Benzyl Isothiocyanate Sensitizes Human Pancreatic Cancer Cells to Radiation by Inducing Apoptosis
Total Page：16
File Type：pdf, Size：1020Kb
INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 28: 1043-1047, 2011 Benzyl isothiocyanate sensitizes human pancreatic cancer cells to radiation by inducing apoptosis MAKI OHARA1,3, SHINICHI KIMURA1, AYA TANAKA1, KEN OHNISHI2, RYUICHI OKAYASU4 and NOBUO KUBOTA1 1Department of Radiological Sciences and 2Department of Biology, Center for Humanity and Sciences, Ibaraki Prefectural University of Health Sciences, Ami, Inashiki-gun, Ibaraki 300-0394; 3Department of Environmental Sciences, Faculty of Science, Ibaraki University, Mito, Ibaraki 310-0035; 4Heavy-Ion Radiobiology Research Group, Research Center for Charged Particle Therapy, National Institute of Radiological Sciences, Inage-ku, Chiba 263-8555, Japan Received June 20, 2011; Accepted July 27, 2011 DOI: 10.3892/ijmm.2011.770 Abstract. Isothiocyanates are a class of naturally occurring Usually, pancreatic cancer is diagnosed at an advanced stage, chemopreventive agents known to suppress proliferation of therefore prognosis is very poor. Less than 5% of those diag- cancer cells in culture. The present study was undertaken in nosed are still alive 5 years after diagnosis. Complete remission order to examine the effects of benzyl isothiocyanate (BITC), is still rather rare. Surgery remains the most effective treatment one of the common dietary isothiocyanates, on the radiosensi- for pancreatic cancers. However, only 10-20% of cancers are tivity of human pancreatic cancer cells and to gain insights into suitable for tumor resection, after which local recurrences are the underlying molecular mechanism of BITC-induced radio- common (2). Furthermore, pancreatic cancer cells have unusual sensitization. Two human pancreatic cancer cell lines, PANC-1 resistance to both chemotherapy and radiotherapy (3,4). These and MIAPaCa-2, were treated with BITC and irradiated with facts warrant development of novel therapeutic radiosensitizers X-rays. Radiation sensitivity, apoptosis, and protein levels were for the treatment of pancreatic cancer. determined by a clonogenic assay, fluorescence microscopic Epidemiological studies have indicated that dietary intake analysis with DAPI staining and Western blotting, respectively. of cruciferous vegetables may be protective against various MIAPaCa-2 cells were relatively more sensitive to BITC treat- types of cancers (5,6). Isothiocyanates are degradation products ment compared with PANC-1 cells. Radiosensitization was of glucosinolates that are common in cruciferous vegetables observed in both PANC-1 and MIAPaCa-2 cells incubated with including broccoli, watercress, cabbage and cauliflower (7). In BITC at 5 to 10 µM and 2.5 to 5 µM for 24 h, respectively. The addition to the chemopreventive properties, recent studies have combination treatments with BITC and X-rays also revealed an shown that certain isothiocyanates can inhibit proliferation of increased percentage of apoptotic cells. In addition, treatment cancer cells by inducing cell cycle arrest, apoptosis induction, with BITC and X-rays resulted in a decrease in the protein and autophagic cell death (8,9). Benzyl isothiocyanate (BITC), levels of the X-linked inhibitor of apoptosis (XIAP), inhibitor of one of the best studied members of the isothiocyanate family, apoptosis (IAP) family protein, and in a marked increase in the is of interest in cancer research due to suppression of cancer apoptosis protease activating factor-1 (Apaf-1), essential for acti- cell viability in association with cell cycle arrest, apoptosis vation of caspase-9 in stress-induced apoptosis. BITC may be induction, and autophagic cell death (10-14). In addition, BITC a useful radiosensitizer for radiotherapy of pancreatic cancers. inhibits angiogenesis in vivo by down-regulation of vascular endothelial growth factor (VEGF) receptor 2 protein levels in Introduction the tumor (15). Based on these observations, it is considered that BITC is a promising candidate for further study in cancer In Japan and the United States, pancreatic cancer is respectively prevention and therapy. Recently, it has been reported that BITC the fifth and fourth leading cause of cancer-related deaths (1). caused a significant decrease in the expression and activity of histone deacetylase (HDAC)1 and HDAC3 in human pancre- atic carcinoma cells, but not in normal cells (16), indicating that BITC acts as a HDAC inhibitor in human pancreatic cells. Many laboratories have shown that HDAC inhibitors, including Correspondence to: Dr Nobuo Kubota, Department of Radiological trichostatin A, MS-275 and Scriptaid sensitize human tumor Sciences, Ibaraki Prefectural University of Health Sciences, 4669-2 Ami-machi, Ami, Inashiki-gun, Ibaraki 300-0394, Japan cells to radiation (17,18). The radiosensitizing effect of BITC E-mail: [email protected] has been reported recently against human pancreatic carci- noma cells (19). However, the mechanistic aspect underlying Key words: human pancreatic cancer cells, benzyl isothiocyanate, radiosensitization by BITC is not