A functional polymorphism in the promoter region of IL-33 is associated with the reduced risk of colorectal cancer.

Aim: We aimed to investigate IL-33 polymorphisms with risk of colorectal cancer (CRC). Materials & methods:IL-33 rs7025417 and rs1332290 were genotyped using a quantitative allelic Taqman assay. The expression of IL-33 mRNA was determined by real-time PCR and promoter activity was assayed using the Dual-Luciferase Reporter Assay. Results: The IL-33 rs7025417 CC genotype and C allele may decrease CRC risk. The IL-33 rs1332290 AC carriers had an increased risk of developing clinical Stage III-IV CRC. Lower levels of IL-33 mRNA were present in individuals with the rs7025417 CC genotype. Moreover, the rs7025417 C allele suppressed promoter activity of IL-33. Conclusion: These data suggest that the rs7025417 CC genotype may downregulate IL-33 mRNA and subsequently reduce the risk of CRC.

Concomitant modulation of PTEN and Livin in gastric cancer treatment.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and Livin are important in the development of gastric cancer (GC). PTEN and Livin are involved in the regulation of tumor cell proliferation, migration and apoptosis. The modulation of PTEN or Livin has been investigated extensively in various cancer models. However, no studies have been performed to evaluate the combined effect of concurrently modulating these two genes on the development of GC. In the present study, the BGC823 human gastric carcinoma cell line was transfected with a dual gene modified vector (pCL-neo-PTEN-siLivin) in parallel with single gene modified vectors (pCL­neo­PTEN or pRNAT­U6.1­siLivin), and an empty control vector. Dual gene modulation (pCL­neo­PTEN­siLivin) had a more marked effect on the inhibition of cell proliferation, induction of apoptosis, and reduction of cell penetration in Matrigel, compared with either single gene alone or empty vector transfection. In a xenograft nude mouse model, the inoculation of pCL­neo­PTEN­siLivin­transfected BGC823 cells led to a markedly reduced tumor burden, compared with that in all other inoculation groups. In conclusion, the overexpression of PTEN concomitant with Livin gene silencing was confirmed as a feasible and effective in vitro and in vivo gene modulation method, which may represent a potential therapeutic strategy for the treatment of GC.

Over-expression of microRNA-940 promotes cell proliferation by targeting GSK3ß and sFRP1 in human pancreatic carcinoma.

Increasing study reports that Wnt/ß-catenin signaling pathway plays an essential role in numerous cancers growth, progression and metastasis. Aberrant miR-940 expression has been studied in gastric and breast cancer. However, the molecular mechanism of miR-940 enhancing proliferation and metastatic ability in human pancreatic carcinoma is far from to know. Real-time PCR was used to quantify miR-940 expression. Luciferase reporter assays here were performed to verify the activity of Wnt/ß-catenin signaling pathway and targeting gene relationships, and immunofluorescence assay was applied to observe ß-catenin expressed intensity. Bioinformatics analysis together with in vivo and vitro functional analysis indicated the potential targeting genes of miR-940. Specimens from 15 pairs of patients with human pancreatic carcinoma were involoved to confirm the relationship between miR-940 expression and the GSK3ß/sFRP1 through real-time PCR and western blot assays. Bioinformatics combined with cell luciferase function researches determined the possible regulation of miR-940 on the 3'-UTR of the GSK3ß and sFRP1 genes, resulting in the Wnt/ß-catenin signaling activation. Further, miR-940 knockdown significantly recovered GSK3ß and sFRP1 expression and relieved Wnt/ß-catenin-mediated cell invasion, migration, metastasis and proliferation. The ectopic up-regulation of miR-940 significantly suppressed GSK3ß/sFRP1 expression and promoted pancreatic carcinoma proliferation and invasion. Our study suggested mechanistic relationship between miR-940 and Wnt/ß-catenin in the development and progression of pancreatic carcinoma through regulation of GSK3ß and sFRP1.

Hyperthermia combined with 5-fluorouracil promoted apoptosis and enhanced thermotolerance in human gastric cancer cell line SGC-7901.

