RESUMO
Urinary nucleosides are associated with many types of cancer. In this study, six targeted urinary nucleosides, namely adenosine, cytidine, 3-methylcytidine, 1-methyladenosine, inosine, and 2-deoxyguanosine, were chosen to evaluate their role as biomarkers of four different types of cancer: lung cancer, gastric cancer, colon cancer, and breast cancer. Urine samples were purified using solid-phase extraction (SPE) and then analyzed using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The Mann-Whitney U test and Principal Component Analysis (PCA) were used to compare differences in urinary nucleosides between patients with one of four types of cancer and healthy controls. The diagnostic sensitivity of single nucleosides for different types of cancer ranged from 14% to 69%. In contrast, the diagnostic sensitivity of a set of six nucleosides ranged from 37% to 69%. The false-positive identification rate associated with the set of six nucleosides in urine was less than 2% compared with that of less than 5% for a single nucleoside. Furthermore, combining the set of six urinary nucleosides with carcinoembryonic antigen improved the diagnostic sensitivity for colon cancer. In summary, the study show that a set of six targeted nucleosides is a good diagnostic marker for breast and colon cancers but not for lung and gastric cancers.
Assuntos
Biomarcadores Tumorais/urina , Neoplasias da Mama/urina , Neoplasias do Colo/urina , Neoplasias Pulmonares/urina , Nucleosídeos/urina , Neoplasias Gástricas/urina , Humanos , TaiwanRESUMO
Fructus gardeniae has long been used by traditional Chinese medical practitioners for its anti-inflammatory, anti-oxidant, anti-tumor and anti-hyperlipidemic characteristics. Here we describe our finding that F. gardeniae greatly reduces anti-enterovirus 71 (EV71) activity, resulting in significant decreases in EV71 virus yields, EV71 infections, and internal ribosome entry site activity. We also found that geniposide, a primary F. gardeniae component, inhibited both EV71 replication and viral IRES activity. Our data suggest the presence of a mechanism that blocks viral protein translation. According to our findings, F. gardeniae and geniposide deserve a closer look as potential chemopreventive agents against EV71 infections.
Assuntos
Antivirais/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Enterovirus Humano A/efeitos dos fármacos , Infecções por Enterovirus/tratamento farmacológico , Gardenia/química , Iridoides/farmacologia , Medicina Tradicional Chinesa , RNA Viral/genética , Ribossomos/metabolismo , Antivirais/química , Linhagem Celular Tumoral , Medicamentos de Ervas Chinesas/química , Infecções por Enterovirus/virologia , Humanos , Iridoides/química , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/genética , RNA Viral/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
BACKGROUND: Urinary homovanillic acid (HVA)/vanillylmandelic acid (VMA), orotic acid (OA), and homogentisic acid (HGA) are diagnostic biomarkers of neuroblastoma, ornithine carbamoyl transferase deficiency (OCTD), and alkaptonuria (AKU), respectively. In this study, a high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for simultaneous quantification of HVA, VMA, OA, and HGA in urine. METHODS: After sample preparation, which involved only the dilution procedure, samples were quantified by LC-MS/MS. Full-scan MS/MS mode enabled the urinary markers to be quantified with a high degree of specificity and sensitivity. Rather than using a separate enzymatic method to normalize the concentration of creatinine in urine, we quantified the level of creatinine in urine in one LC-MS run. RESULTS: The limits of detection were 10 µg/l for HGA, 25 µg/l for HVA/VMA, and 50 µg/l for OA with a single-to-noise ratio of 3; the limits of quantification were 50 µg/l for HVA and HGA, 100 µg/l for VMA, and 250 µg/l for OA. The linear dynamic range for quantification of the analytes covered 2 to 3 orders of magnitude, depending on the analyte. The relative standard deviation of the developed LC-MS/MS method was less than 4% for the intra-day validation and 10% for the inter-day validation. CONCLUSIONS: The results show that our LC-MS/MS technique is a highly sensitive and rapid method for screening for biomarkers that are diagnostic of three metabolic diseases.
Assuntos
Alcaptonúria/diagnóstico , Biomarcadores/análise , Cromatografia Líquida , Neuroblastoma/diagnóstico , Doença da Deficiência de Ornitina Carbomoiltransferase/diagnóstico , Espectrometria de Massas em Tandem , Biomarcadores/urina , Humanos , Limite de Detecção , Fatores de TempoRESUMO
BACKGROUND: Cataracts are the leading cause of blindness worldwide; however, there is no evidence regarding the direct formation of cataracts. At present, there is no treatment method other than surgery to prevent the formation or progression of cataracts. OBJECTIVE: Understanding the protein changes during various stages of cataracts might help realize the mechanism of the formation and progression of cataracts. METHODS: Lens materials were collected from cataract surgery. Cataracts were classified according to lens opacity using the gradation of the Lens Opacities Classification System. Lens proteins were separated by 2-dimensional polyacrylamide gel electrophoresis. Protein spots were visualized by Coomassie blue staining, and expression patterns were analyzed. Protein spots of interest were excised from 2-dimensional polyacrylamide gel electrophoresis gels, digested in situ with trypsin, and analyzed by mass spectrometry and liquid chromatographic tandem mass spectrometry. RESULTS: Crystallin was the major protein in the cataract lens, and αA, ßB1, αB, and ßA4 were the dominant types. Crystallin αB and ßA4 increased with the formation of lens opacity. Moreover, phosphorylation and truncation of these proteins increased with the progression of cataracts. CONCLUSION: Crystallin αB and ßA4 and phosphorylation and truncation of crystallin in the lens might contribute to the formation of cataracts. In contrast, acetylation was not dominant in the progression of cataracts and did not play major role in the formation of cataracts.
