Gastric Cancer Cell Lines BGC-823 Research Articles

Daucosterol inhibits cancer cell proliferation by inducing autophagy through reactive oxygen species-dependent manner.

This study aims to evaluate the anti-cancer effect of daucosterol and explore its possible mechanism. MTT and colony formation assay were performed to determine the effect of daucosterol on cancer cell proliferation in vitro. H22 allograft model was used for the assessment of its anti-cancer activity in vivo. Intracellular generation of reactive oxygen species (ROS) was measured using DCFH-DA probe with flow cytometry system and a laser scanning confocal microscope. LC3 (microtubule-associated protein 1 light chain 3)-II conversion was monitored with immunofluorescence and immunoblotting to demonstrate daucosterol-induced autophagy. We found that daucosterol inhibits the proliferation of human breast cancer cell line MCF-7 and gastric cancer cell lines MGC803, BGC823 and AGS in a dose-dependent manner. Furthermore, daucosterol inhibits murine hepatoma H22 cell growth in ICR mice. Daucosterol treatment induces intracellular ROS generation and autophagy, but not apoptotic cell death. Treatment with ROS scavenger GSH (reduced glutathione), NAC (N-acetyl-l-cysteine) or autophagy inhibitor 3-Methyladenine (3-MA) counteracted daucosterol-induced autophagy and growth inhibition in BGC823 and MCF-7 cancer cells. Daucosterol inhibits cancer cell proliferation by inducing autophagy through ROS-dependent manner and could be potentially developed as an anti-cancer agent.

Engineering of lipid prodrug-based, hyaluronic acid-decorated nanostructured lipid carriers platform for 5-fluorouracil and cisplatin combination gastric cancer therapy

PurposeThe first-line chemotherapy treatment protocol for gastric cancer is combination chemotherapy of 5-fluorouracil (5-FU) and cisplatin (CDDP). The aim of this study was to engineer prodrug-based nanostructured lipid carriers (NLC) platform for codelivery of 5-FU and CDDP to enhance therapy and decrease toxicity.MethodsFirst, 5-FU-stearic acid lipid conjugate was synthesized by two steps. Second, 5-FU-stearic acid prodrug and CDDP were loaded in NLC. Finally, hyaluronic acid (HA) was coated onto NLC surface. Average size, zeta potential, and drug loading capacity of NLC were evaluated. Human gastric cancer cell line BGC823 (BGC823 cells) was used for the testing of in vitro cytotoxicity assays. In vivo antitumor activity of NLC was evaluated in mice bearing BGC823 cells model.ResultsHA-coated 5-FU-stearic acid prodrug and CDDP-loaded NLC (HA-FU/C-NLC) showed a synergistic effect in combination therapy and displayed the greatest antitumor activity than all of the free drugs or uncoated NLC in vitro and in vivo.ConclusionThis work reveals that HA-coated NLC could be used as a novel carrier to code-liver 5-FU and CDDP for gastric cancer therapy. HA-FU/C-NLC could be a promising targeted and combinational therapy in nanomedicine.

Effect and mechanism of Vav3 gene to regulate inhibitor of apoptosis proteins in human gastri cancer cell line BGC823

Objective To examine the expression of Vav3 in gastric cancer and the effect of inhibiting Vav3 gene on apoptosis of human gastri cancer cell line BGC823 cells and the the underlying mechanism. Methods Specific small interfering RNA(siRNA)targeting Vav3 was designed, synthesized, and transfected into BGC823 cells. The expression of Vav3 was detected by real- time quantitative polymerase chain reaction (Real- time PCR) and Western blotting. Cellular viability was measured by methyl thiazol tetrazolium (MTT)assay. Cell apoptosis rate was determined by flow cytometry(FCM). The expression levels of Survivin, c- inhibitor of apoptosis protein 1(IAP1), c- IAP2, XIAP, Livin and Cysteinyl aspartate- specific protease(Caspase)- 3 were also determined. Results As compared with para- carcinoma tissues(0.173± 0.025)and gastric epithelial cell line GES- 1(0.250±0.036), Vav3 level was significantly up- regulated in gastric cancer tissues(0.727±0.101)and gastric cancer cell line BGC823(0.850±0.140,P 0.05).The expression and activity of Caspase-3 were increased after Vav3- siRNA tansfection into BGC823 cells(all P< 0.05). Conclusion Vav3 was overexpressed in gastric cancer. Vav3 gene silencing effectively promoted gastric cancer cell apoptosis, suggesting that inhibiting Vav3 can promot apoptosis of gastric cancer cells by regulating some IAPs. Key words: Stomach cancer; Vav3; Small interfering RNA; Inhibitor of apoptosis proteins; Cysteinyl aspartate-specific protease-3

