Introduction

Breast cancer is a major health problem, being the second deadliest form of cancer in women, behind lung cancer. Great strides have been made in the diagnosis, research, and treatment of breast cancer. However, breast cancer cells that are resistant to radiation, anti-hormonal therapy, and chemotherapy are also resistant to apoptosis. Treatments with antiestrogens are hampered by the development of hormone-refractory disease or hormone resistance (Glinsky et al. 1997; Teixeira et al. 1995).

Apoptosis is a form of cell suicide that is accompanied by characteristic biochemical and morphological changes. It involves DNA fragmentation, cytoplasmic shrinkage, the formation of membrane-bound structures that contain pieces of organelles and cytosol, called apoptotic bodies (Kerr et al. 1972), and activation of lysosomal enzymes (Halaby et al. 1994). Apoptosis plays an important role during development, tissue homeostasis, and growth regulation of differentiated tissues and is now recognized as an important target for the development of novel therapies against diseases such as cancer and AIDS.

Lysosomes contain at least 40 hydrolytic enzymes that are active at acidic pH. These enzymes are implicated in the cell death that occurs during the physiological involution of the uterus (Woessner 1965), the prostate gland (Helminen et al. 1972), and the mammary gland (Helminen and Ericsson, 1968). Lysosomal hydrolases also initiate apoptosis in diverse model systems (Tata 1994; Roberts et al. 1997; Li et al. 1998; Shibata et al. 1998). Finally, lysosomal hydrolases induce apoptosis of breast tumors in mammary carcinomas in rodents (Anton and Brandes 1968; Ball et al. 1982; Beem et al. 1987). In particular, acid phosphatase is the marker enzyme for lysosomes (Pelletier and Novikoff 1972) and has been used to monitor cell death and cell lysis (Heryanto et al. 1977; Beem et al. 1987; Halaby et al. 1994; Halaby et al. 1997).

MCF-7 cells provide an excellent in vitro model system to study human breast cancer. This cell line was established from the pleural effusion of a patient who was diagnosed with adenocarcinoma of the breast (Soule et al. 1973). In addition, the MCF-7 line retains several characteristics of differentiated mammary epithelium including the ability to respond to estrogen via cytoplasmic estrogen receptors (Brandes and Hermonat 1983). Elevated plasma estrogen concentrations are implicated in inducing breast cancer (Gustafsson and Warner 2000; Munster et al. 2001).

We have previously shown that the activity and localization of acid phosphatase can be used to monitor apoptosis (Halaby et al. 1994; Halaby 2000) and that acid phosphatase histochemical staining is marked in biopsies of non-invasive breast cancer, such as ductal carcinoma in situ (Halaby et al. 2001). Furthermore, we have shown that an increase in acid phosphatase activity is amongst the earliest biochemical indicators of apoptosis in two diverse model systems: the labial gland of Manduca Sexta, rat mammary gland, and breast carcinomas (Halaby et al. 1994; Halaby 2001; Halaby et al. 2001). In this report we demonstrate that MCF-7 cells display morphological alterations that are indicative of apoptotic cells when exposed to tumor necrosis factor-a (TNF-a). TNF-a is a cytokine with a wide range of biological functions, including induction of apoptosis in human tumor cells (Sugarman et al. 1985; Fiers 1991). The lysosomal compartment expanded in TNF-a-treated cells, and the activity of acid phosphatase was significantly elevated in experimental cells. Moreover, we show that the execution of the cell death program by TNF-a in MCF-7 cells involves impairment of mitochondrial respiration. Our results indicate that further research is warranted to determine if the deliberate activation of lysosomal enzymes or compromising mitochondrial activity may lead to the death of breast cancer cells.

Methods and Materials

Cell Culture

The human breast cancer cell line MCF-7 was purchased from American Type Culture Collection (Manassas, VA). The cells were routinely maintained in a-Minimally Essential Media (aMEM; BioWhittaker, Walkersville, MD) supplemented with 10 % fetal bovine serum (Gibco, Grand Island, NY), 2 mM glutamine, penicillin (100 Units/mL), and streptomycin (100 mg/mL) in a humidified chamber at 37°C in 5% CO₂/95% O₂. Cells were grown in T-25 flasks (Gibco) to yield 5 X 10⁶ cells/mL. Cells were trypsinized, washed with phosphate buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄×7H₂O, 1.4 mM KH₂PO₄, pH 7.4), resuspended in aMEM, and pooled.


