

Enhanced gap junction formation with LDL and apolipoprotein B

Gap junctions are plasma membrane specializations involved in direct cell-cell communication. Intercellular communication is dependent upon the assembly of gap junction structures and would be influenced by agents which alter the assembly process. We investigated the effects of low density lipoprotein (LDL) on gap junction assembly between cultured Novikoff cells using quantitative dye transfer and freeze-fracture electron microscopic methods. We observed a concentration-dependent increase in dye transfer (maximum effect at 2.5 micrograms/ml) and a sixfold increase in the number of aggregated gap junction particles per cell. Immunoblots of Novikoff cells probed with anti-connexin43 antibody revealed no detectable increase in gap junction protein (connexin) levels. The influence of the different components of LDL on junction formation was also examined. First, we treated cells with cholesterol (0-150 microM) in serum-free BSA media and observed a decrease in junction assembly. Second, we added apolipoprotein-B (apo-B) in phosphatidyl choline vesicles to the cells and observed a concentration-dependent increase in dye transfer (maximum effect at 2.5 micrograms protein/ml) and a fivefold increase in the number of aggregated gap junction particles per cell. The addition of phosphatidyl choline vesicles without apo-B had no effect on gap junction formation. Thus, we demonstrated that gap junction assembly can be modulated by LDL and apo-B treatments.

Lysophosphatidylcholine stabilizes small unilamellar phosphatidylcholine vesicles. Phosphorus-31 NMR evidence for the "wedge" effect

Sonication of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-sn-glycero-3-phosphocholine (lysoPC, up to approximately 30 mol %) produces small unilamellar vesicles (SUV, 250-265 A diameter). Phosphorus-31 NMR of the POPC/lysoPC vesicles gives rise to four distinct peaks for POPC and lysoPC in the outer and in the inner bilayer leaflet which can be used to localize and quantify the phospholipids in both vesicle shells. Addition of paramagnetic ions (3 mM Pr3+) enhances outside/inside chemical shift differences and allows monitoring of membrane integrity by the absence of Pr3+ in the vesicle interior. 31P NMR shows that lysoPC in these highly curved POPC/lysoPC vesicles prefers the outer bilayer leaflet. LysoPC incorporation into POPC SUV furthermore causes a substantial and concentration-dependent decrease in spin-spin relaxations (T*2) of the outside POPC phosphorus signals from 55 ms for pure POPC vesicles (v1/2, 5.8 Hz) to 29.5 ms (v1/2, 10.8 Hz) for POPC/lysoPC vesicles containing 25 mol % lysoPC. Our findings are consistent with the idea of a cone-shaped lysoPC molecule which, for geometric reasons, is preferentially accommodated in the outer bilayer leaflet. LysoPC incorporation into POPC SUV restricts POPC headgroup motion and tightens phospholipid packing, but only in the outer bilayer shell.

Phosphatidylcholine as the choline donor in sphingomyelin synthesis

Sphingomyelin synthesis was studied in cultured Novikoff rat hepatoma cells by following transfer of [14C]choline label into sphingomyelin (SPH). The study was facilitated by the fact that prelabeling of the cells with [Methyl-14C]choline resulted in rapid accumulation of essentially all the label (≈95%) in phosphatidylcholine (PC). The redistribution of PC label during a 15-hr chase was dependent upon the extracellular choline concentration. Under conditions of free choline diffusion (500 μM choline), loss of lable from PC was most pronounced, and the percentage of total radioactivity that became trapped in the extracellular water-soluble choline pool was an order of magnitude greater than in low choline medium (27 μM choline). Despite the significant loss of water-soluble label from the cells in high choline medium, SPH labeling proceeded at essentially the same rate at either choline concentration. During the label chase in 500 μM choline, the specific radioactivity of PC decreased, but the specific radioactivity of SPH continued to increase for 9–12 hr until it reached the specific radioactivity of PC. In the presence of 300 μM neophenoxine (NPO), transfer of label from PC into SPH was stimulated. NPO also decreased the specific radioactivity of PC to about the same extent as that of SPH was increased. Because transfer of choline label from PC to SPH was not affected by loss or dilution of water-soluble precursors, and because the specific radioactivity of PC and SPH, in the absence or presence of NPO, responded in a characteristic precursor product fashion, we conclude that sphingomyelin synthesis in Novikoff cells circumvents the water-soluble choline pool and that phosphatidylcholine serves as the immediate choline source. All our data support the phosphatidylcholine:ceramide cholinephosphotransferase pathway of sphingomyelin synthesis.

