Evaluation of the relationship between [18F]FDG and P-glycoprotein expression: an experimental study

doi:10.1016/j.nucmedbio.2011.12.007

Nuclear Medicine and Biology

Volume 39, Issue 5, July 2012, Pages 671-678

https://doi.org/10.1016/j.nucmedbio.2011.12.007 Get rights and content

Abstract

Introduction

P-glycoprotein (P-gp) is a cell-membrane-associated protein that transports a variety of drug substrates. We sought to evaluate the relationship between 2-[¹⁸F]fluoro-2-deoxy-d-glucose ([¹⁸F]FDG) and P-gp expression using breast carcinoma Bcap37/multidrug resistant (MDR1) and Bcap37 in vitro and in vivo.

Methods

The function of P-gp expressed in Bcap37/MDR1 cells was evaluated using verapamil (VER), a classical inhibitor of P-gp. The accumulation of ^99mTc-methoxyisobutylisonitrile ([^99mTc]MIBI) in vitro was measured. In vivo imaging of severe combined immune deficiency (SCID) mice implanted with Bcap37 and Bcap37/MDR1 cells was performed by scintigraphy and micro-positron emission tomography (PET).

Results

The uptake of [^99mTc]MIBI was 0.62%±0.05% in the Bcap37/MDR1 cells and 2.02%±0.28% in the Bcap37 cells. VER significantly increased the uptake of [^99mTc]MIBI in the Bcap37/MDR1 cells (1.90%±0.09%) but not in the Bcap37 cells (2.15%±0.27%). In vivo, neither the Bcap37 nor Bcap37/MDR1 tumors grown in the SCID mice could be detected by [^99mTc]MIBI scintigraphy. Both the Bcap37 and Bcap37/MDR1 tumors were visible by micro-PET. The mean standardized uptake value (SUV) was significantly higher in the Bcap37 tumors (1.00±0.06) than in the Bcap37/MDR1 (0.67±0.11) tumors. VER significantly increased the mean SUV in the Bcap37/MDR1 tumors (1.02±0.16) but not in the Bcap37 tumors (1.09±0.22).

Conclusions

[¹⁸F]FDG combined with VER may be an effective noninvasive method of determining P-gp expression in tumors.

Introduction

The resistance of malignant tumors to chemotherapy is a major cause of treatment failure. One important mechanism in this resistance is the overexpression of adenosine triphosphate (ATP)-binding cassette transporters. The best-characterized member of this superfamily of transporters is P-glycoprotein (P-gp), which is significantly correlated with protein overexpression and chemoresistance in neuroblastoma, lymphoma, osteosarcoma and hepatocellular carcinoma (HCC) [1], [2], [3], [4]. P-gp is a 170-kDa, 1280-amino-acid product of the multidrug resistant (MDR) 1 gene found on the long arm of chromosome 7. It acts as an energy-dependent efflux pump that transports various chemically and functionally unrelated substances out of tumor cells, including, but not limited to, chemotherapeutic agents.

Positron emission tomography (PET) and PET/computed tomography (CT) are minimally invasive diagnostic tools that are used to detect and stage malignant tumors and to monitor the response of chemotherapeutics. The most commonly used PET radiotracer for in vivo tumor diagnostics is 2-[¹⁸F]fluoro-2-deoxy-d-glucose ([¹⁸F]FDG), which reflects changes in the glucose metabolic rate in tumor tissues. Several reports have shown that [¹⁸F]FDG uptake was inversely correlated with the expression level of P-gp in lung cancer, HCC and intrahepatic cholangiocarcinoma [4], [5], [6]. Lorke et al. [7] reported that [¹⁸F]FDG uptake was reduced in P-gp-positive tumor cells compared with P-gp-negative tumor cells in an animal study. Seo et al. [8] showed that [¹⁸F]FDG accumulation in vitro was reduced in MDR1 cell lines. However, Marian et al. [9] reported that MDR1-resistant tumors cells showed significantly higher [¹⁸F]FDG accumulation than MDR1-sensitive cells in vitro and in vivo studies. These contradictory results may have been due to the use of different tumor cell lines. We therefore chose the human breast cancer cell lines Bcap37 and Bcap37/MDR1, transfected with the human MDR1 (hMDR1) gene, to study the relationship of P-gp expression and [¹⁸F]FDG uptake in vitro and in vivo.

We report the results of [¹⁸F]FDG and ^99mTc-methoxyisobutylisonitrile ([^99mTc]MIBI) imaging in the presence and absence of verapamil (VER) in Bcap37 and Bcap37/MDR1 tumors and cell lines.