This study was designed to investigate the proliferation inhibition and apoptosis-promoting effect under hyperthermia and chemotherapy treatment, at cellular level. Human gastric cancer cell line SGC-7901 was cultivated with 5-fluorouracil at different temperatures. Cell proliferation and apoptosis were determined, and expression of Bcl-2 and HSP70 was measured at different treatments. Cell survival rates and inhibition rates in chemotherapy group, thermotherapy group, and thermo-chemotherapy group were drastically lower than the control group (P<0.05). For tumor cells in the thermo-chemotherapy group, survival rates and inhibition rates at three different temperatures were all significantly lower than those in chemotherapy group and thermotherapy group (P<0.05). 5-Fluorouracil induced apoptosis of SGC-7901 cells with a strong temperature dependence, which increased gradually with increase in temperature. At 37°C and 43°C there were significant differences between the thermotherapy group and chemotherapy group and between the thermo-chemotherapy group and thermotherapy group (P<0.01). The expression of Bcl-2 was downregulated and HSP70 was upregulated, with increase in temperature in all groups. Cell apoptosis was not significant at 46°C (P>0.05), which was probably due to thermotolerance caused by HSP70 accumulation. These results suggested that hyperthermia combined with 5-fluorouracil had a synergistic effect in promoting apoptosis and enhancing thermotolerance in gastric cancer cell line SGC-7901.

Elevated expression of NEDD9 is associated with metastatic activity in gastric cancer.

OBJECTIVE: To investigate the protein and mRNA expression of NEDD9 in gastric cancer (GC) tissues, adjacent atypical hyperplasia tissues, and normal gastric mucosa tissues, and analyze its relationship with the pathological features and prognosis of GC. METHODS: Forty cases of GC tissues, 20 cases of adjacent atypical hyperplasia tissues, and 40 cases of normal gastric mucous tissues were collected. Immunohistochemistry and Western blot were used to examine the expression of NEDD9 protein in various tissues. Situ hybridization and reverse transcription polymerase chain reaction were applied to detect the expression of NEDD9 mRNA in various tissues. The correlation of NEDD9 expression with invasion and metastasis of GC was analyzed. RESULTS: The protein expression level of NEDD9 was significantly higher in GC tissues than in adjacent atypical hyperplasia tissues and normal gastric mucous tissues (P<0.05). The protein expression level of NEDD9 was positively related to the invasion depth of carcinoma and tumor lymph node metastasis (P<0.05), but unrelated to age, sex, tumor size, and clinical classification of cancer (P<0.05). The mRNA expression level of NEDD9 was also significantly higher in GC tissues than in adjacent atypical hyperplasia tissues and normal gastric mucous tissues (P<0.05), and positively related with the tumor lymph node metastasis and invasion depth of carcinoma (P<0.05). CONCLUSION: NEDD9 is involved in the occurrence and development of GC, and it may be an important biological marker of GC metastasis and infiltration.

The role of microRNA-1274a in the tumorigenesis of gastric cancer: accelerating cancer cell proliferation and migration via directly targeting FOXO4.

MicroRNAs (miRNAs) are a series of 18-25 nucleotides length non-coding RNAs, which play critical roles in tumorigenesis. Previous study has shown that microRNA-1274a (miR-1274a) is upregulated in human gastric cancer. However, its role in gastric cancer progression remains poorly understood. Therefore, the current study was aimed to examine the effect of miR-1274a on gastric cancer cells. We found that miR-1274a was overexpressed in gastric cancer tissues or gastric cancer cells including HGC27, MGC803, AGS, and SGC-7901 by qRT-PCR analysis. Transfection of miR-1274a markedly promoted gastric cancer cells proliferation and migration as well as induced epithelial-mesenchymal transition (EMT) of cancer cells. Our further examination identified FOXO4 as a target of miR-1274a, which did not influence FOXO4 mRNA expression but significantly inhibited FOXO4 protein expression. Moreover, miR-1274a overexpression activated PI3K/Akt signaling and upregulated cyclin D1, MMP-2 and MMP-9 expressions. With tumor xenografts in mice models, we also showed that miR-1274a promoted tumorigenesis of gastric cancer in vivo. In all, our study demonstrated that miR-1274a prompted gastric cancer cells growth and migration through dampening FOXO4 expression thus provided a potential target for human gastric cancer therapy.

Tumor M2-pyruvate kinase in stool as a biomarker for diagnosis of colorectal cancer: A meta-analysis.

