Elsevier

Nuclear Medicine and Biology

Volume 38, Issue 7, October 2011, Pages 1067-1077
Nuclear Medicine and Biology

Auger electron emitter against multiple myeloma — targeted endo-radio-therapy with 125I-labeled thymidine analogue 5-iodo-4′-thio-2′-deoxyuridine

https://doi.org/10.1016/j.nucmedbio.2011.02.018Get rights and content

Abstract

Introduction

Multiple myeloma (MM) is a plasma cell malignancy characterized by accumulation of malignant, terminally differentiated B cells in the bone marrow. Despite advances in therapy, MM remains an incurable disease. Novel therapeutic approaches are, therefore, urgently needed. Auger electron-emitting radiopharmaceuticals are attractive for targeted nano-irradiation therapy, given that DNA of malignant cells is selectively addressed. Here we evaluated the antimyeloma potential of the Auger electron-emitting thymidine analogue 125I-labeled 5-iodo-4′-thio-2′-deoxyuridine ([125I]ITdU).

Methods

Cellular uptake and DNA incorporation of [125I]ITdU were determined in fluorodeoxyuridine-pretreated KMS12BM, U266, dexamethasone-sensitive MM1.S and -resistant MM1.R cell lines. The effect of stimulation with interleukin 6 (IL6) or insulin-like growth factor 1 (IGF1) on the intracellular incorporation of [125I]ITdU was investigated in cytokine-sensitive MM1.S and MM1.R cell lines. Apoptotic cells were identified using Annexin V. Cleavage of caspase 3 and PARP was visualized by Western blot. DNA fragmentation was investigated using laddering assay. Therapeutic efficiency of [125I]ITdU was proven by clonogenic assay.

Results

[125I]ITdU was shown to be efficiently incorporated into DNA of malignant cells, providing a promising mechanism for delivering highly toxic Auger radiation emitters into tumor DNA. [125I]ITdU had a potent antimyeloma effect in cell lines representing distinct disease stages and, importantly, in cell lines sensitive or resistant to the conventional therapeutic agent, but was not toxic for normal plasma and bone marrow stromal cells. Furthermore, [125I]ITdU abrogated the protective actions of IL6 and IGF1 on MM cells. [125I]ITdU induced massive damage in the DNA of malignant plasma cells, which resulted in efficient inhibition of clonogenic growth.

Conclusion

These studies may provide a novel treatment strategy for overcoming resistance to conventional therapy in multiple myeloma.

Introduction

Multiple myeloma (MM) is a plasma cell malignancy characterized by accumulation of malignant, terminally differentiated B cells in the bone marrow (BM). Agents such as dexamethasone (Dex), thalidomide, lenalidomide and bortezomib achieve responses in MM patients [1], [2], [3], [4]. However, although the majority of MM patients initially respond to therapy, they subsequently relapse due to development of resistance to chemotherapy [5]. Interestingly, MM cells of patients in active phase of disease (progressive/relapsing) are usually characterized by high proliferative and low apoptotic characteristics, while in the late stage of MM, the majority of cells have a low rate of proliferation, typically with less than 1% of cells synthesizing DNA [6], [7]. For the MM, the tumor cell growth, survival and resistance are enhanced by the BM microenvironment [8]. Cells that have entered the BM interact with stromal cells (BMSCs) through cell–cell contact [9]. This interaction triggers BMSCs to secrete interleukin 6 (IL6) and insulin-like growth factor 1 (IGF1), which mediate growth, survival and resistance in MM cells [10], [11]. The resistance gained from this interaction enables the proliferative clonogenic subpopulation of MM cells to continue uncontrolled regrowth and leads finally to disease relapse.

