A 24 h treatment method of AZ-AHR reporter cells, a HepG2 mobile line harboring a stably transfected XRE-driven reporter gene assemble [29], with rising concentrations of khellin and visnagin (.001 to 20 ) resulted in a dose-dependent enhance of reporter gene exercise (Determine one). A optimum 24fold (for visnagin) and 83-fold (for khellin) induction fee was observed in cells handled with twenty of the respective test compound. Noteworthy, a very first statistical substantial improve in luciferase exercise was previously noticed right after administration of one nM khellin and 10nM visnagin, respectively. Treatment method of the AZ-AHR cells with 5 of the powerful AHR agonist 3MC (positive control) led to a higher, approximately one hundred sixty-fold induction fee of XRE-driven promoter activity (Figure 1). These information place to the concept that equally furanochromones are moderate activators of hepatic AHR signaling. 280744-09-4To additional verify this notion, we analyzed the time-dependent influence of khellin (ten ) and visnagin (ten ) publicity on mRNA expression of CYP1A1 in HepG2 cells. As revealed in Determine 2A, a slight induction of CYP1A1 expression was observed soon after 8 h of remedy, while the peak expression was arrived at more eight h afterwards. At this time stage, visnagin triggered an roughly 160-fold induction of CYP1A1 transcription, whilst khellin increased the expression rate around ninety-fold. This was surprising, because in the reporter gene assays, khellin turned out to be the far more strong activator of the XRE-pushed reporter gene build. Noteworthy, we and some others have previously described very similar discrepancies among the effects received from XRE-pushed Total protein extracts for every single sample were being geared up from 1 well of 6-very well plate dish. Cells had been washed twice with ice-cold PBS and scraped into 1 ml of PBS. The suspension was centrifuged (two,300x g/two min/4) and the pellet was resuspended in 150 of ice-cold lysis buffer (a hundred and fifty mM NaCl ten mM Tris pH 7.2 .one% (w/v) SDS anti-protease cocktail, 1% (v/v) Triton X-one hundred anti-phosphatase cocktail, one% (v/v) sodium deoxycholate 5 mM EDTA). The combination was vortexed and incubated for 10 min on ice and then centrifuged (15,700x g/13 min/4). Supernatant was gathered and the protein information was identified by the Bradford reagent. SDSAGE gels (eight%) ended up operate on a BioRad apparatus according to the common procedure adopted by the protein transfer on to PVDF membrane. The membrane was saturated with five% non-body fat dried milk for 1 h at room temperature. Blots have been probed with primary antibodies against CYP1A1 (goat polyclonal, sc-9828, G-18, diluted 1:five hundred for detection in human hepatocytes rabbit polyclonal, sc-20772, H-70, diluted 1:five hundred for detection in HepG2 cells), CYP1B1 (mouse monoclonal, sc-374228, G-four, 1:one thousand), actin (goat polyclonal sc-1616, one-19, diluted 1:2000), all obtained from Santa Cruz Biotechnology (Santa Cruz, CA, Usa) or GAPDH (rabbit monoclonal, 2118, 14C10, diluted one:a thousand) obtained from Mobile Signaling Technology, overnight at 4. Chemiluminescence detection was performed making use of horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) and Western blotting Luminol kit (Santa Cruz Biotechnology). Densitometric analyses have been Determine 1. Visnagin and khellin encourage XRE-driven luciferase activity in AZ-AHR cells. Stably transfected AZ-AHR cells have been taken care of for 24 h with visnagin (VIS .001 -twenty ), khellin (KHEL, .001 -20 ), 5 3MC and/or automobile (DMSO .one% v/v). Analyses had been carried out in 4 unbiased experiments and are expressed as fold induction in excess of untreated cells. Worth is considerably various from DMSO-treated cells (p < 0.05)reporter gene assays and other indicator tests for AHR activation, such as gel retardation assays [34], EROD assays [10], and gene expression analyses [35]. To prove a direct involvement of the AHR in the observed transcriptional changes, we introduced the specific AHR antagonist 3'-methoxy-4'-nitroflavone (MNF) [36] to our expression analyses (Figure 2B). For this purpose, HepG2 cells were pre-treated for 1 h with MNF (or solvent) and subsequently were co-exposed for 16 h to 10 of either visnagin or khellin. Whereas MNF exposure alone did not significantly alter basal CYP1A1 expression, the visnagin- and khellin-mediated induction of CYP1A1 mRNA was clearly abolished in the co-exposure scenario (Figure 2B). Therefore, it is highly likely that the two furanochromones bind to the AHR and modulate downstream gene expression. Since the induction of mRNA is often correlated with the induction of protein, we performed western blotting analysis for CYP1A1. As a positive control we used 1 3MC and 5 nM TCDD. We observed massive induction of CYP1A1 protein by TCDD but almost unimportant induction by 1 3MC after 48 hrs (Figure 2C). However, both furanochromones induced