Perillic Acid Research Articles

A snapshot of plasma metabolites in first-episode schizophrenia: a capillary electrophoresis time-of-flight mass spectrometry study

Few biomarkers have been known that can easily measure clinical conditions in mental illnesses such as schizophrenia. Capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) is a new method that can measure ionized and low-molecular-weight metabolites. To explore global metabolomic alterations that characterize the onset of schizophrenia and identify biomarkers, we profiled the relative and absolute concentrations of the plasma metabolites from 30 patients with first-episode schizophrenia (FESZ, four drug-naïve samples), 38 healthy controls and 15 individuals with autism spectrum disorders using CE-TOFMS. Five metabolites had robust changes (increased creatine and decreased betaine, nonanoic acid, benzoic acid and perillic acid) in two independent sample sets. Altered levels of these metabolites are consistent with well-known hypotheses regarding abnormalities of the homocysteine metabolism, creatine kinase-emia and oxidative stress. Although it should be considered that most patients with FESZ received medication, these metabolites are candidate biomarkers to improve the determination of diagnosis, severity and clinical stages, especially for FESZ.

Radical-Scavenging Compounds from Olive Tree (Olea europaea L.) Wood

The purpose of this study was to complete knowledge on the chemical composition and radical-scavenging activity of olive tree wood. Two new monoterpene glycosides, (-)-oleuropeic acid 6'-O-α-D-glucopyranosyl ester (6a) and (-)-perillic acid 1'-O-β-D-primeverosyl ester (8), together with the known compounds (-)-oleuropeic acid (1), (-)-olivil (2), the aldehydic form of oleuropein aglycone (3), (+)-1-hydroxypinoresinol 1-O-β-D-glucopyranoside (4), (-)-oleuropeic acid 1'-O-β-D-glucopyranosyl ester (5), (-)-oleuropeic acid 6'-O-β-D-glucopyranosyl ester (6b), and (-)-olivil 4-O-β-D-glucopyranoside (7) were isolated from an ethyl acetate extract. The radical scavengers found (2-4 and 7) were detected and isolated with the help of the online HPLC-DAD-DPPH/ABTS technique. Compounds 2-4 and 7 displayed a higher antioxidative effect against the free radical DPPH than the reference BHT and lower than hydroxytyrosol, whereas compounds 1, 5, 6a, 6b, and 8 showed no activity.

Bioconversion of R-(+)-limonene to perillic acid by the yeast Yarrowia lipolytica

Perillyl derivatives are increasingly important due to their flavouring and antimicrobial properties as well as their potential as anticancer agents. These terpenoid species, which are present in limited amounts in plants, may be obtained via bioconversion of selected monoterpene hydrocarbons. In this study, seventeen yeast strains were screened for their ability to oxidize the exocyclic methyl group in the p-menthene moiety of limonene into perillic acid. Of the yeast tested, the highest efficiency was observed for Yarrowia lipolytica ATCC 18942. The conversion of R (+)-limonene by Y. lipolytica was evaluated by varying the pH (3 to 8) and the temperature (25 to 30 °C) in a reaction medium containing 0.5% v/v limonene and 10 g/L of stationary phase cells (dry weight). The best results, corresponding to 564 mg/L of perillic acid, were obtained in buffered medium at pH 7.1 that was incubated at 25 °C for 48 h. The stepwise addition of limonene increased the perillic acid concentration by over 50%, reaching 855 mg/L, whereas the addition of glucose or surfactant to the reaction medium did not improve the bioconversion process. The use of Y. lipolytica showed promise for ease of further downstream processing, as perillic acid was the sole oxidised product of the bioconversion reaction. Moreover, bioprocesses using safe and easy to cultivate yeast cells have been favoured in industry.

