Negative regulation between the expression levels of receptor for hyaluronic acid-mediated motility and hyaluronan leads to cell migration in pancreatic cancer.

Receptor for hyaluronic acid (HA)-mediated motility (RHAMM) expression is upregulated in pancreatic ductal adenocarcinoma (PDAC). In the present study, small interfering RNA knockdown was used to investigate the regulatory mechanism and function of RHAMM in PDAC cells. Reverse transcription-quantitative PCR was used to measure the mRNA expression levels of RHAMM, hyaluronan synthases (HAS1, HAS2 and HAS3) and hyaluronidases (HYAL1, HYAL2 and HYAL3) in eight PDAC cell lines. Cell migration was assessed using a Transwell assay, while HA concentration was measured using an ELISA. The results revealed that RHAMM-knockdown significantly increased migration in two PDAC cell lines, significantly decreased migration in one cell line and did not affect migration in the other cell lines, and was positively associated with changes in HA production. There was a linear negative correlation between RHAMM mRNA expression and HA concentration in PDAC cells and tissues. The negative correlation between RHAMM mRNA expression and HA concentration was demonstrated in other models, including SUIT2 cells treated with an HA inhibitor or stimulator and a system involving co-culture of SUIT2 cells and stromal fibroblasts. The present findings demonstrated a negative correlation between RHAMM mRNA expression and HA production in a subset of PDAC cell lines. The efficacy of a therapeutic strategy targeting RHAMM should be carefully evaluated in future studies.

4-Methylumbelliferone inhibits enhanced hyaluronan synthesis and cell migration in pancreatic cancer cells in response to tumor-stromal interactions.

Hyaluronic acid (HA) in tumor stroma promotes tumor invasion and progression. 4-Methylumbelliferone (4-MU) is a potent HA synthesis inhibitor. In the present study, the effects of 4-MU on enhanced HA synthesis and cell migration in pancreatic ductal adenocarcinoma (PDAC) cells, in response to co-culture with stromal fibroblasts, was investigated. The HA concentration was determined using ELISA and a Transwell migration assay was used to analyze cell migratory capability. The mRNA expression levels of hyaluronan synthases (HAS1, HAS2 and HAS3) were determined using the quantitative polymerase chain reaction. Co-culture between Panc-1 cells and stromal fibroblasts markedly increased cell migration in association with increasing HA production, which was markedly associated with an increase in HAS3 mRNA expression. Treatment with 4-MU markedly decreased the HA production and cell migration of Panc-1 cells in the co-culture system. The results of the present study suggested that interactions between PDAC cells and stromal fibroblasts increased HA production, resulting in a marked increase in migration of PDAC cells, and 4-MU may be used as a chemotherapeutic agent to inhibit the enhanced migration of PDAC cells in response to tumor-stromal interactions.

KIAA1199/CEMIP/HYBID overexpression predicts poor prognosis in pancreatic ductal adenocarcinoma.

BACKGROUND: KIAA1199 (also known as CEMIP or HYBID), a newly identified protein involved in hyaluronan degradation, has been suggested to play a critical role in cancer progression. The aim of this study was to investigate the expression and functional significance of KIAA1199 in pancreatic ductal adenocarcinoma (PDAC). METHODS: Using quantitative real-time RT-PCR, we analyzed KIAA1199 mRNA expression in 6 PDAC cell lines and frozen tissues from 14 patients with PDAC. We also used immunohistochemistry to analyze KIAA1199 protein expression in formalin-fixed, paraffin-embedded tissues from 98 patients with PDAC. The KIAA1199 expression pattern was then correlated with clinicopathological variables and patient outcome. The effect of KIAA1199 on migratory ability of PDAC cells was determined by KIAA1199 knockdown with small-interfering RNA (siRNA). RESULTS: The KIAA1199 mRNA expression was significantly higher in PDAC tissues than in the corresponding non-tumor tissues (P < 0.0001). Immunohistochemical analysis revealed high expression of KIAA1199 in 26 (26.5%) of 98 PDAC tissues. The overall survival was significantly shorter in patients with high KIAA1199 expression than in patients with low KIAA1199 expression (P = 0.0001). In multivariate analysis, high KIAA1199 expression (P = 0.003) and UICC stage (P = 0.003) were independent factors predicting poor prognosis. Furthermore, the KIAA1199 mRNA expression was higher in most PDAC cell lines and siRNA knockdown of KIAA1199 resulted in decreased migration. CONCLUSION: These findings suggest that overexpression of KIAA1199 may contribute to increased migration of PDAC cells and predict shorter survival after surgical resection.

