Activation of p53 with Nutlin-3a radiosensitizes lung cancer cells via enhancing radiation-induced premature senescence

Abstract

Radiotherapy is routinely used for the treatment of lung cancer. However, the mechanisms underlying ionizing radiation (IR)-induced senescence and its role in lung cancer treatment are poorly understood. Here, we show that IR suppresses the proliferation of human non-small cell lung cancer (NSCLC) cells via an apoptosis-independent mechanism. Further investigations reveal that the anticancer effect of irradiation correlates well with IR-induced premature senescence, as evidenced by increased senescence-associated β-glactosidase (SA-β-gal) staining, decreased BrdU incorporation and elevated expression of p16(INK4a) (p16) in irradiated NSCLC cells. Mechanistic studies indicate that the induction of senescence is associated with activation of the p53-p21 pathway, and that inhibition of p53 transcriptional activity by PFT-α attenuates IR-induced tumor cell killing and senescence. Gain-of-function assays demonstrate that restoration of p53 expression sensitizes H1299 cells to irradiation, whereas knockdown of p53 expression by siRNA inhibits IR-induced senescence in H460 cells. Furthermore, treatment with Nutlin-3a, a small molecule inhibitor of MDM2, enhances IR-induced tumor cell killing and senescence by stabilizing the activation of the p53-p21 signaling pathway. Taken together, these findings demonstrate for the first time that pharmacological activation of p53 by Nutlin-3a can sensitize lung cancer cells to radiation therapy via promoting IR-induced premature senescence.

Keywords: Non-small cell lung cancer; Nutilin-3a; Radiotherapy; Senescence; p53; siRNA.

Fig. 1

IR suppresses the growth of NSCLC cells via an apoptosis-independent mechanism. (A) Clonogenic survival assays show that the number of cancer cell-derived colonies decreases with IR doses. (B) The results of clonogenic assays were normalized to the clonogenic survival of non-irradiated control cells and the survival fractions of A549 and H460 cells were plotted. (C–F) PI staining and flow cytometric analyses were employed to examine apoptosis (Sub G1 cells) and cell cycle distribution in H460 and A549 cells at 7 days after 6Gy of irradiation. (C) Representative flow cytometry graphs of cell cycle analysis of irradiated and control H460 cells. (D) The results of cell cycle analysis of H460 cells are presented as mean ± SEM (n = 3). (E) Representative flow cytometry graphs of cell cycle analysis of irradiated and control A549 cells. (F) The results of cell cycle analysis of A549 cells are presented as mean ± SEM (n = 3). (G and H) Activation of caspase-3 was determined by Western blotting at 24 h after IR or camptothecin (CPT, 5 μM) treatment. *p < 0.05 vs. control; **p <0.01 vs. control.

Fig. 2

IR induces premature senescence in NSCLC cells in a dose-dependent manner. (A) SA-β-gal staining increased (upper panel) and BrdU incorporation decreased (lower panel) with radiation dose in irradiated H460 cells. (B) The percentage of SA-β-gal positive senescent cells is presented as mean ± SEM (n = 3). (C) The percentage of BrdU positive proliferating cells is presented as mean±SEM (n = 3). **p < 0.001 vs. control.

Fig. 3

Activation of the p53-p21 pathway is involved in IR-induced senescence in NSCLC cells. (A) The activation of p53 in irradiated H460 cells was determined by Western blotting using a phosphorylated p53 (P-p53, Ser15) specific antibody. The expression levels of p21 and p16 were examined by Western blotting. Relative density of protein bands was quantified using Image J software (NIH) and normalized to β-actin. (B) The specificity of PFT-α to inhibit the transcriptional activity of p53 in NSCLC cells was determined by Western blotting. (C) H460 cells were pre-incubated with p53 specific inhibitor PFT-α (10 μM) prior to radiation exposure and clonogenic survival assays were performed to determine whether inhibition of p53 by PFT-α affects IR-induced tumor cell killings. (D and E) SA-β-gal staining and BrdU incorporation assays were performed to determine senescent cells in irradiated H460 cells. (F) Knockdown of p53 expression by siRNA was confirmed by Western blotting. (G) The effect of p53 knockdown on IR-induced senescence was determined by SA-β-gal assays. a, p < 0.01 vs. control; b, p < 0.05 vs. IR; #, p < 0.001 vs. control; $, p < 0.01 vs. IR; ***, p < 0.001 vs. NC-siRNA + IR.

Fig. 4

Restoration of p53 expression sensitizes lung cancer cells to IR-induced cell killing. (A) H1299 cells were transfected with pCEP4-p53 or pCEP4 empty vectors as control to restore the expression of p53 as described previously [32]. The expression of p53 in transfected cells was detected by Western blotting. (B) Clonogenic assays were performed to examine IR-induced tumor cell killing. *p < 0.01 vs. H1299/pCEP4 cells.

Fig. 5

Activation of p53 with Nutlin-3a enhances IR-induced tumor cell killing in NSCLC cells. (A) Lung cancer cells were pre-incubated with 3 μM of Nutlin-3a (Nut) for 30min prior to irradiation. The status of p53 activation was determined by p-p53 (Ser15) Western blotting analyses at 24 h after 4Gy of IR. The expression of total p53 and p21 were also determined by Western blotting. (B) Clonogenic assay was employed to detect IR-induced tumor cell killing. (C) SA-β-gal staining was performed to determine senescent cells in irradiated H460 cells. (D) BrdU incorporation assay was used to detect the proliferating cells. a, p < 0.05 vs. control (Ctl); b, p < 0.001 vs. Ctl; c, p < 0.01 vs. IR.