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Apoptosis in human pancreatic cancer cells induced by eicosapentaenoic acid
Nutrition 21 (2005) 1010 –1017 www.elsevier.com/locate/nut
Applied nutritional investigation
Apoptosis in human pancreatic cancer cells induced by eicosapentaenoic acid Tetsuya Shirota, M.D.ⴱ, Seiji Haji, M.D., Ph.D., Mitsuo Yamasaki, M.D., Takuya Iwasaki, M.D., Ph.D., Toshiharu Hidaka, M.D., Yoshifumi Takeyama, M.D., Ph.D., Hitoshi Shiozaki, M.D., Ph.D., and Harumasa Ohyanagi, M.D., Ph.D. Department of Surgery, Kinki University School of Medicine, Osaka, Japan Manuscript received September 21, 2004; accepted December 3, 2004.
Abstract
Objectives: Clinical studies have shown that administration of eicosapentaenoic acid (EPA) to patients who have unresectable pancreatic cancer induces marked attenuation of cachexia. However, the exact mechanisms of the beneficial effect of EPA on pancreatic cancer are unknown. This examined the effect of EPA on proliferation of human pancreatic cancer cell lines and sought to clarify its mechanisms. Methods: The effects of EPA on proliferation of three human pancreatic cancer cell lines (SW1990, AsPC-1, and PANC-1) were assessed. Induction of apoptosis and expressions of apoptosis-related proteins were measured. The effect of EPA on cyclo-oxygenase–2 expression in these cell lines was determined. Results: EPA inhibited proliferation of all three human pancreatic cancer cell lines in a dosedependent fashion. Simultaneously, EPA treatment induced apoptosis and this was associated with caspase-3 activation. EPA treatment was also associated with a decrease in intracellular levels of cyclo-oxygenase–2 protein. Conclusion: We have demonstrated that EPA inhibits human pancreatic cancer cell growth due at least in part to the induction of apoptotic cell death. Such apoptosis is associated with activation of caspase-3 and suppression of cyclo-oxygenase–2 expression. Greater understanding of the molecular events associated with the biological activity of EPA should enhance the therapeutic potential of administration of EPA to patients who have pancreatic cancer. © 2005 Elsevier Inc. All rights reserved.
Keywords:
Eicosapentaenoic acid; -3 Polyunsaturated fatty acids; Pancreatic cancer; Apoptosis; Cyclo-oxygenase–2
Introduction Pancreatic cancer is a devastating disease that currently affects approximately 28 000 people per year in the United States [1]. The 5-y survival rate in patients who have pancreatic cancer is lower than 5%, making this disease the fourth most common cause of cancer-related mortality [1]. Due to the relatively late diagnosis of pancreatic cancer, the tumor frequently metastasizes or invades surrounding tissues on diagnosis, and most patients (approximately 70% to 80%) are not eligible for a curative resection [1]. In addiⴱ Corresponding author. Tel.:⫹81-72-366-0221; fax:⫹81-72-366-0111. E-mail address: [email protected] (T. Shirota). 0899-9007/05/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2004.12.013
tion, non-surgical treatment options such as chemotherapy [2] and/or radiotherapy [3] have shown disappointing results thus far, and reasons for resistance of pancreatic cancer cells to radiation and cytotoxic agents and their aggressive growth are not completely understood. The care of patients who have unresectable pancreatic cancer has focused on relief of symptoms, such as weight loss, pain, jaundice, steatorrhea, nausea, and anemia, in an attempt to optimize quality of life, but care remains unsatisfactory. Polyunsaturated fatty acids (PUFAs) have been recognized as important energy sources and components of cell membranes. PUFAs also play key roles in many cellular events, such as immune function [4], aging [5], neonatal development [6], anti-inflammatory effects [7], and gene
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regulation [8]. No toxicity of PUFAs on normal cell cultures or in whole animals have been reported [9,10]. During recent decades, PUFAs have been suggested as natural products that may play significant roles in modulating cancer development [11]. In general, -6 PUFAs appear to enhance tumor growth and proliferation [12], whereas -3 PUFAs seem to have an inhibitory effect on this process [13]. Eicosapentaenoic acid (EPA; 20:5-3) is an -3 PUFA found abundantly in fish oil. EPA retards the growth and development of breast, colon, and liver cancers and leukemia in vitro and in vivo [9,14,15,16]. In addition, clinical and experimental cancer studies further support the use of EPA to promote weight maintenance and decrease anorexia in patients with unresectable pancreatic cancer [17]. The cellular features of apoptosis are one of the most important issues in cancer research due to the significance of apoptosis in tumor development and chemotherapy [18]. Apoptosis induced by -3 PUFAs has been demonstrated in human colon cancer [19], leukemia [16], and lymphoma [20]. Apoptosis is regulated by several processes involving changes in expression or activity of distinct proteins [21]. Caspases plays a central role in apoptosis. Specifically, caspase-3, a protease located downstream of the caspase-activating mechanism, activates caspase-3–activated DNAse and induces DNA fragmentation when activated. Cyclo-oxygenase is an enzyme associated with the arachidonic acid cascade. The -3 PUFAs may influence inflammation and cancer cell proliferation via cyclo-oxygenase–2 (COX-2). Further, COX-2 is believed to modulate apoptosis, possibly by activation of caspase-3 [22]. However, the molecular mechanisms associated with apoptosis and the antiproliferative effect of human pancreatic cancer cells by EPA have not been well defined. In this study, we determined the effect of EPA on pancreatic cancer cell proliferation and induction of apoptosis.
