Article pubs.acs.org/bc
Zn(II)-bis(cyclen) Complexes and the Imaging of Apoptosis/Necrosis Dorte Oltmanns,† Sabine Zitzmann-Kolbe,‡ Andre Mueller,‡ Ulrike Bauder-Wuest,† Martin Schaefer,† Matthias Eder,† Uwe Haberkorn,§ and Michael Eisenhut*,† †
Department of Radiopharmaceutical Chemistry, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany ‡ Global Drug Discovery, Bayer Healthcare, Berlin, Germany § Department of Nuclear Medicine, University Hospital Heidelberg, Im Neuenheimer Feld 400, 69120 Heidelberg, Germany S Supporting Information *
ABSTRACT: In vivo cell-death imaging is still a challenging issue. Until now, only 99mTc-labeled HYNIC-rh-annexin A5 has been extensively studied in clinical trials. In the ongoing search for an alternative imaging agent, we synthesized a series of fluorescent zinc-cyclen complexes as annexin A5 mimics and studied structural variations on the uptake behavior of cells undergoing apoptosis/necrosis. The number of cyclen chelators was varied and the spacer separating cyclen from the central scaffold was modified. Five zinc-cyclen complexes were labeled with fluorescein for flow cytometric studies and one was labeled with 18F for in vivo applications. Jurkat cells were treated with staurosporine to induce apoptosis/necrosis, incubated with the fluorescein-labeled zinc complexes and analyzed them by flow cytometry. Fluorescent annexin A5 and propidium iodide were applied as reference dyes. Flow cytometry revealed greater accumulation of zinc-cyclen complexes in staurosporine treated cells. The uptake was contingent on the presence of zinc and the fluorescence intensity was dependent on the number of zinc-cyclen groups. Confocal laser scanning microscopy showed the {bis[Zn(cyclen)]}4+ complex distributed throughout the cytosol different to annexin A5. Owing to the structural similarity of the bis-cyclen ligands with CXCR4 binding bis-cyclam derivatives the zinc-cyclen complex uptake was challenged with the meta derivative of AMD3100. Lack of uptake depletion in staurosporine treated cells ruled out measurable CXCR4 interaction. PET imaging using the 18F labeled zinc-cyclen complex revealed significantly higher uptake in an irradiated Dunning R3327-AT1 prostate tumor as compared to the contralateral control tumor. PET imaging of a HelaMatu tumor model additionally showed an increased uptake after taxol treatment. It could be demonstrated that the fluorescent zinc-cyclen complexes offer potential as new agents for flow cytometry and microscopic imaging of cell death. In addition, the 18F labeled analogue holds promise for in vivo applications providing informations about cell death after radiation therapy and cytostatic drug treatment.
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fluorescent annexin A5 was introduced as a tool for flowcytometric detection of apoptosis4 and is now broadly applied in cell biological research.5,6 The in vivo imaging of apoptosis was made possible using 99mTc labeled AnxA5,7 which was later modified to 99mTc-HYNIC-rh-annexin A5 and examined in clinical studies.8 On the basis of PS selectivity and binding strength of AnxA5, searches for small molecules as annexin mimics have been undertaken to further improve the property of apoptosis imaging. In reference to the role of Ca2+ in AnxA5, various Zn2+ complexes providing similar cocomplexation characteristics have been investigated as phosphate selective compounds.
