Gastric Cancer BGC-823 Cells Research Articles

Effects of microRNA-29 family members on proliferation and invasion of gastric cancer cell lines

MicroRNAs have emerged as post-transcriptional regulators that are critically involved in the biologic behavior of cells. This study was designed to investigate the effect of members of the microRNA-29 family on the expression of cell division cycle 42 (Cdc42) and their roles on proliferation, migration, and invasion of gastric cancer cells. We detected microRNA-29s and Cdc42 expression in gastric cancer cells by real-time polymerase chain reaction (PCR) and Western blot analysis. Negative controlled RNA (ncontrol), microRNA-29 family members (microRNA-29a, -29b, and -29c), and Cdc42-specific small interfering RNA (si-Cdc42) were chemically synthesized and transfected into SGC7901 and BGC823 gastric cancer cells, which have a relatively low expression of microRNA-29s and a relatively high expression of Cdc42. The expression of Cdc42 and the phosphorylation of its downstream molecular PAK1 expressions were determined by Western bolt analysis. Cell Counting Kit-8 was used to measure cell proliferation, and wound-healing and invasion assays were used to examine the abilities of migration and invasion. Similar to si-Cdc42, the ectopic expression of microRNA-29 family members significantly reduced the expression of Cdc42 and its downstream molecular PAK1 phosphorylation levels. Consistently, ectopic expression of microRNA-29s inhibited proliferation and migration in gastric cancer cells. Invasive cell counts of the SGC7901, ncontrol/SGC7901, si-Cdc42/SGC7901, microRNA-29a/SGC7901, microRNA-29b/SGC7901, and microRNA-29c/SGC7901 cell groups were 84.0+/-4.2, 71.7+/-4.6, 16.3+/-3.2, 15.7+/-3.8, 16.3+/-3.0, and 16.7+/-3.1, respectively. The invasive cell counts of the BGC823, ncontrol/BGC823, si-Cdc42/BGC823, microRNA-29a/BGC823, microRNA-29b/BGC823, and microRNA-29c/BGC823 cell groups were 199.0+/-10.5, 146.3+/-9.7, 72.7+/-8.2, 86.7+/-8.5, 86.0+/-8.5, and 73.3+/-8.3, respectively (P<0.05). Members of the microRNA-29 family can obviously inhibit cell proliferation, migration, and invasion of gastric cancer cells by targeting Cdc42.

Construction of COL1A1 short hairpin RNA vector and its effect on cell proliferation and migration of gastric cancer cells

To construct COL1A1-targeted short hairpin RNA (shRNA) vector with pSilencer 4.1-CMV neo siRNA expression vector and to evaluate its effect on proliferation and migration of gastric cancer BGC-823 cells in vitro. Three COL1A1-shRNA plasmids (COL1A1-shRNA-1, COL1A1-shRNA-2, COL1A1-shRNA-3), targeting different sites of COL1A1 gene, were constructed using pSilencer 4.1-CMV neo siRNA expression vector and transfected into gastric cancer BGC-823 cells. Real time quantitative RT-PCR and Western blot were performed to detect expression levels of COL1A1. MTT and Transwell migration assays were employed to evaluate the effects of COL1A1 gene silence on cell proliferation and migration. Three recombinant plasmids targeting COL1A1 were constructed successfully. The expressions of COL1A1 in BGC-823 cells, including mRNA and protein levels, were significantly inhibited by the COL1A1-shRNA transfectants, which resulted in a clear reduction of cell proliferation and migration capacity. The COL1A1-shRNA can effectively knock down gene expression and inhibit proliferation and migration of gastric cancer BGC-823 cells.

