Cisplatin and zoledronic acid: two drugs combined in a Pt(II) complex with potential antitumor activity towards bone tumors and metastases.

Treatment of primary bone malignancies comprises surgery, radiotherapy, chemotherapy, and analgesics. Platinum-based chemotherapeutics, such as cisplatin, are commonly used for the treatment of bone cancer but, despite their success, outcomes are limited by toxicity and resistance. Recently, dinuclear Pt complexes with a bridging geminal bisphosphonate ligand proved to be endowed with selective accumulation in bone tumors or metastases leading to improved efficacy and reduced systemic toxicity. Further improvement could be expected by the use of a bisphosphonate ligand with intrinsic pharmacological activity such as zoledronic acid (ZL). In the present work is reported the synthesis and full characterization of the dinuclear Pt(II) complex [{cis-Pt(NH3)2}2(ZL)]HSO4 which combines two drugs with antitumor activity, cisplatin and zoledronic acid. Both drugs, individually, are already approved by the U.S. Food and Drug Administration and the European Medicinal Agency for clinical use. The in vitro cytotoxicity of the new Pt(II)-ZL complex has been tested against a panel of human tumor cell lines.

Crystal Structure of the Human Copper Chaperone ATOX1 Bound to Zinc Ion.

The bioavailability of copper (Cu) in human cells may depend on a complex interplay with zinc (Zn) ions. We investigated the ability of the Zn ion to target the human Cu-chaperone Atox1, a small cytosolic protein capable of anchoring Cu(I), by a conserved surface-exposed Cys-X-X-Cys (CXXC) motif, and deliver it to Cu-transporting ATPases in the trans-Golgi network. The crystal structure of Atox1 loaded with Zn displays the metal ion bridging the CXXC motifs of two Atox1 molecules in a homodimer. The identity and location of the Zn ion were confirmed through the anomalous scattering of the metal by collecting X-ray diffraction data near the Zn K-edge. Furthermore, soaking experiments of the Zn-loaded Atox1 crystals with a strong chelating agent, such as EDTA, caused only limited removal of the metal ion from the tetrahedral coordination cage, suggesting a potential role of Atox1 in Zn metabolism and, more generally, that Cu and Zn transport mechanisms could be interlocked in human cells.

Improvement of Kiteplatin Efficacy by a Benzoato Pt(IV) Prodrug Suitable for Oral Administration.

Kiteplatin, [PtCl2(cis-1,4-DACH)] (DACH = diaminocyclohexane), contains an isomeric form of the oxaliplatin diamine ligand trans-1R,2R-DACH and has been proposed as a valuable drug candidate against cisplatin- and oxaliplatin-resistant tumors, in particular, colorectal cancer. To further improve the activity of kiteplatin, it has been transformed into a Pt(IV) prodrug by the addition of two benzoato groups in the axial positions. The new compound, cis,trans,cis-[PtCl2(OBz)2(cis-1,4-DACH)] (1; OBz = benzoate), showed cytotoxic activity at nanomolar concentration against a wide panel of human cancer cell lines. Based on these very promising results, the investigation has been extended to the in vivo activity of compound 1 in a Lewis Lung Carcinoma (LLC) model and its suitability for oral administration. Compound 1 resulted to be remarkably stable in acidic conditions (pH 1.5 to mimic the stomach environment) undergoing a drop of the initial concentration to ~60% of the initial one only after 72 h incubation at 37 °C; thus resulting amenable for oral administration. Interestingly, in a murine model (2·106 LLC cells implanted i.m. into the right hind leg of 8-week old male and female C57BL mice), a comparable reduction of tumor mass (~75%) was observed by administering compound 1 by oral gavage and the standard drug cisplatin by intraperitoneal injection, thus indicating that, indeed, there is the possibility of oral administration for this dibenzoato prodrug of kiteplatin. Moreover, since the mechanism of action of Pt(IV) prodrugs involves an initial activation by chemical reduction to cytotoxic Pt(II) species, the reduction of 1 by two bioreductants (ascorbic acid/sodium ascorbate and glutathione) was investigated resulting to be rather slow (not complete after 120 h incubation at 37 °C). Finally, the neurotoxicity of 1 was evaluated using an in vitro assay.

Multinuclear Metal-Binding Ability of the N-Terminal Region of Human Copper Transporter Ctr1: Dependence Upon pH and Metal Oxidation State.

