Small-molecule inhibition of APE1 induces apoptosis, pyroptosis, and necroptosis in non-small cell lung cancer

Abstract

Apurinic/apyrimidinic endonuclease 1 (APE1) plays a critical role in the base excision repair (BER) pathway, which is responsible for the excision of apurinic sites (AP sites). In non-small cell lung cancer (NSCLC), APE1 is highly expressed and associated with poor patient prognosis. The suppression of APE1 could lead to the accumulation of unrepaired DNA damage in cells. Therefore, APE1 is viewed as an important marker of malignant tumors and could serve as a potent target for the development of antitumor drugs. In this study, we performed a high-throughput virtual screening of a small-molecule library using the three-dimensional structure of APE1 protein. Using the AP site cleavage assay and a cell survival assay, we identified a small molecular compound, NO.0449-0145, to act as an APE1 inhibitor. Treatment with NO.0449-0145 induced DNA damage, apoptosis, pyroptosis, and necroptosis in the NSCLC cell lines A549 and NCI-H460. This inhibitor was also able to impede cancer progression in an NCI-H460 mouse model. Moreover, NO.0449-0145 overcame both cisplatin- and erlotinib-resistance in NSCLC cell lines. These findings underscore the importance of APE1 as a therapeutic target in NSCLC and offer a paradigm for the development of small-molecule drugs that target key DNA repair proteins for the treatment of NSCLC and other cancers.

Fig. 1. Expression levels of APE1 are positively correlated with NSCLC.

a, b Statistical analysis of APE1 mRNA expression levels in LUAD and LUSC from the TCGA database. c APE1 protein levels in normal lung HELF cell, NSCLC cell lines A549, NCI-H460, and NCI-H1299. d Immunohistochemical staining of APE1 in labeled normal lung tissues or tumor tissues. e Colony-formation assay in control or APE1-KD A549 and NCI-H460 cells. Representative images from one of three experiments are shown. Data are shown as the mean ± SD of three independent experiments. ***p < 0.001.

Fig. 2. Screening of small molecular inhibitor of APE1.

a The schemata of AP site cleavage assay. b Chemical structure of NO.0449-0145. c Cell survival assay showed that NO.0449-0145 treatment induced cell death in A549, NCI-H460, NCI-H1299 cells but not in HELF cell. d Circular dichroism spectroscopy analysis of APE1/NO.0449-0145 binding. e Docking studies identified four high-potential sites of APE1 binding with NO.0449-0145. f Inhibitory effects of NO.0449-0145 on FEN1 mutations. Each experiment was repeated three times. **p < 0.01.

Fig. 3. Inhibition of FEN1 by NO.0449-0145 leads to the accumulation of DNA damage.

a Western blot assay to determine γ-H2AX levels in A549 and NCI-H460 cells following different dose of NO.0449-0145 treatments. b, c Immunofluorescence staining of γ-H2AX foci and 53BP1 foci in cells. The quantification of the foci numbers per cells were showed in right panel. d Comet assay of A549 and NCI-H460 cells treated with or without NO.0449-0145. Representative images for the quantification of the average tail moment reported for each treatment condition. Each experiment was repeated three times. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 4. NO.0449-0145 induces cell death in NCI-H460 and A549 cells.

a Morphological analysis of NCI-H460 and A549 cells with different dose of NO.0449-0145 treatment for 24 h. The red arrows indicate the apoptotic bodies; the black arrows point to bubbling of cells. b Annexin V/PI staining and flow cytometry analysis of NCI-H460 and A549 cells with different dose of NO.0449-0145 treatment for 24 h. c Levels of BAD, BAK, Mcl-1 and Bcl-XL assessed by western blotting for the cells treated with NO.0449-0145 for 12 h. d Representative Hoechst/PI fluorescence images of cells with different treatment. e Western blotting analysis of basic RIPK3, MLKL, phosphorylation of RIPK3 (P-RIPK3), and MLKL (P-MLKL) in A549 and NCI-H460 cells. f Western blotting analysis of caspase-4 and GSDMD in A549 and NCI-H460 cells. g Secreted IL-1β from cells treated with NO.0449-0145 for 24 h. Each experiment was repeated three times. The significance between treated samples and control samples was determined using a t-test. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 5. NO.0449-0145 suppresses NSCLC xenograft tumors.

a–c Tumor volumes and weights monitored from mice (n = 5 per group) treated with vehicle or different dose of NO.0449-0145 (3.125, 6.25, and 12.5 mg/kg) every 2 days. d Mice body weights monitored during treatment. e Representative micrographs of H&E staining and IHC staining for Ki67. f Representative micrographs of Immunofluorescence staining for γ-H2AX and 53BP1 foci. g, h Representative micrographs of IHC staining for BAK, BCL-XL, GSDMD, caspase-4, RIPK3, and phosph-RIPK3. The significance between treated samples and control samples was determined using a t-test. *p < 0.05; ***p < 0.001.

Fig. 6. NO.0449-0145 overcomes the cisplatin resistance of A549 cell.

a Cell survival assay in A549-DDP cells treated with NO.0449-0145 for 48 h. b Morphological analysis of different treated A549-DDP cells. c Representative Hoechst/PI fluorescence images of A549-DDP cells with different treatment. d Western blotting analysis of γ-H2AX in different treated A549-DDP cells. e Representative image of Immunofluorescence staining for γ-H2AX foci. f Drug sensitivity assay of A549-DDP cells following co-treatment with cisplatin and NO.0449-0145 or vehicle. The data are presented as the mean ± SD values, and the error bars represent data from triplicate biological experiments. *p < 0.05; **p < 0.01; ***p < 0.001.