Research On The Purification And Mechanism Of An Anti-Tumor Peptide From Musca Domestica Pupae

Musca domestica is an important resource in the study of insects, which grows in the environment full of pathogens all their lives and spread diseases between humans and animals, but themselves are not infected with pathogens. There is a class of biologically active substances in Musca domestica bodies. Some lectins, which have been isolated and characterized from Musca domestica larvae/pupae could inhibit proliferative of tumor cells. However, the research of peptides and their anti-tumor activities was rare.In this study, an anti-tumor peptide from Musca domestica pupae (MATP) has been purified by gel filtration chromatography and anion-exchange chromatography. MATP was a single-component and its molecular weight was18kDa which was confirmed by Tricine-SDS-PAGE and HPLC. The activity against HepG2cells of MATP was determined with MTT assay, which showed that the anti-tumor process was dose-dependent. The results of HE staining, AO/EB staining and Hoechst33342/PI staining indicated that MATP inducing HepG2cells apoptosis based on the typical apoptotic morphological changes. Flow cytometric analysis (FCM) demonstrated that MATP caused apoptosis of HepG2cells through cells arrested at S phase and the apoptotic rates significantly increased.The results of fluorescence dyeing and Western blot showed that the generation of intracellular reactive oxygen species (ROS) was increased in HepG2cells treated with MATP and ROS induced a sustained activation of the phosphorylation of JNK, but not ERK and p38. Simultaneously, the results of FCM analysis indicated that the apoptosis induced by MATP was reversed by NAC (ROS inhibitor) and SP600125(JNK inhibitor). These results proved that ROS/JNK participated in apoptosis of HepG2treated with MATP. Moreover, the results of Western blot described that MATP induced an obvious inactivation of phosphorylated-AktSer473which prevented IkBα from degeneration and this change did not rely on the production of ROS. Simultaneously, the results of FCM analysis testified that the apoptosis induced by MATP was aggravated by LY294002(Akt inhibitor). These findings suggested that Akt was involved in the apoptosis of HepG2treated with MATP, too.The results of immunofluorescence and Western blot showed that the activation of phosphorylated-JNK together with the inactivation of phosphorylated-AktSer473inhibited NF-kB p65to enter the nucleus. Simultaneously, the results of FCM analysis indicated that the apoptosis induced by MATP was increased by PDTC (NF-kB p65inhibitor). These results proved that the MATP induced apoptosis through a JNK-and Akt-mediated NF-kB pathway. The results of Western blot described that the suppression of NF-kB p65entering the nucleus induced the decreases of Bcl-2. Simultaneously, MATP induced the expression increase of Bax but this mechanism did not dependent on the decrease of NF-kB p65in nucleus. These findings evidenced that the MATP induced apoptosis through the increase of Bax-to-Bcl-2expression ratio. The release of Cytochrome c (Cyto c) from mitochondria which intensified the expression of Caspase-9and Caspase-3was enhanced by the increase of Bax-to-Bcl-2expression ratio. The apoptosis of HepG2cells was induced ultimately by the increase of Caspase-3. Taken together, these findings suggested that the MATP induced apoptosis through a ROS/JNK-and Akt-mediated NF-kB pathway.