Human Gastric Carcinoma BGC-823 Research Articles

Posttranscriptional regulation of the telomerase hTERT by gambogic acid in human gastric carcinoma 823 cells

We previously reported that gambogic acid (GA), a natural product, was an effective telomerase inhibitor by repressing hTERT promoter. In this study, posttranscriptional regulation of the telomerase hTERT by GA was investigated in BGC-823 human gastric carcinoma cells. The telomerase activity was detected by PCR-TRAP assay. RT-PCR assay and Western blot were performed to examine the repression of telomerase hTERT and c-Myc after GA or c-Myc-specific siRNA treatment. The results indicated that GA repressed telomerase activity and hTERT transcriptional activity via down-regulation of c-Myc expression in BGC-823 human gastric carcinoma cells. We further observed that hTERT transcriptional activity was not completely blocked by c-Myc-specific siRNA, suggesting that additional factors are involved in the repression of telomerase activity. The results of Western blot and immunoprecipitation assay revealed that GA inhibits the phosphorylation of Akt. The further results also confirmed that celecoxib, an inhibitor of Akt phosphorylation, could significantly repressed telomerase activity alone and enhance the repression of telomerase activity combined with GA. These data suggested that GA inhibits the posttranslational modification of hTERT by inhibiting the phosphorylation of Akt. Collectively, we suggest that GA represses telomerase activity not only by repressing hTERT transcriptional activity via c-Myc but also by posttranslational modification of hTERT via Akt.

Gambogic acid-induced G2/M phase cell-cycle arrest via disturbing CDK7-mediated phosphorylation of CDC2/p34 in human gastric carcinoma BGC-823 cells

Molecular mechanisms of cell-cycle arrest caused by gambogic acid (GA), a natural product isolated from the gamboge resin of Garcinia hanburryi tree, have been investigated using BGC-823 human gastric carcinoma cells as a model. Based on our 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazoliumbromide (MTT) assay and flow cytometric analysis, treatment of BGC-823 cells with growth suppressive concentrations of GA caused an irreversible arrest in the G(2)/M phase of the cell cycle. Western blot analysis demonstrated that GA-induced cell-cycle arrest in BGC-823 cells was associated with a significant decrease in CDC2/p34 synthesis, which led to the accumulation of phosphorylated-Tyr(15) (inactive) form of CDC2/p34. Real-time PCR, western blot and kinase activity assays revealed that GA-induced reduction of CDC2/p34 expression was mediated through the inhibition of cyclin-dependent kinase (CDK)-activating kinase (CDK7/cyclin H) activity. In addition, GA-treated cells were shown to have a low level of CDK7 kinase-phosphorylated-Thr(161) CDC2/p34 (active). Taken together, our results suggested that the inhibited proliferation of GA-treated BGC-823 cells was associated with the decreased production of CDK7 mRNA and protein, which in turn, resulted in the reduction of CDK7 kinase activity. The reduced CDK7 kinase activity is responsible for the inactivation of CDC2/p34 kinase and the irreversible G(2)/M phase cell-cycle arrest of human gastric carcinoma BGC-823 cells.

Syntheses, Characterization and Antitumour Activities of Rare Earth Metal Complexes with 2-(((4,6-dimethyl)-2-Pyrimidinyl)thio)-Acetic Acid

Eight rare earth metal (III) complexes with 2-(((4,6-dimethyl)-2-pyrimidinyl)thio)-acetic acid, LnL 3- nH 2O [HL = 2-(((4,6-dimethyl)-2-pyrimidinyl)thio)-acetic acid; Ln = La, Ce, Pr, Nd, Sm Eu, Gd, Tb; n = 4 or 5], were prepared and characterized by elemental analysis, complexometric titration, thermal analysis, conductivity, IR and 1H-NMR. The results reveal that carboxyl group of the ligand coordinates with rare earth ions in bidentate mode after deprotonated. The water molecules exist as crystal water in the complexes. The anti-tumour activities of HL and some complexes were tested by both the MTT and SRb methods. The results show that the suppression ratios of some complexes against the tested tumour cells (HL-60 human leukemia cell lines, BGC-823 human gastric carcinoma cell lines, hela human cervix adenocarcinoma cell lines and Bel-7402 human hepatic carcinoma cell lines) are superior to HL.

Anti-tumor effects of Apoptin gene on human gastric carcinoma BGC-823 and its correlation with apoptotic signal transduction

