|dc.description.abstract||Introduction and Aims: Malignant mesothelioma is a rare and aggressive cancer that affects the mesothelial cells of mainly the pleural cavity. The current treatment standard relies on cisplatin/pemetrexed combination chemotherapy, and whilst this is the most effective option currently, it only improves life expectancy by two months. Small-molecule and gene-based therapy has been a potential hot spot in research over the past 20 years. However, significant limitations mean many of these therapies have failed to move past pre-clinical trials. The discovery of the HIV-TAT protein and its ability to translocate across the cell membrane unassisted has created avenues to using cell penetrating peptides for the delivery of biologically active molecules into the cell. The aim of this project was to establish a viable novel treatment option by identifying kinase targets that were essential in mesothelioma cells by use of a siRNA screen, and then utilising cell penetrating peptide drugs to inhibit the activity of these targets.
Methodology: An initial primary siRNA screen was used to identify potential target options in pathological NCI-H28 cell lines, using non-malignant MET5A cell lines as a control. These targets were then validated individually by use of siRNAs. Cell viability and knockdown were quantified using an MTT. Once targets were validated, cell penetrating peptide molecules were used to inhibit the target proteins, and MTT analysis was used to quantify cell viability. One peptide was selected for further analysis. This included introducing two more malignant cell lines, MM-Miller, and IST-Mes2, as well as investigating the activity of its target after treatment using phospho-specific Western blot analysis of the downstream substrates. Finally the uptake efficiency of the selected peptide was tested by conjugating a fluorescent molecule to it and using flow cytometry to analyse the uptake into the cells.
Results: The primary siRNA screen identified four kinases (LMTK3, MAP3K11, PI4K2A, and PI4KB) as targets for treatment of malignant mesothelioma. These results were validated in the individual siRNA experiments which showed the siRNA targeting MAP3K11 resulted in the most significant magnitude of cell death in the NCI-H28 cell lines. From this a series of cell penetrating peptides were generated to target the four identified protein kinases. A peptide targeting MAP3K11, KFF:MAP3K11.H2 resulted in the highest level of cell death for the NCI-H28 cells, however this same level of cell death was not observed in the other two malignant cell line IST-Mes2 and MM-Miller. Western blot analysis of the phosphorylation state of MAP2K4 and JNK, phosphorylation targets of MAP3K11, revealed that treatment with KFF:MAP3K11.H2 resulted in a decreased level of phosphorylation in both MeT-5A and NCI-H28 cells, inferring the treatment peptide was reducing the activity of the target. This was not consistent for the IST-Mes2 and MM-Miller cell lines. Finally uptake analysis display that the peptide was highly efficient at cell internalisation.
Conclusion: KFF:MAP3K11.H2 resulted in a high degree of cell death for the malignant cell line NCI-H28.However, this effect was not similarly potent in the other malignant cell line IST-Mes2, and MM-Miller. This in conjunction with the Western blot data suggests that MAP3K11 inhibition and the downregulation of the MAPK/JNK pathway can result in a decrease in cell viability for the NCI-H28 cells. However, this was not consistent for MM-Miller and IST-Mes2 cells. Finally, KFF3K was shown to have a highly efficient uptake profile, whilst being non-cytotoxic.||en_US