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Prevention of pancreatic cancer

Chapter Number 1

Prevention of Pancreatic Cancer 2

Xia Jiang et al.* 4

Department of Environmental Biochemistry, Graduate School of Medicine, 5

Chiba University, Inohana 1-8-1 Chuo-ku, Chiba, 6

Japan 7

1. Introduction

It is an intriguing problem whether human cells and/or bodies have the physiological 9

function which supervises and selects survival and death fate in response to various kinds 10

of stressors such as radiation. For example, what are the physiological effects of X-ray 11

irradiation for examination of breast conditions including the risk of genetic mutation and 12

cancer occurrence (Fig. 1A).

Based on a protease-activation signal in E.Coli SOS response we have attempted to search for 14

human SOS response, particularly signals which protect stability of genetic information 15

(Fig.1B) by estimation of plasminogen activator-like protease (PA) activity in peripheral blood-16

derived lymphocytes and cultured human RSa cells with hyper-mutability and their variant 17

hypo-mutable cells (Fig.1C) (Suzuki et al.,2005). Levels of PA activity in lymphocytes are 18

changed after X-ray irradiation for breast examination and after exposure in vitro to chemical 19

such as bisphenol A (Takahashi et al.,2000). The activity in variant cells was found to be 20

associated with Heat shock protein (HSP) 27 expression, resulting in enhancement of error-free 21

DNA repair function (Wano et al.,2004). A HSP27-bound protein, Annexin II, was also 22

suggested to play some roles on the error-free function in human cell nuclei (Tong et al., 2008 23

and Jin et al., 2009). We recently developed a method to analyze base substitution mutation of 24

the K-ras codon 12 and found that a decreased mutation frequency accompanied increased 25

GRP78 expression in human RSa cells (Hirano et al.,2008 and Zhai et al.,2005). The repair 26

function seems to suppress base substitution mutation of K-ras codon 12. The base substitution 27

mutation was also found to be regulated by extracellular factors, human interferon and serum 28

factors from cancer patients and stressors-exposed persons (Suzuki et al., 2005, Hirano et al., 29

2008 and Chi et al.,2007). This regulation seems to be mediated by PA activation and the 30

following chaperones expression (Suzuki et al., 2005, Isogai et al., 1994, Takahashi et al., 2003 31

and Kita et al., 2009). Thus, studies on molecular mechanisms to supervise cellular mutability, 32

including frequency of Ras gene mutation, are important for discussion about relationships 33

with a network of proteases and chaperones and/or cytokines.

*Shigeru Sugaya1, Qian Ren1, Tetsuo Sato1, Takeshi Tanaka1, Fujii Katsunori2, Kazuko Kita1

and Nobuo Suzuki1

1Department of Environmental Biochemistry, Graduate School of Medicine, Chiba University, Inohana 1-8-1

Chuo-ku, Chiba, Japan

2Department of Pediatrics, Chiba University Graduate School of Medicine, Chiba, Japan

Pancreatic Cancer Book 1 2 ＡＧｅｎｅｔｉｃ

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1 シャペロン培養ヒト細胞水アーゼ味噌

リンパ球ＳｔｒｅｓｓｏｒＣｕｌｔｕｒｅｄｈｕｍａｎＬｙｍｐｈｏｙｔｅｓ2 Fig. 1. Strategy of the method to search for human SOS response. 3 (A) A question concerning X-ray effects on genetic mutation and the following cancer. (B) A 4 physiological function which supervises protection mechanisms for genetic information 5 stability. (C) Modulation of cellular fate by serum factors and Plasminogen activator-like 6 protease (PA) activation, elucidated by the observation of peripheral blood lymphocytes and 7 cultured human cells after stressors exposure. 8

Incidence of K-ras gene mutation is high in pancreatic carcinomas, and therefore 9 suppression of the mutation could be beneficial for inhibiting pancreatic cancer occurrence 10 (Hirano et al., 2008). We were intrigued by the possibility that this carcinogen-induced 11 mutation could be suppressed, via modulation of GRP78 expression, by agents such as 12 foods. 13

