Ganna V. Kalayda

Platinum-based drugs: past, present and future

Platinum-based drugs cisplatin, carboplatin and oxaliplatin are widely used in the therapy of human neoplasms. Their clinical success is, however, limited due to severe side effects and intrinsic or acquired resistance to the treatment. Much effort has been put into the development of new platinum anticancer complexes, but none of them has reached worldwide clinical application so far. Nedaplatin, lobaplatin and heptaplatin received only regional approval. Some new platinum complexes and platinum drug formulations are undergoing clinical trials. Here, we review the main classes of new platinum drug candidates, such as sterically hindered complexes, monofunctional platinum drugs, complexes with biologically active ligands, trans-configured and polynuclear platinum complexes, platinum(IV) prodrugs and platinum-based drug delivery systems. For each class of compounds, a detailed overview of the mechanism of action is given, the cytotoxicity is compared to that of the clinically used platinum drugs, and the clinical perspectives are discussed. A critical analysis of lessons to be learned is presented. Finally, a general outlook regarding future directions in the field of new platinum drugs is given.

Altered localisation of the copper efflux transporters ATP7A and ATP7B associated with cisplatin resistance in human ovarian carcinoma cells

BackgroundCopper homeostasis proteins ATP7A and ATP7B are assumed to be involved in the intracellular transport of cisplatin. The aim of the present study was to assess the relevance of sub cellular localisation of these transporters for acquired cisplatin resistance in vitro. For this purpose, localisation of ATP7A and ATP7B in A2780 human ovarian carcinoma cells and their cisplatin-resistant variant, A2780cis, was investigated.MethodsSub cellular localisation of ATP7A and ATP7B in sensitive and resistant cells was investigated using confocal fluorescence microscopy after immunohistochemical staining. Co-localisation experiments with a cisplatin analogue modified with a carboxyfluorescein-diacetate residue were performed. Cytotoxicity of the fluorescent cisplatin analogue in A2780 and A2780cis cells was determined using an MTT-based assay. The significance of differences was analysed using Students t test or Mann-Whitney test as appropriate, p values of < 0.05 were considered significant.ResultsIn the sensitive cells, both transporters are mainly localised in the trans-Golgi network, whereas they are sequestrated in more peripherally located vesicles in the resistant cells. Altered localisation of ATP7A and ATP7B in A2780cis cells is likely to be a consequence of major abnormalities in intracellular protein trafficking related to a reduced lysosomal compartment in this cell line. Changes in sub cellular localisation of ATP7A and ATP7B may facilitate sequestration of cisplatin in the vesicular structures of A2780cis cells, which may prevent drug binding to genomic DNA and thereby contribute to cisplatin resistance.ConclusionOur results indicate that alterations in sub cellular localisation of transport proteins may contribute to cisplatin resistance in vitro. Investigation of intracellular protein localisation in primary tumour cell cultures and tumour tissues may help to develop markers of clinically relevant cisplatin resistance. Detection of resistant tumours in patients may in turn enable individualization of the chemotherapy in the early stage of treatment.

Cellular accumulation and cytotoxicity of macromolecular platinum complexes in cisplatin-resistant tumor cells

The development of resistance is one of the major limitations for the use of platinum (Pt) complexes in cancer chemotherapy. As reduced cellular uptake is a well-known resistance mechanism of cisplatin we explored the potential to overcome resistance in cisplatin-resistant A2780 ovarian carcinoma cells by means of macromolecular prodrugs exploiting endocytosis as alternative uptake mechanism. Two Pt-albumin (PL04-HSA, PL07-HSA) complexes and one Pt-polyethylene glycol complex (PEG(10k)-(Mal-Pt-DACH)(2)) were investigated. Intracellular platinum accumulation was quantified by FAAS. Cytotoxic activity was measured using the MTT assay. Endocytosis mechanisms were investigated by co-incubation experiments with bafilomycin A(1) and methyl-beta-cyclodextrin, inhibitors of the clathrin-mediated and caveolae-mediated endocytosis, respectively. Whereas the intracellular accumulation of the low molecular precursors PL04 and PL07 was reduced in the resistant cell variant, no difference between sensitive and resistant cells was observed for the three macromolecular complexes. In the presence of bafilomycin A(1) intracellular accumulation of all investigated macromolecular complexes was decreased whereas methyl-beta-cyclodextrin only affected the Pt-PEG complex. The Pt-PEG complex exhibited a higher cytotoxic activity than the albumin conjugates but also showed cross-resistance with cisplatin. In conclusion, cellular accumulation of macromolecular platinum complexes is not altered in cisplatin-resistant A2780 cells as these complexes enter the cells mainly via endocytotic pathways. Macromolecular platinum complexes specially designed to circumvent reduced cellular accumulation may be a promising approach to overcome cisplatin resistance.

