1383-1391.qxd 29/3/2010 11:48 Ì ™ÂÏ›‰·1383 ONCOLOGY REPORTS 23: 1383-1391, 2010 Profiling of cell cycle genes of breast cells exposed to etodolac D. ROY1,2*, G.A. ARASON2*, B. CHOWDHURY3,4, A. MITRA5 and G.M. CALAF6,7 1Department of Natural Sciences, Hostos College of the City University of New York, Bronx, NY; 2Graduate Biotechnology Program, Manhattan College, Riverdale, NY; 3Human Retrovirus Section, Vaccine Branch, National Cancer Institute, National Institutes of Health, Frederick, MD; 4Department of Cellular Injury, Military Casualty Research, Walter Reed Army Institute of Research, Silver Spring, MD; 5Department of Chemistry, The College of Mount Saint Vincent, Riverdale, NY; 6Center for Radiological Research, Columbia University Medical Center, New York, NY, USA; 7Instituto de Alta Investigación, Universidad de Tarapaca, Arica, Chile Received September 29, 2009; Accepted November 6, 2009 DOI: 10.3892/or_00000775 Abstract. Breast cancer represents the second leading cause Introduction of cancer-related deaths in the world. There is increasing evidence that perturbation of cell cycle regulation is an Breast cancer represents the second leading cause of cancer- important contributing factor to various cancer progression related deaths in the United States and other Western countries; stages. There are key checkpoints in the cell cycle involving accounting for about 30-40% of all newly diagnosed cancers various regulatory proteins. The relationship between these (1,2). Hereditary breast cancer accounts for just 5-10% of cell cycle regulatory proteins and cell cycle arrest by cyclo- cases. It is generally believed that a family history of breast oxygenase (COX) inhibitors during neoplastic progression cancer and hormonal imbalances also contributes to the remains largely unknown. Preclinical studies and epidemio- development of breast cancer (3,4). The etiology of breast logical investigations have consistently shown that non- cancer remains unknown, however, a profound and diverse steroidal anti-inflammatory drugs have some anti-proliferative number of factors including developmental, genetic, nutrition, and anti-oxidative stress response on various tumors. In this chemotherapy, hormones, the environment and other yet study, the effect of etodolac, a 1,8-diethyl-1,3,4,9-tetrahydro- undetermined factors have been implicated as risk factors for pyrano (3,4-ß) indole-1-acetic acid on signaling pathways mammary tumorigenesis (5-8). was investigated by examining the differential expression of The malignant breast cell phenotype develops as a result various cell cycle regulatory protein genes. A human cell of a multi-step process, requiring multiple genetic mutations cycle gene array was used to profile the expression of 96 (9-12). These mutations contribute to a cell that is increas- genes involved in the cell cycle regulation. Differentially ingly characterized by uncontrolled proliferation, deregulated expressed genes were highly altered by etodolac treatment. production of growth factors, non-responsiveness to extra- Twenty-six genes were up- and 20 down-regulated with 0.5 cellular anti-proliferative signals, anchorage independence, and 2 mM etodolac treatment, respectively. Seven genes metastatic potential and resistance to antineoplastic agents. (ATM, BAX, CCNA2, CDC27, RAD50 and p21) were Recent advances in the molecular biology of breast cancer prominently altered, and six (ATM, CCND2, CCNF, have identified various genes associated with tumorigenesis CDC20, CDK1A and RAD50) were commonly altered with (13,14). The development and progression of neoplastic trans- both concentrations. This finding indicated that etodolac formation and the experimental reversal of tumorigenicity could play a critical role on cancer cells by inducing cell are accompanied by complex changes in patterns of gene death. expression. Complicated events are required for normal cells to change their behavior and these events involve the inter- action between genes and possible environmental factors _________________________________________ such as repeated exposure to ultraviolet light, irradiation, chemical pollutants, diet, chemotherapy, tobacco, alcohol Correspondence to: Dr Debasish Roy, Department of Natural among others. These factors seem to modulate the trans- Sciences, The City University of New York, Hostos College formation of genes associated with the cell cycle pathway Campus, no. A-507E, 475 Grand Concourse, Bronx, NY 10451, (12-15). Another potential mechanism through which genetic USA alterations may occur requires that the cellular DNA E-mail: [email protected] replication process becomes error prone. An increase in the error frequency associated with the DNA synthetic *Contributed equally machinery responsible for