Materials and methods Reagents A purified preparation of EPA (⬎98% pure) isolated from fish oil, in ethyl ester form, was kindly provided by Mochida Pharmaceutical Co. (Tokyo, Japan). EPA was dissolved in ethanol before use. Cell lines Human pancreatic cancer cell lines (SW1990, AsPC-1, and PANC-1) were obtained from American Type Culture Collection (Rockville, MD, USA). SW1990 and PANC-1 were cultured in Dulbecco’s Modified Eagle Medium containing 10% heat-inactivated fetal calf serum. AsPC-1 was cultured in RPMI 1640 containing 10% fetal calf serum. Cultures were maintained at 37°C in a humidified atmo-
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sphere of 5% CO2. SW1990 and AsPC-1 are metastatic in behavior, whereas PANC-1 is a non-metastatic pancreatic cancer cell line [23]. Cell proliferation analysis Cell proliferation assays were performed with the CellTiter 96 AQueous One Solution Cell Proliferation Assay kit according to the manufacturer’s instructions (Promega, Madison, WI, USA). Briefly, 5 ⫻ 103 cells/well in medium were placed onto 96-well plates. EPA was added to cellular cultures at final concentrations of 0, 100, 300, and 500 M. Cultures were incubated for 48 h and then added to 20 L/well of CellTiter 96 AQueous One Solution Reagent, which contains a tetrazolium compound, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, an inner salt; MTS was added to the culture medium. After incubating the plate for 1 h in a CO2 incubator, absorbance at 490 nm was recorded with a 96-well plate reader. Apoptosis assays Apoptosis was measured with the terminal deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) method, which used the MEBSTAIN Apoptosis Kit Direct (Medical and Biological Laboratories, Nagoya, Japan) according to the manufacturer’s instructions. Briefly, human pancreatic cancer cell lines exposed to EPA for 48 h were washed twice with phosphate buffered saline containing 0.2% bovine serum albumin (PBS-BSA). Cells were then fixed with buffered paraformaldehyde at 4°C for 30 min, followed by washing twice with PBS-BSA. Cold 70% ethanol was added to the cell pellet, which was incubated for 30 min at ⫺20°C for permeabilization. After washing twice with PBS-BSA, 30 L of terminal deoxynucleotidyl transferase reaction reagent was added to the cell pellet for 1 h at 37°C in the dark. Cells were washed twice with PBS-BSA and resuspended in 500 L of PBS-BSA. Results were analyzed by flow cytometry using CellQuest software and a FACScaliber instrument (BD Pharmingen, San Jose, CA, USA). The resulting histogram (Fig. 2a) indicates background fluorescence as determined by incubation without terminal deoxynucleotidyl transferase enzyme. In this histogram, the area of dUTP-positive cells, which demonstrate higher levels of fluorescence intensity than background levels, were defined. The ratio of dUTP-positive cells was then compared between each group. Electron microscopy Cells were trypsinized from flasks and washed by centrifugation in PBS, and cell pellets were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer. Cell pellets were processed as described previously [24].
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Flow cytometric analysis of active caspase-3
Statistical analysis
Detection of active caspase-3 using a fluorescein isothiocyanate (FITC)-conjugated monoclonal active caspase-3 antibody apoptosis kit (BD Biosciences) was used to assess apoptosis induced by EPA. Briefly, 1 ⫻ 106 cells were cultured in the presence or absence of EPA and harvested after 48 h. Cells were washed twice with PBS and resuspended in Cytofix/Cytoperm solution for 20 min on ice. After two washes with Perm/Wash Buffer at room temperature, pellets were resuspended in Perm/Wash buffer containing the FITC-conjugated monoclonal active caspase-3 antibody and incubated for 30 min at room temperature. Each sample was then washed with Perm/Wash buffer and analyzed by flow cytometry. Background fluorescence was determined by incubation without FITC enzyme. Active caspase-3 antibody-positive cells that demonstrated higher levels of fluorescence intensity than background levels were then defined. The ratio of active caspase-3 antibody-positive cells was then compared between each group.
Data are presented as mean ⫾ standard deviation. Statistical analysis was performed using Student’s t test (StatView, SAS Institute, Cary, NC, USA). Statistical significance was set at P ⬍ 0.05.
Preparation of cell lysates and western blot analysis Cells were trypsinized, scraped, and then washed twice with cold PBS (10 mM, pH 7.4). The cell suspension was centrifuged at 2000g for 10 min at 4°C; The medium was aspirated and then stored at ⫺80°C, followed by freezing in liquid nitrogen. Ice-cold lysis buffer (20 mM Tris-HCl, 100 mM NaCl, 1mM ethylene-diaminetetra-acetic acid, 1% NP40, and 1 mM phenyl methyl sulfonyl fluoride, pH 7.4) was added to the cells, which were then placed on ice for 30 min. The lysates were homogenized to break up cell aggregates. Lysates were cleared at 14 000g for 20 min at 4°C, and the supernatant (total cell lysates) was collected. Protein concentration was determined by DC assay (Bio-Rad Laboratories, Tokyo, Japan) according to the manufacturer’s protocol. Expression of COX-2 was examined by western blotting. In brief, 20 to 40 g of protein was resolved over 4% to 12% polyacrylamide gels. Separated proteins were western blotted onto nitrocellulose membranes (Invitrogen Life Technologies, La Jolla, CA, USA) and blocked in blocking buffer (5% non-fat dry milk and 0.1% Tween 20 in 20 mM triethanolamine buffer saline, pH, 7.6) for 1.5 h at 4°C. Antibodies were diluted to 1:100 (anti–COX-2; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and 1:200 (anti–-actin; Ambion, CA, USA) in the same buffer and incubated overnight at 4°C. The secondary antibodies, conjugated to horseradish peroxidase (Amersham Life Science, Tokyo, Japan), were diluted at 1:5000 and incubated overnight at 4°C. Protein expression was detected by using the Lumi-Light PLUS Western Blotting Substrate (Roche Diagnostics Corp., Tokyo, Japan) and visualized and quantified with the luminescent image analyzer LAS-1000plus in conjunction with ImageGauge 3.12 (Fuji Film Company, Ltd., Tokyo, Japan). Expression levels were normalized by -actin expression.
