Luteolin suppresses gastric cancer progression by reversing epithelial-mesenchymal transition via suppression of the Notch signaling pathway.

BACKGROUND: Gastric cancer (GC) is one of the most malignant tumors and the second leading cause of cancer-related deaths in the world. Luteolin, a flavonoid present in many fruits and green plants, suppresses cancer progression. The effects of luteolin on GC cells and their underlying mechanisms remain unclear. METHODS: Effects of luteolin on cell proliferation, migration, invasion, and apoptosis were examined in vitro and in vivo by cell counting kit-8 (CCK-8), transwell assays, and flow cytometry, respectively. Real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blots were performed to evaluate Notch1 signaling and activation of epithelial-mesenchymal transition (EMT) in GC cells treated with or without luteolin. Immunohistochemistry was performed to examine proliferation and Notch1 expression in xenograft tumors. RESULTS: Luteolin significantly inhibited cell proliferation, invasion, and migration in a dose-dependent and time-dependent manner and promoted cell apoptosis. Luteolin reversed EMT by shrinking the cytoskeleton and by inducing the expression of epithelial biomarker E-cadherin and downregulating the mesenchymal biomarkers N-cadherin, vimentin and Snail. Furthermore, Notch1 signaling was inhibited by luteolin, and downregulation of Notch1 had similar effects as luteolin treatment on cell proliferation, migration, and apoptosis. In addition, luteolin suppressed tumor growth in vivo. A higher expression of Notch1 correlated with a poor overall survival and a poor time to first progression. Furthermore, co-immunoprecipitation analysis revealed that activated Notch1 and ß-catenin formed a complex and regulated cell proliferation, migration, and invasion. CONCLUSIONS: In this study, GC progression was inhibited by luteolin through suppressing Notch1 signaling and reversing EMT, suggesting that luteolin may serve as an effective anti-tumor drug in GC treatment.

Metallopanstimulin-1 regulates invasion and migration of gastric cancer cells partially through integrin ß4.

MPS-1 (metallopanstimulin-1), also known as ribosomal protein S27, was overexpressed in gastric cancer cells. However, how MPS-1 contributes to gastric carcinogenesis has not been well characterized. Here, we show that high expression of MPS-1 was observed in gastric cancer tissues and associated with gastric cancer cell metastasis. Alteration of MPS-1 expression regulates invasion and migration of gastric cancer cells both in vitro and in vivo. Furthermore, by using Signal-Net and cluster analyses of microarray data we identified integrin ß4 (ITGB4) as a downstream target of MPS-1 that mediates its effects on cell metastasis. Knockdown of MPS-1 expression in gastric cancer cells led to significant reduction of ITGB4 expression at both the RNA and protein levels. Mechanically, we found that overexpression of ITGB4 in MPS-1 knockdown cells largely recovers the ability of invasion and migration. Conversely, knockdown of ITGB4 partially reduced cell invading/migrating ability induced by MPS-1 overexpression. Moreover, MPS-1 and ITGB4 expressions are positively correlated in gastric cancer cell lines and tissues. Finally, the survival analyses show that the expression of MPS-1 and ITGB4 is associated with poor outcomes in gastric cancer patients. Collectively, our findings suggest that MPS-1 regulates cell invasiveness and migration partially through ITGB4 and that MPS-1/ITGB4 signaling axis may serve as therapeutic targets in the treatment of gastric cancer.

π-π Stacking mediated drug-drug interactions in human CYP2E1.

Because of having many low molecular mass substrates, CYP2E1 is of particular interests to the pharmaceutical industry. Many evidences showed that this enzyme can adopt multiple substrates to significantly reduce the oxidation rate of the substrates. The detailed mechanism for this observation is still unclear. In the current study, we employed GPU-accelerated molecular dynamics simulations to study the multiple-binding mode of human CYP2E1, with an aim of offering a mechanistic explanation for the unexplained multiple-substrate binding. Our results showed that Thr303 and Phe478 were key factors for the substrate recognition and multiple-substrate binding. The former can form a significant hydrogen bond to recognize and position the substrate in the productive binding orientation in the active site. The latter acted as a mediator for the substrate communications via π-π stacking interactions. In the multiple-binding mode, the aforementioned π-π stacking interactions formed by the aromatic rings of both substrates and Phe478 drove the first substrate far away from the catalytic center, orienting in an additional binding position and going against the substrate metabolism. All these findings could give atomic insights into the detailed mechanism for the multiple-substrate binding in human CYP2E1, providing useful information for the drug metabolism mechanism and personalized use of clinical drugs.

miRNA-199a-3p in plasma as a potential diagnostic biomarker for gastric cancer.

