Paclitaxel Induces Programmed Cell Death in MDAMB-468 Human Breast Cancer Cells1

The ability of paditaxel, one of the most active chemotherapeutic agents against breast cancer, to induce programmed cell death in hormone-independent MDA-MB-468 human breast cancer cells was assessed. Treatment of MDAMB-468 cells led to growth inhibition, high-molecular-weight and oligonucleosomal DNA fragmentation, and apoptosis-associated morphological changes after either 3or 24-h exposure to paditaxel concentrations 1O ni i. Additionally, cleavage products of poly(ADP.ribose) polymerase and lamin B1, two proteins that are cleaved early in the execution phase of programmed cell death, were detected. Quantitative studies mdicated that exposure to paditaxel for 24 h resulted in more DNA fragmentation than did 3-h exposure. Rapid induction of the early-response gene c-jun but not c-myc was associated with paditaxel treatment. The ability of paditaxel to induce highmolecular-weight DNA fragmentation and apoptosis-associated morphological changes in three other breast cancer cell lines was also established. These data suggest that paditaxel, an agent known to stabilize microtubules and prevent cell division but not to act directly on DNA, induces programmed cell death in breast cancer cells. INTRODUCTION Breast cancer is the most common cancer in American women. New approaches to systemic therapy are needed, as age-adjusted mortality has not changed greatly for 50 years (1). Exploitation of pathways of programmed cell death (2-4), a mechanism of cellular suicide, is one potential approach. We have previously used human breast cancer cells to show that hormone-dependent MCF-7 cells growing in vivo undergo programmed cell death upon estrogen withdrawal (5). Furthermore, even hormone-independent breast cancer cells retain the ability to undergo programmed cell death in response to diverse agents, including fluoropyrimidines (6). Received 8/3 1/95; revised 1/25/96; accepted 1/31/96. I This work was supported by NIH Grants 5T32CA0907 1 , RO1 CA57545, UO1CA66084, and CA69008 and the Susan 0. Komen Foundation. S. H. K. is a Leukemia Society of America Scholar. 2 To whom requests for reprints should be addressed, at the Johns Hopkins Oncology Center, Johns Hopkins University School of Medicine, 422 North Bond Street, Baltimore, MD 21231. Phone: (410) 955-8489; Fax: (410) 955-0840. The critical event(s) necessary for activation of programmed cell death pathways have not yet been characterized, and specific targeting of a chemotherapeutic agent to these pathways is not yet possible. Increased understanding of the mechanisms by which existing chemotherapeutic agents act may lead to the development of new drugs or more effective use of the currently available ones. Paclitaxel (Taxo!; Bristol-Myers/Squibb, Wallingford, CT) is one of the most active new agents currently used for the clinical treatment of breast cancer (7-10). It is known for its unique ability to stabilize microtubules, thus preventing chromosome segregation and subsequent cellular division. The goal of these studies was to determine whether paclitaxel, an agent that does not cause direct DNA damage, induces programmed cell death in human breast cancer cells. MATERIALS AND METHODS Cell Line and Culture Conditions. MDA-MB-468 cells were purchased from American Type Culture Collection (Rockville, MD) and maintained in improved minimal essential medium supplemented with 5% fetal bovine serum (Biofluids, Rockville, MD) and 2 mtvt glutamine. MCF-7, MDA-MB-23 1, and Hs578t cells (obtained from Dr. Marc Lippman, Vincent T. Lombardi Cancer Center, Washington, DC) were maintained in DMEM supplemented with 5% fetal bovine serum and 2 mr i glutamine. Cultures were incubated at 37#{176}C in a humidified 5% CO2 atmosphere and passaged every S days. Mycoplasma testing was routinely negative. Paclitaxel. Paclitaxel was a gift from Bristol-Myers/ Squibb. For all experiments, a concentrated paclitaxel solution (10 mM in DMSO, stored at 4#{176}) was diluted in medium to the desired concentration. In each experiment, control cells were exposed to a DMSO concentration equivalent to the highest DMSO concentration present in the paclitaxel-treated cells. Growth Inhibition Assay. Exponentially growing cells were plated in triplicate at 3-5 X l0 cells/cm2 in 24-well plates. After attachment, medium was changed, and cells were incubated in the presence or absence of at least eight paclitaxel concentrations. If drug exposure time was less than 120 h, cells were washed three times with medium ( 30-fold drug dilution with each wash) to ensure that the residual extracellular drug concentration was below that which would have effects over the total time of the experiment. After 120 h, the cells were detached by trypsinization and counted using a Coulter counter. Initial experiments demonstrated that cell numbers determined using a Coulter counter were equivalent to numbers of trypan blue-excluding cells (viable cells) determined by cell counting using a hemocytometer. IC50 values were determined from plots of the percentage of the control cell number versus the logarithm of the drug concentration. All experiments were carried out at Research. on April 20, 2017. © 1996 American Association for Cancer clincancerres.aacrjournals.org Downloaded from 848 Paclitaxel-induced Cell Death 3 The abbreviations used are: PARP, poly(ADP-ribose) polymerase; EGF, epidermal growth factor. least twice, and values reported are means ± SDs of individual determinations from all experiments. Assessment of Morphology. Exponentially growing MDA-MB-468 cells were incubated in the presence or absence of 100 mvt pacitaxel for 3 or 24 h. Cells were then washed and incubated in drug-free medium for the additional periods of time before fixation in methanol. Fixed cells were stained with 0.1 mg/mi Hoechst dye no. 33342 (Sigma Chemical Co., St. Louis, MO) and visualized by fluorescence microscopy using a Zeiss Axioskop microscope (Zeiss, Hanover, MD) with filter set 48179/