Section snippets

Cell culture

The human breast carcinoma cell lines Bcap37 and Bcap37/MDR1 were obtained from the Zhejiang University College of Pharmaceutical Sciences. The cells were cultivated in Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco, Karlsruhe, Germany) under standard conditions (37°C, 100% relative humidity, 5% CO₂/95% air) and in McCoy's 5A medium (Gibco/Life Technologies, Karlsruhe, Germany) supplemented with 10% heat-inactivated fetal calf serum.

Uptake of R123 in vitro

Rhodamine 123 (R123) is a cationic compound that

R123 uptake

R123 is taken up and retained by nonresistant cells but pumped rapidly out of the cytoplasm by the P-gp transporter protein in MDR cells. The Bcap37 and Bcap37/MDR1 cells were incubated in the presence of R123. All Bcap37 cells exhibited a bright fluorescent staining indicating the presence of R123 (Fig. 1A). In contrast, the Bcap37/MDR1 cells showed only a very weak fluorescence (Fig. 1B). Additionally, inhibition of the P-gp function by adding VER to the R123 incubation medium resulted in a

Discussion

This study was designed to assess the relationship between P-gp expression and [¹⁸F]FDG uptake in vitro and in vivo. For that purpose, the MDR breast cancer Bcap37/MDR1 cell line and the Bcap37 nonresistant tumor cell line were used. The P-gp function of the Bcap37/MDR1 cells was assessed by R123-uptake experiments. The P-gp drug transporter activity was assessed by measuring [^99mTc]MIBI accumulation [10].

R123 is a cationic compound that excites at 488 nm and emits at 515–575 nm (green

Conclusion

[¹⁸F]FDG combined with VER may be an effective noninvasive method for the diagnosis of P-gp expression in tumors. Furthermore, [¹⁸F]FDG-PET or PET/CT using the ListMode acquisition mode may have potential clinical value for detecting P-gp in tumors.

Acknowledgments

The authors would like to thank Jiangsu Institute of Nuclear Medicine for the help in the mice data acquisition by micro-PET.

The study was financially supported by the Key Laboratory of Nuclear Medicine Open Subject, Ministry of Health, Jiangsu Institute of Nuclear Medicine, P.R. China.

