Effects of acidic non-steroidal anti-inflammatory drugs on human cytochrome P450 4A11 activity: Roles of carboxylic acid and a sulfur atom in potent inhibition by sulindac sulfide.

Cytochrome P450 4A11 (CYP4A11) has many endogenous and exogenous compounds containing a carboxyl group in their structure as substrates. If drugs with this characteristic potently attenuate the catalytic function of CYP4A11, drug-drug interactions may occur. Acidic non-steroidal anti-inflammatory drugs (NSAIDs) possess a carboxylic acid in their structure. However, it remains unclear whether these drugs inhibit CYP4A11 activity. The present study examined the inhibitory effects of acidic NSAIDs on CYP4A11 activity using human liver microsomes (HLMs) and recombinant CYP4A11. Sulindac sulfide, ibuprofen, and flurbiprofen effectively decreased the luciferin-4A O-demethylase activity of HLMs and recombinant CYP4A11 (inhibition rates of 30-96% at an inhibitor concentration of 100 µM), while salicylic acid, aspirin, diclofenac, mefenamic acid, indomethacin, etodolac, ketoprofen, loxoprofen, S-naproxen, pranoprofen, zaltoprofen, and oxaprozin exhibited weaker inhibitory activity (inhibition rates up to 23%). Among the drugs tested, sulindac sulfide was the most potent inhibitor of CYP4A11 activity. A kinetic analysis of the inhibition of CYP4A11 by sulindac sulfide revealed mixed-type inhibition for HLMs (Ki = 3.38 µM) and recombinant CYP4A11 (Ki = 4.19 µM). Sulindac sulfide is a pharmacologically active metabolite of sulindac (sulfoxide form), which is also oxidized to sulindac sulfone. To elucidate the role of a sulfur atom of sulindac sulfide in the inhibition of CYP4A11, the inhibitory effects of sulindac sulfide and its oxidized forms on CYP4A11 activity were examined. The potency of inhibition against HLMs was greater in the order of sulindac sulfide, sulindac, and sulindac sulfone; IC50 values were 6.16, 52.7, and 71.6 µM, respectively. The present results indicate that sulindac sulfide is a potent inhibitor of CYP4A11. These results and the molecular modeling of CYP4A11 with sulindac sulfide and its oxidized forms suggest that a sulfur atom of sulindac sulfide as well as its carboxylic acid play important roles in the inhibition of CYP4A11.

Tamoxifen resistance alters sensitivity to 5-fluorouracil in a subset of estrogen receptor-positive breast cancer.

Sequential treatment with endocrine or chemotherapy is generally used in the treatment of estrogen receptor (ER)-positive recurrent breast cancer. To date, few studies have investigated the effect of long-term endocrine therapy on the response to subsequent chemotherapy in ER-positive breast cancer. We examined whether a preceding endocrine therapy affects the sensitivity to subsequent chemotherapy in ER-positive breast cancer cells. Three ER-positive breast cancer cell lines (T47D, MCF7, BT474) and tamoxifen-resistant sublines (T47D/T, MCF7/T, BT474/T) were analyzed for sensitivity to 5-fluorouracil, paclitaxel, and doxorubicin. The mRNA levels of factors related to drug sensitivity were analyzed by RT-PCR. MCF7/T cells became more sensitive to 5-fluorouracil than wild-type (wt)-MCF7 cells. In addition, the apoptosis induced by 5-fluorouracil was significantly increased in MCF7/T cells. However, no difference in sensitivity to chemotherapeutic agents was observed in T47D/T and BT474/T cells compared with their wt cells. Dihydropyrimidine dehydrogenase (DPYD) mRNA expression was significantly decreased in MCF7/T cells compared with wt-MCF7 cells. The expression of DPYD mRNA was restored with 5-azacytidine treatment in MCF7/T cells. In addition, DPYD 3'-UTR luciferase activity was significantly reduced in MCF7/T cells. These data indicated that the expression of DPYD mRNA was repressed by methylation of the DPYD promoter region and post-transcriptional regulation by miRNA in MCF7/T cells. In the mouse xenograft model, capecitabine significantly reduced the tumor volume in MCF7/T compared with MCF7. The results of this study indicate that endocrine therapy could alter the sensitivity to chemotherapeutic agents in a subset of breast cancers, and 5-fluorouracil may be effective in tamoxifen-resistant breast cancers.

