A novel role for apatinib in enhancing radiosensitivity in non-small cell lung cancer cells by suppressing the AKT and ERK pathways

AP preparation, cell culture, and ionizing radiation (IR) protocol

AP was obtained from the Jiangsu Hengrui Medicine Company (Jiangsu, China). AP was dissolved in 100% dimethyl sulfoxide (DMSO) as a 100 mM stock solution and diluted with RPMI-1640 medium to the desired concentration.

Cells were cultured as previously described in Li et al. (2021). Specifically, the normal mammary epithelial cell line HBEC and four human NSCLC cell lines, A549, H460, H226, and H522, were purchased from the American Type Culture Collection (Manassas, VA, USA). LK2 cells were obtained from the Cell Bank of the Chinese Academy (Shanghai, China). All cells were cultured in RPMI-1640 medium (Sigma-Aldrich, Darmstadt, Germany) and supplemented with 10% fetal bovine serum (Clark Bioscience, Richmond, VA, USA) and 1% penicillin/streptomycin (Sigma-Aldrich, Darmstadt, Germany) in a humidified incubator at 37 °C in 5% CO₂. A 6-MeV X-ray medical linear accelerator (Elekta Synergy, Elekta, Stockholm, Sweden) was used to irradiate the cells at a dose rate of 300 cGy/min (dose: 0 to 8 Gy) at room temperature.

RNA extraction, reverse transcription, and qRT-PCR

RNA extraction, reverse transcription, and qRT-PCR were conducted as previously described in Li et al. (2021). Specifically, total RNA was extracted from cells using the TRIzol™ Plus Kit (Takara, Osaka, Japan) according to the manufacturer’s instructions. cDNA was synthesized with total RNA (1 µg) using a real-time PCR system (Life Technologies, Carlsbad, CA, USA), and qRT-PCR was performed with total cDNA (100 ng) using an Applied Biosystems 7,500 Real-Time PCR system with SYBR™ Green Master Mix (Takara, Osaka, Japan). The relative expression of VEGFR2 was determined using the 2^−ΔΔCt method after normalization to GAPDH expression. The primers for VEGFR2 were as follows: forward, 5′-GTGATCGGAAATGACACTGGAG-3′ and reverse, 5′-CATGTTGGTCACTAACAGAAGCA-3′.

Flow cytometric analysis

Apoptosis and cell cycle status were conducted as previously described in Li et al. (2021). Specifically, apoptosis was measured using the FITC Annexin V Apoptosis Detection Kit (BD Pharmingen, San Diego, CA, USA) according to the manufacturer’s instructions. Approximately 10 × 10⁵ A549 cells or 15 × 10⁵ LK2 cells were seeded into six-well plates and irradiated with 0 or 8 Gy the next day, cultured for another 48 h, and then incubated with FITC-labeled annexin V and propidium iodide (PI) at room temperature in the dark for 15 min.

Cell cycle analysis was performed using 50 µg/mL PI and 100 µg/mL DNase-free RNase A (Solarbio, Beijing, China). A total of 40 × 10⁵ A549 cells or 60 × 10⁵ LK2 cells were seeded into 25 cm² Cell Culture Flasks. A total of 24 h post-irradiation, cells were harvested with trypsin, washed with phosphate-buffered saline, and fixed in 70% ice-cold ethanol at 4 °C for 12 h. After washing, the cell pellet was resuspended in PI staining buffer and incubated at 37 °C for 30 min in the dark. Apoptosis and cell cycle status were analyzed by flow cytometry (BD FACSCalibur, BD Biosciences, San Jose, CA, USA).

Cell proliferation and colony formation assay

Cells were seeded into plates with 96 wells at 2,000 A549 cells or 4,000 LK2 cells per well. After exposure to a single dose of radiation (8 Gy), cells were incubated for 24, 48, 72, or 96 h, at which point Cell Counting Kit-8 (Beyotime, Shanghai, China) was used to determine cell viability. Absorbance was measured using a microplate reader (BioTek, Winooski, VT, USA) at a wavelength of 450 nm. The IC₅₀ value of each cell line was calculated using the GraphPad Prism 7 software (La Jolla, CA, USA).