Results Effect of EPA on cell proliferation of human pancreatic cancer cell lines Three different human pancreatic cancer cell lines, SW1990, AsPC-1, and PANC-1, were employed to evaluate the effect of EPA on cell proliferation. As seen in Fig. 1, significant and dose-dependent suppression of proliferation was observed in all cell lines after 48 h of incubation. Growth inhibition was confirmed by microscopic observation. Effect of EPA on induction of apoptosis of human pancreatic cancer cell lines Induction of apoptosis was assayed by the TUNEL method. Upon exposure to 500 M EPA, all three cell lines showed an increased ratio of dUTP-positive cells (Fig. 2b), indicating that EPA had significantly induced apoptosis in all three cell lines (Table 1). Effect of EPA on cell ultrastructure in human pancreatic cancer cell lines The effect of EPA on human pancreatic cancer cell lines was also examined by electron microscopy. In control PANC-1 cells (Fig. 3a), the chromatin was floccular and dispersed throughout nuclei; the nuclear membrane was intact. In PANC-1 cells treated with 500 M EPA for 48 h, typical morphologic changes of apoptosis were seen. These early changes included chromatin condensation (Fig. 3b) and margination under the nuclear envelope with damage to the nuclear membrane (Fig. 3c). PANC-1 cells in the later stage of apoptosis are shown in Fig. 3d. Cells in this micrograph demonstrate nuclear condensation and fragmentation with formation of apoptotic bodies. The same results were observed in SW1990 and AsPC-1 cells treated with EPA. Morphologic changes suggesting necrosis, characterized by cell swelling and membrane breakdown, were not observed in any of the cell lines exposed to EPA. Effect of EPA on caspase-3 activity of human pancreatic cancer cell lines Active caspase-3 was assayed in human pancreatic cancer cell lines by flow cytometry. As presented in Table 2,
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Fig. 1. Effect of EPA on cell proliferation of human pancreatic cancer cell lines. EPA treatment (100, 300, and 500 M for 48 h) causes dose-dependent suppression of proliferation. Data are expressed as mean ⫾ standard deviation of three independent experiments (n ⫽ 10). *Values significantly different from 0 M (P ⬍ 0.05). EPA, eicosapentaenoic acid; O.D., optical density.
active caspase-3 increased significantly at 48 h in all three cell lines exposed to EPA. Effect of EPA on COX-2 expression in human pancreatic cancer cell lines Total cell lysates were analyzed by western blotting for COX-2. COX-2 protein, detected as 72- to 74-kDa reactive bands in cell lysates, was downregulated in all three human pancreatic cancer cell lines after incubation with EPA (Fig. 4a). Bands were quantified by ImageGauge software and normalized to actin levels. As shown in Fig. 4b, COX-2 antibody-reactive bands were significantly decreased by EPA in a dose-dependent fashion.
Discussion In this study, we have demonstrated that EPA inhibits net proliferation of human pancreatic cancer cells at least in part by induction of apoptotic cell death. Such apoptosis was associated with activation of caspase-3 and suppression of COX-2. Our findings of significant induction of apoptosis by EPA are consistent with those of Lai et al. [25] and Ross et al. [26]. In contrast, in a study using leukemia cell lines HL-60 and K-562, although EPA inhibited cell proliferation in both types of cells, EPA did not induce apoptosis in K-562 cells [16]. In the present experiments, we observed
that EPA induced apoptosis in all three human pancreatic cancer cell lines (which varied in grade of malignancy). However, for cell lines AsPc-1 and PANC-1, the ratio of apoptotic cells was relatively low when compared with the level of inhibition of cell proliferation induced by EPA. There are several possibilities to explain this discrepancy. One is that a process other than apoptosis, typically necrosis, was responsible for cell death. However, this is unlikely because no necrosis was observed when electron microscopy was performed on the various cell lines treated by EPA. Another possibility is that the sensitivity of the TUNEL method (used to detect apoptotic cells in the present investigation) was relatively low. It is possible that we failed to detect (pre-)apoptotic cells, which nonetheless may have exhibited a decreased uptake of tetrazolium salt in the MTS assay. The molecular mechanisms involved in EPA-induced apoptosis are not fully understood. Caspases are assumed to play an important role in apoptosis; activation of the caspase cascade requires initiator caspases, such as caspase-8 and caspase-9, and effector caspases, such as caspase-3, leading to apoptosis [27]. The caspases are synthesized as inactive enzymes, and once activated by proteolytic cleavage, the apoptotic processes are switched on. To date the correlation between apoptosis and activation of the caspase-3 by EPA in human pancreatic cancer cells has not been evaluated. Using flow cytometry, we demonstrated that cellular activity of caspase-3 increased after incubation with EPA. The
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Fig. 2. Effect of EPA on induction of apoptosis of human pancreatic cancer cell lines. (a) Horizontal line indicates background fluorescence as determined by incubation without terminal deoxynucleotidyl transferase enzyme. In this histogram, the area of dUTP-positive cells, which demonstrate higher levels of fluorescence intensity than do background levels, were defined. (b) Three human pancreatic cancer cells incubated with 500 M EPA for 48 h were fixed and permeabilized, and DNA fragments were revealed by staining with fluorescein isothiocyanate– conjugated dUTP. The ratio of dUTP positive cells are shown. dUTP, deoxyuridine triphosphate; EPA, eicosapentaenoic acid.