Interference of Chkl/2 by RNA Regulates G2/M Arrest and Expressions of Cell Cycle Related Proteins Induced by Diallyl Disulfide*

Chk1 and Chk2 play major role in cell cycle checkpoint signaling pathway, which are mainly involved in G2/M cell cycle checkpoint signal transduction. Firstly, the siRNA targeting at Chk1 or Chk2 gene was transfected into human gastric cancer BGC823 cells for 24 h before 15 mg/L DADS was added Then, the mRNA and protein expression of Chk1 or Chk2 was detected by Real-time PCR and Western-blot respectively Cell cycle rates and expressions of CDC25C and cyclinB1 were determined by FCM and Western blot respectively. The results showed that the Chk1 or Chk2 expression was inhibited in Chk1 or Chk2 siRNA-transfected group, in which the Chk1 or Chk2 expression at mRNA level was reduced 84.7% and 69.0% respectively and the protein expression of Chk1 or Chk2 was reduced 73.4% and 78.5% respectively as compared with that in empty control group (P 0.05). Western blot showed that although DADS decreased expression of CDC25C and cyclinB1 in untransfected cells, inhibition of expression of CDC25C and cyclinB1 treated by DADS was blocked by Chk1 gene silence (P < 0.05). On the contrary.. Chk2 gene silence can not do so. These results suggest Chk1 gene silence could abrogate G2/M arrest induced by DADS in BGC823 cell line, and Chk1/CDC25C/cyclinB1 pathway was involved at the G2/M arrest induced by DADS

Type II cGMP-dependent protein kinase inhibits proliferation of the gastric cancer cell line BGC-823.

Our previous studies have demonstrated that the expression and activity of protein kinase G (PKG) II are significantly lower in human gastric cancer cell lines than in normal cells. This study was designed to investigate the effect of PKG II activation on the proliferation of human cultured BGC-823 gastric cancer cells. An adenoviral construct encoding the PKG II gene (Ad-PKG II) was used to infect BGC-823 cells, and the activity of the enzyme was induced by cGMP analogue 8-pCPT-cGMP. The proliferation-inhibitory effect of PKG II was analyzed by the MTT assay, BrdU incorporation assay and detection of proliferating cell nuclear antigen (PCNA) expression. Colony formation in soft agarose was performed to analyze the effect of PKG II on the anchorage-independent growth of the cells. The effect of PKG II in vivo was investigated in an immunocompromised nude mice model, and its effect on the cell cycle was analyzed by flow cytometry. The results showed that Ad-PKG II infection increased the expression of PKG II in BGC-823 cells. The activation of PKG II by 8-pCPT-cGMP caused a significant decrease in the number of live cells and inhibited DNA synthesis in individual cells. PKG II activation inhibited the EGF-induced increase in PCNA expression. The activation of PKG II also caused a significant inhibition of colony formation in soft agarose and significantly suppressed the in vivo growth of BGC-823 cells in immunocompromised nude mice. There was substantial cell arrest at the G1 phase and a decrease in the number of S phase cells in the Ad-PKG II/8-pCPT-cGMP-treated cells. These data indicate that the activation of PKG II by 8-pCPT-cGMP inhibits the proliferation of human gastric cancer cells.

Growth inhibitory effect and Chk1-dependent signaling involved in G2/M arrest on human gastric cancer cells induced by diallyl disulfide

Diallyl disulfide (DADS) inhibits growth and induces cell cycle G2/M arrest in human gastric cancer MGC803 cells. In this study, 15 mg/L DADS exerted similar effects on growth and cell cycle arrest in human gastric cancer BGC823 cells. Due to the importance of cell cycle redistribution in DADS-mediated anti-carcinogenic effects, we investigated the role of checkpoint kinases (Chk1 and Chk2) during DADS-induced cell cycle arrest. We hypothesized that DADS could mediate G2/M phase arrest through either Chk1 or Chk2 signal transduction pathways. We demonstrated that DADS induced the accumulation of phosphorylated Chk1, but not of Chk2, and that DADS down-regulated Cdc25C and cyclin B1. The expression of mRNA and total protein for Chkl and Chk2 was unchanged. Chk1 is specifically phosphorylated by ATR (ATM-RAD3-related gene). Western blot analysis showed that phospho-ATR was activated by DADS. Taken together, these data suggest that cell cycle G2/M arrest, which was associated with accumulation of the phosphorylated forms of Chk1, but not of Chk2, was involved in the growth inhibition induced by DADS in the human gastric cancer cell line BGC823. Furthermore, the DADS-induced G2/M checkpoint response is mediated by Chk1 signaling through ATR/Chk1/Cdc25C/cyclin B1, and is independent of Chk2.