INTRODUCTION
Evading apoptosis is one of the hallmarks of cancer. Overcoming this resistance is important for the success of therapeutic interventions. Apoptosis is accepted as one of the key readouts for early prediction of anticancer treatment response. Imaging apoptosis, therefore, will provide important information to ascertain the effectiveness of anticancer therapies. Due to the loss of membrane asymmetry at the onset of apoptosis, phosphatidylserine (PS) becomes exposed on the extracellular cell membrane. Annexin A5 (AnxA5), a protein involved in the inhibition of blood coagulation, binds to externalized phosphatidylserine (PS) of apoptotic cells. The binding itself is driven through the formation of a ternary Ca 2+ complex of AnxA5 with the carboxyl-phospholipid headgroup of PS.1,2 This concerted interaction results in a considerable strong binding affinity of Kd 50 GBq/μmol. After evaporation, the complexation of 18F-6 with Zn2+ was carried out in 100 μL PBS for 5 min at 70 °C containing 10 μL of an aqueous 1.5 mM Zn(NO3)2 solution. For imaging experiments, the zinc complex of 18F-6 was diluted with 400 μL PBS and filtered sterile. 2617
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Cell Experiments. Flow Cytometry. Apoptosis was induced with 106 cells/mL using staurosporine (1 μM) during 4 h. The cells were washed with PBS and split into 1 mL samples containing 106 cells. After centrifugation, cells were taken up in 100 μL TES buffer for incubation with complex solutions or in 100 μL annexin binding buffer for annexin experiments. 1.0 μL of a 0.5 mM complex solution was added to the TES buffered cells and 1.5 μL of Alexa Fluor 488 conjugated annexin was added to the cells taken up in annexin binding buffer. After 15 min incubation in the dark, the suspensions were diluted with 300 μL TES or annexin binding buffer. Flow-cytometry measurements were performed with 100 μL cell suspension and 900 μL cold buffer. 6 × 103 cells were counted in each experiment. Where necessary, 10 μL propidium iodide solution (250 μg/mL) was added just before the measurement. Control cells were treated in the same way and measured as a comparison with each experiment. Evidence of CXCR4. The anti-hCXCR-4 antibody was diluted with cold PBS to a concentration of 10 μg/mL. Control and apoptotic Jurkat cells (approximately 106) were suspended in 100 μL antibody solution and incubated for 30 min at 4 °C. The cells were washed three times with cold PBS and taken up in 100 μL PBS solution of FITC-labeled antimouse monoclonal antibody Ig2A. After 30 min incubation at room temperature, the cells were washed again three times with cold PBS and finally dispersed in 400 μL PBS. 100 μL cell suspension was added to 900 μL cold PBS and measured in a flow cytometer. Competition with meta AMD3100. 1.0 μL of a 50 mM aqueous solution of meta AMD3100, synthesized according to the literature24 and 1.0 μL of 0.5 mM complex [Zn21]4+ solution was added to 106 staurosporine treated Jurkat cells in cold TES buffer. The cells were incubated 15 min at room temperature in the dark. The samples were diluted with TES buffer to 400 μL. 100 μL of the suspension were added to 900 μL cold buffer and measured by flow cytometry. Confocal Laser Scanning Microscopy (CLSM). Apoptosis induction of Jurkat cells for CLSM experiments was performed as described above. To prevent free floating under coverslips, the cells were immobilized on polylysine precoated slides. After incubation with [Zn21]4+ for 15 min, the cells were transferred to the object slide and placed under the microscope. Images were taken from the midcell z-plane. Animal Studies. The experiments were approved by the governmental review committee on animal care. All animal experiments were performed in compliance with the current version of the German law concerning animal protection