Inhibition of CYP3A4 by 6′,7′-dihydroxybergamottin in human CYP3A4 over-expressed hepG2 cells
Inhibition of CYP3A4 by 6′,7′-dihydroxybergamottin in human CYP3A4 over-expressed hepG2 cells
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DOI：
10.1111/j.2042-7158.2012.01562.x
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发表时间：
2012-12-01
影响因子：
3.3
通讯作者：
Fujimura, Akio
中科院分区：
文献类型：
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作者：
Araki, Nobutaka;Tsuruoka, Shuichi;Fujimura, Akio
目的建立高表达人CYP3A4和谷氨酰胺合成酶(GS)的肝母细胞瘤细胞系HepG2-GS-3A4。我们进一步报道，这些细胞可用于体外筛选CYP3A4的抑制剂。本研究的目的是利用该细胞研究6‘，7’-二羟基佛手苷(DHB)对西柚汁中主要呋喃香豆素类化合物6‘，7’-DHB的抑制作用。方法检测硝苯地平对HepG2-GS-3A4细胞NADPH-细胞色素还原酶(POR)活性和蛋白表达的影响。同时对细胞微粒体部分的共结合光谱分析进行了评价。主要发现DHB和酮康唑，一种著名的CYP3A4抑制剂，以浓度依赖的方式抑制硝苯地平的氧化。当DHB浓度为3.0 mU m时，足以抑制硝苯地平氧化的POR活性降低，而0.9 mU浓度的酮康唑则不影响POR活性。DHB和酮康唑均不影响细胞POR蛋白的表达。加入DHB或酮康唑后，细胞色素P3A4mRNA和蛋白的表达无明显变化。在CO结合光谱分析中，DHB还降低了450 nm处的吸收速率，而不改变最大吸收波长。酮康唑在450 nm处的平均吸光度略有下降，但差异不显著。结论DHB对细胞色素P3A4活性的抑制包括对POR活性的抑制。HepG2-GS-3A4可能是评价这一机制的一个很好的工具。
Objectives We previously established HepG2-GS-3A4, a cell line from hepatoblastoma with overexpression of human CYP3A4 and glutamine synthetase (GS). We further reported that these cells can be applied for screening inhibitors of CYP3A4 in vitro. The purpose of this study was to determine whether our CYP3A4-overexpresed cell could be applied to evaluate mechanisms of CYP3A4 inhibition by 6',7'-dihydroxybergamottin (DHB), which is one of the major furanocoumarins in grapefruit juice, by using these cells. Methods Nifedipine oxidation, activity and protein expression of NADPH-cytochrome reductase (POR) of HepG2-GS-3A4 cell were measured. CO-binding spectrumassay in microsomal fraction of the cells was also evaluated. Key findings DHB and ketoconazole, a well-known inhibitor of CYP3A4, inhibited nifedipine oxidation in a concentration-dependent manner. DHB at a concentration of 3.0 mu m, sufficient to inhibit the nifedipine oxidation, decreased POR activity; however, ketoconazole at a concentration of 0.9 mu m, sufficient to inhibit the oxidation, did not affect the activity. The expression of POR protein in HepG2-GS-3A4 cells was not changed by either DHB or ketoconazole. The expression of CYP3A4 mRNA and protein was not changed by the addition of DHB or ketoconazole. DHB also reduced the absorption rate at 450 nm in a CO-binding spectrum assay without alteration of the wavelength of maximum absorption. The mean absorption value at 450 nm slightly decreased with ketoconazole; however, the difference was not significant. Conclusions We concluded that inhibition of CYP3A4 activity by DHB includes the inhibition of POR activity. HepG2-GS-3A4 might be a good tool to evaluate the mechanisms.