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The reactivity of the anticancer drug picoplatin ( cis -amminedichlorido(2-methylpyridine)platinum(II) complex) with the model proteins hen egg white lysozyme (HEWL) and bovine pancreatic ribonuclease (RNase A) was investigated by electrospray ionisation mass spectrometry (ESI MS) and X-ray crystallography. The data were compared with those previously obtained for the adducts of these proteins with cisplatin, carboplatin and oxaliplatin under the same experimental conditions. ESI-MS data show binding of Pt to both proteins, with fragments retaining the 2-methylpyridine ligand and, possibly, a chloride ion. X-ray crystallography identifies different binding sites on the two proteins, highlighting a different behaviour of picoplatin in the absence or presence of dimethyl sulfoxide (DMSO). Metal-containing fragments bind to HEWL close to the side chains of His15, Asp18, Asp119 and both Lys1 and Glu7, whereas they bind to RNase A on the side chain of His12, Met29, His48, Asp53, Met79, His105 and His119. The data suggest that the presence of DMSO favours the loss of 2-methylpyridine and alters the ability of the Pt compound to bind to the two proteins. With both proteins, picoplatin appears to behave similarly to cisplatin and carboplatin when dissolved in DMSO, whereas it behaves more like oxaliplatin in the absence of the coordinating solvent. This study provides important insights into the pharmacological profile of picoplatin and supports the conclusion that coordinating solvents should not be used to evaluate the biological activities of Pt-based drugs.

Study Question: What is the impact of low- or moderate-risk gonadotoxic chemotherapy received prior to testicular tissue freezing (TTF), and of the cancer itself, on spermatogonia quantity in testicular tissue from (pre)pubertal boys?, Summary Answer: Vincristine, when associated with alkylating agents, has an additional adverse effect on spermatogonia quantity, while carboplatin has no individual contribution to spermatogonia quantity, in testicular tissue of (pre)pubertal boys, when compared to patients who have received non-alkylating chemotherapy., What Is Known Already: The improved survival rates after cancer treatment necessitate the inclusion of fertility preservation procedures as part of the comprehensive care for patients, taking into consideration their age. Sperm cryopreservation is an established procedure in post-pubertal males while the TTF proposed for (pre)pubertal boys remains experimental. Several studies exploring testicular tissue of (pre)pubertal boys after TTF have examined the tubular fertility index (TFI, percentage of seminiferous tubule cross-sections containing spermatogonia) and the number of spermatogonia per seminiferous tubule cross-section (S/T). All studies have demonstrated that TFI and S/T always decrease after the introduction of chemotherapeutic agents, especially those which carry high gonadotoxic risks such as alkylating agents., Study Design, Size, Duration: Testicular tissue samples from 79 (pre)pubertal boys diagnosed with cancer (from 6 months to 16 years of age) were cryopreserved between May 2009 and June 2014. Their medical diagnoses and previous chemotherapy exposures were recorded. We examined histological sections of (pre)pubertal testicular tissue to elucidate whether the chemotherapy or the primary diagnosis affects mainly TFI and S/T., Participants/materials, Setting, Methods: (Pre)pubertal boys with cancer diagnosis who had been offered TTF prior to conditioning treatment for hematopoietic stem cell transplantation were included in the study. All the patients had previously received chemotherapy with low- or moderate-risk for future fertility. We have selected patients for whom the information on the chemotherapy received was complete. The quantity of spermatogonia and quality of testicular tissue were assessed by both morphological and immunohistochemical analyses., Main Results and the Role of Chance: A significant reduction in the number of spermatogonia was observed in boys treated with alkylating agents. The mean S/T values in boys exposed to alkylating agents were significantly lower compared to boys exposed to non-alkylating agents (P = 0.018). In contrast, no difference was observed for patients treated with carboplatin as the sole administered alkylating agent compared to the group of patients exposed to non-alkylating agents. We observed an increase of S/T with age in the group of patients who did not receive any alkylating agent and a decrease of S/T with age when patients received alkylating agents included in the cyclophosphamide equivalent dose (CED) formula (r = 0.6166, P = 0.0434; r = -0.3759, P = 0.0036, respectively). The TFI and S/T decreased further in the group of patients who received vincristine in combination with alkylating agents (decrease of 22.4%, P = 0.0049 and P < 0.0001, respectively), but in this group the CED was also increased significantly (P < 0.0001). Multivariate analysis, after CED adjustment, showed the persistence of a decrease in TFI correlated with vincristine administration (P = 0.02)., Limitations, Reasons for Caution: This is a descriptive study of testicular tissues obtained from (pre)pubertal boys who were at risk of infertility. The study population is quite heterogeneous, with a small number of patients in each sub-group. Our results are based on comparisons between patients receiving alkylating agents compared to patients receiving non-alkylating agents rather than chemotherapy-naive patients. The French national guidelines for fertility preservation in cancer patients recommend TTF before highly gonadotoxic treatment. Therefore, all the patients had received low- or moderate-risk gonadotoxic chemotherapy before TTF. Access to testicular tissue samples from chemotherapy-naive patients with comparable histological types of cancer was not possible. The functionality of spermatogonia and somatic cells could not be tested by transplantation or in vitro maturation due to limited sample sizes., Wider Implications of the Findings: This study summarizes the spermatogonial quantity of (pre)pubertal boys prior to TTF. We confirmed a negative correlation between the cumulative exposure to alkylating agents and spermatogonial quantity. In addition, the synergistic use of vincristine in combination with alkylating agents showed a cumulative deleterious effect on the TFI. For patients for whom fertility preservation is indicated, TTF should be proposed for chemotherapy with a predicted CED above 4000 mg/m2. However, the data obtained from vincristine and carboplatin use should be confirmed in a subsequent study including more patients., Study Funding/competing Interest(s): This study had financial support from a French national research grant PHRC No. 2008/071/HP obtained by the French Institute of Cancer and the French Healthcare Organization. The sponsors played no role in the study. The authors declare no conflicts of interest., Trial Registration Number: N/A., (© The Author(s) 2023. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Introduction: Testicular germ cell tumor is the most frequent neoplasia in men of reproductive age, with a 5-year survival rate of 95%. Antineoplastic treatments induce sperm DNA fragmentation, especially within the first year post-therapy. Data in the literature are heterogeneous concerning longer follow-up periods, and the large majority is limited to 2 years., Objective: To define the timing for the recovery of sperm DNA damage and the proportion of patients with severe DNA damage at 2 and 3 years from the end of therapy., Materials and Methods: Sperm DNA fragmentation was evaluated in 115 testicular germ cell tumor patients using terminal deoxynucleotidyl transferase dUTP nick end labeling assay coupled with flow cytometry before (T 0 ) and 2 (T 2 ) and 3 (T 3 ) years post-treatment. Patients were divided based on the type of treatment: carboplatin, bleomycin-etoposide-cisplatin, and radiotherapy. For 24 patients, paired sperm DNA fragmentation data were available at all time-points (T 0 -T 2 -T 3 ). Seventy-nine cancer-free, fertile normozoospermic men served as controls. Severe DNA damage was defined as the 95th percentile in controls (sperm DNA fragmentation = 50%)., Results: Comparing patients versus controls, we observed: (i) no differences at T 0 and T 3 and (ii) significantly higher sperm DNA fragmentation levels (p < 0.05) at T 2 in all treatment groups. Comparing pre- and post-therapy in the 115 patients, the median sperm DNA fragmentation values were higher in all groups at T 2 , reaching significance (p < 0.05) only in the carboplatin group. While the median sperm DNA fragmentation values were also higher in the strictly paired cohort at T 2 , about 50% of patients returned to baseline. The proportion of severe DNA damage in the entire cohort was 23.4% and 4.8% of patients at T 2 and T 3 , respectively., Discussion: Currently, testicular germ cell tumor patients are advised to wait 2 years post-therapy before seeking natural pregnancy. Our results suggest that this period may not be sufficient for all patients., Conclusion: The analysis of sperm DNA fragmentation may represent a useful biomarker for pre-conception counseling following cancer treatment., (© 2023 The Authors. Andrology published by Wiley Periodicals LLC on behalf of American Society of Andrology and European Academy of Andrology.)

Luteolin (Lut) has been shown to inhibit gastric cancer (GC); however, its efficacy compared to other clinical drugs has not been examined in human samples. This study aimed to elucidate the antitumor activity of Lut in GC patient-derived organoids (PDOs). PDOs were established from GC cancer tissues, and the characterization of tissues and PDOs was performed using whole-exome sequencing. Drug sensitivity tests were performed by treating PDOs with Lut, norcantharidin (NCTD), and carboplatin (CP). RNA sequencing of PDOs was performed to elucidate the antitumor mechanism of Lut, which was further verified in three GC cell lines. Eleven PDOs were successfully constructed, and were highly consistent with the pathophysiology and genetic changes in the corresponding tumors. The IC50s of Lut, NCTD, and CP of PDOs were 27.19, 23.9, and 37.87 μM, respectively. Lut treatment upregulated FOXO3, DUSP1, and CDKN1A expression and downregulated IL1R1 and FGFR4 expression in GC cell lines, which was consistent with the results of PDOs. We demonstrate that Lut exerted stronger antitumor effects than CP, but a similar effect to that of NCTD, which was obtained in an in vitro PDO system. Additionally, Lut exerted varying degrees of antitumor effects against the PDOs, thereby indicating that PDO may be a useful preclinical drug screening tool for personalized treatment., (© 2023 The Authors. Phytotherapy Research published by John Wiley & Sons Ltd.)