and welfare. Animals were kept under standard laboratory conditions at the German Cancer Research Center or at Bayer Healthcare Research Facilities. Male Copenhagen rats (approximately 180 g) were subcutaneously transplanted on both thighs with 1 × 106 Dunning adeno carcinoma R3327-AT1 cells. Fourteen days after tumor cell implantation, the left tumor was irradiated with a single dose of 50 Gy using an Artiste linear accelerator (Siemens AG, München, Germany). The right tumor served as control. One day before (day 13 after tumor cell inoculation) and six and ten days after irradiation, the rats were imaged using [Zn2(18F-6)]4+. 15−25 MBq in 200 μL PBS was injected into the tail vein. For the PET-scan, the animals were anesthetized with sevoflurane and maintained under anesthesia during the scan (2.5% for induction and 1% for maintenance of anesthesia). PET studies were performed with the Siemens
Inveon small animal PET scanner (Siemens, Knoxville, USA). A transmission scan was done for 10 min prior to tracer administration with two rotating germanium pin sources to obtain cross sections for attenuation correction. PET data were acquired for 1 h in list mode on an Inveon scanner (Siemens Erlangen Germany) using a matrix of 256 × 256 (pixel size 0.3882 × 0.3882 × 0.796 mm3). The images were reconstructed iteratively using the space alternating generalized expectation maximization method (SAGE, 16 subsets, 4 iterations) applying median root prior correction. 1.5 × 106 HelaMatu (human cervix carcinoma) tumor cells were inoculated in a volume of 100 μL (medium/matrigel) into the right shoulder of nude mice (NMRI nu/nu, female, ca. 20 g, obtained from Taconic). At day 12 after inoculation, animals were treated with taxol using 18 mg/kg i.v. or vehicle (5% cremophore, 5% ethanol). PET images were acquired using an Inveon small animal PET/CT scanner (Siemens, Knoxville, USA) at 60 min p.i. for 10 min. After the PET study, animals were sacrificed, and tumors were removed and cut into 18 μM slices (Cryostat Fa Leica CM 3050 S). Tumor slices were exposed to a phosphoimager plate (BAS-SR Imaging plate, Fuji, IP-SR 20 × 25) overnight. Analysis was performed using the BAS-5000 Imaging System (Fuji). Cleaved caspase 3 in the tumor samples was detected by immunohistochemistry (IHC) using a polycolonal antibody against cleaved caspase 3 (rabbit, Cell Signaling, # 9661-S). IHC was performed using established standard protocols.
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RESULTS Syntheses. The syntheses of the title compounds essentially followed the preparation strategy applied to obtain bis(dipicolylamine) derivatives. 12 This route offered the possibility of attaching different fluorescent dyes as well as other markers such as radiolabels in the perspective of in vivo imaging. As outlined in Scheme 1, the core structures were Scheme 1. Syntheses of Bis(cyclen) Scaffolds
(a) Trityl chloride, Et3N, CH2Cl2; (b) CBr4, K2CO3, Ph3P, CH2Cl2; (c) 3,5-Bis(hydroxymethyl)phenol, K2CO3, acetonitrile, reflux; (d) 4Hydroxymethylphenol, K2CO3, acetonitrile, reflux; (e) CBr4, Ph3P, CH2Cl2.
obtained as N-trityl protected 3,5-bis(bromomethyl)- (9) and 4-bromomethyl phenyl (10) derivatives. Tris(Boc)cyclen (Z) was alkylated using precursors 10 and 12 yielding compounds 13 and 19, respectively (Scheme 2). Selective cleavage of the trityl group with hexafluoroisopropa2618
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Scheme 2. Syntheses of Bis(cyclen) Ligands
(a) Tris(Boc)cyclen, K2CO3, KI, acetonitrile, 50 °C; (b) Hexafluoroisopropanol/1% H2O, CH2Cl2; (c) CF-PFP ester, DIPEA, DMF; (d) FITC, DIPEA, DMF; (e) TFA, CH2Cl2; (f) Aminoethyl-tris(Boc)cyclen, K2CO3, acetonitrile, 50 °C; (g) 4-Hydroxymethylphenol, K2CO3, acetonitrile, reflux; (h) CBr4, Ph3P, CH2Cl2; (i) 3,5-Bis(hydroxymethyl)phenol, K2CO3, acetonitrile, reflux.
