| Cancer is one of the leading causes of mortality worldwide. The reason why it has become a hot potato is mainly closely related with its disease characteristics. One of the most troublesome characteristics of cancer cells is that it can migrate and invade normal tissues continually and then formed a new clone. It has showed that the content, composition and molecular size of chondroitin sulfate (CS) are significant different between tumor cell and corresponding normal tissues in the process of tumorigenesis. Usually, the differences in the composition and structure of CS chains are closely related to the occurrence and development of tumors. It has been a focus all over the world that CS may be exploited as an antineoplastic drugs based on the characteristics above. Previous studies have indicated that the anti-tumor activities of CS are determined by its sulfate mode and molecular weight. Among various CS types, CS-E with high sulfation degree and complex sulfation pattern shows a wide spectrum of antitumor activity, indicating that it has a potential druggability. However, at present the commercial CS-E is mainly obtained from Todarodes Pacificus, a wild type small squid, is difficult to mass production and therefore is relatively expensive. Moreover, the resource of CS-E are under serious threat with the increasing of marine pollution. In addition, there is very few studies on the activity of CS-E oligosaccharides against tumors.In this study, we purified a novel E-unit-riched CS (NER-CS) through screening cartilage resources, and establishing and optimizing the separation and purification processes of CS, and analyzed its physicochemical properties. Meanwhile, we prepared E-unit-riched CS oligosaccharides through optimizing the conditions of enzymatic digestion and isolation. In addition, the anti-tumor metastasis activity of the novel CS and its oligosaccharides were analyzed at the molecular and animal levels, and the related mechanism was discussed as well. The details are as follows:1. The preparation and purity analysis of NER-CS:The raw materials used for CS preparation were cartilage of Dosidicus Gigas, which just as waste in food processing. Then the preparation method of CS including crushing, dehydration and skim, enzymatic hydrolysis, removing protein, alcohol precipitation, ion-exchange chromatography, desalination and so on was established. The final yield of NER-CS was 16±2%. To identify the glycosaminoglycan type of the preparation, the commercial Chondroteinase B, Chondroteinase ABC (CSase ABC), Hyaluronidase from Streptomyces Hyalurolyticus, and homemade rHCLase were used to treat the purified NER-CS. According to the substrate specificity of enzymes, the NER-CS polysaccharide was identified as CS rather than other glycosaminoglycan types. The content of CS in the NER-CS sample was determined using the carbazole reaction, and the protein content of the sample was analyzed using a BCA protein assay kit. The results showed that the purity of CS was more than 97% and the amount of protein was under 2%.2. Preliminary analysis of the structure of NER-CS:By gel filtration chromatography, the molecular mass of CS was determined to be～623.2 kDa, using a series of size-defined glucan polysaccharides as marker. To analysis the disaccharide composition of CS, the polysaccharide was completely degraded by CSase ABC and then the digest was analyzed by HPLC. The results demonstrated that the CS was mainly composed of O-unit, C-unit, A-unit and E-unit. When the content of E-unit was up to the highest ratio of 44.5%, O-unit, C-unit and A-unit accounted for 7.1%, 12.6% and 35.8%, respectively. The sulfate degree was 1.37. The above results indicate that the NER-CS was a novel highly sulfated CS.3. Preliminary analysis of the biological activity of NER-CS:The anticoagulant activity of NER-CS was detected using the Aglyco kit.The results showed that the anti-FⅡa activity of NER-CS was 20±2 U/mg, about 10% of that of heparin used as a positive control here, and the anti-FⅩa activity of NER-CS was 0.9 ±0.2 U/mg, only 4% as much as heparin’s. To analyze the anti-tumor metastasis activity of NER-CS, we initially established the mice metastasis model of breast cancer 4T1 cells. The anti-tumor rate of NER-CS with the dosage of 50,100 and 200 μg for each mice was 54.3±2.5%,75.1±3.5%, and 42.9±2.1%, respectively, while the anti-tumor rate of heparin with the dosage of 100 ug for each mice was 74.6±1.8%. To determine the interaction activity of NER-CS with five growth factors and three selectins, the NER-CS was biotinylated and immobilized on a sensor chip precoated with streptavidin. The results showed that NER-CS could strongly interact with HGF and bFGF, and the dissociation equilibrium constants were (3.47±1.73) X 10"6 and (5.39±2.92) X 10-8, respectively.4. Preparation and anti-tumor activity assay of E-unit-riched CS oligosaccharides:To prepare CS oligosaccharides using rHCLase with high activity, the enzyme dosage and reaction time for the CS degradation were optimized by single factor experiment. CS oligosaccharides with different degree of polymerization (DP) were then isolated and purified. The disaccharide composition and anti-tumor metastasis activity and of the prepared oligosaccharides were finally analyzed. Oligosaccharides ranging from DP2 to DP 18 could be obtained. In the optimized process,120 mg of NER-CS was degraded by 40 μg of rHCLase in PBS containing 0.5 M NaCl at 37 ℃ for 8 h. With the dosage of 100 μg, the anti-tumor rate of oligosaccharides of DP8, DP10, DP12, and DP14 were 70.3+3.8%,74.0+2.6%, 83.2±3.6%, and 74.8+4.6%, respectively, while the anti-tumor rate of NER-CS was 75.7±3.7%.In conclusion, NER-CS, a novel E-unit-riched CS, has been identified from Dosidicus Gigas cartilage. NER-CS showed high interaction activity with HGF and bFGF. Moreover, the NER-CS showed high anti-tumor activity while the anticoagulant activity was low. A series of oligosaccharides with different degrees of polymerization were purified from the enzymatic digestion of NER-CS, in which the dodecasaccharide showed the highest antitumor activity.This study has provided a beneficial reference for the research and development of CS associated anti-tumor metastasis drugs and health care products. |
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