Gastric Cancer Cell Lines BGC-823 Research Articles

Activation of Akt and ERK signalling pathways induced by etoposide confer chemoresistance in gastric cancer cells

Aims To identify whether phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase/extracellular-regulated protein kinases signalling pathways are implicated in the chemoresistance of gastric cancer and to explore the possible mechanisms. Methods Gastric cancer cell lines SGC7901 and BGC823 were exposed to etoposide, Wortmannin + etoposide or PD98059 + etoposide. Cell cycle distribution and cell apoptosis were detected using flow cytometry and Hoechst 33258 staining. Cells viability was determined by a colourimetric assay utilising 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). Akt activity was detected using non-radioactive immunoprecipitation-kinase assay. Western blotting was exploited to evaluate the level of phosphorylated ERK1/2 and expressions of c-Myc and p53 protein. Results Etoposide suppressed the viability of SGC7901 and BGC823 cells in a time- and dose-dependent manner; PD98059 and Wortmannin were able to enhance the cytotoxicity of etoposide. The apoptotic levels of cells treated with Wortmannin + etoposide or PD98059 + etoposide were significantly higher than those of cells treated with etoposide only. Phospho-ERK1/2, Akt activity and expression of c-Myc were significantly induced by etoposide in a time-dependent manner; moreover, there was a weak effect on the expression of p53 protein. Both Wortmannin and PD98059 elevated the level of p53 expression strikingly, however, only PD98059 suppressed the up-regulation trend of c-Myc expression induced by etoposide. Conclusion Chemotherapy reagent activated phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase/extracellular-regulated protein kinases signalling pathways, which decreased the chemotherapy sensitivity of gastric cancer cell lines SGC7901 and BGC823 via suppressing the expression of p53 and enhancing the expression of c-Myc. This may be one of the molecular mechanisms of gastric cancer chemoresistance.

Conversion of cadherin isoforms in cultured human gastric carcinoma cells

To explore the expression of cadherin isoforms in cultured human gastric carcinoma cells and its regulation. The expressions of cell adhesion molecules (including E-cadherin, N-cadherin, alpha-catenin, beta-catenin) and cadherin transcription factors including snail, slug and twist were determined by reverse transcriptase-polymerase chain reaction(RT-PCR), immunoblotting and immunofluorescence in SV40-immortalized human gastric cell line Ges-1 and human gastric cancer cell lines MGC-803, BGC-823 and SGC-7901. All cell lines expressed N-cadherin, but not E-cadherin. N-cadherin immunofluorescence was detected at cell membranous adherents junctions where co-localization with immunofluorescent staining of inner surface adhesion proteins alpha- and beta-catenins was observed. The transformed Ges-1 and gastric cancer cell lines all expressed transcription factors (snail, slug and twist) which inhibited the expression of E-cadherin and triggered epithelial-mesenchymal transformation. Cadherin isoforms can change from E-cadherin to N-cadherin in transformed human gastric cancer cells, which is associated with intracellular events of stomach carcinogenesis and high expression of corresponding transcription factors.

A proteomic investigation into a human gastric cancer cell line BGC823 treated with diallyl trisulfide

Garlic is generally used as a therapeutic reagent against various diseases worldwide. Although a great effort is made to understand the pharmaceutical mechanisms of garlic and its derivatives, there are many mysteries to be uncovered. Using proteomic means, herein we have systematically studied the responses of protein expression in BGC823 cells, a gastric cancer cell line, induced by diallyl trisulfide (DATS), a major component of garlic derivatives. A total of 41 unique proteins in BGC823 were detected with significant changes in their expression levels corresponding with DATS administration. Of these proteins, five typical ones, glutathione S-transferase-pi (GST-pi), voltage-dependent anion channel-1 (VDAC-1), Annexin I, Galectin and S100A11, were further examined by Western blotting, resulting in coincident data with the proteomic evidence. Moreover quantitative real-time RT-PCR experiments offered dynamic data of mRNA expression, indicating the responses of Annexin I and GST-pi expression within a short period after DATS treatment. Interestingly, approximately 50% of DATS-sensitive proteins (19/41) in BGC823 are tightly associated with apoptotic pathways. These proteomic results presented, therefore, provide additional support to the hypothesis that garlic is a strong inducer of apoptosis in tumor cells.

