Extracellular zinc promotes cell proliferation and its deficiency leads to impairment of this process, which is particularly important in epithelial cells. We have recently characterized a zinc-sensing receptor (ZnR) linking extracellular zinc to intracellular release of calcium. In the present study, we addressed the role of extracellular zinc, acting via the ZnR, in regulating the MAP kinase pathway and Na Arrested cell proliferation is a hallmark of zinc deficiency. This is particularly true in gastrointestinal cells (1-4), where insufficient dietary zinc attenuates the renewal of the epithelium leading to severe diarrhea (1). Extracellular zinc has been shown to regulate cell proliferation via the MAP 1 kinase pathway in several cell types (5-7). Although the mitogenic and anti-apoptotic effects of zinc are well recognized (8, 9), and the treatment of severe diarrhea by addition of dietary zinc is common, the direct link between this ion and the cellular mechanisms regulating proliferation is not well understood. It has been suggested that a decrease in intracellular zinc may lead directly to a reduction in activity of various metalloenzymes involved in transcription and cell metabolism (1, 10). Several studies, however, indicate that extracellular zinc acts as a signaling molecule. In tracheal cells, for example, extracellular zinc, through activation of Src, leads to transactivation of EGFR and subsequently to activation of ERK1/2 (11). In fibroblasts, extracellular zinc has been shown to trigger the activation of the PI3K pathway, subsequently leading to the activation of AKT and the S6 kinase (12). The signaling pathways linking extracellular zinc to these proteins and to subsequent regulation of cellular ion, pH or volume homeostasis, however, remain poorly understood.We have recently identified and characterized an extracellular zinc-sensing receptor (ZnR) that triggers, upon exposure to extracellular zinc, the release of Ca 2ϩ from intracellular stores by activation of the IP 3 pathway (13). The pharmacological profile of the calcium response triggered by the ZnR, particularly its sensitivity to the PLC inhibitor, U73122, and the inhibitory effect of the IP 3 receptor blocker, 2-APB, indicates that the ZnR is a G q -coupled receptor (GPCR). Both the G␣ and the G␤␥ dimer of various GPCRs have been linked to activation of the MAP kinase via multiple intracellular pathways (14 -16). In intestinal cells, furthermore, the muscarinic receptor has been shown to play a key role in promoting ion transport through activation of MAP and PI 3-kinase signal transduction (17,18). A role for the ZnR in activation of the MAP kinase pathway is demonstrated in this work.The ZnR was initially characterized in the colonocytic cell line, HT29, where a robust calcium signal was generated following activation of the receptor by changes in the concentration of extracellular Zn 2ϩ (13). Importantly, the Ca 2ϩ response induced by the ZnR in HT29 cells is triggered by ϳ80 M zinc, i.e. within the physiological range of zinc conc...

Leptin serum levels are about 5 times higher in obese people than in normal individuals. We aimed at investigating the signaling pathways induced by leptin in the human colonic cell lines LS174T and HM7. Both cells expressed the leptin transmembrane Ob-receptor. Leptin activated the mitogen-activated protein kinase pathway, induced invasion of colonic cells and concomitantly increased the formation of lamellipodial structures. A direct and novel dose-and time-dependent activation of RhoA, Cdc42 and Rac1 by leptin is demonstrated in these aggressive colon cancer cells. The activation of the Rho family of GTPases was amenable to specific inhibition: Wortmannin inhibited leptin-induced Rac1 and Cdc42 activation but did not affect RhoA activation, and inhibited the formation of leptin-induced lamellipodia and cell invasion. The Rac1 inhibitor NSC23766 inhibited only leptininduced Rac1 activation and concomitantly, lamellipodium formation and cell invasion. The Src kinase inhibitor II (SrcKI-II) exerted a positive effect on RhoA activation, inhibited tyrosine phosphorylation of p190RhoGAP and inhibited leptin-induced Cdc42 activation and leptin-induced lamellopodium formation and cell invasion. The specific JAK2 inhibitor AG490 exerted a positive effect on Rac1 and Cdc42 activation by leptin and concomitantly inhibited RhoA activation. AG490 did not inhibit leptin-induced lamellopodium formation or cell invasion. Our findings clearly indicate that leptin activates PI3K and Src kinase pathways in the metastatic colon cancer cells LS174T and HM7. These signaling pathways induce the activation of Rac1 and Cdc42, lamellopodium formation and concomitantly enhanced cell invasion, but leptin activation of RhoA is not associated with enhanced cell locomotion and invasion. Understanding in-depth the pathways involved in leptin-associated enhanced cell locomotion and invasion may contribute with the design of novel therapeutics to treat obesity-associated advanced colorectal cancer.

Allicin (diallyl thiosulfinate) is the best-known biologically active component in freshly crushed garlic extract. We developed a novel, simple method to isolate active allicin, which yielded a stable compound in aqueous solution amenable for use in in vitro and in vivo studies. We focused on the in vitro effects of allicin on cell proliferation of colon cancer cell lines HCT-116, LS174T, HT-29, and Caco-2 and assessed the underlying mechanisms. This allicin preparation exerted a time- and dose-dependent cytostatic effect on these cells at concentrations ranging from 6.2 to 310 μM. Treatment with allicin resulted in HCT-116 apoptotic cell death as demonstrated by enhanced hypodiploid DNA content, decreased levels of B-cell non-Hodgkin lymphoma-2 (Bcl-2), increased levels of bax and increased capability of releasing cytochrome c from mitochondria to the cytosol. Allicin also induced translocation of NF-E2-related factor-2 (Nrf2) to the nuclei of HCT-116 cells. Luciferase reporter gene assay showed that allicin induces Nrf2-mediated luciferase transactivation activity. SiRNA knock down of Nrf2 significantly affected the capacity of allicin to inhibit HCT-116 proliferation. These results suggest that Nrf2 mediates the allicin-induced apoptotic death of colon cancer cells.

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