Celecoxib substituted biotinylated poly(amidoamine) G3 dendrimer as potential treatment for temozolomide resistant glioma therapy and anti-nematode agent

https://doi.org/10.1016/j.ejps.2020.105439Get rights and content

Abstract

Glioblastoma multiforme (GBM) is a one of the most widely diagnosed and difficult to treat type of central nervous system tumors. Resection combined with radiotherapy and temozolomide (TMZ) chemotherapy prolongs patients' survival only for 12 - 15 months after diagnosis. Moreover, many patients develop TMZ resistance, thus important is search for a new therapy regimes including targeted drug delivery. Most types of GBM reveal increased expression of cyclooxygenase-2 (COX-2) and production of prostaglandin E2 (PGE2), that are considered as valuable therapeutic target. In these studies, the anti-tumor properties of the selective COX-2 inhibitor celecoxib (CXB) and biotinylated third generation of the poly(amidoamine) dendrimer substituted with 31 CXB residues (G3BC31) on TMZ –resistant

U-118 MG glioma cell line were examined and compared with the effect of TMZ alone including viability, proliferation, migration and apoptosis, as well as the cellular expression of COX-2, ATP level, and PGE2 production. Confocal microscopy analysis with the fluorescently labeled G3BC31 analogue has shown that the compound was effectively accumulated in U-118 MG cells in time–dependent manner and its localization was confirmed in lysosomes but not nuclei. G3BC31 reveal much higher cytotoxicity for U-118 MG cells at relatively low concentrations in the range of 2–4 µM with compared to CBX alone, active at 50–100 µM. This was due to induction of apoptosis and inhibition of proliferation and migration. Observed effects were concomitant with reduction of PGE2 production but independent of COX-2 expression. We suggest that investigated conjugate may be a promising candidate for therapy of TMZ-resistant glioblastoma multiforme, although applicable in local treatment, since our previous study of G3BC31 did not demonstrate selectivity against glioma cells compared to normal human fibroblasts.

However, it has to be pointed that in our in vivo studies conducted with model organism, Caenorhabditis elegans indicated high anti-nematode activity of G3BC31 in comparison with CXB alone that confirms of usefulness of that organism for estimation of anti-cancer drug toxicity.

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.

References (111)

  • K. Jain et al.

    Dendrimer toxicity: let's meet the challenge

    Int. J. Pharm.

    (2010)
  • V. Jendrossek

    Targeting apoptosis pathways by Celecoxib in cancer

    Cancer Lett. Apoptosis Target. Drugs Cancer

    (2013)
  • J. Jiang et al.

    Prostaglandin E2 Signaling: alternative Target for Glioblastoma?

    Trends Cancer

    (2017)
  • Y.-.Y. Kim et al.

    Anti-cancer effects of celecoxib in head and neck carcinoma

    Mol. Cells

    (2010)
  • S.Y. Lee

    Temozolomide resistance in glioblastoma multiforme

    Genes Dis.

    (2016)
  • F. Lefranc et al.

    Glioblastoma quo vadis: will migration and invasiveness reemerge as therapeutic targets?

    Cancer Treat. Rev.

    (2018)
  • R.J. Levitt et al.

    Growth inhibition of breast epithelial cells by celecoxib is associated with upregulation of insulin-like growth factor binding protein-3 expression

    Biochem. Biophys. Res. Commun.

    (2004)
  • J. Lewis et al.

    Chapter 1 basic culture methods

  • B. Liu et al.

    Celecoxib, a cyclooxygenase‐2 inhibitor, induces apoptosis in human osteosarcoma cell line MG‐63 via down‐regulation of PI3K/Akt

    Cell Biol. Int.

    (2008)
  • H.-.F. Liu et al.

    Celecoxib induces p53-PUMA pathway for apoptosis in human colorectal cancer cells

    Chem. Biol. Interact.

    (2008)
  • D. Lucio et al.

    Cyclodextrin-grafted poly(anhydride) nanoparticles for oral glibenclamide administration. In vivo evaluation using C. elegans

    Int. J. Pharm.

    (2018)
  • J.N. Meyer et al.

    Intracellular uptake and associated toxicity of silver nanoparticles in Caenorhabditis elegans

    Aquat. Toxicol. Aquat. Toxicol. Nanomater.

    (2010)
  • M.M. Monick et al.

    Phosphatidylinositol 3-kinase activity negatively regulates stability of cyclooxygenase 2 mRNA

    J. Biol. Chem.

    (2002)
  • E. Nikolskaya et al.

    Influence of the doxorubicin conjugated PAMAM dendrimer surface charge on cytotoxic effects and intracellular trafficking routes in tumor cells

  • J. Qiu et al.

    Cyclooxygenase-2 in glioblastoma multiforme

    Drug Discov. Today

    (2017)
  • S. Reed et al.

    Celecoxib inhibits STAT3 phosphorylation and suppresses cell migration and colony forming ability in rhabdomyosarcoma cells

    Biochem. Biophys. Res. Commun.

    (2011)
  • G. Russell-Jones et al.

    Vitamin-mediated targeting as a potential mechanism to increase drug uptake by tumours

    J. Inorgan. Biochem.

    (2004)
  • K.-.W. Seo et al.

    Antitumor effects of celecoxib in COX-2 expressing and non-expressing canine melanoma cell lines

    Res. Vet. Sci.

    (2014)
  • Ł. Uram et al.

