A novel mechanism for the anti-cancer activity of aspirin and its analogues

Abstract

Abstract Colorectal cancer (CRC), which includes cancer of the large bowel and rectum is the third most common cancer in men and the second in women and there is a poorer survival rate in less developed regions of the world such as West Africa mainly due to the ‘out of reach’ costs of chemotherapy. Evidence suggests that aspirin, a non-steroidal anti-inflammatory drug (NSAID) has the potential to decrease incidence of, or mortality from a number of cancers including CRC through several mechanisms of action. However, this evidence is dampened by aspirin’s gastrointestinal (GI) toxicity, which have been found to be mostly age-dependent. The search for potential aspirin-related compounds with the same or better cytotoxic effects against cancer cells accompanied by a safer toxicity profile has been ongoing over the years and led to us to synthesise a number of novel aspirin analogues. One of the mechanisms of action suggested for the anticancer property of aspirin is the COX-dependent pathway. In this thesis SW480 cell line, a CRC cell line that is COX-2 negative and mismatch repair (MMR) proficient was used to study the possible COX-independent mechanism of action for aspirin, its analogues and diflunisal at 0.5 mM. Diflunisal was included in this study because it is also a salicylate with reports of having cytotoxic effects. OE33 and FLO1 oesophageal cancer cells were also employed in the epidermal growth factor receptor (EGFR) and synergy experiments to show effects were not just specific to SW480 cells alone. These aspirin analogues were synthesised, identified using nuclear magnetic resonance (NMR) and infra-red (IR) spectroscopy, and tested for purity using thin layer chromatography (TLC) and melting point. The findings of this study suggest that these compounds breakdown into salicylates and perturb epidermal growth factor (EGF) internalization with PN517 (fumaryldiaspirin) and PN590 (ortho-thioaspirin) also driving EGF co-localization with early-endosome antigen-1 (EEA1). The perturbation of the internalization of EGF by aspirin and PN517 was also observed by a time-lapse assay using live confocal imaging. These compounds also had specific effects on different tyrosine phosphorylation sites of the EGFR, with none but PN590 inhibiting 4 phosphorylation at Y1068, and all but PN502 (ortho-aspirin), PN548 (meta-aspirin) and PN549 (para-aspirin) inhibiting phosphorylation at Y1045 and Y1173. Given that the EGF internalization assay involved the cells being treated with compounds for 2 h, cells were also treated for this same time period and probed with pEGFR 1045, which resulted in the compounds having no significant effect on phosphorylation at that site which is responsible for the ubiquitination of the EGFR. Most of these compounds were apoptotic with some showing a combination of apoptosis and necrosis. Aspirin and its isomers drove apoptotic cell death in SW480 cells via the BCL2-BAX pathway while the thioaspirins appear to follow the p21 pathway by decreasing the expression of the protein. In addition, it was shown that PN502 (aspirin), PN517 and PN590 had synergistic effects when used in combination with oxaliplatin at ED50, ED75 and ED90 in SW480 CRC cells. The cytotoxicity of these compounds individually or in combination was determined using MTT assay followed by the use of the CompuSyn and CalcuSyn software to calculate combination index (CI), which indicated whether a drug combination was synergistic, antagonistic or additive. PN517 and PN524 were synergistic when used in combination with cisplatin in OE33 oesophageal cancer cells. Effect of these compounds on the EGFR indicates a delay or disruption of the signalling pathway involved in the proliferation of cancer cells, thus, translating into protection against tumour formation or progression while the synergistic effects of these compounds when used in combination with platinum compounds can provide patients with less toxic chemotherapeutic regimen especially in patients with CRC tumours that harbour mutant TP53 gene and normally resistant to oxaliplatin. It is therefore proposed that the perturbation of EGF internalization is a novel mechanism of action for aspirin and its analogues in cancer therapy. These positive findings shed light on the understanding of the possible mechanism of action for aspirins and gives hope for a more affordable, less toxic therapy for the prevention, treatment and management of cancer.