Chemotherapy-induced mucositis : the role of matrix metalloproteinases and the extracellular matrix.

Abstract

Chemotherapeutic agents, including irinotecan hydrochloride, are highly effective in the treatment of a range of cancers; however, they cause a variety of unwanted toxicities. Mucositis is the term used to describe the damage caused by cytotoxic agents to mucous membranes of the alimentary tract (AT). This condition affects 40-100% of patients depending on dose regimen. There is currently no effective treatment and the underlying molecular mechanisms are not fully understood. Previous research has shown that mucositis encompasses changes in stress response gene expression and subsequently activation of tissue injury and inflammation mediators. Matrix metalloproteinases (MMPs) are a group of zinc-dependent endopeptidases; which have been shown to play a role in tissue injury and inflammation in many gastrointestinal disorders. Furthermore, MMPs mediate these phenomena through the regulation of the extracellular matrix (ECM). This work aims to elucidate whether MMPs contribute to the pathogenesis of mucositis and whether these can be used as biomarkers for mucositis development or be targeted for future treatment strategies. To investigate these aims, studies were performed in an animal model of irinotecan-induced mucositis. A pilot clinical study was also conducted. To investigate the role of MMPs in mucositis pathogenesis, a time-course model of irinotecan-induced mucositis was utilised. Rats were administered with 200mg/kg irinotecan intraperitoneally at 0h and killed 30, 60, 90 min, 2, 6, 12, 24, 48, 72, 144h post- treatment. Sections were embedded in paraffin or frozen for further analysis. To ensure the accuracy of the molecular investigations in this thesis, the appropriateness of a range of housekeeping genes for normalisation of RT-PCR methods was investigated for the first time in this model. Findings indicated that the most suitable combination of genes to use is Ywhaz/UBC in the jejunum and UBC/β-actin in the colon or UBC if restricted to a single housekeeping gene. Subsequent molecular and histological assessments demonstrated a significant alteration in gene expression and tissue levels of MMPs and their inhibitors (TIMPs) following irinotecan (p˂0.05). The increase in MMP-2, -3, -9 and -12 levels was associated with inflammatory infiltrate and maximum tissue damage. In contrast, MMP-1 expression correlated with tissue restitution. Furthermore, histological techniques illustrated a substantial increase in total collagen deposits around crypts from 24h in the jejunum and colon. Fibronectin expression decreased significantly in both regions from 6-24h following treatment. Irinotecan induced a significant alteration in epithelial cell kinetics in both the jejunum and colon (p˂0.05) and this correlated with changes in ECM components. To determine if systemic MMP levels are useful markers of impending toxicity, a pilot clinical study was carried out. Eight patients receiving a variety of chemotherapy regimens were recruited. The most reported toxicity following treatment was diarrhoea. Analysis of patient serum samples revealed a 5.74-fold increase in systemic MMP-3 and a 2-fold increase in systemic MMP-12 levels following the administration of chemotherapy. Analysis of MMP-3 levels with patient symptoms revealed a correlation. Findings from this thesis provide clear evidence demonstrating a role for MMPs and ECM components in the pathogenesis of irinotecan-induced mucositis. Alterations in total collagen deposits and fibronectin levels in the AT following treatment may underlie the dysregulated cell kinetics following treatment hence leading to toxicity. Furthermore, preliminary findings from the pilot clinical study suggest that circulating MMPs are potential biomarkers of gastrointestinal toxicity induced by specific chemotherapy agents.