Wenkai Ni

The expressions and clinical significances of tissue and serum galectin-3 in pancreatic carcinoma.

PurposeGalectin-3, a member of the beta-galactoside-binding protein family, is involved in many biological processes, including cell proliferation, regulating cell cycle, angiogenesis, tumorigenesis, metastasis, etc. The aim of this study is to elucidate the relationship between galectin-3 and clinicopathological variables and to evaluate the clinical significance of serum galectin-3 in the diagnosis of pancreas carcinoma.MethodsGalectin-3 expression in 78 pairs of pancreatic carcinoma tissues and the adjacent nontumorous tissues was tested by immunohistochemistry. The relationship between galectin-3 expression and clinical variables was analyzed. A sensitive method of time-resolved fluorescence immunological assay (TRFIA) for the detection of galectin-3 was established, and serum galectin-3 in cases with different pancreatic diseases was measured by TRFIA and ELISA. Further we compared the sensitivity and specificity of determining galectin-3, carcinoembryonic antigen (CEA) and carbohydrate antigen199 (CA199) for diagnosis of pancreatic carcinoma and assessed the complementary diagnostic value of galectin-3, CEA and CA199 for pancreatic carcinoma.ResultsImmunohistochemistry showed that galectin-3 expression was significantly higher in the human pancreatic carcinoma tissues than in the adjacent nontumorous tissues. The expression levels were correlated with the differentiation degree with the higher expression in poor differentiation tissues. Serum galectin-3 detected by both TRFIA and ELISA was much higher in patients with pancreatic carcinoma than in other groups. Serum galectin-3 was not correlated with CEA and CA199. Combined determination of these three markers has the complementary diagnostic value for human pancreatic carcinoma and may increase the diagnostic sensitivity to 97.5%.ConclusionsGalectin-3 is overexpressed in pancreatic carcinoma tissues, and it is correlated with the tumor differentiation. Serum galectin-3 is higher in cases with pancreatic carcinoma than in benign pancreatic diseases and healthy persons. Combined determination of serum galectin-3, CEA and CA199 may improve the diagnostic power for pancreatic carcinoma.

CtBP2 contributes to malignant development of human esophageal squamous cell carcinoma by regulation of p16INK4A

C‐terminal binding protein‐2 (CtBP2), as a transcriptional co‐repressor, has been shown to mediate the repression of p16INK4A, a tumor suppressor gene product, in primary human cells. Here we aimed to investigate how the correlation between CtBP2 and p16INK4A influenced the development of esophageal squamous cell carcinoma (ESCC). Immunohistochemistry of ESCC tissue sections indicated that the CtBP2 and p16INK4A expressions were inversely correlated to each other with a linear regression coefficient of −0.747 (P < 0.05), and Western blot analysis revealed that CtBP2 was higher expressed in tumorous tissues than in adjacent non‐tumorous tissues. Either CtBP2 or p16INK4A expression was significantly related to histological differentiation (P = 0.016 or 0.001) and to the expression of Ki‐67, a proliferating marker (P = 0.006 or 0.02), and patients with higher CtBP2 and lower p16INK4A expressions had shorter overall survival. We also observed that CtBP2 modulated the cell proliferation and cell cycle in ECA109 cells, an ESCC cell line, by inhibiting p16INK4A. Overexpression or knockdown of CtBP2 in ECA109 cells was found to inhibit or activate the mRNA or protein expression of p16INK4A, which in turn altered the cell proliferation and cell cycle in ECA109 cells, as measured by flow cytometry and cell count assay. Additionally, after ECA109 cells silenced for CtBP2 were treated with cisplatin (an anti‐ESCC agent), the p16INK4A expression was up‐regulated, and the cell apoptosis was promoted, thus confirming the repression of p16INK4A by CtBP2. Collectively, all results suggested that CtBP2 might contribute to the progression of ESCC through a negative transcriptional regulation of p16INK4A. J. Cell. Biochem. 114: 1343–1354, 2013.