BACKGROUND: MicroRNA (miRNA) has been shown the potential of cancer diagnosis. We investigated whether plasma miRNA expression could discriminate between patients with and without gastric cancer. METHODS: This study was divided into three steps: (1) miRNA microarray profiling on plasma samples from 20 gastric cancer patients and 20 healthy controls; (2) miRNA selection by real-time qRT-PCR on 30 pairs of plasma from patients and controls; and (3) qRT-PCR validation on an independent set of plasma from 180 gastric cancer patients, 80 healthy controls, and 20 patients with gastric precancerous diseases. RESULTS: Of the 959 human miRNAs analyzed by microarray, 37 up-regulated miRNAs and seven down-regulated miRNAs were found in gastric cancer plasma. Of the seven discrepant miRNAs validated on the plasma from 30 gastric cancer patients and 30 healthy controls, both miRNA-199a-3p and miRNA-151-5p were significantly elevated (p < 0.05) and were significantly reduced after surgery (p < 0.05) in gastric cancer patients. Further large-scale validation showed that these two miRNAs expressions in plasma were significantly higher in gastric cancer patients than healthy controls and patients with gastric precancerous diseases, respectively. However, only the expression of miRNA-199a-3p in plasma was significantly associated with tumor invasion and with lymph node metastasis and tumor, node, metastasis stage. This marker yielded an area under the receiver operating characteristic curve area of 0.837 with 80 % sensitivity and 74 % specificity in discriminating gastric cancer patients from healthy controls. In gastric cancer tissue, miRNA-199a-3p was expressed in the cytoplasm of tumor cells. CONCLUSIONS: miRNA-199a-3p in plasma could be a novel potential diagnostic biomarker for gastric cancer detection.

MiRNA-199a-3p: A potential circulating diagnostic biomarker for early gastric cancer.

BACKGROUND: Tumor-associated miRNAs have been detected in serum or plasma. We investigated whether plasma miRNA-199a-3p could be a potential circulating biomarker for early gastric cancer (EGC). METHODS: By using real-time qRT-PCR, the expression of miRNA-199a-3p were compared between these pre-operative plasmas from 30 EGC patients and 70 healthy controls, and between these pre-operative and post-operative plasmas. Further validation was on an independent set of plasmas from 50 EGC patients. RESULTS: The expression of miRNA-199a-3p (47.5 ± 6.5) in plasma in EGC patients was significantly higher than that from healthy controls (13.9 ± 2.7, P < 0.001) and gastric precancerous diseases (GPD) patients (19.2 ± 2.5, P = 0.004), respectively. Furthermore, the expression levels of miRNA-199a-3p (11.8 ± 2.9, P = 0.012) in the post-operative plasmas were significantly reduced when compared to the pre-operative plasmas. With respect of clinicopathological characteristics, the expression of miRNA-199a-3p in plasma was not associated with the depth of tumor invasion. Moreover, the AUC of the expression of miRNA-199a-3p in plasma for EGC diagnosis was 0.818, which was significantly higher than that of combined tumor markers (0.556). The sensitivity, specificity and accuracy of miRNA-199a-3p expression in plasma for EGC diagnosis were 76%, 74%, and 75%, respectively. CONCLUSIONS: Plasma miRNA-199a-3p could be a novel potential diagnostic biomarkers for EGC.

Knockdown of metallopanstimulin-1 inhibits NF-κB signaling at different levels: the role of apoptosis induction of gastric cancer cells.