OBJECTIVE: The aim of this meta-analysis was to evaluate the diagnosis value of tumor M2-pyruvate kinase (M2-PK) in stool as a biomarker for diagnosis of colorectal cancer. MATERIALS AND METHODS: By searching the databases of Cochrane Library, PubMed, China national knowledge Information and Wanfang, the diagnosis study related to tumor M2-PK in stool as a biomarker for diagnosis of colorectal cancer were screened and included in this study. The pooled sensitivity, specificity, positive likelihood ratio (+LR), negative likelihood ratio (-LR) and the receiver operating characteristic curve (ROC) were calculated by stata 11.0 software. RESULTS: According to the including criteria, 14 trials including 1990 subjects were finally included in this meta-analysis. The pooled diagnosis sensitivity, specificity, +LR, -LR and area under curve were 0.78 (95% confidence interval [CI]: 0.74-0.81), 0.77 (95% CI: 0.76-0.79), 4.38 (95% CI: 3.27-5.88), 0.28 (95% CI: 0.23-0.34) and 0.86 (95% CI: 0.834-0.89). No statistical publication bias was found in this study. CONCLUSION: Tumor M2-PK in stool can be a useful biomarker in the diagnosis of colorectal cancer with relative high sensitivity and specificity.

Combination of the FGFR4 inhibitor PD173074 and 5-fluorouracil reduces proliferation and promotes apoptosis in gastric cancer.

Our previous findings revealed that FGFR4 may be a novel therapeutic target for gastric cancer. The aim of the present study was to explore the effects of a combination of PD173074 (PD) and 5-fluorouracil (5-Fu) on the biological behavior of gastric cancer cell lines and the relevant mechanisms involved. MKN45, a gastric cancer cell line, was treated with each single agent alone or a combination of FGF19, PD and 5-Fu. Then, a series of functional assays were performed using CCK-8 assay and flow cytometry. Western blot analysis was used to determine the expression of signaling pathway and downstream-related molecules in the MKN45 cells following the different treatments. As the concentration of PD and 5-Fu increased, the cell viability gradually decreased; the viability of the combination group was less than the viability following single administration. Western blot analysis showed that FGFR4 expression was weak in the 5-Fu-treated groups when compared with the control. PD markedly increased the apoptosis rate of MKN45 cells when compared to the control; the apoptosis rate in the cells treated with the combination of PD and 5-Fu was higher than that in the cells following single treatment. Furthermore, PD reduced the expression of p-ERK and Bcl-xl and increased caspase-3 expression. Inhibition of the activity of FGFR4 may be the main mechanisms of PD effect while 5-Fu reduced FGFR4 expression. Furthermore, the effects of the combination of 5-Fu and PD in inhibiting proliferation, increasing apoptosis and arresting cell cycle were superior to these effects following the single agent treatments, suggesting that the two drugs applied in combination may contribute to the effective treatment of gastric cancer.

2R of thymidylate synthase 5'-untranslated enhanced region contributes to gastric cancer risk: a meta-analysis.

BACKGROUND: Studies investigating the association between 2R/3R polymorphisms in the thymidylate synthase 5'-untranslated enhanced region (TYMS 5'-UTR) and gastric cancer risk have generated conflicting results. Thus, a meta-analysis was performed to summarize the data on any association. METHODS: Pubmed, Embase, and CNKI databases were searched for all available studies. The strength of association between TYMS 5'-UTR 2R/3R polymorphism and gastric cancer risk was estimated by odds ratios (ORs) with 95% confidence intervals (CIs). RESULTS: Six individual case-control studies with a total of 1,472 cases and 1,895 controls were included into this meta-analysis. Analyses of total six relevant studies showed that there was no obvious association between the TYMS 5'-UTR 2R/3R polymorphism and gastric cancer risk. Subgroup analyses based on ethnicity showed 2R of TYMS 5'-UTR 2R/3R contributes to gastric cancer risk in the Asian population (ORHomozygote model=1.71, 95%CI 1.19-2.46, P=0.004; ORRecessive genetic model=1.70, 95%CI 1.18-2.43, P=0.004). However, the association in Caucasian populations was uncertain due to the limited studies. CONCLUSIONS: Our meta-analysis suggests that 2R of TYMS 5'-UTR 2R/3R contributes to gastric cancer risk in the Asian population, while this association in Caucasians populations needs further study.

MTHFR C677T polymorphism and colorectal cancer risk in Asians, a meta-analysis of 21 studies.

BACKGROUND: Previous studies concerning the association between methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism and colorectal cancer risk in Asian populations generated conflicting results. A meta-analysis was therefore performed to allow a more reliable estimate of any link. METHODS: Relevant studies concerning the association between the MTHFR C677T polymorphism and risk of colorectal cancer were included into this meta-analysis. The quality of the studies was assessed according to a predefined scale. Odds ratios (ORs) and 95% confidence intervals (CIs) were determined for this gene-disease association using fixed or random effect models according to the heterogeneity between included studies. RESULTS: Finally, 21 studies with a total of 6692 cases and 8266 controls were included. Meta-analyses showed that there was an obvious association of the MTHFR 677T allele with decreased risk of colorectal cancer (OR = 0.91, 95%CI=0.85-0.98, P=0.011). Subgroup analyses by country further identified this association, with dietary folate as the main source of heterogeneity. CONCLUSION: The MTHFR 677T allele is associated with a lower risk of colorectal cancer in Asian populations, and there is effect modification by population plasma folate.

