Research ArticleExperimental Studies
Open Access

Apigenin-induced Apoptosis in Lung Adenocarcinoma A549 Cells: Involvement in IFNA2, TNF, and SPON2 With Different Time Points

RIEKO AIDA, KAZUNORI OKANO, KYOKO NAKATA and HIROMI HAGIWARA
Anticancer Research July 2025, 45 (7) 2781-2790; DOI: https://doi.org/10.21873/anticanres.17647

Abstract

Background/Aim: Apigenin, found in a variety of vegetables and fruits, exhibits anti-oxidant, anti-inflammatory and anticancer effects. Recently, we reported the possibility that apigenin induces apoptosis in human lung adenocarcinoma A549 cells through the miR-34a-5p/SNAI1/caspase-3/-7 pathway. Understanding how apigenin triggers apoptosis in cancer cells will help lay the groundwork for developing effective cancer treatments.

Materials and Methods: The lung adenocarcinoma A549 Cell line was used. To determine whether caspase-8 or caspase-9 is activated, we performed a caspase activity assay. Real-time qRT-PCR was performed to identify mRNAs that may stimulate the upstream pathways, including tumor necrosis factor-a (TNF-a), spondin-2 (SPON2), and interferon-a2 (IFNA2), which are known to be involved in apoptosis in various cancer cell lines.

Results: In apigenin-treated cells, early-stage apoptosis was observed at 24 h, with increased activity of caspase-8 at 18 h and again at 24 h, and caspase-9 at 24 h and further at 48 h. However, mRNA levels of caspase-8 and caspase-9 significantly decreased after 24 h. Real-time RT-qPCR analysis revealed increased mRNA levels of TNF-a, spondin-2, and interferon-a2 after 24 h of apigenin treatment in A549 cells, whereas treatment for 48 h led to decreased expression of SPON2 and IFNA2.

Conclusion: Apigenin promotes apoptosis in A549 cells by modulating various signaling pathways at different time points.

Keywords:

Introduction

Flavonoids work as scavengers of reactive oxygen species (ROS) (1), acting as antioxidants (2). In addition, they have anti-inflammatory (3-5) and anticancer (6-9) properties. Apigenin (5, 7, 4′-trihydroxyflavone), one of the flavones belonging to the flavonoid class, is widely found in various vegetables, fruits, and medical herbs such as chamomile, guava and schizonepeta (10). Several reports describe apigenin-induced apoptosis in various cancer cell lines, with each study primarily highlighting on a specific signaling pathway (11-13). The A549 cell line, derived from human lung adenocarcinoma, is frequently used in a wide range of research applications. Lung adenocarcinoma, classified as non-small cell lung cancer, accounts for approximately 40% of all lung cancers. Therefore, the observation of apoptosis in A549 cell line is important from the perspective of the pathognomonic disease concept and therapeutic strategies. Our previous study reported that apigenin-induced apoptosis via the miR-34a-5p/SNAI1/caspase-3/-7 pathway in human lung adenocarcinoma A549 cell line (14).

Apoptosis is a mechanism of controlled cell death characterized by nuclear aggregation and fragmentation of DNA. Caspases are central regulators of apoptosis. The intrinsic pathway is triggered by various cellular stresses, such as DNA damage, leading to the activation of the initiator caspase-9 via the mitochondrial pathway. On the other hand, the extrinsic pathway is initiated by extracellular death signals such as Fas ligand and tumor necrosis factor (TNF)-a, leading to the activation of initiator caspase-8. Ultimately, both pathways lead to the activation of effector caspase-3/-7.

