Original ArticlesAntitumor activity of nanoliposomes encapsulating the novobiocin analog 6BrCaQ in a triple-negative breast cancer model in mice
Introduction
Hsp90 (Heat shock protein 90) is a well-conserved protein involved in the proper refolding of proteins in cells that are overcoming stress conditions. It is located in the cytosol as Hsp90α and β and possesses isoforms or analogs located in mitochondria (hsp75/Tumor Necrosis Factor Receptor Associated-Protein-1, TRAP-1) and endoplasmic reticulum (Glucose Regulated Protein 94, grp94) [1]. Almost 400 proteins can be repaired or stabilized by hsp90, some of which are involved in the hallmarks of cancer, thus facilitating the oncogenic process [[2], [3], [4]]. Hsp90 has an important role in the cell in physiological conditions, being at the center of a quality-control system together with the ubiquitin ligase system. Targeting hsp90 in the cancer disease is a valid and promising strategy because this one target can influence many oncogenic pathways [5]. Hsp90 is part of a complex machinery and works together with other proteins such as hsp70, hsp40 and many other co-chaperones [6]. Its structure consists of three domains, each one having a specific role: an N-terminal domain involved in ATP hydrolysis, an intermediate domain with high affinity for client proteins and co-chaperones, and a C-terminal domain interacting with other co-chaperons and responsible for dimerization [7]. The existence of another ATP binding site located at this level has been described, stimulating new research efforts to target this domain [8]. Several inhibitors have been synthesized to specifically target these domains but little information is available regarding the co-crystallization of inhibitors and the C-terminal domain of hsp90. N-terminal inhibitors such as geldanamycin have shown very promising results in solid tumors (for example, prostate cancer and metastatic breast cancer) but were often associated with side effects and poor tolerance to the treatment. They were also shown to induce an overexpression of hsp70, in the so-called Heat Shock Response (HSR) [10], which could be responsible for resistance and chemoprotective effects [9]. The triggering of high levels of chaperones (hsp70, hsp90 and hsp27) is due to the activation of transcription factor HSF-1 in response to stress situations including N-terminal-targeted hsp90 inhibition. HSF-1 interacts with hsp90 and is released in these situations, promoting the transcription of chaperone genes [10,11]. This has led to constant efforts to synthesize more efficient compounds with higher clinical tolerability and that do not induce a HSR, including C-terminal modulators which were designed either from in silico studies or by synthesizing novobiocin (nvb) or cyclic peptide analogs [[12], [13], [14]]. Among the nvb derivatives, 6BrCaQ showed promising results in MCF-7 cells in terms of anti-proliferative effects in vitro and client protein degradation but was also found to be very insoluble in aqueous media [15]. Our strategy was then to use nanoparticulate carriers to overcome solubility problems and improve bioavailability of such hydrophobic compounds but also to specifically deliver them to the pathological site thus facilitating their potential transfer to the clinic [16,17]. In a previous study, we encapsulated 6BrCaQ in PEGylated liposomes and studied them in vitro in terms of anti-cancer activity in the PC-3 prostate cancer cell line. Liposomal 6BrCaQ showed pro-apoptotic effects, a G2/M cell cycle blockade and a synergistic effect with doxorubicin [18]. Moreover, liposomal 6BrCaQ retarded cell migration. As a result of their long circulation time, their biocompatibility, their versatility and their structure, PEGylated liposomes are an interesting platform for drug delivery of poorly soluble drugs to solid tumors [19]. As nanosized-carriers, they can take advantage of the enhanced permeation and retention effect (EPR) which is attributed to higher permeability of tumor blood vessels allied with reduced lymphatic drainage resulting in a better accumulation at the cancer site [20]. For these reasons, and because they allowed us to avoid the use of Cremophor® or DMSO (known to induce adverse effects) [21,22] for administration to mice, we focused our study on the anti-cancer effect of 6BrCaQ encapsulated in PEGylated liposomes and, for the first time, on the demonstration of its anti-tumor effect in vivo in an orthotopic breast cancer model.
Section snippets
Chemicals
6BrCaQ was synthesized by the Medicinal Chemistry group at our faculty (BioCIS, Faculté de Pharmacie, Châtenay-Malabry, France) through Pd- or Cu-catalyzed cross coupling methodologies [23,24]. The pure compound was solubilized at 10 mM in chloroform for liposome preparation and in DMSO for use as a free drug. Egg chicken l-α-phosphatidylcholine (Egg PC), Cholesterol (CH), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethyleneglycol)-2000] (DSPE-PEG2000),
Characterization of liposomes
Empty liposomes had a mean diameter of 124 nm and loaded liposomes had a similar diameter of around 121 nm. Both preparations had a PDI<0.2 showing that the distributions were monodisperse. Loaded and unloaded liposomes showed similar surface charge since no difference of zeta potential was observed (both around −13 mV). Encapsulation efficiency was found to be around 13.5% (molar ratio) for 2 mM incorporated 6BrCaQ which corresponds to a drug loading around 1.6% (molar ratio). This result is
Discussion
The aim of our work was to characterize the anti-tumor potential of a new analog of nvb, 6BrCaQ, encapsulated in PEGylated liposomes in a breast cancer model in vivo. This type of liposome is frequently used for drug delivery of anticancer agents because they show reduced liver and spleen accumulation and prolonged residence time in the blood due to the presence of PEG [27,28] (so-called “stealth” liposomes). The hydrophilic PEG chains on the surface of liposomes hinder opsonization by plasma
Conclusion
In this report, we showed, on breast cancer models, that liposomes encapsulating 6BrCaQ exert significant anti-proliferative effects in vitro, inducing cell cycle arrest in the S-G2/M phase and in vivo through tumor growth stabilization observed by luciferase bioluminescence. In vivo, our histological observations revealed a significant decrease in the number of proliferative cells and increased necrotic areas in treated mice. Moreover, no differences between control and treated groups in terms
Conflicts of interest statement
The authors have declared that no competing interest exists.
Acknowledgements
We would like to thank CNRS, Université Paris-Sud, la Ligue contre le Cancer Comité des Hauts de Seine (Grant 4FI10626MIUR) and Fondation ARC (Grant SFI20111203965). F.S received a Ph.D. fellowship from the French MENSR. We also thank Sophie Leboucher (Institut Curie, Orsay, France) for the preparation of histological slides and H&E staining, Florence Giffard from Inserm U1199 for the Ki67 staining and Valérie Nicolas from MIPSIT platform and Claudine Deloménie from TRANSPROT platform of the
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2020, Journal of Controlled ReleaseCitation Excerpt :Sauvage et al. also evaluated cytotoxic effect of 6BrCaQ (an hsp90 inhibitor derived from novobiocin) loaded in liposomes on breast cancer cells using MTS assay. According to their results, drug loaded liposome exhibited lower IC50 than free drug [40]. XTT is another tetrazolium salt converted to formazan in the presence of an electron coupling reagent such as phenazine methosulfate (PMS) or menadione sodium bisulfite (MSB) [41].
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2019, Journal of Colloid and Interface ScienceCitation Excerpt :To this end, we have used two different lipid membrane systems. The first is liposomes of a composition that is often used for drug delivery [22–24] and mitochondrial targeting in-vivo [13–15,17] (egg PC, cholesterol and DSPE-PEG2000 in 65:30:5 M proportions). However, to obtain more detailed physicochemical data, DMPC bilayers were used as a simplified model of the outer mitochondrial membrane [21].