The ribosomal protein S27 (metallopanstimulin-1, MPS-1) has been reported to be a multifunctional protein, with increased expression in a number of cancers. We reported previously that MPS-1 was highly expressed in human gastric cancer. Knockdown of MPS-1 led to spontaneous apoptosis and repressed proliferation of human gastric cancer cells in vitro and in vivo. However, how does MPS-1 regulate these processes is unclear. Here we performed microarray and pathway analyses to investigate possible pathways involved in MPS-1 knockdown-induced apoptosis in gastric cancer cells. Our results showed that knockdown of MPS-1 inhibited NF-κB activity by reducing phosphorylation of p65 at Ser536 and IκBα at Ser32, inhibiting NF-κB nuclear translocation, and down-regulating its DNA binding activity. Furthermore, data-mining the Gene-Regulatory-Network revealed that growth arrest DNA damage inducible gene 45ß (Gadd45ß), a direct NF-κB target gene, played a critical role in MPS-1 knockdown-induced apoptosis. Over-expression of Gadd45ß inhibited MPS-1 knockdown-induced apoptosis via inhibition of JNK phosphorylation. Taken together, these data revealed a novel pathway, the MPS-1/NF-κB/Gadd45ß signal pathway, played an important role in MPS-1 knockdown-induced apoptosis of gastric cancer cells. This study sheds new light on the role of MPS-1/NF-κB in apoptosis and the possible use of MPS-1 targeting strategy in the treatment of gastric cancer.

[Expression of metastasis associated 1 family member 2 (MTA2) in gastric cancer and its relationship with transcription factor Sp1].

OBJECTIVE: To determine the expression patterns of metastasis associated 1 family member 2 (MTA2) in gastric cancer and non-cancerous gastric mucosa, and analyze its relationship with nuclear transcription factor Sp1 expression. METHODS: Tissue samples and clinicopathological information from 83 gastric cancer patients, who underwent surgery, were collected from Shanghai Rui Jin Hospital. All samples included cancer tissue and non-cancerous mucosa which was 5 cm away from the tumor lesion. The expression of MTA2 and Sp1 were detected by immunohistochemistry (IHC) staining. The mRNA of MTA2 was also detected by reverse transcription-polymerase chain reaction (RT-PCR). SPSS software was used for statistical analysis. RESULTS: The expression of MTA2 protein was significantly higher in primary lesions of the gastric cancer than that in non-cancerous mucosa by IHC (31.3% vs 12.0%, P < 0.01). MTA2 expression was closely related with tumor invasion or T staging (χ(2) = 5.677, P < 0.05). Yet, no significant relationship was observed between MTA2 expression and other clinicopathological parameters, including the age, sex, tumor differentiation, Lauren classification, lymph node metastasis, distant metastasis, as well as pathological staging. Furthermore, MTA2 expression was concomitant with Sp1 expression (r = 0.320, P < 0.05). Elevated MTA2 expression was observed in Sp1 positive cancer tissues (χ(2) = 9.565, P < 0.01). RT-PCR results also demonstrated that MTA2 mRNA was also highly expressed in the tissue samples with Sp1 expression. CONCLUSIONS: MTA2 is highly expressed in the primary lesions of gastric cancer than that in adjacent non-cancerous tissues, and is closely related with tumor invasion. MTA2 expression is elevated in Sp1 positive gastric cancer.

MR Imaging of activated hepatic stellate cells in liver injured by CCl4 of rats with integrin-targeted ultrasmall superparamagnetic iron oxide.

OBJECTIVE: To demonstrate the feasibility of the ultrasmall superparamagnetic iron oxide (USPIO) modified by cyclo (Arg-Gly-Asp-Try-Cys) peptide (c(RGDyC)-USPIO) for targeting hepatic stellate cells (HSCs). MATERIALS AND METHODS: A c(RGDyC)-USPIO probe was prepared by conjugating c(RGDyC) with USPIO through a thiol-maleinide interaction. The specificity of c(RGDyC)-USPIO for HSCs was investigated in vitro. In vivo, normal and fibrosis rats were treated with either c(RGDyC)-USPIO or USPIO, and magnetic resonance imaging (MRI) of the rats performed after administration of the probes for 4 h. The T2 relaxation times changes before and after probe injection were analyzed and the locations of probes in normal or injured mice were identified histologically. RESULTS: The hydrodynamic size of c(RGDyC)-USPIO was 13 ± 3 nm. HSCs took up more specific probes than plain ones. The reduction of T2 relaxation times in fibrosis rat by c(RGDyC)-USPIO was much greater than that by USPIO (P < 0.05). Prussian blue staining and transmission electron microscopy of the injured rat liver treated with c(RGDyC) demonstrated that c(RGDyC)-USPIO were specifically engulfed by the activated HSCs. CONCLUSION: In vivo cellular targeted imaging of activated HSCs in liver fibrosis using c(RGDyC)-USPIO targeting α(v)ß(3) integrins was feasible using a clinical 1.5-Tesla MR system.