Type II cGMP-dependent protein kinase inhibits proliferation of the gastric cancer cell line BGC-823

Our previous studies have demonstrated that the expression and activity of protein kinase G (PKG) II are significantly lower in human gastric cancer cell lines than in normal cells. This study was designed to investigate the effect of PKG II activation on the proliferation of human cultured BGC-823 gastric cancer cells. An adenoviral construct encoding the PKG II gene (Ad-PKG II) was used to infect BGC-823 cells, and the activity of the enzyme was induced by cGMP analogue 8-pCPT-cGMP. The proliferation-inhibitory effect of PKG II was analyzed by the MTT assay, BrdU incorporation assay and detection of proliferating cell nuclear antigen (PCNA) expression. Colony formation in soft agarose was performed to analyze the effect of PKG II on the anchorage-independent growth of the cells. The effect of PKG II in vivo was investigated in an immunocompromised nude mice model, and its effect on the cell cycle was analyzed by flow cytometry. The results showed that Ad-PKG II infection increased the expression of PKG II in BGC-823 cells. The activation of PKG II by 8-pCPT-cGMP caused a significant decrease in the number of live cells and inhibited DNA synthesis in individual cells. PKG II activation inhibited the EGF-induced increase in PCNA expression. The activation of PKG II also caused a significant inhibition of colony formation in soft agarose and significantly suppressed the in vivo growth of BGC-823 cells in immunocompromised nude mice. There was substantial cell arrest at the G1 phase and a decrease in the number of S phase cells in the Ad-PKG II/8-pCPT-cGMP-treated cells. These data indicate that the activation of PKG II by 8-pCPT-cGMP inhibits the proliferation of human gastric cancer cells.

Rofecoxib augments anticancer effects by reversing intrinsic multidrug resistance gene expression in BGC‐823 gastric cancer cells

To investigate combined chemotherapeutic effects of rofecoxib in combination with 5-fluorouracil (5-FU), cisplatin (DDP) and etoposide (VP-16) in vitro, and to explore the potential mechanisms in modulating multidrug resistance (MDR) expression. The BGC-823 gastric cancer cell line was incubated for 48 h with 0.1 micromol/L rofecoxib, 5-FU, DDP and VP-16 (1 microg/mL, 10 microg/mL and 100 microg/mL) alone, and combined with rofecoxib, respectively. Methyl-thiazolyl-tetrazolium and the terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-yriphosphate nick-end labeling assays were performed to calculate inhibitory rates and apoptotic index. Middle effects principles (CI values) were used to determine the interaction between rofecoxib and chemotherapeutic agents. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis were employed to determine expression of MDR1, multidrug resistance-associated protein 1 (MRP1), glutathione S-tranferase-pi (GST-pi) mRNA and protein in gastric cancer cells administered by rofecoxib, respectively. Both anticancer drugs such as 5-FU, DDP and VP-16 and rofecoxib inhibited the cells' proliferation and induced apoptosis in a dose-dependent manner, and a more significant inhibition was achieved when the cells were co-treated with anticancer drugs and rofecoxib. There was a synergetic role when different concentrations of chemotherapeutic agents were combined with rofecoxib (all CI < 1, P < 0.01 or 0.05). RT-PCR analyses of MDR gene families in BGC-823 gastric cancer cells revealed a strong expression in MRP1 and GST-pi mRNA, but MDR1 mRNA was undetectable. After administration with different concentrations of rofecoxib (0.1, 1.0, 10 micromol/L), significant downregulation of MRP1 and GST-pi mRNA was observed (MRP1: from 0.984 +/- 0.093-0.513 +/- 0.098; GST-pi: from 1.078 +/- 0.201-0.472 +/- 0.084, P < 0.01 or 0.05). In addition, MRP1 and GST-pi protein expression induced by rofecoxib were also reduced (P < 0.01 or 0.05). Rofecoxib, a specific cyclooxygenase-2 inhibitor, plays a chemotherapeutic sensitizer role in various anticancer agents on the BGC-823 gastric cancer cell line, which could be partly explained by its ability to reverse the intrinsic MRP1 and GST-piin vitro.

