Research ArticleExperimental Studies
Open Access

Magnolol’s Therapeutic Efficacy and Immunomodulatory Effects in Oral Squamous Cell Carcinoma

CHIEN-FU TSENG, HSIN-MING CHEN, TSAI-LAN LIAO, FEI-TING HSU, CHI-JUNG YEH, WEI-TING CHEN and SANG-HENG KOK
In Vivo September 2024, 38 (5) 2152-2164; DOI: https://doi.org/10.21873/invivo.13678

Abstract

Background/Aim: Oral squamous cell carcinoma (OSCC) presents a significant health challenge, requiring effective treatments. Magnolol, a compound with potential anticancer properties, warrants investigation in OSCC treatment. Here, we aimed to assess the efficacy of magnolol in inhibiting progression of OSCC and to explore the underlying mechanisms of its action. Materials and Methods: We evaluated the effect of magnolol on tumor progression using the MOC1-bearing orthotopic model. We examined its impact on pathology and toxicity through hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC), and biochemical analysis. We also investigated the immunoregulatory effects of magnolol in the MOC1-bearing model using flow cytometry. Results: At high doses, magnolol significantly reduced tumor volume (p<0.0001 for comparisons between treated with magnolol and untreated groups) and weight loss by 70% in vivo. It also induced caspase-dependent apoptosis, evidenced by 2.42-, 2-, and 2.2-fold increases in the expression of caspase-3, -8, and -9, respectively, in mouse tumors treated with high 60 mg/kg of magnolol compared to untreated (p<0.0001 for all comparisons). Magnolol demonstrated no toxicity, maintaining body weight and normal biochemical parameters, including liver and kidney function. Pathological evaluations showed no adverse effects on organs in all treatment groups. Moreover, high doses of magnolol enhanced natural killer cells (by 3%), dendritic cells (20-25%), and cytotoxic T cells (20-40%) while reducing myeloid-derived suppressor cells and regulatory T cells by 1.5 times. Conclusion: Magnolol demonstrates potential as a therapeutic agent for OSCC, offering antitumor efficacy and immunomodulatory benefits.

Key Words:

Oral squamous cell carcinoma (OSCC) is a prevalent subtype of malignant tumors in the head and neck region, primarily occurring in the oral cavity and oropharynx. Prolonged exposure to carcinogens, such as tobacco, betel nuts, and alcohol significantly correlates with the development of OSCC (1, 2). Current treatment modalities for OSCC encompass surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy (3). To improve the prognosis of OSCC patients, it is imperative to pursue the development of potentially effective complementary therapies.

Traditional Chinese medicine (TCM) has gained recognition as a complementary approach to elevate therapeutic benefits and mitigate adverse effects associated with cancer treatment modalities in patients (4-6). Lin et al. reported that adjunctive therapy with TCM correlates with a lower mortality rate in patients with head and neck cancer in Taiwan (7). Further investigation is warranted to elucidate the therapeutic effects and mechanisms of TCM in treating head and neck cancer.

It has been discovered that formulations consisting of medicinal plants or compounds extracted from medicinal plants exhibit anti-OSCC properties (8-10). Pristimerin, an active compound found in several medicinal plants, mediates growth inhibition and apoptosis of OSCC cells by initiating endoplasmic reticulum stress (9). Isoliquiritigenin, a natural compound isolated from licorice, suppresses OSCC cells and reduces their resistance to cisplatin by inducing survivin ubiquitination, in vitro (11).

Magnolol, a multifunctional compound of Magnolia officinalis, demonstrates various bioactive properties, including anti-inflammatory, antioxidant, and protective effects against ischemic injury (12, 13). Magnolol also has the potential to exert anti-cancer effects by inducing apoptosis, inhibiting cell cycle progression, disrupting the tumor microenvironment, and boosting antitumor immunity (14-16). Additionally, studies have revealed that magnolol can induce apoptosis through the caspase-dependent pathway and reduce cancer stemness in OSCC cells (17, 18). However, the inhibitory effects of magnolol on OSCC and its mechanisms have not been fully elucidated. Therefore, this study aimed to assess the inhibitory effectiveness and underlying mechanisms against OSCC in vivo.

Materials and Methods

Reagent and antibodies. Most of the reagents and antibodies used in this study are listed in Table I and Table II.

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Table I.

Reagents used in the study.

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Table II.

Primary antibodies used in this study for immunohistochemistry (IHC) staining.

MOC1 cells culture. Mouse oral squamous cell carcinoma cell line (MOC1) was obtained from KERAFAST, INC. (Boston, MA, USA). Our previous study described the culture procedure (19, 20). In brief, cells were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with 5% fetal bovine serum (FBS), 5 mg/l insulin, 40 μg/l hydrocortisone, 5 μg/l epidermal growth factor (EGF), and 1% penicillin/streptomycin.

