Toxicology in Vitro 28 (2014) 693–699

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Palmatine activates AhR and upregulates CYP1A activity in HepG2 cells but not in human hepatocytes Jiri Vrba a,⇑, Marika Havlikova b, Denisa Gerhardova a, Jitka Ulrichova a,b a b

Department of Medical Chemistry and Biochemistry, Faculty of Medicine and Dentistry, Palacky University, Hnevotinska 3, Olomouc 77515, Czech Republic Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Hnevotinska 3, Olomouc 77515, Czech Republic

a r t i c l e

i n f o

Article history: Received 18 November 2013 Accepted 17 February 2014 Available online 27 February 2014 Keywords: Alkaloid Palmatine Cytochrome P450 CYP1A1 Aryl hydrocarbon receptor Herb–drug interactions

a b s t r a c t The protoberberine alkaloid palmatine is present in preparations from medicinal plants such as Coptis chinensis and Corydalis yanhusuo. This study examined whether palmatine affects the expression of cytochromes P450 (CYPs) 1A1 and 1A2 in primary cultures of human hepatocytes and human hepatoma HepG2 cells grown as monolayer or spheroids. Gene reporter assays showed that palmatine significantly activated the aryl hydrocarbon receptor (AhR) and increased the activity of CYP1A1 gene promoter in transiently transfected HepG2 cells. In HepG2 monolayer culture, palmatine also significantly increased mRNA and activity levels of CYP1A1, albeit with considerably less potency than 2,3,7,8-tetrachlorodibenzo-p-dioxin, a prototypical CYP1A inducer. On the other hand, CYP1A activity was not significantly elevated by palmatine in HepG2 spheroids. Moreover, palmatine induced mild or negligible changes in CYP1A1 and CYP1A2 mRNA expression without affecting CYP1A activity levels in primary human hepatocytes. It is concluded that palmatine activates the AhR-CYP1A pathway in HepG2 monolayer, while the potential for CYP1A induction is irrelevant in cell systems which are closer to the in vivo situation, i.e. in HepG2 spheroids and primary cultures of human hepatocytes. Possible induction of CYP1A enzymes by palmatine in vivo remains to be investigated. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction The protoberberine alkaloid palmatine (Fig. 1) is one of the main bioactive components of preparations from medicinal plants such as Coptis chinensis (Ma and Ma, 2013) and Corydalis yanhusuo (Kim et al., 2011). Herbal products containing palmatine and other protoberberine alkaloids are mainly used in traditional medicine in China, Korea and India (Khan et al., 2013) for their antimicrobial, antiinflammatory, antidiabetic, hepatoprotective and analgesic effects (Ma and Ma, 2013; Yi et al., 2013). Palmatine alone has been shown, for instance, to attenuate galactosamine/lipopolysaccharide-induced hepatic failure in mice (Lee et al., 2010) and to protect the heart from ischemia/reperfusion injury in rats (Kim et al., 2009). At the molecular level, the biological activity of palmatine is presumably associated with its ability to interact with proteins (Khan et al., 2013) and nucleic acids (Bhadra and Kumar, 2011). For example, palmatine has been reported to inhibit enzymes including reverse transcriptase (Sethi, 1983) and acetylcholinesterase (Huang et al., 2012), and, to modulate expression of genes such

⇑ Corresponding author. Tel.: +420 585632310; fax: +420 585632302. E-mail address: [email protected] (J. Vrba). http://dx.doi.org/10.1016/j.tiv.2014.02.008 0887-2333/Ó 2014 Elsevier Ltd. All rights reserved.

as those encoding calmodulin 1, Janus kinase 2 and inositol polyphosphate multikinase (Suzuki et al., 2011). Herbal products are usually available without prescription, and hence patients may combine herbal remedies and conventional drugs without medical advice. It is therefore necessary to identify possible drug-drug and/or herb–drug interactions which can arise from the inhibition or induction of drug-metabolizing enzymes, particularly cytochrome P450 (CYP) enzymes (Thummel and Wilkinson, 1998). The potential for herb–drug interactions is generally rather high since phytopreparations contain complex mixtures of pharmacologically active compounds. Palmatine has been found to inhibit the activity of several human CYP enzymes including CYP1A1, CYP1B1 (Lo et al., 2013), CYP2D6 (Han et al., 2011) and CYP3A4 (Su et al., 2007). The effect of palmatine on the expression of drug-metabolizing enzymes has not been studied to date. Nonetheless, it has been shown that berberine (Fig. 1), an alkaloid structurally related to palmatine, is an activator of the aryl hydrocarbon receptor (AhR) (Vrzal et al., 2005), a ligand-dependent transcription factor that regulates the expression of CYP1A1, CYP1A2 and CYP1B1 (Nebert et al., 2004). CYP1A1 and CYP1B1 are primarily extrahepatic enzymes, while CYP1A2 is constitutively expressed in human liver (Chang and Waxman, 1998) where it metabolizes approximately 5% of therapeutic drugs (Ingelman-Sundberg and

