Functional Depletion of HSP72 by siRNA and Quercetin Enhances Vorinostat-Induced Apoptosis in an HSP72-Overexpressing Cutaneous T-Cell Lymphoma Cell Line, Hut78

Abstract

Histone deacetylase inhibitors (HDACis) are one of the therapeutic options for cutaneous T-cell lymphoma (CTCL), but they have limited effects. We previously demonstrated that HSP72 overexpression is associated with chemoresistance to HDACis in lymphoma cells. The purpose of this study was to investigate whether the functional depletion of HSP72 enhances the effect of the HDACi vorinostat. First, we established a stable HSP72-knockdown CTCL cell line and confirmed the influence of HSP72 reduction on the antitumor effects of vorinostat. Next, we studied the effect of quercetin, an inhibitor of HSP72, on the antineoplastic effects of vorinostat. In five CTCL cell lines examined, HSP72 expression was highest in Hut78 cells, and HSP72 knockdown enhanced vorinostat-induced apoptosis in these cells. Low-dose quercetin reduced HSP72 expression, increased HDAC activity, and enhanced vorinostat-induced suppression of Hut78 cell proliferation. A single low dose of quercetin induced G2 arrest and only slightly increased the sub-G1 cell fraction. Quercetin also significantly enhanced vorinostat-induced apoptosis, caspase-3, caspase-8, and caspase-9 activity, and the loss of mitochondrial membrane potential. HSP72 knockdown enhanced vorinostat-induced apoptosis in an HSP72-overexpressing CTCL cell line, and thus, quercetin may be a suitable candidate for combination therapy with vorinostat in clinical settings.

Keywords: HSP72; combination therapy; cutaneous T-cell lymphoma; histone deacetylase inhibitor; quercetin.

Figure 1

Expression of HSP72 protein in five cutaneous T-cell lymphoma (CTCL) cell lines. Expression of HSP72 in each cell line was described as the relative mean fluorescence intensity (rMFI) + SD (n = 3).

Figure 2

Characterization of HSP72-knockdown Hut78 cells. HSP72 mRNA expression for MOCK (gray bar) and HSP72-knockdown (black bar) Hut78 cells, normalized to GAPDH expression levels ((a), n = 2). Flow cytometric analysis of HSP72 protein expression in MOCK (gray bar) and HSP72 knockdown (black bar) cells, showing the relative mean fluorescence intensity (rMFI) + SD ((b), n = 3). Proliferation assay with vorinostat using the WST-1 assay kit ((c), n = 4). ◆: MOCK, and ■: HSP72 knockdown Hut78 cells. Assessment of the influence of HSP72 knockdown on the antitumor effects of vorinostat. Representative images are shown in the left panels, and the summary is shown in the right panel (d). Assessment of apoptosis by annexin-V assays (e). * p < 0.05, ** p < 0.01, by student t-test.

Figure 3

Influence of quercetin on vorinostat-treated Hut78 cells. Cell proliferation was assessed by WST-1 assays ((a), n = 4). ◆: treated by vorinostat, ■: quercetin, and ○: both vorinostat and quercetin (1:40). HSP72 protein expression was measured via FACS (b). HDAC activity was assessed by the HDAC-Glo™ I/II Assays and adjusted with the CellTiter-Fluor^TM Cell Viability Assay (c). Acetylated lysin (d) and acetylated histone H3 (e) were measured via FACS. * p < 0.05, ** p < 0.01 by Bonferroni t-test.

Figure 4

Assessment of the effect of quercetin on the antitumor effects of vorinostat in Hut78 cells. Propidium iodide staining (a). Representative images are shown in the left panels and the summary of the analysis is shown as a bar graph in the right panel. Annexin V assay (b); cleaved PARP assay (c); activated caspase-3/7 (d), caspase-9 (e), and caspase-8 (h) analysis; loss of mitochondrial membrane potential (f); bcl-XL expression (g) as assessed via FACS. * p < 0.05, ** p < 0.01 by Bonferroni t-test.