Elsevier

Polymer

Volume 116, 5 May 2017, Pages 429-438
Polymer

Nitroxide radical-containing nanoparticles as potential candidates for overcoming drug resistance in epidermoid cancers

https://doi.org/10.1016/j.polymer.2017.02.052Get rights and content

Highlights

  • Therapeutic application of radical-containing nanoparticle (RNP) in chemoresistance.

  • RNPs sensitize multidrug resistant cancers to conventional drug, doxorubicin.

  • RNPs modulate ROS-regulated drug efflux proteins, P-gp and MRP1.

Abstract

Multidrug resistance in cancer cells contributes to the failure of conventional chemotherapy in more than 90% of cancer patients (metastatic). This is attributed to reactive oxygen species (ROS)-regulated drug efflux proteins, P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP1). In this study, we focused on overcoming multidrug resistance with a therapeutic application of ROS-scavenging nitroxide radical-containing nanoparticles, RNPN (pH-sensitive) and RNPO (pH-insensitive), in combination with the conventional chemotherapeutic drug, doxorubicin (Dox), in drug-resistant epidermoid cancer cell lines, KB-C2 (P-gp expressing) and KB/MRP (MRP1 expressing). We confirmed that the combination treatment with RNPs increased Dox uptake in multidrug-resistant cancer cells, which further enhanced cell cytotoxicity. The abrogation of the crucial ROS signaling was confirmed with RNP treatment, which deterred ROS-regulated drug efflux protein (P-gp and MRP1) expression, resulting in the sensitization of resistant cells to Dox. These results establish ROS-scavenging RNPs as potential therapeutic candidates to overcome drug resistance in multidrug-resistant cancers.

Introduction

The effectiveness of anticancer drugs is greatly limited owing to the drug-resistant characteristics of tumor cells, which are either innate (primary) or acquired during chemotherapy. Acquired resistance is more challenging to deal with as it is not only limited to the original initiating drug, but also, over time, the tumors acquire cross-resistance to other anticancer drugs from a diverse resistance mechanism, hence further complicating cancer therapeutics [1]. Resistance to chemotherapy has been reported to be the cause of treatment failure in more than 90% of cancer patients (metastatic) [2].

Chemoresistance is attributed to various resistant mechanisms, for instance, drug inactivation [3], [4], alteration of molecular drug targets by mutation [5], [6], repair of drug-damaged DNA [7], [8], evasion of cell death (apoptosis and autophagy pathway) [9], [10], [11], [12], epithelial–mesenchymal transition [13], [14], cancer heterogeneity [15], and drug efflux [16]. Of these, drug efflux is one of the most established drug-resistant mechanisms, which limits the accumulation of drugs by enhancing its efflux. Other than attaining drug resistance in cancer cells, a drug efflux system prevents toxin accumulation in healthy cells, the bile duct, intestines, and the blood–brain barrier [17], [18].

The most extensively studied drug efflux proteins implicated in cancer are members of the ATP-binding cassette (ABC) transporter superfamily, which includes P-glycoprotein (P-gp), a multidrug resistance protein (MDR) also called ABCB1, and multidrug resistance-associated protein-1 (MRP1) encoded by ABCC1 [19]. P-gp is a 170 kDa transmembrane glycoprotein that functions as an ATP-dependent efflux pump [20], [21] and is overexpressed in various cancers owing to drug resistance [22], [23]. P-gp transports hydrophobic compounds and has been implicated in resistance against paclitaxel and doxorubicin (Dox) [24]. On the contrary, MRP1, a 190 kDa transmembrane glycoprotein, not only transports organic anions and hydrophobic compounds but also confers resistance to Dox [25]. P-gp and MRP1 have been reported to be associated with poor clinical outcomes in acute myeloid leukemia and neuroblastoma, respectively [26], [27]. Their co-expression is associated with poor outcomes in acute myeloid leukemia [28], [29].

Since the mechanism of chemoresistance contributed by P-gp and MRP1 have different target substrates and distinct transport pathways [30], a combination therapy may be quite effective to overcome the multidrug resistance. For instance, treatment with one drug to sensitize cancer cells by altering the gene expression of crucial survival proteins followed by a cytotoxic drug treatment for already vulnerable cancer cells. Furthermore, the regulation of P-gp and MRP1 by reactive oxygen species (ROS) in cancers has been reported [31], [32], which may be a crucial target for antioxidant functioning as chemosensitizers in resistant cancers for anticancer drugs.