Liver X receptors (LXRs) are nuclear transcription factors important in the regulation of cholesterol transport, and glucose and fatty acid metabolism. The antiproliferative role of LXRs has been studied in a variety of malignancies and may represent a therapeutic opportunity in cancers lacking targeted therapies, such as triple-negative breast cancer. In this study, we investigated the impact of LXR agonists alone and in combination with carboplatin in preclinical models of breast cancer. In vitro experiments revealed a dose-dependent decrease in tumor cell proliferation in estrogen receptor-positive breast cancer cells, whereas LXR activation in vivo resulted in an increased growth inhibitory effect in a basal-like breast cancer model (in combination with carboplatin). Functional proteomic analysis identified differences in protein expression between responding and nonresponding models related to Akt activity, cell-cycle progression, and DNA repair. Furthermore, pathway analysis suggested that the LXR agonist in combination with carboplatin inhibits the activity of targets of E2F transcription factors and affects cholesterol homeostasis in basal-like breast cancer., (© 2023 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Apoptosis is a natural physiological process that can maintain the homeostasis of the body and immune system. This process plays an important role in the system's resistance to autoimmune development. Because of the dysfunction of cell apoptosis mechanism, the number of autoreactive cells in the peripheral tissue increases along with their accumulation. This will lead to the development of autoimmune diseases, such as multiple sclerosis (MS). MS is an immune-mediated disease of the central nervous system characterized by severe white matter demyelination. Because of the complexity of its pathogenesis, there is no drug to cure it completely. Experimental autoimmune encephalomyelitis (EAE) is an ideal animal model for the study of MS. Carboplatin (CA) is a second-generation platinum anti-tumor drug. In this study, we attempted to assess whether CA could be used to ameliorate EAE. CA reduced spinal cord inflammation, demyelination, and disease scores in mice with EAE. Moreover, the number and proportion of pathogenic T cells especially Th1 and Th17 in the spleen and draining lymph nodes were reduced in CA-treated EAE mice. Proteomic differential enrichment analysis showed that the proteins related to apoptosis signal changed significantly after CA treatment. CFSE experiment showed that CA significantly inhibited the T cell proliferation. Finally, CA also induced apoptosis in activated T cells and MOG-specific T cells in vitro. Overall, our findings indicated that CA plays a protective role in the initiation and progression of EAE and has the potential to be a novel drug in the treatment of MS., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: Previous studies have demonstrated that, in contrast to the properties of food-derived copper, water-derived copper exerts neurotoxic effects and exhibits different speciation during digestion. The cellular uptake efficiencies of different speciation of copper are distinct. However, it is unclear whether these different speciation share the same transport pathway in intestinal epithelial cells. In the present study, the intracellular accumulation of copper derived from copper ion and copper complex solutions was investigated in Caco-2 cells., Results: The cellular accumulation of copper derived from copper ions was higher than that of copper derived from the copper complex. Treatment with carboplatin and Ag + , which are copper transporter receptor 1 (Ctr1, LC31A1) inhibitors, did not inhibit copper accumulation in Caco-2 cells, but inhibited copper accumulation in HepG2 cells. Zinc ion significantly decreased the intracellular copper content from 114 ± 7 μg g -1 protein to 88 ± 4 μg g -1 protein in the copper ion-treated Caco-2 cells, but not in the copper complex-treated Caco-2 cells (84.6 ± 14 μg g -1 protein versus 87.7 ± 20 μg g -1 protein, P > 0.05). Additionally, copper accumulation in Caco-2 and HepG2 cells significantly differed depending on different solvents (Hanks' balanced salt solution and NaNO 3 , P < 0.05)., Conclusion: These results indicate that the intracellular accumulation of copper derived from copper ion and copper complex is mediated by distinct copper transport pathways. Copper speciation may be an important factor that affects copper absorption and toxicity. © 2022 Society of Chemical Industry., (© 2022 Society of Chemical Industry.)

Background: Epithelial ovarian cancer is the most lethal gynaecological cancer worldwide. Chemotherapy resistance represents a significant clinical challenge and is the main reason for poor ovarian cancer prognosis. We identified novel expression of markers related to epithelial mesenchymal transitions (EMT) in a carboplatin resistant ovarian cancer cell line by proteomics. This was validated in the platinum resistant versus sensitive parental cell lines, as well as platinum resistant versus sensitive human ovarian cancer patient samples. The prognostic significance of the different proteomics-identified marker proteins in prognosis prediction on survival as well as their correlative association and influence on immune cell infiltration was determined by public domain data bases., Methods: We explored the proteomic differences between carboplatin-sensitive OVCAR5 cells (parental) and their carboplatin-resistant counterpart, OVCAR5 CBPR cells. qPCR and western blots were performed to validate differentially expressed proteins at the mRNA and protein levels, respectively. Association of the identified proteins with epithelial-mesenchymal transition (EMT) prompted the investigation of cell motility. Cellular bioenergetics and proliferation were studied to delineate any biological adaptations that facilitate cancer progression. Expression of differentially expressed proteins was assessed in ovarian tumors obtained from platinum-sensitive (n = 15) versus platinum-resistant patients (n = 10), as well as matching tumors from patients at initial diagnosis and following relapse (n = 4). Kaplan-Meier plotter and Tumor Immune Estimation Resource (TIMER) databases were used to determine the prognostic significance and influence of the different proteomics-identified proteins on immune cell infiltration in the tumor microenvironment (TME)., Results: Our proteomics study identified 2422 proteins in both cell lines. Of these, 18 proteins were upregulated and 14 were downregulated by ≥ twofold (p < 0.05) in OVCAR5 CBPR cells. Gene ontology enrichment analysis amongst upregulated proteins revealed an overrepresentation of biological processes consistent with EMT in the resistant cell line. Enhanced mRNA and/or protein expression of the identified EMT modulators including ITGA2, TGFBI, AKR1B1, ITGAV, ITGA1, GFPT2, FLNA and G6PD were confirmed in OVCAR5 CBPR cells compared to parental OVCAR5 cell line. Consistent with the altered EMT profile, the OVCAR5 CBPR cells demonstrated enhanced migration and reduced proliferation, glycolysis, and oxidative phosphorylation. The upregulation of G6PD, AKR1B1, ITGAV, and TGFβ1 in OVCAR5 CBPR cells was also identified in the tumors of platinum-resistant compared to platinum-sensitive high grade serous ovarian cancer (HGSOC) patients. Matching tumors of relapsed versus newly diagnosed HGSOC patients also showed enhanced expression of AKR1B1, ITGAV, TGFβ1 and G6PD protein in relapsed tumors. Among the identified proteins, significant enhanced expression of GFPT2, FLNA, TGFBI (CDGG1), ITGA2 predicted unfavorable prognosis in ovarian cancer patients. Further analysis suggested that the expression of TGFBI to correlate positively with the expression of identified and validated proteins such as GFPT2, FLNA, G6PD, ITGAV, ITGA1 and ITGA2; and with the infiltration of CD8 + T cells, macrophages, neutrophils, and dendritic cells in the TME., Conclusions: Our research demonstrates proteomic-based discovery of novel EMT-related markers with an altered metabolic profile in platinum-resistant versus sensitive ovarian cancer cell lines. The study also confirms the expression of selected identified markers in the tumors of platinum-resistant versus sensitive, and in matching relapsed versus newly diagnosed HGSOC patients. The study provides insights into the metabolic adaptation of EMT-induced carboplatin resistant cells that confers on them reduced proliferation to provide effective migratory advantage; and the role of some of these identified proteins in ovarian cancer prognosis. These observations warrant further investigation of these novel target proteins in platinum-resistant patients., (© 2022. The Author(s).)

Scope: Sweet potato (Ipomoea batatas L.) is one of the leading crops worldwide, containing high nutritional components such as fiber and polyphenols. Root tuber of Simon 1 (SIMON), a cultivar of sweet potato, is a folk food in China with a hemostasis function but lacking experimental data support., Methods and Results: Now the protective effect of SIMON on chemotherapy-induced thrombocytopenia (CIT), a serious complication of cancer treatment, is investigated for the first time by a CIT mouse model induced by intraperitoneal injection of carboplatin. As a result, SIMON raises the number of peripheral platelets, white blood cells, and bone marrow nucleated cells in CIT mice significantly. Besides, carboplatin-induced atrophy of the thymus, spleen, and disordered metabolism of the inflammatory immune system and glycerophospholipids are also reversed by SIMON. Phytochemical analysis of SIMON indicates 16 compounds including eight phenolic derivatives, which might be associated with its anti-CIT bioactivity., Conclusion: Sweet potato (SIMON) may be an efficient function food in the prevention of bleeding disorders., (© 2022 Wiley-VCH GmbH.)

