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. 2024 Feb 16;29(4):877.
doi: 10.3390/molecules29040877.

Strophanthidin Induces Apoptosis of Human Lung Adenocarcinoma Cells by Promoting TRAIL-DR5 Signaling

Xiao Tian  1   2 , Liangzhen Gu  2   3 , Fangang Zeng  4 , Xingkai Liu  2 , Yang Zhou  2   3 , Yang Dou  2   3 , Juanjuan Han  2 , Yao Zhao  2   3 , Yanyan Zhang  2 , Qun Luo  2   3 , Fuyi Wang  1   2   3
Affiliations

Strophanthidin Induces Apoptosis of Human Lung Adenocarcinoma Cells by Promoting TRAIL-DR5 Signaling

Xiao Tian et al. Molecules. .

Abstract

Strophanthidin (SPTD), one of the cardiac glycosides, is refined from traditional Chinese medicines such as Semen Lepidii and Antiaris toxicaria, and was initially used for the treatment of heart failure disease in clinic. Recently, SPTD has been shown to be a potential anticancer agent, but the underlying mechanism of action is poorly understood. Herein, we explored the molecular mechanism by which SPTD exerts anticancer effects in A549 human lung adenocarcinoma cells by means of mass spectrometry-based quantitative proteomics in combination with bioinformatics analysis. We revealed that SPTD promoted the expression of tumor necrosis factor (TNF)-related apoptosis-inducing ligand receptor 2 (TRAIL-R2, or DR5) in A549 cells to activate caspase 3/6/8, in particular caspase 3. Consequently, the activated caspases elevated the expression level of apoptotic chromatin condensation inducer in the nucleus (ACIN1) and prelamin-A/C (LMNA), ultimately inducing apoptosis via cooperation with the SPTD-induced overexpressed barrier-to-autointegration factor 1 (Banf1). Moreover, the SPTD-induced DEPs interacted with each other to downregulate the p38 MAPK/ERK signaling, contributing to the SPTD inhibition of the growth of A549 cells. Additionally, the downregulation of collagen COL1A5 by SPTD was another anticancer benefit of SPTD through the modulation of the cell microenvironment.

Keywords: Trail-DR4/5 signaling; anticancer agent; apoptosis; bioinformatics; mass spectrometry; quantitative proteomics; strophanthidin.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
(A) Chemical structure of strophanthidin (SPTD). (B,C) Flow cytometric quantification of viability (Q1), early-stage apoptosis (Q2), late-stage apoptosis (Q3) and necrosis (Q4) of A549 cells without ((B), control) and with ((C), SPTD) treatment of 1.0 μM SPTD at 37 °C for 24 h. (D) Percentage of cells at various states in the control and SPTD groups of A549 cells (n = 3). (E,F) Cell cycle profiles of A549 cells without (E) and with (F) treatment of 1.0 μM SPTD at 37 °C for 24 h. (G) Histograms of population distribution in different cell cycles of A549 cells without (control) and with (SPTD) treatment of 1.0 μM SPTD at 37 °C for 24 h.
Figure 2
Figure 2
Quantitative proteomics analysis. (A) Venn diagram of the numbers of the proteins commonly expressed in A549 cells without (control) and with SPTD (1.0 μM) treatment. (B) The volcanic map of the proteins identified in both control and treated groups of A549 cells with various abundance ratios (ARs, SPTD vs. control) and p-values. Blue dots refer to proteins with statistically insignificant change in expression level between the two groups of cells, while red and green ones refer to proteins with statistically significant change in expression level between the two groups of cells with p ≤ 0.05 (−lg p ≥ 1.3) and log2AR ≥ 0.38, and with p ≤ 0.05 (−lg p ≥ 1.3) and log2AR ≤ −0.38. (C) The heat-map of the differentially expressed proteins (DEPs) with p ≤ 0.05 and |log2AR| ≥ 0.38 subjected to SPTD treatment. (D,E) The fold-change (FC) of the 15 top upregulated DEPs (orange) (D) and the top downregulated DEPs (blue) (E).
Figure 3
Figure 3
Bioinformatics analysis of the differentially expressed proteins (DEPs) with |FC| ≥ 1.3 identified in A549 cells treated with 1.0 μM of SPTD. (A) Top 20 cellular components, (B) top 20 molecular functions.
Figure 4
Figure 4
Bioinformatics analysis of the differentially expressed proteins (DEPs) with |FC| ≥ 1.3 identified in A549 cells treated with 1.0 μM of SPTD. (A) Top 20 involved biological processes and (B) top 20 associated KEGG pathways of the DEPs.
Figure 5
Figure 5
Top 20 associated canonical pathways of the differentially expressed proteins (DEPs) with |FC| ≥ 1.3 identified in A549 cells treated with 1.0 μM of SPTD enriched by IPA.
Figure 6
Figure 6
Schematic diagram generated by IPA of the death receptor signaling pathway with which the DEPs with |FC| ≥ 1.3 identified in A549 cells exposed to 1.0 μM of SPTD are highly associated with. The red color represents a protein (or complex) that is upregulated, green represents downregulation and the fold-change value is noted below the gene name.
Figure 7
Figure 7
Enrichment of the core protein–protein interaction (PPI) networks of DEPs with |FC| ≥ 1.3 identified in A549 cells exposed to SPTD. (A) This PPI network indicates that all of the DEPs involved interact with each other via the core protein LMNA. (B) This PPI network indicates that all DEPs involved downregulate p38 MARK/ART/ERK signaling via interaction with VEGF, PDGF and TGFβ. The red color refers to upregulation of the genes, and green to downregulation, with fold-change value listed below the gene names. The orange color indicates that the gene is predicted by IPA to be upregulated or activated, blue indicates it is downregulated and grey indicates there is no prediction available. The solid line indicates direct interaction between two proteins and the dot line indicates indirect interaction. The asterisk (*) after a gene (or protein) name indicates that multiple identifiers in the dataset file map to a single gene (or protein) in the Global Molecular Network.
Figure 8
Figure 8
Immunofluorescence images of cleaved/activated caspase 3 (A), 6 (B) and 8 (C) in A549 without (control) and with treatment of 1.0 μM SPTD for 3 h at 37 °C. Green fluorescence represents the target protein (λex = 488 nm, λem = 525 nm) and blue fluorescence represents the nucleus stained by DAPI (λex = 360 nm, λem = 460 nm).
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