elongating the DNA could lead to an accumulation of mutations (5). Key words: cell cycle, breast cells, etodolac There is increasing evidence that perturbation of cell cycle regulation is an important contributing factor to various cancer progression stages. There are two key checkpoints in 1383-1391.qxd 29/3/2010 11:48 Ì ™ÂÏ›‰·1384 1384 ROY et al: CELL CYCLE GENES OF BREAST CELLS the cell cycle, the G1-S and G2-M involving various regulatory proteins (16,17). At present, the relationship between these cell cycle regulatory proteins and cell cycle arrest by cyclo- oxygenase (COX) inhibitors during neoplastic progression remains largely unknown. COX, also known as PGHS (prostaglandin G/H synthase), is a key enzyme in catalyzing the conversion of AA (arachidonic acid) to PGs (prostaglandin) and other eicosanoids. Two isoforms of COX, COX-1 and COX-2 have been identified in different tissues. COX-1 is responsible for generating PGs for normal physiological function, whereas COX-2 is an early gene, which is rapidly Figure 1. The chemical structure of etodolac: (1,8-diethyl-1,3,4,9- induced by a variety of agents, including lipopolysaccharides, tetrahydropyrano (3,4-ß) indole-1-acetic acid). cytokines, growth factors and tumor promoters. Many studies have consistently showed that mRNA and protein levels of COX-2, but not COX-1, are markedly elevated during various (Collaborative Research, Bedford, MA) (31). MCF-10F cell neoplastic progressions (18). line was tested from time to time to ensure that it was free Preclinical studies and early epidemiological investi- from any type of mycoplasma contamination. gations have consistently shown that non-steroidal anti- inflammatory drugs (NSAIDs) have some anti-proliferative Drug treatment. The MCF-10F cell line was treated with and anti-oxidative stress response on various tumors, including non-steroidal anti-inflammatory drug (NSAID) 1,8-diethyl- breast, colon and many other types of malignancies (19,20). 1,3,4,9-tetrahydro-pyrano (3,4-ß) indole-1-acetic acid They act either by blocking cyclooxygenase (Cox) enzyme (etodolac) (Sigma Chemical Co.). Etodolac (143.7 mg) activity, thus inhibiting the conversion of arachidonic acid to (Sigma Chemical Co.) was dissolved in 5 ml of 0.01% DMSO prostaglandins or by induction of apoptosis and/or cell-cycle solution. This stock solution (1.5 ml) was exposed to 15 ml arrest (21,22). Among the various NSAIDs, therefore, these of culture media to get 10 mM concentration of etodolac agents appear to be an attractive option for cancer chemo- (32). The culture medium was gently shaken immediately prevention, which involves prevention/rectification of altered after addition to dissolve the drug solution uniformly. Then it expression of cell cycle regulatory proteins. Most studies was incubated at 37˚C for 48 h with 5% CO2. Medium was on etodolac have been performed with various human colon changed with the addition of fresh drug solution of the same and other carcinoma cell lines and few of them focus on non- concentrations after 24 h of incubation period. Only 0.01% malignant proliferative human mammary epithelial cell lines DMSO solution (without addition of drug) was additionally related to cell growth and cell cycle parameters (23-25). used as the solvent control and allowed to grow for the Laboratory experiments with various cancer cell lines reveal same 48 h period with a change after 24 h. Etodolac (0.5 and that etodolac induced a significant reduction in gastro- 2.0 mM) and 48 h growth period was chosen as an optimum intestinal cancer cell line Caco2, HT29 and MKN45 due to activity considering other concentrations and growth periods COX-2 inhibition. Inhibition of cell division and alteration of (31). All experiments were done in duplicate and repeated at the cell cycle distribution were observed in cultured colon least three times with different adjacent passage of cell lines cancer cells, tumor cells were arrested in G0/G1 phase, and to obtain consistent results. finally undergoing apoptosis. Furthermore, etodolac was found to inhibit COX-2 associated PGE2 synthesis in HT-29/Inv3 Isolation and purification of total RNA. Total RNA was cell lines (26,27). In this study, the effect of the NSAID, isolated from both the control (MCF-10F) and the etodolac etodolac, a 1,8-diethyl-1,3,4,9-tetrahydro-pyrano (3,4-ß) treated cell lines with TRIzol reagent (Invitrogen Corp., indole-1-acetic acid (Fig. 1) on signaling pathways was Long Island, NY). Each sample comprising 500 μg of total investigated by examining the differential expression of RNA, was treated with 5 μl of DNAse I (10 U/μl) (Boehringer various cell cycle regulatory protein genes. Mannheim, Indianapolis, IN) for 60 min at 37˚C. Then 10X Termination Mix (0.1 M EDTA at pH 8.0 and