The human LUAD cell line A549 (https://www.cellosaurus.org/CVCL_0023; cat. no. CCL-185; American Type Culture Collection) was cultured in RPMI-1640 (cat. no. 11875093; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS) (cat. no. C04001; Shanghai VivaCell Biosciences, Ltd.) and 1% penicillin-streptomycin at 37°C in a humidified incubator with 5% CO₂. The cells were passaged every two days.

A549 cells (1.5×10⁴ cells/well) were seeded into a 96-well plate and incubated in 2% FBS-RPMI-1640 containing activin A (0–40 ng/ml) (cat. no. 338-AC; R&D Systems, Inc.) for 24 and 48 h, respectively. Next, 10 µl of CCK-8 reagent (cat. no. GK10001; GLPBIO) was added to the culture medium of each well, and the cells were incubated for 2 h at 37°C in 5% CO₂. The absorbance values (A value) were obtained at the wavelengths of 450 and 650 nm. Each experiment was performed in triplicate compound wells and repeated three times. The percentage (%) of cell viability was calculated as follows: (%)=(sample well A value/control well A value) ×100%.

RTCA is a powerful technology for detecting cell fate (15). The RTCA instrument (xCELLigence RTCA S16; ACEA Bioscience, Inc.; Agilent) was used to analyze the proliferation property of A549 cells. The experimental method was as previously described (15). In brief, 50 µl of cell-free culture medium (2% FBS-RPMI-1640) was added to the well of an E16 ×CELLigence microtiter plate to test the background impedance of each well for 1 min. Subsequently, A549 cells (1×10⁴ cells in 50 µl culture medium) were added to the plate and cultured at 37°C in 5% CO₂ for ~2 h, and then treated with different concentrations of activin A (0–40 ng/ml) for 48 h. Cells were monitored at 15 min intervals. Each experiment was set up with double compound holes and repeated thrice. The cell index indirectly represented cell activity.

A549 cells (1.5×10⁴ cells/well) were seeded into a 96-well plate and incubated in 2% FBS-RPMI-1640 containing activin A (0–40 ng/ml) for 24 h. In accordance with a cell proliferation ELISA protocol (cat. no. 11647229001; Merck KGaA), 10 µl of BrdU labeling reagent was added, and incubation was continued for 2 h. After discarding the supernatants, 200 µl/well of FixDenat reagent was added for 30 min to fix the cells and denature the DNA. After discarding the supernatants, 100 µl/well of anti-BrdU-POD was added and followed by incubation for 90 min. After discarding the supernatants and washing, 100 µl/well of substrate solution was added and incubated for 30 min. The absorbance values (A value) were obtained at the wavelengths of 370 and 492 nm. Each experiment was performed in triplicate compound wells. The percentage (%) of cell proliferation was calculated as follows: (%)=(sample well A value/control well A value) ×100%.

A549 cells (1.5×10⁴ cells/well) were seeded into a 96-well plate and cultured in 2% FBS-RPMI-1640 culture medium containing activin A (0–40 ng/ml). Cells were then fixed with 4% paraformaldehyde for 20 min and stained with 10 µg/ml Hoechst 33342 solution (cat. no. 14533; Sigma-Aldrich; Merck KGaA) in the dark for 10 min at room temperature and observed under an inverted fluorescence microscope (IX71; Olympus Corporation). Experiments were repeated three times.

The experiment was performed as previously described (6). A549 cells (1×10⁵ cells/well) were seeded in a 12-well plate and cultured in 2% FBS-RPMI-1640 culture medium containing activin A (0–40 ng/ml) for 24 h. Thereafter, the cells were collected and suspended in 100 µl of 1X Annexin V binding buffer. Next, 1 µl of Annexin V-YF^®488 and 1 µl of PI (cat. no. Y6002; Suzhou Yuheng Biotechnology Co., Ltd.; www.uebio.com) were used to mark the cells for 15 min in the dark. Finally, the labeled cells were analyzed using flow cytometry (guava easyCyte HT; Cytek Biosciences). FlowJo (FlowJo_v10.6.2; FlowJo LLC) was used to collect and analyze the data to obtain the percentage of fluorescent cells. The experiments were repeated three times. BAPTA-AM (2.5 µM; cat. no. HY-100545), calcium agonist ionomycin (500 nm; cat. no. HY-13434; both from MedChemExpress) and ERK inhibitor FR180204 (10 µM; cat. no. abs810541; Absin Bioscience, Inc.) were also used before assessment of cell apoptosis.

