Research ArticleExperimental Studies

Magnolol Suppresses ERK/NF-κB Signaling and Triggers Apoptosis Through Extrinsic/Intrinsic Pathways in Osteosarcoma

CHI-HUAN LI, MING-CHOU KU, KUN-CHING LEE, PO-FU YUEH, FEI-TING HSU, RONG-FONG LIN, CHUNG-CHI YANG, WEI-CHUN WANG, JIANN-HWA CHEN, LI-CHO HSU and YUAN-HAO LEE
Anticancer Research September 2022, 42 (9) 4403-4410; DOI: https://doi.org/10.21873/anticanres.15940

Abstract

Background/Aim: Osteosarcoma is an aggressive primary malignant bone tumor that occurs in childhood. Although the diagnostic and treatment options have been improved, osteosarcoma confers poor prognosis. Magnolol, an active component of Magnoliae officinalis cortex, has been widely applied in herb medicine and has been shown to have multiple pharmacological activities. However, whether magnolol possesses anti-osteosarcoma capacity remains unknown. Materials and Methods: We examined magnolol is cytotoxicity, and whether it regulates apoptosis and oncogene expression using MTT, flow cytometry and Western blotting assays in osteosarcoma cells. Results: Magnolol exerted toxicity towards U-2 OS cells by inducing intrinsic/extrinsic apoptosis pathways. Additionally, treatment of U-2 OS cells with magnolol inhibited MAPK1 mitogen-activated protein kinase 1 (ERK)/Nuclear factor kappa B (NF-Embedded ImageB) signaling involved in tumor progression and reduced the expression of anti-apoptotic and metastasis-associated genes. Conclusion: Magnolol may induce apoptosis and inactivate ERK/NF-Embedded ImageB signal transduction in osteosarcoma cells.

Key Words:

Osteosarcoma (OS), the most common form of malignant bone tumor, often occurs in children, adolescents, and adults older than 50 years (1, 2). Chemotherapy induces DNA damage and interferes with DNA metabolism resulting in cell cycle arrest, cellular senescence, and apoptosis in cancers (3). Chemoresistance and metastasis are major causes of treatment failure in OS (4-6). Mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) is a crucial mediator of malignant phenotypes of OS, including growth, survival, angiogenesis, invasion, metastasis, and chemoresistance through upregulating of oncogenic transcriptional factors. Inhibition of ERK signaling has been reported to suppress OS but also to enhance the anti-OS efficacy of chemotherapy (7, 8). Therefore, ERK has been considered as a promising therapeutic target for the treatment of OS.

Several multikinase inhibitors have been indicated to prolong the survival of patients with relapsed and unresectable high-grade OS after failure of chemotherapy. Both sorafenib and regorafenib are oral multikinase inhibitor that show positive effects on overall survival and progression-free survival in patients with OS (9). Sorafenib and regorafenib induce apoptosis, inhibit growth, reduce anti-apoptotic signaling, and impair invasion ability in OS cells. Suppression of ERK signaling was associated with either sorafenib- or regorafenib-mediated inhibition of OS progression (10, 11).

Natural compounds isolated from medicinal plants exert anti-cancer functions in OS down-regulation of ERK activation (12-14). For instance, amentoflavone, a biflavonoid compound found in Selaginella tamariscina, blocked ERK signaling and inhibited the growth and invasion capacity of OS in vitro and in vivo (2, 15). Magnolol, a bioactive compound of the medicinal plant Magnolia officinalis, alleviated ERK-mediated osteoclastogenesis and resulted in the suppression of ovariectomy induced bone loss (16). In addition, magnolol also induced apoptosis through G0/G1 phase arrest and p53-mediated intrinsic pathway in OS cells (17). However, the anti-OS effect of magnolol has not yet been fully understood. The major goal of this study was to evaluate the anticancer effect and action mechanism of magnolol on OS cells.

