Screening And Purification Of Antimicrobial Peptides With Anti-tumor Cells K562 Activity From The Tenebrio Molitor Linnaeus And Experimental Study Of Anti-K562 In Vitro Of The Antimicrobial Peptide

Objectives:1. To isolate and purify the antimicrobial peptides with anti-tumor cells K562 (human myeloid leukemia cells) activity from the Tenebrio molitor L. larvae.To carry out molecular identification of the purified antimicrobial peptide.2. To observe that whether the antimicrobial peptide with anti-tumor cells K562 activity had the role of anti-normal human cells 293T (human renal epithelial cells).To compare the inhibition of the antimicrobial peptide and that of hydroxyurea on proliferation of K562, and then to determine that whether the effect of anti-tumor of the antimicrobial peptide was better than that of hydroxyurea.3. To observe the effect of the antimicrobial peptide of Tenebrio molitor L. on cell cycle of K562.To observe the effect of the antimicrobial peptide of Tenebrio molitor L. on mitochondrial membrane potential and cysteinyl aspartic proteinase-3 (caspase-3) of K562. Thereby we would explore the mechanism of anti-K562 cells of the antimicrobial peptide.Methods:1. Antimicrobial peptides of Tenebrio molitor L. larvae induced by ultrasonic waves were isolated and purified by trituration, centrifugalization, solid phase extraction (SPE) and reversed-phase high-performance liquid chromatography (RP-HPLC).Then the antimicrobial peptides with anti-K562 cells activity were sieved by methyl thiazolyl tetrazolium (MTT) colorimetric method and light microscope observation. Protein nature of peaks purifid by RP-HPLC was detected using RP-HPLC in 280nm UV wavelength. Purified antimicrobial peptides with anti-K562 cells activity by RP-HPLC was identified with polyacrylamide gel electrophoresis (PAGE).2. Effect of screened peak 9 of antimicrobial peptides on anti-normal human cell 293T was detected by MTT method. We would find the inhibition rate of different concentrations of peak 9 of antimicrobial peptides and hydroxyurea acting on K562 by using MTT colorimetric method, and then find the regression equation and IC50 by using SPSS software according to the concentration and inhibition rate.3. To observe the effect of peak 9 of the antimicrobial peptide of Tenebrio molitor L. on cell cycle of K562 using FCM. The rhodamine 123 was used as a fluorescence probe to label the K562, and the effect of peak 9 of the antimicrobial peptide of Tenebrio molitor L. on mitochondrial membrane potential of K562 was observed using LSCM (laser scanning confocal microscope, LSCM). Caspase-3 enzyme activity was detected by caspase-3 detection kit and microplate reader.Results:1. Supernatant eluted with 10%,30%,80% acetonitrile(ACN) in aqueous solution by solid phase extraction.Analysis of variance(ANOVA) revealed that the differences between components of solid phase extraction and the PBS control group had statistical significance(F=43.502,P<0.01). Comparing each component and control group with the Dunnett-t method, only 80% was active(P<0.01). Relative inhibition rate of cells of the component (named sp8 group) was 65.64%. Light microscopic observation (×100):The field of vision in control group was clean. The entire field of vision in sp8 group was cluttered. They formed a vivid contrast. Light microscopic observation (×400):Cells were almost intact in control group. In sp8 group some cells had been decomposed completely; Only half or more than half of one cell in some cells was observed and the other half or less than half of one cell had been broken down into pieces; Some cell contents were released out through the gap due to the incomplete cell membrane and the formation of the gap; Some cells had deformed and would be broken down.24 peaks of sp8 group appeared after purification by RP-HPLC (214nm wavelength). ANOVA showed that the differences between peaks and the PBS control group had statistical significance (F=12.801,P< 0.01). Comparing each component and control group with the Dunnett-t method, peak1-4 and 9 were active(P<0.01). Relative inhibition rate of cells of the five peaks were 42.21%,50.76%, 63.86%,40.97% and 47.86% respectively. Two strong absorption peaks were found respectively at the retention time of 22.003 min and 15.469 min in the 280nm wavelength, which were consistent with the retention time 21.997 min of peak 9 and the position of peak 4 in the 214nm wavelength.280nm was the wavelength of the characteristic absorption peak of proteins containing aromatic amino acid. So, peak 9 and 4 could be identified as antimicrobial peptides, the others need to be proved. The images of role of 5 peaks of RP-HPLC in the K562 cells were similar in images to sp8 group in the light microscope (×100 and×400). There was also no significant difference between the 5 peak images. Light microscopic observation was the same as above. Clear peak 9 and 4 protein bands were obtained after PAGE. Therefore, Peak 9 and peak 4 were identified as antimicrobial peptides.2. Effect of peak 9 of antimicrobial peptides on anti-normal human cell 293T:t-test showed that the differences between antimicrobial peptides group and the PBS control group had no statistical significance (t=1.395, P=0.236>0.05), which showed that peak 9 had no role of anti-293T. Effect of antimicrobial peptides and hydroxyurea on inhibition of proliferation of K562 cells:regression equation of antimicrobial peptides and hydroxyurea were respectively Y=-12.33+8.35X and Y=-6.98+1.96X, and IC50 of that were respectively 29.98μg/m and 3 644.45μg/ml. Between the IC50 we could see a big difference which was that IC50 value of antimicrobial peptides was much smaller than that of hydroxyurea. The effect of antimicrobial peptides on inhibition of proliferation of K562 cells was significantly greater than effect of hydroxyurea on that.3. Effect of peak 9 of antimicrobial peptide on cell cycle of K562 cell detected by flow cytometry:The ratio of cells'G0/G1 phase and G2/M phase increased, S phase decreased. Comparing G0/G1 phase and S phase group with the PBS control group, the differences on the ratio of cells had statistical significance(P<0.01). It illustrated that the antimicrobial peptide would enable cell cycle block in the G0/G1 phase, thus inhibiting the DNA synthesis of S phase. Effect of peak 9 of the antimicrobial peptide on mitochondrial membrane potential and caspase-3 of K562 cells:ANOVA revealed that the differences on fluorescence value of rhodamine 123 between antimicrobial peptide's concentrations group (16μg/ml,24μg/ml,32μg/ml,40μg/ml) and the PBS control group had statistical significance(F= 16.909, P<0.01). Further analysis with LSD-t test revealed that except fluorescence value of 16μg/ml group was not significantly greater than that of 24μg/ml group, the differences between any other two groups were statistically significant(P<0.05). With the concentration increasing progressively, the number of cells, fluorescence value and fluorescence intensity decreased progressively. It showed that the antimicrobial peptide could reduce the mitochondrial membrane potential of K562 cells. Caspase-3 of the antimicrobial peptide group was significantly higher than that of PBS control group. There was statistical significance between the two groups (t=43.289, P=0.000).Conclusions:1. There are antimicrobial peptides and anti-bacterial substances which have anti-K562 activity in the Tenebrio molitor Linnaeus larvae. We get the peak 9 and 4 of the antimicrobial peptides. There relative molecular mass (Mr) are about 6×103(International standards require that "molecular mass" should be replaced by "relative molecular mass". Therefore we replace the "Da" with unit "1" of "relative molecular mass".). 2. Peak 9 almost had no role of anti-normal cells. This is consistent with most reported literature. Peak 9 is superior to hydroxyurea on inhibition of proliferation of K562 cells, and has good application prospects.3. Peak 9 may play an anti-tumor role by three ways:(1) to effect the cell cycle of K562 by inhibiting the DNA synthesis of S phase; (2) to inhibit the cell respiration by reducing the mitochondrial membrane potential; (3) to induce apoptosis through the channel of mitochondria.