A549 cells were incubated in 2% FBS-RPMI-1640 medium with 4 µmol/l Fluo-4 (cat. no. F14201; Thermo Fisher Scientific, Inc.) and were protected from light for 40 min at 37°C. The cells were then washed twice with 2% FBS-RPMI-1640 medium and seeded into a 24-well plate. Calcium fluorescence signaling was detected using a BioTek Cytation5 cell imager multimode reader (Agilent Technologies, Inc.) after the cells were adherent. The baseline fluorescence signal (F₀) was recorded every 0.25 sec for a total of 3 sec. Next, the cells were treated with 2% FBS-RPMI-1640 and activin A 20 ng/ml, respectively, and the fluorescence signal (F) was immediately detected on the machine for another 3 sec. The experimental data were exported from BioTek Cytation5 software, and the normalized fluorescence intensity was represented by F/F₀. Experiments were repeated three times.

The experiment was performed as previously described (16). Total RNA from A549 cells was extracted using RNAiso Plus reagent (cat. no. 9108; Takara Biomedical Technology Co., Ltd.) and the cDNA synthesis kit (cat. no. CW2020M; CoWin Biosciences) was used to complete the reverse transcription according to the manufacturer's instructions. PCR was performed using 2X Es Taq MasterMix (cat. no. CW0690H; CoWin Biosciences). The qPCR thermocycling conditions were as follows: 95°C for 1.5 min, 94°C for 0.5 min, 56°C for 0.5 min and 72°C for 1 min for 30 cycles, with a final 10-min extension step at 72°C. Finally, 2% agarose gel electrophoresis stained with Super Gelred (cat. no. S2001; Suzhou Yuheng Biotechnology Co., Ltd.; www.uebio.com.) was used to separate PCR products. The PCR bands were visualized using ImageMaster VDS. ImageJ software (version 1.53e; National Institutes of Health) was used to digitalize the bands. Primer sequences are included in Table I.

The experiment was performed as previously described (16). Briefly, A549 cells were lysed using protein extraction reagent (cat. no. 78503; Thermo Fisher Scientific, Inc.) containing protease & phosphatase inhibitor cocktail (cat. no. 87785; Thermo Fisher Scientific, Inc.) and 5 mmol/l EDTA solution (cat. no. R1021; Thermo Scientific, Inc.). According to the instructions, the BCA protein assay kit (cat. no. 23227; Thermo Fisher Scientific, Inc.) was applied to quantify the proteins, which were obtained from centrifugation of 12,000 g at 4°C for 20 min. Then, 10% SDS-PAGE gel electrophoresis was performed to separate 30 µg of protein. After electrophoresis, the product was transferred onto polyvinylidene difluoride membrane (cat. no. IPVH00010; Sigma-Aldrich; Merck KGaA). Next, the membranes were blocked with 5% skim milk in TBS-Tween 20 for 2 h at room temperature (RT), and then incubated with specific primary antibodies overnight at 4°C. The corresponding horseradish peroxidase-conjugated secondary antibodies (1:160,000; Goat anti-mouse IgG for CHOP antibody, cat. no. A3682; Goat anti-rabbit IgG for other antibodies, cat. no. A0545; all from Merck KGaA) were further incubated for 2 h at RT. Finally, the bands attached to the membrane were visualized using an ECL luminescence reagent (cat. no. BL520; Biosharp Life Sciences) using a Tanon 4600 chemiluminescent imaging system. The immunoblots were quantified using ImageJ software, and the levels of proteins were normalized against GAPDH. The following antibodies were used for western blotting: ActRIIA (1:1,000; cat. no. abs149480), GAPDH (1:10,000; cat. no. abs132004; both from Absin Bioscience, Inc.), GADD34 (1:1,000; cat. no. ab131402; Abcam), CHOP (1:1,000; cat. no. 2895), cleaved-caspase3 (1:1,000; cat. no. 9661), ERK1/2 (1:1,000; cat. no. 4695), phosphorylated (p)-ERK1/2 (1:2,000; cat. no. 4370), p-Smad3 (1:1,000; cat. no. 9520), Smad3 (1:1,000; cat. no. 9523; all from Cell Signaling Technology, Inc.), caspase-12 (1:1,000; cat. no. WL03268), JNK (1:1,000; cat. no. WL01295) and p-JNK (1:1,000; cat. no. WL01813; all from Wanleibio Co., Ltd.).

