Elsevier

Methods

Volume 27, Issue 3, July 2002, Pages 228-233
Methods

Dynamics of multidrug resistance: P-glycoprotein analyses with positron emission tomography

https://doi.org/10.1016/S1046-2023(02)00079-8Get rights and content

Abstract

Multidrug resistance (MDR) is characterized by the occurrence of cross-resistance to a broad range of structurally and functionally unrelated drugs. Several mechanisms are involved in MDR. One of the most well-known mechanisms is the overexpression of P-glycoprotein (P-gp), encoded by the MDR1 gene in humans and by the mdr1a and mdr1b genes in rodents. P-gp is extensively expressed in the human body, e.g., in the blood–brain barrier and also in solid tumor tissue. Overexpression of P-gp on tumor membranes might result in MDR of human tumors. To circumvent this resistant phenotype, several P-gp modulators such as cyclosporin A (CsA) are available. Competition between P-gp drugs and modulators results in decreased transport of the drug out of tumor tissue and an increased cellular level of these drugs. For effective clinical treatment it is important to have knowledge about P-gp functionality in tumors. Therefore, we have developed a method to measure the P-gp functionality in vivo with PET and [11C]verapamil as a positron-emitting P-gp substrate. The results obtained in rodents and in cancer patients are described in this article.

Introduction

At present, malignant brain tumors and metastases are still difficult to treat with cytotoxic agents. Even though new chemotherapeutic schedules have improved results of cancer treatment in other parts of the body (e.g., small cell lung cancer, breast cancer), efficacy for brain metastases is poor. For instance, in 10% of all patients with small cell lung cancer (SCLC), brain metastases are detected at the time of presentation. In another 20–25% they are diagnosed later in life and in up to 65% of cases at autopsy [1].

One problem in tumor treatment is the so-called multidrug resistance (MDR). MDR is defined as an acquired or intrinsic resistance to many cytotoxic drugs which are natural products and do not necessarily share chemical structures, such as anthracyclines (doxorubicin), vinca alkaloids (vincristine), epipodophyllotoxins (etoposide), and taxanes (paclitaxel). Different mechanisms behind MDR such as the presence of membrane efflux pumps [P-glycoprotein (P-gp) and the multidrug resistance-associated protein (MRP)], decreased topoisomerase II protein, increased drug detoxification, and overexpression of the 110-kDa lung-resistant related protein (LRP) [2] are reported to play a role in MDR.

The brain is considered as a sanctuary for several drugs because of the presence of the blood–brain barrier (BBB) [3]. The BBB is a complex layer of brain capillary endothelial cells, astroglia, pericytes, and perivacular macrophages within a basal lamina [4]. Its physiological function is to separate the brain from the blood to permit the rigorous control of the cerebral environment that is necessary for complex neural signaling and for brain homeostasis. One of the mechanisms to maintain brain homeostasis is the expression of P-gp and MRP in the blood–brain barrier. The BBB is an impediment not only to the chemotherapeutic treatment of brain tumors, but also to drug therapy of psychiatric and neurodegenerative diseases. Therefore the brain is considered as a sanctuary for P-gp substrates [5]. Intracellular levels of P-gp substrates may be increased by modulation of P-gp, i.e., inhibition of P-gp-facilitated transport with compounds such as cyclosporin A (CsA) and PSC 833 [6].

Positron emission tomography (PET) enables visualization of human physiology by electronic detection of short-lived positron-emitting radiopharmaceuticals. It is the only noninvasive technology that can routinely and quantitatively measure metabolic, biochemical, and functional activity in living tissue. The decay of a positron emitter is schematically represented in Fig. 1. Positrons (ß+) are particles with the mass of an electron, but a positive instead of a negative charge. They are emitted from the nuclei of radioisotopes that are unstable because they have an excessive number of protons and a positive charge. Positron emission stabilizes the nucleus by removing a positive charge through the conversion of a proton to a neutron. During this type of decay, one element is converted to another, the daughter having an atomic number one less than that of the parent. For radioisotopes used in PET, the daughter element is stable (i.e., not radioactive). All radioisotopes used in PET decay by positron emission. A positron (ß+) emitted from a decaying nucleus travels a short distance (i.e., maximally a few millimeters) before colliding with an electron of a nearby atom.

