Article pubs.acs.org/molecularpharmaceutics
Cannabinoid CB2 Receptor as a New Phototherapy Target for the Inhibition of Tumor Growth Ningyang Jia,†,‡,▽ Shaojuan Zhang,‡,§,▽ Pin Shao,‡ Christina Bagia,∥ Jelena M. Janjic,∥ Ying Ding,⊥ and Mingfeng Bai*,‡,#,¶ †
Department of Radiology, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai 200438, P. R. China ‡ Molecular Imaging Laboratory, Department of Radiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15219, United States § Department of Diagnostic Radiology, The First Hospital of Medical School, Xi’an Jiaotong University, Xi’an, Shaanxi 710061, P. R. China ∥ Graduate School of Pharmaceutical Sciences, Mylan School of Pharmacy, Duquesne University, Pittsburgh, Pennsylvania 15282, United States ⊥ Department of Biostatistics, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, United States # University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania 15232, United States ¶ Department of Bioengineering, University of Pittsburgh, Pittsburgh, Pennsylvania 15232, United States S Supporting Information *
ABSTRACT: The success of targeted cancer therapy largely relies upon the selection of target and the development of efficient therapeutic agents that specifically bind to the target. In the current study, we chose a cannabinoid CB2 receptor (CB2R) as a new target and used a CB2R-targeted photosensitizer, IR700DX-mbc94, for phototherapy treatment. IR700DX-mbc94 was prepared by conjugating a photosensitizer, IR700DX, to mbc94, whose binding specificity to CB2R has been previously demonstrated. We found that phototherapy treatment using IR700DX-mbc94 greatly inhibited the growth of CB2R positive tumors but not CB2R negative tumors. In addition, phototherapy treatment with nontargeted IR700DX did not show significant therapeutic effect. Similarly, treatment with IR700DX-mbc94 without light irradiation or light irradiation without the photosensitizer showed no tumor-inhibitory effect. Taken together, IR700DXmbc94 is a promising phototherapy agent with high target-specificity. Moreover, CB2R appears to have great potential as a phototherapeutic target for cancer treatment. KEYWORDS: targeted therapy, phototherapy, CB2 receptor, cancer, cannabinoid
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INTRODUCTION Photodynamic therapy (PDT) has been demonstrated to be a noninvasive and effective therapeutic strategy for cancer treatment. The process of PDT involves the activation of light-sensitive photosensitizers (PS) by light irradiation at specific wavelengths,1 which leads to the production of reactive oxygen species (ROS) that cause cancer cells death.2 PDT has been clinically approved to treat several types of cancers, such as esophageal cancer, nonsmall cell lung cancer, and precancerous changes of Barrett’s esophagus and skin. Moreover, many clinical trials are currently under way to study the potential of PDT in the treatment of brain, head and neck, prostate, bladder, and skin cancers.3 Despite the promise of PDT, most available PDT photosensitizers can also cause phototoxicity to normal tissues due to the lack of tumor specificity. To overcome this limitation, much effort has been invested to develop tumor-targeted photosensitizers.4 For example, Mitsunaga et al. reported photoimmunotherapy © 2014 American Chemical Society
(PIT) based on antibodies against human epidermal growth factor receptor (EGFR) coupled with a phthalocyanine dye IR700DX, which showed phototoxic efficacy when bound to EFGR on the cell membrane.5 In addition to tumor-associated antibodies, small molecules and peptides such as arginine oligopeptide6 and folate2 have also been used in PDT as targeting molecules. In particular, many synthetic peptides have been utilized to develop targeted PDT agents. For example, Taratula et al. attached luteinizing hormone-releasing hormone (LHRH) peptide to a phthalocyanine-encapsulated dendrimer for targeted PDT treatment of ovarian cancer that overexpresses the LHRH receptor.7 In a similar way, Master and coworkers coupled the GE-11 peptide to the surface of Received: Revised: Accepted: Published: 1919
