| Objective: The objective of this part of research was to establish a reliable protein chip SELDI-TOF-MS platform and to optimize the technological conditions in the experiments.Methods: Special protein or peptide spectra was determined by surface enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS)measurement treating the cancer OSC cell lysate sample for KB onto three types of Protein Chip(CM-10, H4, IMAC-3-Cu) for each case. According to the protein profiling results, suitable parameters and protein chips in cell lysate were screened respectively, and the reliability and stability of the technology were assessed.Results: Hundreds of proteins were detected by there chips respcetively. Most of the proteins detected by protein chip range from 1 000 to 30 000 at the Mass/charge(M/Z) value. Analysis of cell lysate protein fingerprints showed CM-10 Chip combined the most proteins, followed by H4 Chip, and less in IMAC-3-Cu Chip. The coefficient variation(CV) of protein intensity and M/Z value was 0.058038 and 0.000121, respectively, with in the same protein chip. The coefficient variation(CV) of protein intensity and M/Z value was 0.071305 and 0.000152, respectively, between the protein chips.Conclusion: SELDI-TOF-MS is an idea technological platform for proteomic research because of high reproducibility and stability. CM-10 protein chip could be suitable for the research of cell proteomics, and Our study is to assess the use of SELDI-TOF-MS to identify multiple cell protein biomarkers for various samples on CM-10 chip. Quality control and standardization of analysis conditions could be key issue for the reliability of outcome. Objective: To screen differentially expressed proteins in human oral squamous carcinoma parental cell line KB and multidrug resistance cell line KBV200 using CM-10 chips and surface-enhanced laserdesorption/ionization-time of flight-mass spectrometry technique. Methods: Detected the IC50 sensitivity of human oral squamous carcinoma parental cell line KB and drug-resistant cell line KBV200 by Methyl thiazolyl tetrazolium (MTT) assay to 6 kinds of chemotherapeutics respectively and calculated the respective resistance index. The total protein extract lysate of human oral squamous carcinoma cell lines KB and KBV200 were extracted respectively, which performed SELDI-TOF-MS to compare the proteins of different protein fingerprints. The detected peaks were filtrated and analyzed by Ciphergen Proteinchip software 3.2.0, then we classified the contrasted ratio of height from(>0.5) as differentially expressed protein.Results: Cell line KBV200 cultured by VCR-induced was resistant to VCR, with its resistance index 56.7 folds higher than KB, and cross-resistance to ADM, DDP, 5FU, but not resistance to cyclophosphamide and ara-C. Ranging from 2 000-50 000(M/Z), 18 potential biomarkers from the CM-10 surface could differentiate between cell lines KB and KBV200 (P < 0.05), whose mass-to-charge ratio (M/Z) were 2289, 2546, 3824, 3997, 4113, 4941, 4968, 5149, 5654, 5971, 6084, 7272, 7879, 9961, 10093, 10289, 10838, 22703, 14 were up-regulated and 4 were down-regulated.Conclusion: With surface-enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS) technique, we effectively screen and identify the specific expressed proteins from KBV200 to KB. Objective: To screen differentially expressed proteins in human oral squamous carcinoma drug-resistant cell line KBV200 before and after treatment of five kinds of traditional Chinese medicine respectively using surface enhanced laser desorption/ionization-time of flight-mass spectrometry technique.Methods: Detected the IC10 sensitivity of human oral squamous carcinoma drug-resistant cell line KBV200 by Methyl thiazolyl tetrazolium (MTT) assay to 5 kinds of traditional Chinese medicine and determined the drug concentration of non-toxic dose, respectively. The different protein expression profiles of KBV200 cells were detected by SELDI-TOF-MS technique combining with CM-10 chip before and after treatment of five kinds of traditional Chinese medicine respectively. Detected protein peaks were filtrated and analyzed using Ciphergen Proteinchip software 3.2.0 and Biomarker Wizard software. Differentially expressed proteins were defined as those whose absolute ratio values were greater than 0.5. Results: Ranging from 