nol was followed by the introduction of fluorescent markers carboxyfluorescein pentafluorophenol ester (5(6)-CF-PFP) and fluorescein isothiocyanate (FITC). Deprotection of the cyclen amino functionalities yielded chelator 1 as well as the mono cyclen 3. In order to introduce a pendant heteroatom into the spacer, tris(Boc)cyclen providing an aminoethyl side arm was alkylated with compound 10. Removal of the trityl group, conjugation with 5(6)-CF-PFP, and Boc deprotection resulted in chelator 2. The cyclen multimers 4 and 5 were obtained via elongation of the central scaffold 10 with 4-hydroxymethylphenol and 3,5-di(hydroxymethyl)phenol. Alkylation of tris(Boc)cyclen using the resulting bromo compounds 23 and 28 together with selective trityl deprotection and reaction with 5(6)-CF-PFP gave compounds 26 and 31, respectively. Final Boc deprotection resulted in the cyclen derivatives 4 and 5 which were then complexed with Zn2+ for biological experiments. The synthesis of the radiofluorination precursor 35 proceeded through alkylation of tris(Boc)cyclen with the TBDMS protected phenol 33 (Scheme 3). After silyl
deprotection, compound 35 was reacted with 2-fluoroethyl tosylate and treated with TFA to cleave the Boc groups forming ligand 6. The radiolabeled congener 18F-6 was prepared accordingly using HPLC purified 2-[18F]fluoroethyl tosylate. The final step implied the formation of the Zn2+ complexes. Aqueous solutions of the fluorescein-cyclen derivatives were heated at 50 °C with 1 equiv Zn2+/cyclen. The resulting complexes were lyophilized and redissolved in H2O to form 0.5 mM solutions. The HPLC purified, noncarrier added radiofluorinated bis(cyclen) 6 was transferred into the zinc complex [Zn2(18F-6)]4+ by adding Zn2+ salt and heating. Flow Cytometry. The influence of the {bis[Zn(cyclen)]}4+ complexes on cell size and granularity was studied with [Zn21]4+ to exclude cell damaging of untreated Jurkat cells during flow cytometry. The cells were, therefore, analyzed using the flow cytometric sideward (SSC) and forward (FSC) scatter signals which allowed the detection of advanced apoptotic and secondary necrotic cells. The cells were incubated in TES buffer with [Zn21]4+ at concentrations in the range 0−50 μM for up 2619
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Bioconjugate Chemistry Scheme 3. Synthesis of Bis(cyclen) 6 and
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F Labeling
(a) CBr4, Ph3P, CH2Cl2; (b) tris(Boc)cyclen, K2CO3, KI, acetonitrile, 50 °C; (c) (n-Bu)4N+ F−, THF; (d) 2-Fluoroethyl tosylate, K2CO3, acetonitrile, reflux; (e) TFA, CH2Cl2; (f) K2CO3, acetonitrile, sealed reaction vial, 30 min/120 °C.
vs 38.5% of apoptotic cells after staurosporine treatment. This value varied between 30% and 60%. Simultaneous binding of annexin A5 and propidium iodide are found in Q2, indicating cells which were in the transition of apoptosis to necrosis (late apoptosis and secondary necrosis).26 Here, 2.5% was obtained for untreated and 19.1% for staurosporine treated cells. Cells in segment Q3 represent unchanged cells and those in Q4 are solely dyed with PI, presumably completely necrotic cells. Lower row of density plots (Figure 3) illustrates flow cytometry results which were obtained with the same experimental setup as described above but in the presence of [Zn21]4+. After staurosporine treatment, 11.9% was found in Q1 and 47.2% in Q2. The latter value indicated [Zn21]4+binding together with PI accumulation. The sum of Q1 and Q2 was about 60% for both AnxA5-AF and the zinc complex. It appears remarkable that after the addition of [Zn21]4+ and PI the SSC and FSC signals of the staurosporine treated cells were shifted into the secondary necrosis fraction (arrow in SI) and that these signals can be attributed to Q2. With the exception of [Zn22]4+, this effect was also observed using the remaining fluorescent zinc-cyclen complexes. The insertion of a pendant ethylene amino group like in [Zn22]4+ led to histograms almost indistinguishable between staurosporine treated and control cells. Both the [Zn22]4+ complex-related fluorescence of treated and control cells appeared in Q1 at 38.2% and 31.9%, respectively, showing similar distribution patterns. The intensity of the green fluorescence of [Zn22]4+ was, however, strongly reduced as compared with the fluorescence of [Zn21]4+ in apoptotic cells. Ligands 3, 4, and 5 were synthesized to study the influence of the number of [Zn(cyclen)]2+ groups on the fluorescence of staurosporine treated cells. While complex [Zn24]4+ showed similar results as obtained with [Zn21]4+, the tetrameric cyclen complex [Zn45]8+ increased the mean fluorescence intensity (MFI) of the green fluorescence signals of treated Jurkat cells by a factor of 2.3. The monomeric [Zn3]2+ produced in turn a
to 72 h. As demonstrated in the Supporting Information, toxicity against vital Jurkat cells proved negligible. Apoptosis was induced in Jurkat cells with 1 μM staurosporine for 4 h.25 Figure 3 shows flow-cytometric density
Figure 3. Flow cytometric density plots of Jurkat cells in the presence of AnxA5-AF/propidium iodide (PI) and [Zn21]4+ (lower panel) after treatment with staurosporine (right). Untreated controls are shown on the left.
