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α1-Acid Glycoprotein Has the Potential to Serve as a Biomimetic Drug Delivery Carrier for Anticancer Agents
Journal of Pharmaceutical Sciences 108 (2019) 3592-3598
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Pharmaceutics, Drug Delivery and Pharmaceutical Technology
a1-Acid Glycoprotein Has the Potential to Serve as a Biomimetic Drug Delivery Carrier for Anticancer Agents Kotaro Matsusaka 1, Yu Ishima 2, Hitoshi Maeda 1, Ryo Kinoshita 1, Shota Ichimizu 1, Kazuaki Taguchi 3, Victor Tuan Giam Chuang 4, Koji Nishi 5, Keishi Yamasaki 5, Masaki Otagiri 5, Hiroshi Watanabe 1, Toru Maruyama 1, * 1
Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan Department of Pharmacokinetics and Biopharmaceutics, Institute of Biomedical Sciences, Tokushima University, 1-78-1, Sho-machi, Tokushima 770-8505, Japan 3 Division of Pharmacodynamics, Keio University Faculty of Pharmacy, 1-5-30, Shibakoen, Minato-ku, Tokyo 105-8512, Japan 4 School of Pharmacy, Monash University Malaysia, Jalan Lagoon Selatan, 47500 Bandar Sunway, Selangor, Malaysia 5 Faculty of Pharmaceutical Sciences, Sojo University, 1-22-4 Ikeda, Nishi-ku, Kumamoto 860-0082, Japan 2
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 May 2019 Revised 25 June 2019 Accepted 2 July 2019 Available online 6 July 2019
Nanosize plasma proteins could be used as a biomimetic drug delivery system (DDS) for cancer treatment when loaded with anticancer drugs based on the fact that plasma proteins can serve as a source of nutrients for cancer cells. This prompted us to investigate the potential of a1-acid glycoprotein (AGP) for this role because it is a nanosize plasma protein and binds a variety of anticancer agents. Pharmacokinetic analyses indicated that AGP is distributed more extensively in tumor tissue than human serum albumin, which was already established as a cancer DDS carrier. AGP is possibly being incorporated into tumor cells via endocytosis pathways. Moreover, a synthetic AGP-derived peptide which possesses a high ability to form an a-helix, as deduced from the primary structure of AGP, was also taken up by the tumor cells. AGP loaded with anticancer agents, such as paclitaxel or nitric oxide, efficiently induced tumor cell death. These results suggest that AGP has the potential to be a novel DDS carrier for anticancer agents. © 2019 Published by Elsevier Inc. on behalf of the American Pharmacists Association.
Keywords: a1-acid glycoprotein orosomucoid DDS carrier cancer anticancer agents
Introduction Cancer treatment has made impressive progress with the development of molecular targeted drugs and therapeutic antibodies in recent years.1 However, an effective treatment for refractory cancers such as metastasis, recurrence, and resistance to treatment continue to be a substantial challenge.2,3 A nanosized drug delivery system (DDS) with organ-specific targeting ability has the potential to overcome such limitations in the field of cancer therapy.4-7 The enhanced permeability and retention (EPR) effect, in which molecules with molecular weights in excess of 40-50 kDa selectively accumulate in tumor tissues to a much greater extent than in normal tissue, is a principle concept for the development of nanosized anticancer therapeutics.8,9 Nutrients play an especially important and essential role in supporting the uncontrolled tumor growth, metastasis, and resistance to therapy.10,11 Plasma proteins have recently received
* Correspondence to: Toru Maruyama (Telephone: þ81-96-371-4150). E-mail address: [email protected] (T. Maruyama).
considerable attention as a source of cancer nutrients, given the fact that they are actively taken up by tumor cells via the endocytic pathway and are then intracellularly digested into basic units that can be utilized as nutrients.12 A typical example is human serum albumin (HSA),13 and we refer this uptake phenomenon by cancer cells as an “endogenous albumin transport system”. We hypothesize that an anticancer drug-loaded nanosized plasma protein may serve as a biomimetic DDS biomaterial for cancer treatment.14 The most successful example of this strategy is paclitaxel-bound HSA (nab-paclitaxel; Abraxane) that has already been approved in many countries and has been used in the treatment of various types of refractory tumors, such as breast cancer, gastric cancer, nonesmallcell lung cancer, and pancreatic cancer.14-16 Human a1-acid glycoprotein (AGP) is an acute phase plasma glycoprotein, and its secondary structure is mainly composed of bsheets.17,18 The molecular weight of AGP is 44 kDa, which is within the molecular size range for a compound to undergo EPR effect. AGP, along with HSA, functions as a drug carrier in plasma.19 Many anticancer drugs bind to AGP with high affinities.20 On the other hand, AGP administered to sarcoma-bearing rats has been found to accumulate in the tumor tissue rather than other organs.21 These
https://doi.org/10.1016/j.xphs.2019.07.002 0022-3549/© 2019 Published by Elsevier Inc. on behalf of the American Pharmacists Association.
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findings suggest that AGP has the potential to function as a new nanosized DDS carrier for anticancer drugs. In this study, the pharmacokinetics of AGP in tumor-bearing mice were investigated before evaluation for the cell-penetrating ability of AGP using 4 different types of tumor cells. Finally, the antitumor activity of AGP loaded with anticancer agents such as paclitaxel and nitric oxide (NO) was examined by using an in vitro cell system. Materials and Methods Materials AGP was a donation from the Chemo-Sero-Therapeutic Research Institute (Kumamoto, Japan). HSA was purchased from the ChemoSero-Therapeutic Research Institute and defatted according to a previously reported method.22 VivoTag-S® 750 and Na125I were purchased from Perkin Elmer Cetus (Norwalk, CT). Fluorescein isothiocyanate (FITC) and 5-(N-ethyl-N-isopropyl) amiloride (EIPA) were purchased from Sigma-Aldrich (St. Louis, MO). N-1Naphthylethylenediamine dihydrochloride, sulfanilamide, HgCl2, and NaNO2 were purchased from Nacalai Tesque (Kyoto, Japan). Chlorpromazine (CPZ), methyl-b-cyclodextrin (Met-b-CyD), and isoamyl nitrite were purchased from Wako Chemicals (Osaka, Japan). Hoechst 33,342 and diethylenetriaminepentaacetic acid (DTPA) and the cell counting kit-8 (WST-8) were purchased from Dojindo Laboratories (Kumamoto, Japan). The FITC-peptide (its amino acid position in AGP; 135-148) was purchased from SCRUM Inc. (Tokyo, Japan). Paclitaxel was purchased from MedChemExpress (Shanghai, China). Traut’s reagent (2-imminothiolane) was purchased from Pierce Chemical Company (Rockford, IL). 4-Amino5-methylamino2’,7’-difluorofluorescein diacetate (DAF-FM DA) was purchased from Sekisui Medical Company, Ltd. (Tokyo, Japan). Cell Cultures Human breast cancer MCF-7 cells and human cervical cancer HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% heat-inactivated fetal bovine serum, 100 U/ mL penicillin, and 100 mg/mL streptomycin. Human liver cancer HepG2 cells and murine colon cancer C26 cells were cultured in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin. A subculture was performed at 3-4 days intervals. Cells were detached from the culture flask by a 0.25% trypsin-EDTA solution from the culture flask for plating and passing the cells. Each cell line was grown on 100 mm dishes and incubated at 37 C under 5% CO2 to approximately 70% confluence. Animal Experiments Eight-week-old male BALB/c nude mice were purchased from Japan SLC (Shizuoka, Japan). The mice were housed in a 12-h light/ dark cycle in a humidity-controlled room. For tumor induction, mice were inoculated with C26 cells (2.0 106 cells/mouse) by subcutaneous injection into the dorsal skin and were evaluated when the tumor tissue distribution in the mice tumor volume reached 150-200 mm3. Tumor volume was calculated using the formula 0.4 (a b2), where “a” is the largest diameter and “b” is the smallest diameter. All animal experiments were approved by the animal experimentation committee of Kumamoto University (Kumamoto, Japan) and were performed in accordance with the ethical guidelines of Kumamoto University for the care and use of laboratory animals.
