Detection and Characterisation of Compounds Inhibiting Stress Granule Formation in Cancer Cells

Abstract

Cancer is one of the leading causes of death worldwide and despite significant improvements to treatment and prevention, cancer cases remain on the rise. Chemotherapy is used to treat patients with cancer, however these do not only kill the cancer cells but also kill normal, healthy cells in the patient. Furthermore, cancer cells have the capacity to become resistant to chemotherapeutic treatment. Therefore, new treatments need to be developed to overcome this problem. One avenue that is being researched is the use of natural products as chemotherapeutic drugs. Over 60% of anticancer drugs used today are either natural products, or their synthetic derivatives and new research is being performed to screen plant and animal secondary metabolites to discover new compounds with anti-cancer therapeutic potential. The research reported in this thesis uses two compounds purified from natural products to explore a novel approach for cancer treatment. Stress granules (SGs) are messenger ribonucleoprotein particles that are produced in the cytoplasm of the cell in response to stress. Stress granules have been linked to the inhibition of apoptosis and development of multiple drug resistance and it has been suggested that cancer cells can hijack stress granules and use their biological activities to enhance cancer cell survival. In a study by Fournier et al the inhibition of stress granules in bortezomib resistant cancer cells allowed these cells to become sensitive to bortezomib treatment and resulted in an increase in cell death from 15% to 75% (Fournier et al., 2010). This suggests that the inhibition of stress granule formation may restore chemo-sensitivity to the cancer cells, however, the full effect has not been explored beyond the cell based experiments described by Fournier et al. This research project was based on the research by Fournier et al, suggesting that SG inhibition can increase the efficacy of bortezomib. The aims of this project were to discover natural products that inhibited SG formation and use these natural products in combination with the chemotherapeutics bortezomib and sorafenib to increase their efficacies. Chapter 3 describes the optimisation and characterisation of SG formation in HEK293, MCF7, T47D, Vero, HeLa, MDAMB231 and MCF7MDR cells for the development of a SG inhibition assay. Chapter 4 describes the screening of 36 compounds from the Davis Open Access Compound Library from which 2 compounds, RAD112 and psammaplysin F were discovered; having activity inhibiting SG formation in the in vitro SG inhibition assay. In chapter 5, the mechanism of action of RAD112 and psammaplysin F were explored. RAD112 belongs to the chalcone class and this class of compound is known for the disruption of microtubules. A microtubule assay was performed analysing the effect of RAD112 against a known microtubule inhibitor, nocodazole and it was confirmed that RAD112 was disrupting microtubules. Psammaplysin F did not cause the disruption of microtubules, therefore, the most common pathway of SG formation, phosphorylation of eIF2α (p-eIF2α) was analysed. It was discovered that psammaplysin F was reducing the amount of p-eIF2α in HEK293, MCF7, Vero and MCF7MDR cells. The mechanism of action studies of both compounds show promising results that warrant further evaluation. Combinational therapies have many advantages over single chemotherapy regimes in breast cancer as it has been shown that it can increase the patients disease free survival rate and reduce the risk of reoccurrence. In chapter 6 RAD112 and psammaplysin F were used in combination with bortezomib or sorafenib and the interaction between RAD112 with bortezomib or sorafenib and psammaplysin F with bortezomib or sorafenib was determined by cell viability assays and analysed using Compusyn software. The increase in efficacy of bortezomib or sorafenib when combined with RAD112 and psammaplysin F was also examined in the in vitro combinational assay. All combinations of the compounds with the drugs resulted in a synergistic interaction in most cell lines. However, psammaplysin F and sorafenib had the strongest synergistic interaction in MCF7MDR cells, with a combination index (CI) value of <0.4. The IC50 of sorafenib was decreased 4 fold in MCF7MDR cells and 7 fold in MCF7 cells when combined with psammaplysin F. The efficacy of bortezomib and sorafenib was increased after treatment with RAD112 and psammaplysin F suggesting that psammaplysin F and bortezomib have the potential to be used in combination with known chemotherapeutics to restore drug efficacy. Altogether, this thesis has identified two compounds that inhibit SG formation, RAD112 and psammaplysin F. The mechanism of action studies has revealed two different mechanisms of action for SG inhibition, microtubule inhibition and inhibition of p-eIF2α for RAD112 and psammaplysin F respectively. Combinational studies has resulted in synergistic interactions between RAD112 and bortezomib or sorafenib and psammaplysin F and bortezomib and sorafenib and increased efficacy of bortezomib and sorafenib. These findings show promise as a new strategy in the battle against cancer and further studies involving the complete mechanism of action of these compounds have to be carried out before they can move onto pre-clinical evaluation.