The inhibitory effect of compounds on the long-term proliferation of NSCLC cells. NSCLC H1299 cells and BEAS-2B human bronchial epithelial cells were treated with the indicated concentrations (from 0.5 to 10 μM) of tested compounds for 14 days. Afterward, the cells were paraformaldehyde-fixed and stained with crystal violet. (a) Chemical structures of 4-HPPP and its structural analogs. (b) Representative results of the colony formation of H1299 and BEAS-2B cells following compound treatment. (c) The quantitative results of (b) were statistically analyzed with one-way ANOVA. ^∗p < 0.05; ^∗∗p < 0.001. Vehicle control vs. 4-HPPP treatments. #1: 4-HPPP; #2: 4-[2356-tetrafluoro-4-(4-hydroxyphenoxy)phenoxy]phenol; #3: 4-[4-(4-aminophenoxy)-2356-tetrafluorophenoxy]aniline; #4: 4-[4-(4-amino-3-nitrophenoxy)phenoxy]-2-nitroaniline.

4-HPPP induces apoptosis of NSCLC H1299 cells. The apoptosis induced by 4-HPPP was assessed using flow cytometry-based annexin V-PI staining. (a) H1299 cells were treated with 4-HPPP for the indicated time course. (b) The quantitative results of (a). p < 0.05 (vehicle vs. 4-HPPP treatment) was considered statistically significant. ^∗p < 0.05; ^∗∗p < 0.001.

The effect of 4-HPPP on Akt phosphorylation changes in NSCLC cells. The phosphorylation changes at serine⁴⁷³ and threonine⁴⁵⁰ of Akt along with the prosurvival factor Bcl-2 were assessed using the Western blotting assay. β-Actin was used as an internal control to ensure equal loading.

The effect of 4-HPPP on cell cycle progression. (a) The accumulation of sub-G₁ and aneuploidy (N>4N) in NSCLC cells. H1299 cells were seeded and treated with different concentrations of 4-HPPP for 48 and 72 h. The treated cells were 70% ethanol-fixed and PI-stained and then subjected to a cell cycle distribution analysis by flow cytometry. Changes in cell cycle progression, the sub-G₁ population, and aneuploidy following 4-HPPP treatment. (b) Quantitative analysis of cell cycle progression. ^∗p < 0.05; ^∗∗p < 0.001.

Assessment of DNA damage induced by 4-HPPP. (a) H1299 cells were administered the indicated concentrations of 4-HPPP for 48 h. Afterward, 4-HPPP-induced DNA damage was detected using a flow cytometry-based γH2AX detection assay. The green fluorescence of FITC (FL1) indicates the γH2AX-positive population (R1 region). The data showed that 4-HPPP increased the phosphorylation of γH2AX, a marker of DNA damage, in a dose-responsive manner. (b) Quantification analysis of (a). γH2AX was observed in H1299 cells following 4-HPPP treatment at concentrations from 0.5 to 10 μM by the Western blotting assay (c) and immunofluorescence assay (d). The data are presented as the mean ± SD. ^∗p < 0.05; ^∗∗p < 0.005; ^∗∗∗p < 0.001. Scale bar: 100 μm.

4-HPPP-induced activation of DNA damage markers in NSCLC cells. (a) H1299 cells were seeded and treated with 4-HPPP for 48 h. Afterward, the DNA damage induced by 4-HPPP was assessed by flow cytometry. The markers of DNA damage, including phosphor-ATR, phosphor-ATM, and DNA-PK, were determined. The results showed that 4-HPPP increased the phosphorylation of ATR and ATM and the protein level of DNA-PK in a dose-responsive manner. (b) The quantitative results of (a). ^∗p < 0.05; ^∗∗p < 0.001.

4-HPPP-induced changes in endogenous ROS and antioxidants in NSCLC cells. (a) H1299 cells were treated with the indicated concentrations of 4-HPPP for 24 h and 48 h. Afterward, intracellular levels of ROS were measured by the flow cytometry-based DCF-DA assay described in Materials and Methods. (b) Quantitative analysis of (a). (c) Changes in endogenous antioxidants SOD1, SOD2, and PRX1 in H1299 cells following 4-HPPP treatment by Western blotting. (d) Quantitative analysis of (c). ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001.

The effect of 4-HPPP on the motility of NSCLC cells. (a) H1299 cells were treated with the indicated concentrations of 4-HPPP for 18 h. Afterward, the cells were stained with 0.1% w/v Giemsa. (b) Quantitative analysis of (a). ^∗p < 0.05 and ^∗∗p < 0.01 for 4-HPPP treatments against vehicle control, respectively.

Effect of 4-HPPP on zebrafish-based xenografted NSCLC cells. (a) Survival rate of zebrafish larvae following 4-HPPP exposure. (b) The results showed that 5 μM and 10 μM 4-HPPP caused body axis bending and edema in zebrafish larvae, indicating that a high concentration of 4-HPPP could cause deformation and toxicity toward zebrafish larvae. (c) The motility of xenografted H1299 cells in the yolk sac of zebrafish larvae. The intensity of red fluorescence is proportional to the xenograft tumor size. For each group, the sample size of larvae (N) > 60. (d, e) Quantitative analysis of (c). The data are presented as the mean ± S.D. ^∗∗p < 0.05 and ^∗∗∗p < 0.001 against vehicle control. (e) The statistical analysis of the migration ability of xenografted H1299 cells using Fisher's exact test.

A proposed mechanism of 4-HPPP-induced antiproliferation and apoptosis in NSCLC cells through modulating the signaling of endogenous antioxidant systems, such as SOD2 and PRX1, causing DNA damage and high-grade aneuploidy. Eventually, the accumulation of DNA damage and hyperaneuploidization induce apoptosis in NSCLC cells.