    Biotinylated PAMAM G3 dendrimer conjugated with celecoxib and/or Fmoc-l-Leucine and its cytotoxicity for normal and cancer human cell lines

    Eur. J. Pharm. Sci.

    (2018)
  • Ł. Uram et al.

    Cellular uptake of glucoheptoamidated poly(amidoamine) PAMAM G3 dendrimer with amide-conjugated biotin, a potential carrier of anticancer drugs

    Bioorg. Med. Chem.

    (2017)
  • Ł. Uram et al.

    The effect of G3 PAMAM dendrimer conjugated with B-group vitamins on cell morphology, motility and ATP level in normal and cancer cells

    Eur. J. Pharm. Sci.

    (2017)
  • M. Vera et al.

    New celecoxib multiparticulate systems to improve glioblastoma treatment

    Int. J. Pharm.

    (2014)
  • L. Albertazzi et al.

    Dendrimer internalization and intracellular trafficking in living cells

    Mol. Pharmaceut.

    (2010)
  • E. Alphandéry

    Glioblastoma treatments: an account of recent industrial developments

    Front. Pharmacol.

    (2018)
  • E.S. Amirian et al.

    Aspirin, NSAIDs, and glioma risk: original data from the glioma international case-control study and a meta-analysis

    Cancer Epidemiol. Biomarkers Prev.

    (2019)
  • Azhar, A., Booker, G., Polyak, S., 2015. Mechanisms of biotin...
  • J. Barar et al.

    Blood-brain barrier transport machineries and targeted therapy of brain diseases

    Bioimpacts

    (2016)
  • L.J. Bischof et al.

    Assays for toxicity studies in C. elegans with Bt crystal proteins

  • U. Böhme et al.

    Counterion condensation and effective charge of PAMAM dendrimers

    Polymers (Basel)

    (2011)
  • R.P. Bruhns et al.

    Survival as a function of nonsteroidal anti-inflammatory drug use in patients with glioblastoma

    Cureus J. Med. Sci.

    (2018)
  • A. Carmo et al.

    Effect of temozolomide on the U-118 glioma cell line

    Oncol. Lett.

    (2011)
  • M.C. Chamberlain

    Temozolomide: therapeutic limitations in the treatment of adult high-grade gliomas

    Expert Rev. Neurother.

    (2010)
  • T.-.T. Ching et al.

    Celecoxib extends C. elegans lifespan via inhibition of insulin-like signaling but not cyclooxygenase-2 activity

    Aging Cell

    (2011)
  • V.A. Cuddapah et al.

    A neurocentric perspective on glioma invasion

    Nat. Rev. Neurosci.

    (2014)
  • M.L.V.da Cunha et al.

    Metastasis from glioblastoma multiforme: a meta-analysis

    Revista da Associação Médica Brasileira

    (2019)
  • Z.-.J. Dai et al.

    Antitumor activity of the selective cyclooxygenase-2 inhibitor, celecoxib, on breast cancer in Vitro and in Vivo

    Cancer Cell Int.

    (2012)
  • S.E. Daugherty et al.

    Nonsteroidal anti-inflammatory drugs and glioma in the NIH-AARP diet and health study cohort

    Cancer Prev. Res. (Phila)

    (2011)
  • F. Giuliano et al.

    Origins of prostaglandin E2: involvements of cyclooxygenase (COX)-1 and COX-2 in human and rat systems

    J. Pharmacol. Exp. Ther.

    (2002)
  • L. Gong et al.

    Celecoxib pathways: pharmacokinetics and pharmacodynamics

    Pharmacogenet. Genomics

    (2012)
  • S. Grösch et al.

    Cyclooxygenase-2 (COX-2)–independent anticarcinogenic effects of selective COX-2 inhibitors

    J. Natl. Cancer Inst.

    (2006)
  • Cited by (18)

    • Dendrimers in the context of targeting central nervous system disorders

      2022, Journal of Drug Delivery Science and Technology
      Citation Excerpt :

      Overexpression of MCT 1 and 4 in glioma cells can be targeted by biotin-conjugated dendrimers. With this hypothesis [123], have developed biotin-conjugated G3 dendrimers substituted with 31 celecoxib (a COX-2 inhibitor) and 20 celecoxib residues to evaluate their accumulation and anticancer activity in TMZ resistant human cell-line of glioblastoma multiforme (U 118-MG) [123]. Celecoxib was taken as an active molecule because in glioma, over expression of COX 2 and high extent of prostaglandin E2 (PGE 2) are observed [124].

    • Dendrimers as carriers for active targeting of brain tumors

      2022, Nanocarriers for Drug-Targeting Brain Tumors
    • Modular design of multifunctional core-shell tecto dendrimers complexed with copper(II) for MR imaging-guided chemodynamic therapy of orthotopic glioma

      2021, Nano Today
      Citation Excerpt :

      Nowadays, a variety of nanoplatforms have been utilized for imaging and therapeutics of glioma [28–31]. Among them, dendrimers, especially poly(amidoamine) (PAMAM) dendrimers, have been used to build up convincing nanosystems for glioma treatment due to their remarkable and unique physicochemical and biological properties [32–37]. For instance, the Jiang group [38] developed the use of peptide HAIYPRH (T7)-conjugated generation 5 (G5) PAMAM dendrimers through a polyethylene glycol (PEG) spacer for effective delivery of MR imaging contrast agents of Gd chelates to achieve specific glioma targeting.

    View all citing articles on Scopus
    View full text