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

Spheroid-Based Approach to Assess Tissue Relevance of Analysis of Dispersed-Settled Tissue Cells by Cytometry of Reaction Rate Constant

File main pdf file (CRRC of dispersed-settled spheroidal cells.pdf) describes experimental results and their interpretation for a study of tissue relevance of analyses of dispersed-settled tissues cells by Cytometry of Reaction Rate Constant (CRRC). CRRC uses time-lapse fluorescence microscopy to measure a rate constant of a catalytic reaction in individual cells and, thus, facilitate accurate size determination for cell subpopulations with distinct efficiencies of this reaction. Practical CRRC requires that a tissue sample be disintegrated into a suspension of dispersed cells and these cells settle on the support surface before being analyzed by CRRC. We call such cells “dispersed-settled” to distinguish them from cells cultured as a monolayer. Studies of the dispersed-settled cells can be tissue-relevant only if the cells maintain their 3D tissue state during the multi-hour CRRC procedure. Here we propose an approach for assessing tissue relevance of the CRRC-based analysis of the dispersed-settled cells. Our approach utilizes cultured multicellular spheroids as a 3D cell model and cultured cell monolayers as a 2D cell model. The CRRC results of the dispersed-settled cells derived from spheroids are compared to those of spheroids and monolayers in order to find if the dispersed-settled cells are representative of the spheroids. To demonstrate its practical use, we applied this approach to a cellular reaction of multi-drug-resistance (MRD) transport which was followed by extrusion of a fluorescent substrate from the cells. The approach proved to be reliable and revealed long-term maintenance of MDR transport in the dispersed-settled cells obtained from cultured ovarian cancer spheroids. Accordingly, CRRC can be used to determine accurately the size of a cell subpopulation with an elevated level of MDR transport in tumor samples, which makes CRRC a suitable method for the development of MDR-based predictors of chemoresistance. The proposed spheroid-based approach for validation of CRRC is applicable to other types of cellular reactions, and, thus, will be an indispensable tool for transforming CRRC from an experimental technique into practical analytical tool. Additional (zip) files contain supporting images, kinetic traces, and histograms. Their detailed descriptions are provided in the main pdf file. </div

Activation of the Janus kinase/signal transducer and activator of transcription pathway in multiple myeloma is not related to point mutations in kinase and pseudokinase domains ofJAK1

Considering the recent impact of tyrosine kinase inhibitors in the treatment of myeloproliferative disorders carrying a recurrent JAK2 mutation not identified in multiple myeloma (MM), this study aimed to search for mutations in kinase and pseudokinase domains of the JAK1 gene in an attempt to define any critical and recurring change that can be used as a therapeutic target. We obtained CD138 + purified cells from 27 bone marrow aspirates of untreated MM, four normal controls and four MM cell lines. After amplification of kinase and pseudokinase domains of JAK1 in cDNA samples, the fragments were automatically sequenced. Seventy-eight percent of MM cases showed at least one polymorphism, all being synonymous single nucleotide polymorphisms (SNPs), with allele frequencies consistent with previous studies in normal European, African American and Asian populations. The four cell lines also showed only synonymous SNPs. Mutations in the kinase and pseudokinase domains of the JAK1 gene do not seem to be important for activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway because we were not able to find any recurrent mutation in a case series of 27 patients and four MM cell lines