DOI:http://dx.doi.org/10.7314/APJCP.2012.13.6.2647 RNA Expression of Cytochrome P450 in Mexican Women with Breast Cancer RESEARCH COMMUNICATION RNA Expression of Cytochrome P450 in Mexican Women with Breast Cancer Cindy Bandala1, E Floriano-Sánchez1,2*, N Cárdenas-Rodríguez1,3, J López- Cruz4, E Lara-Padilla1 Abstract Involvement of cytochrome P450 genes (CYPs) in breast cancer (BCa) may differ between populations, with expression patterns affected by tumorigenesis. This may have an important role in the metabolism of anticancer drugs and in the progression of cancer. The aim of this study was to determine the mRNA expression patterns of four cytochrome P450 genes (CYP2W1, 3A5, 4F11 and 8A1) in Mexican women with breast cancer. Real- time PCR analyses were conducted on 32 sets of human breast tumors and adjacent non-tumor tissues, as well as 20 normal breast tissues. Expression levels were tested for association with clinical and pathological data of patients. We found higher gene expression of CYP2W1, CYP3A5, CYP4F11 in BCa than in adjacent tissues and only low in normal mammary glands in our Mexican population while CYP8A1 was only expressed in BCa and adjacent tissues. We found that Ki67 protein expression was associated with clinicopathological features as well as with CYP2W1, CYP4F11 and CYP8A1 but not with CYP3A5. The results indicated that breast cancer tissues may be better able to metabolize carcinogens and other xenobiotics to active species than normal or adjacent non-tumor tissues. Keywords: CYP2W1 - CYP3A5 - CYP4F11 - CYP8A1 - mRNA - breast cancer Asian Pacific J Cancer Prev, 13, 2647-2653 Introduction Thus, it is important to determine the expression patterns in different populations. Breast cancer (BCa) is the most common malignant Cytochrome P450 2W1 (CYP2W1) has been shown tumor in the 25 years and over Mexican female population to participate in the oxidation of arachidonic acid and with an increase of 30% of cancer-related deaths in the last catalysts the reductive activation of AQ4N to AQ4 (an 20 years (WHO, 2008; SINAIS, 2010). The expression anticancer drug) and in the bioactivation of several pro- of tumors specific proteins in mammary glands may play carcinogens including polycyclic aromatic hydrocarbons a critical role in the development of BCa as well as in the and aflatoxin B1 (Wu et al., 2006; Nishida et al., 2010). success of chemotherapy treatment. To date, very few mRNA expression was detected in tumor tissues from critical markers have been validated for the prediction of colon, adrenal, and lung, while no expression was seen drug efficiency in BCa (Vaclavikova et al., 2007). in a normal tissues like brain, heart, colon, kidney, and Cytochrome P450 (CYP) system is a multigene placenta (Karlgren et al., 2005). Recent data obtained superfamily of enzymes that play a central role in activating by RT-PCR of whole mice RNA indicate that CYP2W1 and detoxifying a wide variety of xenobiotics as well as mRNA is expressed in mouse embryos but not in adult endobiotics (Nebert, 1991). Cytochrome P450s have been animals (Huang et al., 1998), suggesting a role for proposed to be of importance both in carcinogenesis, CYP2W1 in fetal life. CYP2W1 mRNA was here found by activating precarcinogens, and as determinants to be expressed in rat fetal tissues, especially in colon of cancer chemotherapy, where they participate in but also in lung where the expression increased by fetal activation or inactivation of anti-cancer drugs (Oyama age and transiently in brain at day 16. After birth, the et al., 2004; Rooseboom et al., 2004). The relevance of CYP2W1 gene gradually becomes silent in rat and mouse, P450 modulation to cancer risk, tumorigenesis, cancer and in support of such silencing also occurring in human progression and metabolizing of different anticancer drugs is the fact that we found no or only very small amounts has not been well understood in humans. The expression of of CYP2W1 mRNA in all adult non-transformed tissues major P450s is polymorphic and results in the generation examined. It appears that CYP2W1 provides the most of multiple population-specific phenotypes with different specific form of cytochrome P450 for tumor expression drug metabolizing capabilities (Vaclavikova et al., 2007). hitherto found (Karlgren et al., 2005). 