Visualization of multidrug resistance in vivo

Various mechanisms are involved in multidrug resistance (MDR) for chemotherapeutic drugs, such as the drug efflux pumps, P-glycoprotein (Pgp) and multidrug resistance-associated protein (MRP). In this review the mechanisms involved in MDR are described and results are reviewed with particular attention to the in vivo imaging of Pgp and MRP. Various detection assays provide information about the presence of drug efflux pumps at the mRNA and protein levels. However, these methods do not yield information about the dynamic function of Pgp and MRP in vivo. For the study of Pgp- and MRP-mediated transport, single-photon emission tomography (SPET) and positron emission tomography (PET) are available. Technetium-99m sestamibi is a substrate for Pgp and MRP, and has been used in clinical studies for tumour imaging, and to visualize blockade of Pgp-mediated transport after modulation of the Pgp pump. Other ^99mTc radiopharmaceuticals, such as ^99mTc-tetrofosmin and several ⁹⁹Tc-Q complexes, are also substrates for Pgp, but to date only results from in vitro and animal studies are available for these compounds. Several agents, including [¹¹C]colchicine, [¹¹C]verapamil and [¹¹C]daunorubicin, have been evaluated for the quantification of Pgp-mediated transport with PET in vivo. The results suggest that radiolabelled colchicine, verapamil and daunorubicin are feasible substrates with which to image Pgp function in tumours. Uptake of [¹¹C]colchicine and [¹¹C]verapamil is relatively high in the chest area, reducing the value of both tracers for monitoring Pgp-mediated drug transport in tumours located in this region. In addition, it has to be borne in mind that only comparison of Pgp-mediated transport of radioalabelled substrates in the absence and in the presence of Pgp blockade gives quantitative information on Pgp-mediated pharmacokinetics. Leukotrienes are specific substrates for MRP. Therefore, N-[¹¹C]acetyl-leukotriene E₄ provides an opportunity to study MRP function non-invasively. Results obtained in MRP₂ mutated GY/TR rats have demonstrated visualization of MRP-mediated transport. This tracer permits the study of MRP transport function abnormalities in vivo, e.g. in Dubin-Johnson patients, who are MRP₂ gene deficient. Results obtained show the feasibility of using SPET and PET to study the functionality of MDR transporters in vivo.     

In vivo and in vitro multitracer analyses of P-glycoprotein expression-related multidrug resistance

P-glycoprotein (Pgp) is an ABC (ATP binding cassette) transporter that is often overexpressed in tumours, contributing significantly to their multidrug resistance. In this study, we explored whether the radiotracers used in tumour diagnostics can be used for in vivo visualisation of Pgp-related multidrug resistance. We also examined the effects of different Pgp modulators on the accumulation of these radioligands in tumours with or without Pgp expression. In a SCID BC-17 mouse model, cells of the drug-sensitive KB-3-1 (MDR^–) and the KB-V1 Pgp-expressing (MDR⁺) human epidermoid carcinoma cell lines were inoculated to yield tumours in opposite flanks. For in vivo scintigraphic (biodistribution) and positron emission tomography (PET) examinations, the mice were injected with technetium-99m hexakis-2-methoxybutylisonitrile (^99mTc-MIBI), carbon-11 labelled methionine and fluorine-18 fluoro-2-deoxy-d-glucose (¹⁸FDG). For validation, in vitro cell studies with ^99mTc-MIBI, ^99mTc-tetrofosmin, [¹¹C]methionine and ¹⁸FDG were carried out using a gamma counter. The expression and function of the MDR product were proved by immunohistochemistry and spectrofluorimetry. ^99mTc-MIBI uptake was significantly lower in KB-V1 cells as compared with KB-3-1-derived tumours in vivo (Pgp⁺/Pgp^– =0.61±0.13; P<0.01) and cells in vitro (Pgp⁺/Pgp^– =0.08±0.01; P<0.001). Cyclosporin A reversed ^99mTc-MIBI uptake in the Pgp+ cells, while verapamil failed to modify it. ¹⁸FDG uptake was significantly higher in KB-V1 tumours (Pgp⁺/Pgp^– =1.36±0.05; P<0.01) and cells (Pgp⁺/Pgp^– =1.52±0.12; P<0.001). Whereas cyclosporin A eliminated the difference between FDG uptake in MDR⁺ and MDR^– cell lines, verapamil significantly increased it. When the animals were treated with verapamil, the ratio of ^99mTc-MIBI uptake in the MDR⁺ tumours to that in the MDR^– tumours decreased to 0.38±0.05 (P<0.01), while the ratio of ¹⁸FDG uptake increased to 2.1±0.3 (P<0.001). There were no significant differences in the [¹¹C]methionine uptake in the MDR⁺ and MDR^– tumours and cell lines, nor was [¹¹C]methionine accumulation modified by cyclosporin A. Parallel administration of ¹⁸FDG and ^99mTc-MIBI combined with verapamil treatment seems to be a good candidate as a non-invasive marker for the diagnosis of MDR-related Pgp expression in tumours.     

