High-dose radiation causes extensive damage to normal tissue surrounding the tumor, thereby inducing lesions and prolonging toxic side effects 51. Unfortunately, the adverse untargeted effects of radiotherapy include alterations to the microenvironment of the target tissue, induction of metastasis, and worsening of clinical outcome 52. There is an unmet clinical need for delivering low-dose radiation and maintaining a low total radiotherapeutic dose. In our experimental setting, delivery of 0.5 Gy in combination with a high dose rate of 2400 MU/min had minimal cellular radiotoxicity in HEM and HDF while accelerating the killing of melanoma cells. Significant upregulation of apoptotic genes in melanoma cells confirmed these findings. Radiation-mediated DNA damage and cell death of melanoma cells were evident immediately, but the data also indicate that the toxic effect continued, as cell survival determined from colony counts was significantly less than that predicted from the cell count at 7 days after irradiation. In contrast, HEM and HDF showed nonsignificant radiotoxicity. Our data indicate that this radioprotection may be a consequence of overexpression of DNA repair genes and minimal DNA damage (Fig. 2). In addition, the cell proliferation potential of HEM and HDF was not altered by radiation; instead, the cells showed significant upregulation of cyclins to promote cell division for recovery after 7 days. Moreover, the levels of the proteins cyclin D1 and cyclin D2 in melanoma cells 1 day after irradiation (24 Gy/min) were minimally decreased compared with that in nonradiated controls, indicating that the process of apoptosis had begun soon after irradiation, consistent with downregulation of Bcl-2 in the irradiated (24 Gy/min) melanoma samples. These in-vitro data provide evidence that use of the FFF mode, a dose rate of 2400 MU/min, and a low total dose of 0.5 Gy can potentially fulfill clinical needs and enhance clinical outcomes.
The authors thank the radiotherapists and radiation physicists of the John Theurer Cancer Center, Hackensack University Medical Center (Hackensack, New Jersey, USA) for their continuous support and help in carrying out irradiation using TrueBeam, which was required for this study. They also thank Rana, Michael Jones, and Irfan Qureshi for helping with irradiation and preparation of TrueBeam. This study was funded by the John Theurer Cancer Center (Hackensack University Medical Center, New Jersey, USA).
There are no conflicts of interest.
1. Markovic SN, Erickson LA, Rao RD, Weenig RH, Pockaj BA, Bardia A, et al.. Malignant melanoma
in the 21st century, part 2: staging, prognosis, and treatment. Mayo Clin Proc 2007; 82:490–513.
2. Bandarchi B, Jabbari CA, Vedadi A, Navab R. Molecular biology of normal melanocytes and melanoma
cells. J Clin Pathol 2013; 66:644–648.
3. Wadasadawala T, Trivedi S, Gupta T, Epari S, Jalali R. The diagnostic dilemma of primary central nervous system melanoma
. J Clin Neurosci 2010; 17:1014–1017.
4. Dye DE, Medic S, Ziman M, Coombe DR. Melanoma
biomolecules: independently identified but functionally intertwined. Front Oncol 2013; 3:252.
5. Lejeune FJ, Rimoldi D, Speiser D. New approaches in metastatic melanoma
: biological and molecular targeted therapies. Expert Rev Anticancer Ther 2007; 7:701–713.
6. Goulart CR, Mattei TA, Ramina R. Cerebral melanoma
metastases: a critical review on diagnostic methods and therapeutic options. ISRN Surg 2011; 2011:276908.
7. Forschner A, Heinrich V, Pflugfelder A, Meier F, Garbe C. The role of radiotherapy in the overall treatment of melanoma
. Clin Dermatol 2013; 31:282–289.
8. Stevens G, McKay MJ. Dispelling the myths surrounding radiotherapy for treatment of cutaneous melanoma
. Lancet Oncol 2006; 7:575–583.
9. Walls AC, Han J, Li T, Qureshi AA. Host risk factors, ultraviolet index of residence, and incident malignant melanoma
in situ among US women and men. Am J Epidemiol 2013; 177:997–1005.
10. Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, et al.. Mutations of the BRAF gene in human cancer
. Nature 2002; 417:949–954.
11. Kolch W. Meaningful relationships: the regulation of the Ras/Raf/MEK/ERK pathway by protein interactions. Biochem J 2000; 351 Pt 2 (Pt 2):289–305.
12. Calipel A, Lefevre G, Pouponnot C, Mouriaux F, Eychène A, Mascarelli F. Mutation of B-Raf in human choroidal melanoma
cells mediates cell proliferation and transformation through the MEK/ERK pathway. J Biol Chem 2003; 278:42409–42418.