The data of The Cancer Genome Atlas (TCGA)-LUAD were obtained from Genomic Data Commons Data Portal (https://portal.gdc.cancer.gov/projects/TCGA-LUAD). GBM R package (https://www.rdocumentation.org/packages/gbm/versions/2.1.8.1) was used to analyze the data. Cox proportional hazard regression model was used to analyze and determine independent risk factors, and estimate the hazard ratio (HR) and 95% confidence interval.

RNA-sequencing expression profiles and corresponding clinical information for TCGA-LUAD were downloaded from the TCGA dataset. GSE116959 profiles were obtained from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE116959). The GSE116959 dataset was generated using GPL17077 Agilent-039494 SurePrint G3 Human GE v2 8×60K Microarray 039381 platform and included 57 LUAD samples and 11 peritumoral normal lung tissues. The raw data were downloaded as MINiML files and preprocessed via background adjustment, normalization. Limma package (versions 3.18; Bioconductor–limma) was used to analyze the data.

All data are presented as the mean ± standard deviation. Statistical analysis was conducted using SPSS software (version 26; IBM Corp.). Comparisons were performed using unpaired Student's t-test or one-way analysis of variance (ANOVA), followed by the least significant difference (LSD) test. P<0.05 was considered to indicate a statistically significant difference.

To confirm the clinical value of activin signaling activation in LUAD, LUAD data were first obtained from TCGA database and the K-M survival curves of ActRIIA and its downstream signaling proteins in LUAD were analyzed. Although the association did not reach the level of statistical significance, it was found that high expression of ActRIIA was positively associated with the survival of patients with LUAD, which was consistent with the positive effects of mitogen-activated protein kinase 1 (MAPK1; ERK2) and MAPK3 (ERK1) on their survival. By contrast, high expression of Smad3 (HR=1.28 >1) was negatively associated with their survival (Fig. 1A-D). Moreover, when the gene expression profiles of 11 non-tumor samples and 57 LUAD tissue samples from the GEO Series GSE116959 database for the identification of differentially expressed genes were examined, we found significant differences between the LUAD and non-tumor samples (Fig. 1E). In addition, the results of microarray-based data analysis revealed that MAPK1 and MAPK3 were negatively associated with LUAD (Fig. 1F and G), suggesting that MAPK1 and MAPK3 might be signaling molecules positively associated with the survival of patients with lung cancer. These results indicated that ActRIIA-ERK1/2 signaling may have inhibitory effects on LUAD cells.

The results of CCK-8 assay indicated that the viability of A549 cells decreased significantly after stimulation with 20 and 40 ng/ml of activin A for 24 and 48 h, respectively (Fig. 2A). RTCA was then performed, a non-invasive and effective tool for real-time dynamic monitoring of cell growth that is widely used for measuring cell proliferation and adhesion (15,17), to assess the proliferation of A549 cells. The results also demonstrated that activin A significantly inhibited the proliferation of A549 cells (Fig. 2B and C), which was consistent with the observed decrease in cell viability. The proliferation of A549 cells was further evaluated by BrdU incorporation. The results revealed that the proliferation of A549 cells decreased significantly after treatment with activin A, compared with that in the control group (Fig. 2D). These results indicated that activin A can suppress A549 cell proliferation.