In the present chapter, cellular levels of GRP78 in RSa cells that had been cultured with 14 aqueous extracts of Japanese miso and the unfermented ingredients of miso are shown using 15 immunoblotting analysis. The mutability of the treated cells are also evaluated after UVC 16 irradiation using the differential dot-blot hybridization test for K-ras codon 12 mutation. In 17 previous reports, dietary supplementation with long-term fermented miso has been shown to 18 act as a chemopreventive agent against gastric and colon carcinogenesis in rats (Ohara et al., 19 2002a, 2002b, and Ohuchi et al., 2005). Miso is a fermented food that has formed an important 20 part of the Japanese diet for over 1300 years (Yoshikawa et al., 1998). It is prepared by the

21 microbial fermentation of a mixture of raw materials (soybean, wheat, barley and rice) over a 22

Prevention of Pancreatic Cancer 3 1

long period until the ripe miso is obtained (Hesseltine ＆Shibasaki,1961). Little is known 2

about the ability of miso to modulate the mutability of human cells.

2. Human SOS response and suppression of K-ras mutation by japanese

miso possibly via GRP78 expression in human RSa cells

2.1 Effect of miso samples on RSa cell viability

2.1.1 Preparation of miso samples

The Japanese Enbunhikae miso (EM) was purchased from Marui Co., Ltd. (Chino, Japan). 8

Rice-koji, a raw material used in the preparation of miso, was obtained from Hanamaruki 9

Co., Ltd (Nagano, Japan). Aqueous extracts of miso and rice-koji were prepared as follows: 10

each (10 g) was suspended in 20 ml of MilliQ water, and the suspension was heated at 90 °C 11

for 5 minutes and then at 70 °C for 10 minutes. The suspension was centrifuged at 1780 × g 12

and 4 °C for 10 minutes and the supernatant was then filtered through a 0.22 μm membrane.

The dose of sample used in each treatment is shown as a percentage of volume per volume 14

(v/v); 1% is equivalent to 5 mg of miso or rice-koji per ml.

2.1.2 Culture conditions and UVC irradiation

RSa cells were established from human embryo-derived fibroblast cells by double infection 17

with the Simian virus 40 and the Rous sarcoma virus.The cells were confirmed to have high 18

UV sensitivity and low DNA-repair activity (Kuwata et al.,1976, Suzuki ＆Fuse, 1981, 19

Suzuki,1984). Cells were cultured in Eagle's minimal essential medium (EMEM) ( Nissui, 20

Tokyo, Japan) containing 10% calf serum (CS) (Invitrogen, Carlsbad, CA, USA) at 37 °C in a 21

humidified atmosphere containing 5% CO2. UVC was generated by a 6 W National 22

germicidal lamp (Panasonic, Osaka, Japan). The intensity of UVC was 1 J/m2/s, as 23

measured by a UV radiometer, UVR-254 (Tokyo Kogaku Kikai, Tokyo, Japan). Irradiation of 24

the cells with UVC was performed as described previously (Suzuki ＆ Fuse, 1981) and mock 25

irradiation of cells was carried out in the same manner but without UVC irradiation.

2.1.3 Colony survival assay

To determine the optimal concentration of miso sample for use in the culture medium in cell 28

mutability tests, the colony survival capacity of RSa cells cultured with or without EM 29

extracts was examined (Fig. 2). The survival capacity of cells treated with or without miso 30

was measured using a colony survival assay as reported previously (Suzuki et al.,1984).

Logarithmically growing cells were seeded in 100 mm dishes (800 cells/dish), incubated for 32

20 h to allow the cells to attach, and then treated with or without miso extracts. One h after 33

treatment, the cells were grown in fresh medium containing 5% CS in 100 mm dishes for 34

about 14 d and were then stained with 0.2% methylene blue in 30% methanol. The results of 35

the colony survival assay are expressed as percentages of the colony numbers observed for 36

miso extracts-treated cells relative to those of untreated cells. Colony survival rates were 37

over 85% when a miso extract concentration of 1% was used, but at concentrations higher 38

than 1% a decrease of more than 10% in survival rate was observed (Fig. 2). An MTT assay 39

showed that miso extract concentrations of less than 1% were not cytotoxic after 48 h of 40

culture (data not shown).

