Fig 2: Immunoblot analyses of CYP1A1, CYP1B1 and CPR in the control, B(a)P (2 µM)- and berberine (10 µM)-treated MCF-7 cells. The vehicle control cells were exposed to the same amount of DMSO. After exposure to these chemicals, crude membrane fractions were prepared from cell lysate and subjected to electrophoresis. Immunoblot analysis was performed as described in the section of Methods. Panel (A) shows the representative blots; the band intensities of the blots were analyzed and the relative percentage to the control cells is shown in Panel (B). Results represent the mean ± SD of 3 separate experiments. * p < 0.05, compared with the control cells.

Fig 3: Effects of estradiol (A), fulvestrant (B) and ERa knockdown (C) on the mRNA levels of CYP1A1 and CYP1B1 in vehicle control and berberine-exposed MCF-7 cells. In panel (A), cells were cultured in the medium supplemented with 10% FBS or 10% DCCFBS, as indicated. The mRNA levels of CYP1A1 and ß-actin were determined using RT–PCR analysis followed by gel electrophoresis. In panels (B,C), cells were exposed to berberine and/or fulvestrant in a 10% FBS-supplemented medium. In panel (C), cells were transfected with scramble and ERa siRNA in a serum-free medium. After 6 h, the medium was replaced by a FBS-supplemented medium. Cells were cultured in the incubator for 18 h and then exposed to berberine for 24 h. Cell lysates were prepared and subjected to the immunoblot analysis as described in the Materials and Methods. The level of caveolin-1 was determined as the control for loading equal amount of protein of lysate. The representative blot is shown on the top. Data represent the mean ± SD of 3 separate experiments with 4 determinations in each experiment. * p < 0.05, compared with the cells without berberine-treatment.

Fig 4: mRNA and protein expression of CYP enzymes, AOEs and cytokine IL-1ß (A-C). (A) mRNA expression was analyzed in SVG cells (at 24, 48, and 72 h) treated with 1 µM concentration of BaP (daily treatment). (B) Representative western blots of IL-1ß, AOEs (SOD1 and CAT), and CYP enzymes (CYP2A6, CYP1B1, CYP1A1) in SVG cells (Western blot at 24, 48, and 72 h) treated with 1 µM concentration of BaP (daily treatment). Untreated controls were considered as 100% in the densitometry analysis. (C) Densitometry analysis. Statistical analyses were carried out by using Student’s t-test. Results are expressed as means ± S.E.M of n = 4 experiments. **P =0.01, ***P =0.001 in comparison with untreated control.

Fig 5: Effects of the protoberberines on the mRNA levels of CYP1A1, CYP1A2 and CYP1B1 in MCF-7 (A,C) and MB-231 (B) cells. Cells were exposed to B(a)P (2 µM) and protoberberines for 24 h. Total cellular RNA was isolated and subjected to reverse transcriptional reaction. In panels (A,B), the cDNA was subjected to PCR using the primer sets as described in the Materials and Methods. The PCR product was analyzed using gel electrophoresis and relative band intensity was determined. Results show the induction fold of the CYP1 mRNA level normalized by the mRNA level of ß-actin. In panel (C), CYP1A1 mRNA level was determined using real-time PCR analysis and two primer sets were used as described in the Materials and Methods. Results show the induction fold of the CYP1A1 mRNA level normalized by the mRNA level of GAPDH. Data represent the mean ± SD of 3 separate experiments. * p < 0.05, compared with the control cells.