Targeted Cancer Therapy with a 2-Deoxyglucose ... - Cancer Research

Cancer Research

Therapeutics, Targets, and Chemical Biology

Targeted Cancer Therapy with a 2-Deoxyglucose–Based Adriamycin Complex Jie Cao1, Sisi Cui1, Siwen Li1, Changli Du1, Junmei Tian1, Shunan Wan1, Zhiyu Qian2, Yueqing Gu1, Wei R. Chen3, and Guangji Wang4

Abstract Adriamycin (ADM) has been effective against many types of solid tumors in clinical applications. However, its use is limited because of systemic toxicities, primarily cardiotoxicity, and multidrug resistance. In this study, a new active receptor-mediated complex, ADM conjugated with 2-amino-2-deoxy-D-glucose and succinic acid (2DG–SUC–ADM), was designed to target tumor cells through glucose transporter 1 (GLUT1). MTT assay and confocal images showed that the complex had better inhibition rate to tumor cells and low toxicity to normal cells. Most importantly, the complex displayed a potential to reverse overcome multidrug resistance in cancer cells, with more complex transported into the nucleus of tumor cells. Our in vivo experiments also showed that this new complex could significantly decrease organ toxicity and enhance the antitumor efficacy compared with free ADM, indicating a promising drug of 2DG–SUC–ADM for targeted cancer therapy. Cancer Res; 73(4); 1362–73. 2012 AACR.

Introduction Anticancer drugs have been used extensively in cancer therapy over the past 6 decades (1). Most anticancer drugs are small molecules that penetrate into cells by diffusion (2, 3). One of their main drawbacks is that these therapeutic agents cannot specifically target tumor cells within the pathologic sites, which both weakens their anticancer effects and results in serious toxic and side effects (4–7). Adriamycin (ADM) has been effective against many types of solid malignant tumors. However, because of its nontargeting nature, ADM induces severe side effects, such as nephrotoxicity, hepatotoxicity, and baldness, in particularly on the heart and gastrointestinal tract. In addition, multidrug resistance of ADM also limits its clinical applications. To overcome these limitations, loading ADM into macromolecular carriers, such as iron oxide, carboxymethylpullulan, poly (L-lysine citramide), and polyethylene glycol has attracted much attention (8–11). Such delivery systems can Authors' Affiliations: 1Department of Biomedical Engineering, State Key Laboratory of Natural Medicines, School of Life Science and Technology, China Pharmaceutical University; 2Department of Biomedical Engineering, School of Automation, Nanjing University of Aeronautics and Astronautics, Nanjing, China; and 3Department of Engineering and Physics, University of Central Oklahoma, Edmond, Oklahoma; 4State Key Laboratory of Natural Medicines, Key Laboratory of Drug Metabolism & Pharmacokinetics, China Pharmaceutical University, Nanjing 210009, China Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). J. Cao, S. Cui, and S. Li contributed equally to this work. Corresponding Author: Yueqing Gu, Department of Biomedical Engineering, China Pharmaceutical University, Nanjing 210009, Jiangsu Province, China. Phone: 86-25-83271046; Fax: 86-25-83271046; E-mail: [email protected]; Institutional E-mail: cpuyueqing@163. com; doi: 10.1158/0008-5472.CAN-12-2072 2012 American Association for Cancer Research.

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improve ADM anticancer activity and decrease its side effects. However, the drug-loading efficiency of these conjugates is low and the ADM release is usually unsatisfactory (12). A promising approach to increase drug efficiency and selectivity is to covalently conjugate ADM to a targeting ligand that can specifically bind to the tumor cells and form an ADM prodrug (13–15). According to the design concept of drugs, the primary amino group, which is far from the anticancer active anthracence ring of ADM molecule, has higher reactivity than the 13carboxyl group, 3-amino group, a-H, and hydroxyl group. The modification of the primary amino group with other molecules will not affect the antitumor ability of ADM. Thus, it is feasible to covalently bind the amino group with carboxyl groups to form a stable amide bond. In addition, studies have shown that modification of the primary amino group could greatly reduce the cardiotoxicity, which is one of the main objectives to modify ADM (16). Glucose, which plays an important role in human physiology, is an indispensable energy source in the metabolic process. Malignant tumors show an elevated glycolytic rate and a highglucose demand, even under aerobic conditions (17, 18). The uptake of glucose is through glucose transporters (GLUT). Of these transporters, GLUT1 is reported to be the main mediator of glucose uptake (19). Consequently, the increased demand for glucose to fuel energy production in tumors translates to elevated expression and activity of GLUT1, which can be exploited as targeting ligand for drugs (20). Studies show that the glucose analog, 2-amino-2-deoxy-D-glucose (2DG) is the formation of 2-deoxyglucose-6-phosphate by hexokinase phophorylation (21–24), which can be recognized and transported into the cells by GLUT1 on the cell membrane. These findings have been extensively exploited in preclinical and clinical imaging for tumor diagnosis, staging, and monitoring of therapeutic response (25–29). Especially, using radiolabeled 2fluoro-2-deoxyglucose (18FDG) as a targeting ligand to form

2DG-Based Adriamycin Complex

drugs has been studied (30–32). However, up to now, 2DGbased prodrugs have not been carefully studied. In this study, we developed a new active receptor-mediated complex, by conjugating adriamycin with 2-amino-2-deoxy-Dglucose and succinic acid (2DG–SUC–ADM), which modified the primary amine group with 2DG using succinic anhydride as reaction linkage. It was designed to target tumor cells through GLUT1.