Screening for predictive biomarkers of bevacizumab treatment in gastric cancer subcutaneous xenografts

To establish subcutaneous xenograft models of gastric cancer in nude mice and to screen the predictive biomarkers of bevacizumab effectiveness. Subcutaneous xenograft models were established using BGC823 gastric cancer cell line in 20 male 4-week old BALB/C-nu/nu nude mice and were randomly divided into four groups, bevacizumab group(15 mg/kg), 5-FU group(15 mg/kg), combined group and control group, with 5 mice in each group. Bevacizumab and 5-FU were administered intraperitoneally every other day for three weeks. After treatment, tumor size and inhibition rate were calculated. Expression of CD31 was examined by immunohistochemistry for evaluation of microvascular density(MVD). Levels of human vascular endothelial growth factor(VEGF), basic fibroblast growth factor (bFGF), placental growth factor (PIGF) and interleukin 8(IL-8) were tested by enzyme linked immunosorbent assay(ELISA). Compared to the control group, bevacizumab group and combined group had a significantly lower MVD(5.2±1.0 and 4.3±1.2 vs. 13.8±1.6, P<0.05), a smaller tumor volume [(305.6±184.1) mm(3) and (242.2±71.4) mm(3) vs.(1535.2±625.1) mm(3), P<0.05], and lower levels of VEGF and IL-8 in tumor tissues [VEGF:(351.6±84.1) ng/L and (242.2±71.4) ng/L vs. (1256.7±702.1) ng/L, P<0.05); IL-8:(20 903±1485) ng/L and (27 489±6772) ng/L vs. (57 032±2437) ng/L, P<0.05]. The above parameters were not significantly different between 5-FU group and control group(all P>0.05). Levels of bFGF and IGF were not significantly different among four groups as well(all P>0.05). VEGF and IL-8 may be used to be biomarkers candidates to predict bevacizumab effectiveness on human gastric cancer.

Hypoxic Microenvironment Induces EMT and Upgrades Stem-Like Properties of Gastric Cancer Cells.

Hypoxia microenvironment, as a major feature of solid tumors, increases tumors progression and metastasis. To research whether hypoxia influences the epithelial-mesenchymal transition (EMT) and cancer stem cells (CSCs) of gastric cancer cells and their cell biological behaviors. Human gastric cancer cell lines BGC823 and SGC7901 were cultivated in different oxygen tensions for proliferation, colony formation, soft agar formation, migration, and invasion analyses. Markers of EMT (E-cadherin, N-cadherin, Vimentin, and Snail) and markers of CSCs (Sox2, Oct4, and Bmi1) were investigated by real-time polymerase chain reaction, Western blotting, and immunofluorescent analysis. Cultivated at hypoxic condition, BGC823 and SGC7901 cells morphology began to change significantly. The cells pretreated under hypoxia grew faster than those cells always cultivated in normoxia. Meanwhile, hypoxia pretreatment dramatically promoted cell proliferation, migration and invasion, and increased capability of colony and soft agar colony formation. Furthermore, under hypoxia, E-cadherin decreased and N-cadherin, Vimentin, Snail, Sox2, Oct4, and Bmi1 increased both on the level of messenger RNA and protein. We drew a conclusion that the hypoxic microenvironment induced EMT, upgraded stem-like properties of gastric cancer cells, promoted invasion and metastasis, and behaved more malignantly.