RESUMO
BACKGROUND: Breast cancer is the most common female cancer and the fourth leading cause of cancer death among women in Taiwan. We measured urinary nucleoside levels in female breast cancer patients (n=36) to evaluate the diagnostic value of nucleosides as potential tumor markers. METHODS: Purification of urinary nucleosides was performed using a 96-well solid phase extraction (SPE, cation-exchange column) procedure to decrease the variation between the single column preparations and to shorten the pretreatment time. Cation-exchange allows for the comprehensive purification of modified nucleosides, such as 2-deoxynucleosides, that are not purifiable by phenylboronic acid-based SPE. High-performance liquid chromatography (HPLC) coupled with mass spectrometry (MS) in selected reaction monitoring (SRM) mode was used to quantify multiple nucleosides. Tubercidin was used as an internal standard. The qualitative parameters, retention time, and the parent and daughter ions used revealed that the method was more specific and sensitive than traditional UV detection. RESULTS: Urinary levels of 3 nucleosides, cytidine, 3-methylcytidine, and inosine were significantly higher in breast cancer patients than in normal controls (p<0.01). The discriminative powers of cytidine, 3-methylcytidine, and inosine were 58%, 58%, and 62%, respectively. CONCLUSIONS: LC/MS/MS is a highly specific and sensitive method for rapidly screening a large number of urinary nucleosides that may be potential cancer markers. The 3-methylcytidine may be a candidate marker for breast cancer.
Assuntos
Biomarcadores Tumorais/urina , Cromatografia Líquida de Alta Pressão/métodos , Nucleosídeos/urina , Espectrometria de Massas por Ionização por Electrospray/métodos , Estudos de Casos e Controles , Feminino , Humanos , Padrões de ReferênciaRESUMO
Hepatocellular carcinoma (HCC) is a common neoplasm in Taiwan, for which early diagnosis is difficult and the prognosis is usually poor. HCC is usually diagnosed by abdominal sonography and serum alpha-fetoprotein (AFP) detection. Modified nucleosides, regarded as indicators for the whole-body turnover of RNAs, are excreted in abnormal amounts in the urine of patients with malignancies and can serve as tumor markers. We analyzed the excretion patterns of urinary nucleosides from 25 HCC patients and 20 healthy volunteers by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) under optimized conditions. The HPLC/ESI-MS/MS approach with selective reaction monitoring (SRM) allowed for the sensitive determination of nucleosides in human urine samples. The mean levels of the urinary nucleosides adenosine, cytidine, and inosine were significantly higher in HCC patients than healthy volunteers (average of 1.78-, 2.26-, and 1.47-fold, respectively). However, the mean levels of urinary 1-methyladenosine, 3-methylcytidine, uridine, and 2'-deoxyguanosine were not significantly different. Combined with the determination of serum AFP levels, the higher levels of urinary adenosine, cytidine, and inosine may be additional diagnosis markers for HCC in Taiwanese patients.
Assuntos
Biomarcadores Tumorais/urina , Carcinoma Hepatocelular/diagnóstico , Nucleosídeos/urina , Adulto , Idoso , Cromatografia Líquida de Alta Pressão , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
BACKGROUND: Increased levels of modified nucleosides have been observed in urine from patients suffering from several cancers. In this study, we evaluated whether urinary nucleosides can serve as potential tumor markers for colorectal cancer by high performance liquid chromatography-electrospray/tandem mass spectrometry (HPLC/ESI-MS/MS). METHODS: A simple and specific method based on HPLC/ESI-MS/MS was developed to determine the urinary nucleosides from patients with colorectal cancer. We studied the excretion patterns of nucleosides in urine from 26 patients with colorectal cancer and 18 healthy controls. RESULTS: The LC/MS/MS approach with selective reaction monitoring (SRM) allowed for the sensitive determination of nucleosides in human urine samples with colorectal cancer. The mean levels of 5 urinary nucleosides (adenosine, cytidine, N(2),N(2)-dimethylguanine, 8-hydroxy-2'-deoxyguanosine and uridine) were significantly higher in the patients with colorectal cancer than in the healthy adults. CONCLUSIONS: The results indicate that urinary nucleosides determined by LC/MS/MS may be useful as biological markers for colorectal cancer. Our findings suggest that LC/MS/MS is a highly specific and sensitive method for rapidly screening a large number of nucleoside that may be useful as markers for cancer in humans.
Assuntos
Biomarcadores Tumorais/urina , Neoplasias Colorretais/urina , Nucleosídeos/urina , Adulto , Idoso , Idoso de 80 Anos ou mais , Cromatografia Líquida de Alta Pressão , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em TandemRESUMO
Monascus-fermented red mold rice has been extensively used as a folk medicine for thousands of years. Monascus secondary metabolites, including monacolin K, monascorubrin, and ankaflavin, have been reported to have an antiproliferative effect on cancer cells. However, the cell machinery responsible for the antiproliferation of Monascus-fermented red mold rice treatment in cancer cells remains unclear. A proteomic approach using two-dimensional gel electrophoresis, matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry, and tandem mass spectrometry was used to identify proteins with modified expression in Caco-2 cells treated with Monascus-fermented red mold rice extract. A total of 20 proteins were identified with significantly altered expression (P < 0.05) in response to Monascus-fermented red mold rice extract treatment. The deregulated proteins that were identified included heat shock protein 70, protein kinase C epsilon type, clusterin-associated protein 1, and two tumor suppressors (N-chimaerin and calponin-2). Our results suggested the involvement of heat shock protein 70-mediated cytotoxicity in the Caco-2 cells treated with Monascus-fermented red mold rice extract.