Human Breast Tissue Disposition and Bioactivity of Limonene in Women with Early-Stage Breast Cancer

Limonene is a bioactive food component found in citrus peel oil that has shown chemopreventive and chemotherapeutic activities in preclinical studies. We conducted an open-label pilot clinical study to determine the human breast tissue disposition of limonene and its associated bioactivity. We recruited 43 women with newly diagnosed operable breast cancer electing to undergo surgical excision to take 2 grams of limonene daily for two to six weeks before surgery. Blood and breast tissue were collected to determine drug/metabolite concentrations and limonene-induced changes in systemic and tissue biomarkers of breast cancer risk or carcinogenesis. Limonene was found to preferentially concentrate in the breast tissue, reaching high tissue concentration (mean = 41.3 μg/g tissue), whereas the major active circulating metabolite, perillic acid, did not concentrate in the breast tissue. Limonene intervention resulted in a 22% reduction in cyclin D1 expression (P = 0.002) in tumor tissue but minimal changes in tissue Ki67 and cleaved caspase-3 expression. No significant changes in serum leptin, adiponectin, TGF-β1, insulin-like growth factor binding protein-3 (IGFBP-3), and interleukin-6 (IL-6) levels were observed following limonene intervention. There was a small but statistically significant postintervention increase in insulin-like growth factor I (IGF-I) levels. We conclude that limonene distributed extensively to human breast tissue and reduced breast tumor cyclin D1 expression that may lead to cell-cycle arrest and reduced cell proliferation. Furthermore, placebo-controlled clinical trials and translational research are warranted to establish limonene's role for breast cancer prevention or treatment.

Whole‐cell‐based CYP153A6‐catalyzed (S)‐limonene hydroxylation efficiency depends on host background and profits from monoterpene uptake via AlkL

Living microbial cells are considered to be the catalyst of choice for selective terpene functionalization. However, such processes often suffer from side product formation and poor substrate mass transfer into cells. For the hydroxylation of (S)-limonene to (S)-perillyl alcohol by Pseudomonas putida KT2440 (pGEc47ΔB)(pCom8-PFR1500), containing the cytochrome P450 monooxygenase CYP153A6, the side products perillyl aldehyde and perillic acid constituted up to 26% of the total amount of oxidized terpenes. In this study, it is shown that the reaction rate is substrate-limited in the two-liquid phase system used and that host intrinsic dehydrogenases and not CYP153A6 are responsible for the formation of the undesired side products. In contrast to P. putida KT2440, E. coli W3110 was found to catalyze perillyl aldehyde reduction to the alcohol and no oxidation to the acid. Furthermore, E. coli W3110 harboring CYP153A6 showed high limonene hydroxylation activities (7.1 U g CDW-1). The outer membrane protein AlkL was found to enhance hydroxylation activities of E. coli twofold in aqueous single-phase and fivefold in two-liquid phase biotransformations. In the latter system, E. coli harboring CYP153A6 and AlkL produced up to 39.2 mmol (S)-perillyl alcohol L tot-1 within 26 h, whereas no perillic acid and minor amounts of perillyl aldehyde (8% of the total products) were formed. In conclusion, undesired perillyl alcohol oxidation was reduced by choosing E. coli's enzymatic background as a reaction environment and co-expression of the alkL gene in E. coli represents a promising strategy to enhance terpene bioconversion rates.

D-Limonene modulates T lymphocyte activity and viability

d-Limonene, a cyclic terpene that is a major component of several plant essential oils, is used widely as an additive in perfumes, soaps, foods and beverages, and has also been shown to possess chemopreventative and chemotherapeutic activity. A limited number of studies have been conducted investigating the effect of d-limonene on immune system function. We show that d-limonene and its metabolites limonene-1-2-diol and perillic acid inhibit the production by CD3+CD4+ T cells of IFN-γ, IL-2, TNF-α, IL-4 and IL-13, and the production by CD3+CD8+ T cells of IFN-γ, IL-2, and TNF-α. Additionally, the upregulation of CD25, CD69 and CD40L by activated T lymphocytes is modulated by d-limonene, limonene-1-2-diol and perillic acid treatment. Furthermore, high concentrations of d-limonene, limonene-1-2-diol and perillic acid induce T lymphocyte cell death. These data suggest that d-limonene possesses immunomodulatory activity that must be considered when utilizing the compound for therapeutic or commercial purposes.

