Forsythiae Fructus aqueous extract attenuates cisplatin-induced kaolin consumption (pica) by inhibiting NLRP3 inflammasome activation in rats.

The present study was conducted to evaluate the effect of Forsythiae Fructus aqueous extract (FAE) against cisplatin-induced emesis and to explore the antiemetic mechanism of FAE by focusing on NLRP3 inflammasome activation in a rat pica model. Our results showed that FAE significantly ameliorated cisplatin-induced acute and delayed pica in rats. Moreover, FAE improved the gastrointestinal histopathological injury and reduced the levels of serum ROS, IL-1ß, and IL-18 in cisplatin-treated rats. In addition, the expressions of NLRP3, ASC, caspase-1, and IL-1ß and the colocalization of the NLRP3 with ASC or caspase-1 in rat gastric antrum and ileum were also suppressed by FAE. Taken together, our findings indicate that FAE has a therapeutic effect against CINV, which may be related to its inhibition of the activation of NLRP3 inflammasome.

How versatile is the use of ultrafiltration to study biointeractions of therapeutic metallodrugs?

For a representative number of approved or investigational anticancer metallodrugs varying in lipophilicity, unspecific adsorption onto ultracentrifugal filter units was studied. It was found that for fairly hydrophilic compounds, such as cisplatin and oxaliplatin, the binding to filters does not substantially affect their amount measured (by ICP-MS) after ultrafiltration (>95%). In the case of metal complexes with moderate lipophilicity (log P > -0.1), adsorption effects turn out to be substantial. This might impede using ultrafiltration for studying the transformations of such drugs in human serum, unless they are rapidly converted into the protein adducts. The adsorption-suppressing effect of proteins was proved for indazolium trans-[tetrachloridobis(1H-indazole)ruthenate(III)] whose recovery from the filters was 61 and 14% in free and HSA-bound form, respectively.

A multi-methodological inquiry of the behavior of cisplatin-based Pt(IV) derivatives in the presence of bioreductants with a focus on the isolated encounter complexes.

The study of Pt(IV) antitumor prodrugs able to circumvent some drawbacks of the conventional Pt(II) chemotherapeutics is the focus of a lot of attention. This paper reports a thorough study based on experimental methods (reduction kinetics, electrochemistry, tandem mass spectrometry and IR ion spectroscopy) and quantum-mechanical DFT calculations on the reduction mechanism of cisplatin-based Pt(IV) derivatives having two hydroxido (1), one hydroxido and one acetato (2), or two acetato ligands (3) in axial position. The biological reductants glutathione and ascorbic acid were taken into consideration. The presence of a hydroxido ligand resulted to play an important role in the chemical reduction with ascorbic acid, as verified by 15N-NMR kinetic analysis using 15N-enriched complexes. The reactivity trend (1 > 2 > 3) does not reflect the respective reduction peak potentials (1 < 2 < 3), an inverse relationship already documented in similar systems. Turning to a simplified environment, the Pt(IV) complexes associated with a single reductant molecule (corresponding to the encounter complex occurring along the reaction coordinate in bimolecular reactions in solution) were characterized by IR ion spectroscopy and sampled for their reactivity under collision-induced dissociation (CID) conditions. The complexes display a comparable reduction reactivity ordering as that observed in solution. DFT calculations of the free energy pathways for the observed fragmentation reactions provide theoretical support for the CID patterns and the mechanistic hypotheses on the reduction process are corroborated by the observed reaction paths. The bulk of these data offers a clue of the intricate pathways occurring in solution.Graphic abstract.

Zwitterionic hydrophilic interaction liquid chromatography-tandem mass spectrometry with HybridSPE-precipitation for the determination of intact cisplatin in human plasma.

