Gene Cloning, Activities Identification And Primary Study Of Mechanism Of Inducing Cancer Cells Apoptosis Of Hainanenins

Amphibians, as a species resource with high medicinal and food value for sustainable development, has been in people’s lives for long time. Their skin secretion is treasure trove for drug development which recorded on ancient Chinese pharmacopoeia. Chemical composition of amphibian skin secretion could be divided into biogenic amines, alkaloids, peptides and other categories, wherein the content of biogenic amines and peptides are particularly rich, and both of which are the basis for novel drug development. Our country has immense potential space in drug development with plenty species resources, especially abundant endemic species.In this study, two novel types of active peptides were purified from dorsal skin secretion of Amolops hainanensis, one of which had never been reported and belonged to new family. Then a cDNA library of Dorsal skin secretion was constructed, from which31cDNA sequences encoding10active peptides were obtained. Through multiple-sequence alignment, these10peptides belonged to4families respectively, and5peptides of these10represented the prototypes of a novel family, including the deduced nucleotide sequence of purified peptides mentioned above. According to the generic name of the species of origin, they were designated as hainanenin-1～5. Each of them consists of21aa residues with a C-terminal disulphide loop of7residues between Cys15and Cys21.Hainanenin-1and hainanenin-5were selected and synthesized to screen in vitro activities, including antimicrobial, antioxidant activities and hemolytic inhibition of anti-tumor activity. The results revealed that hainanenin-1and hainanenin-5possessed strong and broad-spectrum antimicrobial activities against17kinds of bacteria, and slight antioxidant activities, relatively high hemolytic activities; Hainanenin-1exhibited strong growth inhibition activities to5kinds of cancer cells, IC50ranging from10～13μM, and IC50lowed to6.9μM against breast cancer cell, but within the concentration scope of IC50to cancer cell, hainanenin-1had unobvious influence on normal cell HUVEC.We investigated the mechanism of hainanenin-1inducing MCF-7apoptosis:Scanning electron microscopy (SEM) image of MCF-7incubated with hainanenin-1showed that microvillia retracted on the surface of membrane, membrane foamed and eventually formed pores, the integrity of cell membrane was destructed; AnnexinV-FITC and PI double staining result suggested that hainanenin-1lead an earlyapoptosis of MCF-7, and hochast33258staining result revealed hainanenin-1induce chromatin condensation; Strong activation was observed after treatment with hainanenin-1for activation of caspase3and caspase9, but caspase1had no obvious change. We recommend hainanenin-1as a anticancer peptide through its positive anticancer activity, hainanenin-1together with its character in structure and a superiority in anticancer activity will make it useful in medicinal design.