Contribution of intracellular ATP to cisplatin resistance of tumor cells

Abstract

Decreased cellular accumulation of cisplatin is a frequently observed mechanism of resistance to the drug. Beside passive diffusion, several cellular proteins using ATP hydrolysis as an energy source are assumed to be involved in cisplatin transport in and out of the cell. This investigation aimed at clarifying the contribution of intracellular ATP as an indicator of energy-dependent transport to cisplatin resistance using the A2780 human ovarian adenocarcinoma cell line and its cisplatin-resistant variant A2780cis. Depletion of intracellular ATP with oligomycin significantly decreased cellular platinum accumulation (measured by flameless atomic absorption spectrometry) in sensitive but not in resistant cells, and did not affect cisplatin efflux in both cell lines. Inhibition of Na(+),K(+)-ATPase with ouabain reduced platinum accumulation in A2780 cells but to a lesser extent compared with oligomycin. Western blot analysis revealed lower expression of Na(+),K(+)-ATPase α(1) subunit in resistant cells compared with sensitive counterparts. The basal intracellular ATP level (determined using a bioluminescence-based assay) was significantly higher in A2780cis cells than in A2780 cells. Our results highlight the importance of ATP-dependent transport, among other processes mediated by Na(+),K(+)-ATPase, for cisplatin influx in sensitive cells. Cellular platinum accumulation in resistant cells is reduced and less dependent on energy sources, which may partly result from Na(+),K(+)-ATPase downregulation. Our data suggest the involvement of other ATP-dependent processes beside those regulated by Na(+),K(+)-ATPase. Higher basal ATP level in cisplatin-resistant cells, which appears to be a consequence of enhanced mitochondrial ATP production, may represent a survival mechanism established during development of resistance.

Cisplatin resistance in cell models: evaluation of metallomic and biological predictive biomarkers to address early therapy failure

“…37,43,48 Furthermore, as expected, the A2780cis cells did not show cisplatininduced apoptosis. 17,46 According to all these results, cisplatin-induced adduct levels depended on the intracellular Pt content within each cell line, but not necessarily when all of the cell lines were considered together.…”

“…Intracellular Pt content, cisplatin G-G intra-strand crosslinks, and DNA strand breaks were determined immediately after treatment (AT-0 time) and also one hour later (AT-1 time). In this way, it was possible to estimate not only the Pt influx and efflux 37 but also adduct dynamics (formation versus repair/removal). 38 As described for other human cells, 35 only G-G intra-strand crosslinking adducts were detected in the analyzed cells.…”

“…10,22 Despite similarities in cell viability with the GM04312 cells, the sensitivity of the A2780 cells to cisplatin cannot be linked to a deficiency of DNA repair. However, it might be related to their higher Pt influx, as described, 27,37,42,43 even for other platinum chemicals, 44 which could be related to high levels of the copper transport protein CTR1, 23 although recently the influence of this type of protein was considered controversial. 45 Nevertheless, since high Pt influx was not accompanied by high levels of induced DNA adducts, the drug effects on cell viability in these A2780 cells must be related to cisplatin activity in the cell cytoplasm, and not specifically related to apoptosis, because the apoptotic cell number did not significantly change at different Pt concentrations.…”

“…Furthermore, differences in intracellular cisplatin at AT-0 and AT-1 times revealed a considerable cisplatin efflux after one hour for all the cell lines except A2780cis, which was much higher and faster than those reported before. 14,23,37,48 In the analysis of genomic stability, the Comet assay is the most widely used tool. 34 However, detection of DNA strand breaks after cisplatin treatment is not a straightforward result; some authors reported them in different cells and organisms, 20,26 and others reported their absence.…”

Involvement of Src signaling in the synergistic effect between cisplatin and digoxin on cancer cell viability

“…Currently, a series of articles have described the relationship between cisplatin and Na,K‐ATPase, and this enzyme can be involved in the sensitivity of cells to cisplatin . Studies by Neault et al showed that at low concentrations, cisplatin is capable of binding to the lipid moiety of the enzyme, while high levels of cisplatin bind to the peptide groups of the enzyme through the CO and CN groups with a binding constant of 0.193 mM …”