SRC Tyrosine Kinase Inhibitor and X-rays Combined Effect on Glioblastoma Cell Lines
Abstract
:1. Introduction
2. Results
2.1. c-SRC Inhibition Improves the Efficacy of Radiotherapy on U251-MG Cell Line
2.1.1. Evaluation of Cell Survival from Clonogenic Assay
2.1.2. Radiobiological Meaning of A, B and A/B Ratio Parameters
2.2. c-SRC Inhibition Sustains Radiation-Induced DNA Damage over Time
2.3. c-SRC Inhibition Reduces Cell Migration
3. Discussion
4. Materials and Methods
4.1. Cell Culture and Hypoxia Experiments
4.2. Irradiation and Drug Treatments
4.3. Clonogenic Assay
4.4. Radiobiological Parameters Calculation
4.5. γ-H2AX Immunofluorescence Analysis
4.6. Migration Assay
4.7. Statistical Analyses
5. Conclusions
Author Contributions
Funding
Conflicts of Interest
Abbreviations
GBM | Glioblastoma |
RT | Radiotherapy |
MMP-2 | Matrix metalloproteinase-2 |
MMP-9 | Matrix metalloproteinase-9 |
SFKs | SRC family kinases |
FAK | Focal adhesion kinase |
EGFR | Epidermal growth factor receptor |
SF | Surviving fraction |
PE | Plating efficiency |
DMF | Dose modifying factor |
OER | Oxygen enhancement ratio |
LQ | Linear-quadratic |
mAb | Monoclonal antibodies |
TKi | Tyrosine-kinase inhibitors |
nRTK | Non receptor tyrosine kinase |
ECM | Extracellular matrix |
DMSO | Dimethylsulfoxide |
BSA | Bovine serum albumin |
References
- Wen, P.Y.; Weller, M.; Lee, E.Q.; Alexander, B.A.; Barnholtz-Sloan, J.S.; Barthel, F.P.; Batchelor, T.T.; Bindra, R.S.; Chang, S.M.; Chiocca, E.A.; et al. Glioblastoma in Adults: A Society for Neuro-Oncology (SNO) and European Society of Neuro-Oncology (EANO) Consensus Review on Current Management and Future Directions. Neuro-Oncology 2020. [Google Scholar] [CrossRef] [PubMed]
- Cabrera, A.R.; Kirkpatrick, J.P.; Fiveash, J.B.; Shih, H.A.; Koay, E.J.; Lutz, S.; Petit, J.; Chao, S.T.; Brown, P.D.; Vogelbaum, M.; et al. Radiation therapy for glioblastoma: Executive summary of an American Society for Radiation Oncology Evidence-Based Clinical Practice Guideline. Pract. Radiat. Oncol. 2016, 6, 217–225. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Gerstner, E.R.; Zhang, Z.; Fink, J.R.; Muzi, M.; Hanna, L.; Greco, E.; Prah, M.; Schmainda, K.M.; Mintz, A.; Kostakoglu, L.; et al. ACRIN 6684: Assessment of Tumor Hypoxia in Newly Diagnosed Glioblastoma Using 18F-FMISO PET and MRI. Clin. Cancer Res. 2016, 22, 5079–5086. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Liu, C.; Lin, Q.; Yun, Z. Cellular and molecular mechanisms underlying oxygen-dependent radiosensitivity. Radiat. Res. 2015, 183, 487–496. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Grimes, D.R.; Partridge, M. A mechanistic investigation of the oxygen fixation hypothesis and oxygen enhancement ratio. Biomed. Phys. Eng. Express 2015, 1, 045209. [Google Scholar] [CrossRef] [PubMed]
- Valable, S.; Corroyer-Dulmont, A.; Chakhoyan, A.; Durand, L.; Toutain, J.; Divoux, D.; Barre, L.; MacKenzie, E.T.; Petit, E.; Bernaudin, M.; et al. Imaging of brain oxygenation with magnetic resonance imaging: A validation with positron emission tomography in the healthy and tumoural brain. J. Cereb Blood Flow Metab. 2017, 37, 2584–2597. [Google Scholar] [CrossRef] [Green Version]