CYP1A1 protein to level exceeding DMSO- as well as 3MC-treated cells (Figure 2C). To test if the enhanced CYP1A1 gene expression was translated into corresponding enzyme activities, we assayed the CYP1A1/1A2-mediated 7-ethoxyresorufin-O-dealkylase (EROD) activity in HepG2 cells treated with increasing concentrations of visnagin or khellin (Figure 2D). Treatment of the cells for 16 h and 48 h with 1 3MC resulted in a 4.3and 5.7-fold increase of EROD activity. Exposure to 5 nM TCDD increased CYP1A enzyme activity 7- (16 h) and 23.5fold (48 h), respectively. In contrast to these high-affinity AHR ligands, neither khellin nor visnagin treatment led to a significant induction of CYP1A enzyme activity after 16 h. After 48 h, we observed a slight but significant enhancement of EROD activity, which, at least for visnagin, displayed a reverse dose-response, pointing to a possible inhibition of CYP1A enzyme activities. To verify this, we pretreated HepG2 cells with 5 nM TCDD for 48 h and consequently treated the cells with the substrate mixture containing 7-ethoxy-O-resorufin with or without visnagin or khellin. As shown in Figure 2E, the TCDD-induced catalytic activity was decreased in a dosedependent manner by both compounds. Even though this observation may theoretically also reflect an interference of visnagin/khellin with the cellular uptake of 7-ethoxy-O-resorufin, the most likely explanation is a visnagin/khellin-mediated inhibition of CYP1A catalytic activity. This finding is in accordance with earlier studies on S9-treated Salmonella typhimurium TA98 cultures, showing that khellin exposure reduced the metabolic activation of various pro-mutagenic PAHs [37] and 2-amino-3-methylimidazo(4,5-f)-quinoline, which is mainly activated via CYP1A-mediated N-hydroxylation [38]. In combination with our results, exposure to visnagin and khellin would probably rather inhibit metabolic activation of procarcinogens than inducing it. The other way round, the Figure 2. Effect of visnagin and khellin exposure on CYP1A1 expression and activity in HepG2 cells. A) HepG2 cells were treated with 10 visnagin, 10 khellin, and/or DMSO for 4 h, 8 h, 16 h, and 24 h. The results from PCR are shown as fold of DMSO-treated control cells. The data are mean from three independent experiments and were normalized to beta-actin transcription. - value is significantly different from DMSO-treated cells (p < 0.05). B) HepG2 cells were pre-treated with 20 MNF for 1 h and then exposed to 10 visnagin or 10 khellin for additional 16 h. The results from PCR are shown as fold of DMSO-treated control cells. The data are mean from three independent experiments and were normalized to beta-actin transcription. - value is significantly different from DMSO-treated cells (p < 0.05). - value is significantly reduced in comparison to cells treated with VIS and KHEL, respectively (p < 0.05). C) HepG2 cells were treated with visnagin (VIS 1 -20 ), khellin (KHEL 1 -20 ), 1 3MC, 5 nM TCDD, and/or vehicle (DMSO 0.1% v/v) for 48 h. Thereafter, western blotting analyses for detection of CYP1A1 and actin were performed as described in Materials and Methods section. The representative western blot analysis of two independent experiments (passages) is presented. D) HepG2 cells were treated with visnagin (VIS 1 -20 ), khellin (KHEL 1 -20 ), 1 3MC, 5 nM TCDD, and/or vehicle (DMSO 0.1% v/v) for either 16 h (upper panel) or 48 h (lower panel). EROD activity was determined as described in Materials and Method section. Analyses were performed in three independent experiments and are shown as fold induction over untreated cells. - value is significantly different from DMSO-treated cells (p < 0.05). E) HepG2 cells were treated with TCDD (5 nM) for 48 h. Thereafter, substrate mixture was supplemented with increasing doses of visnagin (VIS 1 nM -20 ) or khellin (KHEL 1 nM -20 ) and EROD activity was determined as described in Materials and Methods section. Data are mean from three independent experiments and are expressed as percentage (%) of TCDD-mediated induction (i.e. induction by TCDD = 100%). - value is significantly different from TCDD-treated cells (p < 0.005).Figure 3. Visnagin and khellin modulate the expression of several AHR target genes in HepG2 cells. HepG2 cells were pretreated for 1 h with 20 MNF or 0.1% (v/v) DMSO and were subsequently exposed to 10 visnagin (VIS) or 10 khellin (KHEL). After 16 h, RNA was isolated and reverse transcribed and the expression of CYP1B1, AhRR, PAI-2 and VEGF was measured by qRT-PCR. The data are mean from three independent experiments and were normalized to beta-actin expression. value is significantly increased compared to DMSO-treated cells - value is significantly reduced in comparison to cells treated with VIS and KHEL, respectively (p < 0.05)inhibition of CYP1A enzyme activities may lead to alterations in the pharmacokinetics of drugs, such as the leukotriene receptor antagonist verlukast [39], the antipsychotic drug clozapine [40], and the calcium channel blocker