Cytotoxicity and P-Glycoprotein Modulating Effects of Quinolones and Indoloquinazolines from the Chinese Herb Evodia rutaecarpa

• Abstract
• Introduction
• Materials and Methods
• Test substances
• Principle of the cell growth inhibition assay
• Principle of the calcein assay
• Isolation of porcine brain capillary endothelial cells (PBCECs)
• Results and Discussion
• Acknowledgements
• References

Abstract

The antimycobacterial quinolones 1-methyl-2-undecyl-4-quinolone, dihydroevocarpine and evocarpine as well as the indoloquinazoline alkaloids rutaecarpine and evodiamine - all from the Chinese medicinal herb Evodia rutaecarpa - were tested in two in vitro assays, for cytotoxicity and interaction with p-glycoprotein (p-gp). Cytotoxicity was measured in a cell proliferation assay against CCRF-CEM leukemia cells and their p-gp over-expressing subline CEM/ADR5000. An assay monitoring the p-gp-dependent accumulation of the dye calcein in porcine brain capillary endothelial cells (PBCECs) was used to study interactions of the test substances with this efflux pump. Rutaecarpine and evodiamine showed quite high toxicity with IC₅₀ values from 2.64 to 4.53 μM and were weak modulators of p-gp activity. The degrees of resistance in CEM/ADR5000 towards the saturated quinolones 1-methyl-2-undecyl-4-quinolone and dihydroevocarpine were between 3 and 4. In the calcein assay, these two quinolones were shown to be moderate modulators of p-gp activity. Evocarpine, on the other side, is not transported by p-gp, and showed only slight toxicity at the highest test concentration of 30 μM.

Introduction

Recently we reported the antimycobacterial effects of quinolones from the fruits of the Chinese medicinal herb Evodia rutaecarpa (Juss.) Benth. (Rutaceae). The most abundant quinolone, evocarpine (3) had been the most active with a minimal inhibitory concentration (MIC) of 2 μg/mL (5.9 μM) against Mycobacterium fortuitum, M. smegmatis and M. phlei test strains [1]. Besides the evocarpine analogues, E. rutaecarpa contains indoloquinazoline alkaloids such as rutaecarpine and evodiamine, flavanoids, limonoids and essential oil [2]. In order to gain more insight into whether these unusual quinolones have potential as leads to therapeutics, we studied possible toxic effects in cell proliferation assays with human CCRF-CEM leukemia cells and their p-glycoprotein (p-gp) over-expressing subline CEM/ADR5000. Additionally, the substances were tested for their modulatory effect on p-gp activity in a microplate scale assay using porcine brain capillary endothelial cells (PBCECs). The blood-brain barrier (BBB), the physical barrier between blood vessels and the central nervous system (CNS), stops many substances from entering the CNS. Because clinically used drugs may show CNS toxicity, it is important to elucidate their distribution in the brain [3], [4]. Some well-studied quinolone antibiotics show very low distribution in the brain, due to ATP-dependent efflux pumps [5] such as p-glycoprotein (p-gp), which transports them back into the blood circulation.

We tested the three quinolones 1-methyl-2-undecyl-4-quinolone (1), dihydroevocarpine (2) and evocarpine (3) together with the indoloquinazoline alkaloids rutaecarpine (4) and evodiamine (5), which are all main constituents in medicinally used E. rutaecarpa fruits [2].

Materials and Methods

Test substances

Compounds 1 - 5 were isolated from the fruits of Evodia rutaecarpa as previously described [6].

