Abstract
Lung cancer is the second most common cancer worldwide with an estimated 2.2 million cases recorded in 2020, accounting for 11.4% of total cancer cases. NSCLC adenocarcinomas are a lung cancer subtype accounting for approximately 40% of lung cancers. The main problem surrounding the treatment of cancers is drug resistance, often leading to disease progression and metastasis. Naturally occurring compounds make up approximately 60% of current cancer therapeutics, indicating a vital role in the cancer drug market. Spiroleucettadine is an alkaloid derived from the Leucetta genus of sea sponge. Spiroleucettadine was tested in a National Cancer Institute screen where it was reported to have the greatest potency in the NSCLC cell line, H522 (IC50 = 0.37 μM). The H522 cell line contains a mutation in the tumour suppressor protein p53, a common occurrence in cancers with a frequency of ~50%.
This study aimed to compare the effect of spiroleucettadine on cell viability in the NSCLC cell lines H522, H3122 and A549. In addition to this, the effects of spiroleucettadine on cell cycle arrest, apoptosis induction and protein expression will be investigated to determine the potential mechanistic action of spiroleucettadine in the NSCLC cell line, H522.
The cytotoxicity of spiroleucettadine was examined using p53 mutated NSCLC (H522) cells, KRAS+ cells (A549) and EML4-ALK+ cells (H3122) using the Sulforhodamine B assay. Cells were treated using a concentration range (0.01 – 100 μM) of spiroleucettadine for 72 hours. Cell cycle arrest and apoptotic induction of H522 cells by spiroleucettadine was measured using flow cytometry analysis. Western blotting was undertaken to determine changes in expression of key cell cycle and apoptotic proteins following spiroleucettadine treatment. For both protocols, H522 cells were treated using 0.5x, 1 x and 2 x EC50 values as determined by prior cell viability assays, for 24 hours.
The EC50 of spiroleucettadine, with regard to cell viability, was 0.72 ± 0.09 μM, 3.60 ± 0.03 μM and 1.59 ± 0.11 μM, in H522, A549 and H3122 cells, respectively.
A significant increase in the percentage of cells in the G2/M phases of the cell cycle was observed following 2 x EC50 treatment of H522 cells with spiroleucettadine when compared to the untreated control. This treatment also resulted in a significant decrease in the percentage of cells in the G0/G1 phase of the cell cycle compared to the control. Therefore, spiroleucettadine produced an arrest in the G2/M phase of the cell cycle. This was further backed up by the increased expression of the inhibitory pCDK1 and decreased cyclin D1 expression. Furthermore, increased cyclin B1 expression was observed, a potential indicator of microtubule instability and damage.
Finally, spiroleucettadine (2 x EC50) induced apoptosis, increased the expression of the pro-apoptotic protein, Bim, and increased cleaved caspase 3 expression, a key indicator of apoptosis.
To conclude, this thesis has exhibited the potential of spiroleucettadine as a new anticancer drug in the NSCLC cell line, H522. Spiroleucettadine inhibited cell proliferation, produced a G2/M phase cell cycle arrest and induced apoptosis at low micromolar concentrations. G2/M cell cycle arrest was achieved via increased pCDK1 expression while apoptosis was induced through increased Bim expression leading to activation of cleaved caspase 3. Further studies should investigate the potential action of spiroleucettadine on microtubules and in vivo activity to further determine the suitability of spiroleucettadine for clinical lung cancer treatment.