results obtained in our experiments suggest that active caspase-3 is involved in EPA-induced apoptosis of human pancreatic cancer cells. In contrast, Puertollano et al. [28] reported that EPA-induced apoptosis in YAC-1 lymphoma cells occurred by a caspase-3–independent mechanism. Such differences might be due to the different phenotype or genotype of the different cell lines tested. Further study with capase-3 inhibitors and initiator caspases is necessary to elucidate the precise molecular mechanisms behind EPA induction of apoptosis. COX-2 is induced in response to inflammation and is not detectable in most normal, non-inflamed tissues. However, COX-2 is overexpressed in several different types of human cancer [29,30], including pancreatic cancer [31]. We evaluated the effect of EPA on COX-2 expression because the relation between EPA-induced antiproliferation and COX-2 expression in human pancreatic cancer cells has not been established. We observed that COX-2 expression was
Table 1 Effects of EPA treatment on induction of apoptosis* Ratio of dUTP-positive cells (%)
0 M EPA (100 M) EPA (300 M) EPA (500 M)
SW1990
AsPC-1
PANC-1
9.3 ⫾ 0.3 30.3 ⫾ 1.4† 55.3 ⫾ 2.8† 85.5 ⫾ 1.1†
3.1 ⫾ 0.4 6.6 ⫾ 0.5† 5.4 ⫾ 0.2† 6.5 ⫾ 0.4†
3.4 ⫾ 0.5 6.4 ⫾ 0.5† 8.1 ⫾ 0.9† 14.3 ⫾ 0.9†
dUTP, deoxyuridine triphosphate; EPA, eicosapentaenoic acid; FITC, fluorescein isothiocyanate; TUNEL, terminal deoxyuridine triphosphate nick end labeling. Data are expressed as mean ⫾ standard deviation of three independent expriments (n ⫽ 5). * Human pancreatic cancer cell lines were incubated in the presence of EPA at concentrations of 100, 300, and 500 M/L for 48 h. Apoptotic cells were determined by the FITC-TUNEL method. The ratio of dUTP-positive cells was calculated as defined in MATERIALS AND METHODS. † Values significantly different from 0 M (P ⬍ 0.05).
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Fig. 3. Effect of EPA on ultrastructure of PANC-1 cells. After treatment with 500 M EPA for 48 h, PANC-1 cells cultured on glass coverslips were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer and examined by electron microscopy. (a) Control cells. (b, c) Early apoptotic changes. (d) Cell in later stage of apoptosis and apoptotic bodies (arrow) are visible. Scale bars ⫽ 1 m. EPA, eicosapentaenoic acid.
downregulated in the cell lines incubated with EPA. Our results were similar to those reported by Hamid et al. [32], whose study focused on breast cancer. It has also been reported that upregulation of COX-2 expression inhibits apoptosis [33] and that COX-2 inhibition induces apoptosis by activating caspase-3 [22], which are findings that correspond with our results. In the present study, COX-2 was suppressed in all three cell lines to a similar extent, although the degree of apoptosis induced by EPA varied. Although the reason for this could not be elucidated in the present study, there might have been differences between cell lines in their sensitivity to changes in COX-2 as part of the potential mechanism that induces apoptosis through caspase-3 activation. The involvement of COX-2 in apoptotic mechanisms is complicated, and intrinsic (by activation of caspase-9) and
Table 2 Caspase-3 activation by EPA treatment* Ratio of active caspase-3 antibody-positive cells (%)
0 M EPA (100 M) EPA (300 M) EPA (500 M)
SW1990
AsPC-1
PANC-1
4.4 ⫾ 0.6 9.5 ⫾ 0.7† 32.4 ⫾ 2.2† 68.0 ⫾ 2.9†
7.2 ⫾ 0.2 9.5 ⫾ 0.4† 12.6 ⫾ 0.5† 17.2 ⫾ 0.5†
8.3 ⫾ 0.4 9.9 ⫾ 0.3† 11.4 ⫾ 0.3† 15.4 ⫾ 0.4†
EPA, eicosapentaenoic acid. Data are expressed as mean ⫾ standard deviation of three independent experiments (n ⫽ 5). * Human pancreatic cancer cell lines were incubated in the presence of EPA at concentrations of 100, 300, and 500 M/L for 48 h. Active caspase-3 antibody-positive cells were determined by flow cytometry. The ratio of active caspase-3 antibody-positive cells was calculated as defined in MATERIALS AND METHODS. † Values significantly different from 0 M (P ⬍ 0.05).
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Fig. 4. Effect of EPA on COX-2 expression in human pancreatic cancer cell lines. Three human pancreatic cancer cell lines, SW1990, AsPC-1, and PANC-1, were incubated in the presence or absence of 100 or 300 M EPA for 48 h. (a) Protein (20 to 40 g) from each cell line was analyzed by western immunoblot analysis. The blot was probed with antibodies to COX-2 and then reprobed with the actin control. (b) Quantification by ImageGauge software of the COX-2 bands, normalized to actin, shows a significant and dose-dependent decrease in the molecular weight forms of COX-2 (n ⫽ 5). *Values significantly different from 0 M (P ⬍ 0.05). Bars represent standard deviations. COX-2, cyco-oxygenase–2; EPA, eicosapentaenoic acid.
extrinsic (by activation of caspase-8) mechanisms in the caspase cascade require further studies. Moreover, it has been shown that cell proliferation can be inhibited as a result of downregulation of COX-2 expression in colorectal cancer cell lines [34], leading to a decrease in eicosanoids (prostaglandin E2) produced from -6 PUFAs. In that regard, involvement of EPA in the arachidonic acid cascade is expected. In the present study, because we did not use other PUFA types, it is unknown whether EPA specifically affects
inhibition of all pancreatic cancer cell lines. However, Falconer et al. [35] reported that -3 PUFAs including EPA suppressed proliferation of pancreatic cancer cell line and that, among -3 PUFAs, EPA showed the strongest effect. With regard to possible clinical applications, the concentration of EPA used in the present study has been reported [9] to have no adverse effects toward normal cells and could therefore be considered for clinical purposes. EPA can be given orally or intravenously. A previous study in vivo
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showed that the action of EPA can be enhanced and adverse effects can be alleviated by a combination of cytotoxic chemotherapy and EPA [36]. These effects could be examined in future clinical trials and provide benefit for the treatment of patients with pancreatic cancer. In summary, we have demonstrated that EPA inhibits human pancreatic cancer cell growth due at least in part to induction of apoptotic cell death. Such apoptosis is associated with activation of caspase-3 and suppression of COX-2 expression.
