Zinc deficiency and excess influence cellular homeostasis and are believed to modulate apoptosis. Zinc also regulates cell growth and proliferation. Understanding of the role of zinc in the mechanisms associated with these changes is limited because of its diverse, complex, and cell-specific effects. Therefore, we investigated the oxidative stress responses and the underlying molecular mechanisms associated with the disruption of intracellular zinc homeostasis in H4IIE rat hepatoma cells. We found that zinc excess (100 μM) and DTPA (diethylenetriaminepentaacetic acid; 50-100 μM) induced zinc deficiency both generate reactive oxygen species (ROS) and decrease viability in H4IIE cells. However, cotreatment with the antioxidant, N-acetyl-L-cysteine (NAC) both reduced ROS production and protected cells from death. We additionally observed an increase in Bax mRNA and cytochrome c release from the mitochondria in DTPA-treated cells and an elevated expression of Fas/Fas ligand mRNA with zinc treatment. Both treatments increased p53 and MdM2 protein concentrations along with caspase 3/7 activity. These results suggest that zinc deficiency stimulates mitochondrial-dependent apoptosis whereas zinc activates the extrinsic-apoptotic pathway. Both decreasing and increasing cellular zinc concentrations modulate ROS mediated apoptosis and warrant further research on zinc mediated cancer chemoprevention in this and other cancer cell lines.
LIV‐1 is an estrogen‐regulated gene encoding a member of the ZIP family of zinc transporters (SLC39A6). It is expressed in both estrogen‐receptor positive and negative breast cancer cells. In estrogen‐receptor positive MCF‐7 breast cancer cells, LIV‐1 expression is induced by both estrogen and insulin. LIV‐1 has also been observed to induce the expression of the cell adhesion protein E‐cadherin, thought to play a role in metastasis. In the present study, we investigated the regulation of LIV‐1 and its relationship to E‐cadherin in MDA‐MB‐231estrogen‐receptor negative breast cancer cells. Cells were treated with insulin and EGF for 24 h and the mRNA concentrations of LIV‐1 and E‐cadherin were analyzed by RT‐PCR. EGF treatment induced LIV‐1 mRNA by about 2‐fold whereas insulin increased expression only to a minor extent. The mRNA expression of E‐cadherin paralleled that of LIV‐1 with these treatments. MDA‐MB‐231 cells were also transfected with an shRNA construct designed to knock down LIV‐1 expression. This procedure reduced LIV‐1 mRNA concentrations by 30% and expression of E‐cadherin was reduced to a similar extent. This association between LIV‐1 and E‐cadherin expression in MDA‐MB‐231 cells suggests that LIV‐1 may be a regulator of E‐cadherin in both estrogen‐receptor positive and negative cells. The presence of LIV‐1 could serve as a prognostic marker for breast cancer patients with invasive tumors.
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