Differentially expressed genes execute zinc-induced apoptosis in precancerous esophageal epithelium of zinc-deficient rats

Abstract

Zinc deficiency (ZD) in rats increases esophageal cell proliferation and the incidence of N-nitrosomethylbenzylamine-induced esophageal tumors. Conversely, zinc replenishment (ZR) rapidly induces apoptosis in esophageal epithelia and reverses cancer development. We investigated gene expression changes in ZR versus ZD esophageal epithelia to identify differentially expressed genes associated with the antitumor effect of ZR. Weanling rats were fed a ZD diet for 6 weeks to establish esophageal cell proliferation or a zinc-sufficient (ZS) diet. Then, 10 ZD rats were treated with zinc gluconate intragastrically and switched to ZS diet; the remaining 10 ZD and ZS animals were treated with saline. All animals were killed 26-28 h later. Using cDNA microarrays, realtime polymerase chain reaction amplification and RNA hybridization techniques, we identified novel differentially expressed genes, including a RNA-binding protein with two RNA recognition motifs and a zinc knuckle (ZD7), and a DNA/RNA helicase with a DEAD box (ZD10) with two splice variants, ZD10a and ZD10b. In situ hybridization detected increased mRNA expression of ZD7, ZD10a and ZD10b in ZR esophageal epithelia, which displayed markedly increased occurrence of apoptotic cells, relative to ZD epithelia. Overexpression of ZD7 in human esophageal cancer cells resulted in induction of apoptosis and activation of caspase-3 and -7, activities that were inhibited by caspase-specific inhibitors. In addition, ZD7 mRNA levels and zinc-induced apoptosis in rat squamous carcinoma cells were reduced by specific small interfering ribonucleic acids. Thus, ZR rapidly induces ZD7 and ZD10 expression, which in turn stimulates apoptosis. These results provide the beginnings of a molecular pathway for zinc-induced apoptosis under conditions that reverse esophageal tumor initiation.

Modulation of Gene Expression in Precancerous Rat Esophagus by Dietary Zinc Deficit and Replenishment

“…2). Consistent with previous reports (16,24), serum zinc content (Ag/100 mL F SD) rose from a zinc-deficient level of 43 F 14 to 727 F 221 and 610 F 255 at 8 and 12 hours after zinc replenishment, respectively. At 48 hours, the serum zinc level had decreased to 286 F 55, approaching the level in zinc-sufficient rats.…”

“…Zinc deficient (ZD ). ZR2, ZR4, ZR8, ZR12, ZR24, and ZR48 represent 2,4,8,12,24, and 48 hours after zinc replenishment. zinc-sufficient levels by 48 hours (Fig.…”

“…Our earlier study identified differentially expressed genes in rat esophagus that are associated with the anticancer effect of zinc replenishment (24). Using cDNA microarray analysis, we found five up-regulated and three down-regulated transcripts in zinc-replenished rat esophageal mucosa f27 hours after the zincdeficient rats were dosed with intragastric zinc versus zincdeficient esophagus.…”