Catherine Armstrong1,2, Jade Huguet1,2, Fleur Gaudette1, François Bélanger1, Danuta Balicki,1,3,4 Jacques Turgeon1,2
1. CRCHUM, Centre Hospitalier de l’Université de Montréal, CHUM-Hôtel-Dieu, 3850 St. Urbain Street, Montreal, Quebec, Canada, H2W 1T7
2. Faculty of pharmacy, Université de Montréal, Montréal, Québec, Canada, H3C 3J7
3. Service de médecine génique, Department of Medicine, CHUM
4. Faculty of medicine, Université de Montréal, Montréal, Québec, Canada, H3C 3J7
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ABSTRACT
Background : Cytochrome P450 (CYP450) enzymes are known to be involved in the metabolism of chemotherapy agents that are commonly used in the treatment of breast cancer. While CYP450s are primarily found in the intestines and liver, where they likely control the systemic exposure to drugs, they are also present at significant levels in other tissues. In such tissues, they would be responsible for the local metabolism of drugs. Hence, variable expression of CYP450 genes at the tissue level, next to their effector site, could explain inter-subject variability in the local metabolism, efficacy and toxicity of drugs. In order to determine if gene expression is variable for these enzymes, their expression level of mRNAs in breast cancer cell lines and functional CYP450 activities were evaluated. Methods: Seven commonly used breast cancer cell lines, (Hs578T, MDA-MB-231, MDA-MB-468, MCF-7, SKBR3, T47D, ZR-75-1) and one benign breast cell line (MCF- 10A) were cultured and then extracted for RNA. The RNA was reverse transcribed to cDNA where the mRNA levels of 19 CYP450 isoenzymes were determined by Quantitative Real-Time PCR. Functional activities of CYP1B1 and 2J2 were determined by incubating whole cells with 7-ethoxyresorufin and ebastine, respectively, and measuring the metabolite produced by LC-MS. Results: The relative mRNA levels of the different CYP450 enzymes showed a large variability between the different cell lines. CYP1B1 mRNA was highly expressed in most of the breast cancer cell lines; CYP2J2 mRNA is the most abundant CYP450 found in SKBR3 and ZR-75-1 cell lines contributing 62.9 and 15.4% of their total CYP450 make-up respectively; CYP3A4/5/7 mRNAs expressions were 41.5, 13.9 and 43.3 times greater in the benign control compared to the mean value; whereas 2C9 mRNAs showed no expression in any cell line analyzed. The metabolism of 7-ethoxyresorufin was observed in 4 of the 8 cell lines, where the Km observed was between 0.05 to 0.1 µM and Vmax ranged from 0.04 to 3.7 pmol/mg protein/min. The metabolism of ebastine was observed in 7 of the 8 cell lines, where the Km was between 0.2 and 3.1 µM and Vmax
74 between 0.58 and 2.6 pmol/mg protein/min. Correlation studies between mRNA expression of CYP1B1 and CYP2J2 and the metabolism of 7-ethoxyresorufin and ebastine, respectively, revealed very strong correlations of 0.98 and 0.99, respectively. Conclusion: The variability in expression levels of CYP450 mRNAs appears to be characteristic of the different cell lines analyzed. We propose that the variable expression of CYP450s in breast cancer cells could explain part of the inter-subject variability in response to chemotherapy agents. The variability of CYP450 mRNAs expression may also identify new targets for the synthesis of anti-cancer agents.
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INTRODUCTION
The cytochrome P450 (CYP450) superfamily is a family of enzymes which work to detoxify the body of many xenobiotic molecules by biotransforming them into more hydrosoluble molecules.[1] In human, there are 57 known CYP450 isoforms which have been classified based on their sequence homology, and have various roles in the metabolism and synthesis of molecules. CYP450s isoforms play an important role in the synthesis and degradation of many endogeneous molecules, such as steroids. CYP1A1, 1A2 and 1B1 have been shown to be implicated in the hydroxylation of progesterone, testosterone and estrogen. [2-5] CYP17A1 and 19A1 are two isoforms which are also important in the formation of androstenedione through the use of aromatase activity. [3,6] Since these steroids have been shown to impact breast cancer survival and treatment, these isoforms will be important to evaluate. [7]
CYP450s are most abundantly found in the liver, where CYP3A4 is considered the most important for drug metabolism. However, the presence of CYP450 enzymes, with tissue specific expression, have been shown in other tissues such as the intestines, kidneys, brain, lungs and heart at varying concentrations. [8-15] The expression of specific enzymes in extra hepatic tissue shows that local metabolism could play an important role in drug metabolism and cause a source of variation in drug effects.
Many chemotherapy agents, both active drug and pro-drug, are known substrates of CYP450s.[16] Therefore, the presence of CYP450s at both the intestinal/hepatic level and the tissular level will influence the response of chemotherapy agents. At the hepatic level, the CYP450s present cause
76 decreased bioavailability of medications. The portion of the medication that reaches the targeted cells would then encounter other CYP450s which would locally metabolize the drug (either for clearance or activation purposes). For active drugs, if the molecule is a substrate of a specific CYP450 highly expressed in the cells of interest, it would be metabolized too quickly from the targeted cell and cause no local effect; whereas a pro-drug which requires CYP450 metabolism could be activated locally due to the presence of a specific enzyme at the targeted site. Since tissue specific expression of CYP450 enzymes are expected so is the metabolism potential.
Inter-subject variability of CYP450 mRNA expression would be expected in breast tissue based on similar results observed in other tissues. This can also be suggested when evaluating the efficacy of chemotherapy agents in breast cancer cell lines. A study performed in 2009 evaluated the effects of three chemotherapy agents in nineteen breast cancer cell lines to determine the efficacy of the chemotherapy agents. The study showed that not every chemotherapy agent was effective in all cell lines analyzed which shows that inter-subject response is present.[17] Since chemotherapy agents are substrates of CYP450 enzymes, the response difference of the cell lines may be partly explained by the presence of differing CYP450 expression.
Therefore, we believe that breast tumors will display variations in CYP450 expression, and this tissue specific expression could cause a variation in local metabolism which is an important phenomenon to consider in breast cancer treatment and survival. To evaluate this point, commonly used cell lines were analyzed for mRNA expression of CYP450 enzymes, which we believe will display inter-cell line variation, which would indicate a potential variation in breast
77 cancer patients. In addition, functional activities of CY1B1 and 2J2, two isoforms which have been shown to be highly expressed through this work, were determined using two specific substrates, 7-ethoxyresorufin and ebastine, respectively. These differences could help identify new pathways for the synthesis of new anti-cancer agents, either as pro-drug or active drugs.
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