Isotype-Specific Inhibition of Histone Deacetylases: Identification of Optimal Targets for Radiosensitization

Abstract

PurposeHistone deacetylase (HDAC) inhibitors radiosensitize tumor cells. To elucidate mechanisms underlying radiosensitization by HDAC inhibition, understanding of differential contributions of HDAC isotypes is needed. The aim of this study was to investigate involvement of known HDAC isotypes in modulation of cellular radiosensitivity.Materials and MethodsBecause pharmacologic HDAC inhibitors lack isotype-specificity, RNA interference against 11 HDAC isotypes was used to inhibit HDAC in an isotype-specific manner. Radiation cell survival was evaluated using a clonogenic assay in SQ20B cells transfected with small interfering RNA specifically targeting HDAC isotypes. Immunocytochemistry was performed for detection of γH2AX foci. Protein expression was measured using Western blotting.ResultsAmong 11 HDAC isotypes tested, specific inhibition of 7 isotypes (HDAC1, HDAC3, HDAC4, HDAC6, HDAC7, HDAC10, and HDAC11) enhanced radiation lethality in SQ20B cells. Radiosensitization by inhibition of these HDAC isotypes was accompanied by delay of DNA double strand break repair. Radiosensitivity of SQ20B cells was not altered by selective inhibition of the remaining four isotypes (HDAC2, HDAC5, HDAC8, and HDAC9). Inhibition of HDAC isotypes resulted in downregulation of various proteins involved in pro-survival and DNA damage repair pathways.ConclusionIsotype-specificity exists in HDAC inhibition-induced radiosensitization. Different HDAC isotypes are differentially involved in modulation of cellular radiosensitivity.

Disulfiram, a Re-positioned Aldehyde Dehydrogenase Inhibitor, Enhances Radiosensitivity of Human Glioblastoma Cells In Vitro

“…Cell survival was measured using clonogenic assay in triplicates as previously reported [19]. Briefly, cells were irradiated with graded doses of 0, 2, 4, 6, and 8 Gy of 6 MV X-ray (Clinac 6EX, Varian Medical Systems, Palo Alto, CA).…”

“…Immunocytochemistry of γH2AX as a marker for detecting DNA damage was assayed as previously reported [19]. After exposure to DSF for 24 hours followed by irradiation with X-ray dose of 6 Gy, cells were subjected to γH2AX immunocytochemistry analysis at 0, 2, 6, and 24 hours after irradiation.…”

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“…For the clonogenic assays, identical numbers of cells were plated across the different treatment groups for each radiation dose as previously reported [ 52 ]. Cells were seeded into each well of six-well culture plates and transfected with miRNA for 18 hours.…”

Histone deacetylase 10, a potential epigenetic target for therapy

“…The elevated radio-sensitization caused by inhibiting HDAC10 involves downregulation of Rad51a, a component of DNA repair pathways (106). In addition, positive HDAC10 expression is correlated with adverse clinical features, such as larger tumor size, poor tumor stage, and more lymph node metastasis (99) (Figure .…”