The present study aims to determine the major metabolites of amentoflavone (AMF) and further evaluate their inhibitory effects on PARP-1. First, different fractions (Frs. 1-9), which were collected according to retention time of AMF metabolites based on UHPLC-QTOF-MS/MS qualitative analysis, were evaluated on their inhibitory effects against PARP-1. Then, two mono-sulfate metabolites in the fractions with potent PARP-1 inhibitory effect were targetedly semi-synthesized. Moreover, three mono-sulfate conjugates (compound 8, 9 and 10), including one disulfate conjugate (compound 10), were isolated and their structures were fully elucidated by UHPLC-QTOF-MS/MS and NMR. Finally, the binding mode of compound 8 (amentoflavone-4‴-O-sulfate) toward PARP-1 and its potentiation on carboplatin (CBP) in A549 cells were investigated. This study was the first report on bioactivity evaluation of AMF metabolites in rat bile on PARP-1 and the potentiation of compound 8 on carboplatin (CBP) in A549 cells in vitro. This paper also provided scientific basis for the AMF metabolites on PARP-1 inhibition and chemosensitization., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: This research was to develop a special method for enriching Circulating tumor cells (CTCs) of Hepatocellular carcinoma (HCC) by Glypican-3 immunoliposomes (GPC3-IML), and to analyze the correlation between the CTCs count and tumor malignancy, as well as to investigate the mutation characteristics of CTC-derived NGS., Results: In this study characterization of physical parameters was performed with the preparation of GPC3-IML. CTCs in peripheral blood of HCC patients were further separated and identified. Immunofluorescence was used to identify CTCs for further counting. By this means, the correlation between CTCs count and clinicopathological features was analyzed, and the genetic mutation characteristics of NGS derived from CTCs were investigated and compared with that of tissue NGS. Results showed that compared with EpCAM and vimentin, GPC-3 had a stronger CTCs separation ability. There was a correlation between "positive" count of CTCs (≥ 5 PV-CTC per 7.5 ml blood) and BCLC stage (P = 0.055). The result of CTC-NGS was consistent with that of tissue-NGS in 60% cases, revealing that KMT2C was a common highly-frequent mutated gene., Conclusion: The combination of immunomagnetic separation of CTCs and anti-tumor marker identification technology can be regarded as a new technology of CTCs detection in peripheral blood of patients with HCC. Trial registration EHBHKY2020-k-024. Registered 17 August 2020-Retrospectively registered.

Prolonged treatment with cisplatin (CDDP) frequently develops chemoresistance. We have previously shown that p22phox, an endoplasmic reticulum (ER) membrane protein, confers CDDP resistance by blocking CDDP nuclear entry in oral squamous cell carcinoma (OSCC) cells; however, the underlying mechanism remains unresolved. Using a fluorescent dye-labeled CDDP, here we show that CDDP can bind to p22phox in both cell-based and cell-free contexts. Subsequent detection of CDDP-peptide interaction by the Tris-Tricine-based electrophoresis revealed that GA-30, a synthetic peptide matching a region of the cytosolic domain of p22phox, could interact with CDDP. These results were further confirmed by liquid chromatography-mass spectrometry (LC-MS) analysis, from which MA-11, an 11-amino acid subdomain of the GA-30 domain, could largely account for the interaction. Amino acid substitutions at Cys50, Met65 and Met73, but not His72, significantly impaired the binding between CDDP and the GA-30 domain, thereby suggesting the potential CDDP-binding residues in p22phox protein. Consistently, the p22phox point mutations at Cys50, Met65 and Met73, but not His72, resensitized OSCC cells to CDDP-induced cytotoxicity and apoptosis. Finally, p22phox might have binding specificity for the platinum drugs, including CDDP, carboplatin and oxaliplatin. Together, we have not only identified p22phox as a novel CDDP-binding protein, but further highlighted the importance of such a drug-protein interaction in drug resistance.

Background: Antitumor agents based on platinum have gained a well-established place in the treatment of several forms of cancer. Their efficiency is hampered by serious toxic effects against healthy tissues as well. Ototoxicity is a serious side effect leading to hearing impairment and represents an important issue affecting the patients' quality of life. The currently used platinum chemotherapeutics exert different toxicity towards cochlear cells. The aim of our study was to answer some questions regarding the differential uptake and cellular pharmacodynamics of Cisplatin (CDDP), Carboplatin (CBDCA) and Oxaliplatin (L-OHP) in the HEI-OC1 cochlear cell line., Methods: We studied the expression of copper transporters CTR1, ATP7A and ATP7B which are presumably involved in the uptake, cellular transport and efflux of platinum compounds by immunofluorescence microscopy and flow-cytometry. The cellular uptake of the compounds was evaluated through the determination of intracellular platinum concentration by atomic absorption spectroscopy. The effects of the treatment of HEI-OC1 cells with platinum compounds were also evaluated: cytotoxicity with the Cell Titer Blue viability test, formation of reactive oxygen species with 2',7' -dichlorofluorescein diacetate, genotoxicity with the comet assay and apoptosis with the cleaved PARP ELISA test., Results: CTR1, ATP7A and ATP7B were all expressed by HEI-OC1 cells. The treatment with the platinum compounds led to a modulation of their expression, manifested in a differential platinum uptake. Treatment with Cisplatin led to the highest intracellular concentration of platinum compared to Oxaliplatin and Carboplatin at the same dose. Treatment with CuSO4 reduced platinum uptake of all the compounds, significantly in the case of Cisplatin and Carboplatin. CDDP was the most cytotoxic against HEI-OC1 cells, with an IC50 = 65.79  μM, compared to 611.7 μM for L-OHP and 882.9 μM for CBDCA, at the same molar concentration. The production of ROS was the most intense after CDDP, followed by L-OHP and CBDCA. In the comet assay, at the 100 μM concentration, L-OHP and CBDCA induced DNA adducts while CDDP induced adducts as well as DNA strand breaks. CBDCA and L-OHP lead to a significant increase of cleaved PARP at 24h (p < 0.001), suggesting an important apoptotic process induced by these compounds at the used concentrations., Conclusions: The results obtained in the current study suggest that the modulation of copper transporters locally may represent a new strategy against platinum drugs ototoxicity., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Purpose: While doses of carboplatin are mostly individualized according to the Calvert equation based on estimated Glomerular Filtration Rate (eGFR), there is still uncertainty regarding the best formula to predict GFR. Since Janowitz et al. recently proposed a new equation predicting GFR in cancer patients, we aimed to compare this equation to other carboplatin clearance (carboCL) predicting formulae., Methods: The actual carboCL of 491 patients was compared to predicted carboCL according to the Calvert formula using several equations to predict GFR (Janowitz, Cockcroft-Gault, MDRD, CKD-EPI, CKD-EPI with cystatin C (CKD-EPI-cysC)); and according to two others that directly predict carboCL (Chatelut and Thomas). The formulae were compared on Mean Percentage Error (MPE), Mean Absolute Percentage Error (MAPE) and percentage of patients with a prediction error above 20% (P20)., Results: The MPE, MAPE and P20 were, respectively, within the ranges - 5.2 to + 5.9%; 14.0-21.2% and 23-46%. The MAPE and P20 of Calvert-CKD-EPI-cysC were the lowest. The performance of Calvert-CKD-EPI was better than that of other creatinine-based formulae although not significantly different from the Calvert-Janowitz formula. Among formulae based on creatinine only, Calvert-CKD-EPI and Calvert-Janowitz are the least influenced by patient characteristics., Conclusion: Whereas CysC improves carboplatin CL prediction, the Calvert-CKD-EPI equation seems the most suitable creatinine-based formula to predict carboCL homogeneously in all subgroups of patients.

Here we report a new study performed at single molecule level on the interaction of the antineoplastic drug Carboplatin and the DNA molecule - the main target of the drug inside cells in cancer chemotherapies. By using optical tweezers, we measure how the mechanical properties of the DNA-Carboplatin complexes changes as a function of the drug concentration in the sample, for two different ionic strengths ([Na] = 150 mM and [Na] = 1 mM). From these measurements, the binding mechanism and the physicochemical (binding) parameters of the interaction were inferred and directly compared to those obtained for the precursor drug Cisplatin under equivalent conditions. As the main conclusion, we show that Carboplatin binds preferentially forming covalent monoadducts in contrast to Cisplatin, which is hydrolyzed easier and presents a higher efficiency in forming covalent diadducts along the double-helix. In addition, we explicitly show that Carboplatin is much less sensitive to ionic strength changes when compared to Cisplatin. These findings provide new insights on the interactions of platinum-based compounds with the DNA molecule, being important to improve the current treatments and in the development of new antineoplastic agents., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Post-chemotherapy cognitive impairment, also known as 'chemobrain,' is a neurological condition in which cognitive function is impaired as a result of cancer chemotherapy treatment. In this work, we used fast-scan cyclic voltammetry (FSCV) to measure electrically evoked dopamine release and uptake in whole brain preparations from zebrafish that have been treated with carboplatin, an agent associated with chemobrain. We administered carboplatin by addition to the fish's tank water or their food. One week of treatment with 100 μM carboplatin in the water was needed to significantly impair dopamine release (∼40 % of control); however, only one day of treatment through the zebrafish's food was needed to cause a similar impairment. Atomic absorption spectroscopy measurements suggested that administration through food resulted in higher initial levels of carboplatin compared to water administration, but water administration resulted in an increase over time. Uptake, determined by modeling stimulated release plots, was unaffected. These results are consistent with our previous findings of diminished neurotransmitter release in rats and support a role for zebrafish in chemobrain-related studies., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Identifying single nucleotide polymorphisms (SNPs) that influence chemotherapy disposition may help to personalize cancer treatment and limit toxicity. Genome-wide approaches are unbiased, compared with candidate gene studies, but usually require large cohorts. As most chemotherapy is given cyclically multiple blood sampling is required to adequately define drug disposition, limiting patient recruitment. We found that carboplatin and paclitaxel disposition are stable phenotypes in ovarian cancer patients and tested a genome-wide association study (GWAS) design to identify SNPs associated with chemotherapy disposition. We found highly significant SNPs in ABCC2, a known carboplatin transporter, associated with carboplatin clearance (asymptotic P = 5.2 × 10 6 , empirical P = 1.4 × 10 -5 ), indicating biological plausibility. We also identified novel SNPs associated with paclitaxel disposition, including rs17130142 with genome-wide significance (asymptotic P = 2.0 × 10 -9 , empirical P = 1.3 × 10 -7 ). Although requiring further validation, our work demonstrated that GWAS of chemotherapeutic drug disposition can be effective, even in relatively small cohorts, and can be adopted in drug development and treatment programs.

