Investigating the toxicity of a novel molecular cancer therapeutic agent as seen in vitro and in vivo

Abstract

Background: Cancer is a leading cause of morbidity and mortality globally, with traditional therapies still leading to relatively low survival rates. The phosphatidyinositol-3-kinase (PI3K)/protein kinase B (Akt)/ mammalian target of rapamycin (mTOR) pathway is a vital signal transduction pathway that regulates cell proliferation, metabolism and survival. Aberrations of the pathway have been found to contribute to the development of several forms of cancer, including Non-Small Cell Lung Cancer (NSCLC) and Osteosarcoma (OS). Voxtalisib (SAR245409) is a novel form of molecular targeted therapy produced by Cayman Chemical©, that acts as a dual PI3K- mTOR inhibitor. One of the main challenges attributed to the development of novel chemotherapeutic drugs, is the requirement for their selective toxicity to cancer cells in preference to physiologically healthy tissue. Despite the fact that their mechanism of action is designed to target selected cancer-cell specific deregulated systems, they cause an unavoidable influence on relevant pathways in healthy cells, resulting in undesired toxicity. It is therefore of prime pharmacotoxicological relevance to study selective toxicities of such novel agents under development. Methodology: The research was divided into two phases. Phase I consisted of toxicity testing via two cell culture experiments, cell viability (PrestoBlue® assays) and wound healing assays, that were conducted on two tumour cell lines which represent the prominent cancer types discussed throughout the dissertation, the H1975 cell line as a model for NSCLC and Saos-2 as a model for OS. The primary aims of the cell culture experiments include: (1) determining the therapeutic inhibitory effects of the drug under study, voxtalisib, on both cell lines in the context of migration and proliferation, and (2) defining the inhibitory concentration for 30% loss in cell viability (IC30). Phase II involved acute toxicity testing on zebrafish (Danio rerio) larvae. The larvae treated via a range of different voxtalisib concentrations, and their mortality rate, morphological abnormalities and behavioural response were recorded for 96 hours after the exposure. Mortality rate and morphology were recorded via a simple external observation, and compared to the controls, while behavioural analysis was recorded via an automated tracking device which determined locomotor activity following light and startle response. The principal objectives of Phase II included obtaining information regarding the drug’s potential to induce adverse effects on healthy organisms and gathering a brief insight into toxic and safe ranges which could be compared to other therapeutic alternatives. Results: When tested in the tumour cell lines, voxtalisib caused a concentration- and time- dependent decrease in both percentage cell viability and migration in the wound healing assays, with an approximated IC30 of around 2.5μM. In the zebrafish larvae, voxtalisib was associated with significant time- and dose- dependent adverse effect, causing a high mortality rate above 10μM and severe malformations at concentration that exceeded 5μM. The approximated LC50 for voxtalisib in zebrafish larvae was around 5μM, and the lowest-observable-adverse-effect-level was estimated to range between 1.25μM and 2.5μM. [...]