This laboratory has recently developed a tissue culture model for studying intestinal iron transport. The model, Caco-2 cells grown in bicameral chambers, allows us to evaluate the uptake of iron from the apical chamber representing the intestinal lumen into the apical surface of the cells, the transport of iron across the cell, and then the transfer of iron from the basolateral surface into the basal chamber representing the intestinal serosa. We have used the system to study the uptake and transport of Fe(ll), Fe(lIl), and heme iron as well as the uptake and transport of Zn and Al. Additionally we have investigated the effects of Al and Zn on the uptake and transport of Fe. In the course of these later studies on the interactions of metals with Fe, we began to examine the transport of Pt using the antitumor drug cis-diamminedichloroplatinum (II) (cis-Pt). Cis-Pt has had a dramatic impact on the response rates and long-term survival of patients with testicular and ovarian tumors. The limitations for the use of cis-Pt include poor activity against common tumors like colorectal cancers and the emergence of drug resistance. Our preliminary studies used the cytotoxicity of the Caco-2 cells to cis-Pt as a measure of cis-Pt uptake and examined the effect of preloading the cells with heme. These studies demonstrated that heme (iron protoporphyrin IX) but not Fe(III) had a dramatic effect of lowering the index of cytotoxicity of cis-Pt while Zn and Zn-protoporphyrin IX (ZnPP) enhanced the Caco-2 cells resistance to cis-Pt. Further Caco-2 cells stably transfected with the metallothionein (Mt) gene (CaMt cells) to over produce Mt were more resistant to cis-Pt than Caco-2 cells and were not effected by heme. These experiments suggest that the heme moiety per se is affecting the cytotoxicity of cis-Pt and that Mt overproduction as seen in the CaMt cells and with Zn induction in the Caco-2 cells has a protective effect on the cytotoxicity of cis-Pt. Clearly, interactions with other metals that would potentiate cis-Pt cytotoxicity would be of clinical benefit. The purpose of the present proposal is to further explore the interaction of cis-Pt with heme and Mt. Investigating: I. The Modulation of cis-Pt Cytotoxicity by Heme and Metallpothionein determining. Specificity of heme and Mt in modulating the cytotoxicity of cis-Pt. The role of heme and Mt on cis-Pt uptake and cellular distribution. The role of cis-Pt in modifying the cellular metabolism of heme with emphasis on cis-Pt effect on the catabolism of heme and on the compartmentalization of heme and heme-Fe. II. Mechanisms for increased cis-Pt toxicity measuring oxidative damage in heme treated cells.