Determination of Cell Viability

Cell viability was assessed using a mitochondrial function assay, the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction assay (Sigma, St. Louis, MO). Stock solutions of MTT (5 mg/mL) were prepared in aMEM. Dissolved MTT is converted to an insoluble purple formazan by cleavage of the tetrazolium ring by dehydrogenase enzymes of living cells (Mosmann 1983). MCF-7 cells (5 X 10⁶ cells/mL) were seeded in 6-well plates containing 2 mL/well of aMEM and treated in the absence or presence of TNF-a (1 ng/mL, 10 ng/mL, or 25 ng/mL) for 24 h at 37°C. After this incubation period, MTT (0.2 mL) was added to each well and the cells were incubated for 4 h at 37°C. The medium was then removed and the converted dye was solubilized with the addition of acidic isopropanol (0.1 N HCl in absolute isopropanol). Absorbance, which was proportional to cell viability, was measured at 570 nm with background subtraction at 660 nm.


Lysosomal Assay

To localize lysosomes in MCF-7 cells, a histochemical assay (Sigma kit 181-A) for acid phosphatase was used. The p-nitrophenol phosphate (substrate) stock solution was prepared by dissolving a 100 mg capsule in 25 ml dH2O. The stock solution was dispensed in 0.5 ml aliquots and stored at -20°C. The cells (5 X 10⁶ cells/mL) were grown on tissue culture treated microscope glass cover slides (Fisher Scientific, Pittsburgh, PA), which were submerged in 6-well plates that contained 2 mL/well aMEM. Cells were allowed to attach to the slides overnight and were treated with or without TNF-a (1 ng/mL) for 24 h at 37°C. Prior to fixation, 0.6 mL sodium nitrite solution were added to 0.6 mL fast garnet GBC solution, mixed by inversion; allowed to stand 2-4 min; and added to 23 mL dH₂O. Three mL acetate solution and 3 mL naphthol AS-BI phosphoric acid solution were added to the previous solution and mixed well. Slides were fixed in a citrate-acetone-formaldehyde solution at room temperature, in Coplin jars for 30 sec, and rinsed with dH₂O for 1 min. Slides were then incubated for 1 h at 37°C in naphthol AS-BI phosphate and fast garnet stain in acetate buffer. Slides were rinsed with tap H₂O for 2 min, dried for 15 min in a fume hood, counterstained with methylene blue for 1 min, rinsed with dH₂O, and mounted in CrystalMount (Biomeda Corp., Foster City, CA). The presence of acid phosphatase was indicated by distinct focal precipitates, which were resolved by light microscopy.


Statistical Analysis

A student's t-test was used for the determination of statistical significance. Absorbance values for TNF-a-treated and control cells were considered statistically different at p < 0.05.

Results

Mitochondrial Function

Mitochondrial dehydrogenases from living cells are able to convert soluble MTT to an insoluble formazan via a reduction reaction. We show by MTT assay (mitochondrial activity) that TNF-a is cytotoxic to MCF-7 cells. The optical density of the converted dye is measured spectrophotometrically. The cell viability of control cells was significantly higher than that of TNF-a-treated cells (Fig. 1). TNF-a killed MCF-7 cells in a dose-dependent fashion. Statistically significant differences were observed between cell viability in experimental cells relative to control cells at all three concentrations of TNF-a (Fig. 1). In increasing concentration order, the values were p < 0.02, p < 0.003, and p < 0.03. This indicates that mitochondrial respiration can be used to quantitate apoptosis in MCF-7 cells that are exposed to TNF-a. The control cells stained more intensely with MTT, indicating that the majority of mitochondria possessed functional enzymes, while the experimental cells did not (data not shown).

Discussion