Relation between DNA damage measured by comet assay and OGG1 Ser326Cys polymorphism in antineoplastic drugs biomonitoring

Antineoplastic drugs are hazardous chemical agents used mostly in the treatment of patients with cancer, however health professionals that handle and administer these drugs can become exposed and develop DNA damage. Comet assay is a standard method for assessing DNA damage in human biomonitoring and, combined with formamidopyrimidine DNA glycosylase (FPG) enzyme, it specifically detects DNA oxidative damage. The aim of this study was to investigate genotoxic effects in workers occupationally exposed to cytostatics (n = 46), as compared to a control group with no exposure (n = 46) at two Portuguese hospitals, by means of the alkaline comet assay. The potential of the OGG1 Ser326Cys polymorphism as a susceptibility biomarker was also investigated. Exposure was evaluated by investigating the contamination of surfaces and genotoxic assessment was done by alkaline comet assay in peripheral blood lymphocytes. OGG1 Ser326Cys (rs1052133) polymorphism was studied by Real Time PCR. As for exposure assessment, there were 121 (37%) positive samples out of a total of 327 samples analysed from both hospitals. No statistically significant differences (Mann-Whitney test, p > 0.05) were found between subjects with and without exposure, regarding DNA damage and oxidative DNA damage, nevertheless the exposed group exhibited higher values. Moreover, there was no consistent trend regarding the variation of both biomarkers as assessed by comet assay with OGG1 polymorphism. Our study was not statistically significant regarding occupational exposure to antineoplastic drugs and genetic damage assessed by comet assay. However, health professionals should be monitored for risk behaviour, in order to ensure that safety measures are applied and protection devices are used correctly.

Comet assay as a human biomonitoring tool: application in occupational exposure to antineoplastic drugs

Antineoplastic drugs are a heterogeneous group of chemicals used in the treatment of cancer, and have been proved by IARC to be mutagens, carcinogens and teratogens agents. In general, chemicals that interact directly with DNA by biding covalently or by intercalating, or indirectly by interfering with DNA synthesis, were among the first chemotherapeutics developed. Also, these drugs can induce reactive oxygen species that can lead to DNA damage and, consequently, mutations. These drugs are often used in combination to achieve synergistic effects on tumour cells resulting from their differing modes of action. However, most if not all of these chemical agents are generally nonselective and, along with tumour cells, normal cells may undergo cytotoxic/genotoxic damage. The in vivo exposure to antineoplastic drugs has been shown to induce different types of lesions in DNA, depending on the particular stage of cell cycle at the time of treatment. Besides the patients that use these drugs as a treatment, workers that handle and/or administer these drugs can be exposed to these substances; namely pharmacy, and nursing personnel in hospital context.

Proteomics in the assessment of the therapeutic response of antineoplastic drugs: strategies and practical applications

Uncovering mechanisms of unknown pathological mechanisms and body response to applied medication are the drive forces toward personalized medicine. In this post-genomic era, all eyes are tuned to proteomic field, searching for the answers and explanations by investigating the final physiological functional units – proteins and their proteoforms. Development of cutting-edge mass spectrometric technologies and powerful bioinformatics tools, allowed life-science community mining of disease-specific proteins as biomarkers, which are often hidden by high complexity of the samples and/or small abundance. Nowadays, there are several proteomics-based approaches to study the proteome. This chapter focuses on gold standard proteomics strategies and related issues towards candidate biomarker discovery, which may have diagnostic/prognostic as well as mechanistic utility.

Task-based approach used on surfaces sampling strategy definition: the case of antineoplastic occupational exposure

Task-based approach implicates identifying all the tasks developed in each workplace aiming to refine the exposure characterization. The starting point of this approach is the recognition that only through a more detailed and comprehensive understanding of tasks is possible to understand, in more detail, the exposure scenario. In addition allows also the most suitable risk management measures identification. This approach can be also used when there is a need of identifying the workplace surfaces for sampling chemicals that have the dermal exposure route as the most important. In this case is possible to identify, through detail observation of tasks performance, the surfaces that involves higher contact (frequency) by the workers and can be contaminated. Identify the surfaces to sample when performing occupational exposure assessment to antineoplasic agents. Surfaces selection done based on the task-based approach.

Antioxidant And Antiproliferative Activities Of Methanolic Extract From A Neglected Agricultural Product: Corn Cobs.