肿瘤多药耐药蛋白抑制剂

肿瘤细胞产生多药耐药性是肿瘤化疗失败的主要原因之一,肿瘤多药耐药性（ｍｕｌｉｄｒｕｇｒｅｓｉｓａｎｃｅ,ＭＤＲ）的产生主要与肿瘤细胞的膜蛋白,Ｐ－糖蛋白（ＰＧ－１７０）多药耐药蛋白（ＭＲＰ－１９０）和肺耐药蛋白（ＬＲＰ）的过多表达有关。在发现维拉帕米,奎尼丁和环孢菌素等药物具有逆转ＭＤＲ作用的基础上,发展了一些新的能够逆转ＭＤＲ现象的多药耐药蛋白抑制剂,如环孢菌素的类似物ＳＤＺＰＳＣ８３３和奎民丁     

分子影像技术在多药耐药监测中的研究进展

肿瘤的多药耐药（MDR）是导致癌症复发和转移的重要因素。ATP结合盒转运蛋白（ABC转运蛋白）在人体广泛存在,并且ABC转运蛋白的过度表达是导致肿瘤细胞产生MDR的主要原因之一。利用分子影像技术对MDR肿瘤进行早期监测,有利于临床医师制定有效的治疗方案,并在一定程度上能改善肿瘤患者的预后。笔者就分子影像技术在监测MDR方面的研究进展进行简要综述。     

Clinical imaging of multidrug resistance in cancer.

The most well-characterized mechanism of multidrug resistance (MDR) involves P-glycoprotein (Pgp), a transmembrane protein acting as an ATP-dependent drug efflux pump. The recognition of 99mTc-Sestamibi and other lipophilic cations as transport substrates for Pgp provided the necessary tool for the clinical assessment of Pgp function in patients with cancer. Many clinical studies from different institutions and trials including a variety of malignancies indicate that both tumor uptake and clearance of 99mTc-Sestamibi are correlated with Pgp expression and may be used for the phenotypic assessment of multidrug resistance. Although both parameters may predict tumor response to chemotherapy, the extraction of efflux rate constants appeared to provide a more direct index of Pgp function as compared to tracer uptake ratio allowing to trace a continuous spectrum of drug transport activity. Preliminary studies reported the use of MDR imaging agents to monitor the modulating ability of revertant compounds. Although the results support the feasibility of this approach, the alteration of tracer pharmacokinetics induced by the modulators certainly constitutes a challenge in the development of a simple functional test suitable in clinical practice. The extension of the acquired imaging methodology to tumors with redundant intrinsic resistant mechanisms such as lung cancer requires further investigations on the relative contribution and clinical relevance of each mechanism. Due to the multifactorial nature of the phenomenon, the development of new tracers with substrate specificity for other known drug transporters would hopefully help to dissect the complex array of cellular mechanisms contributing to treatment failure.     

重组人p53腺病毒在乳腺癌裸鼠体内耐药逆转作用及其对P-糖蛋白表达的影响

目的 研究重组人p53腺病毒（rAd-p53）对人乳腺癌MCF-7/ADR裸鼠移植瘤的耐药逆转作用及其对耐药相关P-糖蛋白（P-glycoprotein,P-gp）表达的影响.方法 建立人乳腺癌MCF-7/ADR裸鼠耐药模型,随机分为对照组、rAd-p53组、阿霉素组、联合组,分别给予不同治疗.观察裸鼠肿瘤体积的变化,western blot检测P-gp蛋白表达情况.结果 成瘤后rAd-p53组、阿霉素组、联合组抑瘤率分别为42.05％、49.21％、69.97％,各治疗组体积与对照组相比差异有统计学意义（P＜0.05）,且联合组显著优于rAd-p53组和阿霉素组（P＜0.05）.对照组、rAd-p53组、阿霉素组、联合组P-gp表达水平分别为（0.5964±0.0806）、（0.5506±0.0127）、（0.5641±0.0055）、（0.4652±0.0982）,联合组P-gp表达水平低于其他各组.结论 Ad-p53对人乳腺癌阿霉素耐药细胞株MCF4/ADR建立的裸鼠移植瘤具有多药耐药的逆转作用,并可下调P-gp的表达.     

