Chemico-Biological Interactions 206 (2013) 394–402

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Curcumin induces radiosensitivity of in vitro and in vivo cancer models by modulating pre-mRNA processing factor 4 (Prp4) Adeeb Shehzad a, Jeen-Woo Park a, Jaetae Lee b, Young Sup Lee a,⇑ a b

School of Life Sciences, College of Natural Sciences, Kyungpook National University, Daegu 702-701, Republic of Korea Department of Nuclear Medicine, Kyungpook National University Hospital, Daegu 700-721, Republic of Korea

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Article history: Received 6 May 2013 Received in revised form 20 September 2013 Accepted 8 October 2013 Available online 19 October 2013 Keywords: Curcumin Radiotherapy ROS Apoptosis Radio-sensitizer Cancer

a b s t r a c t Radiation therapy plays a central role in adjuvant strategies for the treatment of both pre- and postoperative human cancers. However, radiation therapy has low efficacy against cancer cells displaying radio-resistant phenotypes. Ionizing radiation has been shown to enhance ROS generation, which mediates apoptotic cell death. Further, concomitant use of sensitizers with radiation improves the efficiency of radiotherapy against a variety of human cancers. Here, the radio-sensitizing effect of curcumin (a derivative of turmeric) was investigated against growth of HCT-15 cells and tumor induction in C57BL/6J mice. Ionizing radiation induced apoptosis through ROS generation and down-regulation of Prp4K, which was further potentiated by curcumin treatment. Flow cytometry revealed a dose-dependent response for radiation-induced cell death, which was remarkably reversed by transfection of cells with Prp4K clone. Over-expression of Prp4K resulted in a significant decrease in ROS production possibly through activation of an anti-oxidant enzyme system. To elucidate an integrated mechanism, Prp4K knockdown by siRNA ultimately restored radiation-induced ROS generation. Furthermore, B16F10 xenografts in C57BL/6J mice were established in order to investigate the radio-sensitizing effect of curcumin in vivo. Curcumin significantly enhanced the efficacy of radiation therapy and reduced tumor growth as compared to control or radiation alone. Collectively, these results suggest a novel mechanism for curcumin-mediated radiosensitization of cancer based on ROS generation and down-regulation of Prp4K. Ó 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Ionizing radiation (IR) has gained attention as a beneficial modality for cancer treatment, in which cancer patients undergo radiation therapy either alone or in combination with chemical agents, collectively known as radio-chemotherapy [1,2]. Radioresistance remains a basic hurdle for achieving maximal therapeutic efficacy of radiotherapy for the treatment of different cancers [3]. Therefore, a valuable approach for the prevention of radioresistance in patients undergoing radiotherapy is urgently needed. Studies have shown that concomitant treatment of radiotherapy with targeted anti-cancer agents improves the outcomes of cancer patients [4]. However, radio-sensitizers that augment tumor cell killing and reduce the radiation dose–response threshold for cancer cells with less deleterious effects on normal cells are lacking and require further development [5,6]. Reactive oxygen species (ROS) generation plays a central role in cell signaling and considered essential for various biological ⇑ Corresponding author. Address: School of Life Sciences, College of Natural Sciences, Kyungpook National University, 1370 Sangeok-dong, Buk-ku, Daegu 702701, Republic of Korea. Tel.: +82 53 950 6353; fax: +82 53 943 2762. E-mail address: [email protected] (Y.S. Lee). 0009-2797/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cbi.2013.10.007

processes [7]. Biological systems have developed an efficient and complex structure of defense mechanisms for coping with lethal oxidative stress. The most commonly antioxidant enzymes involved in defense mechanisms include superoxide dismutase
(SOD) that catalyzes the disproportion of superoxide anion O 2 to hydrogen peroxide (H2O2) and O2, and peroxidases which decompose H2O2 and hydroperoxides [8,9]. Since ROS alterations mediate IR-induced cell death, elucidation of the factors modulating antioxidant enzymes may be of clinical importance in the protection of cells against IR-induced apoptosis [10]. NADPH is essential for the reconstruction of reduced glutathione (GSH) pool by glutathione reductase and for the activity of NADPH-dependent thioredoxin system [11], both provide protection to cells against oxidative damage. Furthermore, isocitrate dehydrogenase (ICDH) such as mitochondrial NADP+-dependent ICDH (IDPm) and cytosolic ICDH (IDPc), mediate antioxidant function during oxidative stress. It has been reported that mitochondrial ICDH (IDPm) functioning to supply of NADPH needed for GSH production against mitochondrial oxidative damage [12]. Additionally, peroxiredoxins (Prx) is a group of non-seleno thiol-specific peroxidases composed of six isoforms (Prx1–Prx6), widely distributed in the cytosol, mitochondria, peroxisome and plasma, all of which inhibit ROS generation. In addition to their anti-oxidant role, these members also

