Celecoxib substituted biotinylated poly(amidoamine) G3 dendrimer as potential treatment for temozolomide resistant glioma therapy and anti-nematode agent
Graphical abstract
Introduction
Glioblastoma multiforme (GBM), known as astrocytoma grade IV, is the most common primary malignant brain tumor and one of the most lethal. Present treatment for glioblastoma includes surgical resection, followed by radiotherapy in combination with temozolomide (TMZ). In spite of that therapy, median survival rate is estimated at approximately 15 months, and fewer than 5% of patients survive more than 5 years after diagnosis. Poor prognosis and fails of GBM treatment are due to its highly heterogeneous and malignant character. Specific tumor localization, presence of blood-brain barrier (BBB) and high rate of tumor recurrence are challenging factors in development of effective anti-glioblastoma therapy (Lefranc et al., 2018; Qiu et al., 2017; Reardon and Wen, 2015).
Many drugs, acting by various molecular mechanisms, have been proposed for glioma therapy but only few compounds, belonging to DNA alkylating agents, including temozolomide (TMZ), carmustine, lomustine, and procarbazine, are commonly used in clinical treatment. The efficacy of concomitant radiation therapy followed by adjuvant TMZ for 6 months was shown to increase median overall survival and 2-year survival by 2 months and 16%, respectively, as compared to radiotherapy alone (Stupp et al., 2005). However such a treatment induce many deleterious side-effects (Alphandéry, 2018; Chamberlain, 2010; Patil et al., 2013). Therefore, new, more effective therapeutic strategies for glioma treatments are under extensive investigations.
In search for specific molecular markers it was proven that in many types of tumors including gliomas, constitutively elevated expression of cyclooxygenase-2 (COX-2) and high level of its product, prostaglandine PGE2, were present (Grösch et al., 2006; Kardosh et al., 2004; Qiu et al., 2017). PGE2 is known to promote tumorigenesis by stimulation of cell division, inhibition of apoptosis, alteration of cell adhesion leading to metastasis and stimulation of neovascularization (Zhang et al., 2011). COX-2 is a main source of PGE2 since catalyzes the production of prostaglandin H2 (PGH2) from cellular membrane arachidonic acid that is next converted to PGE2 by PGE2 synthase (Qiu et al., 2017). Thus inhibition of that pathway became important molecular target in designing a new therapeutic approaches.
Non-steroidal anti-inflammatory drugs (NSAIDs), beside their anti-inflammatory and analgesic properties, were proven to exert anti-neoplastic action and decrease risk of various types of cancers. However, it has to be pointed out that some contradictory reports are also present (Amirian et al., 2019; Bruhns et al., 2018; Daugherty et al., 2011). Celecoxib (CXB), the NSAIDs family member, was the first COX-2-selective inhibitor known to exert potent anticancer activities against various human cancers, (Dai et al., 2012; Kim et al., 2010; Zhang et al., 2011). CXB has been also reported to elevate radiosensitivity and decrease drug resistance in glioma (Ma et al., 2011; Suzuki et al., 2013). Following studies revealed that anticancer activity of CXB depends not only on down-regulation of COX-2 expression and targeting PGE2 signaling cascade (Grösch et al., 2006; Jiang et al., 2017; Kim et al., 2010; Liu et al., 2012) but is also due to other mechanisms. In human colorectal cancer cells CXB induces apoptosis through increase of p53 gene expression (Liu et al., 2008b) and in glioma cells increases death via DNA damage, leading to p53-dependent G1 cell-cycle arrest and autophagy (Kang et al., 2009). It has been shown also that in glioma CXB suppress the activation of Wnt/β-catenin signaling pathway that regulates key cellular functions including proliferation, differentiation, migration, genetic stability, apoptosis, and stem cell renewal and is involved in pathogenesis of several cancers (Pai et al., 2017; Sareddy et al., 2013).
The point of interest is that celecoxib administered patients with glioma did not exert visible proper results of its effectiveness. That may be due to its low ability to cross the blood-brain barrier (BBB) (Novakova et al., 2014) or to patients low achieved plasma concentrations as compared with its anti-cancer activity in vitro .The effective concentration of CBX causing inhibition of proliferation and/or induction of apoptosis in vitro amounts to 100 µM (Yerokun and Winfield, 2015), whereas therapeutic human plasma concentration reach about 10 µM concentration (Maier et al., 2009). Moreover, systemic administration of high dosage of CXB causes many undesirable side effects, particularly in patients with renal disease and heart failure, that can be diminished by controlled, targeted release systems (Vera et al., 2014).
Therefore, a promising strategy of glioma treatment relies on using specific drug delivery systems e.g. based on polymeric nanocarriers. One of very promising are poly(amidoamine) dendrimers (PAMAMs), which increases solubility, bioavailability, and promote delivery of anticancer drugs into tumor cells (Chauhan et al., 2020). It is documented that due to their unique molecular properties, dendrimers reveal high potential as nanocarriers of different molecules like drugs, nucleic acids or fluorescent markers (Gupta and Perumal, 2014; Madaan et al., 2014). PAMAM G3 dendrimers have well-defined three-dimensional, hyperbranched structure with 3.1 nm diameter and functional 32 amino groups on their surface, which can be modified (Uram et al., 2018). PAMAMs of third generation stand out as relatively low cytotoxic (Jain et al., 2010), reveal high permeability for cellular membranes and ability to accumulate in intracellular compartments such as lysosomes and mitochondria (Uram et al., 2015).
