BLT Stages

Abstract Bachelor Project FBT: Testing novel anti-cancer agents in aggressive B cell lymphomas

The incidence rates of non-Hodgkin lymphoma are among the highest worldwide in the USA and Western Europe. DLBCL is the most common subtype. Although DLBCL is aggressive, more than 50 % of patients are cured. Unfortunately, with approximately one third, refractory disease or relapse arises, which remains a major cause of morbidity and mortality.

Therefore this thesis aimed to test the in vitro effects of two new anti-cancer agents on aggressive lymphoma cells. For Nutlin 3 the effects on protein levels were examined, while for Bruceantin the apoptotic effects were investigated.

Seven human lymphoma cell lines (BL2, Raji, RI-1, U2932, Karpas422, SuDHL4 and Jurkat) were used. These were treated for 24 hours with Nutlin 3 (10 nM) followed by Western blot analysis or treated for 24, 48 and 72 hours with Bruceantin (10 nM, 50 nM) followed by EZ4U assay, apoptotic assays (Annexin V staining and caspase 3/7 activity) and cell cycle analysis (PI staining).

After performing the Western blot it was shown that Nutlin 3 increased the p21 protein expression in BL2, Raji and U2932. A rise in p53 protein expression was observed in BL2, Raji, RI-1 and U2932, while this was decreased in Karpas422. The MDM2 protein in SuDHL4, RI-1 and U2932 was elevated after Nutlin 3 treatment. This induction indicates that the functioning of p53 signalling was influenced by Nutlin 3.

After performing the cell growth assay (EZ4U assay) the IC50 curves of Bruceantin for the different cell lines were created. The cell lines, SuDHL4, BL2, Raji and Karpas422 had low IC50 values and thus were more sensitive to Bruceantin, while U2932, Jurkat and RI-1 had higher IC50 concentrations, so less sensitive. To analyse the in vitro apoptotic effects, the positivity for Annexin V after treatment was tested. This resulted in Annexin V positivity for SuDHL4 and BL2, whereas RI-1, U2932, Karpas422 and Jurkat were less sensitive to Bruceantin. In the next step the determination of caspase 3/7 was performed to confirm the results from Annexin V staining. The same trends were observed. Finally, to confirm the data, cell cycle analysis was performed. The cells of SuDHL4 and BL2 showed an increased subG1 peak, while in U2932, Karpas422, Jurkat and RI-1 this was not detected. Some cell lines showed an effect in other cell cycle phases as well.

The conclusion is that Nutlin 3 increases the expression of p21, p53 and MDM2 in some cell lines, indicating the functioning of p53 signalling. Bruceantin caused higher Annexin V positivity, a higher percentage of cells exhibited a higher caspase 3/7 activity and a cleaved DNA, indicating that Bruceantin possesses pro-apoptotic effects on aggressive lymphoma cells. Both agents are promising to develop novel anti-lymphoma therapies, but more research is necessary.