Neglected agricultural products (NAPs) are defined as discarded material in agricultural production. Corn cobs are a major waste of agriculture maize. Here, a methanolic extract from corn cobs (MEC) was obtained. MEC contains phenolic compounds, protein, carbohydrates (1.4:0.001:0.001). We evaluated the in vitro and in vivo antioxidant potential of MEC. Furthermore, its antiproliferative property against tumor cells was assessed through MTT assays and proteins related to apoptosis in tumor cells were examined by western blot. MEC showed no hydroxyl radical scavenger capacity, but it showed antioxidant activity in Total Antioxidant Capacity and DPPH scavenger ability assays. MEC showed higher Reducing Power than ascorbic acid and exhibited high Superoxide Scavenging activity. In tumor cell culture, MEC increased catalase, metallothionein and superoxide dismutase expression in accordance with the antioxidant tests. In vivo antioxidant test, MEC restored SOD and CAT, decreased malondialdehyde activities and showed high Trolox Equivalent Antioxidant Capacity in animals treated with CCl4. Furthermore, MEC decreased HeLa cells viability by apoptosis due an increase of Bax/Bcl-2 ratio, caspase 3 active. Protein kinase C expression increased was also detected in treated tumor cells. Thus, our findings pointed out the biotechnological potential of corn cobs as a source of molecules with pharmacological activity.

The Effect Of Celastrol, A Triterpene With Antitumorigenic Activity, On Conformational And Functional Aspects Of The Human 90kda Heat Shock Protein Hsp90α, A Chaperone Implicated In The Stabilization Of The Tumor Phenotype.

Hsp90 is a molecular chaperone essential for cell viability in eukaryotes that is associated with the maturation of proteins involved in important cell functions and implicated in the stabilization of the tumor phenotype of various cancers, making this chaperone a notably interesting therapeutic target. Celastrol is a plant-derived pentacyclic triterpenoid compound with potent antioxidant, anti-inflammatory and anticancer activities; however, celastrol's action mode is still elusive. In this work, we investigated the effect of celastrol on the conformational and functional aspects of Hsp90α. Interestingly, celastrol appeared to target Hsp90α directly as the compound induced the oligomerization of the chaperone via the C-terminal domain as demonstrated by experiments using a deletion mutant. The nature of the oligomers was investigated by biophysical tools demonstrating that a two-fold excess of celastrol induced the formation of a decameric Hsp90α bound throughout the C-terminal domain. When bound, celastrol destabilized the C-terminal domain. Surprisingly, standard chaperone functional investigations demonstrated that neither the in vitro chaperone activity of protecting against aggregation nor the ability to bind a TPR co-chaperone, which binds to the C-terminus of Hsp90α, were affected by celastrol. Celastrol interferes with specific biological functions of Hsp90α. Our results suggest a model in which celastrol binds directly to the C-terminal domain of Hsp90α causing oligomerization. However, the ability to protect against protein aggregation (supported by our results) and to bind to TPR co-chaperones are not affected by celastrol. Therefore celastrol may act primarily by inducing specific oligomerization that affects some, but not all, of the functions of Hsp90α. To the best of our knowledge, this study is the first work to use multiple probes to investigate the effect that celastrol has on the stability and oligomerization of Hsp90α and on the binding of this chaperone to Tom70. This work provides a novel mechanism by which celastrol binds Hsp90α.

Predictive Value Of Phase I Trials For Safety In Later Trials And Final Approved Dose: Analysis Of 61 Approved Cancer Drugs.

Phase I trials use a small number of patients to define a maximum tolerated dose (MTD) and the safety of new agents. We compared data from phase I and registration trials to determine whether early trials predicted later safety and final dose. We searched the U.S. Food and Drug Administration (FDA) website for drugs approved in nonpediatric cancers (January 1990-October 2012). The recommended phase II dose (R2PD) and toxicities from phase I were compared with doses and safety in later trials. In 62 of 85 (73%) matched trials, the dose from the later trial was within 20% of the RP2D. In a multivariable analysis, phase I trials of targeted agents were less predictive of the final approved dose (OR, 0.2 for adopting ± 20% of the RP2D for targeted vs. other classes; P = 0.025). Of the 530 clinically relevant toxicities in later trials, 70% (n = 374) were described in phase I. A significant relationship (P = 0.0032) between increasing the number of patients in phase I (up to 60) and the ability to describe future clinically relevant toxicities was observed. Among 28,505 patients in later trials, the death rate that was related to drug was 1.41%. In conclusion, dosing based on phase I trials was associated with a low toxicity-related death rate in later trials. The ability to predict relevant toxicities correlates with the number of patients on the initial phase I trial. The final dose approved was within 20% of the RP2D in 73% of assessed trials.

The anticancer drug tamoxifen is active against Trypanosoma cruzi in vitro but ineffective in the treatment of the acute phase of Chagas disease in mice

The activity of the antineoplastic drug tamoxifen was evaluated against Trypanosoma cruzi. In vitro activity was determined against epimastigote, trypomastigote and amastigote forms of CL14, Y and Y benznidazole resistant T. cruzi strains. Regardless of the strain used, the drug was active against all life-cycle stages of the parasite with a half maximal effective concentration ranging from 0.7-17.9 µM. Two experimental models of acute Chagas disease were used to evaluate the in vivo efficacy of treatment with tamoxifen. No differences in parasitemia and mortality were observed between control mock-treated and tamoxifen-treated mice.

