Proteasome and heat shock protein 70 (HSP70) inhibitors as therapeutic alternative in multiple myeloma

HSP70 connects multiple signaling pathways that work synergistically to protect tumor cells from death by proteotoxic stress and represents a possible target to establish a new approach for multiple myeloma treatment. Therefore, bioluminescent cell lines RPMI8226-LUC-PURO and U266-LUC-PURO were treated with HSP70 (VER155008) and/or proteasome (bortezomib) inhibitors and immunodeficient mice were used for subcutaneous xenograft models to evaluate tumor growth reduction and tumor growth inhibition after treatment. Bioluminescence imaging was used to follow tumor response. Treatment with bortezomib showed similar to 60% of late apoptosis in RPMI8226-LUC-PURO (without additional benefit of VER155008 in this cell line). However, U266-LUC-PURO showed similar to 60% of cell death after treatment with VER155008 (alone or with bortezomib). RPMI8226-LUC-PURO xenograft presented tumor reduction by bioluminescence imaging after treatment with bortezomib, VER155008 or drug combination compared to controls. Treatment with bortezomib, alone or combined with VER155008, showed inhibition of tumor growth assessed by bioluminescence imaging after one week in both RPMI8226-LUC-PURO and U266-LUC-PURO cell lines when compared to controls. In conclusion, our study shows that the combination of proteasome and HSP70 inhibitors induced cell death in tumor cells in vitro (late apoptosis induction) and in vivo (inhibition of tumor growth) with special benefit in U266-LUC-PURO, bearing 17p deletion.Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), BrazilFundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP), Brazil [2010/17668-6]Univ Fed Sao Paulo, UNIFESP, Dept Clin & Expt Oncol, Discipline Hematol & Hemotherapy, Sao Paulo, SP, BrazilUniv Sao Paulo, Fac Med, Canc Inst State Sao Paulo, Ctr Translat Invest Oncol, Sao Paulo, SP, BrazilUniv Fed Sao Paulo, UNIFESP, Dept Pathol, Sao Paulo, SP, BrazilUniv Fed Sao Paulo, UNIFESP, Dept Biochem, Sao Paulo, SP, BrazilUniv Fed Sao Paulo, UNIFESP, Dept Clin & Expt Oncol, Discipline Hematol & Hemotherapy, Sao Paulo, SP, BrazilUniv Fed Sao Paulo, UNIFESP, Dept Pathol, Sao Paulo, SP, BrazilUniv Fed Sao Paulo, UNIFESP, Dept Biochem, Sao Paulo, SP, BrazilFAPESP [2010/17668-6]Web of Scienc

Estudos funcionais da expressão dos genes triap1 e hsp70 em linhagens celulares de mieloma múltiplo