We investigated apigenin-induced apoptosis focused on TNF, SPON2, and IFNA2, and these proteins have been reported to be involved in apoptosis through different signaling pathways. TNF is a gene that codes for TNF-a, a pleiotropic cytokine. The signal is received by either TNF receptor 1 (TNFR1) or TNF receptor 2 (TNFR2). TNF-a plays various critical roles in inflammation, immunity, cell survival, and cell death. When TNF-a binds to TNFR1, the signal-inducing apoptosis (15) is transmitted through the RIP1/caspase-8/caspase-3/-7 pathway (16-20). TNF-a-induced apoptosis is found in cancer cells such as breast cancer (21), lung adenocarcinoma (22), and gastric cancer (23). Notably, cells such as macrophages, lymphocytes, fibroblasts, and keratinocytes are known to produce TNF-a. However, some reports describe the production of TNF-a by epithelial cells (24,25). Spondin-2, translated from SPON2 is a member of the F-spondin family and is secreted as an extracellular matrix protein. Its expression levels are significantly up-regulated in prostate cancer (26), lung adenocarcinoma (27), hepatocellular carcinoma (28), colorectal carcinoma (29), and gastric cancer (30). Moreover, down-regulation of SPON2 decreases cell proliferation, migration, and invasion (27, 28) and induces apoptosis via activated caspase-3 (31). IFNA2 encodes interferon-a2 (IFN-a2), a cytokine belonging to the family of type I IFNs. Importantly, IFN-a2 is secreted by infected cells or dying cells to exert key roles in the innate immune response (32). Moreover, it induces apoptosis through an IFN-a type-2 receptor (IFNAR-2)-dependent signaling pathway (33). In human hepatoma cells, CASP8 is the target gene of IFN-a2 (34), which also up-regulates the activation of the two proapoptotic Bcl-2 family members, Bak and Bax, in human myeloma (35).

In the present study, we investigated the apoptotic signaling pathways induced by apigenin in A549 cells by focusing on TNF, SPON2, and IFNA2. Investigating these pathways will further deepen our understanding of apoptosis.

Materials and Methods

Reagents. Apigenin (purity >98.0%) was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan), and its structure is shown in Figure 1. It was dissolved in dimethyl sulfoxide (DMSO) for cell culture (Sigma-Aldrich Co., St. Louis, MO, USA).

Figure 1.
Figure 1.

The structure of apigenin.

Cell lines and culture. Human lung adenocarcinoma A549 cells were obtained from RIKEN Cell Bank (Tsukuba, Japan) and cultured in Dulbecco’s Modified Eagle’s Medium (low glucose) (DMEM, Sigma-Aldrich), supplemented with 10% fetal bovine serum (Moregate BioTech, Bulimba, Australia), 1% penicillin and streptomycin, and 2% GlutaMax™ (GIBCO, Dublin, Ireland), and incubated in a humidified atmosphere of 5% CO2 at 37°C. For the Annexin-V assay, cells were seeded at a density of 1.6×104 cells/ml in 35-mm glass bottom dishes (IWAKI, Tokyo, Japan) and treated with various concentrations of apigenin. To assess the activity of caspase-8 and caspase-9, cells were seeded at a density of 1×104 cells/well in an EZVIEW Glass Bottom Culture Plate LB 96-well plate (IWAKI). Cells were subcultured for 1 d prior to treatment with 40 μM or 80 μM apigenin for the caspase-8 activity assay. For the caspase-9 activity assay, cells were treated with 60 μM or 100 μM apigenin after being subcultured for 1 d. To extract total RNA, cells were seeded at a density of 1×105 cells/well in 6-well plates and then treated with 80 μM apigenin for 24 h or 50 μM apigenin for 48 h. For controls, cells were treated with dimethyl sulfoxide (DMSO) for 24 h or 48 h. The concentration (50 μM) and exposure time (48 h) were selected based on the 50% lethal dose.

Annexin V apoptosis assay. Cells in the early stages of apoptosis were fluorescently imaged using the Annexin V-FITC Apoptosis Detection Kit (BioVision, Milpitas, CA, USA), following the manufacturer’s instructions. Fluorescence from Annexin V-FITC, propidium iodide (PI), and scattered light were collected at wavelengths of 510 to 540 nm, >570 nm, and within the visible wavelength range, respectively. Images were captured with a scanning laser confocal microscope (Olympus FV300) equipped with a 488 nm excitation laser and a 20× objective (NA. 0.5).

