Evaluation of the radiosensitizing effect of MEK inhibitor KZ-001 on non-small cell lung cancer cells in vitro

A549 NSCLC cells (Chinese Academy of Medical Sciences and Peking Union Medical College) were cultured in F12 (Gibco, C11765500BT) medium supplemented with 10% fetal bovine serum (FBS) (Yeasen, 40130ES76), and H460/H441 was maintained in a 1640 (Gibco) medium supplemented with 10% FBS (Yeasen, 40130ES76). Both culture media contained 1% penicillin and streptomycin (Solarbio, P1400). Cellular proliferation assay analysis was conducted using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) (Solarbio, M1080). In brief, the cells were seeded in 96-well plates (Corning, 3599) at an appropriate density in a 90 μL cell culture medium until attached. Then, KZ-001 (Kechow; the characterization and purity of the KZ-001 are indicated in Figure 1) at 1 mM was gradient-diluted with DMSO at a 1:10 ratio. Finally, the assay was prepared with a 10-fold concentration in 5% DMSO media. The cells were treated either with KZ-001 alone, irradiation at different doses, or KZ-001 combined with irradiation. The irradiation was carried out using a Pantak (Solon) X-ray source at a dose rate of 1.55 Gy/min. After treatment, MTT (0.5 mg/mL) was added to the culture plate and incubated for 4 h. Then, the culture medium was discarded and 150 μL DMSO was added. The absorbance was measured at 570 nm using a microplate reader (Utrao, SM600).

Figure 1.

RIPA buffer (R0020), protein phosphatase inhibitor (all-in-one, 100×) (P1260), and BCA protein assay kit (PC0020) were purchased from Solarbio. In addition, NuPage 10% Bis-Tris Gel (NP0301BOX) was purchased from Invitrogen. PVDF membrane (Immobilon-P) and eECL western blot kit (CW0049) were from Millipore and CWBIO, respectively. Further, anti-p-ERK1/2 antibody (4370) and anti-ERK1/2 (4695) antibody were purchased from Cell Signaling Technology. Anti-Bcl-xl antibody (ab32370) and anti-Bax antibody (ab32503) were purchased from Abcam. Finally, anti-GAPDH antibody (HRP-60004) was purchased from ProteinTech.

The cells were seeded at an appropriate density (80%–90% confluency). After being subject to treatment with 6 Gy irradiation, KZ-001 at 100 nM, or 6 Gy in combination with KZ-001 at 100 nM, respectively, the cell lysates were extracted and collected using radioimmunoprecipitation assay (RIPA) buffer containing protein phosphatase inhibitor (all-in-one, 100×). As a control, the cells were treated with a 0.1% DMSO medium.

Equal quantities of total protein from cell were resolved on NuPage 10% Bis-Tris Gel and then transferred to the PVDF membrane. The proteins of interest were blotted with the indicated antibodies (anti-p-ERK1/2 ab; anti-ERK1/2 ab; anti-Bcl-xl ab; and anti-Bax ab). As a final step, the chemiluminescence was generated with an eECL western blot kit and detected with the use of a Bio-Rad ChemiDoc XRS+ System.

The A549 and H460 (Chinese Academy of Medical Sciences and Peking Union Medical College) cells were trypsinized to generate a single-cell suspension and were then seeded in 6-well plates for 14 h to allow attachment before treatment. Each treatment condition had 3 replicates. After treating by vehicle (dimethylsulfoxide) or KZ-001 (100 nM) for 1–2 h, the cells were treated with 0 Gy, 1 Gy, 2 Gy, 4 Gy, or 6 Gy of radiation. The drugs were washed out after 24 h; next, the cells were maintained for 2 weeks. The colonies thus obtained were fixed and stained with the use of 0.1% crystal violet (Solarbio, C8470) in 20% methanol. All the colonies consisting of at least 50 cells were counted. Finally, the data were analyzed using GraphPad Prism 8.0 (GraphPad Software, Inc.).