AIM: To investigate the antitumor effects of Apoptin gene on human gastric carcinoma BGC-823, and to explore the mechanism of apoptin in the participation of signal transduction pathway.METHODS: Recombinant plasmid pVAX1-Apoptin was transfected into BGC-823 cells by application of liposome in vitro. The antitumor effect on BGC-823 cells was measured throughmethyl thiazolyl tetrazolium (MTT) assay, and the morphological changes of pVAX1-Apoptin-transfected cells were observed by AO/EB staining. Furthermore, the mitochondrial trans-membrane potential (ΔΨm) was analyzed by flow cytometry and the release of cytochrome c (Cyto c) was detected by Western blot assay. Colorimetric assay was used to analyze the activities of Caspase-3/9.RESULTS: pVAX1-Apoptin caused a more powerful cytotoxic effect than pVAX1 did (72 h after 10-μg plasmid transfection, 23.37% vs 99.61%, P 0.01), and apoptosis was a dominant death style in the pVAX1-Apoptin-transfected cells. Furthermore, in pVAX1-Apoptin-transfected cells, an increase of cytosolic cytochrome c con-tent co-occurred with a loss of trans-membrane potential (compared with pVAX1-transfected BGC-823 cells, 46.7% ± 7.26% vs 70.66% ± 5.98%, P 0.01). In addition, a significant difference was found between pVAX1-Apoptin-and pVAX1-transfected cells in Caspase-9 and Cas-pase-3 activities (Caspase-9: 0.181 ± 0.032 vs 0.079 ± 0.013, P 0.01; Caspase-3: 0.242 ± 0.041 vs 0.058 ± 0.023, P 0.01).CONCLUSION: In BGC-823 cells, pVAX1-Apoptin induced collapse of the mitochondrial trans-membrane potential can exert a feedback effect to elicit cytochrome c release, and then lead to the activation. Cytochrome cdependent Caspase-9 activation stimulates the key down-stream factors of apoptotic pathway, exhibiting an inhibitory effect on BGC-823 cells.

Co-expression of heat shock protein 70 and glucose-regulated protein 94 in human gastric carcinoma cell line BGC-823

To investigate the co-expression and significance of heat shock protein 70 (HSP70) and glucose-regulated protein 94 (grp94) in human gastric carcinoma cell line BGC-823. The expression and localization of HSP70 and grp94 in human gastric carcinoma cell line BGC-823 were determined by immunocytochemistry and indirect immunofluorescence cytochemical staining. Flow cytometry was used to analyze the correlation between expression of HSP70, grp94 and cell cycle in BGC-823 cell line. Gastric cancer cell line BGC-823 expressed high level of HSP70 and grp94. The positive rate of HSP70 and grp94 was 84.9+/-4.94% and 79.6+/-5.16%, respectively. Both of them were stained in cell plasma. There was a significant difference compared with control group (1.9+/-0.94%, P<0.01). During the cell cycle, HSP70 and grp94 were continuously expressed in BGC-823. HSP70 and grp94 are highly expressed in human gastric carcinoma BGC-823 cells through the whole cell cycle. There is no relationship between expression of HSP70, grp94 and cell cycle.

Applying proteomic methodologies to analyze the effect of hexamethylene bisacetamide (HMBA) on proliferation and differentiation of human gastric carcinoma BGC-823 cells

Human gastric carcinoma BGC-823 cells underwent morphological differentiation and cell cycle arrest in vitro when treated with 5 mM hexamethylene bisacetamide (HMBA) for 48 h. To further understand the mechanism of HMBA-induced differentiation, proteomic methodologies were applied to screen and identify altered proteins involved in the commitment of BGC-823 cells to differentiate. Five distinct altered proteins were acquired by two-dimensional (2-D) PAGE and were consequently identified as ras-related protein rab-35 (Rab-35), splice truncated isoform of transmembrane protease, serine 3 (serine TADG-12), regulator of G-protein signaling 1 (RGS1), ret finger protein-like 1 (RFPL1) and F-actin capping protein alpha-3 subunit (GSG3) by analysis of mass spectrograph. Of the five proteins, serine TADG-12 down-regulated under the detectable level after HMBA treatment, Rab-35, RGS1 and RFPL1 sharply up-regulated within the HMBA-induced BGC-823 cells, and GSG3, appearing in both treated and untreated cells, remarkably increased within BGC-823 cells after HMBA stimulation. Our results implicate that the molecular mechanism of BGC-823 cell differentiation in response to HMBA may involved in complex processes including a signaling network linking vesicle transport, actin cytoskeleton remodeling except for morphology differentiation, cell cycle G1 arrest.

Applying proteomic methodologies to analyze the effect of hexamethylene bisacetamide (HMBA) on proliferation and differentiation of human gastric carcinoma BGC-823 cells

Human gastric carcinoma BGC-823 cells underwent morphological differentiation and cell cycle arrest in vitro when treated with 5 mM hexamethylene bisacetamide (HMBA) for 48 h. To further understand the mechanism of HMBA-induced differentiation, proteomic methodologies were applied to screen and identify altered proteins involved in the commitment of BGC-823 cells to differentiate. Five distinct altered proteins were acquired by two-dimensional (2-D) PAGE and were consequently identified as ras-related protein rab-35 (Rab-35), splice truncated isoform of transmembrane protease, serine 3 (serine TADG-12), regulator of G-protein signaling 1 (RGS1), ret finger protein-like 1 (RFPL1) and F-actin capping protein alpha-3 subunit (GSG3) by analysis of mass spectrograph. Of the five proteins, serine TADG-12 down-regulated under the detectable level after HMBA treatment, Rab-35, RGS1 and RFPL1 sharply up-regulated within the HMBA-induced BGC-823 cells, and GSG3, appearing in both treated and untreated cells, remarkably increased within BGC-823 cells after HMBA stimulation. Our results implicate that the molecular mechanism of BGC-823 cell differentiation in response to HMBA may involved in complex processes including a signaling network linking vesicle transport, actin cytoskeleton remodeling except for morphology differentiation, cell cycle G1 arrest.

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