Cyclin-dependent kinase inhibitor 1A inhibits pyroptosis to enhance human lung adenocarcinoma cell radioresistance by promoting DNA repair

Abstract

Purpose: One of the best anticancer treatments available is radiotherapy, which can be used either alone or in conjunction with other forms of treatment including chemotherapy and surgery. Nevertheless, a number of biochemical and physiological processes that react to ionizing radiation might provide tumor cells radioresistance, which makes radiotherapy ineffective. It has been found that CDKN1A regulates DNA damage repair, which contributes to tumor radioresistance. However, the precise mechanism is still unknown. Therefore, this study aimed to explore the mechanisms underlying CDKN1A-enhanced radioresistance in tumor cells.

Methods: Cells were irradiated with 4 Gy after CDKN1A overexpression or knockdown. CDKN1A expression was measured using real-time PCR, cell viability was evaluated using cell counting kit-8 and colony formation assays, and cytotoxicity was assessed using a lactate dehydrogenase assay. Pyroptosis in cells was analyzed using caspase-1 activity assay, enzyme-linked immunosorbent assay, and flow cytometry. Inflammation activation was detected through a co-immunoprecipitation assay. Activation of pyroptosis-related proteins was analyzed using immunohistochemistry, Western blot, and immunofluorescence. Tumor radioresistance in vivo was evaluated in a mouse xenograft model.

Results: Radiotherapy upregulated CDKN1A expression, which promoted lung adenocarcinoma cell survival. CDKN1A influenced radiation-induced pyroptosis in A549, which mainly depended on inhibiting the activation of the AIM2 inflammasome by promoting DNA repair. Additionally, CDKN1A upregulation enhanced A549 xenograft tumor radioresistance by inhibiting radiation-induced pyroptosis in vivo.

Conclusions: CDKN1A inhibits pyroptosis to enhance the radioresistance of lung adenocarcinoma cells by promoting DNA repair. This study may serve as a reference for developing novel targeted therapies against cancer.

Keywords: CDKN1A; DNA repair; Inflammasomes; Pyroptosis; Radiotherapy.

Fig. 1

CDKN1A promotes A549 cell survival after radiotherapy. (a) Heat map of gene expression before and after radiotherapy. (b) A549 cells were exposed to 4 Gy radiation after CDKN1A overexpression, and cell viability was assessed using CCK-8 assay (mean ± SD, one-way ANOVA with Tukey's multiple comparison test; third vs. fourth group p < 0.01, n = 3 independent experiments). (c) A549 cells were exposed to 4 Gy radiation after CDKN1A knockdown, and cell viability was assessed using CCK-8 assay (mean ± SD, one-way ANOVA with Tukey's multiple comparison test; third vs. fourth group p < 0.05, n = 3 independent experiments). (d, e) Radiation-induced death of A549 cells with CDKN1A overexpression was monitored using colony formation assays. Representative images (d) and quantitation (e) are shown (mean ± SD, one-way ANOVA with Tukey's multiple comparison test; third vs. fourth group p < 0.01, n = 3 independent experiments). (f, g) Radiation-induced death of CDKN1A-overexpressing A549 cells was detected via flow cytometry. Representative images (f) and quantitation (g) are shown (mean ± SD, one-way ANOVA with Tukey's multiple comparison test; third vs. fourth group p < 0.001, n = 3 independent experiments). (h, i) Radiation-induced death of CDKN1A-knockdown A549 cells was monitored using colony formation assays. Representative images (h) and quantitation (i) are shown (mean ± SD, one-way ANOVA with Tukey's multiple comparison test; third vs. fourth group p < 0.05, n = 3 independent experiments). (j, k) Radiation-induced death of CDKN1A-knockdown A549 cells was detected using flow cytometry. Representative images (j) and quantitation (k) are shown (mean ± SD, one-way ANOVA with Tukey's multiple comparison test; third vs. fourth group p < 0.01, n = 3 independent experiments).

Fig. 2

CDKN1A upregulation inhibits radiation-induced pyroptosis in A549 cells. (a–g) Western blot analysis of indicated proteins (a) and quantification graphs (b–g) in CDKN1A-overexpressing and control cells following radiation treatment. (h) Caspase-1 activity in A549 under different treatment conditions (mean ± SD, one-way ANOVA with Tukey's multiple comparison test; third vs. fourth group p < 0.01, n = 3 independent experiments). (i) IL-18 concentration in the supernatant of A549 cultures under different treatment conditions (mean ± SD, one-way ANOVA with Tukey's multiple comparison test; third vs. fourth group p < 0.01, n = 3 independent experiments). (j) IL-1β concentration in the supernatant of A549 cultures under different treatment conditions (mean ± SD, one-way ANOVA with Tukey's multiple comparison test; third vs. fourth group p < 0.01, n = 3 independent experiments). (k) Cell death assessment based on the amount of LDH released into the supernatant (mean ± SD, one-way ANOVA with Tukey's multiple comparison test; third vs. fourth group p < 0.001, n = 3 independent experiments). (l, m) Irradiation-induced death in A549 cells by 7AAD and Annexin V cytometry assay (l) and quantification graphs (m; mean ± SD, one-way ANOVA with Tukey's multiple comparison test; third vs. fourth group p < 0.001, n = 3 independent experiments). (n–q) Representative blots of caspase-1, p20, IL-18, and IL-1β expression in A549 cells were determined using Western blot (n) and quantification graphs (o–q). (r, s) ASC specks detected by immunofluorescence staining (r) and quantification graphs (s; mean ± SD, one-way ANOVA with Tukey's multiple comparison test; third vs. fourth group p < 0.001, n = 3 independent experiments). Scale bar: 100 μm.