Five new platinum(IV) derivatives of carboplatin each incorporating the histone deacetylase inhibitor 4-phenylbutyrate in axial position were synthesized and characterized by 1 H and 195 Pt NMR spectroscopy, electrospray ionization mass spectrometry and elemental analysis, namely cis,trans-[Pt(CBDCA)(NH 3 ) 2 (PBA)(OH)] (1), cis,trans-[Pt(CBDCA)(NH 3 ) 2 (PBA) 2 ] (2), cis,trans-[Pt(CBDCA)(NH 3 ) 2 (PBA)(bz)] (3), cis,trans-[Pt(CBDCA)(NH 3 ) 2 (PBA)(suc)] (4) and cis,trans-[Pt(CBDCA)(NH 3 ) 2 )(PBA)(ac)] (5) (PBA=4-phenylbutyrate, CBDCA=1,1-cyclobutane dicarboxylate, bz=benzoate, suc=succinate and ac=acetate). The reduction behavior in the presence of ascorbic acid was studied by high performance liquid chromatography. The cytotoxicity against a panel of human tumor cell lines, histone deacetylase (HDAC) inhibitory activity, cellular accumulation and the ability to induce apoptosis were evaluated. The most effective complex, compound 3, was found to be up to ten times more effective than carboplatin and to decrease cellular basal HDAC activity by approximately 18% in A431 human cervical cancer cells., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Development of chemoresistance is a cogent clinical issue in oncology, whereby combination of anticancer drugs is usually preferred also to enhance efficacy. Paclitaxel (PTX), combined with carboplatin, represents the standard first-line chemotherapy for different types of cancers. We here depict a double-edge role of mitochondrial DNA (mtDNA) mutations induced in cancer cells after treatment with platinum. MtDNA mutations were positively selected by PTX, and they determined a decrease in the mitochondrial respiratory function, as well as in proliferative and tumorigenic potential, in terms of migratory and invasive capacity. Moreover, cells bearing mtDNA mutations lacked filamentous tubulin, the main target of PTX, and failed to reorient the Golgi body upon appropriate stimuli. We also show that the bioenergetic and cytoskeletal phenotype were transferred along with mtDNA mutations in transmitochondrial hybrids, and that this also conferred PTX resistance to recipient cells. Overall, our data show that platinum-induced deleterious mtDNA mutations confer resistance to PTX, and confirm what we previously reported in an ovarian cancer patient treated with carboplatin and PTX who developed a quiescent yet resistant tumor mass harboring mtDNA mutations., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Diffusion-weighted magnetic resonance imaging (DW-MRI) was used to evaluate the effects of single-agent and combination treatment regimens in a spheroid-based animal model of ovarian cancer. Ovarian tumour xenografts grown in non-obese diabetic/severe-combined-immunodeficiency (NOD/SCID) mice were treated with carboplatin or paclitaxel, or combination carboplatin/paclitaxel chemotherapy regimens. After 4 weeks of treatment, tumours were extracted and underwent DW-MRI, mechanical testing, immunohistochemical and gene expression analyses. The distribution of the apparent diffusion coefficient (ADC) exhibited an upward shift as a result of each treatment regimen. The 99-th percentile of the ADC distribution ("maximum ADC") exhibited a strong correlation with the tumour size (r 2  = 0.90) and with the inverse of the elastic modulus (r 2  = 0.96). Single-agent paclitaxel (n = 5) and combination carboplatin/paclitaxel (n = 2) treatment regimens were more effective in inducing changes in regions of higher cell density than single-agent carboplatin (n = 3) or the no-treatment control (n = 5). The maximum ADC was a good indicator of treatment-induced cell death and changes in the extracellular matrix (ECM). Comparative analysis of the tumours' ADC distribution, mechanical properties and ECM constituents provides insights into the molecular and cellular response of the ovarian tumour xenografts to chemotherapy. Increased sample sizes are recommended for future studies. We propose experimental approaches to evaluation of the timeline of the tumour's response to treatment.

We report progress on predicting tumor response to platinum-based chemotherapy with a novel mass spectrometry approach. Fourteen bladder cancer patients were administered one diagnostic microdose each of [ 14 C]carboplatin (1% of the therapeutic dose). Carboplatin-DNA adducts were quantified by accelerator mass spectrometry in blood and tumor samples collected within 24 hours, and compared with subsequent chemotherapy response. Patients with the highest adduct levels were responders, but not all responders had high adduct levels. Four patient-derived bladder cancer xenograft mouse models were used to test the possibility that another drug in the regimen could cause a response. The mice were dosed with [ 14 C]carboplatin or [ 14 C]gemcitabine and the resulting drug-DNA adduct levels were compared with tumor response to chemotherapy. At least one of the drugs had to induce high drug-DNA adduct levels or create a synergistic increase in overall adducts to prompt a corresponding therapeutic response, demonstrating proof-of-principle for drug-DNA adducts as predictive biomarkers. Mol Cancer Ther; 16(2); 376-87. ©2016 AACR., (©2016 American Association for Cancer Research.)

Platinum-based drugs (cisplatin, carboplatin, and oxaliplatin) are widely used therapeutic agents for cancer treatment. Even though the platinum (Pt)-drugs are routinely used clinically, a clear picture of their distribution within tumor tissues is lacking. The current methods to image the distribution of Pt drugs are limited and do not enable the discrimination of the drug from its metabolites. In this manuscript, we demonstrate a methodology that enables chemical imaging of a Pt drug and its metabolites simultaneously and specifically. Matrix-Assisted Laser Desorption/Ionization (MALDI) Mass Spectrometry Imaging (MSI) is combined with an on-tissue chemical derivatization using diethyldithiocarbamate (DDTC). DDTC abstracts the Pt atom to generate ionizable complexes that can be imaged by MALDI MSI. We demonstrate that Pt drugs and their metabolites can be specifically imaged. This approach was successfully applied to map the penetration and metabolism of oxaliplatin in hyperthermic intraperitoneal chemotherapy (HIPEC)-like treated 3D colorectal tumor mimics. The distribution of cisplatin and carboplatin was mapped in additional 3D tumor mimics. We demonstrate that the approach can also be used to image the distribution of copper ions in cells. This method has the potential to be used to evaluate the penetration and distribution of a wide range of compounds.

The study of the distribution of the cytostatic drugs cisplatin, carboplatin, and oxaliplatin along the kidney may help to understand their different nephrotoxic behavior. Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) allows the acquisition of trace element images in biological tissues. However, results obtained are affected by several variations concerning the sample matrix and instrumental drifts. In this work, an internal standardization method based on printing an Ir-spiked ink onto the surface of the sample has been developed to evaluate the different distributions and accumulation levels of the aforementioned drugs along the kidney of a rat model. A conventional ink-jet printer was used to print fresh sagittal kidney tissue slices of 4 μm. A reproducible and homogenous deposition of the ink along the tissue was observed. The ink was partially absorbed on top of the tissue. Thus, this approach provides a pseudo-internal standardization, due to the fact that the ablation sample and internal standard take place subsequently and not simultaneously. A satisfactory normalization of LA-ICP-MS bioimages and therefore a reliable comparison of the kidney treated with different Pt-based drugs were achieved even for tissues analyzed on different days. Due to the complete ablation of the sample, the transport of the ablated internal standard and tissue to the inductively coupled plasma-mass spectrometry (ICP-MS) is practically taking place at the same time. Pt accumulation in the kidney was observed in accordance to the dosages administered for each drug. Although the accumulation rate of cisplatin and oxaliplatin is high in both cases, their Pt distributions differ. The strong nephrotoxicity observed for cisplatin and the absence of such side effect in the case of oxaliplatin could explain these distribution differences. The homogeneous distribution of oxaliplatin in the cortical and medullar areas could be related with its higher affinity for cellular transporters such as MATE2-k.