The colony formation assay was conducted as previously described in Li et al. (2021). Specifically, 200 cells were seeded into 6-well plates and irradiated (0, 2, 4, 6, and 8 Gy) the next day. A total of 2 weeks later, the cells were fixed in 4% paraformaldehyde and stained with 0.1% crystal violet, and the number of colonies per dish was counted. The plating efficiency and surviving fraction were calculated as previously described (Sun et al., 2019).

Immunofluorescence detection of phosphorylated histone H2AX (γH2AX)

The immunofluorescence assay was conducted as previously described in Li et al. (2021). Specifically, cells growing on glass coverslips were exposed to 0 or 8 Gy irradiation. A total of 4 h later, the cells were fixed in 4% paraformaldehyde and incubated with the primary phospho-γH2AX antibody (Ser139; Abcam, San Diego, CA, USA) and a secondary antibody conjugated to Cy3 (Beyotime) according to the manufacturer’s protocol. Staining was analyzed using a confocal laser scanning microscope (Nikon, Tokyo, Japan).

Protein extraction and western blotting

Protein extraction and western blotting were conducted as previously described in Li et al. (2021). Specifically, total protein was extracted from cells using radioimmunoprecipitation assay lysis buffer (Beyotime). Total protein concentrations were quantified using a bicinchoninic acid assay kit (Beyotime). Equal amounts (30 µg) of proteins were boiled at 100 °C, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% (w/v) skim milk at room temperature and immunoblotted overnight at 4 °C with primary antibodies against GAPDH (60004-1-Ig; Proteintech, Chicago, IL, USA), VEGFR2 (abs131800; Absin, Shanghai, China), and AKT (#4685; Cell Signaling Technology, Beverly, MA, USA), phospho-AKT (#4060; Cell Signaling Technology, Beverly, MA, USA), ERK (#4695; Cell Signaling Technology, Beverly, MA, USA), and phospho-ERK (#4370, Cell Signaling Technology, Beverly, MA, USA). The membranes were then incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies (HRP-conjugated goat anti-mouse IgG, Zsgb Bio, ZB-2305; HRP-conjugated goat anti-rabbit IgG, Zsgb Bio, ZB-2301).

Statistical analysis

The experimental data were calculated as the mean ± standard deviation of at least three independent experiments. The data were analyzed, and statistical graphs were created using GraphPad Prism 7. Differences between groups were analyzed using the Student’s t-test, one-way analysis, and two-way ANOVA. Statistically significant differences were determined at p < 0.05 (*), <0.01 (**), or <0.001 (***).

Expression of VEGFR2 in NSCLC cells

The A549, H522, H460, LK2, and H226 cell lines were evaluated for VEGFR2 expression by western blotting and qRT-PCR (Figs. 1A and 1B). VEGFR2 protein expression differed among the five NSCLC cell lines. A549 and LK2 cells were selected for further study based on their expression of VEGFR2.

To examine the effects of AP on NSCLC cell growth, the cytotoxicity of AP against NSCLC cell lines was determined using the CCK8 assay. The human lung cancer cell lines A549 and LK2 were cultivated with a series of increasing concentrations of AP (0, 5, 10, 20, 40, and 80 μmol/L) for 24, 48, and 72 h. AP significantly inhibited the proliferation of A549 and LK2 cells in vitro in a dose- and time-dependent manner (Figs. 1C and 1E). After calculation, the IC₅₀ doses of AP after treatment for 72 h in LK2 cells and A549 cells were 30.73 μmol/L and 17.61 μmol/L, respectively (Figs. 1D and 1F). We chose the IC₂₀ concentration at 72 h in subsequent experiments.