verapamil [41]. In addition, visnagin- and khellin administration did not only modulate CYP1A1 expression but also modulated the transcription of CYP1B1 in an AHR-dependent manner. As shown in Figure 3, visnagin and khellin treatment elevated CYP1B1 gene expression, whereas MNF successfully counteracted this induction. CYP1B1 is involved in steroid breakdown [42] and of crucial relevance regarding the carcinogenicity of certain PAHs, especially 7,12dimethylbenz(a) anthracene [43,44]. Moreover, we observed increased level of CYP1B1 protein in the presence of both furanochromones as well (Figure S1). Beside their effect on the expression of the prototype target genes CYP1A1 and CYP1B1, we investigated if khellin and visnagin can influence the transcription of other AHR target genes (Figure 3). To this aim we pretreated HepG2 cells for 1 h with 20 MNF (or solvent) and subsequently exposed them for 16 h to 10 visnagin or 10 khellin, respectively. Likewise CYP1, gene expression of the AHRR is also regulated via functional XRE located in its enhancer/promoter sequence [45,46]. As expected, treatment of HepG2 cells with visnagin and khellin resulted in an increased expression of AHRR, which was also significantly attenuated by MNF pre-treatment. 25383539The AHRR protein is a negative feedback inhibitor of AHR signaling that also dimerizes with ARNT and, due to the lack of a C-terminal transactivation domain, terminates XREdependent transcription [47]. Recently, a study on primary human mammary epithelial cells and human lung cancer cells provided evidence that the AHRR is not just a negative regulator of AHR, but also a critical regulator of cell growth and apoptosis [48]. PAI-2 is an important factor influencing the growth and differentiation of cells by regulating proteolysis of the extracellular matrix [49], and PAI-2 was previously shown to be up-regulated in an AHR-dependent manner [50]. VEGF expression is induced during hypoxia to promote proliferation and migration of endothelial cells and thus is an important trigger for vasculogenesis and angiogenesis during embryonic development, wound healing and tumor growth [51,52]. Exposure of MCF10A cells to dioxin was shown to result in an increased expression of PAI-2 and VEGF, which was blunted in presence of either MNF or PP2, a src kinase inhibitor, indicating that both genes are regulated through the c-srcdependent, non-genomic AHR signaling pathway [53]. As shown in Figure 3, treatment of HepG2 cells with 10 visnagin or khellin also enhanced PAI-2 transcription in an AHR-dependent manner, as indicated by the samples cotreated with MNF. However, only visnagin significantly induced VEGF transcription in the tested concentration. This induction was again blocked by MNF co-exposure. These results strongly indicate that the two furanochromones stimulate both XRE-dependent as well as XRE-independent, non-genomic AHR signaling. This in turn points to the idea that visnagin and khellin may affect cellular functions and pathways beyond CYP-mediated metabolism, and thus may contribute to pathophysiological processes in human hepatic cells. To ensure that the AHR-activating properties identified so far were not restricted to the used hepatocarcinoma cell line, we exposed primary human hepatocytes for 24 h to visnagin and khellin and subsequently investigated CYP1A1 mRNA as well as protein expression. As expected, the AHR agonists used as positive controls, 3MC (1 ) and TCDD (5 nM), significantly induced CYP1A1 mRNA expression (Table 1). The induction varied greatly among the hepatocyte cultures from different donors, pointing to the presence of interindividual differences in CYP1A1 responsiveness, probably due to age- and genderrelated factors [54]. However, this was probably not our case since when we compared basal CYP1A1 mRNA level among hepatocyte cultures in DMSO-treated samples (Figure S2) with gender and age of donors, no correlation was observed. Nevertheless, interindividual differences became also visible after exposure of the primary hepatocytes to visnagin and khellin (1 , 10 , and 20 ). Whereas we observed a clear induction of CYP1A1 transcription in any of the four different hepatocyte cultures, the amplitude of the response varied dramatically from donor to donor (Table 1). Therefore, we decided to further investigate the effect of khellin/visnagin exposure on CYP1A1 expression in primary hepatocytes on protein level. As shown in Figure 4, 10 and 20 of both furanochromones led to a roughly 3- to 6-fold increase in CYP1A1 protein expression after 48 h, which turned out to be statistically significant for all tested concentrations (except the samples treated with 20 khellin). In its band intensity, the furanochromone-induced up-regulation was comparable to that reached upon exposure to 1 3MC (Figure 4). The observed increase in CYP1A1 protein expression upon khellin/visnagin exposure raised the idea of elevated catalytic CYP1A activities.
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