Short hairpin RNA–mediated inhibition of S100A4 promotes apoptosis and suppresses proliferation of BGC823 gastric cancer cells in vitro and in vivo

S100A4 has been found to be over-expressed in gastric cancer and associated with poor prognosis of the patients, yet the exact role and molecular mechanism of S100A4 in gastric cancer has not been determined. We found that S100A4 inhibition by RNAi lead to reduced proliferation and increased apoptosis of gastric cancer cell line BGC823. Intratumoral injection of p S100A4-shRNA suppressed tumor growth in nude mice. We also found that S100A4 inhibition decreased the expression of both NF-κB p65 and phospho( Ser32) -I-κB-α. Our results suggested that S100A4 may be an attractive candidate for the therapeutic targeting of gastric cancer.

Arsenic trioxide induces apoptosis and G2/M phase arrest by inducing Cbl to inhibit PI3K/Akt signaling and thereby regulate p53 activation

Arsenic trioxide (ATO) strongly induces apoptosis in acute promyelocytic leukemia (APL), but it induces cell cycle arrest in most solid tumors. In this study, we investigated the mechanism of ATO action on APL-derived NB4 cells and gastric cancer cell lines. ATO decreased the viability of both cell lines, but gastric cancer cells were much less susceptible. ATO-induced G2/M phase arrest and p53 degradation in gastric cancer MGC803 cells. In contrast, ATO-induced apoptosis in NB4 cells without degradation of p53. Both processes were accompanied by transient activation of Akt. The PI3K/Akt inhibitor LY294002 significantly increased the amount of p53 protein and ATO-induced apoptosis in both cell lines and decreased G2/M phase arrest of MGC803 cells. In addition, ATO up-regulated the expression of Cbl proteins in both cell lines. Inhibition of Cbl with the proteasome inhibitor Ps341 decreased apoptosis in NB4 cells and increased the G2/M phase arrest of MGC803 cells, and it also prolonged the activation of PI3K/Akt by ATO. Consistent results with those in MGC803 cells were showed in gastric cancer cell BGC823 and SGC7901 after ATO treatment. These results demonstrate that inhibition of PI3K/Akt signaling by Cbl is involved in both ATO-induced apoptosis of NB4 cells and ATO-induced G2/M phase arrest of gastric cancer cells. Cbl achieved these effects probably via its regulating PI3K/Akt pathway, and thereby modulated p53 activation.

Concomitant use of Ad5/35 chimeric oncolytic adenovirus with TRAIL gene and taxol produces synergistic cytotoxicity in gastric cancer cells

Chimeric adenoviral vectors possessing fiber derived from human adenovirus subgroup B (Ad35) have been developed for their high infection efficiency in cell types which are refractory to adenovirus serotype 5 (Subgroup C). The present study constructed an E1B-deleted chimeric oncolytic adenovirus, SG235-TRAIL, which carries a human TRAIL gene expression cassette and whose fiber shaft and knob domains are from serotype Ad35. It was found that SG235-TRAIL preferentially replicated in gastric cancer cell lines, SGC-7901 and BGC-823 compared to in normal human fibroblast BJ cells. Also, when compared with a replication-deficient chimeric vector Ad5/35-TRAIL, SG235-TRAIL mediated a higher level of the transgene expression via viral replication in the cancer cells. Further, because of the more efficient cell-entry and infection, SG235-TRAIL induced stronger cell apoptosis than the Ad5 CRAD vector, ZD55-TRAIL. In addition, SG235-TRAIL in combination with the chemotherapeutic drug, taxol, produced a synergistic cytotoxic effect in cancer cells in vitro without causing significant toxicity to normal cells. In the gastric tumor xenograft mouse model, intratumoral SG235-TRAIL injection produced a significant antitumor effect 14 days after treatment. Pathological examination demonstrated TRAIL expression and associated apoptosis in majority of SG235-TRAIL-treated tumor cells. These results suggest that SG235-TRAIL is a potential novel, efficient anti-cancer agent, and in combination with taxol, it would be even more useful with considerably low toxic side effects.

