In vitro drug response and efflux transporters associated with drug resistance in pediatric high grade glioma and diffuse intrinsic pontine glioma

Susanna J E Veringa, Dennis Biesmans, Dannis G van Vuurden, Marc H A Jansen, Laurine E Wedekind, Ilona Horsman, Pieter Wesseling, William Peter Vandertop, David P Noske, GertJan J L Kaspers, Esther Hulleman, Susanna J E Veringa, Dennis Biesmans, Dannis G van Vuurden, Marc H A Jansen, Laurine E Wedekind, Ilona Horsman, Pieter Wesseling, William Peter Vandertop, David P Noske, GertJan J L Kaspers, Esther Hulleman

Abstract

Pediatric high-grade gliomas (pHGG), including diffuse intrinsic pontine gliomas (DIPG), are the leading cause of cancer-related death in children. While it is clear that surgery (if possible), and radiotherapy are beneficial for treatment, the role of chemotherapy for these tumors is still unclear. Therefore, we performed an in vitro drug screen on primary glioma cells, including three DIPG cultures, to determine drug sensitivity of these tumours, without the possible confounding effect of insufficient drug delivery. This screen revealed a high in vitro cytotoxicity for melphalan, doxorubicine, mitoxantrone, and BCNU, and for the novel, targeted agents vandetanib and bortezomib in pHGG and DIPG cells. We subsequently determined the expression of the drug efflux transporters P-gp, BCRP1, and MRP1 in glioma cultures and their corresponding tumor tissues. Results indicate the presence of P-gp, MRP1 and BCRP1 in the tumor vasculature, and expression of MRP1 in the glioma cells themselves. Our results show that pediatric glioma and DIPG tumors per se are not resistant to chemotherapy. Treatment failure observed in clinical trials, may rather be contributed to the presence of drug efflux transporters that constitute a first line of drug resistance located at the blood-brain barrier or other resistance mechanism. As such, we suggest that alternative ways of drug delivery may offer new possibilities for the treatment of pediatric high-grade glioma patients, and DIPG in particular.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. a. Cell survival among primary…
Figure 1. a. Cell survival among primary pHGG cultures exposed to classical chemotherapeutic drugs. b. Cell survival among primary pHGG cultures exposed to novel drugs.
Figure 2. Western blot for detection of…
Figure 2. Western blot for detection of P-gp, MRP1, and BCRP1 in pHGG cultures.
MW represents approximate molecular weight of these proteins, as indicated at the right. The pHGG lanes were loaded with 20 µg of protein, the lanes with positive controls were loaded with 5 µg of protein.
Figure 3. Immunohistochemical staining of ABC-transporters in…
Figure 3. Immunohistochemical staining of ABC-transporters in pHGG sections.
Expression of P-gp (A) and BCRP1 (C) is located to the endothelial cells of the tumor vasculature. Whereas MRP1 (B) expression is visualized mainly in the cytoplasm of tumor cells as well as in the vasculature.

References

    1. Broniscer A, Gajjar A (2004) Supratentorial high-grade astrocytoma and diffuse brainstem glioma: two challenges for the pediatric oncologist. Oncologist 9: 197–206.
    1. Tamber MS, Rutka JT (2003) Pediatric supratentorial high-grade gliomas. Neurosurg Focus 14: e1.
    1. Jansen MHA, van Vuurden DG, Vandertop WP, Kaspers GJL (2012) Diffuse intrinsic pontine gliomas: a systematic update on clinical trials and biology. Cancer Treat Rev 38: 27–35.
    1. Hargrave D, Bartels U, Bouffet E (2006) Diffuse brainstem glioma in children: critical review of clinical trials. Lancet Oncol 7: 241–248.
    1. Broniscer A (2006) Past, present, and future strategies in the treatment of high-grade glioma in children. Cancer Invest 24: 77–81.
    1. Ueno M, Nakagawa T, Wu B, Onodera M, Huang CL, et al. (2010) Transporters in the brain endothelial barrier. Curr Med Chem 17: 1125–1138.
