Parasitol Res (2009) 104:1011–1016 DOI 10.1007/s00436-008-1283-y
Resistance of cholangiocarcinoma cells to parthenolide-induced apoptosis by the excretory–secretory products of Clonorchis sinensis Young Ju Kim & Min-Ho Choi & Sung-Tae Hong & Young Mee Bae
Received: 3 November 2008 / Accepted: 7 November 2008 / Published online: 5 December 2008 # Springer-Verlag 2008
Abstract Infection by Clonorchis sinensis, the Chinese or oriental liver fluke, is a significant risk factor for the development of cholangiocarcinoma, a human epithelial carcinoma of the intrahepatic bile duct. Parthenolide is a sesquiterpene lactone that has strong anticancer properties and is also known to induce apoptosis in cholangiocarcinoma cells. Many investigators have reported that excretory– secretory (ES) products of C. sinensis as well as Opisthorchis viverrini promote the development of cholangiocarcinomas. However, the intrinsic mechanism is not clearly understood. Therefore, we investigated the biological roles of the ES products in a cholangiocarcinoma cell line, HuCCT1. The ES products of C. sinensis increased proliferation of HuCCT1 cells and augmented the expression of cyclooxygenase (COX)-2. To determine whether cells treated with ES products would respond differently to parthenolide, HuCCT1 cells were treated with parthenolide alone or parthenolide after pretreatment with ES products. Cells pretreated with ES products were resistant to parthenolideinduced apoptosis. Because parthenolide has been reported to be a COX-2 inhibitor, we hypothesize that COX-2 might be a key factor that promotes resistance of cholangiocarcinoma cancer cells to parthenolide-induced apoptosis. These results suggest that chemotherapy treatment regimens in Y. J. Kim : M.-H. Choi : S.-T. Hong : Y. M. Bae (*) Department of Parasitology and Tropical Medicine, Seoul National University College of Medicine, 28 Yeongeon-dong, Jongro-gu, Seoul 110-799, South Korea e-mail: [email protected]
Y. J. Kim : M.-H. Choi : S.-T. Hong : Y. M. Bae Institute of Endemic Diseases, Seoul National University Medical Research Center, 28 Yeongeon-dong, Jongro-gu, Seoul 110-799, South Korea
cholangiocarcinoma patients with C. sinensis infection should be modulated to account for ES products excreted by the liver fluke.
Introduction Infection by liver flukes mainly involves Clonorchis sinensis and Opisthorchis viverrini and is regarded as an urgent public health problem in East Asia (Keiser and Utzinger 2005; Sripa et al. 2007). C. sinensis, the Chinese or oriental human liver fluke, is a bile duct parasite and is endemic in various southern parts of Asia (Korea, China, Taiwan) and in the northern part of Vietnam (Hong 2003; Choi et al. 2005; Lim et al. 2006). The main histological features of C. sinensis infection are irregular dilatation of the bile ducts and glandular hyperplasia (Schwartz 1980; Watanapa and Watanapa 2002; Hong 2003; Choi et al. 2005). Moreover, hamsters infected with liver flukes such as C. sinensis or O. viverrini have a greater risk of developing cholangiocarcinomas (Lee et al. 1993; Prempracha et al. 1994), and, with C. sinensis, the risk of cholangiocarcinoma is correlated with the endemicity (Lim et al. 2006). Therefore, C. sinensis is classified as a risk factor (carcinogen group 2A) for hepatobiliary cancer by the International Agency for Research on Cancer (1993). Cholangiocarcinomas are a malignant cancer of epithelial cells that reside in the hepatic biliary tract (Gores 2003; Han et al. 2004) and are often caused by physical damage, chronic inflammation, injury, and epithelial cell proliferation (Han et al. 2004). The symptoms of clonorchiasis induce severe histopathological changes of the intrahepatic bile duct and surrounding liver tissues; these changes include desquamation, proliferation, glandular change, metaplasia of the cholangial epithelium, gall stone forma-