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Applied nutritional investigation
Apoptosis in human pancreatic cancer cells induced by eicosapentaenoic acid Tetsuya Shirota, M.D.ⴱ, Seiji Haji, M.D., Ph.D., Mitsuo Yamasaki, M.D., Takuya Iwasaki, M.D., Ph.D., Toshiharu Hidaka, M.D., Yoshifumi Takeyama, M.D., Ph.D., Hitoshi Shiozaki, M.D., Ph.D., and Harumasa Ohyanagi, M.D., Ph.D. Department of Surgery, Kinki University School of Medicine, Osaka, Japan Manuscript received September 21, 2004; accepted December 3, 2004.
Abstract
Objectives: Clinical studies have shown that administration of eicosapentaenoic acid (EPA) to patients who have unresectable pancreatic cancer induces marked attenuation of cachexia. However, the exact mechanisms of the beneficial effect of EPA on pancreatic cancer are unknown. This examined the effect of EPA on proliferation of human pancreatic cancer cell lines and sought to clarify its mechanisms. Methods: The effects of EPA on proliferation of three human pancreatic cancer cell lines (SW1990, AsPC-1, and PANC-1) were assessed. Induction of apoptosis and expressions of apoptosis-related proteins were measured. The effect of EPA on cyclo-oxygenase–2 expression in these cell lines was determined. Results: EPA inhibited proliferation of all three human pancreatic cancer cell lines in a dosedependent fashion. Simultaneously, EPA treatment induced apoptosis and this was associated with caspase-3 activation. EPA treatment was also associated with a decrease in intracellular levels of cyclo-oxygenase–2 protein. Conclusion: We have demonstrated that EPA inhibits human pancreatic cancer cell growth due at least in part to the induction of apoptotic cell death. Such apoptosis is associated with activation of caspase-3 and suppression of cyclo-oxygenase–2 expression. Greater understanding of the molecular events associated with the biological activity of EPA should enhance the therapeutic potential of administration of EPA to patients who have pancreatic cancer. © 2005 Elsevier Inc. All rights reserved.
Keywords:
Eicosapentaenoic acid; -3 Polyunsaturated fatty acids; Pancreatic cancer; Apoptosis; Cyclo-oxygenase–2
Introduction Pancreatic cancer is a devastating disease that currently affects approximately 28 000 people per year in the United States [1]. The 5-y survival rate in patients who have pancreatic cancer is lower than 5%, making this disease the fourth most common cause of cancer-related mortality [1]. Due to the relatively late diagnosis of pancreatic cancer, the tumor frequently metastasizes or invades surrounding tissues on diagnosis, and most patients (approximately 70% to 80%) are not eligible for a curative resection [1]. In addiⴱ Corresponding author. Tel.:⫹81-72-366-0221; fax:⫹81-72-366-0111. E-mail address: [email protected] (T. Shirota). 0899-9007/05/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2004.12.013
tion, non-surgical treatment options such as chemotherapy [2] and/or radiotherapy [3] have shown disappointing results thus far, and reasons for resistance of pancreatic cancer cells to radiation and cytotoxic agents and their aggressive growth are not completely understood. The care of patients who have unresectable pancreatic cancer has focused on relief of symptoms, such as weight loss, pain, jaundice, steatorrhea, nausea, and anemia, in an attempt to optimize quality of life, but care remains unsatisfactory. Polyunsaturated fatty acids (PUFAs) have been recognized as important energy sources and components of cell membranes. PUFAs also play key roles in many cellular events, such as immune function [4], aging [5], neonatal development [6], anti-inflammatory effects [7], and gene
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regulation [8]. No toxicity of PUFAs on normal cell cultures or in whole animals have been reported [9,10]. During recent decades, PUFAs have been suggested as natural products that may play significant roles in modulating cancer development [11]. In general, -6 PUFAs appear to enhance tumor growth and proliferation [12], whereas -3 PUFAs seem to have an inhibitory effect on this process [13]. Eicosapentaenoic acid (EPA; 20:5-3) is an -3 PUFA found abundantly in fish oil. EPA retards the growth and development of breast, colon, and liver cancers and leukemia in vitro and in vivo [9,14,15,16]. In addition, clinical and experimental cancer studies further support the use of EPA to promote weight maintenance and decrease anorexia in patients with unresectable pancreatic cancer [17]. The cellular features of apoptosis are one of the most important issues in cancer research due to the significance of apoptosis in tumor development and chemotherapy [18]. Apoptosis induced by -3 PUFAs has been demonstrated in human colon cancer [19], leukemia [16], and lymphoma [20]. Apoptosis is regulated by several processes involving changes in expression or activity of distinct proteins [21]. Caspases plays a central role in apoptosis. Specifically, caspase-3, a protease located downstream of the caspase-activating mechanism, activates caspase-3–activated DNAse and induces DNA fragmentation when activated. Cyclo-oxygenase is an enzyme associated with the arachidonic acid cascade. The -3 PUFAs may influence inflammation and cancer cell proliferation via cyclo-oxygenase–2 (COX-2). Further, COX-2 is believed to modulate apoptosis, possibly by activation of caspase-3 [22]. However, the molecular mechanisms associated with apoptosis and the antiproliferative effect of human pancreatic cancer cells by EPA have not been well defined. In this study, we determined the effect of EPA on pancreatic cancer cell proliferation and induction of apoptosis.