Pancreatic Cancer Book 1

4 1 Fig. 2. Effect of EM extracts on cell survival. 2 RSa cells were treated with or without the indicated concentrations of EM extracts for 1 h. 3 After treatment, the cells were cultured with fresh medium containing 5% CS in 100 mm 4 dishes for 14 d and were then stained with 0.2% methylene blue in 30% methanol. 5

2.2 Effect of miso extracts on GRP78 expression and the mutability of RSa cells 6

2.2.1 GRP78 expression 7

GRP78 expression was analyzed by immunoblotting as described previously (Wano et al., 8 2004). Cells were lysed with a lysis buffer containing 20 mM Tris-HCl (pH 7.4), 1 mM EDTA, 9 1 mM EGTA, 0.5% NP-40 and protease inhibitors, 1 mM phenylmethylsulfonyl fluoride, 0.05 10 mM leupeptin, 0.05 mM antipain and 0.05 mM pepstain A. The cell lysates were centrifuged 11 at 15,000 × g and 4 °C for 20 min, and the supernatant was then treated with SDS sample 12 buffer. Detection of the GRP78 protein was performed using mouse monoclonal anti-GRP78 13 (1:2000 dilution; SPA-827; StressGen, Victoria, Canada) antibodies. β-Actin was also 14 analyzed using mouse monoclonal anti-β-actin antibodies (1:30000 dilution; ab40864; 15 Abcam, Cambridge, UK) as a loading control. The antigen-antibody complexes were 16 detected by horseradish peroxidase (HRP) conjugated anti-mouse IgG (Amersham 17 Biosciences, Buckinghamshire, UK) following the ECL system (GE Healthcare, 18 Buckinghamshire, UK). The GRP78 protein was quantified using Multi Gauge Ver2.2 image 19 analysis software (Fuji Foto film, Tokyo, Japan) and expressed relative to the quantity of β-20 actin measured. When RSa cells were cultured with EM extracts at a concentration of 1%, 21 expression of GRP78 was enhanced by over 1.5 fold compared with the expression observed 22 in mock-treated cells (Fig 3A). 23

2.2.2 Mutability 24

Mutations in K-ras codon 12 were detected according to a method described previously 25 (Suzuki N ＆ Suzuki H., 1993). Logarithmically growing cells were inoculated at near 26 confluency (5×l05 cells/100 mm dish) to avoid cell selection by the lethal effects of UVC

27 irradiation, as described elsewhere (Suzuki N ＆ Suzuki H., 1995). Six d after UVC 28

Prevention of Pancreatic Cancer

2 Fig. 3. Effect of EM extracts on GRP78 expression (A) and the mutability (B) of RSa cells.

3 Cells were cultured with or without EM extracts at a concentration of 1% for 2

4 h. (A) Cell 4 lysates were prepared after miso treatment and subjected to immunoblotting analysis of

5 GRP78 and β-actin proteins. (B) After miso treatment, cells were irradiated with UVC or left

6 unirradiated as controls (6 J/m 2). Six d after UVC irradiation, genomic DNA was extracted,

7 and the K-ras codon 12 mutation was detected by PCR and differential dot-blot hybridization,