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OCH3

O O

OCH3

+

N

CH3O OCH3

+

N

CH3O OCH3

Palmatine

Berberine

Fig. 1. Chemical structures of palmatine and berberine.

Rodriguez-Antona, 2005). All three CYP1 enzymes are also involved in metabolic activation of various procarcinogens and their induction may hence represent a severe risk for human health (Shimada and Fujii-Kuriyama, 2004). The structural similarity between palmatine and berberine inspired us to investigate whether palmatine affects the expression of CYP1A enzymes in primary cultures of human hepatocytes and human hepatoma HepG2 cells grown as monolayer or spheroids. We found that palmatine activates the AhR and increases mRNA and activity levels of CYP1A1 in HepG2 monolayer, while CYP1A-inducing activity is irrelevant in HepG2 spheroids and primary human hepatocytes. 2. Materials and methods 2.1. Chemicals Palmatine chloride (No. 361615), berberine chloride (No. B3251) and dimethyl sulfoxide (DMSO) were obtained from Sigma–Aldrich (St. Louis, MO, USA). 2,3,7,8-Tetrachlorodibenzo-pdioxin (TCDD) was obtained from Ultra Scientific (North Kingstown, RI, USA). Stock solutions of TCDD, palmatine and berberine in DMSO were stored at 20 °C. 2.2. HepG2 cell monolayer culture and treatment The human hepatocyte carcinoma HepG2 cell line (No. 85011430, ECACC, Salisbury, UK) was cultured at 37 °C in Dulbecco’s modified Eagle’s medium (No. D5796, Sigma) supplemented with 1% non-essential amino acids, 100 U/mL penicillin, 100 lg/mL streptomycin (Invitrogen, Carlsbad, CA, USA) and 10% fetal bovine serum (Biochrom, Berlin, Germany) in a humidified atmosphere containing 5% CO2. The cells were sub-cultured before confluence. For all experiments on monolayer cultures, with the exception of gene reporter assays, HepG2 cells at passage 4–15 were seeded in the complete culture medium at a density of 1  105 cells/cm2. After overnight stabilization, cells were treated in fresh medium with palmatine, berberine or 5 nM TCDD (positive control). Negative controls were treated with 0.1% (v/v) DMSO only. 2.3. Primary cultures of human hepatocytes Human tissue samples were obtained from multi-organ donors according to the protocols approved by the local ethics committee of the University Hospital, Olomouc, Czech Republic. Hepatocytes were isolated as described previously (Pichard et al., 1990) and seeded on collagen-coated culture dishes at a density of 1.3  105 cells/cm2 in culture medium (Isom et al., 1985) containing 5% bovine serum (Invitrogen). On the following day, the medium was exchanged for serum-free medium. After 24 h of stabilization,