Materials and Methods Materials Adriamycin hydrochloride (ADMHCl, MW 579.99) was purchased from Beijing Huafenglianbo Technology CO. LTD. D(þ)-Glucosamine hydrochloride (MW 164.16), succinic anhydride (SUC, MW 100.07), quercetin (MW 302.23), 1-ethyl-3-(3dimethylaminopropyl) carbodiimide hydrochloride (EDCI, MW 191.07), and N-hydroxy-succinimide (NHS, MW 115.08) were from Aladdin. All reagents and solvents were of analytic or HPLC grade and were used without further purification. RPMI-1640, 3-(4,5-dimethylthialzol-a-yl)-2,5-diphenyltetrazolium bromide (MTT), fetal bovine serum (FBS), penicillin, streptomycin, and trypsin-EDTA were purchased from commercial sources. Synthesis of 2DG–SUC–ADM Synthesis of 2-(3-carboxyl-1-oxopropyl) amino-2-dexoy-Dglucose (2DG–SUC). 2-(3-Carboxyl-1-oxopropyl)amino-2dexoy-D-glucose was synthesized as shown in Fig. 1A. Triethylamine (3.40 mL) and water (7.5 mL) were added in a flask containing D-(þ)-glucosamine hydrochloride (5.00 mg, 2.32 mmol). Acetone solution of succinic anhydride (SUC, 2.50 g) was subsequently added dropwise to the mixture and stirred for 4 hours. The product was collected by filtration, washed with ethanol and diethyl ether. The purified 2DG–SUC was dried in vacuum and obtained as white solid powder (4.68 g) with a 72% yield. Synthesis of 2DG–SUC–ADM. The complex 2DG–SUC– ADM was synthesized in 2 steps, as shown in Fig. 1A. First, 2DG–SUC (7.00 mg) was activated 3 hours by NHS (4.33 mg) and EDCI (6.98 mg) to obtain solution A. Second, ADMHCl (10.00 mg) was dissolved in anhydrous DMF (2.0 mL) at 37 C to form solution B. Afterward, solution B was added in solution A and the pH was adjusted to 7.5. The mixture was stirred for 48 hours in the dark. The product was obtained by lyophilization and purified by silica gel column chromatography. Characterization of 2DG–SUC–ADM Q-TOF Micro Mass Spectrometer (Waters) and Nuclear Magnetic Resonance Spectrometer (BRUKER) were used to confirm the successful synthesis of 2DG–SUC–ADM. UV-Vis absorption spectra of samples were acquired using a 754-PC spectrophotometer at room temperature. In vitro studies Cell culture. Tumor cells (MCF-7, Bel-7402, HepG2, MDAMB-231, U87MG, HELF, SKOV3, and S180) and normal cell HELF were purchased from American Type Culture Collection

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and cultured in RPMI-1640 medium supplemented with 10% (v/v) calf serum, penicillin (100 U/mL), and (100 mg/mL) streptomycin. Cells were maintained at 37 C in a humidified atmosphere containing 5% CO2. In vitro tumor-targeting ability of 2DG–SUC–ADM. Five tumor cell lines (MCF-7, HepG2, Bel-7402, MDA-MB-231, and SKOV3) were used for GLUT1 expression evaluation by reverse transcriptase PCR (sense: 50 -TGCTCATCAACCGCAACGA-30 , antisense: 50 -CACCACAAACAGCGACA CG-30 ). To determine the tumor-targeting ability of 2DG–SUC– ADM to tumor cells, the uptake of 2DG–SUC–ADM and ADM by tumor cells were compared on the mentioned different tumor cell lines. Briefly, different cells were respectively seeded in the confocal dishes and incubated for 24 hours. Then, cells were incubated in a 200-mL solution of 2DG–SUC–ADM or ADM for 0.5 hours. After washing with PBS, the cells were imaged by a laser confocal microscope (Olympus FV1100). To confirm the GLUT1 receptor mediation, in vitro receptor blocking experiments with free 2DG or a GLUT1-inhibitor, quercetin, were conducted on HepG2 cells. After cultured, the HepG2 cells at 37 C for 24 hours, 2DG (25 or 50 mmol/L) or quercetin (25 or 50 mmol/L) was preliminarily added to the cells for 30-minute incubation. Subsequently, 2DG–SUC–ADM was added to the dishes and cultured for another 30 minutes. After washing with PBS, the cells were imaged using laser confocal microscopy. In vitro therapeutic efficacy. Cell viability assays were carried out to evaluate the therapeutic efficacy of 2DG–SUC– ADM in cancer cells (MDA-MB-231 and HepG-2) and normal cells (HELF). ADM-resistance was evaluated in ADM-sensitive and ADM-resistant MCF-7 cells. After 24-hour cultivation, 2DG–SUC–ADM or free ADM of different concentrations (1.625, 3.25, 6.5, 13, 26, and 52 nmol/mL) were added into the cells of the 96 wells (n ¼ 6) and incubated for 48 hours. Then MTT solution (20 mL; 5 mg/mL) was added into each well. The absorbance of the solution in each well was measured at 570 nm with a multiwell plate reader. Animal experiments Animal models. All animal experiments were carried out in compliance with the Animal Management Rules of the Ministry of Health of the People's Republic of China. MCF-7, SKOV3, and S180 cells (5 106) were subcutaneously injected into the upper right axillary fossa in the nude mice or Kunming mice (Charles River Laboratories, n ¼ 10 per group). As the tumors grew up to a diameter of 0.3 0.4 cm, the mice were used for NIR imaging and treatment. In vivo dynamics and targeting ability of 2DG–ICG–Der01. To investigate the dynamic distribution and tumortargeting ability of 2DG–SUC–ADM in nude mice, we replaced the ADM in 2DG–SUC–ADM with an organic dye ICG-Der-01 to form 2DG–ICG–Der-01 for NIR imaging. The 2DG–ICG–Der-01 was intravenously administrated into the subject mice and the fluorescence images of the mice were acquired by our NIR imaging system (33, 34) at different time points postinjection. The tumor/normal tissue ratio (T/N ratio) was analyzed and compared by using the region of


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Figure 1. A, synthetic scheme of 2DG–SUC–ADM, through modification of the primary amine group of ADM with 2DG using succinic anhydride as reaction linkage. B, the absorption spectra of 2DG–SUC–ADM, free ADM, and 2DG. C, fluorescence emission spectra of 2DG–SUC–ADM, free ADM, and 2DG.

interests (ROI) function of the analysis. To confirm that the targeting ability of 2DG–ICG–Der-01 is attributed to the GLUT1 mediation, the in vitro and in vivo blocking experiments were conducted. First, in vitro receptor blocking experiments with free 2DG or GLUT1-inhibitor quercetin (35) were conducted in HepG2 tumor cells. The methods are the same as described earlier, except that the HepG2 cells were cultured in 6-well plates and the fluorescence images were acquired using a NIR imaging system, as our confocal microscope is not equipped with NIR fluorescence detector. For the in vivo blocking study, a mixture of 50 mg/kg 2DG (or 5 mg/kg quercetin) and 10 nmol/L 2DG–ICG-Der–01 was injected into the tumor-bearing mice (n ¼ 5) and the fluorescence images were acquired using the NIR imaging system. Data are expressed as means SD (n ¼ 5).