We report herein the development, functional and molecular characterization of an isogenic, paired bladder cancer cell culture model system for studying platinum drug resistance. The 5637 human bladder cancer cell line was cultured over ten months with stepwise increases in oxaliplatin concentration to generate a drug resistant 5637R sub cell line. The MTT assay was used to measure the cytotoxicity of several bladder cancer drugs. Liquid scintillation counting allowed quantification of cellular drug uptake and efflux of radiolabeled oxaliplatin and carboplatin. The impact of intracellular drug inactivation was assessed by chemical modulation of glutathione levels. Oxaliplatin- and carboplatin-DNA adduct formation and repair was measured using accelerator mass spectrometry. Resistance factors including apoptosis, growth factor signaling and others were assessed with RNAseq of both cell lines and included confirmation of selected transcripts by RT-PCR. Oxaliplatin, carboplatin, cisplatin and gemcitabine were significantly less cytotoxic to 5637R cells compared to the 5637 cells. In contrast, doxorubicin, methotrexate and vinblastine had no cell line dependent difference in cytotoxicity. Upon exposure to therapeutically relevant doses of oxaliplatin, 5637R cells had lower drug-DNA adduct levels than 5637 cells. This difference was partially accounted for by pre-DNA damage mechanisms such as drug uptake and intracellular inactivation by glutathione, as well as faster oxaliplatin-DNA adduct repair. In contrast, both cell lines had no significant differences in carboplatin cell uptake, efflux and drug-DNA adduct formation and repair, suggesting distinct resistance mechanisms for these two closely related drugs. The functional studies were augmented by RNAseq analysis, which demonstrated a significant change in expression of 83 transcripts, including 50 known genes and 22 novel transcripts. Most of the transcripts were not previously associated with bladder cancer chemoresistance. This model system and the associated phenotypic and genotypic data has the potential to identify some novel details of resistance mechanisms of clinical importance to bladder cancer.

This rapid report focuses on the pharmacodynamic mechanism of the carboplatin/paclitaxel combination and correlates it with its cytotoxicity. Consistent with the synergistic to additive antitumor activity (the combination index ranging from 0.53 to 0.94), cells exposed to this combination had significantly increased carboplatin-DNA adduct formation when compared to that of carboplatin alone (450 ± 30 versus 320 ± 120 adducts per 10(8) nucleotides at 2 h, p = 0.004). Removal of paclitaxel increased the repair of carboplatin-DNA adducts: 39.4 versus 33.1 adducts per 10(8) nucleotides per hour in carboplatin alone (p = 0.021). This rapid report provides the first pharmacodynamics data to support the use of carboplatin/paclitaxel combination in the clinic.

An increasing number of genetic variants have been implicated in autism spectrum disorders (ASDs), and the functional study of such variants will be critical for the elucidation of autism pathophysiology. Here, we report a de novo balanced translocation disruption of TRPC6, a cation channel, in a non-syndromic autistic individual. Using multiple models, such as dental pulp cells, induced pluripotent stem cell (iPSC)-derived neuronal cells and mouse models, we demonstrate that TRPC6 reduction or haploinsufficiency leads to altered neuronal development, morphology and function. The observed neuronal phenotypes could then be rescued by TRPC6 complementation and by treatment with insulin-like growth factor-1 or hyperforin, a TRPC6-specific agonist, suggesting that ASD individuals with alterations in this pathway may benefit from these drugs. We also demonstrate that methyl CpG binding protein-2 (MeCP2) levels affect TRPC6 expression. Mutations in MeCP2 cause Rett syndrome, revealing common pathways among ASDs. Genetic sequencing of TRPC6 in 1041 ASD individuals and 2872 controls revealed significantly more nonsynonymous mutations in the ASD population, and identified loss-of-function mutations with incomplete penetrance in two patients. Taken together, these findings suggest that TRPC6 is a novel predisposing gene for ASD that may act in a multiple-hit model. This is the first study to use iPSC-derived human neurons to model non-syndromic ASD and illustrate the potential of modeling genetically complex sporadic diseases using such cells.

Objective: Cervical cancer is the most common solid cancer diagnosed in pregnancy. Platinum is an active drug in the treatment of patients with cervical cancer. In the second and third trimesters, platinum is used to prevent cancer progression until fetal maturity is reached. However, knowledge about the transplacental passage of platinum is very limited., Study Design: Between May 2008 and June 2014, platinum-based neoadjuvant chemotherapy was applied to 21 consecutive patients with cervical cancer diagnosed in their second trimester. At the time of delivery by cesarean delivery, synchronous samples from maternal blood, umbilical cord blood, and amniotic fluid were taken and analyzed for platinum concentrations., Results: The mean week of gestation at cancer diagnosis was 17 (13-23). On average 3 (range, 2-4) cycles of chemotherapy were applied. Cesarean deliveries were carried out between 30.4 and 36.5 weeks of gestation. Twenty-two healthy babies without renal, hepatic, auditory, or hematopoietic impairment were delivered. Platinum concentrations in umbilical cord blood and amniotic fluid were 23-65% and 11-42% of the maternal blood, respectively., Conclusion: This series on in vivo measurement of platinum concentrations in the fetomaternal compartment observed that because of consistently lower platinum values in the fetoplacental unit, a placental filtration mechanism of platinum may be assumed., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Purpose: Preparation of Nanographene oxide (NGO) - Gelatin hybrids for efficient treatment of Neuroblastoma., Methods: Nanohybrids were prepared via non-covalent interactions. Spectroscopic tools have been used to discriminate the chemical states of NGO prior and after gelatin coating, with UV visible spectroscopy revealing the maximum binding capacity of gelatin to NGO. Raman and X-ray photoelectron spectroscopy (XPS) demonstrated NGO and Gelatin&#95;NGO nanohybrids through a new chemical environments produced after noncovalent interaction. Microscopic analyses, atomic force microscopy (AFM) and scanning electron microscopy (SEM) are used to estimate the thickness of samples and the lateral width in the nanoscale, respectively., Results: The cell viability assay validated Gelatin&#95;NGO nanohybrids as a useful nanocarrier for Carboplatin (CP) release and delivery, without obvious signs of toxicity. The nano-sized NGO (200 nm and 300 nm) did not enable CP to kill the cancer cells efficiently, whilst the CP loaded Gel&#95;NGO 100 nm resulted in a synergistic activity through increasing the local concentration of CP inside the cancer cells., Conclusions: The nanohybrids provoked high stability and dispersibility in physiological media, as well as enhanced the anticancer activity of the chemotherapy agent Carboplatin (CP) in human neuroblastoma cells.

Platinum drugs are potent chemotherapeutic agents widely used in cancer therapy. They exert their biological activity by binding to DNA, producing DNA adducts; however, in the cell nucleus, DNA is complexed with histone proteins into a nucleoprotein structure known as chromatin. The aim of this study was to explore the binding affinity of oxaliplatin and carboplatin to chromatin using spectroscopic as well as thermal denaturation and equilibrium dialysis techniques. The results showed that the drugs quenched with chromophores of chromatin and the quenching effect for oxaliplatin (Ksv = 3.156) was higher than carboplatin (Ksv = 0.28). The binding of the drugs exhibited hypochromicity both in thermal denaturation profiles and UV absorbance at 210 nm. The binding was positive cooperation with spontaneous reaction and oxaliplatin (Ka = 5.3 × 10(3) M(-1), n = 1.7) exhibited higher binding constant and number of binding sites than carboplatin (Ka = 0.33 × 10(3) M(-1), n = 1.0) upon binding to chromatin. Also secondary structure of chromatin proteins was altered upon drugs binding. It is concluded that oxaliplatin represents higher binding affinity to chromatin compared to carboplatin. In chromatin where DNA is compacted into nucleosomes structure with histones, the affinity of the platinated drugs is reduced and histone proteins may play a fundamental role in this binding process., (Copyright © 2014. Published by Elsevier Masson SAS.)

Background: Chemotherapy response in ovarian cancer patients is frequently compromised by drug resistance, possibly due to altered drug metabolism. Platinum drugs are metabolised by glutathione S-transferase P1 (GSTP1), which is abundantly, but variably expressed in ovarian tumours. We have created novel ovarian tumour cell line models to investigate the extent to which differential GSTP1 expression influences chemosensitivity., Methods: Glutathione S-transferase P1 was stably deleted in A2780 and expression significantly reduced in cisplatin-resistant A2780DPP cells using Mission shRNA constructs, and MTT assays used to compare chemosensitivity to chemotherapy drugs used to treat ovarian cancer. Differentially expressed genes in GSTP1 knockdown cells were identified by Illumina HT-12 expression arrays and qRT-PCR analysis, and altered pathways predicted by MetaCore (GeneGo) analysis. Cell cycle changes were assessed by FACS analysis of PI-labelled cells and invasion and migration compared in quantitative Boyden chamber-based assays., Results: Glutathione S-transferase P1 knockdown selectively influenced cisplatin and carboplatin chemosensitivity (2.3- and 4.83-fold change in IC50, respectively). Cell cycle progression was unaffected, but cell invasion and migration was significantly reduced. We identified several novel GSTP1 target genes and candidate platinum chemotherapy response biomarkers., Conclusions: Glutathione S-transferase P1 has an important role in cisplatin and carboplatin metabolism in ovarian cancer cells. Inter-tumour differences in GSTP1 expression may therefore influence response to platinum-based chemotherapy in ovarian cancer patients.