Principle of the cell growth inhibition assay

CCRF-CEM, and CEM/ADR5000 cells (kindly provided by Dr. Axel Sauerbrey, Department of Pediatrics, University of Jena, Jena, Germany) were incubated with test substance for seven days, after which they were counted and compared to the cell number of a vehicle-treated (DMSO) control. The results represent the net outcome of cell death and cell proliferation [7]. CEM/ADR5000 cells are a p-gp over-expressing multidrug-resistant subline of CCRF-CEM, resistant to drugs such as doxorubicin, vincristine, and paclitaxel. Briefly, aliquots of 5 × 10⁴ cells/mL were seeded in 24-well plates. Test compounds were added immediately. The number of cells in four 10 μL aliquots from each well was counted visually seven days after treatment, using a cell counting chamber (improved Neubauer chamber, Brand GmbH + Co. KG.: Wertheim, Germany), under a light microscope. The compounds were tested at least three times with different cell preparations at dilutions 0.01, 0.03, 0.1, 0.3, 1, 3 and 10 μg/mL, against both CCRF-CEM and CEM/ADR5000 cells. Mean values and the standard deviations were determined (Microsoft, Excel). IC₅₀ values with fiducial limits were calculated by probit-log analysis using SPSS 6.0 for MS Windows as described for a quantitative biological assay [8]. The degree of cross-resistance of a substance can be calculated by dividing the IC₅₀ of CEM/ADR5000 by the IC₅₀ of CCRF-CEM. Because p-gp limits the accumulation of certain substances in the brain, resistance in p-gp over-expressing CEM/ADR5000 cells compared to CCRF-CEM cells can serve as a model for the transport of compounds across the BBB via p-gp [7].

Principle of the calcein assay

The assay with PBCECs was performed as described by Bauer et al. [9]. Briefly, PBCECs were isolated and cultured in 96-well plates, with calcein acetoxymethyl ester (calcein-AM) in the absence and presence of test substances. Calcein, a fluorescent dye, is formed by intracellular hydrolysis of the non-fluorescent calcein acetoxymethyl ester (calcein-AM), a p-gp substrate. Test substances, which interact with p-gp, either result in an increased intracellular concentration of calcein, by inhibiting p-gp activity or in a reduced amount of fluorescence by inducing p-gp efflux activity. After washing and lysing the PBCECs, the extent of intracellularly accumulating fluorescence was monitored with a fluorescence plate reader (Fluoroskan Askent, Labsystems; Helsinki, Finnland). Verapamil (Knoll AG; Ludwigshafen, Germany) was used as a positive control. In parallel, the toxicity of the substances towards PBCECs was measured by adding alamar blue which is reduced in the environment inside viable cells. No strong reduction (>10 %) of the cell viability was seen even at the highest test concentration of 50 uM (data not shown).

The tests were performed at least three times at the concentrations 0.1, 1, 5, 10, 25 and 50 μM. Data are given as mean values with their standard deviations. Control and treatment groups were compared by either Student’s t-test or one-way analysis of variance, followed by Dunnett’s post hoc test. Differences were considered significant at * p < 0.01. The p-gp modulating effect of a substance in this test design can be distinguished as strong p-gp modifiers (effect > 500 % at < 10 μM), moderate (effect of 250 - 500 % at 10 - 50 μM), and weak modulators (effect of 125 - 250 % at 10 - 50 μM). Substances with an effect < 125 % at 10 - 250 μM were classified as non-modulators [9].

Isolation of porcine brain capillary endothelial cells (PBCECs)

