Transport of cisplatin and bis-acetato-ammine-dichlorocyclohexylamine Platinum(IV) (JM216) in human ovarian carcinoma cell lines: identification of a plasma membrane protein associated with cisplatin resistance.

The mechanisms by which cis-diamminedichloroplatinum(II) (cisplatin) is transported across the plasma membrane (i.e., passive diffusion versus active transport) were investigated in the 41M and CH1 human ovarian carcinoma cell lines and their acquired cisplatin-resistant variants 41McisR6 and CH1cisR6, respectively. Intracellular cisplatin accumulation was significantly reduced (4.0 +/- 1.7-fold) in the parental 41M line at 4 degrees C when compared to incubations at 37 degrees C. However, no significant differences in platinum uptake were observed in the 41McisR6 and in the CH1 pair of lines at 4 degrees C versus 37 degrees C. Similarly, in the presence of ouabain (an inhibitor of Na+,K+-ATPase), there was a marked reduction (2.0 +/- 0.4-fold) in drug accumulation in the sensitive 41M cells only, and no changes in drug uptake were observed in the other cell lines in the absence or presence of ouabain. Platinum accumulation was significantly enhanced in all cell lines in the presence of metabolic inhibitors (NaF and NaN3). These results suggest that in the parental 41M cell line, cisplatin transport may occur via passive diffusion and active/facilitated transport, whereas in the resistant 41McisR6 variant, cisplatin enters cells by passive diffusion only. The orally active drug bis-acetato-ammine-dichloro-cyclohexylamine platinum(IV) (JM216) is a lipophilic platinum(IV) complex that has been shown to circumvent cisplatin resistance in the 41McisR6 by increasing drug uptake. Across the entire range of concentrations used (5-50 microm), intracellular accumulation of JM216 was significantly reduced in 41M and 41McisR6 cells (3.5 +/- 0.7-fold; P < 0.01), and in CH1 and CH1cisR6 cells (14.2 +/- 6.0-fold; p < 0.01) at 4 degrees C when compared to incubations at 37 degrees C. No significant difference in JM216 uptake was observed in the 41M pair of lines in the absence or presence of ouabain. Additional studies have revealed that the fold reduction observed in cis-ammine(cyclohexylamine)dichloroplatinum(II) (JM118) accumulation in the 41M and 41McisR6 cells at 4 degrees C (3.7 +/- 1.9) reflects similar fold reductions to those observed with JM216 uptake at 4 degrees C. These results suggest that the mechanism of JM216 transport across cell membranes is through passive diffusion, predominantly as a result of its enhanced lipophilicity. Notably, an overexpression of a Mr 36,000 plasma membrane protein was observed in the 41McisR variants when compared to the sensitive 41M line. Increased levels of this Mr 36,000 protein may relate to the observed reduction in active transport of cisplatin in the 41McisR6 variant. Tyrosine phosphorylation of the Mr 36,000 protein appeared to be greater in the resistant 41McisR6 variant than in the parental 41M line. In addition, the constitutive levels of the Mr 36,000 protein in the CH1 pair of lines and in two acquired JM216-resistant variants (41M/JM216R and CH1/JM216R), where resistance in these cell lines is not mediated through reduced drug uptake, were similar to those observed in their respective parental lines. These results suggest that the overexpression of this Mr 36,000 protein in the acquired cisplatin-resistant subline 41McisR6 may play a significant role in cisplatin uptake in resistant cells exhibiting reduced drug accumulation as a major mechanism of cisplatin resistance.