March 10, 2014 April 24, 2014 April 29, 2014 April 29, 2014 dx.doi.org/10.1021/mp5001923 | Mol. Pharmaceutics 2014, 11, 1919−1929
Molecular Pharmaceutics
Article
Molecular Probes, a 1 O 2 indicator) according to the manufacturer’s instructions. In addition, 1,3-dimethyl-2-thiourea (DMTU, 50 mM, a free radicals scavenger) or sodium azide (NaN3, 50 mM, a 1O2 quencher) was added with IR700DXmbc94 to quench the production of free radicals and 1O2, respectively. Fluorescence spectra of APF (excitation/emission 480/500 nm) or SOSG (Excitation/emission 500/520 nm) were recorded using SynergyTM H4 Hybrid Multi-Mode Microplate Reader. To demonstrate that IR700DX-mbc94 produces ROS in a light-dose-dependent manner, we measured ROS production with an increasing dose of light illumination (during a period of 20 min at a 5 min interval). We then used APF to determine intracellular ROS generation of IR700DX-mbc94-mediated phototherapy. CB2 mid-DBT cells were treated with 5 μM of IR700DX-mbc94 for 6 h. Unbound IR700DX-mbc94 was removed by washing cells once. ROS production indicated by APF (10 μM) was compared among no treatment, IR700DX-mbc94 alone, and IR700DX-mbc94 + light irradiation (48 J/cm2). Fluorescence intensity was recorded using SynergyTM H4 Hybrid MultiMode Microplate Reader. To study the targeted delivery of IR700DX-mbc94, CB2 midDBT cells were treated with 5 μM IR700DX-mbc94 or IR700DX for 6 h before fluorescence intensity was measured. Unbound IR700DX-mbc94 or IR700DX was removed by washing cells with medium once. The amount of bound IR700DX-mbc94 or IR700DX was measured by reading fluorescence intensity using SynergyTM H4 Hybrid MultiMode Microplate Reader. Furthermore, to study whether DMTU or NaN3 could effectively quench ROS products in living cells, 50 mM DMTU or NaN3 was coincubated with IR700DX-mbc94 for 6 h to inhibit IR700DX-mbc94-induced cell death. Cell death was compared among three groups of CB2-mid DBT cells: cells treated with IR700DX-mbc94 + irradiation (48 J/cm2), cells treated with IR700DX-mbc94 + irradiation (48 J/cm2) + DMTU, and cells treated with IR700DX-mbc94 + irradiation (48 J/cm2) + NaN3. To account for the cell death caused by DMTU and NaN3, cell death was normalized to the corresponding treatment without light irradiation (for example, the cell death caused by IR700DX-mbc94 + irradiation + DMTU was normalized to that caused by IR700DX-mbc94 + DMTU). Cytoxicity was evaluated using a CellTiter-Glo Luminescent Cell Viability Assay kit (Promega) according to the manufacturer’s instructions. The CellTiter Glo Luminescent Cell Viability assay measures the number of viable cells based on the quantitation of the ATP present. The luminescence signal was detected at 528 nm. To study whether enhanced ROS production by IR700DXmbc94 compared to that of IR700DX contributed to the phototherapy effects, we evaluated the photobleaching of IR700DX-mbc94 and IR700DX, respectively. IR700DXmbc94 (5 μM) or IR700DX (5 μM) in phosphate buffered saline (PBS) was exposed to light illumination. Fluorescence intensity was recorded after increasing light irradiation (0, 24, 48, 72, 96, 120, and 144 J/cm2). Furthermore, to demonstrate whether the photobleaching profile changed after IR700DXmbc94 bound to cells, CB2-mid DBT cells were seeded into 96 well plates and treated with 5 μM IR700DX-mbc94 for 6 h. Medium with unbound IR700DX-mbc94 was transferred to new wells, and 100 μL of fresh medium was added into wells with remaining cells. Both cells (with bound IR700DX-mbc94) and medium (with unbound IR700DX-mbc94) were exposed
