Depletion of OLFM4 gene inhibits cell growth and increases sensitization to hydrogen peroxide and tumor necrosis factor-alpha induced-apoptosis in gastric cancer cells
- Rui-hua Liu1, 2,
- Mei-hua Yang†3,
- Hua Xiang2,
- Li-ming Bao1, 4,
- Hua-an Yang5,
- Li-wen Yue6,
- Xue Jiang7,
- Na Ang7,
- Li-ya Wu7 and
- Yi Huang1Email author
© Liu et al; licensee BioMed Central Ltd. 2012
Received: 3 December 2011
Accepted: 3 April 2012
Published: 3 April 2012
Human olfactomedin 4 (OLFM4) gene is a secreted glycoprotein more commonly known as the anti-apoptotic molecule GW112. OLFM4 is found to be frequently up-regulated in many types of human tumors including gastric cancer and it was believed to play significant role in the progression of gastric cancer. Although the function of OLFM4 has been indicated in many studies, recent evidence strongly suggests a cell or tissue type-dependent role of OLFM4 in cell growth and apoptosis. The aim of this study is to examine the role of gastric cancer-specific expression of OLFM4 in cell growth and apoptosis resistance.
OLFM4 expression was eliminated by RNA interference in SGC-7901 and MKN45 cells. Cell proliferation, anchorage-independent growth, cell cycle and apoptosis were characterized in vitro. Tumorigenicity was analyzed in vivo. The apoptosis and caspase-3 activation in response to hydrogen peroxide (H2O2) or tumor necrosis factor-alpha (TNF α) were assessed in the presence or absence of caspase inhibitor Z-VAD-fmk.
The elimination of OLFM4 protein by RNA interference in SGC-7901 and MKN45 cells significantly inhibits tumorigenicity both in vitro and in vivo by induction of cell G1 arrest (all P < 0.01). OLFM4 knockdown did not trigger obvious cell apoptosis but increased H2O2 or TNF α-induced apoptosis and caspase-3 activity (all P < 0.01). Treatment of Z-VAD-fmk attenuated caspase-3 activity and significantly reversed the H2O2 or TNF α-induced apoptosis in OLFM4 knockdown cells (all P < 0.01).
Our study suggests that depletion of OLFM4 significantly inhibits tumorigenicity of the gastric cancer SGC-7901 and MKN45 cells. Blocking OLFM4 expression can sensitize gastric cancer cells to H2O2 or TNF α treatment by increasing caspase-3 dependent apoptosis. A combination strategy based on OLFM4 inhibition and anticancer drugs treatment may provide therapeutic potential in gastric cancer intervention.
KeywordsGastric cancer Olfactomedin 4 RNA interference Cell growth Apoptosis resistance
Human OLFM4 (olfactomedin 4, also known as hGC-1, GW112), originally termed human cloned from myeloid precursor cells after granulocyte colony-stimulating factor stimulation , is a secreted glycoprotein more commonly known as the anti-apoptotic molecule GW112 [2, 3]. OLFM4 is normally expressed in bone marrow, prostate, small intestine, stomach, colon and pancreas [1, 4]. Subsequently, increased OLFM4 levels were also found in the crypt epithelium of inflamed colonic mucosa of inflammatory bowel diseases  and in gastric biopsies infected with Helicobacter pylori [6, 7]. More recently, up-regulated OLFM4 expression has been described in lung and breast , prostatic , gastric [3, 9] and pancreatic cancers [8, 9] as well as in colorectal adenomas [10–14].