PBCECs were isolated as previously described [10]: Briefly, cortical grey matter from fresh porcine brains was minced and digested enzymatically using 0.5 % dispase (Roche; Mannheim, Germany). Cerebral microvessels were obtained after centrifugation in 15 % dextran (Sigma-Aldrich; Taufkirchen, Germany) and were subsequently incubated in buffer containing 1 mg/mL collagenase/dispase (Roche). The resulting cell suspension was supplemented with 10 % horse serum (Biochrom; Berlin, Germany) and filtered through 150 μm nylon mesh, and PBCECs were separated on a discontinuous Percoll® (Sigma) gradient consisting of Percoll® 1.03 g/mL^-1 (20 mL) and 1.07 g/mL^-1 (15mL). Isolated PBCECs were filtered through 80 μm nylon mesh before being seeded, at a density of 250,000 cells/cm², onto collagen (Roche)-coated 96-well cell culture plates. Cells were cultivated at standard conditions in Medium 199 containing 100 μg/mL streptomycin, 100 μg/mL penicillin G, 10 mmol/L HEPES, 10 % heat-inactivated horse serum and 0.8 mmol/L glutamine (all from Biochrom; Berlin, Germany). After reaching confluency, the medium was changed after 6 days in culture and cells were grown in 45 % DMEM, 45 % F12-HAM, 100 μg/mL streptomycin, 100 μg/mL penicillin G, 10 mmol/L HEPES and 2 mmol/L glutamine (all from Biochrom). On the following day, medium was removed from each well and cells were washed twice with a Krebs-Ringer buffer (37 °C) and were subsequently used for transport experiments.

Results and Discussion

In the cell proliferation assay the quinolones showed toxicity dependent on the saturation of their side chain. This can be seen in the difference in absolute toxicity shown in the IC₅₀ values of 1-methyl-2-undecyl-4-quinolone (1) and dihydroevocarpine (2) in both cell lines (Fig. [1]). Evocarpine (3), the only unsaturated quinolone tested, showed only slight toxicity in both cell lines at the highest test concentration of 30 μM (13 % and 0 % inhibition against CCRF-CEM and CEM/ADR5000, respectively), whereas the saturated quinolones 1 and 2 showed clear IC₅₀ values of 4.56 and 14.08 μM, respectively. Furthermore, 1 and 2 showed cross-resistance in the multi-resistant CEM/ADR5000 cells with IC₅₀ values of 17.19 and 33.14 μM, respectively (Fig. [1]). This effect is reproducible and consistent. It is, however, small with resistance quotients of roughly only 3 - 4.

Interaction with p-gp can also be observed in the assay using PBCECs, where 1 and 2 showed no increase in intracellular dye compared to the controls at 0.1 and 1 μM (Fig. [2]). At concentrations of 5, 10 and 25 μM, the fluorescence rose to between twice and three times that of the vehicle-treated control. In the calcein assay compound 1, which had the highest toxicity amongst the quinolones in the cell proliferation assay, showed a fluorescence activity of approximately twice that of the control at 5 μM. This rose to approximately three times that of the control at 10 and 25 μM, before dropping to about 200 % at 50 μM. Substance 2 has a similar pattern with maximum fluorescence activity at 10 μM, and a gradual decrease thereafter (Fig. [2]). Compounds 1 and 2 can be classified as moderate modifiers of p-gp. In both test systems the activity of evocarpine (3) was quite different from the other quinolones, as it was not transported by p-gp in PBCECs.

Against CCRF-CEM the indoloquinazoline alkaloids 4 and 5 exhibited IC₅₀ values of 4.53 and 3.30 μM, respectively, which is little different from their toxicity towards multi-resistant CEM/ADR5000 cells, where they had IC₅₀ values of 2.79 and 2.64 μM. In the calcein assay using PBCECs, 5 showed no significant increase in cellular fluorescence compared to the controls at 0.1 and 1 μM. At higher concentrations of 25 and 50 μM, this rose to about twice that of the vehicle-treated control. Compound 4 did not seem to have much influence on p-gp at 0.1 and 1 μM as well. At 5 μM it had a two-fold higher activity than the control. This peaked at 280 % at 10 μM, before falling to 260 and 210 % at 25 and 50 μM, respectively (Fig. [2]). According to Bauer et al. [10] both 4 and 5 can be classified as weak modulators of p-gp.