P42.3: An abductor of cell cycle.

As a newly discovered tumor-associated gene, p42.3 was originally ascertained in gastric cancer cell line BGC823 and has been confirmed as a cell cycle-dependent gene that is overexpressed in many human tumor cell lines and embryonic tissues. p42.3 can regulate the level of relevant cycle-dependent proteins and promote malignant transformation of cells. A variety of cellular functions, including cell proliferation, cell invasion and cell migration, are under control of p42.3. Our review, namely the introduction of the structure of p42.3, underlying activity regulation mechanisms of p42.3 as well as the role p42.3 plays in malignant cellular transformation process, are accompanied by the presentation of potential directions of further researches of cancer prevention and therapy in which p42.3 is inevitable.

Impact of combined proglumide and celecoxib on cell proliferation and PGE2 secretion in human gastric cancer cell line BGC-823

Impact of combined proglumide and celecoxib on cell proliferation and PGE2 secretion in human gastric cancer cell line BGC-823

JWA reverses cisplatin resistance via the CK2-XRCC1 pathway in human gastric cancer cells.

Gastric cancer is the third most common malignancy in China, with a median 5-year survival of only 20%. Cisplatin has been used in first-line cancer treatment for several types of cancer including gastric cancer. However, patients are often primary resistant or develop acquired resistance resulting in relapse of the cancer and reduced survival. Recently, we demonstrated that the reduced expression of base excision repair protein XRCC1 and its upstream regulator JWA in gastric cancerous tissues correlated with a significant survival benefit of adjuvant first-line platinum-based chemotherapy as well as XRCC1 playing an important role in the DNA repair of cisplatin-resistant gastric cancer cells. In the present study, we demonstrated the role of JWA in cisplatin-induced DNA lesions and aquired cisplatin resistance in five cell-culture models: gastric epithelial cells GES-1, cisplatin-sensitive gastric cancer cell lines BGC823 and SGC7901, and the cisplatin-resistant gastric cancer cell lines BGC823/DDP and SGC7901/DDP. Our results indicated that JWA is required for DNA repair following cisplatin-induced double-strand breaks (DSBs) via XRCC1 in normal gastric epithelial cells. However, in gastric cancer cells, JWA enhanced cisplatin-induced cell death through regulation of DNA damage-induced apoptosis. The protein expression of JWA was significantly decreased in cisplatin-resistant cells and contributed to cisplatin resistance. Interestingly, as JWA upregulated XRCC1 expression in normal cells, JWA downregulated XRCC1 expression through promoting the degradation of XRCC1 in cisplatin-resistant gastric cancer cells. Furthermore, the negative regulation of JWA to XRCC1 was blocked due to the mutation of 518S/519T/523T residues of XRCC1, and indicating that the CK2 activated 518S/519T/523T phosphorylation is a key point in the regulation of JWA to XRCC1. In conclusion, we report for the first time that JWA regulated cisplatin-induced DNA damage and apoptosis through the CK2—P-XRCC1—XRCC1 pathway, indicating a putative drug target for reversing cisplatin resistance in gastric cancer.

Insulin-like growth factor-I induces epithelial to mesenchymal transition via GSK-3β and ZEB2 in the BGC-823 gastric cancer cell line.

Metastasis is the most common cause of mortality in patients with gastric cancer. Epithelial-to-mesenchymal transition (EMT), which may be stimulated by insulin-like growth factor-I (IGF-I) is involved in the metastasis of numerous tumors; however, the molecular mechanism by which IGF-I may induce tumor cell EMT remains to be elucidated in gastric cancer. The present study aimed to investigate the induction of EMT in BGC-823 gastric cancer cells. It was identified that IGF-I induced EMT by upregulating the levels of ZEB2 transcription factor, and this was dependent on the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway in these cells. In addition, glycogen synthase kinase 3β (GSK-3β), an intracellular downstream effector of PI3K/Akt, sustained the epithelial phenotype by repressing ZEB2 expression and the subsequent inhibition of EMT induced by IGF-I, suggesting the involvement of a potential PI3K/Akt-GSK-3β-ZEB2 signaling pathway in IGF-I-induced EMT in gastric cancer BGC-823 cells. Overall, the results of the present study suggest that IGF-I induced EMT by the activation of a PI3K/Akt-GSK-3β-ZEB2 signaling pathway in gastric cancer BGC-823 cells. Therefore, this study may provide more useful information regarding the mechanism of gastric cancer metastasis.