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In vivo antitumor efficacy of 2DG–SUC–ADM. Kunming mice-bearing S180 and nude mice-bearing SKOV3 tumors were randomly assigned into 3 groups (n ¼ 6 per group). The mice in each group were treated every other day for 10 days via tail vein injection with different solutions (0.2 mL): (A) Saline (control group); (B) ADM solution (6 mg/kg); (C) 2DG–SUC–ADM solution (containing ADM 6 mg/kg). The therapeutic efficacies and systematic toxicities of 2DG–SUC–ADM on these tumorbearing mice were assessed by measuring tumor volume and body weight of each mouse every day. Histology examination. To further investigate the side effects of 2DG–SUC–ADM on other organs, histology analysis of hearts and kidneys of the treated mice was conducted. The sliced organs were stained with hematoxylin and eosin and examined under a microscope.

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Statistical analysis Data were expressed as mean SD. Statistical analysis was conducted by using Students t test with statistical significance assigned for P value less than 0.05.

Results Identification and characterization of 2DG–SUC–ADM 2DG–SUC–ADM was synthesized following the procedure as illustrated in Fig. 1A. The absorption and fluorescence spectra of 2DG–SUC–ADM, free ADM, and 2DG were obtained. As shown in Fig. 1B, the absorption peaks of 2DG–SUC–ADM remained almost the same as that of free ADM after the conjugation of ADM and 2DG. As shown in Fig. 1C, an obvious blue shift of the maximum emission peak of 2DG–SUC–ADM (550 nm) was observed compared with the maximum emission peak of free ADM ( 600 nm), possibly due to the fact that the combination had increased the transition energy of the chromophores of ADM. The successful conjugation of 2DG and ADM was further evidenced by mass spectrum and nuclear magnetic resonance (NMR). Mass spectrum (Fig. 1D) indicates that the molecular


weight (MW) of 2DG–SUC–ADM is 804.1, whereas the calculated MW is 804 based on its molecular structure (C37H44N2O18). The peaks in the 1H NMR spectrum (D2O, 500 MHz) of 2DG–SUC–ADM (Fig. 1E) are assigned as follows. d 7.60 (1H, m), 7.32 (2H, m), 5.47 (1H, s), d 5.18 (1H, d, J ¼ 3.0 Hz), 4.85 (2H, s), 4.26 (1H, m), 3.88 (3H, s), 3.76 (1H, m), 3.90 to 3.44 (m, 6H), 2.92 (1H, d, J ¼ 17.9 Hz), 2.67 (t, 2H, J ¼ 6.6 Hz), 2.62 (t, 2H, J ¼ 7.4 Hz), 2.61 (1H, d, J ¼ 18.4 Hz), 2.71(2H, m), 2.07 (3H, m), and 1.34 (3H, d, J ¼ 6.5 Hz). In vitro tumor-targeting ability To assess the correlation between GLUT1 and the targeting ability of 2DG–SUC–ADM, tumor cells (MCF-7, HepG2, SKOV3, Bel-7402, and MDA-MB-231) with different GLUT1 expression levels were investigated (Fig. 2A and B). The GLUT1 expression on the different cancer cells was found to decrease in the following order: HepG2>MDA-MB231>Bel-7402>SKOV3>MCF-7. To investigate the tumor-targeting ability of 2DG–SUC– ADM, confocal images were acquired on MCF-7 cells incubated with free ADM or 2DG–SUC–ADM, as shown in Fig. 2, the


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fluorescence intensity from the cells incubated with 2DG– SUC–ADM is much higher than that of free ADM. In addition, the merged fluorescence images indicate that 2DG–SUC–ADM and ADM are mainly within the cytoplasm region rather than in nuclei after 5-minute incubation, which can be clearly observed by staining the cell nuclei with 40 ,6-diamidino-2phenylindole (DAPI). The enhanced cellular uptake of 2DG– SUC–ADM is a clear evidence of its tumor-targeting ability. The specificity of 2DG–SUC–ADM uptake via GLUTs was conducted and displayed in Fig. 2E–I. As shown in the figures, the uptake of 2DG–SUC–ADM was significantly inhibited by 2DG (25 or 50 mmol/L) or quercetin (25 or 50 mmol/L) in the HepG2 cells. The corresponding mean fluorescence intensity of the HepG2 cells incubated with 2DG–SUC–ADM in the absence and presence of 2DG or quercetin, as shown in Fig. 2J, further indicated that the uptake of 2DG–SUC–ADM was mostly mediated by GLUTs. In vitro antitumor activity of 2DG–SUC–ADM To evaluate the therapeutic efficacy of 2DG–SUC–ADM and its potential cytotoxicity, cell viability assays were carried out in cancer cells (MDA-MB-231 and HepG-2) and normal cells (HELF). The results in Fig. 3 and Supplementary Fig. S1A indicated that 2DG–SUC–ADM effectively reduced the viability of cancer cells while having little cytotoxicity in normal cells compared with free ADM. In addition, free 2DG ligand had no negative effect on cell growth in either cancerous or normal cells. As shown in Fig. 3A and B, 2DG–SUC–ADM had a higher therapeutic efficacy for cancer cells than free ADM. Supplementary Fig. S1A shows that there is a significantly decrease in cell viability of ADM-treated HELF cells, due to the high cytotoxicity of nontargeting ADM. While, 2DG–SUC–ADM has negligible cytotoxicity in HELF normal cells. Moreover, we used 2DG–SUC–ADM and free ADM to treat ADM-sensitive MCF-7 cells and ADM-resistant MCF-7 cells (MCF-7/ADR) to investigate drug resistance. Figure 3C shows that 2DG–SUC– ADM and free ADM have similar antitumor effect on the ADMsensitive MCF-7 cells. Significant drug resistance was observed in MCF-7/ADR cells treated by free ADM, as shown in Fig. 3D. However, 2DG–SUC–ADM showed high antitumor efficacy on MCF-7/ADR cells, indicating that 2DG–SUC–ADM has the potential to reverse overcome ADM-resistance in drug-resistant MCF-7 cell lines. Figure 4 displays the cellular uptake of 2DG–SUC–ADM or free ADM in HepG2 cells at different time points. After incubation with each drug for 5 minute, fluorescence emission of ADM was observed, not only from the cytoplasm but also from cell nuclei. Owing to the tumor-targeting ability of 2DG ligand to GLUT1, 2DG–SUC–ADM is first accumulated in the cell membrane and is subsequently transported inside cells, mainly located in the cytoplasm at 5-minute incubation and gradually entered into the nuclei at 10 minutes. Red emission of 2DG–SUC–ADM was brighter than that of free ADM from the cells at the same time point. After incubation for 10 minutes there are significant morphologic differences between 2DG–SUC–ADM and free ADM-treated cells. The morphologic characteristics of apoptosis cells, such as cell shrinkage, nuclear condensation, and fragmentation were

