Publication Date:
2013-11-15
Description:
Introduction Some evidences show that HSP70 is overexpressed in many cancers, and that high expression of this chaperone correlates with increased tumor grade and/or poor prognosis. Overexpression of HSP70 can provide a selective survival advantage to tumor cells in part due to its ability to inhibit cell death, through APAF-1 and caspase-9 pathway. Currently, some studies have shown that silencing of Hsp70 with antisense RNA induced massive cell death in breast cancer cell lines but was non-toxic to normal breast epithelial cells or normal human fibroblasts. This cell death was not dependent on p53, or inhibited by Bcl2. Other studies indicated that antisense-mediated Hsp70 inhibition in lung, oral, colon, prostate, liver and brain cancer cell lines also causes apoptosis. Despite 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 as a potential target for MM therapy through: 1) Hsp70 stable silencing in MM cell lines; 2) evaluation of the silencing effect of Hsp70 on the cell cycle and apoptosis. Methods MM cell lines (U266, SKO-007, SK-MM2 and RPMI8226) were maintained in RPMI 1640 media supplemented with 10% fetal bovine serum, L-glutamine, MEM NEAA, garamycin, at 37°C in the presence of CO2 atmosphere at 5%. The Hsp70 expression in the four MM cell lines was assessed using real time PCR (qPCR). The relative gene expression was calculated using U266 as control and β-actin as a constitutive gene. Cell lines were submitted to transduction with pLKO lentiviral vector containing the short hairpin RNAs (shRNAs) for Hsp70 silencing (shRNAHsp70). Lentiviral vectors were transduced into the same strains with control sequences (scramble). Apoptosis was analyzed by flow cytometry, using annexin V and propidium iodide (PI), and using APAF-1 and caspase-9 expression by qPCR. Results RPMI8226 and U266 cell lines were chosen for the transduction experiments because their genetic characteristics [the first has normal FISH for 17p and the second has del17p (p53X1)] and good levels of Hsp70 expression. The transduction efficiency, measured in U266 line with pLKO vector with GFP gene, was 70%, demonstrating that there would be no technical restriction to perform this experiment. RPMI8226 and U266 cell lines were subjected to three independent transductions with the pLKO lentiviral vector containing shRNAHsp70 and shRNA scramble, followed by apoptosis evaluation by flow cytometry and qPCR. Hsp70 expression was inhibited in at least 80% of cells in both RPMI8226 and U266 when shRNAHsp70 transduced cell lines were compared with shRNA scramble. Late apoptosis (annexin V+/PI+) analysis, 7 days after transduction, showed statistically significant difference between cell lines transduced with shRNAHsp70 and shRNA scramble (p
Print ISSN:
0006-4971
Electronic ISSN:
1528-0020
Topics:
Biology
,
Medicine
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