Functional Studies Of Hsp70 Gene Expression In Multiple Myeloma Cell Lines Suggest That This Gene Inhibition Can Be a Potential Therapeutic Target, Independent Of 17p/p53 Status
Authors: Alves Veruska Lia Fook , Braga Walter Moises Tobias , Zanatta Daniela Bertolli , de Carvalho Fabricio , de Lourdes Chauffaille Maria , Strauss Bryan E. , Colleoni Gisele Wally Braga 
Published: October 2019

Abstract

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<0.001 for RPMI8226 and p<0.05 for U266) and between cell lines transduced with shRNAHsp70 and wild type (p<0.0001 for RPMI8226 and p<0.01 for U266) (One-Way ANOVA). We also found a statistically significant difference among relative expression of APAF-1 and caspase-9 in both U266 and RPMI8226 transduced with shRNAHsp70 and wild type or the same cell lines transduced with empty vector, 7 days after transduction, confirming the results obtained at cellular level.

Conclusion
In line with recent literature, the results obtained here with the Hsp70 stable silencing in MM cell lines RPMI8226 and U266 show a strong impact on the induction of late apoptosis, at gene and cellular levels, through APAF-1/caspase-9 pathway, suggesting that inhibitors of this heat shock can be explored as potential tools in the treatment of MM, using antisense-mediated Hsp70 inhibition, whatever patient's 17p (p53) FISH status (Support by FAPESP 2010/17668-6).

Disclosures:
No relevant conflicts of interest to declare.