Recently, the therapeutic application of nanoparticles in resistant tumors has become the most sought-after strategy to overcome multidrug resistance in cancers [34]. Nanoparticles, in combination therapy (nanoformulation) or just as a drug carrier, have contributed immensely to enhancing the therapeutic efficacy of drugs in resistant tumors. This is because of their prolonged systemic circulation lifetime, increased intratumoral drug accumulation (enhanced permeation and retention effect), sustained drug release kinetics, targeted delivery (lower adverse effects), and through bypassing the drug efflux mechanism [33], [34], [35], [36].

Hence, we focused on overcoming the multidrug resistance in epidermoid cancer cell lines through ROS-scavenging polymeric micelles, pH-sensitive redox nanoparticles (RNPN) and pH-insensitive redox nanoparticles (RNPO) (Fig. 1). RNPN and RNPO are nitroxide radical-containing nanoparticles, composed of self-assembling amphiphilic block copolymers, poly(ethylene glycol)-b-poly[4-(2,2,6,6-tetramethylpiperidine-1-oxyl)aminomethylstyrene] (MeO-PEG-b-PMNT) and methoxy-poly(ethylene glycol)-b-poly(4-[2,2,6,6-tetramethylpiperidine-1-oxyl]oxymethylstyrene)] (MeO-PEG-b-PMOT), respectively [37], [38]. Antioxidant-mediated therapeutic applications of RNPO and RNPN have been confirmed in various oxidative stress-induced in vivo disease models, such as ischemia reperfusion injuries, intracerebral hemorrhage, ulcerative colitis, and many cancer models [39], [40], [41], [42], [43]. One of the most important features of RNPs is their exceptionally low toxicity as they do not enter healthy cells and, therefore, maintain normal redox reactions; for example, with the electron transport chain to maintain a healthy mitochondrial level [44], [45]. Diverse therapeutic applications of RNPs are not only restricted to being drug carriers like various other nanoparticles, but they also possess additional therapeutic effects [46]. Furthermore, combination therapy involving RNPN and Dox in in vivo cancer models has proven effective at inhibiting tumor growth with negligible adverse effects [47]. Overall, the in vivo therapeutic application of ROS scavenger RNPs either alone, as drug carriers, or in combination have been effective owing to their stable nature, increased bioavailability, and biocompatibility [48].

Considering the dependence of cancers on ROS signaling and ROS-mediated regulation of drug efflux transporters, P-gp and MRP1, we studied the therapeutic application of pH-sensitive RNPN and pH-insensitive RNPO (40 nm in diameter) in epidermoid cancer cell lines, drug-sensitive KB-3-1, drug-resistant P-gp-expressing KB-C2, and MRP1-expressing KB/MRP in combination with the conventional chemotherapeutic drug, Dox (Fig. 1). KB-C2 and KB/MRP epidermoid cancer cell lines have been reported to be resistant to Dox by effluxing the drug out with active ABC transporters, P-gp and MRP1, respectively. In this study, we confirmed an increased Dox uptake in resistant cancer cell lines with the treatment of ROS-scavenging RNPs, which further enhanced cancer cell cytotoxicity. The abrogation of the crucial ROS signaling was also confirmed with the combined treatment of RNPs and Dox by evaluating ROS levels. This, in turn, affected the drug efflux protein regulation resulting in an increased sensitivity to Dox. Interestingly, we also observed a therapeutic effect of RNPs individually in resistant cancer cell lines, hence implicating the vital role of the ROS signaling pathway in these cell lines. Thus, these results establish ROS-scavenging RNPs not only as an adjunct to sensitize resistant cells for chemotherapy but also as a potential therapeutic candidate to overcome drug resistance.

Section snippets

Materials

Dox and colchicine were purchased from Wako Pure Chemical Industries (Osaka, Japan). Dulbecco's modified Eagle's medium (DMEM; 1000 mg/L glucose, l-glutamine, and sodium bicarbonate) and fetal bovine serum (FBS) were purchased from Sigma-Aldrich (St Louis, MO, USA). A Penicillin-Streptomycin-Neomycin (PSN) antibiotic mixture and Hoechst 33258 were procured from Invitrogen (Eugene, OR, USA). Sodium chloride, potassium chloride, tris(hydroxymethyl)aminomethane (Tris base), disodium hydrogen

