Research ArticleExperimental Studies

Magnolol Induces Apoptosis Through Extrinsic/intrinsic Pathways and Attenuates NF-κB/STAT3 Signaling in Non-small-cell Lung Cancer Cells

YANG-CHENG LEE, YUEH-SHAN WENG, HSIAO-YU WANG, FEI-TING HSU, FU-SHIN CHUEH, JENG-YUAN WU, WEI-LUNG CHEN and JIANN-HWA CHEN
Anticancer Research August 2022, 42 (8) 3825-3833; DOI: https://doi.org/10.21873/anticanres.15873

Abstract

Background/Aim: Non-small-cell lung cancer (NSCLC) is the most common type of lung cancer worldwide, and treatment outcomes are still poor. Magnolol, a hydroxylated biphenyl isolated from Magnolia officinalis, was found to be effective against hepatocellular carcinoma via inactivating nuclear-factor-kappa B (NF-Embedded ImageB) signaling. However, whether magnolol targets not only NF-Embedded ImageB but also other factors in NSCLC and may contribute to the suppression of tumor progression is unclear. Materials and Methods: Cell viability, flow cytometry, and western blotting assays were used to identify the mechanism of magnolol action in human lung adenocarcinoma cell lines A549 and CL1-5-F4. Results: Our results indicated that magnolol induced cytotoxicity through extrinsic/intrinsic apoptosis signaling and suppressed phosphorylation of signal transducer and activator of transcription 3 (STAT3)/NF-Embedded ImageB and expression of their downstream proteins. Conclusion: Magnolol not only induced extrinsic and intrinsic apoptosis signaling but also inactivated STAT3/NF-Embedded ImageB and attenuated their signaling of epithelial–mesenchymal transition and metastasis-related protein expression in NSCLC.

Key Words:

Several transcription factors drive tumor formation and progression through induction of downstream oncogene expression. Signal transducer and activator of transcription 3 (STAT3) and nuclear-factor-kappa B (NF-Embedded ImageB) are transcription factors of numerous oncogenes and are crucial mediators in multiple oncogenic processes such, as tumor growth, survival, angiogenesis, epithelial–mesenchymal transition and metastasis (1-5). Radiotherapy, chemotherapy and targeted therapy are used for treatment of non-small-cell lung cancer (NSCLC) (6, 7). As demonstrated in cell and animal models, constitutive activation of STAT3 and NF-Embedded ImageB reduces the anticancer effect of radiation, cisplatin and gefitinib (an epidermal growth factor receptor inhibitor) on NSCLC. Inhibition of STAT3 and NF-Embedded ImageB signaling promotes treatment efficacy in NSCLC (8-11). Development of STAT3 and NF-Embedded ImageB signaling inhibitors may be crucial to increase therapeutic benefits for patients with NSCLC.

Regorafenib, a multiple tyrosine kinase inhibitor, has been shown to mediate regression of NSCLC and enhance chemosensitivity of NSCLC to cisplatin due to its suppression of NF-Embedded ImageB signaling (12, 13). In addition, many bioactive compounds isolated from plants exert an anti-NSCLC effect by blocking STAT3 and NF-Embedded ImageB signaling (8, 14). For instance, sanguinarine (a benzo phenanthridine alkaloid extracted from Sanguinaria canadensis and Fumaria species) and galangin (an active bioflavonoid compound derived from the root of Alpinia galangal) have been shown to trigger inhibition of NSCLC cell growth through STAT3 inactivation (15) and sensitizing NSCLC to cisplatin by suppression of the NF-Embedded ImageB pathway (16), respectively.

In addition to inhibition of transcription factors, effective induction of apoptosis is also associated with tumor suppression. Anticancer agents trigger cellular stresses, such as death ligand–receptor interaction, DNA damage, endoplasmic reticulum stress and mitochondrial dysfunction to mediate extrinsic and intrinsic apoptosis (17). Magnolol, a polyphenolic compound found in the plant Magnolia officinalis, has been shown to induce apoptosis and cell growth inhibition in NSCLC through the initiation of the caspase-independent mitochondrial pathway and inhibition of microtubule polymerization (18-20). However, the precise mechanism of action of magnolol in its effects on apoptosis and STAT3/NF-Embedded ImageB signaling in NSCLC cells has not been fully elucidated. Therefore, the main goal of present study was to verify whether caspase-mediated apoptosis and suppression of STAT3/NF-Embedded ImageB signaling are involved in the anti-NSCLC effect of magnolol.