Increased Expression of HYAL1 in Pancreatic Ductal Adenocarcinoma.

OBJECTIVES: Increased production and processing (degradation) of hyaluronan (HA) is critical for cancer invasion and metastasis. Although HA is known to be overexpressed in pancreatic ductal adenocarcinoma (PDAC), little is known about the expression and biological significance of HA-degrading enzymes, hyaluronidases (HYALs), in PDAC. METHODS: Expression of HYALs mRNA was examined in PDAC cells by quantitative real-time RT-PCR. HYAL1 protein expression was examined in primary PDAC tumors by enzyme-linked immuno-sorbent assay. The migratory ability of PDAC cells was determined by a transwell cell migration assay. RESULTS: Screening of mRNA expression of three major HYAL genes (HYAL1, 2, and 3) identified HYAL1 as a gene overexpressed in PDAC cells. Treatment of PDAC cells with 5-aza-2'-deoxycytidine and/or trichostatin A further increased the HYAL1 expression, suggesting a possible involvement of epigenetic mechanisms in the transcriptional regulation of this gene. HYAL1 protein concentrations were significantly higher in primary PDAC tissues as compared with nontumor pancreatic tissues (P = 0.049). Importantly, inhibition of HYAL activity by dextran sulfate significantly inhibited the migration of PDAC cells showing strong HYAL1 expression (P = 0.002). CONCLUSIONS: These findings suggest that overexpression of HYAL1 is a common mechanism that may contribute to the aggressive phenotype of PDAC.

Targeting hyaluronan for the treatment of pancreatic ductal adenocarcinoma.

Progression of cancer is often associated with interactions between cancer cells and extracellular matrix (ECM) surrounding them. Increasing evidence has suggested that accumulation of hyaluronan (HA), a major component of ECM, provides a favorable microenvironment for cancer progression. Pancreatic ductal adenocarcinoma (PDAC) is characterized typically by a dense desmoplastic stroma with a large amount of HA, making this molecule as an attractive target for therapy. Several studies have shown efficacy of inhibitors of HA synthesis or signaling for the treatment of PDAC. Recent studies have also demonstrated substantial improvements in the effects of chemotherapy by a targeted depletion of stromal HA in PDAC using an enzymatic agent. Thus, targeting HA has been recognized as a promising therapeutic strategy to treat this highly aggressive neoplasm. In this review article, we summarize our current understanding of the role of HA in the progression of PDAC and discuss possible therapeutic approaches targeting HA.

A novel epigenetic mechanism regulating hyaluronan production in pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an abundant stroma enriched with hyaluronan (HA), a major component of extracellular matrix known to play a critical role in tumor progression. The mechanisms that regulate HA synthesis in PDAC are poorly understood. To investigate whether DNA methylation and HA production from PDAC cells are associated, we studied the effect of 5-aza-2'-deoxycitidine (5-aza-dC), an inhibitor of DNA methylation, or DNA methyltransferase 1 (DNMT1) knockdown by small interfering RNA, on the HA production from PDAC cells. HA production into the conditioned medium was evaluated in PDAC cells treated with 5-aza-dC or DNMT1 knockdown. mRNA expression of HA synthase (HAS) genes was investigated by real-time RT-PCR. Treatment of PDAC cells with 5-aza-dC led to a significant increase in the HA production (up to 2.5-fold increase) in all 4 cell lines tested. This enhanced HA production by 5-aza-dC treatment was accompanied by increased mRNA expression of HAS2 and HAS3. Furthermore, increased HA production and HAS2/HAS3 mRNA expression was also observed in PDAC cells by knockdown of DNMT1. These findings provide evidence, for the first time, that epigenetic mechanism is involved in the regulation of HA synthesis in PDAC cells.

Hyaluronan stimulates pancreatic cancer cell motility.

