Efeitos antitumorais do mastoparano e do peptídeo INKKI em modelos de melanomas experimentais “in vitro” e “in vivo”

Abstract

Mastoparan (INLKALAALAKKIL) is a α-helical and amphipathic tetradecapeptide obtained from wasp venom Vespula lewissi. Casoparan is a cationic peptide (INKKI) originated from the hydrolysis of bovine β-casein, exhibit a motif homology with mastoparan. The aim of this study was to evaluate the effect in vitro and in vivo of mastoparan and INKKI on an experimental melanoma model. Cytotoxicity was evaluated on tumor or non-tumor lines by MTT assay. INKKI effects on growth kinetics, adhesion, migration and invasion assay on B16F10 cells were evaluated. Study of the cell cycle phases was analyzed by flow cytometry. Cell signaling was carried out by Western blotting. Metastatic and dorsal model in vivo tumor was standardized using C57BL/6 male mice and NOD/SCID/γcnull mice. Simultaneously, tumor cell death by apoptosis induced by mastoparan was determined using the Annexin V-FITC/PI assay.The loss of mitochondrial membrane potential (∆Ψm) was evaluated by cytometry on staining cells with TMRE probe. Generation of reactive oxygen species (ROS) was performed usingDHE probe. DNA fragmentation was evaluated by electrophoresis in an agarose gel and chromatin condensation was assessed by DAPI staining. In vivo study was performed on dorsal melanoma model. The results showed that mastoparan presented cytotoxic activity and INKKI exhibited antiproliferative effects. In addition, date showed that INKKI was able to inhibit growth kinetics, cell adhesion, invasion and migration. Complement, protein analyzes showed decrease of GSK3β, phosphor Src Tyr 416, phosphor FAK Tyr 925 and 397 levels. Cell cycle analysis showed that the INKKI was able arrest in the G0/G1phases and decreased the proportion of the S phase. These data were confirmed by Western blotting, showing that INKKI reduces of Cyclin D1 and D3, NFК-β and MAPK/Erk1/2 expression. Additionally, the treatment with INKKI induced reducing metastasis, inhibit of melanoma cutaneous growth, increase of survive rate and did not showed antitumor activity on immunocompromised mice. The results with 12 mastoparan showed that peptide was able to induce increase positive cells populations for annexin V. Furthermore, induced decrease of ∆Ψm, increased generation (ROS), induced DNA fragmentation and chromatin condensation. Also, mastoparan increases the expression of cleaved caspases-3, -9 and -12, PARP, Bim, Bak, Cytochrome c as well as decrease of expression of phospho Bad (S112), VDAC, PHB1 and Bcl-2. Most importantly, mastoparan reduced the growth of subcutaneous melanoma and increases of survive. In conclusion, mastoparan and INKK presents different mechanism action. Mastoparan induces apoptosis in melanoma cells through of intrinsic mitochondrial pathway and INKKI induces arrest cell cycle. We also demonstrated that INKKI has antimetastatic effects and inhibited growth of the volume tumor on melanoma cutaneous. Mastoparan presented therapeutic effects in vivo.