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A triphenylphosphonium cation, [^99mTc]Technetium cyclopentadienyltricarbonyl-6-hexanoyl-triphenylphosphonium cation ([^99mTc]3) was prepared to target multidrug resistance (MDR). The radiotracer was evaluated in the MDR-negative MCF-7 and MDR-positive MCF-7/ADR cell lines in vitro, as well as animal models in vivo. [^99mTc]3 was proofed to be a substrate of P-glycoprotein and multidrug resistant protein 1, and showed a higher accumulation in the MDR-negative MCF-7 cells compared to ^99mTc-sestamibi in vitro. The MCF-7 tumor-to-MCF-7/ADR tumor ratio of [^99mTc]3 was ∼3 at 1 h p.i. in the biodistribution study. These results demonstrated the capability of the radiotracer to detect multidrug resistance in tumor cells.
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Citation Excerpt :
These results are in good agreement with our in vivo findings that [18F]FDG is not transported by rodent Abcb1 and suggest that [18F]FDG is neither transported by human nor by rodent ABCB1. If [18F]FDG is not transported by ABCB1 in vivo, other factors than efflux by ABCB1 must have accounted for the lower accumulation of [18F]FDG in ABCB1-overexpressing as compared with non-ABCB1-expressing tumors [9,10]. These factors may include differences in GLUT expression levels, differences in hexokinase activity or differences in vascularization or blood flow between tumors.
Transport of 2-[¹⁸F]fluoro-2-deoxy-d-glucose ([¹⁸F]FDG) by the multidrug efflux transporters P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) at the blood–brain barrier (BBB) may confound the interpretation of [¹⁸F]FDG brain PET data. Aim of this study was to assess the influence of ABCB1 and ABCG2 at the BBB on brain distribution of [¹⁸F]FDG in vivo by performing [¹⁸F]FDG PET scans in wild-type and transporter knockout mice and by evaluating changes in [¹⁸F]FDG brain distribution after transporter inhibition.
Dynamic small-animal PET experiments (60 min) were performed with [¹⁸F]FDG in groups of wild-type and transporter knockout mice (Abcb1a/b^(−/−), Abcg2^(−/−) and Abcb1a/b^(−/−)Abcg2^(−/−)) and in wild-type rats without and with i.v. pretreatment with the known ABCB1 inhibitor tariquidar (15 mg/kg, given at 2 h before PET). Blood was sampled from animals from the orbital sinus vein at the end of the PET scans and measured in a gamma counter. Brain uptake of [¹⁸F]FDG was expressed as the brain-to-blood radioactivity concentration ratio in the last PET time frame (K_b,brain).
K_b,brain values of [¹⁸F]FDG were not significantly different between different mouse types both without and with tariquidar pretreatment. The blood-to-brain transfer rate constant of [¹⁸F]FDG was significantly lower in tariquidar-treated as compared with vehicle-treated rats (0.350 ± 0.025 mL/min/g versus 0.416 ± 0.024 mL/min/g, p = 0.026, paired t-test) but K_b,brain values were not significantly different between both rat groups.
Our results show that [¹⁸F]FDG is not transported by Abcb1 at the mouse and rat BBB in vivo. In addition we found no evidence for Abcg2 transport of [¹⁸F]FDG at the mouse BBB.
Our findings imply that functional activity of ABCB1 and ABCG2 at the BBB does not need to be taken into account when interpreting brain [¹⁸F]FDG PET data.
18FDG a PET tumor diagnostic tracer is not a substrate of the ABC transporter P-glycoprotein
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Citation Excerpt :
Thus, expression of Pgp at high level may increase the ATP demand of cells explaining our observations. Yu et al. (2012) also measured an increased in vitro uptake of 18FDG into the Pgp+ Bcap37/MDR1 human breast carcinoma cell line compared to its Pgp− counterpart (Bcap37) upon short (10 min) incubation. Similarly, in their in vivo 18FDG uptake experiments they also measured higher 18FDG accumulation in the Bcap37/MDR1 tumors compared to the Bcap37 tumors.
2-[¹⁸F]fluoro-2-deoxy-d-glucose (¹⁸FDG) is a tumor diagnostic radiotracer of great importance in both diagnosing primary and metastatic tumors and in monitoring the efficacy of the treatment. P-glycoprotein (Pgp) is an active transporter that is often expressed in various malignancies either intrinsically or appears later upon disease progression or in response to chemotherapy. Several authors reported that the accumulation of ¹⁸FDG in P-glycoprotein (Pgp) expressing cancer cells (Pgp⁺) and tumors is different from the accumulation of the tracer in Pgp nonexpressing (Pgp⁻) ones, therefore we investigated whether ¹⁸FDG is a substrate or modulator of Pgp pump.
Rhodamine 123 (R123) accumulation experiments and ATPase assay were used to detect whether ¹⁸FDG is substrate for Pgp. The accumulation and efflux kinetics of ¹⁸FDG were examined in two different human gynecologic (A2780/A2780AD and KB-3-1/KB-V1) and a mouse fibroblast (3T3 and 3T3MDR1) Pgp⁺ and Pgp⁻ cancer cell line pairs both in cell suspension and monolayer cultures.
We found that ¹⁸FDG and its derivatives did not affect either the R123 accumulation in Pgp⁺ cells or the basal and the substrate stimulated ATPase activity of Pgp supporting that they are not substrates or modulators of the pump. Measuring the accumulation and efflux kinetics of ¹⁸FDG in different Pgp⁺ and Pgp⁻ cell line pairs, we have found that the Pgp⁺ cells exhibited significantly higher (p ⩽ 0.01) ¹⁸FDG accumulation and slightly faster ¹⁸FDG efflux kinetics compared to their Pgp⁻ counterparts. The above data support the idea that expression of Pgp may increase the energy demand of cells resulting in higher ¹⁸FDG accumulation and faster efflux.
We concluded that ¹⁸FDG and its metabolites are not substrates of Pgp.
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Evaluation of the relationship between [18F]FDG and P-glycoprotein expression: an experimental study

Abstract

Introduction

Methods

Results

Conclusions

Introduction

Section snippets

Cell culture

Uptake of R123 in vitro

R123 uptake

Discussion

Conclusion

Acknowledgments

Surgery

J Photochem Photobiol B

J Biol Chem

Cancer Lett

Bioorg Med Chem

Bioorg Med Chem

Bioorg Med Chem Lett

P-glycoprotein expression as a predictor of the outcome of therapy for neuroblastoma

N Engl J Med

Expression of P-glycoprotein in high-grade osteosarcomas in relation to clinical outcome

N Engl J Med

Expression of P-glycoprotein and glutathione-S-transferase in recurrent lymphomas: the possible role of Epstein–Barr virus, immunophenotypes, and other predisposing factors

J Clin Oncol

Fluorine-18 fluorodeoxyglucose positron emission tomography predicts tumor differentiation, P-glycoprotein expression, and outcome after resection in hepatocellular carcinoma

Clin Cancer Res

P-glycoprotein expression is associated with FDG uptake and cell differentiation in patients with untreated lung cancer

Nucl Med Commun

In vitro and in vivo tracer characteristics of an established multidrug-resistant human colon cancer cell line

J Nucl Med

Evaluation of the relationship between [¹⁸F]FDG and P-glycoprotein expression: an experimental study