This approach revealed the late onset of apoptosis in most cell lines pretreated with NVP-AUY922 and 17-DMAG, and to a significantly lesser extent soon after therapy with NVP-BEP800 . Consequently, the radiosensitising activities of NVP-AUY922 and NVP-BEP800 in all Entinostat tested cell lines can not be explained solely by the drug-mediated susceptibility to apoptosis. Functional tumour suppressor protein p53 was apparently not essential for the radiosensitising action of NVP-AUY922 and NVP-BEP800, simply because both drugs radiosensitised all tested cell lines, independent of their p53 status . This discovering is constant together with the current data for two non-small-cell lung cancer cell lines, NCI-H460 and A549 , nevertheless it conflicts with all the results for squamous carcinoma cell lines , indicating that the Hsp90 inhibitor 17-AAG is a much more effective radiosensitiser inside a cell line with p53 wild variety compared with 4 p53-mutated cell lines. Summarising the western blot data shown in Figure three, neither changes in survival markers and apoptosis-associated protein nor alterations in p53 had been important to account for the sensitivity of two out of four tested cell lines to NVP-AUY922 and NVP-BEP800, either as a drug treatment alone or in combination with radiation.
At variance with expectations, the alkaline Comet assay revealed, in all tested cell lines, screening compounds a lower in TM values and thus a reduced DNA fragmentation immediately after combined drug-IR treatment, compared with those induced by IR alone .
The minor DNA fragmentation is often explained by the remarkable adjustments in the cell cycle brought on by Hsp90 inhibitors, that may be, an S-phase depletion and G2/M arrest , which had been apparently linked to big alterations in DNA compactness. As shown elsewhere , cells in the S phase show the highest TM values, whereas the TM values of G2/M cells are even reduce than those within the G1 phase. It ought to be noted that the Comet assay doesn’t give a measure for radiosensitivity within the standard sense, that may be, chromosome breakage, micronucleus formation, reduced growth and cloning survival, or elevated mutation frequency. Rather, the Comet assay evaluates chromatin integrity as a function of time promptly after irradiation. So, variations in chromatin compaction can strongly have an effect on the results from the Comet assay . The recognition of DNA harm by the Comet assay can also be effectively known to rely on numerous factors involved in the release of DNA in the nuclear protein matrix . In view of the above considerations, the observed drug-mediated reduction of IR-induced DNA fragmentation may have resulted in the drug-mediated, cell cycle-related changes in the compactness of chromatin/DNA structure. Regardless of the lower initial DNA fragmentation detected by the Comet assay, the rates of DNA restitution in three cell lines soon after a combined drug-IR treatment have been reduced than those after IR alone.