Î³-Glutamyltranspeptidase-Triggered Intracellular Gadolinium

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#-Glutamyltranspeptidase-Triggered Intracellular Gadolinium Nanoparticle Formation Enhances the T-Weighted MR Contrast of Tumor 2

Zijuan Hai, Yanhan Ni, Dilizhatai Saimi, Hongyi Yang, Haiyang Tong, Kai Zhong, and Gaolin Liang Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.8b05154 • Publication Date (Web): 11 Mar 2019 Downloaded from http://pubs.acs.org on March 11, 2019

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γ-Glutamyltranspeptidase-Triggered

Intracellular

Gadolinium Nanoparticle Formation Enhances the T2Weighted MR Contrast of Tumor Zijuan Hai,†,§,# Yanhan Ni,†,# Dilizhatai Saimi,† Hongyi Yang,‡ Haiyang Tong,‡ Kai Zhong*,‡ and Gaolin Liang*,† †

Hefei National Laboratory of Physical Sciences at Microscale, Department of Chemistry, University of

Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, China ‡

High Magnetic Field Laboratory, Hefei Institutes of Physical Science, Chinese Academy of Sciences, 350

Shushanhu Road, Hefei, Anhui 230031, China §

Institutes of Physical Science and Information Technology, Anhui University, 110 Jiulong Road, Hefei,

Anhui 230601, China

KEYWORDS: γ-glutamyltranspeptidase, gadolinium nanoparticle, T2-weighted magnetic resonance imaging ABSTRACT Magnetic resonance imaging (MRI) is advantageous in the diagnosis of deep internal cancers but contrast agents (CAs) are always needed to improve MRI sensitivity. Gadolinium (Gd)-based agents are routinely used as T1-dominated CAs in clinic but using intracellularly formed Gd nanoparticles to enhance the T2-weighted MRI of tumor in vivo at high magnetic field has not been reported. Herein, we rationally designed a “smart” Gd-based probe Glu-Cys(StBu)-Lys(DOTA-Gd)-CBT (1), which was subjected to γ-glutamyltranspeptidase (GGT) cleavage and an intracellular CBT-Cys condensation reaction to form Gd nanoparticles (i.e., 1-NPs) to enhance the T2‑weighted MR contrast of tumor in vivo at 9.4 T.

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Living cell experiments indicated that, the 1-treated HeLa cells had an r2 value of 27.8 mM-1 s-1 and an r2/r1 ratio of 10.6. MR imaging of HeLa tumor-bearing mice indicated that the T2 MR contrast of the tumor enhanced 28.6% at 2.5 h post intravenous injection of 1. We anticipate that our probe 1 could be employed for T2-weighted MRI diagnosis of GGT-related cancers in the future when high magnetic field is available in clinic. TEXT γ-glutamyltranspeptidase (GGT, E.C. 2.3.2.2) is a cell surface-associated enzyme, which participates in cellular secretion and absorption processes.1 GGT can cleave the γ-glutamyl bond of glutathione (GSH) to yield cysteinyl-glycine which, upon dipeptidase hydrolysis, produces cysteine and glycine.2 This GGT-initiated process plays important role in the survival and growth of cancer cells since cysteine is significantly relied by the cancer cells.3 Indeed, GGT was found overexpressing in several cancer cells such as liver,4 cervical,5 and ovarian cancer cells.6 Hence, detection and imaging of GGT may facilitate the diagnosis of the GGT-related cancers. Conventional analytical methods such as colorimetric assay,7 high-performance liquid chromatography (HPLC),8 and electrochemistry9 have been used to detect GGT activity in vitro. Optical and nuclear techniques have been employed to image GGT activity in superficial xenografted tumor of living subjects.10-12 However, optical imaging with shallow tissue penetration depth is not suit for the diagnosis of abovementioned internal deep GGT-related cancers.13,14 Although nuclear imaging offers high sensitivity, its spatial resolution is poor. Magnetic resonance imaging (MRI) is an imaging technique characterized with its noninvasiveness, unlimited penetration depth, and high spatial resolution.15 But it suffers from poor sensitivity. Thus, in clinic MRI, about 35% of the patients need to be injected with contrast agents (CAs) to enhance the contrast between their normal and pathological tissues.16 According to their impact abilities (i.e., relaxivity, r1 or r2) on the longitudinal (T1) or transversal (T2) relaxation of their vicinity water protons, MR CAs are roughly categorized into T1 and T2 types.17 The r2/r1 ratio is a decisive parameter to predict the type of a MR CA. It was found that low r2/r1 ratio (< 5) brings T1-dominated contrast while higher r2/r1 ratio (> 8) leads to T2dominated contrast of a CA.18,19 As we known, paramagnetic gadolinium(Gd)-based agents are routinely