Materials and methods Reagents A purified preparation of EPA (⬎98% pure) isolated from fish oil, in ethyl ester form, was kindly provided by Mochida Pharmaceutical Co. (Tokyo, Japan). EPA was dissolved in ethanol before use. Cell lines Human pancreatic cancer cell lines (SW1990, AsPC-1, and PANC-1) were obtained from American Type Culture Collection (Rockville, MD, USA). SW1990 and PANC-1 were cultured in Dulbecco’s Modified Eagle Medium containing 10% heat-inactivated fetal calf serum. AsPC-1 was cultured in RPMI 1640 containing 10% fetal calf serum. Cultures were maintained at 37°C in a humidified atmo-
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sphere of 5% CO2. SW1990 and AsPC-1 are metastatic in behavior, whereas PANC-1 is a non-metastatic pancreatic cancer cell line [23]. Cell proliferation analysis Cell proliferation assays were performed with the CellTiter 96 AQueous One Solution Cell Proliferation Assay kit according to the manufacturer’s instructions (Promega, Madison, WI, USA). Briefly, 5 ⫻ 103 cells/well in medium were placed onto 96-well plates. EPA was added to cellular cultures at final concentrations of 0, 100, 300, and 500 M. Cultures were incubated for 48 h and then added to 20 L/well of CellTiter 96 AQueous One Solution Reagent, which contains a tetrazolium compound, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, an inner salt; MTS was added to the culture medium. After incubating the plate for 1 h in a CO2 incubator, absorbance at 490 nm was recorded with a 96-well plate reader. Apoptosis assays Apoptosis was measured with the terminal deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) method, which used the MEBSTAIN Apoptosis Kit Direct (Medical and Biological Laboratories, Nagoya, Japan) according to the manufacturer’s instructions. Briefly, human pancreatic cancer cell lines exposed to EPA for 48 h were washed twice with phosphate buffered saline containing 0.2% bovine serum albumin (PBS-BSA). Cells were then fixed with buffered paraformaldehyde at 4°C for 30 min, followed by washing twice with PBS-BSA. Cold 70% ethanol was added to the cell pellet, which was incubated for 30 min at ⫺20°C for permeabilization. After washing twice with PBS-BSA, 30 L of terminal deoxynucleotidyl transferase reaction reagent was added to the cell pellet for 1 h at 37°C in the dark. Cells were washed twice with PBS-BSA and resuspended in 500 L of PBS-BSA. Results were analyzed by flow cytometry using CellQuest software and a FACScaliber instrument (BD Pharmingen, San Jose, CA, USA). The resulting histogram (Fig. 2a) indicates background fluorescence as determined by incubation without terminal deoxynucleotidyl transferase enzyme. In this histogram, the area of dUTP-positive cells, which demonstrate higher levels of fluorescence intensity than background levels, were defined. The ratio of dUTP-positive cells was then compared between each group. Electron microscopy Cells were trypsinized from flasks and washed by centrifugation in PBS, and cell pellets were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer. Cell pellets were processed as described previously [24].
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Flow cytometric analysis of active caspase-3
Statistical analysis
Detection of active caspase-3 using a fluorescein isothiocyanate (FITC)-conjugated monoclonal active caspase-3 antibody apoptosis kit (BD Biosciences) was used to assess apoptosis induced by EPA. Briefly, 1 ⫻ 106 cells were cultured in the presence or absence of EPA and harvested after 48 h. Cells were washed twice with PBS and resuspended in Cytofix/Cytoperm solution for 20 min on ice. After two washes with Perm/Wash Buffer at room temperature, pellets were resuspended in Perm/Wash buffer containing the FITC-conjugated monoclonal active caspase-3 antibody and incubated for 30 min at room temperature. Each sample was then washed with Perm/Wash buffer and analyzed by flow cytometry. Background fluorescence was determined by incubation without FITC enzyme. Active caspase-3 antibody-positive cells that demonstrated higher levels of fluorescence intensity than background levels were then defined. The ratio of active caspase-3 antibody-positive cells was then compared between each group.
Data are presented as mean ⫾ standard deviation. Statistical analysis was performed using Student’s t test (StatView, SAS Institute, Cary, NC, USA). Statistical significance was set at P ⬍ 0.05.
Preparation of cell lysates and western blot analysis Cells were trypsinized, scraped, and then washed twice with cold PBS (10 mM, pH 7.4). The cell suspension was centrifuged at 2000g for 10 min at 4°C; The medium was aspirated and then stored at ⫺80°C, followed by freezing in liquid nitrogen. Ice-cold lysis buffer (20 mM Tris-HCl, 100 mM NaCl, 1mM ethylene-diaminetetra-acetic acid, 1% NP40, and 1 mM phenyl methyl sulfonyl fluoride, pH 7.4) was added to the cells, which were then placed on ice for 30 min. The lysates were homogenized to break up cell aggregates. Lysates were cleared at 14 000g for 20 min at 4°C, and the supernatant (total cell lysates) was collected. Protein concentration was determined by DC assay (Bio-Rad Laboratories, Tokyo, Japan) according to the manufacturer’s protocol. Expression of COX-2 was examined by western blotting. In brief, 20 to 40 g of protein was resolved over 4% to 12% polyacrylamide gels. Separated proteins were western blotted onto nitrocellulose membranes (Invitrogen Life Technologies, La Jolla, CA, USA) and blocked in blocking buffer (5% non-fat dry milk and 0.1% Tween 20 in 20 mM triethanolamine buffer saline, pH, 7.6) for 1.5 h at 4°C. Antibodies were diluted to 1:100 (anti–COX-2; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and 1:200 (anti–-actin; Ambion, CA, USA) in the same buffer and incubated overnight at 4°C. The secondary antibodies, conjugated to horseradish peroxidase (Amersham Life Science, Tokyo, Japan), were diluted at 1:5000 and incubated overnight at 4°C. Protein expression was detected by using the Lumi-Light PLUS Western Blotting Substrate (Roche Diagnostics Corp., Tokyo, Japan) and visualized and quantified with the luminescent image analyzer LAS-1000plus in conjunction with ImageGauge 3.12 (Fuji Film Company, Ltd., Tokyo, Japan). Expression levels were normalized by -actin expression.
