The Endocytic Receptor Protein LRP-1 Modulate P-glycoprotein Mediated Drug Resistance in MCF-7 Cells
Aubery Henry, Marine Mauperin, Jerome Devy, Stephane Dedieu, Lise Chazee, Cathy Hachet, Christine Terryn, Laurent Duca, Laurent Martiny, Emmanuelle Devarenne-Charpentier, Hassan El Btaouri
Abstract
Multidrug resistance (MDR) is a major obstacle to successful cancer chemotherapy. A typical form of MDR is due to the overexpression of membrane transport proteins., such as Glycoprotein-P (P-gp), resulting in an increased drug efflux preventing drug cytotoxicity. P-gp is mainly localized on the plasma membrane; however, it can also be endocytosed resulting in the trafficking of P-gp in endoplasmic reticulum, Golgi, endosomes, and lysosomes. The lysosomal P-gp has been found to be capable of transporting and sequestering P-gp substrates (e.g., Doxorubicin (Dox)) into lysosomes to protect cells against cytotoxic drugs. Many translational studies have shown that low-density lipoprotein receptor-related protein-1 (LRP-1) is involved in endocytosis and regulation of signalling pathways. LRP-1 mediates the endocytosis of a diverse set of extracellular ligands that play important roles in tumor progression.
Introduction
Drug resistance is an obstacle that impairs the success of cancer therapies. This resistance occurs after repeated cycles of chemotherapy leading to the acquisition of tumor resistance [1]. Multiple mechanisms contribute to drug resistance, such as increased drug efflux, altered drug metabolism and activation of downstream transduction pathways [2, 3]. Recent studies have suggested that immune cells in tumor microenvironment (TME) play important roles in mediating acquired drug resistance [4, 5]. Indeed, reduced number of cytotoxic effector immune cells within the TME drive the tumor-mediated immunosuppression and could trigger acquired resistance development against immune checkpoint inhibitors [6–8]. Additionally, it has also been reported that the extracellular matrix influences tumor chemoresistance [9]. In fact, In fact, several studies confirmed the implication of collagen and fibronectin in chemoresistance induction [10–12]. Our previous study demonstrated that Thrombospondin-1 contribute to the modulation of P-gp drug resistance in thyroid carcinoma FTC-133 cells [13].
Materials and methods
2.1. Materials
MCF-7, a human breast carcinoma cell line derived from the pleural effusion metastasis, weas purchased from ATCC (USA). Dox was purchased from Farmitalia (Italy). MCF-7 resistant cells (MCF-7R) were obtained from MCF-7 parental cells by increased Dox treatment. DMEM/F-12, trypsin and Lipofectamine RNAiMAX were from Invitrogen (France). Bovine fetal serum was from Dutscher (France). LRP-1 polyclonal antibody ‘ab92544’ was from Abcam (France). Caspase-7 ‘#9492’, ERK 1/2 ‘#4695’, Phospho-ERK 1/2 ‘#4370’, LAMP-1 ‘#9091’ and P-gp ‘#13342’ antibodies were from Cell Signaling Technology (France). LRP-1 siRNA kit ‘sc-40101’ was purchased from Santa Cruz Biotechnology (USA). Kit ECL was from Amersham (Germany). U0216, a elective inhibitor of MEK-1 and MEK-2, was from Cell Signaling Technology (France). UptiBlue and BCA kit ‘23225’ were from Uptima (Thermofisher, France). CaspACE assay kit ‘G8091’ was from Promega (France). β-actin antibody ‘A5316’, invertase enzyme ‘I9274’ and all other reagents were from Sigma (USA). Histidine-tagged RAP was purified as previously described [41] and used as an antagonist of LRP-1-dependent endocytosis [41, 42].
Results
To study LRP-1 effects on multidrug resistant cells, two human breast carcinoma cells were used: parental MCF-7 cells sensitive to Dox called MCF-7S and MCF-7 cells resistant to Dox that were obtained by stepwise exposure of MCF-7S cells to increasing doses of Dox and called MCF-7R [47]. No significant difference in the morphology and growth rates between the two-line cells MCF-7S and MCF-7R cells was observed. We first analysed the expression of P-gp and LRP-1 in both cell types and Western blot analysis clearly showed a higher level of P-gp and LRP-1 expression in MCF-7R cells compared to MCF-7S cells (Fig 1).
Discussion
Multidrug-resistance (MDR) is a major obstacle to successful cancer chemotherapy and one important mechanism of MDR involves the plasma membrane glycoprotein P-gp. This transporter confers to cancer cells the ability to resist to cytotoxic drugs by pumping the drug out of the cells and thus reducing its cytotoxicity [21, 54, 55]. Several studies have demonstrated a negative correlation between P-gp expression levels and chemosensitivity in a range of human malignancies [55–57]. It is well established that P-gp actively effluxes cytotoxic substrates such as Dox [22]. Many studies showed that P-gp may traffic in cells through an intracellular endosomal system with protein vesicles transported to endosome compartments and thus forming an intracellular pool [51]. Indeed, P-gp has been reported to bound to the lysosomal membrane thus promoting the accumulation of the chemotherapeutic agent in acidic organelles [58]. This strongly suggested the existence of both spatial and temporal distribution of P-gp and an intracellular traffic between the plasma membrane and endocytic organelles. Our study revealed that P-gp was localized both in and out late endosomes in MCF-7 human breast cancer cells. The localization of P-gp in endosomes has been suggested to serve as an intra-cellular reservoir to P-gp moving to the plasma membrane [17–19] and P-gp was internalized into cells from the plasma membrane to lysosomes via the endocytic pathway [59]. However, the detailed mechanisms of this P-gp intracellular traffic remain unclear.
Acknowledgments
We thank Marie-Line SOWA and Sylvie RICORD for their assistance.
Citation: Henry A, Mauperin M, Devy J, Dedieu S, Chazee L, Hachet C, et al. (2023) The endocytic receptor protein LRP-1 modulate P-glycoprotein mediated drug resistance in MCF-7 cells. PLoS ONE 18(9): e0285834. https://doi.org/10.1371/journal.pone.0285834
Editor: Ming Tan, China Medical University (Taiwan), TAIWAN
Received: June 18, 2022; Accepted: May 2, 2023; Published: September 28, 2023
Copyright: © 2023 Henry et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: Data relevant to this study, including the raw blot/gel test images, are available at https://doi.org/10.6084/m9.figshare.21476841.v1.
Funding: -This work was supported by the University of Reims Champagne Ardennes and the Centre National de la Recherche Scientifique (CNRS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors received no specific funding for this work
Competing interests: The authors have declared that no competing interests exist.
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0285834#abstract0
