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Clinical Cancer Research Vol. 9, 738-742, February 2003
© 2003 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

Caspase 3 in Breast Cancer

Norma O’Donovan, John Crown, Helen Stunell, Arnold D. K. Hill, Enda McDermott, Niall O’Higgins and Michael J. Duffy1

Departments of Medical Oncology [N. O’D., J. C.] and Nuclear Medicine [M. J. D.], St. Vincent’s University Hospital, and Department of Surgery [H. S., A. D. K. H., E. M., N. O’H., M. J. D.] and Conway Institute of Biomolecular and Biomedical Science [A. D. K. H., M. J. D.], University College Dublin, Dublin 4, Ireland


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: An inability to undergo apoptosis is widely thought to contribute to both tumorigenesis and tumor progression. One of the key mediators of apoptosis is the thiol protease caspase 3. In this investigation, caspase 3 mRNA and protein expression in breast cancer was examined.

Experimental Design: Caspase 3 was measured at the mRNA level using reverse transcription-PCR and at the protein level using both Western blotting and activity assays. Levels of apoptosis were determined using an ELISA, which detects nucleosomes released during DNA fragmentation.

Results: Relative levels of caspase 3 mRNA were similar in breast carcinomas (n = 103), fibroadenomas (n = 25), and normal breast tissues (n = 6). However, levels of both the precursor and active forms of caspase 3 were significantly higher in carcinomas compared with both fibroadenomas (P = 0.0188) and normal breast tissues (P = 0.0002). Levels of apoptosis were also highest in the carcinomas and correlated significantly with active caspase 3 levels (r = 0.481; P = 0.0003). In the carcinomas, expression of caspase 3 showed no significant relationship with either tumor size, tumor grade, nodal status, or steroid receptor status but was significantly higher in ductal carcinomas than in lobular carcinomas (P = 0.0188).

Conclusions: We conclude that rates of apoptosis as measured by both caspase 3 activation and nucleosome release are higher in breast cancer than in nonmalignant breast tissue. This finding would appear to conflict with the widely held belief that apoptosis is reduced in malignancy. The proliferation:apoptosis ratio, however, may be higher in carcinomas than in the corresponding normal tissue.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Apoptosis or programmed cell death is important in malignancy for two reasons (1 , 2) . Firstly, suppression of apoptosis appears to be a critical event in both cancer initiation and progression. Secondly, most cytotoxic anticancer agents cause tumor regression, at least in part, by inducing apoptosis. Therefore, defects in apoptosis may cause drug resistance and result in treatment failure.

Although multiple genes are involved in apoptosis, the key mediators of the process are the caspases. Caspases are aspartate-specific cysteine proteases, which cleave their substrates on the carboxyl side of the aspartate residue (3 , 4) . Currently at least 14 different caspases are known to exist, of which two-thirds play a role in apoptosis. The caspases involved in apoptosis can be divided into two main groups, the initiator caspases (e.g., caspases 8, 9, and 10) and the downstream effector caspases (e.g., caspases 2, 3, 6, and 7). It is the members of the latter group that degrade multiple cell proteins and are responsible for the morphological changes in apoptosis.

Caspase 3 is the most widely studied of the effector caspases. It plays a key role in both the death receptor pathway, initiated by caspase 8, and the mitochondrial pathway, involving caspase 9. In addition, several studies have shown that caspase 3 activation is required for apoptosis induction in response to chemotherapeutic drugs e.g., taxanes, 5-fluorouracil, and doxorubicin (5, 6, 7, 8) . Caspase 3 is produced as an inactive 32-kDa proenzyme, which is cleaved at an aspartate residue to yield a 12-kDa and a 17-kDa subunit. Two 12-kDa and two 17-kDa subunits combine to form the active caspase 3 enzyme. Caspase 3 cleaves a wide range of cellular substrates including structural proteins (e.g., lamins) and DNA repair enzymes [e.g., poly(ADP-ribose) polymerase]. It also activates an endonuclease caspase-activated DNAse, which causes the DNA fragmentation that is characteristic of apoptosis (3) .

Compared with other genes involved in apoptosis (e.g., p53 and the Bcl-2 family), relatively little work has been carried out on caspase expression in breast cancer. The aim of this study was therefore to investigate caspase 3 expression and its relationship to apoptosis in breast cancer.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Sample Processing.
Primary breast carcinoma, breast fibroadenoma, and normal breast tissue samples were obtained at the time of surgery. Before processing, necrotic tissue was removed. The tissue samples were then snap frozen in liquid nitrogen and stored at -80°C. Tissue samples were homogenized using a Mikro-Dismembrator U (Braun Biotech International, Melsungen, Germany), to yield a fine powder. An aliquot of the powder was extracted with 50 mM Tris buffer (pH 7.4) containing 1 mM monothioglycerol and assayed for ER2 and PR by ELISA (Abbott Diagnostics, North Chicago, IL). The cutoff point for ER and PR was 250 fmol/g wet weight tissue.

