Expression of Drug Resistance Proteins Pgp, MRP1, MRP3, MRP5 AND GST-π in Human Glioma
Chemotherapy in glioma is poorly effective: the blood–brain barrier and intrinsic and/or acquired drug resistance of tumor cells could partly explain this lack of major effect. We investigated expression of P-glycoprotein (Pgp), multidrug resistance protein (MRP) 1, MRP3, MRP5 and glutathione-S-transferase π (GST-π) in malignant glioma patients. Cytofluorimetric analysis of 48 glioma specimens and 21 primary cultures showed high levels of MRP1, moderate levels of MRP5 and low levels of Pgp, GST-π and MRP3. Immunohistochemistry (25 glioma specimens) showed expression of GST-π (66.7% of cases), MRP1 (51.3%), MRP5 (45.8%), Pgp (34.8%) and MRP3 (29.9%) in tumor cells. Moreover, analysis of tumor samples by real time quantitative PCR showed mRNA expression of all investigated genes. Tumor vasculature, analyzed in glioma specimens and in tumor derived endothelial cells, showed expression of all investigated proteins. Non-tumor brain samples (from a patient with arteriovenous malformation and from one with epilepsy), normal human astrocytes and cultured endothelial cells were also analyzed: astrocytes and endothelial cells expressed the highest levels of the investigated proteins, mainly MRP1 and MRP5. No significant differences in proteins expression were detected between primary or recurrent gliomas, suggesting that glioma chemoresistance is mostly intrinsic. Therefore, we detected, for the first time, the presence of MRP3 and MRP5 on glioma specimens – both in tumor and endothelial cells – and we delineated an expression profile of chemoresistance proteins in glioma. The possible association of inhibitors of drug efflux pumps with chemotherapy could be investigated to improve drugs delivery into the tumor and their cytotoxic effects.
Keywordsastrocytes blood–brain barrier glioma GST-π MRP1 MRP3 MRP5 multidrug resistance P-glycoprotein
Cordon-Cardo, C, O’Brien, JP, Casals, D, Rittman-Grauer, L, Biedler, JL, Melamed, MR, Bertino, JR 1989Multidrug-resistance gene (P-glycoprotein) is expressed by endothelial cells at blood–brain barrier sitesProc Natl Acad Sci USA86695698PubMedGoogle Scholar Tóth, K, Vaughan, MM, Peress, NS, Slocum, HK, Rustum, YM 1996MDR1 P-glycoprotein is expressed by endothelial cells of newly formed capillaries in human gliomas but is not expressed in the neovasculature of other primary tumorsAm J Pathol149853858PubMedGoogle Scholar Sun, H, Dai, H, Shaik, N, Elmquist, WF 2003Drug efflux transporters in the CNSAdv Drug Deliv Rev5583105CrossRefPubMedGoogle Scholar Borst, P, Evers, R, Kool, M, Wijnholds, J 1999The multidrug resistance protein familyBiochim Biophys Acta1461347357PubMedGoogle Scholar Mohri, M, Nitta, H, Yamashita, J 2000Expression of multidrug resistance-associated protein (MRP) in human gliomasJ Neuro-Oncol49105115CrossRefGoogle Scholar Spiegl-Kreinecker, S, Buchroithner, J, Elbling, L, Steiner, E, Wurm, G, Bodenteich, A, Fischer, J, Micksche, M, Berger, W 2002Expression and functional activity of the ABC-transporter proteins P-glycoprotein and multidrug-resistance protein 1 in human brain tumor cells and astrocytesJ Neuro-Oncol572736CrossRefGoogle Scholar Bart, J, Groen, HJM, Hendrikse, NH, Graaf, WTA, Vaalburg, W, Vries, EGE 2000The blood–brain barrier and oncology: new insights into function and modulationCancer Treat Rev26449462CrossRefPubMedGoogle Scholar Ali-Osman, F, Caughlan, J, Gray, GS 1989Decreased DNA interstrand cross-linking and cytotoxicity induced in human brain tumor cells by 1,3-bis(2-chloroethyl)-1-nitrosourea after in vitro reaction with glutathioneCancer Res4959545958PubMedGoogle