Investigating mechanisms involved in the pharmacology of non-small cell lung cancer

Abstract

Introduction: Non-small cell lung cancer is one of the most common cancer types worldwide, having a higher occurrence in males with a mortality rate of 15%. Dysregulation of the PI3K/AKT/mTOR Pathway is what causes this carcinoma. The Akt molecule is a 256 amino-acid protein, weighing 40kDa, leading to multiple phosphorylation events bringing about increased cellular proliferation, survival, and growth, being the specialized properties of cancer cells. Aim: To investigate the efficacy of AZD5363 in binding and inhibiting the Akt protein, from phosphorylating its constituent substrates, leading to cancerous properties within cells, thus having inhibited part of the PI3K/AKT/mTOR Pathway. The levels of free eIF4E were also investigated as these should correlate to the level of inhibition of the Akt protein. Akt inhibits the activation of the TSC1/TSC2 complex which inhibits RHEB from activating mTORC1 leading to an accumulation in free eIF4E levels. Methodology: For this project, H460 cells provided by the Department of Clinical Pharmacology and Therapeutics were utilized. These cells were cultured in vitro and stored under standard human conditions. PrestoBlue, which is a blue-colored cell-permeable chemical, turns red upon reduction within living cells. A PrestoBlue Assay was carried out and analyzed via spectrophotometry, to observe and measure the rate of cellular proliferation and obtain an accurate incubation period for optimum results via the action of the drug. Secondly, the cells were exposed to varying concentrations of AZD5363 at the 24, 48, and 72-hour timepoints. Finally, an ELISA assay was later carried out to obtain an accurate measurement of the concentration concerning free eIF4E. Data Analysis: The raw data obtained from the viability assay was checked for the normality assumption due to having a low population number. Even though most of the data was normally distributed, normality was still assumed as similar studies which had similarly low numbers made use of such parametric tests due to their accuracy in determining statistical relationships. The significance of the data obtained was measured by making use of both the independent sample T-test to observe significance between the results of each timepoint separately as compared to the untreated cells, and an ANOVA with Tukey correction as a post hoc analysis to account for any significance between the results of each timepoint together at the same concentrations. Results: The efficacy of the drug appears to be dependent on both the concentration whichis administered and the time under which the H460 cells are being exposed. The levels of free eIF4E are yet to be measured, however, it is assumed that such levels should correlate with the reduction of Akt levels, thus free eIF4E levels are expected to be low when compared to that in the untreated cells which will be presented for the VIVA. Conclusion: The AZD5363 drug-induced a reduction in the levels of Akt which led to reduced protein synthesis and hence reduced cellular proliferation as is indicated by the viability assay. Statistically, it has been shown that most of the drug concentrations administered gave significant results when compared to the untreated H460 cells. It will later be confirmed as to whether the levels of free eIF4E were also affected, but it is assumed based on various similar studies, that such levels are to be much lower when compared to untreated H460 cells.