plots (upper row) obtained with Alexa Fluor 488 labeled annexin A5 (AnxA5-AF) and propidium iodide (PI). Cells with high binding of Annexin A5 were found in the rectangle Q1, indicating 4.7% of apoptotic cells for the control cell population 2620
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much smaller MFI, 2 vs 34 of [Zn3]2+ and [Zn21]4+or [Zn24]4+, respectively (SI). The relevance of zinc(II) in the cyclen complexes, which as outlined in the introduction was thought to be necessary for the ternary interaction with PS, was tested by comparing the complex with the free ligand. The free cyclen derivative 4 produced an unspecific fluorescence of low intensity, whereas the complex [Zn 2 4]4+ showed strong fluorescence of staurosporine treated cells as described above (SI). All flow cytometric results described so far were obtained in the presence of the respective zinc complexes. Removal of these dyes by two washing steps resulted in the loss of most of the intracellular dye. The reverse result was obtained by increasing the zinc-complex concentration leading to an increase of the mean fluorescence intensity of treated Jurkat cells. Loss and increase of fluorescence intensity indicates reversibility of concentration-dependent accumulation. Figure 4 shows this
Figure 5. CLSM images (λ exc 488 nm/3%; BP 505−530 nm) of staurosporine treated Jurkat cells incubated with [Zn21]4+ (a) and AnxA5-AF (b). The different locations of both compounds are conclusive: cytoplasmic distribution of [Zn21]4+ (a) and outer membrane accumulation of AnxA5-AF (b).
single dose of 50 Gy. The contralateral tumor served as a control. PET images were obtained one hour after intravenous injection of [Zn2(18F-6)]4+ at day six after irradiation (Figure 6a). The irradiated tumor accumulated more radioactivity than the control which showed only faint uptake in the rim area. The time dependence of radioactivity in both tumors indicated modestly elevated initial uptake and retarded washout in the irradiated tumor (SI). With the exception of kidneys and liver, the residual abdominal background activity proved to be low. In addition, [Zn2(18F-6)]4+ was tested with HelaMatu tumor bearing mice 2 days after taxol treatment. PET images were acquired 60 min after injection of [Zn2(18F-6)]4+. The treated animal shown in Figure 6b indicated higher tumor uptake as compared to the control tumor (Figure 6d). In comparison to the rat image, the background radioactivity of the mouse (liver, kidneys, intestine) appeared to be higher. In addition, bone joint activity indicated some defluorination. Quantification of tumor radioactivity in the excised tumors confirmed the stronger accumulation in treated tumors (SI). Additional autoradiography of excised tumor slices after the PET-study confirmed this finding in the treated tumors (Figure 7a versus c) and staining for cleaved caspase 3 proved the presence of apoptotic tissue after taxol treatment (Figure 7b versus d).
