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Auxotrophic recombinant Mycobacterium bovis BCG overexpressing Ag85B enhances cytotoxicity on superficial bladder cancer cells in vitro

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Abstract

BCG therapy remains at the forefront of immunotherapy for treating patients with superficial bladder cancer. The high incidence of local side effects and the presence of non-responder diseases have led to efforts to improve the therapy. Hence, we proposed that an auxotrophic recombinant BCG strain overexpressing Ag85B (BCG ∆leuD/Ag85B), could enhance the cytotoxicity to the human bladder carcinoma cell line 5637. The rBCG was generated using an expression plasmid encoding the mycobacterial antigen Ag85B to transform a BCG ∆leuD strain. The inhibitory effect of BCG ∆leuD/Ag85B on 5637 cells was determined by the MTT method, morphology observation and a LIVE/DEAD assay. Gene expression profiles for apoptotic, cell cycle-related and oxidative stress-related genes were investigated by qRT-PCR. Bax, bcl-2 and p53 induction by BCG ∆leuD/Ag85B treatment was evaluated by Western blotting. BCG ∆leuD/Ag85B revealed a superior cytotoxicity effect compared to the control strains used in this study. The results showed that the expression level of pro-apoptotic and cell cycle-related genes increased after BCG ∆leuD/Ag85B treatment, whereas the mRNA levels of anti-apoptotic genes decreased. Interestingly, BCG ∆leuD/Ag85B also increased the mRNA level of antioxidant enzymes in the bladder cancer cell line. Bax and p53 proteins levels increased following treatment. In conclusion, these results suggest that treatment with BCG ∆leuD/Ag85B enhances cytotoxicity for superficial bladder cancer cells in vitro. Therefore, rBCG therapy may have potential benefits in the treatment of bladder cancer.

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Acknowledgments

This work was supported by the Brazilian funding agencies: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Rio Grande do Sul (FAPERGS). The authors thank Alan McBride for a critical review of the manuscript.

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The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Programa de Pós-Graduação em Biotecnologia (PPGB), Grupo de Pesquisa em Oncologia Celular e Molecular, Biotecnologia/Centro de Desenvolvimento Tecnológico, Universidade Federal de Pelotas, Campus Universitário s/n, Capão do Leão, Pelotas, RS, 96010-900, Brazil

    Karine Rech Begnini, Caroline Rizzi, Vinicius Farias Campos, Sibele Borsuk, Eduarda Schultze, Virginia Campello Yurgel, Fernanda Nedel, Odir Antônio Dellagostin, Tiago Collares & Fabiana Kömmling Seixas

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  1. Karine Rech Begnini
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  2. Caroline Rizzi
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  3. Vinicius Farias Campos
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Correspondence to Fabiana Kömmling Seixas.

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Begnini, K.R., Rizzi, C., Campos, V.F. et al. Auxotrophic recombinant Mycobacterium bovis BCG overexpressing Ag85B enhances cytotoxicity on superficial bladder cancer cells in vitro. Appl Microbiol Biotechnol 97, 1543–1552 (2013). https://doi.org/10.1007/s00253-012-4416-2

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