Ó Springer 2006
Clinical & Experimental Metastasis (2005) 22: 523–532 DOI 10.1007/s10585-005-4919-7
Metastatic progression of pancreatic cancer: Changes in antioxidant enzymes and cell growth Anne Lewis5, Juan Du2, Jingru Liu2, Justine M. Ritchie3, Larry W. Oberley2,3,5 & Joseph J. Cullen1,2,3,4,5 1
Departments of Surgery, 2Radiation Oncology, 3The Holden Comprehensive Cancer Center, Medical Center, 5University of Iowa College of Medicine, Iowa City, IA, USA
4
Veterans Affairs
Received 19 July 2005; accepted in revised form 8 November 2005
Key words: antioxidants, hypoxia, metastasis, pancreatic cancer, tumor growth
Abstract Pancreatic cancer has a dismal prognosis due to the fact that patients present late when metastatic disease is already present. Previous studies have demonstrated that pancreatic cancer cells have decreased levels of MnSOD, which correlates well with increased rates of tumor cell proliferation. Recently, we have found that nude mice injected with MIA PaCa-2 human pancreatic cancer cells in the flank occasionally develop ascites and intra-abdominal metastatic deposits. Mice that developed ascites were sacrificed and the ascites cultured. Necropsy demonstrated metastatic tumors in the retroperitoneum, which were excised, digested, and cultured. Western blots, enzyme activity and enzyme activity gels were performed for manganese superoxide dismutase (MnSOD), copper/zinc (CuZnSOD), catalase, and glutathione peroxidase (GPx) in the ascites cell line, metastatic tumor cell line, MIA PaCa-2 primary pancreatic cancer cell line, and the Capan-1, a metastatic pancreatic cancer cell line. Cell growth, plating efficiency, growth in soft agar and growth in nude mice were determined in the ascites, metastatic tumor, and MIA PaCa-2 cell lines. MnSOD, CuZnSOD, and GPx protein and activity were increased in the ascites, metastatic tumor, and Capan-1 cell lines compared to MIA PaCa-2. The ascites and metastatic tumor cell lines had decreased cell growth, plating efficiency, and growth in soft agar, but the ascites cell line had increased cell growth in 4 and 1% O2 concentrations in vitro and more rapid growth in vivo. Metastatic disease is associated with changes in the content and activity of antioxidant enzymes with an associated change in growth characteristics depending on the O2 concentrations. Introduction At initial presentation, approximately 50% of patients diagnosed with pancreatic cancer will have metastatic disease [1]. Survival for patients with metastatic disease is approximately 3–6 months, whereas patients with non-metastatic, locally advanced pancreatic cancer have a median survival of 6–12 months [2]. Metastases after apparent curative resection contribute to the dismal prognosis of pancreatic cancer. Local recurrence occurs most commonly in up to 73% of the patients who develop recurrence after resection [2]. Hepatic metastases and peritoneal deposits are also common metastatic sites occurring in 62% and 42%, respectively [2]. Malignant ascites from pancreatic cancer is a collection of protein-containing fluid and cancer cells within the peritoneal cavity from intraperitoneal seeding. Neovascularization and increased capillary Correspondence to: Joseph J. Cullen, University of Iowa Hospitals and Clinics, 4605 JCP, Iowa City, IA 52242, USA. Tel: +1-319-353-8297; +1-319-339-5497 (Home); Fax: +1-319-356-8378; E-mail:
[email protected]
permeability, permitting macromolecular transit through the endothelial linings of the newly formed capillary endothelium, compounded with the aggressive invasive phenotype of neoplastic cells and increased potential for metastatic spread, contributes to ascites formation. Lymphatic obstruction caused by tumor invasion of the diaphragmatic lymph channels with the increased fluid production result in malignant fluid accumulation in the peritoneal cavity. As with most other solid tumors, primary pancreatic cancers have low levels of antioxidant enzymes [3]. Immunohistochemistry demonstrated that MnSOD, CuZnSOD, catalase, and GPx are decreased in human pancreatic ductal carcinoma specimens when compared to normal human pancreas. In another study by our group [4], we hypothesized that pancreatic cancer cell lines also had altered levels of antioxidant proteins and antioxidant activities compared to normal pancreas. Immunoblotting, enzyme activities, and enzyme activity gels were performed for MnSOD, CuZnSOD, catalase, and GPx in normal human pancreas and in the human pancreatic cancer cell lines BxPC-3, Capan-1, MIA PaCa-2, and AsPC-1. The primary pancreatic cancer cell
524 lines BxPC-3, MIA PaCa-2, and AsPC-1 had decreased levels of MnSOD immunoreactive protein as well as activity. However, the metastatic pancreatic cancer cell line (Capan-1) had increased levels of MnSOD immunoreactive protein and activity relative to the other pancreatic cancer cell lines and normal pancreas. Thus, our initial studies suggested that primary pancreatic cancer and primary pancreatic cancer cell lines have lowered levels of MnSOD compared to normal pancreas, while metastatic pancreatic cancer cell lines had elevated MnSOD levels. Antioxidant content and growth characteristics of tumor cells may change with progression to metastatic disease. Specifically, increased MnSOD levels may correlate with the development and progression of metastatic disease. Several other groups have demonstrated