The isolation and identification of apolipoprotein C-I in hormone-refractory prostate cancer using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry

Androgens play a central role in prostate cancer pathogenesis, and hence most of the patients respond to androgen deprivation therapies. However, patients tend to relapse with aggressive prostate cancer, which has been termed as hormone refractory. To identify the proteins that mediate progression to the hormone-refractory state, we used protein-chip technology for mass profiling of patients＇ sera. This study included 16 patients with metastatic hormone-refractory prostate cancer who were initially treated with androgen deprivation therapy. Serum samples were collected from each patient at five time points： point A, pre-treatment; point B, at the nadir of the prostate- specific antigen （PSA） level; point C, PSA failure; point D, the early hormone-refractory phase; and point E, the late hormone-refractory phase. Using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry, we performed protein mass profiling of the patients＇ sera and identified a 6 640-Da peak that increased with disease progression. Target proteins were partially purified, and by amino acid sequencing the peak was identified as a fragment of apolipoprotein C-I （ApoC-I）. Serum ApoC-I protein levels increased with disease progression. On immunohistochemical analysis, the ApoC-i protein was found localized to the cytoplasm of the hormone-refractory cancer cells. In this study, we showed an increase in serum ApoC-I protein levels in prostate cancer patients during their progression to the hormone-refractory state, which suggests that ApoC-I protein is related to progression of prostate cancer. However, as the exact role of ApoC-I in prostate cancer pathogenesis is unclear, further research is required.

Effect of surface-enhanced laser desorption/ionization time-of-flight mass spectrometry on identifing biomarkers of endometriosis

Background Endometriosis is a common gynecological disease. This study aimed to screen proteins that were expressed differently in patients with endometriosis versus normal controls using proteomic techniques, surface-enhanced laser desorption/ionization time-of-flight mass spectrometry （SELDI-TOF-MS）.Methods Protein chip SELDI-TOF-MS combines the advantages of microarray and mass spectrometry, and can screen latent markers in sera of patients with endometriosis. Serum samples from patients and normal volunteers were analyzed by SELDI-TOF-MS. Results After comparing the serum protein spectra of 36 patients with 24 normal controls, 24 differently expressed potential biomarkers （P 〈0.01） were identified. Using Biomarker Pattern software, we established a tree model of the 60 serum protein spectra. When using the three bJomarkers to classify the samples, the sensitivity for diagnosing endometriosis was 91.7%, specificity was 95.8%, and coincidence rate was 93.3%. Then we used serum samples from 12 patients and 8 normal controls to validate the tree model and report the sensitivity for diagnosing endometriosis was 91.7%, specificity was 75%, and coincidence rate was 85%. Conclusions SELDI-TOF-MS may be a useful tool in high-risk population screening for endometriosis. The identification and application of the biomarkers need to further study.

Analysis of variabilities of serum proteomic spectra in patients with gastric cancer before and after operation

AIM： To study the variabilities of serum proteomic spectra in patients with gastric cancer before and after operation in order to detect the specific protein markers that can be used for quick diagnosis of gastric cancer. METHODS： Proteomic spectra of 46 serum samples from patients with gastric cancer before and after operation and 40 from normal individuals were generated by IMAC-Cu protein chip and surface-enhanced laser desorption/ionization time of flight mass spectrometry. RESULTS： Fourteen differentially expressed proteins in serum were screened by analysis of proteomic spectra of preoperative patients and normal individuals. We obtained 4 proteins （heat shock protein 27, glucoseregulated protein, prohibitin, protein disulfide isomerase A3） making up marker pattern which was able to class the patient-team and normal-team. These marker patterns yielded 95.7% sensitivity and 92.5% specificity, respectively. The proteins over-expressed in serum of preoperative patients were obviously down-regulated. CONCLUSION： Specific protein markers of gastric cancer can be used for the quick diagnosis of gastric cancer and judgment of prognosis. SELDI-TOF-MS is a useful tool for the detection and identification of new protein markers in serum.

Proteiomic patterns for endometrial cancer using SELDI-TOF-MS

Serum samples from endometrial cancer (EC) patients and healthy females were analyzed using surface-enhanced laser desorption-ionization time-of-flight mass spectrometry (SELDI-TOF-MS) to discover the potential diagnostic biomarker for detection of EC. A preliminary training set of spectra derived from 40 EC patients and 30 healthy women were used to develop a proteomic model that effectively discriminated cancer patients from healthy women. The training set had a specificity of 100% and sensitivity of 92.5% in the EC detection. A blind test set, including 20 new cancer cases and 10 healthy women, was used to validate the sensitivity and specificity of this multivariate model, which had a corresponding results of 60% in specificity and 75% in sensitivity, respectively. The combination of SELDI-TOF-MS with bioinformatics tools could help find new biomarkers and establish the detection of EC with high sensitivity and specificity.

Detection of pancreatic cancer with normal carbohydrate antigen 19-9 using protein chip technology

AIM: To develop a method to differentiate pancreatic cancer patients from healthy or benign individuals when carbohydrate antigen (CA) 19-9 is normal.