Previously, we have reported evocarpine (3) to have an MIC value of 2 μg/mL (5.9 μM) against strains of Mycobacterium fortuitum, M. smegmatis and M. phlei [1]. Against these very sensitive human leukaemia cell lines, however, it showed no cell proliferation inhibiting effects at 10 μM and only very slight cell activity at 30 μM. These in vitro data, therefore, suggest little toxicological threat from the use of the non-saturated quinolone evocarpine (3). In the blood-brain barrier model with PBCECs, 3 is not transported by p-gp activity, quite opposite to the saturated quinolones 1 and 2, which are also somewhat more toxic in CCRF-CEM cells. The indoloquinazolines evodiamine and rutaecarpine, also present in the fruits of Evodia rutaecarpa, were both shown to be toxic and to weakly modulate p-gp. This leads to the conclusion that, from a toxicological point of view, evocarpine should be applied as a pure compound in drug therapy and not in the form of an extract.

Liao et al. [11] described the in vitro and in vivo antitumor activity of evodiamine (5) in human multi-drug resistant breast cancer cells. Dehydroevodiamine attenuates tau hyperphosphorylation and spatial memory deficit induced by activation of glycogen synthase kinase-3 in rats [12]. The ability of indoloquinazolines from E. rutaecarpa to pass the blood-brain barrier would support this effect.

Acknowledgements

M. Adams was supported on a research grant form the Heinrich Jörg Foundation (Graz, Austria) to do these studies.

References

• 1 Adams M, Wube A A, Bucar F, Bauer R, Kunert O, Haslinger E. Quinolone alkaloids from Evodia rutaecarpa: a potent new group of antimycobacterial compounds.  Int J Antimicrob Agents. 2005;  26 262-4.
  PubMedSearch in Google Scholar
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• 5 Tamai I, Yamashita J, Kido Y. Limited distribution of new antibacterial agents into brain caused by multiple efflux transporters at the blood-brain-barrier.  J Pharmacol Exp Ther. 2000;  295 146-52.
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• 6 Adams M, Kunert O, Haslinger E, Bauer R. Inhibition of leukotriene biosynthesis by quinolone alkaloids from the fruits of Evodia rutaecarpa .  Planta Med. 2004;  70 904-8.
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• 7 Efferth T, Davey M, Olbrich A, Ruecker G, Gebhart E, Davey R. Activity of drugs from traditional Chinese medicine toward sensitive and MDR1- or MRP1-overexpressing multidrug-resistant human CCRF-CEM leukemia cells.  Blood Cells Mol Dis. 2001;  28 160-8.
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• 8 Finney J D. Statistical methods in biological assay. London; Charles Griffin 1978: 58-65.
  Search in Google Scholar
• 9 Bauer B, Miller D S, Fricker G. Compound profiling for P-glycoprotein at the blood - brain barrier using a microplate screening system.  Pharm Res. 2003;  20 1170-6.
  CrossrefPubMedSearch in Google Scholar
• 10 Fellner S, Bauer B, Miller D S, Schaffrik M, Fankhanel M, Spruss T. et al . Transport of paclitaxel (Taxol) across the blood-brain barrier in vitro and in vivo .  J Clin Invest. 2002;  110 1309-18.
  CrossrefPubMedSearch in Google Scholar
• 11 Liao C -H, Pan S L, Guh J H, Chang Y L, Pai H C, Lin C H. et al . Antitumor mechanism of evodiamine, a constituent from Chinese herb Evodiae fructus, in human multiple-drug resistant breast cancer NCI/ADR-RES cells in vitro and in vivo .  Carcinogenesis. 2005;  26 968-75.
  PubMedSearch in Google Scholar
• 12 Peng J H, Zhang C E, Wei W, Hong X P, Pan X P, Wang J Z. Dehydroevodiamine attenuates tau hyperphosphorylation and spatial memory deficit induced by activation of glycogen synthase kinase-3 in rats.  Neuropharmacology. 2007;  52 1521-7.
  CrossrefPubMedSearch in Google Scholar

