Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
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Neoplastic cells require Fe to sustain their rapid growth. A number of growth factors including Fe are essential for the proliferation of these cells and as a result restricting their supply may have potential in cancer therapy. The aim of this study was to compare examples of different classes of molecules which have a high affinity for Fe for their potential antineoplastic activity in rat hepatoma cell lines. Two other criteria which were taken into account were their ability to bind preferentially to either ferric or ferrous ions and their membrane permeability

From the results observed in this investigation the most effective antineoplastic chelator was the polyanionic amine DTPA.

Blatt et al.,(1988) in a study on neuroblastoma cells also found DTPA to be an extremely effective chelator producing a dose dependent inhibition of cell growth (IC50 60µM). Similar observations were made on CCL-19 cells (Alcain et al.,1994). DTPA and EDTA are both membrane impermeable chelators, with their sites of action limited to the extra-cellular fluid, plasma membrane and within endosomes. Both of these chelators reduced Fe uptake. Neither can remove Fe from Tf in the plasma, hence the site of action maybe Fe released at the cell surface or within the endocytotic vesicle. The fact that neither of the chelators decreases Fe uptake over short periods would suggest the slow removal of iron from within the endosomal vesicle.

In contrast, the permeable Fe chelators PIH and L1 were immediately effective in reducing Fe uptake by cells. However Fe binding alone does not necessarily inhibit proliferation. EDTA, with a similar structure to DTPA and causing a similar decrease in Fe uptake, had little effect on cell proliferation. Possible reasons for the high activity of DTPA compared to EDTA include binding to other essential cations such as Zn²⁺and Ca²⁺. However EDTA also binds these ions, albeit with a lower affinity. Both DTPA and EDTA bind Fe ²⁺ as well as Fe³⁺. At pH 7.4, DTPA and EDTA form different ionic species. It may be possible that the form of DTPA at physiological pH is a more effective chelator of an Fe pool involved in cell division

Iron on the cell surface has been reported to be involved in transplasma membrane electron transport, specifically a component of transplasma membrane oxidoreductase (Crane et al.,1985, Goldenberg et al.,1990, Alcaine et al.,(1994 a,b, 1997) It is possible that the ferric form of iron is important at the cell membrane as the ferrous impermeable chelator BPS had little effect. However, Alcaine et al., (1994, a,b) found that BPS was an important inhibitor of proliferation only in the absence of FCS.

In contrast, the ferrous chelator DP which can penetrate the plasma membrane and hence has access to more cellular Fe pools, inhibited cell proliferation and Fe uptake to a similar degree. DP is a hydrophobic chelator which partitions into cell membranes and binds iron as it passes through (Nunez et al.,1983. DP is an effective chelator in reticulocytes (Morgan, 1971, 1976, Nunez et al.,1983) and erythroleukemic cells (Bridges and Cudkowicz, 1984) and partially inhibits receptor mediated endocytosis and the recycling of Tf (Goldenberg et al.,1990).

The three membrane permeable chelators PIH, L1 (and DFO) all of which preferentially bind ferric ions markedly inhibited cell proliferation and the uptake of Fe. The lipophilic chelator L1 under some conditions, may remove Fe directly from Tf (Kontoghioghes and Evans, 1985) and chelates Fe from intracellular Fe pools in melanoma cells (Richardson and Baker, 1994). The ability of L1 to remove Fe from ferritin has also reported (Mostert et al.,1987). PIH like L1 is also lipophilic and capable of preventing ⁵⁹ Fe uptake from Tf in hepatocytes (Baker et al.,1985; 1992) indicating that its site of chelation is close to where Fe is released from Tf. A number of PIH analogues have been synthesised and some appear highly potent at inhibiting tumor cell proliferation at much lower IC50 concentrations than PIH itself (Richardson et al.,1995). These analogues were also found to be considerably more effective than DFO at preventing ⁵⁹Fe uptake from ⁵⁹Fe-Tf and in mobilizing Fe from prelabelled cells (Richardson and Milnes, 1997). DFO on the other hand is more hydrophilic and thus does not permeate through membranes as easily as L1 and PIH. Over shorter periods of time (2h) DFO has little effect on ⁵⁹Fe uptake or ⁵⁹Fe mobilization from melanoma cells (Richardson and Baker, 1994), but over longer periods of time (24h) is effective at preventing ⁵⁹Fe uptake from Tf and at chelating intracellular Fe.

With the exception of DP, all chelators showed a similar pattern of inhibition of Fe uptake in hepatocytes and hepatoma cells indicating a lack of selectivity.

However it cannot be assumed that the same mechanisms are involved. It is well established that besides the receptor mediated process of Fe uptake, several other non receptor mediated mechanisms are involved in Fe uptake by hepatocytes (eg Trinder et al., 1986) and some neoplastic cells (eg Richardson and Baker, 1992; 1994, Richardson and Ponka, 1997). DP appears the only chelator to have a far greater effect on hepatoma cells than hepatocytes. The mechanisms behind this apparent specificity are unknown at present.

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