Cationic lipophilic radiotracers for functional imaging of multidrug resistance

The multidrug resistance (MDR) phenotype is frequently associated with the overexpression of transmembrane drug proteins such as the P-glycoprotein (Pgp) and/or multidrug resistance related protein-1 (MRP1). These proteins belong to the superfamily of the so-called ATP-binding cassette superfamily and act as drug efflux pumps of a broad range of chemotherapeutic agents commonly used in the treatment of malignancies. These proteins have been found to be overexpressed in both haematological and solid tumours and are considered as adverse prognostic factors. The ability to obtain in vivo and non-invasively information regarding the functional activity of MDR-related transporters, using probes that mimic the antineoplastic agents, provide a very useful tool in the clinical setting by determining the individual tumour susceptibility to chemotherapy. This previous knowledge could serve as a critical tool for optimizing chemotherapeutic protocols on a patient-specific basis. The emergence of non-invasive molecular imaging techniques using radiolabelled probes provides an interesting approach for functional assessment of the classical mechanism of MDR in cancer patients. Toward this objective, the clinically approved 99mTc-labelled cationic lipophilic complexes (sestamibi and tetrofosmin) have been characterized as transport substrates of Pgp and MRP1 and proposed as surrogate markers of chemotherapeutic agents for functional evaluation of MDR by single-photon emission tomography (SPECT). Here we review the potential applications of these agents in identifying drug resistance mechanisms based on functional assays and their potential as a tool for evaluating the efficacy of MDR inhibitors, using cellular and animal models of chemoresistance.     

分割剂量电离辐射对卵巢癌细胞多药耐药的影响

目的 研究不同分割剂量电离辐射方案对卵巢癌细胞多药耐药性的影响.方法 采用卵巢癌亲本细胞SKOV3及其耐药细胞株SKVCR,分别进行假照射、单次照射(10 Gy)、常规分割照射(2 Gy ×5)和超分割照射(1 Gy×2 ×5).MTT方法检测细胞对4种化疗药物硫酸长春新碱( vincristine,VCR)、依托泊苷(etoposide,VP-16)、盐酸吡柔比星(pirarubicin,THP)和顺铂(cisplatin,DDP)敏感性的变化.Western blot检测P-gp蛋白表达量.结果 与SKOV3相比,SKVCR倍增时间增加为1.8倍,P-gp糖蛋白表达增高,4种药物对SKVCR的IC50浓度明显增加(P＜0.05).在SKOV3中,与假照组相比,单次照射后细胞对THP、DDP药敏性降低,常规分割照射后细胞对VCR、THP、VP-16药敏性降低,超分割照射使VP-16药敏性降低;在SKVCR中,与假照组相比,3个照射组对VCR、VP-16的药敏性增高,对DDP的药敏性无明显改变,单次和分割照射均可降低P-gp表达.结论 单次、分割和超分割照射在SKOV3亲本细胞中诱导多药耐药,在耐药株SKVCR中逆转对VCR、VP-16的耐药性.     

中药逆转白血病多药耐药的研究

多药耐药是白血病化疗失败的主要原因,如何逆转是一重要课题,本文从多药耐药的机制出发,阐述了中药逆转白血病多药耐药的研究现状及进展.     

99Tcm-MIBI显像对肿瘤多药耐药检测的应用

MDR(多药耐药)是目前肿瘤化疗失败的主要原因,对MDR的检测可以帮助化疗决策的制定,从而使肿瘤患者得到更有效的治疗。99Tcm-MIBI(99Tcm-甲氧基异丁基异腈)是mdr1基因编码的P-gp(P-糖蛋白)和MRP(多药耐药相关蛋白)的转运底物,肿瘤细胞内99Tcm-MIBI摄取减低表明其P-gp的高表达,并与MRP的表达相关。因此,99Tcm-MIBI显像可在治疗前预测对化疗的反应,并为选择更有效的化疗策略提供依据。99m     