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Monitoring Tumor Tissue Distribution of AGP by IVIS Imaging System AGP was labeled with VivoTag-S® 750, a N-Hydroxysuccinimide-ester labeling reagent. AGP (1 mg/mL) and VivoTagS® 750 (100 mg/mL) were mixed in 50 mM carbonate/bicarbonate buffer (pH 8.5) for 1 h at room temperature. After the reaction, the nonreacted fluorophore was removed by dialysis and ultrafiltration. The VivoTag-S® 750-labeled AGP in C26 tumor-bearing mice were then monitored at 6 h postinjection by an IVIS imaging system (Summit Pharmaceuticals Int. Co., Tokyo, Japan). Pharmacokinetics and Biodistribution of AGP and HSA AGP and HSA were radiolabeled with 125I according to previously reported procedures23 and purified using a Pharmacia BioGel PD-10 column (GE Healthcare, Madison, WI). The radiolabeled proteins were diluted with nonlabeled protein before carrying out the pharmacokinetic experiments to adjust the dose (mg/kg) of protein in each group. The 125I proteins (1 mg/kg) were injected into the tail vein of mice (1.35 105 cpm/mouse), which were then sacrificed at 6 h postinjection. The organs were rinsed with saline and weighed, and the 125I radioactivity contained in each tissue was determined using a gamma-counter (ARC-5000; Hitachi Aloka Medical, Tokyo, Japan). Cellular Uptake Experiments Using FITC-AGP or FITC-Peptide AGP was labeled with FITC. AGP (final concentration 4 mg/mL) and FITC (final concentration 1 mg/mL) were dissolved in phosphate buffer saline (PBS; pH 7.4), followed by mixing for 2 h at room temperature. The solution was purified by means of a PD-10 col€ttingen, Germany) over the umn and VIVASPIN 2 (Sartorius, Go entire day, and the fluorescence intensity in the supernatant, which contains FITC-AGP and in the filtrate, which contains unbound FITC was then measured. We then calculated the purity of FITC-AGP using the following formula (Purity ¼ fluorescence intensity in the filtrate/fluorescence intensity in the supernatant); we confirmed that the purity was about 99.9%. Tumor cells (4.0 104 cells/well) were seeded in 24-well microplates (Greiner, Kremsmünster, Austria) and cultured for 24 h in serum containing cell culture medium. After complete adhesion, the cells were incubated at 37 C with serum-free cell culture medium for 2 h. The cells were then incubated in serum-free cell culture medium containing FITCAGP or FITC-peptide. After being incubated for 2 h, the tumor cells were washed twice with cold Dulbecco’s PBS (DPBS) and detached by treatment with 0.25% trypsin-EDTA. The resulting cells were analyzed by flow cytometry (guava easyCyte™; Merck Millipore, Billerica, MA) using a 488 nm laser. In the case of 4 C experiments, tumor cells were preincubated in serum-free medium at 37 C for 2 h and then incubated in serum-free cell culture medium containing FITC-AGP or FITC-peptide at 4 C for 2 h. Confocal Microscopy MCF-7 and HepG2 cells (2 104 cells/well) were plated on sterilized 8-well SLIDE & CHAMBER and cultured in DMEM or RPMI1640, respectively, for 24 h. After complete adhesion, the cells were incubated at 37 C with serum-free cell culture medium for 2 h. The cells were then incubated in serum-free cell culture medium containing FITC-AGP. After washing with cold DPBS, the cells were incubated with Hoechst 33,342 for 30 min at 37 C. After washing with cold DPBS, the cells were fixed by treatment with 4% paraformaldehyde. The distribution of the FITC-AGP was then determined by a confocal laser scanning microscope TCS SP5 (Leica, Nussloch, Germany) equipped with a 10 25 eyepiece, a 63/1.4 oil lens, and an electronic Zoom 4.
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Measurement of Circular Dichroism Spectra Circular dichroism (CD) spectra were recorded with a Jasco J-720 spectropolarimeter (JASCO, Tokyo, Japan) using 10 mM FITC-peptide in 20 mM PBS (pH 7.4) or 100% butanol. UV spectra were recorded in 1 mm path length cells for far-UV spectra, as previously reported.24-26 Synthesis of Paclitaxel-AGP and Evaluation of Its Cytotoxicity AGP-paclitaxel was prepared by mixing AGP and paclitaxel at a ratio of 2:1 to minimize the presence of free paclitaxel. The cytotoxicity of paclitaxel-AGP was evaluated by cell viability. The cell viability assay was performed using WST-8, which is based on the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay. MCF7 and HepG2 cells were seeded in 96-well plates at 1.0 104 cells/well and were cultured for 24 h in DMEM or RPMI1640, respectively. The MCF-7 and HepG2 cells were then washed twice with DPBS and incubated with AGP, paclitaxel, or paclitaxel-AGP. After incubating the cultures for 48 h, 100 mL of a WST-8 solution was added to each well, and the cells were incubated for an additional 2 h at 37 C. The number of surviving cells was determined by measuring the absorbance at 450 nm. Cell viability was calculated as the percentage of the control value (only culture medium). Synthesis of Poly-S-Nitrosated AGP and Evaluation of Its Cytotoxicity Terminal sulfhydryl groups were added to the AGP molecule by incubating 0.15 mM AGP with 3 mM Traut’s reagent in 100 mM potassium phosphate buffer containing 0.5 mM DTPA (pH 7.8) for 1 h at room temperature. The resulting modified AGP was then Snitrosylated by incubation with 15 mM isoamyl nitrite at 37 C for 3 h. After the reaction, the resulting poly-S-nitrosated AGP (PolySNO-AGP) was dialyzed and concentrated by VIVASPIN 2. The cytotoxicity of the Poly-SNO-AGP was evaluated by the same method as described above. Determination of the S-Nitrosylation Efficiency The amount of the S-nitroso moieties on poly-SNO-AGP was determined using a 96-well plate. First, 20 mL aliquots of the polySNO-AGP solution and NaNO2 (standard) were incubated with 200 mL of 10 mM sodium acetate buffer (pH 5.5) containing 100 mM NaCl, 0.5 mM DTPA, 0.015% N-1-naphthylethylenediamine dihydrochloride, and 0.15% sulfanilamide in the presence and absence at 0.09 mM HgCl2 for 30 min at room temperature. The absorbance
was then measured at 540 nm. The number of moles of NO per mole of AGP was determined by subtracting the values for the absence of HgCl2 from values in the presence of HgCl2. The value thus obtained was 3.27 mol NO/mol AGP. Detection of Cellular NO MCF-7 and HepG2 cells (1.0 104 cells/well) were seeded in 96well plates in DMEM or RPMI1640, respectively, for 24 h. The cells were washed twice with DPBS, and then incubated for an additional 30 min in DPBS containing 10 mM DAF-FM DA, which is a fluorescent indicator of NO. After washing the cells twice with DPBS, they were cultured in DPBS containing various concentrations of polySNO-AGP for 3 h. After incubation, the fluorescence was measured using a plate reader (excitation wavelength, 485 nm; emission wavelength, 535 nm). The change in fluorescence was calculated by subtracting the fluorescence at 0 h from the fluorescence measured at 3 h. Data Statistical Analysis Data are shown as the mean ± SD for the indicated number of samples. A significant difference test was conducted using Student t-test between the 2 groups. For data over 3 groups, multiple comparison tests were performed using the Tukey-Kramer method. In all tests, the values were judged to be statistically significant when the hazard value was <0.05. Results AGP Distribution in Tumor Tissue of C26-Bearing Mice VivoTag-S® 750 labeled AGP was intravenously administered to mice-bearing murine colon cancer C26 cells (mean size: 200 mm3), and the tumor distribution of AGP was then evaluated by IVIS at 6 h after administration. A marked VivoTag-S® 750-derived fluorescence was observed at the tumor site (Fig. 1a). 125I-labeled AGP was also intravenously administered to the same type of tumor-bearing mice as above. The objective here was to determine the extent that AGP migrates to tumor tissue compared with HSA which has been confirmed to function as a carrier of anticancer drugs in clinic. Similar to the imaging data, a high level of radioactivity was detected in the tumor tissue (Fig. 1b). In addition, the radioactivity of AGP was significantly higher than that of HSA. These collective data indicate that a larger fraction of the administered AGP was distributed to tumor tissue compared with HSA.
Figure 1. Distribution of AGP to tumor tissue. (a) Visualization of the tumor tissue distribution of AGP by optical imaging. VivoTag-S® 750-labeled AGP was intravenously injected into C26 tumor-bearing mice, and its dynamic biodistribution was determined by IVIS at 6 h postinjection. The circle denotes the tumor region. (b) 125I-labeled proteins (AGP and HSA) were intravenously injected into C26 tumor-bearing mice. Subsequently, tumor grafts and other organs were collected at 6 h postinjection for radioactivity determination. Results are the means ± SD (n ¼ 4). *p < 0.05 versus HSA.
