Photodynamic Oncotherapy Mediated by Gonadotropin-releasing

ABSTRACT: Here, we report photodynamic oncotherapies mediated by gonadotropin-releasing hormone (GnRH) receptors. We synthesized conjugates 1 and 2 by...
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Brief Article Cite This: J. Med. Chem. 2017, 60, 8667-8672

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Photodynamic Oncotherapy Mediated by Gonadotropin-Releasing Hormone Receptors Peng Xu,† Yuhua Jia,†,‡ Yongshuai Yang,†,‡ Jincan Chen,† Ping Hu,† Zhuo Chen,*,† and Mingdong Huang*,†,§ †

State Key Laboratory of Structural Chemistry and Danish-Chinese Centre for Proteases and Cancer, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, Fujian 350002, P. R. China ‡ College of Life Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, P. R. China § College of Chemistry, Fuzhou University, Fuzhou, Fujian 350116, P. R. China S Supporting Information *

ABSTRACT: Here, we report photodynamic oncotherapies mediated by gonadotropin-releasing hormone (GnRH) receptors. We synthesized conjugates 1 and 2 by coupling zinc phthalocyanine (ZnPc) to GnRH analogues. Compared to unmodified ZnPc, conjugates 1 and 2 exhibited higher and more specific phototoxicities to breast cancer cells. Furthermore, the two conjugates demonstrated excellent antitumor efficacies in a breast cancer-grafted animal model. Biodistribution study suggested the high biosafety of conjugate 2 because of the low retention in brain and skin.

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INTRODUCTION

their excellent photophysical and photochemical properties, including deep skin penetration and low skin burns.14,15 Here, we report the conjugation of ZnPc and two different GnRHRs-targeting peptides (Figure 1) and the antitumor

Targeted cancer therapies cause low systemic toxicities by precisely interfering molecules involved in the processes of tumor growth, progression, and spread.1 Photodynamic therapy (PDT) is a promising therapeutic modality for various tumors and nonmalignant diseases through generating cytotoxic reactive oxygen species (ROS).2,3 PDT also leads to the shutdown of angiogenesis, facilitating the clearance of tumor cells. In addition, PDT induces immune responses, in contrast to the immunosuppressive nature of typical chemotherapy. Tumor targeted PDT has the advantage of reducing damages on healthy tissues.4,5 Gonadotropin-releasing hormone (GnRH) is a hypothalamic peptidic hormone with a sequence of EHWSYGLRPG, which was responsible for the release of follicle-stimulating hormone and luteinizing hormone from the anterior pituitary via its Gprotein-coupled GnRH receptors (GnRHRs).6 GnRHRs are also aberrantly expressed on the surfaces of sex-hormonedependent tumors, like breast and ovarian tumors,6 and thus can be a therapeutic target. Various cytotoxic agents targeting GnRHRs have been developed to enhance the selectivity of cancer therapies.7−9 However, one concern for such agents is the risk of penetrating blood−brain barrier (BBB) and disturbing the hypothalamic functions. In our previous studies on zinc phthalocyanine (ZnPc) type photosensitizers,10−12 we found no accumulations in brain.13 The ZnPc derivatives have been demonstrated as potent medicinal photosensitizers due to © 2017 American Chemical Society

Figure 1. Chemical structures of conjugates 1 and 2.

efficacies of GnRHRs-mediated PDT in vitro and in vivo. Conjugate 1 used the native GnRH peptide.10 In contrast, conjugate 2 had an improved design using a GnRH analogue (EHWSY[D-Lys]LRPG), providing an anchoring point at side chain of D-Lys6, which substituted the original Gly6. In this case, the integrity of the GnRH moiety was better maintained, and the D-lysine prevents the proteolytic degradation in plasma.9 In addition, a lysosomally cleavable hexapeptide (GGGFLG)16 was selected as a linker between ZnPc and the GnRH moiety. Received: August 22, 2017 Published: October 2, 2017 8667

