The anti-cancer effects of the ethyl acetate (EtOAc) fraction (E-OHS) from the ethanol extract of Opuntia humifusa against A-549 human lung cancer cells were investigated. Selective cytotoxicity was assessed in RAW 264.7 cells and A-549 cells using the MTS assay. After Annexin V-FITC/PI staining, apoptosis induction was confirmed by flow cytometry, and apoptotic nuclear fragmentation was examined using DAPI staining. Furthermore, induction of cell cycle arrest was confirmed by flow cytometry after PI staining, and anti-metastasis was evaluated using wound healing assay. The molecular mechanisms underlying apoptosis, cell cycle arrest, anti-metastasis, and upstream signaling were elucidated using western blot. E-OHS induced anti-cancer activity in a dose-dependent manner in A-549 cells, whereas it did not exhibit cytotoxicity in RAW 264.7 cells. Annexin V-FITC/PI and DAPI assays showed that number of apoptotic bodies increased in a dose-dependent manner. Western blot analysis showed that pro-caspase-9, pro-caspase-3, and PARP decreased, whereas cleaved-caspase-12, Bax, and cleaved PARP increased. This suggests that apoptosis was jointly induced through mitochondrial-mediated intrinsic pathway and endoplasmic reticulum (ER) stress-related pathway. Flow cytometry analysis showed that the distribution of G1/S phase cells increased in the E-OHS-treated group, whereas the distribution of G2/M phase cells decreased, confirming that E-OHS induced cell cycle arrest by inhibiting transition of G1/S phase. Western blot analysis reconfirmed at the molecular level that the cell cycle was arrested in the G1/S phase, as supported by the decrease in CDK2, CDK4, and cyclin A2. Wound-healing assay showed that the migration ability of A-549 cells was inhibited with E-OHS, and western blot analysis confirmed the decrease in integrin β1, MMP-9, and HIF-1α, demonstrating that E-OHS inhibits A-549 cell metastasis. Finally, western blot analysis showed a dose-dependent decrease in ERK1/2, confirming that induction of apoptosis and cell cycle arrest, as well as anti-metastasis, are influenced by upstream signaling pathway.
Lung cancer was the most commonly diagnosed cancer in 2022, with an estimated 2.5 million new cases worldwide, accounting for 1 in 8 cancer cases (12.4% of all cancer cases worldwide). It was followed by female breast cancer (11.6%), colorectal cancer (9.6%), prostate cancer (7.3%), and stomach cancer (4.9%). Lung cancer was also the leading cause of cancer deaths, accounting for an estimated 1.8 million deaths (18.7%). It was followed by colorectal cancer (9.3%), liver cancer (7.8%), female breast cancer (6.9%), and stomach cancer (6.8%) 1. The age-standardized 5-year survival rates for lung cancer were generally low, in the range of 10–20% in both developed and developing countries. In Korean men, lung cancer had the second-lowest 5-year survival rate after pancreatic cancer, and in Korean women, it had the third-lowest survival rate after pancreatic cancer and liver cancer 2. Lung cancer is the leading cause of cancer death in both men and women in the United States, accounting for approximately 20% of all cancer deaths. The overall survival rate is only about 25%, which is significantly lower than other major cancers such as colon cancer (approximately 65%) or breast cancer (approximately 91%). This low survival rate suggests that effective treatment of lung cancer remains a major challenge due to the difficulty of early diagnosis, limited efficacy of existing treatments, and side effects 3. According to the International Agency for Research on Cancer (IARC), among the four major subtypes of lung cancer (adenocarcinoma, squamous cell carcinoma, small cell carcinoma, and large cell carcinoma), adenocarcinoma has become the most common subtype in both men and women. As of 2022, 45.6% of male lung cancer patients were adenocarcinoma, and 59.7% of female lung cancer patients were adenocarcinoma. In 2020, 39.0% of male lung cancer patients and 57.1% of female lung cancer patients were adenocarcinoma. Accordingto IARC, 70% of lung cancer patients in non-smokers were adenocarcinoma 4 5. A-549 cells are a human adenocarcinoma cell line widely used as a representative model of non-small cell lung cancer (NSCLC), which