Cisplatin is a first-line chemotherapeutic for the treatment of a wide variety of cancers since its discovery in the 1960s. Although various techniques have been reported for the measurement of total platinum in biological matrices, such as inductively coupled plasma-mass spectrometry and derivatization procedures, a specific, sensitive and robust assay for the quantification of intact cisplatin is still lacking. Therefore, we present a rapid, selective, sensitive, and reliable UHPLC-MS/MS based method for the determination of intact cisplatin in human plasma in support of a Phase II clinical trial. The optimal chromatographic behavior of cisplatin was achieved on a Syncronis HILIC column (50 × 2.1mm, 1.7µm, zwitterionic stationary phase). The retention behavior of cisplatin on this zwitterion-based stationary phase was well described by an adsorptive interaction model. A simple sample preparation based on protein precipitation combined with the removal of phospholipids by HybridSPE-precipitation was developed. The method was proven to be free of a relative matrix effect. The assay was validated within a range of 20 - 10,000ng/mL using 100µL of plasma sample. The intra and inter-day precisions were all less than 7.6%, and none of the bias was greater than 13.1%, thus corroborating that the developed method is precise and accurate. As a proof of concept, the assay has been successfully applied to plasma samples obtained from different patients who were enrolled in the Phase II trial and were treated with cisplatin.

Molecular biology of testicular germ cell tumors

Testicular germ cell tumors (TGCTs) are the most common solid tumors in young adult men. They constitute a unique pathology because of their embryonic and germ origin and their special behavior. Genetic predisposition, environmental factors involved in their development and genetic aberrations have been under study in many works throughout the last years trying to explain the susceptibility and the transformation mechanism of TGCTs. Despite the high rate of cure in this type of tumors because its particular sensitivity to cisplatin, there are tumors resistant to chemotherapy for which it is needed to find new therapies. In the present work, it has been carried out a literature review on the most important molecular aspects involved in the onset and development of such tumors, as well as a review of the major developments regarding prognostic factors, new prognostic biomarkers and the possibility of new targeted therapies (AU)

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Combining TBP-based rOFFGEL-IEF with FASP and nLC-ESI-LTQ-MS/MS for the analysis of cisplatin-binding proteins in rat kidney.

In this work, a methodology based on a reducing IEF separation in combination with a FASP tryptic digestion able to maintain the integrity of cisplatin-protein complexes has been developed. The method is based on OFFGEL-IEF under conditions provided by the thiol-free reducing agent TBP, which allowed the separation of cisplatin-binding proteins in liquid fractions. The FASP procedure is applied as an intermediate stage between the IEF separation and MS analysis where the proteins are retained and concentrated in a commercially available ultrafiltration device. The filter unit acts as a proteomic reactor for detergent removal, buffer exchange, chemical modification (reduction and alkylation) and protein digestion. Finally, purified peptides are recovered by centrifugation. This procedure provides efficiencies comparable to standard in-solution digestion and the risk of platinum-complexes loss is minimized due to the fact that reagents employed along the process are subsequently eliminated before the following step. The stability of platinum-protein complexes under the FASP tryptic digestion, either using TBP or DTT as reducing agents, was maintained, allowing the identification of several platinum-containing peptides from cisplatin-HSA. This methodology was applied to the separation of platinum-enriched protein fractions obtained by SEC-ICP-MS in a kidney tissue extract from a rat treated with cisplatin, followed by further identification by nLC-ESI-LTQ-MS/MS after FASP tryptic digestion of selected platinum-containing liquid fractions.

Sequential extraction of platinum, cisplatin and carboplatin from environmental samples and pre-concentration/separation using vesicular coacervative extraction and determination by continuum source ETAAS.

A sequential extraction procedure is developed for the separation of trace levels of hexachloroplatinate, cisplatin and carboplatin from soil, which are then, pre-concentrated using a vesicular coacervative cloud point extraction method prior to their determination as platinum by continuum source ETAAS. Sequential extraction of carboplatin, cisplatin and hexachloroplatinate from a specific red soil is achieved by using the 20% HCl, aqua regia at room temperature and by combination of aqua regia and HF with microwave digestion, respectively. The pre-concentration of these species from the extracted solutions is based on the formation of extractable hydrophobic complexes of PtCl6(2-) anionic species with free cationic head groups solubilizing sites of the Triton X-114 co-surfactant stabilized TOMAC (tri-octyl methyl ammonium chloride) vesicles through electrostatic attraction. This process separates the platinum from bulk aqueous solution into a small vesicular rich phase. The parameters affecting the extraction procedures are optimized. Under the optimized conditions, the achieved pre-concentration factor is 20 and detection limit is 0.5 ng g(-1) for soil and 0.02 ng mL(-1) for water samples. The spiked recoveries of hexachloroplatinate, cisplatin and carboplatin in water and soil extracts in the vesicular coacervative extraction are in the range of 96-102% at 0.5-1 ng mL(-1) with relative standard deviation of 1-3%. The accuracy of the method for platinum determination is evaluated by analyzing CCRMP PTC-1a copper-nickel sulfide concentrate and BCR 723 road dust certified reference materials and the obtained results agreed with the certified values with 95% confidence level of student t-test. The results were also compared to mixed-micelle (MM)-CPE method reported in the literature.