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participate in various biological processes such as cell proliferation and apoptosis [13,14]. Therefore, use of compounds that induce ROS generation could be of primary importance in the treatment of cancer [15]. In recent decades, naturally occurring phytochemicals in plants have garnered considerable attention as mediators of cellular redox status as well as a strategy for the treatment of human diseases, including cancer. Curcumin [1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5dione], a ployphenolic turmeric constituent, inhibits cell proliferation and cell cycle transition in colon adenocarcinoma cell lines [16]. Curcumin has been reported to modulate several biological and biochemical signaling pathways, including inhibition of cell growth and induction of apoptosis through ROS generation [17]. Additionally, curcumin together with IR inhibited NF-jB activity in the ARMS cells, and reduced tumor volume in Rh30 and Rh41 ARMS xenografts [18]. Recently, a curcumin analogue T83 has been shown to sensitize human nasopharyngeal carcinoma cells to IR and induced apoptosis through inhibition of Jab1 as well as reduced cell growth and cell cycle progression [19]. Until now, the detailed mechanism of the sensitizing effects of curcumin on colorectal cancer is poorly understood. Colorectal carcinoma and melanoma both are aggressive malignancies as well as resistant to therapeutic strategies, including chemotherapy and radiotherapy. We recently reported that curcumin-induced Prp4B-dependent apoptosis in human colorectal carcinoma (HCT-15) cells [20]. In the present study, we demonstrate that curcumin enhanced the radio-sensitivity of HCT-15 cell lines through ROS generation. Curcumin also inhibited the activation of anti-oxidant enzymes such as IDPm and IDPc, Prx1,-2, and -6, and Pre-mRNA processing factor 4 (Prp4) kinase (Prp4K). Curcumin combined with IR potentiated the efficacy of radiation against melanoma cell implanted-tumors in mice. It is our goal that elucidating the radio-sensitizing effects of curcumin on apoptosis in cancer cells will provide new insights into developing a therapeutic strategy for cancer radiotherapy. 2. Materials and methods 2.1. Materials Curcumin, Hoechst 33342, and propidium iodide (PI) were obtained from Sigma–Aldrich (USA). 20 ,70 -Dichlorofluorescin diacetate (DCFHDA) was purchased from Molecular Probes (Eugene, OR). Antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), Cell Signaling (Beverly, MA) and Ab Frontier (South Korea). Electrophoresis reagents and Bio-Rad protein assay kit were provided by Bio-Rad Laboratories (USA). These chemicals were prepared according to the manufacturer’s instructions. 2.2. Cell culture and treatment HCT-15 cells (CCL-225) were obtained from ATCC, and grown at a density of 1  106 cells per dish in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) in an incubator containing a humidified atmosphere of 95% air and 5% CO2 at 37 °C. Exponentially growing cells were exposed to radiation at room temperature with a 137Cs source (Cis Bio International, Gif-sur Yvette, France) at 1–5 Gy/min and treated with 30 lM curcumin. 2.3. Analysis of cell morphology Morphological analysis of IR-induced apoptosis was performed with fluoresce microscopy after staining using Hoechst 33342 dye. After irradiation, HCT-15 cells were incubated with curcumin for 24 h, fixed in 4% paraformaldehyde and permeabilized with PBS/