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Cell-Penetrating Properties of AGP Into Tumor Cells We next examined whether AGP was taken up into tumor cells using MCF-7 (human breast carcinoma), HepG2 (human hepatocellular carcinoma), C26 (murine colon carcinoma), and Hela (human cervical carcinoma) cells by fluorescence-activated cell sorting analysis. When FITC-labeled AGP (FITC-AGP) was added to tumor cell cultures at 37 C (Fig. 2a), the FITC-derived fluorescence was
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observed in all types of cancer cells that were used in this study. A particularly high fluorescence was observed in MCF-7 and HepG2 cells. To further investigate this observation, intracellular imaging of FITC-AGP was conducted against MCF-7 and HepG2 cells by confocal microscopy. Again, a high level of FITC-derived fluorescence was observed in the interior of MCF-7 and HepG2 cells (Fig. 2b). These results suggest that AGP was taken up by tumor cells at 37 C. To further examine whether intracellular migration of
Figure 2. The incorporation of AGP into tumor cells. (a) Four types of tumor cells were incubated with FITC-AGP (150 mg/mL) for 2 h at 37 C in serum-free medium. The intracellular incorporation of FITC-AGP was determined by flow cytometry. The results are the mean ± SD (n ¼ 4). **p < 0.01 versus FITC-AGP (). (b) MCF-7 and HepG2 cells were incubated with FITC-AGP (150 mg/mL) for 2 h at 37 C in serum-free medium. Hoechst 33,342 (blue) was used to visualize nuclei. Intracellular localization of FITC-AGP was analyzed with confocal microscopy. (c) MCF-7 and HepG2 cells were incubated with FITC-AGP (150 mg/mL) for 0.5, 1, and 2 h at 37 C or 4 C. (d) MCF-7 and HepG2 cells were pretreated with chlorpromazine 10 mg/mL (60 min) or methyl-b-cyclodextrin 5 mM (30 min), EIPA 100 mM (30 min), and subsequently treated with FITC-AGP (150 mg/mL) for 30 min at 37 C. Inhibitor () was used as a control. Results are the mean ± SD (n ¼ 4). **p < 0.01 versus inhibitor (), *p < 0.05 versus inhibitor ().
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Figure 3. A conformational change from b-sheet to a-helix on the migration of AGP to tumor cells. (a) Alignment of the amino acid sequence of AGP. Putative portions of a-helix site deduced from a primary structure are shown in under line. (b) Far UV CD spectra of AGP derived peptide in the PBS (dashed line) or butanol (solid line). (c) MCF-7 and HepG2 cells were incubated with FITC-AGPederived peptide (10 mM) for 2 h at 37 C. Intracellular incorporation of FITC-peptide was analyzed by flow cytometry. Results are the means ± SD (n ¼ 4). **p < 0.01 versus FITC-peptide ().
AGP was energy dependent, the same experiments were carried out at 4 C. In contrast to 37 C, almost no fluorescence intensity was observed at 4 C (Fig. 2c). These data suggest that AGP is incorporated into the tumor cells in an energy-dependent manner, possibly via an endocytosis pathway. We also attempted to identify which endocytosis pathway was involved in the intracellular migration of AGP by using the following endocytosis inhibitors: CPZ, a clathrin-mediated endocytosis inhibitor; methyl-b-cyclodextrin (Met-b-CyD), a caveolae/ lipid raft inhibitor27; and EIPA, a macropinocytosis inhibitor. In the case of MCF-7 cells, the intracellular fluorescence derived from FITC-AGP was decreased to approximately 50% and 77% in the presence of CPZ and EIPA, respectively (Fig. 2d); whereas, the FITCAGP fluorescence was decreased to 28% by the Met-b-CyD treatment in HepG2 cells (Fig. 2e). These results suggest that clathrin and macropinocytosis pathways might be involved in the AGP uptake by MCF-7 cells, whereas the caveolae/lipid raftsemediated pathway could contribute to the endocytosis of AGP in HepG2 cells.
a-helix Formation of the AGP-Derived Peptide and Its Cell Penetration Into Tumor Cells Because AGP undergoes a dynamic conformational change from
b-sheet to a-helix when it interacts with both artificial and bio-
logical membranes,24-26 we hypothesized that this conformational change may contribute to the intracellular transfer of AGP into tumor cells. To explore this possibility further, we selected a peptide sequence from the primary structure of AGP (amino acid position 135-148; KEQLGEFYEALDCL) as a candidate for the synthesis of a cell-penetrating peptide because this primary structure region of AGP was reported previously to possess a high ability to form an a-helix (Fig. 3a).25 This AGP-derived peptide was synthesized with a his-tag and used in the subsequent experiments.
CD spectra of the AGP-derived peptide was measured in both 20 mM PBS (pH7.4) and 100% butanol because the secondary structure of AGP was largely transformed made to a b-sheet to an a-helix structure when it was dissolved in butanol. As shown in Figure 3b, AGP-derived peptide exhibited the peaks of CD spectra at 209 nm and 222 nm in PBS solution, suggesting that this peptide formed ahelix.28 Such a phenomena was enhanced in butanol solution compared with above situation. This suggests that AGP-derived peptide favorably form a-helix in the hydrophobic condition. A fluorescence-activated cell sorting analysis was then performed to evaluate the cell-penetrating ability of the FITC-labeled AGP-derived peptide in MCF-7 and HepG2 cells. The results indicated that FITC-derived fluorescence was observed in both MCF-7 and HepG2 cells (Fig. 3c). Antitumor Activity of Paclitaxel-Loaded AGP Finally, to examine whether AGP functions as a DDS carrier, AGP loaded with anticancer agents was prepared, and its antitumor effects were evaluated using MCF-7 and HepG2 cells. In the case of AGP, the anticancer agents can be loaded by 2 different modes, namely noncovalent complex formation at ligand binding pocket or by covalent attachment to amino acid residues on the surface of the AGP. As an example of the noncovalently binding mode, we examined the effect of paclitaxel which has a high binding affinity to AGP and exerts an antitumor effect when entering cells. In this experiment, paclitaxel was added to an AGP solution at a paclitaxel:AGP ratio of 1:2 to minimize the presence of free paclitaxel. In fact, free paclitaxel was undetectable in the filtrate by HPLC analysis after ultrafiltration was performed on this 1:2 ratio of paclitaxel:AGP mixture. The viability of MCF-7 and HepG2 cells was then measured in the presence of paclitaxel-loaded AGP. In this experiment, paclitaxel alone and AGP alone were used as controls. As previously reported,
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Figure 4. AGP as an anticancer drug carrier (drug loading mode; complexation). Cytotoxicity of paclitaxel-AGP complex on tumor cells. MCF-7 and HepG2 cells were treated with AGP, paclitaxel, or paclitaxel-AGP for 48 h. Results are the means ± SD (n ¼ 4). **p < 0.01 versus control.
incubation with paclitaxel alone caused a marked suppression in the viability of MCF-7 cells (Fig. 4). Interestingly, paclitaxel-loaded AGP also significantly reduced cell viability to nearly the same extent of paclitaxel alone (Fig. 4). By contrast, AGP alone had no effect on the viability of MCF-7 cells. Similar results were also obtained when HepG2 cells were used (Fig. 4).
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chains,17 it is also possible that recognition of AGP by CD169 may mediate the active distribution of AGP to tumor tissue. The results reported herein also confirms that AGP is incorporated into 4 types of tumor cells, via endocytosis pathways including clathrin, macropinocytosis, and caveolae/lipid raft mediated routes, depending on the type of tumor cells. The findings reported here indicate that the contribution of these pathways are dependent on the tumor cell type, where clathrin and macropinocytosis are involved in the intracellular uptake of AGP into MCF-7 cells, whereas AGP migrates into HepG2 cells via a caveolae/ lipid raftemediated pathway. In fact, it was possible to deliver both paclitaxel or NO-loaded AGP to the tumor cells resulting in antitumor activities against both MCF-7 and HepG2 cells. Peptides with a-helix forming ability have recently been used in the DDS field, especially to allow molecules to escape from endosomes to the cytosol compartment.33,34 We previously demonstrated that AGP contains b-sheet rich structure in aqueous solution but transforms to a-helix structure when it interacts with a membrane and this structural transition becomes obvious in an acidic environment.24-26 Such a structural transition is similar to melittin, a major pain producing substance of the honeybee.35 Melittin has recently been evaluated as a cell-penetrating protein for such a process through the formation of an a-helix structure.36,37 Actually, the AGP-derived peptide, which is predicted to possess a propensity
Antitumor Activity of NO-Loaded AGP As an example of a covalently binding mode, we used NO because it exhibits excellent antitumor effects.29,30 We prepared Poly-SNO-AGP in which an average of 3.3 NO molecules were covalently bound to the newly introduced thiol groups on AGP and also examined its antitumor ability against MCF-7 and HepG2 cells. As shown in Figure 5a, Poly-SNO-AGP reduced the viability of both types of tumor cells in a concentration-dependent manner. The intracellular NO levels were also measured under the same conditions using an NO detectable fluorescence probe (DAF-FM DA). As shown in Figure 5b, the intracellular fluorescence intensity increased with increasing levels of Poly-SNO-AGP, indicating that NO was delivered to the cells in a Poly-SNO-AGP dose-dependent manner. Interestingly, there was a negative relationship between cell viability and the fluorescence intensity derived from DAF-FM DA (Fig. 5c). This suggests that intracellularly delivered NO by Poly-SNO-AGP induced tumor cell death.