Relevance of copper transporter 1 for cisplatin resistance in human ovarian carcinoma cells.

Defects in intracellular accumulation of the antitumour drug cisplatin are a commonly observed feature in the cells selected for cisplatin resistance. Copper transporter 1 (CTR1) has been suggested to play an important role in drug uptake and resistance. Here, we describe a detailed investigation of the involvement of CTR1 in cisplatin uptake and its relevance for cisplatin resistance using a well characterised sensitive/cisplatin-resistant cell line pair: A2780 human ovarian carcinoma cell line and its cisplatin-resistant variant A2780cis. A2780cis cells showed decreased cisplatin accumulation and lower CTR1 expression compared to A2780 cells. Co-incubation with copper sulphate affected neither cisplatin accumulation (determined by flameless atomic absorption spectrometry) nor its cytotoxicity (determined using an MTT-assay, MTT=3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide). In both cell lines, CTR1 was localised near the nucleus as found using confocal fluorescence microscopy. The steady-state localisation of the protein in perinuclear region appears to involve its continuous endocytosis from cell surface. In contrast to copper, cisplatin exposure had no influence on the sub cellular localisation of CTR1. Co-localisation between CTR1 and a fluorescent cisplatin analogue labelled with carboxyfluorescein-diacetate could be observed in vesicular structures when continuous retrieval of the protein from cell membrane was inhibited. Our results strongly suggest that CTR1 mediates cisplatin uptake in the cell lines studied. Upon its transport across the plasma membrane by CTR1 the platinum drug is likely to be internalised along with the protein. Our findings imply that reduced CTR1 expression accounts for decreased cisplatin accumulation and represents one of the determinants of cisplatin resistance in A2780cis cell line.

Enhancing lipophilicity as a strategy to overcome resistance against platinum complexes

Decreased influx represents one of the major resistance mechanisms of platinum complexes. In order to address the question if this mechanism of resistance can be overcome by enhancing the lipophilicity of platinum complexes, we investigated the influence of lipophilicity on cellular accumulation and cytotoxicity in a panel of oxaliplatin analogues with different carrier ligands. Cellular accumulation, DNA platination and cytotoxicity were measured in a cisplatin-sensitive and -resistant ovarian carcinoma (A2780/A2780cis) and in an oxaliplatin-sensitive and -resistant ileocecal colorectal adenocarcinoma (HCT-8/HCT-8ox) cell line pair. Platinum concentrations were determined by flameless atomic absorption spectrometry or adsorptive stripping voltammetry. Passive diffusion represented the main influx mechanism of oxaliplatin analogues during the first minutes of incubation as indicated by a correlation between lipophilicity and early influx rate. Afterwards, the predominant influx mechanism was lipophilicity-independent. More lipophilic complexes showed a reduced cytotoxic activity, although the early influx rate was increased. The resistance profiles of the two cell line pairs were found to be different: HCT-8ox cells were less resistant against more lipophilic complexes, whereas A2780cis cells exhibited a comparable degree of resistance against all investigated compounds. However, the reduction in resistance factor of HCT-8ox cells cannot be explained by increased influx suggesting that other resistance mechanisms are circumvented upon exposure to more lipophilic compounds. Though resistance against more lipophilic platinum complexes analogues is lower we conclude that enhancing lipophilicity is not a successful strategy to overcome platinum resistance as higher lipophilicity is also associated with lower cytotoxic activity.

Contribution of intracellular ATP to cisplatin resistance of tumor cells

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 α1 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.