8 as described in Materials and Methods. Data are the means ±SD for three experiments. 9

or mock irradiation, genomic DNA was extracted using a standard proteinase 10 K/SDS/phenol/chloroform procedure. DNA of SW480 cells carrying the K-ras mutation at 11 codon 12 was used as a positive control for genomic DNA. Target sequences of sample DNA 12 were amplified in vitro by PCR using the primers 5’-GACTGAATATAAACTTGTGG-3’ and 13 5’-CTATTGTTGGATCATATTCG-3’, and the amplified DNA (0.25 μg) was dot-blotted onto 14 nylon membranes. After hybridization with digoxigenin-11-dUTP-3’ end-labeled K-ras 15 codon 12 probes, the membranes were reacted with alkaline phosphatase conjugated 16 polyclonal sheep anti-Dig Fab (Boehringer Ingelheim, Mannheim, Germany) and colored 17 with the nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate solution 18 (Boehringer Ingelheim, Mannheim, Germany). As a control probe, the oligonucleotide 5’- 19 GTTGGAGCTGGTGGCGTAGG-3’ was used, and a mixed mutant probe, containing the 20 following oligonucleotides mixed at the same concentration ratios, 21 was used: 5’-GTTGGAGCTAGTGGCGTAGG-3’, 5’-GTTGGAGCTCGTGGCGTAGG-3’,

22 5’-GTTGGAGCTTGTGGCGTAGG-3’, 5’-GTTGGAGCTGATGGCGTAGG-3’, 5’-

23 B A

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6 GTTGGAGCTGCTGGCGTAGG-3’, 5’-GTTGGAGCTGTTGGCGTAGG-3’. Photographs 1 were taken as permanent records of the results. To determine whether miso extracts 2 suppress the mutability of RSa cells, the K-ras codon 12 mutation assay was performed. A 3 black dot, indicating base substitution mutation, was detected after hybridization of the PCR 4 products from genomic DNA of SW480 cells containing the K-ras codon 12 mutation, and 5 this was used as a positive control (Fig. 3B). Under the assay conditions, the intensity of the 6 black dot was clearly enhanced after UVC irradiation in mock-treated RSa cells (Fig. 3B).

7 However, EM extracts-treated RSa cells did not show black dot signals either after UVC or

8 mock irradiation (Fig. 3B). 9

2.3 Effect of GRP78 siRNA on the modulation of UVC cell mutability by miso extracts 10

To further examine whether GRP78 expression levels are causally related to the miso 11 treatment modulation of RSa cells mutability, its expression was inhibited by GRP78 siRNA 12 transfection. Duplex small interfering RNA (siRNA) with Stealth modification against 13 human GRP78 (GRP78 siRNA) was synthesized based on the protein’s nucleotide sequence

14 (Invitrogen), as described previously (Suzuki T et al., 2007). The sequence of the duplex was

17 Fig. 4. Effect of GRP78 siRNA transfection on UVC-induced mutagenicity. 18 (A) After GRP78 or NC siRNA transfection, cell lysates were separated by SDS–PAGE and 19 analyzed by immunoblotting analysis using anti-GRP78 and anti-β-actin (loading control) 20 antibodies. (B) After siRNA transfection, cells were treated with or without EM extracts for 21 24 h and then irradiated with UVC (6 J/m 2). Mutability of RSa cells was determined by the 22 K-ras codon 12 mutation assay using PCR and differential dot-blot hybridization, as

23 described in Materials and Methods.

24 B A

Prevention of Pancreatic Cancer

7 as follows: 5’-UAC CCU UGU CUU CAG CUG UCA CUC G-3’ / 3’-AUG GGA ACA GAA 1 GUC GAC AGU GAG C-5’. Stealth RNAi negative control duplex (NC siRNA), with a GC 2 content similar to that of the above Stealth RNAi, was used as a negative control. The NC 3 siRNA was designed to minimize sequence homology to any known vertebrate transcript 4 and for use in RNA interference (RNAi) experiments as a control for sequence independent 5 effects following Stealth RNAi delivery in any vertebrate cell line. Treatment of cells with 6 siRNA was carried out as described previously (Harborth et al., 2001), with minor 7 modifications. The siRNAs (128 nM) were transfected for 5 h into RSa cells using 8 Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. The cells were 9 detached from the test dish 48 h after transfection and used for each experiment. 10