phthalocyanine-incorporated micelles and evaluated the efficacy of the resulting PDT nanomedicine in a xenograft mouse head and neck tumor model.8 Compared with antibodies, small targeting molecules have the advantage of fast clearance, but attachment to a large photosensitizer often compromises the binding. Despite such advances, PDT with high target specificity still remains challenging as unbound PDT photosensitizers can still cause damage to normal tissues. As such, there is urgent need to improve PDT efficacy by developing highly target-specific PDT photosensitizers. In our recent study, we developed the first type 2 cannabinoid receptor (CB 2 R)-targeted photosensitizer, IR700DX-mbc94, based on a phthalocyanine dye and a functional CB2R-targeted molecule. CB2R is considered as an attractive target for cancer treatment. Under healthy conditions, high CB2R expression is only present in immune cells.9 However, CB2R expression is up-regulated in many types of cancers, such as prostate, skin, liver, brain, colon, and breast cancers.10−14 The up-regulation in cancer cells and low expression in corresponding healthy tissues afford CB2R great therapeutic potential for cancer treatment with low side effects. We found that phototherapy treatment with IR700DX-mbc94 lead to efficient cell death in CB2R positive cancer cells. However, unlike typical PDT agents, unbound IR700DXmbc94 did not cause significant cell death.15 These results indicate the high target-specificity of IR700DX-mbc94. To further evaluate the potential of IR700DX-mbc94 as a PDT agent, here we performed comprehensive in vivo phototherapy study using tumor mouse models. We found that phototherapy treatment with IR700DX-mbc94 efficiently inhibited the growth of CB2R positive (CB2R+) tumors, whereas the same treatment with nontargeted IR700DX did not show significant therapeutic effect. Furthermore, growth of CB2R negative (CB2R-) tumors was not affected after phototherapy with IR700DX-mbc94. These results suggest that IR700DX-mbc94 is a promising PDT agent with high target specificity.
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EXPERIMENTAL SECTION Synthesis of IR700DX-mbc94. IR700DX-mbc94 was synthesized by coupling IR700DX with a conjugable CB2R ligand mbc94 using the previously reported method.15 Cell Culture. CB2-mid DBT (delayed brain tumor) cells as well as wild type (WT) DBT cells were used for both in vitro and in vivo studies. CB2-mid DBT is a transfected mouse malignant astrocytoma cell line that expresses CB2R at the endogenous levels.16 WT-DBT is a DBT cell line without CB2R expression. Both CB2-mid DBT and WT-DBT cells were cultured using the same methods as those described in our previously published work.17 Briefly, cells were cultured in DMEM containing 10% fetal bovine serum, 4 mM glutamine, 100 units/mL penicillin and 100 μg/mL streptomycin. Mechanism Study of Phototherapy Using IR700DXmbc94. We first tested the types of ROS produced in cell-free solution conditions. For phototherapy treatment, 5 μM of IR700DX-mbc94 or free IR700DX dye was irradiated for 20 min with light from an LED light source (L690-66-60, Marubeni America Co.) at wavelengths of 670−710 nm (peak at 690 nm) and a power density of 40 mW/cm2, as measured with an optical power meter (PM100, Thorlabs). ROS production was determined using 10 μM aminophenyl fluorescein (APF, Molecular Probes, a free radical indicator) and 10 μM singlet oxygen (1O2) sensor green (SOSG, 1920
dx.doi.org/10.1021/mp5001923 | Mol. Pharmaceutics 2014, 11, 1919−1929
Molecular Pharmaceutics
Article
Figure 1. Production of ROS in IR700DX-mbc94 or IR700DX mediated phototherapy. IR700DX-mbc94 (5 μM) or IR700DX (5 μM) was irradiated for 20 min with light at wavelengths of 670−710 nm (peak at 690 nm) and a power density of 40 mW/cm2. (A) Production of free radicals from IR700DX-mbc94 was determined using 10 μM of APF. DMTU (50 mM) was used to quench free radicals. IR700DX-mbc94 or light exposure alone was used as the negative control. (B) Production of 1O2 from IR700DX-mbc94 was determined using 10 μM SOSG. NaN3 (50 mM) was used to quench 1O2. IR700DX-mbc94 or light exposure alone was used as the negative control. (C) Production of free radicals from IR700DX was determined using 10 μM APF. DMTU (50 mM) was used to quench free radicals. IR700DX or light exposure alone was used as the negative control. (D) Production of 1O2 from IR700DX was determined using 10 μM SOSG. NaN3 (50 mM) was used to quench 1O2. IR700DX or light exposure alone was used as the negative control.
