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. 2023 Sep 20;26(5):471.
doi: 10.3892/ol.2023.14059. eCollection 2023 Nov.

p21 promotes gemcitabine tolerance in A549 cells by inhibiting DNA damage and altering the cell cycle

Tian Fu  1   2   3 , Xuan Ma  4 , Shen-Lin Du  5 , Zhi-Yin Ke  1   2 , Xue-Chun Wang  1   2 , Hai-Han Yin  1   2 , Wen-Xuan Wang  1   2 , Yong-Jun Liu  1   2 , Ai-Ling Liang  1   2
Affiliations

p21 promotes gemcitabine tolerance in A549 cells by inhibiting DNA damage and altering the cell cycle

Tian Fu et al. Oncol Lett. .

Abstract

Gemcitabine is one of the most widely used chemotherapy drugs for advanced malignant tumors, including non-small cell lung cancer. However, the clinical efficacy of gemcitabine is limited due to drug resistance. The aim of the present study was to investigate the role of p21 in gemcitabine-resistant A549 (A549/G+) lung cancer cells. IC50 values were determined using a Cell Counting Kit-8 (CCK-8) assay. mRNA and protein expression levels of genes were measured by reverse transcription-quantitative PCR and western blotting, respectively. The cell cycle distribution and apoptosis rate were analyzed by flow cytometry. DNA damage in cells was evaluated by single-cell gel electrophoresis. The results of western blot analysis and the CCK-8 assay demonstrated that the expression of p21 was higher in A549/G+ cells than in gemcitabine-sensitive cells. Knockdown of p21 expression in gemcitabine-resistant cells sensitized these cells to gemcitabine (with the IC50 decreasing from 84.2 to 26.7 µM). Cell cycle analysis revealed different changes in the cell cycle distribution in A549/G+ cells treated with the same concentration of gemcitabine, and decreased expression of p21 was shown to promote G1 arrest. The apoptosis assay and comet assay results revealed that decreased p21 expression resulted in accumulation of unrepaired DNA double-strand breaks (DSBs) and induction of apoptosis by gemcitabine. The present study demonstrated that knockout of p21 mRNA expression in A549/G+ cells promotes apoptosis and DNA DSB accumulation, accompanied by G1 arrest. These results indicated that p21 is involved in regulating the response of A549 cells to gemcitabine.

Keywords: A549 cells; DNA damage; drug resistance; gemcitabine; p21.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1.
Figure 1.
p21 is markedly upregulated in gemcitabine-resistant A549 cells. (A) p21 mRNA and (B) protein expression levels in A549/G+ and parental A549 cells. (C) A549 cells were transfected with siRNA, and western blotting was used to verify the interference efficiency of p21. (D) A549/G+ cells were transfected with plasmids, and western blotting was used to verify the efficiency of p21 overexpression. (E) After p21-siRNA or siNC was successfully transfected into A549/G+ cells, the cells were treated with different concentrations of gemcitabine (1, 5, 10, 25, 50, 100, 500 and 1,000 µM), and a CCK8 proliferation assay was performed to detect the IC50. (F) Overexpression of p21 in A549 cells was assessed after treatment with different concentrations of gemcitabine (1, 5, 10, 25, 50, 100, 500 and 1,000 µM), and then a CCK8 proliferation assay was performed to detect the IC50. siRNA, small interfering RNA; NC, negative control; OE, overexpressed; A549/G+, gemcitabine-resistant A549; CCK-8, Cell Counting Kit 8. *P<0.05 and ***P<0.001 vs. respective controls.
Figure 2.
Figure 2.
Changes in the cell cycle distribution of two cell lines following treatment with the same concentration of gemcitabine. (A) A549 and A549/G+ cells were treated with 10.0 µM gemcitabine for 24 h and assessed by PI staining and flow cytometry. (B) A549 cell cycle changes with 10.0 µM gemcitabine. (C) A549/G+ cell cycle changes with 10.0 µM gemcitabine. A549/G+, gemcitabine-resistant A549; Gem, gemcitabine. *P<0.05, **P<0.01 and ***P<0.001 vs. respective controls.
Figure 3.
Figure 3.
Knockdown of p21 promotes gemcitabine-induced G1 arrest. (A) A549/G+ cells were transfected with siNC or siRNA-p21. They were then treated with 10.0 µM gemcitabine for 24 h. The cell cycle distribution was detected by PI staining and is shown in the bar graph as percentages of cells. (B) Cell cycle changes in the untreated siRNA-p21 group. (C) Cell cycle changes in the siRNA-p21 group treated with 10.0 µM gemcitabine. (D) Western blotting detection of cell cycle-related proteins (CCNA2, CCNE1 and CCND1) in siNC (or siRNA-p21)-transfected A549/G+ cells. GAPDH protein was used as an internal control. (E) Western blotting detection of cell cycle-related proteins (CCNA2, CCNE1 and CCND1) in siNC (or siRNA-p21)-transfected A549/G+ cells combined with gemcitabine (10 µM). siRNA, small interfering RNA; NC, negative control; Gem, gemcitabine. *P<0.05, **P<0.01 and ***P<0.001 vs. respective controls.
Figure 4.
Figure 4.
Knockdown of p21 expression results in accumulation of unrepaired DSBs and induction of apoptosis by gemcitabine. (A) Western blotting detection of DNA double-strand replication-related protein (γ-H2AX) in A549 cells and A549/G+ cells coupled with various concentrations of gemcitabine (0, 10 and 100 µM). (B) Western blotting detection of apoptosis and DNA double-strand replication-related protein (γ-H2AX and cleaved caspase-3) in siNC- or siRNA-p21-transfected A549/G+ cells treated with different concentrations of gemcitabine (0 and 10 µM). (C) Western blotting detection of DNA double-strand damage was observed with p21 overexpression. (D) Representative images of the comet assay in A549/G+ cells treated with siRNA-p21 (or siNC) combined with gemcitabine. (E) Flow cytometric analysis of apoptosis in siNC- or siRNA-p21-transfected A549/G+ cells treated with various concentrations of gemcitabine (0.0, 5.0, 50 and 500 µM). Gem, gemcitabine; A549/G+, gemcitabine-resistant A549; siRNA, small interfering RNA; NC, negative control; OE, overexpressed. *P<0.05, **P<0.01 and ***P<0.001 vs. respective controls, #P<0.05 and ##P<0.01 vs. the same drug concentration in A549 cells.
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Grants and funding

The present study was funded by the National Natural Science Foundation of China (grant no. 81071853).