Figure 5.
Figure 5. Cellular uptake of iron complexes of ELT, interactions with DFX and CP40, and proposed mechanisms of interaction of ELT with chelatable cellular iron and effects of second chelator. Iron uptake into HuH7 cells from preformed chelate complexes of ELT or DFX is shown at 6 hours in (A) and (B) of the same experiment. Control iron release with ELT or CP40 (A) or DFX alone (B) are also shown. CP40 was chosen for evaluation because of its lack of iron removal from cells when used as a single agent and its lack of iron donation to cells. After incubation, cells were washed, with the first wash containing DFO at 30 µM IBE and then with 3 PBS washes before intracellular iron concentration was determined, using the ferrozine assay as described in “Materials and methods.” (A) Iron uptake from chelate complexes of ELT is shown, where complexes of ELT were presented either as 1:1 or 1:2 ratios of iron:ELT. CP40 inhibits iron uptake from both FeELT and FeELT2. (B) In contrast to CP40, DXF does not inhibit the net uptake of iron from preformed complexes of ELT. Preformed complexes of DFX (DFX2Fe) donate some iron to cells, but less than from complexes of ELT. (C) Proposed mechanisms of interaction of ELT with cellular iron with or without a second chelator. ELT diffuses into cells, rapidly binding LIP iron and thus decreasing ROS. Iron complexes of ELT then diffuse out of the cell, some of which can subsequently donate iron back to the cell (however establishing a net deironing effect of ELT monotherapy) (A). Diffusion of ELT into cells was not measured directly but has been previously shown in other cells and is consistent with its low molecular weight, its high lipid solubility, and rapid intracellular ROS inhibition. A second chelator (L) can increase intracellular iron chelation, and thus cellular iron release, if it gains direct access to LIP, as is known to occur with DFX, but not with CP40. ELT binds chelatable iron (from citrate) faster than DFX (Figure 4A-B). DFX binds iron from complexes of ELT faster than those bound to citrate (Fe:citrate 10:100) (Figure 4C). A second chelator can also increase net iron release extracellularly by competitive removal of iron from ELT–iron complexes, thus decreasing the donation of iron from ELT–iron complexes to cells. Both intracellular and extracellular donation of iron to a second chelator (L) potentially frees up ELT for a further round of iron chelation.

Cellular uptake of iron complexes of ELT, interactions with DFX and CP40, and proposed mechanisms of interaction of ELT with chelatable cellular iron and effects of second chelator. Iron uptake into HuH7 cells from preformed chelate complexes of ELT or DFX is shown at 6 hours in (A) and (B) of the same experiment. Control iron release with ELT or CP40 (A) or DFX alone (B) are also shown. CP40 was chosen for evaluation because of its lack of iron removal from cells when used as a single agent and its lack of iron donation to cells. After incubation, cells were washed, with the first wash containing DFO at 30 µM IBE and then with 3 PBS washes before intracellular iron concentration was determined, using the ferrozine assay as described in “Materials and methods.” (A) Iron uptake from chelate complexes of ELT is shown, where complexes of ELT were presented either as 1:1 or 1:2 ratios of iron:ELT. CP40 inhibits iron uptake from both FeELT and FeELT2. (B) In contrast to CP40, DXF does not inhibit the net uptake of iron from preformed complexes of ELT. Preformed complexes of DFX (DFX2Fe) donate some iron to cells, but less than from complexes of ELT. (C) Proposed mechanisms of interaction of ELT with cellular iron with or without a second chelator. ELT diffuses into cells, rapidly binding LIP iron and thus decreasing ROS. Iron complexes of ELT then diffuse out of the cell, some of which can subsequently donate iron back to the cell (however establishing a net deironing effect of ELT monotherapy) (A). Diffusion of ELT into cells was not measured directly but has been previously shown in other cells and is consistent with its low molecular weight, its high lipid solubility, and rapid intracellular ROS inhibition. A second chelator (L) can increase intracellular iron chelation, and thus cellular iron release, if it gains direct access to LIP, as is known to occur with DFX, but not with CP40. ELT binds chelatable iron (from citrate) faster than DFX (Figure 4A-B). DFX binds iron from complexes of ELT faster than those bound to citrate (Fe:citrate 10:100) (Figure 4C). A second chelator can also increase net iron release extracellularly by competitive removal of iron from ELT–iron complexes, thus decreasing the donation of iron from ELT–iron complexes to cells. Both intracellular and extracellular donation of iron to a second chelator (L) potentially frees up ELT for a further round of iron chelation.

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