Risk factors for melanoma include dysplastic nevi syndrome, continuous solar ultraviolet radiation exposure, family history of melanoma, sunburns, BRAF gene mutation, and White ancestry 1. Malignant melanoma is highly aggressive, chemoresistant, and poorly radioresponsive, and is responsible for as much as 80% of the mortality among all skin cancers; it has a 5-year survival rate of 14% 2. Melanoma can arise from skin, eyes, mucosa, or the central nervous system 3. Patients diagnosed with thin lesions (<1 mm) have an increased cure rate after surgery, but 5% develop metastatic melanoma, which limits 10-year survival 4. Therapy for metastatic melanoma has improved with the understanding of melanoma signaling pathways and the identification of tumor cell targets in the cell. Identification of small molecules that interfere with key signaling pathways has helped the progress of new therapeutic approaches in melanoma 5.
Among these, radiotherapy treatments reduce the rate of recurrence, improve control of local disease, and limit metastasis to the bone or brain 6. Melanoma metastasizes to the brain in 10–40% of cases 7. Recent management protocols for melanoma incorporate chemotherapy, immunotherapy, and radiotherapy 1,8.
Mutation of the BRAF gene is a common risk factor for melanoma 9. BRAF acts as a mitogen-activated protein kinase kinase kinase 10 in the ERK pathway network 11 and regulates cell growth, differentiation, and survival 12. BRAF V600E is the most common mutation; it occurs in more than 50% of all melanoma cases and leads to hyperactive kinase 13–15. Family atypical multiple mole melanoma syndrome is caused by a familial autosomal dominant gene 16 and is associated with a large number of atypical nevi typically presenting as cutaneous melanoma 17.
Radiotherapy can be an effective treatment for melanoma, but radioresistance of melanoma cells affects clinical outcomes 18. In the past few years, modern linear accelerators operating in a flattening filter-free (FFF) mode and having increased dose rate capabilities have improved radiotherapy, with advantages over conventional radiotherapy including shortened dose delivery time, lower dose delivery outside the field, shorter treatment period, and lower rates of secondary malignancies 19. In addition, improved image guidance, along with volumetric-modulated arc therapy capabilities, has improved target conformity, while reducing exposure of normal tissue surrounding the lesion. The ability to deliver radiation in a concave isodose profile to minimize injury to normal surrounding tissue represents a significant advance in radiotherapy 20. Maintaining a high survival rate among normal cells subsequent to radiation treatment is a crucial component of all radiotherapies, and various in-vitro conditions have been tested 21.
Aberrations in mitochondrial functions resulting in deregulation of cellular aerobic respiration, differentiation, and proliferation have been reported in multiple malignancies including breast, colon, lung, liver, and kidney cancers, and leukemia and lymphoma 22, as well as in many neurological disorders 23. Inhibition of mitochondrial respiration or oxidative phosphorylation increases therapeutic efficiency in some in-vitro and in-vivo models 24, and it has been suggested that an increase in the tumoricidal efficacy of radiotherapy can be achieved by targeting the mitochondria 25.
In a direct in-vitro comparison between a conventional dose rate (400 MU/min) and an unconventional dose rate (2400 MU/min) coupled with a low total dose (0.5 Gy) of 10X FFF 10 MV X-rays, we found a significant improvement in the survival of normal cells and a concurrent increase in apoptosis in melanoma cells. Titrated doses of inhibitors to the mitochondrial respiratory chain increased the radiosensitivity of melanoma cells while maintaining normal melanocyte survival. Treatment of melanoma cells in vitro with the unconventional dose rate (FFF mode at 2400 MU/min) and low dose radiation protocol (0.5 Gy) has not been reported yet. Our radiation delivery protocol in the engineered mode of the intensity-modulated radiotherapy device and associated hardware/software can potentially translate to the clinical setting. Our results support the investigation of novel radiotherapeutic options and open doors to the untested field of radiotherapy research.