Relationship between dopamine-stimulated phospholipid methylation and the single-carbon folate pathway

In a previous study we demonstrated the ability of dopamine (DA) to stimulate phospholipid methylation (PLM) via a novel mechanism involving the D4 dopamine receptor (D4R) in which single‐carbon folates appeared to be the primary source of methyl groups. To further understand the relationship between D4R‐mediated PLM and folate metabolism, we examined the effect of several folate pathway interventions on the level of basal and DA‐stimulated incorporation of [14C]‐labeled formate into phospholipids in cultured SH‐SY5Y neuroblastoma cells. These interventions included: (i) Overexpression of methenyltetrahydrofolate synthetase (MTHFS). (ii) Treatment with 5‐formylTHF. (iii) Treatment with the MTHFS inhibitor 5‐formyltetrahydrohomofolic acid (5‐formylTHHF). (iv) Growth in nucleoside‐free media. 31P‐NMR was also used to follow DA‐induced changes in cell phospholipid composition. MTHFS overexpression and 5‐formylTHHF treatment, both of which lower 5‐methylTHF levels, each reduced basal PLM and its stimulation by DA. In contrast, 5‐formylTHF, which increases 5‐methylTHF, caused a dose‐dependent increase in both basal and DA‐stimulated PLM. Growth in nucleoside‐free media caused time‐dependent changes in PLM, which were due to the absence of purine nucleosides. While basal PLM was maintained at a reduced level, DA‐stimulated PLM was initially increased followed by a later decrease. Together, these findings indicate a close functional relationship between single‐carbon folate metabolism and DA‐stimulated PLM, consistent with a role for 5‐methylTHF as the methyl donor for the D4R‐mediated process.

Increased synthesis of phosphocholine is required for UV-induced AP-1 activation.

Exposure of mammalian cells to UV irradiation stimulates phosphatidylcholine hydrolysis and activates the transcription factor AP-1. Since phosphocholine (PCho), a phospholipid metabolite, is a potential regulator of mitogenesis and carcinogenesis, we examined the effect of UV exposure on the formation of PCho and the possible mediatory role of PCho in UVB-and UVC-induced activation of AP-1 in mouse JB6 epidermal cells. We found that both UVB and UVC irradiation resulted in increased PCho levels. Hemicholinium-3 (HC-3), an inhibitor of choline kinase, strongly inhibited UV-induced AP-1 activity. By contrast, relatively low levels of PCho (80 μM) or choline (20 μM) nearly doubled UV-induced AP-1 activity, while higher (2–20 mM) concentrations of PCho alone stimulated AP-1 activity 6–8-fold. Importantly, HC-3 inhibited only the stimulatory effect of choline, but not of PCho, on AP-1 activity. Of the mitogen-activated protein (MAP) kinases involved in the regulation of AP-1 activity, UVC stimulated the MAP kinase family ERK-1/ERK-2, JNK as well as p38 kinase activity. These UVC effects were all inhibited by HC-3. With UVB, by contrast, only the activation of ERK-1/ERK-2 was inhibited by HC-3. The data suggest that increased formation of PCho is required for UV-induced activation of AP-1 by an ERK-1/ERK-2-dependent mechanism.

Rapid phospholipase A2 stimulation and diacylglycerol cholinephosphotransferase inhibition in baby hamster kidney cells during initiation of dengue virus infection

Abstract Infection of baby hamster kidney (BHK-21) cells by dengue type 2 (DEN-2) virus is accompanied by a rapid but transient stimulation of phospholipase A2 activity which reaches a maximum within 40 min. Thereafter, the phospholipase A2 activity, that is associated with the microsomal fraction, returns to normal levels at an equally rapid pace. In contrast, microsomal phospholipase A1, lysophosphatidylcholine acyltransferase, or lysophospholipase are not altered early in the infection. Microsomal diacylglycerol cholinephosphotransferase was inhibited in phase with phospholipase A2 stimulation. The data obtained on the DEN-2 BHK-21 system suggest that virus-triggered phospholipase A2 activation and ensuing lysophospholipid production may facilitate initial interaction of the virion with the cell plasma membrane and may aid viral penetration.

Lyso platelet activating factor (LysoPAF) and its enantiomer. Total synthesis and carbon-13 NMR spectroscopy.

Described is a reaction sequence for the total synthesis of lyso platelet activating factor (lysoPAF; 1-O-alkyl-sn-glycero-3-phosphocholine) and its enantiomer. The procedure is versatile and yields optically pure isomers of defined chain length. The synthesis is equally suited for the preparation of lysoPAF analogues and its enantiomers with unsaturation in the long aliphatic chain. First,rac-1(3)-O-alkylglycerol is prepared by alkylation ofrac-isopropylideneglycerol with alkyl methanesulfonate followed by acid-catalyzed removal of the ketal group. The primary hydroxy group of alkylglycerol is then protected by tritylation, the secondary hydroxy group is acylated, and the protective trityl group is removed under mild acidic conditions with boric acid on silicic acid, essentially without acyl migration. Condensation of the diradylglycerol with bromoethyl dichlorophosphate in diethyl ether, hydrolysis of the resulting chloride, and nucleophilic displacement of the bromine with trimethylamine givesrac-1-O-alkyl-2-acylglycero-3-phosphocholine in good overall yield. The racemic alkylacylglycerophosphocholine is finally treated with snake venom phospholipase A2 (Ophiophagus hannah) which affords 1-O-alkyl-sn-glycero-3-phosphocholine (lysoPAF) of natural configuration in optically pure form. The “unnatural” 3-O-alkyl-2-O-acyl-sn-glycerol-1-phosphocholine enantiomer, which is not susceptible to phospholipase A2 cleavage, gives 3-O-alkyl-sn-glycero-1-phosphocholine upon deacylation with methanolic sodium hydroxide. Homogeneity and structure of the intermediates and final products were ascertained by carbon-13 nuclear magnetic resonance spectroscopy on monomeric solutions.