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0.5% Triton X-100, after which nuclei were stained for 20 min using Hoechst 33342 dye. 2.4. Determination of apoptosis Cell cycle analysis was performed by flow cytometric staining of permeabilized cells with PI to determine DNA content. Cells were collected by centrifugation, fixed with 70% ethanol, and then stained with PI solution containing 50 lg/mL of PI, 0.1% Triton X100, 0.1 mM EDTA and 50 lg/mL of RNase for 20 min at 4 °C. Stained DNA was analyzed by a flow cytometer (Becton Dickinson), and the percentage of nuclei with sub-G1 content indicated apoptotic cells. 2.5. Measurement of ROS Intracellular ROS concentration was assessed using the oxidantsensitive fluorescent probe-DCFHDA, as shown previously [21]. Cells were exposed to 5 lM DCFHDA for 20 min and then washed with 1 PBS. DCF fluorescence (excitation, 480 nm; emission, 520 nm) was imaged using a laser confocal scanning microscope (DM/R-TCS, Leica) coupled to a microscope (Leitz DM REB). 2.6. Preparation of cytosolic protein fraction Cytosolic fraction of cells was prepared as described previously [21]. Cells were sonicated in buffer A (20 mM Tris, pH 7.5, 250 mM sucrose, 10 mM EGTA, 2 mM EDTA, 1 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 1 lg/mL of aprotinin, 1 lg/mL of leupeptin, and 1 lg/mL of pepstatin) and centrifuged for 10 min at 1000g to remove cell debris. Supernatants were then centrifuged at 13,000g for 5 min and the resulting supernatant was saved as the cytosolic fraction. 2.7. Immunoblot analysis Proteins ware extracted from whole cell lysates, fractionated by SDS–PAGE and electrotransferred onto nitrocellulose membranes. Membranes were then blocked using 5% non-fat dry milk and subsequently probed with primary antibody. Antibody–antigen complexes were visualized using horseradish peroxidase-labeled antirabbit or anti-mouse IgG, followed by detection using enhanced chemiluminescence (Amersham Pharmacia Biotech, UK). 2.8. Production of cDNA and siRNA-Prp4 HCT15 clones Clone cDNA of Prp4K (sc117203) was obtained from OriGene (USA), whereas siRNA-Prp4K (SC-76257) and scrambled siRNA (sc-37007) pools were obtained from Santa Cruz Biotechnology and utilized according to the manufacturer’s instructions. Briefly, HCT-15 cells were cultured at a density of 1  106 cells per dish to 45–55% confluence. Cells were then transfected by using both Lipofectamine LTX and Plus reagent for cDNA, whereas RNAiMAX was used for siRNA-Prp4K (Invitrogen, Carlsbad, CA, USA). After transfection, cells were exposed to radiation and curcumin. The effect of scrambled siRNA (sc-37007) was also evaluated. 2.9. Animal study protocol Male C57BL/6J mice obtained from Hyochang Science (Daegu, Korea) were housed five mice per cage, under conditions of constant temperature of 22 °C, and a light/dark cycle of 12 h. Our experimental protocol was approved by the Institutional Animal Care and Use Committee, and is in agreement with international laws and policies (NIH Guide for the Care and Use of Laboratory Animals, NIH publication No. 85-23, 1985). A total amount of

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1  105 mouse melanoma (B16F10) cells were implanted subcutaneously using an insulin syringe into the shaved flank of each mouse (Fig. 1). After 1 week of implantation, 10 mice at 7-weeksold were randomly divided into three treatment groups; (A) control (vehicle), (B) piperine (50 mg/kg orally/day) and (C) curcumin (500 mg/kg orally/day). After 3 weeks, mice were divided into three additional groups (n = 5) (D) (irradiated with 5 Gy of IR), (E) piperine in combination with 5 Gy of IR, and (F) curcumin in combination with 5 Gy of IR. Animals were irradiated once with 5 Gy/min at 21 day and sacrificed after 1 week. Tumor volumes were measured weekly using Vernier calipers and calculated according to the formula: V = 4/3pW2L (short size2  long size/2 (mm3). The median times were calculated for each cohort, and the various groups were compared using unpaired Student’s t test. Developed tumors in flanks of mice were cut out from mice using scissor, and stored at 80 °C.

was detected in response to IR exposure and curcumin treatment. Importantly, oxidant-induced 20 ,70 -dichlorofluorescein fluorescence was significantly enhanced by combined curcumin and IR treatment as compared to either treatment alone. To understand the morphological characteristics of apoptosis, cells were stained with Hoechst 33342 dye after exposure to c-irradiation, followed by curcumin treatment and imaging by fluorescence microscopy. The apoptotic cell ratio increased in curcumin treated cells with IR, which displayed distinctive features of apoptosis such as cell size reduction, fractional detachment, and rounding or fragmented nuclei (data not shown). These results advocate a potential role for curcumin in enhancing IR-induced cell death.

