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The aim of this study was to investigate effects of irradiations with the therapeutic proton and carbon ion beams in two non-small cell lung cancers, CRL5876 adenocarcinoma and HTB177 large cell lung carcinoma. The DNA damage response dynamics, cell cycle regulation, and cell death pathway activation were followed. Viability of both cell lines was lower after carbon ions compared to the therapeutic proton irradiations. HTB177 cells showed higher recovery than CRL5876 cells seven days following the treatments, but the survival rates of both cell lines were lower after exposure to carbon ions with respect to therapeutic protons. When analyzing cell cycle distribution of both CRL5876 and HTB177 cells, it was noticed that therapeutic protons predominantly induced G1 arrest, while the cells after carbon ions were arrested in G2/M phase. The results illustrated that differences in the levels of phosphorylated H2AX, a double-strand break marker, exist after therapeutic proton and carbon ion irradiations. We also observed dose- and time-dependent increase in the p53 and p21 levels after applied irradiations. Carbon ions caused larger increase in the quantity of p53 and p21 compared to therapeutic protons. These results suggested that various repair mechanisms were induced in the treated cells. Considering the fact that we have not observed any distinct change in the Bax/Bcl-2 ratio following irradiations, it seemed that different types of cell death were involved in the response to the two types of irradiations that were applied.

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 microM), bevacizumab (5 microg/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 microM FM, bevacizumab and protons, have reduced melanoma cell proliferation through the induction of G1 phase arrest. Single FM (250 microM) 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 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 microM, 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 approximately 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.

Analysis of elimination of four human radioresistant malignant cell lines to mono-energetic and non mono-energetic incoming carbon ion beams, characterized by different linear energy transfer (LET) qualities is performed. Comparisons with protons from the middle of the therapeutic spread out Bragg peak (SOBP) and reference γ-rays are also included. HTB140 cells were irradiated at five positions, with different LET, along the 62 MeV carbon pristine Bragg peak. To provide reliable reproducibility of irradiations at INFN-LNS, as the carbon Bragg peak is very narrow, precise positioning of samples for desired LET value is complicated. The peak was slightly widened using two ripple filters. After defining irradiation position and LET at the peak itself where cell killing is almost the highest, irradiation position with the same LET value was found within somewhat broadened peak. HTB140, MCF-7, HTB177 and CRL5876 cells were irradiated at the two described positions. Additionally, irradiations in the middle of 62 MeV proton SOBP and reference γ-rays were performed. Doses ranged from 0.5 to 16 Gy. Cell survival and corresponding radiobiological parameters were assessed seven days after irradiations. When moving irradiation position along the carbon Bragg curve, LET rises from 85 to 747 keV/µm, while surviving fraction at 2 Gy (SF2) for HTB140 cells, falls from 0.72 to 0.57 further rising to 0.73 on the distal fall-off part of the curve. Improved cell radiosensitivity is seen for the doses below 4 Gy. Relative biological effectiveness (RBE) increases from 4.56 to 7.69 and drops to 4.23. Almost the highest cell killing LET, being ∼200 keV/µm, is used to irradiate HTB140, MCF-7, HTB177 and CRL5876 cells within the pristine and slightly broadened Bragg peak. After irradiations with protons of the mid SOBP, carbon ions of the pristine and slightly widened Bragg peak RBE ranges for HTB140 cells from 2.08, 4.81 to 7.06, for MCF-7 from 1.70, 3.28 to 4.17, for HTB177 from 1.98, 4.18 to 5.08 and for CRL5876 from 1.33, 2.57 to 3.51. Significant elimination of HTB140 cells is observed along the carbon Bragg curve. The highest one is achieved by LET that is at the level of already reported. For the same LET, mono-energetic carbon ions provide higher cell elimination than the non mono-energetic. For all cell lines, both carbon ion beams, more the monoenergetic one, express stronger killing rate than protons and especially γ-rays. [ABSTRACT FROM AUTHOR]

Abstract: 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 γ-rays and 62MeV protons, close to the Bragg peak maximum, delivering doses of 8–24Gy. Treatments with fotemustine (FM) and dacarbazine (DTIC) were carried out with concentrations of 0.05–2mM. High radio-resistance of HTB140 cells revealed by a clonogenic assay was confirmed by microtetrasolium and sulforhodamine B, through the surviving fraction at 2Gy (SF2), being 0.961–0.956 for γ-rays and 0.931–0.887 for protons. A better efficiency of protons was illustrated by relative biological effectiveness at 2Gy (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 (IC50), was obtained at 48h, having values of 76μM for DTIC and 145μM for FM. The cytostatic ability of the drugs pointed out that the presence of DTIC at 24h, 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 γ-rays demonstrated a lower level of cell inactivation. [Copyright &y& Elsevier]

Viability of human HTB140 melanoma cells after being exposed to fotemustine (FM) and dacarbazine (DTIC) as well as to proton irradiation was studied. Effects of 100 and 250 μM drugs were assessed after incubation of 6, 24, 48, 72, and 96 h. Irradiations were performed with 62 MeV therapeutic protons, delivering to the cell monolayer single doses of 2, 4, 8, 12, and 16 Gy. Viability was evaluated 7 days after irradiation. Inactivation level was estimated using microtetrasolium (MTT) and sulforhodamine B (SRB) assays. Combined effects of each drug and protons, were carried out using the same drug concentrations. Proton doses applied were those used in therapy, that is, 12 and 16 Gy. With the increase of drug concentration or irradiation dose, level of cell inactivation reached approximately 60%, 48 h after drug treatment or 7 days after irradiation at 16 Gy. Considering the rate of drug concentrations used, as well as the level of doses applied, it appears that HTB140 cells are more resistant to proton irradiation than to alkylating agents tested. The combined treatment with FM or DTIC and protons did not show significant changes of cell viability as compared to the effects of single agents. Since the time point for measuring cumulative effects of drug and irradiation was 48 h post irradiation, it seems that the obtained level of viability could be attributed primarily to the effects of drugs. [ABSTRACT FROM AUTHOR]