Carboplatin is a second-generation platinum anticancer agent used for the treatment of a variety of cancers. Previous X-ray crystallographic studies of carboplatin binding to histidine (in hen egg-white lysozyme; HEWL) showed the partial conversion of carboplatin to cisplatin owing to the high NaCl concentration used in the crystallization conditions. HEWL co-crystallizations with carboplatin in NaBr conditions have now been carried out to confirm whether carboplatin converts to the bromine form and whether this takes place in a similar way to the partial conversion of carboplatin to cisplatin observed previously in NaCl conditions. Here, it is reported that a partial chemical transformation takes place but to a transplatin form. Thus, to attempt to resolve purely carboplatin binding at histidine, this study utilized co-crystallization of HEWL with carboplatin without NaCl to eliminate the partial chemical conversion of carboplatin. Tetragonal HEWL crystals co-crystallized with carboplatin were successfully obtained in four different conditions, each at a different pH value. The structural results obtained show carboplatin bound to either one or both of the N atoms of His15 of HEWL, and this particular variation was dependent on the concentration of anions in the crystallization mixture and the elapsed time, as well as the pH used. The structural details of the bound carboplatin molecule also differed between them. Overall, the most detailed crystal structure showed the majority of the carboplatin atoms bound to the platinum centre; however, the four-carbon ring structure of the cyclobutanedicarboxylate moiety (CBDC) remained elusive. The potential impact of the results for the administration of carboplatin as an anticancer agent are described.

The X-ray structure of the adduct formed between oxaliplatin and the model protein hen egg white lysozyme is reported here. The structure is compared with those of cisplatin and carboplatin derivatives, previously solved. Relevant changes are highlighted among these crystal structures that are suggestive of significant differences in the reactivity of platinum drugs with this protein; possible biological implications are discussed.

Cisplatin, carboplatin, and oxaliplatin represent three generations of platinum based drugs applied successfully for cancer treatment. As a consequence of the employment of platinum based cytostatics in the cancer treatment, it became necessary to study the mechanism of their action. Current accepted opinion is the formation of Pt-DNA adducts, but the mechanism of their formation is still unclear. Nanomaterials, as a progressively developing branch, can offer a tool for studying the interactions of these drugs with DNA. In this study, fluorescent CdTe quantum dots (QDs, λem = 525 nm) were employed to investigate the interactions of platinum cytostatics (cisplatin, carboplatin, and oxaliplatin) with DNA fragment (500 bp, c = 25 μg/mL). Primarily, the fluorescent behavior of QDs in the presence of platinum cytostatics was monitored and major differences in the interaction of QDs with tested drugs were observed. It was found that the presence of carboplatin (c = 0.25 mg/mL) had no significant influence on QDs fluorescence; however cisplatin and oxaliplatin quenched the fluorescence significantly (average decrease of 20%) at the same concentration. Subsequently, the amount of platinum incorporated in DNA was determined by QDs fluorescence quenching. Best results were reached using oxaliplatin (9.4% quenching). Linear trend (R(2) = 0.9811) was observed for DNA platinated by three different concentrations of oxaliplatin (0.250, 0.125, and 0.063 mg/mL). Correlation with differential pulse voltammetric measurements provided linear trend (R(2) = 0.9511). As a conclusion, especially in the case of oxaliplatin-DNA adducts, the quenching was the most significant compared to cisplatin and nonquenching carboplatin., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Dorsal root ganglion (DRG) neurons are affected by platinum-induced neurotoxicity and neurodegenerative processes associated with disturbed copper homeostasis and transport. This study aimed to understand the role of copper transporter 1 (Ctr1) in the uptake and toxicity of copper and platinum drugs in cultured rat DRG neurons, and the functional activities of rat Ctr1 (rCtr1) as a membrane transporter of copper and platinum drugs. Heterologous expression of rCtr1 in HEK293 cells (HEK/rCtr1 cells) increased the uptake and cytotoxicity of copper, oxaliplatin, cisplatin and carboplatin, in comparison to isogenic vector-transfected control cells. Cultured rat DRG neurons endogenously expressed rCtr1 protein on their neuronal cell body plasma membranes and cytoplasm, and displayed substantial capacity for taking up copper, but were resistant to copper toxicity. The uptake of copper by both cultured rat DRG neurons and HEK/rCtr1 cells was saturable and inhibited by cold temperature, silver and zinc, consistent with it being mediated by rCtr1. Cultured rat DRG neurons accumulated platinum during their exposure to oxaliplatin and were sensitive to oxaliplatin cytotoxicity. The accumulation of platinum by both cultured rat DRG neurons and HEK/rCtr1 cells, during oxaliplatin exposure, was saturable and temperature dependent, but was inhibited by copper only in HEK/rCtr1 cells. In conclusion, rCtr1 can transport copper and platinum drugs, and sensitizes cells to their cytotoxicities. DRG neurons display substantial capacity for accumulating copper via a transport process mediated by rCtr1, but appear able to resist copper toxicity and use alternative mechanisms to take up oxaliplatin., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The anticancer agents cisplatin and carboplatin bind to histidine in a protein. This crystal structure study at data-collection temperatures of 100 and 300 K examines their relative binding affinities to a histidine side chain and the effect of a high X-ray radiation dose of up to ∼1.8 MGy on the stability of the subsequent protein-Pt adducts. Cisplatin binding is visible at the histidine residue, but carboplatin binding is not. Five refined X-ray crystal structures are presented: one at 100 K as a reference and four at 300 K. The diffraction resolutions are 1.8, 2.0, 2.8, 2.9 and 3.5 Å.

The interaction of DNA and Carboplatin was studied with DNA labeled gold nanoparticles (AuNPs) based optical nanobiosensor. Carboplatin, a cytotoxic drug, is responsible for producing nephrotoxicity at effective dose. Thus, we have developed optical nanobiosensor for monitoring carboplatin-DNA interaction based on Fluorescence Resonance Energy Transfer (FRET) phenomenon. Paracetamol, an analgesic agent, was used as controlled drug in this study. The DNA labeled AuNPs, exposed to carboplatin, a binding event among the DNA and carboplatin takes place, resulting in a conformational change within the biosensor complex which decreases the distance among the fluorescent molecules or the fluorescent/quencher molecules. As the carboplatin interact with DNA, an increase in fluorescence intensity was observed. So, the major difference in increased fluorescence intensity between carboplatin-DNA and paracetamol-DNA interaction shows significant observations. Results have demonstrated that Optical sensor is able to rapidly and effectively monitor carboplatin-DNA interaction with a detection limit up to 0.45 μg/ml. This suggests that the developed optical nanobiosensor was ideal for monitoring Drug-DNA interaction studies while performing combinatorial synthesis for new drug development., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Purpose: To evaluate the ability of a physiology-based pharmacokinetic (PBPK) model to predict the systemic drug exposure of high- and low-dose etoposide in children from a model developed with adult data., Methods: Simulations were performed with PK-Sim(®) (Bayer Technology Services). Model development was done using data from adult patients receiving etoposide in a conventional and high-dose polychemotherapy regimen before stem cell transplantation. Michaelis-Menten parameters from in vitro experiments reported in the literature were applied to describe the metabolism and excretion processes by P450 enzymes and transporters. The model was scaled down to children and compared to etoposide plasma concentrations in this age group., Results: Simulated plasma concentration-time courses of protein-bound and free etoposide in adults for high- and low-dose schedules agreed with the observed data. Mean simulated total clearance of high- and low-dose etoposide was 0.70 ml/min/kg (Cl(observed): 0.70 ml/min/kg) versus 0.50 ml/min/kg (Cl(observed): 0.60 ml/min/kg), respectively. Integrated Michaelis-Menten kinetics was adequately transformed to age-related pharmacokinetics in children. Predictions of the pharmacokinetics in different age groups were also in good agreement with observed data. Drug interactions triggered by P-glycoprotein inhibitors or nephrotoxic drugs can also be elucidated., Conclusions: The PBPK model matched the pharmacokinetics in different dosing regimens in adults. Furthermore, the scaling procedure from the adult model to children provides useful predictions for paediatric patients. Comedication with drugs influencing the metabolism and excretion has to be taken into account. This approach could be useful for planning pharmacokinetic studies in children.

Despite an increasing understanding of the molecular mechanisms by which platinum drug DNA adducts interact with cellular processes, the relationship between adduct formation in tumours and clinical response remains unclear. We have determined carboplatin-DNA adduct levels in biopsies removed from ovarian cancer patients following treatment. Reliability of DNA adduct measurements in tissues samples were assessed using experimental animals. Platinum-DNA adduct levels were measured using inductively coupled plasma mass spectrometry (ICP-MS) and plasma drug concentrations determined by atomic absorption spectrometry (AAS). Adduct levels in tissues and plasma pharmacokinetics were determined in Balb/c mice exposed to platinum drugs. Comparisons of adduct levels in tumour and normal tissue were made in nu/nu mice carrying human neuroblastoma xenografts. At 30 min post-cisplatin administration, adduct levels in DNA from kidney and liver were approximately 10- and 6-fold higher than spleen or tumour. By 60 min, levels in liver and kidney, but not spleen or tumour, had fallen considerably. Carboplatin showed high adduct levels only in kidney. Adduct levels in tumour xenografts were comparable to those induced in vitro with similar drug exposures. In clinical samples removed 6h after drug administration, adduct levels ranged from 1.9 to 4.3 and 0.2 to 3.6 nmol Pt/g DNA for tumour biopsies and peripheral blood mononuclear cells, respectively. No correlation was apparent between these two data sets. The present results demonstrate that reliable measurements of adducts in clinical tumours are feasible. Future results should provide insight into drug resistance., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The influence of genetic variants on paclitaxel-induced toxicity is of considerable interest for reducing adverse drug reactions. Recently, the genetic variants CYP2C8*3, CYP2C8-HapC, and CYP3A5*3 were associated with paclitaxel-induced neurotoxicity. We, therefore, investigated the impact of CYP2C8-HapC and CYP3A5*3 on paclitaxel/carboplatin-induced myelosuppression and neurotoxicity. Thirty-three patients from a prospective pharmacokinetics study were genotyped using pyrosequencing. Patients with variant alleles of CYP2C8-HapC were found to have significantly lower nadir values of both leukocytes and neutrophils (p < 0.05) than patients with the wild-type genotype. CYP3A5*3/*1 patients were shown to have borderline, significantly lower nadir values of leukocytes (p = 0.07) than *3/*3 patients. Combining the two genotypes resulted in a significant correlation with both leukopenia and neutropenia (p = 0.01). No effect of these genetic variants on neurotoxicity could be shown in this rather small study, but their importance for paclitaxel-induced toxicity could be confirmed., (Copyright © 2011 Wiley-Liss, Inc.)

