Publicação
Discovering and exploiting bacterial proteins as anticancer agents
| Resumo: | Azurin is a low molecular weight protein, produced by Pseudomonas aeruginosa and possesses several antitumor properties, like causing P-cadherin levels to decrease in invasive breast cancer cells. In this work, we studied the effect of lysosome and proteasome inhibitors on P-cadherin level, using a breast cancer cell line, expressing high P-cadherin level (MCF-7/AZ.Pcad), previously treated with azurin. Additionally, we evaluated how a cholesterol-depleting agent (MβCD) affects P-cadherin level. The effects of both inhibitors on P-cadherin were observed by western blot and confirmed that azurin mediates P-cadherin degradation through lysosome and proteasome proteolytic pathways. We also described, for the first time, that MβCD causes P-cadherin level to decrease. Together, these findings have increased our understanding of how the bacterial protein azurin is acting as anti-cancer agent. In this work we have also studied the in vitro cytotoxicity of two other bacterial proteins (MPT 63 and Ndk) against human breast and lung cancer cells. MPT 63 is an antigen secreted by Mycobacterium tuberculosis that induces immunogenic responses in animal models and its cytotoxicity against several tumor cell lines was recently described in a patent. Nucleoside diphosphate kinase (Ndk) is a ubiquitous enzyme which maintains the nucleotide pools within the cells, and can be secreted by P. aeruginosa. A human Ndk, termed Nm23-H1, also showed an anti-metastatic role in different cancer models. In order to test possible antitumor properties of these proteins, MTT cell viability assays were performed in breast and lung cancer models (MCF-7/AZ.Mock and A549) using increasing azurin, MPT 63 and Ndk concentrations, and different exposure times. In addition, matrigel invasion assay was performed in A549 invasive cells treated with Ndk. Both azurin, MPT 63 and Ndk evidenced cytotoxicity against both cancer models in a time and dose dependent manner. Ndk revealed cytotoxic activity and selectivity against tumor cells similar to azurin. We observed a small decrease in cell invasion using this protein. In summary, we promoted a screening of new bacterial proteins that demonstrated antitumor potential, especially Ndk. |
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| Autores principais: | Silva, Gonçalo Emanuel Fialho Mourata da,1989- |
| Assunto: | Cancro Apoptose Azurin Teses de mestrado - 2013 |
| Ano: | 2013 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | Azurin is a low molecular weight protein, produced by Pseudomonas aeruginosa and possesses several antitumor properties, like causing P-cadherin levels to decrease in invasive breast cancer cells. In this work, we studied the effect of lysosome and proteasome inhibitors on P-cadherin level, using a breast cancer cell line, expressing high P-cadherin level (MCF-7/AZ.Pcad), previously treated with azurin. Additionally, we evaluated how a cholesterol-depleting agent (MβCD) affects P-cadherin level. The effects of both inhibitors on P-cadherin were observed by western blot and confirmed that azurin mediates P-cadherin degradation through lysosome and proteasome proteolytic pathways. We also described, for the first time, that MβCD causes P-cadherin level to decrease. Together, these findings have increased our understanding of how the bacterial protein azurin is acting as anti-cancer agent. In this work we have also studied the in vitro cytotoxicity of two other bacterial proteins (MPT 63 and Ndk) against human breast and lung cancer cells. MPT 63 is an antigen secreted by Mycobacterium tuberculosis that induces immunogenic responses in animal models and its cytotoxicity against several tumor cell lines was recently described in a patent. Nucleoside diphosphate kinase (Ndk) is a ubiquitous enzyme which maintains the nucleotide pools within the cells, and can be secreted by P. aeruginosa. A human Ndk, termed Nm23-H1, also showed an anti-metastatic role in different cancer models. In order to test possible antitumor properties of these proteins, MTT cell viability assays were performed in breast and lung cancer models (MCF-7/AZ.Mock and A549) using increasing azurin, MPT 63 and Ndk concentrations, and different exposure times. In addition, matrigel invasion assay was performed in A549 invasive cells treated with Ndk. Both azurin, MPT 63 and Ndk evidenced cytotoxicity against both cancer models in a time and dose dependent manner. Ndk revealed cytotoxic activity and selectivity against tumor cells similar to azurin. We observed a small decrease in cell invasion using this protein. In summary, we promoted a screening of new bacterial proteins that demonstrated antitumor potential, especially Ndk. |
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