Figure 6. Zinc enhances MI-219 mediated p53 transcription that is blocked by TPEN.

[A & B] Semi-confluent HCT-116 and MCF-7 cells grown in triplicate were exposed to either vehicle (DMSO); MI-219 (10 μM); MI-219 (10 μM) + ZnCl₂ (16 μM); MI-219 (10 μM) + ZnCl₂ (16 μM) + TPEN (1 μM); MI-219 (10 μM) + CaCl₂ (15 μM) or MI-219 (10 μM) + CaCl₂ (15 μM) + Bapta-AM (10 μM) for 24 hrs in 6 well plates. Nuclear lysates were isolated and p53 transcription assay was performed in 96 well plates according to manufacturer's protocol (Cayman Chemicals Ann Arbor, MI, USA). ZnCl₂ (16 μM) enhances MI-219 (10 μM) mediated p53 transcription that is blocked by TPEN. Calcium does not induce p53 transcription and the calcium chelator, Bapta-AM has no affect. NiCl₂ treated HELA cell lysates were used as positive control (provided in kit) while MI-10 the inactive analogue of MI-219 was used as negative control. * represents p<0.05 and ** p< 0.01. [C] ZnCl₂ suppresses MDM2 protein expression and enhances MI-219-induced MDM2-p53 disruption. MCF-7 cells were exposed to either vehicle (DMSO); MI-219 (10 μM); MI-219 (10 μM) + ZnCl₂ (16 μM); MI-219 (10 μM) + ZnCl₂ (32 μM); MI-219 (10 μM) + ZnCl₂ (16 and 32 μM) in the presence of TPEN (1 μM) for 24 hrs and protein lysates were subjected to western blot analysis followed by probing with MDM2 antibody. ZnCl₂ (16 and 32 μM) suppressed MDM2 expression [D] Co-immunoprecipitation studies of MCF-7 cells demonstrate reduced disruption of MDM2-p53 interaction in the presence of TPEN. Again ZnCl₂ abrogates the effects of TPEN. Blots are representative of three independent experiments.