RNA Extraction.
RNA extractions were performed using an RNace Total Pure kit (Bioline, Randolph, MA). RNA integrity was assessed by gel electrophoresis, and concentrations were measured spectrophotometrically.

RT-PCR Analysis.
cDNA was synthesized from 1 µg of total RNA, using 50 µM oligo(dT)12–18 primers (Promega, Madison, WI), 0.4 mM deoxynucleotide triphosphates (Promega), 1x Moloney murine leukemia virus buffer, and 100 units of Moloney murine leukemia virus reverse transcriptase (Invitrogen, Carlsbad, CA). PCR was performed using primers for the housekeeping gene GAPDH and caspase 3 (Maxim Biotech, San Francisco, CA). Briefly, the 25-µl reaction mix contained 5 µl of cDNA, 250 µM deoxynucleotide triphosphates, 1x multiple polymerase chain reaction primers, 1x multiple polymerase chain reaction buffer, and 1.25 units of Taq polymerase (Promega). Amplification was performed using 2 cycles of 1 min at 95°C and 4 min at 56°C followed by 28 cycles of 1 min at 94°C and 2.5 min at 56°C, with a final extension of 10 min at 72°C. PCR products were visualized on a 2% agarose gel. Band intensities were measured by densitometry using the Eagle Eye gel documentation system (Stratagene, La Jolla, CA) and expressed as arbitrary units, relative to GAPDH.

Caspase 3 Western Blotting.
Cytosolic protein extracts were prepared by extraction in Tris (pH 7.4) containing 0.1% Triton X-100. Protein concentrations were determined using the BCA protein assay (Pierce, Milwaukee, WI). Fifty µg of protein from each sample were separated on a 12% polyacrylamide gel. After electrophoresis, protein was transferred to a nitrocellulose membrane that was blocked for 1 h at room temperature in 1x Tris-buffered saline containing 5% skimmed milk powder, 1% BSA, and 0.05% Triton X-100. The membrane was then incubated overnight at 4°C in the above blocking solution containing rabbit anti-caspase 3 polyclonal antibody (1:1000; BD Transduction Laboratories, San Diego, CA). Washed membranes were then probed with horseradish peroxidase-conjugated goat antirabbit secondary antibody, and detection was performed using the chemiluminescent substrate Luminol (Santa Cruz Biotechnology, Santa Cruz, CA). Recombinant caspase 3 was used as a positive control, and MCF7 cells, which do not express caspase 3, were used as a negative control. Membranes were reprobed with a mouse anti-ß-actin antibody (Sigma, St. Louis, MO) to control for equal loading of protein. Band intensities were measured by densitometry.

Caspase Activity Assays.
Cytosolic protein extract (100 µg) was incubated with 50 µM Ac-DEVD-AFC (Alexis Corp., San Diego, CA) in caspase activity assay buffer [20 mM PIPES, 100 mM NaCl, 10 mM DTT, 1 mM EDTA, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, and 10% sucrose (pH 7.2)] at 37°C for 100 min. Fluorescence readings were measured on a Fluoroskan Ascent FL (Thermo Labsystems, Helsinki, Finland) using an excitation wavelength of 390 nm and an emission wavelength of 510 nm. A standard curve was constructed using AFC, and caspase 3-like activity values were expressed as nmol product formed (AFC)/mg protein/min.

Apoptosis ELISA.
Levels of apoptosis were measured in tumor samples using the Cell Death Detection ELISA (Roche, Mannheim, Germany), which detects nucleosome fragments released into the cytoplasm during apoptosis (9 , 10) . In brief, 30 µg of cytosolic extracts, in duplicate, were incubated in streptavidin-coated microtiter plates with the biotin-conjugated anti-histone antibody and the peroxidase-conjugated anti-DNA antibody for 2 h at room temperature, followed by washing and colorimetric detection using ABTS substrate. Absorbances were measured at 405 nm (reference wavelength, 490 nm). A standard curve was constructed using the nucleosome solution supplied with the kit. Apoptosis values were expressed as concentration of nucleosomes (µg/mg total protein).