hepatocytes were treated in fresh serum-free medium with 0.1% (v/v) DMSO (control), palmatine or 5 nM TCDD (positive control). Hepatocyte cultures used in this study were prepared from liver samples of four donors: a 46-year-old man (culture LH45), a 65-year-old man (culture LH47), a 17-year-old woman (culture LH48), and a 38-year-old man (culture LH49). 2.4. Cell viability assay After treatment of HepG2 cells and hepatocytes with 0.1% DMSO (control), palmatine or 1.5% (v/v) Triton X-100 (positive control), the cell viability was determined using an MTT reduction assay. In brief, cells were washed with PBS and incubated for 2 h at 37 °C in fresh culture medium containing 0.5 mg/ml MTT (Sigma). After this, culture medium was removed and intracellular formazan produced by active mitochondria was solubilized in DMSO containing 1% ammonia. The absorbance at 540 nm was measured on a spectrophotometric plate reader and used for calculation of relative cell viability where cells treated with DMSO only represented 100% viability. 2.5. Gene reporter assays A p1A1-luc plasmid containing 50 -flanking region (1566 to +73) of human CYP1A1 gene subcloned into the KpnI-HindIII double digested pGL3-Basic vector (Promega, Madison, WI, USA) upstream of the firefly luciferase reporter gene was originally prepared by Dr. R. Barouki (Morel and Barouki, 1998). A pDRE-luc plasmid containing two inverted repeats of the xenobiotic responsive element of mouse cyp1a1 upstream of the thymidine kinase promoter and luciferase reporter gene was a kind gift from Dr. L. Poellinger (Karolinska Institute, Stockholm, Sweden). For gene reporter assays, sub-confluent HepG2 cells were detached by trypsinization and cell suspensions in serum-free medium were transiently transfected using jetPEI transfection reagent (Polyplus Transfection, Illkirch, France) with 210 ng of p1A1-luc or pDRE-luc per 8.3  104 cells/well in 24-well plate. Following overnight incubation, cells were treated in serum-containing medium with 0.1% DMSO (control), palmatine or 5 nM TCDD (positive control). After treatment, cell extracts were prepared and analyzed using the Luciferase Assay System (Promega) on an FB12 luminometer (Berthold Detection Systems, Pforzheim, Germany). The luminescence values were used for calculation of fold changes versus control. 2.6. Quantitative real-time PCR After treatment, total RNA was extracted using the TRI Reagent Solution (Applied Biosystems, Foster City, CA, USA) and the concentration of RNA was determined by spectrophotometry at 260 nm. RNA samples (2 lg) were reverse transcribed using the

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High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Real-time PCR was performed on a LightCycler 480 II system (Roche Diagnostics, Mannheim, Germany) using the TaqMan Universal PCR Master Mix and the TaqMan Gene Expression Assays consisting of specific primers and FAM dye-labeled TaqMan minor groove binder probes (Applied Biosystems). The assay ID for CYP1A1 was Hs00153120_m1; for CYP1A2, Hs01070369_m1; for GAPDH, Hs99999905_m1; and for 18S, Hs99999901_s1, respectively. Amplification conditions were 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles with 95 °C for 15 s and 60 °C for 1 min. Crossing point values, equivalent to CT, were determined using the second derivative maximum analysis. Relative changes in gene expression were calculated by the comparative CT method using the 2DDC T equation with results normalized to GAPDH mRNA or 18S rRNA levels. 2.7. Western blot analysis After treatment, whole cell lysates were prepared and protein levels of CYP1A1/2 and actin were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis using 10% gel as described previously (Vrba et al., 2011). Primary antibodies, including goat polyclonal CYP1A1 (G-18) and goat polyclonal actin (I-19) antibodies, were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). 2.8. CYP1A activity (EROD) assay The activity of CYP1A enzymes in intact HepG2 cell monolayer cultures and primary cultures of human hepatocytes was assessed fluorimetrically as 7-ethoxyresorufin O-deethylase (EROD) activity as described previously (Vrba et al., 2011). 2.9. Organotypic cultures of HepG2 cells (HepG2 spheroids) The organotypic cultures of HepG2 cells were prepared using the Gravity Plus system (InSphero, Zurich, Switzerland). HepG2 cells (passage 4–15), cultured as described in Section 2.2, were detached before confluence by trypsinization and resuspended in fresh complete culture medium. The cell suspension was distributed into the Gravity Plus plate at 2000 cells/well in 40 lL drop. Cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2 with the medium changed by refreshing 70% of the culture volume every 2–3 days. Compact multi-cellular spheroids were observed 2–3 days after the initial seeding. On day 3 or day 4, the spheroids were transferred to the Gravity Trap plate and maintained under the same conditions. All experiments on the spheroids were started on day 10 after the initial seeding. Possible toxicity of palmatine was examined by monitoring the spheroid morphology using a Zeiss Axiovert 40 CFL microscope equipped with a 10 long-working distance objective and connected to AxioVision Rel. 4.8 imaging software (Carl Zeiss MicroImaging, Gottingen, Germany). Triton X-100 (1.5%) was used as a positive control. For the induction of CYP1A, HepG2 spheroids were treated for 24 h with 0.1% DMSO (control), palmatine, 5 nM TCDD (positive control) or 5 lM 3-methylcholanthrene (Sigma; positive control) in serum-containing medium. After treatment, the CYP1A activity was evaluated as 7-ethoxyresorufin O-deethylase (EROD) activity. The spheroids were incubated for 3 h at 37 °C in serum-free medium containing 8 lM 7-ethoxyresorufin and 10 lM dicumarol (Sigma; inhibitor of cytosolic diaphorase). After this, the fluorescence of resorufin released into the medium was read on a spectrophotometric plate reader at excitation and emission wavelengths of 530 and 585 nm, respectively. The fluorescence values were used for calculation of fold changes versus control.