Separate and important issue in glioma therapy is an ability of the therapeutic agents to cross cellular membranes and BBB, and avoidance of chemoresistance mechanisms. Many types of cancer cells including glioma reveal overexpression of biotin receptors and increased biotin uptake, what makes this vitamin useful in targeting chemotherapeutics (Miranda-Gonçalves et al., 2013; Ren et al., 2015; Russell-Jones et al., 2004). Monocarboxylic acid transport (MCT) system realized via transporters such as MCT1 and MCT8 is one of influx system involved in transport of vitamins such as biotin through the BBB into CNS (Barar et al., 2016). Overexpression of MCT1 and MCT4 was observed in 87% of 78 bioptates of glioma (Miranda-Gonçalves et al., 2013). The other route of biotin transport is performed by sodium dependent multivitamin transporter (SMVT/SLC5A6), highly expressed in brain (Azhar et al., 2015). Thus biotinylation of PAMAMs and other nanoparticles improves its cellular uptake and allows BBB penetration (Hemmer et al., 2013; Uram et al., 2017a; Veszelka et al., 2017).
The aim of this study was to investigate anti-glioma activity of biotin targeted PAMAM G3 dendrimer substituted by 31 CXB residues (G3BC31) for the TMZ resistant human cell line of glioblastoma multiforme (U-118 MG) as compared to CXB or TMZ administered alone. Effects of various drug combinations on viability, proliferation, migration and apoptosis, as well as the cellular expression of COX-2, ATP level, and PGE2 production were determined. Cellular accumulation and localization of fluorescently labelled biotinylated PAMAM G3 dendrimer containing 20 residues of CXB (G3BC20F) was also evaluated. In vivo toxicity of dendrimer conjugate (G3BC31) and CXB were tested using model organism nematode Caenorhabditis elegans that has been employed extensively in toxicological studies of many nanoparticles (Lucio et al., 2018; Meyer et al., 2010; Walczynska et al., 2018) and as model organism providing insights into cancer cells metabolism, stem cell reprogramming and dedifferentiation (Kyriakakis et al., 2015). C. elegans is an valuable model for high-throughput anticancer drug screening, because its germline development is tightly regulated by conserved external signaling pathways, including Wnt, Notch and Ras, which are oncogenic signaling pathways in the development of cancer stem cells (Kobet et al., 2014).
Section snippets
Materials
Celecoxib (CXB, PHR1683) and temozolomide (TMZ, PHR1437) was purchased from Fluka (AG, Buchs, Switzerland) then dissolved and diluted in DMSO (Sigma-Aldrich) to 100 mM concentration. The following poly(amidoamine) dendrimers of third generation (G3 PAMAM) with ethylenediamine (EDA) core were synthesized as described (Uram et al., 2018): native G3 PAMAM, G3 PAMAM dendrimer conjugated with one biotin molecule and 31 celecoxib residues (G3BC31) and its fluorescently labeled analog with one biotin
Toxicity
It is known that about 50% of TMZ treated patients do not respond to this drug (Lee, 2016). Therefore in our study we used the U-118 MG glioma cell line, resistant to TMZ. It was confirmed by the XTT assay that viability of that cell line was not changed after 24 h treatment with TMZ up to 200 µM concentration (Fig. 1A). Similarly, Carmo et al. (2011) and Li et al. (2013) indicated that U-118 MG glioma cells were resistant to TMZ up to 100 µM concentration after 24 and 48 h incubation as
Conclusions
PAMAM G3 dendrimers, substituted with targeting biotin and CXB molecules can be a valuable tool for increasing the effectiveness of CXB glioma therapy challenged by limited therapeutic human plasma concentrations of that compound, presence of BBB and undesired CXB side effects. Studied dendrimer conjugate G3BC31 targeted by biotin molecule and carrying 31 CBX residues efficiently penetrated and accumulated in TMZ-resistant glioblastoma U-118 MG cells at relatively low concentrations in the
CRediT authorship contribution statement
Łukasz Uram: Conceptualization, Investigation, Methodology, Validation, Formal analysis, Resources, Writing - original draft, Visualization, Supervision, Project administration, Funding acquisition. Joanna Markowicz: Investigation, Methodology, Formal analysis, Writing - original draft, Visualization. Maria Misiorek: Investigation, Methodology. Aleksandra Filipowicz-Rachwał: Investigation. Stanisław Wołowiec: Investigation, Methodology, Validation. Elżbieta Wałajtys-Rode: Writing - review &
Declaration of Competing Interest
The authors declare no conflict of interest.
Acknowledgements
This research was funded by National Science Centre, Poland, grant 2014/13/D/NZ3/02825. We gratefully acknowledge Prof. Agata Wawrzyniak from Rzeszow University, for kindly enabling us to perform measurements using the confocal microscope.
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