Statistical Analysis.
Nonparametric Spearman rank correlations, Mann-Whitney t tests, and {chi}2 tests were performed using StatView 5.0.1. (SAS Institute Inc., Cary, NC). P < 0.05 was regarded as statistically significant.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Caspase 3 mRNA Analysis.
Table 1Citation shows the results of semiquantitative RT-PCR analysis of caspase 3 mRNA expression in normal breast tissues, fibroadenomas, and primary breast carcinomas. No significant difference in either the frequency or the level of caspase 3 expression was found in the three different tissue types. Caspase 3 mRNA expression in primary breast carcinomas did not correlate with tumor size, nodal status, histological type, or ER status. However, caspase 3 mRNA was detected more frequently and at significantly higher levels in PR-negative tumors than in PR-positive tumors (Mann Whitney test, P = 0.0108).


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  		Table 1 Levels and frequency of caspase 3 mRNA expression in normal breast tissue, fibroadenomas, and primary breast carcinomas, as determined by semiquantitative RT-PCR

 
Caspase 3 Protein Analysis.
Caspase 3 protein expression was measured in breast tissues by Western blotting with an antibody that reacts with both the inactive procaspase 3 (32 kDa) and the large subunit (17-kDa) of active caspase 3 (Fig. 1)Citation . Procaspase 3 was detected in all of the samples tested (Table 2)Citation . However, the levels of procaspase 3 were significantly higher in primary carcinomas than in either fibroadenomas (Mann Whitney test, P = 0.0188) or normal breast tissues (Mann Whitney test, P = 0.0002). Levels of active caspase 3 (17-kDa subunit) also tended to be higher in the primary carcinomas (n = 83; mean = 0.016 absorbance units) than in either fibroadenomas (n = 23; mean = 0.004 absorbance units) or normal breast tissues (n = 11; mean = 0.005 absorbance units), although these differences did not reach statistical significance (Table 2)Citation . In addition, the 17-kDa active subunit of caspase 3 constituted a larger proportion of the total caspase 3 protein in the carcinoma samples (1.85%) and the fibroadenomas (2.32%) than in the normal breast tissues (0.3%).



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  		Fig. 1. Western blot analysis to measure expression of procaspase 3 (32 kDa) and the large subunit of active caspase 3 (17 kDa). Measurement of ß-actin was used to control for equal loading of protein samples.

 

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  		Table 2 Levels and frequency of caspase 3 protein expression in normal breast tissue, fibroadenomas, and primary carcinomas, as determined by Western blotting

 
No significant relationship was observed between caspase 3 protein levels and either tumor size, nodal status, grade, or steroid receptor status (Table 3)Citation . A significant correlation was observed between caspase 3 protein and histological tumor type. Although the numbers of lobular tumors tested were low (n = 6), levels of procaspase 3 were significantly higher in the ductal tumors (n = 67) than in the lobular group (Mann Whitney test, P = 0.0209; Fig. 2Citation ). Furthermore, active caspase 3 was not detected in any of the lobular tumors but was found in 38 of 67 (56.7%) ductal tumors. Caspase 3 protein levels showed no significant relationship with the relative levels of caspase 3 mRNA.


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  		Table 3 Relationship between caspase 3 protein expression and current prognostic indicators for breast cancer

 


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  		Fig. 2. Levels of procaspase 3 protein in ductal and lobular tumors, measured by Western blotting. Boxes represent the 75th percentile with the median indicated, and the bars represent the 90th percentile.

 
Caspase 3 Activity Assays.
Caspase 3-like activity was measured using the fluorometric substrate Ac-DEVD-AFC. Caspase 3 activity was not detected in any of the normal breast tissues (n = 8) or fibroadenomas (n = 20) tested. In contrast, of the 49 primary carcinomas tested, 11 were positive (22.4%) for caspase 3 activity (mean, 0.008; range, 0–0.097). Levels of caspase 3 activity in the primary carcinomas (n = 49) correlated with the levels of both procaspase 3 and active caspase 3 measured by Western blotting [Spearman rank correlations: P = 0.0162 (r = 0.347) and P = 0.0169 (r = 0.345), respectively].

Apoptosis Assays.
Apoptosis levels were measured using the Cell Death Detection ELISA (Roche), which measures nucleosomes released into the cytoplasm when DNA fragmentation occurs during apoptosis. Levels of apoptosis were higher in the primary carcinoma samples (mean = 3.1 µg nucleosomes/mg protein; n = 58) and fibroadenomas (mean = 3.2 µg nucleosomes/mg protein; n = 21) than in the normal breast tissues (mean = 0.7 µg nucleosomes/mg protein; n = 10). No significant correlation was found between either caspase 3 mRNA or procaspase 3 protein and levels of apoptosis. In contrast, active caspase 3, whether measured by Western blotting (r = 0.481; P = 0.0003; n = 63) or an activity assay (r = 0.352; P = 0.0279; n = 40), correlated significantly with apoptosis rates (Fig. 3)Citation . No relationship was observed between the levels of apoptosis and tumor size, nodes, histology, or steroid receptor status.



