Novel Molecular Therapeutic Agents for Hepatocellular Carcinoma

Abstract

Hepatocellular carcinoma (HCC) constitutes the majority of primary liver cancers. Worldwide, liver cancer is the sixth most predominant cancer and the fourth main cause of cancer-related mortality. The current treatments of advanced-stage HCC have demonstrated limited survival benefits; therefore, there is a pressing need to develop new targeted therapeutics that can effectively combat HCC. This project explored the activity of novel molecules belonging to two different classes against HCC in vitro. These molecules were first, a series of 3-arylisoquinolinones and second, a tirapazamine-dopamine/phenylboronate hyaluronic acid conjugate (TPZ-D-HA). The 3-arylisoquinolinones were chosen for this study because the NCI COMPARE analysis had suggested that these agents could interact with cell microtubules which are a recognised target for cancer therapy. Microtubule-targeting agents possess antimitotic, antiangiogenic and vascular disrupting properties, and they have shown a therapeutic promise against HCC. Five differently substituted 3-arylisoquinolinones were examined. The meta-substituted compounds showed substantial cytotoxicity (IC50=0.07–3.1 µM) towards liver, colorectal, lung and breast cancer cells, whereas the para analogues displayed inferior activity (IC50>50 µM). Of the meta compounds, Cpd. 4 was generally the most potent cytotoxic agent with marked less toxicity to normal hepatocytes. Mechanistically, Cpd. 4 inhibited tubulin polymerisation into microtubules, disrupted actin and microtubule networks, caused G2/M cell cycle arrest and induced apoptosis. Also, it inhibited the formation of capillary-like tubes and disrupted the pre-established tubes of endothelial cells. Further, tumour hypoxia is an attractive target for cancer treatment since it promotes the development of aggressive and therapy-resistant phenotypes in most solid tumours including HCC. TPZ is a bioreductive drug that selectively kills hypoxic cells; however, it still causes toxicities towards normal tissues. HA-based drug delivery systems can preferentially accumulate the loaded drug into tumour tissues owing to the passive and active targeting properties of HA. Therefore, TPZ was conjugated with HA in an attempt to ultimately reduce TPZ toxicity and maximise its delivery to tumours. Chemically, the conjugation of TPZ with HA was enabled by forming TPZ-dopamine (TPZ-D) which then bound to HA yielding TPZ-D-HA. The toxicity of the three components of the conjugate (TPZ, TPZ-D and the conjugate itself) was evaluated in liver cancer cell lines. Firstly, it was established that the cells were able to express the cellular reductases (required for the hypoxic activation of TPZ, TPZ-D and TPZ-D-HA) and HA receptors (required for the binding and uptake of TPZ-D-HA). As expected, TPZ showed substantially high toxicity towards the hypoxic versus the normoxic cells (hypoxic cytotoxicity ratios [HCRs] = 22– >>34, 3 hours-treatment). Under normoxia or hypoxia, TPZ-D-HA was equipotent to TPZ-D while greatly more cytotoxic and less hypoxia-selective than TPZ (HCRs= ~3, 3 hours-treatment). Interestingly, there was no correlation between the expression levels of HA receptors and TPZ-D-HA cytotoxicity. In conclusion, the antiproliferative potential of the novel 3-arylisoquinolinones is markedly influenced by a subtle change in the structure (meta versus para). The meta-substituted isoquinolinone Cpd. 4 is a microtubule-targeting agent with potential tumour-selectivity, antiangiogenic and vascular disrupting features in HCC. Moreover, TPZ-D-HA would be able to target aerobic plus hypoxic HCC cells but possibly not the aerobic healthy cells. It may also be worthy to further evaluate the combination between Cpd. 4 and TPZ-D-HA in vivo.

Date of Award	1 Aug 2020
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Ian Stratford (Supervisor) & Katie Finegan (Supervisor)