Overexpression of ABCB1 Transporter Confers Resistance to mTOR Inhibitor WYE-354 in Cancer Cells.

Jingqiu Wang,Zhuo-Xun Wu,Chao-Yun Cai,Linguo Zhao,Qiu-Xu Teng,Dong-Hua Yang,Jing-Quan Wang,Yuqi Yang,Zhe-Sheng Chen,Zi-Ning Lei

doi:10.3390/ijms21041387

Abstract

The overexpressing ABCB1 transporter is one of the key factors leading to multidrug resistance (MDR). Thus, many ABCB1 inhibitors have been found to be able to overcome ABCB1-mediated MDR. However, some inhibitors also work as a substrate of ABCB1, which indicates that in order to achieve an effective reversal dosage, a higher concentration is needed to overcome the pumped function of ABCB1, which may concurrently increase the toxicity. WYE-354 is an effective and specific mTOR (mammalian target of rapamycin) inhibitor, which recently has been reported to reverse ABCB1-mediated MDR. In the current study, 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay was carried out to determine the cell viability and reversal effect of WYE-354 in parental and drug-resistant cells. Drug accumulation was performed to examine the effect of WYE-354 on the cellular accumulation of chemotherapeutic drugs. The ATPase (adenosine triphosphatase) activity of the ABCB1 transporter in the presence or absence of WYE-354 was conducted in order to determine the impact of WYE-354 on ATP hydrolysis. Western blot analysis and immunofluorescence assay were used to investigate the protein molecules related to MDR. In addition, the interaction between the WYE-354 and ABCB1 transporter was investigated via in silico analysis. We demonstrated that WYE-354 is a substrate of ABCB1, that the overexpression of the ABCB1 transporter decreases the efficacy of WYE-354, and that the resistant WYE-354 can be reversed by an ABCB1 inhibitor at a pharmacological achievable concentration. Furthermore, WYE-354 increased the intracellular accumulation of paclitaxel in the ABCB1-mediated MDR cell line, without affecting the corresponding parental cell line, which indicated that WYE-354 could compete with other chemotherapeutic drugs for the ABCB1 transporter substrate binding site. In addition, WYE-354 received a high score in the docking analysis, indicating a strong interaction between WYE-354 and the ABCB1 transporter. The results of the ATPase analysis showed that WYE-354 could stimulate ABCB1 ATPase activity. Treatment with WYE-354 did not affect the protein expression or subcellular localization of the ABCB1. This study provides evidence that WYE-354 is a substrate of the ABCB1 transporter, implicating that WYE-354 should be avoided for use in ABCB1-mediated MDR cancer.

Highlights

The failure of cancer chemotherapy could result from multidrug resistance (MDR)
This result indicated that ABCB1 overexpression is responsible for WYE-354-mediated drug resistance
The chemotherapeutic drugs firstly bind to ABCB1 when crossing the membrane of certain cells, and the ATP hydrolysis provides energy, which results in pumping the drugs out of the cells; this process could reduce the intracellular concentration of drugs, resulting ABCB1-mediated drug resistance [35,36,37]

Summary

Introduction

Exploring compounds to antagonize MDR is of importance to improve the efficiency of chemotherapy [1]. The hydrolysis of ATP provides an energy source for ABCB1 to transport exogenous antineoplastic drugs; as a result, this reduces the intracellular drug concentration and leads to MDR [6]. Because the overexpression of ABCB1 is an important factor of MDR in tumors, inhibiting the expression and/or function of ABCB1 is the most direct way to overcome MDR in tumors. Some of the inhibitors are the substrate of ABCB1, and the inhibition of ABCB1 requires a high serum concentration of drugs, which may be toxic, and limits the application of these inhibitors [13,14,15]. In order to overcome MDR efficiently, it is essential to identify whether the inhibitor functions as a substrate

Methods

Results

Conclusion