Anticancer activity of fish oils against human lung cancer is associated with changes in formation of PGE2 and PGE3 and alteration of Akt phosphorylation.

The beneficial effects of omega-3 fatty acids are believed to be due in part to selective alteration of arachidonate metabolism that involves cyclooxygenase (COX) enzymes. Here we investigated the effect of eicosapentaenoic acid (EPA) on the proliferation of human non-small cell lung cancer A549 (COX-2 over-expressing) and H1299 (COX-2 null) cells as well as their xenograft models. While EPA inhibited 50% of proliferation of A549 cells at 6.05 µM, almost 80 µM of EPA was needed to reach similar levels of inhibition of H1299 cells. The formation of prostaglandin (PG)E3 in A549 cells was almost threefold higher than that of H1299 cells when these cells were treated with EPA (25 µM). Intriguingly, when COX-2 expression was reduced by siRNA or shRNA in A549 cells, the antiproliferative activity of EPA was reduced substantially compared to that of control siRNA or shRNA transfected A549 cells. In line with this, dietary menhaden oil significantly inhibited the growth of A549 tumors by reducing tumor weight by 58.8 ± 7.4%. In contrast, a similar diet did not suppress the development of H1299 xenograft. Interestingly, the ratio of PGE3 to PGE2 in A549 was about 0.16 versus only 0.06 in H1299 xenograft tissues. Furthermore, PGE2 up-regulated expression of pAkt, whereas PGE3 downregulated expression of pAkt in A549 cells. Taken together, the results of our study suggest that the ability of EPA to generate PGE3 through the COX-2 enzyme might be critical for EPA-mediated tumor growth inhibition which is at least partly due to down-regulation of Akt phosphorylation by PGE3.

Discovery of IACS-9439, a Potent, Exquisitely Selective, and Orally Bioavailable Inhibitor of CSF1R.

Tumor-associated macrophages (TAMs) have a significant presence in the tumor stroma across multiple human malignancies and are believed to be beneficial to tumor growth. Targeting CSF1R has been proposed as a potential therapy to reduce TAMs, especially the protumor, immune-suppressive M2 TAMs. Additionally, the high expression of CSF1R on tumor cells has been associated with poor survival in certain cancers, suggesting tumor dependency and therefore a potential therapeutic target. The CSF1-CSF1R signaling pathway modulates the production, differentiation, and function of TAMs; however, the discovery of selective CSF1R inhibitors devoid of type III kinase activity has proven to be challenging. We discovered a potent, highly selective, and orally bioavailable CSF1R inhibitor, IACS-9439 (1). Treatment with 1 led to a dose-dependent reduction in macrophages, promoted macrophage polarization toward the M1 phenotype, and led to tumor growth inhibition in MC38 and PANC02 syngeneic tumor models.

Discovery of IPN60090, a Clinical Stage Selective Glutaminase-1 (GLS-1) Inhibitor with Excellent Pharmacokinetic and Physicochemical Properties.

Inhibition of glutaminase-1 (GLS-1) hampers the proliferation of tumor cells reliant on glutamine. Known glutaminase inhibitors have potential limitations, and in vivo exposures are potentially limited due to poor physicochemical properties. We initiated a GLS-1 inhibitor discovery program focused on optimizing physicochemical and pharmacokinetic properties, and have developed a new selective inhibitor, compound 27 (IPN60090), which is currently in phase 1 clinical trials. Compound 27 attains high oral exposures in preclinical species, with strong in vivo target engagement, and should robustly inhibit glutaminase in humans.

Pharmacokinetics of caffeic acid phenethyl ester and its catechol-ring fluorinated derivative following intravenous administration to rats.

The pharmacokinetic profiles of caffeic acid phenethyl ester (CAPE) and its catechol-ring fluorinated derivative (FCAPE) were determined in rats after intravenous administration of 5, 10 or 20 mg/kg for CAPE and 20 mg/kg for FCAPE, respectively. The plasma concentrations of CAPE and FCAPE were measured using a validated liquid chromatography tandem mass spectrometric method. The pharmacokinetic parameters were estimated using non compartmental analysis (NCA) and biexponential fit. The results showed that the area under the plasma concentration-time curve for CAPE treatment increased in a proportion greater than the increase in dose from 5 to 20 mg/kg of CAPE. Total body clearance values for CAPE ranged from 42.1 to 172 ml/min/kg (NCA) and decreased with the increasing dose of CAPE. Similarly, the volume of distribution values for CAPE ranged from 1555 to 5209 ml/kg, decreasing with increasing dose. The elimination half-life for CAPE ranged from 21.2 to 26.7 min and was independent of dose. That FCAPE was distributed extensively into rat tissues and eliminated rapidly was indicated by a high value of volume of distribution and similar short elimination half-life as that of CAPE.

