br Results br BD inhibited the proliferation
3.1. BD inhibited the proliferation of Non-small cell lung cancer cells
To examine the cytotoxic eﬀects of BD (the chemical structure is shown in Fig. 1A) on Non-small cell lung cancer (NSCLC) cells, CCK-8 assay was conducted. As shown in Fig. 1B and Supplementary Fig. 1, BD inhibited proliferation of the four Non-small cell lung cancer cell lines in a concentration-dependent manner, like that of gemcitabine. Treat-ment of H460 Alprostadil with 0.4 μmol/L BD resulted in approximately 55% of survival fraction compared with untreated cells. In the same ex-perimental panel, the other three cell lines (A549, H1975, PC9) were slightly less sensitive to BD than H460 cells. The IC50 values of BD at 48 h of treatment for four cells were 0.5 μmol/L (H460), 0.6 μmol/L (A549), 1.0 μmol/L (PC9) and 2.7 μmol/L (H1975), respectively. However, BD showed a much less potent cytotoxic eﬀect on BEAS-2B (Human normal lung epithelial cell) cell line, with IC50 values of 4.7 μmol/L, indicating a favorable cytotoxic profle of BD on Non-small cell lung cancer.
As H460 and A549 cells showed desirable sensitivity to BD, these two cell lines were used for the subsequent studies. Given that cancer cells are able to grow and form visible colonies, a clonogenic formation assay was also performed to identify the notably inhibited cell pro-liferation of two NSCLC cells after BD treatment (Fig. 1C). Fig. 1C and D demonstrated that H460 and A549 cells exposed to BD displayed sig-nificantly lower survival. Taken together, these results suggest that BD suppressed cell growth and colony formation in human NSCLC cells.
Cell morphological changes under microscope was examined by DAPI staining. Compared to the uniformly distributed blue fluorescence in control cells, the cells treated with diﬀerent concentrations of BD exhibited chromatin condensation, fragmented or shrunken nuclei. In addition, apoptotic bodies were observed at high BD concentrations both in H460 and A549 cells (Fig. 2A and Supplementary Fig. 2). The annexin V-PI double staining followed by flow cytometry analysis was also employed to detect early and late apoptotic cells after BD treat-ment. BD exerted a dose-dependent apoptotic eﬀect on both H460 and A549 cells (Fig. 2B, D). At the concentrations of 0.5 μmol/L and 1.0 μmol/L, BD caused 31.4% and 33.8% of the H460 and A549 cells to undergo apoptosis within 24 h, respectively. In contrast, non-treated cells showed normal cell viability without significant cell death. The expression of PARP and Caspase 3 was also decreased, while the ex-pression of cleaved-caspase 3 was increased after BD treatment. In addition, The BD treatment also markedly attenuated the expression of the anti-apoptotic protein Bcl-2, while augmented the expression of the pro-apoptotic protein BAX (Fig. 2C, E). These results clearly suggest that BD induced apoptosis in NSCLC cells.
3.3. BD inhibited NSCLC cell growth via JNK pathway
The JNK signaling pathway have important roles in regulating tumor cell apoptosis . To further explore the mechanism underlying BD-induced apoptosis in NSCLC cells, we evaluated whether BD
Fig. 3. Eﬀects of BD on phosphorylation of JNK in human NSCLC cells. H460 and A549 human NSCLC cells were treated with BD (0.5 μmol/L) for various time periods. (A) The expression levels of p-JNK and JNK were evaluated by western blot analysis. Actin was used as a loading control. (B) The ratio of p-JNK/JNK was analyzed with Image J software using actin for normalization. Data are expressed as the mean ± S.D. of three independent experi-ments. Significance was determined by the Student's t-test (*P < 0.05, **P < 0.01, ***P < 0.001 vs. untreated controls).
Fig. 4. Involvement of JNK in the enhanced apoptosis induced by BD in human NSCLC cells. H460 and A549 cells were pretreated with specific JNK inhibitor SP600125, and then treated with or without BD as indicated. Quantification of the apoptotic cells by Annexin V-FITC/PI staining was repeated for three times, and standard deviation was re-presented as error bars. Significance was de-termined by the Student's t-test (**P < 0.01,
modulates the JNK pathway when aﬀecting H460 and A549 cells. BD treatment markedly stimulated the phosphorylation of JNK in a time-dependent manner (Fig. 3A). To further analyze whether the JNK pathway required for BD-induced apoptosis, we inhibited JNK pathway by SP600125. As shown in Fig. 4 and Supplementary Fig. 3, the in-hibition of JNK pathway significantly decreased apoptosis induced by BD in H460 and A549 cells. According to the above results, it suggested that the induction of JNK pathway might contribute to the apoptosis induced by BD.