The 14mer peptide corresponding to the N-terminal region of human copper transporter Ctr1 was used to investigate the intricate mechanism of metal binding to this plasma membrane permease responsible for copper import in eukaryotic cells. The peptide contains a high-affinity ATCUN Cu(II)/Ni(II)-selective motif, a methionine-only MxMxxM Cu(I)/Ag(I)-selective motif and a double histidine HH(M) motif, which can bind both Cu(II) and Cu(I)/Ag(I) ions. Using a combination of NMR spectroscopy and electrospray mass spectrometry, clear evidence was gained that the Ctr1 peptide, at neutral pH, can bind one or two metal ions in the same or different oxidation states. Addition of ascorbate to a neutral solution containing Ctr11-14 and Cu(II) in 1:1 ratio does not cause an appreciable reduction of Cu(II) to Cu(I), which is indicative of a tight binding of Cu(II) to the ATCUN motif. However, by lowering the pH to 3.5, the Cu(II) ion detaches from the peptide and becomes susceptible to reduction to Cu(I) by ascorbate. It is noteworthy that at low pH, unlike Cu(II), Cu(I) stably binds to methionines of the peptide. This redox reaction could take place in the lumen of acidic organelles after Ctr1 internalization. Unlike Ctr11-14-Cu(II), bimetallic Ctr11-14-2Cu(II) is susceptible to partial reduction by ascorbate at neutral pH, which is indicative of a lower binding affinity of the second Cu(II) ion. The reduced copper remains bound to the peptide, most likely to the HH(M) motif. By lowering the pH to 3.5, Cu(I) shifts from HH(M) to methionine-only coordination, an indication that only the pH-insensitive methionine motif is competent for metal binding at low pH. The easy interconversion of monovalent cations between different coordination modes was supported by DFT calculations.

19 F NMR Allows the Investigation of the Fate of Platinum(IV) Prodrugs in Physiological Conditions.

PtIV prodrugs can overcome resistance and side effects of conventional PtII anticancer therapies. By 19 F-labeling of a PtIV prodrug (Pt-FBA, FBA=p-fluorobenzoate), the activation under physiological conditions could be investigated. Unlike single-electron reductants, multi-electron agents can efficiently promote the two electrons reduction of PtIV to PtII . The activation of Pt-FBA in cell lysate is highly dependent upon the type of cancer cells. When administered to E. coli, Pt-FBA is reduced intracellularly and free FBA can shuttle out of the cell. The reduction rate greatly increases by inducing metallothionein overexpression and is lowered by addition of ZnII ions. When injected into mice, Pt-FBA undergoes fast reduction in the bloodstream accompanied by metabolic degradation of FBA; nevertheless, unreduced Pt-FBA can accumulate to detectable levels in liver and kidneys. The 19 F NMR approach has the advantage of avoiding the interference of all background signals.

Effect of chirality on the anticancer activity of Pt(II) and Pt(IV) complexes containing 1R,2R and 1S,2S enantiomers of the trans-1,2-diamino-4-cyclohexene ligand (DACHEX), an analogue of diaminocyclohexane used in oxaliplatin.

Six enantiomerically pure, oxaliplatin-like, platinum compounds (two platinum(II) and four platinum(IV)), all containing unsaturated cyclic diamine trans-1,2-diamino-4-cyclohexene (DACHEX) as a substitute for the trans-1,2-diaminocyclohexane used in oxaliplatin, were investigated. The complexes were characterized by elemental analyses, ESI-MS, and 1H-NMR spectroscopy. For the four Pt(IV) complexes the electrochemical redox behaviour, investigated by cyclic voltammetry, showed that all complexes possess reduction potentials suitable for activation in vivo. The antiproliferative activity was assessed in vitro on human cancer cell lines, also selected for resistance to platinum-based drugs or belonging to the MultiDrug-Resistant (MDR) phenotype. All complexes exhibited antiproliferative activity superior to that of cisplatin and almost equivalent to or better than that of oxaliplatin; moreover, most complexes were also capable of overcoming both the cisplatin- and the oxaliplatin-resistance. By comparing the effectiveness of the enantiomerically pure compounds with the racemic one, the R,R enantiomer emerged as the most effective in the case of Pt(II) complexes whereas the S,S enantiomer was the most effective in the case of the Pt(IV) derivatives. From the results obtained also against 3D spheroid tumor models, cis,trans,cis-[Pt(OXA)(OBz)2(1S,2S-DACHEX)] (OBz = benzoate) emerged as the most promising candidate for further preclinical investigation.

New Oxaliplatin-Pyrophosphato Analogs with Improved In Vitro Cytotoxicity.

