Through the determination of viscosity average molecular weight, enzyme activity, hydrolysis ratio and reducing sugar concentration, the optimal technological conditions of complex enzymes composed of commercial α-amylase, cellulase and pectinase were investigated. Results indicated that complex enzymes could result in low molecular weight chitosan ranged from 1000 to 4000 after enzymatic degradation for 2 h under the condition of zymolyte ratio 1:5(m/m), pH 5.3 and temperature 56℃, and the structure of those products failed to vary obviously by FTIR analysis.Keywords: Chitosan, Low molecular weight chitosan, Complex enzymes, Preparation Chitosan (chitosan, CTS) is a kind of natural polysaccharide bioactive substances containing nitrogen, tends to be the sixth largest life elements excluding the body necessities viz. sugar, protein, fat, fiber and minerals, and exerts an extensive and intensive application in food, cosmetic and pharmaceutical areas (Zhu, 2006, PP. 41-45; Jiang, 2006, PP. 99-102). Due to its large molecular weight and the structure containing abundant hydrogen bonds, chitosan is only soluble in dilute acid, insoluble in water, and tough to be assimilated by human bodies, which hinders its application dramatically. If chitosan was degraded into LMWC (low molecular weight chitosan) with the molecular weigh less than 10000, its water solubility increased greatly, conducive to human intestinal digestion and absorption, and possessed the functions such as promoting to produce splenic antibody, and lowering cholesterol, blood pressure, blood sugar and blood lipid level in serum and liver (Wei, 2003, PP. 614-617; Kim, 2005, PP. 357-368).Recently, studies on the preparation of water soluble LMWC have attracted many researchers' attention all over the world (Huang, 2002, PP. 381-385). Degradation methods initiated by scholars mainly included chemical, physical and enzymatic degradation. Among them, enzymatic degradation has been recognized as one of the most promising approaches to prepare LMWC, because its reaction condition was moderate without byproducts and the degradation process was easy to monitor. However, the specific enzyme for chitosan was expensive, and its application in industry was limited. The applications of single non-specific enzyme in hydrolyzing chitosan such as α-amylase (Yu, 2008, PP. 464-466; Zhang, 2003, PP. 44-48), pectinase (Ipsita, 2003, PP. 582-588; Cabrera, 2005, PP. 165-172), cellulose (Lin, 2009, PP. 47-53; Xia, 2008, PP. 6751-6762; Han, 2007, PP. 98-101;Xie, 2009, PP. 1895-1899, protease (Li, 2005, PP. 441-448) and so on have been documented, but those enzymes showed limited effects on degradation, even if enhancing their amount. Consequently, we investigated effects of complex enzymes containing cellulase, pectinase and α-amylase on chitosan degradation.

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Progression to CRPC represents a major challenge for treatment of advanced PCa. However, underlying mechanisms of this process remain largely unknown. We found that androgen deprivation in mice bearing PCa patient-derived tumors and in PCa LNCaP cells resulted in increased expression of wnt transcriptional factors: TCF4 and LEF1, as well as c-myc and NRP2. Using GenBank and MatInspector software, we identified five potential binding sites for TCF4 in the 5′ flanking promoter region spanning from –3891 to +108 base pairs of the NRP2 gene. Treatment of LNCaP cells with Wnt3a and Wnt 5a conditioned medium increased NRP2 mRNA expression; and inhibition of Wnt signaling by overexpression of secreted Wnt antagonists (WIF1 and Frzb/sFRP3) in two CRPC cell lines: C4-2B and PC3 down-regulated the expression of NRP2 in both in vitro cell cultures and in in vivo xenograft tumors. Chromatin immunoprecipitation/real-time PCR a! nalysis revealed that both WIF1 and Frzb/sFRP3 expression decreased the in vivo binding of TCF4 and beta-catenin to the NRP2 promoter at five predicted sites. Knock-down of NRP2 by short-hairpin RNA in CRPC cell lines markedly reduced cellular invasiveness and migration capacity. Furthermore, we analyzed gene microarray data sets from the UCI SPECS project. The total of 204 samples from 82 patients include 66 tumors, 65 tumor-adjacent stromas, 28 far stromas and 45 normal prostate samples. The mRNA expression levels of the genes of interest were normalized to the average expression levels of these genes from the normal prostate samples. We showed that the percentage of samples with WIF1 down-regulation progressively increased from far stroma to adjacent stroma and tumors. Strikingly, the levels of NRP2 mRNA are up-regulated in all tested adjacent stroma and most of tumor samples. Furthermore, the expression levels of NRP2 in far and adjacent stroma and tumors are inversely related to WIF1 mRNA levels (correlation coefficients are –0.6918, –0. 2349 and –0.5931; Ps<0.01). For a large series of clinical samples, NRP2 expression appears to be associated with WIF1 down-regulation. These results suggest that NRP2 may be a novel Wnt target gene and contribute to progression of CRPC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 440. doi:1538-7445.AM2012-440