The Effect And Preliminary Mechanism Of Antimicrobial Peptides Anoplin And PFR On The Proliferation Of Leukemic Cells

Antimicrobial peptides (AMPs) are peptides generated in the biological defense systemagainst exogenous pathogens, which is an important constituent of the non-specific immunesystem. Studies show that AMPs could kill the Gram-positive bacteria and Gram-negativebacteria significantly, and a small part of them also have the ability to inhibit the proliferation oftumor cells. Anoplin is an AMP isolated from the venom sac of the spider wasp AnopliusSamariensis and is one of the smallest, linear,-helical and amphipathic AMP found naturally todate consisting of only10amino acids. Previous results show that anoplin possesses potentantimicrobial activity against both Gram-positive and Gram-negative bacteria but little hemolyticactivity toward human erythrocytes. The human lactoferrin is an iron ion-binding glycoproteinwith the molecular weight of80kDa which contains a short-helix of the11amino acidsfragment (LF11peptide) in the N-terminus and this fragment has obvious effect againstGram-negative and Gram-positive bacteria."PFR peptide" is a small AMP of9amino acidswhich was designed and synthesized based on the structure-activity relationship of LF11peptide.Considering the fact that the leukemia is one of the top ten high incidence of malignancy,therefore, further studies about the effect of both AMPs mentioned above on the proliferation ofleukemia cells are very important.In this study, three kinds of leukemia cells (human chronic myelogenous leukemia cell lineK562cells, human acute myeloid leukemia cell line HL-60cells and mouse erythroleukemia cellline MEL cells) were used to investigate the effect of both AMPs on leukemia cells, andpaclitaxel was used as a positive control. The inhibitory effect on the proliferation of leukemiccells induced by incubation with AMPs was detected by MTT assay and microscopic observation.The changes of the cell membrane permeability induced by AMPs were investigated using themethod of the propidium iodide (PI) uptake combined with the observation of invertedfluorescent microscope. Additionally, the scanning electron microscopy was used to observe thechanges of the morphology of cell membrane. Moreover, flow cytometry was used to investigatethe cell cycle distribution and apoptosis, as well as the stain assay of Hoechst33342was used toobserve the apoptosis phenomenon. The results of MTT assay and microscopic observation showed that both anoplin and PFRhardly affect the growth of bone marrow cells isolated from mice, but both anoplin and PFR, aswell as paclitaxel, could inhibit the proliferation of leukemia cells in a dose-and time-dependentmanner. The strength order of anoplin on these three kinds of leukemia cell lines wasMEL>K562>HL-60, while the strength order of PFR peptide was MEL>HL-60>K562. Theinhibitory effect of PFR peptide is superior to that of anoplin on the three kinds of leukemia celllines. The results of PI staining showed that the membrane permeability of leukemia cells waschanged obviously, in which the red fluorescence was not detected in the control group while thecells treated by AMPs appeared red fluorescence brightly. The membranes of these leukemiacells were changed obviously under the test of scanning electron microscopy, in which the cellmembranes were corrugativus and ruptured, and there were holes on the surface. The resultsmentioned above indicated that the leukemia cells were killed by the AMPs through thedestruction of the structure of cell membrane firstly. The results of cell cycle distribution testedby flow cytometry showed that the AMPs could block the cell cycle in G0/G1phase significantly.The results of the fluorescent staining using Hoechst33342showed that the nuclei of leukemiacells treated by the AMPs appeared bright blue fluorescence, but there was no typical apoptosisbody. The results of apoptosis tested by flow cytometry showed that there was no typical peak ofearly apoptosis and late apoptosis, which was consistent with the results of fluorescent stainingusing Hoechst33342.In short, the results of this study show that the antimicrobial peptides anoplin and PFR doesnot affect the proliferation of mouse bone marrow cell, but they can inhibit the proliferation ofleukemia cells selectively and the inhibitory effect of PFR is superior to that of anoplin. Inaddition, the mechanism of the AMPs to inhibit the proliferation of leukemia cells may bethrough the destruction of the structure of cell membrane firstly, such as perforation on the cellmembrane. Then the intracellular events are involved, especially the blockade of the cell cycle,which induce the inhibition of the proliferation of leukemia cells.