It has been suggested that OLFM4 is involved in cellular process such as apoptosis and tumor growth . Although the cellular function of OLFM4 has been investigated, these results do not always coincident. Overexpression of OLFM4 has been shown to facilitate mouse prostate tumor Tramp-C1 cells growth in syngeneic C57/BL6 mice  but inhibit human prostate cancer PC-3 cell proliferation . Moreover, up-regulated OLFM4 showed a strong anti-apoptotic activity in mouse lymphoid vein endothelial SVEC cells and human adenocarcinoma HeLa cells [1, 2], whereas recent findings suggested a proapoptotic effect of OLFM4 in human myeloid leukemia HL-60 cells . Evidence from these studies strongly suggests that roles of OLFM4 in cell growth control and apoptosis may depend on the cell or tissue type [10, 13–15]. To date, however, very limited data concerning the role of OLFM4 in the cell growth and apoptosis profiles of gastric cancer cells has been published.
In the present study, we analyzed OLFM4 protein expression in gastric cancer cells and normal human gastric epithelial GES-1 cells by western blotting. Using plasmid-mediated short hairpin RNA (shRNA), we inhibited OLFM4 expression in the gastric cancer SGC-7901 and MKN45 cells to observe cell proliferation, cell cycle phase, apoptosis in vitro and to assess its tumorigenic capacity in vivo. We also explored the apoptosis and caspase-3 activation in response to cytotoxic agents such as H2O2 or TNF α in the presence or absence of caspase inhibitor Z-VAD-fmk between OLFM4 knockdown cells and HK control cells.
Cell culture, reagents and mice
The human gastric cancer cells BGC-823, HGC-27, SGC-7901, MKN28, MKN45 and human normal gastric epithelial GES-1 cells were maintained DMEM medium (GibcoBRL, Gaithersburg, MD) containing 10% fetal bovine serum (FBS, GibcoBRL, USA),100 U/ml of penicillin and 100 μg/ml of streptomycin. H2O2 and TNF-α were obtained from Sigma (St. Louis, MO) and Z-VAD-fmk was purchased from Calbiochem (San Diego, CA). BALB/C nude (nu/nu) mice (4-6 weeks old, SPF degree, 20 ± 3 g) were purchased from Laboratory Animal Center of Chongqing medical University (Chongqing, China). All procedures were conducted according to the internationally accepted ethical guidelines (NIH publication no. 85-23, revised 1985).
Plasmid constructs and stable transfection
shRNA-mediated RNAi plasmid (pGenesil 1.1-siOLFM4) and a scrambled control plasmid (pGenesil 1.1-HK) were constructed to knock down the endogenous OLFM4 in SGC-7901 and MKN45 cells. After transfection and neomycin (G418) selection, OLFM4 knock-down SGC-7901-siOLFM4, MKN45-siOLFM4 cells and scrambled SGC-7901-HK, MKN45-HK control cells were stably obtained, respectively (details shown in Additional file 1: Supplementary data).
RNA extraction and quantitative RT-PCR (qRT-PCR)
Total RNA in various cells or tumor xenografts was extracted using the RNeasy Mini Kit (Qiagen, CA, USA), and was followed by cDNA synthesis using the ReverTra Ace-α-first strand cDNA synthesis system (Toyobo, Osaka, Japan) as previous described . Quantitative real-time PCR was performed using 7500 real-time PCR system (Applied Biosystems) with SYBR-Green as a fluorescent dye (Toyobo, Osaka, Japan) (details shown in Additional file 1: Supplementary data). Fold changes in gene expression were determined using the "2 - ddCT" method .
Cell proliferation assay in vitro and cell viability measurement
Cell proliferation and cell viability were measured using Cell proliferation WST-1 kit (Roche) according to the manufacturer's instructions. For cell proliferation, cells were seeded at a density of 1 × 103 cells per well of a 96-well plates and grown for 5 days. The optical density (450 nm) value was detected using the Microplate Reader (Tacan, Swaziland) per day. Each assay was performed in triplicate. As for measurement of cell viability, cells (1 × 104/well) were seeded at 200 μl of media in 96-well plates. After 12 h incubation, H2O2 or TNF α was treated in indicated concentrations. Relative absorbance was measured as described in cell proliferation.
