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Contribution of Decreased Expression of Ku70 to Enhanced Radiosensitivity by Sodium Butyrate in Glioblastoma Cell Line (U251)*

Yuhui LI (李宇辉)1, 2†, Hongxia ZHOU (周红霞)1†, Enming XING (邢恩明)1, Meera Dassarath1, 3, Jinghua REN (任精华)1, Xiaorong DONG (董晓荣)1, Hongli LIU (刘红利)1, Kunyu YANG (杨坤禹)1#, Gang WU (伍 钢)1 1Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430023, China 2Department of Oncology, Wuhan Central Hospital, Wuhan 430014, China 3Department of Oncology, Queen Victoria Hospital, Candos, Quatre-Bornes, Mauritius © Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2011

Summary: The present study investigated the enhanced radiosensitivity of U-251 cells induced by so-dium butyrate (NaB) and its possible mechanisms. Increased radiosensitivity of U251 cells was exam-ined by clonogenic cell survival assays. The expression of Ku70 mRNA and protein was detected by using RT-PCR and Western blotting respectively. γ-H2AX foci were measured at different time points after ionizing irradiation alone or combined with NaB treatment. The results showed that cell survival rate was significantly reduced, both D0 and Dq values were decreased (D0: 1.43 Gy vs. 1.76 Gy; Dq: 1.22 Gy vs. 2.05 Gy) after the combined treatment as compared with irradiation alone, and sensitivity enhancing ratio (SER) reached 1.23. The average number of γ-H2AX foci per cell receiving the com-bined treatment was significantly increased at different time points, and the expression levels of Ku70 mRNA and protein were suppressed by NaB in a dose-dependent manner. It was concluded that en-hanced radiosensitivity induced by NaB involves an inhibited expression of Ku70 and an increase in γ-H2AX foci, which suggests decreased ability in DSB repair. Key words: sodium butyrate; radiosensitivity; Ku70; DNA double-strand breaks; γ-H2AX

Malignant gliomas are the most common and fatal type of primary brain tumors. The current standard treatment includes aggressive surgery, concurrent chemoradiotherapy, and adjuvant chemotherapy[1]. However, most malignant gliomas still recur within or beside the high-dose irradiated region due to their insuf-ficient radiosensitivity, carrying a poor prognosis for the glioma patients.

Histone deacetylase (HDAC) inhibitors, which rap-idly emerge as a novel class of molecularly targeted anticancer agents, have recently been found to enhance tumor radiosensitivity in some tumors[2–6]. HDAC in-hibitors induce hyperacetylation of core histone proteins (H3 and H4) in chromatin and nonhistone proteins (P53, HSP90, α-tubulin), thereby disrupting their interaction with adjacent DNA and allowing transcription factors to activate certain specific genes[7, 8]. These changes in gene expression may possibly explain the key antitumor ac-tivities of HDAC inhibitors, such as activation of cellular differentiation, inhibition of cell cycle, and the induction of apoptosis[9, 10].

Sodium butyrate (NaB), a natural short-chain fatty acid, is one of the most widely investigated HDAC in-

Yuhui LI, E-mail: liyuhui090718@gmail.com †Both authors contributed equally to this work. #Corresponding author, E-mail: yangkunyu@medmail.com.cn *This project was supported by a grant from National Natural Sciences Foundation of China (No. 30870739).

hibitors[11]. Previous studies have indicated that NaB has the ability to act as a radiation sensitizer in some tumor cells[12, 13]. However, in spite of a growing interest in combining HDAC inhibitors with radiation as a clinical strategy for treating cancers, the exact molecular mecha-nism by which NaB mediates its radiosensitizing effect is unknown. In the present study, we investigated the ra-diosensitizing effect of NaB on a human glioma cell line, U251. We hypothesized that NaB-mediated radiosensiti-zation might involve the inhibition of the repair of radia-tion-induced DNA double-strand breaks (DSBs).

1 MATERIALS AND METHODS 1.1 Cell Culture

U251 glioma cells were purchased from Shanghai Cell Bank (China). The U251 cells were routinely grown in Dulbecco’s modified Eagle’s medium containing 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin, and 10% nonessential amino acid. Cells were maintained at 37°C in a humidi-fied incubator with 5% CO2 and 95% room air. 1.2 NaB Toxicity Test

U251 cells were inoculated in 96-well plates, and treated with NaB at the following concentrations: 0, 1, 3, 5, 7 and 9 mmol/L. Twenty-four h later, the cell viability was tested by using a MTT assay. Twenty μL of 5 mg/mL MTT solution was added to every pore. Cells were incubated at 37°C for 4 h to allow color develop-ment and thereafter 200 μL DMSO was added. The ab-

31(3):359-364,2011J Huazhong Univ Sci Technol［Med Sci］ DOI 10.1007/s11596-011-0381-8

