RESUMO
Mammary tumors are the most frequent type of neoplasms in intact female dogs. New therapies that target neoplastic cells without affecting normal cells are highly sought. The Bacillus anthracis toxin has been reengineered to target tumor cells that express urokinase plasminogen activators and metalloproteinases. In previous studies carried out in our laboratory, the reengineered anthrax toxin had inhibitory effects on canine oral mucosal melanoma and canine osteosarcoma cells. In this study, five canine neoplastic epithelial cell lines (four adenocarcinomas and one adenoma) and one non-neoplastic canine mammary epithelial cell line were treated with different concentrations of reengineered anthrax toxin components. Cell viability was quantified using an MTT assay and half-maximal inhibitory concentration (IC50) values. Cell lines were considered sensitive when the IC50 was lower than 5000 ng/ml. One canine mammary adenocarcinoma cell line and one mammary adenoma cell line showed significantly decreased viability after treatment, whereas the non-neoplastic cell line was resistant. We conclude that the reengineered anthrax toxin may be considered a targeted therapy for canine mammary neoplasms while preserving normal canine mammary epithelial cells.
Assuntos
Antígenos de Bactérias , Toxinas Bacterianas , Doenças do Cão , Neoplasias Mamárias Animais , Animais , Cães , Neoplasias Mamárias Animais/tratamento farmacológico , Toxinas Bacterianas/farmacologia , Feminino , Antígenos de Bactérias/farmacologia , Linhagem Celular Tumoral , Doenças do Cão/tratamento farmacológico , Células Epiteliais/efeitos dos fármacos , Adenocarcinoma/veterinária , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/patologia , Sobrevivência Celular/efeitos dos fármacos , Adenoma/veterinária , Adenoma/tratamento farmacológico , Adenoma/patologiaRESUMO
Canine and human osteosarcomas (OSA) share similarities. Novel therapies are necessary for these tumours. The Bacillus anthracis toxin was reengineered to target and kill cells with high expressions of matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA). Since canine OSA express MMPs and uPA, we assessed whether the reengineered toxin could show efficacy against these tumours. Two OSA cell lines (canine D17 and human MG63) and a non-neoplastic canine osteoblastic cell line (COBS) were used. Cells were treated with different concentrations of the reengineered anthrax toxin and cell viability was quantified using MTT assay. The cell cycle, apoptosis, and necrosis were analysed by flow cytometry. The wound-healing assay was performed to quantify the migration capacity of treated cells. D17 and MG63 cells had significantly decreased viability after 24 h of treatment. Cell cycle analysis revealed that OSA cells underwent apoptosis when treated with the toxin, whereas COBS cells arrested in the G1 phase. The wound-healing assay showed that D17 and MG63 cells had a significantly reduced migration capacity after treatment. These results point for the first time towards the in vitro inhibitory effects of the reengineered anthrax toxin on OSA cells; this reengineered toxin could be further tested as a new therapy for OSA.
Assuntos
Antígenos de Bactérias/farmacologia , Antineoplásicos/farmacologia , Toxinas Bacterianas/farmacologia , Neoplasias Ósseas/tratamento farmacológico , Osteossarcoma/tratamento farmacológico , Adolescente , Animais , Antígenos de Bactérias/genética , Apoptose/efeitos dos fármacos , Toxinas Bacterianas/genética , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Cães , Relação Dose-Resposta a Droga , Feminino , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Masculino , Metaloproteinases da Matriz/metabolismo , Proteínas de Membrana/metabolismo , Invasividade Neoplásica , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Engenharia de ProteínasRESUMO
Canine oral mucosal melanomas (OMM) are the most common oral malignancy in dogs and few treatments are available. Thus, new treatment modalities are needed for this disease. Bacillus anthracis (anthrax) toxin has been reengineered to target tumor cells that express urokinase plasminogen activator (uPA) and metalloproteinases (MMP-2), and has shown antineoplastic effects both, in vitro and in vivo. This study aimed to evaluate the effects of a reengineered anthrax toxin on canine OMM. Five dogs bearing OMM without lung metastasis were included in the clinical study. Tumor tissue was analyzed by immunohistochemistry for expression of uPA, uPA receptor, MMP-2, MT1-MMP and TIMP-2. Animals received either three or six intratumoral injections of the reengineered anthrax toxin prior to surgical tumor excision. OMM samples from the five dogs were positive for all antibodies. After intratumoral treatment, all dogs showed stable disease according to the canine Response Evaluation Criteria in Solid Tumors (cRECIST), and tumors had decreased bleeding. Histopathology has shown necrosis of tumor cells and blood vessel walls after treatment. No significant systemic side effects were noted. In conclusion, the reengineered anthrax toxin exerted inhibitory effects when administered intratumorally, and systemic administration of this toxin is a promising therapy for canine OMM.
Assuntos
Antígenos de Bactérias/uso terapêutico , Antineoplásicos/uso terapêutico , Toxinas Bacterianas/uso terapêutico , Doenças do Cão/tratamento farmacológico , Melanoma/tratamento farmacológico , Neoplasias Bucais/tratamento farmacológico , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/farmacologia , Antineoplásicos/farmacologia , Toxinas Bacterianas/genética , Toxinas Bacterianas/farmacologia , Doenças do Cão/metabolismo , Doenças do Cão/patologia , Cães , Feminino , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Melanoma/metabolismo , Melanoma/patologia , Melanoma/veterinária , Mucosa Bucal/efeitos dos fármacos , Mucosa Bucal/metabolismo , Mucosa Bucal/patologia , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Neoplasias Bucais/veterinária , Engenharia de Proteínas , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
Pulmonary tuberculosis (TB) is characterized by oxidative stress and lung tissue destruction by matrix metalloproteinases (MMPs). The interplay between these distinct pathological processes and the implications for TB diagnosis and disease staging are poorly understood. Heme oxygenase-1 (HO-1) levels were previously shown to distinguish active from latent TB, as well as successfully treated Mycobacterium tuberculosis infection. MMP-1 expression is also associated with active TB. In this study, we measured plasma levels of these two important biomarkers in distinct TB cohorts from India and Brazil. Patients with active TB expressed either very high levels of HO-1 and low levels of MMP-1 or the converse. Moreover, TB patients with either high HO-1 or MMP-1 levels displayed distinct clinical presentations, as well as plasma inflammatory marker profiles. In contrast, in an exploratory North American study, inversely correlated expression of HO-1 and MMP-1 was not observed in patients with other nontuberculous lung diseases. To assess possible regulatory interactions in the biosynthesis of these two enzymes at the cellular level, we studied the expression of HO-1 and MMP-1 in M. tuberculosis-infected human and murine macrophages. We found that infection of macrophages with live virulent M. tuberculosis is required for robust induction of high levels of HO-1 but not MMP-1. In addition, we observed that CO, a product of M. tuberculosis-induced HO-1 activity, inhibits MMP-1 expression by suppressing c-Jun/AP-1 activation. These findings reveal a mechanistic link between oxidative stress and tissue remodeling that may find applicability in the clinical staging of TB patients.