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observed after the cells were incubated with 2DG–SUC– ADM for 10 minutes. Similar results were observed in other cell lines (Bel-7402, MDA-MB-231, and MCF-7) after incubation with ADM and 2DG–SUC–ADM (Supplementary Fig. S1B–S1D). Figure 4B shows the mean fluorescence intensities of 2DG–SUC–ADM and ADM in various tumor cells detected at different time points, with obvious higher fluorescence in the 2DG–SUC–ADM–treated cells than that of ADM treated. The mean fluorescence emission intensities of 2DG–SUC–ADM–treated cells follows the order of HepG2>MDA-MB-231Bel7402>MCF7, which is consistent with the GLUT1 expression (Fig. 2A and B). Moreover, tumor cells were dead after treatment with 2DG–SUC–ADM for 20 minutes and the fluorescence emission from the cells disappeared. However, the mean fluorescence emission from different tumor cells treated by free ADM gradually increased and then disappeared after 30-minute incubation. To assess the antitumor efficacy and antidrug resistance of 2DG–SUC–ADM, MCF-7/ADR cells were treated with 2DG– SUC–ADM or free ADM. As shown in Fig. 5A, emission from MCF-7/ADR cells incubated with 2DG–SUC–ADM was observed, primarily due to the tumor-targeting capacity of the prodrug with the 2DG ligand. The fluorescence emission from 2DG–SUC–ADM–treated cells (both ADM sensitive and ADM resistant) was much higher than that from free ADM-treated cells (Fig. 5B), which suggests that 2DG–SUC–ADM has the potential to prevent overcome ADM-resistance in treating multidrug-resistant tumor cells. On the basis of cell viability assays, 2DG–SUC–ADM has prominent antitumor effect on MCF-7 cells at the dose of 12 mmol/L. HELF cells were treated with the same concentration of the prodrug to investigate its potential cytotoxicity to normal cells. Supplementary Fig. S1E shows no obvious morphologic changes in the HELF cells. However, MCF-7 cells with the same treatment showed significant characteristics of apoptosis (Fig. 5C). These results show that 2DG–SUC–ADM has a negligible side effect on normal cells and a high specificity for tumor cells. In vivo dynamic distribution and tumor-targeting ability of 2DG–ICG–Der-01 Before studying in vivo tumor-targeting ability of 2DG, we verified that 2DG–ICG–Der-01 was transported into cells in the same manner as 2DG–SUC–ADM using in vitro receptor blocking experiments. Results shown in Fig. 6A and B showed that the fluorescence intensity in cells incubated with 2DG– ICG–Der-01 with blocking dose of 2DG or quercetin were much weaker than that of 2DG–ICG–Der-01 without blocking substance, which confirmed that the uptake of 2DG–ICG–Der-01 was mediated by GLUT. In vivo dynamic processes of 2DG–ICG–Der-01 were shown in Supplementary Fig. S2A. 2DG–ICG–Der-01 was quickly spread in the whole body at about 30-minute postinjection and accumulated in the liver at 6-hour postinjection. After 24 hours, it was mostly cleared from the body. To investigate the tumor-targeting ability of 2DG ligand, nude mice-bearing MCF-7/estradiol or U87MG tumors that overexpress GLUT1 were used. Fluorescence images at different time points after

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Figure 2. A, GLUT1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expressions in MCF-7, SKOV3, MDA-MB-231, HepG2, and Bel7402 cancer cells. B, quantitative analysis of GLUT1/GAPDH in MCF7, SKOV3, MDA-MB-231, HepG2, and Bel-7402 cancer cells. C and D, laser confocal fluorescence microscopy images of MCF-7 cells, incubated at 37 C for 0.5 hours with free ADM (C) and 2DG–SUC–ADM (D); the cells were counterstained with DAPI for the cell nucleus. Bar, 20 mm. E–I, laser confocal fluorescence microscopy images of HepG2 cells, incubated at 37 C with 2DG–SUC–ADM in the absence (E) and presence of 25 mmol/L (F) and 50 mmol/L (G) of 2DG or 25 mmol/L (H) and 50 mmol/L (I) of quercetin. Bar, 30 mm. J, mean fluorescence intensity of HepG2 cells, incubated with 2DG–SUC–ADM in the absence or presence of 2DG or quercetin. Data are given as mean SD (n ¼ 5). , P < 0.05. DIC, differential interference contrast.

