Cytochrome P450 2E1: Its Role in Disease and Drug Metabolism by Aparajita Dey

Author:Aparajita Dey
Language: eng
Format: epub
Publisher: Springer Netherlands, Dordrecht

To study whether upregulation of the antioxidant proteins GCSc, HO-1, and catalase in E47 cells is mediated by Nrf2, SiRNA-Nrf2 was used to block the effects of Nrf2. The non-target siRNA, siRNA-control(Si-c) was used as a control. The transfection efficiency of siRNA-Nrf2 and siRNA-control in C34 and E47 cells was similar (50.1  ±  4.6%). After transfection of siRNA-Nrf2, Nrf2 mRNA levels were decreased in C34 cells, and more dramatically in E47 cells compared with C34 or E47 cells transfected with siRNA-control. GCSc and HO-1 mRNA levels also showed some decreases in C34 cells when transfected with siRNA-Nrf2. Importantly, the increased GCSc and HO-1 mRNA expression in E47 cells was completely blocked after transfection with siRNA-Nrf2. Although catalase mRNA was induced in E47 cells, siRNA-Nrf2 had no effect on catalase mRNA levels in both C34 and E47 cells, indicating that Nrf2 is not a critical transcriptional factor for expression of catalase in the HepG2 cells as it is for the expression of GCS or HO-1. As found with the mRNA levels, Nrf2, GCSc, and HO-1 protein levels were decreased in a time-dependent manner in C34 cells and more dramatically in E47 cells by siRNA-Nrf2 (immunoblots shown in Fig. 2.3a and quantified in Fig. 2.3b). The decline in GCSc and HO-1 proteins parallels the decline in Nrf2 protein in both cell lines, suggesting an association between Nrf2 levels and these two antioxidant proteins. Catalase protein level in C34 and E47 cells transfected with siRNA-Nrf2 was unchanged compared with siRNA-control (Fig. 2.3a, b). CYP2E1 protein levels in the E47 cells were unchanged by transfection of siRNA-control or siRNA-Nrf2 (Fig. 2.3a).

Fig. 2.3SiRNA against Nrf2 decreases the elevated protein levels of Nrf2, GCS (GCLC) and HO-1 but not catalase in E47 cells. The SiRNA against Nrf2 had no effect on CYP2E1 protein levels in the E47 cells. 2.5 million E47 or C34 cells per well were transfected with either Si-C or Si-Nrf2. After 30 h, cells were collected, washed, lysed and immunoblotted for detection of the indicated enzymes

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