Anchorage-independent growth assay
Anchorage-independent growth was performed on soft agar to reflect in vitro clonogenicity. Briefly, cells (5 × 102) from each colony were suspended in 0.3% agar in DMEM and then plated on solidified agar (0.5%) in 6-well dishes. Cells were incubated for 2 weeks at 37°C in 5% CO2 before the colonies was measured. Number of colonies was counted at 200 × magnification for five random fields. Each assay was performed in triplicate.
Flow cytometry analysis
Flow cytometry (FCM) analysis was performed to assess cell cycle progression or apoptosis. (details shown in Additional file 1: Supplementary data)
Enzymatic activity of caspase-3 and -9 was measured using a fluorometric assay according to a method described previously .
Western blot analysis
Western blotting was performed as described previously . The following antibodies were used for western blotting: anti-OLFM4 (Abcam, Cambridge, UK) and anti-β-actin (Santa Cruz, CA, USA) The relative quantity of proteins was analyzed using Quantity One software (Bio-Rad, Hercules, CA, USA) and normalized to that of β-actin (details shown in Additional file 1: Supplementary data).
Xenograft tumor model
Fourty nude mice were divided into four groups randomly. Each group was injected subcutaneously in the backs with the suspension of 200 μl containing 2 × 106 cells above mentioned, respectively. The volume of xenografts was serially measured. The mice were sacrificed after 35 days. The xenografts were excised and weighed. The inhibition rates of xenografts were calculated according to the formula: inhibition rate (%) = 1-mean weight (OLFM4 knock down cells-injected group or HK control cells-injected group)/mean weight (HK control cells-injected group) × 100%. Then, the tumor tissue was subject to total RNA isolation or immunohistochemistry detection.
OLFM4 proteins in tumor xenografts were analyzed by IHC using rabbit-anti OLFM4 (Abcam, Cambridge, UK) (details shown in Additional file 1: Supplementary data).
Data from independent experiments were expressed as the mean ± S.D. of at least three experiments. Comparisons between groups were analyzed by two-tailed Student's t-test or ANOVA, as appropriate, and p values < 0.05 were considered to be statistically significant.
Efficient knock down of OLFM4 gene by plasmid-mediated siRNA in gastric cancer cells
To down-regulate OLFM4 expression, a plasmid-mediated shRNA targeting OLFM4 gene was constructed to stably knock down OLFM4 expression in SGC-7901 and MKN45 cells. As shown in Figure 1B, OLFM4 mRNA levels were significantly reduced in SGC-7901-siOLFM4 and MKN45-siOLFM4 cells compared to their HK control or parental cells (P < 0.01). These results were further confirmed by western blotting analysis (Figure 1C). No significant difference in OLFM4 expression was observed between parental and HK control cells. The levels of mRNA and protein for the β-actin were similar among the different groups. These results suggest that a plasmid-mediated OLFM4-siRNA can specifically and efficiently knock down OLFM4 level in gastric cancer cells. Given the analysis stated above, therefore, OLFM4 knock down cells and HK control cells were chosen for further investigation.
OLFM4 knockdown inhibits gastric cancer cell proliferation and anchorage-independent growth in vitro
Growth-inhibitory effect of decreased OLFM4 in gastric tumor xenografts
Moreover, we also performed subcutaneous tumor formative assay in nude mice to evaluate the growth suppression effect of down-regulated OLFM4 in vivo. Nude mice were subcutaneously injected with OLFM4 knockdown or HK control cells. Tumor volumes per 4 days and tumor weight at 5 weeks were measured respectively after subcutaneous injection. As shown in Figure 2C-D, whether tumor volume or tumor weight produced by OLFM4 knock down SGC-7901 and MKN45 cells had significantly reduced growth compared with tumors produced in mice injected with HK control-transfected cells (P < 0.01). The inhibitory rate of SGC-7901 and MKN45 knock down cells-injected group on tumor growth was 40.29% and 37.48% respectively (Figure 2E).