Materials and Methods

Reagents and antibodies. Magnolol was purchased from Wuhan ChemFaces Biochemical Co., Ltd. (Wuhan, Hubei, PR China). Dimethyl sulfoxide (DMSO), crystal violet, and 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). McCoy’s 5A medium, fetal bovine serum (FBS), and penicillin-streptomycin (PS) were all purchased from GIBCO®/Invitrogen Life Technologies (Carlsbad, CA, USA). Primary antibodies against myeloid cell leukemia 1 (MCL-1) (D35A5) [1:1,000, Cell Signaling Technology (CST), Danvers, MA, USA], X-linked inhibitor of apoptosis protein (XIAP, 1:1,000, CST), cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein (c-FLIP, 1:1,000, CST), matrix metallopeptidase 2 (MMP2, 1:1,000, Invitrogen), matrix metallopeptidase 9 (MMP9, PA5-13199, 1:1,000, Invitrogen), vascular endothelial growth factor (VEGF, ab46154, 1:1,000, Abcam, Cambridge, UK), urokinase-type plasminogen activator (uPA, 1:1,000, GenTex, Irvine, CA, USA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (D16H11) XP® (1:1,000, CST), extracellular signal-regulated kinase (ERK) Thr202/Try204 (1:1,000, CST), ERK (1:1,000, CST), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-Embedded ImageB) Ser536 (93H1) (1:1,000, CST), and NF-Embedded ImageB (D14E12) XP® (1:1,000, CST) were purchased from different companies as listed. Secondary antibodies for western blotting, including peroxidase affiniPure Goat Anti-Mouse IgG and Goat Anti-Rabbit IgG were all obtained from Jackson Immunoresearch Laboratories Inc. (West Grove, PA, USA).

Cell culture. U-2 OS cells were purchased from Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan, ROC), and maintained in complete McCoy’s 5A medium (10% Fetal Bovine Serum, 1% Penicillin-Streptomycin). Cells were subcultured following trypsinization using 1× trypsin EDTA solution C (SARTORIUS, Göttingen, Germany), collected, resuspended to adequate plates, and incubated in a 37°C humidified incubator in a 5% CO2 and 95% air mixture (2).

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. U-2 OS cells were plated in 96 well plates at 5×103 cells per well and incubated overnight. Then cells were treated with various concentrations of magnolol (0-200 μM) for 24 or 48 h. After treatment, the medium was discarded and replaced by MTT solution (0.5 mg/ml MTT in PBS) for another 2 h incubation at 37°C. The MTT solution was removed, 100 μl DMSO were added in each well and the optical density (OD) was measured at 570 mm absorbance wavelength. The blank value was defined as baseline (+/−0.01) (18).

Transwell assay (migration and invasion). U-2 OS cells were plated in 6 well plates at 5×104 cells per well and incubated overnight. Then, cells were treated with various concentrations of magnolol (0, 75, 100 μM) for 48 h. For the migration test, 8 μm pore transwells (BD Biosciences, Franklin Lakes, NJ, USA) were inserted in 24-well plate. For the invasion test, the upper inserts of transwells were coated with a matrigel mixture (matrigel:medium=1:1), placed in the 24 well plate and incubated in 37°C. The treated cells were then trypsinized, seeded at 5×104 cells (in serum-free medium) in the upper chambers of the transwells and the bottom chambers were filled with 500 μl complete medium and incubated for another 24 h. After incubation, transwells were fixed with fixation solution (methanol: acetic=3:1) and the cells were stained with 0.1% crystal violet. Bright images were captured using a light microscope. The density of migration and invasion cells were calculated using Image J software version 1.50 (National Institutes of Health, Bethesda, MD, USA) (19).