Overexpressed nuclear BAG‐1 in human hepatocellular carcinoma is associated with poor prognosis and resistance to doxorubicin

Bcl‐2‐associated athanogene‐1 (BAG‐1) is a multifunctional anti‐apoptotic protein which regulates an array of cellular processes, including apoptosis, signaling, proliferation, transcription, and cell motility and has been reported to be over‐expressed in a number of human malignancies. To investigate the possible involvement of BAG‐1 in tumorigenesis of hepatocellular carcinoma (HCC), we performed Western blot analysis in eight paired samples of HCC and adjacent peritumoral tissues and immunohistochemistry in 65 paraffin sections of HCC, which both showed an enhanced expression of nuclear BAG‐1 isoform in HCC tissues. Statistical analysis confirmed that overexpression of nuclear BAG‐1 in HCC tissues was significantly associated with histological grading (P < 0.001), poor prognosis (P = 0.004), and was found to be an independent prognostic indicator for HCC (P = 0.023). We also noted that BAG‐1 was overexpressed in four HCC cell lines compared with a normal hepatocyte cell line, and BAG‐1 overexpression increased resistance of HCC cells to doxorubicin, a common chemotherapeutic agent for HCC. Furthermore, we observed that knock down of BAG‐1 with siRNA in HepG2 cells increased the chemosensitivity of cells, a process mediated through inhibition of doxorubicin‐triggered NF‐κB activation; and knock down of BAG‐1 suppressed proliferation and cell cycle transition of HepG2 cells. In consequence, our results for the first time indicated that BAG‐1 was dysregulated in HCC and suppression of BAG‐1 expression which resulted in inhibiting of NF‐κB signaling might be developed into a new strategy in HCC therapy. J. Cell. Biochem. 114: 2120–2130, 2013.

Combined analysis of serum γ-glutamyl transferase isoenzyme II, α-L-fucosidase and α-fetoprotein detected using a commercial kit in the diagnosis of hepatocellular carcinoma.

γ-glutamyl transferase isoenzyme II (GGT-II) is a sensitive biomarker of hepatocellular carcinoma (HCC). However, numerous disadvantages of the traditional manual method affected its application. The commercial kit provided a convenient and fast method for the determination of GGT-II levels. The purposes of the present study were to compare the reproducibility and sensitivity between the manual and commercial kit methods and to evaluate the diagnostic efficiency for HCC with the combined analysis of GGT-II, α-L-fucosidase (AFU) and α-fetoprotein (AFP). In patients with various liver diseases (HCC, liver cirrhosis and chronic hepatitis) and normal subjects, GGT-II was detected by manual and commercial polyacrylamide gel electrophoresis (PAGE). The levels of AFU and AFP were assayed by colorimetry and a chemiluminescence immunoassay, respectively. The commercial PAGE had equal diagnostic efficiency with traditional manual PAGE and no significant differences were observed in intra- and average-gel reproducibility and GGT-II sensitivities between the manual and commercial PAGE (P>0.05). The incidence of GGT-II detected by commercial PAGE in HCC patients was 84.1% and <8% in benign liver disease. The levels of AFU and AFP in the benign liver diseases and normal subjects were lower than those in HCC. According to the cut-off value obtained by receiver operating characteristic curves, a total of 56.6 and 59.3% of HCC patients (64 out of 113 and 67 out of 113) had AFU >636.5 μmol/l h and AFP >44.0 μg/l, respectively. There were no significant correlations between GGT-II and AFU or AFP. Combined detection of GGT-II with AFU or AFP increased the diagnostic sensitivity to 92.9 and 93.8%, respectively. These results suggest that commercial PAGE provides a simple and reproducible method for GGT-II detection. Combined determination of GGT-II with AFU or AFP exhibited superior sensitivity and specificity for the diagnosis of HCC.

Decreased Expression and Prognostic Role of Mitogen-Activated Protein Kinase Phosphatase 4 in Hepatocellular Carcinoma