New antitumoral agents I: In vitro anticancer activity and in vivo acute toxicity of synthetic 1,5-bis(4-hydroxy-3-methoxyphenyl)-1,4-pentadien-3-one and derivatives

This paper describes a new method for the preparation of 1,5-bis(4-hydroxy-3-methoxyphenyl)-1,4-pentadien-3-one 1 and its derivatives 2-5. This set of synthetic compounds exhibited high antitumoral activities regarding in vitro screening against several human tumor cell lines as lung carcinoma NCI-460, melanoma UACC-62, breast MCF-7, colon HT-29, renal 786-O, ovarian OVCAR-03 and ovarian expressing the resistance phenotype for adriamycin NCI-ADR/ RES, prostate PC-3, and leukemia K-562. Compounds were also tested against murine tumor cell line B16F10 melanoma and lymphocytic leukemia L1210 as well as to their effect toward normal macrophages. Specific activity against colon cancer cells HT-29 was observed for all tested compounds and suggests further studies with models of colon cancer. Compounds 1, 2, and 4 showed significant cytotoxic activity with IC(50) values <= 2.3 mu M for all human cancer cell lines. Intraperitoneal acute administration of compound 1 and 2 showed very low toxicity rate. (C) 2010 Elsevier Ltd. All rights reserved.

Permeation of Large Antineoplastics into Wild Strain of Saccharomyces cerevisiae

Saccharomyces cerevisiae has been used in genotoxicity and cytotoxicity assays for several years before the Ames Test approach. However the cell permeability of yeast has been considered a limitant factor to this kind of assay and many researchers have been introducing genetic modifications into wild strains to improve the sensitivity to chemical compounds. In our study, we used Saccharomyces cerevisiae ATCC 9763, well known and very common strain in antibiotic assays, and we evaluated the cytotoxicity of some antineoplastic agents (etoposide, epirubicin, carboplatin, cisplatin and mitoxantrone). Each culture was observed under the light of microscope and photographed. Neither genetic modification nor addition of permeation inducers, as dimethylsulfoxide (DMSO), were introduced during the assays and the cells presented good sensitivity to those compounds, demonstrating that other potential strains and characteristics of cells should be reconsidered to improve these assays apart from the cellular permeability.

Stereoselective synthesis of the dolastatin units by organotrifluoroborates additions to alpha-amino aldehydes

Dolastatin units were synthesized from the 1,2-addition reactions of potassium allyl or crotyltrifluoroborate salts to aldehyde derivatives from natural amino acids. The reactions were carried out in presence of a phase-transfer catalyst in a biphasic medium at room temperature and excellent yields (>89-93%) and stereoselective (>90:10 to 98:2) were obtained. The dolastatin units 8 and 14a-b were obtained after three steps in good overall yields (50-62%). (C) 2007 Elsevier Ltd. All rights reserved.

The Ruthenium Complex cis-(Dichloro)tetraammineruthenium(III) Chloride Presents Selective Cytotoxicity Against Murine B Cell Lymphoma (A-20), Murine Ascitic Sarcoma 180 (S-180), Human Breast Adenocarcinoma (SK-BR-3), and Human T Cell Leukemia (Jurkat) Tumor Cell Lines

The aim of present study was to verify the in vitro antitumor activity of a ruthenium complex, cis-(dichloro)tetraammineruthenium(III) chloride (cis-[RuCl(2)(NH(3))(4)]Cl) toward different tumor cell lines. The antitumor studies showed that ruthenium(III) complex presents a relevant cytotoxic activity against murine B cell lymphoma (A-20), murine ascitic sarcoma 180 (S-180), human breast adenocarcinoma (SK-BR-3), and human T cell leukemia (Jurkat) cell lines and a very low cytotoxicity toward human peripheral blood mononuclear cells. The ruthenium(III) complex decreased the fraction of tumor cells in G0/G1 and/or G2-M phases, indicating that this compound may act on resting/early entering G0/G1 cells and/or precycling G2-M cells. The cytotoxic activity of a high concentration (2 mg mL(-1)) of cis-[RuCl(2)(NH(3))(4)]Cl toward Jurkat cells correlated with an increased number of annexin V-positive cells and also the presence of DNA fragmentation, suggesting that this compound induces apoptosis in tumor cells. The development of new antineoplastic medications demands adequate knowledge in order to avoid inefficient or toxic treatments. Thus, a mechanistic understanding of how metal complexes achieve their activities is crucial to their clinical success and to the rational design of new compounds with improved potency.