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used as T1-dominated CAs at both low and high magnetic fields.20,21 Recently, we found that, when a Gdbased CA self-assembles into Gd-nanofiber, its r2 value and r2/r1 ratio increase with the increase of magnetic field strength, suggesting a Gd-based nanostructure could be used as a T2-weighted CA at high magnetic field.22 However, there has been no report of using in situ formed Gd nanoparticles to enhance the T2weighted MR contrast of tumor in vivo at high magnetic field so far. Encouraged by above pioneering studies and using a CBT-Cys click condensation reaction,23,24 in this work, we rationally designed a Gd-based probe which was subjected to GGT-triggered Gd nanoparticle formation in cancer cells to enhance the T2‑weighted MR contrast of tumor in vivo at high magnetic field. This Gd-based probe Glu-Cys(StBu)-Lys(DOTA-Gd)-CBT (1) was designed to contain three components (Figure 1 and Scheme S1): (1) a Glu structure for GGT cleavage; (2) the disulfided Cys and 2-cyanobenzothiazole (CBT) structures for reduction-controlled CBT-Cys condensation; (3) a DOTA-Gd structure for MRI. As illustrated in Figure 1A, once being uptaken by cancer cells, 1 is firstly cleaved by GGT on the cell membrane to yield 1-Cleaved. Then the disulfide bond of 1-Cleaved is reduced by intracellular GSH to expose the 1,2-aminothiol structure which instantly condenses with the cyano group of CBT to yield the cyclized dimer 1-Dimer. And the amphiphilic 1-Dimer self-assembles into Gd-based nanoparticles (i.e., 1-NPs) via π-π stacking inside cells with high r2 value (27.8 mM-1 s-1 in this work) and r2/r1 ratio (10.6) for enhanced T2‑weighted MRI of GGT-overexpressing tumor at 9.4 T. For comparison, a control probe Ac-Cys(StBu)-Lys(DOTA-Gd)-CBT (1-Ctrl), which can be reduced by intracellular GSH but cannot be cleaved by GGT, was synthesized for parallel studies (Figure 1B).


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Figure 1. Schematic illustrations of (A) GGT-triggered formation of 1-NPs inside cells from 1 to enhance the T2 MR contrast of tumor at high magnetic field and (B) GSH-triggered reduction of 1-Ctrl to yield 1Ctrl-Cleaved inside cells. GGT-Triggered 1-NPs Formation In Vitro. We began the study with the syntheses and characterizations of 1 and 1-Ctrl, and their respective precursors Glu-Cys(StBu)-Lys(DOTA)-CBT (E) and Ac-Cys(StBu)-Lys(DOTA)-CBT (K) (Schemes S1-S2, Figures S1-S6). To ensure the purity of products, we used high performance liquid chromatography (HPLC) to purify the product at each step. After synthesis, we firstly tested the stability of 1 in vitro. Neither the UV-vis spectra of 1 within 7 d nor the HPLC traces of 1 within 28 d in phosphate-buffered saline (PBS) at 37 °C showed obvious change (Figure S7), suggesting very high physicochemical stability of 1. Then we used HPLC to monitor the conversion of 200 μM 1 to 1-Cleaved by GGT as a function of GGT concentration. As shown in Figure S8, after 2 h incubation with GGT at 37 °C in PBS, with the increase of GGT concentration, the HPLC peak of 1 at retention time of 20.5 min decreased while a new peak at retention time of 17.9 min increased. This new peak was identified as 1-Cleaved by high resolution matrix-assisted laser desorption/ionization mass (HR-