SNPs of excision repair cross complementing group 5 and gastric cancer risk in Chinese populations.

We conducted a case-control study to determine the association between several potential SNPs of excision repair cross complementing group 5 (XPG) and gastric cancer susceptibility, and roles of XPG polymorphisms in combination with H.pylori infection in determining risk of gastric cancer. In our study, we collected 337 newly diagnosed gastric cancer cases and 347 health controls. Three SNPs of XPG, rs2296147T>C, rs2094258C>T and rs873601G>A, were genotyped using the Taqman real-time PCR method with a 7900 HT sequence detector system. H. pylori infection was diagnosed by ELISA. By multivariate logistic regression analysis, the rs2296147 CC genotype was associated with a decreased risk of gastric cancer (OR=0.52, 95% CI=0.27-0.97), and rs2094258 TT was associated with elevated risk (OR=2.13, 95% CI=1.22-3.35). Positive H.pylori individuals with rs2094258 TT genotypes demonstrated increased risk of gastric cancer (OR=2.13, 95% CI=1.22-3.35), while rs2296147 CC was associated with lower risk among patients with negative H.pylori (OR=0.45, 95%CI=0.22-0.89). Our findings suggested that XPG polymorphisms might contribute to risk of gastric cancer among Chinese populations, but the effect needs to be further validated by larger sample size studies.

[Expression of mammalian target of rapamycin in colon cancer and its effect on proliferation and apoptosis of human colon cancer HT-29 cells].

OBJECTIVE: To explore the expression of mammalian target of rapamycin (mTOR) in colon cancer and the inhibitory effect of mTOR siRNA on the proliferation and apoptosis of human colon cancer HT-29 cells. METHODS: Immunohistochemistry was employed to detect the expression of phosphorylated mTOR (p-mTOR) in colon cancer tissues (n = 50) and normal colon tissues (n = 50) from First Affiliated Hospital of Zhengzhou University (October 2009 to June 2010). The correlations were examined between the expression of p-mTOR and such clinical pathological data as colon cancer staging and lymph node metastasis. The oligonucleotide templates of mTOR siRNA were designed and employed to transfect HT-29. The nonsense control groups were established accordingly by siRNA with an insignificant order. And the blank group had no transfection. The protein level of mTOR was measured by Western blotting. The method of MTT was used to detect the cell proliferation. And the method of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was employed to detect the apoptosis. RESULTS: The expression of p-mTOR was higher in the colon cancer group than the control group [60% (30 cases) vs 14% (7 cases), P < 0.05]. And the expression level was correlated with the degree of lymph node metastasis and tumor differentiation (r = 0.311, 0.427, both P < 0.05). The expression of mTOR protein in the transfection group was lower than the blank and the nonsense control groups (0.39 ± 0.25 vs 1.18 ± 0.05, 1.46 ± 0.09, both P < 0.05). The proliferation was lower than those of the blank and the nonsense control groups (0.275 ± 0.033 vs 0.460 ± 0.028, 0.450 ± 0.037, both P < 0.05). The apoptotic indices were higher than those of the blank and control groups (12.33 ± 1.53 vs 0.33 ± 0.31, 1.67 ± 0.58, both P < 0.05). CONCLUSION: The expression of mTOR was higher in colon cancer tissues than that in normal colon tissues. The RNA interference of mTOR in HT-29 cell line can effectively knock down the expression of mTOR so as to significantly inhibit cell proliferation and promote cell apoptosis.

[Construction and identification of gene vector expressing PTEN while simultaneously silencing Livin].