Tea polyphenols modulate antioxidant redox system on cisplatin-induced reactive oxygen species generation in a human breast cancer cell.

Tea polyphenols (TPP) have potent antioxidant and anticancer properties, particularly in patients undergoing radiation or chemotherapy. However, few studies have been conducted on treatments using a combination of TPP and the conventional chemical anticancer drug cisplatin (CP). This study was designed to investigate the mechanism of the cytotoxicity of total TPP and CP, which may synergistically induce cell death in cancer cells. Here, breast cancer cells (MCF-7) were treated with various concentrations of TPP alone or in combination with the chemotherapeutic drug CP. The effect of TPP on cell growth, intracellular reactive oxygen species (ROS) level, apoptosis and gene expression of caspase-3, caspase-8 and caspase-9 and p53 was investigated. The MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay revealed that the MCF-7 cells were less sensitive to growth inhibition by TPP treatment than either CP or the combination therapy. Propidium iodide nuclear staining indicated that exposure to this combination increased the proportion of apoptotic nuclei compared with a single-agent treatment. Flow cytometry analysis was used to quantify changes in intracellular ROS. Detection of activated caspases by fluorescently labelled inhibitors of caspases (FLICA) combined with the plasma membrane permeability assay demonstrated that the percentage of early and late apoptotic/secondary necrotic cells was higher in the cells treated with the combination than in those treated with either TPP or CP alone. The combined TPP and CP treatment synergistically induced apoptosis through both caspase-8 and caspase-9 activation and p53 over-expression. This suggests that TPP plus CP may be used as an efficient antioxidant-based combination therapy for estrogen receptor (ER)-positive and p53-positive breast cancer.

OFFGEL isoelectric focusing and polyacrylamide gel electrophoresis separation of platinum-binding proteins.

In this work a 2D electrophoretic separation procedure able to maintain the integrity of platinum-protein bonds has been developed. The method is based on the use of sequential OFFGEL isoelectric focussing (IEF) and PAGE. A systematic study of the reagents used for PAGE, for OFFGEL-IEF separation, and post-separation treatment of gels (such as enzymatic digestion and sample preparation for MS analysis) was tackled regarding their suitability for the identification of platinum binding proteins using standard proteins incubated with cisplatin. The distribution of platinum in high and low molecular weight fractions (separated by cut-off filters) was determined by ICP-MS, which allows evaluating platinum-protein bond stability under the conditions studied. SDS-PAGE in the absence of ß-mercaptoethanol or dithiotreitol preserved the platinum-protein bonds. In addition, neither the influence of the electric field during the electrophoretic separation, nor the processes of fixing, staining and destaining of proteins in the gel did result in the loss of platinum from platinum binding proteins. SDS-PAGE under non-reducing conditions provides separation of platinum-binding proteins in very narrow bands with quantitative recoveries. Different amounts of platinum-bound proteins covering the range 0.3-2.0 µg were separated and mineralised for platinum determination, showing good platinum linearity. Limits of detection for a mixture of five standard proteins incubated with cisplatin were between the range of 2.4 and 13.9 pg of platinum, which were satisfactory for their application to biological samples. Regarding OFFGEL-IEF, a denaturing solution without thiourea and without dithiotreitol is recommended. The suitability of the OFFGEL-IEF for the separation of platinum binding proteins of a kidney cytosol was demonstrated.

CE in anticancer metallodrug research--an update.

