Chrysosplenetin, in the absence and presence of artemsininin, alters breast cancer resistance protein-mediated transport activity in Caco-2 cell monolayers using aristolochic acid I as a specific probe substrate

Zhang, Yuanyuan; Zhang, Chenxu; Chen, Jie; Ma, Liping; Yang, Bei; Wang, Jianhuan; Wu, Xiuli; Chen, Jing

doi:10.1016/j.bjp.2017.10.006

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Short communications • Rev. bras. farmacogn. 27 (6) • Nov-Dec 2017 • https://doi.org/10.1016/j.bjp.2017.10.006 copy

Chrysosplenetin, in the absence and presence of artemsininin, alters breast cancer resistance protein-mediated transport activity in Caco-2 cell monolayers using aristolochic acid I as a specific probe substrate

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ABSTRACT

The present study describes the impact of chrysosplenetin, in the absence and presence of artemisinin, on in vitro breast cancer resistance protein-mediated transport activity in Caco-2 cell monolayers using aristolochic acid I as a specific probe substrate. We observed that novobiocin, a known breast cancer resistance protein active inhibitor, increased P_{app (AP-BL)} of aristolochic acid I 3.13 fold (p < 0.05) but had no effect on P_{app (BL-AP)}. Efflux ratio (P_BA/P_AB) declined 4.44 fold (p < 0.05). Novobiocin, consequently, showed a direct facilitation on the uptake of AAI instead of its excretion. Oppositely, both artemisinin and chrysosplenetin alone at dose of 10 µM significantly decreased P_{app (BL-AP)} instead of P_{app (AP-BL)}. Chrysosplenetin alone attenuated the efflux ratio, which was suggestive of being as a potential breast cancer resistance protein suppressant. Oddly, P_{app (BL-AP)} as well as efflux ratio were respectively enhanced 2.52 and 2.58 fold (p < 0.05), when co-used with artemisinin and chrysosplenetin in ratio of 1:2. The potential reason remains unclear; it might be relative to binding sites competition between artemisinin and chrysosplenetin or the homodimer/oligomer formation of breast cancer resistance protein bridged by disulfide bonds, leading to an altered in vitro breast cancer resistance protein-mediated efflux transport function.

Keywords:
Chrysosplenetin; Artemisinin; Aristolochic acid I; Breast cancer resistance protein; Caco-2 cells monolayers

No.	Fluorescence intensity	Concentration (µg/ml)	P_app (cm/s)
1	103.26	0.012	4.04 × 10^-7
2	102.68	0.009	3.03 × 10^-7
3	101.97	0.005	1.68 × 10^-7

Theoretical concentration^a a Selected concentrations represent the low, medium and high (LQC, MQC, and HQC, respectively) concentrations. Intra-day and inter day accuracy and precision were determined with n = 5 for each concentration. %RE (% relative error) = [(Measured concentration - theoretical concentration)/theoretical concentration] × 100. %CV (% coefficient of variation) = (SD/mean) × 100. (µmol/l)	Accuracy and precision						Average extraction recoveries (%)
	Intra-day			Inter-day
	Measured concentration (µg/l)	Precision (%CV)	Accuracy (%RE)	Measured concentration (µg/l)	Precision (%CV)	Accuracy (%RE)
5	5.04 ± 0.03	0.60	0.80	5.16 ± 0.16	3.10	3.20	70.80 ± 0.32
20	20.13 ± 0.21	1.04	0.65	20.33 ± 0.43	2.12	1.65	72.40 ± 0.07
100	99.68 ± 0.90	0.90	-0.32	100.15 ± 1.90	1.90	1.50	78.30 ± 0.13

Condition	Quality control ^a a Selected concentrations represent the low, medium and high (LQC, MQC, and HQC, respectively) concentrations. Data were expressed as mean ± SD (n = 5). %RE (% relative error) = [(Measured concentration - theoretical concentration)/theoretical concentration] × 100. %CV (% coefficient of variation) = (SD/mean) × 100. (µM)	Measured concentration (ng/ml)	Precision (%CV)	Accuracy (%RE)
37 °C for 3 h	5	5.08 ± 0.24	4.72	1.60
	20	20.40 ± 1.35	6.62	2.00
	100	110.35 ± 3.56	3.23	10.35
Room temperature for 4 h	5	5.19 ± 0.32	6.17	3.80
	20	20.35 ± 2.02	9.93	1.75
	100	111.45 ± 4.15	3.72	11.45
-20 °C for 7 days	5	4.78 ± 0.22	4.60	-4.40
	20	18.43 ± 0.35	1.90	-7.85
	100	89.18 ± 3.23	3.62	-10.82

Drugs	Dosage (µM)	P_app (×10^-5cm/s)		P_BA/P_AB (efflux ratio)
		AP-BL	BL-AP
Negative control	-	3.35 ± 0.11	22.78 ± 0.00	6.80 ± 0.21
Novobiocin (positive control)	100	10.5 ± 1.46^a a p < 0.05 vs negative control. ↑	16.6 ± 0.28	1.53 ± 0.19^a a p < 0.05 vs negative control. ↓
Artemisinin alone	10	2.49 ± 0.12	15.2 ± 1.41^a a p < 0.05 vs negative control. ↓	6.09 ± 0.87
Chrysosplenetin alone	10	3.08 ± 0.06	15.5 ± 0.28^a a p < 0.05 vs negative control. ↓	5.04 ± 0.01^a a p < 0.05 vs negative control. ↓
Artemisinin:Chrysosplenetin (1:2)	10:20	3.28 ± 0.03	57.5 ± 8.62^a a p < 0.05 vs negative control. ↑	17.52 ± 2.58^a a p < 0.05 vs negative control. ↑

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[1] * Corresponding authors. xiuli2005@163.com chenjing@nxmu.edu.cn