Abstract 3558: A pilot clinical study of limonene in women with early stage breast cancer: Effects of short-term treatment on tissue limonene disposition and serum markers

Abstract Background: Limonene is a highly lipophilic monoterpene found in citrus peel oil that has demonstrated anticancer properties in preclinical studies. The purpose of this study was to evaluate whether limonene and its primary metabolite perillic acid (PA) would distribute extensively to the breast tissue and reach an effective drug concentration. Secondary endpoints included evaluation of changes in cancer-related biomarkers in plasma and tissue. Methods: Participants (N = 40) with newly diagnosed stage 0 - 2 breast cancer took 2 grams daily of oral limonene for 2 - 6 weeks (21.5 ± 8.8 days) prior to planned surgical tumor resection. Blood was drawn pre/post intervention for measurement of serum concentration of limonene, PA, and protein biomarkers, as well as to assess toxicity profiles. A small piece of breast tissue adjacent to the tumor mass was also collected for analysis of limonene and PA levels. Limonene and PA levels in tissue and serum were analyzed using specific chromatography-based assays. Results: Limonene was found to preferentially concentrate in breast tissue (332.3 + 336.1 uM) versus plasma (0.49 + 0.67 uM) with tissue-to-plasma concentration ratio (TPCR) of 1,667 + 312.5 (P<0.001). PA did not concentrate in breast tissue (5.73 + 10.28 uM) versus plasma (3.89 + 6.81 uM) with TPCR of 1.4 + 0.56. Post-intervention serum levels of leptin, adiponectin, TGF-beta1, IGFBP-3 and IL-6 were unchanged from baseline. There was a small but statistically significant post-intervention increase in IGF-1, however the change was no longer significant after adjustment for number of days on agent. Possibly or probably related adverse events were primarily GI-related and were not dose-limiting. No clinically significant changes in complete blood count, renal, hepatic or other blood chemistry studies were noted. Analysis of the intervention effects on tissue biomarkers of proliferation, apoptosis, and cell cycle regulation is ongoing. Discussion: Oral limonene preferentially concentrates in the breast tissue and has a favorable side effect profile. PA does not readily concentrate in breast tissue when administered as oral limonene. Short-term limonene intervention resulted in minimal changes in systemic biomarkers. Further clinical trials with a longer intervention are necessary to establish limonene's potential role as a chemopreventive agent in breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3558. doi:1538-7445.AM2012-3558

Influence of the Active Compounds of Perilla frutescens Leaves on Lipid Membranes

The leaves of the annual plant Perilla frutescens are used widely as a spice and a preservative in Asian food as well as in traditional medicine. The active compounds in the leaves are the cyclic monoterpene limonene (1) and its bio-oxidation products, perillaldehyde (2), perillyl alcohol (3), and perillic acid (4). These compounds are known to be biologically active and exhibit antimicrobial, anticancer, and anti-inflammatory effects that could all be membrane mediated. In order to assess the possible biophysical effects of these compounds on membranes quantitatively, the influence of limonene and its bio-oxidation products has been investigated on a membrane model composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) using differential scanning calorimetry (DSC), isothermal titration calorimetry (ITC), and electron paramagnetic resonance spectroscopy (EPR). It was found that limonene (1), perillyl alcohol (2), and perillaldehyde (3) partitioned into the DMPC membrane, whereas perillic acid (4) did not. The DSC results demonstrated that all the partitioning compounds strongly perturbed the phase transition of DMPC, whereas no perturbation of the local membrane order was detected by EPR spectroscopy. The results of the study showed that limonene (1) and its bio-oxidation products affect membranes in rather subtle ways.

P3-11-03: A Phase I Trial of D-Limonene in Newly Diagnosed Breast Cancer Patients: A Chemoprevention Study.

