Babaodan inhibits cell growth by inducing autophagy through the PI3K/AKT/mTOR pathway and enhances antitumor effects of cisplatin in NSCLC cells

Abstract

Babaodan capsule (BBD), a traditional Chinese (TCM) formula, has been widely used as an alternative remedy for multiple types of malignancies, clinically. However, the underlying mechanisms behind the efficacy of BBD remain poorly understood, particularly in regard to lung cancer. Herein, we demonstrate that BBD induced autophagic death in A549 and A549DDP cells without apoptosis. Treatment with autophagic inhibitor 3-MA, Baf-A1 and PI3K agonist, IGF-1, fully proved our conclusion, as well as uncovered the potential downregulated signaling pathway, PI3K/AKT/mTOR. The study additionally found that BBD could downregulate the expression of MDR1 and increase the chemosensitivity of cisplatin. Collectively, our results, both in vivo and in vitro, demonstrate that BBD leads to autophagic cell death through downregulating the PI3K/AKT/mTOR signaling pathway and improved the antitumor effects of cisplatin in non-small cell lung cancer (NSCLC).

Keywords: Babaodan capsule (BBD); PI3K/AKT/mTOR pathway; autophagy; non-small cell lung cancer (NSCLC).

Conflict of interest statement

None.

Figures

Figure 1

BBD inhibited cell growth in a dose-dependent manner in A549 and A549DDP cells. A. IC50 value of cisplatin was determined by CCK8 assay. B. Cell viability after BBD treatment for 24 hours or 48 hours was determined through CCK8 assay. C. Cells were incubated in the same concentrations of BBD for similar 24 hours, cell viability was detected and compared. D. Photographs of crystal violet-stained colonies of BBD-treated both cells growing in 6-well plates for 12-14 days after infection, and the number of cells in each colony was counted. *, P

Figure 2

BBD induced cytotoxicity in A549 and A549DDP primarily by autophagy. A549 (A) and A549DDP (B) cells were treated with different concentrations of BBD, the cellular proteins Bcl-2, Bax, caspase-cleaved PARP were determined by Western-blot analysis. A549 (C) and A549DDP (D) cells were pretreated with 3-MA, and then treated for another 24 hours with BBD, cell viability was then determined by CCK8 assay and compared to the BBD treatment alone. (E, F) Western blot analysis of LC3II expression after BBD administration for 24 hours in both cells. The corresponding expression levels are shown as bar graphs. *, P

Figure 3

BBD increased autophagic flux. A549 and A549DDP cells were treated with Baf-A1, BBD, BBD + Baf-A1, as well as untreated control group. (A, B) Western blot analysis revealed the expression of LC3II in both cells. (C) TEM evaluated autophagy in A549 cells. A549 (D) and A549DDP (E) cells were treated with 3-MA, BBD, BBD + 3-MA, as well as untreated control group. Similarly the LC3II status was determined by Western blot. The corresponding expression levels are shown as bar graphs. **, P

Figure 4

BBD induced autophagy via downregulating the PI3K/AKT/mTOR signaling pathway. The expression of PI3K, p-PI3K, AKT, p-AKT, mTOR, p-mTOR were examined by Western blot after BBD (A) or IGF-1 (B) treated for 24 hours. A549 (C) and A549DDP (D) cells were treated with IGF-1, BBD, BBD + IGF-1, as well as untreated control group, LC3II expression was determined by Western blot. **, P

Figure 5

BBD potentiated the chemosensitivity of cisplatin in A549 and A549DDP. (A) IC50 value of cisplatin with or without BBD-pretreatment. (B) Wound healing assay exhibited the migration ability of BBD + DDP treated cells, compared with DDP alone group. (C) Transwell assay revealed the invasion ability of BBD + DDP treated cells, compared with DDP alone group. qRT-PCR assay (D) and Western blot assay (E) determined the expression of MDR1. *, P

Figure 6

BBD exerted anti-tumor activity in A549 xenografts. A549 cells were injected into the nude mice, and BBD, DDP, and control PBS administered as indicated. A. Representative images of tumors from mice in each group. B. Tumor volume was measured every 4 days. *, P