Because of the requirement for a continuous supply of deoxynucleotides, DNA replication presents a potential target for therapeutic intervention. Several thymidine and uridine analogues labeled with γ or positron emitters were synthesized and evaluated for noninvasive imaging to address tumor proliferation in patients [12], [13], [14], [15]. Moreover, the efficiency of radiolabeled 5-iodo-2′-deoxyuridine (IdUrd) for tumor imaging in vivo was successfully shown [12], [16], [17], [18]. However, a potential therapeutic use of IdUrd is limited by its fast enzymatic degradation by thymidylate phosphorylase (TP) leading to the low rate of [125I]IdUrd DNA incorporation. To circumvent the rapid in vivo catabolism and to obtain preferential incorporation of radiohalogenated nucleoside analogues into DNA through the salvage pathway, Toyohara et al. synthesized a new thymidine analogue, 5-iodo-4′-thio-2′-deoxyuridine (ITdU) [19]. The relative enzymatic degradation of ITdU by TP is about 3.6% compared with that of IdUrd [20]. Moreover, ITdU was shown to be efficiently incorporated into DNA through the salvage pathway, providing a promising mechanism for delivering Auger electrons emitters such as 123I and 125I into tumor DNA [21]. Auger electrons emitted within DNA are extremely toxic to the cell [22]. This effect is due to generation of high-ionization-density clusters that primarily induce double-strand breaks, which are highly cytotoxic forms of DNA damage. The accumulating damage increases genetic instability and reduces the apoptotic threshold of the cell [23]. The properties of Auger electrons (short path length with particularly low energy resulting in a very high linear energy transfer, LET) limit the cytotoxic effects to a sphere of a few nanometers in the immediate vicinity of the site of decay. Cytosolic and extracellular decay of Auger emitters is 10–100-fold less radiotoxic than decay within DNA, presenting multiple advantages for targeted radiotherapy using Auger electron-emitting drugs [24]. Previously, we reported preferential tumor targeting and a high incorporation rate of ITdU into DNA of leukemia cells growing subcutaneously in CB17ICR SCID mice [20]. Pretreatment with a nontoxic dose of fluorodeoxyuridine (FdUrd) resulted in increased incorporation of ITdU by blocking the activity of thymidylate synthase (TS) and depleting the cellular Thd-triphosphate pool [20], [21]. The most efficient increase in ITdU incorporation was observed in tumor tissue leading to extensive tumor damage, while only moderate injury occurred in normal tissues [20].

Based on previous studies, we undertook a preclinical study of [125I]ITdU as a novel agent for treatment of MM. We evaluated the activity of [125I]ITdU against MM cell lines representing distinct differentiation stages of the disease. In addition, we examined the antitumor potency in drug-resistant MM cells. 125I-labeled ITdU selectively induced apoptosis in immature and terminally differentiated malignant plasma cells but not in normal plasma and BM stromal cells. Importantly, [125I]ITdU provoked cell death in MM cells stimulated with IL6 or IGF1, proliferative and survival factors in MM cells. We showed that [125I]ITdU has a potent antimyeloma activity and overcomes drug resistance in MM cells.

Section snippets

Chemicals

Chemicals and solvents were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Merck (Darmstadt, Germany) or otherwise as indicated. All reagents and solvents were of the highest commercially available grade. No-carrier-added sodium 125I-iodide was obtained from GE Healthcare (Chalfont St. Giles, UK). The 5-trimethylstannyl precursor of ITdU and the unlabeled ITdU were synthesized according to previously reported methods [19], [25].

Radiochemistry

ITdU was labeled with 125I as previously described [20].

Phenotypic analysis of MM cell lines

Because phenotypes of MM cells have been described with respect to their differentiation stage and resistance to drug-induced apoptosis, we included MM cell lines with different expression patterns of typical CD markers as in vitro models of distinct disease stages [26]. The expression of each CD marker was assessed by the percentage of positive cells and by mean fluorescence intensity (MFI). This analysis indicated an immature character of the U266 cell line (CD38CD45++CD138). The majority

Discussion

In this study, we present evidence indicating that the Auger electron-emitting thymidine analogue [125I]ITdU has potent in vitro antimyeloma activity. The exposure of MM cells to [125I]ITdU resulted in activation of the intrinsic apoptosis pathway and extensive DNA damage leading to efficient tumor cell killing. [125I]ITdU efficiently inhibited survival in MM cell lines representing different MM phenotypes, including cells sensitive or resistant to Dex. Importantly, no cytotoxicity was detected

Acknowledgments

This work was supported by grant KFO120 from Deutsche Forschungsgemeinschaft and grant DJCLS R 08/21v from Deutsche Jose Carreras-Leukämie Stiftung.

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