Melanoma cell lines (WC00046, WC00060, and WC00081; Fig. 1a) were purchased from Coriell Institute (Camden, New Jersey, USA). Cells were cultured in RPMI containing 10% fetal bovine serum and 1% penicillin/streptomycin (Invitrogen, Grand Island, New York, USA). All melanoma cell lines were positive for BRAF mutation and negative for NRAS mutation, and the WC00046 and WC00081 cell lines were positive for p53 (Fig. 6a). Primary human epidermal melanocytes (HEM) and culture media were purchased from ScienCell (Carlsbad, California, USA), and normal human dermal fibroblasts (HDF) were prepared as previously described 26–28. Oligomycin and rotenone (50 nmol/l; Sigma Aldrich, St. Louis, Missouri, USA) were used to block mitochondrial respiration.
Cells were seeded (5×105) in T-25 culture flasks (BD-Falcon; BD-Biosciences, Durham, North Carolina, USA), allowed to adhere overnight, and irradiated with 10 MV X-rays at dose rates of 400 or 2400 MU/min using TrueBeam (Varian Medical Systems, Palo Alto, California, USA) with 10X FFF. The total dose range of 10 MV X-rays was 0.25–8 Gy (100–1000 Gy data not shown) for multiple experiments.
Colony formation assays
One day after irradiation, HEM and melanoma cells were treated with trypsin, resuspended, counted using a Beckman Coulter Counter (Beckman Coulter, Brea, California, USA), and serially diluted (1 : 100, 1 : 1000, and 1 : 10 000) in complete medium for seeding in culture dishes (BD Falcon, Corning, New York, USA). Colonized cells were stained with hematoxylin for 30 min, fixed with 100% ethanol for 30 min, washed in water for clearing the background, dried overnight, and counted 29,30.
RNA isolation and qRT-PCR
RNA was extracted using TRIzol (Invitrogen), and selected genes (Figs 1d and e, 2d and e and 4b were amplified by quantitative real-time reverse transcriptase PCR (qRT-PCR) with SYBR green (Qiagen, Germantown, Maryland, USA) on a FAST Model 7900HT (Applied Biosystems, Foster City, California, USA) as previously published 31. Primer sequences are given in Table 1. Data were analyzed on SDS 7900HT software v2.2.2 (Applied Biosystems, Grand Island, New York, USA) using the comparative threshold cycle (Ct) method (
) for calculating fold changes and SDs 32.
A collagen-based cell migration assay (QCM TM 24-well kit; Millipore, Billerica, Massachusetts, USA) was used as described by the manufacturer. Cells (5×105 cells/well) were seeded and allowed to migrate for 24 h. Cells from the bottom of the insert chamber were collected by trypsin treatment and counted on a Beckman Coulter counter.
An MTS assay (Promega, Madison, Wisconsin, USA) was used to measure proliferation of irradiated cells (BioTek Synergy HT, Winooski, Vermont, USA) as described by the manufacturer.
Cells were treated with mitotracker Red CMXRos (Invitrogen) and mitochondrial activity was analyzed under a Zeiss Fluorescent microscope (Zeiss, Thornwood, New York, USA) before and after irradiation. Fluorescence was quantified by using ImageJ software.
An EpiQuik in-situ kit (Epigentek, Farmingdale, New York, USA) was used and the cells were grown after irradiation and fixed and permeabilized according to the manufacturer’s protocol and previously published literature 33.
Western blotting experiments were carried out 34 using lysates prepared from cell lines (Pierce IP lysis buffer cat# 87788; Invitrogen). SDS-PAGE gels (12%, Criterion TGX Precast Gels) were used to fractionate cell lysates.
Whole-cell extracts were fractionated by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane using a Bio-Rad transfer apparatus (Hercules, California, USA). The membrane was blocked using 5% nonfat dry milk in TBST (10 mmol/l Tris, pH 8.0, 150 mmol/l NaCl, 0.5% Tween 20) for 60 min, it was then washed and incubated with primary antibodies against Bcl-2 (1 : 1000, #7973; Abcam, Cambridge, Massachusetts, USA), caspase-3 (1 : 2000, H-277), PARP 1 (1 : 2000, #SC-7148, SC- 8007; Santa Cruz Biotechnology Inc., Santa Cruz, California, USA) and Actin (1 : 10 000, AB-6276) at 4°C overnight. The membranes were then washed and incubated with a 1 : 5000 dilution of horseradish peroxidase-conjugated donkey anti-mouse IgGs. The blots were washed and developed using the ECL system.