Analysis on the mechanism of Helicobacter pylori-induced apoptosis in gastric cancer cell line BGC-823.

Helicobacter pylori (Hp) can induce apoptosis of gastric cancer cells. The mechanism of the process still needs further elucidating. This study was aimed to analyse the mechanism through which Hp induce apoptosis in human gastric cancer cell line BGC-823. The extract from VacA(+) and CagA(+) Helicobacter pylori strain NCTC11637 was applied to induce apoptosis. The expression, breakdown, and phosphorylation of proteins were probed by Western blotting with specific antibodies. Apoptosis of the cells was detected by flow cytometry. The results showed that incubating the cells with Hp extract caused the breakdown of both caspase-3 and -1. The breakdown was dose-dependent and correlated with the occurrence of the Hp extract-induced apoptosis. Among the substrates of caspase-3, DNA fragment factor (DFF) was degraded during incubation with Hp extract and a small fragment was released. However, poly(ADP-ribose) polymerase (PARP) did not break down during the incubation. Tyrosine kinase inhibitor Genistein prevented both the break down of caspase-3 and the apoptosis induced by Hp extract. MAPK/ERK inhibitor PD98059 did not prevent the apoptosis induced by Hp extract. The expression and activity of JNK, and the expression of Bcl-2 and Fas proteins did not change during the incubation with Hp extract. The results suggested that Hp extract initiated apoptosis in BGC-823 cells through activating tyrosine kinase, caspase-1, -3, and DFF.

Association between endogenous gene expression and growth regulation induced by TGF-β1 in human gastric cancer cells

To investigate the association between endogenous gene expression and growth regulation including proliferation and apoptosis induced by transforming growth factor-beta1 (TGF-beta1) in human gastric cancer (GC) cells. Reverse transcription polymerase chain reaction (RT-PCR) was performed to detect the main components of the TGF-beta1/Smads signal pathway in human poorly differentiated GC cell line BGC-823. Localization of Smad proteins was also determined using immunofluorescence. Then, the BGC-823 cells were cultured in the presence or absence of TGF-beta1 (10 ng/mL) for 24 and 48 h, and the effects of TGF-beta1 on proliferation and apoptosis were measured by cell growth curve and flow cytometry (FCM) analysis. The ultrastructural features of BGC-823 cells with or without TGF-beta1 treatment were observed under transmission electron microscope. The apoptotic cells were visualized by means of the terminal deoxynucleotidyl transferase (TdT)-mediated dTUP in situ nick end-labeling (TUNEL) method. Meanwhile, the expression levels of endogenous p15, p21 and Smad7 mRNA and the corresponding proteins in the cells were detected at 1, 2 and 3 h after culture in the presence or absence of TGF-beta1 (10 ng/mL) by semi-quantitative RT-PCR and Western blot, respectively. The TGF-beta1/Smad signaling was found to be intact and functional in BGC-823 cells. The growth curve revealed the most evident inhibition of cell proliferation by TGF-beta1 at 48 h, and FCM assay showed G1 arrest accompanied with apoptosis induced by TGF-beta1. The typical morphological changes of apoptosis were observed in cells exposed to TGF-beta1. The apoptosis index (AI) in TGF-beta1-treated cells was significantly higher than that in the untreated controls (10.7+/-1.3% vs 0.32+/-0.06%, P<0.01). The levels of p15, p21 and Smad7 mRNA and corresponding proteins in cells were significantly up-regulated at 1 h, but gradually returned to basal levels at 3 h following TGF-beta1 (10 ng/mL) treatment. TGF-beta1 affects both proliferation and apoptosis of GC cells through the regulation of p15 and p21, and induces transient expression of Smad 7 as a negative feedback modulation of TGF-beta1 signal. Our results suggest a novel functional role of p21 as an accelerant of TGF-beta1-mediated apoptosis in GC cells.

Matrix metalloproteinase 11 depletion inhibits cell proliferation in gastric cancer cells

Our previous study has shown that matrix metalloproteinase 11 (MMP11) is highly expressed in tumor cell lines and primary tumor of gastric cancer (GC). In order to reveal the correlation between expression of MMP11 and biological features of GC cell, we have constructed the recombinant plasmids producing hairpin small interfering RNA (siRNA) to target MMP11 mRNA using a vector-based RNA interference technology. Stable transfection of recombinants into GC cell line BGC823 specifically depleted the mRNA and protein of MMP11 as demonstrated by RT-PCR and Western blotting analysis. The siRNA-treated cells exhibited significantly decreased growth ability compared with mock transfectants and parental BGC823 cells. Furthermore, colony formation of MMP11 deficient cells was dramatically inhibited in soft agar and tumorigenicity was reduced in nude mice, respectively. These results provide new insights into the function of MMP11 and suggest that MMP11 may play an important role in the control of cell proliferation and tumor development in GC.

