EZH2 inhibition promotes ANGPTL4/CREB1 to suppress the progression of ulcerative colitis.

AIMS: Enhancer of zeste homolog 2 (EZH2) is associated with ulcerative colitis development. However, the mechanism of EZH2 in ulcerative colitis progression remains unclear. MAIN METHODS: Lipopolysaccharide (LPS)-treated Caco-2 cells and dextran sodium sulfate (DSS)-treated mice were used as model of ulcerative colitis. The levels of EZH2, angiopoietin-like 4 (ANGPTL4) and cyclic adenosine monophosphate response element-binding protein 1 (CREB1) were tested via quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. Cell viability and apoptosis was measured via 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide or flow cytometry. The abundances of inflammatory cytokines were examined via qRT-PCR and enzyme-linked immunosorbent assay. The association between EZH2 and ANGPTL4 was explored via chromatin immunoprecipitation. The colon damage in DSS-treated mice was investigated by colon length, histological analysis, inflammatory response and apoptosis. KEY FINDINGS: LPS induced viability inhibition, inflammatory response and apoptosis in Caco-2 cells. EZH2 expression was increased but ANGPTL4 and CREB1 levels were decreased in LPS-challenged Caco-2 cells. Overexpression of ANGPTL4 or CREB1 suppressed LPS-induced damage in Caco-2 cells. EZH2 could target ANGPTL4 to mediate CREB1 expression. Inhibition of EZH2 suppressed LPS-caused injury. Moreover, knockdown of ANNGPTL4 or CREB1 attenuated the role of EZH2 inhibition. DSS caused the reduced colon length and increased inflammatory response as well as apoptosis. EZH2 expression was up-regulated but ANGPTL4 and CREB1 expression were down-regulated in DSS-treated mice. SIGNIFICANCE: Inhibition of EZH2 declined LPS-induced injury in Caco-2 cells by mediating ANGPTL4 and CREB1, indicating the potential of EZH2 in treatment of ulcerative colitis.

Peptide-Conjugated Quantum Dots Act as the Target Marker for Human Pancreatic Carcinoma Cells.

BACKGROUND/AIMS: In the present study, we describe a novel and straightforward approach to produce a cyclic- arginine-glycine-aspartic (RGD)-peptide-conjugated quantum dot (QD) probe as an ideal target tumor biomarker. Due to its specific structure, the probe can be used for targeted imaging of pancreatic carcinoma cells. METHODS: Pancreatic carcinoma cells were routinely cultured and marked with QD-RGD probe. The QD-RGD probe on the fluorescence-labeled cancer cell was observed by fluorescence microscopy and laser confocal microscopy. Cancer cell viability was detected by MTT assay after culturing with QD-RGD probe. RESULTS: Fluorescence microscopy and laser confocal microscopy displayed that 10nmol/L QD-RGD probe was able to effectively mark pancreatic carcinoma cells. In comparison with organic dyes and fluorescent proteins, the quantum dot-RGD probe had unique optical and electronic properties. CONCLUSION: QD-RGD probe has a low cytotoxicity with an excellent optical property and biocompatibility. These findings support further evaluation of QD-RGD probes for the early detection of pancreatic cancer.

XPD Lys751Gln polymorphisms and the risk of esophageal cancer: an updated meta-analysis.

OBJECTIVE: Published data regarding the association between xeroderma pigmentosum group D XPD Lys751Gln polymorphisms and esophageal cancer (EC) cancer remain controversial. The present meta-analysis aimed to obtain a more precise estimation of the relationship between XPD Lys751Gln polymorphisms and the risk of EC. METHODS: All eligible case-control studies of Lys751Gln polymorphisms and susceptibility to EC were selected from PubMed, Web of Science and CNKI up to October 2013. The data were extracted, and pooled odds ratios (ORs) with 95% confidence intervals (CIs) were calculated. RESULTS: A total of 21 case-control studies from 19 reports were assessed in this meta-analysis, including 6,581 cases and 8,251 controls. There was a significant association between the XPD Lys751Gln polymorphism and the risk of esophageal cancer in the overall population (Dominant model: OR=1.30, 95%CI: 1.07-1.57, p<0.05; Lys/Gln vs. Gln/Gln: OR=1.20, 95%CI: 1.05-137, p<0.05; Gln/Gln vs. Lys/Lys: OR=1.76, 95%CI: 1.08-2.85, p=0.02; Recessive model: OR=1.48, 95%CI: 1.06-2.07, p=0.02). Similar results were found when stratified according to the cancer type, ethnicity and control source. However, no associations were found among smokers or drinkers. CONCLUSION: The results of this meta-analysis suggest that XPD Lys751Gln polymorphisms contribute to susceptibility to EC.

