Inhibition of IKK-NFκB pathway sensitizes lung cancer cell lines to radiation

Abstract

Objective: : Cancer cell radioresistance is a stumbling block in radiation therapy. The activity in the nuclear factor kappa B (NFκB) pathway correlates with anti-apoptotic mechanisms and increased radioresistance. The IKK complex plays a major role in NFκB activation upon numerous signals. In this study, we examined the interaction between ionizing radiation (IR) and different members of the IKK-NFκB pathway, as well as upstream activators, RAF1, ERK, and AKT1.

Methods: : The effect of 4 Gy of IR on the expression of the RAF1-ERK-IKK-NFκB pathway was examined in A549 and H1299 lung cancer cell lines using Western blot analysis and confocal microscopy. We examined changes in radiation sensitivity using gene silencing or pharmacological inhibitors of ERK and IKKβ.

Results: : IKKα, IKKγ, and IκBα increased upon exposure to IR, thereby affecting nuclear levels of NFκB (phospho-p65). ERK inhibition or siRNA-mediated down-regulation of RAF1 suppressed the post-irradiation survival of the examined lung cancer cell lines. A similar effect was detected on survival upon silencing IKKα/IKKγ or inhibiting IKKβ.

Conclusions: : Exposure of lung cancer cells to IR results in NFκB activation via IKK. The genetic or pharmacological blockage of the RAF1-ERK-IKK-NFκB pathway sensitizes cells to therapeutic doses of radiation. Therefore, the IKK pathway is a promising target for therapeutic intervention in combination with radiotherapy.

Keywords: IKK; NFκΒ; ionising radiation; lung cancer; radiosensitization.

1

IR affects protein levels of the IKK members, upstream activators, and phosphorylation of the downstream p65 NFκB in A549 and H1299 lung cancer cell lines. A549 and H1299 cells were treated with 4 Gy IR, and cytoplasmic (IKKα, pIKKα, IKKγ, IκBα, pp65 NFκB, RAF1, and pAKT1S1) or nuclear fractions (pp65 NFκB) of cell lysates were collected 30 min, 4 and 24 h, and 7 days post-irradiation and analyzed with Western blot. Non-irradiated samples were used as an untreated control (denoted as 0), and β-actin and lamin B1 were used as loading controls for cytoplasmic and nuclear fractions, respectively.

2

IR affects protein levels of IKK members, upstream activators, and phosphorylation of the downstream p65 NFκB in A549 and H1299 lung cancer cell lines. Cells were collected for the confocal microscopy immunofluorescence analysis at the same time points as for the Western blot analysis (Figure 1) and fixed and stained with the relevant antibodies. Representative images demonstrate antibody staining at the indicated time points. Hoechst 33342 was used as a nuclear marker. Scale bar denotes 10 μM.

S1

Constitutive over-expression of nuclear NFκB pp65 in NSCLC cell lines. Nuclear fractions from cells of two normal human lung tissues (denoted as NL1 and NL2) and two NSCLC cell lines, A549 and H1299, were compared regarding their endogenous expression of NFκB pp65. Lamin B1 was used as a loading control.

3

Down-regulation of IKK family members and upstream activator Raf1 increases radiosensitivity of A549 and H1299 lung cancer cell lines. (A) Immunoblot analysis demonstrates the effective down-regulation of RAF1, IKKα, and IKKγ in A549 and H1299 cell lines after a 48 h incubation of cells with the designated pools of siRNAs. si-scrambled treated cells were used as a control, and β-actin was used as a loading control. (B) Immunoblot analysis shows NFκB pp65 nuclear protein levels after a 48 h incubation of cells with the designated siRNA pools against RAF1, IKKα, and IKKγ. si-scrambled treated cells were used as control, and lamin B1 was used as a loading control. C-D) A549 and H1299 cells were transfected with siRNA pools against RAF1, IKKα, and IKKγ, and dose-response viability curves are plotted after treatment with 2, 4, 6 or 8 Gy IR.

4

Pharmacological inhibition of IKKβ and upstream activator ERK decreases pp65 protein accumulation in the cell nuclei and increases radiosensitivity of A549 and H1299 lung cancer cell lines. (A) Immunoblot analysis demonstrates the protein levels of pp65 NFκB at 30 min after treating A549 and H1299 cells with 4 Gy IR in the presence or absence of 75 μM of the ERK inhibitor (upper panel) or 20 μM of the IKKβ inhibitor (lower panel). Lamin B1 was used as a loading control, and con denotes the untreated control. (B) A549 and H1299 cells were pre-treated for 24 h with 75 μM of ERK or 20 μM of IKKβ inhibitor and irradiated with 2, 4, 6 or 8 Gy IR. Cell viability was monitored 7 days later. Pairwise comparisons were performed for all cases, and their P values are shown.