Studies On Purification And Anti-tumor Activity From Nocardiopsis Actinomycetes

Actinomycetes is a main kind of terrestrial survival prokaryotes that is both hyphae grow and Spore propagation. Actinomycetes has becomme a class of microorganism resources which have important economic value and biologial function owing the ability to produce large amounts of antibiotics,enzymes,inhibitors and other biologically active substances. We isolated 49 strains of Actinomycetes from the soil samples collected around the country. For each strain screening of antitumor activity, one of them has broad-spectrum anti-tumor activity, and combined with the morphology observation and physiological, biochemical characteristics,16s rRNA genes Blast homologous sequence alignment analysis to determine a novel Nocardiopsis.sp. The Nocardiopsis.sp had highly toxicity to tumor cell, then was named Nocardiopsis.sp X032, hereinafter referred to as Nocardiopsis.sp X032 (referred NX032,CCTCC NO:M 2015361). Small molecule and protein having anti-tumor activity was obtained from NX032 by multi-step separation and purification.After cultivation for 12 Day in fermentation medium, the molecule crude extraction was obtained by DM-301 macroreticular resin and was fractionated by HPLC(Agilent 1290 Infinity),the Peakl with antitumor activity was obtained; the crude protein with antitumor activity was obtained by ammonium sulfate precipitation and separation by Sephadex G-75, SDS-PAGE identified protein A molecular weigh was 30.2 kDa. The antitumor activity of Peakl and protein A were identified by LC-MS/MS showed that the molecular weight of Peakl was 158.03 KDa and protein A reveal its a ATP synthase subunit a.Active Peak1 (NZF1) and protein A(NZP1)acting on the B16,Hela, Hep-3B and HUVEC respectively. MTT assay found actived NZF1 and NZP1 had a highly toxicity to B16,Hep-3B and Hela,and inhibited the proliferation of three tumors in a dose-dependent manner and time-dependent manner, but had no obvious effect on HUVEC cell proliferation. Submicroscopic structure of B16,Hep-3B and Hela was impaired, nucleus shrinked and diminished, tubulin decreased and rounded up on the nucleus, then cell rounded, mitochondria fluorescence intensity weaken, but HUVEC was not obvious damaged by confocal using Hochest33342、tubulin Alexa Fluor 488 and Mito Tracker Red CMVRos. B16 treated on Peakl and proteinA was detected DNA Ladder by gel electrophoresis. Flow cytometry showed NZF1and NZP1 induced B16 apoptosis, and cell was blocked in sub-S phase and sub-G2 phase,respectively. Western blot found that Bax protein and PARP shear protein expression up-regulation and Bcl-2 protein expression down-regulation.There results above indicated that NZF1 and NZP1 had a highly toxicity to B16 and impaired submicroscopic structure of cell, induced B16 cell apoptosis by mitochondrial pathway and cell was blocked in sub-S and sub-G2 phase,resepectively. But had little toxicity to HUVEC obviously. This provided strain resources and laied theory foundation to the application of Nocardiopsis.sp as well as the research on mechanism of antitumor and new anti-cancer drugs.