| Melanoma is a highly deteriorate and invasive skin tumor. Melanoma progresses rapidly with poor prognosis and is generally resistant to normal chemotherapy, therefore lack of effective treatments. Recently, cationic antimicrobial peptides with anti-cancer activity gains increasingly widespread attention because of its unique mechanism of action. Our previous studies have found that Temporin-1CEa, a cationic antimicrobial peptide from the skin secretion of Chinese forest frog, specifically kills cancer cells with a broad anticancer-spectrum. In our present study, the selective anticancer activity of Temporin-1CEa and its six peptidic analogues against A375 melanoma cells was evaluated. Their anti-tumor angiogenesis and tumor metastasis and invasion inhibitory activities were further tested.We firstly evaluated the selective cytotoxicity of Temporin-1CEa and its peptides analogues against melanoma using human A375 melanoma cells and immortalized human epidermal cell Hacat cells by MTT method. The results showed that temporin-1CEa and its analogues exerted cytotoxicity against A375 cells with sequencing as LK2(6)AN(2L)>LK2(6)A(L)>Temporin-1CEa>LK1>LK3>LK2(6)>LK2(5), wherein Temporin-1CEa, LK1 and LK2(6)A(L) showed better cancer selectivity. In addition, by using Annexin V-FITC staining and flow cytometry analysis, we found A375 cells showed abundant membrane negatively charged components phosphatidylserine(PS) than Hacat cells, which might be one of the mechanisms underlying the selective anticancer activity of Temporin-1CEa and its analogues against A375 cells. Additionally, our results also found that NaClO3, which suppressing the expression of cell surface sulfate groups, can significantly enhance the anti-tumor activity of Temporin-1CEa, LK1 and LK2(6)A(L), suggesting that cell surface sulfate groups can block interactions between peptides and tumor cells.After demonstrating the selective cytotoxicity of Temporin-1CEa and its analogues against human melanoma peptide A375 cells, their anti-melanoma metastasis and invasion activity were further studied. The transwell migration/invasion assay showed that Temporin-1CEa, LK1 and LK2(6)A(L) significantly inhibited A375 cell migration and invasion, with an order as Temporin-1CEa>LK2(6)A(L)>LK1. In addition, the data from ELISA analysis revealed that Temporin-1CEa, LK1 and LK2(6)A(L) significantly inhibited the release of human matrix metalloproteinase-2(MMP-2) and vascular endothelial growthfactor(VEGF) with a dose-dependent manner, suggesting the tumor metastasis and invasion inhibitory activities of Temporin-1CEa and its analogues might be associated with reduced melanoma cell MMP-2 and VEGF expression and release.The tumor angiogenesis is also closely related to migration and invasion, therefore, we next tested the anti-angiogenic properties of Temporin-1CEa and its analogues. The results indicated that Temporin-1CEa, LK1 and LK2(6)A(L) can significantly inhibit the proliferation of human umbilical vein endothelial cells HUVEC in a dose-dependent manner;The cells scratches test also suggested that temporin-1CEa and its analogues significantly inhibited scratches recovery in HUVEC cells, with an order as LK2(6)A(L)>Temporin-1CEa>LK1. Meanwhile, Temporin-1CEa, LK2(6)A(L) and LK1 also has a significant inhibitory effect on HUVEC cells Transwell migration/invasion and Matrigel tube formation, with the potency order as Temporin-1CEa>LK2(6)A(L)>LK1. Moreover, the chicken chorioallantoic membrane angiogenesis experiment showed that Temporin-1CEa,LK1 and LK2(6)A(L) can inhibit angiogenesis in vivo.In summary, Temporin-1CEa and its peptidic analogues show selective cytotoxicity against A375 cells, and can inhibit the invasion and metastasis of tumor cells via MMP-2 and VEGF-related mechanisms, and has significant anti-angiogenic activity. These findings may be helpful for further structural transformation of antimicrobial peptide Temporin-1CEa to develop candidates for the clinical treatment of melanoma. |
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