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MALDI/MS) analysis (Figure S9). At the concentration 400 U/L of GGT, probe 1 was completely converted to 1-Cleaved. Therefore, we chose 400 U/L of GGT for in vitro studies. Kinetic study indicated that 1 has a Michaelis constant (Km) 25.9 μM and a turnover number (kcat) 38.4 min-1 towards GGT (Figure S10), which are comparable to those of other reported GGT probes (Table S1). After that, we used HPLC to trace the process of GGT-triggered 1-NPs formation and transmission electron microscopy (TEM) to characterize 1-NPs in vitro. In the presence of 2 mM GSH, 1-Cleaved in above reaction mixture at 2 h was converted to a new product at retention time of 14.4 min which was identified as 1-Dimer by HRMALDI/MS analysis (Figure 2A and Figure S11), suggesting that the disulfide bond of 1-Cleaved was reduced by GSH and the reduced intermediate underwent the CBT-Cys condensation reaction to yield 1Dimer. Time-course dynamic light scattering (DLS) measurements showed the size of 1-NPs increased over time and stabilized at 2 h and on with a mean hydrated diameter of 59.9  5.7 nm (Figure S12). TEM image of the GSH-containing incubation mixture at 2 h clearly showed that the nanoparticles 1-NPs had an average diameter of 48.3  5.1 nm (Figures 2B and Figure S13). Energy-dispersive X-ray spectrum (EDS) of 1-NPs clearly indicated the existence of Gd in the nanoparticles (Figure S14). Above results verified that, in the presence of reducing agent, GGT could trigger 1 to form the nanoparticles 1-NPs. For the control probe 1-Ctrl, after 2 h incubation with 400 U/L GGT and 2 mM GSH at 37 °C in PBS, HPLC and HRMALDI/MS analyses indicated that it was only reduced by GSH to yield 1-Ctrl-Red but insusceptible to GGT cleavage for nanoparticle formation (Figures S15-S16). Therefore, 1-Ctrl was used for further parallel studies. In Vitro Formation of 1-NPs Enhances T2 MR Imaging in Phantoms of GGT. After verifying that GGT could trigger 1-NPs formation in vitro, we measured the r2 value of 1-NPs (i.e., 1+GGT+GSH) and compared that with those of 1, 1-Ctrl-Red (i.e., 1-Ctrl+GGT+GSH), and clinically used CA Gd(III)diethylenediaminepentaacetic acid (Gd-DTPA) at 9.4 T. Before that, we measured the r1 values of these four groups at low magnetic field (3 T) and found that formation of 1-NPs increased the r1 value of 1 by 1.55 folds (Figure S17), as we previously reported.20 T2- and T1-weighted MR phantom images of four groups were obtained on a 9.4 T MR scanner: experimental Group (i.e., 200 μM 1+GGT+GSH), control ACS Paragon Plus Environment

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Group I (i.e., 200 μM 1), control Group II (i.e., 200 μM 1-Ctrl +GGT+GSH), and control Group III (i.e., 200 μM Gd-DTPA). As shown in Figure S18A, T2 relaxation time of 1+GGT+GSH was measured to be 159.5 ms, much shorter than that 373.1 ms of 1, 384.6 ms of 1-Ctrl+GGT+GSH, or that 418.4 ms of GdDTPA. From the slopes of fitted calibration curves of relaxation rate vs. concentration, the r2 values of the four groups were calculated to be 25.1 mM-1 s-1 for 1+GGT+GSH, 5.79 mM-1 s-1 for 1, 5.30 mM-1 s-1 for 1Ctrl+GGT+GSH, and 4.26 mM-1 s-1 for Gd-DTPA at 9.4 T, respectively (Figure 2C). These results suggested that GGT-triggered formation of 1-NPs in vitro enhanced the r2 value of 1 by 4.34-fold at 9.4 T. The r1 values of four groups at 9.4 T were measured to be 3.02 mM-1 s-1 for 1+GGT+GSH, 2.89 mM-1 s-1 for 1, 2.65 mM-1 s-1 for 1-Ctrl, and 3.60 mM-1 s-1 for Gd-DTPA, respectively (Figure 2D), suggesting that formation of 1-NPs almost did not affect the longitudinal relaxivity (i.e., r1) of 1 at high magnetic field. Thus, the r2/r1 ratio of 1+GGT+TCEP was calculated to be 8.31, suggesting that GGT-triggered formation of 1-NPs could be used as excellent T2-dominated CAs with high r2 value at high magnetic field. This enhanced T2 effect could be ascribed to the introduced larger susceptibility gradient by Gd in 1-NPs at high magnetic field.25,26

Figure 2. (A) HPLC traces of 200 μM 1 in PBS buffer (black), incubated with 400 U/L GGT (red), or incubated with 400 U/L GGT and 2 mM GSH (blue) at 37 °C for 2 h. (B) TEM image of 1-NPs in PBS buffer. Transverse relaxation rates (1/T2) (C) and longitudinal relaxation rates (1/T1) (D) of 1+GGT+GSH,


