Investigation of the role of protein kinase C (PKC) in cytostasis induced by tumour promoting phorbol esters and related compounds

Protein kinase C (PKC) is considered to be the major receptor for tumour promoting phorbol esters such as 12-0- tetradecanoylphorbol-13-acetate (TPA). These agents evoke a plethora of biological effects on cells in culture. The growth of A549 human lung carcinoma cells maintained in medium fortified with 10% foetal calf serum (FCS) is arrested for 6 days by TPA and other biologically active phorbol esters. In the work described in this thesis, the hypothesis was tested that modulation of PKC activity is closely related to events pivotal for cytostasis to occur. The effect of several phorbol esters, of newly synthesized analogues of diacylglycerols (DAG) and of bryostatins (bryos) on cell growth and ability to modulate activity of PKC has been investigated.Determination of the subcellular distribution of PKC following treatment of cells with TPA and partial enzyme purification by non-denaturing poly-acrylamide gel electrophoresis revealed translocation of enzyme activity from cytosoUc to paniculate fraction. Chronic exposure of cells to TPA resulted in a time and concentration dependent degradation of enzyme activity. Synthetic DAG and DAG analogues, unable to arrest the growth of cells at non-toxic concentrations, were neither able to affect subcellular PKC distribution nor compete effectively for phorbol ester binding sites at physiologically relevant concentrations. Bryos 1,2,4 and 5, natural products, possessing antineoplastic activity in mice, elicited transient arrest of A549 cell growth in vitro. They successfully competed for phorbol ester receptors in A549 cells with exquisite affinity and induced a shift in sub-cellular PKC distribution, though not to the same extent as PTA. Enzyme down-regulation resulted from prolonged exposure of cells to nanomolar concentrations of bryos. In vivo studies demonstrated that neither PDBu nor bryo 1 was able to inhibit A549 xenograft growth in athymic mice. The growth of A549 cell populations cultured under conditions of serum-deprivation was inhibited only transiently by biologically active phorbol esters. Fortification of serum-free medium with EGF or fetuin was able to partially restore sensitivity to maintained growth arrest by PTA. PKC translocation to the paniculate cellular fraction and subsequent enzyme down-regulation, induced by TPA, occurred in a manner similar to that observed in serum-supplemented cells. However, total PKC activity and cytosolic phorbol ester binding potential were greatly reduced in the serum-deprived cell population. Western blot analysis using monospecific monoclonal antibodies revealed the presence of PKC-a in both A549 cell populations, with significantly reduced protein levels in serum- deprived cells. PKC-/9 was not detected in either cell population.

Investigation of the antiproliferative properties of tumour promoting phorbol esters and related compounds

Tumour promoting phorbol esters such as 12-0-tetradecanoylphorbol-13-acetate (TPA) exert a multitude of biological effects on many cellular systems, many of which are believed to be mediated via the activation of the enzyme protein kinase C (PKC). TPA and other biologically active phorbol esters inhibited the proliferation of the A549 human lung carcinoma cell line. However, after 5-6 days culture in the continued presence of the phorbol ester cells began to proliferate at a rate similar to that of untreated cells. Resistance to TPA was lost following subculturing, although subculture in the presence of 10 nM TPA for more than 9 weeks resulted in a more resistant phenotype. The selection of a TPA-resistant subpopulation was not responsible for the observed resistance. The antiproliferative properties of other PKC activators were investigated. Mezerein induced the same antiproliferative effects as TPA but synthetic diacylglycerols (DAGs), the presumed physiological ligands of PKC, exerted only a non-specific cytotoxic influence on growth. Bryostatins 1 and 2 were able to induce transient growth arrest of A549 cells in a manner similar to phorbol esters at nanomolar concentrations, but at higher concentrations blocked both their own antiproliferative action and also that of phorbol esters and mezerein. Fourteen compounds synthesized to mimic features of the phorbol ester pharmacophore and/or DAGs did not mimic the antiproliferative properties of TPA in A549 cells and exerted only a DAG-like non-specific cytotoxicity at high concentrations. The subcellular distribution and activity of PKC was determined following partial purification by non-denaturing polyacrylamide gel electrophoresis. Treatment with TPA, mezerein or bryostatins resulted in a concentration-dependent shift of PKC activity from the cytosol to cellular membranes within 30 min. Significant translocation was not observed on treatment with DAGs. Chronic exposure of cells to TPA caused a time- and concentration dependent down-regulation of functional PKC activity. A complete loss of PKC activity was also observed on treatment with growth-inhibitory concentrations of bryostatins. No PKC activity was detected in cells resistant to the growth-inhibitory influence of TPA. Measurement of intracellular Ca2+ concentrations using A549 cells cultured on Cytodex 1 microcarrier beads revealed that TPA, mezerein and the bryostatins induced a similar rapid rise in intracellular Ca2+ levels.