Arsenic trioxide inhibits metastatic potential of mouse hepatoma H22 cells in vitro and in vivo

BACKGROUND:It has been pointed out that only low-dose arsenic trioxide(ATO)presents therapeutic benefits outweighing the toxic side effects.Low-dose ATO can effectively alleviate acute promyelocytic leukemia(APL). However,it is quite challenging in treating solid tumors. The purpose of this study was to investigate the effect of ATO at low concentrations on the metastatic potential of mouse hepatoma H22 cells and the anti-metastatic mechanism of ATO. METHODS:The metastatic potential of H22 cells was evaluated by adhesion,migration and invasion assays after exposure to a low dose of ATO in vitro.The mouse lung metastatic model induced by injection of H22 cells via the tail vein was adopted for the evaluation of metastatic potential. Different proteins in the lysate of H22 cells exposed to ATO at different concentrations were investigated by surface- enhanced laser desorption and ionization time-of-flight mass spectrometry(SELDI-TOF-MS).Finally,Western blotting analyses were made to detect the expression pattern of MMP-2 and nm23-M1 proteins. RESULTS:Significant cell death started at ATO concentrations above 2μmol/L.The growth and adhesion potential of H22 cells was inhibited in a time-and dose- dependent manner,and the migration and invasion potential of H22 cells was inhibited in a dose-dependent manner while ATO concentration was below 2μmol/L. Mice injected with ATO at a dose of 0.5 mg/kg had fewer lung metastases.However,mice injected with ATO at a dose of 2 mg/kg or 4 mg/kg had a high mortality rate and more liver injuries.A total of 15 different protein peaks were identified between the lysate of H22 cells treated with ATO and controls.Two proteins that peaked atm/z 5302 and 17207 coincided with MMP-2(fragment) and nm23-M1,respectively.Western blotting analyses demonstrated that MMP-2 and MMP-2 fragments were down-regulated and nm23-M1 was up-regulated in H22 cells treated with 2μmol/L ATO for 48 hours. CONCLUSIONS:ATO at a low dose inhibits the metastatic potential of mouse hepatoma H22 cells in vitro and in vivo, and involves down-regulation of MMP-2 and up-regulation of nm23-M1.

Identification of differentially expressed proteins in human stage Ⅰ lung adenocarcinoma and tumor-adjacent tissues with two-dimension gel electrophoresis profiling

Objective： The aim of this study was to establish reproducible two-dimensional electrophoretic assay used for profiling and identification of differentially expressed proteins in human stage I lung adenocarcinoma and paired normal tumor-adjacent tissue. Methods： The proteins from 12 human stage I lung adenocarcinoma tissues and normal tumor-adjacent tissues were separated using isoelectric focusing electrophoresis （the first dimension） and the subsequent homogeneous SDS-polyacrylamide gel electrophoresis （SDS-PAGE） （the second dimension）. The differentially expressed proteins were determined with PDQuest image analysis software, and identified using matrix-assisted laser desorption/ionization time of flight mass spectrometry （MALDI-TOF-MS） and database searching. Results： The well-reproducible 2-DE gel patterns of human stage I lung adenocarcinoma and normal tumor-adjacent tissues were profiled and 26 differentially expressed proteins uncovered. Nine of these 26 protein spots were cut out from the preparation gels and determined with MALDI-TOF-MS. Searching against the protein database, four candidate proteins were identified. They were 60S acidic ribosomal protein P2, Cathepsin B1, Apolipoprotein A-I precursor, and La 4.1 protein. Conclusion： In this study, high reproducible 2-DE gel protein images of human stage I lung adenocarcinoma and paired normal tumor-adjacent tissues were achieved successfully, and 4 differentially expressed proteins were revealed. These data will be helpful for screen of early biomarker and study of molecular mechanisms of human lung adenocarcinoma.

Applied research on serum protein fingerprints for prediction of Qi deficiency syndrome and phlegm and blood stasis in patients with non-small cell lung cancer

OBJECTIVE:This study screened serum tumor biomarkers by surface enhanced laser desorption/ionization time-of-flight mass spectrometry(SELDI-TOF-MS) to establish a subset which could be used for the prediction of Qi deficiency syndrome and phlegm and blood stasis in patients with non-small cell lung cancer;and as diagnostic model of Chinese medicine.METHODS:Serum samples from 63 lung cancer patients with Qi deficiency syndrome and phlegm and blood stasis,and 28 lung cancer patients with non-Qi deficiency syndrome and phlegm and blood stasis were analyzed using SELDI-TOF-MS with a PBS II-C protein chip reader.Protein profiles were generated using immobilized metal affinity capture(IMAC3) protein chips.Differentially-expressed proteins were screened.Protein peak clustering and classification analyses were performed using Biomarker Wizard and Biomarker Pattern software packages,respectively.RESULTS:A total of 268 effective protein peaks were detected in the 1,000-10,000 Da molecular range for the 15 serum proteins screened(P<0.05).The decision tree model was M 2284.97,with a sensitivity of 96.2% and a specificity of 66.7%.CONCLUSION:SELDI-TOF-MS techniques,combined with a decision tree model,can help identify serum proteomic biomarkers related to Qi deficiency syndrome and phlegm and blood stasis in lung cancer patients;and the predictive model can be used to discriminate between Chinese medicine diagnostic models of disease.