Hyaluronan (HA) accumulates in pancreatic ductal adenocarcinoma (PDAC), but functional significance of HA in the aggressive phenotype remains unknown. We used different models to investigate the effect of HA on PDAC cell motility by wound healing and transwell migration assay. Changes in cell motility were examined in 8 PDAC cell lines in response to inhibition of HA production by treatment with 4-methylumbelliferone (4-MU) and to promotion by treatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or by co-culture with tumor-derived stromal fibroblasts. We also investigated changes in cell motility by adding exogenous HA. Additionally, mRNA expressions of hyaluronan synthases and hyaluronidases were examined using real time RT-PCR. Inhibition of HA by 4-MU significantly decreased the migration, whereas promotion of HA by TPA or co-culture with tumor-derived fibroblasts significantly increased the migration of PDAC cells. The changes in HA production by these treatments tended to be associated with changes in HAS3 mRNA expression. Furthermore, addition of exogenous HA, especially low-molecular-weight HA, significantly increased the migration of PDAC cells. These findings suggest that HA stimulates PDAC cell migration and thus represents an ideal therapeutic target to prevent invasion and metastasis.

Receptor for Hyaluronic Acid-Mediated Motility is Associated with Poor Survival in Pancreatic Ductal Adenocarcinoma.

Receptor for hyaluronic acid (HA)-mediated motility (RHAMM) is a nonintegral cell surface receptor involved in the aggressive phenotype in a wide spectrum of human malignancies, but the significance of RHAMM in pancreatic ductal adenocarcinoma (PDAC) remains unknown. In this study, we investigated the expression of RHAMM and its clinical relevance in PDAC. RHAMM mRNA expression was examined in 8 PDAC cell lines and in primary pancreatic cancer and adjacent non-tumor tissues from 14 patients using real-time RT-PCR. Western blotting was carried out to analyze the expression of RHAMM protein in PDAC cell lines. We also investigated the expression patterns of RHAMM protein in tissue samples from 70 PDAC patients using immunohistochemistry. The RHAMM mRNA expression was increased in some PDAC cell lines as compared to a non-tumorous pancreatic epithelial cell line HPDE. The RHAMM mRNA expression was significantly higher in PDAC tissues as compared to corresponding non-tumorous pancreatic tissues (P < 0.0001). The RHAMM protein expression was higher in the vast majority of PDAC cell lines relative to the expression in HPDE. The immunohistochemical analysis revealed strong expression of RHAMM in 52 (74%) PDAC tissues. Strong expression of RHAMM was significantly associated with a shorter survival time (P = 0.038). In multivariate analysis, tumor stage (P = 0.039), residual tumor (P = 0.015), and strong RHAMM expression (P = 0.034) were independent factors predicting poor survival. Strong expression of RHAMM may predict poor survival in PDAC patients and may provide prognostic and, possibly, therapeutic value.

Prognostic impact of hyaluronan and its regulators in pancreatic ductal adenocarcinoma.

BACKGROUND: Although pancreatic ductal adenocarcinoma is characterized by an abundant stroma enriched with hyaluronan (HA), the prognostic impact of HA and its regulators remains unknown. METHODS: Using immunohistochemistry, expression patterns of HA and its regulators, including a synthesizing enzyme (HAS2), and a degrading enzyme (HYAL1) were investigated in patients who received surgical resection. The prognostic significance of these markers and other clinicopathological variables was determined using univariate and multivariate analyses. The HA levels were determined quantitatively by enzyme-linked immunosorbent assay (ELISA). RESULTS: We found that strong expressions of HA (P=0.008) and HAS2 (P=0.022) were significantly associated with shorter survival time after surgery. By contrast, weak expression of HYAL1 was significantly associated with poor survival (P=0.001). In multivariate analysis, tumor stage (hazard ratio (HR)=2.76, 95% confidence interval (CI): 1.14-6.66 P=0.024), strong HA expression (HR=6.04, 95%CI: 1.42-25.69 P=0.015), and weak HYAL1 expression (HR=3.16, 95%CI: 1.19-8.40 P=0.021) were independent factors predicting poor survival. ELISA revealed higher concentration of HA in pancreatic cancer tissues than in normal pancreatic tissues (P=0.001). CONCLUSION: These findings suggest, for the first time, that HA and its regulators may have prognostic impact in patients with pancreatic cancer.

[Effect of health education intervention in schools of Yanrui Town, Yushan County].