accounts for approximately 85% of all lung cancers. These cells are characterized by an epithelial-like morphology and harbor a well-defined genetic background, including wild type TP53 and oncogenic KRAS mutations 6. Due to their consistent responsiveness to various anti-cancer agents, A-549 cells serve as reliable In Vitro model for evaluating cellular responses such as apoptosis and cell cycle arrest. Therefore, in the present study, A-549 cells were selected as an appropriate model to investigate the therapeutic potential of ethyl acetate (EtOAc) fraction (E-OHS) of ethanol extracts from Opuntia humifusa stem in human lung cancer. O. humifusa is a plant belonging to the Cactaceae family. In Korea, this plant and its fruit are called cheonnyeoncho, and in English, it is called creeping prickly-pear. It is native to the eastern United States, northeastern Mexico, and southeastern Canada, and is currently reported to inhabit southern Europe, South Africa, Australia, and Japan. O. humifusa has anti-oxidant, anti-bacterial, and anti-cancer activities, and research on its various physiological activities is actively being conducted 7 8 9 10 11 However, there is no study on the effect of O. humifusa on lung cancer. Therefore, this study aimed to investigate the anti-cancer effect of the E-OHS obtained from O. humifusa on A-549 human lung cancer cells. The E-OHS contains flavonoids and their derivatives, so it is expected to have anti-oxidant, anti-bacterial, anti-inflammatory, and anti-cancer activities and can be a good candidate for developing an effective anti-cancer agent 12 13. In this study, the E-OHS will be treated to A-549 cells, a human lung cancer cell line, to systematically investigate induction of apoptosis and cell cycle arrest and inhibition of metastasis using MTS assay, flow cytometry, DAPI staining, wound-healing assay, and western blot analysis. The MTS assay measures cell viability by assessing the metabolic activity of living cells. This is achieved by quantifying the absorbance of formazan, which is produced when cellular enzymes reduce the MTS reagent 14 15. Apoptosis is quantitatively evaluated using Annexin V-FITC and Propidium Iodide (PI) staining followed by flow cytometry, which detects changes in phosphatidylserine exposure and membrane integrity 16 17. Cell cycle distribution is also analyzed via flow cytometry after staining nuclear DNA with PI, allowing assessment of cell cycle arrest at specific phase 18. DAPI staining, a method that utilizes a fluorescent dye binding to nuclear DNA, is employed to visualize nuclear condensation and fragmentation associated with apoptosis under a fluorescence microscope 19. The wound-healing assay is performed to evaluate cell migration by monitoring the closure of an artificially introduced gap in a confluent cell monolayer over time 20. Finally, western blot analysis is used to measure the levels of proteins involved in apoptosis, cell cycle arrest, metastasis inhibition, and upstream signaling pathways. Proteins are separated by SDS-PAGE, transferred to a membrane, and probed with specific primary and secondary antibodies 21.
The preparation of the E-OHS from O. humifusa followed the method used in the previous study 22.
2.2. Cell Lines and ReagentsA-549 human lung cancer cells Paraformaldehyde Solution in PBS (GeneAll, Korea) and AntiFade Mounting Medium (with DAPI) (MedChem Express, USA) were used.
2.3. Cell CultureFor culture of A-549 RPMI 1640, FBS, and penicillin were used. For culture of RAW 264.7, DMEM, FBS, and penicillin were used. The A-549 cells were cultured in an incubator at 37℃ and 5% CO2, and the medium composition was RPMI 1640 90%, FBS 10%and penicillin 1%. The A-549 cells were subcultured every week at 1:3 split ratios using 0.05% trypsin, and every 3 days, medium was changed. The cells were treated with 0.1% DMSO or various concentrations of E-OHS derived from O. humifusa. Normal RAW 264.7 cells were cultured in an incubator at 37°C, 5% CO2, and the medium contained 90% DMEM, 10% FBS, and 1% penicillin. The RAW 264.7 cells were subcultured every week at 1:4 split ratios using 0.05% trypsin, and every 2 days, medium was changed. The cells were treated with 0.1% DMSO or various concentrations of E-OHS derived from O. humifusa.