2.10. Statistical analysis Results were expressed as means ± standard deviation (SD). The differences in means were analyzed by the Student t-test. One-way ANOVA was used to evaluate dose-dependence. A p value of less than 0.05 was considered as statistically significant. 3. Results 3.1. Palmatine induces AhR activity and CYP1A1 transcription in transfected HepG2 cells First, the possible cytotoxicity of palmatine in HepG2 cells grown as monolayer and primary cultures of human hepatocytes was examined using the MTT reduction assay. Palmatine was tested at concentrations of 1 to 50 lM since at these concentrations berberine, an alkaloid structurally similar to palmatine, induced AhR-dependent gene expression in vitro (Vrzal et al., 2005). After treatment of HepG2 cells for 24 h with palmatine, a small non-significant decrease in cell viability was found only at the highest concentration tested with viability reduced to 92% by 50 lM palmatine. Under the same experimental conditions, palmatine had no substantial effect on viability of human hepatocytes (Table 1). The study further examined the effect of palmatine on AhR activity and CYP1A1 transcription using HepG2 cells transiently transfected with pDRE-luc plasmid containing only binding sites for AhR or with p1A1-luc plasmid containing human CYP1A1 gene promoter. Cell exposure for 24 h to 5 nM TCDD, a potent AhR activator (Stejskalova et al., 2011), resulted in a 48-fold activation of pDRE-luc and 32-fold activation of p1A1-luc compared with the control (Fig. 2). Palmatine was also found to activate both plasmids after 24 h of treatment. The effect of palmatine was dosedependent but much weaker than that of TCDD. At a concentration of 50 lM, palmatine significantly increased the activities of pDREluc and p1A1-luc to 2.2-fold and 2.7-fold, respectively, compared with the control (Fig. 2). 3.2. Effect of palmatine on CYP1A1 and CYP1A2 mRNA levels in HepG2 monolayers and primary human hepatocytes The human CYP1A subfamily comprises two members, CYP1A1 and CYP1A2 (Ingelman-Sundberg and Rodriguez-Antona, 2005). In HepG2 monolayer cultures, only the effect of palmatine on CYP1A1 gene expression was examined since the expression of CYP1A2 gene is extremely low in the HepG2 cell line (Wilkening et al., 2003). Using quantitative real-time PCR, palmatine was found to increase CYP1A1 mRNA levels in HepG2 monolayers. Palmatine was, however, a less potent inducer of CYP1A1 gene expression than both TCDD and berberine. After 8 h of exposure, the levels of CYP1A1 mRNA induced by 50 lM palmatine, 50 lM berberine

Table 1 Effect of palmatine on the viability of HepG2 cell monolayer cultures and primary cultures of human hepatocytes. Substance

Concentration

DMSO Triton X-100 Palmatine

0.1% 1.5% 1 lM 10 lM 25 lM 50 lM

Viability (% of control) HepG2 cells

Human hepatocytes

100 0 103.7 ± 7.4 104.8 ± 6.8 99.8 ± 5.5 92.0 ± 5.2

100 0 107.1 ± 5.3 106.9 ± 3.4 108.0 ± 3.3 100.8 ± 3.6

HepG2 cells and human hepatocytes were treated for 24 h with 0.1% DMSO (control), 1–50 lM palmatine or 1.5% Triton X-100 (positive control) and the cell viability was determined by the MTT assay. Data are means ± SD of three experiments.

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Luciferase activity (fold of control)

696 70

*

60 50

pDRE-luc p1A1-luc

*

40 30 20 4 3

*

*

2

*

*

CYP1A2 mRNA levels in hepatocyte cultures LH45 and LH49. Cultures LH47 and LH48 appeared to be more responsive to palmatine. Nonetheless, the maximal levels of CYP1A1/GAPDH and CYP1A2/ GAPDH mRNA were only 2-fold to 3-fold compared with the control (Table 2). Similar changes in both CYP1A1 and CYP1A2 mRNA levels were also found when the results were normalized to 18S rRNA (data not shown).

