RESUMO
In cancer therapy, exosomes efflux enhances resistance of cancer cells toward anticancer agents through mediating the transport of anticancer drugs outside the cells. In this study, a rapid, simple and highly sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the determination of Doxorubicin (DOX) in exosomes of cancer cells and human plasma using Ketotifen as an internal standard (IS). Plasma samples spiked with DOX and two cancer cell lines (A549 & MCF-7) were incubated with different concentrations of DOX and IS. The analytes were then extracted with methanol after protein precipitation and the chromatographic separation was carried out using a C18 column, with a mixture of acetonitrile-water- formic acid (85:15:0.1%, v/v/v) as mobile phase. Multiple reaction monitoring (MRM) was utilized to monitor the protonated precursor to product ion transitions of m/z 544.25â¯>â¯397.16 and m/z 310.08â¯>â¯96.97 for the quantification of DOX and IS, respectively. The method was linear over ranges of 1-1000â¯ng/mL for DOX in plasma and 2-1000â¯ng/mL for DOX in exosome samples. The lower limit of quantification of this method was 1â¯ng/mL, 2â¯ng/mL and 2â¯ng/mL in human plasma, A549 & MCF-7 cells respectively. Intra- and inter day precision of all quality control concentrations were less than 10.33% and the accuracy values ranged from -4.82 to 12.60%. The optimized UPLC-MS/MS method proved to be fast, specific, simple and highly sensitive and was successfully applied for the estimation of DOX in the exosomes of cancer cells and plasma.
RESUMO
PURPOSE: To investigate the relationship between late tissue response after radiotherapy, cellular sensitivity and DNA repair capacity measured in dermal fibroblasts and chromosomal aberrations measured in lymphocytes. The study was in particular designed to compare cellular parameters of patients with maximum differences in late tissue reactions. MATERIALS AND METHODS: The study was performed with 16 pair-wise matched head and neck cancer patients 2-7 years after curative therapy exhibiting maximum differences (grade 1 vs. grade 3) in late normal tissue reactions. Clinical endpoints were fibrosis, telangiectasia, mucositis and xerostomia using the radiation therapy oncology group score. Patients with grade 3 reactions were tested for mutations in ataxia telangiectasia (AT), Nijmegen Breakage Syndrome (NBS), MRE11, RAD50 and DNA ligase IV genes by means of polymerase chain reaction-single-strand conformation polymorphism and sequencing analysis. Skin fibroblasts obtained from biopsies were used to determine the cellular sensitivity by colony formation and the induction and repair of DNA double-strand breaks (dsb) using constant-field gel electrophoresis. Lymphocytes were taken to measure chromosomal damage either in metaphase using conventional chromosome analysis or in G(0) using premature chromosome condensation (PCC)-technique. RESULTS: Patients with extreme late reactions (grade 3) showed no evidence for an AT, NBS, MRE11 or RAD50 mutation. Studies with fibroblasts revealed that extreme late reactions were associated neither with a pronounced cellular radiosensitivity nor with a difference in dsb repair capacity. In contrast, there was a significant difference in chromosomal damage measured in lymphocytes. After in vitro irradiation with 6Gy, lymphocytes taken from overreacting patients showed on average a significantly higher number of lethal aberrations than lymphocytes isolated from patients with mild reactions (7.2+/-0.8 vs. 5.0+/-0.3). Similar differences were found for PCC fragments. CONCLUSION: This study suggests that lymphocytes are more promising than fibroblasts to predict patient's normal tissue response after radiotherapy.