Discussion The characteristics of an effective DDS carrier for cancer therapeutics include the following 4 characteristics: (1) biocompatibility, (2) drug loading ability, (3) tumor tissue distribution, and (4) tumor cell-penetrating ability (for drugs that act inside tumor cells). In this research, we examined the suitability of AGP as a DDS carrier of anticancer drugs, because it is a drug carrier in plasma. As mentioned in the introduction section, intravenous administration of exogenous AGP has been reported to result in higher AGP distribution to tumor tissue than other organs in rats bearing the sarcoma cells.21 The present study also found that AGP was preferentially distributed to tumor tissue on mice-bearing C26 cells than HSA which currently in use as a DDS carrier for anticancer drugs.13 Because the molecular weight of AGP is approximately 44 kDa, its size is sufficient to allow it to be transported via the EPR effect.8 It is possible that the EPR effect is involved in the tumor tissue distribution of AGP. In addition, a membrane surface lectin (CD169) that strongly binds to sialic acid units as an endogenous substrate in the tumor tissue has been reported recently.31,32 Because AGP contains a large number of sialic acids at its glycan
Figure 5. AGP as an anticancer drug carrier (drug loading mode; Chemical modification). (a) Cytotoxicity of Poly-SNO-AGP on tumor cells. MCF-7 and HepG2 cells were treated with various concentration of Poly-SNO-AGP for 48 h. Results are the mean ± SD (n ¼ 4). **p < 0.01 versus 0 mM NO. (b) Intracellular delivery of NO by Poly-SNO-AGP on tumor cells. MCF-7 and HepG2 cells were pretreated with DAF-FM DA (10 mM) for 30 min and treated with various concentrations of Poly-SNO-AGP for 2 h. Results are the means ± SD (n ¼ 4). **p < 0.01 versus 0 mM NO. (c) Relationship between cell viability and fluorescence intensity.
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to form an a-helix structure, adopted an a-helix structure when present in a hydrophobic environment such as butanol solution and was transferred into the MCF-7 and HepG2 cells. These findings suggest that in addition to endocytosis pathway, the cellpenetrating property of AGP can be partially attributed to the peptide regions of the molecule with a-helix forming ability as like melittin. Moreover, such a cell-penetrating mechanism may also contribute to the endosomal escape of such a molecule because the b-sheet to a-helix transition of AGP is facilitated by the interaction with the negatively charged membrane.24 Among the anticancer drugs that are currently in use, nabpaclitaxel is a DDS product in which paclitaxel is bound to HSA.20 Because nab-paclitaxel exerts excellent antitumor effects, it is widely used in the treatment of various types of refractory tumors, including breast cancer, gastric cancer, nonesmall-cell lung cancer, and pancreatic cancer. However, the development of a new type of therapeutic which is superior to nab-paclitaxel is urgently needed for the treatment of refractory tumors. One solution for this problem is to explore and develop a new DDS carrier for paclitaxel. Because the properties of AGP include a higher tumor distribution and higher affinity for paclitaxel than HSA, a paclitaxel-loaded nanoparticle based on AGP might show better therapeutic effects than nab-paclitaxel. Moreover, polymerization and PEGylation of AGP or a nanoparticle derived from it could facilitate further tumor distribution via enhancing the EPR effect. Alternatively, it would be expected that the more active targeting of AGP to tumor tissue could be achieved by the attachment of a homing substance that selectively recognizes the components of tumor tissue. In addition, recent basic and clinical studies suggest that AGP itself has antitumor activities, including antimetastasis and antiinvasive actions.38,39 For example, the expression level of AGP decreased in tumor tissue in patients with hepatocellular cancer and was negatively correlated with disease progression in patients with intrahepatic metastasis.39 These findings indicate that utilizing AGP as a DDS carrier has the potential for exerting dual functions with anticancer activity and would allow the efficient delivery of anticancer agents to tumor tissue. Conclusion In the present study, we found that AGP migrated to tumor tissue compared with HSA which has already been used as carrier of anticancer drug in the clinic and also has the ability to migrate into tumor cells. Consequently, AGP loaded with anticancer agents caused the tumor cell death. These findings indicate that AGP has the potential to function as a DDS carrier of anticancer drugs although there is a need for further in vivo experiments. Acknowledgments This study was funded by the Research Foundation for Pharmaceutical Sciences, a Grant-in-Aid Scientific Research from the Japan Society for the Promotion of Science (KAKENHI 15H04758), and the Takeda Science Foundation, Japan. References 1. Dobbelstein M, Moll U. Targeting tumour-supportive cellular machineries in anticancer drug development. Nat Rev Drug Discov. 2014;13(3):179-196. 2. Wan L, Pantel K, Kang Y. Tumor metastasis: moving new biological insights into the clinic. Nat Med. 2013;19(11):1450-1464. 3. Niero EL, Rocha-Sales B, Lauand C, et al. The multiple facets of drug resistance: one history, different approaches. J Exp Clin Cancer Res. 2014;33:37. 4. Minko T, Dharap SS, Pakunlu RI, Wang Y. Molecular targeting of drug delivery systems to cancer. Curr Drug Targets. 2004;5(4):389-406. 5. Jones T, Saba N. Nanotechnology and drug delivery: an update in oncology. Pharmaceutics. 2011;3(2):171-185.
6. Yao VJ, D'Angelo S, Butler KS, et al. Ligand-targeted theranostic nanomedicines against cancer. J Control Release. 2016;240:267-286. 7. Blanco E, Hsiao A, Mann AP, Landry MG, Meric-Bernstam F, Ferrari M. Nanomedicine in cancer therapy: innovative trends and prospects. Cancer Sci. 2011;102(7):1247-1252. 8. Maeda H, Bharate GY, Daruwalla J. Polymeric drugs for efficient tumor-targeted drug delivery based on EPR-effect. Eur J Pharm Biopharm. 2009;71(3):409-419. 9. Maeda H. The enhanced permeability and retention (EPR) effect in tumor vasculature: the key role of tumor-selective macromolecular drug targeting. Adv Enzyme Regul. 2001;41:189-207. 10. Boroughs LK, DeBerardinis RJ. Metabolic pathways promoting cancer cell survival and growth. Nat Cell Biol. 2015;17(4):351-359. 11. Wise DR, Thompson CB. Glutamine addiction: a new therapeutic target in cancer. Trends Biochem Sci. 2010;35(8):427-433. 12. Kamphorst JJ, Nofal M, Commisso C, et al. Human pancreatic cancer tumors are nutrient poor and tumor cells actively scavenge extracellular protein. Cancer Res. 2015;75(3):544-553. 13. Shahzad Y, Khan IU, Hussain T, et al. Bioactive albumin-based carriers for tumour chemotherapy. Curr Cancer Drug Targets. 2014;14(8):752-763. 14. Kratz F, Elsadek B. Clinical impact of serum proteins on drug delivery. J Control Release. 2012;161(2):429-445. 15. Yardley DA. nab-Paclitaxel mechanisms of action and delivery. J Control Release. 2013;170(3):365-372. ndez-García I, Carvalho FL, Vera R, Hidalgo M. Nab16. Viúdez A, Ramírez N, Herna paclitaxel: a flattering facelift. Crit Rev Oncol Hematol. 2014;92(3):166-180. 17. Fournier T, Medjoubi NN, Porquet D. Alpha-1-acid glycoprotein. Biochim Biophys Acta. 2000;1482(1-2):157-171. 18. Luo Z, Lei H, Sun Y, Liu X, Su DF. Orosomucoid, an acute response protein with multiple modulating activities. J Physiol Biochem. 2015;71(2):329-340. 19. Israili ZH, Dayton PG. Human alpha-1-glycoprotein and its interactions with drugs. Drug Metab Rev. 2001;33(2):161-235. 20. Bohnert T, Gan LS. Plasma protein binding: from discovery to development. J Pharm Sci. 2013;102(9):2953-2994. 21. Shibata K, Okubo H, Ishibashi H, Tsuda-Kawamura K, Yanase T. Rat alpha 1-acid glycoprotein: uptake by inflammatory and tumour tissues. Br J Exp Pathol. 1978;59(6):601-608. 