tion, and cholangial carcinoma (Schwartz 1980; Watanapa and Watanapa 2002; Hong 2003; Choi et al. 2005). In cholangiocarcinoma cells, cyclooxygenase-2 (COX-2) is known to be upregulated and to increase the intracellular levels of prostaglandins—lipid inflammatory mediators that promote proliferation of tumor cells (Chariyalertsak et al. 2001; Hayashi et al. 2001; Endo et al. 2002; Han et al. 2004). C. sinensis infection has been related with aberrant COX-2 expression (Kim et al. 2003), and upregulated COX-2 has been shown to be associated with increased proliferation of tumor cells (Schmitz et al. 2007). The sesquiterpene lactone, parthenolide, is the principal active component in feverfew (Tanacetum parthenium) and is used to treat fever (Knight 1995), migraines (Johnson et al. 1985; Murphy et al. 1988), inflammation (Hehner et al. 1998; Lyss et al. 1998), and tumors (Kupchan et al. 1971; Woynarowski and Konopa 1981). Parthenolide is known to induce apoptosis of cholangiocarcinoma cells in a dosedependent manner (Nzeako et al. 2002; Kim et al. 2005) and to inhibit nuclear factor κB (Hwang et al. 1996; Bork et al. 1997). Recently, we reported that ES products from C. sinensis induced proliferation of a human epithelial cell line through the upregulation of cell cycle proteins including E2F1 (Kim et al. 2008a, b). Furthermore, Davis et al. (2005) showed that COX-2 expression is positively regulated by E2F1 activity. Therefore, we questioned whether ES products from C. sinensis affect the sensitivity of the cholangiocarcinoma cell line, HuCCT-1, to parthenolide. In the current study, we show that cholangiocarcinoma cells treated with ES products were resistant to parthenolide-induced apoptosis, which may be mediated via the COX-2 pathway. In light of these results, we suggest that chemotherapeutic regimens in cholangiocarcinoma patients with C. sinensis may need to be modulated to account for the antiapoptotic effects of liver fluke by-products.
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medium was centrifuged for 30 min at 15,000 rpm and then filtered with a syringe-driven 0.2-μm filter unit (Choi et al. 2003). The amount of protein in the extract was measured using the Bradford assay (Bio-Rad, Hercules, CA, USA). Cells and reagents The cholangiocarcinoma cell line, HuCCT1, was maintained in RPMI1640 medium (Gibco) containing 10% fetal bovine serum (FBS; Gibco), 2 mM L-glutamine, 100 μg/ml penicillin, and 100 U/ml streptomycin at 37°C under a humidified atmosphere of 5% CO2. Polyclonal or monoclonal antibodies were used to detect the cell cycle, including anti-cyclin-E (sc-247), anti-cyclin-B1 (sc-245), anti-E2F1 (sc-193), anti-CDK2 (sc-163), anti-CDC2 (sc-53), and antiCDK4 (sc-260). All antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and used at 1:1,000 dilutions. The antibody against calnexin (BD 610523) was purchased from Transduction Laboratories (BD Biosciences, San Jose, CA, USA) and used at a 1:2,000 dilution. Antimouse and antirabbit immunoglobulin G antisera conjugated with horseradish peroxidase (HRP) were purchased from DAKO (Glostrup, Denmark). Cell proliferation assay
Materials and methods
We used the XTT formazan method to measure cell proliferation. XTT (1 mg/ml) was dissolved in warm medium (without phenol red), and 1.25 mM phenazine methosulfate (PMS) was prepared in PBS. The cells were grown in tissue-culture-grade, 96-well, flat-bottomed microtiter plates with 100 μl of culture medium per well. After incubating for the indicated periods, 50 μl of the XTT–PMS mixture (final XTT concentration, 0.3 mg/ml) was added to each well. The microtiter plates were incubated for 4 h at 37°C, and the formazan product was quantified by measuring the absorbance at 492 and 690 nm on a microtiter plate reader (Martens et al. 1998; Kuo et al. 2006). All experiments were performed in triplicate.