13. Khan MK, Khan N, Almasan A, Macklis R. Future of radiation therapy for malignant melanoma
in an era of newer, more effective biological agents. Onco Targets Ther 2011; 4:137–148.
14. Colombino M, Capone M, Lissia A, Cossu A, Rubino C, De Giorgi V, et al.. BRAF/NRAS mutation frequencies among primary tumors and metastases in patients with melanoma
. J Clin Oncol 2012; 30:2522–2529.
15. McCubrey JA, Steelman LS, Abrams SL, Lee JT, Chang F, Bertrand FE, et al.. Roles of the RAF/MEK/ERK and PI3K/PTEN/AKT pathways in malignant transformation and drug resistance. Adv Enzyme Regul 2006; 46:249–279.
16. Eckerle Mize D, Bishop M, Resse E, Sluzevich J. Riegert-Johnson DL, Boardman LA, Hefferon T, Roberts M. Familial atypical multiple mole melanoma
Syndromes. Bethesda, MD: National Centre for Biotechnology Information (US); 2009.
17. Peralta-Leal A, Rodríguez MI, Oliver FJ. Poly(ADP-ribose)polymerase-1 (PARP-1) in carcinogenesis: potential role of PARP inhibitors in cancer
treatment. Clin Transl Oncol 2008; 10:318–323.
18. Barranco SC, Romsdahl MM, Humphrey RM. The radiation response of human malignant melanoma
cells grown in vitro. Cancer
Res 1971; 31:830–833.
19. Sharma SD. Unflattened photon beams from the standard flattening filter free accelerators for radiotherapy: advantages, limitations and challenges. J Med Phys 2011; 36:123–125.
20. Rana S. Intensity modulated radiation therapy versus volumetric intensity modulated arc therapy. J Med Radiat Sci 2013; 60:81–83.
21. Balcer-Kubiczek EK. Apoptosis
in radiation therapy: a double-edged sword. Exp Oncol 2012; 34:277–285.
22. Xu X, Duan S, Yi F, Ocampo A, Liu GH, Izpisua Belmonte JC. Mitochondrial regulation in pluripotent stem cells. Cell Metab 2013; 18:325–332.
23. Dhillon VS, Fenech M. Mutations that affect mitochondrial functions and their association with neurodegenerative diseases. Mutat Res Rev Mutat Res 2014; 759:1–13.
24. Gogvadze V, Orrenius S, Zhivotovsky B. Mitochondria in cancer
cells: what is so special about them? Trends Cell Biol 2008; 18:165–173.
25. Kam WW, Banati RB. Effects of ionizing radiation on mitochondria. Free Radic Biol Med 2013; 65:607–619.
26. Suh KS, Mutoh M, Mutoh T, Li L, Ryscavage A, Crutchley JM, et al.. CLIC4 mediates and is required for Ca2+
-induced keratinocyte differentiation. J Cell Sci 2007; 120 (Pt 15):2631–2640.
27. Pajoum Shariati SR, Shokrgozar MA, Vossoughi M, Eslamifar A. In vitro co-culture of human skin keratinocytes and fibroblasts on a biocompatible and biodegradable scaffold. Iran Biomed J 2009; 13:169–177.
28. Godwin LS, Castle JT, Kohli JS, Goff PS, Cairney CJ, Keith WN, et al.. Isolation, culture, and transfection of melanocytes. Curr Protoc Cell Biol 2014; 63:1.8.1–1.8.20.
29. Franken NA, Rodermond HM, Stap J, Haveman J, van Bree C. Clonogenic assay of cells in vitro. Nat Protoc 2006; 1:2315–2319.
30. Munshi A, Hobbs M, Meyn RE. Clonogenic cell survival assay. Methods Mol Med 2005; 110:21–28.
31. Zhu S, Oremo JA, Li S, Zhen M, Tang Y, Du Y. Synergistic antitumor activities of docetaxel and octreotide associated with apoptotic-upregulation in castration-resistant prostate cancer
. PLoS One 2014; 9:e91817.
32. Lu R, Gao H, Wang H, Cao L, Bai J, Zhang Y. Overexpression of the Notch3 receptor and its ligand Jagged1 in human clinically non-functioning pituitary adenomas. Oncol Lett 2013; 5:845–851.
33. Zhao J, Xiang Y, Xiao C, Guo P, Wang D, Liu Y, Shen Y. AKR1C3 overexpression mediates methotrexate resistance in choriocarcinoma cells. Int J Med Sci 2014; 11:1089–1097.