yet completely understood. radiosensitization, apoptosis Thus, the present study was undertaken to investigate the radio- sensitization effect of BITC using PANC-1 and MIAPaCa-2 1044 ΟΗΑRΑ et al: RADIOSENSITIZATION BY BITC human pancreatic cancer cells, in order to demonstrate that BITC enhances the responses of both cell lines to radiation by inducing apoptosis through caspase activation. Materials and methods Cell culture and chemicals. The human pancreatic tumor cell lines, PANC-1 and MIAPaCa-2, were maintained in α-minimum essential medium (α-MEM) supplemented with 20 mM 4-(2-hydroxyethyl) piperazine ethane sulfonic acid (HEPES), 8 mM NaHCO3, 50 µg/ml streptomycin, 50 U/ml penicillin and 10% fetal calf serum (20). Cells were cultured in a humidified incubator at 37˚C with a mixture of 98% air Figure 1. Effect of BITC on cell survival of PANC-1 (●) and MIAPaCa-2 and 2% CO2. BITC was purchased from LKT Laboratories (St. (◼) cells. Cells were exposed to various concentrations of BITC for 24 h at Paul, MN, USA). 37˚C, and cell survival was measured by the colony formation method. Data points and bars are the average and standard errors, respectively, from three Irradiation. Cells were irradiated with 10 MV X-rays from separate experiments. a linear accelerator (Mitsubishi Electric, Tokyo, Japan) at a dose rate of 4 Gy/min. Doses were measured using an Innax Dosemaster (NE Technology, Berks, UK) before irradiation. ferred to polyvinylidene difluoride (PVDF) membranes using a semi-dry transfer system. The membranes were blocked in Clonogenic assay. Cell survival for PANC-1 and MIAPaCa-2 5% non-fat milk for 1 h at room temperature, then incubated cells was measured by a colony formation assay (21). Briefly, overnight at 4˚C with a primary antibody specifically recog- 105 cells were seeded in 24 cm2 flasks and incubated for nizing cleaved PARP, Bcl-2, Bax, apoptosis protease activating approximately 24 h before treatment with BITC. Cells were factor-1 (Apaf-1), X-linked inhibitor of apoptosis (XIAP) (Cell exposed to BITC or DMSO (control) for 16 h, irradiated with Signaling Technology), and actin (Chemicon International, X-rays, and incubated in the presence of drug for a further 8 h. Inc., Temecula, CA, USA). Primary antibodies were detected After treatment with BITC for 24 h, cells were washed with using the horseradish peroxidase (HRP)-conjugated second Dulbecco's phosphate-buffered saline (PBS) and dispersed antibodies and an enhanced chemiluminescence (ECL) detec- with 0.05% trypsin containing 0.02% EDTA. Single cells were tion system (GE Healthcare, Buckinghamshire, UK). counted using a cell counter, diluted, and plated in 60-mm dishes at various cell densities. The dishes were incubated in a Results CO2 incubator for 12 days. Colonies were stained with crystal violet dissolved in 20% methanol. Colonies of over 50 cells Effect of BITC on cell survival of human pancreatic tumor were counted as survivors. cells. To determine the effect of BITC on cell survival, PANC-1 and MIAPaCa-2 cells, 2 human pancreatic cancer cell lines, Determination of apoptosis. Apoptosis induction was measured were treated with increasing concentrations of BITC for 24 h, by detecting apoptotic bodies (22). PANC-1 and MIAPaCa-2 and cell survival was assessed by a colony formation method. cells were exposed to BITC or DMSO for 16 h, irradiated with As shown in Fig. 1, BITC treatment resulted in concentration- 6 Gy of X-rays, and incubated in the presence of the drug for a dependent cell death in both cell lines. further 8 h. The medium was then removed and replaced with fresh medium free of drug. At 24 h after X-ray irradiation, both Radiosensitization by BITC. To assess the effect of BITC attached and floating cells were collected by trypsinization and treatment for 24 h on the radiosensitivity of PANC-1 and centrifugation, resuspended in a fixative solution containing MIAPaCa-2 cells, concentrations of 5 and 10 µM, and 2.5 3% paraformaldehyde in PBS and stained with DAPI. The cells and 5 µM were used for PANC-1 and MIAPaCa-2 cells, were placed on microscope slides and covered with glass cover- respectively. Cell survival curves were constructed for both slips. Then, the cells were photographed using a fluorescence cell lines after X-ray irradiation in the presence or absence microscope, and the number of apoptotic cells was counted. of BITC. Actively growing cells were exposed to BITC for 16 h at 37˚C, irradiated with X-rays, and incubated for an Western blot analysis. Cells were exposed to BITC or DMSO additional 8 h. Both cell lines showed significant increases for 16 h, irradiated with 6 Gy of X-rays, and incubated in the in radiation-induced cell killing when combined with BITC presence of the drug for a further 8 h. Cells were washed with treatment (Figs. 2 and 3). The radiosensitivity enhancement ice-cold PBS, collected, and pelleted by centrifugation. Cells ratios measured at a survival rate of 10% were 1.19 at 5 µM were lysed in lysis buffer (Cell Signaling Technology, Beverly, and 1.33 at 10 µM in PANC-1 cells, and 1.24 at 2.5 µM and MA, USA) and then frozen and thawed three times.