Biobanking of fresh-frozen human colon tissues: impact of tissue ex-vivo ischemia times and storage periods on RNA quality.

BACKGROUND: Biobanking plays an important role in translational cancer research. The impact of tissue ex-vivo ischemia time and storage period on RNA integrity is not well documented. METHODS: Fresh-frozen colon tissues were collected in Taizhou Hospital of Zhejiang Province in China since 2004. Fifty-one colon cancer tissues with tumor cell content higher than 70 % and matched normal tissues during four storage periods (less than 15 months, 16-20 months, 21-25 months, and 26-40 months) were chosen to detect RNA quality. Fresh colon cancer tissues from 5 patients were cut into pieces and kept at room temperature or on ice for 0.5, 1, 2, and 4 h before snap freezing. RNA integrity was determined by microcapillary electrophoresis by the RNA integrity number (RIN) algorithm. RESULTS: Sixty-seven percent of normal colon tissues and 94 % of colon cancer specimens yielded RNA with a RIN of ≥7. Matched colon cancer and normal tissues showed significant difference in RNA quality. RNA remained stable in colon cancer tissues kept at room temperature and on ice for up to 4 h, and long-term storage of banked colon specimens did not negatively influence RNA quality (RNA with RIN of ≥7 banked less than 15 months, 83 %; 16-20 months, 78 %; 21-25 months, 77 %; 26-40 months, 90 %). CONCLUSIONS: Frozen colon tissues yield high-quality RNA in approximately 80 % of specimens. Ex-vivo ischemia times and storage periods did not adversely affect RNA quality. This study showed that standard operation protocols and the maintenance of high-quality tissue repositories were the keys to translational medicine research.

MiRNA expression signature for potentially predicting the prognosis of ovarian serous carcinoma.

One of the best prognostic predictors for patients with epithelial ovarian cancer is the Federation of Obstetrics and Gynecology (FIGO) stage at diagnosis. Advanced-stage ovarian serous carcinoma (OSC) generally have poor prognosis. The goal of this study is to develop and validate a miRNA expression profile that can differentiate the OSC at early and advanced stages and study its correlation with the prognosis of OSC. To identify a unique microRNA (miRNA) pattern associated with the progression of OSC at early and advanced stages, a miRNA microarray was performed using Chinese tumor bank specimens of patients with OSC stage I or III in a retrospective analysis. The expression of four dysregulated miRNAs was validated using quantitative real-time polymerase chain reaction (qRT-PCR) in an external cohort of 51 cases of OSC samples at stages I and III. Kaplan-Meier analysis was performed to analyze the correlation between the expression of some miRNAs and prognosis. Of the 768 miRNAs analyzed in the microarray, 26 miRNAs were significantly either up- or downregulated, with at least a 2-fold difference, in OSC stage I compared with stage III. The qRT-PCR results showed that miR-510, miR-509-5p, and miR-508-3p were significantly downregulated and that miR-483-5p was upregulated in stage III OSC compared with stage I, which was consistent with the microarray results. Kaplan-Meier analysis showed low miR-510 expression, low miR-509-5p expression, and advanced FIGO stage, and chemotherapy resistance were significantly associated with poorer overall survival (P < 0.05). Our results suggest that miRNAs may play a role in the progression of OSC, and miR-510 and miR-509-5p may be considered novel-candidate clinical biomarkers for predicting OSC outcome.

Synchronous detection of miRNAs, their targets and downstream proteins in transferred FFPE sections: applications in clinical and basic research.

After discovering new miRNAs, it is often difficult to determine their targets and effects on downstream protein expression. In situ hybridization (ISH) and immunohistochemistry (IHC) are two commonly used methods for clinical diagnosis and basic research. We used an optimized technique that simultaneously detects miRNAs, their binding targets and corresponding proteins on transferred serial formalin fixed paraffin embedded (FFPE) sections from patients. Combined with bioinformatics, this method was used to validate the reciprocal expression of specific miRNAs and targets that were detected by ISH, as well as the expression of downstream proteins that were detected by IHC. A complete analysis was performed using a limited number of transferred serial FFPE sections that had been stored for 1-4 years at room temperature. Some sections had even been previously stained with H&E. We identified a miRNA that regulates epithelial ovarian cancer, along with its candidate target and related downstream protein. These findings were directly validated using sub-cellular components obtained from the same patient sample. In addition, the expression of Nephrin (a podocyte marker) and Stmn1 (a recently identified marker related to glomerular development) were confirmed in transferred FFPE sections of mouse kidney. This procedure may be adapted for clinical diagnosis and basic research, providing a qualitative and efficient method to dissect the detailed spatial expression patterns of miRNA pathways in FFPE tissue, especially in cases where only a small biopsy sample can be obtained.

