Bifunctional modulating effects of an indigo dimer (bisindigotin) to CYP1A1 induction in H4IIE cells

In this study, we measured and characterized the bifunctional effects of a newly identified natural compound-bisindigotin (SLY-1), isolated from leaf extracts of Isatis indigotica, to CYP1A1/EROD activities in H4IIE cells. The compound, SLY-1 (1muM) elicited a transitory and significant induction of CYP1A1 RNA/protein levels and EROD activities in the cells. Maximum levels of CYP1A1 expression and EROD induction were attained at 8 and 12h of post-treatment, respectively. Thereafter the induction decreased significantly. Similar profile of CYP1A2 and CYP1B1 mRNA induction was observed. In contrast TCDD elicited CYP1A1/EROD induction was persistent. The transitory effect by SLY-1 is most likely due to the clearance of SLY-1 by cellular metabolism. Taken together the observation indicated that SLY-1 is an Ah receptor agonist for CYP1A1/CYP1A2/CYP1B1/EROD induction. Interestingly in the TCDD/SLY-1 cotreatment study, although synergistic effects on CYP1A1 expression and EROD induction were observed at 4-8h, significant inhibitory effects to TCDD induced CYP1A1 protein and EROD activity were detected at 12-24h of post-treatment. Because there was no significant reduction of CYP1A1, CYP1A2 or CYP1B1 transcript levels between TCDD- and TCDD/SLY-1 treated cells, the data pointed to the translational and/or post-translational inhibitory effect. The cellular signal transduction system may be modulated following exposure to SLY-1. To investigate the possible mechanisms involved, various specific kinase inhibitors or activators (chelerythrin, PD98059, U0126, ZM336372, SB202190, PKA inhibitor PKI (6-22) amide, and dbcAMP) were used for the assessment. Chelerythrine, PD98059 or dbcAMP treatment in TCDD induced cells showed significant inhibitory effects on CYP1A1 mRNA/protein expressions and EROD activities. U0126 had no observable EROD inhibitory effect. ZM336372 or SB202190 showed inhibition only at EROD activities. The results indicated that the SLY-1 inhibitory effect was possibly not mediated by the cAMP/PKA, PKC or MEK pathways. Nevertheless our results indicate that SLY-1 is not only an inducer of the CYP1A1 system, but also a potent inhibitor of CYP1A1 enzyme.