Alphastatin-C a new inhibitor of endothelial cell activation is a pro-arteriogenic agent in vivo and retards B16-F10 melanoma growth in a preclinical model

Most characterized angiogenic modulators are proteolytic fragments of structural plasma and/or matrix components. Herein, we have identified a novel anti-angiogenic peptide generated by the in vitro hydrolysis of the C-terminal moiety of the fibrinogen alpha chain, produced by the snake venom metalloprotease bothropasin (SVMP), a hemorrhagic proteinase in Bothrops jararaca venom. The 14-amino acids peptide (alphastatin-C) is a potent antagonist of basic fibroblast growth factor, induced endothelial cell (HUVEC-CS) proliferation, migration and capillary tube formation in matrigel. It also inhibits cell adhesion to fibronectin. The basis of the antagonism between bFGF and alphastatin-C is elucidated by the inhibition of various bFGF induced signaling pathways and their molecular components modification, whenever the combination of the stimuli is provided, in comparison to the treatment with bFGF only. To corroborate to the potential therapeutic use of alphastatin-C, we have chosen to perform in vivo assays in two distinct angiogenic settings. In chick model, alphastatin-C inhibits chorioallantoic membrane angiogenesis. In mouse, it efficiently reduces tumor number and volume in a melanoma model, due to the impairment of tumor neovascularization in treated mice. In contrast, we show that the alphastatin-C peptide induces arteriogenesis, increasing pial collateral density in neonate mice. alphastatin-C is an efficient new antiangiogenic FGF-associated agent in vitro, it is an inhibitor of embryonic and tumor vascularization in vivo while, it is an arteriogenic agent. The results also suggest that SVMPs can be used as in vitro biochemical tools to process plasma and/or matrix macromolecular components unraveling new angiostatic peptides.

Identification of very small cancer stem cells expressing hallmarks of pluripotency in B16F10 melanoma cells and their reoccurrence in B16F10-derived clones

Melanoma is characterized by high heterogeneity and plasticity, most likely due to the presence of mutated melanocyte stem cells or immature progenitor cells in the skin that serves as precursors to melanoma. In the present study, for the first time, we identified rare cells in the murine melanoma B16F10, and human A2058 and SK-MEL-28?cell lines that express pluripotency markers, including Oct4, Nanog, Sox2 and a marker of melanoma cancer cells (ALDH1/2). These cells are very small with round morphology and they grow onto melanoma cells, thereby demonstrating feeder layer dependence similar to that of other pluripotent cells. These cells underwent self-renewal, symmetric and asymmetric division. We called these cells murine very small cancer stem cells (VSCSC). VSCSC were also found in B16F10-derived clones after 3–5 consecutive passages, where they occur as single cells or as small colonies, nevertheless, always using melanoma cells as feeders. These cells formed melanospheres enriched with Oct4-and ALDH1/2-positive cells. We also evaluated the possible effect of VSCSC that presented in the parental cell line (B16F10) and in clones based on their functional characteristics. We found that VCSCS present in the B16F10?cell line reappearing in their clones were required for continuous tumor growth and were responsible for melanoma cell heterogeneity and plasticity rather than directly affecting functional characteristics of melanoma cells. Our data, together with those of previous reports suggested the existence of melanoma-competent melanocyte stem cells, which corroborate the hypothesis of the existence of tumor-initiating cells and cancer stem cell hierarchies, at least in melanoma

Identification of very small cancer stem cells expressing hallmarks of pluripotency in B16F10 melanoma cells and their reoccurrence in B16F10-derived clones

Melanoma is characterized by high heterogeneity and plasticity, most likely due to the presence of mutated melanocyte stem cells or immature progenitor cells in the skin that serves as precursors to melanoma. In the present study, for the first time, we identified rare cells in the murine melanoma B16F10, and human A2058 and SK-MEL-28?cell lines that express pluripotency markers, including Oct4, Nanog, Sox2 and a marker of melanoma cancer cells (ALDH1/2). These cells are very small with round morphology and they grow onto melanoma cells, thereby demonstrating feeder layer dependence similar to that of other pluripotent cells. These cells underwent self-renewal, symmetric and asymmetric division. We called these cells murine very small cancer stem cells (VSCSC). VSCSC were also found in B16F10-derived clones after 3–5 consecutive passages, where they occur as single cells or as small colonies, nevertheless, always using melanoma cells as feeders. These cells formed melanospheres enriched with Oct4-and ALDH1/2-positive cells. We also evaluated the possible effect of VSCSC that presented in the parental cell line (B16F10) and in clones based on their functional characteristics. We found that VCSCS present in the B16F10?cell line reappearing in their clones were required for continuous tumor growth and were responsible for melanoma cell heterogeneity and plasticity rather than directly affecting functional characteristics of melanoma cells. Our data, together with those of previous reports suggested the existence of melanoma-competent melanocyte stem cells, which corroborate the hypothesis of the existence of tumor-initiating cells and cancer stem cell hierarchies, at least in melanoma

