Isolation Of Antitumor Peptide From Ciona Intestinalis L. And Study Of Its Antitumor Mechanism

Malignant tumor is one of the most serious diseases which threaten the health ofpeople. Beside inhibiting the growth of cancer cells, most of current anti-canceragents act against normal mammalian cells, consequently causing severe side effects.So it become an important assignment to find a kind of anti-tumor drugs withefficiency and low side effects. But the traditional drugs were limited on the land, andanother huge reservoir---marine organisms had been ignored for a long time. Manyinspiring achievement had been made by scientist on the finding of marinecompounds with anti-tumor activity. Marine organisms represented an essentiallyunexploited reservoir for products with potentially biological and pharmacologicalinterest. So far, many natural products derived from marine organisms in literatureswere characterized by low cytotoxity and new mechanisms. Ascidian is a kind ofmarine animal with an important value in the marine anti-tumor pharmacology field.Many compounds with bioactive, especially antitumor, activity have been discoveredfrom it. In the present study, an anti-tumor polypeptide was purified from Cionaintestinalis L., and the possible anticancer pharmacological action modes were alsoinvestigated.In the present study, a novel polypepetide with potent cytotoxicity was purified.The purification procedure consisted basically of70%acetone fractionation,ultra-filtration, DEAE Sepharose Fast Flow ion-exchange chromatography, gelchromatography and HPLC. The anti-tumor peptide(PCI5931) was obtained from theCiona intestinalis L. finally, and its molecular weight which was detected with mass spectroscopy was5931Dalton. N-terminal amino acid sequence analysis revealed thatthe N-terminal amino acid sequence of PCI5931is Met-Val-Val-Pro-Pro-Asp-Gly-Gln-Ser-Glu-Cys-Pro-Asp-Gly-Asn. Cell proliferation assay in vitro, using MTTmethod showed that the PCI5931can inhibit several cancer cells growth potently.Multiple approaches, including scanning electron microscopy, AO/EB dual staining,DAPI staining, cell cycle arrest assay, mitochondrial membrane potential detectionand western blotting, were employed to investigate the possible pharmacologicalanti-tumor action modes of PCI5931. The results showed that the growth inhibitioneffect was perhaps related with the arrest of these cells in G2/M phase. And PCI5931treatment could disrupt the mitochondrial membrane potential. The result of westernblotting suggested that mitoehondria pathway was involved in cell apoptosis whichwas induced by PCI5931.Our observations indicated that Ciona intestinalis L. is an important source ofantitumor compounds. The new polypeptide, PCI5931, can inhibit the growth ofseveral cancer cells potently. In contrast, the peptide displayed very weak cytotoxicityto benign cells. PCI5931, as a kind of candidate antitumor drug, is promising to bedeveloped as a novel kind of anti-tumor agent. Additionaly, the polypeptide inducedcell apoptosis through mitoehondria pathway. However, the precise mechanismunderlining the PC1593induced cell death is not clear. Further study is needed toaddress how the polypeptide interact with signal molecules.