22. Chen RF. Removal of fatty acids from serum albumin by charcoal treatment. J Biol Chem. 1967;242(2):173-181. 23. Watanabe H, Yamasaki K, Kragh-Hansen U, et al. In vitro and in vivo properties of recombinant human serum albumin from Pichia pastoris purified by a method of short processing time. Pharm Res. 2001;18(12):1775-1781. 24. Nishi K, Sakai N, Komine Y, Maruyama T, Halsall HB, Otagiri M. Structural and drug-binding properties of alpha(1)-acid glycoprotein in reverse micelles. Biochim Biophys Acta. 2002;1601(2):185-191. 25. Nishi K, Komine Y, Fukunaga N, Maruyama T, Suenaga A, Otagiri M. Involvement of disulfide bonds and histidine 172 in a unique beta-sheet to alpha-helix transition of alpha 1-acid glycoprotein at the biomembrane interface. Proteins. 2006;63(3):611-620. 26. Nishi K, Komine Y, Sakai N, Maruyama T, Otagiri M. Cooperative effect of hydrophobic and electrostatic forces on alcohol-induced alpha-helix formation of alpha1-acid glycoprotein. FEBS Lett. 2005;579(17):3596-3600. 27. Chatterjee M, Ben-Josef E, Robb R, et al. Caveolae-mediated endocytosis is critical for albumin cellular uptake and response to albumin-bound chemotherapy. Cancer Res. 2017;77(21):5925-5937. 28. Kelly SM, Price NC. The use of circular dichroism in the investigation of protein structure and function. Curr Protein Pept Sci. 2000;1(4):349-384. 29. Beckman JS, Crow JP. Pathological implications of nitric oxide, superoxide and peroxynitrite formation. Biochem Soc Trans. 1993;21(2):330-334. 30. Brüne B, von Knethen A, Sandau KB. Nitric oxide (NO): an effector of apoptosis. Cell Death Differ. 1999;6(10):969-975. 31. Komohara Y, Ohnishi K, Takeya M. Possible functions of CD169-positive sinus macrophages in lymph nodes in anti-tumor immune responses. Cancer Sci. 2017;108(3):290-295. 32. Asano K, Nabeyama A, Miyake Y, et al. CD169-positive macrophages dominate antitumor immunity by crosspresenting dead cell-associated antigens. Immunity. 2011;34(1):85-95. 33. Kakudo T, Chaki S, Futaki S, et al. Transferrin-modified liposomes equipped with a pH-sensitive fusogenic peptide: an artificial viral-like delivery system. Biochemistry. 2004;43(19):5618-5628. 34. Hayashi Y, Hatakeyama H, Kajimoto K, Hyodo M, Akita H, Harashima H. Multifunctional envelope-type nano device: evolution from nonselective to active targeting system. Bioconjug Chem. 2015;26(7):1266-1276. 35. Raghuraman H, Chattopadhyay A. Melittin: a membrane-active peptide with diverse functions. Biosci Rep. 2007;27(4-5):189-223. 36. Ladokhin AS, White SH. Folding of amphipathic alpha-helices on membranes: energetics of helix formation by melittin. J Mol Biol. 1999;285(4):1363-1369. 37. Hou KK, Pan H, Schlesinger PH, Wickline SA. A role for peptides in overcoming endosomal entrapment in siRNA delivery - a focus on melittin. Biotechnol Adv. 2015;33(6 Pt 1):931-940. 38. Lee SY, Lim JW, Kim YM. Effect of alpha1-acid glycoprotein expressed in cancer cells on malignant characteristics. Mol Cells. 2001;11(3):341-345. 39. Fang T, Cui M, Sun J, et al. Orosomucoid 2 inhibits tumor metastasis and is upregulated by CCAAT/enhancer binding protein b in hepatocellular carcinomas. Oncotarget. 2015;6(18):16106-16119.
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Pharmaceutics, Drug Delivery and Pharmaceutical Technology
a1-Acid Glycoprotein Has the Potential to Serve as a Biomimetic Drug Delivery Carrier for Anticancer Agents Kotaro Matsusaka 1, Yu Ishima 2, Hitoshi Maeda 1, Ryo Kinoshita 1, Shota Ichimizu 1, Kazuaki Taguchi 3, Victor Tuan Giam Chuang 4, Koji Nishi 5, Keishi Yamasaki 5, Masaki Otagiri 5, Hiroshi Watanabe 1, Toru Maruyama 1, * 1
Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan Department of Pharmacokinetics and Biopharmaceutics, Institute of Biomedical Sciences, Tokushima University, 1-78-1, Sho-machi, Tokushima 770-8505, Japan 3 Division of Pharmacodynamics, Keio University Faculty of Pharmacy, 1-5-30, Shibakoen, Minato-ku, Tokyo 105-8512, Japan 4 School of Pharmacy, Monash University Malaysia, Jalan Lagoon Selatan, 47500 Bandar Sunway, Selangor, Malaysia 5 Faculty of Pharmaceutical Sciences, Sojo University, 1-22-4 Ikeda, Nishi-ku, Kumamoto 860-0082, Japan 2
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 May 2019 Revised 25 June 2019 Accepted 2 July 2019 Available online 6 July 2019
Nanosize plasma proteins could be used as a biomimetic drug delivery system (DDS) for cancer treatment when loaded with anticancer drugs based on the fact that plasma proteins can serve as a source of nutrients for cancer cells. This prompted us to investigate the potential of a1-acid glycoprotein (AGP) for this role because it is a nanosize plasma protein and binds a variety of anticancer agents. Pharmacokinetic analyses indicated that AGP is distributed more extensively in tumor tissue than human serum albumin, which was already established as a cancer DDS carrier. AGP is possibly being incorporated into tumor cells via endocytosis pathways. Moreover, a synthetic AGP-derived peptide which possesses a high ability to form an a-helix, as deduced from the primary structure of AGP, was also taken up by the tumor cells. AGP loaded with anticancer agents, such as paclitaxel or nitric oxide, efficiently induced tumor cell death. These results suggest that AGP has the potential to be a novel DDS carrier for anticancer agents. © 2019 Published by Elsevier Inc. on behalf of the American Pharmacists Association.
Keywords: a1-acid glycoprotein orosomucoid DDS carrier cancer anticancer agents
Introduction Cancer treatment has made impressive progress with the development of molecular targeted drugs and therapeutic antibodies in recent years.1 However, an effective treatment for refractory cancers such as metastasis, recurrence, and resistance to treatment continue to be a substantial challenge.2,3 A nanosized drug delivery system (DDS) with organ-specific targeting ability has the potential to overcome such limitations in the field of cancer therapy.4-7 The enhanced permeability and retention (EPR) effect, in which molecules with molecular weights in excess of 40-50 kDa selectively accumulate in tumor tissues to a much greater extent than in normal tissue, is a principle concept for the development of nanosized anticancer therapeutics.8,9 Nutrients play an especially important and essential role in supporting the uncontrolled tumor growth, metastasis, and resistance to therapy.10,11 Plasma proteins have recently received
* Correspondence to: Toru Maruyama (Telephone: þ81-96-371-4150). E-mail address: [email protected] (T. Maruyama).
considerable attention as a source of cancer nutrients, given the fact that they are actively taken up by tumor cells via the endocytic pathway and are then intracellularly digested into basic units that can be utilized as nutrients.12 A typical example is human serum albumin (HSA),13 and we refer this uptake phenomenon by cancer cells as an “endogenous albumin transport system”. We hypothesize that an anticancer drug-loaded nanosized plasma protein may serve as a biomimetic DDS biomaterial for cancer treatment.14 The most successful example of this strategy is paclitaxel-bound HSA (nab-paclitaxel; Abraxane) that has already been approved in many countries and has been used in the treatment of various types of refractory tumors, such as breast cancer, gastric cancer, nonesmallcell lung cancer, and pancreatic cancer.14-16 Human a1-acid glycoprotein (AGP) is an acute phase plasma glycoprotein, and its secondary structure is mainly composed of bsheets.17,18 The molecular weight of AGP is 44 kDa, which is within the molecular size range for a compound to undergo EPR effect. AGP, along with HSA, functions as a drug carrier in plasma.19 Many anticancer drugs bind to AGP with high affinities.20 On the other hand, AGP administered to sarcoma-bearing rats has been found to accumulate in the tumor tissue rather than other organs.21 These
https://doi.org/10.1016/j.xphs.2019.07.002 0022-3549/© 2019 Published by Elsevier Inc. on behalf of the American Pharmacists Association.