Effects of acid and lactone forms of statins on S-warfarin 7-hydroxylation catalyzed by human liver microsomes and recombinant CYP2C9 variants (CYP2C9.1 and CYP2C9.3).

The inhibition of CYP2C9-mediated warfarin metabolism by acid or lactone forms of statin converted in the body and effects of CYP2C9 genetic variants on their inhibition are not fully understood. Here, the effects of acid and lactone forms of statins on S-warfarin 7-hydroxylation were investigated in vitro. S-Warfarin 7-hydroxylase activities of human liver microsomes (HLMs), recombinant CYP2C9.1 (rCYP2C9.1), and rCYP2C9.3 (Ile359Leu variant) in the presence of statins were determined by high-performance liquid chromatography. Lactone forms of atorvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin inhibited the activity of HLMs more potently than the corresponding acid forms, whereas fluvastatin acid showed stronger inhibition than fluvastatin lactone. When the effects of statins on rCYP2C9 variants were examined, inhibition profiles of acid versus lactone forms of statins except for fluvastatin were similar between rCYP2C9.1 and rCYP2C9.3. However, the degrees of inhibition by atorvastatin lactone, fluvastatin acid, fluvastatin lactone, lovastatin lactone, and pitavastatin lactone (Ki values) were significantly different between these variants. These results indicated that lactone forms of statins other than fluvastatin showed more potent inhibition of CYP2C9-catalyzed S-warfarin 7-hydroxylation than the corresponding acid forms. Furthermore, our results indicated that Ile359Leu substitution in CYP2C9 affected the inhibitory potencies of statins.

Expression profile of cytochrome P450s and effects of polycyclic aromatic hydrocarbons and antiepileptic drugs on CYP1 expression in MOG-G-CCM cells.

AIMS: This study was performed to investigate the expression profile of cytochrome P450 (CYP) isoforms and effects of polycyclic aromatic hydrocarbons (PAHs) and antiepileptic drugs on CYP1 expression in human astrocytoma MOG-G-CCM cells. MAIN METHODS: CYP1A1 and CYP1B1 expression were determined by quantitative real-time polymerase chain reaction, Western blotting, and immunocytochemistry. KEY FINDINGS: MOG-G-CCM cells expressed various CYP isoforms. Among the CYP isoforms analyzed, CYP1B1 showed the highest expression level, followed by CYP1A1. Furthermore, CYP1B1 was localized in both the endoplasmic reticulum and mitochondria. 3-Methylcholanthrene (3-MC), benz[a]anthracene (B[a]A), benzo[a]pyrene (B[a]P), and valproic acid (VPA) increased the expression of CYP1B1 and CYP1A1. The potent aryl hydrocarbon receptor antagonist GNF351 significantly suppressed the 3-MC- and VPA-mediated upregulation of CYP1B1 and CYP1A1. In addition, VPA potentiated the induction of CYP1B1 and CYP1A1 by 3-MC, B[a]A, and B[a]P, although the augmentation of CYP1A1 was more remarkable than that of CYP1B1. In contrast, other antiepileptic drugs (carbamazepine, lamotrigine, levetiracetam, phenytoin) did not affect the 3-MC-mediated upregulation of CYP1B1 and CYP1A1. VPA is known to act as a histone deacetylase (HDAC) inhibitor. Therefore, the effects of trichostatin A, a representative HDAC inhibitor, on CYP1 induction by 3-MC were examined. Trichostatin A enhanced the 3-MC-mediated upregulation of CYP1A1 but not CYP1B1. SIGNIFICANCE: These results partially indicated that VPA may augment the PAH-mediated induction of CYP1B1 and CYP1A1 through the activation of transcription by HDAC inhibition.

In Vitro Inhibitory Effects of Sesamin on CYP4F2 Activity.

Sesamin is a major lignan in sesame seeds, and a recent meta-analysis of controlled trials indicated that sesamin intake decreases blood pressure. The antihypertensive effect of sesamin has been suggested to be due to sesamin-mediated suppression of 20-hydroxyeicosatetraenoic acid production catalyzed by CYP4F2. However, the detailed mechanism underlying inhibition of CYP4F2 function by sesamin remains unclear. In this study, the effects of sesamin on catalytic activity of CYP4F2 were investigated in vitro. Sesamin inhibited luciferin-4F2/3 O-dealkylase activity of recombinant human CYP4F2 with an IC50 value of 0.381 µM. When preincubated in the presence of reduced nicotinamide adenine dinucleotide phosphate (NADPH) for 20 min, sesamin potentiated the inhibition of CYP4F2 activity. Moreover, kinetic analysis of the inactivation revealed that sesamin showed a preincubation time- and concentration-dependent inhibition of CYP4F2 activity yielding a maximal inactivation rate constant (kinact) value of 0.354 min-1 and half-maximal inhibitory concentration (KI) value of 1.12 µM. The inactivation of CYP4F2 by sesamin required NADPH. These results indicated that sesamin is a mechanism-based inactivator of human CYP4F2.

