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Molecular targeted cancer therapy is a promising treatment strategy. Considering the central role of the epidermal growth factor receptor in cell proliferation and survival, there are indications that targeted agents like tyrosine kinase inhibitors, i. e., erlotinib, may enhance the antitumor treatment by radiation. The aim of this study is to analyze the inactivation effects of g-rays and to test the radiosensitizing potential of erlotinib on human lung adenocarcinoma cells in vitro. Irradiations were performed with doses ranging from 1 Gy to 8 Gy. In order to increase the radiosensitivity of CRL-5876 lung adenocarcinoma cells, the cells were treated with a clinically relevant concentration of 2 µM erlotinib. The effects of single and combined treatments were monitored using clonogenic survival, cell viability and proliferation assays at different time points. For the detection and visualization of the phosphorylated histone H2AX (γ-H2AX), an important biological marker of DNA double-strand break formation, fluorescence immunocytochemistry, was performed. The response to the treatment was monitored at four time points: 30 min, 2, 6, and 24 h. Irradiations with g-rays resulted in significant cell inactivation regarding all analyzed biological endpoints. Combined treatments revealed consistent cell inactivation. Moreover, compared to g-rays alone, elevated levels of g-H2AX foci were observed after pretreatment with erlotinib, indicating radiosensitization through impaired DNA repair. [Projekat Ministarstva nauke Republike Srbije, br. 173046 i br. 171019]

This study was conducted in order to evaluate the ability of carbon ions to induce DNA double-strand breaks and apoptosis in the radio-resistant human HTB140 melanoma cells. The cells were irradiated with 12C ions having the linear energy transfer of 258 keV/mm. Irradiations were performed in the dose range from 2 to 16 Gy. Induction of DNA double-strand breaks was evaluated 2 hour after irradiation through expression of gH2AX protein. Increased level of gH2AX detected in irradiated samples was especially high after irradiation with 12 and 16 Gy. Dose dependent increase of apoptosis was detected 48 hour after irradiation by flow-cytometry, with the maximum value of 20.4% after irradiation with 16 Gy, and the apoptotic index of 9.3. Pro-apoptotic effects of carbon ion beams were confirmed by changes of key molecules of the mitochondrial apoptotic pathway, p53 protein expression, Bax/Bcl-2 ratio and caspase-3 activation.

Abstract: In order to contribute to the understanding of mechanisms by which regulatory proteins recognize genetic information stored in DNA, analyses of their interaction with specific nucleotides are usually performed. In this study, the electrophoretic mobility shift assay (EMSA) was applied to analyze the interaction of nuclear proteins from the liver of rats of different age i.e., young (3-month-old), middle- aged (12-month-old) and aged (24-month-old), with radioactively labelled synthetic oligonucleotide analogues, corresponding to GRE. The levels of GRE binding activity were assessed by quantitative densitometric scanning of the autoradiograms. The results showed statistically significant decreasing values of up to 78% and 49% in middle aged and old animals, respectively, compared to young animals (p < 0.05). The specificity of the nuclear proteins-GRE interaction was demonstrated by competition experiments with unlabelled GRE. In a supershift assay, using the antibody BuGR2, it was shown that the GR proteins present in nuclear extracts have a high affinity for the GRE probe. The stabilities of the protein-DNA complexes were analysed and it was concluded that they changed during ageing. .

The complex relationship between linear energy transfer (LET) and cellular response to radiation is not yet fully elucidated. To better characterize DNA damage after irradiations with therapeutic protons, we monitored formation and disappearance of DNA double-strand breaks (DNA DSB) as a function of LET and time. Comparisons with conventional γ-rays and high LET carbon ions were also performed. In the present work, we performed immunofluorescence-based assay to determine the amount of DNA DSB induced by different LET values along the 62 MeV therapeutic proton Spread out Bragg peak (SOBP) in three cancer cell lines, i.e. HTB140 melanoma, MCF-7 breast adenocarcinoma and HTB177 non-small lung cancer cells. Time dependence of foci formation was followed as well. To determine irradiation positions, corresponding to the desired LET values, numerical simulations were carried out using Geant4 toolkit. We compared γ-H2AX foci persistence after irradiations with protons to that of γ-rays and carbon ions. With the rise of LET values along the therapeutic proton SOBP, the increase of γ-H2AX foci number is detected in the three cell lines up to the distal end of the SOBP, while there is a decrease on its distal fall-off part. With the prolonged incubation time, the number of foci gradually drops tending to attain the residual level. For the maximum number of DNA DSB, irradiation with protons attain higher level than that of γ-rays. Carbon ions produce more DNA DSB than protons but not substantially. The number of residual foci produced by γ-rays is significantly lower than that of protons and particularly carbon ions. Carbon ions do not produce considerably higher number of foci than protons, as it could be expected due to their physical properties. In situ visualization of γ-H2AX foci reveal creation of more lesions in the three cell lines by clinically relevant proton SOBP than γ-rays. The lack of significant differences in the number of γ-H2AX foci between the proton and carbon ion-irradiated samples suggests an increased complexity of DNA lesions and slower repair kinetics after carbon ions compared to protons. For all three irradiation types, there is no major difference between the three cell lines shortly after irradiations, while later on, the formation of residual foci starts to express the inherent nature of tested cells, therefore increasing discrepancy between them. [ABSTRACT FROM AUTHOR]

Abstract 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.