Figure 4. Mean fluorescence intensity (MFI) of staurosporine treated Jurkat cells in the presence of increasing amounts of [Zn21]4+ (Data are expressed as mean ± SD; n = 3.)
dependence, which was obtained with [Zn21]4+ and staurosporine treated Jurkat cells. The mean fluorescence intensity increased within the 0−100 μM range without reaching saturation. Confocal Laser Scanning Microscopy (CLSM). Clues about the intracellular location of the {bis[Zn(cyclen)]} 4+ complexes were obtained using confocal laser scanning microscopy (CLSM). Staurosporine treated Jurkat cells were incubated with a zinc-cyclen complex and placed under the microscope. Figure 5a (and SI) exemplarily shows staurosporine treated cells which deposited [Zn21]4+ throughout the cytoplasm. The membrane binding of AnxA5-AF on the other hand is obvious (Figure 5b). Influence of the CXCR4 Receptor. The presence of CXCR4 on Jurkat cells was confirmed on control and staurosporine-treated cells using the mouse anti-CXCR4 antibody mAb12G5. Flow cytometry revealed a strong expression of ca. 80% for both cell types. The mean fluorescence intensity was comparable, which means that there is no significant difference in the exposure of CXCR4 on treated and control cells. In addition, competition with the meta derivative of AMD3100 was performed which is known to bind CXCR4 in the low nanomolar range. Even with 100-fold molar excess, no depletion of the [Zn21]4+ fluorescence was observed with apoptotic cells (SI). PET Imaging. The in vivo behavior of the {bis[Zn(cyclen)]}4+ compounds was studied with [Zn2(18F-6)]4+. Male Copenhagen rats bilaterally transplanted with Dunning R3327-AT1 prostate tumors were irradiated on one side with a
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DISCUSSION
Among other approaches, search for the replacement of the apoptosis imaging agent [99mTc(HYNIC-rh-annexin A5)] has been focused on metal complexes which were also attractive as phosphate sensors in cell biology. Especially, zinc-cyclen complexes have been found to bind phosphate or other anionic groups.18 Consequently, a series of mono-, di-, and tetrameric cyclen ligands have been synthesized, capable of stabilizing Zn 2+ for in vivo applications and of functioning as carriers of fluorescent or radioactive reporter probes. The syntheses of the complex ligands summarized in Schemes 1−3 were followed by zinc complexation as the final reaction step. Flow cytometry of Jurkat cells which are incubated with fluorescent annexin A5, like AnxA5-AF, represents an established method to determine the degree of apoptosis after contact with cytostatic agents.4−6 The inhibitory activity of staurosporine on protein kinases of Jurkat cells is known to induce apoptosis.25 Owing to the disposition of apoptotic cells, 2621
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Figure 6. One hour coronal PET image of a rat with a bilaterally transplanted Dunning prostate adenocarcinoma 6 d after 50 Gy radiation therapy (a). The irradiated tumor is indicated by the arrow. One hour PET image of a mouse with HelaMatu tumors (arrows) two days after treatment with taxol (b,c) and the untreated control (d). Transaxial slices are shown in (b) and (d), and a coronal slice is shown in (c).
Figure 7. Autoradiography of HeLaMatu tumor slices taken from animals of the PET study (a,c) and IHC images (frames) stained for cleaved caspase 3 (b,d). Upper panel: Tumor samples from control animals. Lower panel: Tumor samples from taxol treated animals. The radioactive uptake (frames) corresponds with the presence of cleaved caspase 3 IHC.