OLFM4 knock-down delays G1 to S transition but does not trigger obvious apoptosis in gastric cancer cells
To determine whether apoptosis is involved in this growth inhibition, we next performed cell apoptosis analysis. Interestingly, reduced OLFM4 expression could not result in significant changes in apoptosis (Figure 4B), which is consistent with the cell cycle analysis showing no apparent sub-G1 phase in the tested cells. To further examine alterations of apoptotic signals, caspase-3/-9 activity was also identified by colorimetric activation assays. Both caspase-3 and -9 activations showed no significant changes after knockdown of OLFM4 in SGC-7901 and MKN45 cells (Figure 4C). These results suggest that down-regulation of OLFM4 may exert an inhibitory effect on cell growth by regulating cell cycle progression not involving apoptosis in gastric cancer cells.
Deletion of OLFM4 sensitizes gastric cancer cells to H2O2 or TNF α-induced apoptosis
Caspase-3 activity is involved in H2O2 or TNF α-induced apoptosis in OLFM4 knock-down cells
Recently accumulating data demonstrated OLFM4 is frequently overexpressed in many types of human tumors including gastric cancer, and it was believed to play a crucial role in the development and progression of gastric carcinogenesis [19, 20]. Although previous studies have shown that OLFM4 is involved in apoptosis and tumor growth, recent observations also suggest that cell or tissue-specific effects may exist for the OLFM4 gene. Relatively little is known regarding the tumor growth and apoptosis underlying gastric cancer-specific OLFM4 expression. To gain a better understanding of this role for the altered OLFM4 in human gastric cancer, experimental support is required to validate the role of the OLFM4 gene in gastric cancer.
It has been shown that abnormal expression of hGC-1 may be regulated at the transcriptional or posttranscriptional level . In our present works, we directly investigated OLFM4 protein expression pattern in gastric cancer cells and normal GES-1 cells. SGC-7901 and MKN45 cells expressing relative high OLFM4 levels were chosen for this study. Since reducing the target gene expression by genetic means in established cell lines is helpful for a better understanding of its role in maintaining the malignant phenotype particularly in analyzing genes that are essential for cellular survival , we generated stable clone pools of SGC-7901 and MKN45 expressing OLFM4-siRNA or scrambled HK control by the plasmid-based siRNA approach and confirmed knock-down efficiency of OLFM4 gene at mRNA and protein levels by qRT-PCR and western blotting.
Our present works demonstrate that OLFM4 plays an essential role in gastric cancer tumorigenesis. Knockdown of OLFM4 inhibits cell proliferation and anchorage-independent growth ability in vitro. Xenograft tumor model in vivo also implies that decreased OLFM4 can inhibit the tumor growth of human gastric cancer cells. These results indicate that OLFM4 plays a crucial role in cell proliferation of gastric cancer cells. Our results also observed that knock-down of OLFM4 did not influence the rate of apoptosis and caspase-3 and 9 activations in OLFM4 knockdown cells, suggesting that apoptosis might not be the mechanism underlying the inhibition of tumor growth. Thus, we postulate that OLFM4 expression is not essential for cancer cell survival, which is in accordance with a recent observation that genetic knock-out mice for OLFM4 show normal development and hematopoietic phenotypes . To further characterize the mechanism underlying growth inhibition, we performed cell cycle analysis and demonstrated that inhibition of OLFM4 expression induced gastric cancer cells to accumulate in G1 phase of the cell cycle, suggesting that down-regulated OLFM4 may exert an inhibitory effect on cell growth by a mechanism regulating cell cycle progression not involving apoptosis in gastric cancer cells.
Resistance of tumor cells to the induction of apoptosis is one of the main factors responsible for the failure of many conventional anticancer therapies that use anticancer agents and radiation. Therefore, apoptosis control in cancer cells is of critical biological and clinical importance [22, 23]. Anti-apoptotic activity is another important function of OLFM4 gene . In particular, H2O2-induced cellular apoptosis was attenuated by overexpressed OLFM4 in a prostatic cancer cell line . Moreover, MKN45 cells have been shown an ability of resistance to TNF α-induced apoptosis . Given these findings, we hypothesize that knockdown of OLFM4 expression might enhance H2O2 or TNF α-induced apoptosis in gastric cancer cells. Here, we showed that knock-down of OLFM4 effectively enhanced cell apoptosis in response to H2O2 or TNF α stimulation in MKN45 as well as SGC-7901 cells, suggesting blocking OLFM4 may increase the susceptibility of gastric cancer cells to the presence of H2O2 or TNF α.