    1. Bredel M (2001) Anticancer drug resistance in primary human brain tumors. Brain Res Brain Res Rev 35: 161–204.
    1. Nies AT (2007) The role of membrane transporters in drug delivery to brain tumors. Cancer Lett 254: 11–29.
    1. Decleves X, Amiel A, Delattre JY, Scherrmann JM (2006) Role of ABC transporters in the chemoresistance of human gliomas. Curr Cancer Drug Targets 6: 433–445.
    1. Doyle LA, Yang W, Abruzzo LV, Krogmann T, Gao Y, et al. (1998) A multidrug resistance transporter from human MCF-7 breast cancer cells. Proc Natl Acad Sci U S A 95: 15665–15670.
    1. Whelan RD, Waring CJ, Wolf CR, Hayes JD, Hosking LK, et al. (1992) Over-expression of P-glycoprotein and glutathione S-transferase pi in MCF-7 cells selected for vincristine resistance in vitro. Int J Cancer 52: 241–246.
    1. Hooijberg JH, Peters GJ, Assaraf YG, Kathmann I, Priest DG, et al. (2003) The role of multidrug resistance proteins MRP1, MRP2 and MRP3 in cellular folate homeostasis. Biochem Pharmacol 65: 765–771.
    1. Dixit S, Hingorani M, Achawal S, Scott I (2011) The sequential use of carmustine wafers (Gliadel(R)) and post-operative radiotherapy with concomitant temozolomide followed by adjuvant temozolomide: a clinical review. Br J Neurosurg 25: 459–469.
    1. Hart MG, Grant R, Garside R, Rogers G, Somerville M, et al... (2011) Chemotherapy wafers for high grade glioma. Cochrane Database Syst Rev CD007294.
    1. Marquez-Rivas J, Ramirez G, Ollero-Ortiz A, Gimenez-Pando J, Emmerich J, et al. (2010) Initial experience involving treatment and retreatment with carmustine wafers in combination with oral temozolomide: long-term survival in a child with relapsed glioblastoma multiforme. J Pediatr Hematol Oncol 32: e202–e206.
    1. Paugh BS, Qu C, Jones C, Liu Z, Damowicz-Brice M, et al. (2010) Integrated molecular genetic profiling of pediatric high-grade gliomas reveals key differences with the adult disease. J Clin Oncol 28: 3061–3068.
    1. Wolff J, Brown R, Buryanek J, Pfister S, Vats T, et al... (2011) Preliminary experience with personalized and targeted therapy for pediatric brain tumors. Pediatr Blood Cancer.
    1. Stommel JM, Kimmelman AC, Ying H, Nabioullin R, Ponugoti AH, et al. (2007) Coactivation of receptor tyrosine kinases affects the response of tumor cells to targeted therapies. Science 318: 287–290.
    1. Horton TM, Pati D, Plon SE, Thompson PA, Bomgaars LR, et al. (2007) A phase 1 study of the proteasome inhibitor bortezomib in pediatric patients with refractory leukemia: a Children's Oncology Group study. Clin Cancer Res 13: 1516–1522.
    1. Phuphanich S, Supko JG, Carson KA, Grossman SA, Burt Nabors L, et al. (2010) Phase 1 clinical trial of bortezomib in adults with recurrent malignant glioma. J Neurooncol 100: 95–103.
    1. Suri V, Das P, Pathak P, Jain A, Sharma MC, et al. (2009) Pediatric glioblastomas: a histopathological and molecular genetic study. Neuro Oncol 11: 274–280.
    1. Qu HQ, Jacob K, Fatet S, Ge B, Barnett D, et al. (2010) Genome-wide profiling using single-nucleotide polymorphism arrays identifies novel chromosomal imbalances in pediatric glioblastomas. Neuro Oncol 12: 153–163.
    1. Paugh BS, Qu C, Jones C, Liu Z, Damowicz-Brice M, et al. (2010) Integrated molecular genetic profiling of pediatric high-grade gliomas reveals key differences with the adult disease. J Clin Oncol 28: 3061–3068.