3.3. Palmatine increases CYP1A enzyme activity in HepG2 monolayer but not in human hepatocytes and HepG2 spheroids

1 0 1

DMSO

TCDD

10

25

50

Palmatine (µM)

Fig. 2. Effect of palmatine on transcriptional activation of pDRE-luc and p1A1-luc plasmids in HepG2 cells. HepG2 cells were transiently transfected with pDRE-luc or p1A1-luc plasmids and subsequently treated for 24 h with 0.1% DMSO (control), palmatine or 5 nM TCDD (positive control). After treatment, cells were lysed and luciferase activity was determined luminometrically. Data are means ± SD of three experiments. *p < 0.05, significantly increased versus control.

and 5 nM TCDD reached 2.6-fold, 8.5-fold and 12.6-fold, respectively, when normalized to GAPDH mRNA (Fig. 3A). Similar results were also obtained when the expression of CYP1A1 mRNA was normalized to 18S rRNA (data not shown). The induction of CYP1A1 mRNA by palmatine was both time- and dose-dependent in HepG2 cells. After 24 h of exposure, the levels of CYP1A1 mRNA were significantly increased by palmatine at concentrations starting from 10 lM. At concentrations of 10, 25 and 50 lM, palmatine elevated the levels of CYP1A1/GAPDH mRNA to 3.1-fold, 4.5-fold and 6.8-fold, respectively, compared with the control (Fig. 3B). Treatment of HepG2 cells for 24 h with 50 lM berberine or 5 nM TCDD resulted in 517-fold and 484-fold increase in the levels of CYP1A1/GAPDH mRNA, respectively (Fig. 3B). As expected, TCDD induced the expression of both CYP1A1 and CYP1A2 genes in primary cultures of human hepatocytes. The levels of CYP1A1 mRNA induced by 24 h exposure to 5 nM TCDD varied between 119-fold and 167-fold while CYP1A2 mRNA levels reached 55-fold to 82-fold when normalized to GAPDH mRNA (Table 2). The effect of palmatine on CYP1A mRNA levels in human hepatocytes was weaker than that on HepG2 cells and differed according to hepatocyte culture. At a concentration of 50 lM, palmatine had negligible effect on both CYP1A1 and CYP1A2 mRNA levels after 8 h of exposure (data not shown). After 24 h, 1–50 lM palmatine caused mild or negligible changes in CYP1A1 and

The effect of palmatine on protein and activity levels of CYP1A enzymes in HepG2 cell monolayers and human hepatocytes was examined by the western blot and EROD assays. Using antibody detecting both CYP1A1 and CYP1A2, we observed an obvious increase in CYP1A protein levels in both cell types after 24 h of exposure to 5 nM TCDD (Fig. 4A). The same treatment with TCDD also caused 52-fold and 31-fold elevation in EROD activity in HepG2 monolayers and human hepatocytes, respectively, compared with the control (Fig. 4B). After 24 h of treatment with 1 to 50 lM palmatine, western blot analyses did not reveal any substantial changes in the protein levels of CYP1A enzymes in either HepG2 cell monolayers or human hepatocytes (Fig. 4A). In human hepatocytes, palmatine consistently showed negligible effect on EROD activity levels (Fig. 4B). On the other hand, a dose-dependent upregulation of EROD activity was produced by palmatine in HepG2 monolayers after 24 h of exposure. Palmatine induced a weak but significant elevation in EROD activity at as low as 1 lM concentration with the activity increased to 1.4-fold compared with the control. At the highest concentration of palmatine (50 lM), EROD activity in HepG2 cells increased 2.6-fold compared with the control (Fig. 4B). The O-deethylation of 7-ethoxyresorufin is catalyzed by three isoenzymes, namely, CYP1A1, CYP1A2 and CYP1B1 (Chang and Waxman, 1998). In case of HepG2 cells that express neither CYP1A2 (Wilkening et al., 2003) nor CYP1B1 (Kress and Greenlee, 1997), the EROD activity may be ascribed solely to the activity of CYP1A1. The study finally examined the effect of palmatine on EROD activity in HepG2 cells grown as multi-cellular spheroids which represent a more complex system than conventional cell monolayers (Mueller et al., 2011). As shown in Fig. 5A, 24 h exposure to palmatine did not impair either the structure or the compactness of HepG2 spheroids. These results agree with those showing negligible cytotoxicity of palmatine in HepG2 monolayers (see Section 3.1). In contrast to monolayer cultures, palmatine did not