Abstract Background: D-limonene (DL) is a highly lipophilic monoterpene found naturally in citrus that has been demonstrated in preclinical studies to have anticancer properties. Early phase clinical trials support the investigation of DL in the chemoprevention of breast cancer. We sought to evaluate whether DL and its presumed active metabolite perillic acid (PA) would distribute extensively to the breast tissue and reach an effective drug concentration. We hypothesized that the mechanism of DL is related to the induction of apoptosis in cancer cells. Materials and Methods: We enrolled 40 patients with newly diagnosed Stage 0–2 breast cancer to take 2 grams daily of oral DL for 2–6 weeks prior to planned surgical intervention. Blood was drawn pre/post intervention to assess for toxicity and plasma concentration of DL and PA. Adverse effects related or possibly related to the study drug were noted. A small piece of breast tissue adjacent to the tumor mass was used to measure drug concentration for each patient. DL and PA levels in breast tissue were analyzed by gas chromatography and liquid chromatography, respectively, in tandem with mass spectrometry. Analysis of variance testing was used to determine if DL or PA preferentially concentrated in the breast tissue compared to plasma. We tested for a possible modulation of the biomarkers estrogen receptor (ER), progesterone receptor, HER2, Ki67 or grade pre/post treatment by comparing results from the core biopsy to the surgical pathology and applying pairwise Student's T tests. The caspase 3 and the annexin V assays were performed by plating 10,000 cells per well for the cell lines MCF7, MDA-231, BT474, and T47D and separately administering DL and PA acid in serial dilutions in their treatment concentration ranges in triplicate and read by microplate. Results: DL was found to preferentially concentrate in breast tissue versus plasma (tissue/plasma concentration ratio (TPCR) of 1297, p<0.001) while PA did not concentrate (TPCR of 1.4, p=0.9). 20 patients (50%) reported Grade 1 eructation, which was the most common adverse effect. A slight decrease in white count from a mean of 7.1 to 6.6 (p=0.03) and a slight increase in ALT from a mean of 22.5 to 26.9 (p=0.03) were noted. No other statistically significant changes in laboratory values related to serum complete blood count, renal, hepatic or other studies were noted. No change in tissue biomarkers were noted post-treatment. DL produced a dose dependent increase in the apoptotic markers cleaved caspase 3 and annexin V for the ER positive cell lines MCF7, BT474, and T47D, however, no such response was noted for the triple negative cell line MDA-231. A PA dose response trend was noted with the annexin V but not the caspase 3 assay for each cell line. Discussion: D-limonene preferentially concentrates in the breast tissue and is a candidate chemopreventive agent based on its favorable side effect profile. PA does not readily concentrate in the breast when administered as oral DL. Our correlative studies establish that DL induces apoptosis in ER positive breast cancer cell lines; no definite relationship between PA and apoptosis was found. Further clinical trials of DL are necessary to establish its potential role as a chemopreventive agent in breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-11-03.

Bilayer-Modifying Potential of Limonene and its Metabolites

Membrane protein function depends on lipid bilayer properties, which can be modified by small molecules. This may account for some of the undesired clinical effects of amphiphatic drugs that modify bilayer properties. In this study, we assessed whether D-limonene and its metabolites (perillyl alcohol, perillaldehyde, and perillic acid) alter lipid bilayer properties, as sensed by bilayer-spanning gramicidin (gA) channels, using a fluorescence assay and single-channel electrophysiology. D-limonene and its metabolites are terpenes that are found in a variety of foods, particularly citrus oils. They have been shown as potential cancer prevention and treatment agents and as antimicrobials. These terpenes are also used as solvents and flavor and fragrance agents. Given the wide range of biological functions of these hydrophobic/amphiphilic compounds, it becomes important to determine to what extent they alter bilayer properties. Using the fluorescence assay, we find that at micromolar concentrations (nominal concentrations): D-limonene decreases gA channel activity, perillyl alcohol and perillaldehyde increase activity, and perillic acid has no apparent effect. When examined using single-channel electrophysiology, each terpene increased gA channel lifetime and appearance rate. In the case of perillaldehyde and perillyl alcohol there is agreement between the two assays but not for D-limonene and perillic acid. We are exploring the basis for these differences. The changes in gA channel function (bilayer properties) were observed at low membrane concentrations (mole fraction ∼0.02-0.03), indicating that these terpenes are potent bilayer modifiers.

