抗菌肽Temporin-1CEa及其改造肽抑制血管新生和黑色素瘤转移侵袭的作用研究

Study on inhibitory effect of antimicrobial peptide Temporin-1CEa and its analogues inhibit metastasis and invasion of human melanoma A375 cells and reduce angiogenesis

目的筛选抗菌肽Temporin-1CEa及其6种改造肽对人黑色素瘤A375细胞增殖的抑制作用,并考察其在体外对A375细胞转移侵袭和血管新生的影响。方法 MTT法检测细胞生长抑制率;细胞划痕实验和Transwell实验检测细胞的迁移/侵袭能力;体外基质胶小管实验检测血管生成,ELISA实验检测细胞MMP-2和VEGF的释放量。结果 Temporin-1CEa及其改造肽LK1,LK2(6)A(L),对A375细胞增殖具有较强的抑制活性,而对人永生化表皮细胞Ha Cat的细胞毒作用较弱;同时,这3种肽对人脐静脉血管内皮细胞HUVEC的增殖亦具有较强的抑制作用,提示其可能具有血管新生抑制活性。HUVEC细胞划痕实验、Transwell实验和体外基质胶小管实验,进一步证实这3种肽确实能够有效的抑制HUVEC细胞的迁移,并对血管新生产生浓度依赖性的抑制作用。A375细胞Transwell实验结果表明,这3种肽还能够显著地抑制A375细胞的迁移和侵袭。ELISA实验结果表明,这3种肽的上述作用可能与其显著降低A375细胞的MMP-2和VEGF释放量有关。结论 Temporin-1CEa及其改造肽LK1和LK2(6)A(L)不仅对黑色素瘤A375细胞具有较强的选择性细胞毒作用,并抑制其迁移和侵袭,还具有较好的血管新生抑制活性,有望成为新型的抗黑色素瘤治疗药物。

Objective To screen the inhibitory effects of antimicrobial peptide Temporin-lCEa and its analogues on human melanoma A375 cells proliferation. And to evaluate their impacts on A375 cells metastasis and invasion and their angiogenesis reduction activity in vitro further. Methods Cell proliferation inhibition ratio was detected by MTT assay. The ability of migration/invasion was determined by wound/healing test and transwell test. Tubule formation assay was performed to investigate the effects of peptides on angiogenesis. The release of MMP-2 and VEGF was detected by ELISAI Results MTT results indicated that Temporin-1CEa and two of its analogues, LK1 and LK2 （6）A （L）, significantly inhibited A375 cells proliferation without Significant eytotoxieity to human keratinocyte HaCaT cells. Moreover, the proliferation of human umbilical vein endothelial cells （HUVEC） was also inhibited by these peptides, suggesting an angiogenesis inhibitory activity. The data from HUVEC wound/healing test, transwell test and tubule formation assay further confirmed the migration inhibition and angiogenesis reduction activities of these three peptides, in a dose-dependent manner. In addition, these peptides also inhibited the migration of A375 cells in transwell test. Consistently, the ELISA data further supported that the metastasis/ invasion inhibitory activities and angiogenesis reduction potential of tempofin-1CEa and their analogues on A375 cells might be due to the decreased MMP-2 and VEGF secretion. Conclusion Tempofin-lCEa and its analogues not only exerte potent and selective cytotoxicity to A375 cells, but also inhibite A375 cells metastasis and invasion and reduce angiogenesis, therefore, have the potential to be a new type anti-melanoma drugs.