2.10. Statistical analysis

Prp4K is a serine–threonine protein kinase that plays a central role in cell signaling and proliferation. Herein, we report a possible role for Prp4K in the resistance of HCT-15 cells to IR-induced apoptosis. Cells were transfected with cDNA-Prp4K pool and exposed to 5 Gy of c-irradiation. Transfection of cells with cDNA-Prp4K prevented IR-mediated cell death (Fig. 3A). To further determine the role of Prp4K in IR-induced apoptosis, HCT-15 cells were stably transfected with a siRNA-Prp4K. The results were analyzed by MTT and imaged by confocal microscopy. As shown in Fig. 3B, cell death decreased after transfection with Prp4K clone as compared to exposure to 5 Gy of IR without cDNA probe. However, silencing of Ppr4K with siRNA enhanced IR-induced cell death of HCT-15 cells. This result demonstrates that Prp4K plays a major role in IR-induced cell death. Conventional evidence has demonstrated a role for ROS generation in cell death. To determine whether or not Prp4K over-expression reverses IR-induced oxidative stress apoptosis, we measured intracellular oxidation status using DCFHDA, a specific oxidation-sensitive fluorescent probe. As shown in Fig. 3C, transfection with Prp4K clone diminished IR-induced ROS generation. To confirm that this specific decrease in DCF-detectable ROS generation was associated with Prp4K, cells were transfected with siRNA-Prp4K followed by exposure to IR. It was observed that silencing of Prp4K restored IR-induced ROS generation (Fig. 3C). Furthermore, HCT-15 cells transfected with scrambled siRNA showed similar effects as control cells without silencing (Fig. 3C). Taken together, these results demonstrate that Prp4K over-expression provides protection against IR-induced cell death and induce resistance to IR by reducing steady-state levels of intracellular oxidants.

All data are presented as mean ± standard deviation (SD) of three separate experiments. Data were evaluated using SPSS for Student’s t-test and subjected to one-way or two way analysis of variance. Significance of the difference between the means was determined and considered significant at ⁄P 6 0.05. 3. Results The present study was conducted to investigate whether or not curcumin can sensitize in vitro and in vivo cancer models to radiation as well as to explore the mechanism of radio-resistance. 3.1. Curcumin potentiates IR-induced apoptosis in HCT-15 cells We investigated the effects of curcumin on cellular markers of apoptosis in HCT-15 cells exposed to IR. Irradiated cells were incubated with curcumin for 24 h, and the sub-G1 fraction from fixed nuclei of cells was measured by PI staining and FACS analysis. Cell death increased distinctly in HCT-15 cells treated with both curcumin and IR as compared to IR-treated cells alone (Fig. 2A). IR-induced cell death dose dependently, which was further potentiated by curcumin treatment (Supplementary Fig. 1). To confirm an association between apoptotic cell death and ROS generation, intracellular oxidation levels in cancer cells were measured by fluorescence microscopy using a specific cell-permeable fluorescent dye, DCFHDA. As shown in Fig. 2B, ROS generation

3.2. Prp4K involves in IR resistance

Fig. 1. Schematic representation of experimental protocol as described in Section 2. Group treatments were as follows: (A) control was treated only with vehicle, (B) piperine (50 mg/kg orally/day), (C) curcumin (500 mg/kg orally/day), (D) irradiated with 5 Gy of IR, (E) piperine in combination with 5 Gy of IR, and (F) curcumin in combination with 5 Gy of IR (n = 5).