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findings suggest that AGP has the potential to function as a new nanosized DDS carrier for anticancer drugs. In this study, the pharmacokinetics of AGP in tumor-bearing mice were investigated before evaluation for the cell-penetrating ability of AGP using 4 different types of tumor cells. Finally, the antitumor activity of AGP loaded with anticancer agents such as paclitaxel and nitric oxide (NO) was examined by using an in vitro cell system. Materials and Methods Materials AGP was a donation from the Chemo-Sero-Therapeutic Research Institute (Kumamoto, Japan). HSA was purchased from the ChemoSero-Therapeutic Research Institute and defatted according to a previously reported method.22 VivoTag-S® 750 and Na125I were purchased from Perkin Elmer Cetus (Norwalk, CT). Fluorescein isothiocyanate (FITC) and 5-(N-ethyl-N-isopropyl) amiloride (EIPA) were purchased from Sigma-Aldrich (St. Louis, MO). N-1Naphthylethylenediamine dihydrochloride, sulfanilamide, HgCl2, and NaNO2 were purchased from Nacalai Tesque (Kyoto, Japan). Chlorpromazine (CPZ), methyl-b-cyclodextrin (Met-b-CyD), and isoamyl nitrite were purchased from Wako Chemicals (Osaka, Japan). Hoechst 33,342 and diethylenetriaminepentaacetic acid (DTPA) and the cell counting kit-8 (WST-8) were purchased from Dojindo Laboratories (Kumamoto, Japan). The FITC-peptide (its amino acid position in AGP; 135-148) was purchased from SCRUM Inc. (Tokyo, Japan). Paclitaxel was purchased from MedChemExpress (Shanghai, China). Traut’s reagent (2-imminothiolane) was purchased from Pierce Chemical Company (Rockford, IL). 4-Amino5-methylamino2’,7’-difluorofluorescein diacetate (DAF-FM DA) was purchased from Sekisui Medical Company, Ltd. (Tokyo, Japan). Cell Cultures Human breast cancer MCF-7 cells and human cervical cancer HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% heat-inactivated fetal bovine serum, 100 U/ mL penicillin, and 100 mg/mL streptomycin. Human liver cancer HepG2 cells and murine colon cancer C26 cells were cultured in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin. A subculture was performed at 3-4 days intervals. Cells were detached from the culture flask by a 0.25% trypsin-EDTA solution from the culture flask for plating and passing the cells. Each cell line was grown on 100 mm dishes and incubated at 37 C under 5% CO2 to approximately 70% confluence. Animal Experiments Eight-week-old male BALB/c nude mice were purchased from Japan SLC (Shizuoka, Japan). The mice were housed in a 12-h light/ dark cycle in a humidity-controlled room. For tumor induction, mice were inoculated with C26 cells (2.0 106 cells/mouse) by subcutaneous injection into the dorsal skin and were evaluated when the tumor tissue distribution in the mice tumor volume reached 150-200 mm3. Tumor volume was calculated using the formula 0.4 (a b2), where “a” is the largest diameter and “b” is the smallest diameter. All animal experiments were approved by the animal experimentation committee of Kumamoto University (Kumamoto, Japan) and were performed in accordance with the ethical guidelines of Kumamoto University for the care and use of laboratory animals.
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Monitoring Tumor Tissue Distribution of AGP by IVIS Imaging System AGP was labeled with VivoTag-S® 750, a N-Hydroxysuccinimide-ester labeling reagent. AGP (1 mg/mL) and VivoTagS® 750 (100 mg/mL) were mixed in 50 mM carbonate/bicarbonate buffer (pH 8.5) for 1 h at room temperature. After the reaction, the nonreacted fluorophore was removed by dialysis and ultrafiltration. The VivoTag-S® 750-labeled AGP in C26 tumor-bearing mice were then monitored at 6 h postinjection by an IVIS imaging system (Summit Pharmaceuticals Int. Co., Tokyo, Japan). Pharmacokinetics and Biodistribution of AGP and HSA AGP and HSA were radiolabeled with 125I according to previously reported procedures23 and purified using a Pharmacia BioGel PD-10 column (GE Healthcare, Madison, WI). The radiolabeled proteins were diluted with nonlabeled protein before carrying out the pharmacokinetic experiments to adjust the dose (mg/kg) of protein in each group. The 125I proteins (1 mg/kg) were injected into the tail vein of mice (1.35 105 cpm/mouse), which were then sacrificed at 6 h postinjection. The organs were rinsed with saline and weighed, and the 125I radioactivity contained in each tissue was determined using a gamma-counter (ARC-5000; Hitachi Aloka Medical, Tokyo, Japan). Cellular Uptake Experiments Using FITC-AGP or FITC-Peptide AGP was labeled with FITC. AGP (final concentration 4 mg/mL) and FITC (final concentration 1 mg/mL) were dissolved in phosphate buffer saline (PBS; pH 7.4), followed by mixing for 2 h at room temperature. The solution was purified by means of a PD-10 col€ttingen, Germany) over the umn and VIVASPIN 2 (Sartorius, Go entire day, and the fluorescence intensity in the supernatant, which contains FITC-AGP and in the filtrate, which contains unbound FITC was then measured. We then calculated the purity of FITC-AGP using the following formula (Purity ¼ fluorescence intensity in the filtrate/fluorescence intensity in the supernatant); we confirmed that the purity was about 99.9%. Tumor cells (4.0 104 cells/well) were seeded in 24-well microplates (Greiner, Kremsmünster, Austria) and cultured for 24 h in serum containing cell culture medium. After complete adhesion, the cells were incubated at 37 C with serum-free cell culture medium for 2 h. The cells were then incubated in serum-free cell culture medium containing FITCAGP or FITC-peptide. After being incubated for 2 h, the tumor cells were washed twice with cold Dulbecco’s PBS (DPBS) and detached by treatment with 0.25% trypsin-EDTA. The resulting cells were analyzed by flow cytometry (guava easyCyte™; Merck Millipore, Billerica, MA) using a 488 nm laser. In the case of 4 C experiments, tumor cells were preincubated in serum-free medium at 37 C for 2 h and then incubated in serum-free cell culture medium containing FITC-AGP or FITC-peptide at 4 C for 2 h. Confocal Microscopy MCF-7 and HepG2 cells (2 104 cells/well) were plated on sterilized 8-well SLIDE & CHAMBER and cultured in DMEM or RPMI1640, respectively, for 24 h. After complete adhesion, the cells were incubated at 37 C with serum-free cell culture medium for 2 h. The cells were then incubated in serum-free cell culture medium containing FITC-AGP. After washing with cold DPBS, the cells were incubated with Hoechst 33,342 for 30 min at 37 C. After washing with cold DPBS, the cells were fixed by treatment with 4% paraformaldehyde. The distribution of the FITC-AGP was then determined by a confocal laser scanning microscope TCS SP5 (Leica, Nussloch, Germany) equipped with a 10 25 eyepiece, a 63/1.4 oil lens, and an electronic Zoom 4.
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Measurement of Circular Dichroism Spectra Circular dichroism (CD) spectra were recorded with a Jasco J-720 spectropolarimeter (JASCO, Tokyo, Japan) using 10 mM FITC-peptide in 20 mM PBS (pH 7.4) or 100% butanol. UV spectra were recorded in 1 mm path length cells for far-UV spectra, as previously reported.24-26 Synthesis of Paclitaxel-AGP and Evaluation of Its Cytotoxicity AGP-paclitaxel was prepared by mixing AGP and paclitaxel at a ratio of 2:1 to minimize the presence of free paclitaxel. The cytotoxicity of paclitaxel-AGP was evaluated by cell viability. The cell viability assay was performed using WST-8, which is based on the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay. MCF7 and HepG2 cells were seeded in 96-well plates at 1.0 104 cells/well and were cultured for 24 h in DMEM or RPMI1640, respectively. The MCF-7 and HepG2 cells were then washed twice with DPBS and incubated with AGP, paclitaxel, or paclitaxel-AGP. After incubating the cultures for 48 h, 100 mL of a WST-8 solution was added to each well, and the cells were incubated for an additional 2 h at 37 C. The number of surviving cells was determined by measuring the absorbance at 450 nm. Cell viability was calculated as the percentage of the control value (only culture medium). Synthesis of Poly-S-Nitrosated AGP and Evaluation of Its Cytotoxicity Terminal sulfhydryl groups were added to the AGP molecule by incubating 0.15 mM AGP with 3 mM Traut’s reagent in 100 mM potassium phosphate buffer containing 0.5 mM DTPA (pH 7.8) for 1 h at room temperature. The resulting modified AGP was then Snitrosylated by incubation with 15 mM isoamyl nitrite at 37 C for 3 h. After the reaction, the resulting poly-S-nitrosated AGP (PolySNO-AGP) was dialyzed and concentrated by VIVASPIN 2. The cytotoxicity of the Poly-SNO-AGP was evaluated by the same method as described above. Determination of the S-Nitrosylation Efficiency The amount of the S-nitroso moieties on poly-SNO-AGP was determined using a 96-well plate. First, 20 mL aliquots of the polySNO-AGP solution and NaNO2 (standard) were incubated with 200 mL of 10 mM sodium acetate buffer (pH 5.5) containing 100 mM NaCl, 0.5 mM DTPA, 0.015% N-1-naphthylethylenediamine dihydrochloride, and 0.15% sulfanilamide in the presence and absence at 0.09 mM HgCl2 for 30 min at room temperature. The absorbance
was then measured at 540 nm. The number of moles of NO per mole of AGP was determined by subtracting the values for the absence of HgCl2 from values in the presence of HgCl2. The value thus obtained was 3.27 mol NO/mol AGP. Detection of Cellular NO MCF-7 and HepG2 cells (1.0 104 cells/well) were seeded in 96well plates in DMEM or RPMI1640, respectively, for 24 h. The cells were washed twice with DPBS, and then incubated for an additional 30 min in DPBS containing 10 mM DAF-FM DA, which is a fluorescent indicator of NO. After washing the cells twice with DPBS, they were cultured in DPBS containing various concentrations of polySNO-AGP for 3 h. After incubation, the fluorescence was measured using a plate reader (excitation wavelength, 485 nm; emission wavelength, 535 nm). The change in fluorescence was calculated by subtracting the fluorescence at 0 h from the fluorescence measured at 3 h. Data Statistical Analysis Data are shown as the mean ± SD for the indicated number of samples. A significant difference test was conducted using Student t-test between the 2 groups. For data over 3 groups, multiple comparison tests were performed using the Tukey-Kramer method. In all tests, the values were judged to be statistically significant when the hazard value was <0.05. Results AGP Distribution in Tumor Tissue of C26-Bearing Mice VivoTag-S® 750 labeled AGP was intravenously administered to mice-bearing murine colon cancer C26 cells (mean size: 200 mm3), and the tumor distribution of AGP was then evaluated by IVIS at 6 h after administration. A marked VivoTag-S® 750-derived fluorescence was observed at the tumor site (Fig. 1a). 125I-labeled AGP was also intravenously administered to the same type of tumor-bearing mice as above. The objective here was to determine the extent that AGP migrates to tumor tissue compared with HSA which has been confirmed to function as a carrier of anticancer drugs in clinic. Similar to the imaging data, a high level of radioactivity was detected in the tumor tissue (Fig. 1b). In addition, the radioactivity of AGP was significantly higher than that of HSA. These collective data indicate that a larger fraction of the administered AGP was distributed to tumor tissue compared with HSA.