The Role of protein kinase C modulation in the antiproliferative effects of bistratene A, bryostatin 1 and phorbol esters

PKC-mediated signalling pathways are important in cell growth and differentiation, and aberrations in these pathways are implicated in tumourigenesis. The objective of this project was to clarify the link between cell growth inhibition and PKC modulation.The PKC activators bryostatin 1 and 12-0-tetradecanoylphorbol-13-acetate (TPA) inhibited growth in A549 and MCF-7 adenocarcinoma cells with great potency, and induced HL-60 leukaemia cell differentiation. Bistratene A affected these cells similarly. Experiments were conducted to test the hypotheses that bistratene A exerts its effects via PKC modulation and that characteristics of cytostasis induced by bryostatin 1 and TPA depend upon PKC isozyme-specific events. After incubation of A549 cells with TPA or bistratene A, 2D phosphoprotein electrophoretograrns revealed three proteins phosphorylated by both agents. However, bistratene A was unable to induce the formation of cellular networks on the basement membrane substitute Matrigel, and staurosporine was unable to reverse bistratene A-induced [3H]thymidine uptake inhibition, unlike TPA. Bistratene A did not induce PKC translocation or downregulation, activate or inhibit A549 and MCF-7 cell cytosolic PKC or compete for phorbol ester receptors. Western blot analysis and hydroxylapatite chromatography identified PKC α, ε and ζ in these cells. Bistratene A was unable to activate any of these isoforms. Therefore the agent does not exert its antiproliferative effects by modulation of PKC activity. The abilities of bryostatin 1 and TPA (10nM-1μM) to induce PKC isoform translocation and downregulation were compared with antiproliferative effects. Both agents induced dose-dependent downregulation and translocation of PKC α and ε to particulate and nuclear cell fractions. PKC ζ was translocated to the particulate fraction by both agents in MCF-7 cells. The similarity of PKC isoform redistribution by these agents did not explain their divergent effects on cell growth, and the role of nuclear translocation of PKC in cytostasis was not confirmed by these studies. Alternative factors governing the characteristics of growth inhibition induced by these agents are discussed.

Some reactions of hydroperoxides in the presence of cyclic phosphate esters

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Controlled growth of poly(2-(diethylamino)ethyl methacrylate) brushes via atom transfer radical polymerisation on planar silicon surfaces

Progress in making pH-responsive polyelectrolyte brushes with a range of different grafting densities is reported. Polymer brushes of poly(2-(diethylamino)ethyl methacrylate) were synthesised via atom transfer radical polymerisation on silicon wafers using a 'grafted from' approach. The [11-(2-bromo-2-methyl) propionyloxy]undecyl trichlorosilane initiator was covalently attached to the silicon via silylation, from which the brushes were grown using a catalytic system of copper(I) chloride and pentamethyldiethylenetriamine in tetrahydrofuran at 80°C. X-ray reflectivity was used to assess the initiator surfaces and an upper limit on the grafting density of the polymer was determined. The quality of the brushes produced was analysed using ellipsometry and atomic force microscopy, which is also discussed.

Conformal sulfated zirconia monolayer catalysts for the one-pot synthesis of ethyl levulinate from glucose

Here we describe a simple route to creating conformal sulphated zirconia monolayers throughout an SBA-15 architecture that confers efficient acid-catalysed one-pot conversion of glucose to ethyl levulinate.

Mechanistic studies on the degradation of gasoline oxygenates by advanced oxidation technologies and the reaction of 4-methyl-1,2,4-triazoline-3,5-dione with tetracyclopropylethylene