Materials and Methods

Cell lines. To carefully confirm the effects of magnolol, we used two human lung adenocarcinoma cell lines in this study. Human lung adenocarcinoma cell line, A549, and CL1-5-F4, derived from metastatic lung tumor, were maintained in Dulbecco’s modified Eagle’s medium/F-12 and F12-K medium (Thermo Fisher Scientific, Fremont, CA, USA) with 10% fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin and 100 μg/ml streptomycin (Thermo Fisher Scientific) in an incubator at 37°C with 5% CO2 and 95% humidity (21, 22).

Reagents and antibodies. The chemical reagents were all purchased from Sigma (St. Louis, MO, USA) and were as follows: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), magnolol, dimethyl sulfoxide. 3,3’-dihexyloxacarbocyanine iodide (DiOC6) was purchased from Enzo Life Sciences (Farmingdale, New York, NY, USA). Primary antibodies against NF-Embedded ImageB, matrix metallopeptidase 9 (MMP9), cyclin D1, urokinase-type plasminogen activator (uPA), and β-actin were obtained from Elabsicence (Houston, TX, USA). Antibodies recognizing MMP2 were purchased from Proteintech (Rosemont, IL, USA),and those against NF-Embedded ImageB ser536, vascular endothelial growth factor A (VEGFA), STAT3, STAT3 Tyr705, N-cadherin, zinc finger protein SNAI1 (SNAIL1), zinc finger protein SNAI2 (SLUG), TWIST and zinc finger E-box binding homeobox 1 (ZEB1) were from Cell Signaling (Danvers, MA, USA).

Cell viability (MTT assay). A549 and CL1-5-F4 cells were seeded in 96-well plates, at a density of 5×103 cells/well overnight and then treated with 10, 25, 50, 75 or 100 μM magnolol for 48 h. The medium was replaced by 100 μl MTT reagent (dissolved with medium at a final concentration of 0.5 mg/ml) for 2 h. Then the MTT medium was removed and replaced with 100 μl dimethyl sulfoxide. The absorbance was detected by a Multiskan FC microplate reader (Thermo Fisher Scientific) at 570 nm (21). Viability was represented by the average OD value of each group expressed as a percentage of that of the untreated control.

Analysis of cleaved caspase-3, -8 and -9. A549 and CL1-5-F4 cells were plated in 6-well plates, at a density of 1×105 cells/well overnight and treated with 50 or 75 μM magnolol for 48 h. A549 and CL1-5-F4 cells were then stained with CaspGLOW™ fluorescein staining kit (BioVision, Milpitas, CA, USA) for detection of cleaved caspase-3, caspase-8 and caspase-9 for 30 min at 37°C. The activation of cleaved caspase-3 and caspase-9 after treatment were analyzed by the FL-1 channel and that of cleaved caspase-8 was determined by the FL-2 channel using a NovoCyte flow cytometer with NovoExpress® software (Agilent Technologies Inc.) and FlowJo (version 7.6.1) software for quantification (23).

Analysis of Fas cell surface death receptor (FAS) and Fas cell surface death receptor ligand (FASL). A549 and CL1-5-F4 cells were plated in 6-well plates at a density of 1×105 cells/well overnight and treated with 50 or 75 μM magnolol for 48 h. After treatment, cells were collected and stained with fluorescein isothiocyanate-labeled anti-FAS (BioLegend, San Diego, CA, USA) or phycoerythrin-labeled anti-FASL (BioLegend) in 100 μl binding buffer and incubated for 40 min in the dark at room temperature. FAS and FASL were detected by the FL-1 and FL-2 channels, respectively, using NovoCyte flow cytometer with NovoExpress® software and FlowJo 7.6.1 software for quantification (22, 23).

Analysis of mitochondria membrane potential (ΔΨm). A549 and CL1-5-F4 cells were plated in 6-well plates at a density of 1×105 cells/well overnight and treated with 50 or 75 μM magnolol for 48 h. Cells were collected and stained with 4 μM DiOC6 in 500 μl phosphate-buffered saline for 30 min at 37°C. The DiOC6 signal was detected by the FL-1 channel using NovoCyte flow cytometry with NovoExpress® software and FlowJo 7.6.1 software for quantification (23).