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Fig. 2. Curcumin potentiates IR-induced cell death. (A) HCT-15 cells were harvested and stained with PI, and their DNA contents were analyzed by flow cytometry. Sub-G1 region (presented as M1) includes cells undergoing apoptosis. Sub-G1 fractions of cells (%) were plotted against curcumin or IR treatment alone as well as in combination, for 24 h. (B) Intracellular oxidation level was determined by exposing HCT-15 cells to 5 Gy of c-irradiation, followed by treatment with 30 lM curcumin for 24 h and further incubation with DCFHDA for 20 min. Accumulation of probe in cells was measured by increased emission at 520 nm after the sample was excited at 480 nm. Images of DCFloaded cells were obtained under a fluorescence microscope. Data represent three independent experiments and for each bar. ⁄P 6 0.05.

We also investigated pro-caspase-3 activity and Prp4K expression after transfection with Prp4K clone by using Western blotting. Caspase-3 is activated by proteolytic processing of its 32 kD form into two smaller subunits, and it can be detected by reduction of its pro-enzyme level [22]. As shown in Fig. 3D, IR failed to increase caspase-3 activation as well as down-regulation of Prp4K at the protein level. In the current study, b-actin expression remained consistent upon cDNA-Prp4K transfection and IR exposure and was therefore used as a loading control in Western blotting (Fig. 3D). Summing up, these results demonstrate that Prp4K regulation shares a major role in the survival of HCT-15 cells against IRinduced cell death. 3.3. Curcumin inhibits Prp4K-induced activation of anti-oxidant enzymes in HCT-15 cells To confirm an association between apoptosis and ROS generation in IR-induced cell death, the effects of Prp4K on anti-oxidant

enzyme expression and apoptosis were determined. Anti-oxidant enzymes such as IDPm and IDPc, Prx1, -2, and -6 are known to prevent oxidative damage by eliminating H2O2 and hydroperoxides. Western blot analysis of cDNA-Prp4K-transfected cells showed increased levels of anti-oxidant enzymes as compared with nontransfected control (Fig. 4A). Exposure of HCT-15 cells to 5 Gy of c-irradiation after transfection with Prp4K plasmid did not alter the levels of IDPm, IDPc, Prx1, -2, and -6 (Fig. 4A). Further, Prp4K-transfected cells were exposed to IR and the cytosolic fraction separated. As shown in Fig. 4B, IR exposure had no effect on anti-oxidant enzyme activation. After this, we exposed Prp4K-trasfected cells to IR, followed by curcumin treatment for 24 h. Neither curcumin nor IR treatment alone reduced the expression of IDPm or IDPc, and Prx1, -2, and -6, whereas concomitant treatment inhibited anti-oxidant enzymes activation in whole cell lysates as well as in the cytoplasm (Fig. 4C and D). These results indicate that over-expression of Prp4K inhibits IR-induced apoptosis possibly by decreasing ROS and anti-oxidant enzyme activation.

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Fig. 3. Over-expression of Prp4K restricts IR-induced cell death. (A) HCT-15 cells were transfected with Prp4K clone, exposed to various doses of IR (1–5 Gy), and analyzed by MTT assay. Data represent the average ± SD of three independent experiments for each bar. ⁄P 6 0.05. (B) HCT-15 cells were transfected with cDNA-Prp4K or siRNA-Prp4K and then exposed to 1–5 Gy of c-irradiation. Cell viability was determined by MTT assay. Data represent the average ± SD of three independent experiments for each bar. ⁄ P 6 0.05. (C) HCT-15 cells were transfected with cDNA-Prp4K and siRNA-Prp4K, and then exposed to 5 Gy of c-irradiation for the indicated time, intracellular oxidation status was observed by DCFHDA fluorescence. Transfection with siRNA-Prp4K diminished the protective effect of Prp4K and induced ROS generation as shown by DCFHDA fluorescence. Scrambled siRNA displayed similar effects as control. (D) Cells were transfected with Prp4K clone and then exposed to 1–5 Gy of c-irradiation. Total protein content was isolated from cells and analyzed by Western blotting with anti-Prp4K, pro-caspase-3, or anti-b-actin antibody.

