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Background: Metastatic melanoma is one of the most aggressive tumours and is also very resistant to current therapeutic approaches. The aim of this investigation was the in vitro study of the anti-proliferative effects of fotemustine (FM; 100 and 250 mu M), bevacizumab (5 mu g/ml) and proton irradiation (12 and 16 Gy) on resistant HTB140 human melanoma cells. Methods: Viability was estimated by sulphorhodamine B assay, while cell proliferation was analyzed by 5-bromo-2-deoxyuridine assay. Cell cycle distribution and apoptosis were examined using flow cytometry. Results: Cell viability and proliferation were reduced after all applied treatments. The level of apoptosis significantly increased after treatment with FM, protons or a combination of all agents, while the apoptotic index ranged from 1.2 to 9.2. Proton irradiation, as well as combined treatment with bevacizumab and protons or 100 mu M FM, bevacizumab and protons, have reduced melanoma cell proliferation through the induction of G1 phase arrest. Single FM (250 mu M) or bevacizumab treatment and their combination, as well as the joint application of these 2 agents with protons, reduced cell proliferation and provoked G2 phase accumulation. Conclusion: The analyzed treatments reduced cell viability and proliferation, triggered G1 or G2 cell cycle phase accumulation and stimulated apoptotic cell death. Copyright (C) 2010 S. Karger AG, Basel

Background: Considering that HTB140 melanoma cells have shown a poor response to either protons or alkylating agents, the effects of a combined use of these agents have been analysed. Methods: Cells were irradiated in the middle of the therapeutic 62 MeV proton spread out Bragg peak (SOBP). Irradiation doses were 12 or 16 Gy and are those frequently used in proton therapy. Four days after irradiation cells were treated with fotemustine (FM) or dacarbazine (DTIC). Drug concentrations were 100 and 250 mu M, values close to those that produce 50% of growth inhibition. Cell viability, proliferation, survival and cell cycle distribution were assessed 7 days after irradiation that corresponds to more than six doubling times of HTB140 cells. In this way incubation periods providing the best single effects of drugs (3 days) and protons (7 days) coincided at the same time. Results: Single proton irradiations have reduced the number of cells to similar to 50%. FM caused stronger cell inactivation due to its high toxicity, while the effectiveness of DTIC, that was important at short term, almost vanished with the incubation of 7 days. Cellular mechanisms triggered by proton irradiation differently influenced the final effects of combined treatments. Combination of protons and FM did not improve cell inactivation level achieved by single treatments. A low efficiency of the single DTIC treatment was overcome when DTIC was introduced following proton irradiation, giving better inhibitory effects with respect to the single treatments. Most of the analysed cells were in G1/S phase, viable, active and able to replicate DNA. Conclusion: The obtained results are the consequence of a high resistance of HTB140 melanoma cells to protons and/or drugs. The inactivation level of the HTB140 human melanoma cells after protons, FM or DTIC treatments was not enhanced by their combined application.

The correlation between time dependent viabilities, after applying two radiation qualities and two alkylating agents on HTB140 melanoma cells, has been studied. Irradiations were performed with gamma-rays and 62 MeV protons, close to the Bragg peak maximum, delivering doses of 8-24 Gy. Treatments with fotemustine (FM) and dacarbazine (DTIC) were carried out with concentrations of 0.05-2 mM. High radio-resistance of HTB140 cells revealed by a clonogenic assay was confirmed by microtetrasolium and sulforhodamine B, through the surviving fraction at 2 Gy (SF2), being 0.961-0.956 for gamma-rays and 0.931-0.887 for protons. A better efficiency of protons was illustrated by relative biological effectiveness at 2 Gy (RBE), ranging from 1.69 to 1.89. A kinetic study of concentration dependent cytotoxicity indicated that the best effect of the drugs, estimated as the concentration that produces 50% of growth inhibition (IC(50)), was obtained at 48 h, having values of 76 mu M for DTIC and 145 mu M for FM. The cytostatic ability of the drugs pointed out that the presence of DTIC at 24 h, compared to FM, was insufficient to produce an effect. Protons and FM demonstrated their pro apoptotic capacity. Cross-resistance between treatments applied to the HTB140 cells was observed, protons being the most efficient, while DTIC, FM and gamma-rays demonstrated a lower level of cell inactivation. (C) 2008 Associazione Italiana di Fisica Medica. Published by Elsevier Ltd. ALL rights reserved.

