Abstract
Background: The therapeutic effects of oral capecitabine are proposed to be determined by the equilibrium of two intratumoral metabolizing enzymes, namely thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD). The present study aims to evaluate this hypothesis by in vivo experiments and immunohistochemical analysis in 31 cases of human breast carcinoma. Materials and Methods: The effects of capecitabine on two breast carcinoma cell lines were evaluated by the status of both cell proliferation and apoptosis and mRNA levels of TP and DPD were also determined. TP and DPD status was determined by immunohistochemistry in 31 cases of breast carcinoma tissues and the results were compared with their clinicopathological parameters. Results: The therapeutic efficacy of capecitabine in two cell lines was not related to the levels of TP and DPD mRNA expression. No statistically significant association was detected between the status of these enzymes and the clinicopathological factors. Conclusion: In vitro study demonstrated that capecitabine was effective against the BT-483 and MB-MDA-231 breast carcinoma cell lines used but the significance of the status of intratumoral TP and DPD in determining its therapeutic efficacy needs further studies.
Breast carcinoma is the most common malignancy of women in the great majority of developed countries and is also a leading cause of female mortality, with over 300,000 deaths annually (1). capecitabine is a relatively novel orally administered fluoropyrimidine carbonate primarily designed for the treatment of breast and colorectal carcinomas. In contrast to parenterally administered 5-fluorouracil (5-FU), oral capecitabine concentrates predominantly in tumor tissue compared to adjacent non-neoplastic tissues and plasma (2). Therefore, orally administered capecitabine enables physicians treating breast carcinoma to mimic the effects of continuous infusion 5-FU but in a convenient outpatient setting (3). Capecitabine has been recently proposed as a potential alternative for 5-FU therapy (4) because of reduced toxicity and ease of administration (5). The monotherapy regimen of capecitabine also demonstrated a favorable tolerability profile, with a notably lower incidence of myelosuppression in the patients treated with this agent as a monotherapy compared to the single use of other chemotherapeutic agents (6). Capecitabine, a TP-activated fluoropyrimidine, was rationally designed to generate 5-FU preferentially in situ or at the tumor site as a prodrug (7). 5-FU is well-known to be either catabolized by DPD or anabolized by TP (8). The former enzyme is responsible for detoxification and subsequent elimination of the agent, while the anabolic pathway forms the compounds responsible for cytotoxic activity toward carcinoma cells (8). TP has been demonstrated to be overexpressed in various human tumors and also to play an important role in angiogenesis, tumor growth, invasion and metastasis (9, 10). DPD is the initial and rate-limiting enzyme in the (fluoro) pyrimidine catabolic pathway. Tumor DPD levels were postulated to predict the clinical response to 5-FU-based therapy (11). Therefore, the purpose of our present study investigates the possible correlation between the levels of TP/DPD and therapeutic efficacy of capecitabine as a single agent in human breast carcinoma cell lines. In addition, the significance of TP/DPD status in human breast carcinoma was also evaluated by studying their correlation with clinicopathological parameters of the patients with invasive ductal carcinoma of the breast.
Materials and Methods
Preparation of chemotherapeutic drugs. The powder form of active capecitabine was kindly donated by Roche Company (Basel, Switzerland). It was dissolved in methanol and diluted with Hank's balanced salt solution (HBSS; Invitrogen, USA) prior to use.
Cell proliferation rate measured by WST-1 for capecitabine in breast carcinoma cells. Two breast cancer cell lines BT-483 and MB-MDA-231 (American Type Culture Collection (ATCC), Manassas, USA) were cultured. BT-483 is an estrogen-responsive cell line, whereas MB-MDA-231 is non-estrogen responsive. The culture medium and conditions were based on the ATCC instructions. The concentration for capecitabine was determined on the basis of the preliminary or pilot studies in order to determine the optimal and minimal inhibitory concentrations (data not presented). Hence, the optimal concentration of pre-activated capecitabine was 1 μg/ml (14). The cells were cultured in a 4-well slide in different groups. Cytotoxicity of capecitabine was assessed using cell proliferation reagent WST-1 (Roche Diagnostics, Mannheim, Germany). Cells were cultured at a density of 4,000 cells per well in 96-well microtitre plates. After the treatment with 1 μg/ml pre-activated capecitabine for 24 hours, WST-1 was applied and cells were incubated according to the instructions of the manufacturer. The optical density (OD) was read at 450 nm by a microplate reader (Sunrise, TECAN, Austria).
In situ TUNEL assay. After treatment with 1 μg/ml pre-activated capecitabine for 24 hours, a designated harvest time-points, cells were then fixed with 4% paraformaldehyde overnight at 4°C prior to the application of Apoptag Peroxidase In Situ Apoptosis Detection Kit (Intergen, NY, USA). The assay was performed according to the manufacturer's instructions. The slides were finally counterstained with hematoxylin. The positive control slides included in the kit were stained as described. Negative controls were evaluated by substituting TdT with PBS in the process of staining. The evaluation of TUNEL assay was performed by counting numbers of positively stained cells from 200 cells in each of 10 different fields. Results were subsequently analyzed by Coolsnap Capture system (Roper Scientific Inc., USA), and Image analyzer (Metamorph-Winshell, Universal Imaging Corporation, USA).