In vitro effects of recombinant human growth hormone on growth of human gastric cancer cell line BGC823 cells.

To study the effects of recombinant human growth hormone (rhGH) on growth of human gastric cancer cell line in vitro. Experiment was divided into control group, rhGH group, oxaliplatin (L-OHP) group and rhGH+L-OHP group. Cell inhibitory rate, cell cycle, cell proliferation index (PI) and DNA inhibitory rate of human gastric cancer line BGC823, at different concentrations of rhGH treatment were studied by cell culture, MTT assay and flow cytometry. The distinctly accelerated effects of rhGH on multiplication of BGC823 cell line were not found in vitro. There was no statistical significance between rhGH group and control group, or between rhGH+L-OHP group and L-OHP group (P>0.05). The cell growth curve did not rise. Cell inhibitory rate and cells arrested in G(0)-G(1) phase were obviously increased. Meanwhile, cells in S phase and PI were distinctly decreased and DNA inhibitory rate was obviously increased in rhGH+L-OHP group in comparison with control group and rhGH group, respectively (P<0.01). Cell inhibitory rate showed an increasing trend and PI showed a decreasing trend in rhGH+L-OHP group compared with L-OHP group. In vitro rhGH does not accelerate the multiplication of human gastric cancer cells. It may increase the therapeutic efficacy when it is used in combination with anticancer drugs.

PKCa translocation from mitochondria to nucleus is closely related to induction of apoptosis in gastric cancer cells

PKCs have been implicated in the regulation of cellular differentiation, proliferation, apoptosis and signal transduction. It was demonstrated in this study that PKCalpha was located both at mitochondria and in cytosol in gastric cancer cell line BGC-823. Treatment of cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in the translocation of PKCalpha from both mitochondria and cytosol to nucleus as clearly shown by laserscanningconfocal microscopy, while the protein level of PKCalpha was not changed by TPA treatment as detected by Western blot. The results also revealed that TPA-induced translocation of PKCalpha was in close association with apoptosis induction, and such association was further affirmed by other experiments where various apoptotic stimuli and specific inhibitors of PKC were used. Taken together, these findings indicate that translocation of PKCalpha from both mitochondria and cytosol to nucleus in gastric cancer cell is accompanied by induction of apoptosis, and may imply a new mechanism of the potential linking between cell apoptosis and PKCalpha translocation.

The specific photodynamic effects of monoclonal antibodies conjugated with hematoporphyrin derivative on gastric cancer in vitro and in vivo

A murine monoclonal antibody (MoAb) BB4.3 raised against human gastric cancer cell line BGC823 was purified with Protein A-Sepharose CL-4B affinity chromatography and identified as IgG2a. The MoAb BB 4.3 was conjugated with hematoporphyrin derivative (HPD) by using carbodiimide. The conjugate HPD-BB4.3 retained both antibody activity and photochemical activity of HPD. In vitro the phototoxic effect of HPD-BB4.3 conjugate on target cells was about 15 times higher than that of free HPD. The target-selective photocytotoxicity of HPD-BB 4.3 was verified by the fact that this conjugate showed greater cytotoxicity than the corresponding normal mouse IgG (NIgG) conjugated with HPD, and that it showed less cytotoxicity to BB4.3 negative reaction cell line B-80 than to BGC 823; and moreover, its cytotoxicity to BGC823 cells could be blocked specifically by excess BB4.3 antibody. In an in vivo study, nude mice inoculated with 2 X 10(5) BGC823 cells were administered HPD-BB4.3, HPD, HPD-NIgG, HPD plus BB4.3 and PBS respectively; and were then exposed to light. Out of six animals treated with HPD-BB4.3 conjugate, four remained tumor-free for a long time. There was significant difference between HPD-BB 4.3 treated group and all control groups. The HPD-BB4.3 conjugate was also shown more than 40% inhibition of growth of established tumors in comparison with the control groups.