Effect of cobalt chloride on &lt;I&gt;S100A4&lt;/I&gt; expression in human gastric cancer cell BGC823

S100A4 is an important metastasis-associated gene. Researches have confirmed the close correlation between overexpression of S100A4 gene and gastric cancer's infiltration, lymph node metastasis and in vitro invasiveness of gastric cancer cells. In order to investigate the mechanism of overexpression of S100A4 gene, hypoxia mimetic cobalt chloride (CoCl2) was used to treat gastric cancer cell BGC823, and then the expression of S100A4 mRNA and protein in BGC823 cells were detected by RT-PCR, immunohistochemistry, immunofluorescence, and Western blotting analysis. After treatment with CoCl2, the expression of S100A4 mRNA and protein in BGC823 cell was increased. These results suggested that hypoxia mimetic cobalt chloride could increase the expression of S100A4 gene in gastric cancer cell BGC823.

Short hairpin RNA-mediated silencing of TROP2 gene in gastric cancer BGC-823 cells

Short hairpin RNA-mediated silencing of TROP2 gene in gastric cancer BGC-823 cells

Molecular basis of the effect of midkine on tumor growth in human gastric cancer cell BGC823

Hyperphosphorylation of extracellular signal-regulated protein kinases-1/2 (ERK1/2) is known to promote cancer cell proliferation. We therefore investigated the constitutive phosphorylation levels of ERK1/2 and the expression of its downstream targets c-Fos, c-Jun, and cyclooxygenase-2 (COX-2) in biopsied human gastric cancer tissues. Results showed that ERK1/2 phosphorylation and c-Jun expression were significantly lowered in gastric cancer compared with the non-cancer adjacent tissues. The expression of c-Fos, however, was not altered while COX-2 was significantly up-regulated. To conclude, we demonstrate that hypophosphorylation of ERK1/2 may occur in gastric cancer. Such discovery may have implication in the application of pathway-directed therapy for this malignant disease.

Expression of pituitary homeobox 1 gene in human gastric carcinogenesis and its clinicopathological significance

To investigate the effect of pituitary homeobox 1 (PITX1) expression in cases of human gastric cancer on cancer differentiation and progression, and carcinogenesis. Using polyclonal PITX1 antibodies, we studied the expression of PITX1 in normal gastric mucosa, atypical hyperplasia, intestinal metaplasia, and cancer tissue samples from 83 gastric cancer patients by immunohistochemistry. Moreover, semi-reverse transcription polymerase chain reaction (semi-RT-PCR) was performed to detect the mRNA level of PITX1 in three gastric cancer cell lines and a normal gastric epithelial cell line. Subsequently, somatic mutations of the PITX1 gene in 71 gastric cancer patients were analyzed by a combination of denaturing high performance liquid chromatography (DHPLC) and DNA sequencing. Immunohistochemistry showed that PITX1 was strongly or moderately expressed in the parietal cells of normal gastric mucosa (100%), while 55 (66.3%) out of 83 samples of gastric cancers showed decreased PITX1 expression. Moreover, PITX1 expression was reduced in 20 out of 28 cases (71.5%) of intestinal metaplasia, but in only 1 out of 9 cases (11%) of atypical hyperplasia. More importantly, PITX1 expression was significantly associated with the differentiation, position and invasion depth of gastric cancers (r = -0.316, P < 0.01; r = 0.213, P < 0.05; r = -0.259, P < 0.05, respectively). Similarly, levels of PITX1 mRNA were significantly decreased in 2 gastric cancer cell lines, BGC-823 and SGC-7901, compared with the normal gastric epithelial cell line GES-1 (0.306 +/- 0.060 vs 0.722 +/- 0.102, P < 0.05; 0.356 +/- 0.081 vs 0.722 +/- 0.102, P < 0.05, respectively). Nevertheless, no somatic mutation of PITX1 gene was found in 71 samples of gastric cancer by DHPLC analysis followed by sequencing. Down-regulation of PITX1 may be a frequent molecular event in gastric carcinogenesis. Aberrant levels of PITX1 expression may be closely correlated with the progression and differentiation of gastric cancer.

