A series of 28 flavonoid derivatives containing a N-benzylpiperazine chain have been synthesized and tested for their ability to modulate multidrug resistance (MDR) in vitro. At 5 microM, most compounds potentiated doxorubicin cytotoxicity on resistant K562/DOX cells. They were also able to increase the intracellular accumulation of JC-1, a fluorescent molecule recently described as a probe of P-glycoprotein-mediated MDR. This suggests that these compounds act, at least in part, by inhibiting P-glycoprotein activity. As in other studies, lipophilicity was shown to influence MDR-modulating activity but was not the only determinant. Diverse di- and trimethoxy substitutions on N-benzyl were examined and found to affect the activity differently. The most active compounds had a 2,3, 4-trimethoxybenzylpiperazine chain attached to either a flavone or a flavanone moiety (13, 19, 33, and 37) and were found to be more potent than verapamil.

Folate compound transport into Fisher rat 3T3 (FR3T3) cells at physiological pH occurs predominantly by an acid pH-dependent, mobile carrier system. However, influx of [(3)H]MTX by this system is 3-4-fold higher at pH 6 than at pH 7.5, the optimum for RFC-1-mediated folate compound transport. This acid pH dependency reflects an alteration of influx V(max) rather than of influx K(m) in these cells at different pH. Acid pH-dependent folate compound transport interacts effectively with MTX, 5lLCHO-folateH(4), 5lLCH(3)-folateH(4) and folic acid as permeants (influx Ki = 2.7-5.3 microM). The relative inhibition of influx of [(3)H]MTX by the organic anions, probenecid, and PO(4) was different than for RFC-1 mediated influx. The folate requirements for growth in culture of FR3T3 cells and cytotoxicity of MTX compared to L1210 cells reflects the interactions of these folate compounds with acid pH-dependent folate transport. 5lLCHO-folateH(4) and PO(4) act as exchange anions for this system but their transpositioning has variable effects on transport. 5lLCHO-folateH(4) inhibits influx (decelerative equilibrium exchange) but stimulates efflux of [(3)H]MTX (accelerative equilibrium exchange) while PO(4) inhibits efflux. In FR3T3 cells transfected with cmyc and Hras, influx V(max) for [(3)H]MTX is downregulated 4-fold and 9-fold, respectively. At the same time, RFC-1 expression, which is detectable in FR3T3 cells at the level of its mRNA and RFC-1 mediated folate compound transport, is increased 3-5-fold in these transfectants. The increase in RFC-1 expression in FR3T3Hras cells appears to result from a higher rate of transcription of the gene in these cells as determined by a luciferase reporter gene assay of RFC-1 promoter activity. This downregulation of the acid pH dependent system and concomitant upregulation of the RFC-1 mediated system markedly altered pH dependency for influx of [(3)H]MTX in these transfectants compared to that seen in untransfected cells. We conclude that the major route for internalization at a physiological pH of folate compounds in FR3T3 cells is by an acid pH-dependent carrier-mediated system independent of RFC-1 expression and is downregulated by oncogene expression.

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