Two new Pt(II)-pyrophosphato complexes containing the carrier ligands cis-1,3-diaminocyclohexane (cis-1,3-DACH) and trans-1,2-diamine-4-cyclohexene (1,2-DACHEX), variants of the 1R,2R-diaminocyclohexane ligand present in the clinically used Pt-drug oxaliplatin, have been synthesized with the aim of developing new potential antitumor drugs with high bone tropism. The complexes are more stable at physiological pH than in acid conditions, with Na2[Pt(pyrophosphato)(cis-1,3-DACH)] (1) slightly more stable than [Pt(dihydrogenpyrophosphato)(1,2-DACHEX)] (2). The greater reactivity at acidic pH ensures a greater efficacy at the tumor site. Preliminary NMR studies indicate that 1 and 2 react slowly with 5'-GMP (used as a model of nucleic acids), releasing the pyrophosphate ligand and affording the bis 5'-GMP adduct. In vitro cytotoxicity assays performed against a panel of four human cancer cell lines have shown that both compounds are more active than oxaliplatin. Flow cytometry studies on HCT116 cells showed that the pyrophosphato compounds with the non-classical 1,3- and 1,4-diaminocyclohexane ligands (1 and 4) are the most capable to induce cells' death by apoptosis and necrosis.

Interference between copper transport systems and platinum drugs.

Cisplatin, or cis-diamminedichloridoplatinum(II) cis-[PtCl2(NH3)2], is a platinum-based anticancer drug largely used for the treatment of various types of cancers, including testicular, ovarian and colorectal carcinomas, sarcomas, and lymphomas. Together with other platinum-based drugs, cisplatin triggers malignant cell death by binding to nuclear DNA, which appears to be the ultimate target. In addition to passive diffusion across the cell membrane, other transport systems, including endocytosis and some active or facilitated transport mechanisms, are currently proposed to play a pivotal role in the uptake of platinum-based drugs. In this review, an updated view of the current literature regarding the intracellular transport and processing of cisplatin will be presented, with special emphasis on the plasma membrane copper permease CTR1, the Cu-transporting ATPases, ATP7A and ATP7B, located in the trans-Golgi network, and the soluble copper chaperone ATOX1. Their role in eliciting cisplatin efficacy and their exploitation as pharmacological targets will be addressed.

Platinum(IV) Complexes of trans-1,2-diamino-4-cyclohexene: Prodrugs Affording an Oxaliplatin Analogue that Overcomes Cancer Resistance.

Six platinum(IV) compounds derived from an oxaliplatin analogue containing the unsaturated cyclic diamine trans-1,2-diamino-4-cyclohexene (DACHEX), in place of the 1,2-diaminocyclohexane, and a range of axial ligands, were synthesized and characterized. The derivatives with at least one axial chlorido ligand demonstrated solvent-assisted photoreduction. The electrochemical redox behavior was investigated by cyclic voltammetry; all compounds showed reduction potentials suitable for activation in vivo. X-ray photoelectron spectroscopy (XPS) data indicated an X-ray-induced surface reduction of the Pt(IV) substrates, which correlates with the reduction potentials measured by cyclic voltammetry. The cytotoxic activity was assessed in vitro on a panel of human cancer cell lines, also including oxaliplatin-resistant cancer cells, and compared with that of the reference compounds cisplatin and oxaliplatin; all IC50 values were remarkably lower than those elicited by cisplatin and somewhat lower than those of oxaliplatin. Compared to the other Pt(IV) compounds of the series, the bis-benzoate derivative was by far (5-8 times) the most cytotoxic showing that low reduction potential and high lipophilicity are essential for good cytotoxicity. Interestingly, all the complexes proved to be more active than cisplatin and oxaliplatin even in three-dimensional spheroids of A431 human cervical cancer cells.

Oxidation of Human Copper Chaperone Atox1 and Disulfide Bond Cleavage by Cisplatin and Glutathione.