Interaction of Salicylate and a Terpenoid Plant Extract with Model Membranes: Reconciling Experiments and Simulations

We investigate the effects of two structurally similar small cyclic molecules: salicylic acid and perillic acid on a zwitterionic model lipid bilayer, and show that both molecules might have biological activity related to membrane thinning. Salicylic acid is a nonsteroidal antiinflammatory drug, some of the pharmacological properties of which arise from its interaction with the lipid bilayer component of the plasma membrane. Prior simulations show that salicylate orders zwitterionic lipid membranes. However, this is in conflict with Raman scattering and vesicle fluctuation analysis data, which suggest the opposite. We show using extensive molecular dynamics simulations, cumulatively >2.5 μs, that salicylic acid indeed disorders membranes with concomitant membrane thinning and that the conflict arose because prior simulations suffered from artifacts related to the sodium-ion induced condensation of zwitterionic lipids modeled by the Berger force field. Perillic acid is a terpenoid plant extract that has antiinfective and anticancer properties, and is extensively used in eastern medicine. We found that perillic acid causes large-scale membrane thinning and could therefore exert its antimicrobial properties via a membrane-lytic mechanism reminiscent of antimicrobial peptides. Being more amphipathic, perillic acid is more potent in disrupting lipid headgroup packing, and significantly modifies headgroup dipole orientation. Like salicylate, the membrane thinning effect of perillic acid is masked by the presence of sodium ions. As an alternative to sodium cations, we advocate the straightforward solution of using larger countercations like potassium or tetra-methyl-ammonium that will maintain electroneutrality but not interact strongly with, and thus not condense, the lipid bilayer.

Protective Role of Perillic Acid Against Radiation−Induced Oxidative Stress, Cytokine Profile, DNA Damage, and Intestinal Toxicity in Mice

The radioprotective effect of perillic acid was studied using an in vivo mouse model. Whole-body exposure of Swiss albino mice to γ-rays (6 Gy) reduced the total white blood cell count to 1035 ± 378 cells/mm3 on the 9th day, which was significantly elevated to 2196 ± 382 cells/mm3 by the administration of perillic acid (50 μmoles/kg body weight, intraperitoneally) on the same day. The number of bone marrow cells and α-esterase positive cells in control animals after 11 days of irradiation was reduced to 12.5 ± 0.8 × 106 cells/femur and 674 ± 45.2/4000 cells, respectively. In perillic acid treated animals, bone marrow cellularity was increased to 14.8 ± 1.8 × 106 cells/femur and α-esterase positive cells were 941 ± 56.5 /4000 cells, similar to normal level (935 ± 51.4/4000 cells). Administration of perillic acid could reduce the radiation-induced elevated levels of alkaline phosphatase (ALP), glutathione-pyruvate transferase (GPT) and lipid peroxidation (LPO) in both serum and liver of irradiated animals. Perillic acid could significantly enhance the glutathione (GSH) content in liver and intestinal mucosa of irradiated animals. Histopathological analysis of small intestine also suggests that perillic acid could reduce the radiation-induced intestinal damage. The level of proinflammatory cytokines such as IL-1β, TNF-α and CRP, which were elevated during irradiation, was significantly reduced by the Perillic acid administration. Perillic acid treatment could also stimulate the production of other cytokines such as GM-CSF and IFN-γ in animals exposed to whole-body gamma irradiation. Agarose gel electrophoresis of DNA isolated from bone marrow of mice exposed to gamma radiation showed heavy damage that was reduced by treatment with perillic acid.