that MnSOD levels may be utilized as independent prognostic parameters to evaluate the clinical outcome of patients with other cancers including esophageal [5], colorectal [6–8], and gastric cancers [5, 7, 9]. Malafa et al. has shown that MnSOD expression is increased in 93% of metastatic compared to 44% of non-metastatic gastric tumors [9]. These studies suggest a direct correlation between increased MnSOD expression and metastatic invasion. Studies delineating the mechanism of MnSOD expression and development of metastatic disease have focused on the generation of peroxides leading to expression of matrix metalloproteinases, major contributors to stromal degradation involved in tumor invasion [10]. Previous studies have demonstrated that overexpression of MnSOD increases the levels of intracellular peroxides in a number of cell lines [11–13] while highly metastatic MCF-7 breast cancer cells and gastric tumors both show high levels of MnSOD compared to their nonmetastatic counterparts [9, 14, 15]. Further studies have suggested that MnSOD-dependent generation of H2O2 leads to increased expression of MMP-1 and may explain the enhanced invasive capacity of tumors with elevated levels of MnSOD [10, 11]. Previous studies from our laboratory have utilized heterotopic implantation of human pancreatic tumor cells in the nude mouse model and measured tumor size with various treatments to document the efficacy of these various treatments on tumor progression. Recently our laboratory has found that nude mice injected with MIA PaCa-2 human pancreatic cancer cells in the flank occasionally develop ascites and intra-abdominal metastatic deposits. The aims of our study were to determine the growth characteristics, antioxidant content, and malignant phenotype of metastatic cells from the intra-abdominal deposits and ascitic fluid. Our findings demonstrate that all the antioxidant proteins were increased while MnSOD and GPx protein and activity were increased to a greater degree in the pancreatic tumor cells from ascites compared to the primary tumor. The pancreatic tumor cells from ascites and metastatic tumors also had decreased cell growth, plating efficiency, and
A. Lewis et al. growth in soft agar, but tumor cells from ascites had increased cell growth in 4% and 1% O2 concentrations in vitro and more rapid growth in vivo.
Materials and methods Previous studies in our lab resulted in the development of ascites in three nude mice injected subcutaneously with MIA PaCa-2 human pancreatic cancer cells [16]. The ascites from these mice were drained and the mice were sacrificed, and metastatic retroperitoneal tumors were removed. Tumors and ascites were cultured as described below. Cell culture MIA PaCa-2 cells were purchased from American Type Culture Collection (Manassas, VA) and are human primary pancreatic adenocarcinoma cells derived from tumor tissue of the pancreas obtained from a 65-year-old male. Tumor and ascites cells were obtained from nude mice as described above and cultured according to the method of Tobacman [17]. The cell cultures were maintained at 37 °C in Dulbecco’s modified Eagle medium (DMEM; Gibco, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum and 2.5% horse serum. Cell growth Although cell lines are routinely cultured in 21% oxygen concentration, tumors grow in a relatively hypoxic environment. Thus, metastatic tumor cells, ascites cells, and MIA PaCa-2 cells (1104) were plated in triplicate in 1.5 ml complete media in 24-well plates and maintained in 1, 4, or 21% oxygen. Cells were trypsinized and then counted on alternate days for 2 weeks using a hemocytometer. Cell population doubling time in hours (DT) was determined using the following equation: DTðhoursÞ ¼ 0:693ðt to Þ= lnðNt =No Þ where to = time at which exponential growth began, t = time in hours, Nt = cell number at time t, and No = initial cell number [16]. Plating efficiency Metastatic tumor cells, ascites cells and MIA PaCa-2 cells were plated in triplicate into 60-mm dishes in complete media. The dishes were maintained in the incubator at 1, 4, or 21% oxygen for 6 days to allow colony formation. The colonies were then fixed and stained with 0.1% crystal violet and 2.1% citric acid, and those colonies containing greater than 50 cells were scored.
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Metastatic pancreatic cancer Anchorage-independent growth in soft agar
SOD activity assay
MIA PaCa-2 pancreatic cancer cells, metastatic tumor cells, and ascites cells (5 103) were suspended in 3 ml of complete medium in a solution of 6% agar in doubledistilled H2O so that the final concentration of the agar was 0.3%. This suspension was then plated over 3 ml of complete medium made using a 6% agar solution in double-distilled H2O so that the final concentration of the bottom agar was 0.5%. After 16 days, colonies of >0.1 mm in diameter were scored. The clonogenic fraction was determined using the following equation:
SOD activity was measured using an indirect competition assay between SOD and an indicator molecule, nitroblue tetrazolium. This was performed in the crude homogenate according to the method of Spitz and Oberley [18]. Sodium cyanide (5 mM) inhibits CuZnSOD; therefore, activity measured in the crude homogenate in the presence of sodium cyanide indicates only MnSOD activity. Specific activity was reported as units per mg protein.