Fig. 3

CDKN1A upregulation inhibits the activation of the AIM2 and NLRP3 inflammasomes in A549 cells after radiation. (a–c) Activation of AIM2 and NLRP3 inflammasomes detected using CO-IP (a) and quantification graphs (b,c). (d, e) AIM2 specks detected through immunofluorescence staining (d) and quantification graphs (e) (mean ± SD, one-way ANOVA with Tukey's multiple comparison test; third vs. fourth group p < 0.001, n = 3 independent experiments; scale bar: 100 μm). (f, g) NLRP3 specks detected using immunofluorescence staining (f) and quantification graphs (g; mean ± SD, one-way ANOVA with Tukey's multiple comparison test; third vs. fourth group p < 0.05, n = 3 independent experiments).

Fig. 4

Palbociclib inhibits the activation of the AIM2 inflammasomes but not NLRP3 inflammasomes in A549 cells after radiation. (a–c) Activation of AIM2 and NLRP3 inflammasomes detected using CO-IP (a) and quantification graphs (b, c) after palbociclib treatment. (d, e) After palbociclib treatment, AIM2 specks were detected through immunofluorescence staining (d), and quantification graphs are shown (e; mean ± SD, one-way ANOVA with Tukey's multiple comparison test; third vs. fourth group p < 0.001, n = 3 independent experiments; scale bar: 100 μm). (f, g) After palbociclib treatment, NLRP3 specks were detected using immunofluorescence staining (f), and quantification graphs are shown (g; mean ± SD, one-way ANOVA with Tukey's multiple comparison test; third vs. fourth group p = 0.8351, n = 3 independent experiments). Scale bar: 100 μm.

Fig. 5

CDKN1A affects DNA repair after radiotherapy. (a, b) A549 cells were exposed to 4 Gy radiation after CDKN1A overexpression, and γH2AX expression was detected by Western blot (a) and the quantification graphs (b). (c, d) A549 cells were exposed to 4 Gy radiation after CDKN1A overexpression, and P53BP1 expression was detected by Western blot (c) and the quantification graphs (d). (e, f) A549 cells were exposed to 4 Gy radiation after CDKN1A overexpression, and DNA repair capacity was assessed using comet assay. Representative images (e) and quantitation (f) are shown (mean ± SD, one-way ANOVA with Tukey's multiple comparison test; the third vs fourth group p < 0.001, n = 3 independent experiments). Scale bar: 100 μm. (g) A549 cells were exposed to 4 Gy radiation after CDKN1A overexpression, and γH2AX was detected by immunofluorescence. Scale bar: 50 μm. (h, i) CDKN1A-overexpressing A549 cells were irradiated with 4 Gy after CDKN1A knockdown, and γH2AX was detected by WB (h) and the quantification graphs (i). (j, k) CDKN1A-overexpressing A549 cells were irradiated with 4 Gy after CDKN1A knockdown, and P53BP1 was detected by WB (j) and the quantification graphs (k). (l, m) CDKN1A-overexpressing A549 cells were irradiated with 4 Gy after CDKN1A knockdown, and DNA repair capacity was assessed by comet assay. Representative images (l) and quantitation (m) are shown (mean ± SD, one-way ANOVA with Tukey's multiple comparison test; the third vs fourth group p < 0.001, n = 3 independent experiments). Scale bar: 100 μm. (n) CDKN1A-overexpressing A549 cells were irradiated with 4 Gy after CDKN1A knockdown, and γH2AX was detected by immunofluorescence. Scale bar: 50 μm.

Fig. 6

CDKN1A upregulation enhances A549 xenograft tumor radioresistance in vivo. (a) Images of nude mice with subcutaneous xenograft tumors. (b) Xenograft tumors isolated from sacrificed mice at the end of experiment. (c) Volumes of subcutaneous xenograft tumors (mean ± SD, two-way ANOVA with Tukey's multiple comparison test; third vs. fourth group p < 0.05, each group with four mice). (d) Average weights of xenograft tumors (mean ± SD, one-way ANOVA with Tukey's multiple comparison test; third vs. fourth group p < 0.05, each group with four mice). (e–i) Representative blots of caspase-1 p20, IL-18, IL-1β, and γH2AX expression in xenograft tumors determined by Western blot (e) and quantification graphs (f–i). (j) IL-18 concentration in serum (mean ± SD, one-way ANOVA with Tukey's multiple comparison test; third vs. fourth group p < 0.05, n = 3 independent experiments). (k) IL-1β concentration in serum (mean ± SD, one-way ANOVA with Tukey's multiple comparison test; third vs. fourth group p < 0.05, n = 3 independent experiments). (l) Representative images of xenograft tumors stained with hematoxylin–eosin, and IL-18 and IL-1β levels determined by immunohistochemistry. Scale bar: 100 μm. (m) γH2AX was detected by immunofluorescence in xenograft tumors. Scale bar: 50 μm.