Nachweis residueller DNA-Doppelstrangbrüche in Zellen des nicht-kleinzelligen Lungenkarzinoms nach Bestrahlung mit Kohlenstoffionen vergleichend zu Photonen unter Inhibition des Phosphatidylinositol-3- Kinase/mammalian-Target-of-Rapamycin- sowie des Mitogen-aktivierte-Proteinkinase-Signalwegs
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Abstract
Both photon and carbon ion radiotherapy primarily achieve tumour cell killing through the
induction of DNA double-strand breaks (DSBs). The repair of these breaks involves, among
others, the PI3K/mTOR and MAPK pathways. Inhibition of these pathways has already been
shown in research to achieve radiosensitisation. Therefore it should be investigated, wether the
inhibition of these signalling pathways leads to radiosensitisation through inhibition of DNA
double-strand break repair, and wether dual targeting of both pathways can lead to a further
increase in radiosensitisation.
The experiments were conducted on four NSCLC cell lines (A549, H1299, H661, H1975). The
cells were irradiated using 3 Gy Photons or 1 Gy carbon ions, assuming a RBE of 3. For inhibition
of PI3K/mTOR, the dual inhibitor NVP-BEZ235 (50 nM) was used, and for inhibition of the
MAPK pathway, the MEK-Inhibitor PD98059 (50 μM) was applied. Residual 53BP1 foci in
G1 phase were detected 24 hours after irradiation using 53BP1-CenpF-immunofluorescence
staining.
The following results were observed in the experiments:
Irradiation with either photons or carbon ions resulted in a significant increase in the number
of residual 53BP1 foci in all cases.
No relevant effects were observed in cell cycle distribution; the majority of cells consistently
remained in the G1 phase.
In unirradiated cells, the inhibitors PD98059 and NVP-BEZ235, despite showing activity on
protein expression, had no effect on the mean number of residual 53BP1 foci.
Inhibition of the MAPK pathway did not result in any further increase in the mean number
of residual 53BP1 foci following irradiation with either photons or carbon ions.Inhibition of the PI3K/mTOR pathway led to a significant increase in the mean number of
residual 53BP1 foci in all cell lines after irradiation with either photons or carbon ions.
Dual targeting of both pathways led to a further significant increase in residual 53BP1 foci
following photon irradiation in A549, H1975 and H1200 cells, compared with the inhibition
of PI3K/mTOR alone.
Following carbon ion irradiation, dual targeting led to a significant increase in residual DSBs
only in A549 cells, compared with PI3K/mTOR inhibition alone.
Inhibition of the PI3K/mTOR pathway with NVP-BEZ235 prior to irradiation could further
increase the number of residual DNA double-strand breaks. This is presumably due to the major
role of the PI3K/mTOR pathway in DNA double-strand break repair via non-homologous end
joining. Inhibition of the MAPK pathway with PD98059 alone did not produce such an effect.
Dual targeting of both pathways simultaneously, compared with inhibition of PI3K/mTOR
alone, had a heterogeneous effect depending on irradiation type and cell line. A549 cells consist-
ently showed a significantly positive effect from dual targeting.
The experiments clearly demonstrated, that inhibition of the PI3K/mTOR pathway – depending
on the tumour’s mutation profile, potentially in combination with inhibition of the MAPK
pathway – is a promising target for agents aimed at specific radiosensitisation. This could si-
gnificantly enhance the efficacy of existing radiotherapy, or enable better normal tissue sparing
due to dose reduction. However, the molecular mechanisms governing the effectiveness of dual
targeting still require further investigation.
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This item has been published with the following license: In Copyright