Cancer cells cope with high oxidative stress levels, characterized by a shift toward the oxidized form (GSSG) of glutathione (GSH) in the redox couple GSSG/2GSH. Under these conditions, the cytosolic copper chaperone Atox1, which delivers Cu(I) to the secretory pathway, gets oxidized, i.e., a disulfide bond is formed between the cysteine residues of the Cu(I)-binding CxxC motif. Switching to the covalently-linked form, sulfur atoms are not able to bind the Cu(I) ion and Atox1 cannot play an antioxidant role. Atox1 has also been implicated in the resistance to platinum chemotherapy. In the presence of excess GSH, the anticancer drug cisplatin binds to Cu(I)-Atox1 but not to the reduced apoprotein. With the aim to investigate the interaction of cisplatin with the disulfide form of the protein, we performed a structural characterization in solution and in the solid state of oxidized human Atox1 and explored its ability to bind cisplatin under conditions mimicking an oxidizing environment. Cisplatin targets a methionine residue of oxidized Atox1; however, in the presence of GSH as reducing agent, the drug binds irreversibly to the protein with ammine ligands trans to Cys12 and Cys15. The results are discussed with reference to the available literature data and a mechanism is proposed connecting platinum drug processing to redox and copper homeostasis.

Mechanistic and Structural Basis for Inhibition of Copper Trafficking by Platinum Anticancer Drugs.

Copper (Cu) is required for maturation of cuproenzymes, cell proliferation, and angiogenesis, and its transport entails highly specific protein-protein interactions. In humans, the Cu chaperone Atox1 mediates Cu(I) delivery to P-type ATPases Atp7a and Atp7b (the Menkes and Wilson disease proteins, respectively), which are responsible for Cu release to the secretory pathway and excess Cu efflux. Cu(I) handover is believed to occur through the formation of three-coordinate intermediates where the metal ion is simultaneously linked to Atox1 and to a soluble domain of Cu-ATPases, both sharing a CxxC dithiol motif. The ultrahigh thermodynamic stability of chelating S-donor ligands secures the redox-active and potentially toxic Cu(I) ion, while their kinetic lability allows facile metal transfer. The same CxxC motifs can interact with and mediate the biological response to antitumor platinum drugs, which are among the most used chemotherapeutics. We show that cisplatin and an oxaliplatin analogue can specifically bind to the heterodimeric complex Atox1-Cu(I)-Mnk1 (Mnk1 is the first soluble domain of Atp7a), thus leading to a kinetically stable adduct that has been structurally characterized by solution NMR and X-ray crystallography. Of the two possible binding configurations of the Cu(I) ion in the cage made by the CxxC motifs of the two proteins, one (bidentate Atox1 and monodentate Mnk1) is less stable and more reactive toward cis-Pt(II) compounds, as shown by using mutated proteins. A Cu(I) ion can be retained at the Pt(II) coordination site but can be released to glutathione (a physiological thiol) or to other complexing agents. The Pt(II)-supported heterodimeric complex does not form if Zn(II) is used in place of Cu(I) and transplatin instead of cisplatin. The results indicate that Pt(II) drugs can specifically affect Cu(I) homeostasis by interfering with the rapid exchange of Cu(I) between Atox1 and Cu-ATPases with consequences on cancer cell viability and migration.

Reaction of Histone H1 with trans-Platinum Complexes and the Effect on DNA Platination.

Transplatin is an inactive platinum drug; however, a number of analogues, such as trans-EE and trans-PtTz, demonstrate promising antitumor activity in vitro and in vivo. Although the ultimate target is nuclear DNA, increasing evidence indicate that proteins also play important roles in the display of antitumor activity. The linker histone H1 is situated by the portal between the unwrapped DNA and the nucleosome core. Our recent study revealed that H1 can readily react with cisplatin, and the adducts tend to form ternary complexes with DNA. In this work, we have investigated the reaction of histone H1 with two antitumor-active trans-oriented complexes, trans-EE and trans-PtTz, and the effect of H1 upon the platination of DNA. The results show that trans-platinum drugs are much more reactive than cisplatin toward H1. Interestingly, in addition to the expected bidentate adducts (by displacement of the two labile chlorido ligands), also a tridentate adduct can be formed by displacement of one nonlabile carrier ligand of trans-EE or trans-PtTz. The trans-Pt/H1 adducts can then react with DNA and generate protein-Pt-DNA ternary complexes. Additionally, platinum can be transferred from trans-Pt/H1 adducts to DNA, generating binary trans-Pt/DNA complexes. Such a transfer of the platinum agent to DNA was not observed in the reaction of cisplatin. Furthermore, the detailed investigation carried out on a model peptide indicates that H1 promotes the DNA platination by trans- EE, while it reduces that of trans-PtTz and cisplatin. These results suggest that H1 can play a key role in the DNA platination and modulate the efficacy of different platinum agents.

Cisplatin reacts with histone H1 and the adduct forms a ternary complex with DNA.