Analysis of the expression of cyclin D1 and cyclin D2 (sc-20044 PE, sc-53637 PE; Santa Cruz Biotechnology Inc.) in melanoma cell lines was carried out using Flow cytometry (Gallios; Beckman Coulter) 35,36. The Per Fix-nc kit (Cat #B3116; Beckman Coulter) was used for fixing, permeabilizing, and preparing the cells for the assay.
All experiments were performed a minimum of three times, and the data represent the results of assays performed in triplicate or quadruplicate. Error bars represent 95% confidence intervals. All statistics were based on continuous variables and were obtained using STAT View software (Stat View, Cary, North Carolina, USA). P-values less than 0.05 were considered statistically significant. Student’s t-test was used for comparisons between two groups.
At a total dose of 0.5 Gy, a dose rate of 2400 MU/min killed on average five-fold more melanoma cells than a dose rate of 400 MU/min and preserved 80% survival of normal melanocytes. Three melanoma cell lines (WC00046, WC00060, and WC00081) were chosen on the basis of tumor stage, pathology, and metastatic status (Fig. 1a). The in-vitro migration potential of each cell line was verified as corresponding to the original reported tumor (Fig. 1b). On the basis of cell counts from 7 days after radiation exposure, in-vitro radiation with low total doses (0.25, 0.50, 0.75, and 1 Gy) at a dose rate 2400 MU/min induced significantly higher apoptosis in melanoma cell lines than did a dose rate of 400 MU/min (P<0.005; Fig. 1c). As the total dose increased from 2 to 8 Gy, the difference in cell counts between the two dose rates was reduced significantly for WC00046 and WC00081 cell lines but not for WC00060 cell line. At 7 days after radiation exposure, the maximum apoptotic differential between 400 and 2400 MU/min occurred at a total dose of 0.5 Gy (Fig. 1c, arrows). Under these conditions, cell killing was 2.1-fold greater for WC00046, three-fold greater for WC00060, and 2.4 fold greater for WC00081 (Fig. 1c). Up to 90% of normal melanocytes survived both radiation dose rates (Fig. 1c). qRT-PCR analysis of apoptotic genes (Casp 3, Casp 9, Casp8, AIF, FAS, FASL and PTEN) indicated that Fas-mediated extrinsic cell death signaling was most active in irradiated cells. A dose rate of 2400 MU/min induced an average of 10-fold more apoptosis compared with the control and three-fold more apoptosis compared with the 400 MU/min dose rate (Fig. 1d). Endoplasmic reticular (ER)-stress genes (BBC-3, NOXA, PERK) were upregulated in all cells tested (Fig. 1e). The expression of a radioprotection gene (SOD2) 37 was significantly upregulated in the 2400 MU/min group compared with nonirradiated controls among both melanoma and normal cells. Similar to HEM, normal human fibroblasts maintained higher survival when treated at 2400 MU/min. Cells showed complete recovery from the radiation treatment and continued to proliferate 7 days after irradiation, suggesting that a total dose of 0.5 Gy administered at 2400 MU/min was relatively harmless to HEM and HDF.
