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3Department of Medical Genetics, College of Medicine, Hallym University, Gangwon-do 200–702, ... tosis of HIT-T15 cells, suggesting that SF might pro-.

J BIOCHEM MOLECULAR TOXICOLOGY Volume 25, Number 4, 2011

Silk Fibroin Has a Protective Effect against High Glucose Induced Apoptosis in HIT-T15 Cells Jun Hong Park,1 YoonYi Nam,2 So-Young Park,3 Jin-Kyung Kim,1 Nong-Hoon Choe,4 Jae-Young Lee,2,5 Yang-Seok Oh,1,3 and Jun Gyo Suh2,3 1

Center for Efficacy Assessment and Development of Functional Foods and Drugs, Hallym University, Gangwon-do 200–702, Republic of Korea Institute of Natural Medicine, Hallym University, Gangwon-do 200–702, Republic of Korea; E-mail: [email protected] 3 Department of Medical Genetics, College of Medicine, Hallym University, Gangwon-do 200–702, Republic of Korea 4 College of Veterinary Medicine, Konkuk University, Seoul 143–701, Republic of Korea 5 Department of Biochemistry, College of Medicine, Hallym University, Gangwondo 200–702, Republic of Korea 2

Received 4 March 2010; revised 4 June 2010; accepted 12 June 2010

ABSTRACT: High glucose levels induce cell death in many cell types, including pancreatic β-cells. Although protective agents against glucotoxicity have been searched for extensively, so far none have been found. In this report, we tested silk fibroin (SF) as a candidate material for antiglucotoxicity in the pancreatic β-cell (HIT-T15 cell) line. Approximately 50% of cells were killed after treatment with 80 mg/mL glucose. This reduction of cell number was recovered by the addition of SF at 50 mg/mL. SF treatment also decreased cellular reactive oxygen species (ROS) and increased proliferating cellular nuclear antigen (PCNA) immunoreactivity. In addition, TUNEL assays demonstrated that SF protects against glucose-induced apoptosis of HIT-T15 cells, suggesting that SF might protect cells from cell death by lowering cellular ROS levels. SF also induced expression of the insulin-like growth factor-1 (IGF-1) gene, and IGF-1 expression may be the cause of SF-induced protection against glucose toxicity. Taken together, these results suggest that SF could serve as a potential therapeutic agent to treat the hyperglycemia-induced death of pancreC 2010 Wiley Periodicals, Inc. J Biochem atic β-cells.  Mol Toxicol 25:238–243, 2011; View this article online at wileyonlinelibrary.com. DOI 10:1002/jbt.20381

KEYWORDS: Silk

Fibroin; Glucotoxicity; Apoptosis; Reactive Oxygen Species; Proliferating Cellular Nuclear Antigen; Insulin-Like Growth Factor-1

Correspondence to: Jun Gyo Suh. Jun Hong Park and YoonYi Nam have contributed equally to this work. Contract Grant Sponsor: Priority Research Centers Program through the National Research Foundation of Korea founded by the Ministry of Education, Science and Technology. Contract Grant Number: 2009-0094074. c 2010 Wiley Periodicals, Inc. 

INTRODUCTION Hyperglycemia has an harmful effect on the function and development of pancreatic β-cells as a result of a process referred to as glucotoxicity [1,2]. This is the primary cause of apoptosis of HIT-T15 cells in type II diabetes [3]. Therefore, it is important to prevent HITT15 cell death and/or reverse hyperglycemia-mediated abnormalities associated with diabetes mellitus. Silk-related materials including silkworm water extract and the root bark, fruits, and leaves of the mulberry tree have long been used as an antidiabetic remedy in oriental countries. Silk is composed of two major polypeptides, sericin and fibroin. SF is a core silk protein composed of 18 different kinds of natural amino acids, with a molecular weight of 3.5–3.6 × 105 Da [4]. It is primarily composed of glycine, alanine, serine, and tyrosine [5]. In a previous study, we reported that treatment with fibroin hydrolysates reduced blood glucose level and increased the insulin level in ob/ob mice [6]. In addition, fibroin peptides prevented DNA damage [7] and enhanced insulin sensitivity and glucose metabolism in 3T3-L1 adipocytes [8]. However, the precise mechanism of its protective effect in HIT-T15 cells under high glucose conditions has not explored. In this study, we investigated the effect of SF on glucotoxicity in the hyperglycemia-induced HIT-T15 cells.