P-gp糖蛋白及其编码的基因在受照射鼻 咽癌细胞株中的表达

目的模拟临床放射方法对人鼻咽鳞癌CNE细胞分次照射，检测照射前、后CNE细胞肿瘤多药耐药蛋白P-gp及其编码的基因MDR1的表达和功能。方法对人鼻咽鳞癌CNE细胞进行体外培养，待细胞进入指数生长期后模拟临床放疗方法进行X射线分割照射，2Gy／（d·f^-1），连续5d，总剂量10Gy。于照射前、照射后4、8、13、17和21d分别收取标本进行检测。利用RT-PCR、免疫细胞化学检测照射前、后人鼻咽鳞癌CNE细胞MDR1及P-gp的表达；MTT法检测其耐药指数（RI）的变化，评价P-gp的功能。结果人鼻咽鳞癌CNE细胞MDR1基因照射前呈弱表达，照后4d过度表达，8d到21d表达逐渐降低，与照射前比较，差异有统计学意义（P〈0．05）；其编码的蛋白产物P-gp照射前、照射后4和8d呈弱表达，13至21d表达增高，P-gp迟于MDR1基因高表达。MTT法检测耐药指数结果显示：照射前、照射后4和8d RI值均为1，13、17和21d RI分别增高为8、10和11．2，RI值变化的情况与P-gp的变化情况相一致。结论人鼻咽鳞癌CNE细胞照射前MDR1及P-gp呈弱表达，有原始的低度耐药性；照射后MDR1及功能性P-gp高表达并且持续一段时间。     

人类多药耐药基因转录调控研究进展

多药耐药(MDR)是肿瘤化疗失败的主要原因之一，而人类多药耐药基因(hmdrl)及其编码产物P—糖蛋白(P—gp)的超表达，是引起MDR表型的主要原因。研究表明P—gp的超表达主要在转录水平上进行调控。最近的研究主要集中在明显hmdrl启动子中新的未了解区域以及相应的信号传导通路。这些研究为了解MDR的机制提供了新思路。故就hmdrl的调控研究进展做一综述。     

Evaluation of tumour metabolism and multidrug resistance in patients with treated malignant lymphomas

The management of patients with treated malignant lymphomas requires functional methods to differentiate a residual soft tissue mass. Patients with treated Hodgkin's lymphoma (HL,n = 20, 68 malignant lesions, three benign lesions) or non-Hodgkin's lymphoma (NHL,n = 26, 46 malignant lesions, one benign lesion) were studied with positron emission tomography (PET) and fluorine-18 deoxyglucose (FDG). Oxygen-15 labelled water was used (n = 14, 25 lesions) in addition to FDG in order to obtain information on the tissue perfusion. Long-term follow-up studies with PET and FDG were performed in nine patients up to 511 days after the initiation of second-line therapy. Fourteen patients underwent single-photon emission tomography (SPET) with technetium-99m sestamibi immediately prior to the first PET examination. PET with FDG displays a high sensitivity for the detection of viable tumour tissue, all the malignant lesions being correctly classified in this study. The possible limitations are inflammatory processes, which may obscure tumour detection due to increased FDG uptake, and malignant lesions with low FDG uptake due to reduced perfusion. Difficulties exist in the prognosis of long-term response, since the change in FDG uptake may be variable. Long-term therapy outcome was correlated with the slope values obtained from the standardized integral uptake (SIU) data, which provides a new approach for the evaluation of PET follow-up studies.^99mTc-sestamibi, which should reflect the multidrug resistance, was evaluated with respect to therapy outcome. A high uptake of^99m-Tc-sestamibi was observed in patients with stable disease or better. The data support the hypothesis that sestamibi may reflect multidrug resistance. Due to technical limitations of the SPET technique, the use of a positron-labelled compound would be superior to SPET for clinical application.     

Functional imaging of multidrug resistance in an orthotopic model of osteosarcoma using 99mTc-sestamibi

