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    Estudos funcionais da expressão dos genes triap1 e hsp70 em linhagens celulares de mieloma múltiplo
    (Universidade Federal de São Paulo (UNIFESP), 2014-08-28) Alves, Veruska Lia Fook [UNIFESP]; Colleoni, Gisele Wally Braga Colleoni [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    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.