MOC1 bearing mice establishment. The animal experiments received approval from the Animal Care and Use Committee at China Medical University (approval number: CMUIACUC-2023-269). Male C57BL/6J mice aged six weeks (n=16) were acquired from the National Laboratory Animal Center in Taipei, Taiwan, to establish the orthotopic MOC1 bearing model. All experiments were conducted twice. The model establishment has been detailed in work by Chiang and Oweida et al. (19, 21). In brief, MOC1 (1×106 cells in 10 μl PBS) was injected into the right cheek of C57BL/6. Tumor progression was assessed using caliper measurements. Once the tumor volume reached 50-55 mm3, the mice were divided into three groups, receiving daily gavage doses of 0, 40, and 60 mg/kg magnolol for 22 days.

Extraction of immune related organs. Spleenocytes (SP), bone marrow (BM), tumor-draining lymph nodes (TDLN), and tumor-infiltrating lymphocytes (TIL) (22) from MOC1 bearing mice were isolated on day 23 following anesthesia overdose with isoflurane (>3%). The extracted immune cells from these organs were filtered through a 40 μm strainer twice, and red blood cells were lysed using ACK buffer (Thermo Fisher Scientific, Waltham, MA, USA). Subsequently, the cells were neutralized with Hank’s Balanced Salt Solution and prepared for antibody staining (23, 24).

Flow cytometric analysis of immune cells. This study analyzed various immune cell populations using flow cytometry, as previously described (25). Cytotoxic T cells (CTLs) from spleen (SP), tumor-draining lymph nodes (TDLN), or tumor-infiltrating lymphocytes (TIL) were stained with CD8 for 15 min, followed by staining for IFN-γ and IL-2 after fixation and permeabilization. M1/M2 macrophages from bone marrow (BM), SP, or TIL were identified by staining for CD11b (#553310), CD86 (M1) (#553692), and CD206 (M2) (#565250) in the dark for 15 min. Dendritic cells (DCs) from SP were stained for CD11c (#550260), CD83 (#742263), and MHCII (#557000), while natural killer cells (NKs) from TDLN were stained for CD3 (#555274), CD49b (#560628), and NK1.1(#551114) in dark for 15 min. Regulatory T cells (Tregs) from SP were stained CD4 (#553051) and CD25 (#553866) for 15 min, followed by staining for FOXP3 (#560403) after fixation and permeabilization, and myeloid-derived suppressor cells (MDSCs) from BM were stained for CD11b and Gr-1 (#553128) in dark for 15 min. Flow cytometry analysis was performed using NovoCyte flow cytometer and NovoExpress® software for quantification (Agilent Technologies Inc., Santa Clara, CA, USA). All antibodies were purchased from BD Biosciences (Franklin Lakes, NJ, USA).

Biochemistry analysis. On day 23, after anesthesia overdose with isoflurane (>3%), mice serum was isolated from heart blood. The collected serum was utilized to assess aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyltransferase (γGT), and creatinine (CREA) levels, indicative of liver and kidney function.

Hematoxylin and eosin (H&E) staining. Heart, kidney, liver, and small intestine, tissues from MOC1-bearing mice were harvested and sectioned for H&E staining. Bio-Check Laboratories Ltd. (New Taipei City, Taiwan) carried out the staining process. Tissue images were captured using the TissueFAXS platform (TissueGnostics, Vienna, Austria) at 200× magnification.

Immunohistochemical (IHC) staining. After 22 days of treatment, the mice were sacrificed, and the tumors were removed and fixed with 10% paraformaldehyde at room temperature. Tumor sections obtained from MOC1-bearing mice were subjected to IHC staining using EMD Millipore IHC Select® HRP/DAB reagents (Merck KGaA, Darmstadt, Germany). Following staining, images of the tumor-stained sections were captured at 200× magnification using the TissueFAXS platform (TissueGnostics) and quantified using StrataQuest software with the BF Area total 2 markers tool. Antibodies used in this section are listed in Table III.

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Table III.

Statistical analysis of MOC1 tumor volume across various treatment groups and dates.

Statistical analysis. Quantitative data were presented as means±SD. The group means of the magnolol-treated and untreated groups were compared using either one-way analysis of variance (ANOVA) or two-way ANOVA for multiple comparisons. Tukey’s HSD post-hoc test was employed to determine specific differences between groups following ANOVA analyses. Statistical significance was set at p<0.05.