Helicobacter pylori induces promoter hypermethylation and downregulates gene expression of IRX1 transcription factor on human gastric mucosa.

BACKGROUND AND AIM: Gene silence of IRX1 tumor suppressor by promoter CpG methylation combined with loss of heterozygosity (LOH) has been identified in human gastric cancer. This study investigated the association between methylation of IRX1 and Helicobacter pylori infection in gastric mucosa tissues and cell line. METHODS: IRX1 methylation was studied by methylation specific polymerase chain reaction (MSP) and bisulfate sequencing polymerase chain reaction (BSP) methods in gastric mucosa tissues from H. pylori-positive chronic gastritis patients or H. pylori-negative chronic gastritis patients. Promoter activity, methylation status and gene expressing level of IRX1 were evaluated by persistent infecting H. pylori on human gastric cells GES-1 in vitro. Electron microscopy was used to observe the effect of H. pylori infection on GES-1 gastric mucosa cells. RESULTS: The methylation level of IRX1 promoter in H. pylori positive chronic gastritis and H. pylori negative chronic gastritis was 55.30%±13.17 versus 5.20%±6.31, respectively (P<0.01). H. pylori infection stimulated increased microvillus, and mucous secretion on GES-1 cells. Infection of H. pylori induced IRX1 promoter methylation and downregulation of the promoter activity as well as gene expression significantly. CONCLUSIONS: This study firstly demonstrated that H. pylori infection contributes to IRX1 promoter methylation on gastric mucosa.

mTOR activation in well differentiated pancreatic neuroendocrine tumors: a retrospective study on 34 cases.

BACKGROUND/AIMS: Pancreatic neuroendocrine tumors (PNET) are heterogeneous tumors with dramatically different prognosis. The survival is poor in patients with locally advanced or metastatic tumors. This study investigated expression and activation of mTOR in well differentiated PNET, and the relationship between mTOR activation and patients' clinicopathological characteristics. METHODOLOGY: Thirty-four samples were collected from patients who underwent surgery and had pathologically confirmed well differentiated PNET in Ruijin hospital from 2003 to 2009. All patients were classified based on 2010 WHO classification criteria and followed-up for survival. mTOR and phosphorylated-mTOR (p-mTOR) expressions were investigated by immunohistochemistry. RESULTS: All 34 PNET patients received radical surgical resection, with 1 patient receiving adjuvant chemotherapy. Twenty-five patients were classified as NET G1, and 9 were NET G2. mTOR and p-mTOR expression rates were 70.6% and 61.8%, respectively. p-mTOR positive rate correlated with the WHO classification, the patients classified as NET G2 had higher p-mTOR positive rates (p=0.012). However, a significant correlation between p-mTOR positivity and selected clinicopathological characteristics, was not observed. Twenty-six patients were followed-up for a median time of 16.8 months, but a significant correlation between mTOR activation and patients' prognosis was also not observed. CONCLUSIONS: The expression of mTOR and p-mTOR is extensive in well differentiated PNET patients. Patients classified as NET G2 had a higher mTOR activation rate.

Cell cycle dependent genes from the gastric cancer cell MKN45 can affect tumorigenesis.

BACKGROUND/AIMS: To investigate the cell cycle dependent genes involved in gastric tumorigenesis, possibly determining the relationship between the cell cycle and tumorigenesis. METHODOLOGY: MKN45 cells were collected every hour from Oh to 12h after release from G2/M and G1/S blocks. Nine samples (a-i), chosen at key times of the cell cycle, were prepared for RNA isolation and cDNA microarray analysis. RESULTS: In 2001 viable clones, 959 genes showed periodic variations during the cell cycle. Among 2001 genes that were clustered, a series of up-regulated genes were assigned to different cell cycle phases. Many periodically dependent genes in the cell cycle were ubiquitously expressed and participated in various cell physiological functions, such as transcription, translation, ubiquitination and signal transduction. These cell cycle dependent genes could affect cancer cell proliferation, apoptosis, activation of oncogenes and inactivation of tumor suppressor genes. CONCLUSIONS: We provided a comprehensive understanding of the gene expression profile involved in gastric cancer cell cycles and laid a foundation for further research on mechanisms of gastric tumorigenesis.