The Pleckstrin and Sec7 domain-containing gene as a novel epigenetic modification marker in human gastric cancer and its clinical significance.

The Pleckstrin and Sec7 domain-containing (PSD) gene has been recently found to participate in the progression of several types of cancer. In the present study, we identified PSD as a candidate tumor suppressor gene silenced through epigenetic modification in gastric cancer (GC). PSD mRNA expression and DNA methylation were evaluated by real-time reverse-transcriptase polymerase chain reaction (RT-PCR) and methylation-specific PCR in GC cell lines and tissue samples. Involvement of histone modification in GC cell lines was examined by chromatin immunoprecipitation assay. We also used an siRNA-mediated approach to knock down the PSD gene in SGC7901 cells, which was utilized to detect the role of PSD in GC progression, followed by analysis of cell apoptosis and invasion. PSD gene expression was reduced in all GC cell lines compared with GES1 (an immortalized normal gastric cell line). In addition, PSD expression was markedly downregulated in gastric carcinoma tissues when compared to adjacent non-tumor tissues. Our data also indicated that PSD mRNA and protein levels were associated with tumor differentiation and lymph node metastasis. Aberrant DNA methylation status and histone modification were also found in GC cell lines. Enhanced gene expression was detected when the HGC27, AGS and BGC823 GC cell lines were treated with the DNA-demethylating agent 5-aza-2'-deoxycytidine. However, treatment with trichostatin A, a histone deacetylase inhibitor, had no effect on PSD expression in any of the GC cell lines. Suppression of PSD by siRNA led to enhanced SGC7901 cell invasion. The depletion of PSD expression inhibited cell proliferation and decreased apoptosis in SGC7901 cell lines. Knockdown of the PSD expression decreased caspase-3 and -7 protein levels in SGC7901 cells. PSD gene may function as a tumor suppressor in GC suggesting a vital role for DNA methylation and histone modification in PSD silencing. PSD expression might be a useful biomarker for epigenetic-based GC early diagnosis and may lead to the identification of new targets for pharmacological intervention.

Abstract 1369: miR-645 inhibits apoptosis of AGEJ

Abstract Background: An increasing body of evidence indicates that miRNAs have a critical role in carcinogenesis and cancer progression; however, the role of miRNAs in the tumorigenesis of adencarcinoma of gastric esophageal junction(AGEJ) remains largely unclear.Methods: The SGC7901 and BGC-823 gastric cancer cell lines were used. The expressions of miR-645 and IFIT2 were examined by qRT-PCR. The expressions of IFIT2 was examined by western blotting and immunohistochemistry assay. The cell apoptosis was determined by FACS. MiR-645 inhibitor, mimics and plasmid-IFIT2 transfections were performed to study the loss- and gain-function. Caspase-3/7 activity was examined by caspase-3/7 assay. Results: In the present study, we have reported an increased expression of miR-645 in AGEJ clinical specimens compared with paired non-cancerous tissues. We also observed a significant miR-645 up-regulation in two gastric cancer ( GC ) cell lines, SGC7901 and BGC-823, which were used as cell models because there was no available AGEJ cell lines established to date. We found that inhibition of miR-645 could sensitize dramatically SGC7901 and BGC-823 cells to both serum starvation- and chemotherapeutic drug-induced apoptosis by up-regulating IFIT2, a mediator of apoptosis via a mitochondrial pathway, with a potential binding site for miR-645 in its mRNA's 3′UTR. Further investigation exhibited that IFIT2 expression decreases in SGC7901 and BGC-823 cells and AGEJ tissues. IFIT2 ectopic expression leads to promotion of cell apoptosis, indicating that IFIT2 may function as a suppressor in the development of AGEJ. Furthermore, inhibition of miR-645 induces up-regulation of IFIT2 and increased caspase-3/7 activity compared with control groups. Conclusions: Our data suggest that miR-645 functions as an oncogene in human AGEJ by, at least partially through, targeting IFIT2. &amp;lt;!–EndFragment–&amp;gt; Note: This abstract was not presented at the meeting. Citation Format: Xiaoshan Feng, Ying Wang, Zhikun Ma, Ruina Yang, Mengxi Zhang, Shuo Liang, Shuoguo Li, Gang Liu, Fanming Dai, Shegan Gao. miR-645 inhibits apoptosis of AGEJ. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1369. doi:10.1158/1538-7445.AM2014-1369