PurposeThis study aimed to investigate the potential role and prognostic significance of mitogen-activated protein kinase phosphatase 4 (MKP-4) in the pathology of hepatocellular carcinoma (HCC).MethodsWestern blot analysis and quantitative real-time polymerase chain reaction were performed to detect MKP-4 expression in HCC tissues, pericarcinomatous liver (PCL) tissues, and proliferating HCC cells. The detailed role of MKP-4 was further explored by MKP-4 downregulation in HepG2 cells using small interfering RNA (siRNA). Specimens of 134 HCC patients who had undergone hepatic resection were immunohistochemically evaluated for MKP-4 expression.ResultsMKP-4 protein and mRNA levels were significantly lower in HCC tissues than in PCL tissues. In vitro, its expression was gradually reduced following release of HepG2 cells from serum starvation. The cell counting kit-8 assay and Annexin-V-Fluos staining indicated that MKP-4 knockdown by siRNA in HCC cells enhanced cell survival and inhibited apoptosis. Univariate and multivariate analyses revealed that MKP-4 was a significant predictor for overall survival (OS) and time to recurrence (TTR). High MKP-4 expression was well correlated with prognosis independent of Edmondson grade and microvascular invasion (P < 0.001).ConclusionsMKP-4 expression was downregulated in HCC tissues and proliferating HCC cells and correlated with OS and TTR, which suggested that MKP-4 is a candidate prognostic marker for HCC.

Downregulated DYRK2 expression is associated with poor prognosis and Oxaliplatin resistance in hepatocellular carcinoma.

We aimed to investigate the molecular mechanisms of DYRK2 and the HCC sensitivity to Oxaliplatin in DYRK2-depleted HCC cells. HCC tissue specimens were obtained from 86 HCC patients during hepatectomy. We used immunohistochemistry and western blot to analyze DYRK2 expression in HCC tissues and cell lines, and used siRNA transfection to decrease DYRK2 expression in HCC cells. Flow cytometry and CCK-8 assay were detected in cell cycle progression, cell proliferation and the efficacy of Oxaliplatin, DYRK2 was down-regulated in HCC tissues, compared with adjacent nontumor ones. The significant correlation between DYRK2 expression and clinicopathologic factors was apparently shown in the immunohistochemical and statistical analyses. The expression of DYRK2 was significantly associated with histological grade of HCC patients. Univariate and multivariate survival analyses revealed that DYRK2 was a significant predictor for overall survival of HCC patients. The depletion of DYRK2 promoted HCC cell proliferation, and increased resistance to Oxaliplatin. These data showed that the downregulated expression of DYRK2 in HCC tumor tissues could promote the proliferation of HCC cells. In addition, reducing DYRK2 expression was associated with poor prognosis and Oxaliplatin resistance in HCC.

Upregulated HOXC8 Expression Is Associated with Poor Prognosis and Oxaliplatin Resistance in Hepatocellular Carcinoma.

BackgroundHepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide. It is indispensable to understanding molecular mechanisms of HCC progression and to developing clinically useful biomarkers for this disease.AimIn this article, we examined whether HOXC8 was associated with the poor prognosis of hepatocellular carcinoma and explored the possible underlying mechanism.MethodsThe HOXC8 and Ki67 expression levels in 86 patients with hepatocellular carcinoma were examined using immunohistochemistry. HOXC8 levels in HCC cells were downregulated by siRNA transfection. The cycle progression and cell proliferation status of HCC cells and the oxaliplatin effectiveness were evaluated by flow cytometry and CCK-8 assay. HOXC8, CyclinD1, PCNA, Nkd2, and cleaved caspase-3 levels were detected by western blot.ResultsHOXC8 was upregulated in HCC tissues, compared with adjacent non-tumor ones. HOXC8 expression levels in 86 patients with hepatocellular carcinoma were positively correlated with histological grade. Univariate and multivariate survival analysis revealed that HOXC8 was a significant predictor for overall survival of HCC patients. HOXC8 siRNA knockdown delayed the G1–S phase transition, inhibited cell proliferation, and attenuated resistance to oxaliplatin.ConclusionsHOXC8 promoted HCC proliferation and predicted poor prognosis. Furthermore, upregulated HOXC8 expression was associated with oxaliplatin resistance in hepatocellular carcinoma.

Expression and Clinical Role of Cdc5L as a Novel Cell Cycle Protein in Hepatocellular Carcinoma.