BMI1 and Mel-18 oppositely regulate carcinogenesis and progression of gastric cancer.

BACKGROUND: The BMI1 oncogene is overexpressed in several human malignancies including gastric cancer. In addition to BMI1, mammalian cells also express Mel-18, which is closely related to BMI1. We have reported that Mel-18 functions as a potential tumor suppressor by repressing the expression of BMI1 and consequent downregulation of activated AKT in breast cancer cells. However, the mechanisms of BMI1 overexpression and the role of Mel-18 in other cancers are still not clear. The purpose of this study is to investigate the role of BMI1 and Mel-18 in gastric cancer. RESULTS: BMI1 was found to be overexpressed in gastric cancer cell lines and gastric tumors. Overexpression of BMI1 correlated with advanced clinical stage and lymph node metastasis; while the expression of Mel-18 negatively correlated with BMI1. BMI1 but not Mel-18 was found to be an independent prognostic factor. Downregulation of BMI1 by Mel-18 overexpression or knockdown of BMI1 expression in gastric cancer cell lines led to upregulation of p16 (p16INK4a or CDKN2A) in p16 positive cell lines and reduction of phospho-AKT in both p16-positive and p16-negative cell lines. Downregulation of BMI1 was also accompanied by decreased transformed phenotype and migration in both p16- positive and p16-negative gastric cancer cell lines. CONCLUSIONS: In the context of gastric cancer, BMI1 acts as an oncogene and Mel-18 functions as a tumor suppressor via downregulation of BMI1. Mel-18 and BMI1 may regulate tumorigenesis, cell migration and cancer metastasis via both p16- and AKT-dependent growth regulatory pathways.

[Discovery and verification of matrix gla protein, a TNM staging and prognosis-related biomarker for gastric cancer].

OBJECTIVE: To analyze microarray datasets deposited in the public database and to identify TNM associated genes in gastric cancers. METHODS: Microarray datasets of gastric cancer were selected from GEO database. Differentially expressed genes related to TNM staging were evaluated by significant analysis of the microarray using MultiExperiment Viewer (MEV) platform. Candidate gene expressions in gastric cancer tissues and cell lines were verified by reverse transcriptase polymerase chain reaction (RT-PCR), quantitative RT-PCR, Western blot and immunohistochemistry. RESULTS: GES4007 dataset was re-analyzed leading to the identification of 14 genes associated with TNM staging. Over-expression of matrix gla protein (MGP) was confirmed in gastric cancer cell lines and tumor tissues by quantitative RT-PCR, Western blot and immunohistochemistry. Increased MGP expression was found in 22 of 54 cases of (40.7%) gastric cancer specimens compared to the normal gastric tissues. The up-regulation of MGP mRNA expression closely correlated with TNM stage (P = 0.001) and prognosis (P = 0.006). CONCLUSIONS: Public databases of microarray studies are the valuable resources for data mining. MGP has been identified and confirmed as a novel biomarker for the TNM stage and prognosis of gastric cancer.

[Survival benefit of an herbal formula for invigorating spleen for elderly patients with gastric cancer].

