1. Metformin suppresses CYP1A1 and CYP1B1 expression in breast
1.1. Metformin inhibits CYP1A1 and CYP1B1 expression
An initial experiment was conducted to investigate effects of metformin on CYP1A1 and CYP1B1 expression in breast cancer cells. MCF-7 cells were treated with metformin (1–5 mM) for 24 h and Western blotting was performed to assess the levels of CYP1A1 and CYP1B1 proteins in cell lysates. As shown in Fig. 1A, metformin treatment down-regulated CYP1A1 and CYP1B1 protein levels in a dose-dependent manner. Subsequently, the time-course effect was investigated in cells treated with 2.5 mM metformin for 6, 12, and 24 h. Western blotting data indicated that metformin inhibited expression of CYP1A1 and CYP1B1 protein in a time-dependent manner (Fig. 1B). To evaluate whether the inhibitory effects of metformin on CYP1A1 and CYP1B1 expression were modulated at the level of transcription, MCF-7 cells were treated with metformin under the same conditions, and mRNA expression was analysed by using qRT-PCR.
Consistent with the Western blotting results, metformin strongly suppressed
dependent manners (Fig. 1C and D). To determine whether metformin inhibited the transcription of CYP1B1 mRNA, MCF-7 cells were transfected with CYP1B1-Luc promoter constructs followed by metformin treatment (1–
5 mM) for 24 h. Metformin significantly suppressed CYP1B1 promoter luciferase activity (Fig. 1E). Notably, TCDD treatment strongly increased CYP1A1 and CYP1B1 protein levels, and TCDD-induced CYP1A1 and CYP1B1 induction was abolished by metformin pre-treatment in MCF-7 cells (Fig. 1F). These results indicated that metformin strongly down- regulated CYP1A1 and CYP1B1 expression in breast cancer cells regardless of the presence of xenobiotics.
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Fig. 1. Metformin down-regulates CYP1A1 and CYP1B1 transcription in MCF-7 breast cancer cells. CYP1A1, CYP1B1 and -actin in cell lysates were analysed by Western blotting; results suggested that metformin suppressed CYP1A1 and CYP1B1 protein levels in a dose- (A) and time- (B) dependent manner. CYP1A1 and CYP1B1 mRNAs were analysed by qRT- PCR; results suggested that metformin repressed CYP1A1 and CYP1B1 mRNA levels in a dose- (C) and time- (D) dependent manner. All experiments were performed in triplicate. Bars indicate mean ± SD. *P <
0.05 vs. the control. (E) Metformin inhibits CYP1B1 luciferase promoter activity. All experiments were performed in triplicate. Bars indicate mean ± SD. *P < 0.05 vs. the control. (F) Western blotting analysis showed that metformin attenuated the TCDD-induced CYP1A1 and CYP1B1 protein levels.
1.2. Down-regulation of AhR expression is required for the suppression of CYP1A1 and CYP1B1 by metformin
Previous studies have reported that AhR plays a crucial role in regulation of CYP1A1 and CYP1B1 transcription (Tsuchiya et al., 2003). Therefore, further experiments were conducted to determine the effects of metformin on AhR expression under basal conditions in breast cancer cells. MCF-7 cells were treated with metformin (1–5 mM) for 24 h, and AhR protein in cell lysates was analysed by Western blotting. Metformin suppressed AhR protein levels in whole-cell lysates in a dose-dependent manner (Fig. 2A).
Additionally, metformin reduced AhR protein levels in time-dependent manner (Fig. 2B, upper panel). Moreover, the nuclear protein extraction was performed to analyze the expression of nuclear AhR protein. Metformin markedly inhibited nuclear AhR accumulation (Fig. 2B, lower panel).
Notably, other partners of AhR for its stability, heat-shock protein 90 (Hsp90) and activation ARNT proteins, were not affected by metformin treatment (Fig. 2A). These results suggest that metformin may suppress AhR expression by affecting its transcription rather than by degradation. This was addressed by treating MCF-7 cells with metformin at the same dose and time. Metformin strongly reduced AhR mRNA in a dose- and time- dependent manner in breast cancer cells (Fig. 2C and D), which supports the
hypothesis that metformin down-regulates AhR expression at the transcriptional level.
Metformin treatment alone down-regulated AhR expression and suppressed CYP1B1 promoter luciferase activity in breast cancer cells. To confirm whether the transcriptional factor AhR plays an important role in the down-regulation of CYP1A1 and CYP1B1 by metformin, MCF-7 cells were transiently transfected with a XRE promoter luciferase construct and treated with metformin (1–5 mM) for 24 h. Metformin treatment significantly suppressed XRE luciferase activity. Moreover, pre-treatment of metformin for 1 h markedly attenuated TCDD-induced XRE luciferase activity in breast cancer cells (Fig. 3A). Additional evidence was provided by siRNA- mediated knockdown of AhR expression. As indicated in Fig. 3B, AhR siRNA markedly reduced constitutive AhR, CYP1A1 and CYP1B1 protein levels. These results demonstrated that AhR down-regulation plays a crucial role in metformin-mediated suppression of CYP1A1 and CYP1B1 expression.
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Fig. 2. Metformin down-regulates AhR expression in MCF-7 breast cancer cells. (A) Effects of metformin on AhR protein expression. AhR, Hsp90, ARNT and -actin protein levels in cell lysates were assayed by Western blotting. (B) Time course of the effects of metformin on AhR protein expression. MCF-7 cells were treated with 2.5 mM metformin for indicated time (upper panel). Western blotting analysis of AhR, -actin and lamin B1 proteins from cytosolic or nuclear fractions in MCF-7 breast cancer cells (lower panel). qRT-PCR analysis showed that metformin reduces AhR mRNA levels in a dose- (C) and time- (D) dependent manner. All experiments were performed in triplicate. Bars indicate mean ± SD. *P <
0.05 vs. the control.
Fig. 3. Down-regulation of AhR expression is required for the reduction of CYP1A1 and CYP1B1 by metformin in MCF-7 cells. (A) Metformin inhibits constitutive and TCDD-induced XRE luciferase promoter activity. MCF-7 cells were transiently transfected with a XRE promoter luciferase construct and pre-treated with metformin (1–5 mM) for 1 h, followed by exposure to TCDD for an additional 24 h. All experiments were performed in triplicate.
Bars indicate mean ± SD. *P < 0.05 vs. the control. #P < 0.05 vs. cells treated TCDD alone. (B) AhR siRNA suppresses CYP1A1 and CYP1B1 protein levels. MCF-7 cells were transfected with AhR siRNA or control siRNA for 48 h, and the AhR, CYP1A1, CYP1B1 and -actin protein levels were analyzed by Western blotting.
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