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administration of 2DG–ICG–Der-01 are shown in Supplementary Fig. S2B and Fig. 6C. 2DG–ICG–Der-01 initially was spread in the whole animal after 30 minutes. The tumor sites were identifiable at 2-hour postinjection and the probe was gradually accumulated in the tumor sites. Strong fluorescence emission from the tumors was observed even at 48-h postinjection. To further confirm the in vivo tumor-targeting ability of 2DG–SUC–ADM, block experiments were conducted using nude mice-bearing HepG2 tumors. As shown in Fig. 6C3 and C4, free 2DG or quercetin successfully reduced tumor accumulation of 2DG–ICG–Der-01. Tumor contrast as quantified by ROI analysis of optical imaging shown in Fig. 6D indicated that the Tumor/Normal Tissue ratio was reduced from 3.8 0.27 to 1.72 0.14 and 2.03 0.20 for 2DG–ICG–Der-01 with free 2DG and quercetin, respectively. In vivo antitumor therapeutic efficacy of 2DG–SUC–ADM In vivo antitumor efficacy of 2DG–SUC–ADM was evaluated in mice-bearing S180 and SKOV3 tumors by measuring the tumor growth rate and the body weight of the mice. As shown


in Fig. 7A and D, the tumors of saline-treated mice grew faster than that of the free ADM or 2DG–SUC–ADM–treated mice. It is noted that the growth of S180 tumor is inhibited with a rate of 64.6% after the administration of 2DG–SUC–ADM, which is higher than that of free ADM (about 50%). The inhibition rate of 2DG–SUC–ADM (68.8%)-treated SKOV3 tumor is about 21% higher than that of ADM-treated tumor (47.1%). Furthermore, the body weight of mice-bearing S180 tumors in 2DG–SUC–ADM and saline-treated groups gradually increased during the treatment period (Fig. 7B). However, a significant decrease of body weight (10 g) in the free ADMtreated group was observed at the end of the treatment period. Body weight of ADM-treated mice-bearing SKOV3 tumors reduced dramatically and all mice in this group died within 17 days (Fig. 7E), whereas 2DG–SUC–ADM–treated mice only slightly reduced. The 22-day survival rates of mice in the 2DG– SUC–ADM and free ADM groups were 67%, whereas the survival rate of the control group drastically decreased to 10% (Fig. 7C). Heart and kidney are the main organs affected by free ADM. To evaluate the toxicity of 2DG–SUC–ADM, hearts (n ¼ 7) and


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Figure 3. In vitro antitumor efficacy and cytotoxicity of 2DG–SUC– ADM. Cell viability of MDA-MB231 cells (A) and HepG-2 cells (B) incubated with either free ADM or 2DG–SUC–ADM. Cell viability of 2DG–SUC–ADM and ADM-treated ADM-sensitive MCF-7 cells (C) and ADM-resistant MCF-7 cells (D). Data are given as mean SD (n ¼ 5).

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kidneys (n ¼ 14) from mice in each group were collected at the fourth day and histologic examination was conducted. Compared with the control group, no distinct pathologic changes were found in the hearts and kidneys of the mice injected with

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Figure 4. A, fluorescence images of HepG2 cells after incubation with 2DG–SUC–ADM or free ADM at different time points. B, mean fluorescence emission intensities of ADM or 2DG–SUC–ADM at different time points from different tumor cells: Bel-7402, HepG2, MDA-MB-231, and MCF-7 cells. Bar, 20 mm. DIC, differential interference contrast microscopy.

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Figure 5. A, fluorescence images of MCF-7/ADR cells incubated with free ADM or 2DG–SUC–ADM. B, fluorescence intensity analysis of MCF-7 and MCF-7/ADR cells after incubation with 2DG–SUC–ADM or ADM. C, cytotoxicity of 2DG–SUC–ADM–treated breast cancer MCF-7 cells. Bar, 30 mm.

Histologic examination on hearts and kidneys from mice treated by 2DG–SUC–ADM showed negligible side effects. These results indicate that although the survival rates of 2DG–SUC–ADM and free ADM groups are the same, the toxicity and drug resistance of 2DG–SUC–ADM is far superior to free ADM.

Discussion Despite the hope and promises of ADM as an effective anthracycline for cancer therapy, serious drawbacks hamper ADM's clinical usefulness. In particular, its primary amine group has induced irreversible cardiotoxicity. Modification to this primary amine group has been reported to reduce its toxicity on cardiovasculature (36). In addition, multidrug resistance induced by frequent use of ADM is another problem that significantly limits its clinical application. To overcome these obstacles, we developed a new active receptor-mediated complex, 2DG–SUC–ADM, which modified the primary amine