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sorbance (A) values at 570 nm were determined using a microplate reader. Data generated were used to plot a dose-response curve from which the IC50 was calcu-lated. 1.3 Clonogenic Cell Survival in vitro

Clonogenic cell survival assays were performed as previously described[14]. Cultures were trypsinized to generate a single-cell suspension and appropriate number of cells was seeded into culture dishes. After 16 h, cells were treated with NaB at a concentration of 3 mmol/L for 24 h in the NaB plus ionizing irradiation group, then irradiated at different dose levels (0, 2, 4, 6, 8 Gy) using a 60Cobalt machine at a dose rate of 2.5 Gy/min. The control was irradiated alone at the same time point and at the same dose levels[15]. Twelve to 14 days after seeding, dishes were stained with crystal violet, and surviving colonies of >50 cells were scored under a dissection mi-croscope. The surviving fraction for a given treatment dose was calculated as the plating efficiency of the irra-diated samples relative to that of the unirradiated ones. For each dose level in both groups, three independent experiments were done. Multi-target click model of Sigmaplot 9.0 (Systat Software, USA) was used to fit cell survival curves and calculate sensitization enhancing ratio (SER). 1.4 Reverse Transcription-PCR

Four groups of U251 cells were cultured in RPMI 1640 medium containing NaB of 0, 1, 3, 5 mmol/L, re-spectively, at 37°C for 24 h. Total RNA was isolated with RNeasy (Qiagen, Germany), and quantified by de-termining ultraviolet A values at 260 nm. RNA was re-versely transcribed using a first strand cDNA synthesis kit (FSK-100, TOYOBO, Japan) into cDNA at 42°C for 1 h, and then incubated at 95°C for 5 min to heat-inactivate the enzymes. PCR was performed using Go Taq polymerase (Promega, USA) with the following primers: Ku70 (462 bp), 5'-ATGGCAACTCCAGAGCA GGTG-3' and 5'-AGTGCTTGGTGAGGGCTTCCA-3'; β-actin (434 bp), 5'-CGGGAAATCGTGCGT-3' and 5'-TGGAAGGTAAACAGC-3'. Temperature cycling for the PCR run comprised 37 cycles of DNA denaturation for 30 s at 95°C, primer annealing for 45 s at 57°C and elongation for 1 min at 72°C. The PCR mixtures were cooled to 4°C for 5 min, and analyzed with 2% agarose gel electrophoresis. The A ratio of Ku70/β-actin was used as the relative magnitude of Ku70 analyzed by gel imag-ing analyzer. 1.5 Western Blotting

U251 cells were rinsed in ice-cold PBS, and lysed in lysis buffer. The lysates were centrifuged at 12 000 r/min for 5 min. Protein concentrations of the lysates were determined by the Branford protein assay system. Each lane was loaded with 50 µg protein. Protein was transferred to polyvinylidene difluoride membrane and blocked with 5% BSA for 1 h at room temperature. The membrane was incubated with primary anti-ku70 rabbit polyclonal antibody (Catalog No. AB1358, Millipore, USA) overnight at 4°C and further incubated with the appropriate HRP-labeled goat anti-rabbit secondary an-tibody (Catalog No. 656120, Invitrogen CA, USA) for 1 h at room temperature. Finally, the immunoreactive bands on the membranes were detected with a standard enhanced chemiluminescence (ECL Plus Western Blot-

ting Detection System, Amersham Bioscience, UK). Density of bands in each film was scanned and analyzed using Quantity One 4.6.2 software (Bio-Rad Laborato-ries, USA). 1.6 Immunofluorescent Staining

U251 cells were treated with 3 mmol/L NaB for 24 h and then irradiated with 2 Gy in 6-well plates. At spe-cific time points (0 h, 30 min, 8 h, 24 h after irradiation), the medium was aspirated and cells were fixed in 4% paraformaldehyde for 10 min at room temperature. The cells were penetrated in 0.2% TritonX-100 for 15 min at 4°C, and blocked with 4% bovine serum albumin in PBS for 30 min. Anti-γ-H2AX rabbit polyclonal antibody (Catalog No. 07-164, Millipore, USA) at a dilution of 1:1000 in 1% bovine serum albumin, FITC-labeled sec-ondary antibody (Catalog No, A-11008, Invitrogen, USA) at a dilution of 1:400 in 1% bovine serum albumin in PBS, and 4’6-diamidino-2-phenylindole (1 µg/mL) in PBS with an antifade solution (Molecular Probes, USA) for 5 min were added and incubated accordingly. Cover slips were finally mounted, and slides were examined and captured by Olympus Laser scanning confocal mi-croscopy. γ-H2AX foci were counted in at least 100 cells from the stored images. 1.7 Statistical Analysis

Data were summarized as ±s. All statistical com-parisons were performed using Student’s unpaired two-sided t-test. The statistical calculations were per-formed by SPSS 13.0 statistical software. The criterion for statistical significance was P<0.05.