Introduction: Some evidences suggest that heat shock protein 70 (HSP70) is overexpressed in many types of cancer, and that high expression of this chaperone is linked with increasing tumor grade and/or poor prognosis. The overexpression of HSP70 may provide a selective advantage for tumor cell survival, due in part, to their ability to inhibit cell death via APAF1 (apoptosis protease activating factor 1) and Caspase 9. The TP53 Regulated Inhibitor Of Apoptosis 1 (TRIAP1) gene can modulate apoptotic pathways by interaction with HSP70. Although there are several studies on the role of HSP70 gene in apoptosis and drug resistance, there is a lack of information about this gene in multiple myeloma (MM). Objectives: To analyze the importance of HSP70 and TRIAP1 as potential targets for MM therapy through: 1) stable silencing of HSP70 and TRIAP1 in MM cell lines; 2) evaluation of each gene silencing effect on cell cycle and apoptosis. Methods:. The expression of HSP70 and P53CSV genes in MM cell lines (U266, SKO-007, SK-MM2 and RPMI8226) was examined by quantitative real time PCR (qPCR). Cell lines were submitted to transduction with pLKO lentiviral vector containing short hairpin RNAs (shRNAs) for silencing the target genes (shRNAHSP70 and shRNATRIAP1). Lentiviral vectors with control sequences (scramble) were used to transduce the same cell lines. Apoptosis was assessed by flow cytometry after annexin V and propidium iodide (PI) staining. We also evaluated APAF1 and Caspase9 gene expression by qPCR and Caspase 9 and Caspase 3/7 protein activity. Results:. The cell lines RPMI8226 (without deletion of TP53 by FISH) and U266 (deletion of one allele of TP53 by FISH) were chosen for the transduction experiments because they showed significant levels of expression of HSP70 and TRIAP1. The efficiency of transduction, as measured in both cell lines transduced with the pLKO vector containing the GFP reporter gene, was 70%. RPMI8226 and U266 were submitted to three independent transductions, in triplicate, with the lentiviral vector containing the constructs pLKO shRNAHSP70, shRNATRIAP1 and shRNAscramble. We obtained the stable silencing of HSP70 and TRIAP1 in both MM cell lines. Silencing was confirmed by relative quantitative PCR and Western blotting (for HSP70 only). Inhibition of TRIAP1 increased the percentage of cells in late apoptosis and was accompanied by increased expression of Caspase9 in both MM cell lines. Furthermore, the inhibition of TRIAP1 resulted in accumulation of hypodiploid cells after 24 hours of transduction in U266 cell line. Inhibition of HSP70 showed no significant changes in the cell cycle in both MM cell lines. However, we observed an increment in late apoptosis after inhibition of this gene in the two cell lines and the results were confirmed by increased activity of Caspase3/7. Conclusion: Stable silencing of HSP70 and TRIAP1 in MM cell lines showed a strong impact of this method on the induction of late apoptosis, through APAF1/Caspase9 pathway, suggesting that inhibitors of both genes could be exploited as potential targets for the treatment of MM, helping patients whatever TP53 status assessed by FISH.Introdução: Algumas evidências mostram que muitos tipos de câncer apresentam aumento de expressão da heat shock protein 70 (HSP70) e que a alta expressão desta chaperona está relacionada com a agressividade do tumor e/ou mau prognóstico. O aumento da expressão da HSP70 pode proporcionar vantagem seletiva para a sobrevivência de células tumorais, em parte devido à sua capacidade de inibir a morte celular via APAF-1 (apoptosis protease activating factor 1) e caspase-9. O gene TP53 Regulated Inhibitor of Apoptosis 1 (TRIAP1) pode modular vias apoptóticas pela interação com a HSP70, inibindo a ativação da caspase-9 e prevenindo a indução da apoptose. Apesar de existirem vários estudos sobre o papel do HSP70 na inibição da apoptose e resistência às drogas em câncer, não há informação sobre este gene no mieloma múltiplo (MM). Objetivos: Analisar a importância do HSP70 e TRIAP1 como potenciais alvos para a terapia do MM através de: 1) obtenção de anticorpo policlonal anti-TRIAP1 em modelo murino; 2) silenciamento estável do HSP70 e TRIAP1 em linhagens celulares de MM; 3) avaliação do efeito de silenciamento desses genes em relação ao ciclo celular e apoptose. Métodos: A expressão dos genes TRIAP1 e HSP70 foram avaliadas nas linhagens celulares de MM (U266, SKO-007, SK-MM2 e RPMI8226) por PCR quantitativo em tempo real (qPCR). As linhagens celulares selecionadas foram submetidas à transdução com vetor lentiviral pLKO contendo short hairpin RNAs (shRNAs) para silenciamento dos genes alvos (shRNATRIAP1e shRNAHSP70). Vetores lentivirais com seqüências controle (scramble) foram utilizados para transduzir as mesmas linhagens celulares. A apoptose foi avaliada por citometria de fluxo, utilizando anexina V e iodeto de propídio (PI). Também avaliamos a expressão de APAF1 e Caspase9 por qPCR e a atividade das proteínas Caspase9 e Caspase3/7. Resultados: As linhagens celulares RPMI8226 (ausência de deleção do gene P53 detectada por FISH) e U266 (deleção de um alelo do P53 detectada por FISH) foram escolhidas para realização dos experimentos de transdução, pois apresentaram bons níveis de expressão dos genes TRIAP1 e HSP70. A eficiência de transdução, medida nas duas linhagens selecionadas transduzidas com o vetor pLKO contendo o gene reporter GFP, foi de 70%. As linhagens RPMI8226 e U266 foram submetidas a três transduções independentes, em triplicata, com o vetor lentiviral pLKO contendo as construções shRNATRIAP1, shRNAHSP70 e shRNAscramble. O silenciamento estável de TRIAP1 e HSP70 foi obtido nas duas linhagens celulares de MM. O silenciamento foi confirmado por qPCR e Western Blotting (apenas para HSP70). A inibição do TRIAP1 aumentou o número de células em apoptose tardia (anexina V+/PI+), o que foi acompanhado pelo aumento da expressão de Caspase9, nas duas linhagens celulares de MM. Além disso, a inibição do TRIAP1 resultou no acúmulo de células hipodiplóides, na linhagem U266, após 24 horas da transdução. A inibição do gene HSP70 nas duas linhagens de MM não mostrou mudanças significativas em nenhuma das fases do ciclo celular. No entanto, observamos aumento do número de células em apoptose tardia após inibição de HSP70 nas duas linhagens celulares analisadas e esses resultados foram confirmados pelo aumento da atividade da Caspase3/7. Conclusão: O silenciamento estável de TRIAP1 e HSP70 em linhagens celulares de MM mostra um forte impacto dessa técnica sobre a indução de apoptose tardia, através da via APAF-1/caspase-9, sugerindo que inibidores desses dois genes podem ser explorados como potenciais alvos para o tratamento de MM, privilegiando pacientes independente do status do FISH para P53. .Dados abertos - Sucupira - Teses e dissertações (2013 a 2016

Cancer/Testis Antigens MAGE-C1/CT7 and MAGE-C2/CT10 Are Expressed in 90% of Multiple Myeloma Samples and Can Be Explored in Combined Immunotherapy for This Malignancy

Univ Fed Sao Paulo, Dept Oncol Clin & Expt, Disciplina Hematol & Hemoterapia, UNIFESP EPM, Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Oncol Clin & Expt, Disciplina Hematol & Hemoterapia, UNIFESP EPM, Sao Paulo, BrazilWeb of Scienc