Apoptosis determination by annexin-V/propidium iodide (PI) staining. A549 and CL1-5-F4 cells were plated in 6-well plates at a density of 1×105 cells/well overnight then treated with 50 or 75 μM magnolol for 48 h. FITC Annexin-V Apoptosis Detection Kit I (BD Pharmingen, San Diego, CA, USA) was then used for detection of apoptosis of A549 and CL1-5-F4 cells. After harvesting, cells were double stained with 1 μl annexin-V-fluorescein isothiocyanate and 2 μl PI solution in 100 μl binding buffer for 15 min at room temperature. Annexin V was determined by the FL-1 channel and PI was determined by the FL-2 channel using NovoCyte flow cytometry with NovoExpress® software (Agilent Technologies Inc-) and FlowJo software (version 7.6.1) for quantification. The extent of early apoptosis and late apoptosis were measured from the percentages of annexin V+/PI– and annexin V+/PI+ cells, respectively.

Invasion and migration assay. A549 and CL1-5-F4 cells were plated in 6-well plates, at a density of 1×105 cells/well overnight and treated with 50 or 75 μM magnolol for 48 h. Then the upper channel of a transwell insert was seeded with 5×104 cells and migration and invasion were allowed for another 24 h. For the invasion assay, Transwell inserts were coated with Matrigel 1 day before cells were seeded. Finally, transwell were fixed and stained as previously described (21, 24). The invasion and area were quantified by Image J (National Institutes of Health, Bethesda, MD, USA) and expressed as a percentage of that of the treatment group.

Western blotting assay. A549 and CL1-5-F4 cells were placed in a 10-cm plate at 1×106 cells overnight and then treated with 50 or 75 μM magnolol for 48 h. After treatment, cells were collected and total proteins were extracted by NP-40 lysis buffer containing proteinase inhibitor cocktail and phosphatase inhibitor (Sigma-Aldrich). The protein concentration was measured by the Bradford method (Bio-Rad Laboratories, Hercules, CA, USA). In total, 50 μg protein extract was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (EMD Millipore, Bedford, MA, USA). Membranes were then blocked with blocking buffer (5% non-fat dry milk) and hybridized with different primary antibodies mentioned above and revealed with horseradish peroxidase-conjugated secondary antibody. Chemiluminescence images were detected by a chemiluminescent image system (MultiGel-21, Taipei, Taiwan) with TOPBIO Capture software (Taipei, Taiwan). The intensity of each specific band was normalized with that of the housekeeping protein (β-actin) and then divided by the average value of the 0 μM magnolol control.

Statistical analysis. Results are all expressed as the mean±standard deviation. Statistical significance was measured by one-way analysis of variance. All analyses were performed with GraphPad Prism 7.0 (GraphPad Software Inc., San Diego, CA, USA). Significant difference was defined as a value of p<0.05 between treated and non-treated cells. All experiments were repeated three times independently.

Results

Magnolol markedly induced cytotoxicity and apoptosis of NSCLC cells. As indicated in Figure 1A, the cytotoxicity of magnolol towards both A549 and CL1-5-F4 cells increased with increasing dose. Here, we selected 50 and 75 μM magnolol for further examination, which were estimated to be the 40% and 60% inhibitory concentrations, respectively. Next, we checked whether magnolol-induced cytotoxicity involves the apoptosis of NSCLC cells. Figure 1B shows the annexin-V/PI staining pattern and quantification results of treatment without and with magnolol, and shows apoptosis induction with increasing dose of magnolol. Early apoptosis, represented by annexin V+/PI– cells, was increased by magnolol. Additionally, the expression of cleaved caspase-3 was also increased by magnolol treatment (Figure 1C). Almost 20-25% of cleaved caspase-3 activation, was found in cells treated with 75 μM magnolol. These results indicate that magnolol may induce cytotoxicity of NSCLC cells in association with the activation of the caspase-dependent apoptosis pathway.

Figure 1.
Figure 1.

Cytotoxicity and apoptosis of non-small-cell lung cancer cells was induced by magnolol. A: A549 and CL1-5-F4 cells were treated with 0-100 μM magnolol and assayed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. B: A549 and CL1-5-F4 cells were treated with magnolol and then assayed using annexin-V/propidium iodide (PI) staining in flow cytometry (left) for apoptosis. Results were then quantified (right). C: A549 and CL1-5-F4 cells were treated with magnolol and then assayed cytometrically by staining for cleaved caspase-3 (left). Results were then quantified (right). Significantly different at: *p<0.05, ** p<0.01 and ***p<0.001 vs. 0 μM magnolol; $p<0.05, $$p<0.01 and $$$p<0.001 vs. 50 μM magnolol.