MATERIALS AND METHODS Chemicals and Reagents All reagents were obtained from Sigma-Aldrich (St. Louis, MO) unless otherwise indicated. CellTiter 238

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96® AQueous One Solution and DeadEnd colorimetric kits for TUNEL assays were purchased from Promega (Madison, WI). Silk Fibroin (SF) was obtained from Shindo Biosilk (Seoul, Korea). RPMI 1640 medium, fetal bovine serum (FBS), penicillin, and streptomycin were obtained from Hyclone (Logan, UT). HIT-T15 cells were obtained from the American-Type Culture Collection (ATCC, Manassas, VA).

Cell Culture and Viability Assay We used HIT-T15 cells as a model to study pancreatic β-cells. HIT-T15 cells were cultured in RPMI 1640 medium containing 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37◦ C in 5% CO2 . (1 × 104 cells/well) were seeded in a 96-well plate and preincubated for 24 h. They were treated with SF (0, 0.1, 2, 5, 10, and 20 mg/mL) for 24 h. Cell viability was evaluated by the colorimetric MTT assay, using the CellTiter 96® AQueous One solution according to the manufacturer’s instructions. Death of HIT-T15 cells was induced by treatment with a high concentration of glucose [8]. Briefly, cells (1 × 106 cells/well) were seeded in a six-well plate and preincubated for 24 h, then treated with glucose (0, 10, 20, 40, 80, 160, and 320 mg/mL) for 24 h. The number of viable cells was counted using the trypan blue method. To evaluate the effect of SF on the viability of HIT-T15 cells under hyperglycemic conditions, cells (1 × 105 cells/well) were seeded in a 96-well plate and preincubated for 24 h. The cells were treated with SF (0, 1, 5, 10, 30, and 50 mg/mL) and/or glucose (80 mg/mL). After incubation for 24 h, cell viability was evaluated by the CellTiter 96® AQueous One solution assay.

TUNEL Assays Cells (1 × 104 cells/well) were seeded in a labTek II four-well chamber slide (Nalge Nunc International, Roskilde, Denmark) preincubated for 24 h, then treated with SF (0, 10, 30, and 50 mg/mL) and glucose (80 mg/mL). After incubation for 24 h, a TUNEL assay was performed according to the manufacturer’s instructions and visualized using a Zeiss Axio imager microscope (Zeiss, Jena, Germany).

Measurement of ROS by H2 DCFDA Fluorescence The ROS was detected by H2 DCFDA (Molecular Probes, Eugene, OR). Cells were loaded with 10 uM H2 DCFDA for 30 min at 37◦ C in 5% CO2 in PBS. Cells were washed and returned to media for a 30-min reJ Biochem Molecular Toxicology

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covery period. Samples were illuminated by a mercury lamp and viewed with a FITC filter on a Zeiss Axio imager microscope.

Immunohistochemistry Analysis Cells (1 × 104 cells/well) were seeded in a lab-Tek II four well chamber slide and preincubated for 24 h, then treated with SF (0, 10, 30 and 50 mg/mL) and glucose (80 mg/mL) for 24 h. Cells were then incubated in the presence of PCNA (1:150; Vector, Burlingame, CA) and IGF-1 (1:150; Santa Cruz Biotechnology, Santa Cruz, CA) antibodies. After washing with phosphate buffered saline (PBS), the cells were incubated sequentially in Texas Red conjugated antimouse IgG (1:150; Vector, Burlingame, CA) and antigoat IgG (1:150; Santa Cruz Biotechnology, Santa Cruz, CA). Mounting was conducted using VECTORSHIELD mounting solution with DAPI purchased from VECTOR (Burlingame, CA). All images were visualized using a Zeiss Axio imager microscope.

Reverse Transcription and Polymerase Chain Reaction (RT-PCR) Analysis Total RNA was extracted from cultured HIT-T15 cells using an RNeasy kit (Qiagen) according to the manufacturer’s instructions. For cDNA synthesis, reverse transcription of RNA was performed using a Promega kit (Promega, Madison, WI). The genespecific primers used were as follows: IGF-1, F, 5 — GAA AAT CAG CAG TCT TCC AAC—3 , R, 5 —TCC GGA AGC AAC ACT CAT—3 ; α-tubulin, F, 5 —CTC GCA TCC ACT TCC CTC—3 , R, 5 —ATG CCC TCA CCC ACG TAC—3 . PCR was performed using an Eppendorf Master-cycler PCR machine (Eppendrof, Hamburg, Germany), and the amplified products were visualized on a 1.5% agarose gel under UV light.