Evaluation of antioxidant and immunity-enhancing activities of Sargassum pallidum aqueous extract in gastric cancer rats.

The effect of Sargassum pallidum (brown seaweed) aqueous extract on the immunity function and antioxidant activities in was studied gastric cancer rats. Treatment with Sargassum pallidum aqueous extract at oral doses 400, 600 or 800 mg/kg body weight was found to provide a dose-dependent protection against N-methyl-N'-nitro-Nnitrosoguanidine (MNNG)-induced immunity damage and oxidative injury by enhancing serum interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-10 (IL-10) levels, decreasing interleukin-6 (IL-6), interleukin-1ß (IL-1ß), tumor necrosis factor-alpha (TNF-α) levels, preserving normal antioxidant enzymes activities, and by inhibiting lipid peroxidation in gastric mucosa. It can be concluded that Sargassum pallidum aqueous extract may enhance the immunity and antioxidant activities in gastric cancer rats.

Antioxidant and immunity activities of Fufang Kushen Injection Liquid.

We investigated the effects of Fufang Kushen Injection Liquid (FFKSIL) on gastric immunity and oxidant-antioxidant status during N-methyl-N'-nitro-N-nitroso-guanidine (MNNG)-induced gastric carcinogenesis. The extent of lipid peroxidation and the levels of reduced glutathione (GSH) and activities of the GSH-dependent enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) were used to monitor the peroxidative balance. Enhanced lipid peroxidation in the gastric cancer animals was accompanied by significant decreases in the activities of GSH, GPx, GST and GR. Administration of FFKSIL significantly enhanced serum IgA, IgG, IgM, IL-2, IL-4 and IL-10 levels, decreased serum IL-6 and TNF-α levels, lowered the levels of lipid peroxides and enhanced GSH levels and activities of GSH-dependent enzymes. Our results suggest that FFKSIL blocks experimental gastric carcinogenesis by protecting against carcinogen-induced oxidative damage and improving immunity activity.

[The expression of nicotinamide phosphoribosyl transferase and vascular endothelial growth factor-A in gastric carcinoma and their clinical significance].

OBJECTIVES: To study the expression of nicotinamide phosphoribosyl transferase (Nampt) and vascular endothelial growth factor-A (VEGF-A) in gastric carcinoma and investigate their correlations to clinicopathologic features and prognostic significance. METHODS: The proteins of Nampt and VEGF-A in 68 specimens of gastric carcinoma and 59 specimens normal gastric tissue were detected by immunohistochemistry during January 2000 to December 2004, and the 68 patients were followed up. RESULTS: Nampt protein was detected in the cytoplasm of both tissues, and Nampt in gastric carcinoma (13 ± 5) were significantly higher than that in normal gastric tissue (6 ± 3) (t = 7.46, P < 0.01). The expression of Nampt was correlated to invasive depth (F = 4.693, P = 0.034), lymph node metastasis (F = 4.027, P = 0.049), clinical TNM stage (F = 9.979, P = 0.002), but not to gender, age, tumor location, tumor size, differentiation (P > 0.05). The expression of Nampt is correlated with survival of patients that underwent surgical resection for gastric cancer. The survival rate of patients in negative of Nampt was very higher than that of the positive patients, and its co-expression with VEGF-A showed a trend towards poorer survival. The positive correlation was found between the expression of Nampt and VEGF-A in gastric carcinoma (r = 0.293, P = 0.015). CONCLUSIONS: The expression of Nampt is positively correlated to that of VEGF-A in gastric carcinoma. The correlation between the expression of Nampt and VEGF-A in gastric carcinoma plays an important role cooperatively in carcinogenesis, development and metastasis of gastric carcinoma.