- Ponte, K.F.; Berro, D.H.; Collet, S.; Constans, J.M.; Emery, E.; Valable, S.; Guillamo, J.S. In Vivo Relationship Between Hypoxia and Angiogenesis in Human Glioblastoma: A Multimodal Imaging Study. J. Nucl. Med. 2017, 58, 1574–1579. [Google Scholar] [CrossRef] [Green Version]
- Persano, L.; Rampazzo, E.; Della Puppa, A.; Pistollato, F.; Basso, G. The three-layer concentric model of glioblastoma: Cancer stem cells, microenvironmental regulation, and therapeutic implications. Sci. World J. 2011, 11, 1829–1841. [Google Scholar] [CrossRef]
- Peres, E.A.; Gerault, A.N.; Valable, S.; Roussel, S.; Toutain, J.; Divoux, D.; Guillamo, J.S.; Sanson, M.; Bernaudin, M.; Petit, E. Silencing erythropoietin receptor on glioma cells reinforces efficacy of temozolomide and X-rays through senescence and mitotic catastrophe. Oncotarget 2015, 6, 2101–2119. [Google Scholar] [CrossRef] [Green Version]
- Ahluwalia, M.S.; de Groot, J.; Liu, W.M.; Gladson, C.L. Targeting SRC in glioblastoma tumors and brain metastases: Rationale and preclinical studies. Cancer Lett. 2010, 298, 139–149. [Google Scholar] [CrossRef] [Green Version]
- Keller, S.; Schmidt, M.H.H. EGFR and EGFRvIII Promote Angiogenesis and Cell Invasion in Glioblastoma: Combination Therapies for an Effective Treatment. Int. J. Mol. Sci. 2017, 18. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Liu, Z.; Han, L.; Dong, Y.; Tan, Y.; Li, Y.; Zhao, M.; Xie, H.; Ju, H.; Wang, H.; Zhao, Y.; et al. EGFRvIII/integrin beta3 interaction in hypoxic and vitronectinenriching microenvironment promote GBM progression and metastasis. Oncotarget 2016, 7, 4680–4694. [Google Scholar] [CrossRef] [PubMed]
- Schenone, S.; Radi, M.; Musumeci, F.; Brullo, C.; Botta, M. Biologically driven synthesis of pyrazolo[3,4-d]pyrimidines as protein kinase inhibitors: An old scaffold as a new tool for medicinal chemistry and chemical biology studies. Chem. Rev. 2014, 114, 7189–7238. [Google Scholar] [CrossRef] [PubMed]
- Calgani, A.; Vignaroli, G.; Zamperini, C.; Coniglio, F.; Festuccia, C.; Di Cesare, E.; Gravina, G.L.; Mattei, C.; Vitale, F.; Schenone, S.; et al. Suppression of SRC Signaling Is Effective in Reducing Synergy between Glioblastoma and Stromal Cells. Mol. Cancer Ther. 2016, 15, 1535–1544. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- O’Rourke, S.F.; McAneney, H.; Hillen, T. Linear quadratic and tumour control probability modelling in external beam radiotherapy. J. Math. Biol. 2009, 58, 799–817. [Google Scholar] [CrossRef]
- Hu, G.; Fang, W.; Liu, N.; Li, C. Effects of mir-128a on the invasion and proliferation of glioma U251 cells. Oncol. Lett. 2019, 17, 891–896. [Google Scholar] [CrossRef]
- Stupp, R.; Mason, W.P.; van den Bent, M.J.; Weller, M.; Fisher, B.; Taphoorn, M.J.; Belanger, K.; Brandes, A.A.; Marosi, C.; Bogdahn, U.; et al. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N. Engl. J. Med. 2005, 352, 987–996. [Google Scholar] [CrossRef]