BackgroundCell division cycle 5-like (Cdc5L), as a pre-mRNA splicing factor, is a regulator of mitotic progression. Previous study found that deletion of endogenous Cdc5L decreases the cell viability via dramatic mitotic arrest, while the role of Cdc5L in cancer biology remains under debate.AimsTo investigate the involvement of Cdc5L in the progression of hepatocellular carcinoma (HCC).MethodsIn this study, the expression of Cdc5L was evaluated by Western blot in 8 paired fresh HCC tissues and immunohistochemistry on 116 paraffin-embedded slices. We treated HCC cells by nocodazole to analyze the role of Cdc5L in mitotic progress. To determine whether Cdc5L could regulate the proliferation of HCC cells, we increased endogenous Cdc5L and analyzed the proliferation of HCC cells using Western blot, CCK8, flow cytometry assays, and colony formation analyses. Furthermore, Cdc5L-siRNA oligos were used to confirm that Cdc5L plays an essential role in HCC development.ResultsCdc5L was highly expressed in HCC and significantly associated with multiple clinicopathological factors, including AJCC stage, tumor size, and Ki-67. Besides, univariate and multivariate survival analyses demonstrated that high Cdc5L expression was an independent prognostic factor for HCC patients’ poor survival. Overexpression of Cdc5L favors cell cycle progress of HCC cells, while downregulation of Cdc5L results in cell cycle arrest at G2/M phase and reduced cell proliferation of HCC cells.ConclusionsOur findings suggested that Cdc5L could play an important role in the tumorigenesis of HCC and thus be a potential therapeutical target to prevent HCC progression.

S100A11 promotes human pancreatic cancer PANC‑1 cell proliferation and is involved in the PI3K/AKT signaling pathway

S100A11, a member of S100 calcium-binding protein family, is associated with the numerous processes of tumorigenesis and metastasis. In the present study, the role of S100A11, and its possible underlying mechanisms in cell proliferation, apoptosis and cell cycle distribution in human pancreatic cancer were explored. Immunohistochemical analyses of S100A11 and phosphorylated (p)-AKT serine/threonine kinase (AKT) were performed in 30 resected specimens from patients with pancreatic cancer. PANC-1 cells were transfected with pcDNA3.1-S100A11 or treated with 50 µmol/l LY294002 for 48 h. Cell proliferation was determined using a cell counting kit-8 assay, whereas apoptosis and cell cycle distribution were determined by flow cytometry analysis. The mRNA and protein levels of S100A11, and AKT were determined using semi quantitative reverse transcription-polymerase chain reaction and western blot analyses, respectively. Pearson correlation analysis revealed that the expression levels of S100A11 and p-AKT were positively correlated (r, 0.802; P<0.05). Compared with the control group, S100A11 overexpression significantly promoted PANC-1 cell proliferation and reduced the percentage of early apoptotic cells. Flow cytometric analysis indicated that the proportion of PANC-1 cells in the S phase was significantly elevated and cell percentage in the G0/G1 phase declined in response to S100A11 overexpression (all P<0.05). S100A11 overexpression also significantly increased AKT mRNA and p-AKT protein expression levels (both P<0.05). The phosphoinositide 3-kinase (PI3K) inhibitor, LY294002, significantly inhibited PANC-1 cell proliferation, promoted apoptosis and caused G1/S phase arrest in PANC-1 cells (all P<0.05). These findings together suggest that S100A11 promotes the viability and proliferation of human pancreatic cancer PANC-1 cells through the upregulation of the PI3K/AKT signaling pathway. Thus, S100A11 may be considered as a novel drug target for targeted therapy of pancreatic cancer.

Upregulated TRIM32 correlates with enhanced cell proliferation and poor prognosis in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a major type of primary liver cancer and the sixth most prevalent human malignancies worldwide. However, the molecular mechanisms underlying hepatocarcinogenesis remain unclear. For HCC patients, there is not only a lack of effective therapeutic targets but also a lack of predictive or prognostic biomarkers. In this article, we reported that TRIM32 was obviously upregulated in HCC tumor tissues and HCC cell lines. Its expression patterns were positively correlated with histological grade, tumor sizes, and HBsAg of HCC patients. TRIM32 expression was a significant predictor for the overall survival time of HCC patients. Moreover, the overexpression of TRIM32 in cells accelerated the G1-S phase transition, promoted cell proliferation rates, and induced the resistance of HCC patients to oxaliplatin. All these findings suggest that TRIM32 might play important roles in the hepatocarcinogenesis. TRIM32 could be a novel direction to explore the mechanism underlying HCC pathogenesis.