With the current demographic development and the knowledge that the probability to be diagnosed with cancer increases with age, the search for new treatment options in cancer chemotherapy is of utmost importance for the society. Capillary electrophoretic methods have been applied in the last few years for studying the properties of metal-based drugs and drug candidates. Especially, the elucidation of the mode of action of such compounds could contribute significantly to design new drugs for overcoming the threat of cancer. This review article highlights the developments in metallodrug research applying CE during the last 4 years and follows a review from 2003 (Hartinger, C. G., Timerbaev, A. R., Keppler, B. K., Electrophoresis 2003, 24, 2023-2037). Most importantly the broadening of application areas of CE must be noted: especially the binding studies of metal complexes toward proteins (including the determination of association and rate constants), following redox reactions of metal complexes and their influence on the reactivity toward biotargets, etc. are important development areas of the last few years. In parallel with these new applications goes the usage of new or modified separation methods including microemulsion EKC or ACE, or the advantageous use of equipping the CE system with mass spectrometric detectors such as inductively coupled plasma (ICP) or ESI mass spectrometers (MS) for determining the degree of metallation of a protein or characterizing the adducts. Finally, upcoming requirements for expanding the method's application area are discussed including studies on new targets in the cell, analyzing real-world samples, methodological development, and contributions to improve the design of new anticancer agents.

Reducing toxicity of cisplatin in cancer chemotherapy by extracorporeal removal of excess cisplatin.

Cisplatin and its derivatives are today among the most frequently used agents for treatment of malignancies, the dose, however, is limited by side effects. When an organ or extremity with tumor has a single, well defined artery, cisplatin can be delivered into the tumor, and cisplatin leaving the tumor through the venous drainage can be removed before it empties into the systemic circulation. We developed a hollow fiber device with an immobilized platinum chelator for extracorporeal removal of cisplatin, without the chelator entering the blood. When blood from melanoma patients, obtained during cisplatin infusion, was circulated through the device, 80% of the cisplatin was removed. In experiment using dogs cisplatin (100 mg/m2) was infused into the femoral vein, thus restricting cisplatin to the area to be treated. This method allows for applying high cisplatin doses locally, while reducing systemic side effects.

A case of massive cisplatin overdose managed by plasmapheresis.

OBJECTIVES: Accidental cisplatin overdose occurs with increasing frequency despite the safeguards taken in prescription and administration, since cisplatin has been used increasingly for the treatment of numerous malignancies. Accidentally, a 59-year-old male received massive cisplatin overdose of 300mg/m2. METHODS: Laboratory documentation included measurement of cisplatin concentrations by flameless atomic absorption spectroscopy (Varian, Spectra AA 300). RESULTS: Toxicities included severe emesis, myelosuppression, renal failure, mental deterioration with hallucination, dim vision and hepatic toxicity. Plasmapheresis was effective in lowering the platinum concentration from greatest 2979 ng/ml to 185 ng/ml and appeared to be of clinical benefit. Granulocyte-macrophage colony stimulating factor (GM-CSF) was used to ameliorate myelosuppression. The patient's renal function was restored 3 months later and partial response of esophageal cancer was obtained. CONCLUSIONS: Plasmapheresis was effective in lowering the platinum concentration in massive cisplatin overdose. This case heightens awareness to the possibility of accidental cisplatin overdose and the benefits of prompt management.

Interaction between cisplatin-modified DNA and the HMG boxes of HMG 1: DNase I footprinting and circular dichroism.

The interactions between the two boxes A and B of HMG 1 and cis-diamminedichloroplatinum(II)-modified DNA containing a single intrastrand cross-link at the d(GpG) site were studied by DNase I footprinting and circular dichroism. The DNAase I cleavage patterns of the HMG box-platinated DNA complexes are identical, the two boxes inhibiting the DNase I cutting over at least 15 and 12 nucleotide residues in the platinated strand and the complementary strand, respectively. As judged by circular dichroism, the two boxes have the same alpha-helical content (56%) and they induce the same conformational changes in the platinated DNA.

Electrospray ionization mass spectrometry of covalent ligand-oligonucleotide adducts: evidence for specific duplex ion formation.

Electrospray ionization mass spectrometry (ESI-MS) has been used to examine the covalent binding of the anti-tumour agents cisplatin and hedamycin with self-complementary oligonucleotides 5'-TACGTA-3', 5'-CACGTG-3', 5'-AGGCCT-3' or 5'-CGTACG-3' as models for binding to cellular DNA. The observation of duplex forms of oligonucleotide adducts of these compounds in the gas phase has been found to correlate with the stability of the adducts in solution. Hedamycin, which both intercalates into and alkylates DNA, enhances the stability of duplex ions in ESI mass spectra. In contrast, the binding of cisplatin is known to destabilise duplexes in solution and only weak double-stranded peaks are observed in the ESI spectra of cisplatin-oligonucleotide adducts. Results of titration experiments with the hedamycin-5'-CACGTG-3' adduct and complementary and non-complementary oligo-nucleotides provide strong evidence that the observed duplex ions are the result of specific base-paired associations in the gas phase, rather than non-specific interactions. Finally, estimates of the extent of cisplatin binding to different sequences based on ESI mass spectra of crude reaction mixtures are found to correlate well with data obtained by reversed-phase high-performance liquid chromatography. This work demonstrates the considerable potential of ESI-MS as a tool for characterization of the interactions of antitumour agents with DNA.

