Combining radiation with PI3K isoform-selective inhibitor administration increases radiosensitivity and suppresses tumor growth in non-small cell lung cancer

Abstract

Non-small cell lung cancer (NSCLC) is a malignant lung tumor with a dismal prognosis. The activation of the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway is common in many tumor types including NSCLC, which results in radioresistance and changes in the tumor microenvironment. Although pan-PI3K inhibitors have been tested in clinical trials to overcome radioresistance, concerns regarding their excessive side effects led to the consideration of selective inhibition of PI3K isoforms. In this study, we assessed whether combining radiation with the administration of the PI3K isoform-selective inhibitors reduces radioresistance and tumor growth in NSCLC. Inhibition of the PI3K/AKT pathway enhanced radiosensitivity substantially, and PI3K-α inhibitor showed superior radiosensitizing effect similar to PI3K pan-inhibitor, both in vitro and in vivo. Additionally, a significant increase in DNA double-strand breaks (DSB) and a decrease in migration ability were observed. Our study revealed that combining radiation and the PI3K-α isoform improved radiosensitivity that resulted in a significant delay in tumor growth and improved survival rate.

Keywords: PI3K isoform; non-small cell lung cancer (NSCLC); radiation; radioresistance; radiosensitivity.

Fig. 1

Radioresistance in lung cancer cells is increased by activation of the PI3K/AKT signaling pathway. (A) Expression of phospho-AKT(ser473) and phospho-γ-H2AX(ser139) I at the protein level and (B) Quantification of phospho-AKT and phospho-γ-H2AX protein expression in normal lung tissues and LLC1 tumor tissues after irradiation. N = normal, T = Tumor.

Fig. 2

Effect of irradiation on lung carcinoma cells. (A) Dose-survival curves derived from clonogenic assays (A549 P < 0.0001, LLC1 P < 0.0001). (B) Representative immunofluorescence images of the γ-H2AX expression in A549 and LLC1 cells 6 h after irradiation.

Fig. 3

PI3K isoform-selective inhibitors are effective in inhibiting lung carcinoma cell growth and AKT activation. A549 and LLC1 cells were treated with different concentrations of PI3K isoform-selective inhibitors for 24 h (ranging from 0.1 to 10 μM). (A) MTT cell proliferation assay. (B) Representative Western blots of PI3K/AKT signaling activation in A549 and LLC1 cells treated with PI3K isoform-selective inhibitors for 24 h. PC = Positive Control. A P-value <0.05 was considered as statistically significant. ^*P < 0.05, ^**P < 0.01 and ^***P < 0.001

Fig. 4

Combining radiation therapy with PI3K isoform-selective inhibitors increases radiosensitivity. (A) Results of clonogenic assays. A549 and LLC1 were treated with PI3K isoform-selective inhibitors (GDC0941 0.1 μM, GDC0032 0.1 μM, CAL101 1 μM, IPI145 1 μM) 3 h after irradiation of 2 Gy. (B) Activation of PI3K/AKT signaling pathway in A549 and LLC1 cells. (C) Representative immunofluorescence images of the γ-H2AX expression in A549 and LLC1 cells after the combination therapy. A P-value <0.05 was considered as statistically significant. ^*P < 0.05, ^**P < 0.01 and ^***P < 0.001

Fig. 5

Combining radiation with PI3K isoform-selective inhibitors regulates EMT-associated signaling in NSCLC cell lines. (A) The ability of migration and (B) expression of EMT markers in treated and untreated A549 and LLC1 cells with PI3K isoform-selective inhibitors 24 h after irradiation. IR = irradiation.

Fig. 6

Combining radiation with the PI3K-α isoform inhibitor suppressed lung tumor growth. After subcutaneous injection of LLC1 cells, irradiation (5 Gy) with or without treatment of the GDC0032 (5 mg/Kg body weight) was carried out. (A) Tumor volume growth was measured from 1–25 days. (B) Expression of EMT markers in the lung tumor tissue. IR = irradiation. A P-value <0.05 was considered as statistically significant. ^*P < 0.05, ^**P < 0.01 and ^***P < 0.001.