Formation and repair of platinum (Pt)-induced DNA adducts is a critical step in Pt drug-mediated cytotoxicity. Measurement of Pt-DNA adduct kinetics in tumors may be useful for better understanding chemoresistance and therapeutic response. However, this concept has yet to be rigorously tested because of technical challenges in measuring the adducts at low concentrations and consistent access to sufficient tumor biopsy material. Ultrasensitive accelerator mass spectrometry was used to detect [(14)C]carboplatin-DNA monoadducts at the attomole level, which are the precursors to Pt-DNA crosslink formation, in six cancer cell lines as a proof-of-concept. The most resistant cells had the lowest monoadduct levels at all time points over 24 hr. [(14)C]Carboplatin "microdoses" (1/100th the pharmacologically effective concentration) had nearly identical adduct formation and repair kinetics compared to therapeutically relevant doses, suggesting that the microdosing approach can potentially be used to determine the pharmacological effects of therapeutic treatment. Some of the possible chemoresistance mechanisms were also studied, such as drug uptake/efflux, intracellular inactivation and DNA repair in selected cell lines. Intracellular inactivation and efficient DNA repair each contributed significantly to the suppression of DNA monoadduct formation in the most resistant cell line compared to the most sensitive cell line studied (p < 0.001). Nucleotide excision repair (NER)-deficient and -proficient cells showed substantial differences in carboplatin monoadduct concentrations over 24 hr that likely contributed to chemoresistance. The data support the utility of carboplatin microdosing as a translatable approach for defining carboplatin-DNA monoadduct formation and repair, possibly by NER, which may be useful for characterizing chemoresistance in vivo., (Copyright © 2010 UICC.)

The platination processes of DNA bases with second- and third-generation Pt(II) anticancer drugs have been investigated using density functional theory (DFT) combined with the conductor-like dielectric continuum model (CPCM) approach, in order to describe their binding mechanisms and to obtain detailed data on the reaction energy profiles. Although there is no doubt that a Pt-N7 bond forms during initial attack, the energetic profiles for the formation of the monofunctional adducts are not known. Herein, a direct comparison between the rate of formation of the monofunctional adducts of the second- and third-generation anticancer drugs with guanine (G) and adenine (A) DNA bases has been made in order to spotlight possible common or different behavior. The guanine as target for platination process is confirmed to be preferred over adenine for all the investigated compounds and for both the hydrolyzed forms considered in our investigation. The preference for G purine base is dominated by electronic factors and promoted by a more favorable hydrogen-bonds pattern, confirming the important role played by H-bonds in determining both structural and kinetic control on the purine platination process., (© 2011 American Chemical Society)

We present highly sensitive, rapid methods for the determination of Pt originating from carboplatin in human urine and canine urine, feces, and oral fluid. The methods are based on the quantification of Pt by inductively coupled plasma mass spectrometry, and allow quantification of 7.50 ng/L Pt in human and canine urine (in 15 μL of matrix), 15.0 ng/L Pt in canine oral fluid (in 15 μL of matrix), and 0.105 ng/g Pt in canine feces (in 5 μg of matrix). Sample pretreatment mainly involved dilution with appropriate diluents. The performance of the methods fulfilled the most recent FDA guidelines for bioanalytical method validation. Validated ranges of quantification were 7.50 to 1.00 × 10(4) ng/L Pt in human and canine urine, 0.105-30.0 ng/g Pt in canine feces, and 15.0 to 1.00 × 10(4) ng/L Pt in canine oral fluid. Canine urine and oral fluid cannot be easily obtained. Therefore, we also investigated the validity of the usage of human matrix samples for the preparation of calibration standards and quality control samples as alternatives, to be used in future clinical studies. The assays are used to support biomonitoring studies and pharmacokinetic studies in pet dogs treated with carboplatin.

Platinum agents have been widely used in cancer chemotherapy for a long time. Cisplatin, carboplatin, oxaliplatin and nedaplatin have a common chemical structure consisting of platinum, carrier groups and leaving groups, and undergo the similar mechanism of cytotoxicity. However, each agent differs in its efficacy and adverse effects, although the molecular mechanism involved is unclear. Recently, it was reported that organic cation transporter OCT/SLC22A, and multidrug and toxin extrusion MATE/SLC47A play a role in the pharmacokinetics of platinum agents. Only cisplatin induces nephrotoxicity and the toxicity is kidney-specific. Kidney-specific OCT2 mediates the transport of cisplatin and is the determinant of cisplatin-induced nephrotoxicity. In addition, cisplatin and oxaliplatin are substrates for these transporters, but carboplatin and nedaplatin are not. Substrate specificity could regulate the features of platinum agents. In this commentary, we will discuss the characteristics of OCT and MATE, and demonstrate the recent topics about the relationship between the transport of platinum agents by organic cation transporters and their pharmacological characteristics., (Copyright © 2010 Elsevier Inc. All rights reserved.)

This trial aimed to define a recommended safe dose (RSD) of weekly paclitaxel and irinotecan combined with carboplatin in patients with advanced cancer. Patients with advanced cancer were eligible for this trial. Dose-limiting toxicity (DLT) was considered to be any grade greater than or equal to 3 (G> or =3) nonhematological toxicity except nausea/vomiting, G4 hematological toxicity of more than 4 days without recombinant human granulocyte colony-stimulating factor support, concurrent diarrhea G> or =2 and neutropenia G> or =3, and a treatment delay for more than 14 days because of toxicity. Patients were given carboplatin area under the curve (AUC) 5 mg*min/ml on day 1 combined with irinotecan and paclitaxel on days 1 and 8, every 3 weeks. The starting dose of both irinotecan and paclitaxel was 50 mg/m and a toxicity-guided escalation/de-escalation was planned by 10 mg/m steps. Sixteen patients were enrolled. DLTs occurred in three of the four patients treated at the starting dose level, which defined that dose as the maximum tolerated dose. Accrual continued with irinotecan and paclitaxel doses, which were de-escalated by one step. At this dose level, two of the 12 patients developed DLT, which defined that dose as the RSD. We concluded that the maximum tolerated dose of weekly irinotecan and paclitaxel when given in combination with carboplatin AUC 5 mg*min/ml was 50 mg/m and the RSD 40 mg/m. DLTs were febrile neutropenia, concurrent neutropenia (G3) and diarrhea (G3), and prolonged treatment delay because of toxicity. The most common non-DLT G3/G4 toxicity was leukopenia and neutropenia (18%), and thrombocytopenia and diarrhea (6%). A patient with metastatic endometrial carcinoma treated at the RSD had a compete response of retroperitoneal lymph node metastases, lasting for more than 3 years. Two other patients had their minimal tumor shrinkage documented. Paclitaxel (40 mg/m) and irinotecan (40 mg/m) can safely be administered on days 1 and 8 in combination with carboplatin AUC 5 mg*min/ml given on day 1. At the recommended doses this is a well-tolerated regimen with noticeable antitumor activity and warrants further investigation in phase II studies.

We have investigated the processing of site-specific Pt-DNA cross-links in live mammalian cells to enhance our understanding of the mechanism of action of platinum-based anticancer drugs. The activity of platinum drugs against cancer is mediated by a combination of processes including cell entry, drug activation, DNA-binding, and transcription inhibition. These drugs bind nuclear DNA to form Pt-DNA cross-links, which arrest key cellular functions, including transcription, and trigger a variety of responses, such as repair. Mechanistic investigations into the processing of specific Pt-DNA cross-links are critical for understanding the effects of platinum-DNA damage, but conventional in vitro techniques do not adequately account for the complex and intricate environment within a live cell. With this limitation in mind, we developed a strategy to study platinum cross-links on plasmid DNAs transfected into live mammalian cells based on luciferase reporter vectors containing defined platinum-DNA lesions that are either globally or site-specifically incorporated. Using cells with either competent or deficient nucleotide excision repair systems, we demonstrate that Pt-DNA cross-links impede transcription by blocking passage of the RNA polymerase complex and that nucleotide excision repair can remove the block and restore transcription. Results are presented for approximately 3800-base pair plasmids that are either globally platinated or carry a single 1,2-d(GpG) or 1,3-d(GpTpG) intrastrand cross-link formed by either cis-{Pt(NH(3))(2)}(2+) or cis-{Pt(R,R-dach)}(2+), where {Pt(NH(3))(2)}(2+) is the platinum unit conveyed by cisplatin and carboplatin and R,R-dach is the oxaliplatin ligand, R,R-1,2-diaminocyclohexane.