DOI: 10.1021/acs.jmedchem.7b01216 J. Med. Chem. 2017, 60, 8667−8672

Journal of Medicinal Chemistry

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Brief Article

RESULTS AND DISCUSSION The photophysical and photochemical properties of the unmodified ZnPc conjugate 1 and conjugate 2 were demonstrated in Table 1. Conjugates 1 and 2 showed similar

conjugates 1 and 2 showed similar phototoxicities to the healthy HELF cells and showed significantly enhanced phototoxicities to the three breast cancer cell lines, indicating that the conjugation with GnRH-peptides specifically enhanced the phototoxicities to breast cancer cells (Figure 2A−D). Notably, conjugate 2 exhibited higher phototoxicities to all the breast cancer cell lines than conjugate 1 did. We next determined the cellular uptake of conjugates 1 and 2 in 4T1 cells and found that conjugate 2 showed approximately 2-fold higher time- and dose- dependent cellular-uptake (Figure S7), likely due to the higher affinity of conjugate 2 to GnRHRs on tumor surfaces. Besides, we also determined the ROS generation of conjugates 1 and 2 by using 2′,7′-dichlorofluorescin diacetate (DCFH-DA) as an ROS sensitizer.17 Conjugate 2 (Figure 2F) released >2.5-fold higher amount of ROS than conjugate 1 did at 10 μM (Figure 2E). Furthermore, the higher ROS amount was mostly due to the higher generation of free radicals, which have been reported as the most efficient cytotoxic agents among all species of ROS.18−20 Thus, the higher phototoxicity of conjugate 2 to 4T1 breast cancer cell line was due to the combination of three reasons: (1) higher cellular uptake, (2) higher ROS generation, and (3) higher percentage of free radicals in ROS. Subcellular localization determination showed that conjugate 2 did not penetrate the nuclei membrane (Figure 3A), which favors the genomic safety during PDT. In addition, conjugate 2 showed accumulation in mitochondria and lysosomes (Figure 3B and Figure 3C), but the accumulation on mitochondria was much higher than that on lysosomes (Figure S8). Photosensitizers in mitochondria are suggested to trigger apoptotic cascades21,22 and perform much higher antitumor efficacies than photosensitizers in other organelles do.23,24 Next, we used the in vivo imaging analysis to study the tumor-targeting capabilities of conjugates 1 and 2 in 4T1 cancer-bearing mice (Figure 4). To avoid the interruption of the autofluorescence in hearts and lungs, tumor cells were xenografted subcutaneously at the backs of mice. The

Table 1. Photodynamic Parameters, Fluorescence Quantum Yields (Φf), Fluorescence Decay Time (τs), and Singlet Oxygen (1O2) Quantum Yields (ΦΔ) of Unmodified ZnPc and Conjugates 1 and 2 in DMSOa ZnPc 1 2

Φf

τs (ns)

ΦΔ

0.20 0.19 0.21

3.6 3.1 3.3

0.67 0.72 ± 0.02 0.78 ± 0.04

a

Paremeters were determined and calculated as described in Figure S4, Figure S5, and Table S1. ΦΔ values of conjugate 1 and 2 were presented as the mean ± SD according to four independent experiments with four concentrations of photosensitizers. The data of unmodified ZnPc were according to the reference.10

fluorescence quantum yields (Φf) and fluorescence decay time (τs) compared to the unmodified ZnPc, indicating the conjugation of GnRH-like peptides did not impair the fluorescence generation. However, conjugate 2 showed slightly higher singlet oxygen (1O2) quantum yield (ΦΔ) than unmodified ZnPc and conjugate 1 did. We then studied the antitumor efficacies of the unmodified ZnPc, conjugate 1, and conjugate 2 in a murine breast cancer cell line 4T1, human breast cancer cell lines, MDA-MB-231 and MCF-7, and a healthy cell line, human embryo lung fibroblast (HELF), in vitro. In all the cell lines, none of the three photosensitizers showed measurable dark toxicity (Figure S6 in Supporting Information). With the illumination of 1.5 J/cm2 670 nm laser, unmodified ZnPc showed mostly identical phototoxicities to all the 4 cell lines, indicating the nonselectivity among cells with or without GnRHRs expression (Figure 2). However, in contrast to the unmodified ZnPc,

