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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 μM, 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 ∼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. [ABSTRACT FROM AUTHOR]