Magnolol induced extrinsic and intrinsic apoptosis pathways in NSCLC cells. After confirming the apoptosis-inducing effect of magnolol, we investigated the mechanism of apoptosis more specifically. Here, we first identified whether death receptor dependent apoptosis was affected by magnolol. As illustrated in Figure 2A, the expression of FAS was increased to 20-30% by magnolol in both A549 and CL1-5-F4 cells as compared to non-treated control. In addition, FASL also displayed a similar, dose-dependent pattern of induction by magnolol in the two NSCLC cell lines (Figure 2B).

Figure 2.
Figure 2.

Magnolol induced extrinsic and intrinsic apoptosis pathways in NSCLC cells. A549 and CL1-5-F4 cells were treated with 0, 50, 75 μM magnolol and assayed by for Fas cell surface death receptor (FAS) (A), Fas cell surface death receptor ligand (FASL) (B) cleaved caspase-8 (C), change in mitochondrial membrane potential (Ψm) (D) and cleaved caspase-9 (E) Representative cytograms are shown on the left, with quantification on the right. Significantly different at: *p<0.05, **p<0.01 and ***p<0.001 vs. 0 μM magnolol; $p<0.05, $$p<0.01 and $$$p<0.001 vs. 50 μM magnolol.

We then assessed an important indicator of death receptor-dependent apoptosis, caspase-8. The cleavage of caspase-8 was also increased by magnolol (Figure 2C), indicating activation of the extrinsic apoptosis pathway. We also investigated whether the mitochondria-dependent intrinsic apoptosis pathway is triggered by magnolol. In Figure 2D, loss of ΔΨm was found to be increased by magnolol in both A549 and CL1-5-F4 cells. Magnolol also induced the cleavage of caspase-9, an indicator of mitochondria-dependent apoptosis (Figure 2E).

These results indicate that magnolol-induced apoptosis is associated with the activation of both extrinsic and intrinsic apoptosis pathways.

Magnolol reduced NSCLC cell invasion/migration, epithelial–mesenchymal transition (EMT) and expression of metastasis-related proteins. To identify whether magnolol may affect tumor invasion and migration potential, we used transwell assays to investigate alterations in cell behavior after magnolol treatment. The number of invading cells was markedly reduced in both A549 and CL1-5-F4 cells by magnolol treatment (Figure 3A, left). In addition, migration was also suppressed by magnolol in a dose-dependent manner (Figure 3A, right).

Figure 3.
Figure 3.

Magnolol suppressed invasion and migration ability of non-small-cell lung cancer cells, and expression of epithelial–mesenchymal transition- and metastasis-related proteins of NSCLC cells through suppression of nuclear factor kappa B (NF-Embedded ImageB) and signal transducer and activator of transcription 3 (STAT3) signaling. A: A549 and CL1-5-F4 cells were treated with 0, 50, 75 μM magnolol to examine migration and invasion in Transwell assays. Representative images are shown on the left, with quantification on the right. B: Expression of NF-Embedded ImageB, STAT3, N-cadherin, zinc finger E-box binding homeobox 1 (ZEB1), zinc finger protein SNAI1 (SNAIL1), zinc finger protein SNAI2 (SLUG), TWIST, cyclin D1, matrix metalloproteinase-2 (MMP2), MMP2, vascular endothelial growth factor A (VEGFA) and urokinase-type plasminogen activator (uPA) was defined by western blotting assay. Relative quantification data are means from three independent samples. Significantly different at: ***p<0.001 vs. 0 μM magnolol; $$$p<0.001 vs. 50 μM magnolol.

Next, we investigated the effect on EMT-related proteins and found that the phosphorylation of NF-Embedded ImageB and STAT3 was reduced by magnolol treatment in both A549 and CL1-5-F4 cells (Figure 3B). Both NF-Embedded ImageB and STAT3 play a role in EMT and metastasis of NSCLC cells (25, 26); therefore, we determined whether EMT and metastasis-related proteins were affected by magnolol treatment. As indicated in Figure 3B, the expression of N-cadherin, ZEB1, SNAIL, SLUG and TWIST were all down-regulated by magnolol. These factors all play important roles in regulating EMT of tumor cells (27). Furthermore, the protein expression of MMP2, MMP9, VEGFA, and uPA, which are involved in altering NSCLC metastasis, was also suppressed by magnolol (Figure 3B). Additionally, cyclin D1, which controls cell proliferation, was also reduced by magnolol treatment. These results indicate that invasion/migration, and expression of EMT and metastasis-associated proteins were all inhibited by magnolol.