A549 and H460 cells at a density of 2 × 10⁵/well were inoculated into the wells of 6-well plates and cultured overnight. Next, 100 nM KZ-001 or vehicle was added to the indicated wells. Each treatment was triplicated. Subsequently, 100 nM KZ-001 was added to each well, and the vehicle control group was treated with a 0.5% DMSO culture medium. Then, cells were exposed to IR at a dose of 6 Gy. After 24 h, 48 h, and 72 h of treatment, the cells were digested and stained with fluorescein isothiocyanate (FITC), annexin V, and propediene iodide (PI) as per the instructions of apoptosis detection kit (YEASEN, 40301ES50) and analyzed with the use of flow cytometry (BD Biosciences, FACSCelesta).

A549 cells at a density of 1 × 10⁵ cells/mL were seeded into wells of the 6-well plates, cultured overnight, and then pretreated with 100 nM KZ-001 for 1 h. The vehicle control group was treated with a 0.5% DMSO culture medium. Then, these pretreated cells were exposed to IR at a 6 Gy dose. After 48 h incubation, the cells were digested and washed with PBS, fixed in 70% ice-cold ethanol at 4 °C overnight, and then processed in compliance with the instructions accompanying the cell cycle analysis kit (Yeasen, 40301). Ultimately, the fluorescence intensity of each sample was measured using flow cytometry (BD Bioscience, FACSCelesta) and analyzed with ModFit LT software.

A549 cells at a density of 5 × 10⁵/well were seeded into wells of 12-well plates with coverslips (Solarbio, YA0351) and cultured overnight. The next day, the cells were pretreated with 100 nM KZ-001 or 0.5% DMSO for 1 h, and then irradiated with a 6 Gy dose. After 48 h, the cells were fixed with a 10% neutral buffered formalin (Solarbio, G2161) at 37 °C for 30 min and permeabilized with Triton X-100 (Solarbio, P1080) for 30 min at room temperature. After being blocked with PBS containing 1% bovine serum albumin (Solarbio, A8010) at room temperature for 60 min, the samples were incubated with primary rabbit anti-gamma H2AX (phospho S139) antibody (Abcam, ab81299; 1:250) at 4 °C overnight followed by the anti-rabbit-PE secondary antibody (Proteintech, SA00008-2; 1:50) at room temperature for 1.5 h in the dark.

Statistical analyses were performed with the use of GraphPad Prism 8.0 (GraphPad Software, Inc.), using t test analysis between 2 independent groups. P values were calculated to visually indicate the statistical results, and the significance was defined as follows: *P < 0.05, **P < 0.01, and ***P < 0.001.

We used different concentrations (0.5–1,000 nM) of KZ-001 to treat 3 different NSCLC cells for 24 h, 48 h, and 72 h. The results illustrated that KZ-001 had a strong inhibitory effect on the A549 and H460 proliferation for 72 h (Figures 2A and 2C). As observed, 100 nM KZ-001 inhibited 50% cell viability A549 and H460 after 72 h incubation. Hence, 100 nM was selected as the incubation concentration of KZ-001 for the subsequent irradiation experiments.

Figure 2.

Next, we used the MTT method for a preliminary exploration of the radiosensitivity of the cells. Figures 2B and 2D illustrate that compared to the irradiation monotherapy group, the KZ-001 significantly decreased A549 cell viability at 6 Gy and 8 Gy radiation doses; however, KZ-001 could not reduce the viability of irradiated H460 cells. Hence, for subsequent experiments, we chose 6 Gy as the irradiation dose for NSCLC.

Two NSCLC cell lines, A549 and H460, were pretreated with 100 nM KZ-001 for 1 h before irradiation. After 2 weeks, the number of clones was counted. Results showed that KZ-001 caused the radiosensitivity under 1 Gy, 4 Gy, and 6 Gy irradiation doses. However, KZ-001 did not aggravate the radiation impairment of H460 under any irradiation doses (Figures 2E and 2F). The results are consistent with the results of MTT analysis.

We used annexin V and PI double staining method after exposure to 6 Gy irradiation dose with or without KZ-001 treatment to detect A549 and H460 apoptosis. Figures 2G–I illustrate that compared to 24 h and 48 h, the A549 apoptosis rate reached a relatively high level after 72 h incubation. Interestingly, the A549 apoptosis ratio induced by IR combined with KZ-001 increased significantly in comparison with that by IR monotherapy (10.57% vs. 6.23%, P = 0.0055). However, a time-dependent manner was not observed in the H460 apoptosis (Figures 2J–L). The apoptotic rate under the IR and KZ-001 treatment for 72 h was 5.37%, which was significantly higher than the 2.80% apoptotic rate under the IR treatment group. This data may have statistical significance, but it lacks biological significance compared to the A549 apoptotic rate. In summary, we consider that KZ-001 can increase the radiation-mediated apoptotic rate of the 2 NSCLC cell lines, and A549 had a significant apoptotic response to KZ-001.