OBJECTIVE: To evaluate the effect of a new mode of health education in schools. METHODS: In the Zhaiqian Primary School, Yanrui Town, Yushan County in a hilly schistosomiasis endemic area, a new mode of health education intervention, i. e. "rewards and punishment + advise others by using one's experience + teachers' participation" was carried out, and the knowledge, attitude and practice of schistosomiasis prevention of the pupils, and the schistosome infection rates of the pupils were investigated and the results were analyzed and compared before and after the intervention. RESULTS: Among 204 pupils investigated, the awareness rate of schistosomiasis prevention knowledge increased from 26.47% before the intervention to 86.76% and 99.51% one and two years after the intervention, respectively; the rate of correct attitude increased from 17.04% to 73.04% and 100%, respectively; the rate of the infested water contact decreased from 83.33% to 26.96% and 0, respectively; the schistosome infection rate decreased from 2.94% to 0.49% and 0, respectively. CONCLUSION: The health education of schistosomiasis prevention in schools is necessary, and the new mode of health education, "rewards and punishment + advise others by using one's experience + teachers' participation" is effective in the prevention from schistosome infection in pupils.

New sludge pretreatment method to improve dewaterability of waste activated sludge.

The enhancements of electrolysis-pretreated conditioning were investigated in this study. Normalized capillary suction time (CST) was used to evaluate sludge dewaterability. Extracellular polymeric substance (EPS) concentration, viscosity and scanning electron microscopy (SEM) were determined to explain the observed changes in conditioning process. It indicated that pretreatment at 50 v and 5 min with Ti/RuO(2) anode was determined to be the optimal condition, which generated the lowest normalized CST and optimal soluble EPS concentration, leading to the decreasing of viscosity. EPS had positive correlation with the normalized CST. Subjecting to a combination of electrolysis pretreatment and flocculants conditioning, 50% dosage of cationic polyacrylamide (PAM) could be reduced. When co-conditioned with electrolysis and polymerization ferric sulfate (PFS), it did not present any clear advantages over PFS conditioning alone. Furthermore, SEM investigation indicated that electrolysis pretreatment could rupture sludge, release the interstitial water and extracellular substances, especially protein and polysaccharide, and consequently enhance its dewaterability.

[Molluscicidal effect and toxicity of META-Li on Oncomelania snails in the mountain area].

The molluscicidal effect and toxicity of META-Li were evaluated and compared with niclosamide in Yushan county of Jiangxi Province in August 2008. There are four groups named as META-Li ridge group (1 g/m2), META-Li field group (1 g/m2), niclosamide group (2 g/m2) and control group. At 3 d, 7 d, 15 d and 2 months after drugs sprayed, the corrected mortalities of snails in the 3 groups were considerably higher than those of control (P<0.05). The corrected mortalities in META-Li field group were significantly lower than those of META-Li ridge group and niclosamide group (P<0.05). No adverse effect was observed to non-target organisms in META-Li groups, while fishes, snails and frogs all died at the first day after treated with niclosamide.

[Reconstruction of vertebral lamina with skull titanium plate and autograft in the treatment of thoracolumbar vertebral fracture].

OBJECTIVE: To discuss the surgical skill, treatment effect and indications for reconstruction of vertebral lamina with skull titanium plate and autograft in the treatment of thoracolumbar vertebrae fracture. METHODS: From March 1999 to April 2007, 33 patients with thoracolumbar vertebrae fracture combined nerve injury were treated by reconstruction of vertebral lamina with skull titanium plate and autograft including 30 males and 3 females with an average age of 41 years ranging from 21 to 66. The fracture involved 3 cases in T11, 9 in T12, 16 in L1, 3 in L2 and 2 in L3. There were 12 cases with flexion fracture, 8 with extension fracture, and 13 with burst fracture. Five cases performed emergency operation and in the others the time from injury to operation was 5 to 12 days. GSS fixation was applied in 25 cases and AF in 8. The volume of vertebral canal, the stability of the spine, the height of vertebrae, and the nerve function were observed before and after operation. RESULTS: These 33 patients were followed up for 1 to 3 years (means 25 months). The height of anterior column of the involved vertebrae changed from 58% to 96%, the cobb angle return from average of 26 degree to 2 degree, the volume of vertebral canal extended from 43% to 92%, respectively, when compared between preoperatively and postoperatively. CONCLUSION: Reconstruction of vertebral lamina with skull titanium plate and autograft which is a simple and safe treatment for thoracolumbar vertebrae fracture can increase the stability of spine and avoid second canal stenosis.