Thesis

Quantifying the Anti-Proliferation Effects of Selected FDA-Approved Drugs Identified As Potential MDM2 Inhibitors for Cancer Therapy

The protein p53 is a transcription factor that is activated in response to cellular stress such as DNA damage. It promotes cell cycle arrest or apoptosis preventing further proliferation. In most human cancers, the integrity of p53 pathway is compromised. At an overall frequency of 7% of cancers, p53 retains its wild-type status but is inactivated by abnormally high levels of its main negative regulator, the MDM2 protein, which binds to and blocks p53. Well-studied molecules called Nutlins have shown antitumorigenic activity by binding to a hydrophobic cleft in MDM2 resulting in blockage of the interaction between p53 and MDM2. An alternative method for identifying anti-MDM2 drugs, called Computational Conformer Selection, was developed. In this method FDA – approved drugs were identified that show structural similarities to Nutlin 3a. In this study, the dose – dependent anti-proliferation effects of a lead subset of ranked FDA-approved drugs (bepridil, azelastine, and Protirelin / Thyrel (TRH)) were examined on SJSA – 1 (Tp53 wild-type, MDM2 amplified), SW480 (Tp53 mutant), and SaOs-2 (Tp53 deleted) cancer cell lines. Cell viability after drug treatment was measured with the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The incubation of SJSA-1 cells with the candidate drugs showed a dose-dependent decrease in cell viability. The determined median inhibitory concentration (IC50) values for Nutlin 3a, bepridil, and azelastine were 0.8 µM, 23 µM, and 25 µM, respectively. TRH inhibited cell growth by 56 % at 100 µM. Treatment of SW480 cells with the drugs revealed that the IC50 values for Nutlin 3a, Bepridil, and Azelastine were 0.3 µM, 5 µM, and 11 µM, respectively. TRH did not have a significant inhibitory effect on SW480 cells in the concentration range tested. For SaOs-2 cells, Nutlin 3a was not effective with an IC50 of 25 µM. The IC50 values for Bepridil, and Azelastine on SaOs-2 cells were 10 µM and 15 µM, respectively. TRH was not an effective inhibitor of SaOs-2 cell proliferation. To determine whether the anti-proliferation effects of the candidate drugs were due to the activation of p53 signaling pathway, the cells were exposed to drugs at their respective IC20, IC50, and IC80 for 12 hours and relative protein levels of p53, and its downstream targets MDM2 and p21 were determined by Western blot analysis. In SJSA-1 cells, the p53 protein could not be detected, likely due to its degradation by high levels of MDM2. However, Nutlin 3a activated the p53 signaling pathway in a dose-dependent as observed by increased MDM2 and p21 protein levels. In contrast, there was little to no change in MDM2 and p21 protein levels in SJSA-1 cells exposed to the candidate drugs. As expected, analysis of SW480 cells revealed that the levels of mutant p53 were not altered by drug treatment, and MDM2 and p21 proteins were not detected. In SaOs-2 cells no p53 and MDM2 protein was detected, and although p21 protein was present, it did not show drug dose-dependent increase in its levels. In conclusion, the FDA-approved drugs Bepridil and Azelastine, but not TRH have shown dose a dependent anti-proliferative activity on all three tested cell lines indicating this activity was p53 independent. Western blot analysis further established that the candidate drugs did not activate the p53 signaling pathway. Although the tested drugs work through different mechanisms, their anti-proliferation properties are promising and these drugs may be potentially developed for anti-cancer treatment.

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