Transport of Cisplatin and Bis-acetato-ammine-dichiorocyclohexyl- amine 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 (Le., passive diffusion versus active transport) were investigated in the 4!M and CH1 human ovarian carcinoma cell lines and their acquired cisplatin-resistant variants 4!McisR6 and CH!cisR6, respectively. Intracellular cisplatin accumuhation was significantly reduced (4.0 ± 1.7-fold) in the parental 41M line at 4#{176}C when compared to incubations at 37#{176}C. However, no significant differences in platinum uptake were observed in the 4lMcisR6 and in the CH! pair of lines at 4#{176}C versus 37#{176}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, cisphatin transport may occur via passive diffusion and active/facilitated transport, whereas in the resistant 4lMcisR6 variant, cisplatin enters cebls by passive diffusion only. The orally active drug bis-acetatoammine-dichloro-cychohexylamine platinum(IV) (JM2!6) is a lipophilic platinum(IV) complex that has been shown to circumvent cisplatin resistance in the 4!McisR6 by increasing drug uptake. Across the entire range of concentrations used (5-50 ELM), intracellular accumulation of JM216 was significantly reduced in 4!M and 4!McisR6 cells (3.5 ± 0.7-fold; P < 0.0!), and in CH1 and CHlcisR6 cells (14.2 ± 6.0-fold; p < 0.01) at 4#{176}C when compared to incubations at 37#{176}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)dichlorophatinum(II) (JM1!8) accumulation in the 4!M and 4!McisR6 cells at 4#{176}C (3.7 ± 1.9) reflects similar fold reductions to those observed with JM216 uptake at 4#{176}C. These results suggest that the mechanism of JM2!6 transport across cell membranes is through passive diffusion, predominanthy as a result of its enhanced lipophilicity. Notably, an overexpression of a Mr 36 000 plasma membrane protein was observed in the 4!McisR 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 4lMcisR6 variant. Tyrosine phosphorylation of the Mr 36 000 protein appeared to be greater in the resistant 4lMcisR6 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 JM2!6-resistant variants (41M/JM2!6R and CH1J JM2!6R), 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 cisplatm-resistant subline 4lMcisR6 may play a significant role in cisplatin uptake in resistant cells exhibiting reduced drug accumulation as a major mechanism of cisphatin resistance. INTRODUCTION Since the introduction of cisplatin3 into the clinic in 1971, studies on the development of platinum-based drugs have been aimed at modulating the toxic side effects of cisplatin (primarily nephrotoxicity) and at circumventing both intrinsic and acquired cisplatin resistance. The second generation drug carboplatin has significantly reduced the unfavorable toxicity profile of the parent drug (1). However, both drugs show a similar spectrum of antitumor activity and are iv. preparations (2). Hence, there is an urgent need to develop a third generation of platinumbased analogues that are superior to cisplatin/carboplatin and are capable of overcoming resistance. Cisplatin resistance is multifactorial, mainly involving reduced intracellular drug accumulation, enhanced cytoplasmic detoxification (via increased levels of GSH and metalbothioneins), and increased repair/tolerance of platinum-DNA adducts Received 3/20/95; accepted 4/4/95. I This work was supported by grants to the Institute of Cancer Research from the Cancer Research Campaign (United Kingdom), the Medical Research Council, the Johnson Matthey Technology Centre, and BristolMyers Squibb Oncology. 2 To whom requests for reprints should be addressed. 3 The abbreviations used are: GSH, glutathione; cisplatin, cis-diamminedichlorophatinum(II); JM216; bis-acetato-ammine-dichloro-cyclohexylamine platinum(IV); JM1 18, cis-ammine(cyclohexyhamine)dichloroplatinum(II). Research. on April 13, 2017. © 1995 American Association for Cancer clincancerres.aacrjournals.org Downloaded from