ABSTRACT
Zinc has a crucial role in the biology of p53 in that p53 binds to DNA through a structurally complex domain stabilized by zinc atom. The p53 negative regulator MDM2 protein also carries a C-terminal RING domain that coordinates two zinc atoms, which are responsible for p53 nuclear export and proteasomal degradation. In this clinically translatable study, we explored the critical role of zinc on p53 reactivation by MDM2 inhibitor, MI-219, in colon and breast cancer cells. ZnCl(2) enhanced MI-219 activity (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), apoptosis and colony formation), and chelation of zinc not only blocked the activity of MI-219, but also suppressed reactivation of the p53 and its downstream effector molecules p21(WAF1) and Bax. N,N,N'N'-tetrakis(-)[2-pyridylmethyl]-ethylenediamine (TPEN), a specific zinc chelator, but not 1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (Bapta-AM), a calcium chelator, blocked MI-219-induced apoptosis. Nuclear localization is a prerequisite for proper functioning of p53 and our results confirm that TPEN, and not Bapta-AM, could abrogate p53 nuclear localization and it interfered with p53 transcriptional activation. Addition of zinc suppressed the known p53 feedback MDM2 activation, which could be restored by TPEN. Co-immunoprecipitation studies verified that MI-219-mediated MDM2-p53 disruption could be suppressed by TPEN and restored by zinc. As such, single-agent therapies that target MDM2 inhibition, without supplemental zinc, may not be optimal in certain patients owing to the less recognized mild zinc deficiency among the 'at-risk population' as in the elderly who are more prone to cancers. Therefore, use of supplemental zinc with MI-219 will benefit the overall efficacy of MIs and this potent combination warrants further investigation.
Subject(s)
Chlorides/pharmacology , Enzyme Inhibitors/pharmacology , Proto-Oncogene Proteins c-mdm2/antagonists & inhibitors , Proto-Oncogene Proteins c-mdm2/metabolism , Zinc Compounds/pharmacology , Apoptosis/drug effects , Breast Neoplasms/metabolism , Cell Growth Processes/drug effects , Cell Line, Tumor , Cell Nucleus/metabolism , Chelating Agents/pharmacology , Colonic Neoplasms/metabolism , Drug Synergism , HCT116 Cells , Humans , Transcription, Genetic , Transfection , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
Zinc (Zn) has significant anti-oxidant and anti-inflammatory activity. Zn deficiency can occur in subsets of patients with cystic fibrosis (CF) especially those with malabsorption and impaired growth. Although supplemental Zn has significantly reduced infections in various disorders, its efficacy has not been thoroughly investigated in CF. We performed a double blind placebo controlled pilot study to investigate the effect of daily 30 mg elemental Zn for 1 year on the rate of respiratory tract infections (RTIs), use of antibiotics and plasma cytokines in 26 children with CF (ages 7-18 years). Plasma Zn, Cu, inflammatory cytokines and ex vivo generation of IL-2 were measured at baseline and at the end of the study. The number of days of oral antibiotics was lower in Zn treated patients compared to placebo (P = 0.05). However, compared to placebo, the effect of Zn was greater in patients who exhibited low plasma Zn at baseline (P = 0.02) than those who had plasma Zn levels identical to normal subjects (P = 0.55). Zn supplementation was marginally effective in reducing percentage increase in plasma IL-6 and IL-8 while increasing the percentage change in ex vivo generation of IL-2 in isolated mononuclear cell. In conclusion, oral intake of 30 mg/day of Zn reduced the number of days of oral antibiotics used to treat RTIs in children with CF. A higher daily Zn dose may be needed to decrease RTIs and modify immune responses.
