Possible role of membrane gamma-glutamyltransferase activity in the facilitation of transferrin-dependent and -independent iron uptake by cancer cells

Abstract

BACKGROUND: The molecular mechanisms by which iron is physiologically transported trough the cellular membranes are still only partially understood. Several studies indicate that a reduction step of ferric iron to ferrous is necessary, both in the case of transferrin-mediated and transferrin-independent iron uptake. Recent studies from our laboratory described gamma-glutamyltransferase activity (GGT) as a factor capable to effect iron reduction in the cell microenvironment. GGT is located on the outer aspect of plasma membrane of most cell types, and is often expressed at high levels in malignant tumors and their metastases. The present study was aimed at verifying the possibility that GGT-mediated iron reduction may participate in the process of cellular iron uptake. RESULTS: Four distinct human tumor cell lines, exhibiting different levels of GGT activity, were studied. The uptake of transferrin-bound iron was investigated by using 55Fe-loaded transferrin, as well as by monitoring fluorimetrically the intracellular iron levels in calcein-preloaded cells. Transferrin-independent iron uptake was investigated using 55Fe complexed by nitrilotriacetic acid (55Fe-NTA complex).The stimulation of GGT activity, by administration to cells of the substrates glutathione and glycyl-glycine, was generally reflected in a facilitation of transferrin-bound iron uptake. The extent of such facilitation was correlated with the intrinsic levels of the enzyme present in each cell line. Accordingly, inhibition of GGT activity by means of two independent inhibitors, acivicin and serine/boric acid complex, resulted in a decreased uptake of transferrin-bound iron. With Fe-NTA complex, the inhibitory effect - but not the stimulatory one - was also observed. CONCLUSION: It is concluded that membrane GGT can represent a facilitating factor in iron uptake by GGT-expressing cancer cells, thus providing them with a selective growth advantage over clones that do not possess the enzyme.

Figure 1

Effects of modulation of membrane GGT activity on transferrin-mediated uptake of ⁵⁵Fe by 4 human cancer cell lines expressing different levels of GGT activity. Data shown are means ± SEM of 2–5 experiments. Panel A: U937 histiocytoma cells (*significantly different from control value, P < 0.05; **significantly different from the "+GSH+gly-gly" value, P < 0.05). Panel B: Me665/2/60 melanoma cells (*significantly different from control value, P < 0.05). Panel C: K562 erythroleukemia cells (**significantly different from the "+GSH+gly-gly" value, P < 0.05). Panel D: Me665/2/21 melanoma cells (differences not statistically significant).

Figure 2

Correlation between membrane GGT activity of 4 human tumor cell lines and the corresponding GGT-stimulatable uptake of transferrin-bound iron. Data shown are means ± SEM of 3–5 experiments. The logarithmic correlation curve shown is statistically significant (r = 0.99).

Figure 3

Effects of GGT inhibition on transferrin-bound iron uptake in K562 cells. Online fluorimetric monitoring of intracellular iron levels. Transferrin (>70% iron-saturated) was added where indicated, and the decrease in fluorescence of intracellular calcein was recorded (cfr. Methods for details of the procedure). One representative experiment out of five is reported.

Figure 4

Effects of modulation of membrane GGT activity on the uptake of ⁵⁵Fe-nitrilotriacetate complex by GGT-rich U937 (A) and Me665/2/60 cells (B). Data shown are means ± SEM of 3–5 experiments. * Significantly different from control value, P < 0.05.