Liensinine Inhibits Cell Growth and Blocks Autophagic Flux in Nonsmall-Cell Lung Cancer

Abstract

Liensinine is a bioactive component of Plumula Nelumbinis extracted from the green embryo of the mature seeds of Nelumbonaceae and exhibits therapeutic functions and noteworthy anti-tumor effects in recent studies. However, the potential anti-tumor property and the underlying mechanisms of liensinine in nonsmall-cell lung cancer (NSCLC) have not been illustrated. In this study, we demonstrated that liensinine has the potential anti-tumor property, and it could inhibit growth of NSCLC in vitro and in vivo. In addition, we found that although it induced significant accumulation of autophagosomes, liensinine could quench them for degradation and blocked autophagic flux. Importantly, we observed that liensinine inhibited the normal function of mitochondrial energy supply and impaired the lysosomal function. This research firstly provides a possibility insight that liensinine could be a novel therapeutic strategy for NSCLC.

Figure 1

Liensinine exerts cytotoxicity and induces apoptosis in vitro. (a) A549, H520, and SPC-A1 cells were treated with the indicated concentrations of liensinine for 24h and 48h, respectively, and then CCK-8 assay was performed. (b) A549, H520, and SPC-A1 cells were treated with the indicated concentrations of liensinine for 48h and then incubated with normal culture for 14 days. (c) A549, H520, and SPC-A1 cells were treated with indicated concentrations of liensinine for 48h, and apoptosis assays were performed using annexin V and PI staining. (d) A549 and SPC-A1 cells were treated with 20 μM liensinine with or without Z-VAD, and then apoptosis assay was performed. Three independent experiments were performed. The results were shown as means ± SD, ^∗∗∗∗P < 0.0001.

Figure 2

Liensinine inhibits NSCLC proliferation in vivo. (a) A brief flowchart of the experiment design in vivo. A549 xenograft tumor was established and divided into the following groups (control, 5 mg/kg/d, 20 mg/kg/d) and received 25 days of treatment. (b) The image of tumors from different groups. (c) The curves of tumor growth. (d) The weight of tumors. (e) The body weight of mice. The results were shown as means ± SD, ^∗∗P < 0.01, ^∗∗∗P < 0.001.

Figure 3

Liensinine promotes mitochondrial dysfunction. (a) A549, H520, and SPC-A1 cells were treated with the indicated concentrations of liensinine for 48h and stained with COX IV antibody. Scale bars: 10 μm. (b–d) A549 and SPC-A1 cells were treated with liensinine or DMSO, and the mitochondria structure was observed by transmission electron microscope. The shape and area of mitochondria were measured and calculated. Scale bars: 0.4 μm. (e–f) A549, H520, and SPC-A1 cells were treated with the indicated concentrations of liensinine for 48h and then detected using JC-1 flow cytometry. Three independent experiments were performed. The results were shown as means ± SD, ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001.

Figure 4

Liensinine induces autophagosome accumulation. (a) A549, H520, and SPC-A1 cells were treated with the indicated concentrations of liensinine for 48h, and Western blot assays demonstrated the LC3B expression. (b–c) LC3B puncta were observed by confocal laser microscopy after staining with LC3B antibody, and autophagic vacuoles were calculated. Scale bars: 10 μm. (d–e) A549 cells were treated with liensinine with or without Baf.A1 and then detected the LC3B puncta by immunofluorescence. LC3B puncta number per cell was quantified. (f) A549 cells were treated with liensinine with or without Baf.A1, and then Western blot assays demonstrated the LC3B expression. (g–h) A549 and H520 cells were treated with liensinine as indicated concentrations and then stained with SQSTM1 antibody. SQSTM1 puncta number per cell was quantified. Scale bars: 10 μm. Three independent experiments were performed. The results were shown as means ± SD, ^∗P < 0.05, ^∗∗∗∗P < 0.0001.

Figure 5

Liensinine blocks autophagic flux. (a–b) A549 and SPC-A1 cells transfected with mRFP-GFP-LC3 virus were treated with EBSS, Baf.A1(100 nm), or liensinine (20 μM), and observed by confocal microscopy. The percentage of GFP in mRFP signals was calculated. Scale bars: 10 μm. (c) A549 and SPC-A1 cells were treated with liensinine as indicated concentrations, and Western blot assays demonstrated the SQSTM1, LAMP1, and LAMP2 expression. Three independent experiments were performed. The results were shown as means ± SD, ns = no significant.

Figure 6

Liensinine reduces mitochondrial bioenergetic activity. (a) A549 and SPC-A1 cells were treated with liensinine as indicated concentrations, and then ATP assay kit was used to evaluate the ATP level. (b-c) A549 and SPC-A1 cells were treated with liensinine as indicated concentrations, and the genomic DNA was extracted and performed LR-PCR and q-PCR to evaluate the mitochondria lesions. Details were shown in methods. (d) A heat map was generated after analysis. The relative expression of metabolites with significant differences was shown in red (upregulation) versus green (downregulation). (e) KEGG pathway analysis for the differential metabolites. (f) A549 and SPC-A1 cells were treated with liensinine as indicated concentrations, and Western blot assays demonstrated the mTOR, p-mTOR, AMPK, p-AMPK, Beclin1, and ULK1 expression. The results were shown as means ± SD, ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001, ^∗∗∗∗P < 0.0001.