7a inhibits A549 proliferation
The inhibitory effect of 7a on the proliferation of A549 cells was measured by using CCK-8. A549 cells were treated with different concentrations of 7A for 24, 48, and 72 h. The viability of A549 cells was determined, and the growth curves of A549 cells were drawn. As shown in Figure 2, 7a could inhibit the proliferation and growth of A549 in a time-dose-dependent manner. The IC50 value of 7a against A549 cells was calculated according to Figure 2A. The results showed that the IC50 of 7a on A549 cell growth was 65.72±4.20, 33.46±4.11 and 19.86±2.33μM at 24, 48 and 72h, respectively. These results indicated that 7a could inhibit the proliferation of A549 cells at higher concentration and longer time.
On the other hand, we examined the toxic effects of 7a on two types of normal human tissue cells. We selected normal human lung epithelial cell BEAS-2B and human venous endothelial cell HUVEC. CCK-8 test results showed that 7a had no significant proliferation inhibition effect on these two kinds of cells. Among them, although HUVECs were relatively sensitive to 7a, the maximum proliferation inhibition rate was less than 20% at 40μM, 72h. These results indicated that 7a had a selective killing effect on tumor cells, but had no obvious toxic and side effects on normal cells, so it had a good potential for clinical application.
7a inhibits A549 cell migration and invasion
According to the results of CCK-8 experiment, we selected 2.5 and 5μM as the non-cytotoxic concentration as the drug concentration in the invasion and metastasis experiment. Firstly, we tested the effect of 7a on the migration ability of A549 cells by cell scratch assay, and the results showed that 7a could significantly inhibit the migration ability of A549 cells. The migration inhibition rate was decreased from 43.61% to 21.30% and 17.52% at 18h, and from 61.50% to 31.22% and 22.10% at 36h (Figure 3A). On the other hand, we also examined the effect of 7a on the invasion ability of A549 cells by transwell invasion assay. Compared with the NC group, the number of A549 cells passing through the compartment was reduced in the 7a group at 48h, especially the high concentration (5μM) group (Figure 3B). These results demonstrated that 7a could markedly inhibit the migration and invasion of A549 cells at non-cytotoxic concentration.
To understand the mechanism of 7a inhibiting the migration and invasion of A549 cells, we further detected the expression of cell metastasis-related proteins. As shown in Figure 3C, 7a could significantly increase the expression of E-cadherin, while at the same time decrease the expression of metalloproteinase-9 (MM-9) and metalloproteinase-2 (MMP-2), suggesting that the inhibition of migration and invasion by 7a might be due to the regulation of the expression of these proteins.
7a inhibits vasculogenic mimicry
Highly invasive tumor cells, such as non-small cell lung cancer, can form mimicry vessels, that is, tumor cells show some characteristics similar to vascular endothelial cells and can degrade the basement membrane to connect to form a network structure [11,12]. Therefore, in vitro mimicry angiogenesis experiments, we used matrigel to simulate the basement membrane and observed the simulated angiogenesis of tumor cells in each group. The experimental results are shown in Figure 4A. In the NC group, A549 cells were connected to each other to form multiple grid structures. However, in the 7a dosing group, the branch length of mimetic vessels was significantly reduced, especially after treatment with 10μM 7a for 8h, the cells were dispersed and almost no tubules were formed, and the inhibition rate of mimetic vessel formation was as high as 82.89%. These results indicated that 7a could significantly inhibit the formation of mimicry vessels in A549 cells in a concentration-dependent manner.
As a pro-angiogenic factor, vascular endothelial growth factor (VEGF) not only plays an important role in angiogenesis, but also has been reported to be related to tumor cells [13]. Western blot and ELISA were used to detect the effect of 7a on the expression and secretion of VEGF in A549 cells and culture media. As shown in Figure 4B, 7a inhibited the expression of VEGF in A549 cells compared with the NC group. Next step, ELISA results (Figure 4C) showed that A549 cells were treated with different concentrations of 7a for 24h, and VEGF secretion was significantly reduced in a concentration-dependent manner. The results suggested that 7a could significantly inhibit the expression and secretion of VEGF in 549 cells.
7a inhibits A549 cells adhesion to HUVECs
Heterogeneous adhesion of tumor cells is a pivotal step in tumor metastasis. A549 cells treated with different concentrations of 7a were stained with DIO dye and then added to 24-well plates that covered with a single layer of HUVECs, which could mimic the adhesion of tumor cells to the lining of blood vessels. We examined the adhesion of A549 cells to HUVECs by incubation with different concentrations of 7a for 48 h. The fluorescent cells above the HUVECs monolayer are adhered A549 cells. The results showed that 2.5, 5, 10μM 7a significantly reduced A549 cells adhesion to HUVECs by 3.12%, 13.8%, and 34.4%, respectively (Figure 5A). The results suggested that 7a could inhibit the adhesion of A549 cells to HUVECs in a concentration-dependent manner.
Next step, we explored the molecular mechanism by which 7a regulates the adhesion of A549 cells to vascular endothelial cells. The relevant adhesion protein levels were detected by western blot analysis. As showed in Figure 5B, the expression of N-cadherin and E-selectin were significantly reduced following treatment with different concentrations of 7a. These two adhesion molecules have been proved to involve in adhesion between tumor cells and HUVECs [10,14]. The adhesion could then promote tumor cell aggregation in the blood vessels, which could avoid anoikis and increase tumor metastasis potential. Therefore, 7a may inhibit A549 cell metastasis by inhibiting the expression of N-cadherin and E-selectin.
7a promotes A549 cells apoptosis. The effects of different concentrations of 7a on apoptosis of A549 cells were quantitatively determined by Annexin V-FITC/PI double staining. The normal living cells, early apoptotic cells, necrotic cell and late apoptotic cells could be distinguished by flow cytometry. As shown in Figure 6A, the apoptosis rate of A549 cells in NC group was 5.4%. The total apoptosis rate of A549 cells increased to 9.6%, 16%, and 28.2% after treatment with 5, 10, and 20μM 7a for 48h. To further explore the mechanism of 7a inducing apoptosis in A549 cells, the effect of 7a on the expression of mitochondrial apoptosis-related proteins was detected by western blot. As shown in Figure 6B, the expression levels of proapoptotic proteins Bcl-2, Bax, cleavated PARP, and Caspase-3 were significantly increased, while the expression of antiapoptotic protein Bcl-2 was significantly decreased. This result indicated that the promotion of apoptosis of A549 cells by 7a might be related to the mitochondrial apoptosis pathway.
7a inhibits the activation of the PI3K/AKT signaling pathway
A variety of biological processes in a variety of tumor cells are regulated by the PI3K/Akt signaling pathway, including cell proliferation, apoptosis, survival, growth, and movement. Akt and mTOR proteins have been reported to serve an important role in tumor cell viability and metastasis [15]. In the present study, we detected the relevant proteins in PI3K/AKT signaling pathway by utilizing western blot analysis. As shown in Figure 7A, the phosphorylation levels of PI3K, Akt, and mTOR in the 7a dosing group were significantly decreased, and the expression levels of their downstream proteins NF-κB were decreased. Additionally, we further separately examined the NF-κB level in the cytoplasm and nucleus. As showed in Figure 7B, NF-κB level in the cytoplasm of 7a dosing group were slightly decreased, while NF-κB level in the nucleus was markedly decreased compared to NC group. These results demonstrated that 7a may inhibit the PI3K/AKT/NF-κB signaling pathway.