- Buglione, M.; Triggiani, L.; Borghetti, P.; Pedretti, S.; Pasinetti, N.; Magrini, S.M. The “Radioresistance” of Glioblastoma in the Clinical Setting, and the Present Therapeutic Options. In Radiobiology of Glioblastoma; Pirtoli, L., Gravina, G., Giordano, A., Eds.; Springer: Jersey City, NJ, USA, 2016; pp. 15–27. [Google Scholar]
- Mukhopadhyay, D.; Tsiokas, L.; Zhou, X.M.; Foster, D.; Brugge, J.S.; Sukhatme, V.P. Hypoxic induction of human vascular endothelial growth factor expression through c-Src activation. Nature 1995, 375, 577–581. [Google Scholar] [CrossRef]
- Valle-Casuso, J.C.; Gonzalez-Sanchez, A.; Medina, J.M.; Tabernero, A. Hif-1 and C-Src Mediate Increased Glucose Uptake Induced by Endothelin-1 and Connexin43 In Astrocytes. PLoS ONE 2012, 7, e32448. [Google Scholar] [CrossRef] [Green Version]
- Cammarata, F.P.; Torrisi, F.; Forte, G.I.; Minafra, L.; Bravata, V.; Pisciotta, P.; Savoca, G.; Calvaruso, M.; Petringa, G.; Cirrone, G.A.P.; et al. Proton Therapy and Src Family Kinase Inhibitor Combined Treatments on U87 Human Glioblastoma Multiforme Cell Line. Int. J. Mol. Sci. 2019, 20, 4745. [Google Scholar] [CrossRef] [Green Version]
- Choi, J.; Kang, J.O. Basics of Particle Therapy Ii: Relative Biological Effectiveness. Radiat. Oncol. J. 2012, 30, 1–13. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Plaks, V.; Kong, N.; Werb, Z. The Cancer Stem Cell Niche: How Essential Is The Niche in Regulating Stemness of Tumor Cells? Cell Stem Cell 2015, 16, 225–238. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Skuli, N.; Monferran, S.; Delmas, C.; Favre, G.; Bonnet, J.; Toulas, C.; Cohen-Jonathan Moyal, E. Alphavbeta3/Alphavbeta5 Integrins-Fak-Rhob: A Novel Pathway for Hypoxia Regulation in Glioblastoma. Cancer Res. 2009, 69, 3308–3316. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Park, C.M.; Park, M.J.; Kwak, H.J.; Lee, H.C.; Kim, M.S.; Lee, S.H.; Park, I.C.; Rhee, C.H.; Hong, S.I. Ionizing Radiation Enhances Matrix Metalloproteinase-2 Secretion and Invasion of Glioma Cells Through Src/Epidermal Growth Factor Receptor-Mediated P38/Akt And Phosphatidylinositol 3-Kinase/Akt Signaling Pathways. Cancer Res. 2006, 66, 8511–8519. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Roos, A.; Ding, Z.; Loftus, J.C.; Tran, N.L. Molecular and Microenvironmental Determinants of Glioma Stem-Like Cell Survival and Invasion. Front. Oncol. 2017, 7, 120. [Google Scholar] [CrossRef]
- Brown, J.M.; Wilson, W.R. Exploiting Tumour Hypoxia in Cancer Treatment. Nat. Rev. Cancer 2004, 4, 437–447. [Google Scholar] [CrossRef]
- Eom, K.Y.; Cho, B.J.; Choi, E.J.; Kim, J.H.; Chie, E.K.; Wu, H.G.; Kim, I.H.; Paek, S.H.; Kim, J.S.; Kim, I.A. The Effect of Chemoradiotherapy with SRC Tyrosine Kinase Inhibitor, PP2 and Temozolomide on Malignant Glioma Cells In Vitro and In Vivo. Cancer Res. Treat. 2016, 48, 687–697. [Google Scholar] [CrossRef] [Green Version]
- Carraro, F.; Naldini, A.; Pucci, A.; Locatelli, G.A.; Maga, G.; Schenone, S.; Bruno, O.; Ranise, A.; Bondavalli, F.; Brullo, C.; et al. Pyrazolo[3,4-D]Pyrimidines as Potent Antiproliferative and Proapoptotic Agents Toward A431 And 8701-Bc Cells in Culture Via Inhibition Of C-Src Phosphorylation. J. Med. Chem. 2006, 49, 1549–1561. [Google Scholar] [CrossRef]