2.4. The Cytotoxic Effect of E-OHS on RAW 264.7 Normal CellsTo evaluate the cytotoxicity of E-OHS against normal cells viability was assessed in RAW 264.7 macrophage cells. A total of 1х105 cells/well were seeded into 96-well plates and incubated for 24 h to allow cell stabilization. The cells were then treated with E-OHS at concentrations 0, 0.1% DMSO, 25, 50, 75, and 100μg/mL (0.1% DMSO as the vehicle control) and further incubated for 12 h. After treatment, 20μL of MTS solution (Cell Titer 96® Aqueous One Solution, [3-(4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl)-2H-tetrazolium, inner salt) to each well. The plate was then incubated for an additional 2 h at 37℃ in a CO2 incubator. Finally, absorbance was measured at 490 nm using the cell imaging multi-mode reader (BioTek, USA).
2.5. The Cytotoxic Effect of E-OHS on A-549 Cancer CellsA total of 1х105 cells/well were seeded into 96-well plates and allowed to adhere for 24 h. The E-OHS was then applied at concentrations of 0, 0.1% DMSO, 25, 50, 75, and 100μg/mL (0.1% DMSO as the vehicle control), and the cells were incubated for 12 h. Subsequently, 20μL of MTS solution (Cell Titer 96® Aqueous One Solution) was added to each well, followed by 2 h incubation at 37℃ in a CO2 incubator. Absorbance was measured at 490 nm using a cell imaging multi-mode plate reader (BioTek, USA).
2.6. Apoptosis AnalysisA-549 cells treated with various concentrations of E-OHS (0, 0.1% DMSO, 50, 75, 100 μg/mL) for 12 h were resuspended using 0.05% trypsin, centrifuged at 5,000 rpm for 5 min using a centrifuge (Hanil, Korea), washed with PBS, and centrifuged again. The supernatant was discarded, and 200 μL of Annexin V binding buffer was added to resuspend the cells. Annexin V-FITC and PI (5 μL each) were added, and the samples were incubated in the dark at room temperature for 15 min. Apoptosis was analyzed using a flow cytometer (NovocyteAdvanteon, Agilent).
2.7. Analysis of Nuclear Changes Using 4', 6-Diamidino-2-phenylindole (DAPI) StainingTo observe the morphological changes of the nucleus, A-549 cells were seeded on sterile coverslips in 6-well plates and treated with various concentrations of E-OHS (0, 0.1% DMSO, 25, 50, 75, 100 μg/mL) for 12 h, and then the supernatant was removed with a suction device (LabTek, Korea). The cells were then washed with PBS and fixed with PBS containing 4% paraformaldehyde for 20 min at room temperature. Finally, the cells were permeabilized with -20°C cold 100% methanol for 20 min and stained with DAPI mounting medium (abcam, United Kingdom). The morphological changes of the nucleus were observed using a cell imaging multi-mode reader (BioTek, USA).
2.8. Cell Cycle AnalysisA-549 cells were treated with various concentrations of E-OHS (0, 0.1% DMSO, 50, 75, 100 μg/mL) for 12 h, then harvested using trypsin 0.05% and washed with cell-based assay buffer. Cells were resuspended at a concentration of 4 × 105 cells/mL, fixed in cold fixative, and incubated at -20°C for at least 2 h. After centrifugation at 5,000 rpm for 5 min using a centrifuge (Hanil, Korea), the fixative was removed and resuspended in staining buffer containing RNase A and PI. The mixture was incubated for 30 min at room temperature in the dark. Cell cycle distribution was analyzed using a flow cytometer.
2.9. Wound-healing AnalysisA-549 cells were seeded into 6-well plates at a density of 5×105 cells/mL and cultured until 90% confluency. After stabilization for 24 h, the medium was replaced with a serum-free medium containing various concentrations of E-OHS (0, 0.1% DMSO, 25, 50, 75, 100μg/mL). A linear scratch was introduced in the center each well using a sterile 200μL pipette tip (0 h). The wound closure was monitored using a cell reader at 0 and 12 h post-treatment. The migration rate was calculated by measuring the change in wound width over time.
