Avermectin induces P-glycoprotein expression in S2 cells via the calcium/calmodulin/NF-jB pathway Liang Luo a,b , Yin-Jian Sun c , Lin Yang a , Shile Huang d,⇑ , Yi-Jun Wu a,⇑ a Laboratory of Molecular Toxicology, State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, PR China b Graduate University of the Chinese Academy of Sciences, Beijing 100039, PR China c Department of Veterinary Medicine and Animal Science, Beijing University of Agriculture, Beijing 102206, PR China d Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, LA 71130, USA article info Article history: Received 30 November 2012 Received in revised form 11 February 2013 Accepted 9 March 2013 Available online 22 March 2013 Keywords: P-glycoprotein Avermectin Calcium Chloride NF-jB abstract Avermectin (AVM) is a macrocyclic lactone agent widely used as a nematicide, acaricide and insecticide in veterinary medicine and plant protection. P-glycoprotein (P-gp) is an ATP-dependent drug efflux pump for xenobiotic compounds, and is involved in multidrug resistance. To understand the development of AVM resistance in invertebrates, we investigated the mechanisms by which AVM affected P-gp expression in Drosophila S2 cells. We found that AVM induced upregulation of P-gp protein expression, increased P-gp ATPase activity and enhanced cellular efflux of the P-gp substrate rhodamine 123 from cells. Further- more, we observed that AVM-induced expression of P-gp was due to elevation of intracellular calcium concentration ([Ca 2+ ] i ). This occurred both directly, by activating calcium ion channels, and indirectly, by activating chloride ion channels. These results are supported by our observations that verapamil, a Ca 2+ channel blocker, and niflumic acid, a chloride channel antagonist, significantly attenuated AVM- induced [Ca 2+ ] i elevation, thereby reducing P-gp expression. Inhibition of P-gp with anti-P-gp antibody or cyclosporine A (a P-gp inhibitor) reduced the AVM-induced elevation of [Ca 2+ ] i , implying that P-gp and [Ca 2+ ] i regulate each other. Finally, we found that trifluoperazine, a calmodulin inhibitor, and pyrrol- idine dithiocarbamic acid, an NF- jB inhibitor, attenuated the AVM-induced expression of P-gp, suggesting that AVM induces P-gp protein expression via the calmodulin/Relish (NF-jB) signaling pathway. Ó 2013 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Avermectins (AVMs) are 16-membered macrocyclic lactone derivatives produced by the soil bacteria actinomycete (Streptomy- ces avermitilis ). They display strong nematicidal, insecticidal, and acaricidal activities [1,2] and are thought to bind to the gluta- mate-gated and c-aminobutyric acid (GABA)-gated chloride chan- nel in cells, resulting in the hyperpolarization of neuromuscular cells and muscle paralysis [3,4]. Avermectin B1, the main compo- nent of avermectins, has been found to be a broad-spectrum anthelmintic drug and is widely used in veterinary medicine and plant protection [2]. Consequently, the widespread resistance of nematodes to avermectin B1 has global implications for agriculture and public health [5–7]. To date, little is known about the molecu- lar mechanism of AVM resistance in nematodes and insects. P-glycoprotein (P-gp) (also abbreviated as ABCB1), originally found in human cancer cells [8], belongs to a family of ATP-binding cassette transporters and is very highly conserved in vertebrates and invertebrates [9]. P-gp functions as an ATP-dependent efflux pump, and plays an important role in drug efflux and lipid trans- port in normal cells [10,11]. Studies have demonstrated that the level of P-gp expression predicts multidrug resistance in cancer chemotherapy [12]. Re- cently, elevated expression of P-gp homologs has been docu- mented in avermectin-resistant nematodes and insects [13,14]. P-gp expression has also been found to contribute to the develop- ment of resistance to avermectins in medical nematodes [15]. Moreover, AVMs are good substrates and potent inhibitors of P- gp [16,17]. Ivermectin, a derivative of avermectin B1, can induce P-gp mRNA overexpression in murine hepatocyte cells [18]. How- ever, it is not known if AVMs affect P-gp expression at the protein level. It has been reported that P-gp expression related with calcium or chloride ions. Calcium is a ubiquitous second messenger that regulates a variety of cell physiological functions. P-gp in mamma- lian cells was probably associated with elevated intracellular cal- 0009-2797/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cbi.2013.03.009 ⇑ Corresponding authors. Tel.: +1 318 675 7759 (S. Huang), Address: Institute of Zoology, CAS, 1-5 Beichenxi Road, Beijing 100101, PR China. Tel.: +86 10 64807251; fax: +86 10 64807099 (Y.-J. Wu) E-mail addresses: SHuan1@lsuhsc.edu (S. Huang), wuyj@ioz.ac.cn (Y.-J. Wu). Chemico-Biological Interactions 203 (2013) 430–439 Contents lists available at SciVerse ScienceDirect Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint