Browsing by Author "Blanckenberg, Angelique"
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- ItemThe palladacycle complex AJ-5 induces apoptotic cell death while reducing autophagic flux in rhabdomyosarcoma cells(Springer Nature, 2019-01-28) Bleloch, Jenna Susan; du Toit, Andre; Gibhard, Liezl; Kiman, Serah; Ballim, Reyna Deeya; Lee, Minkyu; Blanckenberg, Angelique; Mapolie, Selwyn; Wiesner, Lubbe; Loos, Ben; Prince, SharonRhabdomyosarcoma (RMS) forms in skeletal muscle and is the most common soft tissue sarcoma in children and adolescents. Current treatment is associated with debilitating side effects and treatment outcomes for patients with metastatic disease are dismal. Recently, a novel binuclear palladacycle, AJ-5, was shown to exert potent cytotoxicity in melanoma and breast cancer and to present with negligible adverse effects in mice. This study investigates the anticancer activity of AJ-5 in alveolar and embryonal RMS. IC50 values of ≤ 0.2 µM were determined for AJ-5 and it displayed a favourable selectivity index of >2. Clonogenic and migration assays showed that AJ-5 inhibited the ability of RMS cells to survive and migrate, respectively. Western blotting revealed that AJ-5 induced levels of key DNA damage response proteins (γH2AX, p-ATM and p-Chk2) and the p38/MAPK stress pathway. This correlated with an upregulation of p21 and a G1 cell cycle arrest. Annexin V-FITC/propidium iodide staining revealed that AJ-5 induced apoptosis and necrosis. Apoptosis was confirmed by the detection of cleaved PARP and increased levels and activity of cleaved caspases-3, -7, -8 and -9. Furthermore, AJ-5 reduced autophagic flux as shown by reduced LC3II accumulation in the presence of bafilomycin A1 and a significant reduction in autophagosome flux J. Finally, pharmacokinetic studies in mice show that AJ-5 has a promising half-life and that its volume of distribution is high, its clearance low and its intraperitoneal absorption is good. Together these findings suggest that AJ-5 may be an effective chemotherapeutic with a desirable mechanism of action for treating drug-resistant and advanced sarcomas.
- ItemPreparation and characterization of novel palladacycles and their evaluation as anticancer agents(Stellenbosch : Stellenbosch University, 2016-12) Blanckenberg, Angelique; Mapolie, Selwyn Frank; Stellenbosch University. Faculty of Science. Dept. of Chemistry and Polymer Science.ENGLISH ABSTRACT: Mononuclear and binuclear (unsubstituted and substituted) palladacycles based on benzylidene-2,6-diisopropylphenylamine, were designed and synthesised for evaluation as anti-cancer agents. The design of these complexes was based on the structure and properties of AJ5, a binuclear palladacycle with significant anti-cancer activity, but poor solubility. In an attempt to improve water solubility the Schiff base ligands, employed to prepare the palladacycles, were modified by introducing hydrophilic functional groups on the aldehyde starting material. Furthermore, the µ-chloro bridged palladacycles were cleaved with either the hydrophilic tertiary phosphine, 1,3,5-triaza-7-phosphaadamantane (PTA), or bis(tertiary phosphine) ligands as a further attempt at improving the water-solubility, whilst maintaining the anti-cancer activity. The synthesised complexes were fully characterised by a range of analytical techniques. The solubilities of the palladacycles were determined in water, dimethyl sulfoxide and water/dimethyl sulfoxide mixtures. None of the complexes were entirely watersoluble, however, the substituted binuclear palladacycles were found to show the highest solubility in dimethyl sulfoxide, with some analogues exhibiting improved solubility, compared to AJ5. NMR spectra of the mononuclear palladacycles showed that these PTA-cleaved, mononuclear palladacycles exhibit fluxional behaviour, especially with regard to the isopropyl substituents on the imine ligand. This fluxionality manifests itself as varying degrees of resolution of the isopropyl signals in the proton NMR spectroscopy, leading to this signal having different shapes depending on the nature of the imine ligand. The variety of peak shapes observed for the methyl signals of the isopropyl groups were thought to be due to a reversible symmetric site-exchange and fortunately this could be simulated by way of a computational model developed in-house. The free energy of activation (ΔG# ) and other thermodynamic parameters for the site-exchange process were calculated and found to correspond with aryl ring rotation. Slight deviations in the expected chemical shifts were attributed to concentration-dependent chemical exchange processes. Both molecular aggregation via PTA self-association and dimerisation by imine dissociation were proposed and shown to be plausible. Furthermore, we were able to show that the “ortho effect” accounts for the influence of the ortho-substituents on the free energy of activation. We were also able to attribute the ability for these species to undergo the proposed chemical exchange processes to the phosphorous ligand, PTA, specifically its small cone angle and nitrogen donor atoms. In vitro evaluation of the mono- and binuclear palladacycles was performed to determine their biological activity as anti-cancer agents against human breast adenocarcinoma MCF7 (estrogen receptor positive) and MDA-MB231 (estrogen receptor negative) cell lines. These studies show that most of the complexes are cytotoxic, with the binuclear palladacycles showing better activity than the mononuclear palladacycles. IC50 values of some of the palladacycles were better than those of cisplatin (below 20 μM) and the IC50 values for the substituted-binuclear palladacycle, BTC2, were found to be comparable to those of AJ5. The complexes were found to induce DNA damage and apoptosis. DNA binding studies were carried out on the most active palladacycle in each series to determine the mode of action. DNA binding studies by electrophoresis, ultraviolet-visible-, circular dichroism- and nuclear magnetic resonance spectroscopy suggest that the palladacycles bind to DNA non-covalently, by a mode different to that of cisplatin. The most likely mode of DNA binding was identified as an electrostatic binding mode. Thus, the formation of an aquated cationic species was proposed to form via hydrolysis of the Pd-Cl bond.