3.4. Curcumin potentiates IR effect in vitro and in vivo We evaluated the combined effects of curcumin and IR against the growth of subcutaneous B16F10 xenograft tumors. After implantation with tumor cells, mice were randomized into six groups as described in Section 2. Curcumin and piperine treatments were initiated after randomization and continued up to 21 days. Mice (n = 5) were irradiated at 21 day once with 5 Gy and maintained for 7 days. As shown in Fig. 5A, combined administration of curcumin and IR resulted in tumor growth inhibition as compared to combined piperine and IR treatment or other treatments alone. Tumor volume was measured according to the formula described in Section 2 and represented in a bar graph. Our results show the expression of anti-oxidant enzymes such as IDPm, IDPc, Prx1, -2, and -6 after treatments with IR, piperine, or curcumin alone, as well as in combination with IR. We also

confirmed caspase-3 activation and Prp4K expression. For the in vitro study, cells were irradiated at 5 Gy and incubated with piperine and curcumin for 24 h. As shown in Fig. 5B, IR in combination with curcumin activated caspase-3 activity, whereas the expression levels of Prp4K and anti-oxidant enzymes IDPm, IDPc, Prx1, -2, and -6, which are known to reverse IR-induced apoptosis, were reduced. To explain the greater augmentation required for in vivo tumor growth compared to the in vitro studies, we conducted Western blotting of isolated tumor tissue from each group of mice. As shown in Fig. 5C, curcumin not only potentiated the effects of IR in vitro but also reduced the activation of anti-oxidant enzymes in vivo. In mice treated with curcumin and IR, activation of caspase-3 and down-regulation of Prp4K were greater than in other groups. Based on these results, it could be suggested that lower concentrations of curcumin combined with IR may exert potent anti-tumorigenic effects compared to traditional radio-sensitizers

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Fig. 4. Prp4K over-expression activates anti-oxidant enzymes in HCT-15 cell lines. (A) HCT-15 cells were transfected with cDNA-Prp4K and exposed to 1–5 Gy of IR. Total protein content was isolated from cells and analyzed by Western blotting. (B) Cells were transfected with Prp4K clone and then irradiated with 1–5 Gy of IR. Cells were fractionated into cytosolic fragments, and equal amounts of lysates were used for immunoblotting with various antibodies. (C) Transfectant HCT-15 cells were exposed to 5 Gy of c-irradiation and treated with 30 lM curcumin for 24 h. Total protein content was isolated from cells and analyzed by Western blotting. (D). Trasfectant cells were fractionated into cytosolic fragments after exposure to IR and curcumin, and equal amounts of lysates were used for immunoblotting. b-Actin expression remained constant in all experiments.

such as piperine. Thus, curcumin may have sensitized the xenograft mouse model to radiation through down-regulation of Prp4K and reduction of anti-oxidant enzyme activation. 4. Discussion The present study evaluated the radio-sensitizing effects of curcumin in vitro against colorectal carcinoma cell lines and in vivo melanoma cells implanted into a xenograft mouse model. Regulation of IR-induced apoptosis has a potential role in cancer treatment, but this common modality of cancer treatment loses its efficacy as soon as cancer cells become resistant to repeated exposure of radiation [23]. Therefore, enhancement of IR using sensitizers will improve the therapeutic efficacy of radiotherapy [24]. Our results indicate that curcumin enhances the anti-tumor effects of radiation therapy both in vitro and in vivo through down-regulation of Prp4K and by modulating cellular redox status through suppression of the antioxidant enzyme system. The empirical effects of siRNA-Prp4K on HCT-15 cells treated with curcumin observed in this study promote the use of therapeutic modifiers in radiation therapy.

The molecular response of tumors to IR is mediated through multiple interrelated mechanisms. One such mechanism includes ROS, which are by-products of cellular metabolism as well as produced in response to various oxidative stresses. ROS play a central role in cell growth, survival, apoptosis, and tumor development [25]. IR has been shown to enhance ROS generation, consequently induce apoptotic cell death. It has been reported that ROS generation initiates cellular signal transduction pathways that either subject cells to oxidative stress resulting from radiation or induce cellular damage [26]. Apoptosis is regarded as programmed cell death, and induction of apoptosis by ROS generation shares a major mechanism with anti-cancer modalities, including chemotherapy and c-irradiation [27]. Therefore, enrichment of ROS-mediated tumor cell apoptosis by IR in combination with natural phytochemicals may be a useful strategy for cancer treatment. Curcumin, a natural polyphenolic compound, exerts anti-tumor effects by mediating cellular redox status [28]. Removal of introns from pre-mRNA is catalyzed by the spliceosome, which is considered as an important mediator for the generation of functional proteins. Prp4 plays a central role in signal transduction and is essential for pre-mRNA processing by spliceo-

