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Purpose: To investigate the effect of TAP7f, a penicillin derivative previously characterized as a potent antitumor agent that promotes ER stress and apoptosis, in combination with thapsigargin, an ER stress inducer, on melanoma cells., Methods: The synergistic antiproliferative effect of TAP7f in combination with thapsigargin was studied in vitro in murine B16-F0 melanoma cells, and in human A375 and SB2 melanoma cells. In vivo assays were performed with C57BL/6J mice challenged with B16-F0 cells. Immunofluorescence and Western blot assays were carried out to characterize the induction of ER stress and apoptosis. Necrotic tumor areas and the potential toxicity of the combined therapy were examined by histological analysis of tissue sections after hematoxylin-eosin staining., Results: In vitro, the combination of TAP7f with thapsigargin synergistically inhibited the proliferation of murine B16-F0, and human A375 and SB2 melanoma cells. When non-inhibitory doses of each drug were simultaneously administered to C57BL/6J mice challenged with B16-F0 cells, a 50% reduction in tumor volumes was obtained in the combined group. An apoptotic response characterized by higher expression levels of Baxenhanced PARP-1 cleavage and the presence of active caspase 3 was observed in tumors from the combined treatment. In addition, higher expression levels of GADD153/CHOP and ATF4 were found in tumors of mice treated with both drugs with respect to each drug used alone, indicating the induction of an ER stress response. No signs of tissue toxicity were observed in histological sections of different organs extracted from mice receiving the combination., Conclusion: The synergistic and effective antitumor action of TAP7f in combination with thapsigargin could be considered as a potential therapeutic strategy for melanoma treatment., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

We have previously examined the in vitro and in vivo antitumor action of TAP7f, a synthetic triazolylpeptidyl penicillin, on murine melanoma cells. In this work, we explored the signal transduction pathways modulated by TAP7f in murine B16-F0 and human A375 melanoma cells, and the contribution of some intracellular signals to the apoptotic cell death. TAP7f decreased ERK1/2 phosphorylation and increased phospho-p38, phospho-JNK and phospho-Akt levels. ERK1/2 blockage suppressed cell growth, while inhibition of p38, JNK and PI3K-I pathways reduced the antitumor effect of TAP7f. Pharmacological inhibition of p38 and JNK, or blockage of PI3K-I/Akt cascade with a dominant negative PI3K-I mutant diminished Bax expression levels and PARP-1 cleavage, indicating the involvement of these pathways in apoptosis. PI3K-I/Akt inhibition also favored an autophagic response, as evidenced by the higher expression levels of Beclin-1 and LC3-II detected in transfected cells exposed to TAP7f. However, although PI3K-I/Akt blockage promoted an autophagic survival response, this mechanism appears not to be critical for TAP7f antitumor action. It was also shown that TAP7f induced ER stress by enhancing the expression of ER stress-related genes and proteins. Downregulation of CHOP protein with specific siRNA increased cell growth and decreased cleavage of PARP-1, supporting its role in apoptosis. Furthermore, it was found that activation of p38, JNK and Akt occurred downstream ER perturbation. In summary, our results showed that TAP7f triggers an apoptotic cell death in melanoma cells through induction of ER stress and activation of p38, JNK and PI3K-I/Akt pathways., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Previously , we demonstrated that the non-antibiotic penicillin derivative TAP7f inhibited melanoma metastasis in vitro and in vivo through the downregulation of β-catenin and integrin αVβ3. As angiogenesis is required for tumor growth and metastasis, we decided to explore the possible antiangiogenic effect of TAP7f. We found that TAP7f inhibited proliferation, migration, tube formation, and actin cytoskeleton organization of human endothelial cells. In a gel plug assay, an in vivo model for angiogenesis, TAP7f also blocked vascular formation induced by fibroblast growth factor 2. Furthermore, when murine B16-F10 melanoma cells pre-treated with TAP7f were injected intradermally in mice, we observed a decrease in the number and thickness of the capillaries surrounding the tumor. Additionally, TAP7f downregulated vascular endothelial growth factor (VEGF) and platelet-derived growth factor-B (PDGF-B) expression in B16-F10 cells and VEGF receptor expression in HMEC-1 endothelial cells. When the antitumor effect of TAP7f was studied in C57BL/6 J mice challenged with B16-F10 melanoma cells, a significant reduction of tumor growth was observed. Furthermore, a decreased expression of VEGF, PDGF-B, and the endothelial cell marker CD34 was observed in tumors from TAP7f-treated mice. Together, our results suggest that the antiangiogenic activity of TAP7f contributes to its antitumor and antimetastatic action and positions this penicillin derivative as an alternative or complementary agent for the treatment of melanoma. Key messages: • TAP7f inhibits proliferation, migration, tube formation, and actin cytoskeleton organization of endothelial cells. • TAP7f downregulates VEGF receptor expression in endothelial cells. • TAP7f downregulates VEGF and PDGF expression in melanoma cells. • TAP7f inhibits angiogenesis in vivo. [ABSTRACT FROM AUTHOR]

The synthetic triazolylpeptidyl penicillin derivative, named TAP7f, has been previously characterized as an effective antitumor agent in vitro and in vivo against B16-F0 melanoma cells. In this study, we investigated the anti-metastatic potential of this compound on highly metastatic murine B16-F10 and human A375 melanoma cells. We found that TAP7f inhibited cell adhesion, migration and invasion in a dose-dependent manner. Additionally, we demonstrated that TAP7f downregulated integrin αvβ3 expression and Wnt/β-catenin pathway, a signaling cascade commonly related to tumor invasion and metastasis. Thus, TAP7f reduced both the enzymatic activity and the expression levels of matrix-metalloproteinases-2 and -9 in a time dependent manner. Moreover, TAP7f inhibited the expression of the transcription factor Snail and the mesenchymal markers vimentin, and N-cadherin, and up-regulated the expression of the epithelial marker E-cadherin, suggesting that the penicillin derivative affects epithelial–mesenchymal transition. Results obtained in vitro were supported by those obtained in a B16-F10-bearing mice metastatic model, that showed a significant TAP7f inhibition of lung metastasis. These findings suggest the potential of TAP7f as a chemotherapeutic agent for the treatment of metastatic melanoma. [ABSTRACT FROM AUTHOR]