    1. Stupp R, Hegi ME, Mason WP, van den Bent MJ, Taphoorn MJB, et al. (2009) Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial. Lancet Oncol 10: 459–466.
    1. Cohen KJ, Pollack IF, Zhou T, Buxton A, Holmes EJ, et al. (2011) Temozolomide in the treatment of high-grade gliomas in children: a report from the Children's Oncology Group. Neuro Oncol 13: 317–323.
    1. Cohen KJ, Heideman RL, Zhou T, Holmes EJ, Lavey RS, et al. (2011) Temozolomide in the treatment of children with newly diagnosed diffuse intrinsic pontine gliomas: a report from the Children's Oncology Group. Neuro Oncol 13: 410–416.
    1. Bredel M, Zentner J (2002) Brain-tumour drug resistance: the bare essentials. Lancet Oncol 3: 397–406.
    1. Cordon-Cardo C, O'Brien JP, Casals D, Rittman-Grauer L, Biedler JL, et al. (1989) Multidrug-resistance gene (P-glycoprotein) is expressed by endothelial cells at blood-brain barrier sites. Proc Natl Acad Sci U S A 86: 695–698.
    1. Daood M, Tsai C, Hdab-Barmada M, Watchko JF (2008) ABC transporter (P-gp/ABCB1, MRP1/ABCC1, BCRP/ABCG2) expression in the developing human CNS. Neuropediatrics 39: 211–218.
    1. Bahr O, Rieger J, Duffner F, Meyermann R, Weller M, et al. (2003) P-glycoprotein and multidrug resistance-associated protein mediate specific patterns of multidrug resistance in malignant glioma cell lines, but not in primary glioma cells. Brain Pathol 13: 482–494.
    1. Kirches E, Oda Y, Von Bossanyi P, Diete S, Schneider T, et al. (1997) Mdr1 mRNA expression differs between grade III astrocytomas and glioblastomas. Clin Neuropathol 16: 34–36.
    1. Henson JW, Cordon-Cardo C, Posner JB (1992) P-glycoprotein expression in brain tumors. J Neurooncol 14: 37–43.
    1. Ni Z, Bikadi Z, Rosenberg MF, Mao Q (2010) Structure and function of the human breast cancer resistance protein (BCRP/ABCG2). Curr Drug Metab 11: 603–617.
    1. Litman T, Jensen U, Hansen A, Covitz KM, Zhan Z, et al. (2002) Use of peptide antibodies to probe for the mitoxantrone resistance-associated protein MXR/BCRP/ABCP/ABCG2. Biochim Biophys Acta 1565: 6–16.
    1. Bredel M (2001) Anticancer drug resistance in primary human brain tumors. Brain Res Brain Res Rev 35: 161–204.
    1. Henson JW, Cordon-Cardo C, Posner JB (1992) P-glycoprotein expression in brain tumors. J Neurooncol 14: 37–43.
    1. Sawada T, Kato Y, Sakayori N, Takekawa Y, Kobayashi M (1999) Expression of the multidrug-resistance P-glycoprotein (Pgp, MDR-1) by endothelial cells of the neovasculature in central nervous system tumors. Brain Tumor Pathol 16: 23–27.
    1. Shukla S, Wu CP, Ambudkar SV (2008) Development of inhibitors of ATP-binding cassette drug transporters: present status and challenges. Expert Opin Drug Metab Toxicol 4: 205–223.
    1. Azzariti A, Porcelli L, Simone GM, Quatrale AE, Colabufo NA, et al. (2010) Tyrosine kinase inhibitors and multidrug resistance proteins: interactions and biological consequences. Cancer Chemother Pharmacol 65: 335–346.
    1. Shi Z, Peng XX, Kim IW, Shukla S, Si QS, et al. (2007) Erlotinib (Tarceva, OSI-774) antagonizes ATP-binding cassette subfamily B member 1 and ATP-binding cassette subfamily G member 2-mediated drug resistance. Cancer Res 67: 11012–11020.