Caspase activity assay. Caspase-8 and caspase-9 activities in A549 cells were visualized using the CellMeter™ Caspase 8 and CellMeter™ Caspase 9 Activity Apoptosis Assay Kits (AAT Bioquest, Pleasanton, CA, USA), respectively, according to the manufacturer’s protocol. The peptide sequences Ile-Glu-Thr-Asp (IETD) and Leu-Glu-His-Asp (LEHD) target caspase-8 and caspase-9, respectively. Caspase-8 produces fluorescent R110 by cleaving (Ac-IETD)2-R110, while caspase-9 yields fluorescent R110 by cleaving (Ac-LEHD)2-R110. Fluorescence was monitored using a Cy-5 dichroic filter set at an emission wavelength of approximately 535 nm and an excitation wavelength of 485 nm through a band-path filter, emitted from a halogen lump. Staurosporine at a concentration of 10 μM served as a positive control.

Real-time qRT-PCR. mRNA expression levels in A549 cells treated with 50 μM apigenin for 48 h or 80 μM apigenin for 24 h were examined by real-time reverse transcription polymerase chain reaction (qRT-PCR). Total RNA (1 μg) was reverse-transcribed using a Roche Transcriptor First Strand cDNA Synthesis Kit (Roche, Basel, Switzerland) according to the manufacturer’s protocol. The qRT-PCR analysis was performed using a Roche LightCycler 480 SYBR Green I Master Mix (Roche) with a LightCycler 480 System II (Roche). The relative mRNA expression levels of each gene were compared to that of β-actin (internal control) by the 2−ΔΔCt method. Specific primers for real-time qRT-PCR were designed using the website of primer3plus and are as follows: CASP8: sense 5′-AAG CAA ACC TCG GGG ATA CT-3′ and antisense 5′-GGG GCT TGA TCT CAA AAT GA-3′, CASP9: sense 5′-AGG CCC CAT ATG ATC GAG GA-3′ and antisense 5′-TCG ACA ACT TTG CTG CTT GC-3′, TNF: sense 5′-AGT ATC CAT GCT CTT GAC CTT GTA G-3′ and antisense 5′-CCC GTA ATT GCT CCA ATC TG-3′, SPON2: sense 5′-ACG GTG ACC GAG ATA ACG TC-3′ and antisense 5′-GGA ACT GAG GCG CTG TCT AC-3′, IFNA2: sense 5′-GCA AGT CAA GCT GCT CTG TG-3′, and antisense 5′-GAT GGT TTC AGC CTT TTG GA-3′, NOTCH1: sense 5′-ACT GTG AGG ACC TGG TGG AC-3′ and antisense 5′-TTG TAG GTG TTG GGG AGG TC-3′, NOTCH3: sense 5′-TGT GGA CGA GTG CTC TAT CG-3′ and antisense 5′-AAT GTC CAC CTC GCA ATA GG-3′, RELA: sense 5′-GCG AGA GGA GCA CAG ATA CC-3′ and antisense 5′-CTG ATA GCC TGC TCC AGG TC-3′, RELB: sense 5′-TCC CAA CCA GGA TGT CTA GC-3′ and antisense 5′-AGC CAT GTC CCT TTT CCT CT-3′, CYLD: sense 5′-AAT GCT ACG ACG ATC CGG AC-3′ and antisense 5′-GCA ACG TTA GGA CTC TGC CT-3′, BAX: sense 5′-CTG CAG AGG ATG ATT GCC G-3′ and antisense 5′-TGC CAC TCG GAA AAA GAC CT-3′, BCL2: sense 5′-ACA GGG TAC GAT AAC CGG GA-3′ and antisense 5′-CAT CCC AGC CTC CGT TAT CC-3′, BAK: sense 5′-GGG TCT ATG TTC CCC AGG AT-3′ and antisense 5′-GCA GGG GTA GAG TTG AGC AG-3′, and β-actin: sense 5′-GGA CTT CGA GCA AGA GAT GG-3′ and antisense 5′-AGC ACT GTG TTG GCT TAC AG-3′ (Eurofins Genomics, Tokyo, Japan).