Discussion

Extrinsic apoptotic signaling can be initiated by FAS/FASL interaction, while intrinsic apoptotic signaling is elicited by the loss of Ψm. Caspase-8 and caspase-9 mediate extrinsic and intrinsic apoptosis through cleavage of downstream executioner caspases, respectively. Caspase-3 is a major executioner caspase involved in DNA fragmentation and cleavage of poly (ADP-ribose) polymerase 1 (17, 28). Our results show that magnolol effectively induced apoptosis and up-regulated FAS/FASL interaction and loss of Ψm. The expression of cleaved caspase-3, -8 and -9 was significantly induced by treatment with magnolol in both A549 and CL1-5-F4 cells (Figure 1C and Figure 2). According to these data, it is suggested that magnolol-induced extrinsic and intrinsic apoptosis through the caspase pathway in NSCLC cells.

EMT-related proteins such as N-cadherin, ZEB1, SNAIL1, SLUG, and TWIST potentiate tumor invasiveness and metastasis through converting the phenotype from non-invasive epithelial to invasive mesenchymal cell state (29, 30). MMP2, MMP9, VEGFA, and uPA are invasion-related proteins which mediate extracellular matrix degradation and angiogenesis, thereby contributing to tumor metastasis and growth (4, 31). Cyclin D1 is crucial cell-cycle regulator involved in promoting tumor growth, mainly through its ability to enhance cell-cycle progression (32). Inhibition of cyclin D1, EMT, and metastasis-related proteins leads to suppression of proliferation and invasion of NSCLC cells (12, 33-35). Our results indicate that magnolol effectively inhibited the migratory and invasive abilities of both A549 and CL1-5-F4 cells (Figure 3A). The levels of cyclin D1, and of EMT- and metastasis-related proteins were attenuated by treatment with magnolol (Figure 3C). In addition, we also found that the magnolol triggered the inhibition of N-cadherin.

Both NF-Embedded ImageB and STAT3 are critical mediators of cancer hallmarks required for tumor progression. Constitutive NF-Embedded ImageB and STAT3 signaling are involved in induction of proliferation, EMT, and expression of invasion-related proteins (36-38). Increased NF-Embedded ImageB and STAT3 activity are associated with poor outcome of patients with NSCLC (12, 39). Inactivation of NF-Embedded ImageB and STAT3 suppresses tumor growth, invasion, and EMT by reducing expression of proliferation-, EMT- and invasion-related proteins in NSCLC cells (12, 26, 40, 41). Our data showed that protein levels of STAT3 (Tyr705) and NF-Embedded ImageB (Ser536) were reduced by treatment with magnolol (Figure 3B). Suppression of NF-Embedded ImageB and STAT3 activity may be correlated with magnolol-induced reduction of proliferation-, EMT- and metastasis-related proteins in NSCLC cells.

In conclusion, this study indicates that caspase-mediated apoptosis and suppression of NF-Embedded ImageB/STAT3 signaling may be associated with magnolol-induced suppression of growth, metastasis, and EMT of NSCLC cells.

Acknowledgements

The Authors thank the Medical Research Core Facilities Center, Office of Research and Development at China Medical University (Taichung, Taiwan, R.O.C) for their technical support. This study was supported by Tainan Municipal Hospital, Tainan, Taiwan (ID: RD-108-03), Chang Bing Show Chwan Memorial Hospital, Changhua, Taiwan (ID: BRD-109029), Taichung Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Taichung, Taiwan (ID: TTCRD111-13), Cathay General Hospital, Taipei, Taiwan (ID: CGH-MR-A11013), respectively.

Footnotes

  • ↵*$ These Authors contributed equally to this study.

  • Authors’ Contributions

    YCL, YSW, HYW, and FTH performed the experiments. YCL, YSW, FTH, FSC and JYW prepared the initial version of the article. FTH, WLC and JHC designed the study, performed the literature review, and prepared the final versions of the article.

  • Conflicts of Interest

    The Authors declare no competing financial interests regarding this study.

  • Received May 18, 2022.
  • Revision received June 19, 2022.
  • Accepted June 21, 2022.

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Magnolol Induces Apoptosis Through Extrinsic/intrinsic Pathways and Attenuates NF-κB/STAT3 Signaling in Non-small-cell Lung Cancer Cells
YANG-CHENG LEE, YUEH-SHAN WENG, HSIAO-YU WANG, FEI-TING HSU, FU-SHIN CHUEH, JENG-YUAN WU, WEI-LUNG CHEN, JIANN-HWA CHEN
Anticancer Research Aug 2022, 42 (8) 3825-3833; DOI: 10.21873/anticanres.15873
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