Anticancer Activity Of Temporin,Antibacterial And Antioxidant Activity Of Lc-NK-lysin-1a

According to the reports of Chinese cancer registry annual in 2017,compared with 2012,the number of people diagnosed with cancer continues to rise from 3.58 million to 3.68 million by 3%.At the same time,the world's new cases were about 14.9 million.New cancer cases in China account for 25%of all cases in the world.At present,the most common methods of cancer treatment are surgery,chemotherapy and radiation therapy.Those treatments are often accompanied by serious side effects while achieving therapeutic effects,so these treatment strategies are not very successful.In addition,multidrug-resistant tumor cells may also appear which will affect the therapeutic effect.Therefore,the research of new antitumor drugs has become an urgent demand.Antibacterial peptides(AMPs)are highly effective and play an important role in the innate immune system against pathogens.It was found that AMPs had inhibitory effect on bacteria,and further more,they had good antitumor activity as well.Nowadays,there are more than 2950 antimicrobial peptides that have been reported,of which more than 210 of them have antitumor activity,and these peptides are also called anticancer peptides.The antibacterial peptide temporin-GHa and temporin-GHb cloned from Hainan Rana guentheri skin were found broad-spectrum,fast and efficient antibacterial activity and antifungal activity.The MTT experiment showed that the antibacterial peptide temporin-GHb also has broad spectrum and high effective antitumor activity.In the study,the antibacterial peptides temporin-GHa and temporin-GHb were synthesized by solid phase synthesis,purified and lyophilized.Finally,the antibacterial peptide was obtained with purity up to 95%.The structure of antimicrobial peptides in different solutions was analyzed by CD spectrum.The anti-tumor activity of temporin-GHa and temporin-GHb against several cancer cell lines(Human epithelial carcinoma cell line Hela,Human hepatocellular liver carcinoma cell line HepG2,Human colon cancer cell line HCT116,Human breast tumor cell line MDA-MB-231,Mouse glioma cells GL261)were studied by MTT cytotoxicity assay.The IC50 of temporin-GHb was 39.91 ?M,46.53 ?M,38.01 ?M,31.59 ?M and 39.21 ?M,respectively.The antitumor activity of temporin-GHb was selective and showed low toxicity to human embryonic kidney cell line,293FT.The study was also analysed the effect of temporin-GHa and temporin-GHb on migration of human breast cancer cells through wound scratch test.The results showed that temporin-GHb has a good inhibitory effect on the migration of breast cancer cells in 16.95 and 33.91 ?M after 24 h treatment.And the inhibition rate of cell migration reached to 36.38%and 93.19%respectively.However,the temporin-GHa could not inhibit cell migration at the same concertration.It has been found that temporin-GHb can induce apoptosis of MDA-MB-231 cells,through the analysis on the cell morphology observation,DNA changes and cell membrane phosphatidylserine changes.And the apoptosis rate reached 34.37%after the cells were treated with 33.91 ?M for 15 h.In the study of the apoptosis mechanism,it has been found that temporin-GHb can increase the intracellular ROS level,depolarize the mitochondrial membrane potential and destroy the physiological function of mitochondria.In addition,the result of q-PCR showed that temporin-GHb can promote the expression of Bax and Caspase-3 genes and inhibit the expression of Bcl-2 gene.Though Lc-NK-lysin-1a has no anti-tumor activity,it has been found that Lc-NK-lysin-la has good antibacterial and antioxidant activity through agar plate drilling experiment and DPPH radical scavenging test.Lc-NK-lysin-1a was effective to inhibit E.coli and S.aureus growth.And the MIC of Lc-NK-lysin-1a is 11.46-22.91?M.Lc-NK-lysin-la showed a concentration-dependent killing on bacteria,and leading bacteria to die by destroying the membrane of cell.Lc-NK-lysin-la can remove 66.72%free radicals of DPPH,when the concention of Lc-NK-lysin-la was 1.47 mM.The results showed that Lc-NKlysin-la can protect 293FT cells from H2O2 injure.It was found that the antibacterial activity and antioxidant activity had good stability after the treatment with different factors on Lc-NK-lysin-1a.