AbstractOxygen is a requirement for almost all living organisms and adaptations to oxygen shortage are essential for surviving periods of oxygen deprivation, known as hypoxia. Cells have evolved a range of mechanisms which increase the supply of oxygen and facilitate metabolic alterations that enable the cell and the organism to maintain functionality under hypoxic conditions. Hypoxia is a hallmark of solid tumours and is associated with increased malignancy and mortality in hepatocellular carcinoma (HCC). Transarterial chemoembolisation therapy (TACE) using doxorubicin is the current standard of care for intermediate HCC, although response rates are poor. Drug eluting bead transarterial chemoembolisation (DEB-TACE) shows improved response rates over TACE. More recently, rapamycin has come under scrutiny as an effective therapy against HCC. Embolisation therapies have been shown to induce hypoxia in HCC, leading to the escape of hypoxia-adapted cancer cells from therapy. The principal transcription factor which orchestrates responses to hypoxia is hypoxia inducible factor 1 (HIF-1). Laboratory and clinical evidence support the hypothesis that HIF-1 activity contributes to cancer progression and increased mortality. Targeting HIF-1 therefore presents an opportunity for improving outcomes of cancer therapy.
A hypoxic model of HCC was established, and used to characterise the responses of the cell line HepG2 to chemotherapeutic agents in both normoxic and hypoxic conditions. Firstly, the time and concentration dependent effects of doxorubicin, rapamycin and both drugs in combination on the viability of HepG2 cells cultured under both normoxic and hypoxic conditions were investigated. SDS-PAGE and Western Blotting was then used to evaluate the responses of HIF-1α, NFkB, S6K and Akt expression to doxorubicin, rapamycin and both drugs in combination in cells cultured under both normoxic and hypoxic conditions. Finally, the anti-tumour effects of doxorubicin, rapamycin and both drugs in combination were investigated in vivo using an ectopic xenograft murine model of HCC.
The in vitro evidence presented in this thesis demonstrates that a concentration of doxorubicin relevant to clinical concentrations following DEB-TACE effectively inhibits the viability of both normoxic and hypoxic liver cancer cells. Also presented is in vitro evidence that low dose rapamycin inhibits the viability of both normoxic, and to a lesser extent, hypoxic liver cancer cells. The addition of low dose rapamycin to doxorubicin was consistently observed to have an additive effect on the inhibition of cell viability. Protein analysis demonstrated that low dose rapamycin inhibits the hypoxia stimulated accumulation of HIF-1α, as does high dose doxorubicin. However, inhibition of HIF-1α was attenuated when the two drugs were used in combination. Cytotoxic effects are not, therefore, wholly dependent on inhibition of HIF-1α. Inhibition of HIF-1α by each drug alone appears to be due to different mechanisms. This study also showed in vivo that combinations of doxorubicin DEB-TACE with either rapamycin DEB-TACE or oral rapamycin are more effective than either treatment alone at reducing tumour burden in a mouse model of HCC. Two clinical trials are now underway to investigate the combination of doxorubicin DEB-TACE and low dose oral rapamycin to treat HCC.
|Date of Award||Sep 2011|
|Sponsors||Biocompatibles UK Ltd|
|Supervisor||Andrew L. Lewis (Supervisor), Andrew W Lloyd (Supervisor), Gary J Phillips (Supervisor) & Wendy Macfarlane (Supervisor)|