Capsaicin-like analogue induced selective apoptosis in A2058 melanoma cells: design, synthesis and molecular modeling

The use of molecules inspired by natural scaffolds has proven to be a very promising and efficient method of drug discovery. In this work, capsaicin, a natural product from Capsicum peppers with antitumor properties, was used as a prototype to obtain urea and thiourea analogues. Among the most promising compounds, the thiourea compound 6g exhibited significant cytotoxic activity against human melanoma A2058 cells that was twice as high as that of capsaicin. Compound 6g induced significant and dose-dependent G0/G1 cell cycle arrest in A2058 cells triggering cell death by apoptosis. Our results suggest that 6g modulates the RAF/MEK/ERK pathway, inducing important morphological changes, such as formation of apoptotic bodies and increased levels of cleaved caspase-3. Compared to capsaicin, 6g had no significant TRPV1/6 agonist effect or irritant effects on mice. Molecular modeling studies corroborate the biological findings and suggest that 6g, besides being a more reactive molecule towards its target, may also present a better pharmacokinetic profile than capsaicin. Inverse virtual screening strategy found MEK1 as a possible biological target for 6g. Consistent with these findings, our observations suggested that 6g could be developed as a potential anticancer agent.

Capsaicin-like analogue induced selective apoptosis in A2058 melanoma cells: design, synthesis and molecular modeling

The use of molecules inspired by natural scaffolds has proven to be a very promising and efficient method of drug discovery. In this work, capsaicin, a natural product from Capsicum peppers with antitumor properties, was used as a prototype to obtain urea and thiourea analogues. Among the most promising compounds, the thiourea compound 6g exhibited significant cytotoxic activity against human melanoma A2058 cells that was twice as high as that of capsaicin. Compound 6g induced significant and dose-dependent G0/G1 cell cycle arrest in A2058 cells triggering cell death by apoptosis. Our results suggest that 6g modulates the RAF/MEK/ERK pathway, inducing important morphological changes, such as formation of apoptotic bodies and increased levels of cleaved caspase-3. Compared to capsaicin, 6g had no significant TRPV1/6 agonist effect or irritant effects on mice. Molecular modeling studies corroborate the biological findings and suggest that 6g, besides being a more reactive molecule towards its target, may also present a better pharmacokinetic profile than capsaicin. Inverse virtual screening strategy found MEK1 as a possible biological target for 6g. Consistent with these findings, our observations suggested that 6g could be developed as a potential anticancer agent.

Murine melanoma cells incomplete reprogramming using non-viral vector

Objectives: The reprogramming of cancer cells into induced pluripotent stem cells or less aggressive cancer cells can provide a modern platform to study cancer-related genes and their interactions with cell environment before and after reprogramming. Herein, we aimed to investigate the reprogramming capacity of murine melanoma B16F10 cells. Materials and methods: The B16F10 was transfected using non-viral circular DNA plasmid containing the genes Sox-2, Oct4, Nanog, Lin28 and green fluorescent protein (GFP). These cells were characterized by immunofluorescence, analysis RT-PCR and cell cycle. Results: Our results demonstrated for the first time that reprogramming of B16F10 may be induced using non-viral minicircle DNA containing the four reprogramming factors Oct4, Sox2, Lin 28, Nanog (OSLN) and the GFP reporter gene. The resulting clones are composed by epithelioid cells. These cells display characteristics of cancer stem cells, thus expressing pluripotent stem cell markers and dividing asymmetrically and symmetrically. Reprogrammed B16F10 cells did not form teratomas; however, they showed the suppression of tumourigenic abilities characterized by a reduced tumour size, when compared with parental B16F10 cell line. In contrast to parental cell line that showed accumulation of the cells in S phase of cell cycle, the cells of reprogrammed clones are accumulated in G1 phase. Long-term cultivation of reprogrammed B16F10 cells induces regression of their reprogramming. Conclusions: Our data imply that in result of reprogramming of B16F10 cells less aggressive Murine Melanoma Reprogrammed Cancer Cells may be obtained. These cells represent an interesting model to study mechanism of cells malignancy as well as provide a novel tool for anti-cancer drugs screening.