Quantitative determination of fluorinated caffeic acid phenethyl ester derivative from rat blood plasma by liquid chromatography-electrospray ionization tandem mass spectrometry.

The quantitative determination of caffeic acid phenethyl ester (CAPE) and its fluorinated derivative (FCAPE) from rat plasma using ultra-performance liquid chromatography with electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) is reported. CAPE and FCAPE were extracted using ethyl acetate in the presence of methyl caffeate (MC) as internal standard. Separation was achieved using a C(18) column (2.1 mm x 50 mm, 1.7 microm) and gradient elution with water and acetonitrile containing 0.2% and 0.1% formic acid, respectively. A non-linear response over a broad concentration range (1-1000 ng/ml, r(2)>0.995 using a quadratic regression model and 1/concentration weighting) was obtained. The inter-day and intra-day variability for CAPE and FCAPE were found to be less than 14.2% and 9.5%, respectively. Data are presented to illustrate the practicality of the method for the pharmacokinetic evaluation of CAPE and FCAPE after intravenous administration to rats.

Liquid chromatography mass spectrometry for quantifying plasma lysophospholipids: potential biomarkers for cancer diagnosis.

Cancer is a complex disease with many genetic and epigenetic aberrations that result in development of tumorigenic phenotypes. While many factors contribute to the etiology of cancer, emerging data implicate lysophospholipids acting through specific cell-surface, and potentially intracellular, receptors in acquiring the transformed phenotype propagated during disease. Lysophospholipids bind to and activate specific cell-surface G protein-coupled receptors (GPCRs) that initiate cell growth, proliferation, and survival pathways, and show altered expression in cancer cells. In addition, a number of enzymes that increase lysophospholipid production are elevated in particular cell lineages and cancer patients' cells, whereas in a subset of patients, the enzymes degrading lysophospholipids are decreased. Thus, ideal conditions are established to increase lysophospholipids in the tumor microenvironment. Indeed, ascites from ovarian cancer patients, which reflects both the tumor environment and a tumor-conditioned media, exhibits markedly elevated levels of specific lysophospholipids as well as one of the enzymes involved in production of lysophospholipids: autotaxin (ATX). The potential sources of lysophospholipids in the tumor microenvironment include tumor cells and stroma, such as mesothelial cells, as well as inflammatory cells and platelets activated by the proinflammatory tumor environment. If lysophospholipids diffuse from the tumor microenvironment into the bloodstream and persist, they have the potential to serve as early diagnostic markers as well as potential monitors of tumor response to therapy. Many scientific and technical challenges need to be resolved to determine whether lysophospholipids or the enzymes producing lysophospholipids alone or in combination with other markers have the potential to contribute to early diagnosis. Breast cancer is the most frequently diagnosed cancer among women. Mammography is associated with morbidity and has a high false positive and false negative rate. Thus, there is a critical need for biomarkers that can contribute to reduced false positive and false negative diagnoses, and to identify, stage, and/or predict prognosis of this disease to improve patient management. Here we describe a technical approach that can be applied to human blood plasma to measure the concentration of growth factor-like lysophospholipids contained in circulation. Using liquid chromatography mass spectrometry (LC/MS/MS), we quantified the amount of lysophosphatidic acid (16:0, 18:0, 18:1, 18:2, and 20:4), lysophosphatidylinositol (18:0), lysophosphatidylserine (18:1), lysophosphatidylcholine (16:0, 18:0, 18:1, 18:2, and 20:4), sphingosine-1-phosphate, and sphingosylphosphorylcholine species from human female plasma samples with malignant, benign, or no breast tumor present. Other methods described here include handling patient blood samples, lipid extraction, and factors that affect lysophospholipid production and loss during sample handling.

The Liebermann-Burchard reaction: sulfonation, desaturation, and rearrangment of cholesterol in acid.