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1, 1-Ctrl+GGT+GSH, or Gd-DTPA in PBS at different Gd concentrations. Each error bar represents the standard deviation of three independent experiments. Intracellular Formation of 1-NPs Enhances T2 MR Imaging in Phantoms of HeLa Cells. Before applying 1 and 1-Ctrl for MRI of GGT activity in living cells, we studied their cytotoxicity on human cervical cancer (HeLa) cells which overexpress GGT.10,27 As shown in Figure S19, 82% or 84% of the cells survived up to 12 h after incubation with 400 μM 1 or 1-Ctrl, indicating that 200 μM 1 or 1-Ctrl is safe for MRI of living cells. HeLa cells were divided into five groups: experimental Group 1 was the cells incubated with 200 μM 1 for 4 h; control Group DON+1 was the cells pretreated with 2 mM 6-diazo-5-oxo-Lnorleucine (DON, a GGT inhibitor)28 for 0.5 h then incubated with 200 μM 1 for 4 h; control Group 1-Ctrl was the cells incubated with 200 μM 1-Ctrl for 4 h; control Group Gd-DTPA was the cells incubated with 200 μM Gd-DTPA for 4 h; Blank Group was cells without any treatment. After treatment, the cells were prepared for TEM observation. As shown in Figure 3A and Figure S20, large area of 1-NPs was found in the cells of the experimental Group 1 but not in the cells of other four control groups. High magnification TEM image indicated that the 1-NPs inside cells had an average diameter of 56.3  4.8 nm (Figure 3B and Figure S21), slightly larger than that of 1-NPs formed in vitro. For T2- and T1-weighted MR phantom images study, HeLa cells in above four groups (experimental group and three control groups) were diluted into four concentrations and resuspended in serum-free culture medium with 1 wt% agarose gel. Inductively coupled plasma-mass spectrometry (ICP-MS) was used to quantitate the Gd concentrations in HeLa cells of these four groups. The Gd concentrations of experimental Group 1, control Group DON+1, and control Group 1-Ctrl were 39.9 μM, 32.7 μM, and 37.1 μM, respectively, which were higher than that 27.6 μM of control Group Gd-DTPA, suggesting higher uptake efficiency of 1 (19.9%) and 1-Ctrl (18.6%) than GdDTPA (13.8%) by HeLa cells. As shown in Figure S22A, compared to control Groups DON+1, 1-Ctrl, and Gd-DTPA, the T2-weighted MR phantom images of the experimental Group 1 showed obviously lower grey value. Similarly, the r2 values were calculated to be 27.8 mM-1 s-1 for 1, 5.20 mM-1 s-1 for DON+1, 4.81 mM-1 s-1 for 1-Ctrl, and 4.11 mM-1 s-1 for Gd-DTPA in living HeLa cells at 9.4 T, respectively (Figure 3C). These results suggested that intracellular GGT-triggered formation of 1-NPs significantly enhanced


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the r2 of 1 by 5.35-fold at 9.4 T. The r1 values of four groups were measured to be 2.62 mM-1 s-1 for 1, 2.45 mM-1 s-1 for DON+1, 2.32 mM-1 s-1 for 1-Ctrl, and 3.96 mM-1 s-1 for Gd-DTPA, respectively (Figure 3D). The r2/r1 ratio of experimental Group 1 was calculated to be 10.6, much higher than that 2.12 of control Group DON+1, suggesting that the intracellularly formed 1-NPs are excellent T2-dominated CAs for imaging intracellular GGT activity at high magnetic field. To further verify that the enhanced r2 value indeed correlated to the GGT levels in cells, we chose human umbilical vein endothelial (HUVEC) cells which express low levels of GGT as a control cell line.13,29 Gd concentrations in HUVEC cells of experimental Group 1 (35.2 μM), control Group 1-Ctrl (32.4 μM), and control Group Gd-DTPA (28.6 μM) were similar to those of these three groups in HeLa cells, indicating comparable probe-uptaking efficiency between HeLa and HUVEC cells. As shown in Figure S23, in HUVEC cells, both the T2-weighted MR phantom image and the r2 value of experimental group 1 did not show obvious difference to those of other two control groups. These results suggested that the obviously increased r2 value of 1 in HeLa cells was ascribed to the GGT-triggered 1-NPs formation.

Figure 3. (A) Low magnification TEM image of HeLa cells in experimental Group 1 which incubated with 200 μM 1 for 4 h. (B) High magnification TEM image of the white rectangle area in A. 1/T2 (C) and 1/T1 (D) of HeLa cells in experimental Group 1, control Group DON+1, control Group 1-Ctrl, and control Group Gd-DTPA at different Gd concentrations. Each error bar represents the standard deviation of three independent experiments.