The Ig V-H complementarity-determining region 3-containing Rb9 peptide, inhibits melanoma cells migration and invasion by interactions with Hsp90 and an adhesion G-protein coupled receptor

The present work aims at investigating the mechanism of action of the Rb9 peptide, which contains the VHCDR 3 sequence of anti-sodium-dependent phosphate transport protein 2B (NaPi2B) monoclonal antibody RebMab200 and displayed antitumor properties. Short peptides corresponding to the hyper variable complementarity-determining regions (CDRs) of immunoglobulins have been associated with antimicrobial, antiviral, immunomodulatory and antitumor activities regardless of the specificity of the antibody. We have shown that the CDR derived peptide Rb9 induced substrate hyperadherence, inhibition of cell migration and matrix invasion in melanoma and other tumor cell lines. Rb9 also inhibited metastasis of murine melanoma in a syngeneic mouse model. We found that Rb9 binds to and interferes with Hsp90 chaperone activity causing attenuation of FAK-Src signaling and downregulation of active Rac1 in B16F10-Next melanoma cells. The peptide also bound to an adhesion G-protein coupled receptor, triggering a concentration-dependent synthesis of cAMP and activation of PKA and VASP signaling as well as IP-3 dependent Ca2+ release. Hsp90 is highly expressed on the cell surface of melanoma cells, and synthetic agents that target Hsp90 are promising cancer therapeutic drugs. Based on their remarkable antitumor effects, the CDR-H3-derived peptides from RebMab200, and particularly the highly soluble and stable Rb9, are novel candidates to be further studied as potential antitumor drugs, selectively acting on cancer cell motility and invasion. (C) 2016 Elsevier Inc. All rights reserved.

BFD-22 a new potential inhibitor of BRAF inhibits the metastasis of B16F10 melanoma cells and simultaneously increased the tumor immunogenicity

Benzofuroxan is an interesting ring system, which has shown a wide spectrum of biological responses against tumor cell lines. We investigated, herein, the antitumor effects of benzofuroxan derivatives (BFDs) in vitro and in a melanoma mouse model. Cytotoxic effects of twenty-two BFDs were determined by MIT assay. Effects of BFD-22 in apoptosis and cell proliferation were evaluated using Annexin V-FITC/PI and CFSE staining. In addition, the effects in the cell cycle were assessed. Flow cytometry, western blot, and fluorescence microscopy analysis were employed to investigate the apoptosis-related proteins and the BRAF signaling. Cell motility was also exploited through cell invasion and migration assays. Molecular docking approach was performed in order to verify the BFD-22 binding mode into the ATP catalytic site of BRAF kinase. Moreover, the BFD-22 antitumor effects were evaluated in a melanoma murine model using B16F10. BFD-22 was identified as a potential hit against melanoma cells. BFD-22 induced apoptosis and inhibited cell proliferation of B16F10 cells. BFD-22 has suppressed, indeed, the migratory and invasive behavior of B16F10 cells. Cyclin D1 and CDK4 expression were reduced leading to cell cycle arrest at G0/G1 phase. Of note, phosphorylation of BRAF at Ser338 was strongly down-regulated by BFD-22 in B16F10 cells. The accommodation/orientation into the binding site of BRAF was similar of BAY43-9006 (co-crystallized inhibitor of BRAF, sorafenib). Importantly, BFD-22 presented in vivo antimetastatic effects and showed better therapeutic efficacy than sorafenib and taxol. BFD-22 can be considered as a new lead compound and, then, can be helpful for the designing of novel drug candidates to treat melanoma. (C) 2016 Elsevier Inc. All rights reserved.

Apoptotic effect of eugenol envolves G2/M phase abrogation accompanied by mitochondrial damage and clastogenic effect on cancer cell in vitro

Background: Eugenol (EUG) is a major phenolic compound present in clove whose anti-cancer properties have been demonstrated previously. These anti-cancer properties may involves the modulation of different mechanisms, including alpha-estrogen receptor (alpha ER) in luminal breast cancer cells, COX-2 inhibition in melanoma cells or p53 and caspase-3 activation in colon cancer cells. Hypothesis: EUG promotes a burst in ROS production causing cell-cycle perturbations, mitochondria toxicity and clastogenesis triggering apoptosis in melanoma breast-and cervix-cancer cells in vitro. Methods: Morphological changes were evaluated through the light-and electronic-microscopy. Cell-cycle, ROS, PCNA and Apoptosis was detected by flow cytometry and clastogenicity was evaluated by Comet-assay. Results: The results obtained herein pointed out that EUG promotes, increasing ROS production leading to abrogation of G2/M of phase of cell-cycle, and consecutively, clastogenesis in vitro. In addition, EUG induces Proliferation Cell Nuclear Antigen (PCNA) downregulation and decreasing in mitochondria potential (Delta Psi m). Of note, a Bax up-regulation was also observed on cells treated with EUG. All of these findings cooperate in order to induce apoptosis in cancer cells. Conclusion: These promising results presented herein shed new light on the mechanisms of action of EUG suggesting a possible applicability of this phenylpropanoid as adjuvant in anti-cancer therapy. (C) 2016 Elsevier GmbH. All rights reserved.