Background: Glomerular filtration rate (GFR) is often used to determine initial dosing of renally excreted cancer drugs. GFR can be calculated using the Cockcroft-Gault (CG) or the modified diet in renal diseases (MDRD) study formulas, both of which are based on serum creatinine levels. The MDRD formula is more accurate in noncancer patients, does not require patient weight, and is reported automatically by all laboratories in British Columbia, Canada. We previously showed that the CG and MDRD formulas have similar accuracy for carboplatin dosing in patients with gynecological malignancies. We now examine dosing of all renally excreted cancer drugs in the general cancer population. Since this setting does not include routine measurement of GFR, we report the concordance of estimates of GFR derived from the CG and MDRD formulas., Methods: Patient data were collected retrospectively at the BC Cancer Agency. The primary outcome was the proportion of patients who would have received a different initial dose due to difference in the GFR. Each patient's dose was determined from dose adjustment tables stated in specific treatment protocols. The secondary outcome was concordance of the GFR derived from CG and MDRD, using the method of Bland and Altman. A difference of >30% was assumed to be clinically significant because this difference would usually lead to dose adjustment based on reclassification of renal function., Results: A total of 313 patients were evaluated, with 40% male. The median age was 56 years, weight 67.5 kg, height 166 cm, and serum creatinine 74 micromol/L (0.84 mg/dL). The median GFR derived from the CG and MDRD formulas were 86.8 mL/min (mean 91 mL/min, SD +/- 30 mL/min) and 87.6 mL/min (mean 88 mL/min, SD +/- 26 mL/min), respectively. A total of 8.6% (27/313) of patients would have received a different dose due to difference in the GFR; of these, 67% (18/27) would have received a higher dose. A difference of >30% in GFR was found in 17.9% (56/313) of patients., Conclusions: There is good concordance of the GFR derived from the CG and MDRD formulas for most cancer patients, with less than 10% of patients expected to receive a different initial dose of chemotherapy. The MDRD formula may be a reasonable alternative to the CG formula for dosing of cancer drugs which are renally excreted or nephrotoxic.

Roche Holding AG, and its subsidiaries Genentech Inc and Chugai Pharmaceutical Co Ltd, are developing trastuzumab emtansine (trastuzumab-DM1) for the treatment of HER2+ metastatic breast cancer. Trastuzumab emtansine is a tumor-activated prodrug resulting from the conjugation of the humanized anti-HER2 mAb trastuzumab, which has been used in the treatment of breast cancer for over 10 years, with ImmunoGen Inc's cytotoxic and antimitotic maytansine derivative DM1. The maytansinoids bind microtubules in a manner similar to the vinca alkaloids; however, maytansinoids have been recognized to be 20- to 100-fold more potent at blocking mitosis. Nevertheless, the use of these compounds as single agents is limited by toxicity. By conjugating DM1 with trastuzumab, the delivery of the cytotoxic agent to target cells is more specific and reduces the safety concerns. In preclinical studies, the conjugation was effective in breast cancer cell lines resistant to trastuzumab, and demonstrated complete tumor regression in SCID mice bearing KPL4 breast cancer xenografts. Clinically, trastuzumab emtansine exhibited efficacy in patients with HER2+ metastatic breast cancer who had progressed on previous chemotherapy regimens or with trastuzumab therapy. Furthermore, preclinical studies have reported that trastuzumab emtansine potentiates the effect of a number of chemotherapeutic agents (including carboplatin, 5-fluorouracil and docetaxel), other antibodies, receptor tyrosine kinase inhibitors and PI3K inhibitors, and many of these combinations are set to be tested in humans. Trastuzumab emtansine offers an exciting new option for the treatment of patients with refractory, metastatic breast cancer.

Background: Solute carriers (SLCs), in particular organic cation transporters (OCTs), have been implicated in the cellular uptake of platinum-containing anticancer compounds. The activity of these carriers may determine the pharmacokinetics and the severity of side effects, including neuro- and nephrotoxicity of platinum-based chemotherapy. As decreased drug accumulation is a key mechanism of platinum resistance, SLCs may also contribute to the development of resistance. Here, we define the role of hSLC22A2 (OCT2) in the cellular uptake of platinum compounds., Experimental Approach: Human embryonic kidney (HEK) 293 cells stably expressing the hSLC22A2 gene (HEK293/hSLC22A2) were used in platinum accumulation studies. Following a 2 h exposure to various platinum compounds (100 microM), intracellular platinum levels were determined by flameless atomic absorption spectrometry., Key Results: HEK293/hSLC22A2 cells, compared with HEK293/Neo control cells, displayed significant increases in oxaliplatin (28.6-fold), Pt[DACH]Cl(2) (20.6-fold), ormaplatin (8.1-fold), tetraplatin (4.5-fold), transplatin (3.7-fold) and cisplatin (1.3-fold), but not carboplatin. SLC22A2-mediated transport could be inhibited by 1-methyl-4-phenylpyridinium. Furthermore, hSLC22A2-mediated oxaliplatin and cisplatin accumulation was time- and concentration-dependent, but non-saturable. Expression of hSLC22A2 in HEK293 cells resulted in enhanced sensitivity to oxaliplatin (12-fold) and cisplatin (1.8-fold). Although, hSLC22A2 mRNA expression was frequently found in ovarian cancer cell lines, its expression in clinical ovarian cancer specimens (n= 80) was low and did not correlate with the treatment outcome of platinum-based regimens., Conclusions and Implications: The hSLC22A2 drug transporter is a critical determinant in the uptake and cytotoxicity of various platinum compounds, particularly oxaliplatin.

Besides various side effects caused by platinum anticancer drugs, they are not efficiently absorbed by the tumor cells. Two Pt-peptide conjugates; cyclic mPeg-CNGRC-Pt (7) and cyclic mPeg-CNGRC-Pten (8) bearing the Asn-Gly-Arg (NGR) targeting sequence, a malonoyl linker, and low molecular weight miniPEG groups have been synthesized. The platinum ligand was attached to the peptide via the carboxylic end of the malonate group at the end of the peptide. The pegylated peptide is nontoxic and highly soluble in water. Platinum conjugates synthesized using the pegylated peptides are also water-soluble with reduced or eliminated peptide immunogenicity. The choice of carboplatin as our untargeted platinum complex was due to the fact that the malonate linker chelates platinum in a manner similar to that of carboplatin. Cell toxicity assay and competition assay on the PC-3 cells (CD13 positive receptors) revealed selective delivery and destruction of PC-3 cells using targeted Pt-peptide conjugates 7 and 8 significantly more than untargeted carboplatin. Platinum uptake on PC-3 cells was 12-fold more for conjugate 7 and 3-fold more for conjugate 8 compared to that of the untargeted carboplatin, indicating selective activation of the CD13 receptors and delivery of the conjugates to CD13 positive cells. Further analysis on effects of conjugates 7 and 8 on PC-3 cells using caspase-3/7, fluorescence microscopy, and DNA fragmentation confirmed that the cells were dying by apoptosis.

Objective: To determine whether a glomerular filtration rate (GFR) assay based on serum iohexol clearance can be used to predict carboplatin clearance in cats., Animals: 10 cats with tumors., Procedures: GFR was measured concurrently by use of plasma clearance of technetium Tc 99m-labeled diethylenetriaminepentaacetic acid ((99m)Tc-DTPA) to yield GFR(99mTc-DTPA) and serum clearance of iohexol to yield GFR(Iohexol). A single dose of carboplatin was administered IV as a bolus. Dose was calculated by use of a target value for the area under the plasma platinum concentration-versus-time curve (AUC(Target)) and estimation of platinum clearance (CL(PT)) derived from GFR(99mTc-DTPA) as follows: dose = AUC(Target) x 2.6 x GFR(99mTc-DTPA) x body weight, where AUC(Target) is 2.75 min.mg.mL(-1). Plasma platinum concentrations were measured via atomic absorption spectrophotometry. Values for GFR(99mTc-DTPA) and GFR(Iohexol) were compared by use of least-squares regression and Bland-Altman analysis. Least-squares regression was used to determine whether CL(PT) could be predicted from GFR(99mTc-DTPA) or GFR(Iohexol) (or both)., Results: GFR(99mTc-DTPA) and GFR(Iohexol) were strongly correlated (r = 0.90), but GFR(Iohexol) values were significantly larger by a factor of approximately 1.4. Platinum clearance had a significant linear relationship to GFR(99mTc-DTPA) (CL(PT) = 2.5 x GFR(99mTc-DTPA)) and to GFR(Iohexol) (CL(PT) = [1.3 x GFR(Iohexol)] + 1.4)., Conclusions and Clinical Relevance: In cats, serum iohexol clearance was an accurate predictor of CL(PT) and can be used to calculate the carboplatin dose as follows: dose = AUC(Target) x ([1.3 x GFR(Iohexol)] + 1.4) x body weight.