Statistical analysis. Numerical data are expressed as the mean±S.D. from three observations. The significance of the differences was analyzed using a two-sided Student’s t-test. Statistical significance was set at p<0.05. The experiments were repeated independently in triplicate, and the results were qualitatively identical in every case.

Results

Annexin V assay after apigenin-treatment for 24 h. We conducted an Annexin V assay on A549 cells cultured for 24 h in a medium containing apigenin (Figure 2). Green fluorescence indicative of Annexin V binding was observed in A549 cells treated with 30 μM and 60 μM of apigenin. In contrast, no fluorescence was detected in cells not exposed to apigenin. This provided evidence of a cytoplasmic membrane phospholipid flip-flop. Additionally, no fluorescence from PI was observed, indicating the integrity of the cytoplasmic membrane and the separation of the cytoplasm from the ambient space.

Figure 2.
Figure 2.

Annexin V apoptosis assay. Plasma membrane condition of A549 was assayed one day after treatment with 0 (A), 30 (B) and 60 (C) μM of apigenin. Flip-flop of plasma membrane is observed by annexin V-FITC (I). The plasma membrane integrity and cell morphology were checked by PI exclusion test (II) and scatter images (III), respectively. Scale bars indicate 100 μm. All the images were collected by scanning laser confocal microscopy (Olympus FV300, excitation laser; 488 nm) with 20× objective (NA. 0.5). Fluorescence from annexin V-FITC, PI and scattered light were collected at 510-540 nm, >570 nm and visible wave range.

Effects of apigenin on the activity and expression of caspase-8 and caspase-9. We investigated the activities of caspase-8 and caspase-9 in A549 cells treated with apigenin. As depicted in Figure 3A and B, 80 μM apigenin increased caspase-8 activity at both 18 h (1.37-fold compared to vehicle, n=3) and 24 h (1.49-fold compared to vehicle, n=3). Conversely, 40 μM apigenin did not enhance the caspase-8 activity in A549 cells. Caspase-9 activity was elevated at 24 h (2.70-fold compared to vehicle, n=3) and 48 h (2.30-fold compared to vehicle, n=3). However, caspase-9 activity decreased at a concentration of 100 μM in 24 h. Furthermore, we conducted real-time qRT-PCR to evaluate caspase-8 and caspase-9 mRNA levels in apigenin-treated A549 cells. Caspase-8 and caspase-9 mRNA levels in cells treated with 80 μM apigenin at 24 h were 0.29±0.23-fold (n=3) and 0.12±0.10-fold (n=3) compared to vehicle (DMSO), respectively (Figure 3C). In contrast, after 48 h of treatment with 50 μM apigenin, caspase-8 and caspase-9 mRNA expression was approximately the same as that in the vehicle (n=3).

Figure 3.
Figure 3.

Effects of apigenin on activity and expression of caspase-8 and caspase-9. Caspase-8 activity in A549 cells treated with 40 mM or 80 μM apigenin for 18 h or 24 h (A). Caspase-9 activity in A549 cells treated with 60 mM or 100 μM apigenin for 24 h or 48 h (B). (C) mRNA expression levels of caspase-8 and caspase-9 in A549 cells treated with 80 μM apigenin for 24 h (black column) or 50 μM apigenin for 48 h (white column) were examined by real-time qRT-PCR (n=3). *p<0.05 vs. vehicle (DMSO). **p<0.01 vs. vehicle (DMSO).

