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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 0.05-2mM. 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 microM for DTIC and 145 microM 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 gamma-rays demonstrated a lower level of cell inactivation.

Novel antineoplastic agents, 8-chloroadenosine 3',5'-monophosphate (8-Cl-cAMP) and tiazofurin (TR), have been shown to be effective against different malignant cells. Through specific mechanisms of action they modulate the cellular signal transduction pathway, thereby causing growth inhibition, cell differentiation, and apoptosis. The aim of this work was the in vitro study of either 8-Cl-cAMP or TR effects on B16/F10 and B16/C3 mouse melanoma cell growth and cell death. Significant cell growth inhibition was obtained after the application of 8-Cl-cAMP or TR. The presence and number of apoptotic cells was evaluated using agarose gel electrophoresis and flow cytometry. The number of apoptotic nuclei, after treatment with antineoplastic agents, did not significantly change in B16/F10 cells, although it did show a significant increase in B16/C3 cells. The expression of c-myc did not significantly change in B16/F10 cells after treatment with 8-Cl-cAMP or TR. The same results were obtained in B16/C3 cells after treatment with 8-Cl-cAMP. The level of c-myc expression showed a significant increase in B16/C3 cells after treatment with TR. Concerning the effects that the analyzed agents exhibited on melanoma cells and other cancer cells, further preclinical studies of these drugs will potentially lead to better understanding of the molecular mechanisms of their action and finally more efficient therapeutic approaches to malignant diseases.

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.

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 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 ∼⃒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]