Zinc Gluconate Induces Potentially Cancer Chemopreventive Activity in Barrett's Esophagus: A Phase 1 Pilot Study

Abstract

Background: Chemopreventive effects of zinc for esophageal cancer have been well documented in animal models. This prospective study explores if a similar, potentially chemopreventive action can be seen in Barrett's esophagus (BE) in humans.

Aims: To determine if molecular evidence can be obtained potentially indicating zinc's chemopreventive action in Barrett's metaplasia.

Methods: Patients with a prior BE diagnosis were placed on oral zinc gluconate (14 days of 26.4 mg zinc BID) or a sodium gluconate placebo, prior to their surveillance endoscopy procedure. Biopsies of Barrett's mucosa were then obtained for miRNA and mRNA microarrays, or protein analyses.

Results: Zinc-induced mRNA changes were observed for a large number of transcripts. These included downregulation of transcripts encoding proinflammatory proteins (IL32, IL1β, IL15, IL7R, IL2R, IL15R, IL3R), upregulation of anti-inflammatory mediators (IL1RA), downregulation of transcripts mediating epithelial-to-mesenchymal transition (EMT) (LIF, MYB, LYN, MTA1, SRC, SNAIL1, and TWIST1), and upregulation of transcripts that oppose EMT (BMP7, MTSS1, TRIB3, GRHL1). miRNA arrays showed significant upregulation of seven miRs with tumor suppressor activity (-125b-5P, -132-3P, -548z, -551a, -504, -518, and -34a-5P). Of proteins analyzed by Western blot, increased expression of the pro-apoptotic protein, BAX, and the tight junctional protein, CLAUDIN-7, along with decreased expression of BCL-2 and VEGF-R2 were noteworthy.

Conclusions: When these mRNA, miRNA, and protein molecular data are considered collectively, a cancer chemopreventive action by zinc in Barrett's metaplasia may be possible for this precancerous esophageal tissue. These results and the extensive prior animal model studies argue for a future prospective clinical trial for this safe, easily-administered, and inexpensive micronutrient, that could determine if a chemopreventive action truly exists.

Keywords: Chemoprevention; Claudin; Epithelial-to-mesenchymal transition; Esophageal adenocarcinoma; Inflammation; microRNA.

Fig. 1

Heatmap showing a sampling of genes whose expression was significantly altered by zinc treatment and which have documented associations or roles in BE progression to neoplasia. Gene expression data are shown for 5 placebo-treated patients (P) and 5 zinc-treated patients (Z). a Genes downregulated as a result of zinc treatment; b genes upregulated as a result of zinc treatment. Data include average fold-change in the mRNA transcript level and the P value for the statistical confidence. One of the placebo patients (P5) showed characteristics of the zinc treatment group for reasons unknown. Genes whose up- or downregulation evidences an anti-inflammatory action of zinc treatment are highlighted in yellow. Genes whose up- or downregulation suggests zinc-induced inhibition of epithelial-to-mesenchymal transition (EMT) signaling are highlighted in green

Fig. 2

Effect of zinc on miRNA species present in Barrett’s epithelia. Seven miRNA species that were significantly upregulated in BE biopsy samples of the zinc treatment group (P < 0.05, Student’s t test, one-tailed) are shown as vertical point plots. Each data point represents the miRNA abundance for a specific patient (n = 6 zinc-treated patients and 6 placebo patients per miRNA analyzed). RNA purification and miRNA analyses are described in Methods

Fig. 3

Effect of zinc on the pro-apoptotic protein, BAX. a Western blot bands for BAX are shown for biopsy tissue from four zinc-treated patients (Z) and four placebo-treated patients (P), with cell lysates prepared and PAGE and immunoblots performed as described in Methods section, b densitometry of above bands expressed as mean ± SEM (P < 0.1, Student’s t test, one-tailed, n = 4). Densitometry that was conducted on a Memcode stain of total protein served as the loading control for each band (lane)

Fig. 4

Effect of zinc on the tight junctional protein, CLAUDIN-7, and the VEGF receptor protein, VEGF-R2, in biopsy tissue samples confirmed histologically as secretory intestinal metaplasia. Densitometry of Western blot bands for the proteins, CLAUDIN-7 and VEGF-R2, expressed as vertical point plots. Each data point represents the Western blot band density obtained from PAGE and immunoblots performed on whole cell lysates of biopsy tissue from a single patient. Patients were zinc-treated or placebo-treated as described in Methods, but the patient groups here consisted only of BE cases confirmed histologically as secretory intestinal metaplasia. P values represent Student’s t tests, one-tailed, n = 5. Densitometry conducted on a Memcode stain of total protein served as the loading control for each band (lane)