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The glycosylation of proteins is responsible for their structural and functional roles in many cellular activities. This work describes a strategy that combines an efficient release, labeling and liquid chromatography–mass spectral analysis with the use of a comprehensive database to analyze N-glycans. The analytical method described relies on a recently commercialized kit in which quick deglycosylation is followed by rapid labeling and cleanup of labeled glycans. This greatly improves the separation, mass spectrometry (MS) analysis and fluorescence detection of N-glycans. A hypothetical database, constructed using GlycResoft, provides all compositional possibilities of N-glycans based on the common sugar residues found in N-glycans. In the initial version this database contains >8,700 N-glycans, and is compatible with MS instrument software and expandable. N-glycans from four different well-studied glycoproteins were analyzed by this strategy. The results provided much more accurate and comprehensive data than that had been previously reported. This strategy was then used to analyze the N-glycans present on the membrane glycoproteins of gastric carcinoma cells with different degrees of differentiation. Accurate and comprehensive N-glycan data from those cells was obtained efficiently and their differences were compared corresponding to their differentiation states. Thus, the novel strategy developed greatly improves accuracy, efficiency and comprehensiveness of N-glycan analysis.
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Objective:To investigate the reversal effect of MDR1 and MRP antisense oligodooxynucleotide (ASODN) on adriamycin-resistant SGC7901/ADM cells. Methods:SGC7901/ADM cells were transfected by MDR1 or MRP ASODNs or the combination of two. Then the mRNA expressions of proteins MDR1 and MRP were checked by RT-PCR,and the expressions of P-gp and MRP by immunocytochemistry;the intracellular fluorescence intensity of Rhodamine 123 in these cells were determined by flow cytometry;and the sensitivity of these cells to adriamycin,carboplatin and other anticancer drugs were determined by MTT assay. Results:The expression of MDR1mRNA and MRP mRNA in SGC7901/ADM cells decreased at 12h after transfection of ASODNs,decreased to the lowest level at 24h and returned to the level before transfection after 48h. The expression of P-gp and MRP decreased significantly at 48h after transfection with ASODNs compared with the control cells. The retention of Rhodamine 123 in SGC7901/ADM cells was significantly higher than that before transfection, and the intracellular fluorescence intensity significantly increased in ASODNs cotransfection SGC7901/ADM cells, compared with those transfected by MDR1 or MRP ASODNs respectively;In addition,the sensitivity of SGC7901/ADM cells to adriamycin, carboplatin and other anticancer drugs obviously increased after cotransfection of MDR1 and MRP ASODNs compared with transfection of MDR1 or MRP ASODN respectively. Conclusion:The transfection of MDR1 or MRP ASODN can partly reverse the multidrug resistance of gastric glandular carcinoma cells SGC7901/ADM,while cotransfection of MDR1 and MRP ASODNs can significantly reverse drug-resistance of these tumor cells.
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Purpose:To investigate the roles of artesunate on the proliferation and apoptosis of gastric carcinoma cells,also to explore it s possible mechanism of anticancer effects. Methods:The inhibition on the proliferation of cultured stomach carcinoma cell line(MGC-803) was observed by using MTT analysis.and apo ptosis was assessed by flow cytometry together with transmission electron micros copy.Using reverse transcriptase polymerase chain reaction,the expression of sur vivin mRNA was evaluated before and after action of artesunate on malignant cell s. Results:The proliferation of MGC-803 was inhibited notably by artesunate with a dosage-dependant character(P
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Aim To evaluate the efficacy of TFU as a precursor of 5-FU on the growth inhibition of human gastric carcinoma cell lines SGC-7901 and MKN-45.Methods In vitro experiments,cell growth inhibition was measured by MTT assay.The rates were compared in the presence and Absence of liver microsomal enzymes.The morphology of apoptotic cells was detected by observation with a fluorescence microscope after staining by using adridine orange/ethidium bromide solution.DNA fragmentation was analyzed by agarose gel electrophoresis and flow cytometry respectively.Western blot was employed to analyze the expression of Bcl-2 and Bax.The in vivo efficacy of TFU was assessed in nude mice bearing tumours.The specimens were re-moved and the in situ cell apoptosis detection kit was employed for TUNEL staining.Results Growth of SGC-7901 and MKN-45 cells was remarkably suppressed by treatment with TFU in the presence of liver microsomal enzymes in vitro,suggesting that TFU might be converted to 5-FU by the enzymes.Similar treatment of TFU induced apoptosis of the cells,which was deduced from typical apoptotic features such as morphology,the formation of characteristic ladder pattern of DNA migration and the accumulation of sub-G1 phase.Furthermore,a significant inhibition of Bcl-2 expression and the up-regulation of Bax were observed after treatment with TFU in the presence of liver microsomal enzymes.Growth of human gastric carcinoma cells was significantly delayed by oral administration of TFU with low side effects.Apoptosis in xenografts was also observed by means of TUNEL staining method.Conclusion Treatment of TFU in the presence of liver microsomal enzymes could promote the inhibition of gastric carcinoma cell proliferation.TFU might sustain release of 5-FU mediated by liver microsomal enzymes.Low dose of 5-FU might trigger the carcinoma cells apoptosis via regulation of Bax and Bcl-2.
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Objective: To study the effects of genistein on the proliferation and cell cycle progression of human gastric carcinoma cells. Methods: 3H TdR incorporation test was used to investigate the cell proliferation. Flow cytometry was used to analyze the cell cycle arrest. Immunocytochemistry technique and Western blotting were used to observe the cyclin B and P21 waf1/cip1 protein expression. Results: Genistein inhibited the proliferation of tumor cells significantly, arrested cell cycle progression at G 2/M phase, and enhanced cyclin B and P21 waf1/cip1 protein expression in dose dependent manner. Conclusion:Proliferatory inhibition and G 2/M arrest of human gastric carcinoma cells after treated with genistein may be due to increased stability of cyclin B protein and the expression of P21 waf1/cip1 .
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Objective To explore the effect of phytic acid on apoptosis of human gastric carcinoma SGC-7901 cells and its mechanism. Method The inhibiting action of phytic acid on SGC-7901 cells was examined by MTT assay; the morphology alteration of SGC-7901 cells was examined by reverse discrepancy microscope; and the expression of c-myc protein was detected by immunohistochemisty method and Western blot method. Results Phytic acid inhibited the growth of human gastric carcinoma SGC-7901 cells in dose and time dependent manner. The growth of cells in test groups were inhibited higher than in control group. The expression of c-myc protein in phytic acid group was lower compared with control group,and reduced in a dose-dependent manner. Conclusion Phytic acid can induce apoptosis of human gastric carcinoma SGC-7901 cells,and the mechanism may be related to apoptosis associated gene c-myc.