MicroRNA-645, up-regulated in human adencarcinoma of gastric esophageal junction, inhibits apoptosis by targeting tumor suppressor IFIT2.

BackgroundAn increasing body of evidence indicates that miRNAs have a critical role in carcinogenesis and cancer progression; however, the role of miRNAs in the tumorigenesis of adencarcinoma of gastric esophageal junction (AGEJ) remains largely unclear.MethodsThe SGC7901 and BGC-823 gastric cancer cell lines were used. The expressions of miR-645 and IFIT2 (Interferon-induced protein with tetratricopeptide repeats 2) were examined by qRT-PCR, The expressions of IFIT2 was examined by western blotting and immunohistochemistry assay. The cell apoptosis was determined by FACS. MiR-645 inhibitor, mimics and plasmid-IFIT2 transfections were performed to study the loss- and gain-function. Caspase-3/7 activity was examined by caspase-3/7 assay.ResultsIn the present study, we have reported an increased expression of miR-645 in AGEJ clinical specimens compared with paired non-cancerous tissues. We also observed a significant miR-645 up-regulation in two gastric cancer (GC) cell lines, SGC7901 and BGC-823, which were used as cell models because there was no available AGEJ cell lines established to date. We found that inhibition of miR-645 could sensitize dramatically SGC7901 and BGC-823 cells to both serum starvation– and chemotherapeutic drug–induced apoptosis by up-regulating IFIT2, a mediator of apoptosis via a mitochondrial pathway, with a potential binding site for miR-645 in its mRNA’s 3′UTR. Further investigation exhibited that IFIT2 expression decreases in SGC7901 and BGC-823 cells and AGEJ tissues. IFIT2 ectopic expression leads to promotion of cell apoptosis, indicating that IFIT2 may function as a suppressor in the development of AGEJ. Furthermore, inhibition of miR-645 induces up-regulation of IFIT2 and increased caspase-3/7 activity compared with control groups.ConclusionsOur data suggest that miR-645 functions as an oncogene in human AGEJ by, at least partially through, targeting IFIT2.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-633) contains supplementary material, which is available to authorized users.

MiR-129-1-3p promote BGC-823 cell proliferation by targeting PDCD2.

MicroRNAs (miRNAs) are the class of small noncoding RNAs, and play an important role in the regulation of gene expression at the posttranscriptional level. In this study, we explored the effect of miR-129-1-3p on the growth and cell cycle of human gastric cancer cell line BGC-823. The miR-129-1-3p mimics or inhibitors were transfected into the BGC-823 cell line, and the cell cycle and cell growth was measured by flow cytometry and real-time cell analyzer, respectively. The possible targets of miR-129-1-3p were analyzed by quantitative real time-PCR (QRT-PCR), Western blotting and Luciferase reporter assay. The results showed that miR-129-1-3p could promote the growth and cell cycle of BGC-823 cells. Although protein expression of programmed cell death 2 (PDCD2) was not changed with miR-129-1-3p, QRT-PCR showed that expression of PDCD2 mRNA was negatively related to the miR-129-1-3p. Luciferase reporter assay revealed that PDCD2 is one of the targets of miR-129-1-3p. Our results indicated that miR-129-1-3p might promote proliferation of BGC-823 cells by targeting PDCD2.