Figure 1. Distribution of AGP to tumor tissue. (a) Visualization of the tumor tissue distribution of AGP by optical imaging. VivoTag-S® 750-labeled AGP was intravenously injected into C26 tumor-bearing mice, and its dynamic biodistribution was determined by IVIS at 6 h postinjection. The circle denotes the tumor region. (b) 125I-labeled proteins (AGP and HSA) were intravenously injected into C26 tumor-bearing mice. Subsequently, tumor grafts and other organs were collected at 6 h postinjection for radioactivity determination. Results are the means ± SD (n ¼ 4). *p < 0.05 versus HSA.
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Cell-Penetrating Properties of AGP Into Tumor Cells We next examined whether AGP was taken up into tumor cells using MCF-7 (human breast carcinoma), HepG2 (human hepatocellular carcinoma), C26 (murine colon carcinoma), and Hela (human cervical carcinoma) cells by fluorescence-activated cell sorting analysis. When FITC-labeled AGP (FITC-AGP) was added to tumor cell cultures at 37 C (Fig. 2a), the FITC-derived fluorescence was
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observed in all types of cancer cells that were used in this study. A particularly high fluorescence was observed in MCF-7 and HepG2 cells. To further investigate this observation, intracellular imaging of FITC-AGP was conducted against MCF-7 and HepG2 cells by confocal microscopy. Again, a high level of FITC-derived fluorescence was observed in the interior of MCF-7 and HepG2 cells (Fig. 2b). These results suggest that AGP was taken up by tumor cells at 37 C. To further examine whether intracellular migration of
Figure 2. The incorporation of AGP into tumor cells. (a) Four types of tumor cells were incubated with FITC-AGP (150 mg/mL) for 2 h at 37 C in serum-free medium. The intracellular incorporation of FITC-AGP was determined by flow cytometry. The results are the mean ± SD (n ¼ 4). **p < 0.01 versus FITC-AGP (). (b) MCF-7 and HepG2 cells were incubated with FITC-AGP (150 mg/mL) for 2 h at 37 C in serum-free medium. Hoechst 33,342 (blue) was used to visualize nuclei. Intracellular localization of FITC-AGP was analyzed with confocal microscopy. (c) MCF-7 and HepG2 cells were incubated with FITC-AGP (150 mg/mL) for 0.5, 1, and 2 h at 37 C or 4 C. (d) MCF-7 and HepG2 cells were pretreated with chlorpromazine 10 mg/mL (60 min) or methyl-b-cyclodextrin 5 mM (30 min), EIPA 100 mM (30 min), and subsequently treated with FITC-AGP (150 mg/mL) for 30 min at 37 C. Inhibitor () was used as a control. Results are the mean ± SD (n ¼ 4). **p < 0.01 versus inhibitor (), *p < 0.05 versus inhibitor ().
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Figure 3. A conformational change from b-sheet to a-helix on the migration of AGP to tumor cells. (a) Alignment of the amino acid sequence of AGP. Putative portions of a-helix site deduced from a primary structure are shown in under line. (b) Far UV CD spectra of AGP derived peptide in the PBS (dashed line) or butanol (solid line). (c) MCF-7 and HepG2 cells were incubated with FITC-AGPederived peptide (10 mM) for 2 h at 37 C. Intracellular incorporation of FITC-peptide was analyzed by flow cytometry. Results are the means ± SD (n ¼ 4). **p < 0.01 versus FITC-peptide ().
AGP was energy dependent, the same experiments were carried out at 4 C. In contrast to 37 C, almost no fluorescence intensity was observed at 4 C (Fig. 2c). These data suggest that AGP is incorporated into the tumor cells in an energy-dependent manner, possibly via an endocytosis pathway. We also attempted to identify which endocytosis pathway was involved in the intracellular migration of AGP by using the following endocytosis inhibitors: CPZ, a clathrin-mediated endocytosis inhibitor; methyl-b-cyclodextrin (Met-b-CyD), a caveolae/ lipid raft inhibitor27; and EIPA, a macropinocytosis inhibitor. In the case of MCF-7 cells, the intracellular fluorescence derived from FITC-AGP was decreased to approximately 50% and 77% in the presence of CPZ and EIPA, respectively (Fig. 2d); whereas, the FITCAGP fluorescence was decreased to 28% by the Met-b-CyD treatment in HepG2 cells (Fig. 2e). These results suggest that clathrin and macropinocytosis pathways might be involved in the AGP uptake by MCF-7 cells, whereas the caveolae/lipid raftsemediated pathway could contribute to the endocytosis of AGP in HepG2 cells.
a-helix Formation of the AGP-Derived Peptide and Its Cell Penetration Into Tumor Cells Because AGP undergoes a dynamic conformational change from
b-sheet to a-helix when it interacts with both artificial and bio-
logical membranes,24-26 we hypothesized that this conformational change may contribute to the intracellular transfer of AGP into tumor cells. To explore this possibility further, we selected a peptide sequence from the primary structure of AGP (amino acid position 135-148; KEQLGEFYEALDCL) as a candidate for the synthesis of a cell-penetrating peptide because this primary structure region of AGP was reported previously to possess a high ability to form an a-helix (Fig. 3a).25 This AGP-derived peptide was synthesized with a his-tag and used in the subsequent experiments.
CD spectra of the AGP-derived peptide was measured in both 20 mM PBS (pH7.4) and 100% butanol because the secondary structure of AGP was largely transformed made to a b-sheet to an a-helix structure when it was dissolved in butanol. As shown in Figure 3b, AGP-derived peptide exhibited the peaks of CD spectra at 209 nm and 222 nm in PBS solution, suggesting that this peptide formed ahelix.28 Such a phenomena was enhanced in butanol solution compared with above situation. This suggests that AGP-derived peptide favorably form a-helix in the hydrophobic condition. A fluorescence-activated cell sorting analysis was then performed to evaluate the cell-penetrating ability of the FITC-labeled AGP-derived peptide in MCF-7 and HepG2 cells. The results indicated that FITC-derived fluorescence was observed in both MCF-7 and HepG2 cells (Fig. 3c). Antitumor Activity of Paclitaxel-Loaded AGP Finally, to examine whether AGP functions as a DDS carrier, AGP loaded with anticancer agents was prepared, and its antitumor effects were evaluated using MCF-7 and HepG2 cells. In the case of AGP, the anticancer agents can be loaded by 2 different modes, namely noncovalent complex formation at ligand binding pocket or by covalent attachment to amino acid residues on the surface of the AGP. As an example of the noncovalently binding mode, we examined the effect of paclitaxel which has a high binding affinity to AGP and exerts an antitumor effect when entering cells. In this experiment, paclitaxel was added to an AGP solution at a paclitaxel:AGP ratio of 1:2 to minimize the presence of free paclitaxel. In fact, free paclitaxel was undetectable in the filtrate by HPLC analysis after ultrafiltration was performed on this 1:2 ratio of paclitaxel:AGP mixture. The viability of MCF-7 and HepG2 cells was then measured in the presence of paclitaxel-loaded AGP. In this experiment, paclitaxel alone and AGP alone were used as controls. As previously reported,
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Figure 4. AGP as an anticancer drug carrier (drug loading mode; complexation). Cytotoxicity of paclitaxel-AGP complex on tumor cells. MCF-7 and HepG2 cells were treated with AGP, paclitaxel, or paclitaxel-AGP for 48 h. Results are the means ± SD (n ¼ 4). **p < 0.01 versus control.
incubation with paclitaxel alone caused a marked suppression in the viability of MCF-7 cells (Fig. 4). Interestingly, paclitaxel-loaded AGP also significantly reduced cell viability to nearly the same extent of paclitaxel alone (Fig. 4). By contrast, AGP alone had no effect on the viability of MCF-7 cells. Similar results were also obtained when HepG2 cells were used (Fig. 4).