Scholar Grant, R, Ironside, J 1995Glutathione S-transferases and cytochrome P450 detoxifying enzyme distribution in human cerebral gliomaJ Neuro-Oncol2517CrossRefGoogle Scholar Esteller, M, Garcia-Foncillas, J, Andion, E, Goodman, SN, Hidalgo, OF, Vanaclocha, V, Baylin, SB, Herman, JG 2000Inactivation of the DNA-repair gene MGMT and the clinical response of gliomas to alkylating agentsN Engl J Med34313501354CrossRefPubMedGoogle Scholar Tanaka, S, Kobayashi, I, Utsuki, S, Oka, H, Fujii, K, Watanabe, T, Nagashima, T, Hori, T 2003O6-methylguanine-DNA methyltransferase gene expression in gliomas by means of real-time quantitative RT-PCR and clinical response to nitrosoureasInt J Cancer1036772CrossRefPubMedGoogle Scholar Ali-Osman, F, Brunner, JM, Kutluk, TM, Hess, K 1997Prognostic significance of glutathione S-transferase π expression and subcellular localization in human gliomasClin Cancer Res322532261PubMedGoogle Scholar Bossanyi, P, Diete, S, Dietzmann, K, Warich-Kirches, M, Kirches, E 1997Immunohistochemical expression of P-glycoprotein and glutathione S-transferases in cerebral gliomas and response to chemotherapyActa Neoropathol94605611CrossRefGoogle Scholar Korshunov, A, Golanov, A, Sycheva, R 2002Immunohistochemical markers for prognosis of cerebral glioblastomasJ Neuro-Oncol58217236CrossRefGoogle Scholar Billson, AL, Palmer, JB, Walker, DA, Lowe, J 1994Multidrug resistance gene (MDR 1) expression in neuro-axial tumors of children and young adultsBr J Neurosurg8585591PubMedGoogle Scholar Reavey-Cantwell, J, Haroun, RI, Zahurak, M, Clatterbuck, RE, Parker, RJ, Mehta, R, Fruehauf, JP, Brem, H 2001The prognostic value of tumors markers in patients with glioblastoma multiforme: analysis of 32 patients and review of the literatureJ Neuro-Oncol55195204CrossRefGoogle Scholar Rieger, L, Rieger, J, Winter, S, Streffer, J, Esser, P, Dichgans, J, Meyermann, R, Weller, M 2000Evidence for a constitutive, verapamil-sensitive, non-P-glycoprotein multidrug resistance phenotype in malignant glioma that is unaltered by radiochemotheraphy in vivoActa neuropathol99555562CrossRefPubMedGoogle Scholar Jaffe, EA, Nachman, RL, Becker, CG, Minick, CR 1973Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteriaJ Clin Invest5227452756PubMedGoogle Scholar Mrsulia, BB, Mrsulia, BT, Fujimoto, T, Klatzo, I, Spatz, M 1976Isolation of brain capillaries: a simplified techniqueBrain Res110361365CrossRefPubMedGoogle Scholar Vescovi, AL, Parati, EA, Gritti, A, Poulin, P, Ferrario, M, Wanke, E, Frolichsthal-Schoeller, P, Cova, L, Arcellana-Panlilio, M, Colombo, A, Galli, R 1999Isolation and cloning of multipotential stem cells from the embryonic human CNS and establishment of transplantable human neural stem cell lines by epigenetic stimulationExp Neurol1567183CrossRefPubMedGoogle Scholar Stewart, LA 2002Chemotherapy in adult high-grade glioma: a systematic review and meta-analysis of individual patient data from 12 randomised trialsLancet35910111018PubMedGoogle Scholar Henderson, CJ, McLaren, AW, Moffat, GJ, Bacon, EJ, Wolf, CR 1998Pi-class glutathione S-transferase: regulation and functionChem Biol Interact111-1126982CrossRefPubMedGoogle Scholar Kvackajova-Kisucka, J, Barancik, M, Breier, A, Kvackajova-Kisucka, J, Barancik, M, Breier, A 2001Drug transporters and their role in multidrug resistance of neoplastic cellsGen Physiol Biophys20215237PubMedGoogle Scholar Bahr, O, Rieger, J, Duffner, F, Meyermann, R, Weller, M, Wick, W 2003P-glycoprotein and multidrug resistance-associated protein mediate specific patterns of multidrug resistance in malignant glioma cell lines, but not in primary glioma cellsBrain Pathol13482494PubMedGoogle Scholar