MiR-421 regulates apoptosis of BGC-823 gastric cancer cells by targeting caspase-3.

MicroRNAs might act as oncogenes or tumor suppressors in cancer. Recent studies have shown that miR-421 is up-regulated in human gastric cancer. Here, we found that miR-421 was over-expressed in gastric cancer tissues and cell lines. Bioinformatics analysis predicted that the caspase-3 gene was a target of miR-421. Caspase-3 was negatively regulated by miR-421 at the post-transcriptional level. Bax and Bcl-2 were also regulated by miR-421. Moreover, tumor necrosis factor receptor-I and -II, death receptors in the apoptosis pathway, were up-regulated by miR-421. The over-expression of miR-421 promoted gastric cancer cell growth and inhibited apoptosis of the BGC-823 gastric cancer cell line. These observations indicate that miR-421 acts as a tumor promoter by targeting the caspase-3 gene and preventing apoptosis of gastric cancer cells through inhibition of caspase-3 expression. These findings contribute to our understanding of the functions of miR-421 in gastric cancer.

Piezo1 is as a novel trefoil factor family 1 binding protein that promotes gastric cancer cell mobility in vitro.

Trefoil factor family 1 (TFF1) is a member of the TFF-domain peptide family involved in epithelial restitution and cell motility. Recently, we screened Piezo1 as a candidate TFF1-binding protein. We aimed to confirm Piezo1 as a novel TFF1 binding protein and to assess the role of this interaction in mediating gastric cancer cell mobility. This interaction was confirmed by co-immunoprecipitation and co-localisation of TFF1 and Piezo1 in GES-1 cells. We used stable RNA interference to knockdown Piezo1 protein expression and restored the expression of TFF1 in the gastric cancer cell lines SGC-7901 and BGC-823. Cell motility was evaluated using invasion assay and migration assay in vitro. The expression levels of the integrin subunits β1, β5, α1 as well as the expression of β-catenin and E-cadherin were detected by Western blot. We demonstrate that TFF1, but not TFF2 or TFF3, bind to and co-localize with Piezo1 in the cytoplasm in vitro. TFF1 interacts with the C-terminal portion of the Piezo1 protein. Wound healing and trans-well assays demonstrated that the restored expression of TFF1 promoted cell mobility in gastric cancer cells, and this effect was attenuated by the knockdown of Piezo1. Western blots demonstrated the decreased expression of integrin β1 in Piezo1-knockdown cells. Our data demonstrate that Piezo1 is a novel TFF1 binding protein that is important for TFF1-mediated cell migration and suggest that this interaction may be a therapeutic target in the invasion and metastasis of gastric cancer.

Decreased core-fucosylation contributes to malignancy in gastric cancer.

The object of the study is to identify N-glycan profiling changes associated with gastric cancer and explore the impact of core-fucosylation on biological behaviors of human gastric cancer cells. A total of 244 subjects including gastric cancer, gastric ulcer and healthy control were recruited. N-glycan profiling from serum and total proteins in gastric tissues was analyzed by DNA sequencer-assisted fluorophore-assisted capillary electrophoresis. The abundance of total core-fucosylated residues and the expression of enzymes involved in core-fucosylation were analyzed with lectin blot, quantitative reverse transcription-polymerase chain reaction, western blot, Immunohistochemical staining and lectin-histochemical staining. The recombinant plasmids of GDP-fucose transporter and α-1,6-fucosyltransferase (Fut8) were constructed and transfected into gastric cancer cell lines BGC-823 and SGC-7901. CCK-8 and wound healing assay were used to assess the functional impact of core-fucosylation modulation on cell proliferation and migration. Characteristic serum N-glycan profiles were found in gastric cancer. Compared with the healthy control, a trianntenary structure abundance, peak 9 (NA3Fb), was increased significantly in gastric cancer, while the total abundance of core-fucosylated residues (sumfuc) was decreased. Core-fucosylated structures, peak6(NA2F) and peak7(NA2FB) were deceased in gastric tumor tissues when compared with that in adjacent non-tumor tissues. Consistently, lens culinaris agglutinin (LCA)-binding proteins were decreased significantly in sera of gastric cancer, and protein level of Fut8 was decreased significantly in gastric tumor tissues compared with that in adjacent non-tumor tissues. Upregulation of GDP-Tr and Fut8 could inhibit proliferation, but had no significant influence on migration of BGC-823 and SGC-7901 cells. Core-fucosylation is down regulated in gastric cancer. Upregulation of core-fucosylation could inhibit proliferation of the human gastric cancer cells.