Results Effect of EPA on cell proliferation of human pancreatic cancer cell lines Three different human pancreatic cancer cell lines, SW1990, AsPC-1, and PANC-1, were employed to evaluate the effect of EPA on cell proliferation. As seen in Fig. 1, significant and dose-dependent suppression of proliferation was observed in all cell lines after 48 h of incubation. Growth inhibition was confirmed by microscopic observation. Effect of EPA on induction of apoptosis of human pancreatic cancer cell lines Induction of apoptosis was assayed by the TUNEL method. Upon exposure to 500 M EPA, all three cell lines showed an increased ratio of dUTP-positive cells (Fig. 2b), indicating that EPA had significantly induced apoptosis in all three cell lines (Table 1). Effect of EPA on cell ultrastructure in human pancreatic cancer cell lines The effect of EPA on human pancreatic cancer cell lines was also examined by electron microscopy. In control PANC-1 cells (Fig. 3a), the chromatin was floccular and dispersed throughout nuclei; the nuclear membrane was intact. In PANC-1 cells treated with 500 M EPA for 48 h, typical morphologic changes of apoptosis were seen. These early changes included chromatin condensation (Fig. 3b) and margination under the nuclear envelope with damage to the nuclear membrane (Fig. 3c). PANC-1 cells in the later stage of apoptosis are shown in Fig. 3d. Cells in this micrograph demonstrate nuclear condensation and fragmentation with formation of apoptotic bodies. The same results were observed in SW1990 and AsPC-1 cells treated with EPA. Morphologic changes suggesting necrosis, characterized by cell swelling and membrane breakdown, were not observed in any of the cell lines exposed to EPA. Effect of EPA on caspase-3 activity of human pancreatic cancer cell lines Active caspase-3 was assayed in human pancreatic cancer cell lines by flow cytometry. As presented in Table 2,
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Fig. 1. Effect of EPA on cell proliferation of human pancreatic cancer cell lines. EPA treatment (100, 300, and 500 M for 48 h) causes dose-dependent suppression of proliferation. Data are expressed as mean ⫾ standard deviation of three independent experiments (n ⫽ 10). *Values significantly different from 0 M (P ⬍ 0.05). EPA, eicosapentaenoic acid; O.D., optical density.
active caspase-3 increased significantly at 48 h in all three cell lines exposed to EPA. Effect of EPA on COX-2 expression in human pancreatic cancer cell lines Total cell lysates were analyzed by western blotting for COX-2. COX-2 protein, detected as 72- to 74-kDa reactive bands in cell lysates, was downregulated in all three human pancreatic cancer cell lines after incubation with EPA (Fig. 4a). Bands were quantified by ImageGauge software and normalized to actin levels. As shown in Fig. 4b, COX-2 antibody-reactive bands were significantly decreased by EPA in a dose-dependent fashion.
Discussion In this study, we have demonstrated that EPA inhibits net proliferation of human pancreatic cancer cells at least in part by induction of apoptotic cell death. Such apoptosis was associated with activation of caspase-3 and suppression of COX-2. Our findings of significant induction of apoptosis by EPA are consistent with those of Lai et al. [25] and Ross et al. [26]. In contrast, in a study using leukemia cell lines HL-60 and K-562, although EPA inhibited cell proliferation in both types of cells, EPA did not induce apoptosis in K-562 cells [16]. In the present experiments, we observed
that EPA induced apoptosis in all three human pancreatic cancer cell lines (which varied in grade of malignancy). However, for cell lines AsPc-1 and PANC-1, the ratio of apoptotic cells was relatively low when compared with the level of inhibition of cell proliferation induced by EPA. There are several possibilities to explain this discrepancy. One is that a process other than apoptosis, typically necrosis, was responsible for cell death. However, this is unlikely because no necrosis was observed when electron microscopy was performed on the various cell lines treated by EPA. Another possibility is that the sensitivity of the TUNEL method (used to detect apoptotic cells in the present investigation) was relatively low. It is possible that we failed to detect (pre-)apoptotic cells, which nonetheless may have exhibited a decreased uptake of tetrazolium salt in the MTS assay. The molecular mechanisms involved in EPA-induced apoptosis are not fully understood. Caspases are assumed to play an important role in apoptosis; activation of the caspase cascade requires initiator caspases, such as caspase-8 and caspase-9, and effector caspases, such as caspase-3, leading to apoptosis [27]. The caspases are synthesized as inactive enzymes, and once activated by proteolytic cleavage, the apoptotic processes are switched on. To date the correlation between apoptosis and activation of the caspase-3 by EPA in human pancreatic cancer cells has not been evaluated. Using flow cytometry, we demonstrated that cellular activity of caspase-3 increased after incubation with EPA. The
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Fig. 2. Effect of EPA on induction of apoptosis of human pancreatic cancer cell lines. (a) Horizontal line indicates background fluorescence as determined by incubation without terminal deoxynucleotidyl transferase enzyme. In this histogram, the area of dUTP-positive cells, which demonstrate higher levels of fluorescence intensity than do background levels, were defined. (b) Three human pancreatic cancer cells incubated with 500 M EPA for 48 h were fixed and permeabilized, and DNA fragments were revealed by staining with fluorescein isothiocyanate– conjugated dUTP. The ratio of dUTP positive cells are shown. dUTP, deoxyuridine triphosphate; EPA, eicosapentaenoic acid.
results obtained in our experiments suggest that active caspase-3 is involved in EPA-induced apoptosis of human pancreatic cancer cells. In contrast, Puertollano et al. [28] reported that EPA-induced apoptosis in YAC-1 lymphoma cells occurred by a caspase-3–independent mechanism. Such differences might be due to the different phenotype or genotype of the different cell lines tested. Further study with capase-3 inhibitors and initiator caspases is necessary to elucidate the precise molecular mechanisms behind EPA induction of apoptosis. COX-2 is induced in response to inflammation and is not detectable in most normal, non-inflamed tissues. However, COX-2 is overexpressed in several different types of human cancer [29,30], including pancreatic cancer [31]. We evaluated the effect of EPA on COX-2 expression because the relation between EPA-induced antiproliferation and COX-2 expression in human pancreatic cancer cells has not been established. We observed that COX-2 expression was
Table 1 Effects of EPA treatment on induction of apoptosis* Ratio of dUTP-positive cells (%)
0 M EPA (100 M) EPA (300 M) EPA (500 M)
SW1990
AsPC-1
PANC-1
9.3 ⫾ 0.3 30.3 ⫾ 1.4† 55.3 ⫾ 2.8† 85.5 ⫾ 1.1†
3.1 ⫾ 0.4 6.6 ⫾ 0.5† 5.4 ⫾ 0.2† 6.5 ⫾ 0.4†
3.4 ⫾ 0.5 6.4 ⫾ 0.5† 8.1 ⫾ 0.9† 14.3 ⫾ 0.9†
dUTP, deoxyuridine triphosphate; EPA, eicosapentaenoic acid; FITC, fluorescein isothiocyanate; TUNEL, terminal deoxyuridine triphosphate nick end labeling. Data are expressed as mean ⫾ standard deviation of three independent expriments (n ⫽ 5). * Human pancreatic cancer cell lines were incubated in the presence of EPA at concentrations of 100, 300, and 500 M/L for 48 h. Apoptotic cells were determined by the FITC-TUNEL method. The ratio of dUTP-positive cells was calculated as defined in MATERIALS AND METHODS. † Values significantly different from 0 M (P ⬍ 0.05).
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Fig. 3. Effect of EPA on ultrastructure of PANC-1 cells. After treatment with 500 M EPA for 48 h, PANC-1 cells cultured on glass coverslips were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer and examined by electron microscopy. (a) Control cells. (b, c) Early apoptotic changes. (d) Cell in later stage of apoptosis and apoptotic bodies (arrow) are visible. Scale bars ⫽ 1 m. EPA, eicosapentaenoic acid.
downregulated in the cell lines incubated with EPA. Our results were similar to those reported by Hamid et al. [32], whose study focused on breast cancer. It has also been reported that upregulation of COX-2 expression inhibits apoptosis [33] and that COX-2 inhibition induces apoptosis by activating caspase-3 [22], which are findings that correspond with our results. In the present study, COX-2 was suppressed in all three cell lines to a similar extent, although the degree of apoptosis induced by EPA varied. Although the reason for this could not be elucidated in the present study, there might have been differences between cell lines in their sensitivity to changes in COX-2 as part of the potential mechanism that induces apoptosis through caspase-3 activation. The involvement of COX-2 in apoptotic mechanisms is complicated, and intrinsic (by activation of caspase-9) and
Table 2 Caspase-3 activation by EPA treatment* Ratio of active caspase-3 antibody-positive cells (%)
0 M EPA (100 M) EPA (300 M) EPA (500 M)
SW1990
AsPC-1
PANC-1
4.4 ⫾ 0.6 9.5 ⫾ 0.7† 32.4 ⫾ 2.2† 68.0 ⫾ 2.9†
7.2 ⫾ 0.2 9.5 ⫾ 0.4† 12.6 ⫾ 0.5† 17.2 ⫾ 0.5†
8.3 ⫾ 0.4 9.9 ⫾ 0.3† 11.4 ⫾ 0.3† 15.4 ⫾ 0.4†
EPA, eicosapentaenoic acid. Data are expressed as mean ⫾ standard deviation of three independent experiments (n ⫽ 5). * Human pancreatic cancer cell lines were incubated in the presence of EPA at concentrations of 100, 300, and 500 M/L for 48 h. Active caspase-3 antibody-positive cells were determined by flow cytometry. The ratio of active caspase-3 antibody-positive cells was calculated as defined in MATERIALS AND METHODS. † Values significantly different from 0 M (P ⬍ 0.05).
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Fig. 4. Effect of EPA on COX-2 expression in human pancreatic cancer cell lines. Three human pancreatic cancer cell lines, SW1990, AsPC-1, and PANC-1, were incubated in the presence or absence of 100 or 300 M EPA for 48 h. (a) Protein (20 to 40 g) from each cell line was analyzed by western immunoblot analysis. The blot was probed with antibodies to COX-2 and then reprobed with the actin control. (b) Quantification by ImageGauge software of the COX-2 bands, normalized to actin, shows a significant and dose-dependent decrease in the molecular weight forms of COX-2 (n ⫽ 5). *Values significantly different from 0 M (P ⬍ 0.05). Bars represent standard deviations. COX-2, cyco-oxygenase–2; EPA, eicosapentaenoic acid.
extrinsic (by activation of caspase-8) mechanisms in the caspase cascade require further studies. Moreover, it has been shown that cell proliferation can be inhibited as a result of downregulation of COX-2 expression in colorectal cancer cell lines [34], leading to a decrease in eicosanoids (prostaglandin E2) produced from -6 PUFAs. In that regard, involvement of EPA in the arachidonic acid cascade is expected. In the present study, because we did not use other PUFA types, it is unknown whether EPA specifically affects
inhibition of all pancreatic cancer cell lines. However, Falconer et al. [35] reported that -3 PUFAs including EPA suppressed proliferation of pancreatic cancer cell line and that, among -3 PUFAs, EPA showed the strongest effect. With regard to possible clinical applications, the concentration of EPA used in the present study has been reported [9] to have no adverse effects toward normal cells and could therefore be considered for clinical purposes. EPA can be given orally or intravenously. A previous study in vivo
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showed that the action of EPA can be enhanced and adverse effects can be alleviated by a combination of cytotoxic chemotherapy and EPA [36]. These effects could be examined in future clinical trials and provide benefit for the treatment of patients with pancreatic cancer. In summary, we have demonstrated that EPA inhibits human pancreatic cancer cell growth due at least in part to induction of apoptotic cell death. Such apoptosis is associated with activation of caspase-3 and suppression of COX-2 expression.
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