Supplementary Materialsmolecules-24-02152-s001. overview, we established a new tool for testing the impact of small molecule compounds on the EXT1 activity of p53 and used it to identify the action of benzimidazoles in melanoma cells. The drugs promoted the stability and transcriptional activity of wild-type p53 via downregulation of its negative regulators Mdm2 and MdmX in cells overexpressing these proteins. The results indicate the potential for repurposing the benzimidazole anthelmintics for the treatment of cancers overexpressing p53 negative regulators. 0.01. 2.2. Mechanism of ABZ and FBZ Action The benzimidazoles effects, particularly on p53, its major downstream target p21, and two main negative p53 regulators Mdm2 and MdmX were investigated by western blot analysis. Western blot analysis of A375 cells treated for 24 h with DINA (40 nM), MK-8745 ABZ (1, 2, and 4 M) and FBZ (1 and 2 M) showed approximately a 2.5-fold increase in p53 protein levels, while FBZ at the highest concentration (4 M) had only a slight effect (1.3 fold) (Figure 3A). The level MK-8745 of p53 downstream effector p21 was the most significantly increased in samples treated with FBZ at concentration 1 M (2.8 fold), while FBZ at concentration 2 M (1.7 fold) and ABZ at concentration 1 M (1.9 fold) had a weaker effect. This result indicated p53 activation, especially at lower concentrations of benzimidazoles (Figure 3B). Next, we studied the mechanism of p53 activation by examining the protein levels of the two main negative p53 regulators Mdm2 and MdmX. Significantly decreased levels MK-8745 of Mdm2 were observed only in samples treated with FBZ (2 and 4 M, 0.1 fold). Interestingly, the effect on MdmX was much more pronounced, the levels of MdmX were significantly decreased upon the treatment with DINA and with both benzimidazoles (0.1C0.2 fold) (Figure 3C,D). Open in a separate window Figure 3 Effect of benzimidazoles on p53 and related proteins levels. DINA (40 nM) was used as a positive control. Solvent (DMSO)-treated cells were used as a negative control (CTRL). (ACD) WB analysis of A375 cells. (A) The A375 cells treated 24 h with ABZ (1, 2, and 4 M), FBZ (1 and 2 MK-8745 M), and DINA (40 nM) revealed p53 stabilization. (B) ABZ (1 M) and FBZ (1 M) increased the level of p21. (C) FBZ (2 M and 4 M) decreased the level of Mdm2, ABZ at all concentrations and FBZ (1 M) had a weaker effect, and DINA did not affect p21. (D) The level of MdmX was decreased upon the treatment with DINA (40 nM), ABZ, and FBZ at concentrations 1, 2, and 4 M. (ECF) Similar results were also obtained MK-8745 with MCF7 breast carcinoma cells. (E) p53 stabilization, a rise of lower and p21 of Mdm2 amounts was recognized in DINA, ABZ (1, 2, and 4 M) and FBZ (2 and 4 M). (F) The loss of MdmX amounts was most pronounced in response to DINA, much less after ABZ and FBZ (1, 2, and 4 M) treatment. (G) In noncancerous HFF cells, the response was milder, p53 was somewhat stabilized upon ABZ (1 M) treatment. The amount of Mdm2 was reduced in ABZ (1 and 4 M), and FBZ (1, 2, and 4 M). MdmX amounts were below the recognition limit within the control HFF cells even. Total cell lysates had been separated on 12.5% SDS gel. Proliferating cell nuclear antigen (PCNA) amounts served like a launching control. Numeric ideals represent the percentage of music group densities from the protein appealing normalized towards the related PCNA as well as the control normalized towards the related PCNA. Much like melanoma cells, the.