In the Liebermann-Burchard (LB) colorimetric assay, treatment of cholesterol with sulfuric acid, acetic anhydride, and acetic acid elicits a blue color. We studied the reactivity of cholesterol under LB conditions and provide definitive NMR characterization for approximately 20 products, whose structure and distribution suggest the following mechanistic picture. The major reaction pathways do not involve cholestadienes, i-steroids, or cholesterol dimers, as proposed previously. Instead, cholesterol and its acetate and sulfate derivatives undergo sulfonation at a variety of positions, often with skeletal rearrangements. Elimination of an SO(3)H group as H(2)SO(3) generates a new double bond. Repetition of this desaturation process leads to polyenes and ultimately to aromatic steroids. Linearly conjugated polyene cations can appear blue but form too slowly to account for the LB color response, whose chemical origin remains unidentified. Nevertheless, the classical polyene cation model is not excluded for Salkowski conditions (sulfuric acid), which immediately generate considerable amounts of cholesta-3,5-diene. Some rearrangements of cholesterol in H(2)SO(4) resemble the diagenesis pathways of sterols and may furnish useful lipid biomarkers for characterizing geological systems.

Detection of histone deacetylase inhibition by noninvasive magnetic resonance spectroscopy.

Histone deacetylase (HDAC) inhibitors are new and promising antineoplastic agents. Current methods for monitoring early response rely on invasive biopsies or indirect blood-derived markers. Our goal was to develop a magnetic resonance spectroscopy (MRS)-based method to detect HDAC inhibition. The fluorinated lysine derivative Boc-Lys-(Tfa)-OH (BLT) was investigated as a (19)F MRS molecular marker of HDAC activity together with (31)P MRS of endogenous metabolites. In silico modeling of the BLT-HDAC interaction and in vitro MRS studies of BLT cleavage by HDAC confirmed BLT as a HDAC substrate. BLT did not affect cell viability or HDAC activity in PC3 prostate cancer cells. PC3 cells were treated, in the presence of BLT, with the HDAC inhibitor p-fluoro-suberoylanilide hydroxamic acid (FSAHA) over the range of 0 to 10 micromol/L, and HDAC activity and MRS spectra were monitored. Following FSAHA treatment, HDAC activity dropped, reaching 53% of control at 10 micromol/L FSAHA. In parallel, a steady increase in intracellular BLT from 14 to 32 fmol/cell was observed. BLT levels negatively correlated with HDAC activity consistent with higher levels of uncleaved BLT in cells with inhibited HDAC. Phosphocholine, detected by (31)P MRS, increased from 7 to 16 fmol/cell following treatment with FSAHA and also negatively correlated with HDAC activity. Increased phosphocholine is probably due to heat shock protein 90 inhibition as indicated by depletion of client proteins. In summary, (19)F MRS of BLT, combined with (31)P MRS, can be used to monitor HDAC activity in cells. In principle, this could be applied in vivo to noninvasively monitor HDAC activity.

Chromatographic behavior of oxygenated derivatives of cholesterol.

Oxygenated derivatives of cholesterol have important functions in many biochemical processes. These oxysterols are difficult to study because of their low physiological concentrations, the facile formation of cholesterol autoxidation artifacts, and lack of information on their chromatographic behavior. Focusing on metabolites and autoxidation products of cholesterol, we have documented the chromatographic mobilities of 35 oxysterols under a variety of conditions: eight solvent systems for thin-layer chromatography on silica gel, several mobile phases for reversed-phase high-performance liquid chromatography (HPLC), and two types of stationary phase for capillary gas chromatography (GC) using trimethylsilyl derivatives. Notable differences in selectivity could be obtained by modifying the stationary or mobile phases. Separations of oxysterol pairs isomeric at side-chain carbons or C-7 were achieved on normal-phase, reversed-phase, chiral, or silver-ion HPLC columns. Chromatographic behavior is also described for side-chain hexadeuterated and heptafluorinated oxysterols, which are useful as standards in isotope dilution analyses and autoxidation studies, respectively. The overall results are relevant to many problems of oxysterol analysis, including the initial separation of oxysterols from cholesterol, determination of highly polar and nonpolar oxysterols, separation of isomeric pairs, selection of derivatization conditions for GC analysis, and quantitation of the extent of cholesterol autoxidation.

Chemical synthesis of 7- and 8-dehydro derivatives of pregnane-3,17alpha,20-triols, potential steroid metabolites in Smith-Lemli-Opitz syndrome.

