Purpose: Three enzymatic pathways of arachidonic acid metabolism, involving cyclooxygenases, lipooxygenases, and cytochrome P450 (CYP) epoxygenases have been identified in mammalian cells, but the latter pathway has been not been as well characterized in cancer. The aim of this study is to determine whether CYP enzymes induce cancer cell proliferation. Experimental Design: RT PCR was used to measure CYP3A4 and 3A5 levels in ER+ and ER- breast cancer lines. siRNA was used to knock down the level of CYP3A4 and 3A5, to determine effects on cell proliferation and apoptosis. Effects of pharmacological inhibition of CYP3A4 and 3A5 by the HIV protease inhibitor ritonavir and the epoxygenase inhibitor 17-ODYA were also determined. Results: CYP3A4 mRNA is expressed in the breast cancer lines T47D, MCF7 and MDA-MB-231 and, whereas CYP3A5 mRNA is expressed in the T47D and MCF7 lines, but not in MDA-MB-231. Although the corresponding proteins cannot be detected by western blot, inhibition of CYP3A4 by siRNA, verified by reduction of mRNA levels, inhibits the proliferation of each of the lines by 30% (P<0.05). CYP3A5 siRNA also blocks the proliferation of the T47D and MCF7 lines, but not MDA-MB-231. Ritonavir, which is a potent CYP inhibitor, inhibits the proliferation of the T47D, MCF7, and MDA-MB-231 lines, exhibiting IC50 values of 15, 30 and 45 microM for the MCF7, T47D, and MDA-MB-231 respectively. CYP 3A4 siRNA sensitizes all three lines to ritonavir, lowering the IC50 values to 7, 10 and 25 microM, for the T47D, MCF7 and MDA-MB-231 lines, respectively. Similarly, 17-ODYA, a CYP3A inhibitor, exhibits an IC50 of 7 microM for proliferation in the MDA231 line under serum-free conditions. Rifampicin, which is a potent CYP3A inducer, increased the mRNA expression of the CYP3A4 and 3A5 isoforms in all the lines and induced proliferation. The addition of 25 microM rifampicin increases the proliferation of MCF7 line 1.8-fold. Because CYP3A4 can synthesize epoxyeicosatrienoic acids (EET’s), we asked whether ritonavir inhibition of breast cancer lines may be abrogated by exogenous EET addition. The addition of regioisomers 8,9 and 11,12 EET to the MCF7 line bypassed the effects of ritonavir addition and restored proliferation at the ritonavir IC50 by 85% and 14,15 EET restored proliferation to 130% of control. Conclusions: CYP3A4 and CYP3A5 mRNA expression is important for the proliferation of breast cancer lines. Certain drugs that inhibit CYP3A4, such as ritonavir, inhibit breast cancer cell proliferation, while others, such as rifampicin, which induce CYP3A4 may induce proliferation. At least some of the inhibition due to ritonavir can by bypassed by addition of CYP3A4 EET regioisomers. These results suggest that drugs that modulate CYP3A4 may influence breast cancer proliferation, in part, through modulation of CYP epoxygenase activity.
[Proc Amer Assoc Cancer Res, Volume 47, 2006]