Fig. 3. Relative changes in CYP1A1 gene expression by palmatine and berberine in HepG2 cell monolayers. (A) HepG2 cells were treated for 8 h with 0.1% DMSO (control), 5 nM TCDD (positive control), 50 lM palmatine or 50 lM berberine. (B) HepG2 cells were treated for 24 h with 0.1% DMSO (control), 5 nM TCDD (positive control), 1–50 lM palmatine or 50 lM berberine. The levels of CYP1A1 mRNA were determined by quantitative real-time PCR with results normalized to GAPDH mRNA. Data are means ± SD of three experiments. *p < 0.05; **p < 0.01, significantly increased versus control.

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J. Vrba et al. / Toxicology in Vitro 28 (2014) 693–699 Table 2 Relative changes in CYP1A1 and CYP1A2 gene expression by palmatine in primary cultures of human hepatocytes. Substance

Concentration

DMSO TCDD Palmatine

0.1% 5 nM 1 lM 10 lM 25 lM 50 lM

CYP1A1 mRNA (fold induction)

CYP1A2 mRNA (fold induction)

LH45

LH47

LH48

LH49

LH45

LH47

LH48

LH49

1.0 144.0 0.9 1.2 1.3 1.1

1.0 158.7 1.3 1.9 2.0 2.2

1.0 119.4 1.2 1.4 2.4 3.2

1.0 166.6 0.7 0.7 0.9 1.0

1.0 59.3 1.0 1.2 1.4 1.5

1.0 82.1 1.2 1.6 1.9 2.2

1.0 67.6 1.2 1.9 2.0 2.4

1.0 55.3 1.1 1.0 1.4 1.5

Four different cultures of human hepatocytes were treated for 24 h with 0.1% DMSO (control), 1–50 lM palmatine or 5 nM TCDD (positive control). The levels of CYP1A1 and CYP1A2 mRNA were determined by quantitative real-time PCR with results normalized to GAPDH mRNA. Data are means of duplicate measurements.

A

B

CYP1A1 Actin Hepatocyte culture LH47 CYP1A1/2 Actin DMSO

TCDD

1

10

25

50

Palmatine (µM)

EROD activity (fold of control)

HepG2 cells

80

*

70

HepG2 cells

60

Human hepatocytes

50

*

40 30 20 4 3

*

2 1

*

*

*

0 1

DMSO

TCDD

10

25

50

Palmatine (µM)

Fig. 4. Effect of palmatine on CYP1A protein and activity levels in HepG2 cell monolayers and primary cultures of human hepatocytes. HepG2 cells or human hepatocytes (as indicated) were treated for 24 h with 0.1% DMSO (control), palmatine or 5 nM TCDD (positive control). (A) After treatment, proteins in the whole cell lysates (30 lg/lane) were analyzed by western blotting and CYP1A1/2 and actin were visualized by chemiluminescent detection. Representative results are shown. (B) After treatment, CYP1A enzyme activity of intact cells was determined by the EROD assay. Data are means ± SD of three experiments. *p < 0.05, significantly increased versus control.

significantly elevate the levels of EROD activity in HepG2 spheroids after 24 h of exposure. A small non-significant increase in EROD activity was found only at the highest concentration of palmatine (50 lM) where the activity was 1.3 (±0.2)-fold compared with the control (Fig. 5B). However, HepG2 spheroids were also less responsive to TCDD in comparison with both HepG2 monolayers and human hepatocytes. After 24 h of exposure, 5 nM TCDD increased EROD activity only 3.0-fold compared with the control. Similarly, another potent CYP1A inducer 3-methylcholanthrene (Stejskalova et al., 2011) at 5 lM concentration and 24 h exposure caused a 4.4-fold increase in EROD activity in HepG2 spheroids

DMSO

Palmatine 50 µM

Palmatine 10 µM

Triton X-100

4. Discussion This study was designed to examine whether palmatine affects the AhR-CYP1A pathway with primary cultures of human hepatocytes and HepG2 cells grown as monolayer or spheroids used as in vitro models. The AhR is a ligand-dependent transcription factor with a number of both endogenous and exogenous agonists

B EROD activity (fold of control)

A

(Fig. 5B). EROD activities induced by TCDD or 3-methylcholanthrene showed no further increase when the exposure time was prolonged to 48 or 72 h (data not shown).