In GRP78 siRNA-transfected cells, cellular levels of GRP78 protein were decreased in EM 11 extracts-treated cells as well as in mock-treated cells, while no decrease was observed in NC 12 siRNA-transfected cells (Fig. 4A). The decrease observed was up to 50% of the NC siRNA 13 control (Fig. 4A). In RSa cells with GRP78 siRNA transfection that had been treated with 14 miso extracts, the dot signal of K-ras codon 12 mutation was enhanced after UVC irradiation 15 (Fig. 4B). 16

2.4 Effect of miso components on cell mutability 17

The effect of rice-koji, a component of miso, on the mutability of RSa cells was examined. 18 The survival rate, measured by a colony survival assay (Fig. 5) and an MTT assay (data not 19 shown), of cells treated with rice-koji at concentrations of less than 10% were more than 20 80%. On the basis of these results, we used 1% rice-koji in subsequent experiments to allow

21 direct comparison with experiments using EM extracts.

22 23 Fig. 5. Effect of rice-koji on cell survival. 24 RSa cells were treated at the indicated concentrations with rice-koji. After rice-koji treatment 25 (1h) cells were grown in fresh medium containing 5% CS in 100 mm dishes for about 14 d, 26 and then stained with 0.2% methylene blue in 30% methanol. Data are the means ±SD for

27 three experiments.

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8 Expression levels of GRP78 in rice-koji-treated RSa cells were slightly increased, at about 1.5 1 fold of the untreated cells (Fig. 6A). Rice-koji-treated cells showed no detectable black dot 2 signal in the K-ras codon 12 mutation test with or without UVC irradiation treatment (Fig.

3 6B).

6 Fig. 6. Effect of rice-koji on GRP78 expression and mutability.

7 (A) Cells were treated with rice-koji (1%) for 24 h, and the cell lysates were subjected to

8 immunoblotting analysis of GRP78 and β-actin proteins. (B) After rice-koji treatment, cells

9 were irradiated with UVC (6 J/m 2). Six d after UVC irradiation genomic DNA was 10 extracted, and the K-ras codon 12 mutation was detected using PCR and differential dot-blot 11 hybridization. 12

We also examined whether GRP78 siRNA transfection affected UVC mutagenicity in rice-13 koji-treated RSa cells. In GRP78 siRNA-transfected cells, GRP78 expression levels decreased 14 to about 40% of those in NC siRNA-transfected cells (Fig. 7A). Cells transfected with GRP78 15 siRNA and then treated with rice-koji, showed black dot signals indicating UVC-induced K-16 ras codon 12 mutation, similar to the signal observed in cells treated with UVC irradiation

17 alone, and in SW480 cells (Fig. 7B).

18 B A

Prevention of Pancreatic Cancer

1 2 Fig. 7. Effect of GRP78 siRNA transfection on UVC-induced mutagenicity in rice-koji treated 3 cells. 4 (A) After GRP78 or NC siRNA transfection, cell lysates were separated by SDS–PAGE and 5 analyzed by immunoblotting analysis using anti-GRP78 and anti-β-actin (loading control) 6 antibodies. (B) After siRNA transfection, cells were treated with or without rice-koji extracts 7 for 24 h and then irradiated with UVC (6 J/m 2). Mutability of RSa cells was determined by 8 the K-ras codon 12 mutation assay using PCR and differential dot-blot hybridization. 9

3. Conclussion 10

The K-ras base substitution mutation is thought to be a cancer-causing DNA sequence 11 change in human cells, and its incidence is particularly high in pancreatic carcinomas. We 12 previously found that proteases are activated in stress-exposed cells and/or human 13 bodies, leading to modulation of chaperone expression and cellular mutability (so-called 14 SOS response). Increased levels of glucose-regulated protein 78 (GRP78) expression are 15 associated with the suppression of the K-ras mutation in human RSa cells irradiated with

16 ultraviolet C (UVC). RSa cells are hyper-mutable and are used to examine the modulation

17 B

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of cell mutability using various agents. Here, we describe investigations into the effect of 2

RSa cell treatment with Japanese miso on GRP78 expression and the suppression of UVC 3

mutagenicity. Aqueous extracts of miso and its components were tested. Miso treatment 4

was found to increase GRP78 levels, as estimated by immunoblotting analysis, and to 5

decrease UVC-induced K-ras codon 12 base substitution mutation frequency. Increases in 6

GRP78 expression and decreases in mutation frequency were not observed in cells whose 7

GRP78 levels had been down-regulated using GRP78 siRNA transfection. This suggests 8

that miso extracts suppress UVC mutagenicity by increasing GRP78 expression in human 9

cells.

