HDAC inhibitor, valproic acid, induces p53-dependent radiosensitization of colon cancer cells

Abstract

Agents that inhibit histone deacetylases (HDAC inhibitors) have been shown to enhance radiation response. The aim of this study was to evaluate the effects of low, minimally cytotoxic concentrations of the HDAC inhibitor, valproic acid (VPA), on radiation response of colorectal cancer cells. Cell lines LS174T and an isogenic pair of HCT116, which differed only for the presence of wild-type p53, were exposed to ionizing radiation (IR) alone, VPA alone, or the combination. Clonogenic survival, gamma-H2AX induction, apoptosis, changes in mitochondrial membrane potential, and mitochondrial levels of p53 and Bcl-2 family proteins were assessed. In vivo studies monitored tumor growth suppression after therapy in mice bearing HCT116/p53(+/+) and HCT116/p53(-/-) tumor xenografts. VPA led to radiosensitization, which was dependent on p53 status. A decrease in clonogenic survival, an increase in apoptosis, and an increase in levels of gamma-H2AX were observed after VPA+IR, compared to IR alone, in wild-type p53 cells (LS174T and HCT116/p53(+/+)), as opposed to p53 null cells (HCT116/p53(-/-)). Exposure to VPA resulted in enhancement of IR-induced mitochondrial localizations of Bax and Bcl-xL, mitochondrial membrane potential, and cytochrome c release only in wild-type p53 cell lines. VPA also enhanced tumor growth suppression after IR only in wild-type p53 xenografts. These data suggest that VPA may have an important role in enhancing radiotherapy response in colorectal cancer, particularly in tumors with the wild-type p53 genotype.

FIG. 1.

Changes in histone acetylation after valproic acid (VPA) exposure. LS174T and HCT116 cells were exposed to varying concentrations of VPA for 16 hours. Cellular protein extracts were prepared, as described in Materials and Methods, and analyzed by immunoblot assay with antibody against acetylated histone H4 (acetyl-H4). β-actin was included as a control to show equivalent protein loading.

FIG. 2.

Clonogenic survival after valproic acid (VPA) and ionizing radiation (IR) exposure. Log-phase cells were trypsinized and plated as single cells. After 6 hours of incubation to allow for cell attachment, cells were pretreated with 500 μM of VPA for 16 hours and then exposed to different doses of IR. Colony survival was determined 14–20 days later. Values represent the mean from three to four independent experiments. Error bars indicate one standard deviation. There was a significant reduction in clonogenic survival with the addition of VPA for LS174T and HCT116/p53^+/+, but not for HCT116/p53^−/−.

FIG. 3.

Effects of valproic acid (VPA) on ionizing radiation (IR)-induced sub-G₁ cell-cycle distribution and expression of p53, p21, and cyclin B1. Cells were pretreated with or without 500 μM of VPA for 16 hours before γ-radiation (4 Gy). (A) Cells were then stained with propidium iodide and cell-cycle distribution analyzed by flow cytometry at 0, 8, 16, 24, and 32 hours after IR. The data from the two separate experiments were averaged and plotted. Because the percentage of cells in sub-G₁ followed more closely an exponential curve than a linear one, a linear regression was performed on the logarithm of the percentages. The slopes of the curves for IR were compared to that of IR+VPA, using a t statistic, and the pooled variances for the pair-wise regressions. (B) Total protein extracts were also prepared at 0, 2, 8, and 24 hours after IR and analyzed by immunoblot assay with antibodies against p53, p21, and cyclin B1. β-actin was included as a control to show equivalent protein loading.

FIG. 4.

Effects of valproic acid (VPA) on ionizing radiation (IR)-induced γ-H2AX duration and apoptosis. (A) LS174T, HCT116/p53^+/+, and HCT116/p53^−/− cells were pretreated with 500 μM of VPA or no VPA for 16 hours prior to 4-Gy irradiation. Cells were then harvested at indicated times, and cell lysates were prepared for immunoblot analysis of γ-H2AX. β-actin was included to show equivalent protein loading. The data are representative of three independent experiments. (B) Apoptosis after IR and VPA. Cells were first exposed to 500 μM of VPA or left untreated for 16 hours before irradiation (4 Gy). Cells were then collected 48 hours after irradiation and apoptosis assayed by Annexin-V–fluorescein isothiocyanate (FITC) staining and flow cytometric analysis, as described in Materials and Methods. Data represent the average of three experiments. Error bars represent one standard deviation.

FIG. 5.

Changes in expression of apoptosis-related proteins after valproic acid (VPA) and ionizing radiation (IR). LS174T, HCT116/p53^+/+ and HCT116/p53^−/− cells were pretreated with 500 μM of VPA or left untreated for 16 hours, followed by 4-Gy irradiation. Proteins were extracted and lysed in RIPA buffer 24 hours later. Immunoblot assays were performed to detect the expression of DSB-related proteins (KU70, KU86, DNA-pK, and Rad51) and Bcl-2 family proteins (Bax and Bcl-xL) in cells after VPA and IR. β-actin was included to show equivalent protein loading. DSB, double-strand break.

FIG. 6.

Effects of valproic acid (VPA) exposure on ionizing radiation (IR)-induced changes in mitochondrial membrane potential and mitochondrial accumulation of p53 and Bcl-2 family membrane proteins. (A) Cells were pretreated with or without 500 μM of VPA for 16 hours before 4-Gy IR. Cells were then collected and stained with JC-1, as described in Materials and Methods. Mitochondrial membrane potential was quantified by the measurement of JC-1 fluorescence intensity. Data represent the average of three independent assays. Error bars represent one standard deviation. (B) Cytochrome c release and mitochondrial accumulation of p53 and Bcl-2 family proteins in response to VPA and IR exposure. Cells were pretreated with or without 500 μM of VPA for 16 hours before 4-Gy IR. Cytosolic (CF) and mitochondrial (MF) fractions were prepared 24 hours after irradiation. Immunoblot assay with antibodies against cytochrome c, p53, Bax, and Bcl-xL was then performed. β-actin and mt-Hsp70 were included to show equivalent protein loading. The data are representative of three independent experiments. To assess the integrity of purified CFs and MFs, equal amounts of proteins (10 μg) were loaded, and mitochondrial proteins cytochrome c (cyto c), HSP70, and the cytosolic/nuclear protein proliferating cell nuclear antigen (PCNA) were assessed by immnunoblotting (right panel).

FIG. 7.

Tumor-growth delay after valproic acid (VPA) and ionizing radiation (IR) exposure in vivo. Athymic nude mice bearing isogenic HCT 116/p53^+/+ and HCT 116/p53^−/− xenograft tumors were treated with VPA (300 mg/kg × 6) administered intraperitoneally every 12 hours for 3 days and/or 10-Gy irradiation. In the combined treatment group, IR was delivered after the third injection of VPA. The growth curves represent the average value in each group of 5–8 mice. Error bars represent one standard error.