34. Mahmood T, Yang PC. Western blot: technique, theory, and trouble shooting. N Am J Med Sci 2012; 4:429–434.
35. Kotecha N, Krutzik PO, Irish JM. Web-based analysis and publication of flow cytometry experiments. Curr Protoc Cytom 2010; Chapter 10:Unit10.17.
37. Murley JS, Kataoka Y, Baker KL, Diamond AM, Morgan WF, Grdina DJ. Manganese superoxide dismutase (SOD2)-mediated delayed radioprotection induced by the free thiol form of amifostine and tumor necrosis factor alpha. Radiat Res 2007; 167:465–474.
38. Leach JK, Black SM, Schmidt-Ullrich RK, Mikkelsen RB. Activation of constitutive nitric-oxide synthase activity is an early signaling event induced by ionizing radiation. J Biol Chem 2002; 277:15400–15406.
39. Wallace DC. Mitochondria and cancer
. Nat Rev Cancer
40. Baraldi MM, Alemi AA, Sethna JP, Caracciolo S, La Porta CA M, Zapperi Stefano. Growth and form of melanoma
cell colonies. J Stat Mech 2013; 2013:P02032.
41. Siegel R, Ma J, Zou Z, Jemal A. Cancer
statistics, 2014. CA Cancer
J Clin 2014; 64:9–29.
42. Rofstad EK. Radiation sensitivity in vitro of primary tumors and metastatic lesions of malignant melanoma
Res 1992; 52:4453–4457.
43. Rofstad EK. Radiation biology of malignant melanoma
. Acta Radiol Oncol 1986; 25:1–10.
44. Strojan P. Role of radiotherapy in melanoma
management. Radiol Oncol 2010; 44:1–12.
45. Sambade MJ, Peters EC, Thomas NE, Kaufmann WK, Kimple RJ, Shields JM. Melanoma
cells show a heterogeneous range of sensitivity to ionizing radiation and are radiosensitized by inhibition of B-RAF with PLX-4032. Radiother Oncol 2011; 98:394–399.
46. Rao NG, Yu HH, Trotti A 3rd, Sondak VK. The role of radiation therapy in the management of cutaneous melanoma
. Surg Oncol Clin N Am 2011; 20:115–131.
47. Olivier KR, Schild SE, Morris CG, Brown PD, Markovic SN. A higher radiotherapy dose is associated with more durable palliation and longer survival in patients with metastatic melanoma
48. Berk LB. Radiation therapy as primary and adjuvant treatment for local and regional melanoma
Control 2008; 15:233–238.
49. Lengua RE, Gonzalez MF, Barahona K, Ixquiac ME, Lucero JF, Montenegro E, et al.. Toxicity outcome in patients treated with modulated arc radiotherapy for localized prostate cancer
. Rep Pract Oncol Radiother 2014; 19:234–238.
50. Combs SE, Konkel S, Thilmann C, Debus J, Schulz-Ertner D. Local high-dose radiotherapy and sparing of normal tissue using intensity-modulated radiotherapy (IMRT) for mucosal melanoma
of the nasal cavity and paranasal sinuses. Strahlenther Onkol 2007; 183:63–68.
51. Chapel A, Francois S, Douay L, Benderitter M, Voswinkel J. Fifteen years of preclinical and clinical experiences about biotherapy treatment of lesions induced by accidental irradiation and radiotherapy. World J Stem Cells 2013; 5:68–72.
52. Shin JW, Son JY, Raghavendran HR, Chung WK, Kim HG, Park HJ, et al.. High-dose ionizing radiation-induced hematotoxicity and metastasis in mice model. Clin Exp Metastasis 2011; 28:803–810.
53. Maes H, Agostinis P. Autophagy and mitophagy interplay in melanoma
progression. Mitochondrion 2014; 19 Pt A:58–68.
54. Gaude E, Frezza C. Defects in mitochondrial metabolism and cancer
Metab 2014; 2:10.
55. Gembarska A, Luciani F, Fedele C, Russell EA, Dewaele M, Villar S, et al.. MDM4 is a key therapeutic target in cutaneous melanoma
. Nat Med 2012; 18:1239–1247.
56. Perez CA, Mutic S. Advances and future of radiation oncology. Rep Pract Oncol Radiother 2013; 18:329–332.
57. Ramsay EE, Hogg PJ, Dilda PJ. Mitochondrial metabolism inhibitors for cancer
therapy. Pharm Res 2011; 28:2731–2744.
58. Shukuwa T, Katayama I, Koji T. Fas-mediated apoptosis
cells and infiltrating lymphocytes in human malignant melanomas. Mod Pathol 2002; 15:387–396.