Preparation Of Anti-tumor Bioactive Peptide From Yellow-margined Box Turtle (Cistoclemmys Flavomarginata) And Its Anti-cancer Activity

Curoa flavomarginata(synonymy: Cistoclemmys flavomarginata),as a kind of terrestrial amphibian widely distributed in the edges of humid forests or bushy areas not far from water(nevertheless,the turtle is rare in more aquatic habitats,such as paddy field,ponds,and river valleys)in continental China,is popular for its highly nutritious and unique medicinal value.Its blood,shell,meat and heart have long been used by ancient physicians as a precious raw material of traditional Chinese medicine(TCM),such as turtle shell pill and injection(TSPI)as a common TCM was extensively utilized to prevent and treat tuberculosis,hemorrhoids fistula and it could also be helpful to promote the level of reduced leukopenia caused by chemotherapy.Furthermore,C.flavomarginata has also been used as bioactive complex mixture in Chinese folk medicine for preventing carcinomas.An important question is whether its biological activity can be largely or exclusively ascribed to one or more individual compounds present in this TCM.To address this question,we prepared CFM-2 from C.flavomarginata and studied its effects.Therefore,it is of great research value to extract anti-tumor bioactive substances from C.flavomarginata with the technology of separation and purification.The results are as follows:Firstly,water-soluble bioactive components derived from C.flavomarginata meat,blood and shell were obtained by utilizing water-extraction method combined with homogenization,ultrasonic wave,microfiltration and ultrafiltration.3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide(MTT)assay showed that Cf-1,a component produced from aqueous extracts from C.flavomarginata meat with a molecular weight of less than 10 k Da(<10 k Da),exhibited superior anti-tumor activity in vitro.Meanwhile,human hepatocarcinoma Hep G-2 cells treated with Cf-1 obviously displayed an increased number of detached cells in round and shrunken shape.Results of murine H22 hepatocarcinoma model indicated that tumor growth inhibition rate could be as high as 83.8% when treated with high dose of Cf-1(300 mg/kg).Therefore,Cf-1 demonstrated notable anti-tumor activity against H22 hepatocellular carcinoma.In contrast to the positive control group,5-Fluorouracil(5-Fu),group Cf-1 represented no distinct reduction in liver weight,while an increasement was observed in spleen weight(P<0.05),which meaned that Cf-1 did not cause liver damage,but might increase the immunity in murine H22 hepatocarcinoma-bearing mice.In order to further exploit the anti-tumor constituents from C.flavomarginata,MTT cytotoxicity assay was utilized to evaluate the cytotoxity effects of extracted peptides on human hepatocarcinoma(Hep G-2).A peptide coded as CFM-2 was isolated from the meat of C.flavomarginata using ultrafiltration(UF),Sephadex G-25,ion exchange chromatography and RP-HPLC.The molecular weight of the highly purified polypeptide was 2989.6 k Da as determined by Mass Spectrometry and N-terminal amino acid sequence was also determined,the corresponding amino acid sequence is CRIGFGPIPFSLPEGLPKIPSVSTHIKV.The effect of CFM-2 on viability of tumor cell lines as well as benign cell lines was assessed by MTT assay,CFM-2 exhibited significant cytotoxicity to Hep G-2 cells.However,almost no inhibitory effect was found when treating normal fibroblast cell L929.Treatment of Hep G-2 cells with CFM-2 resulted in a significant inhibition of cell proliferation in vitro(half maximal inhibitory concentration IC50=129.57±7.85 ?g/m L for 24 h).The inhibition of cancer cell transfer ability was confirmed by a cell scratch assay and transwell assay.The results showed that CFM-2 can significantly inhibit the horizontal and vertical migration of Hep G-2 cells.Then,cell cycle distribution was examined by flow cytometry.According to these results,CFM-2 obviously blocked G0/G1 phase of Hep G-2 cells.Finally,apoptosis was evaluated by apoptotic morphology analysis with DAPI staining,scanning electron microscopy(SEM),transmission electron microscopy(TEM)and flow cytometric analysis following Annexin V/propidium iodide staining.The results indicated that CFM-2 could destroy Hep G-2 cell nucleus,organelle and cell membrane to induce cell death.CFM-2 may,therefore,be a potential compound for use in the development of novel anti-tumor drug.