this effect is accompanied by the formation of late apoptotic and secondary necrotic cells.26 As illustrated in Figure 3, most staurosporine treated cells were recorded in the Q1 rectangle where cell signals accumulate resulting from annexin A5 binding with externalized PS (see also Figure 5b). In Q2, cell signals from AnxA5-AF and PI binding are collected, which owing to PI uptake indicate leakiness of the damaged cell membrane. This area of flowcytometric measurements (33% of Q1+Q2) represents the above-mentioned secondary necrosis state of dying cells. Replacing AnxA5-AF with the bis(cyclen) complex [Zn21]4+, most cells with green fluorescence also showed PI uptake after staurosporine treatment. Although the cell suspensions were taken from the same cell pool, [Zn21]4+ stained the majority of
cells in the secondary necrotic population Q2 (80% of Q1+Q2) (Figure 3; lower panel). The larger Q2 fraction is obviously related to the common permeability of both dyes [Zn21]4+ and PI. Remarkably, these Q2 signals belong to the SSC and FSC signals indicating changes of size and granularity associated with the onset of cell death (arrow in respective SSC/FSC plot, SI). The question whether intracellular accumulating [Zn21]4+ is responsible for this effect can be related to the observation of Kimura et al. that {bis[Zn(cyclen)]}4+ complexes are able to form ternary complexes with DNA bases like thymidine.27 This interference is, however, only in effect if apoptosis has been triggered by staurosporine and the zinc complex is internalized. Any cell 2622
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indicated no involvement of this receptor in the [Zn21]4+ uptake (SI). The capacity of {bis[Zn(cyclen)]}4+ complexes to transport fluorescent dyes into apoptotic and secondary necrotic cells was also investigated using a radiolabeled complex. High-dose radiation therapy was used to induce cell death which is known to force tumors mainly into necrosis. Rats with Dunning prostate adenocarcinoma transplants were, therefore, irradiated using a single gamma dose of 50 Gy. The rats were imaged with [Zn2(18F-6)]4+ six days after treatment. As illustrated in Figure 6a, the irradiated tumor clearly showed significant uptake while the untreated control was negative. This was due to the higher initial uptake just after injection of [Zn2(18F-6)]4+ and the delayed washout as compared with the contralateral control (SI). The radioactivity of the excretion organs, kidney and liver, was in the expected range, while the residual background activity was low. The accumulated radioactivity in the Dunning tumor is certainly due to massive cell death after such a high radiation dose.29 An investigation into the imaging kinetics as a response to therapy has been performed for ten days after the onset of therapy. The data indicate maximal accumulation of [Zn2(18F6)]4+ at day six while decrease of intensity was observed at day ten (SI). The contralateral control tumor meanwhile increased the uptake indicating cell death due to short tumor doubling time of 5.6 ± 0.4 days.30 A second imaging experiment using [Zn2(18F-6)]4+ was carried out with taxol treated HelaMatu tumor bearing mice. The transaxial PET images of a taxol treated animal (Figure 6b) indicated higher tumor uptake in comparison to the control (Figure 6d). Quantification of the mean tumor uptake values showed an increase by 30% in the treatment group while the tumor size decreased by 15% in comparison to the control group (SI). Autoradiograms of tumor tissue slices shown in Figure 7a,c confirmed the result of the PET study, and immunohistochemistry (Figure 7b,d) validated the presence of apoptotic tissue which corresponded with radioactivity accumulation after taxol treatment. The perspective of having radiolabeled markers of cell death either produced by apoptosis and/or by secondary necrosis is attractive, because therapy control is one of the main areas of interest during the follow-up of cancer patients. Currently, a series of conventional investigations, including radiography, CT, sonography, and MRI, acquire this information covering the extent and morphology of tumor masses and, to some degree, alterations in tumor perfusion. In contrast, nuclear medicine has at its disposal a series of surrogate markers that are used for the assessment of the therapy response. Radiofluorinated bis[Zn(cyclen)]4+ complexes might be more specific for the imaging of therapy response providing information about cell death in treated tumors. Further work is underway to optimize the pharmacokinetics of zinc cyclen complexes and to understand more about the uptake mechanism.