Synergistic antitumor effect of puerarin combined with 5-fluorouracil on gastric carcinoma.

Combination chemotherapy is a crucial method in the treatment of gastric cancer. The aim of the present study was to investigate the inhibitory effects of puerarin and 5­fluorouracil (5­FU) on BGC­823 gastric cancer cells in vitro and in vivo. The in vitro growth inhibition of puerarin or 5­FU alone or combined on BGC­823 cells was determined using a cell counting kit 8 (CCK­8) on living cells. Apoptotic morphological features and proteins expression levels were detected by Hoechst 33258 staining, an Annexin V/propidium iodide apoptosis kit and western blot analysis, respectively. Tumor xenografts were established in nude mice and the inhibitory effects and side effects were detected. Results of the CCK­8, Hoechst 33258 staining and flow cytometry revealed that the combined treatment was more effective than the separate treatments. The tumor volume was 90.65% of that of the controls and the mean tumor weight was only 0.125 g at the end of the experiment in the combination group compared with the control group (0.822 g). In addition, it was determined that liver and renal toxicity did not increase in combined treatment. These findings showed that puerarin and 5­FU produced a significant synergic effect on gastric cancer cells, while there was no increase in side effects.

XPD Asp312Asn polymorphism and esophageal cancer risk: an update meta-analysis based on 3928 cases and 6012 controls.

BACKGROUND: Although xeroderma pigmentosum group D (XPD) was reported to be related with esophageal cancer (EC) risk, the results remained inconsistent. The aim of this meta-analysis was to make a more precise estimation of the relationship between XPD Asp312Asn polymorphism and EC risk. METHODS: We searched PubMed, Web of Science, Embase, Medline, CNKI and Chinese Biomedical database, covering all publications (up to May, 2014). Statistical analyses were performed with Stata software (version 12.0, USA) and RevMan 5.1 (Copenhagen, 2008). The calculation of odds ratios (ORs) with 95% confidence intervals (CI) was calculated to assess the strength of the association. RESULTS: A total of 15 case-control studies from 13 literatures including 3928 cases and 6012 controls described Asp312Asn genotypes and EC risk. A significant association between XPD Asp312Asn polymorphism and EC risk was found when all the eligible studies were pooled into this meta-analysis. It's also the same result in subgroup analysis of smokers in dominant model (OR=1.63, 95% CI: 1.06-2.50, P=0.03). However, in the stratified analysis by ethnicity and source of population controls, no association between them was discovered. CONCLUSION: The XPD Asp312Asn polymorphism was proved to contribute to the risk of EC in this meta-analysis. Data showed that tobacco consumption may increase the susceptibility of EC.

Potential effects of Mina53 on tumor growth in human pancreatic cancer.

Myc-induced nuclear antigen (Mina53) is a protein with a molecular weight of 53 kDa expression of which is induced by c-Myc. Increased expression of Mina53 is documented in some human carcinomas. In this study, we found markedly increased Mina53 expression in pancreatic cancer tissue specimens. This expression did not correlate with clinicopathological characteristics, such as sex, age, and presence of distant metastasis. However, there was a statistically significant association with histological differentiation, TNM stage, and lymph node metastases. To study functional role of Mina53, we silenced its expression by siRNA in PANC-1 cells. These cells were arrested in the G2/M phase, and apoptosis rates were increased. In conclusion, increased expression of Mina53 may play an important role in the development of human pancreatic cancer. Mina53 can be used as a marker for pancreatic cancer and may potentially be exploited as a target for treatment of pancreatic cancer.

Clinical significance of EBP50 overexpression assessed by quantum dot analysis in gastric cancer.

Ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) is a postsynaptic density-95/disc-large/zonula occludens-1 (PDZ) homologous domain-containing protein that is involved in cell signaling. EBP50 regulates cell apoptosis, proliferation and invasion. In the present study, the prognostic impact factor of EBP50 expression was evaluated using a quantum dot (QD)-based assay and immunohistochemistry (IHC). The EBP50 protein expression in gastric cancer (GC) tissues was evaluated using IHC and QD-IHC. The study included 101 patients with GC (29 females and 72 males, aged 24-81 years), diagnosed and treated at the General Surgery Department of Renmin Hospital of Wuhan University (Wuhan, China) between 2000 and 2005. The survival rate was calculated using the Kaplan-Meier method and log-rank tests. IHC and QD analyses of 101 GC tissue specimens revealed that EBP50-positive tumor cells were frequently present in GC. Increased EBP50 immunostaining was observed in 63 specimens (62.4%). The EBP50 expression levels were correlated with increased tumor size and the male gender. EBP50 was well distributed in the cytoplasm and nuclei of the GC cells. However, EBP50 protein expression exhibited no correlation with age, differentiation, stage or lymph node metastasis. There were no associations between the expression of EBP50 and the mean survival rates (IHC, 50.5 vs. 58.1 months, P>0.05; QD, 55.4 vs. 63.2 months, P>0.05). These findings suggest that EBP50 protein expression is not correlated with the prognosis of patients with GC. QD-IHC and IHC have similar advantages for the detection of EBP50 protein expression.

Noscapine induces mitochondria-mediated apoptosis in human colon cancer cells in vivo and in vitro.

Noscapine, a phthalide isoquinoline alkaloid derived from opium, has been widely used as a cough suppressant for decades. Noscapine has recently been shown to potentiate the anti-cancer effects of several therapies by inducing apoptosis in various malignant cells without any detectable toxicity in cells or tissues. However, the mechanism by which noscapine induces apoptosis in colon cancer cells remains unclear. The signaling pathways by which noscapine induces apoptosis were investigated in colon cancer cell lines treated with various noscapine concentrations for 72 h, and a dose-dependent inhibition of cell viability was observed. Noscapine effectively inhibited the proliferation of LoVo cells in vitro (IC(50)=75 µM). This cytotoxicity was reflected by cell cycle arrest at G(2)/M and subsequent apoptosis, as indicated by increased chromatin condensation and fragmentation, the upregulation of Bax and cytochrome c (Cyt-c), the downregulation of survivin and Bcl-2, and the activation of caspase-3 and caspase-9. Moreover, in a xenograft tumor model in mice, noscapine injection clearly inhibited tumor growth via the induction of apoptosis, which was demonstrated using a TUNEL assay. These results suggest that noscapine induces apoptosis in colon cancer cells via mitochondrial pathways. Noscapine may be a safe and effective chemotherapeutic agent for the treatment of human colon cancer.

The detection of EBP50 expression using quantum dot immunohistochemistry in pancreatic cancer tissue and down-regulated EBP50 effect on PC-2 cells.

Ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) is a putative tumor suppressor that is correlated with many human cancers. However, the function of EBP50 in pancreatic cancer (PC) has not been described. In this paper, the EBP50 expression level in PC tissues was characterized. In vitro, the effects of EBP50 down-regulation by siRNA in PC-2 and MiaPaCa-2 cells were evaluated. In addition, possible mechanisms that mediate the influence of EBP50 were examined. Our results show that the EBP50 expression pattern changes during transformation as there is a loss of the normal apical membrane distribution and an ectopic cytoplasmic over-expression of EBP50; furthermore, the EBP50 expression level is subsequently decreased during malignant progression. Down-regulation of EBP50 promoted cancer cell proliferation, increased the colony-forming ability of cells and accelerated the G1-to-S progression. Additionally, the loss of EBP50 accentuated ß-catenin activity, increased cyclin E and phosphorylated Rb expression, and attenuated p27 expression compared to control cells. Our results suggest that EBP50 may function as a potential tumor suppressor.

Overexpression of Dickkopf-3 induces apoptosis through mitochondrial pathway in human colon cancer.

AIM: To investigate the mechanisms of the biological roles of Dickkopf-3 (Dkk-3) in cell invasion, survival and apoptosis in colon cancer cells. METHODS: Three human colon cancer cell lines, i.e., HT-29, LoVo and SW480, were used. Overexpression of Dkk-3 induced by pEGFP-N1-Dkk-3-GFP plasmid in LoVo cells was performed using Lipofectamine 2000 reagent. Reverse transcription polymerase chain reaction and Western blotting were performed to determine the mRNA and protein expression levels of Dkk-3, respectively. Cell proliferation assay, cell cycle analysis, hoechst 33258 assay and Matrigel invasion assay were performed on Dkk-3 overexpressing transfectants. RESULTS: The mRNA and protein expressions of Dkk-3 in HT-29 (mRNA: 0.06 ± 0.02, protein: 0.06 ± 0.01) and LoVo (mRNA: 0.07 ± 0.02, protein: 0.07 ± 0.02) cells were significantly lower than that in SW480 cells (mRNA: 0.92 ± 0.04, protein: 0.69 ± 0.13; all P < 0.05), and the greatest levels of invasiveness was in LoVo cells. Dkk-3 overexpression inhibited the proliferation and invasion of LoVo cells and induced cell cycle arrest at G(0)/G(1) phase and subsequent apoptosis, as indicated by increased chromatin condensation and fragments, upregulated Bax and cytochrome c protein, downregulated survivin and Bcl-2 protein, and the activation of caspase-3 and caspase-9. Furthermore, Dkk-3 overexpression reduced the accumulation of cytosolic fraction of ß-catenin. CONCLUSION: Dkk-3 overexpression induced apoptosis in human colon cancer possibly through the mitochondrial pathway. Dkk-3 may be involved in the Wnt/ß-catenin signaling pathways in colon cancer.