group with 2DG using SUC as reaction linkage. The synthesized complex in this study exhibited significantly high tumor-targeting capability, high anticancer efficiency, as well as reduced ADM resistance. The successful synthesis of 2DG–SUC–ADM was confirmed by the absorption and fluorescence spectra, mass spectrum, NMR, and FTIR (Fig. 1B–E). Previously, studies have shown that because 2DG lacks a hydroxyl group in the 2 position, after transported into the cells, subsequent isomerization by the next enzyme in the metabolic pathway is precluded, thereby inhibiting further metabolism of the 2deoxyglucose-6-phosphate and leading to intracellular retention of the phosphorylated molecule. Our study has shown that 2DG-based probes had high selectivity as well as high accumulation and retention rates in tumors (37), with the targeting ability corresponding to the GLUT1 expression levels in cancer cells (Figs. 2A and B and 4B). In vitro tumortargeting ability of 2DG–SUC–ADM was studied by fluorescence imaging of MCF-7 cells (Fig. 2C and D) and HELF cells (data not shown) incubated with free ADM or 2DG–SUC– ADM. The results using MCF-7 cells showed that 2DG–SUC– ADM was translocated into the cytoplasm after incubation of 5 minutes, whereas only a very low level of free ADM was observed inside the cells. In contrast, the uptake of ADM in HELF cells was higher than that of 2DG–SUC–ADM, due to the fact that the uptake of free ADM was mainly through molecular diffusion but the uptake of 2DG–SUC–ADM was mostly mediated by GLUT1. In vitro blocking and inhibition results using 2DG or quercetin (Fig. 2E–I), showed successful inhibition of 2DG–SUC–ADM uptake, confirmed the uptake of 2DG–SUC–ADM was through GLUTs. These results further support that the GLUT1 is involved in the accelerated glucose uptake in tumors. It could be an important player in several stages of cancer progression. The high tumor-targeting ability of the 2DG-based complex has also been shown in tumor-bearing mice (Fig. 6). In vitro and in vivo targeting experiments indicated that 2DG-based complex had great potential for targeted cancer therapy. The toxic side effect of ADM is an obstacle in clinical applications. In this study, MTT assay was conducted to evaluate the therapeutic efficacy of ADM in the 2DG-based complex on cancer cells and its potential toxicity to normal cells. The results indicate that 2DG–SUC–ADM has higher therapeutic efficacy against cancer cells than free ADM (Fig. 3), showing the selective toxicity of our novel complex construct. No obvious toxicity to the normal cell (HELF) was observed. In contrast, ADM showed high cytotoxicity to HELF cells due to its nontargeting nature. Corresponding to the MTT results, the confocal images of HepG2 cells (Fig. 4) show that the cell membrane became round and shed off, indicating the inhibition of HepG2 cancer cells by 2DG–SUC–ADM. On the contrary, the morphology of HELF cells had no obvious change under 2DG–SUC–ADM treatment, further confirming that the complex is relatively nontoxic to normal cells. Moreover, histologic studies on animal hearts and kidneys showed that the 2DG–SUC–ADM did not induce noticeable toxicity, as shown in Fig. 7F. The histologic results are consistent with our in vitro results.


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Figure 6. A, fluorescence images of HepG2 cells incubated in 6-well plates with 2DG–ICG–Der-01 in the absence or presence of 25 and 50 mmol/L of 2DG or 25 and 50 mmol/L of quercetin. The images were acquired using a NIR imaging system. B, mean fluorescence intensity of HepG2 cells incubated with 2DG–SUC– ICG-Der-01 in the absence or presence of 2DG or quercetin. C, dynamics and tumor-targeting ability of ICG-Der-01 and 2DG–ICG–Der-01 in nude mice. C1 and C2, fluorescence images of nude mice-bearing U87MG tumors after the administration of ICG–Der-01 or 2DG–ICG–Der-01 within 48 hours. C3 and C4, fluorescence images of nude mice-bearing HepG2 tumors after the injection of 2DG–ICG–Der-01 with blocking dose of 2DG or quercetin. D, tumor/normal tissue ratio (T/N ratio ¼ [tumor signal background signal]/[normal signal (muscle) background signal] 100%) calculated from the ROIs at 4-hour postinjection of ICG–Der-01 or 2DG–ICG–Der-01 with and without blocking dose of 2DG or quercetin. Data are given as mean SD (n ¼ 5). , P < 0.05.

Multidrug resistance of ADM is another key issue that hampers its clinical application. We investigated the inhibition rates of multidrug-resistant MCF-7/ADR cells and drug-sensitive MCF-7 cells using 2DG–SUC–ADM. Results in Fig. 3C and D showed that 2DG–SUC–ADM inhibited cell growth in both cell lines, with higher anticancer efficiency than free ADM. After being modified with 2DG, the complex has targeted the tumor cells and to a certain degree escaped the effluxing of P-glycoprotein, which is the molecular alteration found to be most consistently associated with the MDR phenotype and has been correlated with the degree of resistance (38). Thus, special conjugation of ADM to 2DG resulted in its increased anticancer efficiency. Confocal

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images of MCF-7/ADR and drug-sensitive MCF-7 cells incubated with free ADM or 2DG–SUC–ADM show a higher level of 2DG–SUC–ADM uptake than that of free ADM after the same incubation time (Fig. 5A and B), which confirmed the reduction of drug resistance by 2DG–SUC–ADM. It should be noted that despite the fact that the mean fluorescence emission intensity of 2DG–SUC–ADM in MCF-7/ADR cells is much lower than that in drug-sensitive MCF-7 cells (Fig. 5A and B), the intensity ratio of 2DG–SUC–ADM to free ADM is much higher in MCF-7/ADR cells (9.6) than that in MCF-7 cells (6.2). These results have shown that 2DG–SUC–ADM had the potential to prevent ADM resistance in treating multidrug-resistant tumor cells.

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Figure 7. Therapeutic efficacy of 2DG–SUC–ADM in treating Kunming mice-bearing S180 tumors or nude mice-bearing SKOV3 tumors. A, tumor volumes of micebearing S180 tumors under different treatments (saline, free ADM, or 2DG–SUC–ADM, n ¼ 6/group). B, body weights of mice-bearing S180 tumors in different groups. C, the 22day survival rates of mice after administration of saline, free ADM, or 2DG–SUC–ADM. D, tumor volumes of mice-bearing SKOV3 tumors under different treatments (n ¼ 6/group). E, body weights of mice-bearing SKOV3 tumors in different groups. F, hematoxylin and eosin-stained hearts and kidneys of saline-treated mice, ADM-treated mice, or 2DG–SUC–ADM–treated mice. Data are given as mean SD (n ¼ 5). , P < 0.05.