5

***

4 3

***

2 1 0 1

DMSO TCDD 3-MC

10

25

50

Palmatine (µM)

200 µm

Fig. 5. Effect of palmatine on morphology and CYP1A activity levels in HepG2 spheroids. (A) HepG2 spheroids were treated for 24 h with 0.1% DMSO (control), palmatine or 1.5% Triton X-100 (positive control), and the morphology of spheroids was examined by microscopy. Representative figures are shown. Scale bar represents 200 lm. (B) HepG2 spheroids were treated for 24 h with 0.1% DMSO (control), palmatine, 5 nM TCDD (positive control) or 5 lM 3-methylcholanthrene (3-MC; positive control). After treatment, CYP1A enzyme activity of intact spheroids was determined by the EROD assay. Data are means ± SD of four experiments. ***p < 0.001, significantly increased versus control.

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identified to date. Classic exogenous compounds that bind and activate the AhR are hydrophobic, planar or coplanar, polycyclic molecules such as polycyclic aromatic hydrocarbons and halogenated dioxins. Upon ligand binding, the AhR translocates from cytosol to nucleus where it associates with the AhR nuclear translocator (ARNT). The ligand-activated AhR/ARNT complex binds to the DNA at xenobiotic responsive elements and triggers the expression of AhR target genes including those encoding CYP1A1 and CYP1A2 (Stejskalova et al., 2011). CYP1A1 is present in extrahepatic tissues but it is inducible in the liver as well. In contrast, CYP1A2 is a constitutively expressed hepatic enzyme (Chang and Waxman, 1998). The CYP1A enzymes are known to metabolize both endogenous compounds, e.g. eicosanoids, and xenobiotics such as acetaminophen, theophylline and other drugs (Nebert and Karp, 2008). This aside, both enzymes have been recognized to play a negative role in chemical carcinogenesis due to their involvement in metabolic activation of procarcinogens including polycyclic aromatic hydrocarbons and aromatic amines (Shimada and Fujii-Kuriyama, 2004). In this study, we showed, using gene reporter assays, that palmatine at non-cytotoxic concentrations significantly activated the AhR and increased the activity of CYP1A1 gene promoter in transfected HepG2 cells. At concentrations of 10 lM and above, palmatine also significantly elevated CYP1A1 mRNA levels in HepG2 cell monolayer cultures. However, these effects were much lower than those induced by TCDD, a prototypical activator of the AhR-CYP1A pathway. These findings suggest that palmatine, like other plantderived AhR activators (Moon et al., 2006; Vrba et al., 2012), might act as a partial agonist of the AhR. The effect of palmatine on CYP1A1 gene expression was also weaker than that of berberine. We may presume that the affinity of palmatine to AhR is lower than that of berberine since palmatine comprises, on the same tetracyclic structure, two extra methoxy groups instead of one methylenedioxy group which is part of a nearly planar 1,3-dioxole ring in the molecule of berberine (Behre et al., 2012). Although no changes in protein levels of CYP1A1 were detected in HepG2 monolayers by western blotting, presumably due to the low sensitivity of this technique, we found that the induction of CYP1A1 mRNA expression by palmatine was accompanied by modest upregulation of CYP1A1 activity. The increased CYP1A1 activity was observed, despite the fact that palmatine was reported to suppress EROD activity of human recombinant CYP1A1 with IC50 value of 8.7 lM (Lo et al., 2013). This indicates that the intracellular concentrations of palmatine after 24 h of exposure do not reach the values required for efficient enzyme inhibition in intact cells. In contrast to HepG2 cells grown in monolayer, the activity levels of CYP1A were not significantly increased by palmatine in HepG2 spheroids. This three-dimensional in vitro system is considered to represent an organotypic HepG2 cell culture that better reflects in vivo situation than conventional monolayer culture. For instance, HepG2 cells in spheroids have higher drug efflux activity than cells in monolayer (Mueller et al., 2011). Moreover, HepG2 spheroids also have higher CYP1A activity and higher expression levels of genes encoding xenobiotic-metabolizing enzymes, including CYP1A1 and CYP1A2, compared with HepG2 monolayer (Nakamura et al., 2011a,b). Similarly, human hepatocytes, which represent a fully metabolically competent in vitro cell model, also show higher expression of CYP1A1 and CYP1A2 genes than HepG2 cells (Wilkening et al., 2003). In this study, we used four different primary cultures of human hepatocytes responsive to TCDD but palmatine was found to induce only mild or negligible changes in both CYP1A1 and CYP1A2 mRNA levels. This aside, palmatine affected neither protein nor activity levels of CYP1A enzymes in human hepatocytes. Our results therefore suggest that the weak CYP1A-inducing activity of palmatine may be apparent only in cell systems with low levels of CYP1A expression, e.g. in HepG2