Integrated bioprocess for the oxidation of limonene to perillic acid with Pseudomonas putida DSM 12264

An efficient integrated bioprocess for the oxidation of limonene to perillic acid with Pseudomonas putida DSM 12264 was developed. Perillic acid is a promising candidate for natural preservation and pharmaceutical application. At elevated concentrations the monoterpenoic acid inhibits growth and biotransformation activity of P. putida DSM 12264. The maximum growth rate showed a linear decrease from μ = 1.43 h −1 in the absence of perillic acid to complete inhibition at 165 ± 7 mM perillic acid. The maximum specific activity of limonene-transforming resting cells revealed an exponential decrease from almost 8 U/g cdw without perillic acid to <0.5 U/g cdw at >25 mM perillic acid. A method for in situ product recovery (ISPR) based on anion exchange resin was established to overcome product inhibition. A column containing a fluidized bed of Amberlite IRA 410 Cl was coupled to the bioreactor and enabled product removal by continuously recirculating the unfiltered broth through the ISPR unit. This led to a cumulative perillic acid concentration of 187 mM (31 g/L) after 7 days, which represents the highest product concentration achieved in a microbial monoterpene oxyfunctionalization so far. The ISPR approach reduced the further downstream processing steps needed which yielded a 93% pure product with a loss of 2%.

Rosuvastatin induces delayed preconditioning against oxygen-glucose deprivation in cultured cortical neurons

We tested whether rosuvastatin (RST) protected against oxygen-glucose deprivation (OGD)-induced cell death in primary rat cortical neuronal cultures. OGD reduced neuronal viability (%naive controls, mean +/- SE, n = 24-96, P < 0.05) to 44 +/- 1%, but 3-day pretreatment with RST (5 microM) increased survival to 82 +/- 2% (P < 0.05). One-day RST treatment was not protective. RST-induced neuroprotection was abolished by mevalonate or geranylgeranyl pyrophosphate (GGPP), but not by cholesterol coapplication. Furthermore, RST-induced decreases in neuronal cholesterol levels were abolished by mevalonate but not by GGPP. Reactive oxygen species (ROS) levels were reduced in RST-preconditioned neurons after OGD, and this effect was also reversed by both mevalonate and GGPP. These data suggested that GGPP, but not cholesterol depletion, were responsible for the induction of neuroprotection. Therefore, we tested whether 3-day treatments with perillic acid, a nonspecific inhibitor of both geranylgeranyl transferase (GGT) GGT 1 and Rab GGT, and the GGT 1-specific inhibitor GGTI-286 would reproduce the effects of RST. Perillic acid, but not GGTI-286, elicited robust neuronal preconditioning against OGD. RST, GGTI-286, and perillic acid all decreased mitochondrial membrane potential and lactate dehydrogenase activity in the cultured neurons, but only RST and perillic acid reduced neuronal ATP and membrane Rab3a protein levels. In conclusion, RST preconditions cultured neurons against OGD via depletion of GGPP, leading to decreased geranylgeranylation of proteins that are probably not isoprenylated by GGT 1. Reduced neuronal ATP levels and ROS production after OGD may be directly involved in the mechanism of neuroprotection.

Effect of flavour components in lemon essential oil on physical or psychological stress

AbstractCitrus essential oils, including lemon essential oil, have long been used widely in aromatherapy and alternative medicine. This study was designed to assess the effects of flavour components in lemon essential oil on physical and psychological stress. In this evaluation, acute cold stress and communication box techniques were used to apply stress after intraperitoneal administration of essential oil components such as limonene, γ‐terpinene and citral. Serum corticosterone and monoamines in brain tissues were then determined. In the present study, it found the presence of perillic acid, a limonene metabolite, at concentrations of 1.5–2.5 μg/mL in serum and 0.4–0.6 μg/g in brain tissue collected 3 h after administration. The research also showed that the lemon components R‐limonene, citral and γ‐terpinene inhibited elevation of serum corticosterone levels and cerebral monoamine levels. S‐limonene, a stereoisomer of R‐limonene, seemed to have stronger effect than other monoterpenes and inhibited brain monoamines elevation on psychological stress. These findings suggest that limonenes, and particularly S‐limonene, have a potent stress‐alleviating effect, and the possibility that different stereoisomers of limonene have different levels of activity in their effect on stress responses. These results suggest a possibility that ingestion of lemon essential oil containing components such as limonene and citral alleviates both physical and psychological stress. Copyright © 2007 John Wiley &amp; Sons, Ltd.