Effectiveness of chemical agents in removing platinum from DNA isolated from cisplatin-treated HL-60 cells.

Human promyelocytic leukemia cells, HL-60, were treated with cisplatin [cis-diamminedichloroplatinum (II)] (2 mM, 1 h). DNA-cisplatin-protein complexes were isolated and exposed to thiourea (1 M), NaCN (100 mM), diethyldithio-carbamate (500 mM), or N-methyl-D-glucamine dithiocarbamate (500 mM) for 12 h. The release of platinum was measured by atomic absorption spectroscopy. Sodium cyanide was the most effective agent, releasing about 90% of the DNA-bound platinum. Thiourea was the least effective agent, while dithiocarbamates exhibited an intermediate. The ability of the same group of agents to split the proteins off from the protein-cisplatin-DNA complex was also evaluated and similarly dithiocarbamate were also the most effective.

High performance liquid chromatographic separation of cisplatin and its hydrolysis products on alumina and application to studies of their interaction with cysteine.

An alumina stationary phase has been assessed in the study of the retention behaviour of the anticancer drug cisplatin and its major hydrolysis products. Parameters such as buffer concentration in the mobile phase, pH, organic modifier and competing ion have been investigated in order to optimize chromatographic separation with ultraviolet detection. The separation scheme developed has been used to monitor the hydrolysis of cisplatin in aqueous and saline media, and to monitor the interaction of hydrolysed solutions of cisplatin with the amino acid cysteine. A new peak was observed in the chromatograms of such mixtures when they had been allowed to stand for periods of greater than 16 h and, from analysis of the data obtained, it was concluded that this new peak was due to a complex formed between the mono-aquo hydrolysis product of cisplatin and the amino acid.

Microscale synthesis of nitrogen-13-labeled cisplatin.

A microscale synthesis of [13N]cisplatin (cis-dichlorodiammineplatinum(II), cis-DDP) from cyclotron-produced [13N]ammonia is presented. Temperature, reaction time, ratios, and concentration of reactants have been optimized for each step of the synthesis. Purification is performed by ion exchange chromatography. Radiochemical purity and optimization processes are controlled by high performance liquid chromatography and high performance thin layer chromatography--22 mCi [13N]cisplatin in 10 ml of solution is produced. The entire procedure takes approximately 15 min and the specific activity is approximately 300 mCi/mumole at EOB.

Isolation of the adducts of platinum complexes and nucleic acid bases on the Dowex 50 W column.

The method presented here enables the isolation of three monofunctional adducts. These are: platination of cytosine, adenine and guanosine. The three detected bifunctional complexes were: guanine N7 to N7, adenine N7 to N7 and adenine N7 to N1. A mixed bifunctional complex of guanine and adenosine and the product of polymerization of three adenines and two moieties of cis DDP were also detected. In the peak eluted separately from the guanine standard, no Pt was detected. The sample eluted in this peak had UV spectrum different from the standard and may represent the degraded product of guanine and polymerized Pt.

Isolation of the products resulting from the reaction of cis and trans diaminedichloroplatinum [II] with DNA and chromatin on the Dowex 50 W column.

The production of platinated derivatives of nucleic acid bases resulting from the reaction of cis and trans DDP with DNA and chromatin was studied. Bifunctional complex of guanine appeared to be the major product of the interaction of cis isomer with both DNA and chromatin, although other bifunctional adducts of A-Pt-G and A-Pt-A were also isolated. The main product of the interaction of trans DDP with DNA was a monofunctional adduct of guanine. Small amounts of the bifunctional complexes were also isolated. When ssDNA was incubated with trans DDP more bifunctional complexes appeared, what suggests that geometric constrains of double helix prevent formation of these complexes. Trans isomer reacts more easily with chromosomal proteins than cis DDP does. Therefore after the reaction of trans DDP with chromatin less platination occurs on DNA moieties.