Prof. Dr. Rudolf Bauer

Institut für Pharmazeutische Wissenschaften

Abteilung für Pharmakognosie

Universitätsplatz

8010 Graz

Austria

Phone: +43-(0)316-380-8700

Fax: +43-(0)316-380-9860

Email: rudolf.bauer@uni-graz.at

References

• 1 Adams M, Wube A A, Bucar F, Bauer R, Kunert O, Haslinger E. Quinolone alkaloids from Evodia rutaecarpa: a potent new group of antimycobacterial compounds.  Int J Antimicrob Agents. 2005;  26 262-4.
  PubMedSearch in Google Scholar
• 2 Chuang W C, Cheng C M, Chang H C, Chen Y P, Sheu S J. Contents of constituents in mature and immature fruits of Evodia species.  Planta Med. 1999;  65 567-71.
  Thieme ConnectPubMedSearch in Google Scholar
• 3 Ambudkar S V, Dey S, Hrycyna C A, Ramachandra M, Pastan I, Gottesman M M. Biochemical, cellular and pharmacological aspects of the multidrug transporter.  Ann Rev Pharmacol Toxicol. 1999;  39 361-98.
  CrossrefPubMedSearch in Google Scholar
• 4 Schinkel A H. P-glycoprotein, a gatekeeper in the blood-brain barrier.  Adv Drug Deliv Rev. 1999;  36 179-94.
  CrossrefPubMedSearch in Google Scholar
• 5 Tamai I, Yamashita J, Kido Y. Limited distribution of new antibacterial agents into brain caused by multiple efflux transporters at the blood-brain-barrier.  J Pharmacol Exp Ther. 2000;  295 146-52.
  PubMedSearch in Google Scholar
• 6 Adams M, Kunert O, Haslinger E, Bauer R. Inhibition of leukotriene biosynthesis by quinolone alkaloids from the fruits of Evodia rutaecarpa .  Planta Med. 2004;  70 904-8.
  Thieme ConnectPubMedSearch in Google Scholar
• 7 Efferth T, Davey M, Olbrich A, Ruecker G, Gebhart E, Davey R. Activity of drugs from traditional Chinese medicine toward sensitive and MDR1- or MRP1-overexpressing multidrug-resistant human CCRF-CEM leukemia cells.  Blood Cells Mol Dis. 2001;  28 160-8.
  PubMedSearch in Google Scholar
• 8 Finney J D. Statistical methods in biological assay. London; Charles Griffin 1978: 58-65.
  Search in Google Scholar
• 9 Bauer B, Miller D S, Fricker G. Compound profiling for P-glycoprotein at the blood - brain barrier using a microplate screening system.  Pharm Res. 2003;  20 1170-6.
  CrossrefPubMedSearch in Google Scholar
• 10 Fellner S, Bauer B, Miller D S, Schaffrik M, Fankhanel M, Spruss T. et al . Transport of paclitaxel (Taxol) across the blood-brain barrier in vitro and in vivo .  J Clin Invest. 2002;  110 1309-18.
  CrossrefPubMedSearch in Google Scholar
• 11 Liao C -H, Pan S L, Guh J H, Chang Y L, Pai H C, Lin C H. et al . Antitumor mechanism of evodiamine, a constituent from Chinese herb Evodiae fructus, in human multiple-drug resistant breast cancer NCI/ADR-RES cells in vitro and in vivo .  Carcinogenesis. 2005;  26 968-75.
  PubMedSearch in Google Scholar
• 12 Peng J H, Zhang C E, Wei W, Hong X P, Pan X P, Wang J Z. Dehydroevodiamine attenuates tau hyperphosphorylation and spatial memory deficit induced by activation of glycogen synthase kinase-3 in rats.  Neuropharmacology. 2007;  52 1521-7.
  CrossrefPubMedSearch in Google Scholar

Prof. Dr. Rudolf Bauer

Institut für Pharmazeutische Wissenschaften

Abteilung für Pharmakognosie

Universitätsplatz

8010 Graz

Austria

Phone: +43-(0)316-380-8700

Fax: +43-(0)316-380-9860

Email: rudolf.bauer@uni-graz.at