S100P dissociates myosin IIA filaments and focal adhesion sites to reduce cell adhesion and enhance cell migration

S100 proteins promote cancer cell migration and metastasis. To investigate their roles in the process of migration we have constructed inducible systems for S100P in rat mammary and human HeLa cells that show a linear relationship between its intracellular levels and cell migration. S100P, like S100A4, differentially interacts with the isoforms of nonmuscle myosin II (NMIIA, K(d) = 0.5 µm; IIB, K(d) = 8 µm; IIC, K(d) = 1.0 µm). Accordingly, S100P dissociates NMIIA and IIC filaments but not IIB in vitro. NMIIA knockdown increases migration in non-induced cells and there is no further increase upon induction of S100P, whereas NMIIB knockdown reduces cell migration whether or not S100P is induced. NMIIC knockdown does not affect S100P-enhanced cell migration. Further study shows that NMIIA physically interacts with S100P in living cells. In the cytoplasm, S100P occurs in discrete nodules along NMIIA-containing filaments. Induction of S100P causes more peripheral distribution of NMIIA filaments. This change is paralleled by a significant drop in vinculin-containing, actin-terminating focal adhesion sites (FAS) per cell. The induction of S100P, consequently, causes significant reduction in cellular adhesion. Addition of a focal adhesion kinase (FAK) inhibitor reduces disassembly of FAS and thereby suppresses S100P-enhanced cell migration. In conclusion, this work has demonstrated a mechanism whereby the S100P-induced dissociation of NMIIA filaments leads to a weakening of FAS, reduced cell adhesion, and enhanced cell migration, the first major step in the metastatic cascade.

Disruption of the interaction between PMCA2 and calcineurin triggers apoptosis and enhances paclitaxel-induced cytotoxicity in breast cancer cells

Cancer is caused by defects in the signalling mechanisms that govern cell proliferation and apoptosis. It is well known that calcium-dependent signalling pathways play a critical role in cell regulation. A tight control of calcium homeostasis by transporters and channel proteins is required to assure a proper functioning of the calcium-sensitive signal transduction pathways that regulate cell growth and apoptosis. The Plasma Membrane Calcium ATPase 2 (PMCA2) has been recently identified as a negative regulator of apoptosis that can play a significant role in cancer progression by conferring cells resistance to apoptosis. We have previously reported an inhibitory interaction between PMCA2 and the calcium-activated signalling molecule calcineurin in breast cancer cells. Here we demonstrate that disruption of the PMCA2/calcineurin interaction in a variety of human breast cancer cells results in activation of the calcineurin/NFAT pathway, up-regulation in the expression of the pro-apoptotic protein Fas Ligand, and in a concomitant loss of cell viability. Reduction in cell viability is the consequence of an increase in cell apoptosis. Impairment of the PMCA2/calcineurin interaction enhances paclitaxel-mediated cytotoxicity of breast tumoral cells. Our results suggest that therapeutic modulation of the PMCA2/calcineurin interaction might have important clinical applications to improve current treatments for breast cancer patients.

Synthesis and screening of potential antimicrobial compounds

Tuberculosis (TB), an infection caused by human pathogen Mycobacterium tuberculosis, continues to kill millions each year and is as prevalent as it was in the pre-antimicrobial era. With the emergence of continuously-evolving multi-drug resistant strains (MDR) and the implications of the HIV epidemic, it is crucial that new drugs with better efficacy and affordable cost are developed to treat TB. With this in mind, the first part of this thesis discusses the synthesis of libraries of derivatives of pyridine carboxamidrazones, along with cyclised (1,2,4-triazole and 1,2,4-oxadiazole) and fluorinated analogues. Microbiological screening against M. tuberculosis was carried out at the TAACF, NIAID and IDRI (USA). This confirmed the earlier findings that 2-pyridyl-substituted carboxamidrazones were more active than the 4-pyridyl-substituted carboxamidrazones. Another important observation was that upon cyclisation of these carboxamidrazones, a small number of the triazoles retained their activity while in most of the remaining compounds the activity was diminished. This might be attributed to the significant increase in logP value caused by cyclisation of these linear carboxamidrazones, resulting in high lipophilicity and decreased permeability. Another reason might be that the rigidity conferred upon the compound due to cyclisation, results in failure of the compound to fit into the active site of the putative target enzyme. In order to investigate the potential change to the compounds’ metabolism in the organism and/or host, the most active compounds were selected and a fluorine atom was introduced in the pyridine ring. The microbiological results shows a drastic improvement in the activity of the fluorinated carboxamidrazone amides as compared to their non fluorinated counterpart. This improvement in the activity could possibly be the result of the increased cell permeability caused by the fluorine. In a subsidiary strand, a selection of long-chain , -unsaturated carboxylic esters, -keto, -hydroxy carboxylic esters and -keto, -hydroxy carboxylic esters, structurally similar to mycolic acids, were synthesised. The microbiological data revealed that one of the open chain compound was active against the Mycobacterium tuberculosis H37Rv strain and some resistant isolates. The possible compound activity could be its potential to disrupt mycobacterial cell wall synthesis by interfering with the FAS-II pathway.

The effects of the fungicides fenhexamid and myclobutanil on SH-SY5Y and U-251 MG human cell lines

Mixtures of pesticides in foodstuffs and the environment are ubiquitous in the developed world and although agents are usually exhaustively tested individually, the toxicological implications of pesticide mixtures are underreported. In this study, the effects of two fungicides, fenhexamid and myclobutanil were investigated individually and in combination on two human cell lines, SH-SY5Y neuronal cells and U-251 MG glial cells. After 48. h of incubation with increasing concentrations of pesticides ranging from 1 to 1000. μM, gene expression profiles were studied in addition to toxicity end points, including cell viability, mitochondrial depolarisation as well as cellular glutathione maintenance. There were no significant differences between the susceptibility of the two cell lines in terms of cell viability assessment or mitochondrial membrane potential, when agents were administered either individually or in combination. By contrast, in the presence of the fungicides, the SH-SY5Y cells showed significantly greater susceptibility to oxidative stress in terms of total thiol depletion in comparison with the astrocytic cells. Treatment with the two pesticides led to significant changes in the cell lines' expression of several genes which regulate cell cycle control and growth (RB1, TIMP1) as well as responses to DNA attrition (ATM and CDA25A) and control of apoptosis (FAS). There was no evidence in this study that the combination of fenhexamid and myclobutanil was significantly more toxic than individual exposure, although gene expression changes suggested there may be differences in the sub-lethal response of both cell lines to both individual and combined exposure.