- Tintori, C.; Fallacara, A.L.; Radi, M.; Zamperini, C.; Dreassi, E.; Crespan, E.; Maga, G.; Schenone, S.; Musumeci, F.; Brullo, C.; et al. Combining X-Ray Crystallography and Molecular Modeling Toward the Optimization of Pyrazolo[3,4-D]Pyrimidines as Potent C-Src Inhibitors Active in Vivo Against Neuroblastoma. J. Med. Chem. 2015, 58, 347–361. [Google Scholar] [CrossRef]
- Lassman, A.B.; Pugh, S.L.; Gilbert, M.R.; Aldape, K.D.; Geinoz, S.; Beumer, J.H.; Christner, S.M.; Komaki, R.; DeAngelis, L.M.; Gaur, R.; et al. Phase 2 Trial of Dasatinib in Target-Selected Patients With Recurrent Glioblastoma (Rtog 0627). Neuro-Oncology 2015, 17, 992–998. [Google Scholar] [CrossRef] [Green Version]
- Galanis, E.; Anderson, S.K.; Twohy, E.L.; Carrero, X.W.; Dixon, J.G.; Tran, D.D.; Jeyapalan, S.A.; Anderson, D.M.; Kaufmann, T.J.; Feathers, R.W.; et al. A phase 1 and randomized, placebo-controlled phase 2 trial of bevacizumab plus dasatinib in patients with recurrent glioblastoma: Alliance/North Central Cancer Treatment Group N0872. Cancer 2019, 125, 3790–3800. [Google Scholar] [CrossRef] [PubMed]
- Reardon, D.A.; Vredenburgh, J.J.; Desjardins, A.; Peters, K.B.; Sathornsumetee, S.; Threatt, S.; Sampson, J.H.; Herndon, J.E., 2nd; Coan, A.; McSherry, F.; et al. Phase 1 trial of dasatinib plus erlotinib in adults with recurrent malignant glioma. J. Neuro-Oncol. 2012, 108, 499–506. [Google Scholar] [CrossRef] [Green Version]
- Franceschi, E.; Stupp, R.; van den Bent, M.J.; van Herpen, C.; Laigle Donadey, F.; Gorlia, T.; Hegi, M.; Lhermitte, B.; Strauss, L.C.; Allgeier, A.; et al. EORTC 26083 phase I/II trial of dasatinib in combination with CCNU in patients with recurrent glioblastoma. Neuro-Oncology 2012, 14, 1503–1510. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Laack, N.N.; Galanis, E.; Anderson, S.K.; Leinweber, C.; Buckner, J.C.; Giannini, C.; Geoffroy, F.J.; Johnson, D.R.; Lesser, G.J.; Jaeckle, K.A.; et al. Randomized, placebo-controlled, phase II study of dasatinib with standard chemo-radiotherapy for newly diagnosed glioblastoma (GBM), NCCTG N0877 (Alliance). J. Clin. Oncol. 2017, 33, 2013. [Google Scholar] [CrossRef]
- Agarwal, S.; Mittapalli, R.K.; Zellmer, D.M.; Gallardo, J.L.; Donelson, R.; Seiler, C.; Decker, S.A.; Santacruz, K.S.; Pokorny, J.L.; Sarkaria, J.N.; et al. Active efflux of Dasatinib from the brain limits efficacy against murine glioblastoma: Broad implications for the clinical use of molecularly targeted agents. Mol. Cancer Ther. 2012, 11, 2183–2192. [Google Scholar] [CrossRef] [Green Version]
- Fallacara, A.L.; Zamperini, C.; Podolski-Renic, A.; Dinic, J.; Stankovic, T.; Stepanovic, M.; Mancini, A.; Rango, E.; Iovenitti, G.; Molinari, A.; et al. A New Strategy for Glioblastoma Treatment: In Vitro and In Vivo Preclinical Characterization of Si306, a Pyrazolo[3,4-d]Pyrimidine Dual Src/P-Glycoprotein Inhibitor. Cancers 2019, 11, 848. [Google Scholar] [CrossRef] [Green Version]
- Chapman, J.D. Can the Two Mechanisms of Tumor Cell Killing by Radiation Be Exploited for Therapeutic Gain? J. Radiat. Res. 2014, 55, 2–9. [Google Scholar] [CrossRef]