2.10. Western Blot AnalysisA-549 cells were treated with E-OHS (0, 0.1% DMSO, 50, 100μg/mL) for 12 h and washed with PBS. Total proteins were extracted on ice using lysis buffer containing RIPA buffer, phosphatase inhibitor, and protease inhibitor. Protein concentrations were determined by the Brad-ford assay (Bio-Rad). Proteins were separated via 12% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked in 5% non-fat milk in TBST and incubated overnight at 4℃ with primary antibodies diluted in either 3% BSA in TBST or 5% non-fat milk in TBST, according to the datasheet. After washing, membranes were incubated with secondary antibody at room temperature for 1 h. Immunoreactive bands was visualized using an WesternBright ECL kit (Advansta, USA).
2.11. Statistical AnalysisThe differences between the control group and the experimental group were evaluated using Student's t-test, and statistical significance was considered as P< 0.05. The results were expressed as the mean ± standard deviation, and statistical significance was considered as *P< 0.05, **P< 0.01, ***P< 0.001.
To evaluate the cytotoxicity of E-OHS, RAW 264.7 macrophages were treated with various concentrations of E-OHS (0, 0.1% DMSO, 25, 50, 75, and 100 μg/mL) for 12 h. As shown in Figure 1, no cytotoxic effects were observed in any E-OHS treatment group compared to the control group. Based on these results, 100 μg/mL was selected as the highest treatment concentration in subsequent experiments.
A-549 cells were treated with various concentrations of E-OHS (0, 0.1% DMSO, 25, 50, 75, and 100 µg/mL) for 12 h, and cytotoxic effects were assessed using MTS assay. As shown in Figure 2, E-OHS decreased A-549 cell viability in a concentration-dependent manner. In particular, E-OHS at concentrations of 75 µg/mL and 100 µg/mL significantly reduced cell viability.
3.3. Flow Cytometric Analysis of E-OHS-induced Apoptosis in A-549 CellsTo further analyze the apoptotic effects of E-OHS, A-549 cells were treated with various concentrations of E-OHS (0, 0.1% DMSO, 50, 75, and 100 μg/mL) for 12 h. The cells were then stained with Annexin V-FITC and PI, followed by flow cytometry. Annexin V binds to phosphatidylserine (PS) exposed in early apoptotic cells, whereas PI identifies late apoptotic or necrotic cells. Based on quadrant analysis (LL: live cells, LU: necrotic cells, RL: early apoptotic cells, and RU: late apoptotic cells) (Figure 3), E-OHS treatment significantly increased early and late apoptosis in a dose-dependent manner. These results indicate that E-OHS effectively induces apoptosis in A-549 cells.
3.4. DAPI staining Analysis of Apoptotic Features in A-549 Cells Treated with E-OHSTo examine nuclear morphological changes in A-549 cells exposed to different concentrations of E-OHS (0, 0.1% DMSO, 50, 75, and 100 μg/mL), DAPI staining was performed. As shown in Figure 4, E-OHS-treated cells exhibited characteristic apoptotic features, including chromatin condensation and apoptotic body formation. The concentration-dependent increase in the intensity and frequency of these features supports the apoptosis-inducing potential of E-OHS.
3.5. Western Blot Analysis of Apoptosis-related Protein Levels in A-549 CellsTo elucidate the apoptosis-related signaling pathway, A-549 cells were treated with different concentrations of E-OHS (0, 0.1% DMSO, 50, and 100 μg/mL) for 12 h, followed by western blot analysis. In E-OHS-treated cells, the levels of pro-caspase-9 and pro-caspase-3 were significantly reduced or eliminated (Figures 5(A) and 5(B)). The level of cleaved caspase-12 increased in a dose-dependent manner (Figure 5(C)). As shown in Figure 5(D), Bax expression increased in a dose-dependent manner. Finally, as shown in Figure 5(E), higher concentrations of E-OHS decreased PARP levels, while C-PARP levels increased. Taken together, these results suggest that E-OHS induces apoptosis in A-549 cells via the mitochondrial intrinsic pathway and ER stress-related pathway.