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The antitumor efficiency of 2DG–SUC–ADM was confirmed both in vitro and in vivo. In vitro study has shown that 2DG– SUC–ADM had a higher tumor cell inhibition rate compared with the free ADM. The cellular uptake and apoptosis-inducing effect of 2DG–SUC–ADM was conducted by confocal microscopy. After 5-minute incubation, 2DG–SUC–ADM was mostly accumulated on the cell membrane, and the fluorescence emission intensity was much higher than that of ADM (Fig. 4B). However, after 10-minute incubation, all the complex was translocated to the cell nuclei, resulting in cell apoptosis (Fig. 4A and Supplementary Fig. S1B–S1D). The impressive therapeutic efficacy of the complex may be due to the fact that 2DG– SUC–ADM was able to deliver more drugs to the tumor site by glucose uptake. However, the timing of apoptosis of different cancer cells varied, mainly due to different expressions of GLUT1. The in vivo study indicated that the anticancer effect of 2DG–SUC–ADM was significantly greater than that of free ADM. Furthermore, mice treated with 2DG–SUC–ADM showed an increase in body weight, whereas mice treated with free ADM exhibited a significant weight loss, as shown in (Fig. 7B and D). It should be noted that there is a significant difference of body weight change between mice-bearing S180 tumors and SKOV3 tumors, especially in the ADM group. It could be due to the differences in the animal types. The mice


in the SKOV3 group were nude mice with immunodeficiency, which may have less resistance to toxic drug compared with the immune competent Kunming mice in the S180 group. Besides, GLUT1 expression in SKOV3 nude mice was higher than that in S180 mice, and the treatment outcomes were better in SKOV3 mice, which corresponded to the changes of tumor volume in Fig. 7A and D. The body weight change may also be correlated with other reasons such as age, which need to be further investigated. These results have shown that the 2DG–SUC–ADM greatly reduced the systemic toxicity and enhanced the antitumor activity of ADM. In this article, we have conducted in vitro and in vivo studies of 2DG–SUC–ADM anticancer capability to show the proof of concept. Before applying this compound for human trials, numerous experiments must be carried out. For instance, we need to investigate the formulation of this compound for appropriate administration and to further study the pharmacokinetics in different animals. We also need in the future to conjugate this novel compound with specific antibodies to improve its targeting efficiency. Dose limitation, protein binding, and other related issues will also need to be investigated.

Conclusion 2DG–SUC–ADM increased tumor-targeting capability of ADM through glucose uptake. It had much lower toxicity to


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normal cells, and a potential reversal of multidrug resistance in cancer cells. In vitro and in vivo studies have shown that 2DG– SUC–ADM induced a higher level of cancer cell apoptosis and higher inhibition rates in MCF-7 and HepG2 tumors than free ADM. Our results indicated that the complex could significantly decrease toxicity to normal cells and vital organs compared with free ADM. The 2DG–SUC–ADM could become a promising drug for targeted cancer therapy. Disclosure of Potential Conflicts of Interest The authors declare that they have no conflict of interest.

Authors' Contributions Conception and design: Y. Gu Development of methodology: S. Li Writing, review, and/or revision of the manuscript: J. Cao, S. Cui, W.R. Chen Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): C. Du, J. Tian, S. Wan

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): W.R. Chen Study supervision: Z. Qian

Acknowledgments The authors thank the support of the US Fulbright Scholarship for the 2011 to 2012 academic year.

Grant Support

This work was financially supported by the National Natural Science Foundation of China (nos. 30970776, 31050110123, 81071194, 81000666, 81171395, and 81220108012), the Graduate Innovation Project of Jiangsu Province (CXZZ11_0816 and CXLX11_0795), and Program for the Doctoral Tip-top Innovative Talents of China Pharmaceutical University (2011-BPY05). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received May 31, 2012; revised October 25, 2012; accepted November 14, 2012; published OnlineFirst February 8, 2013.

References 1. 2. 3. 4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14. 15. 16.

1372

Emmenegger U, Kerbel RS. Cancer: chemotherapy counteracted. Nature 2010;468:637–8. Bildstein L, Dubernet C, Couvreur P. Prodrug-based intracellular delivery of anticancer agents. Adv Drug Deliv Rev 2011;63:3–23. Nussbaumer S, Bonnabry P, Veuthey JL, Fleury-Souverain S. Analysis of anticancer drugs: a review. Talanta 2011;85:2265–89. Perrino C, Schiattarella GG, Magliulo F, IIardi F, Carotenuto G, Gargiulo G, et al. Cardiac side effects of chemotherapy: state of art and strategies for a correct management. Curr Vasc Pharmacol. 2012 May 8. PMID:22563720. Kim JH, Sung NY, Raghavendran H, Yoon Y, Song BS, Choi J. Gamma irradiation reduces the immunological toxicity of doxorubicin, anticancer drug. Radiat Phys Chem 2009;78:425–28. Raschi E, Vasina V, Ursino MG, Boriani G, Martoni A, De Ponti F. Anticancer drugs and cardiotoxicity: insights and perspectives in the era of targeted therapy. Pharmacol Ther 2010;125:196–218. Pathania D, Millard M, Neamati N. Opportunities in discovery and delivery of anticancer drugs targeting mitochondria and cancer cell metabolism. Adv Drug Deliv Rev 2009;61:1250–75. Kievit FM, Wang FY, Fang C, Mok H, Wang K, Silber JR, et al. Doxorubicin loaded iron oxide nanoparticles overcome multidrug resistance in cancer in vitro. J Control Release 2011;152:76–83. Zhang H, Li F, Yi J, Gu C, Fan L, Qiao Y, et al. Folate-decorated maleilated pullulan-doxorubicin conjugate for active tumor-targeted drug delivery. Eur J Pharm Sci 2011;42:517–26. Zheng C, Xu J, Yao X, Qiu L. Polyphosphazene nanoparticles for cytoplasmic release of doxorubicin with improved cytotoxicity against Dox-resistant tumor cells. J Colloid Interface Sci 2011;355:374–82. Cho HJ, Yoon IS, Yoon HY, Koo H, Jin YJ, Ko SH, et al. Polyethylene glycol-conjugated hyaluronic acid-ceramide self-assembled nanoparticles for targeted delivery of doxorubicin. Biomaterials 2012;33: 1190–200. Ogawara K, Un K, Tanaka K, Higaki K, Kimura T. In vivo anti-tumor effect of PEG liposomal doxorubicin (DOX) in DOX-resistant tumorbearing mice: involvement of cytotoxic effect on vascular endothelial cells. J Control Release 2009;133:4–10. Hu Z, Jiang X, Albright CF, Graciani N, Yue E, Zhang M, et al. Discovery of matrix metalloproteases selective and activated peptide-doxorubicin prodrugs as anti-tumor agents. Bioorg Med Chem Lett 2010; 20:853–6. Mahato R, Tai W, Cheng K. Prodrugs for improving tumor targetability and efficiency. Adv Drug Deliv Rev 2011;63:659–70. Bono JS, Ashworth A. Translating cancer research into targeted therapeutics. Nature 2010;467:543–9. Stepankova J, Studenovsky M, Malina J, Kasparkova J, Liskova B, Novakova O, et al. DNA interactions of 2-pyrrolinodoxorubicin, a


17. 18.

