Pleiotropic resistance to diverse antimalarials in actinomycin D-resistant Plasmodium falciparum

The development and spread of multidrug-resistant Plasmodium falciparum are major health concerns. The molecular mechanisms of multidrug resistance, including resistance to many quinoline-based antimalarials, are largely unknown. In this study, we report on the isolation and partial characterization of actinomycin D (actD)-resistant P. falciparum (3D7(R)/actD2.3) from a chloroquine-susceptible strain, 3D7. The stepwise selection of an actD-resistant clone (3D7(R)/actD2.3) led to the isolation and cloning of P. falciparum that grew in the presence of 2 ng/mL of actD. The parental isolate (3D7) did not grow in the presence of a 10-fold lower drug concentration (0.2 ng/mL). The latter estimate of parasite growth was determined by direct counting of parasites in infected red blood cells. Estimates of drug resistance levels to actD, using a [(3)H]hypoxanthine uptake and incorporation method, showed a 3-fold difference in the IC(50) between 3D7 and 3D7(R)/actD2.3. Interestingly, 3D7(R)/actD2.3 P. falciparum parasites were less sensitive to several antimalarials (chloroquine, mefloquine, quinidine, and artemisinin) and to the mitochondrial specific dye Rhodamine 123. Drug transport studies using [(3)H]actD showed that 3D7(R)/actD2.3 accumulated less drug than 3D7. Moreover, the accumulation of [(3)H]actD was energy dependent. To determine if Pfmdr1 expression, previously implicated in drug resistance to certain antimalarials, mediated the resistance phenotype of 3D7(R)/actD2.3, Pfmdr1 levels in 3D7 and 3D7(R)/actD2.3 were compared by Southern and northern blot analyses. Our results revealed no differences in Pfmdr1 copy number or mRNA levels between 3D7 and 3D7(R)/actD2.3. Furthermore, comparison of Pfmdr1 sequences between 3D7 and 3D7(R)/actD2.3 showed no differences. In addition, verapamil, which reverses P-glycoprotein-mediated drug resistance in mammalian cells, did not reverse the resistance of 3D7(R)/actD2.3 to actD or chloroquine. Taken together, the findings of this study demonstrated that in vitro selection of P. falciparum for resistance to actD leads to decreased sensitivity to diverse drugs and that this pleiotropic drug resistance is associated with reduced drug accumulation not mediated by Pfmdr1.