RASAL1 influences the proliferation and invasion of gastric cancer cells by regulating the RAS/ERK signaling pathway

The aim of this study was to investigate the biological characteristics of the RASAL1 gene in a well-differentiated gastric cancer cell line MKN-28 and a poorly differentiated gastric cancer cell line BGC-823 cells, using RNA interference and gene transfection technology, respectively. MKN-28 cells were transfected with the shRNA of RASAL1 and BGC-823 cells were transfected with the pcDNA 3.1 plasmid vector containing RASAL1. RT-PCR and western blotting were then used to detect the expression of RASAL1 mRNA and protein. The activities of RAS and extracellular signal-regulated kinase 1/2 were analyzed by the pull-down method and western blotting. The proliferate capacity, apoptosis rate, invasive and migratory potentials of MKN-28 or BGC-823 cells were also measured by Cell Counting Kit-8 cell proliferation assay, propidium iodide/Annexin V staining coupled with flow cytometry, and transwell chamber assays, respectively. Measurement of RASAL1 mRNA and protein expression in two cells revealed successful transfection of the shRNA of RASAL1 and RASAL1-pcDNA3.1 plasmid into these two cells. Moreover, decreased expression of RASAL1 in MKN-28 cells resulted in increased expression of RAS-GTP and p-ERK1/2. Interestingly, decreased expression of RASAL1 inhibited apoptosis and facilitated cell proliferation, invasion and migration. The increased expression of RASAL1 in BGC-823 cells caused declined expression of RAS-GTP and p-ERK1/2, as well as promotedapoptosis and restrained cell proliferation, invasion and migration. The down-regulation of RASAL1 promoted the proliferation, invasion and migration of gastric cancer MKN-28 cells, and up-regulation of RASAL1 inhibited the proliferation, invasion and migration of BGC-823 gastric cancer cells by regulating the RAS/ERK signaling pathway. Thus, our results suggest that RASAL1 may play an important role as a tumor suppressor gene in gastric cancer.

MiR-148a regulates MEG3 in gastric cancer by targeting DNA methyltransferase 1

The long non-coding RNA MEG3 has been reported to be a tumor suppressor in a number of malignant tumors including gastric cancer. Several studies have shown that the regulation of MEG3 may attribute to the promoter hypermethylation. However, the mechanism of MEG3 regulation in gastric cancer is still not well understood. MiR-148a can suppress gastric tumorigenesis through regulating the expression of target genes such as DNA methyltransferase 1(DNMT-1). We examined the expression of MEG3 in 52 gastric cancer samples using quantitative real-time PCR and found the down-regulation of MEG3 in both gastric cancer tissues and cell lines. The positive correlation of MEG3 and miR-148a was further confirmed in SGC-7901 and BGC-823 gastric cancer cell lines. Hypermethylation of MEG3 differentially methylated regions was identified by methylation-specific PCR, and MEG3 expression was increased with the inhibition of methylation with siRNA to DNMT-1 in gastric cancer cells. In addition, transfection of MEG3 siRNA into gastric cancer cells diminished the suppression of proliferation induced by overexpression of miR-148a. Our results suggest that the suppression of miR-148a may contribute to the down-regulation of MEG3 in gastric cancer by modulation of DNMT-1.