Combination of a positron and an electron results in an annihilation reaction, after formation of a positronium as an intermediate. The total mass of the two particles is converted i.e., two 511-keV γ rays that are emitted in 180° opposite directions. These photons easily escape from the human body and can be recorded by external detectors. The line connecting two detectors that are virtually simultaneously hit by a 511-keV photon is called a coincidence line. Coincidence lines provide a unique opportunity to construct tomographic images (Fig. 1).

Several techniques are available to study the presence of P-gp and MRP drug transporters at the DNA level, the mRNA level, and the protein level. However, the presence of P-gp and/or MRP in organs does not answer the question about functionality of these drug efflux pumps. For clinical purposes, noninvasive P-gp transport function measurements should be performed in vivo. In principle, this can be achieved by the application of PET and a radiolabeled P-gp substrate as tracer. We studied the P-gp function in solid tumors with [11C]verapamil as a specific P-gp substrate.

Section snippets

Animal model

Nude rats were injected with human small cell lung carcinoma cells (GLC4, 107 cells/0.1 ml RPMI/fetal calf serum (FCS) 10%) in the left flank and P-gp-expressing human small cell lung carcinoma cells (GLC4/P-gp, 107 cells/0.1 ml RPMI/FCS 10%) in the right flank.

Tumor histology

For histological examinations, the tumors were excised and rapidly frozen in liquid nitrogen. From the frozen tumor tissue 4-μm slices were cut. Cell viability and cell density were measured in hematoxylin–eosin-stained slices. Cell

Tumor histology

A semiquantative assessment demonstrated that more than 80–90% of the cells in both tumor types were viable. The GLC4 and the GLC4/P-gp tumors consisted of tumor cells, blood vessels, and rat stroma. Histology revealed that the cell density of GLC4 tumors was lower than that of GLC4/P-gp tumors (P=0.0008, n=3). The cell density of GLC4 tumors was 8.7×109±0.7×109 cells/ml tumor, and that of GLC4/P-gp tumors, 13×109±0.4×109 cells/ml tumor. GLC4 tumor tissue showed no P-gp expression and GLC4/P-gp

Discussion

Multidrug resistance is characterized by the occurrence of cross-resistance to a broad range of structurally and functionally unrelated drugs [7]. Drugs that are involved in MDR belong to the group of drugs, such as the anthracyclines (doxorubicin, daunorubicin), vinca alkaloids (vincristine, vinblastine), epipodophyllotoxins (etoposide), and taxanes (paclitaxel) [8], [9]. One of the most well-known mechanism is the overexpression of P-gp.

P-gp is extensively expressed in the human body, namely,

References (23)

  • A.H Schinkel et al.

    Cell

    (1994)
  • J Lankelma et al.

    Biochim. Biophys. Acta

    (1990)
  • J.L Nugent et al.

    Cancer

    (1979)
  • G.L Scheffer et al.

    Nat. Med.

    (1995)
  • C Cordon-Cardo et al.

    Proc. Natl. Acad. Sci. USA

    (1989)
  • N.J Abbot et al.

    Cerebrovasc. Brain Metast. Rev.

    (1991)
  • D.J Stewart et al.

    J. Neurooncol.

    (1983)
  • D Damiani et al.

    Leukemia

    (1998)
  • L.J Goldstein et al.

    J. Natl. Cancer Inst.

    (1989)
  • J.G Zijlstra et al.

    Cancer Res.

    (1987)
  • C.F Higgins

    Annu. Rev. Cell. Biol.

    (1992)
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