2 RESULTS

2.1 Toxicity of NaB on U251 Cells

U251 cells were treated with increased concentra-tions of NaB for 24 h, followed by a MTT test. As shown in fig. 1, the inhibition rate of U251 cells was elevated with the increase of NaB concentrations, and the IC50 value was 16.12 mmol/L.

Fig. 1 Inhibition activity of NaB on U251 cells measured by

MTT assay 2.2 Enhanced Radiosensitivity by NaB in U251 Cells

U251 cells were exposed to NaB at a concentration of 3 mmol/L for 24 h. Cells were irradiated with various doses and plated for cell survival assay. As shown in fig.

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2, the shoulder area of the survival curve was signifi-cantly narrowed and the surviving fractions at each dose decreased in the NaB plus ionizing irradiation group, compared to the control group. The values of SF2, D0, Dq, and N were all lower in cells pre-treated with NaB than radiation alone. SER was 1.23 (table 1).

Fig. 2 Effects of NaB on tumor cell radiosensitivity by clono-

genic survival assays

Table 1 The main parameters of cell survival curves after ionizing radiation

Groups SF2 D0 (Gy)

Dq (Gy)

N values

Control 0.71 1.76 2.05 3.20 NaB plus ionizing irradiation

0.49 1.43 1.22 2.36

SF2: surviving fraction at 2 Gy; D0: mean lethal dose; Dq: quasi-threshold dose; N: extrapolation number

2.3 Modulation of Ku70 mRNA and Protein Expres-sion by NaB

U251 cells were exposed to the designated concen-trations of NaB for 24 h, and collected for RT-PCR and Western blotting analysis for Ku70 mRNA and protein.

There was a significant reduction in the Ku70 mRNA level in U251 cells after treatment with NaB for 24 h (fig. 3A). There was a dose-dependent decrease in the levels of Ku70 mRNA (control group: 0.217±0.042, NaB plus ionizing irradiation group: 0.153±0.012, 0.085±0.015,

0.053±0.008 for 1, 3, 5 mmol/L NaB respectively), and P value was 0.04, 0.03, and 0.01 respectively.

The expression of Ku70 protein was decreased fol-lowing NaB pretreatment in a similar dose-dependent manner as mRNA (fig. 3B), and was lower in each NaB pretreatment group than in control group (1, 3, 5 mmol/L NaB treatment group: 0.663±0.112, 0.498±0.096, 0.313±0.088 respectively; control group: 0.832±0.142). Differences were all statistically significant (P=0.03, P=0.02, P=0.01, respectively).

Fig. 3 RT-PCR and Western blotting analysis of Ku70 mRNA

(A) and protein (B) in U251 cells treated with NaB at different concentrations M: Marker; 1: Control group; 2, 3, 4: 1, 3 and 5 mmol/L NaB respectively

2.4 Expression of γ-H2AX Foci in NaB-induced U251 Cells

To test the hypothesis that NaB impairs the repair of damaged DNA, γ-H2AX foci were assessed as an indi-cator of DNA damage. U251 cells were pretreated with 3 mmol/L NaB for 24 h, irradiated (2 Gy), and fixed at specific time points for immunocytochemical analysis of γ-H2AX foci. As shown in fig. 4 A and B, γ-H2AX foci could be clearly identified after radiation. The average number of γ-H2AX foci per cell in U251 cells receiving the combined NaB/radiation treatment was 2, 60, 28 and 13, respectively, which was significantly higher than in control group at 0, 30 min, 8 h and 24 h (P<0.05) (fig. 4 A, B and C). This increase in γ-H2AX levels following the combined treatment indicated an inhibition of the DNA DSBs repair induced by NaB.

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Fig. 4 Immunocytochemical analysis of γ-H2AX foci in NaB-induced U251 cells

A: γ-H2AX foci in U251 cells irradiated at a total dose of 2 Gy for 0, 30 min, 8 and 24 h; B: γ-H2AX foci in U251 cells pre-treated with 3 mmol/L NaB for 24 h and then irradiated at a total dose of 2 Gy for 0, 30 min, 8 and 24 h; C: Quantitative analy-sis of γ-H2AX foci in the cells subjected to different treatments [1: Control group; 2: NaB group; 3: Irradiation group (2 Gy for 30 min); 4: NaB+irradiation group (2 Gy for 30 min); 5: Irradiation group (2 Gy for 8 h); 6: NaB+irradiation group (2 Gy for 8 h); 7: Irradiation group (2 Gy for 24 h); 8: NaB+irradiation group (2 Gy for 24 h)]

3 DISCUSSION

Although HDAC inhibitors have been shown to

sensitize some tumor cell lines and rodent tumors to ion-izing radiation, the radiosensitizing effect of NaB on U251 cells and the possible mechanism are still not clear. In our present study, we found that NaB down-regulated the expression of Ku70 (a key component of DSB repair complex), and inhibited the process of DNA repair, thereby enhancing radiosensitivity of U251 cells.