3.6. Flow Cytometric Analysis of Cell Cycle Arrest in E-OHS-Treated A-549 CellsDNA contents and cell counts were assessed in A-549 cells treated with various concentrations of E-OHS (0, 0.1% DMSO, 50, 75, and 100 μg/mL) using PI staining and flow cytometry. As shown in Figure 6, most cells in the control group (untreated and 0.1% DMSO) were distributed in the G2/M phase, whereas in the E-OHS-treated group, most cells were distributed primarily in the G1/S phase in a dose-dependent manner.
Western blot analysis was performed to elucidate changes in the levels of cell cycle arrest-related mediators in A-549 cells treated with different concentrations of E-OHS (0, 0.1% DMSO, 50, and 100 μg/mL) for 12 h. As shown in Figure 7(A) and Figure 7(B), the levels of CDK2, CDK4, and cyclin A2 decreased in a dose-dependent manner, with a significant decrease observed at 100 μg/mL. CDK2 and CDK4 are protein kinases that play a crucial role in the cell cycle, particularly during the G1/S transition, and cyclin A2, in complex with CDK2, is essential for initiating and regulating DNA synthesis during the S phase. Therefore, these results support the induction of cell cycle arrest at the G1/S phase.
3.8. Anti-metastatic Effect of E-OHS on A-549 Cells Assessed by Wound-healing AssayTo investigate the anti-metastatic potential of E-OHS, a wound healing assay was performed on A-549 cells treated with various concentrations of E-OHS (0, 0.1% DMSO, 25, 50, 75, and 100 μg/mL). As shown in Figure 8(A), migration was evaluated based on the wound healing area at 0 h. After 12 h of treatment, E-OHS significantly inhibited cell migration in a concentration-dependent manner (Figure 8(B)). Compared with the control group, the E-OHS-treated group showed a decreased motility as indicated by decreased wound closure. As shown in Figure 8(C), the open wound area (%) in cells treated with E-OHS was significantly increased in a dose-dependent manner compared with the control group (C). Specifically, wound closure was significantly inhibited at concentrations of 50, 75, and 100 μg/mL of E-OHS, with more than 100% inhibition during the initial culture period. The DMSO control (D) showed a slight increase in open wound area compared to the untreated control, but this effect was significantly increased by E-OHS treatment. These results suggest that E-OHS has a potential anti-metastatic effect by inhibiting the migration ability of A-549 cells.
3.9. Western Blot Analysis of Anti-metastasis-related Protein Levels in A-549 CellsTo further confirm the anti-metastatic activity observed in the wound healing assay, the levels of HIF-1α, integrin β1, and MMP-9 proteins, which are mediators of anti-metastasis, were measured by western blot analysis in A-549 cells treated with different concentrations of E-OHS (0, 0.1% DMSO, 50, and 100 μg/mL) for 12 h. HIF-1α plays a significant role in cancer metastasis by promoting cancer cell migration, survival, and proliferation. Integrin β1 plays a significant role in cancer metastasis by facilitating cancer cell adhesion, invasion, and migration. MMP-9 plays a significant role in cancer metastasis by breaking down the extracellular matrix (ECM) and facilitating cancer cell invasion and migration. As shown in Figures 9(A), 9(B), and 9(C), the concentrations of HIF-1α, integrin β1, and MMP-9 decreased in a dose-dependent manner, particularly at a concentration of 100 μg/mL. Therefore, these results support that E-OHS effectively induces anti-metastasis in A-549 cells.
3.10. Western Blot Analysis of Upstream Signaling-related Protein Levels in A-549 CellsTo investigate the levels of mediators involved in the upstream signaling pathway in A-549 cells treated with different concentrations of E-OHS (0, 0.1% DMSO, 50, and 100 μg/mL) for 12 h, western blot analysis was performed. As shown in Figure 10, ERK1/2 was decreased in a dose-dependent manner in A-549 cells treated with E-OHS. This suggests that the upstream signaling pathway involving ERK1/2 may affect E-OHS-induced apoptosis, cell cycle arrest, and anti-metastatic activity in A-549 cells.