Effect of valproate acid sodium on apoptosis of human gastric cancer cells and its mechanism study

To study the effect of valproate acid sodium(VPA) on apoptosis of human gastric cancer cell BGC-823 and to explore its possible mechanism. Cell growth inhibition was examined by MTT assay. Apoptosis rate was detected by FCM with Annexin V/PI staining. The activities and protein expression levels of caspase 3, caspase 8 and caspase 9 were examined by spectrophotometry and indirect immunofluorescence technique respectively. The growth inhibition rate and apoptosis rate of human gastric cancer cells, treated with 0.75-4.00 mmol/L VPA for 24 h and 48 h, elevated in time- and dose-dependent manner. Apoptosis rates of VPA 0.75 mmol/L 24 h and 48 h were (7.2 +/- 0.5)% and (9.2 +/- 1.0)%, of VPA 4.00 mmol/L 24 h and 48 h were (16.7 +/- 2.2)% and (20.4 +/- 1.6)% respectively, which were significantly different as compared to the control [24 h, (4.9 +/- 0.2)%, 48 h, (5.1 +/- 0.8)%] (P< 0.001). The activities and protein expression levels of caspase 3 and caspase 9 were up-regulated compared with the control group (P< 0.001), meanwhile the activity and protein expression of caspase 8 enhanced slightly after VPA treatment for 48 h. VPA can inhibit the growth and induce the apoptosis of BGC-823 cells mainly through the activation of caspase 9 pathway.

Protein kinase B inhibitor enhance sensitivity of gastric cancer cell to etoposide

To observe the effects of etoposide on protein kinase B (PKB) activity in distinct differentiated gastric cancer cell lines and the change of sensitivity to etoposide after pretreatment by wortmannin, a PKB inhibitor. To explore the relationship between PKB activity in gastric cancer cells and their sensitivity to etoposide chemotherapy. Four distinct differentiated gastric cancer cell lines, including MKN-28 (well differentiated), SGC-7901 (moderate differentiated), BGC-823 (poorly differentiated) and HGC-27 (undifferentiated), were studied. The PKB activities of these cell lines were detected by nonradioactive protein-kinase assay at different time points after etoposide treatment for 0,3,6,12,24 h with or without wortmannin pretreatment. Cell viabilities were assayed by MTT and cell apoptosis was analyzed by flow cytometry. Poorer differentiated gastric cancer cell lines had higher PKB activities. Etoposide treatment resulted in increase in PKB activity and apoptosis rate,and decrease in cell survival rate in a time-dependent manner in gastric cancer cell lines. Wortmannin pretreatment abolished PKB activity completely in gastric cancer cells,and decreased survival rate and increased apoptosis rate in SGC-7901, BGC-823, and HGC-27 cell lines. Etoposide can induce the PKB activity in gastric cancer cell lines. Wortmannin pretreatment enhances sensitivity of median and low differentiated gastric cancer cells to etoposide chemotherapy.

Effects of all‐trans‐retinoic on human gastric cancer cells BGC‐823

To determine the inhibitory effects of all-trans-retinoic acid (ATRA) on cell growth, cell cycle and vascular endothelial growth factor (VEGF) expression in the human gastric cancer cell line BGC-823 in vitro. Human gastric cancer BGC-823 cells were treated with various concentrations of ATRA and the cell growth was then determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide viability assay. The cell cycle distribution was analyzed using a flow cytometer. The VEGF mRNA and protein expression were analyzed by semi-quantitative RT-PCR and Western blotting, respectively. ATRA at concentrations of 0.1-10 micromol/L inhibited the growth of BGC-823 cells grown in culture; a time- and dose-dependent inhibitory influence was found. ATRA arrested BGC-823 cells at the G0/G1 phase in a dose-dependent way. Both VEGF mRNA and protein were decreased by ATRA in a dose-dependent way. The anti-tumor effects of ATRA on human gastric cancer cells are associated with G0/G1 phase arrest and decreased VEGF expression.

