| Bromocriptine (BC) resistance is one of the most important reasons for prolactinomas treatment failure and relapse. Exploring the possible mechanisms of BC resistance and making accurate diagnosis for bromocriptine-resistant prolactinoma patients could facilitate the implementation of personalized medicine and improvement of therapeutic efficacy. In this study, a sensitive and rapid quantitative bioanalytical method has been established by using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the determination of BC in both human plasma and prolactinoma tissue. Then the established LC-MS/MS method was employed to investigate the differences in plasma and prolactinoma tissue distribution of BC between bromocriptine-sensitive and-resistant prolactinoma patients. The results revealed that BC could be effectively absorbed and transported into prolactinoma and sustain a certain concentration in bromocriptine-resistant patients. Therefore, the reseaon for failure of BC treatment in bromocriptine-resistant patients was "intrinsic" tumor cell resistence. Based on the above research, BC resistance was investigated using plasma of prolactinoma patients and rat prolactinoma cells by LC-MS/MS-based metabolomics approach to find potential biomarkers for diagnosis of bromocriptine-resistant prolactinoma. Many potential biomarkers have been found and the BC resistance-related metabolic pathways have been identified. The results of this study will not only provide molecular basis of diagnosis and treatment for bromocriptine-resistant prolactinomas but also help to illuminate the mechanism of BC resistance in prolactinomas.1. Quantitative analysis of bromocriptine in plasma and tumor tissue of prolactinoma patientsA quantitative analytical LC-MS/MS method for the BC has been develped and validated. The performance characteristic of the method in terms of sensitivity, rapidness and specificity showed that this approach was adequate for the quantitative determination of BC in both human plasma and prolactinoma tissue. Compared to bromocriptine-sensitive prolactinoma patients, the plasma BC concentrations in bromocriptine-resistant prolactinoma patients at 2 h,4 h and 6 h after oral administration of BC were significantly higher. The tumor BC concentrations in bromocriptine-resistant prolactinoma patients administered BC for 3 months were also significantly higher than those of bromocriptine-sensitive prolactinoma patients. These differences in drug distribution indicated that BC could also be effectively transported to prolactinoma and sustain a certain concentration in bromocriptine-resistant patients. The reseaon for failure of BC treatment in bromocriptine-resistant patients was "intrinsic" tumor cell resistence.2. Plasma metabolomics study on bromocriptine-resistant prolactinomasA metabolomics study using plasma samples from bromocriptine-resistant and-sensitive prolactinoma patients was carried out by LC-MS/MS analysis method.64 metabolites were found to be significantly altered in plasma of bromocriptine-resistant prolactinoma patients compared with bromocriptine-sensitive prolactinoma patients.21 potential biomarkers have been identified by using high resolution MS and MS/MS analysis, as well as isotopic abundance ratio, "nitrogen rule", which included amino acids, sphingolipids and fatty acids, et al. Receiver operating characteristic analysis (ROC) was exploited to evaluate the diagnostic power of the potential biomarkers.9 potential biomarkers (sphinganine-1-phosphate, C16 sphingosine,1-deoxy-tetradecasphinganine, 1-deoxy-sphinganine, (4E,8E, 10E-d18:3) sphingosine, glycerol 3-phosphate, phytosphingosine, C16 sphinganine, C16 sphinganine-1-phosphate) yielded satisfactory accuracy (AUC>0.9) in distinguishing bromocriptine-resistant patients from bromocriptine-sensitive patients. Metabolic pathway analysis was then performed to identify the BC resistance-related metabolic pathways. It was found that 19 metabolic pathways were perturbed in bromocriptine-resistant prolactinoma patients, including sphingolipid metabolism, lysine biosynthesis, aminoacyl-tRNA biosynthesis, alanine, aspartate and glutamate metabolism, lysine degradation, et al.3. Cell metabolomics study on bromocriptine-resistant prolactinomasA cell metabolomics study was carried out using rat prolactinoma GH3 and MMQ cells. Firstly, the cells were quenched in liquid nitrogen and extracted three times with methanol, methanol and water. Systematic analysis of the effects of cell number and re-dissolution solvents on the number and intensity of chromatographic peaks confirmed that 5×106 cells and 80% methanol-water were optimal. Secondly, prolactin (PRL) secretion-inhibiting effects of BC on GH3 and MMQ cells were evaluated by using enzyme-linked immunosorbent assay (ELISA) in control cells and cells treated with BC for 6 h,12 h,24 h,36 h and 48 h. The dynamic changes of PRL in culture medium of GH3 cell provided guidance for screening of potential diagnostic biomarkers. Thirdly, MMQ and GH3 cells untreated with BC were analyzed by the LC-MS/MS method used in plasma metabolomics study.60 potential biomarkers associated with the diagnosis of bromocriptine-resistant prolactinoma were found based on the established multivariate statistical data analysis (MVDA) and correlation analysis.24 potential biomarkers have been identified, including cholines, amino acids, carnitines, nucleosides, et al. The results from metabolic pathways analysis revealed that 14 metabolic pathways were perturbed in GH3 cells, including glycerophospholipid metabolism, valine, leucine and isoleucine biosynthesis, inositol phosphate metabolism, glutathione metabolism, et al. Moreover,5 common perturbed pathways (glycerophospholipid metabolism, pantothenate and CoA biosynthesis, cysteine and methionine metabolism, glycine, serine and threonine metabolism, aminoacyl-tRNA biosynthesis) have been found in both GH3 cells and bromocriptine-resistant prolactinoma patients. |
PDF Full Text Request