To further explore the mechanism of apoptosis at the molecular level, we detected the anti-apoptotic marker Bcl-XL and the apoptotic marker Bax in the A549 cells (Figure 2M) cells and found that after a single IR, the expression of Bcl-XL was not significantly decreased, but KZ-001 could weaken its expression. Furthermore, the expression of Bcl-XL in the irradiation group plus KZ-001 also decreased compared to that in the single irradiation group. This result indicates that KZ-001 can reverse the tolerance of A549 to IR and progress to apoptosis 24 h after irradiation. At the same time, the lack of significant changes in Bax suggests that the time span of 24 h may be too short to observe significant apoptosis. KZ-001 could significantly downregulate the expression of the anti-apoptotic protein Bcl-XL in H460 cells after irradiation (Figure 2N), that is, it could promote cell apoptosis. This was consistent with the significantly higher rate of apoptosis of H460 cells after 24 h compared to the IR group. On the other hand, through the analysis of the apoptosis marker Bax, we found that the expression of Bax in the IR combined with KZ-001 group decreased, suggesting that the progress of apoptosis was blocked, which could also explain why the H460 apoptosis rate increased only about 1.5% compared to the IR group.

Generally, one common property of KRAS-mutant NSCLC is the existence of the predominant KRAS-GTP in cytosol, which persistently activates downstream kinases, with the RAF/MEK/MAPK being the most important. ERK is a significant protein to regulate cell proliferation and survival. KZ-001 as a highly potent and selective MEK inhibitor can inhibit the ERK1/2 phosphorylation to a lower level; however, previous experiments found that KZ-001 could not inhibit the viability of irradiated H460 cells to a lower level, which raised our doubt about the KZ-001 inhibitory effect. Therefore, we tested the ERK1/2 expression under the dual treatment of IR and KZ-001. The results showed that both IR alone and IR combined with KZ-001 can block the H460 ERK1/2 phosphorylation, whereas IR cannot inhibit A549 ERK1/2 phosphorylation; besides, IR combined with KZ-001 can significantly inhibit ERK activation (Figures 2M and 2N). Therefore, KZ-001 can cause radiosensitization in the A549 cell line but not in the H460 cell line. This may be the reason why A549 cells treated with KZ-001 were more sensitive to irradiation.

Since it was observed that the combination of KZ-001 with IR promoted the radiosensitivity on A549, we further investigated whether KZ-001 can affect the cell cycle arrest. In the experiment, A549 was pretreated with 100 nM KZ-001 for 1 h and then exposed to 6 Gy irradiation dose. After 48 h, the cells were collected, stained with PI, and examined for changes in the cell cycle distribution using a flow cytometer. The outcome showed that compared to control, the irradiation alone increased the proportion of cells in the G2 phase from 9.14% to 21.24%, and the combination of IR and KZ-001 could further increase the proportion to 32.22% (Figures 2O–R). The IR plus KZ-001 treatment caused A549 arrest in the G2 phase, the main period of homologous recombination repair after DNA damage. Therefore, we speculated that KZ-001 aggravated the IR-mediated DNA damage.

IR could cause DNA damages to the tumor cells, and DNA double-strand breaks (DSBs) are the most crucial form of this damage. DSBs subsequently trigger the DNA repair mechanism. Activated γH2AX is a significant biomarker for the DNA repair process; hence, γH2AX (the number of phosphorylated γH2AX nuclear aggregates) activation extent can measure the degree of DNA damage caused by IR. Detection results of the phosphorylated γH2AX in cells showed that compared to the irradiation group, the KZ-001 could significantly increase the positive cell ratio to 33.4% (P < 0.05) (Figures 2S–W), suggesting the KZ-001 can aggravate the degree of DNA damage, thereby achieving the effect of increased radiosensitivity.