Immunoblot Analysis For the immunoblot assay, cells were lysed using pro-prep protein extraction buffer purchased from Intron biotechnology (Seongnam, Korea). The protein concentration of each lysate was determined using a BCA protein assay kit (Thermo Fisher Scientific, IL). For western blot analysis, aliquots containing 25 μg of protein were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Millipore, MA). IGF-1 antibodies were used at a dilution of 1:1,000. Membranes were subjected to immunoblot analysis, and proteins were visualized by

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enhanced chemiluminescence (Amersham Pharmacia, Little Chalfont, Buckinghamshire, UK).

Data Analysis Data are presented as the mean ± SEM of at least three independent experiments. To determine the statistical significance, data were analyzed by oneway ANOVA using the GraphPad Prism 4.0 program (GraphPad Software, La Jolla, CA).

RESULTS SF Protects HIT-T15 Cells from Glucose-Induced Cell Death High glucose conditions have been implicated in the death of pancreatic β-cells [9]. We sought to identify the IC-50 concentration of glucose in HIT-T15 cells. Many glucose levels were tested, and live cell counting results identified 80 mg/mL of glucose induced 50% of HIT-T15 cells to die in 24 h. Cell death occurred in a glucose dose-dependent manner (Figure 1A). To evaluate the effect of SF on the viability of HIT-T15 cells in high glucose conditions, we added 80 mg/mL glucose

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to HIT-T15 cells at different SF concentrations. When HIT-T15 cells were treated with culture medium containing SF (0, 1, 5, 10, 30, and 50 mg/mL) and glucose (80 mg/mL) for 24 h, the viability of the cells increased in a dose-dependent manner (Figure 1B).

SF Reduces Cellular ROS and Increases Proliferation of HIT-T15 Cells We used H2 DCFDA to investigate whether SF decreases ROS levels in high glucose conditions. SF treatments decreased H2 DCFDA fluorescence in a dosedependent manner under high glucose conditions (Figure 2A). Based on our immunohistochemical analysis, SF also increased PCNA positive cells in a dosedependent manner (Figure 2B). Non-SF-treated groups had little or no live cells or PCNA positive cells.

SF Reduces Glucose-Induced Apoptosis in HIT-T15 Cells To examine whether SF protected HIT-T15 cells from high glucose induced apoptosis, we measured the number of apoptotic cells using the TUNEL assay. SF treatment significantly reduced the number of

FIGURE 1. Protective effect of silk fibroin against glucose-induced cell death in a pancreatic beta cell line, HIT-T15. (A) Cells were treated with varying levels of glucose for 24 h. Values represent the mean ± SEM (N = 3). ∗ P < 0.05 and ∗∗ P < 0.01 versus glucose 10 mg/mL treated group. (B) Cells were treated with varying levels of SF and/or glucose (80 mg/mL) for 24 h. Values represent the mean ± SEM (N = 3). ∗∗∗ P < 0.001 versus SF (0 mg/mL) treated group.

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FIGURE 2. SF-mediated reduction of the cellular ROS level and an increase in the PCNA level. (A) SF decreased H2 DCFDA fluorescence in a dose-dependent manner (a: 10 mg/mL, b: 10 mg/mL, c: 30 mg/mL, d: 50 mg/mL), ×200. (B) HIT-T15 cells were treated with the anti-PCNA antibody (red) and DAPI (blue). The PCNA signal increased in response to treatment with SF in a dose-dependent manner (a: 10 mg/mL, b: 10 mg/mL, c: 30 mg/mL, d: 50 mg/mL). ×400.

TUNEL-positive cells in a dose-dependent manner (Figure 3A). After 24 h of exposure to 10, 30, or 50 mg/mL of SF and 80 mg/mL of glucose, the number of TUNEL positive cells was decreased to 87%, 72%, and 56%, respectively, of that controls (Figure 3B).