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]

Purpose: Investigation of effects on DNA of γ-irradiated human cancer cells pretreated with free radical scavengers is aimed to create reference data which would enable assessment of the relative efficiency of high linear energy transfer (LET) radiations used in hadron therapy, i.e. protons and carbon ions. Materials and methods: MCF-7 breast and HTB177 lung cancer cells are irradiated with γ-rays. To minimize indirect effects of irradiation, dimethyl sulfoxide (DMSO) or glycerol are applied as free radical scavengers. Biological response to irradiation is evaluated through clonogenic cell survival, immunocytochemical and cell cycle analysis, as well as expression of proteins involved in DNA damage response. Results: Examined cell lines reveal similar level of radioresistance. Application of scavengers leads to the rise of cell survival and decreases the number of DNA double strand breaks in irradiated cells. Differences in cell cycle and protein expression between the two cell lines are probably caused by different DNA damage repair mechanisms that are activated. Conclusion: The obtained results show that DMSO and glycerol have good scavenging capacity, and may be used to minimize DNA damage induced by free radicals. Therefore, they will be used as the reference for comparison with high LET irradiations, as well as good experimental data suitable for validation of numerical simulations. [ABSTRACT FROM AUTHOR]

Response of human HTB140 melanoma cells to proton irradiation in combination with fotemustine (FM) was investigated. Effects of these agents were analysed on cell proliferation and induction of apoptosis. Cells pretreated with 100- or 250-µM of FM were irradiated in the middle of the therapeutic 62-MeV proton spread-out Bragg peak, with a dose of 16 Gy. All treatments reduced proliferation and survival of melanoma cells. The most pronounced effects of the combined treatment were obtained for cell survivals. The level of apoptosis increased after all applied treatments. Particularly good pro-apoptotic effect was achieved when proton irradiation was combined with 250 μM of FM. This was followed by the increased expression of p53 gene. The obtained results have shown that combined application of FM and protons significantly reduced growth of this resistant melanoma cell line. [ABSTRACT FROM PUBLISHER]

Purpose: To analyse changes of cell inactivation and proliferation under therapeutic irradiation conditions along the proton spread out Bragg peak (SOBP) with particular emphasis on its distal declining edge. Materials and methods: HTB140 cells were irradiated at four positions: plateau, middle, distal end and distal declining edge of the 62 MeV proton SOBP. Doses ranged from 2–16 Gy. They were normalised in the middle of SOBP and delivered following the axial physical dose profile. Survival, proliferation and cell cycle were assessed seven days after irradiation. Results: Moving from proximal to distal irradiation position surviving fractions at 2 Gy (SF2) decreased from 0.88–0.59. Increased radiosensitivity of the cells was noticed for the doses below 4 Gy, resulting in two gradients of cell inactivation, stronger for lower and weaker for higher doses. Relative biological effectiveness (RBE) increased from 1.68–2.84 at the distal end of SOBP. A further rise of RBE reaching 7.14 was at its distal declining edge. Following the axial physical dose profile of SOBP the strongest inactivation was attained at its distal end and was comparable to that at its declining edge. Conclusions: Survival data confirmed very high radioresistance of HTB140 cells. An effect similar to low-dose hyper radiosensitivity (HRS) was observed for order of magnitude larger doses. Better response of cells to protons than to γ-rays was illustrated by rather high RBE. Strong killing ability at the SOBP distal declining edge was the consequence of increasing proton linear energy transfer. [ABSTRACT FROM AUTHOR]

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 μM), bevacizumab (5 μg/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 μM FM, bevacizumab and protons, have reduced melanoma cell proliferation through the induction of G1 phase arrest. Single FM (250 μM) 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 [ABSTRACT FROM AUTHOR]

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]

Purpose: To measure the ability of protons and γ-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 60 Co γ-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 γ-rays with a relative biological effectiveness (RBE) of &nvsim;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 γ-rays induced apoptosis. Conclusions: It appears that protons reduce the number of HTB140 cells by apoptosis as well as by severe DNA damage, while γ-rays eliminate viable cells primarily by the production of irreparable DNA damage. Protons have an increased RBE relative to γ-rays. [ABSTRACT FROM AUTHOR]

The aim of the present study was to investigate effects of pomegranate peel (PP) extract on different human cancer cell lines. MTT was performed to estimate cytotoxic effects of PP extract against HTB140, HTB177, MCF7, HCT116 human cancer cell lines and MRC-5 normal fibroblasts. Clonogenic assay was used to reveal cell survival after the treatment with PP extract. Cell cycle analysis was done using flow cytometry. Wound healing assay was applied to estimate inhibitory effects of PP extract on migration of cancer cells. The results showed that PP extract expressed selective cytotoxicity for cancer cells compared to normal cell line. Analyzed cancer cell lines displayed individual variations in sensitivity to PP extract reflected through changes in clonogenic survival, cell cycle distribution and migration, which may be due to the specific nature of each tested cell line. In conclusion, PP extract exhibits good inhibitory effects on tested cancer cell lines. [ABSTRACT FROM AUTHOR]