Knockdown of SAR1B suppresses proliferation and induces apoptosis of RKO colorectal cancer cells.

Colorectal cancer (CRC) is the third most commonly diagnosed cancer worldwide. SAR1 gene homolog B (SAR1B) is a GTPase that has been reported to have a central role in the regulation of lipid homeostasis and is associated with numerous diseases. However, its role in cancer, particularly in CRC, remains unclear. The present study revealed that SAR1B was overexpressed in CRC samples and this was associated with shorter overall survival time in patients with CRC. Colony formation, cell proliferation and flow cytometry assays were conducted to evaluate the functions of SAR1B in CRC. It was reported that SAR1B may be associated with tumorigenesis of CRC. Knockdown of SAR1B suppressed cell proliferation and induced significant apoptosis of RKO cells. Furthermore, microarray analysis was performed to identify the potential targets of SAR1B in CRC. Bioinformatics analysis revealed that SAR1B was significantly involved in regulating 'TGF-ß signaling', 'paxillin signaling', 'cell cycle regulation by BTG family proteins' and 'IGF-1 signaling'. These results suggested that SAR1B may be considered a potential prognostic biomarker and therapeutic target for CRC.

Seasonality of Hg dynamics in the Ebrié Lagoon (Côte d'Ivoire) ecosystem: influence of biogeochemical factors.

This study addresses the different biogeochemical parameters that control the dynamics of Hg, which is a less-studied metal in the Ebrié Lagoon. During two hydrological seasons, the dry season and the rainy season, we regularly sampled and analysed various compartments (e.g. sediments and fishes (Tilapia sp.)) of the lagoon. Thus, the physicochemical parameters were measured in situ (e.g. temperature, pH, salinity, redox potential and dissolved oxygen, total dissolved organic carbon, nitrates and sulphates), and the microbiological parameters (e.g. cultivable cells, total enzymatic activity and catabolic activity) were measured to establish the seasonal variations in the links between Hg and biogeochemical parameters through multivariate statistical analyses. The bioavailability of Hg from an unpolluted site was studied by comparing the ratios of fish and sediment. The results indicated that the seasons influenced the different biogeochemical factors, although for some factors, the variations were not significant. This influence was more pronounced in the dry season than in the rainy season. The impact of microbial activities and organic matter on Hg dynamics was observed in all seasons. However, other factors, such as pH, temperature, salinity, Eh and sulphates, influenced the dynamics of Hg only in the dry season.

Expression of NAMPT is associated with breast invasive ductal carcinoma development and prognosis.

Nicotinamide phosphoribosyltransferase (NAMPT) possesses various functions in human cells, and altered NAMPT expression is associated with human carcinogenesis. The present study detected the expression of NAMPT in normal and cancerous breast tissues from 83 patients using immunohistochemistry, and analyzed its association with the clinicopathological and survival data of the patients. NAMPT was significantly overexpressed in the breast invasive ductal carcinoma tissues compared with adjacent normal mammary gland tissues. Upregulated NAMPT expression was associated with a larger tumor size, lymph node metastasis, advanced clinical tumor-node-metastasis stages, and estrogen receptor and progesterone receptor expression. Furthermore, NAMPT expression was associated with poor overall and disease-free survival in patients with breast cancer. In conclusion, NAMPT increased protein expression in tumor cells may contribute to the development and progression of breast invasive ductal carcinoma. Thus, detection of NAMPT expression might be useful as a biomarker for the early detection and prognosis prediction of breast cancer.

Importance of the plasma soluble HLA-G levels for prognostic stratification with traditional prognosticators in colorectal cancer.