Pregnane-3,17 alpha,20-triols bearing unsaturation at delta(7), delta(8), delta(5,7), or delta(5,8) have been tentatively identified as steroid metabolites in Smith-Lemli-Opitz syndrome (SLOS). Starting with 17 alpha-hydroxypregnenolone diacetate, we have synthesized 13 unsaturated C(21) triols by four different routes in one to four steps. These multifunctional steroids were prepared by a series of regio- and stereoselective transformations chosen to minimize facile olefin isomerization and 17-deoxygenation. The results include a study of stereoselectivity in the reduction of 17 alpha-hydroxy-20-ketosteroids, an alternative method for reducing diethyl azodicarboxylate adducts of delta(5,7) steroids, and an efficient oxidation-isomerization of a delta(5,7) steroid using cholesterol oxidase. The 13 triols and their synthetic precursors were fully characterized by 1H and 13C nuclear magnetic resonance (NMR) spectroscopy. The NMR data, together with molecular modeling, indicated unanticipated conformational heterogeneity for two synthetic intermediates, 17 alpha-hydroxypregna-4,7-diene-3,20-dione and 17 alpha-hydroxy-5 beta-pregn-7-ene-3,20-dione. The unsaturated C(21) triols are useful as reference standards to study adrenal steroid production in SLOS and to develop methods for pre- and postnatal diagnosis of this congenital disorder.

Alternative pathways of sterol synthesis in yeast. Use of C(27) sterol tracers to study aberrant double-bond migrations and evaluate their relative importance.

Yeast produce traces of aberrant sterols by minor alternative pathways, which can become significant when normal metabolism is blocked by inhibitors or mutations. We studied sterols generated in the absence of the delta(8)-delta(7) isomerase (Erg2p) or delta(5) desaturase (Erg3p) by incubating three mutant strains of Saccharomyces cerevisiae with 5 alpha-cholest-8-en-3beta-ol, 8-dehydrocholesterol (delta(5,8) sterol), or isodehydrocholesterol (delta(6,8) sterol), together with the corresponding 3 alpha-3H isotopomer. Nine different incubations gave altogether 16 sterol metabolites, including seven delta(22E) sterols formed by action of the yeast C-22 desaturase (Erg5p). These products were separated by silver-ion high performance liquid chromatography (Ag(+)-HPLC) and identified by gas chromatography-mass spectrometry, nuclear magnetic resonance spectroscopy, and radio-Ag(+)-HPLC. When delta(8)-delta(7) isomerization was blocked, exogenous delta(8) sterol underwent desaturation to delta(5,8), delta(6,8), and delta(8,14) sterols. Formation of delta(5,8) sterol was strongly favored over delta(6,8) sterol, but both pathways are essentially dormant under normal conditions of sterol synthesis. The delta(5,8) sterol was metabolically almost inert except for delta(22) desaturation, whereas the delta(6,8) sterol was readily converted to delta(5,7), delta(5,7,9(11)), and delta(7,9(11)) sterols. The combined results indicate aberrant metabolic pathways similar to those in mammalian systems. However, delta(5,7) sterol undergoes only slight isomerization or desaturation in yeast, an observation that accounts for the lower levels of delta(5,8) and delta(5,7,9(11)) sterols in wild-type yeast compared to Smith-Lemli-Opitz individuals.

Programming of initial steps in bile acid synthesis by breast-feeding vs. formula-feeding in the baboon.

We tested the hypothesis that breast- vs. formula-feeding differentially affects the enzymatic activity of three sterol hydroxylases critical in the initial steps of bile acid formation. Thirty baboons were either breast-fed or formula-fed for the first 14 wk of life before weaning to baboon chow. At 14 and 34 wk of age, liver biopsies were assayed for cholesterol 7alpha-hydroxylase (CYP7A1), 27-hydroxycholesterol-7alpha-hydroxylase (CYP7B1), and cholesterol 27-hydroxylase (CYP27A1). We also determined the kinetics of 3H-27-hydroxycholesterol (27-OHC) turnover in vivo at both ages. At 14 wk of age, hepatic CYP7A1 activity was low but sevenfold higher among formula-fed vs. breast-fed baboons. By 34 wk, CYP7A1 activity had increased nearly 10-fold in both infant diet groups, and the sevenfold difference in CYP7A1 between previously breast- and formula-fed animals persisted. There were no differences in CYP7B1 activities between infant diet groups at either 14 or 34 wk of age although the activity increased in both groups by about 50% from 14 to 34 wk. CYP27A1 activity also increased between 14 and 34 wk of age, and, compared with CYP7A1, relatively small differences in CYP27A1 activity due to infant diet were observed at each age. Plasma 27-OHC turnover had a half-time of 2-4 min. We had previously reported that after weaning, the total bile acid synthesis rate was higher among baboons that were formula-fed than among breast-fed animals. The present results suggest that this difference is most likely due to significantly higher CYP7A1 activity among formula-fed vs. breast-fed animals.