Soft agar plating efficiency (PE) ¼ (colonies formed cells/seeded)100. Cell homogenization and protein determination Cells were washed thrice in PBS (pH 7.0), scraped from the dishes using a rubber policeman and then collected in potassium phosphate buffer (pH 7.8). This was followed by sonic disruption on ice for 30 s in 10-s bursts using a VibraCell sonicator with a cup horn (Sonics and Materials Inc., Danbury, CT) at 100% power. Protein concentration was determined using the Bio-Rad Bradford dye binding protein assay kit (Hercules, CA) according to the manufacturer’s instructions.
Glutathione peroxidase activity Aliquots of sample were assayed according to an established procedure [20] in potassium phosphate buffer, pH 7, containing glutathione, glutathione reductase, NaN3 to inhibit catalase, and NADPH. Hydrogen peroxide was used as the substrate. Catalase activity Catalase activity was measured by the method of Beers and Sizer [21] with the analysis of Aebi [22]. Briefly, this is a spectrophotometric procedure that measures peroxide removal. SOD activity gels
Western blotting Immunoreactive protein corresponding to antioxidant enzymes was identified and quantified from total cell protein by the specific reaction of the immobilized protein with its antibody. Total protein was electrophoresed in a 12.5% SDS-polyacrylamide running gel and a 5% stacking gel. The proteins were then electotransferred to nitrocellulose sheets. After blocking in 20% fetal bovine serum for 1 h, the sheets were washed and then treated with antisera to either MnSOD (1:1000), CuZnSOD (1:500), catalase (1:1000) or GPx (1:250) for 1 h. GPx immunoreactive protein levels were measured using an 8% native protein gel rather than an SDS gel and transferred for 2 h. Polyclonal rabbit-anti-human antibodies to MnSOD, CuZnSOD, and GPx have been prepared and characterized in our laboratory [18]. These have been shown to react with the appropriate protein in a variety of species, including hamster and human [18, 19]. The antibody for catalase was purchased from Athens, Inc. (Athens, GA). The blots were incubated with horseradish peroxidase-conjugated goat-anti-rabbit (Sigma) IgG (1:10,000) for 1 h at room temperature. The washed blot was then treated with ECL Western blot detection solution (Amersham Life Science, Buckinghamshire, United Kingdom) and exposed to X-ray film. All Western blots were performed in triplicate.
To determine if the ascites and metastatic tumor cell lines were derived from the parent MIA PaCa-2 human pancreatic cancer cell line, a SOD activity gel was performed. Mouse SOD has a different electrophoretic mobility compared to human SOD, which can be detected by this technique. Non dissociating slab gels were run essentially by the method of Davis [23] with ammonium persulfate used as the initiator in the running gel (12.0%) and riboflavin-light in the stacking gel (5%). Once run, the gels were stained for SOD activity by the method of Beauchamp and Fridovich [24]. CuZnSOD and MnSOD were differentiated by the presence of sodium cyanide in the staining solution, which inhibits CuZnSOD. Clonogenic assay Previous reports indicate that contrary to metastatic pancreatic cancer cells, MIA PaCa-2 human pancreatic cancer cells are sensitive to various chemotherapeutic drugs such as 2-methoxyestradiol [25, 26]. To determine the effect of 2-methoxyestradiol (5 lM for 24 h), a clonogenic survival assay was performed on metastatic tumor cells, ascites cells, and MIA PaCa-2 cells. After treatment, drug was removed and replaced with media in full serum. Two weeks later, colonies >50 cells were stained and counted. The percent survival was determined using the following equation:
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A. Lewis et al. Clonogenic survival ¼ (colonies formed/cells seeded) 100:
Nude mice Thirty-days-old athymic nude mice were obtained from Harlan Sprague Dawley (Indianapolis, IN). The nude mice protocol was reviewed and approved by the Animal Care and Use Committee of the University of Iowa on July 5, 2001. The animals were housed four to a cage and were fed a sterile commercial stock diet and tap water ad libitum. Animals were allowed to acclimate in the unit for one week before any manipulations were performed. Ascites, metastatic tumor cells, or MIA PaCa-2 cells (2106) were delivered subcutaneously into the flank region of nude mice from a 1-cc tuberculin syringe equipped with a 25-gauge needle. Xenografts of Capan1 metastatic pancreatic cancer cells were also attempted in the in vivo part of this study; however, they failed to form tumors in the nude mice. Tumor size was measured every 7 days by means of a vernier caliper, and tumor volume was estimated according to the following formula: tumor volume = p/6LW2, where L is the greatest dimension of the tumor, and W is the dimension of the tumor in the perpendicular direction [16]. Upon the tumors reaching a predetermined size of 1010 mm, the mice were sacrificed via CO2 asphyxiation.
Results Cell growth To determine cell growth within different oxygen concentrations, MIA PaCa-2, tumor, and ascites cells were incubated in either 1, 4, or 21% oxygen, or the cell counts and doubling times for each cell type were determined. Although cell growth was increased in the MIA PaCa-2 pancreatic cancer cell line at 21% oxygen, it was significantly reduced at 4 and 1% oxygen content when compared with tumor and ascites cells. Cell count was 446 104 cells in the MIA PaCa-2 cells on day 5 at 21% oxygen, compared with 375 104 cells (P