An increased peripheral soluble HLA-G (sHLA-G) expression has been observed in various malignancies while its prognostic significance was rather limited. In this study, the prognostic value of plasma sHLA-G in 178 colorectal cancer (CRC) patients was investigated. sHLA-G levels were analyzed by specific enzyme-linked immunosorbent assay. Data showed sHLA-G levels were significantly increased in CRC patients compared with normal controls (36.8 U/ml vs 25.4 U/ml, p = 0.009). sHLA-G in the died were obviously higher than that of alive CRC patients (46.8 U/ml vs 27.4 U/ml, p = 0.012). Patients with sHLA-G above median levels (≥ 36.8 U/ml, sHLA-Ghigh) had a significantly shorter survival time than those with sHLA-Glow (< 36.8 U/ml, p < 0.001), and sHLA-G could be an independent prognostic factor for CRC patients. With stratification of clinical parameters in survival by sHLA-Glow and sHLA-Ghigh, sHLA-G exhibited a significant predictive value for CRC patients of the female (p = 0.036), the elder (p = 0.009), advanced tumor burden (T3 + 4, p = 0.038), regional lymph node status (N0, p = 0.041), both metastasis status (M0, p = 0.014) and (M1, p=0.018), and clinical stage (I + II, p = 0.018), respectively. Summary, our data demonstrated for the first time that sHLA-G levels is an independent prognosis factor and improves the prognostic stratification offered by traditional prognosticators in CRC patients.

Vulnerability and adaptation to climate change in the Comoe River Basin (West Africa).

Climate change is impacting water users in many sectors: water supply, farming, industry, hydropower, fishing, housing, navigation and health. Existing situations, like population growth, movement of populations from rural to urban areas, poverty and pollution can aggravate the impacts of climate change. The aim of the study is to evaluate the vulnerability of different water user groups to climate change and define communities' adaptation strategies in the Comoe River Basin. Information was collected on communities' concerns and perception on changes in climate and potential adaptation measures and strategies. Results show that 95 % of the sample in the study communities had heard of it and are aware that climate change is occurring. They have been experiencing changes in economic activity and cropping pattern, reduced water level in rivers, crop failure, delay in cropping season, new pests and diseases, food insecurity, drop in income and decline in crop yield. Results also show that communities employ various adaptation strategies including crops diversification, substitution and calendar redefinition, agroforestry, borrowing from friends and money lenders and increasing fertilizer application.

The expression of miR-30a* and miR-30e* is associated with a dualistic model for grading ovarian papillary serious carcinoma.

Histological grade has already been recognized as a very important prognostic factor for ovarian papillary serous carcinoma (OPSC). On the basis of pathogenetic mechanisms, recent findings suggest a dualistic model of OPSC consisting of types I (low-grade) and II (high-grade) cancers. High-grade OPSC is responsible for most ovarian cancer deaths. The goal of our investigation was to identify the differences in key miRNAs and possible regulators through miRNA microarray chip analysis, as well as functional target prediction and clinical outcome between the low and high-grade OPSC patients. The pathogenic basis in differentiation of ovarian cancer subtypes was studied to provide insight into diagnosis and therapy for high-grade cases. Through microarray analysis, we found that miR-30a* and miR-30e* were the top 2 significantly different miRNAs between type I and type II OPSC patients, and both were remarkably downregulated in the latter type. ATF3 and MYC were indicated as potential co-targets of miR-30a* and miR-30e*, and showed a significant upregulation in type II patients. As ATF3 and MYC are often associated with aggressive behavior and poor differentiation, especially in human cancers, these results are in good agreement with our findings and point toward a regulating differentiation function of the miR-30a* and miR-30e* genes. Further analysis using leave­one-out cross predictions and Kaplan-Meier survival analysis strongly suggested that miR-30a* and miR-30e* can be used as biomarkers to tailor histological grade before starting the regimen, and they showed important roles in ovarian cancer differentiation resulting in poorer prognosis. In general, miR-30a* and miR-30e* coupled with expression data that reveal pathogenic regulation to predict histological differentiation, may operate to direct the formation of early detection and therapeutic approaches to individual OPSC patients, especially differentiation therapy to high-grade cases.

Expression of miR-224-5p is associated with the original cisplatin resistance of ovarian papillary serous carcinoma.