- Joiner, M.; van der Kogel, A. Basic Clinical Radiobiology, 4th ed.; Taylor & Fransis Group: London, UK, 2009; p. 375. [Google Scholar]
- Vicario, N.; Bernstock, J.D.; Spitale, F.M.; Giallongo, C.; Giunta, M.A.S.; Li Volti, G.; Gulisano, M.; Leanza, G.; Tibullo, D.; Parenti, R.; et al. Clobetasol Modulates Adult Neural Stem Cell Growth via Canonical Hedgehog Pathway Activation. Int. J. Mol. Sci. 2019, 20. [Google Scholar] [CrossRef] [Green Version]
- Mauri, E.; Sacchetti, A.; Vicario, N.; Peruzzotti-Jametti, L.; Rossi, F.; Pluchino, S. Evaluation of RGD functionalization in hybrid hydrogels as 3D neural stem cell culture systems. Biomater. Sci. 2018, 6, 501–510. [Google Scholar] [CrossRef]
- Gulino, R.; Vicario, N.; Giunta, M.A.S.; Spoto, G.; Calabrese, G.; Vecchio, M.; Gulisano, M.; Leanza, G.; Parenti, R. Neuromuscular Plasticity in a Mouse Neurotoxic Model of Spinal Motoneuronal Loss. Int. J. Mol. Sci. 2019, 20. [Google Scholar] [CrossRef] [Green Version]
Treatment | Normoxia SF50% (Gy) | Hypoxia SF50% (Gy) | Normoxia DMF | Hypoxia DMF | OER |
---|---|---|---|---|---|
X-rays + vehicle | 4.09 | 5.18 | 1 | 1 | 1.27 |
X-rays + 10 μM Si306 | 3.86 | 4.53 | 1.05 | 1.15 | 1.17 |
X-rays+ 20 μM Si306 | 2.54 | 2.67 | 1.38 | 1.94 | 1.05 |
Treatment Normoxia | α (Gy-1) | β (Gy-2) | α/β (Gy) |
---|---|---|---|
X-rays + vehicle | 0.037 ± 0.011 | 0.036 ± 0.009 | 1.03 |
X-ray s+ 10 μM Si306 | 0.060 ± 0.039 | 0.035 ± 0.009 | 1.71 |
X-rays+ 20 μM Si306 | 0.077 ± 0.009 | 0.052 ± 0.005 | 1.48 |
Treatment Hypoxia | α (Gy-1) | β (Gy-2) | α/β (Gy) |
---|---|---|---|
X-rays + vehicle | 0.037 ± 0.024 | 0.020 ± 0.005 | 1.85 |
X-rays + 10 μM Si306 | 0.092 ± 0.010 | 0.013 ± 0.002 | 7.07 |
X-rays + 20 μM Si306 | 0.219 ± 0.025 | 0.014 ± 0.005 | 15.64 |
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Torrisi, F.; Minafra, L.; Cammarata, F.P.; Savoca, G.; Calvaruso, M.; Vicario, N.; Maccari, L.; Pérès, E.A.; Özçelik, H.; Bernaudin, M.; et al. SRC Tyrosine Kinase Inhibitor and X-rays Combined Effect on Glioblastoma Cell Lines. Int. J. Mol. Sci. 2020, 21, 3917. https://doi.org/10.3390/ijms21113917
Torrisi F, Minafra L, Cammarata FP, Savoca G, Calvaruso M, Vicario N, Maccari L, Pérès EA, Özçelik H, Bernaudin M, et al. SRC Tyrosine Kinase Inhibitor and X-rays Combined Effect on Glioblastoma Cell Lines. International Journal of Molecular Sciences. 2020; 21(11):3917. https://doi.org/10.3390/ijms21113917
Chicago/Turabian StyleTorrisi, Filippo, Luigi Minafra, Francesco P. Cammarata, Gaetano Savoca, Marco Calvaruso, Nunzio Vicario, Laura Maccari, Elodie A. Pérès, Hayriye Özçelik, Myriam Bernaudin, and et al. 2020. "SRC Tyrosine Kinase Inhibitor and X-rays Combined Effect on Glioblastoma Cell Lines" International Journal of Molecular Sciences 21, no. 11: 3917. https://doi.org/10.3390/ijms21113917
APA StyleTorrisi, F., Minafra, L., Cammarata, F. P., Savoca, G., Calvaruso, M., Vicario, N., Maccari, L., Pérès, E. A., Özçelik, H., Bernaudin, M., Botta, L., Russo, G., Parenti, R., & Valable, S. (2020). SRC Tyrosine Kinase Inhibitor and X-rays Combined Effect on Glioblastoma Cell Lines. International Journal of Molecular Sciences, 21(11), 3917. https://doi.org/10.3390/ijms21113917