BACKGROUND: Gastric cancer is one of the most common malignant tumors. Traditional Chinese medicine (TCM) has been widely used in treatment of gastric cancer, but still lacking large sample controlled trial to evaluate its efficacy. OBJECTIVE: To analyze the prognostic factors of 220 elderly patients with gastric cancer, and to further study the efficacy of an herbal formula for invigorating spleen and it modifications based on syndrome differentiation of TCM in treatment of gastric cancer in elderly patients and the influence on prognosis. DESIGN, SETTING, PARTICIPANTS AND INTERVENTIONS: A total of 220 elderly patients aged 65 years or over with gastric cancer from Longhua Hospital of Shanghai University of Traditional Chinese Medicine, and Renji Hospital and Ruijin Hospital of Shanghai Jiao Tong University Medical College were prospectively enrolled. All patients were assigned to either traditional Chinese herbal medicine (TCHM) group (89 cases) or non-TCHM group (131 cases). Patients in the TCHM group were treated with an herbal formula for invigorating spleen plus chemotherapy, while patients in the non-TCHM group were only treated with chemotherapy. MAIN OUTCOME MEASURES: Univariate and Cox regression analyses were performed to determine all the potential prognostic factors. Kaplan-Meier curves were used to assess the differences in survival time between TCHM group and non-TCHM group after stratification for TNM stage, surgery or chemotherapy. RESULTS: The 220 eligible patients were histologically confirmed adenocarcinoma of the stomach from 2001 to 2007. Eighty-nine cases in the TCHM group received three or more months of TCHM treatment, and 131 cases in the non-TCHM group did not receive TCHM treatment. Cox regression analysis suggested that the TNM stage, radical resection, three or more treatment cycles of chemotherapy, and TCHM treatment were independent prognostic factors (P<0.01). The patients receiving TCHM treatment demonstrated better prognosis than the other prognostic factors in multivariate analysis; the odds ratio [Exp(beta)] of overall group was 0.322, and 95% confidence interval (CI) was from 0.212 to 0.489. Median overall survival of TCHM group was 41.129 months, and one-, three-, and five-year survival rates were 85.2%, 55.6% and 45.7% respectively. Median overall survival of non-TCHM group was 17.195 months, and one-, three-, and five-year survival rates were 63.9%, 26.9% and 21.9% respectively. In stratification analysis of stage for 96 patients who did not accepted radical resection or suffered from recurrence and metastasis (36 cases in the TCHM group, and 60 cases in the non-TCHM group), Cox regression analysis suggested that three or more treatment cycles of chemotherapy and TCHM treatment were independent prognostic factors for improving survival respectively (P<0.01). The hazard ratio [Exp(beta)] of TCHM in stratification for late stage was 0.421, and 95% confidence interval was from 0.255 to 0.693. Median overall survivals were 17.819 months for TCHM group and 8.548 months for non-TCHM group. In stratification analysis of surgery and chemotherapy for 102 patients with Ib-IV (M0) who accepted radical resection (R0 resection) and three or more treatment cycles of chemotherapy (33 cases in the TCHM group, and 69 cases in the non-TCHM group), the disease-free survival and overall survival did not reach the median at the time of analysis. In the TCHM group, one-, three-, and five-year disease-free survival rates were 97.0%, 59.9% and 50.4%, and one-, three-, and five-year survival rates were 100.0%, 74.1% and 61.4%, respectively. In the non-TCHM group, one-, three-, and five-year disease-free survival rates were 82.6%, 51.1% and 51.1%, and one-, three-, and five-year survival rates were 86.9%, 55.6% and 55.6%, respectively. CONCLUSION: The herbal formula for invigorating spleen has an important value for improving the prognosis of elderly patients with gastric cancer. This herbal formula show survival benefit for advanced gastric cancer in elderly patients. The influence of TCHM on disease-free survival and overall survival of postoperative gastric cancer in elderly patients need to be further evaluated.

Value of multidetector-row computed tomography in the preoperative T and N staging of gastric carcinoma: a large-scale Chinese study.

OBJECTIVES: To investigate the value of multidetector-row computed tomography (MDCT) in the preoperative T and N staging of gastric carcinoma and to further investigate the clinicopathological factors affecting the diagnostic accuracy. METHODS: Seven hundred ninety gastric carcinoma patients underwent preoperative MDCT examination. The results of MDCT were compared with surgical and pathological findings. RESULTS: Early gastric carcinoma patients whose primary tumor was detected by MDCT had higher incidence of lymph node metastasis, larger tumor size, and deeper invasion. The overall accuracy of MDCT in determining T stage of gastric carcinoma was 73.80% (T1 45.93%, T2 53.03%, T3 86.49%, and T4 85.79%). The overall accuracy of MDCT in preoperative N staging was 75.22% (N0 76.17%, N1 68.81%, and N2 80.63%). The overall diagnostic sensitivity, specificity, and accuracy of MDCT for determining lymph node metastasis was 86.26%, 76.17%, and 82.09%, respectively. Multivariate analysis showed that the diagnostic sensitivity of MDCT in determining lymph node metastasis related with tumor size, N stage, and number of metastatic lymph nodes. CONCLUSIONS: The clinical value of MDCT in the preoperative T and N staging of gastric carcinoma is relatively high. MDCT can be the first choice for the preoperative evaluation of patients with gastric carcinoma.