1Section of Research and Graduate Studies, Instituto Politécnico Nacional, 2Laboratory of Biochemistry and Molecular Biology, Escuela Médico Militar, SEDENA, 3Laboratory of Neurochemistry, Instituto Nacional de Pediatría, 4Service of Pathology, Clínica de Especialidades de la Mujer, SEDENA, México, *For correspondence: [email protected] Asian Pacific Journal of Cancer Prevention, Vol 13, 2012 2647 Bandala C et al Cytochrome P450 3A5 (CYP3A5) has been shown possible association between CYP expression patterns to play a role in the 16α-hydroxylation of estrogens in with some clinic-pathological risk factors. humans. Since 16-hydroxy-estrogen 1 (16-OHE1) is a putative breast carcinogen, knowledge of the enzymes Materials and Methods involved in its synthesis provides a basis for blocking its synthesis in vivo (Huang et al., 1998). CYP3A4 Biological Samples and CYP3A5 are both expressed in human female All samples of human mammary carcinomas and paired breast tissue, but not in all individuals (Huang et al., adjacent normal tissue without morphological signs of 1996), these results also provide a basis for selection of carcinoma were obtained from 31 BCa patients diagnosed potentially susceptible individuals. Recent study found at the Pathology Service, Clínica de Especialidades de la that expression of CYP3A4⁄5 was significantly associated Mujer, Secretaría de la Defensa Nacional (SEDENA) in with lymph node metastases in BCa (Murray et al., 2010). Mexico City. Normal tissues of the mammary gland were Cytochrome P450 4F11 (CYP4F11) showed catalytic obtained for reduction of mammary gland in the Service activity against endogenous eicosanoids (leukotriene of Plastic Surgery, Hospital Central Militar (SEDENA). B4) arachidonic acid, prostaglandins and lipoxins, and Tissue samples were collected during surgery and frozen hydroxyeicosatetraenoic acids and commonly used drugs at -80°C. The histological classification of the carcinomas, (erythromycin, benzphetamine, and chlorpromazine) as well as the evaluation of non-tumor breast lesions, were (Kalsotra et al., 2004; Choudhary et al., 2005). CYP4F11 made according to standard diagnostic procedures and is thought to be primarily involved in the metabolism of confirm by four pathologists. Non tumor samples were fatty acids (3-hydroxypalmitate) and arachidonic acid without morphologically detected tumor cells. Patients metabolites (Dhar et al., 2008; Uno et al., 2011). mRNA were asked to read and sign an Informed Consent in is mostly found in liver and kidney; minor expression was agreement with requirements of the Ethical Commission noted in skeletal muscle, placenta, and heart (Cui et al., of the National Institute of Public Health in México. This 2000). project has the approval of Hospital Research Ethical Cytochrome P450 8A1 (CYP8A1; Prostacyclin I2 commission (ref. no. SI-378). synthase; PGIS) gene encodes for an enzyme that acts as an isomerase and rearranges PGH2 to PGI2. PGIS Isolation of total RNA from human tumor and its adjacent is considered to be an atypical CYP because it does not normal tissues possess oxygenase activity (Ullrich et al., 1981; Smith et Approximately 100 mg of human tumor, adjacent al., 1995). PGI2 has many important biological functions. normal tissue and mammary normal tissues were separated It is the most important endogenous inhibitor of platelet and homogenized individually in a Trizol reagent (TRI aggregation discovered to date, and also causes vascular Reagent® Solution, RNA/DNA/Protein Isolation Reagent, relaxation (Moncada et al., 1976; Cathcart et al., 2010). Ambion). Total cellular RNA was extracted according PGI2 is also anti-mitogenic and inhibits DNA synthesis in to the manufacture’s protocol. The concentration of smooth muscle cells (Libby et al., 1988). The presence of total RNA in each sample was measured by Nanodrop PGIS at the nuclear and endoplasmic reticular membrane Spectrophotometer (Delaware, USA) using the ratio of suggests multiple signaling pathways for this enzyme via 260-nm/280-nm. The quality of the isolated RNA was PGI2 generation, involving both cell surface and nuclear accessed based on the integrity of the 28S, 18S, and 5S receptors. However, the cellular signaling initiated by bands after ethidium bromide staining electrophoresis of this class of compounds is probably the least understood a 1% formaldehyde denaturating gel and visualized under of all the primary prostanoids (Bos et al., 2004; Cathcart a UV transilluminator (EDAS 290 KODAK, New Haven, et al., 2010). A number of studies have demonstrated CT). Total RNA (1 μg) was successively supplemented that the prostanoid biosynthesis profile of malignant with 0.5 µL RNase inhibitor (Boehringer Mannheim cells is different compared to normal tissues (Wang and Gmbh Germany). All extracted RNAs were stored at Chen, 1996; Yokoyama et al., 1996). CYP8A1 signaling -80°C. through arachidonic acid metabolism affects a number of tumor cell survival pathways including cell proliferation Quantitative real-time PCR assays and apoptosis as well as, tumor cell invasion, metastasis Specific oligonucleotides of the CYP2W1, CYP3A5, and angiogenesis. PGI2 is a potent antimitogenic and CYP4F11 and CYP8A1 genes and for the reference