Patient selection and tissue collection for TP and DPD analysis. From 2001 to 2004, a total of 31 surgical pathology specimens of the breast invasive ductal carcinoma were retrieved from surgical pathology or archival files of Tohoku University Hospital and Tohoku Kosai Hospital (Sendai, Japan) (Tables I and II). The Ethics Committees at Tohoku University School of Medicine and Tohoku Kosai Hospital approved the research protocols (#2005-68), with informed consent being obtained from these patients before surgery.
Immunohistochemistry. Mouse anti-human TP monoclonal antibody (1C6-203) and rat anti-human DPD monoclonal antibody (2H9-1b) were selected (Chugai Pharmaceutical Co., Tokyo, Japan). A Histofine Kit (Nichirei, Tokyo, Japan), which employs the streptavidin-biotin amplification method (12), was used for the identification of TP, ER, PR, Ki-67, and HER-2/neu immunoreactive staining (12), whereas DAKO CSA System (DAKO) was used for DPD immunohistochemical analysis (13). Antigen retrieval was performed according to the manufacturers' instructions. The dilutions of the primary antibodies were as follows: 1/1,000 TP; 1/50 ER; 1/30 PR; 1/50 Ki-67 and 1/200 HER-2/neu. Staining was completed by a 5-minute incubation with diaminobenzidine tetrahydrochloride. Finally, slides were counterstained with hematoxylin, and mounted with coverlips.
Scoring of immunoreactivity in TP and DPD analysis. TP and DPD immunoreactivity was detected in the cytoplasm of carcinoma cells. ER, PR, Ki-67 and HER-2/neu immunoreactivity was detected in the nuclei of carcinoma cells. The immunoreactivity was evaluated in more than 1,000 carcinoma cells for each case. Cases associated with a labeling index of less than 10% were tentatively designated negative.
Total RNA extraction and qRT-PCR for evaluation of TP and DPD mRNA expression. BT-483 and MB-MDA-231 cells were used as described in addition to the cases examined for immunohistochemistry. Breast carcinoma specimens had been immediately frozen in liquid nitrogen in the operating theatre and stored at -80°C until RNA isolation. RNA was extracted from 31 cases in which immunohistochemistry of TP and DPD was performed, as well as from the two breast carcinoma cell lines. Total RNA was extracted by homogenizing frozen tissue samples or breast carcinoma cell lines in 1 ml TRIzol reagent (Life Technologies, Inc., USA) followed by a phenol-chloroform phase extraction and isopropanol precipitation. All RNA specimens were quantified by spectrophotometry and processed for reverse-transcription (RT). All the specimens were tested by one-step TP and DPD PCR assays using LightCycler TP mRNA Quantification KitPLUS and LightCycler DPD mRNA Quantification KitPLUS with LightCycler system (Roche Diagnostics). The reference gene used was Ribosomal protein L13a (RPL13A), for the correct normalization of gene expression analysis (15). The amplicon sizes were: TP=108 bp, DPD=148 bp and RPL13A=125 bp according to the manufacturer's instructions. A positive control RNA (calibrator, from the LC-mRNA quantification kits for TP and DPD) was also employed in the assay. The mRNA levels of TP and DPD in each case are normalized as a ratio of calibrator (arbitrary units).
Statistical analysis. Statistical significance of data obtained were analyzed by SPSS 15.0 (Chicago, Illinois, USA).
Results
Cell proliferation rate measured by WST-1 in breast carcinoma cells treated with capecitabine. Results obtained were essentially the same in the two cell lines. The proliferation rates for the capecitabine-treated groups examined were less than those of the controls, and were statistically significantly different (Table III).
In situ TUNEL assay. Under light microscopic evaluation, apoptosis-related morphological changes including cell shrinkage and condensation of cytoplasm and chromatin were detected. Apoptotic bodies, i.e. small cell fragments containing fragmented nuclei, were also detected. The mean percentage of apoptotic cells determined by TUNEL assay in the capecitabine-treated group was 11.18% in MB-MDA-231 cells and 8.46±0.34% in BT-483 cells.
Immunohistochemistry of TP and DPD in human breast carcinoma specimens. TP immunoreactivity was detected in the cytoplasm of carcinoma cells and was detected in 13 out of 31 breast carcinomas examined in this study (Figure 1). TP immunoreactivity was also partially detected in morphologically non-neoplastic ductal epithelial cells (Figure 1). TP immunoreactivity was not detected in other components of the tissue such as intratumoral stromal cells and adipocytes of the specimens (Figure 1). No statistically significant association was detected between the status of TP immunoreactivity and clinicopathological factors examined (Table I).
Association between TP immunoreactivity and clinicopathological parameters in 31 breast carcinoma cases.
