This study was conducted in order to validate the radiosensitization effect of valproic acid, a biologically available histone deacetylase inhibitor, for fractionated radiation.
Radiosensitization effect of valproic acid was tested for the A549 cell line and U87MG cell line
Radiosensitization effect of valproic acid was found for both cell lines; A549 at 1.5 mM and 3.0 mM concentration and U87MG at 3.0 mM concentration. In growth delay analysis, a statistically significant radiosensitization effect was observed for both tumors (p < 0.001 for both tumors). Difference for change in slope for control and valproic acid versus radiotherapy and radiotherapy plus valproic acid showed borderline significance for the U87MG cell line (p=0.065), indicating beyond additive effect, whereas this difference was statistically insignificant for A549 tumor (p=0.951), indicating additive effect.
Results of this study indicate that a radiosensitizing effect for fractionated radiotherapy of valproic acid for A549 and U87MG tumors
Histone deacetylase (HDAC) inhibitor is considered a promising anti-cancer agent with an epigenetic modulation effect [
Valproic acid (VA), originally an antiepileptic drug, has recently been shown to directly inhibit the enzymatic activity of HDAC [
In this study,
A human lung cancer cell line, A549 (Korean Cell Line Bank, Seoul, Korea), was cultured in Dulbecco's Modified Eagle's medium (Welgene, Daegu, Korea) supplemented with 10% fetal bovine serum and 12.5 μg/mL of gentamicin. A human glioblastoma cell line, U87MG (Korean Cell Line Bank), was cultured at 37°C and 5% CO2 in culture media RPMI 1640 (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (Gibco) and 12.5 μg/mL of gentamicin (Gibco).
Cells were trypsinized from the exponentially growing monolayer cultures. The pre-determined numbers of cells were seeded into T25 flasks, followed by incubation for 24 hours prior to treatment. Combined cytotoxic effect of VA and radiation was compared with that of radiation alone. Both A549 and U87MG cells were exposed to 1.5 mM and 3 mM of VA. After exposure to VA for 18 hours prior to radiation, cells were irradiated using a 4-MV X-ray from a linear accelerator (Clinac 4/100, Varian Medical Systems, Palo Alto, CA) at a dose rate of 2.46 Gy/min. Graded radiation doses of 0, 2, 4, 6, and 8 Gy were used. After radiation, cells were incubated in drug free medium for 12 days for colony formation. The formed colonies were fixed with methanol and stained with 0.5% crystal violet; the number of colonies containing at least 50 cells was determined, and the surviving fraction was then calculated.
A549 and U87MG cells, 5×106 in number, prepared in 15% fetal calf serum and 0.05 mL Waymouth’ media were administered by intradermal injection into the back of 6-week female BALB/c-nude mice (Orient, Seoul, Korea) weighing 15-25 g under anesthesia. Ketamine hydrochloride (Ketara, Yuhan Yanghang, Seoul, Korea) and xylazine hydrochloride (Rompun, Bayer Korea, Seoul, Korea) were mixed at a ratio of 5:1. Mixed solution was then diluted with normal saline at a ratio of 3:7. Prepared solution, 0.1 mL per 10 g weight of mice, was administered intraperitoneally to mice for pre-procedure anesthesia. Mice were then kept for a period of time until estimated tumor volume reached 250 mm3. Tumor volume was estimated using the formula (length× width×width)/2.
Tumor bearing mice were randomized into four groups; control, VA, irradiation (IR), and IR+VA, with eight mice in each group. Vehicle, which was phosphate buffered saline (PBS) in the current study, was administered intraperitoneally twice per day, 12 hours apart for 6 days for mice in the control group and the IR group. VA dissolved in PBS was administered intraperitoneally twice per day, 12 hours apart for 6 days for mice in the VA group and IR+VA group. Dosage used for VA was 150 mg/kg, mouse.
Irradiation was performed using a linear accelerator at a dose rate of 2.46 Gy/min. In the IR group and IR+VA group, 12 Gy in four fractions were delivered to the tumor harboring back of mice with a 1 cm bolus. Mice in the control group and VA group also underwent sham IR. Mice were irradiated for four consecutive days from the second day of administration of either vehicle or VA. The vehicle/VA and IR administration schedule is summarized in
The experiment was repeated three times for validation. In vivo experiments were approved by the Institutional Animal Care and Use Committee of Seoul National University Hospital, Clinical Research Institute (IACUC No. 06142).
Kaleidagraph ver. 3.51 (Synergy Software, Reading, PA) was used to fit the survival data of irradiated cells into a linear quadratic model. Chi-square test was used for statistical analysis of in vitro experiments. Dose enhancement factor (DEF), defined as ratio of dose with radiation alone over radiation with drug for the same biologic effect was calculated. SAS program ver. 9.1 (SAS Institute, Cary, NC) was used for statistical analysis of
Probability values less than 0.05 were considered statistically significant and less than 0.1 were considered of borderline significance.
Survival curves of A549 and U87MG cells treated with VA and radiation were compared with those of cells treated with radiation alone (
Tumor volume curves for A549 and U87MG cells for the control, VA, IR, and IR+VA groups are shown (
In recent decades, a new paradigm of "epigenetic changes", which are heritable changes in gene expression that are not caused by alterations in the gene nucleotide sequence, in relation to cancer development has evolved [
Effect of HDAC-I is exerted differently depending on the concentration. At a higher concentration, HDAC-I’s show tumor cytotoxicity. Mechanisms underlying this phenomenon are cell cycle redistribution, induction of apoptosis, and downregulation of surviving signals. However, radiosensitization effect is present at non-toxic low concentration and details of molecular mechanisms mediating radiosensitization by HDAC-I’s are less clear. It has been attributed, at least in part, to acetylation-induced changes leading to altered double strand break formation and repair [
Various types of HDAC-I’s have been identified so far. These can be grouped according to four categories; short-chain fatty acids, benzamides, hydroxamates, and cyclic peptides. VA is a short chain fatty acid which has long been used for treatment of epilepsy. Various cell lines, including human glioma, erythroleukemia, and colon cancer cell lines, have been tested for radiosensitizing effect of VA [
In this study,
In
Result from
Second aspect related to results from
Finally, study of the differential effect of combined treatment on both cell lines in
Various HDAC-I’s have been tested in clinical trials. A pioneering study was a phase II study on the combination of VA, temozolomide, and radiotherapy (RT) for patients with high grade brain tumor. Currently, 11 studies utilizing VA and RT are listed on the
Result of this study indicate that a radiosensitizing effect for fractionated radiotherapy of VA for A549 and U87MG cells
Conflict of interest relevant to this article was not reported.
This study was in part supported by Seoul National University Hospital Research Fund (grant No. 04-2012-028-0), National R&D Program for Cancer Control by National Cancer Center Korea (grant No. 1320220), and the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP) (No.2013M2A2A7043683).
Summary of vehicle/valproic acid and radiation administration schedule. Vehicle, phosphate buffered saline; VA, valproic acid 150 mg/kg (mouse, intraperitoneal injection); IR, irradiation.
Survival curves of A549 and U87MG cells treated with valproic acid at various concentrations. (A) A549 cell treated with valproic acid at 1.5 mM. (B) A549 cell treated with valproic acid at 3.0 mM. (C) U87MG cell treated with valproic acid at 1.5 mM. (D) U87MG cell treated with valproic acid at 3.0 mM. Points, mean for three independent experiments; bars, standard error. *p < 0.05, versus control.
Tumor growth delay by valproic acid (VA), irradiation (IR), and IR+VA in A549 tumor (A) and U87MG tumor (B).