Efeitos antitumorais do mastoparano e do peptídeo INKKI em modelos de melanomas experimentais “in vitro” e “in vivo” / Antitumor effects of mastoparan and INKKI peptide on “in vitro” and “in vivo” experimental model melanoma

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.

O mastoparano (INLKALAALAKKIL) é um tetradecapeptídeo α-hélice e anfipático obtido do veneno da Vespula lewissi. O casoparano é um peptídeo catiônico (INKKI) isolado a partir da hidrólise da β-caseina bovina e apresenta motivo homólogo ao matoparano. O objetivo deste trabalho foi avaliar o efeito antitumoral “in vitro” e “in vivo” do mastoparano e do INKKI em modelo de melanoma experimental. A viabilidade foi avaliada em linhagens tumorais e não tumorigênicas através do método MTT. Adicionalmente, os efeitos do INKKI foram avaliados sobre a cinética de crescimento, adesão, migração e invasão celular em culturas de B16F10. O estudo das fases do ciclo celular foi analisado por citometria de fluxo. A via de sinalização foi padronizada por Western blotting. Os modelos metastáticos e dorsais in vivo foram padronizados em animais C57BL/6 e em modelo NOD/SCID/γc null . Simultaneamente, a morte celular por apoptose induzida pelo mastoparano foi determinada pelo ensaio de Anexina V-FITC/PI. A perda do potencial de membrana mitocondrial (∆Ψm) foi avaliada por citometria em células marcadas com sonda TMRE. A geração da espécie reativa de oxigênio (ROS) foi padronizada usando a sonda DHE. A fragmentação do DNA foi avaliada por eletroforese em gel de agarose e a condensação da cormatina foi analisada pela marcação nuclear com DAPI. O estudo in vivo foi padronizado sobre o modelo de melanoma dorsal. Os resultados mostraram que o mastoparano apresentou atividade citotóxica e o INKKI apresentou atividade antiproliferativa. Em adição, os dados mostraram que o INKKI foi capaz de inibir o crescimento celular, a adesão, a migração e invasão. Em complemento, a análise das proteínas mostraram redução nos níveis de GSK3β, N-caderina, fosfo Src Tyr 416 e fosfo FAK Tyr 925 e 397. A análise do ciclo celular mostrou que peptídeo INKKI foi capaz de aumentar a porcentagem da população na fase G1 e diminuição da proporção da fase S. Esses dados foram confirmados por Western blotting onde o tratamento mostrou redução nos níveis das ciclinas D1, D3, NFк-β e Erk1/2. Adicionalmente, os resultados dos ensaios in vivo mostraram que o INKKI reduziu a formação de metástases, inibiu o crescimento do melanoma cutâneo, aumentou a taxa de sobrevida, porém, não apresentou efeito em camundongos imunocomprometidos. Os resultados com o mastoparano mostraram que o peptídeo foi capaz de induzir aumento da população de células positivas para Anexina V. Além disso, induziu diminuição do potencial de membrana mitocondrial, aumentou a geração de ROS, induziu fragmentação do DNA e condensação da cromatina. Também, o mastoprano aumentou a expressão das caspases clivadas 3, 9 e 12, PARP, Bim, Bak, citocromo c, bem como redução da expressão de fosfo BAD 10 (S112), VDAC, PHB1 e Bcl-XL. Mais importante, o mastoparano reduziu o crescimento do melanoma cutâneo e aumentou a sobrevida. Em conclusão, o mastorparano e INKKI apresentam diferentes mecanismos de ação. O mastoparano induz morte celular através da via mitocondrial intrínseca e o peptídeo INKKI induz parada no ciclo celular. Nós também demonstramos que o INKKI teve um efeito antimetastático e inibiu o volume tumoral in vivo. O mastoparano apresentou efeito terapêutico in vivo.

Avaliacao da atividade proliferativa, antitumoral e hematologica dos peptideos derivados da caseina INKKI e YPVEPFTE no melanoma experimental / Evaluation of the activity proliferate antitumor and hematological of the peptides derivatives casein INKKI and YPVEPFTE in the experimental melanoma

Os peptideos INKKI e YPQPFTE foram isolados a patir da hidrolise da B caseina bovina e correspondem as sequencias 26-30 e 114-121 respectivamente. Trabalhos anteriores mostram que estes peptideos apresentaram atividades potenciadoras da bradicinina ...

Peptides INKKI and YPQPFTE were isolated from the bovine B casein after this hidrolysis corresponding to the 23-30 and 114-121 sequence respectively. Previous works had shown that these peptides presented bradkynin potentialing activity ...