Role of platelet-activating factor in the pathogenesis of 5-fluorouracil-induced intestinal mucositis in mice

Gastrointestinal mucositis is a common side effect of cancer chemotherapy. Platelet-activating factor (PAF) is produced during gut inflammation. There is no evidence that PAF participates in antineoplastic-induced intestinal mucositis. This study evaluated the role of PAF in 5-fluorouracil (5-FU)-induced intestinal mucositis using a pharmacological approach and PAF receptor knockout mice (PAFR(-/-)). Wild-type mice or PAFR(-/-) mice were treated with 5-FU (450 mg/kg, i.p.). Other mice were treated with saline or BN52021 (20 mg/kg, s.c.), an antagonist of the PAF receptor, once daily followed by 5-FU administration. After the third day of treatment, animals were sacrificed and tissue samples from the duodenum were removed for morphologic evaluation. In addition, myeloperoxidase activity and the cytokine concentration were measured. 5-FU treatment decreased the duodenal villus height/crypt depth ratio, increased MPO activity, and increased the concentration of TNF-alpha, IL-1 beta and KC in comparison with saline-treated animals. In PAFR(-/-) mice and PAFR antagonist-treated mice, 5-FU-dependent intestinal damage was reduced and a decrease in duodenal villus height/crypt depth ratio was attenuated. However, the 5-FU-dependent increase in duodenum MPO activity was not affected. Without PAFR activation, 5-FU treatment did not increase the TNF-alpha, IL-1 beta and KC concentration. In conclusion, our study establishes the role of PAFR activation in 5-FU-induced intestinal mucositis. This study implicates treatment with PAFR antagonists as novel therapeutic strategy for this condition.

Cell cycle arrest and apoptosis in TP53 subtypes of bladder carcinoma cell lines treated with cisplatin and gemcitabine

Currently, the combination of cisplatin and gemcitabine is considered a standard chemotherapeutic protocol for bladder cancer. However, the mechanism by which these drugs act on tumor cells is not completely understood. The aim of the present study was to investigate the effects of these two antineoplastic drugs on the apoptotic index and cell cycle kinetics of urinary bladder transitional carcinoma cell lines with wild-type or mutant TP53 (RT4: wild type for TP53; 5637 and T24: mutated TP53). Cytotoxicity, cell survival assays, clonogenic survival assays and flow cytometric analyses for cell cycle kinetics and apoptosis detection were performed with three cell lines treated with different concentrations of cisplatin and gemcitabine. G(1) cell cycle arrest was observed in the three cell lines after treatment with gemcitabine and gemcitabine plus cisplatin. A significant increase in cell death was also detected in all cell lines treated with cisplatin or gemcitabine. Lower survival rates occurred with the combined drug protocol independent of TP53 status. TP53-wild type cells (RT4) were more sensitive to apoptosis than were mutated TP53 cells when treated with cisplatin or gemcitabine. Concurrent treatment with cisplatin and gemcitabine was more effective on transitional carcinoma cell lines than either drug alone; the drug combination led to a decreased cell survival that was independent of TP53 status. Therefore, the synergy between low concentrations of cisplatin and gemcitabine may have clinical relevance, as high concentrations of each individual drug are toxic to whole organisms.

Structural effects of lipophilic methotrexate conjugates on model phospholipid biomembranes

Lipophilic conjugates of the antitumor drug methotrexate (MTX) with lipoamino acids (LAAs) have been previously described as a tool to enhance MTX passive entrance into cells, overcoming a form of transport resistance which makes tumour cells insensitive to the antimetabolite. A knowledge of the mechanisms of interaction of such lipophilic derivatives with cell membranes could be useful for planning further lipophilic MTX derivatives with an optimal antitumour activity. To this aim, a calorimetric study was undertaken using a biomembrane model made from synthetic 1,2-dipalmitoyl-glycero-3-phosphocholine (DPPC) multilamellar liposomes. The effects of MTX and conjugates on the phase transition of liposomes were investigated using differential scanning calorimetry. The interaction of pure MTX with the liposomes was limited to the outer part of the phospholipid bilayers, due to the polar nature of the drug. Conversely, its lipophilic conjugates showed a hydrophobic kind of interaction, perturbing the packing order of DPPC bilayers. In particular, a reduction of the enthalpy of transition from the gel to the liquid crystal phase of DPPC membranes was observed. Such an effect was related to the structure and mole fraction of the conjugates in the liposomes. The antitumour activity of MTX conjugates was evaluated against cultures of a CCRF-CEM human leukemic T-cell line and a related MTX resistant sub-line. The in vitro cell growth inhibitory activity was higher for bis(tetradecyl) conjugates than for both the other shorter- and longer-chain derivatives. The biological effectiveness of the various MTX derivatives correlated very well with the thermotropic effects observed on the phase transition of DPPC biomembranes. (C), 2001 Elsevier Science B.V All rights reserved.