Preliminary Experimental Anticancer Mechanism Of Domestica Larva Antimicrobial Peptides To Tumor Cells K562

Objectives:1. To isolate and purify the antimicrobial peptides from the Musca domestica larvae withinhibition activity to K562 cells in chronic myelocytic leukemia, and to observe whether damagethe normal-human cells 293T (human renal epithelial cells).2. To observation the influence of the antimicrobial peptides purified to the K562 cellviability and the growth curve and to observation the influence of the antimicrobial peptidespurified to the permeability of K562 cell membrane, in order to evaluate the optimalconcentration of active play and time, and to evaluate the degree of the K562 cell membranedamage by the the antimicrobial peptides purified, and if the mechanism maybe directly killing;3. To observation the influence of the antimicrobial peptides purified to the nucleus andmitochondrial membrane potential and apoptotic proteins caspase (caspase-3) of K562 cells, andto explore the mechanism of antimicrobial peptides inhibiting K562 whether maybe to induceapoptosising.Methods1. Antimicrobial peptides of Musca domestica larvae with inhibition activity to K562 cellswere sieved, through some processes that induced by acupuncture with E.coli induction, isolatedand purified by trituration, centrifuga1ization, solid phase extraction (SPE) and reversed-phasehigh-performance liquid chromatography (RP-HPLC), to detect the activity to anti-tumor cellsK562 by MTT colorimetric method and observed light microscope. To identify the proteinnature of the peaks puried by comparing the RP-HPLC chromatogram when the wavelength areUV 214 nm and 280 nm, and to detect the effect to normal-human cells 293T by MTTcolorimetric method.2. Recording the survival and the relative inhibition rate to make growth curve, just aftertreating with different concentrations of the antimicrobial peptides and different time to K562,stained by trypan blue exclusion counting. To observe the best concentration and time playingthe role of inhibit; also to observe the changes of cell morphology.3. Small molecule fluorescein FDA (Fluorescein Diacetate) labeled ,to detect theFluorescein leakage in the cell membrane by using Fluorescence spectrophotometer;Macromolecular fluorescent probe Dextran-FITC (40KD) labeled,to observ the K562 cell membrane permeability of macromolecules by using Laser scanning confocal microscope (laserscanning confocal microscope, LSCM) .4. To observ the fluorescence intensity of the nucleus with fluorescence microscope, usinghoechst33258 fluorescent labeled, after the antimicrobial peptide of Musca domestica Larvaepeaks 5 and 8 effect the K562 cells; then to observ the fluorescence intensity of the rhodamine123 in K562 cells with LSCM (laser scanning confocal microscope, LSCM), the intracellularfluorescence intensity indicating the mitochondrial membrane potential and the rhodamine123 asa fluorescence probe to label the K562；finally,to detect the enzyme activity of K562 cells'caspase-3 with caspase-3 immuosorbent and microplate.Results:1. Supernatant eluted with 10%, 30%, 80%acetonitrile (ACN) in aqueous solution by solidphase extraction. Analysis of variance (ANOVA) revealed that the differences betweencomponents of solid phase extraction and the control group had statistical significance (P<0.05).Comparing each component and the control group with the Dunnett-t method, the differenceshad statistically significant (P<0.05). Relative inhibition rate of cells of the component (namedsp8 group) was 84.98%.The sp8 fraction was purified by RP-HPLC, to observe the chromatograms under thecondition of the wavelength were 214nm and 280nm, in addition, the 280nm wavelength is thecharacterisitic wavelength of proteins containing aromatic amino acid absorption, so that thepeaks 5~8 compenent are antimicrobial peptides. According ti the criteria of separation, becausethe separation of the four peaks were more than 1.5, which meeting the requirements ofquantitative analysis, so these four peaks were selected to experiments follw-up. Comparing eachthe component of peaks 5~8 with control group by the Dounnett-t method, the differences ofeach had inhibit activity and the K562 cells. The peaks 5 and 8 had the better relative inhibitoryrate when the concentration was 100μg/ml and the time was 24 