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Fig. 5. Curcumin sensitizes melanoma-induced tumors to radiation in vitro and in vivo. (A) Photographs of mice bearing B16F10-induced melanoma tumors on day 31 after treatment with IR, piperine, or curcumin alone, as well as in combination with IR. Curcumin and IR together reduced tumor size as compared to other treatment groups. Tumor volumes were measured using Vernier calipers, calculated as described in Section 2, and represented in a bar chart, for each ⁄P 6 0.05 (n = 5). (B) HCT-15 cells were exposed to 5 Gy of c-irradiation, after which total protein content was isolated from cells and analyzed by Western blotting with various antibodies. (C) Curcumin inhibited anti-oxidant enzymes in B16F10-induced tumor mice. Developed tumors in flanks of mice were cutout, and tissues were homogenized with tissue lysis buffer. Total protein content was isolated from cells and analyzed by Western blotting. b-Actin was used as a reference in both experiments.

some activation [29]. Prp4K has been reported to be essential for stable tri-snRNP association during spliceosomal B complex formation in pre-mRNA splicing [30], but the exact molecular mechanism by which Prp4K mediates apoptosis upon curcumin treatment is not yet established. It is also known that Prp4K can phosphorylate itself, which is important for coordination of premRNA processing and transcriptional regulation. Mutation of Prp4 leads to pre-mRNA accumulation and impairment of G1-S progression of the cell cycle [31,32]. Our results demonstrate that over-expression of Prp4K prevents IR-induced cell death possibly through reduction of ROS generation and caspase-3 activation. Caspase-3 is a well known apoptotic factor for curcumin-induced apoptosis [33]. HCT-15 cells treated with IR exhibited a profound reduction (8.55%) in their proliferation rate as compared with controls (3.49%). Curcumin treatment also induced cell death (13.68%), whereas combined treatment of curcumin and IR potentiated HCT15 cell death (19.79%) (Fig. 2A). However, transfection of cells with Prp4K clone induced IR resistance and suppressed IR-induced cell death (Fig. 3A). This could be mediated, at least in part, by the activation of anti-oxidant enzymes following Prp4K expression, resulting in suppression of oxidative stress and ROS-induced cell death. However, we found that concomitant administration of curcumin and IR along with silencing of Prp4K by siRNA, enhances the potency of IR-induced apoptosis by down-regulating Prp4K expression and subsequently modulating cellular redox status. Therefore,

it is likely that Prp4K plays a role in preventing apoptosis and developing resistance against IR in cells. Recently, a number of studies have reported inherent limitations associated with the specificity of fluorogenic probes in detecting ROS levels [34–36]. Thus, the fluorescence observed by fluorescence microscopy under condition of intracellular oxidative stress in cells may be the outcome of several factors including, but not limited to increase ROS levels, reduced GSH levels, altered iron uptake, improved peroxidase activity, and cytochrome c release from mitochondria [34–36]. In this study, the fluorescence probes used for the detection of ROS possibly reflect general oxidative stress in the HCT-15 cells. In addition to this, we have presented anti-oxidant enzymes as indicative of the modulation of the cellular redox status. Over-expression of IDPm, IDPc, Prx1, -2, and -6 prevents oxidative stress-induced cell death. IDPm is a major producer of mitochondrial NADPH, subsequently leading to increased mitochondrial GSH pool needed for the defense against ROS-mediated oxidative stress [11,12]. On the other hand, Prx 1, -2, and -6 enzymes each catalyzed the H2O2-dependent oxidation of NADPH in the presence of the Trx system. The oxidation of NADPH required this protein components (Prx), being negligible in the absence of any one of the three. Transient over-expression of Prxs in cultured cells showed that they were able to eliminate the intracellular H2O2 generated in response stress [13,37]. This means that IR perturbed cellular redox balance and shows that Prxs may shift