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  		Fig. 3. Levels of the 17-kDa subunit of active caspase 3 in apoptosis-negative and apoptosis-positive primary carcinomas. Boxes represent the 75th percentile with the median indicated, and the bars represent the 90th percentile. Median levels of active caspase 3 in apoptosis-negative samples were zero.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
A failure to undergo apoptosis is widely believed to be a key event in cancer formation and progression (1 , 2) . Using two indices of apoptosis, i.e., caspase 3 protein levels and nucleosome concentration, we showed that breast carcinomas exhibited higher rates of apoptosis than either fibroadenomas or normal breast tissue. Other investigators have also reported increased rates of apoptosis in breast carcinomas compared with control nonmalignant breast tissue. Using the terminal deoxynucleotidyl transferase-mediated nick end labeling method to measure apoptotic rates, Wong et al. (11) found that the mean apoptotic index in cancer was 4.3%, whereas the corresponding value for the adjacent normal ductal cells was 0.04%. Also using the terminal deoxynucleotidyl transferase-mediated nick end labeling assay, Vakkala et al. (12) reported that the mean apoptotic index was 0.14 in benign epithelial hyperplasias, 0.17 in atypical hyperplasias, 0.61 in in situ carcinomas, and 0.94 in invasive carcinomas.

Whereas apoptosis rates appear higher in invasive breast cancers than in corresponding nonmalignant breast tissue, proliferation rates are also generally increased in the malignant tissues (13) . Indeed, Wong et al. (11) found a significant correlation between the apoptotic index and proliferation rates in invasive breast carcinomas. Furthermore, Mommers et al. (14) showed that whereas both mitotic and apoptotic indices were higher in invasive breast cancer compared with either hyperplasias or well-differentiated in situ cancers, the mitotic/apoptotic index was increased in the invasive carcinomas. Recently, Zhao et al. (15) also showed that both the mitotic index and the apoptotic index are increased in ductal carcinoma in situ. Parton et al. (16) also found a significant correlation between apoptosis and proliferation in breast tumors before treatment. These studies, when taken together, suggest that both apoptosis and proliferation are increased in breast cancer.

Although higher levels of caspase 3 protein were found in malignant versus nonmalignant breast tissue in the present investigation, no significant relationship was found between caspase 3 protein levels and either tumor size, nodal status, or steroid receptor status. Using immunohistochemistry, Vakkala et al. (12) found no significant association between caspase 3 and either tumor grade, ER status, or PR status. However, Mommers et al. (14) reported that the mean apoptotic index was approximately 3-fold higher in poorly differentiated breast cancers than in well-differentiated breast cancers. In this study, higher levels of caspase 3 protein were found in ductal cancers than in lobular breast cancers. Previously, lobular cancers were shown to have a lower apoptotic index than ductal tumors (17) . These findings suggest that the mechanisms of apoptosis may be different in ductal and lobular breast carcinomas.

Our results show that measurement of active caspase 3, whether by Western blotting or by a catalytic activity assay, correlates with apoptosis rates as detected by an ELISA for nucleosome fragments. In contrast, neither procaspase 3 nor caspase 3 mRNA levels correlated with nucleosome fragment levels. These results suggest that whereas measurements of caspase 3 at either the mRNA or precursor protein level may indicate a potential for apoptosis, measurement of the active protein is necessary to detect ongoing apoptosis.

Because caspase 3 is a critical mediator of apoptosis (18) and correlates with apoptotic rates in breast cancer, it is a potential marker for predicting response or resistance to chemotherapeutic agents in breast cancer. Indeed, recent data from breast cancer cell lines support this hypothesis. For example, Blanc et al. (19) showed that caspase 3 was essential for procaspase 9 processing and cisplatin-induced apoptosis in MCF7 breast cancer cells. Using the same cell lines, Yang et al. (20) reported that transfection with cDNA for caspase 3 led to doxorubicin- and etoposide-induced apoptosis. Future work should therefore investigate whether caspase 3 levels in breast carcinomas can predict clinical response/resistance to these agents.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed. Phone: 353-1-2094607; Fax: 353-1-2696018; E-mail: Michael.J.Duffy{at}ucd.ie Back

2 The abbreviations used are: ER, estrogen receptor; PR, progesterone receptor; RT-PCR, reverse transcription-PCR; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; AFC, aminotrifluoromethyl-coumarin. Back

Received 3/23/02; revised 9/10/02; accepted 10/ 9/02.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
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