The results of the current study suggest that E OHS possesses a multifaceted anticancer activity capable of contributing to the development of a safe and a natural therapeutic agent. While previous research has investigated the biological activities of O. humifusa, particularly its anti-inflammatory and antibacterial properties, its therapeutic potential in oncology remains insufficiently elucidated 9 10 23 24 25 26 27 28. Recently, E-OHS has been reported to exhibit a biological activity against PANC 1 pancreatic cancer cells 22. The present study expands the field of natural product based anticancer research by demonstrating that E OHS exerts selective cytotoxicity toward A 549 lung cancer cells while sparing normal macrophage cells 29. This study, also, supports the multifaceted anticancer effects of E-OHS by providing a comprehensive analysis that goes beyond simple antiproliferative activity assessment to include molecular mechanism elucidation and metastasis inhibition. Crucially, this study goes beyond simply highlighting the biological activities of O. humifusa and provides a foundation for its development as a candidate anticancer agent by elucidating the molecular pathways through which E-OHS exerts its therapeutic effects.
It is noteworthy that no cytotoxicity was observed in RAW 264.7 macrophages 30 31 32. Cytotoxicity of E-OHS on A-549 cells was analyzed using MTS assay, and cell viability was decreased in a dose-dependent manner. To determine whether the cell death occurring in these cells was apoptosis or necrosis, flow cytometry was used. Results showed that both early and late apoptosis increased in a dose-dependent manner in E-OHS-treated cells, confirming that apoptosis was the cause of cell death. Subsequently, western blot analysis was performed to identify the involved apoptotic pathways. One of the most notable aspects of E-OHS is its ability to induce apoptosis via both the intrinsic mitochondrial pathway and the endoplasmic reticulum (ER) stress-mediated pathway. This pattern has also been confirmed in other studies 33 34 35. The mitochondrial pathway is characterized by the activation of the pro-apoptotic protein Bax and the sequential activation of pro-caspase-9 and pro-caspase-3, which is well known as a classical mechanism of intrinsic apoptosis 36 37 38 39. PARP plays a crucial role in DNA damage detection and repair, and an increase in cleaved PARP is known to inhibit cancer cell proliferation and DNA repair capacity. The decrease in PARP and the increase in cleaved PARP indirectly support the functional role of activated caspase-3 in inducing apoptosis. This pattern has also been reported in other studies 40 41 42 43. Meanwhile, the induction of apoptosis through caspase-12 activation suggests that E-OHS may induce apoptosis under ER stress by exacerbating the unfolded protein response (UPR) or oxidative stress 44 45.
Another important implication of these findings is the effect of E-OHS on cell cycle regulation. G1/S transition inhibition is closely linked to tumor growth suppression. Impaired G1/S transition inhibition is a common feature in many cancers and contributes to uncontrolled cell proliferation 46 47 48 CDK4 and CDK2 play complementary roles in cell cycle regulation. CDK4 controls the early phase (G1) of the cell cycle, while CDK2 controls the late phase. The Cyclin A2-CDK2 complex is involved in the initiation and progression of DNA synthesis 49. The decrease in CDK4, CDK2, and cyclin A2 observed in western blot analysis, coupled with the inhibition of G1/S transition observed in flow cytometry, suggest that cell cycle arrest primarily occurs in the G1/S phase. Meanwhile, other finding obtained by treating A-549 cells with the Orostachys japonicus EtOAc fraction showed that transition inhibition occurs primarily in the G2/M phase, whereas the results of this study showed a distinct difference that transition inhibition occurs primarily in the G1/S phase 50.
Furthermore, wound healing assays confirmed that E-OHS dose-dependently inhibited A-549 cancer cell migration. Subsequently, western blot analysis confirmed that E-OHS dose-dependently reduced metastasis-related mediator proteins, such as HIF-1α, integrin β1, and MMP-9, in A-549 cancer cells. These results complementarily support the anti-metastatic effect of E-OHS in A-549 cancer cells. HIF-1α is a transcription factor strongly induced in a hypoxic tumor microenvironment, and its expression is particularly significant in lung cancer, which has a poor prognosis. HIF-1α is a transcription factor that is strongly induced in a hypoxic tumor microenvironment, and is particularly significant in lung cancer, which has a poor prognosis. HIF-1α acts as a transcription factor that binds to DNA and is involved in various cellular processes such as angiogenesis, metabolism, and cell survival 51 52. Integrin β1, one of the downstream targets of HIF-1α, mediates the interaction between cells and the extracellular matrix (ECM) to promote cancer cell adhesion and invasion. In particular, the β1 subunit combines with various α subunits to form various integrin complexes, which are involved in cell adhesion, migration, and differentiation 53 54. MMP-9 is an ECM-degrading enzyme that specifically targets collagen IV, disrupting the basement membrane and ECM structure, thereby promoting invasion, metastasis, tumor growth, and angiogenesis. MMP-9 activity is closely related to the progression of various malignancies, and inhibition of this signaling axis can effectively limit tumor progression by attenuating cell motility, angiogenesis, and invasion 55 56.