Characterization of RNPO and RNPN

Amphiphilic PEG-b-PMOT and PEG-b-PMNT block copolymers were synthesized as previously reported [37], [38]. Micelles, RNPO and RNPN, were prepared by self-assembling block copolymers, PEG-b-PMOT and PEG-b-PMNT, by using the dialysis method against water (Fig. 2a-1 and b-1). Micelle formation and TEMPO concentrations were confirmed with an ESR by using a free TEMPO radical linear regression standard curve. One milligram/milliliter of polymer weight of RNPO comprised 0.17 mg/mL of TEMPO (0.99 mmol

Conclusion

The continuous administration of anticancer drugs has been reported to confer an innate or acquired drug resistance in tumors, contributing to a poor drug response and reduced overall survival in patients with metastatic cancer. Chemoresistance in tumors is attributed to ABC transporters, P-gp and MRP1, which are regulated by ROS. The drug efflux mechanism is different for various transporters. Therefore, to overcome chemoresistance, combination therapy holds great potential in cancer

Conflict of interest

The authors declare no competing financial interest.

Acknowledgments

This study was partially supported by a Grant-in-Aid for Scientific Research (S) (25220203) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT). We appreciate Ms. Umeko Horiuchi for her technical assistance.

References (51)

  • R.B. Kim

    Transporters and xenobiotic disposition

    Toxicology

    (2002)
  • D.B. Longley et al.

    Molecular mechanisms of drug resistance

    J. Pathol.

    (2005)
  • H. Zahreddine et al.

    Mechanisms and insights into drug resistance in cancer

    Front. Pharmacol.

    (2013)
  • M. Michael et al.

    Tumoral drug metabolism: overview and its implications for cancer therapy

    J. Clin. Oncol.

    (2005)
  • K. Mehta et al.
  • A.A. Stavrovskaya

    Cellular mechanisms of multidrug resistance of tumor cells

    Biochem. (Mosc.)

    (2000)
  • K. Olaussen et al.

    DNA repair by ERCC1 in non-small-cell lung cancer and cisplatin-based adjuvant chemotherapy

    N. Engl. J. Med.

    (2006)
  • L. Bonanno et al.

    Platinum drugs and DNA repair mechanism in lung cancer

    Anticancer Res.

    (2014)
  • T. Miyashita et al.

    bcl-2 gene transfer increases relative resistance of S49.1 and WEHI7.2 lymphoid cells to cell death and DNA fragmentation induced by glucocorticoids and multiple chemotherapeutic drugs

    Cancer Res.

    (1992)
  • C. Sakakura et al.

    Overexpression of bax sensitizes breast cancer MCF-7 cells to cisplatin and etoposide

    Surg. Today

    (1997)
  • K. Sasaki et al.

    Chloroquine potentiates the anticancer effect of 5-fluorouracil on colon cancer cells

    BMC Cancer

    (2010)
  • K.L. Cook et al.

    Hydroxychloroquine inhibits autophagy to potentiate antiestrogen responsiveness in ER+ breast cancer

    Clin. Cancer Res.

    (2014)
  • A. Singh et al.

    Cancer stem cells and drug resistance: an emerging axis of evil in the war on cancer

    Oncogene

    (2010)
  • Y. Shang et al.

    Roles of epithelial-mesenchymal transition in cancer drug resistance

    Curr. Cancer Drug Targets

    (2013)
  • N. Navin et al.

    Inferring tumor progression from genomic heterogeneity

    Genome Res.

    (2010)
  • Cited by (20)

    • Palladium nanoparticles decorated Chitosan-Pectin modified Kaolin: It's catalytic activity for Suzuki-Miyaura coupling reaction, reduction of the 4-nitrophenol, and treatment of lung cancer

      2022, Inorganic Chemistry Communications
      Citation Excerpt :

      Due to the adverse effects of oxidized fats on human health, the use of antioxidants, which are compounds that neutralize them by reacting with free radicals and thus prevent or reduce their destructive effects in the body, to prevent the formation of these compounds, it seems necessary. Antioxidants can be synthetic or of natural origin [49–51]. The most commonly used chemical oxidants in industry and food are TBHQ, BHT, BHA, propyl galate, which have been identified as carcinogenic and adverse effects on human health.

    View all citing articles on Scopus
    1

    Current address: Faculty of Pharmaceutical Sciences, Tokyo University of Science, Yamazaki 2641, Noda, Chiba 278-8510, Japan.

    View full text