Perillyl alcohol and perillic acid induced cell cycle arrest and apoptosis in non small cell lung cancer cells

Plant products such as perillyl alcohol have been reported to possess anti-tumor activities against a number of human cancers though the mechanism of action has not yet been elucidated. The effects of perillyl alcohol (POH) and its metabolite perillic acid (PA) on the proliferation of non small cell lung cancer (NSCLC, A549, and H520) cells were investigated. Both POH and PA elicited dose-dependent cytotoxicity, induced cell cycle arrest and apoptosis with increasing expression of bax, p21 and caspase-3 activity in both the cell lines. Combination studies revealed that exposing the cells to an IC50 concentration of POH or PA sensitized the cells to cisplatin and radiation in a dose-dependent manner. These results indicate that POH and PA in combination therapy may have chemotherapeutic value against NSCLC.

722 POSTER Antitumor activity and pharmacokinetics of a novel PEGylated irinotecan in irinotecan-resistant colorectal tumours implanted in mice

A number of proteins post-translationally modified by the covalent attachment of mevalonate-derived isoprene groups farnesol (FOH) or geranylgeraniol (GGOH), play a role in cell proliferation. For this reason, protein farnesyltransferase (PFTase) and protein geranylgeranyltransferases (PGGTases) I and II have gained attention as novel targets for the development of antiproliferative agents. Monoterpenes [limonene, perillic acid (PA) and its derivatives] have been shown to inhibit cell growth and protein prenylation in cancer cells. In the present study, we evaluated the effect of S(−) PA on diploid rat aorta smooth muscle cell (SMC) proliferation as related to protein prenylation. S(−) PA (1–3.5 mM) decreased, in a concentration-dependent manner, rat SMC proliferation as evaluated by cell counting and DNA synthesis. Morphological criteria and flow cytometry analysis excluded the induction of apoptosis as a potential antiproliferative mechanism of S(−) PA on SMC and confirmed a block of the cell cycle progression in G0/G1 phase. The antiproliferative effect of S(−) PA could not be prevented by the addition of mevalonate, FOH, and GGOH to the culture medium and was independent of cholesterol biosynthesis. Densitometric analysis of fluorographed gels, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the cell lysates, further supported that S(−) PA (1–3.5 mM), under the same experimental conditions, concentration-dependently inhibited FOH (up to 70%) and GGOH (up to 70%) incorporation into cellular proteins. We provide evidence that S(−) PA affects protein prenylation, an effect that may contribute to its inhibition of SMC proliferation.

Activation of calcium-dependent kinases and epidermal growth factor receptor regulate muscarinic acetylcholine receptor-mediated MAPK/ERK activation in thyroid epithelial cells