Additionally, when western blot analysis was performed to identify the upstream signaling pathway, E-OHS dose-dependently decreased ERK1/2 protein in cancer cells. ERK1/2 is an important member of the MAPK family and is activated by the Ras-Raf-MEK-ERK signaling pathway, which is widely involved in cell proliferation, differentiation, survival, apoptosis, cell cycle, and metastasis 57 58 59. Therefore, the results of the present study suggest that E-OHS can suppress the proliferation and survival of A-549 lung cancer cells while inducing apoptosis, cell cycle arrest, and metastasis inhibition by controlling the expression of ERK1/2, an upstream signaling mediator.
Collectively, these findings show that E-OHS exerts anti-cancer effects through three complementary mechanisms: induction of apoptosis, cell cycle arrest, and metastasis inhibition in A-549 lung cancer cells.
In conclusion, this study demonstrated that E-OHS derived from O. humifusa exhibited significant anti-cancer effects on A-549 human lung cancer cells, while not exhibiting cytotoxicity in normal RAW 264.7 macrophages. Specifically, E-OHS induced apoptosis via the mitochondrial intrinsic pathway and ER stress-mediated pathways, as evidenced by upregulation of Bax and cleaved caspase-12, downregulation of pro-caspase-9 and pro-caspase-3, and increased cleaved PARP. Furthermore, E-OHS was demonstrated to induce cell cycle arrest by downregulating the expression of CDK4, CDK2, and cyclin A2, thereby inhibiting the G1/S phase transition and subsequent S phase progression. Furthermore, E-OHS was demonstrated to induce anti-metastasis by downregulating the expression of pro-metastatic signaling mediators including HIF-1α, integrin β1, and MMP-9. Finally, it was, also, demonstrated that E-OHS could induce apoptosis, cell cycle arrest, and metastasis inhibition by downregulating the expression of ERK1/2, an upstream signaling mediator. Collectively, this study suggests that E-OHS has the potential as a natural agent with anti-lung cancer activity.
Authors declare no conflict of interest.
EJY was involved in designing the work, data collection, data analysis, interpretation, and drafting the article. MCC was involved in data analysis and interpretation. DSL was involved in designing the work, data analysis, interpretation, and drafting the article.
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| In article | View Article | ||
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Published with license by Science and Education Publishing, Copyright © 2025 Eon Ji Yeo, Myung Chul Cha and Dong Seok Lee
This work is licensed under a Creative Commons Attribution 4.0 International License. To view a copy of this license, visit
http://creativecommons.org/licenses/by/4.0/
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| In article | View Article | ||
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| In article | View Article | ||
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| In article | View Article | ||
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| In article | View Article | ||
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| In article | View Article | ||
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| In article | View Article | ||
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| In article | View Article PubMed | ||
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| In article | View Article | ||
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| In article | View Article PubMed | ||
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| In article | View Article PubMed | ||
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| In article | View Article | ||
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| In article | View Article PubMed | ||
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| In article | View Article PubMed | ||
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| In article | View Article PubMed | ||
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| In article | View Article PubMed | ||
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| In article | View Article PubMed | ||
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| In article | View Article PubMed | ||
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| In article | View Article PubMed | ||
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| In article | View Article PubMed | ||
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| In article | View Article PubMed | ||
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| In article | View Article PubMed | ||
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| In article | View Article PubMed | ||
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| In article | View Article PubMed | ||
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| In article | View Article | ||
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| In article | View Article PubMed | ||
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