As it is well known that caspase-3, a key executive molecule of the apoptotic pathway, plays a critical role in apoptotic processes in a variety of cells. We further examined caspase-3 activation in OLFM4 knockdown and HK control cells in the presence or absence of caspase inhibitor Z-VAD-fmk. We observed that treatment of H2O2 or TNF α significantly up-regulated Caspase-3 activity in OLFM4 knockdown cells than HK control cells while pre-treatment with Z-VAD-fmk reversed caspase-3 activity as well as H2O2 or TNF α-induced apoptosis. The results indicate that H2O2 or TNF α-induced apoptosis in OLFM4 knockdown cells is caspase-3 dependent. Based on the present data, it is conceivable that up-regulated OLFM4 enables gastric cancer cells to resist apoptosis induction by decreasing the susceptibility to anticancer drugs.
In fact, antagonists of OLFM4 have been reported to inhibit the proliferation in others types of cancer cells such as human pancreatic cancer PANC-1 cells  and human lung caner SBC-1 cells . However, controversial results have also been observed. Decreased OLFM4 mRNA inhibit PANC-1 cells proliferation by S to G2/M phase arrest , which is different from our results showing delayed G1 phase progress in gastric cancer. Certain important details (or reasons) might explain this discrepancy. First, as recent studies suggested, a cell or tissue specific role of OLFM4 (gastric cancer cells and pancreatic cancer cells) may be a persuasive explanation for this discrepancy. The most noteworthy is, OLFM4 expression at mRNA level but not protein level in PANC-1 cells was successfully measured using RT-PCR  whereas the most recent report by Kim et al. showed that PANC-1 cells has no OLFM4 mRNA expression , indicating the expression pattern and role of OLFM4 gene in PANC-1 cells need further confirmation.
Despite OLFM4 silencing was shown to an inhibitory effect on cell growth and a decreased resistance to H2O2 or TNF α treatment in gastric cancer cells, it is not eliminated that other mechanisms may also be regulated by OLFM4 and contributes to growth inhibition and apoptosis control signaling, considering the crosstalk of the network. Indeed, OLFM4, a target gene of NF-κB pathway [16, 17, 25], has also shown a negative feedback effect on H. pylori infection-induced NF-κB activation in HEK 293 T cells , indicating the regulatory pathways controlled by OLFM4 in gastric cancer could be involved in a very complex and intricate network. Therefore, complex interactions between OLFM4 and other signaling intermediates are needed to be more extensive investigation in our future studies.
Taken together, the present study provides evidences that the elimination of OLFM4 expression in gastric cancer SGC-7901 and MKN45 cells inhibits tumorigenicity both in vitro and in vivo by regulating cell cycle progression not involving apoptosis. Moreover, suppression of OLFM4 enhances caspase-3 dependent apoptosis in response to H2O2 or TNF α in human gastric cancer cells. By understanding the role of OLFM4 in tumor growth and apoptosis resistance, it may be possible to develop a perspective strategy based on a combination of OLFM4 inhibition and anticancer drugs treatment in gastric cancer intervention.
- TNF α:
Tumor necrosis factor-alpha
Short hairpin RNA
This research was supported by the National Natural Science Foundation of China No.30701004 and 81001017, the Foundation for Sci & Tech Research Project of Chongqing (CSTC2011AC5200) and the health bureau of Chongqing (2010-2-175).