We previously showed that stimulation of muscarinic acetylcholine receptors (mAChR) by carbachol (Cch) caused a time- and dose-dependent increase of mitogen-activated protein kinase/extracellular signal-regulated kinases (MAPK/ERK) phosphorylation in thyroid epithelial cells. In this study, we demonstrated that mAChR stimulation also induced a time-dependent increase in the tyrosine phosphorylation of proline-rich tyrosine kinase 2 (Pyk2), which was prevented by pretreatment of thyroid epithelial cells with the specific Src-family tyrosine kinase inhibitor PP2. Besides, phosphorylation of Pyk2 was attenuated by chelation of extracellular Ca2+ or inhibition of phospholipase C (PLC), and was evoked by thapsigargin, a specific microsomal Ca2+-ATPase inhibitor. Incorporation of Pyk2 antisense oligonucleotides in thyroid epithelial cells to down-regulated Pyk2 expression or pretreatment of cells with the Ca2+/calmodulin protein kinase II (CaM kinase II) inhibitor KN-62 significantly reduced Cch-induced MAPK/ERK phosphorylation. In addition, Cch-induced MAPK/ERK phosphorylation was partially inhibited by LY294002 and wortmannin, two selective inhibitors of phosphatidylinositol 3-kinase (PI3K), tyrphostin AG1478, a specific inhibitor of epidermal growth factor receptor (EGFR) kinase, and (−)-perillic acid, a post-translational inhibitor of small G-proteins isoprenylation. Taken together, our data suggest that Pyk2, CaM kinase II and Src-family tyrosine kinases are key molecules for the activation of MAPK/ERK cascade through the EGFR/Ras/Raf pathway in thyroid epithelial cells in response to mAChR stimulation.

Modulatory effect of interleukin-1β on rat isolated basilar artery contraction

An increased level of cytokine interleukin-1 (IL-1) has been detected around the site of stroke. However, the effect of IL-1β on the basilar artery has received little attention. We evaluated the effects of IL-1β on the contractile response of rat isolated basilar artery by measuring isometric tension change. IL-1β (10 ng/ml) and phenylephrine (0.1 nM) markedly enhanced U46619 (30 and 100 nM)-induced basilar artery contraction. The IL-1β-mediated potentiation was partly suppressed by zinc protoporphyrin (3 μM) and was abolished by tetrodotoxin (TTX, 100 nM), (−)-perillic acid (1 μM), PD98059 (0.3 μM), SB203580 (1 μM) and prazosin (1 μM). Our data suggest that IL-1β (10 ng/ml) causes an enhancement of U46619-mediated basilar artery contraction that probably involves TTX-sensitive neuronal release of an α 1-adrenoceptor agonist and activation of p42/p44 and p38 mitogen-activated protein kinases/p21 ras pathways.

P2Y Receptors Activate MAPK/ERK Through a Pathway Involving PI3K/PDK1/PKC-ζ in Human Vein Endothelial Cells

Aims: In this study we investigated the effects of P2receptors in the regulation of mitogen-activatedprotein kinase/extracellular signal-regulated kinase(MAPK/ERK) in human umbilical vein endothelial cells(HUVEC). Methods: Cytosolic Ca²⁺ concentration([Ca²⁺]_i) was measured using fura-2/AM, and MAPK/ERK phosphorylation using Western blot analysis.Results: ATP, 2-meSATP, UTP and UDP cause a rapidand transitory increase in the phosphorylation ofMAPK/ERK. In contrast, negligible response was seenfor a,ß-meATP, a general P2X receptors agonist. ATPdependentactivation of MAPK/ERK was preventedby pretreatment of HUVEC with pertussis toxin or MEKinhibitor PD98059. In addition, activation of the MAPK/ERK cascade by ATP was blocked in cells pretreatedwith wortmannin and LY294002, but not by U73122,BAPTA or a Ca²⁺-free medium. Furthermore, aninhibition of ATP-dependent MAPK/ERKphosphorylation was observed in HUVEC pretreatedwith high doses of GF109203X or myristoylated PKC-ζ pseudosubstrate. Similar results were observedwhen cells were pretreated with the Src tyrosine kinaseinhibitor PP2. However, ATP-stimulated MAPK/ERKactivation was unaffected in cells pretreated withAG1478 or perillic acid. We also found that ATPstimulates both the phosphorylation of 3-phosphoinositide-dependent protein kinase-1 (PDK1)and its translocation to plasma membrane in a timedependentmanner. Conclusion: These observationssuggest that the effects mediated by ATP in HUVECoccur via PTX-sensitive G-protein-coupled P2Yreceptors through PI3K-dependent mechanisms, inwhich PDK1 and PKC-ζ are two key molecules withinsignal cascade leading to MAPK/ERK activation.