Increased 14-3-3ζ Expression In The Multidrug-Resistant Leukemia Cell Line HL-60/Vcr As Compared To HL-60 Cell Line Mediates Cell Growth and Apoptosis

Objectives Acute myeloid leukemia (AML) recurrence is largely a result of multidrug resistance (MDR). Because vincristine-resistant HL-60 cells (HL-60/VCR) express greater levels of 14-3-3¦AE, which is associated with apoptosis and chemosensitivity in other cancers, this study sought to examine its role in AML chemosensitivity using HL-60 and HL-60/VCR cells. Methods The mRNA and protein expression of 14-3-3¦AE, mdr1, Pgp, BCL-2 and MCL-1 were examined using semi-quantitative RT-PCR and Western blot analyses, respectively. The subcellular location of 14-3-3¦AE protein in HL-60 and HL-60/VCR cells was determined using immunofluorescence and confocal microscopy. After siRNA-mediated silencing of 14-3-3¦AE in HL-60 AND HL-60/VCR cells, cell growth and cell cycle progression were determined by direct counting and flow cytometry, respectively. The effect of 14-3-3¦AE siRNA on topotecan (TPT)-induced apoptosis was evaluated using acridine orange/ethidium bromide and Annexin V staining as well as TUNEL analysis. NF-¦EB-DNA biding was also assessed using electrophoretic mobility shift assay . Results As compared to HL-60 cells, increased 14-3-3¦AE mRNA and protein expression was observed in HL-60/VCR cells. In addition, increased mdr-1 mRNA as well as Pgp, Bcl-2, and Mcl-1 protein expression were observed in HL-60/VCR cells. In both HL-60 and HL-60/VCR cells, 14-3-3¦AE was observed in the cytoplasm and nuclear compartments. 14-3-3¦AE siRNA significantly reduced HL-60 and HL-60/VCR cell growth after 48 h and increased the proportion of cells in the G0/G1 phase. Moreover, 14-3-3¦AE siRNA significantly increased the sensitivity of both HL-60 and HL-60/VCR cells to TPT possibly through inhibition of Bcl-2, Mcl-1 and Pgp protein expression. Conversely, increased Bad and Noxa protein expression was observed with 14-3-3¦AE siRNA. NF-ĸB DNA binding was reduced with 14-3-3¦AE siRNA. Conclusions Silencing of 14-3-3¦AE increased the sensitivity of both sensitive and resistant HL-60 cells to TPT-induced apoptosis possibly through altering the expression of apoptosis-associated proteins, suggesting that it may be a potential target for MDR AML. Disclosures: No relevant conflicts of interest to declare.