- Zhang J, Liu WL, Tang DC, Chen L, Wang M, Pack SD, Zhuang Z, Rodgers GP: Identification and characterization of a novel member of olfactomedin-related protein family, hGC-1, expressed during myeloid lineage development. Gene. 2002, 283: 83-93. 10.1016/S0378-1119(01)00763-6.View ArticlePubMedGoogle Scholar
- Zhang X, Huang Q, Yang Z, Li Y, Li CY: GW112, a novel antiapoptotic protein that promotes tumor growth. Cancer Res. 2004, 64: 2474-2481. 10.1158/0008-5472.CAN-03-3443.View ArticlePubMedGoogle Scholar
- Kulkarni NH, Karavanich CA, Atchley WR, Anholt RR: Characterization and differential expression of a human gene family of olfactomedin-related proteins. Genet Res. 2000, 76: 41-50. 10.1017/S0016672300004584.View ArticlePubMedGoogle Scholar
- Shinozaki S, Nakamura T, Iimura M, Kato Y, Iizuka B, Kobayashi M, Hayashi N: Upregulation of Reg 1alpha and GW112 in the epithelium of inflamed colonic mucosa. Gut. 2001, 48: 623-629. 10.1136/gut.48.5.623.PubMed CentralView ArticlePubMedGoogle Scholar
- Mannick EE, Schurr JR, Zapata A, Lentz JJ, Gastanaduy M, Cote RL, Delgado A, Correa P, Correa H: Gene expression in gastric biopsies from patients infected with Helicobacter pylori. Scand J Gastroenterol. 2004, 39: 1192-1200. 10.1080/00365520410003588.View ArticlePubMedGoogle Scholar
- Liu W, Yan M, Liu Y, Wang R, Li C, Deng C, Singh A, Coleman WG, Rodgers GP: Olfactomedin 4 down-regulates innate immunity against Helicobacter pylori infection. Proc Natl Acad Sci USA. 2010, 107: 11056-11061. 10.1073/pnas.1001269107.PubMed CentralView ArticlePubMedGoogle Scholar
- Grutzmann R, Pilarsky C, Staub E, Schmitt AO, Foerder M, Specht T, Hinzmann B, Dahl E, Alldinger I, Rosenthal A, Ockert D, Saeger HD: Systematic isolation of genes differentially expressed in normal and cancerous tissue of the pancreas. Pancreatology. 2003, 3: 169-178. 10.1159/000070087.View ArticlePubMedGoogle Scholar
- Kobayashi D, Koshida S, Moriai R, Tsuji N, Watanabe N: Olfactomedin 4 promotes S-phase transition in proliferation of pancreatic cancer cells. Cancer Sci. 2007, 98: 334-340. 10.1111/j.1349-7006.2007.00397.x.View ArticlePubMedGoogle Scholar
- Koshida S, Kobayashi D, Moriai R, Tsuji N, Watanabe N: Specific overexpression of OLFM4(GW112/HGC-1) mRNA in colon, breast and lung cancer tissues detected using quantitative analysis. Cancer Sci. 2007, 98: 315-320. 10.1111/j.1349-7006.2006.00383.x.View ArticlePubMedGoogle Scholar
- Liu W, Zhu J, Cao L, Rodgers GP: Expression of hGC-1 is correlated with differentiation of gastric carcinoma. Histopathology. 2007, 51: 157-165. 10.1111/j.1365-2559.2007.02763.x.View ArticlePubMedGoogle Scholar
- Li SR, Dorudi S, Bustin SA: Identification of differentially expressed genes associated with colorectal cancer liver metastasis. Eur Surg Res. 2003, 35: 327-336.View ArticlePubMedGoogle Scholar
- Wentzensen N, Wilz B, Findeisen P, Wagner R, Dippold W, von Knebel Doeberitz M, Gebert J: Identification of differentially expressed genes in colorectal adenoma compared to normal tissue by suppression subtractive hybridization. Int J Oncol. 2004, 24: 987-994.PubMedGoogle Scholar