Gasoline oxygenates (MTBE, methyl tert-butyl ether; DIPE, di-isopropyl ether; ETBE, ethyl tert-butyl ether; TAME, tert-amyl ether) are added to gasoline to boost octane and enhance combustion. The combination of large scale use, high water solubility and only minor biodegradability has now resulted in a significant gasoline oxygenate contamination occurring in surface, ground, and drinking water systems. Combination of hydroxyl radical formation and the pyrolytic environment generated by ultrasonic irradiation (665 kHz) leads to the rapid degradation of MTBE and other gasoline oxygenates in aqueous media. ^ The presence of oxygen promotes the degradation processes by rapid reaction with carbon centered radicals indicating radical processes involving O 2 are significant pathways. A number of the oxidation products were identified. The formation of products (alcohols, ketones, aldehydes, esters, peroxides, etc) could be rationalized by mechanisms which involve hydrogen abstraction by OH radical and/or pyrolysis to form carboncentered radicals which react with oxygen and follow standard oxidation chain processes. ^ The reactions of N-substituted R-triazolinediones (RTAD; R = CH 3 or phenyl) have attracted considerable interest because they exhibit a number of unusual mechanistic characteristics that are analogous to the reactions of singlet oxygen (1O2) and offer an easy way to provide C-N bond(s) formation. The reactions of triazolinedione with olefins have been widely studied and aziridinium imides are generally accepted to be the reactive intermediates. ^ We observed the rapid formation of an unusual intermediate upon mixing tetracyclopropylethylene with 4-methyl-1,2,4-triazoline-3,5-dione in CDCl 3. Detailed characterization by NMR (proton, 13C, 2-D NMRs) indicates the intermediate is 5,5,6,6-tetracyclopropyl-3-methyl-5,6-dihydro-oxazolo[3,2- b][1,2,4]-triazolium-2-olate. Such products are extremely rare and have not been studied. Upon warming the intermediate is converted to 2 + 2 diazetidine (major) and ene product (minor). ^ To further explore the kinetics and dynamics of the reaction activation energies were obtained using Arrhenius plots. Activation energies for the formation of the intermediate from reactants, and 2+2 adduct from the intermediate were determined as 7.48 kcal moll and 19.8 kcal mol−1 with their pre-exponential values of 2.24 × 105 dm 3 mol−1 sec−1 and 2.75 × 108 sec−1, respectively, meaning net slow reactions because of low pre-exponential values caused by steric hindrance. ^

Calculations on the end-group conformational barrier in carotenoids and a study of the bioavailability of xanthophyll esters in humans

The present study measures the increase in serum carotenoid concentration in 30 healthy individuals after supplementation with a low dose xanthophyll ester (3 and 6 mg of lutein equivalent/per day) when compared to a placebo. Serum levels of carotenoids were measured using HPLC and showed an increase in the concentration of lutein, zeaxanthin and four lutein metabolites proportional to dose. In order to further assess the importance of the end-group structure in carotenoids we have investigated the influence of the end-group type and functionality on the conformational energy barrier. We used the density functional method implemented on GAUSSIAN 98 to calculate the conformational energy curves for rotation of the P-ring or the E-ring relative to short polyene chains around the C6-C7 single bond. A large barrier is observed for the interconversion of conformers in the E-rings (8 kcal/mol) when compared to beta rings (2.3-3 kcal/mol).

Coordination networks of Cu2+ ions with 1,3-bis[2-(4-pyridyl)ethyl]benzene : strong structure-directing role of the counter ion (nitrate, acetate and sulphate), leading to clusters, sheets and chains

Date of Acceptance: 16/10/2015

Coordination networks of Cu2+ ions with 1,3-bis[2-(4-pyridyl)ethyl]benzene : strong structure-directing role of the counter ion (nitrate, acetate and sulphate), leading to clusters, sheets and chains

Date of Acceptance: 16/10/2015

Novel Synthesis of Carbocycles and Heterocycles Employing Zwitterions Derived from Allenic Esters

Carbon-carbon and carbon-heteroatom bond formations constitute the central events in organic synthesis. In view of this, much of the research in organic synthesis has been focused on devising novel and efficient methods for such bond constructions. In general, polar, pericyclic and radical methodologies are employed for this purpose. The polar and radical reactions proceed via reactive intermediates such as carbanions, enols/enolates, enamines, carbocations, radical cations, radical anions, carbenes, zwitterions etc. In recent years, there has been enormous interest in the chemistry of zwitterionic species largely from the standpoint of their applications in multicomponent reactions (MCRs) and organocatalytic reactions. Zwitterions formed by the addition of nucleophiles to electrophilic π-systems such as acetylenic esters and azoesters have been the subject of extensive investigations; their synthetic utility, however, remained largely unexplored. Investigations in a number of laboratories, including our own, have shown that zwitterions of the type mentioned above on reaction with electrophiles give rise to carbo- and heterocyclic products by 1,3- or 1,4-dipolar cycloadditions. Recently, allenoates, another class of active π-systems were introduced to this field. Against this background, a systematic investigation of the reactions of various zwitterions derived from allenoates with different electrophiles especially 1,2-diones, were carried out. The results of these studies are embodied in the thesis entitled “Novel Synthesis of Carbocycles and Heterocycles Employing Zwitterions Derived from Allenic Esters”.