Flow cytometry. U-2 OS cells were plated in 6 well plates at a density of 5×104 cells per well and incubated overnight. Then, the cells were treated with various concentrations of magnolol (0, 75, 100, 125 μM) for 48 h. After treatment, cells were trypsinized, stained by FITC-DEVD-FMK (cleaved caspase-3), FITC-IETD-FMK (cleaved caspase-8), FITC-VAD-FMK (cleaved caspase-9), CD95 (Fas) Alexa fluor™ 488 (#13-0951-85, Invitrogen), CD178 (Fas-L) PE (#12-9919-42, Invitrogen), PARP-1 (cleaved Asp214) Alexa fluor™ 488 (#53-6668-42, Invitrogen), DiOC6 (3,3′-Dihexyloxacarbocyanine Iodide, ab189808, abcam) for MMP loss detection, and apoptosis analysis using an FITC Annexin V Apoptosis Detection Kit (BD Bioscience) for apoptosis detection, respectively. After cell staining processes, stained cells were washed by PBS and measured the fluorescence intensity by flow cytometry (NovoCyte, Agilent Technologies, ACEA US). The results were analyzed and quantified by FlowJo software (version 7.6.1; FlowJo LLC, Ashland, OR, USA) (20).

Western blotting. U-2 OS cells were plated in 6 well plates at a density of 5×104 cells per well and incubated overnight. Then, cells were treated with various concentrations of magnolol (0, 75, 100, 125 μM) for 48 h. The treated cells were lysed using RIPA lysis buffer containing proteinase and phosphatase inhibitors to collect whole cell protein on ice, and then cellular proteins were separated using 8-12% SDS-PAGE gel. The separated proteins were transferred onto polyvinylidene difluoride (PVDF) membranes and blocked with 5% non-fat milk for one hour at room temperature. The PVDF membranes were incubated with primary and secondary antibodies to detect target proteins and reacted with Immobilon Western Chemiluminescent HRP Substrate (Pierce, Rockford, IL, USA). The target protein band signals were detected using the UVP ChemiDoc-It™ image station (Analytik Jena, Jena, Germany) and quantified using the software VisionWorks (AnalytikJena) (21).

Statistical analysis. The comparison between untreated and treated group was calculated by one-way ANOVA using Microsoft excel 2016 version. A p-value less than 0.05 was considered as significant difference. Each value in this study is displayed as mean±standard error. Statistical difference symbols between groups are defined in each figure legend.

Results

Magnolol induced U-2 OS cell cytotoxicity and activated apoptosis pathways. To reveal the anti-osteosarcoma effect of magnolol, we first used the MTT assay to test the viability of human osteosarcoma U-2 OS cells after treatment with different concentrations of magnolol. It was demonstrated that magnolol significantly reduced U-2 OS cells viability at 125 and 200 μM after 24 h treatment, and markedly reduced cell viability at 75, 100, 125, and 200 μM after 48 h treatment (Figure 1A). We then further investigated whether magnolol-induced cytotoxicity is related to apoptosis induction. We stained cells with multiple cells apoptosis markers and evaluated their levels/activation by flow cytometry. As indicated in Figure 1B and C, fluorescence intensities of both cleaved-caspase-3 and cleaved-PARP-1 were increased after 75, 100, and 125 μM of magnolol treatment. Additionally, U-2 OS cells were double-stained with annexin-V (FITC) and PI (22), which is used to identify apoptosis status of cells. The result showed that 75, 100, and 125 μM of magnolol greatly induced U-2 OS annexin-V activation, which was interpreted as apoptosis induction (Figure 1D). These results indicated that magnolol may not only induce cytotoxicity of human osteosarcoma cells in a dose- and time-dependent manner, but also activated annexin-V to initiate apoptosis cell death.

Figure 1.
Figure 1.

Magnolol induced cytotoxicity and activated apoptosis in U-2 OS cells. (A) Viability U-2 OS cells treated with magnolol is tested using the MTT assay. U-2 OS cells treated with 0, 75, 100, and 125 μM magnolol for 48 h are stained with (B) FITC-DEVD-FMK, (C) PARP-1 (cleaved Asp214) Alexa fluor™ 488, (D) and Annexin V Apoptosis detection kit for flow cytometry measurement (a1 p-value<0.05, a2 p-value<0.01. a3 p-value <0.005 vs. 0 μM magnolol).