Chemoresistance is a major challenge to successful chemotherapy of ovarian cancer, which represents the leading cause of mortality from gynecologic malignancies. We demonstrated that overexpression of miR-224-5p in ovarian cancer patients is associated with platinum-based chemoresistance using miRNA microarray analysis and quantitative real-time polymerase chain reaction (qRT-PCR) validation in vivo, as well as in 4 human ovarian cancer cell lines (C13/OV2008; A2780CP/A2780S) in vitro. In the present study, we aimed to clarify the role of miR-224-5p in regulating the chemoresistance of ovarian cancer. By using the sensitive miRNA transient transfection, we demonstrated expression and bioactivity of miR-224-5p in ovarian cancer cell lines. It is of note that enforced expression of miR-224-5p enhanced chemoresistance to cisplatin in ovarian cancer cells through apoptosis reversion. We predicted and identified the PRKCD gene as one of the targets of miR-224-5p in mediating the primary chemoresistance of ovarian cancer patients. We showed reciprocal expression of miR-224-5p and PRKCD by quantitative analysis in complete response and incomplete response patients in vivo, and 2 pairs of cisplatin resistance and sensitive cell lines in vitro, after either miR-224-5p overexpression or knockdown transfection. Additionally, miR-224-5p and PRKCD can serve as novel predictors and prognostic biomarkers for ovarian papillary serous carcinoma (OPSC) patient response to overall disease-specific survival. Our findings suggest that miR-224-5p may function as an oncogene and induce platinum resistance in OPSC at least in part by downregulating PRKCD, thereby providing a biomarker for predicting chemosensitivity to cisplatin in patients with ovarian cancer.

Extraction optimization of carbohydrate compound from Huangqi using orthogonal design.

The effect of extraction time, extraction temperature and time on the yield of Huangqi carbohydrate compound (HQCC) was investigated using single factor and orthogonal experiment design. The influence by the parameters on the extraction yields of carbohydrate compound decreased in the order of: C (extraction number)>A (extraction time)>B (extraction temperature) according to the R values. Based on this analysis, and considering the carbohydrate compound extraction efficiency, the cost of energy and the feasibility of experiment, the optimum conditions of extraction were therefore determined as follows: extraction time 120min, extraction temperature 80°C, and extraction number 4. Oral administration of HQCC reduced lipid peroxidation level and enhanced antioxidant enzymes activities in gastric mucosa. In addition, HQCC reduced the serum IL-8 and TNF-α levels. In conclusion, these data reveal that HQCC promotes regeneration of damaged gastric mucosa, probably through its antioxidative mechanism.

Overexpression of Nampt in gastric cancer and chemopotentiating effects of the Nampt inhibitor FK866 in combination with fluorouracil.

Nicotinamide phosphoribosyltransferase (Nampt), an enzyme involved in the NAD⁺ salvage pathway, is over-expressed and important in the carcinogenesis in several types of cancers. The expression of Nampt and its role in gastric cancer remain largely unknown. In this study, using real-time PCR and Western blotting we found that Nampt was overexpressed at the mRNA and protein levels, respectively, in established gastric cancer cells and human gastric cancer tissues. The specific Nampt inhibitor FK866 repressed gastric cancer cell proliferation, as assessed by MTT assay. Using transwell and soft agar clonogenic assays, we also found that FK866 suppressed gastric cancer cell migration and anchorage-independent growth, respectively. These inhibitory effects of FK866 were accompanied by significantly decreased expression of VEGF, MMP2, MMP9 and NF-κB. As determined by MTT assay and flow cytometry, FK866 also increased the chemo-sensitivity of gastric cancer cells to fluorouracil by greater inhibition of cell proliferation and the induction of apoptosis. Our findings indicate that Nampt may be a new therapeutic target for gastric cancer.

Nampt/PBEF/visfatin and cancer.

Nampt/PBEF/visfatin is the rate-limiting enzyme that catalyzes the first step in NAD biosynthesis from nicotinamide and regulates growth, apoptosis and angiogenesis of mammalian cells. This enzyme was originally cloned as a putative cytokine shown to enhance the B cell precursor maturation in the presence of IL-7 and stem cell factor. A number of cancers have increased expression of Nampt/PBEF/visfatin, which regulates a variety of different signaling pathways such as PI3K/Akt, ERK1/2 and STAT3. FK866/APO866 and CHS828/GMX1777 are two known inhibitors of Nampt/PBEF/visfatin and have been evaluated as anticancer agents in the clinic. This review will focus on its role in carcinogenesis and cancer progression and its inhibitors as therapeutic target for cancer treatment.