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

Activation of endogenous p53 by combined p19Arf gene transfer and nutlin-3 drug treatment modalities in the murine cell lines B16 and C6

<p>Abstract</p> <p>Background</p> <p>Reactivation of p53 by either gene transfer or pharmacologic approaches may compensate for loss of p19Arf or excess mdm2 expression, common events in melanoma and glioma. In our previous work, we constructed the pCLPG retroviral vector where transgene expression is controlled by p53 through a p53-responsive promoter. The use of this vector to introduce p19Arf into tumor cells that harbor p53wt should yield viral expression of p19Arf which, in turn, would activate the endogenous p53 and result in enhanced vector expression and tumor suppression. Since nutlin-3 can activate p53 by blocking its interaction with mdm2, we explored the possibility that the combination of p19Arf gene transfer and nutlin-3 drug treatment may provide an additive benefit in stimulating p53 function.</p> <p>Methods</p> <p>B16 (mouse melanoma) and C6 (rat glioma) cell lines, which harbor p53wt, were transduced with pCLPGp19 and these were additionally treated with nutlin-3 or the DNA damaging agent, doxorubicin. Viral expression was confirmed by Western, Northern and immunofluorescence assays. p53 function was assessed by reporter gene activity provided by a p53-responsive construct. Alterations in proliferation and viability were measured by colony formation, growth curve, cell cycle and MTT assays. In an animal model, B16 cells were treated with the pCLPGp19 virus and/or drugs before subcutaneous injection in C57BL/6 mice, observation of tumor progression and histopathologic analyses.</p> <p>Results</p> <p>Here we show that the functional activation of endogenous p53wt in B16 was particularly challenging, but accomplished when combined gene transfer and drug treatments were applied, resulting in increased transactivation by p53, marked cell cycle alteration and reduced viability in culture. In an animal model, B16 cells treated with both p19Arf and nutlin-3 yielded increased necrosis and decreased BrdU marking. In comparison, C6 cells were quite susceptible to either treatment, yet p53 was further activated by the combination of p19Arf and nutlin-3.</p> <p>Conclusions</p> <p>To the best of our knowledge, this is the first study to apply both p19Arf and nutlin-3 for the stimulation of p53 activity. These results support the notion that a p53 responsive vector may prove to be an interesting gene transfer tool, especially when combined with p53-activating agents, for the treatment of tumors that retain wild-type p53.</p

Genetic barcode sequencing for screening altered population dynamics of hematopoietic stem cells transduced with lentivirus

Insertional mutagenesis has been associated with malignant cell transformation in gene therapy protocols, leading to discussions about vector security. Therefore, clonal analysis is important for the assessment of vector safety and its impact on patient health. Here, we report a unique approach to assess dynamic changes in clonality of lentivirus transduced cells upon Sanger sequence analysis of a specially designed genetic barcode. In our approach, changes in the electropherogram peaks are measured and compared between successive time points, revealing alteration in the cell population. After in vitro validation, barcoded lentiviral libraries carrying IL2RG or LMO2 transgenes, or empty vector were used to transduce mouse hematopoietic (ckit+) stem cells, which were subsequently transplanted in recipient mice. We found that neither the empty nor IL2RG encoding vector had an effect on cell dynamics. In sharp contrast, the LMO2 oncogene was associated with altered cell dynamics even though hematologic counts remained unchanged, suggesting that the barcode could reveal changes in cell populations not observed by the frontline clinical assay. We describe a simple and sensitive method for the analysis of clonality, which could be easily used by any laboratory for the assessment of cellular behavior upon lentiviral transduction

Magnetic diatomite(Kieselguhr)/Fe2O3/TiO2 composite as an efficient photo-Fenton system for dye degradation

We explored the potential use of diatomite/Fe2O3/TiO2 composites as catalysts for heterogeneous photo-Fenton degradation of methylene blue under neutral pH. Such system consists in magnetic solids synthesized by co-precipitation with Fe2+/Fe3+ in the presence of diatomite, followed by impregnation of TiO2. The results showed that the optimal amount of the catalyst was 2.0 g L−1, since aggregation phenomena become significant above this concentration, which decreases the photodegradation activity. The catalyst is highly efficient in the degradation of methylene blue and shows an easy recovery by an external magnetic field. This allows for an effective catalyst reuse without significant loss of activity in catalytic cycles, which is a highly interesting prospect for recyclable dye degradation systems721420CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPNão temNão tem2013/01669-

BONE MARROW STEM CELLS in FACIAL NERVE REGENERATION FROM ISOLATED STUMPS

IntroductionSevere lesions in the facial nerve may have extensive axonal loss and leave isolated stumps that impose technical difficulties for nerve grafting. Methods: We evaluated bone marrow stem cells (BMSC) in a silicone conduit for rat facial nerve regeneration from isolated stumps. Group A utilized empty silicone tubes; in groups B-D, the tube was filled with acellular gel; and, in groups C and D, undifferentiated BMSC (uBMSC) or Schwann-like cells differentiated from BMSC (dBMSC) were added, respectively. Compound muscle action potentials (CMAPs) were measured, and histology was evaluated. Results: Groups C and D had the highest CMAP amplitudes. Group C had shorter CMAP durations than groups A, B, and D. Distal axonal number and density were increased in group C compared with groups A and B. Conclusions: Regeneration of the facial nerve was improved by both uBMSC and dBMSC in rats, yet uBMSC was associated with superior functional results.INCT Program Project of the National Council for Scientific and Technological DevelopmentConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Univ São Paulo, Dept Otorhinolaryngol, Sch Med, BR-05403000 São Paulo, BrazilUniv São Paulo, Dept Genet & Evolutionary Biol, Biosci Inst, BR-05403000 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biochem, São Paulo, BrazilUniv São Paulo, Dept Cell & Dev Biol, Inst Biomed Sci, BR-05403000 São Paulo, BrazilUniv São Paulo, Viral Vector Lab, Mol Cardiol Lab LIM 13, Heart Inst,Med Sch, BR-05403000 São Paulo, BrazilDepartment of Biochemistry, Federal University of São Paulo, São Paulo, BrazilCNPq: 573633/2008-8FAPESP: CEPID 1998/14254-2FAPESP: 2008/53857-8FAPESP: 2008/00458-9Web of Scienc