EBP50 gene transfection promotes 5-fluorouracil-induced apoptosis in gastric cancer cells through Bax- and Bcl-2-triggered mitochondrial pathways.

5-Fluorouracil (5-FU) plays an important role in the chemotherapy of advanced gastric cancer. However, genetic factors that affect therapeutic efficacy of 5-FU warrant further investigation. In the present study, using stable transfection of the ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) gene, we explored the genetic influences on 5-FU-induced apoptosis of human gastric cancer cells. Stable overexpression of the EBP50 gene was determined by reverse transcription polymerase chain reaction (RT-PCR) assay and western blot analysis. After treatment with 5-FU, cell growth activities in vitro were investigated by MTT assay. Cell apoptosis was evaluated by Hoechst 33258 staining and flow cytometry of Annexin V-FITC/PI staining. Compared with the BGC823 or BGC823/neo cells, EBP50 mRNA and protein levels in the BGC823/EBP50 cells (EBP50-transfected BGC823 cells) were markedly higher. Chemosensitivity and apoptosis rates of the BGC823/EBP50 cells were higher compared to the BGC823 and BGC823/neo cells following treatment with 5-FU. Stable overexpression of extrinsic EBP50 distinctly increases the 5-FU-induced apoptosis of gastric cancer cells, and is a novel strategy by which to improve the chemosensitivity of gastric cancer to 5-FU.

Noscapine induces mitochondria-mediated apoptosis in gastric cancer cells in vitro and in vivo.

PURPOSE: Noscapine plays an important role in the regulation of cell growth and death. It has been reported to potentiate the anti-tumor effect by inducing apoptosis in various malignant cells. However, the mechanism of inducing apoptosis in gastric cancer cells by this agent remains to be clarified. METHODS: In the study, we investigated the signaling pathways by which noscapine induces apoptosis in gastric cancer cell lines. Apoptosis of four human gastric cancer cell lines was induced by treatment with noscapine. RESULTS: Our results indicate that noscapine induced a dose-dependent apoptosis of these cells. The treatment with noscapine upregulated Bax and Cytochrome c (Cyt-c) protein, downregulated Bcl-2 protein. Caspase-3 and caspase-9 were activated, suggesting that the apoptosis is mediated by mitochondrial pathways. Moreover, in xenograft tumor mouse model, noscapine injection successfully inhibited the tumor growth via apoptosis induction which was demonstrated by TUNEL assay. CONCLUSIONS: These data of the study suggest that noscapine induces apoptosis in gastric cancer cells via mitochondrial pathways.

[Expression of Dickkopf-3 in esophageal squamous cell carcinoma].

OBJECTIVE: To investigate the expression of Dickkopf-3 (Dkk-3) in esophageal cancer and normal esophageal tissue and the relationship between Dkk-3 expression and the biological behavior of esophageal cancer. METHODS: Immunohistochemical method of S-P was used to examine Dkk-3 expression in 69 cases of esophageal squamous cell carcinoma and 5 cases of normal esophageal tissue with non-tumor tissue microarray and the results were analyzed and correlated with their clinical and pathological features. RESULTS: Positive Dkk-3 expression was observed in 65.7% (44/67) of the esophageal squamous cell carcinoma cases, but only one of the five cases with normal esophageal tissue showed positive microvascular expression of Dkk-3. In cases with positive expression of Dkk-3 significant differences were found in fiber membrane infiltration, depth of invasion, lymph node metastasis and TNM staging (P < 0.05), while no significant differences were found in the age, gender and pathological grading (P > 0.05). CONCLUSIONS: The upregulation of Dkk-3 expression in esophageal squamous cell carcinoma may contribute to tumor invasion and metastasis.