Cisplatin is an anticancer drug widely used in clinics; it induces the apoptosis of cancer cells by targeting DNA. However, its interaction with proteins has been found to be crucial in modulating the pre and post-target activity. Nuclear DNA is tightly assembled with histone proteins to form nucleosomes in chromatin; this can impede the drug to access DNA. On the other hand, the linker histone H1 is considered 'the gate to nucleosomal DNA' due to its exposed location and dynamic conformation; therefore, this protein can influence the platination of DNA. In this study, we performed a reaction of cisplatin with histone H1 and investigated the interaction of the H1/cisplatin adduct with DNA. The reactions were conducted on the N-terminal domains of H1.4 (sequence 1-90, H1N90) and H1.0 (sequence 1-7, H1N7). The results show that H1 readily reacts with cisplatin and generates bidentate and tridentate adducts, with methionine and glutamate residues as the preferential binding sites. Chromatographic and NMR analyses show that the platination rate of H1 is slightly higher than that of DNA and the platinated H1 can form H1-cisplatin-DNA ternary complexes. Interestingly, cisplatin is more prone to form H1-Pt-DNA ternary complexes than trans-oriented platinum agents. The formation of H1-cisplatin-DNA ternary complexes and their preference for cis- over trans-oriented platinum agents suggest an important role of histone H1 in the mechanism of action of cisplatin.

A minimal structural variation can overcome tumour resistance of oxaliplatin: the case of 4,5-dehydrogenation of the cyclohexane ring.

A new family of anticancer compounds has been derived from oxaliplatin by inserting a double-bond between carbons 4 and 5 of the 1,2-diaminocyclohexane ring. Testing against a panel of human tumour cell lines including cervical (A431), ovarian (2008), and colon carcinomas (HCT-15 and LoVo), and two oxaliplatin-resistant clones (LoVo OXP and LoVo MDR) has shown that the new compounds have, in general, equal if not better cytotoxic activity and are able to overcome the oxaliplatin-resistance. Moreover, the oxalato derivative induced lipid droplets increase in LoVo OXP cells thus suggesting the involvement of metabolism stress in its mechanism of action.

Cytotoxic phenanthroline derivatives alter metallostasis and redox homeostasis in neuroblastoma cells.

Copper homeostasis is generally investigated focusing on a single component of the metallostasis network. Here we address several of the factors controlling the metallostasis for neuroblastoma cells (SH-SY5Y) upon treatment with 2,9-dimethyl-1,10-phenanthroline-5,6-dione (phendione) and 2,9-dimethyl-1,10-phenanthroline (cuproindione). These compounds bind and transport copper inside cells, exert their cytotoxic activity through the induction of oxidative stress, causing apoptosis and alteration of the cellular redox and copper homeostasis network. The intracellular pathway ensured by copper transporters (Ctr1, ATP7A), chaperones (CCS, ATOX, COX 17, Sco1, Sco2), small molecules (GSH) and transcription factors (p53) is scrutinised.

Effect of in vivo post-translational modifications of the HMGB1 protein upon binding to platinated DNA: a molecular simulation study.

Cisplatin is one of the most widely used anticancer drugs. Its efficiency is unfortunately severely hampered by resistance. The High Mobility Group Box (HMGB) proteins may sensitize tumor cells to cisplatin by specifically binding to platinated DNA (PtDNA) lesions. In vivo, the HMGB/PtDNA binding is regulated by multisite post-translational modifications (PTMs). The impact of PTMs on the HMGB/PtDNA complex at atomistic level is here investigated by enhanced sampling molecular simulations. The PTMs turn out to affect the structure of the complex, the mobility of several regions (including the platinated site), and the nature of the protein/PtDNA non-covalent interactions. Overall, the multisite PTMs increase significantly the apparent synchrony of all the contacts between the protein and PtDNA. Consequently, the hydrophobic anchoring of the side chain of F37 between the two cross-linked guanines at the platinated site-a key element of the complexes formation - is more stable than in the complex without PTM. These differences can account for the experimentally measured greater affinity for PtDNA of the protein isoforms with PTMs. The collective behavior of multisite PTMs, as revealed here by the synchrony of contacts, may have a general significance for the modulation of intermolecular recognitions occurring in vivo.

Multi-Acting Mitochondria-Targeted Platinum(IV) Prodrugs of Kiteplatin with α-Lipoic Acid in the Axial Positions.

