| Melanoma is a highly malignant skin tumour,which is facing significant challenges in developing therapy drugs.Antimicrobial peptides(AMPs)are a cationic polypeptide that inhibits tumor growth through several mechanisms of action.In recent years,antimicrobial peptides such as LTX-315 and LL-37 have entered clinical trials as melanoma therapeutics.Polybia-CP(CP)is an α-helical antimicrobial peptide containing 12 amino acids isolated and purified from wasp venom.In the preliminary work,our laboratory substituted amino acids for CP and introduced mitochondrial targeting group TPP to obtain a series of analogues.The lead compound CP-59 with high anti-tumor activity was screened out by preliminary activity evaluation.A single intratumoral administration of CP-59(1mg/ piece)resulted in a 62.5% tumor cure rate and no recurrence within 60 days.The therapeutic effect of CP-59 was significantly higher than LTX-315,which is an antimicrobial peptide that has entered clinical trials.The antitumor mechanism of CP-59 and CP-15 was studied in this paper.Firstly,we studied the membrane lysis activity of antimicrobial peptides.The results showed that CP-59 could rapidly cause B16F10 cells to rounding,swelling and rupturing at high concentration(40 μM).When cells died with typical necrotic characteristics,PI entered the cells.At lower concentrations,CP-59 takes longer to kill cells.Although CP-15 showed high antitumor activity after prolonged incubation,the antitumor activity was significantly lower than that of CP-59 in the short term.This may be because the high hydrophobicity leads to the decrease of the membrane cracking activity of CP-15.Secondly,we studied mitochondrial damage caused by CP-15 and CP-59.The results of mitochondrial swelling showed that both CP-15 and CP-59 could damage the isolated mitochondria.Flow cytometry results showed that CP-15 and CP-59 labeled by FITC could enter into cells at low concentrations(5 μM),and CP-59 had higher membrane penetration efficiency.The results of laser confocal experiments showed that FITC-labeled CP-59 could aggregate in mitochondria.This suggests that CP-59 can target intracellular mitochondria.When CP-59 acted on B16F10 cells,it can lead to the decrease of mitochondrial membrane potential,the increase of mitochondrial ROS and cytoplasmic ROS,and the decrease of cytoplasmic ATP.Mitochondrial damage may induce cell apoptosis.Therefore,the effects of CP-15 and CP-59 on intracellular Caspase-3 activity were studied in this paper.The results showed that CP-59 could improve the activity of caspase-3 more than CP-15,and its antagonist Z-VAD-FMK could reduce the cytotoxicity induced by CP-59.This suggests that CP-59 can induce mitochondria-dependent apoptosis.In addition,Nec-1,an inhibitor of RIP1,did not significantly reduce the cytotoxicity of CP-15 and CP-59,suggesting that necroptosis was not the main cause of cytotoxicity of CP-15 and CP-59.Finally,we studied the antitumor mechanism of CP-59 in vivo.In a C57BL/6melanoma model,results showed that treatment with CP-59 on the right tumor significantly inhibted the growth of the left tumor.This suggests that CP-59 can activate the systemic antitumor immune response and inhibit tumor growth through the abscopal effect.In the present study,the anticancer activity and mechanism of action of the antimicrobial peptide CP-15 and 59 were explored.The experimental results of this paper show that CP-59 can rapidly destroy cell membrane and lead to cell necrosis at high concentration.In addition,CP-59 can enter the mitochondria of cells at low concentration and induce apoptosis by damaging mitochondria,but did not cause obvious cell necroptosis.The results of in vivo anti-tumor experiments showed that CP-59 could not only directly kill cells and inhibit the growth of melanoma,but also inhibit the growth of tumor by activating the systemic antitumor immune response.The results of this study further demonstrate that CP-59 can be a potential lead compound for tumor therapy. |
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