DPD immunoreactivity was detected in the cytoplasm of both carcinoma and intratumoral stromal cells (Figure 1). DPD immunoreactivity was detected in 15 out of 31 breast carcinoma cases examined. DPD immunoreactivity was absent from adipocytes and non-neoplastic ductal epithelial cells (Figure 1). No statistically significant association was detected between the status of DPD immunoreactivity in both carcinoma cells and intratumoral stromal cells, and any of the clinicopathological factors examined (Table II). There were no correlations between the status of TP and DPD immunoreactivity in the 31 breast carcinoma specimens examined (Table I).
mRNA expression of TP and DPD in breast carcinoma cell lines. mRNA expression of TP and DPD in BT-483 and MB-MDA-231 was determined by quantitative RT-PCR analysis. The relative level of TP mRNA expression (arbitrary unit, AU) in BT-483 cells (0.405AU) was higher than that in MDA-MB-231 cells (0.0606 AU). The relative level of DPD mRNA in MDA-MB-231 cells (0.158 AU) was higher than that in BT-483 cells (0.0152 AU).
Association between DPD immunoreactivity and clinicopathological parameters in 31 breast carcinomas.
The proliferation of MB-MDA-231 and BT-483 cell lines treated with capecitabine evaluated by WST-1 assay with comparison to control group.
Discussion
Capecitabine is a useful antitumor agent against breast carcinoma cells and human fresh tissues on their inhibition of cell proliferation and metabolic rate, respectively (14, 16). Capecitabine effectively inhibited the cell proliferation of breast tumor cells in vitro as a single agent by the following mechanisms: induction of apoptosis of breast carcinoma cells, as well as an inhibition of cell proliferation, as confirmed by TUNEL analyses. Several previous studies suggested that tumoral TP and DPD status could be considered as a good response factor in the carcinoma patients exposed to fluoropyrimidine drugs (17-19). In particular, TP expression in tumor tissue has been considered to clinically predict efficacy of capecitabine purely based on a theoretical background (10). However, this report is the first in vitro study evaluating the correlation between therapeutic efficacy of capecitabine and the levels of TP/DPD in breast carcinoma cells.
Representative images of immunohistochemistry for TP and DPD in invasive ductal carcinoma of the breast. A, B: Negative cases of TP (A) and DPD (B) in breast carcinoma cases. There were no or low (<10%) TP or DPD immunoreactivities in breast carcinoma and intratumoral stromal cells, respectively. C, D: Positive cases of TP (C) and DPD (D) in breast carcinoma cases. There were high (<10%) immunoreactivities in breast carcinoma cells but not in intratumoral stromal cells. E, F: TP and DPD immunoreactivities in intratumoral stromal cells and in morphologically normal mammary glands. Immunoreactivity for TP (E) was partially detected in the cytoplasm of epithelial cells (†, arrow heads) but not stromal cells (*). Immunoreactivity for DPD (F) was detected in the cytoplasm of stromal cells (*) but not epithelial cells (†). Bar, 10 μm.
The results of our present study demonstrated that there were no correlations between the levels of TP and DPD mRNA levels and therapeutic efficacy of capecitabine in the treatment of BT-483 and MB-MDA-231 cells at all. These results suggest that at least in these two cells lines of human breast carcinoma, the status of the metabolizing enzymes TP and DPD in the carcinoma cells do not influence the therapeutic efficacy of capecitabine. It then becomes important to study the possible correlation between the enzyme levels of the carcinoma tissues and therapeutic efficacy of capecitabine in the specimens of neoadjuvant study. However, in our present study, the direct comparison between the status of these two enzymes and clinical response or efficacy of capecitabine could not be evaluated because the cases in which the specimens were available had not necessarily been administered capecitabine as a neoadjuvant treatment. In previously published studies of immunohistochemistry, TP was reported to be expressed at a significantly higher level in tumor tissue than in normal tissue of human breast (17, 18). Our findings of immunohistochemical study of TP in human breast cancer specimens (Figure 1A and 1B) were consistent with these results. In our present study, no statistically significant associations were detected between the status of TP and/or DPD immunoreactivity in carcinoma cells and intratumoral stromal cells and any of the clinicopathological factors of the patients examined. There was a statistically significant positive correlation between TP mRNA and protein levels in breast carcinoma tissues but none was detected for DPD. Takenoue et al. also reported a poor correlation between DPD mRNA and protein levels (20). The amount of DPD protein level in tumor and normal tissues was indeed very similar. These findings may explain the absence of any correlation between the results of immunohistochemical and RT-PCR studies of DPD because RT-PCR analysis in principle treated the tissues as a mass. Therefore, the presence of possible contamination or inclusion of non-neoplastic compartments in the specimens may have resulted in higher mRNA values of DPD. Oguri et al. also reported the absence of correlation between TP and the effects of 5-FU in non-small cell lung cancer (NSCLC) cell lines, although a significant positive correlation was reported in DPD status (21).
The lack of correlation between TP/DPD status and effects of capecitabine in vitro in cell lines and clinicopathological parameters in the patients also certainly cast reasonable doubts on the clinical significance of the intratumoral status of these two enzymes in breast carcinoma patients as potential surrogate markers of capecitabine treatment.
- Received February 2, 2009.
- Revision received May 8, 2009.
- Accepted May 13, 2009.
- Copyright© 2009 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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