TXNL1-XRCC1 pathway regulates cisplatin-induced cell death and contributes to resistance in human gastric cancer

Cisplatin is a cytotoxic platinum compound that triggers DNA crosslinking induced cell death, and is one of the reference drugs used in the treatment of several types of human cancers including gastric cancer. However, intrinsic or acquired drug resistance to cisplatin is very common, and leading to treatment failure. We have recently shown that reduced expression of base excision repair protein XRCC1 (X-ray repair cross complementing group1) in gastric cancerous tissues correlates with a significant survival benefit from adjuvant first-line platinum-based chemotherapy. In this study, we demonstrated the role of XRCC1 in repair of cisplatin-induced DNA lesions and acquired cisplatin resistance in gastric cancer by using cisplatin-sensitive gastric cancer cell lines BGC823 and the cisplatin-resistant gastric cancer cell lines BGC823/cis-diamminedichloridoplatinum(II) (DDP). Our results indicated that the protein expression of XRCC1 was significantly increased in cisplatin-resistant cells and independently contributed to cisplatin resistance. Irinotecan, another chemotherapeutic agent to induce DNA damaging used to treat patients with advanced gastric cancer that progressed on cisplatin, was found to inhibit the expression of XRCC1 effectively, and leading to an increase in the sensitivity of resistant cells to cisplatin. Our proteomic studies further identified a cofactor of 26S proteasome, the thioredoxin-like protein 1 (TXNL1) that downregulated XRCC1 in BGC823/DDP cells via the ubiquitin-proteasome pathway. In conclusion, the TXNL1-XRCC1 is a novel regulatory pathway that has an independent role in cisplatin resistance, indicating a putative drug target for reversing cisplatin resistance in gastric cancer.

ZNF139 promotes tumor metastasis by increasing migration and invasion in human gastric cancer cells.

Zinc finger protein 139(ZNF139), a member of zinc finger protein family, is a transcription factor. Our previous research showed ZNF139 was overexpressed in gastric cancer cells. The purpose of present study is to explore impact and mechanism of ZNF139 on metastasis by regulating invasive ability of gastric cancer cells. Quantitative RT-PCR(QRT-PCR) and Western blot were applied for detection of ZNF139 expression in gastric cancer tissues, adjacent cancer tissues, metastatic lymph nodes, gastric cancer cell lines SGC7901 and BGC823 and gastric epithelial cell line GES-1; siRNA specific to ZNF139 was synthesized and then transfected into gastric cancer cell line BGC823; wound healing assay and Transwell assay were used to observe impact of ZNF139-siRNA after being transfected into BGC823 on its invasion and migration; changes in expression of invasion and migration-related genes MMP-2, MMP-9, ICAM-1 and TIMP1 were detected before and after transfection. Gelatin zymogrphy assay were applied to determine the MMP activities. Statistical analysis was based on the SPSS11.5 software.Expression of ZNF139 in gastric adenocarcinoma tissues and cells was significantly higher than the expression in the adjacent cancer tissues, but lower than the expression in the metastatic lymph nodes; ZNF139 expression was present in gastric cancer cell lines, and the expression level was higher than that in normal gastric epithelial cells lines. ZNF139-siRNA significantly inhibited the invasion and migration activity of gastric cancer cell line BGC823. 48h after ZNF139-siRNA was transfected into gastric cancer cell line BGC823, expression and activity of invasion-related genes MMP-2, MMP-9, ICAM-1 mRNA and protein were significantly inhibited, while expressions of TIMP-1 mRNA and protein were significantly increased. At the same time, the gelatinase activities of MMP2 and MMP9 were decreased by ZNF139 interference.ZNF139 was overexpressed in gastric cancer cells, and the expression was further enhanced in the metastasis process. Knocking down ZNF139 expression in gastric cancer cells could effectively reduce gastric cancer cell invasion and migration ability, and this process might play a role by regulating MMP-TIMP balance.