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In Vivo Formation of 1-NPs Enhances T2 MR Imaging of Tumor. After in vitro and living cell imaging, then we conducted T2- and T1-weighted coronal MRI of tumor in mice. Each nude mouse was subcutaneously implanted with two million of HeLa cells in the right thigh. Until the tumors grew to 8-10 mm in diameter, we firstly measured their GGT activity and tested whether the tumors did work with 1. Using a commercially available kit, GGT activity in HeLa tumor lysates was measured to be 533.4 ± 54.4 U/L (Table S2). Then the HeLa tumor lysates were incubated with 200 μM 1 at 37 °C for 2.5 h. HPLC trace clearly showed a main peak of 1-Dimer at retention time of 14.4 min (Figure S24). These results suggesting that GGT activity in HeLa tumors was high enough to coonvert 1 into its nanoparticles 1-NPs. Then the nude mice were randomly divided into three groups (N = 3 for each group): 3 mice intravenously (i.v.) injected with 0.08 mmol/kg 1 through tail vein were designated as the experimental Group 1; 3 mice i.v. pre-injected with 0.25 mmol/kg DON for 0.5 h followed by i.v. injection of 0.08 mmol/kg 1 were designated as control Group DON+1; 3 mice i.v. injected with 0.08 mmol/kg Gd-DTPA as control Group Gd-DTPA. Dynamic T2- and T1-weighted coronal MR images of the three group mice on a 9.4 T MR scanner were shown in Figure S25 and Figure S26, respectively. Typical T2-weighted MR images of the tumors in mice were shown in Figure 4A. Quantitative analyses of the relative tumor-to-muscle (T/M) contrast ratios of T2 values of the time course T2-weighted MR images were shown in Figure 4B. From Figure 4 we could see that, the T2-weight MR contrast of tumors in 1-injected mice reached its maximum at 2.5 h post injection (the T/M ratio of T2 values at 2.5 h was 71.4% of that at 0 h). In contrast, the T2-weight MR contrast of tumor in DON-pretreated mice increased very slightly (the T/M ratio of T2 values at 2.5 h was 94.9% of that at 0 h). As expected, the T/M ratio of tumor in Gd-DTPA-injected mice increased slightly (the T/M ratio of T2 values at 2.5 h was 105.1% of that at 0 h). Above results suggested that GGT-triggered intracellular Gd nanoparticle (i.e., 1-NPs) from 1 largely and specifically enhanced the T2 MR contrast of the GGT-overexpressing tumor. T1-weight MRI of the three groups mice and quantitative analyses of the relative T/M ratios of T1 values indicated that, while Gd-DTPA could enhance the T1 MR contrast of the tumors as expected (the highest T/M ratio of T1 values was 121.5% at 1 h of that at 0 h), neither 1 nor DON+1 showed obvious enhanced T1 MR contrast of the tumors (the highest T/M ratio of T1 values were


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103.4% at 0.5 h for 1 and 106.6% at 1 h for DON+1) (Figure S27). To further chemically verify that the enhanced T2 MR contrast of tumor was due to the formation of 1-NPs, we used HPLC to analyze the tumor lysates of the experimental Group 1 after MRI at 3.5 h. As shown in Figure S28, a main peak at retention time of 14.4 min, which was identified as 1-Dimer, appeared on the HPLC trace. At last, the Gd contents in tumors and main organs (spleens, hearts, lungs, kidneys, and livers) of the three groups of mice were measured with ICP-MS after MRI at 3.5 h. Interestingly, as shown in Table S3, only tumors but not other organs contained significantly different contents of Gd. Specifically, tumors in 1-injected mice had an average Gd content of 5.89 μg/g, 2.20-fold higher than that of tumors in DON-pretreated mice (2.68 μg/g) or 4.33-fold higher than that of tumors in Gd-DTPA-injected mice (1.36 μg/g), respectively. This result suggested that the formation of 1-NPs with longer retention time increased the Gd contents in tumors of the 1-injected mice. Kidneys had the highest Gd concentrations among the organs studied (Figure S29).