AP promoted cell death and enhanced radiosensitivity in NSCLC cells

To explore the efficacy of AP in combination with radiotherapy, the CCK8 assay was conducted to determine whether the inhibitory effect of IR was enhanced by AP. LK2 and A549 cells were pretreated with AP or DMSO for 24, 48, 72, or 96 h and exposed to 8 Gy irradiation. Compared to IR alone, cell growth was remarkably suppressed in the cells pretreated with AP before IR at all of the time points examined, although this effect was particularly visible at 96 h. In addition, cells treated with both AP and IR had less proliferation than cells treated with IR or AP alone (Figs. 2A and 2B). These results suggest that AP suppresses the proliferation of NSCLC cells in vitro and enhances the radiosensitivity of NSCLC cells.

To further confirm the radiosensitivity effects of AP, a colony formation assay was performed. The results revealed that treatment with AP plus IR significantly reduced the survival rate in both NSCLC cell lines compared with cells treated with AP or IR alone. The sensitivity enhancement ratios (SERs) were 1.95 and 2.15 in LK2 and A549 cells, respectively (Table 1). These data suggest that AP sensitizes NSCLC cell lines to IR in vitro.

AP and radiation combination therapy affected apoptosis and cell cycle progression of NSCLC cells

Flow cytometry was used to determine whether AP could induce NSCLC cell apoptosis and enhance the radiosensitivity of A549 and LK2 cells. Annexin V and PI staining were used to detect the percentage of cell death in A549 and LK2 cells treated with AP after 48 h with or without IR. The percentages of apoptotic A549 and LK2 cells were significantly higher in cells treated with AP than in control cells. Further, AP combined with IR remarkably enhanced apoptosis compared with control cells and cells treated with either AP or IR (Fig. 3A). Taken together, these results demonstrate that AP enhances radiation-induced cell apoptosis.

To further investigate the effect of AP in NSCLC cells after IR, the cell cycle distribution after treatment was analyzed by flow cytometry. Radiation-induced DNA damage triggers G1 or G2 cell cycle arrest, allowing cells to repair DNA damage. AP has been reported to hamper cell cycle progression, leading to G0/G1 or G2/M arrest. As shown in Fig. 3B, in A549 cells, radiation-induced DNA damage triggered G1 and G2 arrest, and AP induced G2/M arrest. Cell cycle arrest was significantly enhanced by IR. Cell cycle analysis showed that A549 cells treated with both AP and IR had the greatest increase in the G2/M phase proportion, and this proportion was significantly higher than that of control cells (p < 0.01). The same effect was observed in the LK2 cells. Therefore, AP improves the efficiency of IR by causing G2/M arrest. These results indicate that AP enhances radiation-induced apoptosis and G2/M arrest.

Radiosensitization by AP was associated with delayed DNA-double-strand break (DSB) repair

IR induces DNA-DSBs and triggers DNA damage repair responses. The ability to repair DNA-DSBs reflects the radiosensitivity of cells. γ-H2AX was used to monitor the presence of DNA-DSBs to determine whether AP pretreatment enhances radiation-induced DNA damage and interferes with DNA damage repair. Immunofluorescence staining was used to detect the number of γ-H2AX-positive nuclei. There were notably more γ-H2AX-positive cells among cells treated with both AP and IR than in cells treated with IR alone at 4 h post-IR (Figs. 4A and 4B), which reflected inefficient repair of DSBs in cells treated with the combination. These results indicate that AP significantly increases the number of γ-H2AX-positive nuclei and suppresses repair of radiation-induced DNA-DSBs after IR.

AP blocks AKT and ERK signaling to sensitize lung cancer cells to radiation

The AKT and ERK pathways are known to be activated by IR; this activation might play a role in radioresistance in NSCLC cells. Our previous results showed that downregulation of the AKT and ERK signaling enhanced radiosensitivity (Sun et al., 2019). In order to determine the potential molecular mechanisms by which AP exerts enhanced antitumor effects and regulates radiosensitivity, we examined the phosphorylation of AKT and ERK by western blotting. The results showed that phosphorylated AKT (p-AKT) and ERK (p-ERK) were reduced after AP treatment in both A549 and LK2 cells. The combination of AP and radiation significantly decreased the activities of AKT and ERK in A549 and LK2 cells (Figs. 5A and 5B). In summary, these data indicate that AP enhances radiosensitivity by decreasing AKT and ERK signaling in NSCLC cells.