To ascertain whether activin A inhibits A549 cell proliferation through apoptosis, Hoechst 33342 staining was performed to observe alterations in cellular morphology. The results showed that the number of apoptotic cells in the activin A-stimulated group increased obviously compared with the control group (Fig. 3A). Flow cytometry was then used to detect apoptotic ratio of A549 cells. The results revealed that, compared with the control medium, the apoptotic rate of A549 cells stimulated with 20 ng/ml and 40 ng/ml of activin A increased significantly (Fig. 3B), suggesting that activin A can induce A549 cell apoptosis.

To explore whether activin A mediates apoptosis of A549 cells via the ER stress pathway, western blotting was performed to detect the expression of proteins associated with the ER stress pathway in A549 cells. The results revealed that the levels of GADD34, CHOP, caspase-12 and cleaved-caspase-3 proteins in A549 cells were significantly upregulated by activin A (Fig. 4), indicating that activin A may induce apoptosis of A549 cells through the ER stress pathway.

Calcium signaling is associated with various biological behaviors such as cell proliferation, differentiation and migration (18,19), and exerts a crucial impact on both ER stress-induced apoptosis and mitochondria-mediated apoptosis (20). When the calcium fluorescence probe Fluo-4 AM was used to measure intracellular calcium levels in A549 cells, it was identified that the fluorescence signal intensity of calcium in A549 cells was significantly increased by activin A treatment (Fig. 5A and B). To clarify whether calcium signaling has a role in the activin A-induced cell apoptosis process, A549 cells were treated with intracellular calcium ion chelator BAPTA-AM and calcium agonist ionomycin, before assessing cell apoptosis by flow cytometry. The present results revealed that BAPTA-AM reversed activin A-mediated apoptosis of A549 cells, whereas ionomycin increased the rate of apoptosis induced by activin A (Fig. 5C). These findings indicated that activin A induces apoptosis of A549 cells by promoting cellular calcium influx.

Activin A can regulate cell activity through the canonical Smad3 dependent or non-Smad3 signaling pathways. Initially, RT-qPCR was performed to determine the levels of activin receptor and Smad3 mRNA expression in A549 cells. Whereas ActRIA, ActRIB, ActRIIB and Smad3 mRNA levels in A549 cells did not significantly change with activin A treatment, ActRIIA mRNA level significantly increased (Fig. 6A). Similarly, activin A significantly upregulated ActRIIA protein levels in A549 cells (Fig. 6B). The results of western blotting indicated that activin A did neither significantly affect the p-Smad3 and p-JNK protein levels, nor the p-JNK/JNK or p-Smad3/Smad3 ratio in A549 cells. However, the p-ERK1/2 protein level and p-ERK/ERK ratio were significantly elevated by activin A (Fig. 6C). The aforementioned findings indicated that activin A may regulate ER stress pathway-triggered A549 cell apoptosis via the Smad-independent ERK1/2 signaling.

To confirm that ERK1/2 is involved in the ER stress pathway-mediated apoptosis of A549 cells triggered by activin A, the ERK inhibitor FR180204 was used to pretreat A549 cells for further experiments. It was found that administration of FR180204 resulted in a significant reduction in the p-ERK protein level and the p-ERK/ERK ratio in activin A-treated cells (Fig. 7A) as well as in the apoptotic rate of activin A-induced A549 cells (Fig. 7B). In the next experiment, it was identified that FR180204 significantly downregulated caspase-12 and CHOP protein levels in activin A-treated A549 cells (Fig. 7C). These findings indicated that the ERK signaling pathway participates in activin A-induced A549 cell apoptosis through the ER stress pathway.