The drugs of nitrosourea type, such as FM, express high cytotoxicity through the formation of interstrand cross-links in DNA [33]. The

dominating mechanism of chemoresistance to alkylating agents is the repair of DNA adducts by the enzyme O6-methylguanine DNA-methyltransferase [3]. Ionizing radiation also induces activity of this enzyme [34]. In melanoma cells exposed to the alkylating agents or ionizing radiation the level of O6-methylguanine DNA-methyltransferase may increase, resulting in a resistance to such treatments. Some melanoma cell lines inherently express high level of O6-methylguanine buy GSK1120212 DNA-methyltransferase [5]. The weak effect of combined treatments is due to the relatively high level of O6-methylguanine DNA-methyltransferase that might be intrinsically present in the HTB140 cells and/or triggered by proton irradiation. Another possible reason for such a limited effectiveness BVD-523 supplier of the combination of protons and drugs is the nuclear transcription factor kappa B (NF-κB) that is constitutively expressed in melanoma cells [35]. NF-κB is an important feature in the development and progression of malignancies

by targeting genes that promote cell proliferation, survival, metastasis and angiogenesis. NF-κB also regulates apoptosis by controlling the transcription of genes that block cell death. Activation of NF-κB induces overexpression of bcl-xl, bcl-2, vascular endothelial growth factor and interleukin-8. This may affect resistance to apoptosis induced by radiation and chemotherapy [36]. Alkylating agents as well as ionizing radiation can induce cell death through the activation of apoptosis [21, 28,

37]. However, the described mechanism can cause defects in apoptotic pathways, leading to a high cellular resistance [35]. In the HTB140 cells proton irradiation induced G1 phase arrest, while FM as well as combined treatments provoked significant G2 arrest (Figure 3A). After ionizing radiation a delay in G2 phase is the most frequent event, but significant delays could also occur in G1 and S phase [38]. These results are in agreement Florfenicol with the high radioresistance of HTB140 cells [16]. FM generally produces a G2/M block in the cell cycle, while higher drug concentrations could induce S phase accumulation [39]. In samples exposed to FM or in combined treatments the cell proliferation (Figure 1B) was in agreement with the S phase (Figure 3A). Combined treatment with protons and DTIC, did not induce major changes in the cell cycle as compared to the control or single DTIC treatment (Figure 3B). Similar cell cycle arrest in S and G2/M phase buy Sepantronium caused by DTIC was also reported for other melanoma cells [40]. Compared to protons, after combined treatment there was a slight reduction of G1 phase and an increase of S phase. Most of the analysed cells were in G1/S phase, thus being viable and able to replicate DNA.