Melanoma is an aggressive skin cancer that kills more than 9000 people in the USA annually, and it was estimated that more than 76 000 new cases would be diagnosed in 2014 41. Melanoma is frequently radioresistant, and radiation protocols are effective only as palliative therapy. However, recent studies using external beam radiation therapy both in vitro and in vivo have demonstrated radioresponsiveness and a heterogeneous range of radiation sensitivities of melanoma 42–45. Radiation therapy is infrequently used for treating primary tumors, but recent randomized data generated from high-risk patients suggest the importance of adjuvant radiotherapy 46. Development of the FFF mode has improved radiotherapy for various cancers, including metastasized melanoma, with respect to both local control and palliation in patients 47,48. To achieve better disease control and prevent progression of melanoma, we investigated the use of the FFF mode of irradiation to accelerate cell death in metastatic and malignant melanoma cell lines in an in-vitro setting. Our data demonstrated that the combination of an unconventional high dose rate (2400 MU/min) with a low total dose (0.5 Gy) in the FFF mode produced greater induction of apoptosis in melanoma cells than that obtained with a conventional clinical dose rate (400 MU/min). This protocol produced an average of five-fold more cell death in the in-vitro setting, implying that a similar clinical protocol could be derived that would minimize long-term toxicities resulting from high radiation doses 49. Minimizing toxicities is a major goal of anticancer therapy. With our protocol using a low overall dose but a high dose rate, survival of HEM and HDF were preserved above 80% (Figs 1 and 5). Recently, intensity-modulated radiotherapy for mucosal melanoma yielded a 3-year survival rate and mild toxicity for 75% of patients 50, implying that our unconventional radiation protocol has translational potential to the clinical setting.
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.
Our results demonstrate that radiation at a dose rate of 2400 MU/min enhances apoptosis in melanoma cells through a Fas-mediated apoptotic pathway (Fig. 1d). The activation of Fas and a cascade of several apoptotic genes triggers apoptosis in melanoma cells. Fas-mediated apoptotic signaling in melanoma cells has been documented previously 58. The underlying mechanism for the activation Fas signaling by a high dose rate of radiation is not known, and further study is required. Downregulation of antiapoptotic genes and upregulation of apoptotic and stress genes in ER and cell-death pathways support the high cell-kill efficiency of the 2400 MU/min dose rate compared with the conventional clinical dose rate of 400 MU/min. The absence of differential expression of these genes in primary skin cells suggests that the total dose of 0.5 Gy under both dose rates was relatively harmless.
In summary, this study demonstrates a potential anti-melanoma therapy by using a combination of high dose rate and low total dose (2400 MU/min/0.5 Gy) to enhance the radiosensitivity and apoptotic rates in melanoma cells while preserving the survival of primary skin cells. The radiosensitivity can be further increased by inhibiting the activities of mitochondrial respiration chains. Melanoma cells upregulate mitochondrial respiration to partly overcome the damage caused by radiation treatment. The combinatorial use of a dose rate 2400 MU/min, a low dose of 0.5 Gy, and blockers of mitochondrial respiration activity can potentially introduce innovative antimelanoma therapeutic options in the clinical setting.
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 syndrome. Cancer 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 2012; 12:685–698.
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. Cancer 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. Cancer 2007; 110:1791–1795.
48. Berk LB. Radiation therapy as primary and adjuvant treatment for local and regional melanoma. Cancer 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. 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 of melanoma cells and infiltrating lymphocytes in human malignant melanomas. Mod Pathol 2002; 15:387–396.