damaging effects of the zinc complex on vital cells were not observed (SI). As reviewed in the introduction, binding of the zinc complexes on apoptotic cells through the coordination of externalized PS was thought to lead to membrane binding. However, the fluorescence of [Zn21]4+ was found exclusively in the cytosol of staurosporine affected cells. This finding and the clear membrane fluorescence of AnxA5-AF are apparent in the CLSM images shown in Figure 5. A similar intracellular distribution was reported with bis(zinc(II)-dipicolylamine) complexes.9,12,13 Microscopic fluorescence images indicate that PSS-480 (Figure 1) stains most prominently those apoptotic Jurkat cells which are also positive with 7-aminoactinomycin, a PI like DNA intercalator, known to dye secondary necrotic cells.13 The assumption that zinc-cyclen derivatives can profit from multimerization was demonstrated with [Zn45]8+. With staurosporine treated cells, this tetramer showed a more than doubled fluorescence intensity as compared to the two comparable dimeric complexes [Zn21]4+ and [Zn24]4+. The monocyclen complex [Zn3]2+, on the other hand, produced only signals with reduced intensity. The increase of the fluorescence intensity was obviously mediated by the additional zinc-cyclen functionalities. It may, therefore, be speculated that the uptake initially begins through binding at externalized PS which transports the resulting ternary complexes with the help of aminophospholipid translocase into the cytosol. On the other hand, concentration driven diffusion through a damaged cell membrane may also be conceivable. The absence of zinc, however, inhibits fluorescent signals in Q1 and Q2 (complex ligand 4, SI) which also points to translocase assisted internalization. An additional observation supporting this notion is given by the introduction of an exocyclic pendant nitrogen. Complex [Zn22]4+ contains an extra heteroatom able to coordinate zinc and to compete with PS thereby disturbing PS related membrane penetration. The comparable green, but strongly reduced fluorescences of [Zn22]4+ in treated as well as in control cells may be explained with unspecific binding (SI). A saturation experiment was performed with staurosporine treated cells in order to evaluate an uptake limit. Figure 4 illustrates the increase of fluorescence with increasing concentration of [Zn21]4+. Above 20 μM and up to 100 μM, the uptake of [Zn21]4+ followed a straight relationship without signs of saturation. It was also evident that the removal of these complexes from the medium resulted in a strong reduction of the intracellular fluorescence. These findings indicate a rather weak intracellular interaction of {bis[Zn(cyclen)]}4+ complexes. Owing to the structural similarity of the title compounds, with several bis-cyclam derivatives potential CXCR4 interactions were scrutinized. It is known that the bis-cyclam AMD3100 and its meta derivative including the respective Cu2+, Zn2+, or Ni2+ complexes have anti-HIV activity through binding at the chemokine receptor CXCR4.28 The expression of CXCR4 on Jurkat J6 cells is known and proven to be positive on both staurosporine treated and control cells. Consequently, CXCR4 dependent differences between treated and control cells, which were obtained with the zinc-cyclen complexes, appear to be unlikely. To further exclude the participation of CXCR4 on [Zn21]4+ uptake, in staurosporine treated cells competition with a 100-fold molar excess of meta AMD3100 was performed. The similarity of flow-cytometry results
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ASSOCIATED CONTENT
S Supporting Information *
Flow-cytometry results, material for the role of CXCR4, additional CLSM data, cytotoxicity, and PET data. This material is available free of charge via the Internet at http:// pubs.acs.org. 2623
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AUTHOR INFORMATION Corresponding Author *E-mail:
[email protected]; T +49-6221-422443. ACKNOWLEDGMENTS We would like to thank Dr. H. Spring for performing CLSM, Prof. Dr. P. Huber, and Dr. P. Peschke for providing rats with Dunning carcinoma transplants and the radiation experiment, K. Leota for μPET, and U. Schierbaum for animal care, as well as U. Wagner (all DKFZ) for assistance in chemical syntheses. D.O. is grateful for financial support from Bayer Healthcare. There is no other financial conflict of interest dealing with this subject.
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dx.doi.org/10.1021/bc200457b | Bioconjugate Chem. 2011, 22, 2611−2624