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

Gongchao Huang
Gongchao Huang
, 
Wenqin Zhang
Wenqin Zhang
 und 
Hongqi Tian
Hongqi Tian
   | 26. Okt. 2023
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Article Category: Original article
Online veröffentlicht: 26. Okt. 2023
Seitenbereich: 230 - 237
DOI: https://doi.org/10.2478/abm-2023-0064
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© 2023 Gongchao Huang et al., published by Sciendo
This work is licensed under the Creative Commons Attribution 4.0 International License.

Figure 1.

Characterization and purity of the KZ-001. (A) 1H NMR spectra of KZ-001; (B) 13C NMR spectra of KZ-001; (C) MS spectra of KZ-001; and (D) HPLC spectra of KZ-001.
Characterization and purity of the KZ-001. (A) 1H NMR spectra of KZ-001; (B) 13C NMR spectra of KZ-001; (C) MS spectra of KZ-001; and (D) HPLC spectra of KZ-001.

Figure 2.

Cell viability under the treatment of IR and KZ-001 (A–D). Concentration screening of KZ-001 in A549 cells (A) and in H460 cells (C); irradiated A549 (B) and H460 (D) cell viability comparison between vehicle and KZ-001 treated cells. KZ-001 + IR vs. IR group, *P < 0.05. (E, F) Evaluation of the KZ-001 effect on the proliferation of irradiated A549 (E) and H460 cells (F) by colony formation assay. A549 and H460 cells were seeded and then treated as indicated. For A549 cells, KZ-001 made the percentage of survival significantly decrease under radiation at the dose of 1 Gy, 4 GY, and 6 Gy, whereas the KZ-001 treated H460 cells did not show a decrease in viability compared with the vehicle control group. KZ-001 + IR vs. IR group, *P < 0.05, **P < 0.01, ***P < 0.001. (G–L) Effects of KZ-001 on the apoptosis level of irradiated A549 (G–I) and H460 (J–L) cells at different times. A549 and H460 cells were inoculated into 6-well plates at a proper density, and cells were cultured overnight, followed by exposure to 6 Gy IR with or without KZ-001 at 100 nM. After KZ-001 treatment for 24 h, 48 h, and 72 h, cells were harvested and tested with the use of an apoptosis detection kit as the kit instruction indicated. KZ-001 + IR vs. IR group, *P < 0.05, **P < 0.01. (M, N) Effect of KZ-001 on the expression of pERK and apoptosis markers in irradiated A549 (M) and H460 (N) cells. Cells were seeded into 6-well plates at a proper density, then cells were treated with 6 Gy, KZ-001 100 nM, or 6 Gy combined with KZ-001 100 nM. After 24 h, cell lysate was extracted and analyzed. (O–R) Effect of KZ-001 on the cell cycle of an irradiated A549 cell line. A549 cells were seeded into wells of the 6-well plates, cultured overnight, and then pretreated with KZ-001. Then, cells were exposed to IR at a 6 Gy dose. After 48 h incubation, the cells were digested and processed in compliance with the cell cycle analysis kit (Yeasen, 40301) instructions. (S–W) Effect of KZ-001 on the level of DNA double-strand damage markers in irradiated A549 cells. A549 cells were seeded into wells of a 12-well plate with coverslips and cultured overnight. Then, cells were pretreated with 100 nM KZ-001 or 0.5% DMSO for 1 h, and then irradiated with a 6 Gy dose. After incubation for 48 h, each group of cells was fixed and stained with DSB-related antibodies. (S) Untreated A549 cells; (T) A549 cells was treated with KZ-001 at 100 nM for 48 h; (U) A549 cells was exposed to radiation at 6 Gy; the small red dots to which the green arrows point indicate that DNA DSB took place and that γH2AX was detected with antibody; (V) A549 cells was exposed to radiation at 6 Gy, and then treated with KZ-001 at 100 nM for 48 h. The small red dots became larger than that of the radiation group. (W) The bar chart shows the percentage of γH2AX-carried cells in each group, and the positive rate of the KZ-001 irradiation group was significantly higher than that of the irradiation group alone. DSB, double-strand break; ERK, extracellular regulated protein kinases.
Cell viability under the treatment of IR and KZ-001 (A–D). Concentration screening of KZ-001 in A549 cells (A) and in H460 cells (C); irradiated A549 (B) and H460 (D) cell viability comparison between vehicle and KZ-001 treated cells. KZ-001 + IR vs. IR group, *P < 0.05. (E, F) Evaluation of the KZ-001 effect on the proliferation of irradiated A549 (E) and H460 cells (F) by colony formation assay. A549 and H460 cells were seeded and then treated as indicated. For A549 cells, KZ-001 made the percentage of survival significantly decrease under radiation at the dose of 1 Gy, 4 GY, and 6 Gy, whereas the KZ-001 treated H460 cells did not show a decrease in viability compared with the vehicle control group. KZ-001 + IR vs. IR group, *P < 0.05, **P < 0.01, ***P < 0.001. (G–L) Effects of KZ-001 on the apoptosis level of irradiated A549 (G–I) and H460 (J–L) cells at different times. A549 and H460 cells were inoculated into 6-well plates at a proper density, and cells were cultured overnight, followed by exposure to 6 Gy IR with or without KZ-001 at 100 nM. After KZ-001 treatment for 24 h, 48 h, and 72 h, cells were harvested and tested with the use of an apoptosis detection kit as the kit instruction indicated. KZ-001 + IR vs. IR group, *P < 0.05, **P < 0.01. (M, N) Effect of KZ-001 on the expression of pERK and apoptosis markers in irradiated A549 (M) and H460 (N) cells. Cells were seeded into 6-well plates at a proper density, then cells were treated with 6 Gy, KZ-001 100 nM, or 6 Gy combined with KZ-001 100 nM. After 24 h, cell lysate was extracted and analyzed. (O–R) Effect of KZ-001 on the cell cycle of an irradiated A549 cell line. A549 cells were seeded into wells of the 6-well plates, cultured overnight, and then pretreated with KZ-001. Then, cells were exposed to IR at a 6 Gy dose. After 48 h incubation, the cells were digested and processed in compliance with the cell cycle analysis kit (Yeasen, 40301) instructions. (S–W) Effect of KZ-001 on the level of DNA double-strand damage markers in irradiated A549 cells. A549 cells were seeded into wells of a 12-well plate with coverslips and cultured overnight. Then, cells were pretreated with 100 nM KZ-001 or 0.5% DMSO for 1 h, and then irradiated with a 6 Gy dose. After incubation for 48 h, each group of cells was fixed and stained with DSB-related antibodies. (S) Untreated A549 cells; (T) A549 cells was treated with KZ-001 at 100 nM for 48 h; (U) A549 cells was exposed to radiation at 6 Gy; the small red dots to which the green arrows point indicate that DNA DSB took place and that γH2AX was detected with antibody; (V) A549 cells was exposed to radiation at 6 Gy, and then treated with KZ-001 at 100 nM for 48 h. The small red dots became larger than that of the radiation group. (W) The bar chart shows the percentage of γH2AX-carried cells in each group, and the positive rate of the KZ-001 irradiation group was significantly higher than that of the irradiation group alone. DSB, double-strand break; ERK, extracellular regulated protein kinases.
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