    1. Mi Yj, Liang Yj, Huang HB, Zhao HY, Wu CP, et al. (2010) Apatinib (YN968D1) reverses multidrug resistance by inhibiting the efflux function of multiple ATP-binding cassette transporters. Cancer Res 70: 7981–7991.
    1. Zhang J, Chung T, Oldenburg K (1999) A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. J Biomol Screen 4: 67–73.
    1. Fruehauf JP, Brem H, Brem S, Sloan A, Barger G, et al. (2006) In vitro drug response and molecular markers associated with drug resistance in malignant gliomas. Clin Cancer Res 12: 4523–4532.
    1. Wolff JE, Trilling T, Molenkamp G, Egeler RM, Jurgens H (1999) Chemosensitivity of glioma cells in vitro: a meta analysis. J Cancer Res Clin Oncol 125: 481–486.
    1. Ng WH, Wan GQ, Too HP (2007) Higher glioblastoma tumour burden reduces efficacy of chemotherapeutic agents: in vitro evidence. J Clin Neurosci 14: 261–266.
    1. Wolff JE, Trilling T, Molenkamp G, Egeler RM, Jurgens H (1999) Chemosensitivity of glioma cells in vitro: a meta analysis. J Cancer Res Clin Oncol 125: 481–486.
    1. Riccardi R, Riccardi A, Lasorella A, Di Rocco C, Carelli G, et al. (1994) Clinical pharmacokinetics of carboplatin in children. Cancer Chemother Pharmacol 33: 477–483.
    1. Wolff JE, Trilling T, Molenkamp G, Egeler RM, Jurgens H (1999) Chemosensitivity of glioma cells in vitro: a meta analysis. J Cancer Res Clin Oncol 125: 481–486.
    1. Hempel G, Flege S, Wurthwein G, Boos J (2002) Peak plasma concentrations of doxorubicin in children with acute lymphoblastic leukemia or non-Hodgkin lymphoma. Cancer Chemother Pharmacol 49: 133–141.
    1. Fruehauf JP, Brem H, Brem S, Sloan A, Barger G, et al. (2006) In vitro drug response and molecular markers associated with drug resistance in malignant gliomas. Clin Cancer Res 12: 4523–4532.
    1. Kato Y, Nishimura Si, Sakura N, Ueda K (2003) Pharmacokinetics of etoposide with intravenous drug administration in children and adolescents. Pediatr Int 45: 74–79.
    1. Souid AK, Dubowy RL, Blaney SM, Hershon L, Sullivan J, et al. (2003) Phase I clinical and pharmacologic study of weekly cisplatin and irinotecan combined with amifostine for refractory solid tumors. Clin Cancer Res 9: 703–710.
    1. Kupczyk-Subotkowska L, Siahaan TJ, Basile AS, Friedman HS, Higgins PE, et al. (1997) Modulation of melphalan resistance in glioma cells with a peripheral benzodiazepine receptor ligand-melphalan conjugate. J Med Chem 40: 1726–1730.
    1. Wolff JE, Trilling T, Molenkamp G, Egeler RM, Jurgens H (1999) Chemosensitivity of glioma cells in vitro: a meta analysis. J Cancer Res Clin Oncol 125: 481–486.
    1. Jiang X, Xin H, Sha X, Gu J, Jiang Y, et al. (2011) PEGylated poly(trimethylene carbonate) nanoparticles loaded with paclitaxel for the treatment of advanced glioma: in vitro and in vivo evaluation. Int J Pharm 420: 385–394.
    1. Doz F, Gentet JC, Pein F, Frappaz D, Chastagner P, et al. (2001) Phase I trial and pharmacological study of a 3-hour paclitaxel infusion in children with refractory solid tumours: a SFOP study. Br J Cancer 84: 604–610.
    1. Fruehauf JP, Brem H, Brem S, Sloan A, Barger G, et al. (2006) In vitro drug response and molecular markers associated with drug resistance in malignant gliomas. Clin Cancer Res 12: 4523–4532.
    1. Sankar A, Thomas DG, Darling JL (1999) Sensitivity of short-term cultures derived from human malignant glioma to the anti-cancer drug temozolomide. Anticancer Drugs 10: 179–185.