Figure 2. Antitumor effects of conjugates 1 and 2 in vitro. (A−D) Phototoxicities of unmodified ZnPc (green), conjugate 1 (blue), and conjugate 2 (red) to breast cancer cell lines: 4T1 (A), MDA-MB-231 (B), MCF-7 (C), and healthy cell line HELF (D). (E, F) ROS (red) and free radicals (blue) generation by illuminating conjugates 1 (E) and 2 (F). Values represent the mean of three independent experiments; bars represent the standard deviations (SD). 8668

DOI: 10.1021/acs.jmedchem.7b01216 J. Med. Chem. 2017, 60, 8667−8672

Journal of Medicinal Chemistry

Brief Article

concentrations of photosensitizers in tumor and healthy tissues at the symmetric sites were imaged by capturing the fluorescence signals at 6, 24, 48, and 72 h after intravenous injection. Both conjugates rapidly accumulated in tumor tissues within 6 h and reached the maximum after 24 h. Slight decrease was observed at the time point of 48 h. After 72 h, the concentrations of both conjugates significantly decreased. Compared to conjugate 1, conjugate 2 showed comparable accumulation in tumor tissues but much lower retention in healthy tissues (Figure 4C), indicating the higher selectivity to tumors. We also studied the biodistribution of conjugate 2 (Figure 4D and Figure 4E) and found a consistent low retention in brain (2-fold higher cellular uptake in 4T1 cells in vitro. Furthermore, conjugate 2 performed a 66-fold higher phototoxicity to 4T1 cells than conjugate 1 did in vitro, but the superiority of the antitumor efficacy of conjugate 2 in vivo was much milder. This inconsistency can be explained by the much higher complexity of the PDT processes in vivo. First, tumor cells were exposed in conditioned media with photosensitizers in cellular experiments. Conjugates 1 and 2 directly interacted with the GnRHRs on the tumor surface. In contrast, in animal experiments, photosensitizers were injected into the circulating systems through the tail vein leading to the inevitable clearance by liver and urinary system. Furthermore, other factors that do not exist in cellular levels may also significantly influence the antitumor effects in vivo, like enhanced permeability and retention (EPR) effect, immune responses, and shutdown of the angiogenesis.

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CONCLUSIONS To find high-potency and high-specificity photosensitizers for breast cancers, we covalently conjugated two GnRH analogues to ZnPc in order to target GnRHRs on tumor cells. Both conjugates had remarkable phototoxicity to breast cancer cells in vitro, but conjugate 2 exhibited much higher phototoxicity mainly due to the higher ROS generation. Subcellular localization analysis demonstrated that conjugate 2 majorly located in mitochondria rather than nuclei or lysosomes. Through a breast cancer animal model, the in vivo imaging analysis indicated that both conjugates successfully targeted the tumor tissues, but conjugate 2 showed higher selectivity to 8670