Platinum(II) drugs are activated intracellularly by aquation of the leaving groups and then bind to DNA, forming DNA adducts capable to activate various signal-transduction pathways. Mostly explored in recent years are Pt(IV) complexes which allow the presence of two additional ligands in the axial positions suitable for the attachment of other cancer-targeting ligands. Here we have extended this strategy by coordinating in the axial positions of kiteplatin ([PtCl2(cis-1,4-DACH)], DACH = Diaminocyclohexane) and its CBDCA (1,1-cyclobutanedicarboxylate) analogue the antioxidant α-Lipoic acid (ALA), an inhibitor of the mitochondrial pyruvate dehydrogenase kinase (PDK). The new compounds (cis,trans,cis-[Pt(CBDCA)(ALA)2(cis-1,4-DACH)], 2, and cis,trans,cis-[PtCl2(ALA)2(cis-1,4-DACH)], 3), after intracellular reduction, release the precursor Pt(II) species and two molecules of ALA. The Pt residue is able to target DNA, while ALA could act on mitochondria as activator of the pyruvate dehydrogenase complex, thus suppressing anaerobic glycolysis. Compounds 2 and 3 were tested in vitro on a panel of five human cancer cell lines and compared to cisplatin, oxaliplatin, and kiteplatin. They proved to be much more effective than the reference compounds, with complex 3 most effective in 3D spheroid tumor cultures. Notably, treatment of human A431 carcinoma cells with 2 and 3 did not determine increase of cellular ROS (usually correlated to inhibition of mitochondrial PDK) and did not induce a significant depolarization of the mitochondrial membrane or alteration of other morphological mitochondrial parameters.

Tetrathiomolybdate inhibits the reaction of cisplatin with human copper chaperone Atox1.

Cisplatin is a widely used anticancer drug in clinic, and ammonium tetrathiomolybdate ([(NH4)2MoS4], TM) is a copper chelator used in clinic for the treatment of Wilson's disease. Recently, TM has been found to enhance the therapeutic effect of cisplatin; however, the origin of this effect is not clear. Here we found that TM can inhibit the reaction of cisplatin with Cu-Atox1 and prevent the protein unfolding and aggregation induced by cisplatin. Although Ag(i) binds to Atox1 in a way similar to Cu(i)-Atox1, TM does not prevent the reaction of Ag-Atox1 with cisplatin. This result indicates that the formation of a Mo-centered trimeric protein cluster in the TM-Cu-Atox1 system plays a role in the inhibitory effect. This work provides new insights into the mechanism by which TM enhances the cytotoxic efficacy of cisplatin and helps to circumvent cisplatin resistance of tumor cells.

Dual-acting antitumor Pt(iv) prodrugs of kiteplatin with dichloroacetate axial ligands.

With the aim to obtain dual acting drugs able to target both nuclear DNA and mitochondria, Pt(iv) kiteplatin derivatives having dichloroacetate (DCA) ligands in axial positions have been synthesized. The rather fast hydrolysis (t1/2 of ca. 1 h) and reduction (by ascorbic acid) of these Pt(iv) derivatives did not impede a potent pharmacological effect on tumor cells. Moreover, similarly to kiteplatin, also the Pt(iv)-DCA compounds proved to be capable of overcoming oxaliplatin-resistance, which is particularly important in view of the fact that metastatic colorectal cancer is the third most common cancer in males and the second in females. The possible role of DCA released by the Pt(iv) compounds in eliciting the antiproliferative activity has also been investigated. Pt(iv)-DCA compounds determine a substantial increase of ROS production, blockage of oxidative phosphorylation, hypopolarization of the mitochondrial membrane, and caspase-3/7 mediated apoptotic cell death.

Differential Reactivity of Metal Binding Domains of Copper ATPases towards Cisplatin and Colocalization of Copper and Platinum.

The Menkes (MNK) and Wilson (WLN) disease proteins are two P-type ATPases responsible for active Cu efflux. These ATPases are also associated with resistance to cisplatin. In this work, different metal-binding domains (MBDs) of ATPases (9 out of 12 domains) were compared based on their reactivity towards cisplatin. The reaction rates of the MBDs can be largely different; the reaction of MNK6 is about six times faster than that of WLN2. Copper coordination favors the platination of the MBDs to different extents. The rate of platination was generally greater for holo-MBDs than for apo-MBDS (particularly in the case of WLN4 and WLN2); however, it was negligibly affected in the case of MNK6. Interestingly, the platinum binding weakens the CuI coordination, but does not expel the copper ion from MBDs. The latter results nicely explain the inhibitory effect of Cu upon the cisplatin translocation promoted by Cu-ATPases and can help in understanding how copper levels can modulate the sensitivity of cancer cells to platinum chemotherapy.