Inhibitory effects of antihypertensive drugs on human cytochrome P450 2J2 activity: Potent inhibition by azelnidipine and manidipine.

The inhibitory effects of antihypertensive drugs (dihydropyridine calcium channel blockers, angiotensin II receptor blockers, and angiotensin-converting enzyme inhibitors) on cytochrome P450 2J2 (CYP2J2) activity were examined. Amlodipine, azelnidipine, barnidipine, benidipine, cilnidipine, efonidipine, felodipine, manidipine, nicardipine, nifedipine, nilvadipine, nisoldipine, nitrendipine, telmisartan, delapril, and quinapril inhibited luciferin-2J2/4F12 O-dealkylase activity of recombinant human CYP2J2 in a concentration-dependent manner (IC50 = 0.116-9.19 µM). Kinetic analyses of the inhibition indicated that azelnidipine, barnidipine, benidipine, cilnidipine, efonidipine, manidipine, nicardipine, telmisartan, delapril, and quinapril competitively inhibited CYP2J2 activity, while amlodipine, felodipine, nifedipine, nilvadipine, nisoldipine, and nitrendipine showed mixed inhibition. Among these drugs, manidipine showed the strongest reversible inhibition with Ki value of 0.0294 µM. The docking simulation data supported the potent inhibition of CYP2J2 by these drugs. Next, the effect of preincubation on CYP2J2 inhibition was investigated to determine whether these antihypertensive drugs inhibited CYP2J2 activity in a metabolism-dependent manner. A 20-min preincubation of azelnidipine and felodipine in the presence of NADPH potentiated the inhibition of CYP2J2. Furthermore, kinetic analysis of the inactivation showed that azelnidipine caused a preincubation time- and concentration-dependent decrease in CYP2J2 activity yielding kinact/KI value of 105 l/mmol/min, although felodipine showed no preincubation time-dependent inhibition. The azelnidipine-mediated inactivation required NADPH. These results indicated that manidipine is a potent competitive reversible inhibitor while azelnidipine is a potent mechanism-based inactivator of human CYP2J2.

A Specific Probe Substrate for Evaluation of CYP4A11 Activity in Human Tissue Microsomes and a Highly Selective CYP4A11 Inhibitor: Luciferin-4A and Epalrestat.

The specificity of cytochrome P450 4A11 (CYP4A11) against luciferin-4A O-demethylation in human liver microsomes (HLMs) and human renal microsomes (HRMs) and selectivity of CYP4A11 inhibition by epalrestat were investigated. Kinetic analysis of luciferin-4A O-demethylation yielded Vmax and S50 values of 39.7 pmol/min per milligram protein and 43.2 µM for HLMs (Hill coefficient 1.24) and 39.4 pmol/min per milligram protein and 33.8 µM for HRMs (Hill coefficient 1.34), respectively. Among the selective CYP inhibitors tested, HET0016 (CYP4 inhibitor) exclusively inhibited luciferin-4A O-demethylation by HLMs and HRMs. Furthermore, anti-CYP4A11 antibody nearly abolished the activity of both tissue microsomes. Luciferin-4A O-demethylase activity of HLMs was significantly correlated with lauric acid ω-hydroxylase activity, a marker of CYP4A11 activity (r = 0.904, P < 0.0001). Next, effects of epalrestat on CYP-mediated drug oxidations were examined. Epalrestat showed the most potent inhibition against CYP4A11 (IC50 = 1.82 µM) among the 17 recombinant enzymes tested. The inhibitory effect of epalrestat on CYP4A11 was at least 10-fold stronger than those on CYP4F2, CYP4F3B, and CYP4F12. For known CYP4 inhibitors, in contrast, HET0016 inhibited the activities of CYP4A11 and CYP4F2 (IC50 = 0.0137-0.0182 µM); 17-octadecynoic acid reduced activities of CYP4A11, CYP4F2, CYP4F3B, and CYP4F12 to a similar extent (IC50 = 5.70-17.7 µM). Epalrestat selectively and effectively inhibited the CYP4A11 activity of HLMs (IC50 = 0.913 µM) and HRMs (IC50 = 0.659 µM). These results indicated that luciferin-4A O-demethylase activity is a good CYP4A11 marker of HLMs and HRMs, and that epalrestat is a more selective CYP4A11 inhibitor compared with known CYP4 inhibitors.