19. 20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

distinctively more potent daunosamine-modified analogue of doxorubicin. Biochem Pharmacol 2011;82:227–35. Warburg O. On the origin of cancer cells. Science 1956;123:309–14. Ganapathy V, Thangaraju M, Prasad PD. Nutrient transporters in cancer: relevance to Warburg hypothesis and beyond. Pharmacol Ther 2009;121:29–40. Hediger MA, Rhoads DB. Molecular physiology of sodium-glucose cotransporters. Physiol Rev 1994;74:993–1026. Amann T, Maegdefrau U, Hartmann A, Agaimy A, Marienhagen J, Weiss TS, et al. GLUT1 expression is increased in hepatocellular carcinoma and promotes tumorigenesis. Am J Pathol 2009;174: 1544–52. Simons AL, Ahmad IM, Mattson DM, Dornfeld KJ, Spitz DR. 2-DeoxyD-glucose combined with cisplatin enhances cytotoxicity via metabolic oxidative stress in human head and neck cancer cells. Cancer Res 2007;67:3364–70. Mohanti BK, Rath GK, Anantha N, Kannan V, Das BS, Chandramouli BA, et al. Improving cancer radiotherapy with 2-deoxy-D-glucose: phase I/II clinical trials on human cerebral gliomas. Int J Radiat Oncol Biol Phys 1996;35:103–11. Simons AL, Fath MA, Mattson DM, Smith BJ, Walsh SA, Graham MM, et al. Enhanced response of human head and neck cancer xenograft tumors to cisplatin combined with 2-deoxy-D-glucose correlates with increased 18F-FDG uptake as determined by PET imaging. Int J Radiat Oncol Biol Phys 2007;69:1222–30. Maschek G, Savaraj N, Priebe W, Braunschweiger P, Hamilton K, Tidmarsh GF, et al. 2-Deoxy-D-glucose increases the efficacy of adriamycin and paclitaxel in human osteosarcoma and non–small cell lung cancers in vivo. Cancer Res 2004;64:31–4. Gallagher BM, Fowler JS, Gutterson NI, MacGregor RR, Wan CN, Wolf AP. Metabolic trapping as a principle of oradiopharmaceutical design: some factors resposible for the biodistribution of [18F] 2-deoxy-2fluoro-D-glucose. J Nucl Med 1978;19:1154–61. Yalcin A, Telang S, Clem B, Chesney J. Regulation of glucose metabolism by 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases in cancer. Exp Mol Pathol 2009;86:174–9. Song YS, Lee WW, Chung JH, Park SY, Kim YK, Kim SE. Correlation between FDG uptake and glucose transporter type 1 expression in neuroendocrine tumors of the lung. Lung Cancer 2008;61:54–60. van Baardwijk A, Dooms C, van Suylen RJ, Verbeken E, Hochstenbag M, Dehing-Oberije C, et al. The maximum uptake of 18F-deoxyglucose on positron emission tomography scan correlates with survival, hypoxia inducible factor-1alpha and GLUT-1 in non–small cell lung cancer. Eur J Cancer 2007;43:1392–8. Suzuki H, Fukuyama R, Hasegawa Y, Tamaki T, Nishio M, Nakashima T, et al. Tumor thickness, depth of invasion, and Bcl-2 expression are

Cancer Research


30.

31.

32.

33.

correlated with FDG-uptake in oral squamous cell carcinomas. Oral Oncol 2009;45:891–7. Daumar P, Decombat C, Chezal JM, Debiton E, Madesclaire M, Coudert P, et al. Design, synthesis and in vitro drug release investigation of new potential 5-FU prodrugs. Eur J Med Chem 2011;46: 2867–79. Reux B, Weber V, Galmier MJ, Borel M, Madesclaire M, Madelmont JC, et al. Synthesis and cytotoxic properties of new fluorodeoxyglucosecoupled chlorambucil derivatives. Bioorg Med Chem 2008;16: 5004–20. Miot-Noirault E, Reux B, Debiton E, Madelmont JC, Chezal JM, Coudert P, et al. Preclinical investigation of tolerance and antitumour activity of new fluorodeoxyglucose-coupled chlorambucil alkylating agents. Invest New Drugs 2011;29:424–33. Zhang J, Chen H, Xu L, Gu Y. The targeted behavior of thermally responsive nanohydrogel evaluated by NIR system in mouse model. J Control Release 2008;131:34–40.


34. Shan L, Cui S, Du C, Wan S, Qian Z, Achilefu S, et al. A paclitaxelconjugated adenovirus vector for targeted drug delivery for tumor therapy. Biomaterials 2012;33:146–62. squez FV, Rivas CI, Zhang RH, Strobel P, et al. 35. Vera JC, Reyes AM, Vela Direct inhibition of the hexose transporter GLUT1 by tyrosine kinase inhibitors. Biochemistry 2001;40:777–90. 36. Tang XH, Xie P, Ding Y, Chu LY, Hou JP, Yang JL, et al. Synthesis, characterization, and in vitro and in vivo evaluation of a novel pectinadriamycin conjugate. Bioorg Med Chem 2010;18:1599–609. 37. Guo J, Du C, Shan L, Zhu H, Xue B, Qian Z, et al. Comparison of near-infrared fluorescent deoxyglucose probes with different dyes for tumor diagnosis in vivo. Contrast Media Mol Imaging 2012; 7:289–301. 38. Wang F, Wang YC, Dou S, Xiong MH, Sun TM, Wang J. Doxorubicintethered reponsive gold nanoparticles facilitate intracellular drug delivery for overcoming multidrug resistance in cancer cells. ACS Nano 2011;5:3679–92.


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