irradiation. CB2 mid-DBT cells were treated with 0, 1.56, 3.125, 6.25, 12.5, 25, 50, an 100 μM IR700DX-mbc94 for 24 h in the absence of light irradiation. A CellTiter Glo Luminescent Cell Viability Assay kit was used for measuring cell viability. We also evaluated cell viability upon the treatment of total IR700DXmbc94 as well as bound IR700DX-mbc94 (unbound IR700DXmbc94 was removed by washing cells with medium once). Tumor Mouse Model. The animal studies have been approved by The University of Pittsburgh Institutional Animal Care and Use Committee (IACUC). Female athymic nude mice at 6 to 8 weeks old were purchased from Jackson Laboratories. One million CB2-mid DBT or WT-DBT cells were injected subcutaneously into the right flank of the mice. The tumor sizes were measured18 by a caliper and calculated as the volume = (tumor length) × (tumor width)2/2. Tumor volume was monitored every day. In Vivo CB2 Receptor-Targeted Phototherapy Study. In vivo phototherapy experiments were carried out at approximately 7 days after cell injection. Tumors reaching approximately 40−50 mm3 in volume were selected for study. To determine the optimal interval between the PS administration and light exposure, 7 tumor bearing mice were i.v. injected with 10 nmol of IR700DX-mbc94. At preinjection and 30 min, 1 h, 2 h, 6 h, 12 h, and 24 h postinjection, mice were euthanized, and tumors were removed. Similarly, other 5 tumor-bearing mice were i.v. injected with 10 nmol of IR700DX free photosensitizer and euthanized at preinjection and 1 min, 30 min, 2 h, and 6 h postinjection to harvest tumors. An Odyssey infrared imaging system (LI-COR Biosciences) was used to quantify fluorescence signal in the tumors. Fluorescence signal normalized by tumor weight was compared
under light irradiation. Fluorescence intensity was measured after increasing light irradiation (0, 24, 48, 72, 96, 120, and 144 J/cm2). The photobleaching curve with time was fitted, and the slope was calculated using Prism software (GraphPad, San Diego, CA). Cell Death Induced by IR700DX-mbc94 Phototherapy. CB2-mid DBT cells were seeded into 35 mm MatTek dishes (MatTek Corporation). Cells were treated with IR700DX-mbc94-mediated phototherapy, IR700DX-mbc94 alone, light irradiation alone, and IR700DX-mediated phototherapy. For the treatment of IR700DX-mbc94 or IR700DXmediated phototherapy, cells were incubated with 5 μM IR700DX-mbc94 or IR700DX for 6 h and 1 μg/mL of DAPI for 30 min. Cells were washed 2 times before being exposed to continuous light illumination (48 J/cm2). The morphological changes in living cells were recorded by continuous videos using a Zeiss Axio Observer fluorescence microscopy system. Cells were kept at room temperature during the video recording. Treatment of IR700DX-mbc94 alone, light irradiation alone, and IR700DX-mediated phototherapy were used as the negative controls. At the starting and ending point of videos, fluorescence and differential interference contrast (DIC) images were captured. Fluorescence images with IR700DX-mbc94 were collected with a Cy5 filter set (excitation/emission: 625−655 nm/665−715 nm). Nuclear staining was obtained with a DAPI filter set (excitation/ emission: 335−383 nm/420−470 nm). DIC images were obtained through Trans light DIC. Determination of the Safe Dose of IR700DX-mbc94 for in Vivo Phototherapy Study. We carried out an in vitro cytotoxicity study for IR700DX-mbc94 in the absence of light 1921
dx.doi.org/10.1021/mp5001923 | Mol. Pharmaceutics 2014, 11, 1919−1929
Molecular Pharmaceutics
Article
Figure 2. IR700DX-mbc94 produced ROS in a light-dose-dependent manner. (A) Light-dose-dependent production of free radicals. (B) Light dosedependent production of 1O2. (C) CB2-mid DBT cells treated with DMTU (50 mM) or NaN3 (50 mM) significantly inhibited IR700DX-mbc94 induced phototoxicity. Data points in C represent the mean ± SEM based on triplicate samples. ** p < 0.01 and *** p < 0.001.
Statistical Analysis. All of the data are given as the mean ± standard error of the mean (SEM). Statistical analysis was performed using a two-tailed two sample Student’s t test (comparing 2 groups) if measurements were taken at only one time point or repeated measures ANOVA (Analysis of Variance) if measurements were taken at multiple time points, with p values