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this balance to the antioxidant state and protects cells against prooxidant IR [12,38]. Our present results strengthen the hypothesis that activation of anti-oxidant enzymes plays a protective role against IR-induced cell death in HCT-15 cells. Prp4K mediates this effect, at least in part, by enhancing anti-oxidant enzyme activation, which in turn suppresses ROS generation and inhibits the entire apoptotic pathway. The radio-sensitizing effect of curcumin was previously reported in vivo. Specifically, administration of 1 g/ kg of curcumin has been shown to inhibit inflammatory markers and reduce tumor volume [39]. The aforementioned description clearly shows that lower concentrations of curcumin in combination with IR enhance the efficacy of radiotherapy and may exert anti-tumorigenic effects. In conclusion, our results show that curcumin enhances IRmediated apoptotic cell death in HCT-15 cells through ROS generation. Over-expression of Prp4K leads to increased resistance against IR-induced cell death possibly by reducing ROS generation through anti-oxidant enzyme activation. Therefore, curcumin-induced sensitization to IR-induced cell death may have therapeutic applications for cancer treatment, especially, in overcoming radioresistance to IR during consecutive radiotherapy. Further studies will identify the signaling pathways mediated by Prp4K overexpression. Finally, concomitant administration of curcumin and IR may provide a bridge for improving the efficacy of radiotherapy. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgments This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea Government (MEST) (NRF-2009-0078234). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cbi.2013.10.007. References [1] C. Bianco, R. Bianco, G. Tortora, V. Damiano, P. Guerrieri, P. Montemaggi, J. Mendelsohn, S. De Placido, A.R. Bianco, F. Ciardiello, Antitumor activity of combined treatment of human cancer cells with ionizing radiation and antiepidermal growth factor receptor monoclonal antibody C225 plus type I protein kinase A antisense oligonucleotide, Clin. Cancer Res. 6 (2000) 4343– 4350. [2] W.Y. Koh, K. Lim, J. Tey, K.M. Lee, G.H. Lim, B.A. Choo, Outcome of 6 fractions of 5.3Gray HDR brachytherapy in combination with external beam radiotherapy for treatment of cervical cancer, Gynecol. Oncol. (2013), http://dx.doi.org/ 10.1016/j.ygyno.2013.07.102. [3] C.M. Bradbury, S. Markovina, S.J. Wei, L.M. Rene, S. Karimpour, C. Hunt, D.R. Spitz, D. Gius, Increased activator protein 1 activity as well as resistance to heat-induced radiosensitization, hydrogen peroxide, and cisplatin are inhibited by indomethacin in oxidative stress-resistant cells, Cancer Res. 61 (2001) 3486–3492. [4] K. Camphausen, P.J. Tofilon, Combining radiation and molecular targeting in cancer therapy, Cancer Biol. Ther. 3 (2004) 247–250. [5] B.J. Moeller, M.R. Dreher, Z.N. Rabbani, T. Schroeder, Y. Cao, C.Y. Li, M.W. Dewhirst, Pleiotropic effects of HIF-1 blockade on tumor radiosensitivity, Cancer Cell 8 (2004) 99–110. [6] W. Li, B. Li, N.J. Giacalone, A. Torossian, Y. Sun, K. Niu, O. Lin-Tsai, B. Lu, BV6, an IAP antagonist, activates apoptosis and enhances radiosensitization of nonsmall cell lung carcinoma in vitro, J. Thorac. Oncol. 6 (2011) 1801–1809. [7] L. Packer, E. Cadenas, Oxidants and antioxidants revisited. New concepts of oxidative stress, Free Radic. Res. 41 (2007) 951–952. [8] J.H. Lee, J.W. Park, Oxalomalate regulates ionizing radiation-induced apoptosis in mice, Free Radic. Biol. Med. 42 (2007) 44–51. [9] J. Wang, J. Yi, Cancer cell killing via ROS: to increase or decrease, that is the question, Cancer Biol. Ther. 7 (2008) 1875–1884. [10] T.M. Buttke, P.A. Sandstrom, Oxidative stress as a mediator of apoptosis, Immunol. Today 15 (1994) 7–10.

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