Purpose The purpose of this work was the development of an orthotopic model of osteosarcoma based on luciferase-expressing tumour cells for the in vivo imaging of multidrug resistance (MDR) with ^99mTc-sestamibi. Methods Doxorubicin-sensitive (143B-luc⁺) and resistant (MNNG/HOS-luc⁺) osteosarcoma cell lines expressing different levels of P-glycoprotein and carrying a luciferase reporter gene were inoculated into the tibia of nude mice. Local tumour growth was monitored weekly by bioluminescence imaging and X-ray. After tumour growth, a ^99mTc-sestamibi dynamic study was performed. A subset of animals was pre-treated with an MDR inhibitor (PSC833). Images were analysed for calculation of ^99mTc-sestamibi washout half-life (t _1/2), percentage washout rate (%WR) and tumour/non-tumour (T/NT) ratio. Results A progressively increasing bioluminescent signal was detected in the proximal tibia after 2 weeks. The t _1/2 of ^99mTc-sestamibi was significantly shorter (p < 0.05) in drug-resistant MNNG/HOS-luc⁺ tumours (t _1/2 = 87.3 ± 15.7 min) than in drug-sensitive 143B-luc⁺ tumours (t _1/2 = 161.0 ± 47.4 min) and decreased significantly with PSC833 (t _1/2 = 173.0 ± 24.5 min, p < 0.05). No significant effects of PSC833 were observed in 143B-luc⁺ tumours. The T/NT ratio was significantly lower (p < 0.05) in MNNG/HOS-luc⁺ tumours than in 143B-luc⁺ tumours at early (1.55 ± 0.22 vs 2.14 ± 0.36) and delayed times (1.12 ± 0.11 vs 1.62 ± 0.33). PSC833 had no significant effects on the T/NT ratios of either tumour. Conclusion The orthotopic injection of tumour cells provides an animal model suitable for functional imaging of MDR. In vivo bioluminescence imaging allows the non-invasive monitoring of tumour growth. The kinetic analysis of ^99mTc-sestamibi washout provides information on the functional activity of MDR related to P-glycoprotein expression and its pharmacological inhibition in osteosarcoma.     

恶性骨肿瘤多药耐药的99Tcm-MIBI体层显像研究

大多数肿瘤都有多药耐药-1(MDR-1)基因的过度表达,其表达产物P-糖蛋白(P-gp)在肿瘤的耐药机制中起着关键作用,对P-gp表达的预测在化疗中至关重要,99Tcm-甲氧基异丁基异腈(99Tcm-MIBI)转运分析可作为探测低水平的P-gp表达和定量评价调节转运的敏感指标,指导应用调节剂改善化疗效果.     

Imaging multidrug resistance with 4-[18F]fluoropaclitaxel

Multidrug resistance (MDR) is a cause of treatment failure in many cancer patients. MDR refers to a phenotype whereby a tumor is resistant to a large number of natural chemotherapeutic drugs. Having prior knowledge of the presence of such resistance would decrease morbidity from unsuccessful therapy and allow for the selection of individuals who may benefit from the coadministration of MDR-inhibiting drugs. The Tc-99m-labeled single-photon-emitting radiotracers sestamibi and tetrofosmin have shown some predictive value. However, positron-emitting radiotracers, which allow for dynamic quantitative imaging, hold promise for a more accurate and specific identification of MDRtumors.MDR-expressing tumors are resistant to paclitaxel, which is commonly used as a chemotherapeutic agent. 4-[18F]Fluoropaclitaxel (FPAC) is a PET-radiolabeled analogue of paclitaxel. Preclinical studies have shown the uptake of FPAC to be inversely proportional to tumor MDR expression. FPAC PET imaging in normal volunteers shows biodistribution to be similar to that in nonhuman primates. Imaging in a breast cancer patient showed FPAC localization in a primary tumor that responded to chemotherapy, while failure to localize in mediastinal disease corresponded with only partial response.FPAC PET imaging shows promise for the noninvasive pretreatment identification of MDR-expressing tumors. While much additional work is needed, this work represents a step toward image-guided personalized medicine.     

In-vivo identification of tumor multidrug resistance with 3H-colchicine [corrected]

Multidrug resistance (MDR) is a major obstacle in the clinical treatment of cancer with natural-product anticancer agents. Identification of MDR in vivo could be important in the design of chemotherapeutic regimens. As a first step in developing radiolabeled drugs to detect MDR, we measured the in vivo distribution of radiolabel from [ring C, methoxy-3H]-colchicine ([3H]-CHC) in immunosuppressed mice bearing xenografts of colchicine-resistant and sensitive tumor cell lines. Experiments were done at trace (1 microgram/kg) and LD50 (4 mg/kg) dose levels. Activity concentration/injected dose was more than twice as great in sensitive as in resistant tumors (p less than 0.01) at 60 min following retroorbital injection of [3H]-CHC. There was no significant difference in activity distribution between trace- and high-dose injections for any of the tissues sampled. Chromatographic analysis of plasma and tumor extracts demonstrated extensive extravascular metabolic degradation of [3H]-CHC. The ratio of [3H]-CHC concentration of injected dose between sensitive and resistant tumors was 3:1 (p less than 0.05), due primarily to protein-bound [3H]-CHC. This preliminary study demonstrates that it is possible to distinguish multidrug resistant from sensitive tumors in vivo on the basis of radiolabel uptake from an injected MDR drug. Colchicine, labeled with 11C at the [ring C]-methoxy group, may be useful as a radiopharmaceutical for quantitative identification of MDR in human tumors using PET.