In silico and in vitro identification of secoisolariciresinol as a re-sensitizer of P-glycoprotein-dependent doxorubicin-resistance NCI/ADR-RES cancer cells.

Mohamed A Morsy,Katharigatta N Venugopala,Azza A.K El-Sheikh,Ahmed R.N Ibrahim,Mahmoud Kandeel

doi:10.7717/peerj.9163

Abstract

P-glycoprotein (P-gp) is one of the highly expressed cancer cell efflux transporters that cause the failure of chemotherapy. To reverse P-gp induced multidrug resistance, we employed a flaxseed-derived lignan; secoisolariciresinol (SECO) that acts as an inhibitor of breast cancer resistance protein; another efflux transporter that shares some substrate/inhibitor specificity with P-gp. Molecular dynamics (MD) simulation identified SECO as a possible P-gp inhibitor. Comparing root mean square deviation (RMSD) of P-gp bound with SECO with that bound to its standard inhibitor verapamil showed that fluctuations in RMSD were lower in P-gp bound to SECO demonstrating higher stability of the complex of P-gp with SECO. In addition, the superimposition of P-gp structures after MD simulation showed that the nucleotide-binding domains of P-gp bound to SECO undertook a more central closer position compared with that bound to verapamil. Using rhodamine efflux assay on NCI/ADR-RES cancer cells, SECO was confirmed as a P-gp inhibitor, where cells treated with 25 or 50 µM of SECO showed significantly higher fluorescence intensity compared to control. Using MTT assay, SECO alone showed dose-dependent cytotoxicity, where 25 or 50 µM of SECO caused significantly less NCI/ADR-RES cellular viability compared to control. Furthermore, when 50 µM of SECO was added to doxorubicin (DOX), an anticancer drug, SECO significantly enhanced DOX-induced cytotoxicity compared to DOX alone. The combination index calculated by CompuSyn software indicated synergism between DOX and SECO. Our results suggest SECO as a novel P-gp inhibitor that can re-sensitize cancer cells during DOX chemotherapy.

Highlights

Cancer is one of the leading causes of death worldwide, especially in developed countries, where nearly 4 million newly diagnosed cancer cases are anticipated in Europe in only the year 2018, with breast cancer the most prevalent (Ferlay et al, 2018)
Molecular dynamics (MD) comparison of SECO with verapamil as inhibitors of P-gp A comparison between root mean square deviation (RMSD) of P-gp bound with SECO (P-gpSECO), P-gp bound to its standard inhibitor verapamil (P-gpVerapamil) and the empty form of P-gp without binding to any ligands (ApoP-gp) has been performed (Fig. 1)
P-gpVerapamil reached stabilization at an earlier stage at 32 ns compared to P-gpSECO, which showed a gradual increase in RMSD until 62 ns

Summary

Introduction

Cancer is one of the leading causes of death worldwide, especially in developed countries, where nearly 4 million newly diagnosed cancer cases are anticipated in Europe in only the year 2018, with breast cancer the most prevalent (Ferlay et al, 2018). One of the major problems facing the treatment of cancer is multidrug resistance (MDR) caused, at least in part, by the expression of efflux protein pumps that extrude chemotherapeutic drugs outside cancer cells, decreasing medication concentration and causing treatment failure (Zhou, Wang & Li, 2018). One of these efflux pumps is P-glycoprotein (P-gp) that was reported in several in vitro and in vivo studies to be expressed in cancer cells, especially those of the breast (Pokharel et al, 2016; Tulsyan, Mittal & Mittal, 2016; BadowskaKozakiewicz, Sobol & Patera, 2017; Babaer et al, 2018). During the transition of P-gp from inward open to outward open conformation, these hydrophobic residues change their orientation so their affinity to the substrates decreases to release the substrate to outside the cells (Esser et al, 2017)

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