Preparation of ES products
Metacercariae of C. sinensis were collected from naturally infected freshwater crayfish (Pseudorasbora parva) at an endemic site in Korea. Pepsin–HCl was used to digest the flesh of the host crayfish to obtain the metacercariae, which were then introduced into 4- to 6-week-old Sprague– Dawley rats. After 3 months, the adult worms of C. sinensis were collected from the bile ducts and washed several times with phosphate-buffered saline (PBS) containing 100 μg/ml penicillin and 100 U/ml streptomycin. Freshly isolated adult worms were incubated in sterile PBS for 24 h under an atmosphere of 5% CO2 at 37°C. After incubation, the
Cells were washed in PBS and solubilized using 1% Nonidet P-40 in a buffer containing 150 mM NaCl, 10 mM NaF, 1 mM PMSF, 200 μM Na3VO4, and 50 mM HEPES, pH 7.4. After the insoluble pellets were removed by centrifugation, the lysates were separated by 6%, 8%, 10%, or 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (Immobilon; Millipore, Billerica, MA, USA). The membranes were incubated with primary antibodies for cell-cycle-related proteins. Immune complexes were tagged using goat antirabbit or rabbit antimouse antibodies
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conjugated with HRP and then visualized using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Flow cytometry HuCCT1 cells were plated in six-well cell culture plates at 2×105 cells per well in 2 ml of RPMI1640 containing 10% FBS. After 24 h, the medium was replaced with RPMI1640 containing 0.5% FBS (low-serum medium). The cells were incubated with the ES products from C. sinensis for an additional 24 h, followed by annexin V/7AAD staining for analysis of apoptosis. Annexin-V/7AAD-stained cells were analyzed using a FACSCalibur multicolor flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA), and the data were analyzed using CellQuest software (Becton-Dickinson).
Results and discussion Effect of ES products on cell proliferation C. sinensis infection has been shown to stimulate the proliferation and differentiation of biliary epithelia (Lee et al. 1993, 1994, 1997). To investigate the biological role of ES products from C. sinensis in cholangiocarcinoma cells, we analyzed the proliferation of HuCCT1 cells using the XTT assay. The cells were incubated with ES products in low-serum medium to preclude interactions with serum components. Cells treated with ES products demonstrated a 20–40% increase in cell growth compared to control cells treated with vehicle (PBS; Fig. 1). These results are in agreement with our previous report that showed that ES
products regulate cell cycle progression in 293 and 293T cells via E2F1 (Kim et al. 2008a, b). Expression of cell-cycle-related proteins after treatment with ES products We next asked whether the observed stimulation of cell proliferation could be due to cell cycle progression. We checked several cell-cycle-related proteins and used calnexin as a loading control. After HuCCT1 cells were treated with the indicated concentrations of ES products for 24 h, the cells were subjected to immunoblotting. As shown in Fig. 2, ES products dose-dependently increased the expression of proteins involved in cell cycle progression. In particular, cyclins E and B were remarkably upregulated, indicating that cell cycle progression to the G2/M phase had occurred. In addition, the levels of cyclin-dependent kinases (CDK2 and cdc2) were also increased, while CDK4 expression was slightly increased in the 5-μg ES product treatment group (Fig. 2). This finding further implies that ES products from C. sinensis promote S to G2/M progression. Expression of COX-2 after treatment with ES products The COX pathway has been implicated in hepatic tumorigenesis (Chariyalertsak et al. 2001; Hayashi et al. 2001; Endo et al. 2002; Han et al. 2004). Expression of COX-2 was also shown to be elevated in hepatocellular carcinomas and may correlate with the degree of tumor differentiation (Ralstin et al. 2006). To determine the effect of ES products on COX-2 expression in HuCCT1 cells, protein extracts from cells treated with ES products were subjected to immunoblotting using a COX-2 antibody. As shown in Fig. 3, the expression of COX-2 increased dramatically with increasing doses of ES products. We also investigated the effect of ES products on COX-2 expression in nonmalignant HEK293 and HEK 293T cells. COX-2 expression in these cells was minimal and unchanged by treatment with ES products. This result implies that the effect of ES products from C. sinensis on COX-2 is restricted to malignant cells, at least to HuCCT1 cells in vitro. Restoration of proliferation in parthenolide-treated cells by ES products
Fig. 1 Effect of excretory–secretory products (ESP) on cell proliferation in HuCCT1 cholangiocarcinoma cells. Cells were plated in 96well plates (1.5×104 cells per well). After incubation for 24 h, the medium was replaced with low-serum medium (0.5% FBS-RPMI1640 without phenol red). The cells were incubated in PBS (vehicle) or with various concentrations of ESP in PBS for 24 h. Cell proliferation in each group was determined using the XTT assay. The histograms represent cell proliferation as a percentage of the control±standard deviation (n=4)