DOI: 10.1021/acs.jmedchem.7b01216 J. Med. Chem. 2017, 60, 8667−8672

Journal of Medicinal Chemistry

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Subcellular Imaging. Intracellular distribution of conjugate 2 was determined in MDA-MB-231 cells with an Olympus FluoView FV100 cofocal laser scanning microscope (Melville, NY, USA) coupled with an inverted microscope with a 60× differential interference contrast oil immersion objective lens. 105 cells were seeded in confocal chamber slides (NEST) for 24 h and consequently incubated with 2 mL of Complete medium containing 10−5.5 M conjugate 2 for 2 h. After being washed with serum free medium, the cells were then stained with the nuclei dye, DAPI, the lysosomes dye, LysoTracker Red, and the mitochondria dye, MitoTracker Green, respectively, according to the operation manuals. Through the confocal microscope, conjugate 2 was imaged with ex633 nm and em680/100 nm; DAPI was imaged with ex405 nm and em435/55 nm, MitoTracker Green and LysoTracker Red were imaged with ex488 nm and em500/100 nm. Optical sections were collected at 0.5 μm intervals with a 2.2 mm pinhole aperture. Digitized images of 512 × 512 pixels were obtained with a pixel size of 0.41 μm. Laser line intensity, photometric gain, FMT (fluorescence molecular tomography) setting, and filter attenuation were kept constant throughout the experiments. In Vivo Imaging Analysis. The 4T1 tumor-bearing mice were randomly divided into three groups (four mice per group) with the equivalent average starting tumor sizes of 350−400 mm3. Conjugates 1 and 2 at dosage of 0.2 μmol/kg in a volume of 8 mL/kg dissolved in saline containing 1% DMSO were treated through tail intravenous injection. A saline-treated group was set as a negative control. After dosing, mice in each group were anesthetized with isoflurane, and the accumulations of conjugates 1 and 2 were monitored at different time points (6, 24, 48, and 72 h) by using a fluorescent molecular tomography FMT 2500TM LX instrument (PerkinElmer, Waltham, MA, U.S.). A 680 nm laser diode was used to excite the photosensitizers, and the regions of interest (ROIs) were scanned with default scan parameters. Fluorescence emission with longer wavelength (690−740 nm) was collected. For quantitation, 1 μM of each conjugate 1 or 2 was used for calibration. The collected images were reconstructed by the software TrueQuant version 3.0 (PerkinElmer. Waltham, MA, USA) to three dimensions and to derive quantitative information in terms of the ZnPc concentrations by creating ROIs around the tumor tissues and healthy tissues. A graphic of a 4T1-bearing mouse treated with saline (1% DMSO) was set as a negative control and compared to a graphic of a mouse treated with conjugate 2 after 24 h in Figure S9. Biodistribution. Kunming mice were randomly divided into four groups (five mice per group) injected intravenously via the caudal vein with conjugate 2 at the dosage of 2 mg/kg in a volume of 0.2 mL of saline containing 1% DMSO per mouse. At 6, 24, 48, 72 h after dosing, mice were sacrificed, and the organs and tissues of interest were resected, washed with saline, and dried. 100 mg of each organ or tissue was homogenized and sonicated with 2.5 mL of lysis buffer (1% NaOH and 1% SDS) for 10 min. The homogenates were centrifuged at 4 °C, and the photosensitizer concentration in the supernatant was determined by the fluorescence of ZnPc (λex = 610 nm and λem = 690 nm). Antitumor Effects in Vivo. The 4T1 tumor-bearing mice were randomly divided into three groups (seven mice per group) with equivalent average starting tumor size (around 150 mm3). Two groups were treated with conjugates 1 and 2 (0.144 and 0.188 mg/kg, 0.08 μmol/kg) in saline containing 1% DMSO via tail intravenous injection. The saline (1% DMSO) treated group was set as the negative control. 24 h after dosing, the mice were illuminated once with a LED light source (670 nm, 300 mW, Sundynamic Inc., Qingdao, China), resulting in a total light dosage of 30 J/cm2. The body weights and tumor volumes were recorded daily throughout the 8-day monitor period. Upon completion of the 8-day PDT treatment, the mice were sacrificed, and the tumors were resected and weighed.

Brief Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b01216. Additional experimental procedures, synthetic scheme, purification and characterization data, dark toxicities results, cellular uptake results, determination of photophysical parameters, supplementary graphics of in vivo imaging, daily measurements of body weights, and photographs of resected tumors (PDF) Molecular formula strings and some data (CSV)

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AUTHOR INFORMATION

Corresponding Authors

*Z.C.: e-mail, [email protected]. *M.H.: e-mail, [email protected]. ORCID

Mingdong Huang: 0000-0002-1377-6786 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Grants 21708043, 81572944, U1405229); CAS/SAFEA International Partnership Program for Creative Research Teams; the Strategic Priority Research; Program and Scientific Equipment Development Project of the CAS (Grant XDA09030307); CAS Cross-Disciplinary & Collaborative Research Team Program.

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ABBREVIATIONS USED PDT, photodynamic therapy; GnRH, gonadotropin-releasing hormone; ZnPc, zinc phthalocyanine; ROS, reactive oxygen species; FMT, fluorescence molecular tomography

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REFERENCES

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DOI: 10.1021/acs.jmedchem.7b01216 J. Med. Chem. 2017, 60, 8667−8672