Publication Date:
2014-12-06
Description:
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 its ability in inhibiting cell death via APAF-1 (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. Some studies have shown that HSP70 gene silencing using antisense RNA, abundantly induced cell death in breast cancer lines and it was not toxic to normal breast epithelial cells or human fibroblasts. This cell death was not dependent on P53status or inhibited by Bcl2 pathway. Other studies using HSP70 antisense RNA have also indicated apoptosis of tumor cells in lung cancer, oral cavity, colon, prostate, liver, and brain cell lines. 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 TRIAP1 and HSP70 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 APAF-1 and Caspase 9 gene expression by qPCR and Caspase 9 and Caspase 3/7 protein activity. Results: The cell lines RPMI8226 (without deletion of P53 by FISH) and U266 (deletion of one allele of P53 by FISH) were chosen for the transduction experiments because they showed relevant expression of TRIAP1 and HSP70. The efficiency of transduction, as measured in both cell lines transduced with the pLKO vector containing the GFP reporter gene, was 70%, demonstrating that there would be no technical restriction to perform this experiment. RPMI8226 and U266 were submitted to three independent transductions, in triplicate, with the lentiviral vector containing the constructs pLKO shRNATRIAP1, shRNAHSP70 and shRNAscramble. We obtained the silencing of TRIAP1 and HSP70 genes in both MM cell lines when the transduced cell lines were compared with shRNAscramble. Silencing was confirmed by relative qPCR and Western blotting (for HSP70 only). Inhibition of TRIAP1 expression significantly increased the percentage of cells in late apoptosis (annexin V+/Propidium Iodide+) analysis (p
Print ISSN:
0006-4971
Electronic ISSN:
1528-0020
Topics:
Biology
,
Medicine
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