@masterthesis{ViyofFul, type = {Bachelor Thesis}, author = {Viyof Ful, Flavian}, title = {The effects of P53 stabilitization in SMARCB1-deficent, deficent TP53- wildtype epitheliod sarcoma cells}, school = {Hochschule Rhein-Waal}, abstract = {Epithelioid sarcomas (ES) which are a rare form of soft tissue sarcomas are known to afflict both males and females with a slight bias for males. They are characterised by a defective SMARCB1 gene as well as a TP53 wildtype. Owing to this distinctive trait, it was hypothesised that reactivating p53 protein could hold therapeutic advantage in these type of tumours since p53 is a transcription factor at the crossroads between cell survival or death. P53 downstream target genes regulate the cell cycle or promote apoptosis. Since p53 is inactivated by its natural inhibitor, MDM2, in these types of tumours (Claudia Schaffner et al. 1999), using an MDM2 inhibitor to disrupt the P53-MDM2 interaction and stabilise p53 offered a sensible rationale to explore. It is often the case that cancer cells develop a resistance to a particular chemotherapy over time. Therefore, using a combination of two different inhibitors with completely different mechanisms of action to antagonise the tumour cells and prevent a possible recurrence was also considered. Two epithelioid sarcoma cell lines, VAESBJ and EPI544, were used for these experiments. HDM201, which is an MDM2 inhibitor, and doxorubicin, the current standard in threating epithelioid sarcomas, were both employed to be used as single agent treatments and in combination. Firstly, it had to be established that both cells are sensitive to the inhibitors in a dose dependent fashion. This was done using the MTS assay which quantifies cell viability based on the metabolism of tetrazolium salts and its eventual conversion to a purple soluble formazan residue by viable cells. Next, to further investigate the downstream effects of reactivated p53 in the cell populations, indirect immunofluorescence (IF) was performed to demonstrate that both inhibitors were capable of individually stabilising p53 in both cell lines. To further support the results from the indirect IF, western blot analysis was done to prove that p53 accumulates within the cell. Furthermore, quantitative polymerase chain reaction (PCR) was also performed to explore the extent to which p53's stabilisation influences the fate of the treated cells. The results obtained from the MTS assay experiments showed that the cell lines used were indeed sensitive to both inhibitors as single agent treatments (EPI544 cells IC50 (HDM)= 0.212nM, IC50 (Dox)= 259.9nM) and VAESBJ IC50 (HDM)= 1.52µM, IC50 (Dox)= 17.5nM). At their respective IC50 values in combination, both drugs acted synergistically to halt cell viability in VAESBJ cell line whereas they were antagonistic against EPI544 cell line. However, these discrepancies were due to the dose response of the cells to the drugs. Moreover, from the IF analysis, p53 was clearly stabilised and as a result affected cell viability in all cell lines for both drugs. There was a considerable amount of p53 accumulation in treated cells when analysed by western blot. Lastly, the qPCR data showed that upon stabilisation, p53 upregulated the cell cycle checkpoint protein p21 as well as its natural regulator MDM2 because of a negative feedback loop meant to regulate the amount of p53 in normal cells. BCL2 which encodes an anti-apoptotic protein is also downregulated in all treated cells. Pro-apoptotic TP53I3 gene is significantly upregulated in the cell populations treated with the combination. In a nutshell, the results support the rationale that targeting MDM2 in ES tumours holds therapeutic value. They also highlight the usefulness of combination therapies as a better approach at targeting cancer cells. Although p53 is stabilised, its accumulation and downstream effects are largely dependent on whether it was achieved through posttranslational modifications or not. These results necessitate future research to characterise the effects of a combination of HDM201 and Doxorubicin on ES tumours in vivo.}, language = {en} }