OBJECTIVE: To study the effect of simultaneously increasing PTEN gene expression and inhibiting Livin gene expression on the gastric carcinoma cell line (BGC823) and construct a recombinant vector expressing PTEN while simultaneously silencing Livin. METHODS: The siRNA expression unit against Livin gene (siLivin) was cleaved from pRNAT-U6.1-Livin vector and then inserted into pCL-neo-PTE to construct the recombinant vector pCL-neo-PTEN-siLivin. Then pCL-neo-PTEN-siLivinp, pCL-neo-PTEN, pRNAT-U6.1-Livin, pCL-neo and pRNAT-U6.1 were respectively transfected into the gastric carcinoma cell line (BGC823) with LipofectAMINE(TM) 2000. The mRNA and protein expression level of PTEN and Livin genes in each cell group was detected by fluorescent quantitative RT-PCR and Western blot. RESULTS: Recombinant vectors of pCL-neo-PTEN, pRNAT-U6.1-Livin and pCL-neo-PTEN-siLivin were constructed successfully. After transfection with pCL-neo-PTEN-siLivin, the mRNA and protein expression level of PTEN (0.897±0.112) rose in BGC823 cells while Livin gene became silenced. And the characterization of regulated cell bioactivity improved. There were significant differences between transfected and control groups (P<0.05). And the inhibiting effect on the proliferation and metastasis of BGC823 cell by increasing PTEN expression and silencing Livin simultaneously was better than that only by regulating PTEN genes or Livin genes alternatively. CONCLUSION: The recombinant vector of expressing PTEN and silencing Livin gene simultaneously is successfully constructed.

[Expression and significance of B7-H1 and its receptor PD-1 in human gastric carcinoma].

OBJECTIVE: The B7-H1/PD-1 co-signaling pathway has recently been found to play a pivotal role in the immune evasion of tumor cells from host immune system. The aim of this study was to examine the B7-H1 and PD-1 expression and TILs status in gastric cancer and to elucidate the clinical relevance of B7-H1 and PD-1 to the pathogenesis of gastric carcinoma. METHODS: Immunohistochemistry and ANAE histochemical staining were used to investigate the in situ expression of B7-H1 and PD-1 and TILs status in the gastric tissues. RT-PCR was used to explore B7-H1 and PD-1 expression at the transcriptional level. The B7-H1 expression at protein level was detected by Western blot. RESULTS: Expression of B7-H1 and PD-1 was found to be increased in gastric carcinoma, but absent in normal gastric tissue. B7-H1 expression in gastric carcinoma was inversely correlated with TILs infiltration. B7-H1 but not PD-1 expression in tumor tissue was significantly correlated with some clinicopathhological variables including depth of invasion, lymph node metastasis and distant metastasis. CONCLUSION: B7-H1 and PD-1 expressions are increased in gastric carcinoma. This signaling pathway may inhibit antitumor immune responses in gastric carcinoma. B7-H1 expression plays a critical role in the pathogenesis of human gastric carcinoma,and might be a promising prognostic marker and therapeutic target in the treatment of this disease.

Beta-adrenoceptors, but not dopamine receptors, mediate dopamine-induced ion transport in late distal colon of rats.

Dopamine, an important modulator in the gastrointestinal system, induces concentration-dependent transepithelial ion transport in the distal colon of the rat, as shown by a decrease in the short-circuit current, and acts in a segmentally dependent manner. However, the receptor(s) that mediates dopamine-induced ion transport is unknown. We have investigated the receptor mechanisms underlying dopamine-induced colonic ion transport by means of short-circuit current recording, real-time polymerase chain reaction, and Western blotting analysis, plus gene transfection and enzyme-linked immunosorbance assay. mRNA transcripts of adrenoceptors (alpha, beta) and dopaminergic receptors (D(1) and D(2)) were detected in the rat late distal colonic mucosa, with beta(2) displaying the highest expression. A similar result was found in human colorectal mucosa (equivalent of late distal colon in rat). Pretreatment with a beta(1)-adrenoceptor antagonist (CGP-20712A) and a beta(2)-adrenoceptor antagonist (ICI 118,551) inhibited the dopamine-induced short-circuit current response by 52.59% and 92.51%, respectively. However, neither dopamine D(1) receptor antagonist SCH-23390 nor dopamine D(2) receptor antagonist sulpiride blocked the effect of dopamine. Protein expression of both beta(1)- and beta(2)-adrenoceptors was found in the mucosa of rat distal colon and human sigmoid colon and rectum. Dopamine significantly increased intracellular cAMP levels in COS-7 cells transfected with beta(1)- or beta(2)-adrenoceptors. Thus, beta-adrenoceptors (mainly beta(2)-adrenoceptors), but not dopamine receptors, mediate dopamine-induced ion transport in the late distal colon of the rat. This extends our knowledge of the late distal colon (rats) or colorectum (human) and provides further experimental evidence that might aid the prevention, diagnosis, and clinical therapy of human colorectal diseases.