monolayer. Since monolayer cultures of HepG2 cells are known to have only limited metabolic activity (Wilkening et al., 2003), the lack of the CYP1A-inducing activity in HepG2 spheroids and primary human hepatocytes could also be associated with metabolic inactivation of palmatine. Metabolic studies on palmatine in humans are not available but extensive metabolism of the alkaloid was found in rats with phase I and phase II metabolites identified in plasma, urine and feces (Yang et al., 2009; Zhu et al., 2007). It has been shown that the intestinal microflora and liver microsomes are responsible for the metabolism of palmatine in rats but individual enzymes involved remain unknown (Yang et al., 2009). Pharmacokinetic data for palmatine in humans have not been published but animal studies suggest that plasma concentrations of palmatine do not reach the values at which the alkaloid induces the expression of CYP1A1 in HepG2 cells. A pharmacokinetic study in rats after oral administration of a multi-herbal extract, equivalent to 90 mg of palmatine per kg of body weight, showed that the maximum plasma concentration of palmatine (the parent compound) reached 76.3 lg/L, i.e. 0.22 lM (Zhu et al., 2013). In other pharmacokinetic studies, the maximum concentrations of palmatine found in rat plasma were even lower (Feng et al., 2010; Ma et al., 2009). However, it may be assumed that concentrations of palmatine, for instance, in the intestine and in the liver are higher than those found in plasma, and hence possible upregulation of CYP1A enzymes in vivo cannot be ruled out. Taken together, we conclude that palmatine activates the AhR-CYP1A pathway in HepG2 cell monolayer, while the potential for CYP1A induction is irrelevant in cell systems which are closer to the in vivo situation, i.e. in HepG2 spheroids and primary cultures of human hepatocytes. The safety of palmatine in terms of possible induction of CYP1A enzymes in vivo remains to be evaluated. Conflict of Interest The authors declare that there are no conflicts of interest. Transparency Document The Transparency document associated with this article can be found in the online version.

Acknowledgements We thank Dr. Alexander Oulton for providing linguistic assistance. This work was supported by grant from the Ministry of Education, Youth and Sports of the Czech Republic (No. CZ.1.07/2.3.00/ 30.0004) and by grant from Palacky University (No. LF_2013_008). References Behre, J., Voigt, R., Althofer, I., Schuster, S., 2012. On the evolutionary significance of the size and planarity of the proline ring. Naturwissenschaften 99, 789–799. Bhadra, K., Kumar, G.S., 2011. Therapeutic potential of nucleic acid-binding isoquinoline alkaloids: binding aspects and implications for drug design. Med. Res. Rev. 31, 821–862. Chang, T.K., Waxman, D.J., 1998. Enzymatic analysis of cDNA-expressed human CYP1A1, CYP1A2, and CYP1B1 with 7-ethoxyresorufin as substrate. Methods Mol. Biol. 107, 103–109. Feng, J., Xu, W., Tao, X., Wei, H., Cai, F., Jiang, B., Chen, W., 2010. Simultaneous determination of baicalin, baicalein, wogonin, berberine, palmatine and jatrorrhizine in rat plasma by liquid chromatography-tandem mass spectrometry and application in pharmacokinetic studies after oral administration of traditional Chinese medicinal preparations containing scutellaria-coptis herb couple. J. Pharm. Biomed. Anal. 53, 591–598. Han, Y.L., Yu, H.L., Li, D., Meng, X.L., Zhou, Z.Y., Yu, Q., Zhang, X.Y., Wang, F.J., Guo, C., 2011. In vitro inhibition of Huanglian [Rhizoma coptidis (L.)] and its six active alkaloids on six cytochrome P450 isoforms in human liver microsomes. Phytother. Res. 25, 1660–1665.

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