Figure 4. (A) T2-weighted coronal MR images of HeLa tumor-bearing mice intravenously injected with 0.08 mmol/kg 1 (top row), 0.25 mmol/kg DON for 0.5 h and then 0.08 mmol/kg 1 (middle row), and 0.08 mmol/kg Gd-DTPA (bottom row) at 0 h (left column) and 2.5 h (right column). (B) Normalized time course tumor-to-muscle (T/M) contrast ratios of T2 values in Figure 4A and Figure S25. Each error bar represents


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the standard deviation of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant; analyzed by Student’s t-test. White circles indicate the tumors. In summary, we rationally designed a “smart” Gd-based probe 1, which could subject to GGT-triggered intracellular Gd nanoparticles (i.e., 1-NPs) formation, to enhance the T2‑weighted MR contrast of GGToverexpressing tumor in vivo at high magnetic field. In vitro experiments indicated that, in the presence of GSH, 1 underwent GGT cleavage and a CBT-Cys condensation reaction to form 1-NPs with an average diameter of 48.3 nm. Living cell experiments indicated that, at 9.4 T, the HeLa cells in experimental Group 1 had an r2 value of 27.8 mM-1 s-1 and an r2/r1 ratio of 10.6, while the control Group DON+1 had an r2 value of 5.20 mM-1 s-1 and an r2/r1 ratio of 2.12. MR imaging of HeLa tumor-bearing mice indicated that, at 2.5 h post i.v. injection of 1, T2-weighted MR contrast of the tumors in 1-injected mice enhanced 28.6% while that of GGT inhibitor-pretreated mice only enhanced 5.1% at 9.4 T. Compared with clinical paramagnetic gadolinium (Gd)-based contrast agents (GBCAs), 1 had higher cell uptake efficiency and could be used at lower doses due to that it could form nanoparticles in cancer cells. But, in clinic, the magnetic field for disease diagnosis is not higher than 3 T. At low magnetic field, 1 might not have such good T2-weight contrast effect. Thus, we anticipate that our “smart” probe 1 could be employed for T2-weighted MRI diagnosis of GGT-related cancers in the future when higher magnetic field is available in clinic. ASSOCIATED CONTENT Supporting Information. General methods; Syntheses and characterizations of 1 and 1-Ctrl; Schemes S1-S2; Figures S1-S29; Tables S1-S4. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *Tel.: +86-187-5513-7193. Email: [email protected]. *Tel.: +86-551-6360-7935. Email: [email protected]. ACS Paragon Plus Environment

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Author Contributions #

These authors contributed equally.

Notes The authors declare no competing financial interests. ACKNOWLEDGMENT This work was supported by Ministry of Science and Technology of China (Grants 2016YFA0400904 and 2016YFC1300503), and the National Natural Science Foundation of China (Grants 21725505, 81821001, 91649101, and 21675145). REFERENCES (1) Hanigan, M. H.; Ricketts, W. A. Extracellular Glutathione Is a Source of Cysteine for Cells That Express Gamma-Glutamyl Transpeptidase. Biochemistry 1993, 32, 6302-6306. (2) Borud, O.; Mortensen, B.; Mikkelsen, I. M.; Leroy, P.; Wellman, M.; Huseby, N. E. Regulation of γglutamyltransferase in cisplatin-resistant and -sensitive colon carcinoma cells after acute cisplatin and oxidative stress exposures. Int. J. Cancer 2000, 88, 464-468. (3) Pompella, A.; De Tata, V.; Paolicchi, A.; Zunino, F. Expression of γ-glutamyltransferase in cancer cells and its significance in drug resistance. Biochem. Pharmacol. 2006, 71, 231-238. (4) Yao, D. F.; Jiang, D. R.; Huang, Z. W.; Lu, J. X.; Tao, Q. Y.; Yu, Z. J.; Meng, X. Y. Abnormal expression of hepatoma specific γ-glutamyl transferase and alteration of γ-glutamyl transferase gene methylation status in patients with hepatocellular carcinoma. Cancer 2000, 88, 761-769. (5) Strasak, A. M.; Goebel, G.; Concin, H.; Pfeiffer, R. M.; Brant, L. J.; Nagel, G.; Oberaigner, W.; Concin, N.; Diem, G.; Ruttmann, E.; Gruber-Moesenbacher, U.; Offner, F.; Pompella, A.; Pfeiffer, K. P.; Ulmer, H.; Grp, V. P. S. Prospective Study of the Association of Serum γ-Glutamyltransferase with Cervical Intraepithelial Neoplasia III and Invasive Cervical Cancer. Cancer Res. 2010, 70, 3586-3593. (6) Hanigan, M. H.; Frierson, H. F.; Brown, J. E.; Lovell, M. A.; Taylor, P. T. Human Ovarian-Tumors


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Î³-Glutamyltranspeptidase-Triggered Intracellular Gadolinium

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