- Liu W, Liu Y, Zhu J, Wright E, Ding I, Rodgers GP: Reduced hGC-1 protein expression is associated with malignant progression of colon carcinoma. Clin Cancer Res. 2008, 14: 1041-1049. 10.1158/1078-0432.CCR-07-4125.View ArticlePubMedGoogle Scholar
- Conrotto P, Roesli C, Rybak J, Kischel P, Waltregny D, Neri D, Castronovo V: Identification of new accessible tumor antigens in human colon cancer by ex vivo protein biotinylation and comparative mass spectrometry analysis. Int J Cancer. 2008, 123: 2856-2864. 10.1002/ijc.23861.View ArticlePubMedGoogle Scholar
- Chen L, Li H, Liu W, Zhu J, Zhao X, Wright E, Cao L, Ding I, Rodgers GP: Olfactomedin 4 suppresses prostate cancer cell growth and metastasis via negative interaction with cathepsin D and SDF-1. Carcinogenesis. 2011, 32: 986-994. 10.1093/carcin/bgr065.PubMed CentralView ArticlePubMedGoogle Scholar
- Liu W, Lee HW, Liu Y, Wang R, Rodgers GP: Olfactomedin 4 is a novel target gene of retinoic acids and 5-aza-2'-deoxycytidine involved in human myeloid leukemia cell growth, differentiation, and apoptosis. Blood. 2010, 116: 4938-4947. 10.1182/blood-2009-10-246439.PubMed CentralView ArticlePubMedGoogle Scholar
- Huang Y, Yang M, Yang H, Zeng Z: Upregulation of the GRIM-19 gene suppresses invasion and metastasis of human gastric cancer SGC-7901 cell line. Exp Cell Res. 2010, 316: 2061-2070. 10.1016/j.yexcr.2010.05.010.View ArticlePubMedGoogle Scholar
- Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001, 25: 402-408. 10.1006/meth.2001.1262.View ArticlePubMedGoogle Scholar
- Yasui W, Oue N, Aung PP, Matsumura S, Shutoh M, Nakayama H: Molecular-pathological prognostic factors of gastric cancer: a review. Gastric Cancer. 2005, 8: 86-94. 10.1007/s10120-005-0320-0.View ArticlePubMedGoogle Scholar
- Aung PP, Oue N, Mitani Y, Nakayama H, Yoshida K, Noguchi T, Bosserhoff AK, Yasui W: Systematic search for gastric cancer-specific genes based on SAGE data: melanoma inhibitory activity and matrix metaloproteinase-10 are novel prognostic factors in patients with gastric cancer. Oncogene. 2006, 25: 2546-2557. 10.1038/sj.onc.1209279.View ArticlePubMedGoogle Scholar
- Matsukura S, Jones PA, Takai D: Establishment of conditional vectors for hairpin siRNA knockdowns. Nucleic Acids Res. 2003, 31: e77-10.1093/nar/gng077.PubMed CentralView ArticlePubMedGoogle Scholar
- Altieri DC: Validating survivin as a cancer therapeutic target. Nat Rev Cancer. 2003, 3: 46-54. 10.1038/nrc968.View ArticlePubMedGoogle Scholar
- Chene P: Inhibiting the p53-MDM2 interaction: an important target for cancer therapy. Nat Rev Cancer. 2003, 3: 102-109. 10.1038/nrc991.View ArticlePubMedGoogle Scholar
- Ueda M, Kokura S, Imamoto E, Naito Y, Handa O, Takagi T, Yoshida N, Yoshikawa T: Blocking of NF-kappaB activation enhances the tumor necrosis factor alpha-induced apoptosis of a human gastric cancer cell line. Cancer Lett. 2003, 193: 177-182. 10.1016/S0304-3835(03)00002-8.View ArticlePubMedGoogle Scholar
- Kim KK, Park KS, Song SB, Kim KE: Up regulation of GW112 gene by NFkB promotes an antiapoptotic property in gastric cancer cells. Mol Carcinog. 2010, 49: 259-270.PubMedGoogle Scholar
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