Date of Award

12-31-2025

Thesis Type

Masters

Document Type

Dissertation

Divisions

Faculty of Medicine

Department

Department of Molecular Medicine

Institution

Universiti Malaya

Abstract

Hypoxia, or oxygen deprivation, is a common pathological feature found in hepatocellular carcinoma (HCC). Hypoxia-inducible factor (HIF) is the master transcription factor of oxygen homeostasis, eliciting a wide range of cellular adaptive responses to either enhance oxygen delivery or reduce oxygen consumption. HIF is primarily regulated by two types of oxygen sensors, the prolyl hydroxylase domain-containing proteins (PHDs) and factor-inhibiting HIF (FIH). In tumours, HIF functions as a double-edged sword whereby it could either promote or hinder tumour progression. This is mainly contributed by the pleiotropic nature of hypoxia and tumour heterogeneity. Recently, there is a growing interest in the development of better therapeutics for HCC as existing treatments are still showing unsatisfying outcomes. The highly prevalent hypoxia and it’s unclear roles in tumours prompt the study of HIF in HCC. With the use of pharmacological compounds, this study aimed to investigate the effects of HIF pathway activation on HCC tumour progression. Human HepG2 cell line was employed as the in vitro cellular model of HCC. This study was undertaken by treating the cells with three hypoxia-mimicking agents (HMAs) which target the HIF pathway, including the non-selective deferoxamine (DFO), dimethyloxalylglycine (DMOG) and the selective IOX2, thereby allowing us to study the corresponding effects on aspects including cell proliferation, oxidative stress and epithelial-mesenchymal transition (EMT). To achieve this, cell-based assays including the trypan blue cell proliferation assay, 2’,7’-dichlorofluorescein diacetate (2’,7’-DCFDA) oxidative stress assay and immunoblotting were employed. The results showed differential effects upon pharmacological hypoxia pathway activation in HepG2. DFO reduced HepG2 cell proliferation in a concentration- and time-dependent manner, decreasing cell numbers 1.5-fold with 1.6 μM DFO over 72 hours. Despite that, DFO increased E-cadherin (E-cad) by up to 55% and decreased in alpha-smooth muscle actin (α-SMA) proteins levels by 26% after 48 hours, suggesting that it promotes EMT in HepG2. DMOG and IOX2 induced HepG2 cell proliferation, albeit up to a certain concentration. Notably, 250 μM DMOG and 31.3 μM IOX2 induced cell proliferation by 0.5-fold and 0.4-fold, respectively. Whilst both DMOG and IOX2 reduced α-SMA levels at 24 hours, IOX2-treated cells showed a 60% increase at 48 hours, suggesting that persistent HIF pathway activation by IOX2 may promote EMT. Furthermore, 250 μM DMOG reduced reactive oxygen species (ROS) production, whilst IOX2 had no significant effect on ROS levels in HepG2. This contrast with the clinical PHD inhibitors, Roxadustat and Daprodustat, which increased ROS production at 200 μM. In conclusion, the data suggest a context-dependent effect where non-selective HMAs (DFO and DMOG) generally facilitates pro-tumorigenic functions, whilst the selective PHD2 inhibitor, IOX2, could minimise tumour growth but may enhance migratory capabilities in HCC. Together with the effects of clinical PHD inhibitors, these results partially support the anti-tumorigenic functions of PHD inhibition in HCC and highlight the potential implications of PHD inhibitors as novel antitumour agents.

Initial

khm

Additional Information

Dissertation (M.A.) – Faculty of Medicine, Universiti Malaya, 2025.

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