A dose rate of 2400 MU/min induced greater DNA damage and reduced cell proliferation compared with a dose rate of 400 MU/min at a total dose 0.5 Gy, but HEM and HDF were relatively unharmed. Quantification of DNA damage in WC00046 cells showed that, at a total dose of 0.5 Gy, the dose rate 2400 MU/min caused seven-fold more DNA damage than in nonirradiated controls and nearly two-fold more DNA damage than a 400 MU/min dose rate (P<0.002; Fig. 2a). WC00060 and WC00081 cell lines showed similar sensitivities (data not shown), suggesting a direct correlation between the dose rate and DNA damage. HEM showed no significant DNA damage and HDF showed slightly higher DNA damage only at the 400 MU/min dose rate (Fig. 2a). When cell proliferation was analyzed at 7 days after radiation, melanoma cells (only WC00046 data shown) irradiated at a rate of 2400 MU/min showed a 2.2-fold reduction in proliferation compared with nonirradiated controls and a 1.7-fold reduction compared with cells irradiated at the 400 MU/min dose rate (Fig. 2b). DNA repair genes (MSH-2, MDM2, MDM4, PARP 1, PARP2) were upregulated approximately three-fold in the WC00046 cell line treated at 2400 MU/min, which was correlated with DNA damage; however, these genes were upregulated more than 10-fold in HEM compared with controls (Fig. 2c), indicating very active DNA damage control in normal melanocytes. The cell cycle genes CCND1 and CCND2 were downregulated in melanoma cells (only WC00046 data shown; WC00060 and WC00081 showed similar results), but both genes were upregulated six-fold in HEM compared with controls (Fig. 2e). Neither radiation dose rate significantly affected proliferation of HEM (Fig. 2d) or HDF, and the cells continued to maintain normal physiological morphologies. These cells exhibited complete recovery from the radiation treatment and continued to proliferate 7 days after irradiation, suggesting that a total dose of 0.5 Gy administered at 2400 MU/min was relatively harmless to HEM and HDF.
Radiation induced upregulation of mitochondrial respiration by a post-transcriptional mechanism in both normal and cancer cells. Previous reports have indicated that mitochondrial respiration activities were altered after irradiation 38. To investigate respiration, irradiated melanoma cells were stained with the Mitotracker dye 7 days after irradiation, and fluorescence was qualitatively analyzed (Fig. 3a). Irradiated WC00046 cells showed markedly more fluorescence than nonirradiated controls, and the intensity of staining among cells irradiated at 2400 MU/min was visibly higher than that among cells irradiated at 400 MU/min, suggesting that the radiation directly triggered upregulation of mitochondrial respiration. WC00060 and WC00081 cells gave results similar to WC00046 cells (data not shown). Semiquantitative analysis of the relative fluorescent intensity showed a 1.3-fold increase in cells treated at 400 MU/min and a 2.3-fold increase in cells treated at 2400 MU/min compared with controls, indicating that the increase in fluorescence was directly associated with the radiation dose rate (Fig. 3b). Normal HEM did not show significant upregulation of respiration at either dose rate but HDF showed upregulation. In this setting, images of WC00046 cells that were irradiated and treated with inhibitors showed no significant change in morphology compared with controls (Fig. 3c).
Radiation and mitochondrial respiration inhibitors synergize to induce greater apoptosis in melanoma cells. To investigate whether targeting mitochondrial respiration increased apoptosis in a synergistic manner with radiation, a minimum lethal dose of oligomycin or rotenone was used to pretreat the cells before irradiation. Cell counts 7 days after irradiation showed that apoptosis increased in the presence of the inhibitors for all melanoma cells at both dose rates (Fig. 4a). On average, 76% cell death was observed among cells treated at 2400 MU/min compared with 59% among cells treated at a 400 MU/min dose rate, and cell death among cells treated at 2400 MU/min was 3.6-fold that among nonirradiated controls. In melanocytes, however, the dose of inhibitor used caused apoptosis in less than 20% of cells. In this setting, the expression of major mitochondrial respiratory genes (NDUFS4, SDHC, UQCR10, ATF6, COX412, and ATPAF2) was generally upregulated (Fig. 4b), suggesting that elevated respiratory activity subsequent to radiation treatment was partly a post-transcriptional event. When cell proliferation was quantified, irradiated WC00046 cells that were pretreated with inhibitors showed significant differences [11% for (+)O and 15% for (+)R] between the two dose rates; however, HEM cells did not show differences (Fig. 4c). Neither inhibitor significantly affected HEM survival, indicating that blocking mitochondrial respiration selectively enhances apoptosis of melanoma cells.