Cellular accumulation and DNA platination of two new platinum(II) anticancer compounds based on anthracene derivatives as carrier ligands

The anticancer properties of two new fluorescent platinum(II) compounds, cis-[Pt(A9opy)Cl(2)] and cis-[Pt(A9pyp)(dmso)Cl(2)] are described. These compounds are highly active against several human tumor cell lines, including human ovarian carcinoma sensitive and cisplatin-resistant cell lines (A2780 and A2780R). To study the cellular processing of these new compounds, a series of in vitro studies have been performed, including the investigation of intracellular platinum accumulation and DNA-platination experiments in A2780 and A2780R cells. Compared to cisplatin, both compounds are accumulated highly in both sensitive and resistant cell lines, and more platinum has been found to bind to the nuclear DNA. Interestingly, cis-[Pt(A9opy)Cl(2)] shows high accumulation and DNA adduct formation in the resistant cell line A2780R, as compared to the sensitive counterpart A2780 cell line. This suggests that cis-[Pt(A9opy)Cl(2)] is able to overcome some of the well-known resistance mechanisms in this cell line, such as decreased cellular uptake and increased DNA repair.

Optimized sample preparation strategy for the analysis of low molecular mass adducts of a fluorescent cisplatin analogue in cancer cell lines by CE‐dual‐LIF

Pt‐based anticancer drugs, such as cisplatin, are known to undergo several (bio‐)chemical transformation steps after administration. Hydrolysis and adduct formation with small nucleophiles and larger proteins are their most relevant reactions on the way to the final reaction site (DNA), but there are still many open questions regarding the identity and pharmacological relevance of various proposed adducts and intermediates. Furthermore, the role of buffer components or additives, which are inevitably added to samples during any type of analytical measurement, has been frequently neglected in previous studies. Here, we report on adduct formation reactions of the fluorescent cisplatin analogue carboxyfluorescein diacetate platinum (CFDA‐Pt) in commonly used buffers and cell culture medium. Our results indicate that chelation reactions with noninnocent buffers (e.g., Tris) and components of the cell culture/cell lysis medium must be taken into account when interpreting results. Adduct formation kinetics was followed up to 60 h at nanomolar concentrations of CFDA‐Pt by using CE‐LIF. CE‐MS enabled the online identification of such unexpected adducts down to the nanomolar concentration range. By using an optimized sample preparation strategy, unwanted adducts can be avoided and several fluorescent adducts of CFDA‐Pt are detectable in sensitive and cisplatin‐resistant cancer cell lines. By processing samples rapidly after incubation, we could even identify the initial, but transient, Pt species in the cells as deacetylated CFDA‐Pt with unaltered complexing environment at Pt. Overall, the proposed procedure enables a very sensitive and accurate analysis of low molecular mass Pt species in cancer cells, involving a fast CE‐LIF detection within 5 min.

Synergistic interaction between cisplatin and gemcitabine in neuroblastoma cell lines and multicellular tumor spheroids

The efficacy and mechanism of action of cisplatin and gemcitabine were investigated in a panel of neuroblastoma cell lines and multicellular tumor spheroids. In neuroblastoma spheroids, the combination of cisplatin and gemcitabine induced a complete cytostasis at clinical relevant concentrations. A synergistic effect was observed when cells were coincubated with both drugs or preincubated with gemcitabine first. These administration sequences resulted in NASS cells in decreased ERCC1 and XPA expression, two key proteins of the NER DNA repair system, and increased platinum adduct formation in DNA. Most of these phenomena were not observed in SJNB8 cells which might explain the lack of synergy between cisplatin and gemcitabine in SJNB8 cells. Our results showed favorable interactions between cisplatin and gemcitabine in 4 out of 5 cell lines. Therefore, we feel that inclusion of gemcitabine into cisplatin-containing regiments might be a promising new strategy for the treatment of neuroblastoma.

Relevance of the leaving group for antitumor activity of new platinum(II) compounds containing anthracene derivatives as a carrier ligand

A new anticancer-active platinum(II) compound [Pt(A9pyp)(dmso)(cbdca)], containing the E-1-(9-anthryl)-3-(2-pyridyl)-2-propenone ligand (abbreviated as A9pyp) has been synthesized by the replacement of the anionic chloride ligands in cis-[Pt(A9pyp)(dmso)Cl(2)] by the dianionic chelating cyclobutanedicarboxylate ligand (abbreviated as cbdca). The in vitro relevance of the leaving group of these new platinum(II) compounds has been investigated. Measurements of the time-dependent intracellular accumulation of both compounds in human ovarian carcinoma cell lines show that the leaving group affects their cellular uptake. In addition, the leaving group also influences DNA platination, and, therefore, has an effect on the biological activity against a pair of human ovarian carcinoma cell lines, i.e. sensitive and resistant to cisplatin.