hours, respectively 48.52% and65.80%. Sp8 and peaks 5 to 8components, respectively, compared with the control group byt-test, the differences were not statistically significant (P<0.05), which showed that theantimicrobial peptides sp8 component and the peaks 5~8 components had not inhibit effect tohuman-normal cells 293T.2. It was found that each component had inhibit activity on K562, but not a linearrelationship, after 24 hours treating with different concentrations sp8 and peaks 5~8 of theantimicrobial peptides to K562, stained by trypan blue dye resist counting. There's almost noeffect to K562 calls when the concentration was 25μg/ml,and it's lower when 50μg/ml, it'schangedfrom less than half to more than half of the inhibited when100ug/ml,especially thegroups of peaks 5 and 8. The difference between the lower concentration groups (<25μg/ml) and the control group (0μg/ml) were not statistically significant (P>0.05); the difference between thehigher concentration group (>50μg/ml) and the lower groups (<25μg/ml) were statisticallysignificant (P<0.05). When the concentration were 50μg/ml and 100μg/ml, the groups of peaks5 and 8 with the groups of peaks 6 and 7, respectively, the difference were statistically significant(P<0.05).With treated by the concentration 100μg/ml and 24 hours, K562 cells were observed lightmicroscopic observation(×100): cells were almost intact in control group, some cells had beendecompose completely, another were broken dowen into pieces, else cells'concents werereleased out through the gap due to the incomplete cell membrane and the formation of the gap,others had deformed and would be broken down；the field of vision in group was clean.Theentire field of vision in the groups of sp8 and peaks 5 and 8 were cluttered. When theconcentration of each compenents of sp8 and peaks 5~8 was 50μg/ml and 100μg/ml, with theincreasing of reaction time, the inhibition rate to K562 cells was increased gradually, about 24hours later reached a peak and then decreased , when the medium was changed after 48 hours,the inhibition rate was gradually increased again, and then decline; when the concentration was200μg/ml, with the reaction time increasing, the inhibition rates to K562 cells continued rising,when the mediun was changed after 48 hours, the inhibition rate increased significantly and thencontinued to rise. In addition, the inhibition rate of K562 cells showed no linear relationship,bother before and after treatment at 48 hours.3.After the K562 cells were treated with different concentration of peaks 5 and 8, therelative fluorescence intensity was between (18.95±0.05) and (22.49±0.68) which groups'concentration was below 50μg/ml, the relative fluorescence intensity was between (62.77±4.08)and (70.81±0.18) which groups'concentration was more than 50μg/ml.The single factor analysisof variance, respectively, the relative fluorescence intensity of each group between the higherconcentration groups (>50μg/ml), and the lower concentration (<50μg/ml) were pariwisecomparied by LSD comparision method, the difference were significant (P<0.05).After 2 hourstreated with peaks 5and 8, the small molecule fluorescein of FDA wasleakaged from the K562cell membrane.Conclusion:1. There are some antimicrobial peptides and anti-bacterial sustances which have anti-K562activity in the Musca domestica larva. The antimicrobial peptides of the peaks 5 ~8 were puried,in which the peaks 5 and 8 had the higher activity, and had no effect to human-normal cells 293T,just similar with the papers reported.2. There exists a concentration threshold of Musca domestica larva antimicrobial peptideson K562 cell membrane. The lower concentrations can couse the membrane leakage increasing of cell membrane, the higher concentrations can lead to severe damage of cell membrane,respectively. The antimicrobial peptides inhibit the growth of K562 cells through directly killingthe cells, and the mechanism is that firstly damage the membrane function, then may eapand theholes or may inhibit the growth by other ways after entering the cells.3. The antimicrobial peptides of peaks 5 and 8 can damage the nucles of K562, and lead toapoptosising of K562, the mechanism is reducing the mitochondrial membrane potential of K562cells, activating the caspase-3, disrupting the physiological fuction and accelerating theapoptosising.