Colony survival of melanoma cells following irradiation at 2400 MU/min is significantly less than that after irradiation at 400 MU/min; however, HEM survival is maintained at 80%. To investigate the differences between cell-count data and end-point colony formation, irradiated cells were serially diluted and allowed to form colonies (Fig. 5a). The colony assay has been widely used in radiation biology to assess cytotoxicity and the effects of ionizing radiation on cells, as well as to study radiosensitizers 39,40. Decreases in survival (%) of melanoma cells were observed from cell (Fig. 1c) and colony counts (Fig. 5b), suggesting that the apoptotic effect of ionizing radiation continued past 7 days after irradiation. In contrast to cancer cells, the colony and cell counts of HEM did not show significant differences, indicating that the normal cells recovered and proliferated immediately after radiation treatment. This was consistent with the reduction in or the absence of DNA damage after irradiation (Fig. 2). The colony formation assay indicated that cell killing efficiency following irradiation was three-fold greater for WC00046 cells, seven-fold greater for WC00060 cells, and two-fold greater for WC00081 cells at 2400 MU/min compared with 400 MU/min (Fig. 5b). HEM colonies maintained greater than 80% survival at both dose rates (Fig. 5b). Pretreatment with oligomycin and rotenone before radiation treatment further reduced survival of melanoma cells (average 21% and average 17%, respectively; Fig. 5c).
Protein expression analyses were carried out using anti-Bcl-2, anti-caspase-3 (cleaved), and anti-PARP 1 (cleaved) on untreated and irradiated melanoma cells for antiapoptotic and apoptotic signals, respectively. Expression of Bcl-2 was downregulated in the irradiated melanoma cell lines (24 Gy/min), whereas untreated cells maintained their base level expressions (Fig. 6b). However, no significant changes were observed in the expression levels of caspase-3 between control and irradiated (24 Gy/min) cells (Fig. 6b). PARP 1 protein levels in irradiated melanoma cells were minimally downregulated when compared with nonradiated controls. Flow cytometric analyses of cyclin D1 and cyclin D2 in melanoma cell lines 24 h after irradiation showed a minimal decrease in the expression of the cyclins (Fig. 6c).
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.
Mitochondrial function is directly related to cancer progression 53,54. Our data showed that radiation caused melanoma cells to upregulate mitochondrial respiration, and this active energy production may be associated with a radioprotective mechanism (Fig. 3). Furthermore, proteinalysis of caspase-3 (cleaved) and PARP 1 (cleaved) showed no significant difference in melanoma cells in both nonradiated and irradiated (24 Gy/min) samples (Fig. 6b). Respiratory activity was directly correlated with dose rate in cancer cells up to 7 days after irradiation; however, this was not observed in HEM and HDF, suggesting apoptotic pressure on cancer cells but not on primary normal cells. Radiation-mediated mitochondrial respiratory activities in melanoma cells can be efficiently blocked by oligomycin or rotenone in dose windows that are minimally toxic to primary skin cells (Fig. 3), demonstrating potential efficacy for managing melanoma in combination with selected radiotherapy protocols. Previous studies have shown that sensitization of mitochondrial pathways in melanoma induces responses to chemotherapeutics 55. Furthermore, the metabolic pathways of normal cells depend on oxidative phosphorylation, but cancer cells rely on aerobic glycolysis, which depends on mitochondrial respiration 56. The results of this in-vitro study give a new perspective to melanoma therapy – that is, the combinatorial effect of high dose rate and low total dose has higher apoptotic potential in melanoma cells but minimal deleterious effects on surrounding HEM. Moreover, the addition of mitochondrial inhibitors further enhances apoptosis in melanoma while retaining high survival percentages of HEM, indicating a beneficial role of mitochondrial inhibitors in adjuvant therapy for metastatic and malignant melanomas. Innovative technology developments help deliver the required doses to the tumor and minimize damage to the surrounding tissue 57. In this study, mitochondrial inhibitors were shown to have a significant influence on melanoma radiation therapy, reducing survival of metastatic melanoma cells to 25% in colony assays and 44% in cell-count assays, and maintaining 80% survival of HEM.
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).
Conflicts of interest
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.