[EGFR-blockade by antibody Cetuximab inhibits the growth of human gastric cancer xenograft in nude mice and its possible mechanism].

OBJECTIVE: EGFR-mediated tumor proliferation plays an important role in the development of cancer, and is a key candidate for targeted therapy. The aim of this study is to evaluate the impact of EGFR monoclonal antibody Cetuximab (C225) on the growth, proliferation and apoptsis of gastric cancer xenograft in nude mice, and its possible mechanisms. METHODS: A gastric cancer cell line SGC-7901 with high EGFR expression level was screened from 7 gastric cancer cell lines. Gastric cancer xenografts in nude mice were established, and randomly divided into C225 treatment group and PBS control group. Tumor growth curves were calculated, the impact of C225 on the tumor growth, proliferation and angiogenesis was evaluated by immunohistochemical (IHC) staining Ki67 and CD34, respectively. The effect of C225 on apoptosis in the gastric cancer cells was evaluated by TUNEL assay. The expression levels of EGFR and its transcription factor Sp1 were detected by IHC staining and Western blot. RESULTS: After C225 treatment, the proliferation and growth of gastric cancer xenograft in nude mice were significantly decreased. In the contrast, the apopotic indexes in C225 treatment group and PBS control group were (16.4% +/- 0.3%) and (3.1% +/- 0.9%), respectively, with a significant difference (P < 0.001). There was no significant difference of the densities of CD34-positive microvessels between C225 treatment group and control group. Elevated expression of EGFR and Sp1 after C225 treatment was observed by IHC staining and Western blot assay. CONCLUSION: EGFR monoclonal antibody cetuximab (C225) can effectively inhibit the growth of gastric cancer xenografts in nude mice, and trigger its apoptosis. Yet, C225 treatment may upregulate the expression of EGFR and its transcription factor Sp1. A "block-transcription activation-compensation" mechanism may exist to explain the molecular mechanism of acquired resistance of a single target blockade treatment.

[Galectin-9 isoforms influence the adhesion between colon carcinoma LoVo cells and human umbilical vein endothelial cells in vitro by regulating the expression of E-selectin in LoVo cells].

OBJECTIVE: To study the regulatory effect of galectin-9 isoforms on some molecules involved in cell adhesion/invasion, and the influence of this regulation action on the adhesion between colon carcinoma LoVo cells and human umbilical vein endothelial cells (HUVECs) in vitro. METHODS: Various expression vectors of galectin-9 isoforms were transfected into LoVo cells. 24 h after transfection, the expression of integrin-beta1, E-cadherin, E-selectin, ICAM-1, CD44 and MMP-9 was detected by RT-PCR and Western blot analysis. LoVo cell-HUVEC adhesion assay was performed under conditions of galectin-9 transfection, galectin-9 transfection + galectin-9 antibody, galectin-9 transfection + E-selectin antibody and galectin-9 transfection + beta-lactose, respectively. RESULTS: Galectin-9L down-regulates the mRNA and protein levels of E-selectin while galectin-9M and galectin-9S up-regulate the expression of E-selectin. In LoVo cell-HUVEC adhesion assay, the average fluorescence intensity of vector transfection group, galectin-9L transfection group, galectin-9M transfection group and galectin-9S transfection group was 0.90 +/- 0.20, 0.94 +/- 0.24, 1.60 +/- 0.11 and 1.45 +/- 0.13, respectively, indicating that galectin-9M and galectin-9S facilitated the adherence of LoVo cells to HUVECs (P < 0.05). E-selectin antibody, galectin-9 antibody or beta-lactose inhibited that effect. CONCLUSION: Galectin-9 isoforms regulate the E-selectin expression in LoVo cells differently and thus influence the adhesion between LoVo cells and HUVECs in vitro in different modes. The mechanisms through which galectin-9 isoforms participate in the metastasis process of colon cancer may not be the same.

Aberrant expression and demethylation of gamma-synuclein in colorectal cancer, correlated with progression of the disease.