Magnolol activated both the extrinsic and the intrinsic apoptosis pathways in U-2 OS cells. Next, whether magnolol may trigger the extrinsic and/or the intrinsic apoptosis pathways in U-2 OS cells were also tested. The levels of the mediator of the extrinsic apoptosis pathway, cleaved-caspase-8, were strongly increased by magnolol treatment (Figure 2A). The levels of cell death receptor and its ligand, Fas as well as Fas-L, were both effectively induced by magnolol (Figure 2B and C). However, the levels of the intrinsic pathway marker, cleaved-caspase-9, were also increased by magnolol treatment (Figure 2D). Furthermore, the mitochondrial membrane potential was lost by magnolol treatment (Figure 2E). Taken together, we found that magnolol-induced cytotoxicity of human osteosarcoma U-2 OS is correlated to the activation of the extrinsic and the intrinsic apoptosis pathways.

Figure 2.
Figure 2.

Magnolol activated both the extrinsic and the intrinsic apoptosis pathways in U-2 OS cells. U-2 OS cells treated with 0, 75, 100, and 125 μM magnolol for 48h are stained with (A) FITC-IETD-FMK, (B) CD95 (Fas) Alexa fluor™ 488, (C) CD178 (Fas-L) PE, (D) FITC-VAD-FMK, and (E) DiOC6 for flow cytometry measurement (a1 p-value <0.05, a2 p-value <0.01, a3 p-value <0.005 vs. 0 μM magnolol).

Magnolol suppressed U-2 OS cell migration, invasion ability and tumor progression related protein expression. In addition, we performed western blotting to examine the regulation of various oncogene proteins in U-2 OS cells after magnolol treatment, including anti-apoptosis markers (MCL-1, XIAP, and C-FLIP), angiogenesis markers (VEGF), and migration/invasion related markers (MMP2, MMP9, and uPA). The levels of all the above-mentioned proteins were decreased by magnolol in a dose-dependent manner (Figure 3A). Furthermore, we used 8 μm transwell assays to identify whether the migration and invasion abilities of U-2 OS cells were inhibited by magnolol treatment. The results demonstrated that magnolol may inhibit U-2 OS cells migration and invasion capacity (Figure 3B). Taken together, magnolol may effectively suppress tumor progression associated protein expression and thus inhibit the growth and invasion/migration potential of U-2 OS cells.

Figure 3.
Figure 3.

Magnolol suppressed U-2 OS cell migration and invasion and the expression of tumor progression related proteins. (A) U-2 OS cells are treated with 0, 75, 100, and 125 μM of magnolol for 48 h and expression of several oncogenic proteins (MCL-1, XIAP, C-FLIP, MMP-2, MMP-9, VEGF, uPA) and of housekeeping gene GAPDH were examined using western blotting. (B) U-2 OS cells are pre-treated with 0, 75, and 100 μM magnolol for 48 h, and their migration/invasion abilities are examined using transwell assays (a3 p-value <0.005 vs. 0 μM magnolol).

Magnolol inhibited U-2 OS cell is associated with the inactivaiton of ERK/NF-Embedded ImageB pathway. To investigate the upstream regulator of these oncogenes, we further examined the phosphorylation of several key regulators of tumor progression. Multiple studies have demonstrated that the phosphorylation of ERK and NF-Embedded ImageB in osteosarcoma will trigger their growth. Western blot analysis indicated that magnolol greatly suppressed the phosphorylation of ERK as well as the phosphorylation of NF-Embedded ImageB (Figure 4). Hence, we suggest that magnolol inhibits U-2 OS cells growth via the inactivation of ERK/NF-Embedded ImageB pathway.

Figure 4.
Figure 4.

Magnolol inhibited the ERK/NF-Embedded ImageB pathway. U-2 OS cells are treated with 0, 75, 100, and 125 μM of magnolol for 48 h and the expression of ERK/NF-Embedded ImageB pathway related proteins, such as ERK (Thr202/Tyr204), ERK, NF-Embedded ImageB (Ser536), NF-Embedded ImageB and of the housekeeping gene GAPDH are assayed using western blotting.

