Suitability of GnRH Receptors for Targeted Photodynamic Therapy in Head and Neck Cancers
Abstract
:1. Introduction
2. Results
2.1. GnRH receptor (GnRH-R) Expression in Patient-Derived Head and Neck Squamous Cell Carcinoma (HNSCC) Samples
2.2. Peptide Synthesis
2.3. GnRH-Protoporphyrin IX Conjugate Synthesis
2.4. Receptor Binding Potency of the Novel Compounds
2.5. UV-Vis Absorbance
2.6. GnRH-R Expression of Detroit-562 Human Pharyngeal Carcinoma Cells
2.7. Optimization of the Irradiation and Incubation Time
2.8. In Vitro Cell Viability Inhibition Effect of the Peptide-PpIX Conjugates
3. Discussion
4. Materials and Methods
4.1. Chemicals
4.2. Patients
4.3. Tissue Microarray (TMA) and Immunohistochemistry
4.4. Statistical Analysis
4.5. Peptide Synthesis
4.5.1. GnRH-I[6D-Lys] (Compound 1)
4.5.2. GnRH-I[4Lys(Bu),6D-Lys] (Compound 2) and GnRH-II[4Lys(Bu),6D-Lys] (Compound 3)
4.5.3. GnRH-III[4Lys(Bu),6Asp(OMe)] (Compound 4)
4.6. Conjugate Synthesis (Compounds 5–8)
4.7. RP-HPLC
4.8. Mass Spectrometry
4.9. Radioligand Binding Assay
4.10. UV-Vis Spectroscopy
4.11. Cell Cultures
4.12. Confocal Laser Scanning Microscopy
4.13. In Vitro Cell Viability Assay
4.14. Statistical Evaluation of In Vitro Viability Data
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
ALA | δ-aminolevulinic acid |
BSA | Bovine serum albumin |
Dau | Daunorubicin |
DBU | 1,8-Diazabicyclo[5.4.0]undec-7-ene |
DCM | Dichloromethane |
Dde | N-(1-(4,4-Dimethyl-2,6-dioxocyclohexylidene)ethyl) |
DIC | N,N′-Diisopropylcarbodiimide |
DIPEA | Diisopropylethylamine |
DMF | N,N-Dimethylformamide |
DSS | Disease-specific survival |
<E | pyroglutamic acid |
EGFR | Epidermal growth factor receptor |
EMEM | Eagle’s Minimum Essential Medium |
FBS | Fetal bovine serum |
Fmoc | 9-Fluorenylmethyloxycarbonyl |
FSH | Follicle stimulating hormone |
GnRH | Gonadotropin-releasing hormone |
GnRH-R | Gonadotropin-releasing hormone receptor |
HOBt | 1-Hydroxybenzotriazole hydrate |
LH | Luteinizing hormone |
Mtt | 4-Methyltrityl |
MTT | 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide |
NHSCC | Head and neck squamous cell carcinomas |
PBS | Phosphate-buffered saline |
PDT | Photodynamic therapy |
PpIX | Protoporphyrin IX |
PyBOP | Benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate |
TFA | Trifluoroacetic acid |
TIS | Triisopropylsilane |
TMA | Tissue Microarray |
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Code | Peptide | Code | Conjugate |
---|---|---|---|
1 | GnRH-I[6D-Lys] | 5 | GnRH-I[6D-Lys(PpIX)] |
2 | GnRH-I[4Lys(Bu), 6D-Lys] | 6 | GnRH-I[4Lys(Bu), 6D-Lys(PpIX)] |
3 | GnRH-II[4Lys(Bu), 6D-Lys] | 7 | GnRH-II[4Lys(Bu), 6D-Lys(PpIX)] |
4 | GnRH-III[4Lys(Bu), 6Asp(OMe)] | 8 | GnRH-III[4Lys(Bu), 6Asp(OMe), 8Lys(PpIX)] |
Code | Compound | IC50 Values/nM | |
---|---|---|---|
Human Pituitary | Human Prostate Cancer | ||
1 | GnRH-I[6D-Lys] | 6.44 ± 0.95 | 4.31 ± 0.83 |
2 | GnRH-I[4Lys(Bu), 6D-Lys] | 9.70 ± 1.07 | 8.61 ± 1.08 |
3 | GnRH-II[4Lys(Bu), 6D-Lys] | 5.56 ± 1.07 | 4.65 ± 0.31 |
4 | GnRH-III[4Lys(Bu), 6Asp(OMe)] | 7.27 ± 0.92 | 6.56 ± 1.11 |
5 | GnRH-I[6D-Lys(PpIX)] | 36.29 ± 3.17 | 42.67 ± 7.04 |
6 | GnRH-I[4Lys(Bu), 6D-Lys(PpIX)] | 42.30 ± 4.27 | 41.80 ± 5.74 |
7 | GnRH-II[4Lys(Bu), 6D-Lys(PpIX)] | 79.40 ± 8.88 | 84.50 ± 10.30 |
8 | GnRH-III[4Lys(Bu), 6Asp(OMe), 8Lys(PpIX)] | 288.6 ± 17.3 | 341.4 ± 21.1 |
Code | Compound | IC50 Values/nM |
---|---|---|
5 | GnRH-I[6D-Lys(PpIX)] | 62.3 ± 5.9 |
6 | GnRH-I[4Lys(Bu), 6D-Lys(PpIX)] | 89.8 ± 19.4 |
7 | GnRH-II[4Lys(Bu), 6D-Lys(PpIX)] | 71.1 ± 6.2 |
8 | GnRH-III[4Lys(Bu), 6Asp(OMe), 8Lys(PpIX)] | 81.8 ± 12.6 |
PpIX | Protoporphyrin IX | 209.3 ± 119.5 |
Variable | No. of Patients |
---|---|
Total no. of patients | 60 |
Sex | |
Male | 51 |
Female | 9 |
Age (year) | |
Mean | 58.45 (41–77) |
Localization | |
Oropharynx | 19 |
Larynx | 24 |
Hypopharynx | 16 |
Lingua | 1 |
TNM 1 stage | |
I | 6 |
II | 11 |
III | 15 |
IV A | 20 |
IV B | 3 |
IV C | 5 |
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Pethő, L.; Murányi, J.; Pénzes, K.; Gurbi, B.; Brauswetter, D.; Halmos, G.; Csík, G.; Mező, G. Suitability of GnRH Receptors for Targeted Photodynamic Therapy in Head and Neck Cancers. Int. J. Mol. Sci. 2019, 20, 5027. https://doi.org/10.3390/ijms20205027
Pethő L, Murányi J, Pénzes K, Gurbi B, Brauswetter D, Halmos G, Csík G, Mező G. Suitability of GnRH Receptors for Targeted Photodynamic Therapy in Head and Neck Cancers. International Journal of Molecular Sciences. 2019; 20(20):5027. https://doi.org/10.3390/ijms20205027
Chicago/Turabian StylePethő, Lilla, József Murányi, Kinga Pénzes, Bianka Gurbi, Diána Brauswetter, Gábor Halmos, Gabriella Csík, and Gábor Mező. 2019. "Suitability of GnRH Receptors for Targeted Photodynamic Therapy in Head and Neck Cancers" International Journal of Molecular Sciences 20, no. 20: 5027. https://doi.org/10.3390/ijms20205027