2000-50000(M/Z), after treatment of EGCG, 13 differentially expressed proteins were identified in KBV200 cells(P < 0.05), the five protein peaks 3997 Da,4941 Da,4968 Da,5971 Da,6084 Da expressed at low in KB cell, up-regulated obviously(P < 0.05)in KBV200 cells, but after treatment of EGCG these proteins expression were down-regulated(P < 0.05). After treatment of emodin, 6 differentially expressed proteins were identified in KBV200 cells(P < 0.05), and the protein peak 5971 Da expressed at low in KB cell, up-regulated obviously(P < 0.05)in KBV200 cells, but after treatment of emodin these proteins expression were down-regulated(P < 0.05). After treatment of nitidine, 7 differentially expressed proteins were identified in KBV200 cells(P < 0.05), but there were no differentially expressed protein among KB cell, KBV200 with before and after treatment of nitidine simultaneously. And there were no differentially expressed proteins on before and after treatment of Radix Isatidis and meadowrueleaf corydalis root concentration.Conclusion: During molecular weight range of 2 000-50 000 Da, compared with KB, we find that there are differentially expressed proteins can be captured before and after treatment of EGCG, emodin and nitidine. The differentially expressed proteins possiblly associate with multidrug resistangce reversal of KBV200 cell line in vitro. Objective: Experiments were carried out to preliminary investigate reversal mechanism of multidrug resistance of human oral squamous carcinoma cells KBV200 by four kinds of tranditional Chinese medicine in vitro.Methods: Cell cytotoxicity test in vitro and dose-dependent experiment of four kinds of traditional Chinese medicine reversal effects on KBV200 were detected by MTT assay. Western blot and immunocytochemistry were used to detect the expression of resistance-associated proteins P-gp and LRP, the activity of GST-π, TOPO-Ⅱ, and the tumor cell apoptosis proteins P53 and ratio changes of Bcl-2/Bax. Results: When treated with VCR(200 ng·mL-1) in combination, 0.7, 1, 1.4μg·mL-1 emodin reversed the MDR by 1.44, 2.87, 1.76 folds respectively, 0.3, 0.6, 1.2μg·mL-1 nitidine reversed the MDR by 2.44, 5.54, 1.14 folds respectively, 0.05, 0.1, 0.2μg·mL-1 Radix Isatidis concentration reversed the MDR by 2.56, 3.30, 5.25 folds respectively, 0.4, 0.75, 1.5μg·mL-1 meadowrueleaf corydalis root concentration reversed the MDR by 2.05, 2.67, 4.31 folds respectively, as compared with VRP(μg·mL-1) 1.83 fold.The four kinds of traditional Chinese medicine played the role of cytotoxic sensitizer, up-regulated the expression of P53, down-regulated the expression of P-gp but emodin (P < 0.05). The expression of LRP were lower(P < 0.05) after treatment of emodin and Radix Isatidis concentration. Radix Isatidis concentration also could reduce the activity of GST-π. The ratio of Bcl-2/Bax was lower after treatment of emodin, the others were ineffective. There was no change between KB and KBV200 about TOPO-Ⅱactivity. However, the activity of TOPO-Ⅱin KBV200 was higher after treatment of the four kinds of traditional Chinese medicine.Conclusion: The results suggest that four kinds of traditional Chinese medicine are potent MDR-reversing agents in vitro. Emodin can reverse the multidrug resistance of KBV200 in vitro. The mechanism is probably associated with down-regulating the expression of P-gp and LRP. The mechanism of overcoming MDR is associated with down-regulating the expression of Bcl-2 and up-regulating the expression of P53 to promote apoptosis of KBV200. Nitidine is associated with up-regulating the expression of P53 to promote apoptosis of KBV200. Radix Isatidis concentration also can reverse the multidrug resistance of KBV200 in vitro. The mechanism is probably associated with down-regulating the expression of P-gp and LRP. The mechanism of overcoming MDR is associated with up-regulating the expression of P53 to promote apoptosis of KBV200. And reduces the the activity of GST-π. Meadowrueleaf corydalis root concentration also can reverse the multidrug resistance of KBV200 in vitro. The mechanism is probably associated with down-regulating the expression of P-gp. The mechanism of overcoming MDR is associated with up-regulating the expression of P53 to promote apoptosis of KBV200.
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