Discussion

Anti-apoptotic proteins, such as MCL-1, XIAP, and C-FLIP, mediate acquired resistance of OS cells to chemotherapy and their inhibition sensitizes OS cells to methotrexate and cisplatin (23-25). Ideal apoptotic inducers restrain tumor growth and survival through effective induction of apoptosis and down-regulation of anti-apoptotic protein expression (26). Our results showed magnolol triggered apoptosis while reduced protein levels of MCL-1, XIAP, and C-FLIP in U-2 OS cells (Figure 1B-D and Figure 3A). Zhou et al. presented that magnolol induced apoptosis through the p53-mediated intrinsic apoptosis pathway (17). In addition to the stimulation of intrinsic apoptotic signaling, we also found that extrinsic apoptotic signaling such as activation of Fas, FasL, and cleaved-caspase-8 was effectively triggered by treatment with magnolol (Figure 2).

Metastasis-associated proteins such as MMP-9, MMP-2, and VEGF mediate extracellular matrix degradation and angiogenesis, which enhance tumor metastasis. Furthermore, their over-expression is associated with metastasis and worse survival of patients with OS (1, 20, 27, 28). Our results indicated that magnolol triggered inhibition of metastasis-associated proteins and eliminated the invasion ability of U-2 OS cells (Figure 3). Activation of NF-Embedded ImageB, an oncogenic transcription factor, may be involved in the expression of anti-apoptotic and metastasis-associated proteins. NF-Embedded ImageB is constitutively activated by upstream kinases, such as AKT and MAPKs, in cancers (29, 30). Previous studies showed that PD98059 (the ERK inhibitor) and QNZ (the NF-Embedded ImageB inhibitor) effectively reduced endogenous NF-Embedded ImageB signaling, and the above-mentioned antiapoptotic and metastasis-associated protein expression in OS cells (2, 11). Our data indicated that phosphorylation of both ERK and NF-Embedded ImageB was abolished by treatment with magnolol (Figure 4).

In conclusion, the extrinsic and intrinsic pathways participated in magnolol-induced apoptosis. In addition, magnolol also suppressed ERK/NF-Embedded ImageB-mediated anti-apoptosis and invasion/migration. We suggested that the induction of apoptosis and the suppression of ERK/NF-Embedded ImageB signaling are associated with magnolol-inhibited growth, survival, and invasion/migration of OS cells.

Acknowledgements

The Authors thank the Medical Research Core Facilities Center, and the Office of Research and Development at China Medical University (Taichung, Taiwan, ROC) for their technical support. This study was also supported by the following institutes: National Yang Ming Chiao Tung University Hospital, Yilan, Taiwan (ID: RD2022-010 and RD2022-021), Chang Bing Show Chwan Memorial Hospital, Changhua, Taiwan (ID: BRD-109028), Cathay General Hospital, Taipei, Taiwan (ID: CGH-MR-A11026).

Footnotes

  • ↵*,# These Authors contributed equally to this study.

  • Authors’ Contributions

    CHL, MCK, KCL, PFY and RFL performed all of the experiments. CHL, FTH, RFL, CCY and WCW prepared the first draft of the article. FTH, JHC and YHL participate in the design of this 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 June 23, 2022.
  • Revision received July 12, 2022.
  • Accepted July 13, 2022.

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Magnolol Suppresses ERK/NF-κB Signaling and Triggers Apoptosis Through Extrinsic/Intrinsic Pathways in Osteosarcoma
CHI-HUAN LI, MING-CHOU KU, KUN-CHING LEE, PO-FU YUEH, FEI-TING HSU, RONG-FONG LIN, CHUNG-CHI YANG, WEI-CHUN WANG, JIANN-HWA CHEN, LI-CHO HSU, YUAN-HAO LEE
Anticancer Research Sep 2022, 42 (9) 4403-4410; DOI: 10.21873/anticanres.15940
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