Decleves, X, Fajac, A, Lehmann-Che, J, Tardy, M, Mercier, C, Hurbain, I, Laplanche, JL, Bernaudin, JF, Scherrmann, JM 2002Molecular and functional MDR1-Pgp and MRPs expression in human glioblastoma multiforme cell linesInt J Cancer98173180CrossRefPubMedGoogle Scholar Haga, S, Hinoshita, E, Ikezaki, K, Fukui, M, Scheffer, GL, Uchiumi, T, Kuwano, M 2001Involvement of the multidrug resistance protein 3 in drug sensitivity and its expression in human gliomaJpn J Cancer Res92211219PubMedGoogle Scholar Abe, T, Mori, T, Wakabayashi, Y, Nakagawa, M, Cole, SP, Koike, K, Kuwano, M, Hori, S 1998Expression of multidrug resistance protein gene in patients with glioma after chemotherapyJ Neuro-Oncol401118CrossRefGoogle Scholar Benyahia, B, Huguet, S, Decleves, X, Mokhtari, K, Criniere, E, Bernaudin, JF, Scherrmann, JM, Delattre, JY 2004Multidrug resistance-associated protein MRP1 expression in human gliomas: chemosensitization to vincristine and etoposide by indomethacin in human glioma cell lines overexpressing MRP1J Neurooncol666570CrossRefPubMedGoogle Scholar Abe, T, Hasegawa, S, Taniguchi, K, Yokomizo, A, Kuwano, T, Ono, M, Mori, T, Hori, S, Kohno, K, Kuwano, M 1994Possible involvement of multidrug-resistance-associated protein (MRP) gene expression in spontaneous drug resistance to vincristine, etoposide and adriamycin in human glioma cellsInt J Cancer58860864PubMedGoogle Scholar Kiwit, JC, Hertel, A, Matuschek, AE 1994Reversal of chemoresistance in malignant gliomas by calcium antagonists: correlation with the expression of multidrug-resistant p-glycoproteinJ Neurosurg81587594PubMedGoogle Scholar Dombrowski, SM, Desai, SY, Marroni, M, Cucullo, L, Goodrich, K, Bingaman, W, Mayberg, MR, Bengez, L, Janigro, D 2001Overexpression of multiple drug resistance genes in endothelial cells from patients with refractory epilepsyEpilepsia4215011506CrossRefPubMedGoogle Scholar Oguri, T, Isobe, T, Suzuki, T, Nishio, K, Fujiwara, Y, Katoh, O, Yamakido, M 2000Increased expression of the MRP5 gene is associated with exposure to platinum drugs in lung cancerInt J Cancer8695100CrossRefPubMedGoogle Scholar Kool, M, Haas, M, Scheffer, GL, Scheper, RJ, Eijk, MJ, Juijn, JA, Baas, F, Borst, P 1997Analysis of expression of cMOAT (MRP2), MRP3, MRP4, and MRP5, homologues of the multidrug resistance-associated protein gene (MRP1), in human cancer cell linesCancer Res5735373547PubMedGoogle Scholar Hirrlinger, J, Konig, J, Dringen, R 2002Expression of mRNAs of multidrug resistance proteins (Mrps) in cultured rat astrocytes, oligodendrocytes, microglial cells and neuronesJ Neurochem82716719CrossRefPubMedGoogle Scholar Terrier, P, Townsend, AJ, Coindre, JM, Triche, TJ, Cowan, KH 1990An immunohistochemical study of pi class glutathione S-transferase expression in normal human tissueAm J Pathol137845853PubMedGoogle Scholar Beaulieu, E, Demeule, M, Ghitescu, L, Beliveau, R 1997P-glycoprotein is strongly expressed in the luminal membranes of the endothelium of blood vessels in the brainBiochem J326539544PubMedGoogle Scholar Megard, I, Garrigues, A, Orlowski, S, Jorajuria, S, Clayette, P, Ezan, E, Mabondzo, A 2002A co-culture-based model of human blood–brain barrier: application to active transport of indinavir and in vivo–in vitro correlationBrain Res927153167CrossRefPubMedGoogle Scholar Gaillard, PJ, Sandt, IC, Voorwinden, LH, Vu, D, Nielsen, JL, Boer, AG, Breimer, DD 2000Astrocytes increase the functional expression of P-glycoprotein in an in vitro model of the blood–brain barrierPharm Res1711981205CrossRefPubMedGoogle Scholar Goldman, B 2003Multidrug resistance: can new drugs help chemotherapy score against cancer?J Natl Cancer Inst95255257PubMedGoogle Scholar