In the present study Japanese miso was tested concerning the modulation activity of genetic 11

mutation, K-ras codon 12 base substitution mutation, in addition of human cytokines and 12

serum factors. Several dietary factors have been postulated to act as risk factors for human 13

carcinogenesis (Sugimura, 2000 and Mirvish, 1983). In Japan, intensive studies of the causal 14

relationship between diet and cancer incidence have focused on stomach, lung and 15

colorectal cancers (Masaki et al., 2003, Ngoan et al., 2002, Takezaki et al., 2001, Ozasa et al., 16

2001, Tajima ＆ Tominaga, 1985). The association of dietary factors with pancreatic cancer 17

has been significantly less well studied. In this study, the modulation of cell mutability via 18

the GRP78 chaperone was examined by measuring GRP78 expression in Japanese miso-19

treated human RSa cells. It was found that levels of GRP78 expression increased upon 20

treatment of RSa cells with EM and rice-koji extracts (Figs. 3A and 6A). The pretreatment of 21

cells with these extracts was also found to suppress UVC mutagenicity (Figs. 3B and 6B). An 22

intimate relationship between GRP78 up-regulation and hypo-mutability was also 23

suggested by the results of experiments using GRP78 silencing (Figs. 4 and 7).

We reported that the down-regulation of GRP78 in RSa cells reduces DNA repair capacity 25

in the nucleotide excision repair pathway. Nucleotide excision repair, a highly conserved 26

DNA repair system in human cells, is essential for protection against UVC-induced DNA 27

damage leading to, for example, (6-4)-photoproducts and cyclobutane thymine dimmers 28

(Batty ＆Wood, 2000, de Laat et al.,1999). Thus, one plausible mechanism for the 29

observed hypo-mutable change in RSa cells pre-cultured with miso and rice-koji extracts 30

may be the enhancement of cellular DNA repair function by the up-regulation of GRP78 31

expression.

The k-ras point mutation-enhancing activity of conditioned medium is detected from 33

culture of human pancreatic cancer cells (Hirano et al.,2008), suggesting involvement of 34

extracellular factors from pancreatic cancer cells in tumor-worsening process. Extracellular 35

materials released from cancer cells play crucial roles in development of cancers and 36

resistance to anticancer treatment (Hidalgo et al.,2010). Pancreas carcinoma shows 37

resistance to chemotherapeutic agents (Gong et al.,2010). Thus, we tried to search for the 38

above materials in conditioned medium from pancreatic cancer cells, and identified one of 39

HSP27-bound proteins, annexin II, by molecular mass analysis. Although the functions of 40

extracellular annexin II are not fully understood, annexin II is known to act as a cell surface 41

receptor for extracellular ligands and is suggested to play roles in regulation of proteolytic 42

cascades including PA activities (Hajjar et al., 1994), signal transduction (Singh, 2007), and 43

tumor invasion and metastasis (Chung et al., 1996, Esposito et al., 2006, Singh et al., 2007, Mai

Prevention of Pancreatic Cancer 11 1

et al., 2000). Studies on the mechanisms of PA-involved SOS functions are required for 2

prevention of pancreatic cancer.

4. Acknowledgments

We thank Y. Sumiya for technical assistance. This work was supported in part by a grant 5

from the Miso Central Institute in Japan, and in part by grants-in-aid from the following 6

organizations: the Smoking Research Foundation, the Tokyu Foundation for a Better 7

Environment, the Tsuchiya Foundation, the Goho Life Science International Foundation, the 8

Ministry of Health, Labour and Welfare for the Treatment of Intractable Diseases Research 9

Program and the Japan Society for the Promotion of Science (Japan).

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