Parthenolide has been shown to be an effective anticancer agent. It promotes growth arrest and apoptosis in cholangiocarcinoma and sarcomatous hepatocellular carcinoma cells in a dose-dependent manner (Wen et al. 2002; Kim et al. 2005). Therefore, we studied the effects of ES products in HuCCT1 cells in combination with parthenolide treatment. HuCCT1 cells were incubated with ES products for
Parasitol Res (2009) 104:1011–1016 Pn:
0 µM M
15 µM M
20 µM M
Cell proliferation (%)
150 125 100 75 50 25 0
Concentration of ESP (µg/ml)
Fig. 2 Expression of cell-cycle-related proteins after treatment with excretory–secretory products (ESP). HuCCT1 cells were incubated with either phosphate-buffered saline (vehicle) or various concentrations of ESP for 24 h, and the cells were collected for protein extraction. Approximately 30−50 μg of protein extract from each sample was subjected to immunoblot analysis and probed with the indicated antibodies
24 h and then treated with the indicated concentrations of parthenolide. By the XTT assay, we found that the antiproliferative effect of parthenolide was inhibited by ES products (Fig. 4). As shown in Fig. 4, proliferation of HuCCT1 cells was inhibited by >50% in the parthenolidetreated groups. However, pretreatment of the cells with ES products partially restored the proliferation of HuCCT1 cells. This could have been caused by an increase in COX-2 expression due to ES treatment; COX-2 expression is known to be associated with cancer cell proliferation (Kim et al. 2003; Schmitz et al. 2007). Anticancer drugs have both cytostatic and cytolytic activity. We next investigated whether ES products affect parthenolide-induced apoptosis in HuCCT1 cells; parthenolide is known to induce apoptosis of cholangiocarcinoma cells (Kim et al. 2005). After pretreatment of cells with 5 μg
Fig. 3 Expression of cyclooxygenase (COX)-2 after treatment with excretory–secretory products (ESP). HuCCT1 cells were incubated with either phosphate-buffered saline (vehicle) or various concentrations of ESP for 24 h, and the cells were collected for protein extraction. Protein extract (30 μg) was subjected to immunoblot analysis and probed with the anti-COX-2 antibodies
Fig. 4 Effect of parthenolide on cell proliferation after treatment with excretory–secretory products (ESP). HuCCT1 cells were plated in 96well plates (1.5×104 cells per well). After a 24-h incubation period, the medium was replaced with low-serum medium (0.5% fetal bovine serum–RPMI1640 without phenol red). The cells were incubated in phosphate-buffered saline (vehicle) or with 2 or 5 μg/ml ESP in PBS for 6 h. Cell proliferation in each group was determined using the XTT assay. The histograms represent cell proliferation as a percentage of the control±standard deviation (n=3)
of ES products for 24 h, cells were incubated for 6 h with 15 μM parthenolide and then analyzed by annexin V/7AAD staining. As shown in Fig. 5, cells pretreated with ES products alone showed a 25–35% decrease in annexin V/7AAD staining compared to controls. These data demonstrate that ES products from C. sinensis abolish the anticancer effects of parthenolide in HuCCT1 cells, possibly through COX-2 upregulation. Cholangiocarcinomas are destructive malignant tumors and are known to be caused by chronic infection by liver flukes (Gores 2003; Han et al. 2004). The present report demonstrates that ES products from C. sinensis induce COX-2 expression, increase the proliferation and S to G2/M transition of cholangiocarcinoma cells in vitro, and
Fig. 5 Effect of parthenolide on apoptosis in HuCCT1 cells. Cells were treated with 5 μg/ml excretory–secretory products (ESP) for 24 h followed by 15 μM parthenolide. Apoptotic cells were analyzed by annexin V/7AAD staining assay. The histograms represent cell proliferation as a percentage of the control (CTL)±standard deviation (n=3)
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promote cellular phenotypic changes that counteract the anticancer effects of parthenolide. With knowledge of the human genome and the development of new technology, cancer therapy is progressively moving from a standard “trial-and-error” approach to more personalized treatment regimens (Duffy and Crown 2008). Although more research is required, parasite involvement should be considered in the treatment of cholangiocarcinomas. Therefore, we suggest that parasite-induced cancers such as cholangiocarcinomas may require alternate strategies for effective treatment. Acknowledgments These studies were supported by grants from the Korea Center for Disease Control and Prevention (grant number 80020080583) and the Seoul National University Hospital Research Fund (grant number 04-2008-060-0).
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