Effusanin B Inhibits Lung Cancer by Prompting Apoptosis and Inhibiting Angiogenesis

Abstract

Cancer is one of the deadliest human diseases, causing high rates of illness and death. Lung cancer has the highest mortality rate among all malignancies worldwide. Effusanin B, a diterpenoid derived from Isodon serra, showed therapeutic potential in treating non-small-cell lung cancer (NSCLC). Further research on the mechanism indicated that effusanin B inhibited the proliferation and migration of A549 cells both in vivo and in vitro. The in vitro activity assay demonstrated that effusanin B exhibited significant anticancer activity. Effusanin B induced apoptosis, promoted cell cycle arrest, increased the production of reactive oxygen species (ROS), and altered the mitochondrial membrane potential (MMP). Based on mechanistic studies, effusanin B was found to inhibit the proliferation and migration of A549 cells by affecting the signal transducer and activator of transcription 3 (STAT3) and focal adhesion kinase (FAK) pathways. Moreover, effusanin B inhibited tumor growth and spread in a zebrafish xenograft model and demonstrated anti-angiogenic effects in a transgenic zebrafish model.

Keywords: FAK; STAT3; angiogenesis; anti-tumor; diterpenoid; effusanin B; zebrafish.

Figure 1

Chemical structure of effusanin B.

Figure 2

Effusanin B induced apoptosis in A549 cells. A549 cells were exposed to effusanin B (6, 12, and 24 μM) for 48 h. The cells labeled with propidium iodide (PI) and Annexin V were assessed using flow cytometry. (A) Flow cytometric analysis of A549 cells treated with various concentrations of effusanin B. (B) Histogram showing the proportions of apoptotic cells at 48 h with the treatment of effusanin B. The results are expressed as means ± SD. *** p < 0.001 versus the control group.

Figure 3

Effusanin B arrested the A549 cells in the S phase. A549 cells were treated with effusanin B (6, 12, and 24 μM) for 48 h. Subsequently, propidium iodide (PI) was used to label the cells, and the cell cycle distribution was analyzed using flow cytometry. (A) Flow cytometric analysis of A549 cells after being treated with effusanin B. (B) Histogram showing the distribution of cell cycle phases.

Figure 4

Effusanin B affected MMP levels in A549 cells. A549 cells were treated with effusanin B (6, 12, and 24 μM) for 48 h. Then, the cells were stained with JC-1, and the mitochondrial membrane potential was measured using flow cytometry. (A) Flow cytometric analysis of A549 cells after being treated with effusanin B. (B) Histogram of the ratio of JC-1 polymer/monomer. (C) Histogram of the population of JC-1 polymer and monomer. The results are expressed as means ± SD. ** p < 0.01 and *** p < 0.001 versus control group.

Figure 5

Effusanin B increased ROS levels of A549 cells. A549 cells were treated with effusanin B (6, 12, and 24 μM) for 48 h. PI was used to label the cells, and flow cytometry was used to assess the results. (A) Flow cytometric analysis of A549 cells after being treated with different concentrations of effusanin B. (B) Histogram of relative ROS level compared with the control group. The results are expressed as means ± SD. *** p < 0.001 versus the control group.

Figure 6

Effusanin B regulated proteins related to apoptosis. (A) Western blotting analysis was performed to evaluate the impact of effusanin B on the expression of Bax, Bcl-2, Mcl-1, Cyclin D1, and caspase-3. (B) A histogram depicting the relative protein expression levels compared to the control group. The results are expressed as means ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001 versus control group.

Figure 7

Effusanin B regulated the STAT3 signaling pathway. (A) Western blotting analysis was conducted to evaluate the effects of effusanin B on the expression of STAT3 and p-STAT3. (B) Histogram showing the relative expression level of the protein compared with the control group. The results are expressed as means ± SD. *** p < 0.001 versus the control group.

Figure 8

Effusanin B inhibited the tumor migration and regulated the FAK signaling pathway. (A) A549 cells were photographed at 0 h and 48 h. (B) Data on migration rates (%) are shown in the histogram. (C) Western blotting analysis was used to evaluate the effects of effusanin B on the expression of FAK and p-FAK. (D) Histogram of the relative protein expression level compared with the control group. The results are expressed as means ± SD. *** p < 0.001 versus the control group.

Figure 9

Effusanin B inhibited tumor proliferation and migration in vivo. A549 cells were stained with CM-DiI and then microinjected into 2 dpf zebrafish embryos. After 4 h, the embryos mentioned above were treated with effusanin B (1, 3, and 10 μM) and etoposide (10 μM) for 48 h (15 embryos/group). (A) The intensity and distribution of the red fluorescence in disseminated foci in zebrafish were imaged under a confocal microscope. (B) The proliferation was quantified using ImageJ software. (C) The metastasis of A549 cells was quantified using ImageJ software. All results are expressed as the mean ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001 versus the control group.

Figure 10

The effects of effusanin B on transgenic zebrafish angiogenesis. The embryos were obtained from transgenic zebrafish Tg(fli1:EGFP). Effusanin B and sunitinib malate (2 μM) were used to treat the embryos mentioned above for 48 h. (A) The development of ISVs was observed under a confocal microscope. The red arrows indicated the condition of angiogenesis inhibition. (B) The average length of ISVs of zebrafish after treatment with effusanin B (10, 20, and 40 μM) and sunitinib malate (15 embryos/group). All results are expressed as the mean ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001 versus control group.