2004 Dr. Gary J. Becker Young Investigator Award: Relative thermosensitivity of cytotoxic drugs used in transcatheter arterial chemoembolization.

PURPOSE: Large hepatocellular carcinoma tumors are being treated increasingly with a combination of transcatheter arterial chemoembolization (TACE) and radiofrequency (RF) ablation. However, the high temperatures reached during RF ablation may reduce the cytotoxic effects of antineoplastic agents, but this has not been studied. Therefore, in the present study, the relative thermosensitivity of cytotoxic drugs commonly used in TACE was studied. MATERIALS AND METHODS: The relative cytotoxic effects of cisplatin, doxorubicin HCl, and mitomycin on the growth of human colon HT29 and lung A549 adenocarcinoma cells before and after heating each drug in solution was determined from the standpoints of different durations of exposure (15, 30, 60, 90, and 120 minutes) at a fixed temperature (120 degrees C) and exposure to different temperatures (60 degrees C, 80 degrees C, 100 degrees C, and 120 degrees C) for a fixed period of time (2 hours). After 72 hours of exposure of the cells to each drug, relative cell growth inhibition was assessed by MTT assay, and 50% inhibitory concentration (IC(50)) values were calculated for each cytotoxic agent. Finally, the heat-dependent degradation of mitomycin and doxorubicin was analyzed with use of tandem electrospray mass spectrometry. RESULTS: The relative cytotoxic activities (shown by cell growth inhibition and IC(50) values) of cisplatin, doxorubicin, and mitomycin heated to 120 degrees C for 2 hours decreased by factors of 1.35 (range, 1-1.75), 9.5 (range, 8.5-10.5), and 7.05 (range, 3.5-12), respectively. The cytotoxic activities of doxorubicin and mitomycin continued to decrease with incremental increases in temperature. Similarly, with incremental increases in the duration of exposure to heat, the cytotoxic activities of doxorubicin and mitomycin decreased. Mass spectrometric analysis of residual drug content showed that a 2-hour exposure to a temperature of 120 degrees C caused doxorubicin and mitomycin to degrade by 95% and 84%, respectively. CONCLUSIONS: The cytotoxicity of cisplatin is not affected by heat. The cytotoxicities of doxorubicin and mitomycin are reduced by high temperature and duration of exposure to heat. Although degradation of cytotoxicity starts at 60 degrees C and after 30 minutes of exposure to heat, statistically significant changes are encountered at 100 degrees C and after 90 minutes of exposure.

Cloning and functional characterization of a beta-pinene synthase from Artemisia annua that shows a circadian pattern of expression.

Artemisia annua plants produce a broad range of volatile compounds, including monoterpenes, which contribute to the characteristic fragrance of this medicinal species. A cDNA clone, QH6, contained an open reading frame encoding a 582-amino acid protein that showed high sequence identity to plant monoterpene synthases. The prokaryotically expressed QH6 fusion protein converted geranyl diphosphate to (-)-beta-pinene and (-)-alpha-pinene in a 94:6 ratio. QH6 was predominantly expressed in juvenile leaves 2 weeks postsprouting. QH6 transcript levels were transiently reduced following mechanical wounding or fungal elicitor treatment, suggesting that this gene is not directly involved in defense reaction induced by either of these treatments. Under a photoperiod of 12 h/12 h (light/dark), the abundance of QH6 transcripts fluctuated in a diurnal pattern that ebbed around 3 h before daybreak (9th h in the dark phase) and peaked after 9 h in light (9th h in the light phase). The contents of (-)-beta-pinene in juvenile leaves and in emitted volatiles also varied in a diurnal rhythm, correlating strongly with mRNA accumulation. When A. annua was entrained by constant light or constant dark conditions, QH6 transcript accumulation continued to fluctuate with circadian rhythms. Under constant light, advanced cycles of fluctuation of QH6 transcript levels were observed, and under constant dark, the cycle was delayed. However, the original diurnal pattern could be regained when the plants were returned to the normal light/dark (12 h/12 h) photoperiod. This is the first report that monoterpene biosynthesis is transcriptionally regulated in a circadian pattern.