Radiosensitization of Non-Small Cell Lung Cancer Cells by Inhibition of TGF-β1 Signaling With SB431542 Is Dependent on p53 Status

Abstract

Although medically inoperable patients with stage I non-small cell lung cancer cells (NSCLC) are often treated with stereotactic body radiation therapy, its efficacy can be compromised due to poor radiosensitivity of cancer cells. Inhibition of transforming growth factor-β1 (TGF-β1) using LY364947 and LY2109761 has been demonstrated to radiosensitize cancer cells such as breast cancer, glioblastoma, and lung cancer. Our previous results have demonstrated that another potent and selective inhibitor of TGF-β1 receptor kinases, SB431542, could radiosensitize H460 cells both in vitro and in vivo. In the present study, we investigated whether SB431542 could radiosensitize other NSCLC cell lines, trying to explore the potential implication of this TGF-β1 inhibitor in radiotherapy for NSCLC patients. The results showed that A549 cells were significantly radiosensitized by SB431542, whereas no radiosensitizing effect was observed in H1299 cells. Interestingly, both H460 and A549 cells have wild-type p53, while H1299 cells have deficient p53. To study whether the radiosensitizing effect of SB431542 was associated with p53 status of cancer cells, the p53 of H460 cells was silenced using shRNA transfection. Then it was found that the radiosensitizing effect of SB431542 on H460 cells was not observed in H460 cells with silenced p53. Moreover, X-irradiation caused rapid Smad2 activation in H460 and A549 cells but not in H1299 and H460 cells with silenced p53. The Smad2 activation postirradiation could be abolished by SB431542. This may explain the lack of radiosensitizing effect of SB431542 in H1299 and H460 cells with silenced p53. Thus, we concluded that the radiosensitizing effect of inhibition of TGF-β1 signaling in NSCLC cells by SB431542 was p53 dependent, suggesting that using TGF-β1 inhibitor in radiotherapy may be more complicated than previously thought and may need further investigation.

Figure 1

The effect of SB431542 at different concentrations on cell proliferation. (A) A549 cells and (B) H1299 cells.

Figure 2

SB431542 radiosensitized A549 cells but not H1299 cells. Clonogenic survival of NSCLC cells after irradiation with/without SB431542 pretreatment. (A) The effect of SB431542 at 10 µM on clonogenic survival of NSCLC cells (*p < 0.05 compared with the corresponding control). (B) Survival curves of A549 cells after irradiation with/without SB431542 pretreatment, which fit with linear-quadratic model (ANOVA interaction, p < 0.001). (C) Survival curves of H1299 cells after irradiation with/without SB431542 pretreatment, which fit with linear-quadratic model (p = 0.997).

Figure 3

X-irradiation induced Smad2 activation in H460 and A549 cells but not in H1299 cells. Smad2 phosphorylation in NSCLC cells after irradiation was determined by Western blotting. (A) H460 cells, (B) A549 cells, and (C) H1299 cells.

Figure 4

p53 silencing in H460 cells abolished radiosensitization effect of SB431542 and inhibited Smad2 activation upon irradiation. (A) Silence effect of p53 shRNA. H460 P represents H460 parental cells, H460 NC represents H460 cells transfected with negative control plasmids, H460 p53⁻ represents H460 cells with silenced p53. (B) Survival curves of H460 NC after irradiation with/without SB431542 pretreatment, which fit with linear-quadratic model (p = 0.00687). (C) Survival curves of H460 p53⁻ after irradiation with/without SB431542 pretreatment, which fit with linear-quadratic model (p = 0.520). (D) Smad2 activation in H460 NC upon irradiation. (E) No Smad2 activation in H460 p53⁻ upon irradiation. (F) Inhibition effect of SB431542 on Smad2 activation in H460 NC induced by irradiation.