Synergistic interaction between tetrandrine and chemotherapeutic agents and influence of tetrandrine on chemotherapeutic agent-associated genes in human gastric cancer cell lines

Tetrandrine (Tet), a bis-benzylisoquinoline alkaloid that was isolated from the dried root of Hang-Fang-Chi (Stephania tetrandra S. Moore), is well known as processing a marked antitumor effect in vitro and in vivo. The aim of this study was to assess the interaction between tetrandrine and chemotherapeutic agents widely used in gastric cancer treatment, and to investigate the influence of tetrandrine on chemotherapeutic agent-associated gene expression and apoptosis. Synergistic interaction on human gastric cancer BGC-823 and MKN-28 cells was evaluated using the combination index (CI) method. The double staining with both Annexin-V-FITC and PI was employed to distinguish the apoptotic cells from living cells. Expression of chemotherapeutic agent-associated genes, i.e., excision repair cross-complementing 1 (ERCC1), thymidylate synthase (TS), class III beta-tubulin (beta-tubulin III) and tau, of BGC-823 cells with or without tetrandrine treatment were measured by real-time quantitative PCR. Tetrandrine had a synergistic effect on the cytotoxicity of chemotherapeutic agents in both two gastric cancer cell lines. The combination of tetrandrine and chemotherapeutic agents could also induce apoptosis in a synergistic manner. Tetrandrine could suppress the mRNA expression of ERCC1, TS, beta-tubulin III and tau. Most prominently, ERCC1, TS and beta-tubulin III mRNA levels were markedly suppressed at 0.29-, 0.12- and 0.60-fold, respectively, by the presentation of tetrandrine. Tetrandrine appears a promising candidate for combining with three chemotherapeutic agents. The possible mechanisms might be the synergistic apoptotic effect and the downregulation of chemotherapeutic agent-associated genes.

Effect of B-RAF-specific RNA interference on gastric cancer BGC823 cell line

To investigate the influence of B-RAF-specific RNA interference on the proliferation and apoptosis of gastric cancer BGC823 cell line. B-RAF-siRNA and scramble-siRNA were synthesized and transfected into BGC823 cells by TransMessenger. The expression of B-RAF gene and Bcl-2 gene in BGC823 cells was detected by RT-PCR and the level of apoptosis was evaluated in transfected cells by flow cytometry. Results showed that TransMessenger could effectively transfect B-RAF-siRNA and scramble-siRNA into BGC823 cells. B-RAF-siRNA significantly inhibited the expression of B-RAF gene and Bcl-2 gene in BGC823 cells by more than 90% to 100%. B-RAF-siRNA inhibited BGC823 cell prolifera-tion and induced apoptosis (P < 0.01). In conclusion, B-RAF-siRNA can effectively inhibit the expression of B-RAF gene and Bcl-2 gene, induce cell apoptosis and inhibit the proliferation of gastric cancer BGC823 cells.

A forskolin derivative, FSK88, induces apoptosis in human gastric cancer BGC823 cells through caspase activation involving regulation of Bcl-2 family gene expression, dissipation of mitochondrial membrane potential and cytochrome c release

FSK88, a forskolin derivative, was extracted and purified from cultured tropical plant roots, Coleus forskohlii. Our previous studies have demonstrated that FSK88 can inhibit HL-60 cell proliferation and induce the differentiation of HL-60 cells to monocyte macrophages. In this study, we showed that FSK88 can induce apoptotic death of human gastric cancer BGC823 cells in a dose- and time-dependent manner. Results showed that FSK88-induced apoptosis was accompanied by the mitochondrial release of cytochrome c and activation of caspase-3 in BGC823 cells. Furthermore, treatment with caspase-3 inhibitor (z-DEVD-fmk) was capable of preventing the FSK88-induced caspase-3 activity and apoptosis. FSK88-induced apoptosis in human gastric cancer BGC823 cells was also accompanied by the up-regulation of Bax, Bad and down-regulation of Bcl-2. Theses results clearly demonstrated that the induction of apoptosis by FSK88 involved multiple cellular and molecular pathways and strongly suggest that pro- and anti-apoptotic Bcl-2 family genes, mitochondrial membrane potential (Deltapsi(m)), cytochrome c, and caspase-3, participate in the FSK88-induced apoptotic process in human gastric cancer BGC823 cells.