Effects of single irradiation with gamma rays and protons on human HTB140 melanoma cell growth were compared. Exponentially growing cells were irradiated close to the Bragg peak maximum of the unmodulated 62 MeV protons, as well as with Co-60 gamma rays. Applied doses ranged from 8 to 24 Gy. Viability of cells and proliferation capacity were assessed 7 days after irradiation. Induction of apoptosis and cell cycle phase redistribution were observed 6 and 48 h after irradiation. Significant inhibitory effects of both irradiation qualities were detected 7 days after irradiation. Important reduction of HTB140 cell viability was observed after irradiation with protons. Almost linear and highly significant (P LT 0.001) decrease of cell proliferation was observed 7 days after irradiation with gamma rays and protons, as compared to nonirradiated controls. Protons induced apoptosis, both 6 and 48 h after irradiation. With the increase of post-irradiation incubation time, number of apoptotic cells decreased. Exposure of HTB140 cells to gamma rays did not provoke apoptotic cell death. Important number of cells in G1-S phase, detected by the cell cycle phase redistribution analyses, suggested high metabolic activity of irradiated melanoma cells within the first 48 h. Both irradiation qualities caused modest G2-M arrest 6 and 48 h after irradiation, thus supporting results that illustrated high radioresistance of HTB140 cells.

Purpose: To measure the ability of protons and gamma-rays to effect cell viability and cell survival of human HTB140 melanoma cells. Materials and methods: Exponentially growing HTB140 cells were irradiated close to the Bragg peak maximum of the 62 MeV protons or with Co-60 gamma-rays with single doses, ranging from 8-24 Gy. Cell viability using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay was evaluated at 6 h, 24 h, 48 h or 7 days after irradiation and clonogenic survival was assessed at 7 days after irradiation. Cell cycle phase redistribution and the level of apoptosis were evaluated at 6 h and 48 h after irradiation. Results: The study of cell viability as a function of time (cell survival progression) and cell survival, using a clonal assay, demonstrated the considerably stronger inactivation effect of protons compared to gamma-rays with a relative biological effectiveness (RBE) of similar to 1.64. Cell cycle phase distribution and apoptosis levels with time enabled us to investigate the development and the character of the damage induced by irradiation. Due to the high radio-resistance of HTB140 cells, cell cycle phase redistribution exhibited only a modest cell accumulation in G2/M phase. Protons but not gamma-rays induced apoptosis. Conclusions: It appears that protons reduce the number of HTB140 cells by apoptosis as well as by severe DNA damage, while gamma-rays eliminate viable cells primarily by the production of irreparable DNA damage. Protons have an increased RBE relative to gamma-rays.

The objective of this study was to determine whether apoptosis and cell cycle redistribution were influenced by high-LET irradiation. Exponentially growing HTB140 cells were exposed to an unmodulated 62 MeV proton beam, within the Bragg peak, delivered over the single dose range from 8 Gy to 24 Gy. At 6 h post-irradiation, there was a low level of early apoptosis. At 48 h irradiated cells were more damaged, showing the increase in number of apoptotic nuclei. The dose dependent cell cycle phase distribution was detected at 48 h post-irradiation. The cell population exhibited phase redistribution toward G2/M phase.

The aim of this study was to determine the initial inactivation of cells induced by high-energy proton beam designed for the treatment of eye melanoma. Exponentially growing HTB140 cells were exposed to an unmodulated 62 MeV proton beam delivered over the single dose range from 8 Gy to 24 Gy. Position of samples was in the zone of the Bragg peak, having high LET values. Surviving fractions were evaluated at 6, 24 and 48 h post-irradiation. The survival curves exhibited a well-known shoulder, decreasing for doses higher than 8 Gy. Therefore, a significant dose dependent early cell inactivation after single delivery of 16 Gy to 24 Gy to the cell monolayer was observed. With the increase of the post-irradiation incubation time, a better killing effect, as the consequence of clonogenic survival, was detected.