Pennogenin-3- O-α- L-Rhamnopyranosyl-(1→2)-[α- L-Rhamnopyranosyl-(1→3)]-β- D-Glucopyranoside (Spiroconazol A) Isolated from Dioscorea bulbifera L. var. sativa Induces Autophagic Cell Death by p38 MAPK Activation in NSCLC Cells

Abstract

In our previous study, we reported the isolation of pennogenin-3-O-α-L-rhamnopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→3)]-β-D-glucopyranoside (spiroconazol A), a steroidal saponin, from the flowers of Dioscorea bulbifera L. var. sativa. In the present study, we aimed to investigate the effects of spiroconazol A on autophagy and its underlying mechanisms in A549 and NCI-H358 human non-small cell lung cancer (NSCLC) cells. Spiroconazol A inhibited the proliferation of NSCLC cells in a concentration- and time-dependent manner. To determine the type of programmed cell death induced by spiroconazol A, we performed a characterization of apoptosis in spiroconazol A-treated A549 cells. Our results showed that spiroconazol A significantly suppressed A549 cell viability but did not influence cell apoptosis because phosphatidylserine and caspase activation were not detected. Furthermore, spiroconazol A treatment upregulated the expression of LC3-II and autophagy-related Beclin-1 protein, suggesting that spiroconazol A induces autophagy in A549 cells. Moreover, spiroconazol A activated the phosphorylation of p38 mitogen-activated protein kinase (MAPK) but did not affect the phosphorylation of Janus kinase or ERK1/2. Notably, SB203580, a p38 MAPK inhibitor, had a significant inhibitory effect on spiroconazol A-induced autophagic cell death in A549 cells. Our results indicated that spiroconazol A-induced autophagy is dependent on p38 MAPK signaling and has potential as a therapeutic target in NSCLC.

Keywords: autophagy; cell death; non-small cell lung cancer (NSCLC); p38 MAPK; spiroconazol A.

Figure 1

Non-apoptotic features were presented by spiroconazol A in A549 cells. (A) Cells were exposed to spiroconazol A (2 μM) and were stained with the PI solution. For detection of sub-G_1, indicating cell death, cells were determined by flow cytometry. (B) After treatment with spiroconazol A (2 μM) for the indicated times, cells were stained with FITC-conjugated Annexin V and PI (Cis: cisplatin) and then detected by flow cytometry. (C) After treatment with 50 μM z-VAD-fmk (broad caspase inhibitor) for 1 h, cells were treated with spiroconazol A (2 μM) for 24 h. Cells were stained with the PI solution and then examined by flow cytometry. Experiments were repeated at least three times, and data are expressed as mean ± S.D. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. control group by the Student’s t-test.

Figure 2

Autophagy induced by spiroconazol A in NSCLC cells. (A) Representative images showed the morphological changes in spiroconazol A (2 μM)-treated NSCLC cells using an OLYMPUS IX51 inverted microscope (Southend-on-Sea, Essex, UK). (B) Protein expression of cells exposed to 2 μM of spiroconazol A, using Western blot analysis. β-actin was utilized as an internal control. The relative optical density ratio was determined using a densitometric analysis program (Bio-Rad Quantity One^® Software, version 4.6.3 (Basic), Bio-Rad Laboratories Inc., CA, USA), normalized to the internal control. * p < 0.05, ** p < 0.01 vs. untreated A549 cells, by the Student’s t-test.

Figure 3

Cell death by spiroconazol A-induced autophagy. (A) The pEGFP-LC3-II-transfected A549 cells were treated with 2 μM of spiroconazol A for 24 h; immunofluorescence of pEGFP-LC3-II was detected by confocal fluorescence microscopy. After pretreatment with 10 mM of 3-MA for 1 h, cells were treated with 2 μM of spiroconazol A for 24 h and then examined by (B) Western blot analysis and (C) PI staining, respectively. Experiments were repeated at least three times, and data are expressed as mean ± S.D. The relative optical density ratio was determined using a densitometric analysis program (Bio-Rad Quantity One^® Software, version 4.6.3 (Basic), Bio-Rad Laboratories Inc., CA, USA), normalized to the internal control. * p < 0.05, *** p < 0.001 vs. untreated A549 cells and ^# p < 0.05, ^### p < 0.001 vs. spiroconazol A-treated A549 cells, by the Student’s t-test.

Figure 4

The p38 MAPK kinase signaling pathway is required in spiroconazol A-induced autophagic cell death. (A) After treatment with 2 μM of spiroconazol A for the indicated times, cells were examined by Western blot analysis. After pretreatment with 40 μM of SB203580 (p38 MAPK inhibitor), cells were treated with spiroconazol A for 24 h and then examined by (B) Western blot analysis and (C) PI staining, respectively. Experiments were repeated at least three times, and data are expressed as mean ± S.D. The relative optical density ratio was determined using a densitometric analysis program (Bio-Rad Quantity One^® Software, version 4.6.3 (Basic), Bio-Rad Laboratories Inc., CA, USA), normalized to the internal control. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. untreated A549 cells and ^# p < 0.05, ^### p < 0.001 vs. spiroconazol A-treated A549 cells, by the Student’s t-test.

Figure 5

Chemical structures of pennogenin (1), mannioside A (2), and spiroconazol A (3) isolated from D. bulbifera L. var. sativa.