Recent evidence suggests that gamma-synuclein is abnormally expressed in a high percentage of tumor tissues of diversified cancer types, but rarely expressed in tumor-matched non-neoplastic adjacent tissues (NNAT). The molecular mechanism of CpG island demethylation may underlie aberrant gamma-synuclein expression. To fully understand the roles of aberrant gamma-synuclein expression and demethylation in the development of colorectal cancer (CRC), we examined the expression and methylation status of gamma-synuclein in 67 CRC samples, 30 NNAT samples, and five CRC cell lines as well. By using reverse transcription-polymerase chain reaction (RT-PCR), western blot, and immunohistochemistry analyses, gamma-synuclein expression was detected in both HT-29 and HCT116 cells, and was much higher in CRC samples than in NNAT samples (P < 0.05). The demethylating agent, 5-aza-2 cent-deoxycytidine, can induce re-expression of gamma-synuclein in COLO205, LoVo, and SW480 cells. Unmethylated gamma-synuclein alleles were detected in HT-29, HCT116, and LoVo cells by nested methylation-specific PCR, and the demethylated status of gamma-synuclein was much higher in CRC samples than in NNAT samples by real-time quantitative methylation-specific PCR (P < 0.05). The results of genomic bisulfite DNA sequencing further confirmed that the aberrant gamma-synuclein expression in CRC was primarily attributed to the demethylation of CpG island. The protein expression and demethylation status of gamma-synuclein in 67 CRC samples correlated with clinical stage, lymph node involvement, and distant metastasis. These findings suggest an involvement of aberrant gamma-synuclein expression and demethylation in progression of CRC, especially in advanced stages.

Comparative proteomics analysis of human gastric cancer.

AIM: To isolate and identify differentially expressed proteins between cancer and normal tissues of gastric cancer by two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). METHODS: Soluble fraction proteins of gastric cancer tissues and paired normal tissues were separated by 2-DE. The differentially expressed proteins were selected and identified by MALDI-TOF-MS and database search. RESULTS: 2-DE profiles with high resolution and reproducibility were obtained. Twenty-three protein spots were excised from sliver staining gel and digested in gel by trypsin, in which fifteen protein spots were identified successfully. Among the identified proteins, there were ten over-expressed and five under-expressed proteins in stomach cancer tissues compared with normal tissues. CONCLUSION: In this study, the well-resolved, reproducible 2-DE patterns of human gastric cancer tissue and paired normal tissue were established and optimized and certain differentially-expressed proteins were identified. The combined use of 2-DE and MS provides an effective approach to screen for potential tumor markers.

Differential networking meta-analysis of gastric cancer across Asian and American racial groups.

BACKGROUND: Gastric Carcinoma is one of the most lethal cancer around the world, and is also the most common cancers in Eastern Asia. A lot of differentially expressed genes have been detected as being associated with Gastric Carcinoma (GC) progression, however, little is known about the underlying dysfunctional regulation mechanisms. To address this problem, we previously developed a differential networking approach that is characterized by involving differential coexpression analysis (DCEA), stage-specific gene regulatory network (GRN) modelling and differential regulation networking (DRN) analysis. RESULT: In order to implement differential networking meta-analysis, we developed a novel framework which integrated the following steps. Considering the complexity and diversity of gastric carcinogenesis, we first collected three datasets (GSE54129, GSE24375 and TCGA-STAD) for Chinese, Korean and American, and aimed to investigate the common dysregulation mechanisms of gastric carcinogenesis across racial groups. Then, we constructed conditional GRNs for gastric cancer corresponding to normal and carcinoma, and prioritized differentially regulated genes (DRGs) and gene links (DRLs) from three datasets separately by using our previously developed differential networking method. Based on our integrated differential regulation information from three datasets and prior knowledge (e.g., transcription factor (TF)-target regulatory relationships and known signaling pathways), we eventually generated testable hypotheses on the regulation mechanisms of two genes, XBP1 and GIF, out of 16 common cross-racial DRGs in gastric carcinogenesis. CONCLUSION: The current cross-racial integrative study from the viewpoint of differential regulation networking provided useful clues for understanding the common dysfunctional regulation mechanisms of gastric cancer progression and discovering new universal drug targets or biomarkers for gastric cancer.