Inhibition of NF-κB by combination therapy with parthenolide and hyperthermia and kinetics of apoptosis induction and cell cycle arrest in human lung adenocarcinoma cells

We investigated the mechanisms of thermosensitization related to combination therapy with sesquiterpene lactone parthenolide (PTL), a nuclear factor-κB (NF-κB) inhibitor, and hyperthermia using human lung adenocarcinoma cells A549. The kinetics of apoptosis induction and cell cycle of cells treated with PTL, heating, and combined treatment were examined by flow cytometric analysis. The flow cytometric distribution was calculated and expressed as a percentage. The ratios of the sub-G₁ division, used to determine the induction of apoptosis, increased significantly with the combination therapy. Furthermore, the ratios of G₂/M division increased and the ratios of G₀/G₁ division decreased, indicating cell cycle arrest in G₂/M. The cell phase response to PTL by A549 cells synchronized in the G₁/S border with hydroxyurea was also analyzed. PTL showed remarkable cytotoxicity at the S phase of the cell cycle in A549 cells at all concentrations as well as with hyperthermia, thus PTL reduced the number of cells in the proliferation phase. Inhibition of intracellular transcription factor NF-κB activation in A549 cells with various incubation periods after treatments with PTL, heating and combined treatment was examined by Western blot analysis. Unexpectedly, PTL alone did not inhibit NF-κB activation in cells stimulated with TNF-α, while heating alone inhibited NF-κB early after treatment and that effect faded over time. In contrast, PTL combined with heating completely inhibited NF-κB activation. Our results demonstrated that PTL and heating in combination cause significant thermosensitization of A549 cells via induction of apoptosis or cell cycle arrest in G₂/M by inhibiting NF-κB activation in a synergistic manner.