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chains,17 it is also possible that recognition of AGP by CD169 may mediate the active distribution of AGP to tumor tissue. The results reported herein also confirms that AGP is incorporated into 4 types of tumor cells, via endocytosis pathways including clathrin, macropinocytosis, and caveolae/lipid raft mediated routes, depending on the type of tumor cells. The findings reported here indicate that the contribution of these pathways are dependent on the tumor cell type, where clathrin and macropinocytosis are involved in the intracellular uptake of AGP into MCF-7 cells, whereas AGP migrates into HepG2 cells via a caveolae/ lipid raftemediated pathway. In fact, it was possible to deliver both paclitaxel or NO-loaded AGP to the tumor cells resulting in antitumor activities against both MCF-7 and HepG2 cells. Peptides with a-helix forming ability have recently been used in the DDS field, especially to allow molecules to escape from endosomes to the cytosol compartment.33,34 We previously demonstrated that AGP contains b-sheet rich structure in aqueous solution but transforms to a-helix structure when it interacts with a membrane and this structural transition becomes obvious in an acidic environment.24-26 Such a structural transition is similar to melittin, a major pain producing substance of the honeybee.35 Melittin has recently been evaluated as a cell-penetrating protein for such a process through the formation of an a-helix structure.36,37 Actually, the AGP-derived peptide, which is predicted to possess a propensity
Antitumor Activity of NO-Loaded AGP As an example of a covalently binding mode, we used NO because it exhibits excellent antitumor effects.29,30 We prepared Poly-SNO-AGP in which an average of 3.3 NO molecules were covalently bound to the newly introduced thiol groups on AGP and also examined its antitumor ability against MCF-7 and HepG2 cells. As shown in Figure 5a, Poly-SNO-AGP reduced the viability of both types of tumor cells in a concentration-dependent manner. The intracellular NO levels were also measured under the same conditions using an NO detectable fluorescence probe (DAF-FM DA). As shown in Figure 5b, the intracellular fluorescence intensity increased with increasing levels of Poly-SNO-AGP, indicating that NO was delivered to the cells in a Poly-SNO-AGP dose-dependent manner. Interestingly, there was a negative relationship between cell viability and the fluorescence intensity derived from DAF-FM DA (Fig. 5c). This suggests that intracellularly delivered NO by Poly-SNO-AGP induced tumor cell death.
Discussion The characteristics of an effective DDS carrier for cancer therapeutics include the following 4 characteristics: (1) biocompatibility, (2) drug loading ability, (3) tumor tissue distribution, and (4) tumor cell-penetrating ability (for drugs that act inside tumor cells). In this research, we examined the suitability of AGP as a DDS carrier of anticancer drugs, because it is a drug carrier in plasma. As mentioned in the introduction section, intravenous administration of exogenous AGP has been reported to result in higher AGP distribution to tumor tissue than other organs in rats bearing the sarcoma cells.21 The present study also found that AGP was preferentially distributed to tumor tissue on mice-bearing C26 cells than HSA which currently in use as a DDS carrier for anticancer drugs.13 Because the molecular weight of AGP is approximately 44 kDa, its size is sufficient to allow it to be transported via the EPR effect.8 It is possible that the EPR effect is involved in the tumor tissue distribution of AGP. In addition, a membrane surface lectin (CD169) that strongly binds to sialic acid units as an endogenous substrate in the tumor tissue has been reported recently.31,32 Because AGP contains a large number of sialic acids at its glycan
Figure 5. AGP as an anticancer drug carrier (drug loading mode; Chemical modification). (a) Cytotoxicity of Poly-SNO-AGP on tumor cells. MCF-7 and HepG2 cells were treated with various concentration of Poly-SNO-AGP for 48 h. Results are the mean ± SD (n ¼ 4). **p < 0.01 versus 0 mM NO. (b) Intracellular delivery of NO by Poly-SNO-AGP on tumor cells. MCF-7 and HepG2 cells were pretreated with DAF-FM DA (10 mM) for 30 min and treated with various concentrations of Poly-SNO-AGP for 2 h. Results are the means ± SD (n ¼ 4). **p < 0.01 versus 0 mM NO. (c) Relationship between cell viability and fluorescence intensity.
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to form an a-helix structure, adopted an a-helix structure when present in a hydrophobic environment such as butanol solution and was transferred into the MCF-7 and HepG2 cells. These findings suggest that in addition to endocytosis pathway, the cellpenetrating property of AGP can be partially attributed to the peptide regions of the molecule with a-helix forming ability as like melittin. Moreover, such a cell-penetrating mechanism may also contribute to the endosomal escape of such a molecule because the b-sheet to a-helix transition of AGP is facilitated by the interaction with the negatively charged membrane.24 Among the anticancer drugs that are currently in use, nabpaclitaxel is a DDS product in which paclitaxel is bound to HSA.20 Because nab-paclitaxel exerts excellent antitumor effects, it is widely used in the treatment of various types of refractory tumors, including breast cancer, gastric cancer, nonesmall-cell lung cancer, and pancreatic cancer. However, the development of a new type of therapeutic which is superior to nab-paclitaxel is urgently needed for the treatment of refractory tumors. One solution for this problem is to explore and develop a new DDS carrier for paclitaxel. Because the properties of AGP include a higher tumor distribution and higher affinity for paclitaxel than HSA, a paclitaxel-loaded nanoparticle based on AGP might show better therapeutic effects than nab-paclitaxel. Moreover, polymerization and PEGylation of AGP or a nanoparticle derived from it could facilitate further tumor distribution via enhancing the EPR effect. Alternatively, it would be expected that the more active targeting of AGP to tumor tissue could be achieved by the attachment of a homing substance that selectively recognizes the components of tumor tissue. In addition, recent basic and clinical studies suggest that AGP itself has antitumor activities, including antimetastasis and antiinvasive actions.38,39 For example, the expression level of AGP decreased in tumor tissue in patients with hepatocellular cancer and was negatively correlated with disease progression in patients with intrahepatic metastasis.39 These findings indicate that utilizing AGP as a DDS carrier has the potential for exerting dual functions with anticancer activity and would allow the efficient delivery of anticancer agents to tumor tissue. Conclusion In the present study, we found that AGP migrated to tumor tissue compared with HSA which has already been used as carrier of anticancer drug in the clinic and also has the ability to migrate into tumor cells. Consequently, AGP loaded with anticancer agents caused the tumor cell death. These findings indicate that AGP has the potential to function as a DDS carrier of anticancer drugs although there is a need for further in vivo experiments. Acknowledgments This study was funded by the Research Foundation for Pharmaceutical Sciences, a Grant-in-Aid Scientific Research from the Japan Society for the Promotion of Science (KAKENHI 15H04758), and the Takeda Science Foundation, Japan. References 1. Dobbelstein M, Moll U. Targeting tumour-supportive cellular machineries in anticancer drug development. Nat Rev Drug Discov. 2014;13(3):179-196. 2. Wan L, Pantel K, Kang Y. Tumor metastasis: moving new biological insights into the clinic. Nat Med. 2013;19(11):1450-1464. 3. Niero EL, Rocha-Sales B, Lauand C, et al. The multiple facets of drug resistance: one history, different approaches. J Exp Clin Cancer Res. 2014;33:37. 4. Minko T, Dharap SS, Pakunlu RI, Wang Y. Molecular targeting of drug delivery systems to cancer. Curr Drug Targets. 2004;5(4):389-406. 5. Jones T, Saba N. Nanotechnology and drug delivery: an update in oncology. Pharmaceutics. 2011;3(2):171-185.
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