Expression of TNF, SPON2, and IFNA2 by Apigenin. Next, we investigated the expression of the genes upstream of caspase-8 and caspase-9. The real-time qRT-PCR was performed to evaluate the expression levels of mRNAs at apigenin concentration of 80 μM for 24 h and 50 μM for 48 h. As shown in Figure 4, the expression levels of TNF, SPON2, and IFNA2 significantly increased 2.34±0.37-fold (n=3), 1.40±0.21-fold (n=3), and 2.53±0.72-fold (n=3) against vehicle (DMSO), respectively, at 24 h. By contrast, SPON2 and IFNA2 decreased significantly by 0.56±0.03-fold (n=3) and 0.42±0.02-fold (n=3) against vehicle (DMSO), respectively, at 48 h.

Figure 4.
Figure 4.

Expression of TNF, SPON2, and IFNA2 by apigenin. mRNA expression levels of TNF-α, spondin-2, and interferon-α2 in A549 cells treated with 80 μM apigenin for 24 h (A) or 50 μM apigenin for 48 h (B) was examined by real-time qRT-PCR (n=3). *p<0.05 vs. vehicle (DMSO). **p<0.01 vs. vehicle (DMSO).

Expression of the genes of upstream and downstream TNF, SPON2 and IFNA2. The expression levels of BAX and BAK were also examined. As shown in Figure 5, both BAX (2.10±0.37-fold, n=3) and BAK (1.64±0.44-fold, n=3) mRNA expression was increased in apigenin-treated A549 cells at 48 h. However, there were almost no changes in their expression at 24 h. Although we also examined the mRNA expression levels of NOTCH1/3 (activator of SPON2) (30), RELA and RELB (subunits of NF-κB which targets TNF) (36), and CYLD (cylindromatosis, deubiquitinase involved in the formation of caspase-8 complexes during TNF-a-induced apoptosis) (37), the results could not be evaluated as too small of expression levels.

Figure 5.
Figure 5.

Expression levels of Bax and Bak mRNA. The expression level of Bax and Bak mRNA in A549 cells treated with 80 μM apigenin for 24 h or 50 μM apigenin for 48 h was examined by real-time qRT-PCR (n=3). *p<0.05 vs. vehicle (DMSO). **p<0.01 vs. vehicle (DMSO).

Discussion

In the present study, we investigated the timing and signaling pathways involved in apigenin-induced apoptosis. At 24 h, in contrast to the florescent of annexin V and PI, most cells undergoing apoptosis were in the early stages of the process. In our previous study (14), the activity of caspase-3/-7 was up-regulated at 72 h in apigenin-treated A549 cells. To identify which signaling pathway, extrinsic or intrinsic, activates caspase-3/-7, we examined the activities of caspase-8 and caspase-9. Our results indicated an increase in caspase-8 activity at 24 h and an elevation in caspase-9 activity at both 24 h and 48 h post-treatment.

Enhancement of TNF and IFNA2 and suppression of SPON2 are known to occur upstream of caspase-8 or caspase-9 (Figure 6). Therefore, we explored the expression levels of TNF, IFNA2, and SPON2 using real-time qRT-PCR, revealing a decrease in SPON2 expression by half at 48 h. Several reports revealed that the expression levels of SPON2 were fairly high in various cancer cells (26-29) and that the suppression of SPON2 decreased cell proliferation, migration, and invasion abilities (30) and induced apoptosis via activating caspase-3 (31). In apigenin-treated A549 cells, decreasing SPON2 expression may induce apoptosis. It occurred at 48 h but not at 24 h; thus, apoptosis via the SPON2 signaling pathway might have occurred from 48 h onward. Additionally, Lu et al. reported that the expression levels of BAX and BAK were increased, whereas those of BCL2 were decreased by SPON2 knockdown (31). Based on these results and those reported in the literature, it is possible to induce apoptosis by suppressing SPON2 via the mitochondrial pathway.