NaB has been found to inhibit growth, modulate gene expression, induce apoptosis and produce differen-tiation responses in a variety of cultured tumor cell lines[16–18]. Previous work in different laboratories indi-cated that treatment with NaB alone significantly in-duced cell cycle arrest and apoptosis[16, 19]. This finding was also confirmed by our result that NaB inhibited the growth of U251 cells in a dose-dependent fashion. Due to its significant cytotoxic effects on tumor cells, NaB has been used in combination with various inducers of cell death, such as ionizing radiation and anticancer drugs. Toyooka et al demonstrated that a single treatment with psoralen plus UVA (PUVA) or NaB did not greatly affect cell survival, but the combined treatment with NaB and PUVA induced marked apoptosis with 24 h[20]. Wei et al observed a remarkable reduction in the growth rate of HCT 116 cells in NaB plus radiation combined group compared to that of the single-treatment group. They found that growth arrest and cell death were enhanced in the combined treatment group[21]. In addition to its po-tential as a single-modality anticancer agent, NaB is ef-fective in combination with radiotherapy[13].

In our study, we also found that U251 cell survival rate was significantly lower after exposure to a combined treatment of NaB and radiation than treatment with ra-diation alone, and the SER was 1.23. Possible mecha-nisms underlying HDAC inhibitors-mediated radiosensi-tization in different types of tumor cells have been pro-posed. Firstly, histone acetylation has an open chromatin structure, thereby increasing the efficiency of anticancer agents targeting DNA; secondly, there is some evidence

suggesting that HDAC inhibitors can suppress endoge-nous DNA repair process[22–24]. Also, HDAC inhibitors may induce tumor cells redistribution to a more radio-sensitive cell cycle phase, because both P21WAF1 and P27KIP1 accumulations have been reported after treatment with several HDAC inhibitors[25–27]; Finally, HDAC in-hibitors may decrease the apoptotic threshold of some tumor cells through altered expression of key apoptotic regulators such as bax, bak and bcl-2[28].

Among different types of DNA damage, DSBs are the main lethal lesions induced by ionizing radiation. DSBs can be repaired by homologous recombination (HR) and non-homologous end joining (NHEJ). In mammalian cells, NHEJ is especially important for re-pairing radiation-induced DSBs that are responsible for loss of clonogenic survival[29]. DNA dependent kinase (DNA-PK) is a main component of DNA repair complex, and consists of two components, a 460-kD catalytic subunit (DNA-PKcs) and a ku protein subunit which is a heterodimer comprised of a 70-kD protein (Ku70) and 86-kD protein (Ku80). It has been reported that abnormal expression of Ku70 may alter radiosensitivity of some tumor cells[30–33]. Decreased expression of Ku70 protein increases radiosensitivity of some tumor cells. For ex-ample, Omori et al introduced an antisense Ku70 nucleic acid into a human lung squamous cell carcinoma cell line, and they found that cells with antisense Ku70 construct had lower expression of Ku70 protein and were more radio- and chemo-sensitive than the wild-type cells[34]. In our study, we found that the expression of Ku70 mRNA and protein was both down-regulated in U251 cells after treatment with NaB in a dose-dependent manner.

Quantitative detection of γ-H2AX foci is now con-sidered a gold standard for DSBs analysis[35]. Studies have shown that the ratio of DSBs to visible γ-H2AX foci is close to 1:1[36, 37], and the dephosphorylation of γ-H2AX and elimination of γ-H2AX foci can serve as a useful marker of DSBs repair and correlate with cellular radiosensitivity[38–40]. In this study, we quantitatively detected γ-H2AX foci to study the kinetics of DSBs re-pair in U251 cells at 0, 30 min, 8 h and 24 h after radia-tion. We found that the number of γ-H2AX foci per cell

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in U251 cells receiving combined treatment with NaB and radiation was significantly increased as compared with radiation alone at all time points, which was consis-tent with decreased expression of Ku70 and lowered clonogenic survival. Therefore, down-regulation of the expression of ku70 could explain an interaction between NaB and radiation. Nevertheless, a recent study showed a different result that Scriptaid, a novel HDAC inhibitor, inhibited the process of DNA double-strand break repair in radioresistant SQ-20B cells by suppressing the ex-pression of ku80 but not ku70, thereby conferring to ra-diosensitivity[41]. This difference may contribute to in-trinsic differences between cancer cells, such as different histologic origin and status of ATM or P53[42].

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(Received Oct. 18, 2010)