SF Induces Expression of Insulin-Like Growth Factor-1 Gene To assess whether SF changes levels of pancreatic β-cell function related genes in high glucose conditions, HIT-T15 cells were cultured for 24 h, and the gene expression level was determined by RT-RCR. Among J Biochem Molecular Toxicology

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FIGURE 3. Protective effect of SF in glucose-induced apoptosis. (A) TUNEL-positive cells (arrow) decreased in response to treatment with SF in a dose-dependent manner (a: 10 mg/mL, b: 10 mg/mL, c: 30 mg/mL, d: 50 mg/mL), ×200. (B) The ratio of TUNEL-positive cells to total cells decreased in response to treatment with SF in a dose-dependent manner. ∗∗ P < 0.01 and ∗∗∗ P < 0.001 versus control (N = 3).

the various genes that regulate pancreatic β-cell function, we found that expression of the IGF-1 gene was increased (Figure 4A). To confirm the RT-PCR result, we carried out immunoblotting. Immunoblot analysis confirmed our RT-PCR results, as the expression of IGF-1 protein was increased in SF-treated groups (Figure 4B). Immunohistochemistry also demonstrated that SF treatment induced IGF-1 expression (Figure 4C).

DISCUSSION In this study, we tested whether SF could serve as a candidate material for antiglucotoxicity in HIT-T15

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FIGURE 4. Induction of IGF-1 gene expression by SF: (A) RT-PCR analysis of the IGF-1 gene, (B) western blot analysis of IGF-1 protein. β-actin was used as an internal control, and (C) immunostaining of IGF-1. HIT-T15 cells were treated with the anti-IGF-1 antibody (green) and DAPI (blue). The IGF-1 signal increased in response to treatment with SF in a dose-dependent manner (a: 10 mg/mL, b: 10 mg/mL, c: 30 mg/mL, d: 50 mg/mL). ×400.

cells. We found that 50 mg/mL SF exhibited a protective effect against glucose-induced cell death. Since pancreatic islets contain relatively small quantities of the antioxidant enzymes, pancreatic βcells are at greater risk for oxidative damage [10]. Treatment with super oxide dismutase (SOD) prevents alloxan-induced diabetes in mice [11]. Several investigators have also shown that hyperglycemia induces ROS and causes oxidative stress [12–17]. Thus, to investigate mechanism underlying the effect of SF on glucose-induced cell death, we measured ROS levels and cell proliferation. SF reduced cellular ROS levels

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(Figure 2A) and increased proliferation of HIT-T15 cells (Figure 2B) during exposure to high glucose. These observations are in agreement with those of Ihara et al, who reported that treatment with antioxidants prevented glucose-induced cell death in islets [18]. Some hypoglycemic substances have been isolated from silk-related materials. For example, Moran A, Moran 20K, and moranoline (1-deoxynojirimycin) were isolated from mulberry root bark [19–21], and Ncontaining sugars were isolated from mulberry leaves [22]. IGF-1 is well known as a β-cell growth factor, has significant structural homology to proinsulin [23], and binds to insulin receptor, stimulating insulin-like activity and enhancing insulin sensitivity [24]. In particular, treatment with somatolactogenic hormones (prolactin and growth hormone) and IGF-1 has been shown to increase the number of replicating β-cells in pancreatic islets of rodents by up to 6% [25–28]. Recent studies have demonstrated that SF enhances insulin sensitivity and glucose metabolism in 3T3-L1 cells [8]. Treatment with SF upregulated expression of IGF-1 in our study (Figure 4), suggesting that IGF-1 expression may be the cause of SF-induced protection from glucose toxicity. The SF used in this study contains a mixture of different peptides and individual amino acids. Therefore, there exists the possibility that single amino acids may be the cause of the protective effect we see. To exclude this possibility, a solution containing a similar composition of free amino acids was applied to HITT15 cells. These amino acids did not protect against glucose-induced cell death in pancreatic beta cells (data not shown). This data indicate that SF has a functional effect at the peptide level. We expect that a single active peptide is the cause of this protective effect. Further research will utilize fractionation and biochemical isolation to identify the specific functional peptide of SF. In conclusion, SF treatment reduced cell death and increased proliferation of HIT-T15 cells during high glucose exposure. SF protects against the glucoseinduced apoptosis of HIT-T15 cells, suggesting that it might protect cells from cell death by lowering cellular ROS level. Furthermore, treatment with SF induced expression of IGF-1 gene, which is well known as a β-cell growth factor. Taken together, our results suggest that SF could serve as a potential therapeutic agent for the treatment hyperglycemia-induced death in pancreatic β-cells.

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