    1. Estlin EJ, Lashford L, Ablett S, Price L, Gowing R, et al. (1998) Phase I study of temozolomide in paediatric patients with advanced cancer. United Kingdom Children's Cancer Study Group. Br J Cancer 78: 652–661.
    1. Kletzel M, Kearns GL, Wells TG, Thompson HCJ (1992) Pharmacokinetics of high dose thiotepa in children undergoing autologous bone marrow transplantation. Bone Marrow Transplant 10: 171–175.
    1. Wolff JE, Trilling T, Molenkamp G, Egeler RM, Jurgens H (1999) Chemosensitivity of glioma cells in vitro: a meta analysis. J Cancer Res Clin Oncol 125: 481–486.
    1. Moore AS, Norris R, Price G, Nguyen T, Ni M, et al. (2011) Vincristine pharmacodynamics and pharmacogenetics in children with cancer: A limited-sampling, population modelling approach. J Paediatr Child Health 47: 875–882.
    1. Styczynski J, Olszewska-Slonina D, Kolodziej B, Napieraj M, Wysocki M (2006) Activity of bortezomib in glioblastoma. Anticancer Res 26: 4499–4503.
    1. Coluccia AML, Benati D, Dekhil H, De Filippo A, Lan C, et al. (2006) SKI-606 decreases growth and motility of colorectal cancer cells by preventing pp60(c-Src)-dependent tyrosine phosphorylation of beta-catenin and its nuclear signaling. Cancer Res 66: 2279–2286.
    1. Homsi J, Cubitt CL, Zhang S, Munster PN, Yu H, et al. (2009) Src activation in melanoma and Src inhibitors as therapeutic agents in melanoma. Melanoma Res 19: 167–175.
    1. Premkumar DR, Jane EP, Agostino NR, Scialabba JL, Pollack IF (2010) Dasatinib synergizes with JSI-124 to inhibit growth and migration and induce apoptosis of malignant human glioma cells. J Carcinog 9.
    1. Wang MY, Lu KV, Zhu S, Dia EQ, Vivanco I, et al. (2006) Mammalian target of rapamycin inhibition promotes response to epidermal growth factor receptor kinase inhibitors in PTEN-deficient and PTEN-intact glioblastoma cells. Cancer Res 66: 7864–7869.
    1. Alonso MM, Jiang H, Yokoyama T, Xu J, Bekele NB, et al. (2008) Delta-24-RGD in combination with RAD001 induces enhanced anti-glioma effect via autophagic cell death. Mol Ther 16: 487–493.
    1. van Vuurden D, Hulleman E, Meijer O, Wedekind L, Kool M, et al... (2011) PARP inhibition sensitizes childhood high grade glioma, medulloblastoma and ependymoma to radiation. Oncotarget.
    1. Hjelmeland MD, Hjelmeland AB, Sathornsumetee S, Reese ED, Herbstreith MH, et al. (2004) SB-431542, a small molecule transforming growth factor-beta-receptor antagonist, inhibits human glioma cell line proliferation and motility. Mol Cancer Ther 3: 737–745.
    1. Jane EP, Premkumar DR, Pollack IF (2006) Coadministration of sorafenib with rottlerin potently inhibits cell proliferation and migration in human malignant glioma cells. J Pharmacol Exp Ther 319: 1070–1080.
    1. Geoerger B, Kerr K, Tang CB, Fung KM, Powell B, et al. (2001) Antitumor activity of the rapamycin analog CCI-779 in human primitive neuroectodermal tumor/medulloblastoma models as single agent and in combination chemotherapy. Cancer Res 61: 1527–1532.
    1. Rich JN, Sathornsumetee S, Keir ST, Kieran MW, Laforme A, et al. (2005) ZD6474, a novel tyrosine kinase inhibitor of vascular endothelial growth factor receptor and epidermal growth factor receptor, inhibits tumor growth of multiple nervous system tumors. Clin Cancer Res 11: 8145–8157.

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