Alteration of Y-box binding protein-1 expression modifies the response to endocrine therapy in estrogen receptor-positive breast cancer.

Y-box binding protein-1 (YB-1) plays an important role in tumor progression and drug resistance. This study examined whether YB-1 is involved in the alteration of response to endocrine therapy in estrogen receptor (ER)-positive breast cancer cells. MCF7 cells that stably expressed YB-1 (MCF7-YB-1) and vector control cells (MCF7-vector) were established. These cells were used to analyze the expression of the factors related to ER and growth factor receptor signaling pathways and responses to antiestrogens (tamoxifen and fulvestrant) and estrogen responsive element (ERE) activity. The effect of knocking down endogenous YB-1 expression was tested in wild-type MCF7 cells. In addition, the expression of YB-1 and the factors related to ER and growth factor receptor signaling pathways were evaluated in clinical breast cancers treated with preoperative chemotherapy. The expression of HER2, AIB1, p-Erk, and c-Myc was increased in MCF7-YB-1 cells. In contrast, knocking down of YB-1 decreased the expression of these factors but increased the expression of ERα in wild-type MCF7 cells. Furthermore, sensitivity to antiestrogens was decreased in the MCF7-YB-1 in comparison to that in MCF7-vector cells. The introduction of YB-1 into MCF7 cells inhibited apoptosis and cell cycle arrest at G1 phase induced by antiestrogens. In MCF7-YB-1 cells, the expression levels of p-Erk and c-Myc were continuously upregulated when cells were treated with either tamoxifen or fulvestrant. The ERE activity was reduced in MCF7-YB-1 cells in comparison to MCF7-vector cells, and the ERE activity in MCF7-YB-1 cells was inhibited by fulvestrant at a lower concentration than that which inhibited the ERE activity in MCF7-vector cells. In ER-positive clinical breast cancers treated with preoperative chemotherapy, significantly more number of specimens that showed increased or positive YB-1 expression after chemotherapy was positive for HER2 expression. These data suggest that alteration of YB-1 may modify the crosstalk between the ER pathway and HER2 pathway in ER-positive breast cancer cells, and consequently, may alter the response to endocrine therapy in ER-positive breast cancer cells.

Upregulation of the apoptosis-related inflammasome in cardiac allograft rejection.

BACKGROUND: Inflammation is a major factor in cardiac allograft rejection. Accumulating reports have demonstrated an important role of the inflammation-induced adaptor complex, called the inflammasome, in the field of immunology. The apoptosis-associated, speck-like protein containing a caspase recruitment domain (ASC) is an adaptor protein that forms the inflammasome and regulates caspase-1-dependent generation of inflammatory cytokines. The aim of the present study was to determine how ASC is associated with the development of cardiac allograft rejection. METHODS: We used a murine heterotopic cardiac transplantation model between fully incompatible strains. Donor hearts (n = 9 for each time-point) were harvested for examination on Days 1, 4, 7 and 12 after transplantation. Histopathologic findings of cardiac grafts were evaluated using rejection scores. The expression of ASC and inflammatory cytokines in cardiac grafts were analyzed by immunohistochemistry and real-time reverse transcript-polymerase chain reaction (RT-PCR). RESULTS: Expression levels of both ASC and IL-1 beta were higher in the myocardial interstitium of allografts in parallel to the progress of cardiac rejection during the acute phase after transplantation. In contrast, expression of ASC and IL-1 beta remained low in isografts. Cardiac allografts treated with tacrolimus showed decreased expression of both ASC and IL-1 beta similar to that seen in isografts. Real-time RT-PCR demonstrated similar alteration of ASC and IL-1 beta mRNA expression in cardiac grafts during the acute phase. CONCLUSIONS: Our results demonstrate a novel finding showing that upregulation of ASC is closely associated with the inflammation induced in cardiac grafts after transplantation in the mouse.