Figure 6.
Figure 6.

Schematic diagram illustrating how genes regulate apoptosis with apigenin in A549 cells. Blue arrows and inhibition arrows indicate reaction that occurs in 48 h. The reaction of orange arrows occurs in 24 h.

TNF-a plays a multifaceted role, including cell survival, apoptosis, inflammatory response, and cell differentiation. TNF-a binds as a death ligand to TNF-R1 (14, 15), inducing apoptosis via the caspase-8/caspase-3/-7 signal pathway (15, 16, 18-20). Here, the expression level of TNF increased two-fold within 24 h, and the activity of caspase-8 increased over the same period. Therefore, it is supposed that TNF-a induced early apoptosis by activating the caspase-8/caspase-3/-7 signaling pathway in apigenin-treated A549 cells. Immune cells, such as macrophages, are primarily recognized as producers of sTNF-a; moreover, epithelial cells also possess the capability to produce it (25).

IFN-α2 is a cytokine that binds to the IFNAR1 or IFNAR2 receptor to perform its functions, including antiviral activity, inhibition of cell proliferation, immune modulation, and anticancer effects. Regarding its anticancer effects, IFN-a2 induces apoptosis by binding to the IFNAR2 receptor (33), subsequently activating either the intrinsic or extrinsic inducing apoptosis signaling pathway. The intrinsic pathway is primarily associated with the mitochondria (34, 35, 38), although some reports suggest the involvement of the ER stress pathway (39). Meanwhile, the extrinsic apoptosis signaling pathway, activated by Fas ligand or TNF-a, is also induced by IFN-a2 (40). Real-time RT-qPCR revealed that IFNA2 expression increased after 24 h in apigenin-treated A549 cells. Furthermore, considering that the activity of caspase-8 and caspase-9 also increased in this study, it would be suggested that either or both signaling pathways for apoptosis were activated by IFN-a2.

Figure 6 illustrates the correlation between the timing and the pathways involved in the observed outcomes. Apigenin triggered the activation of caspase-3/-7 in A549 cells, initiating apoptosis from 24 to 72 h. Additionally, upstream of this activation, SPON2 expression was diminished at 48 h, whereas IFNA2 expression was initially elevated at 24 h but then reduced at 48 h. TNF expression escalated at 24 h. The activity of caspase-8, situated downstream of TNF and IFNA2, augmented at 18 and 24 h. In parallel, caspase-9 activity, potentially linked to the down-regulated SPON2 or involved with IFNA2, rose at 24 and 48 h. Moreover, BAX and BAK, deemed upstream of caspase-9 activation, demonstrated enhanced expression at 48 h. These findings indicate that TNF-a, IFN-a2 and spondin-2 may act as factors promoting apoptosis at various intervals in apigenin-treated A549 cells.

Conclusion

Based on the results presented, we propose that TNF-a, IFN-a, and spondin 2 may act as upstream regulators of apoptosis – activating either caspase-8 or caspase-9 at different time points – in apigenin-treated A549 cells.

Footnotes

  • Authors’ Contributions

    R.A. and K.N. carried out majority of the experiments. K.O. performed and analyzed the annexin V assay. R.A. wrote first manuscript. R.A. K.O. and H.H. edited and revised the manuscript. All Authors reviewed the manuscript.

  • Conflicts of Interest

    The Authors declare no competing interests in relation to this study.

  • Received February 17, 2025.
  • Revision received March 24, 2025.
  • Accepted April 2, 2025.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

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Apigenin-induced Apoptosis in Lung Adenocarcinoma A549 Cells: Involvement in IFNA2, TNF, and SPON2 With Different Time Points
RIEKO AIDA, KAZUNORI OKANO, KYOKO NAKATA, HIROMI HAGIWARA
Anticancer Research Jul 2025, 45 (7) 2781-2790; DOI: 10.21873/anticanres.17647
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