Results

Magnolol demonstrated significant inhibition of OSCC progression in vivo. To assess the therapeutic efficacy of magnolol in oral squamous cell carcinoma (OSCC), we established a MOC1-bearing animal model (Figure 1A). Mice were divided into three groups, and tumor volume was measured using calipers. On day 22, mice were euthanized, and tumors and immune-related organs were isolated for further analysis. As depicted in Figure 1B, magnolol significantly suppressed tumor volume in a dose-dependent manner, after day 12 of treatment (p<0.0001 for comparisons between treated with magnolol and untreated groups). Additionally, the tumor progression pattern for each group of mice showed superior control of tumor growth after administration of magnolol; this effect was also dose-dependent (Figure 1C). The mean tumor growth time in the magnolol 60 mg/kg group was 4.25 times lower than in the untreated control group (Table IV). The tumor weight was significantly reduced in the 40 mg/kg magnolol-treated group (p=0.0045) and the 60 mg/kg magnolol-treated group (p<0.0001) as compared to the untreated group (Figure 1D). In Figure 1E, the expression level of caspase-3 was 2.42 times higher in the magnolol-treated group (60 mg/kg) compared to the untreated control (p<0.0001). Moreover, both mitochondria-dependent caspase-8 and death receptor-dependent caspase-9 factors were increased by magnolol in tumor tissue by 2 (p<0.0001) and 2.2 (p<0.0001) times, respectively (Figure 1E). In summary, magnolol markedly suppressed OSCC progression. Magnolol’s inhibition of OSCC progression was also associated with caspase-dependent apoptosis induction.

Figure 1.
Figure 1.

Treatment efficacy of magnolol on MOC1-bearing mice. (A) Experimental flow chart of MOC1-bearing mice. (B) Average tumor volume of each treatment group from day 0 to 22. (C) Tumor progression of each mouse and each treatment group from day 0 to 22. (D) Tumor weight of each treatment group on day 22. (E) The immunohistochemistry images and quantification results of cleaved-caspase3, -8, and -9 in tumor tissues. (Scale bar=100 μm)

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Table IV.

Mean tumor growth time, delay time, and inhibition rate in MOC1 tumor-bearing mice after treatment with different conditions are shown.

Magnolol exhibited no alteration of pathology or general toxicity in MOC1-bearing mice. To ascertain that magnolol does not induce toxicity during a 22-day treatment regimen, we conducted body weight measurements, biochemical analyses, and pathology evaluations by experienced veterinary professionals. As depicted in Figure 2A, the body weight remained consistent in all treatment groups over the 22-day treatment period. The levels of AST (normal range=30-130 U/l) and ALT (normal range=50-350 U/l) were within the normal range for C57 mice models (Figure 2B). The levels of γGT (3-8 U/l) also showed no significant changes in treatment groups compared to the untreated group (Table V). However, CREA levels, normally ranging from 0.2 to 0.5 mg/dl, were elevated in the untreated group, while the treated group remained within the normal range. In Figure 2D, the pathology of normal organs, including the heart, lungs, liver, kidneys, and small intestine, did not exhibit noticeable changes between the untreated control and magnolol-treated groups. Furthermore, the severity of pathology was assessed using four defined severity scores: 0—regular tissue, 1—mild changes, 2—moderate changes, 3—significant changes. As detailed in Table VI, no severity changes were observed between the untreated control and magnolol-treated groups. In summary, magnolol is unlikely to induce general toxicity or tissue damage.

Figure 2.
Figure 2.

General toxicity evaluation of magnolol on MOC1-bearing mice. (A) Body weight of each treatment group from day 0 to 22. (B) Aspartate transaminase (AST) and alanine transaminase (ALT) level in mice serum of each treatment group on day 22. (C) Creatinine (CREA) level in mice serum of each treatment group on day 22. (D) H&E staining images of the heart, kidney, liver, and small intestine of each treatment group on day 22. (Scale bar=100 μm)

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Table V.

The serum level of gamma-glutamyl transpeptidase (γGT) from MOC1 tumor-bearing mice is displayed.

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Table VI.

Severity scores for pathological alterations following different treatments in MOC1-bearing mice.

Magnolol markedly activated accumulation of positive immunoregulation cells in OSCC. In addition to tumor inhibition, we discovered that magnolol possessed immunoregulatory capabilities. We isolated immune-related organs from mice and measured several immune-related markers as follows. Firstly, we investigated the interaction of magnolol with natural killer cells (NKs), an innate immunity marker associated with cancer defense. In Figure 3A, it is shown that the accumulation of NKs (CD3−/CD49b+/NK1.1+) was significantly increased in the tumor-draining lymph nodes (TDLN) in the magnolol-treated group.

Figure 3.
Figure 3.
Figure 3.

Activating positive immunoregulation by magnolol on MOC1-bearing mice. (A) The expression pattern and quantification results of CD3−/CD49b+/NK1.1+ expressed NKs in mice TDLN. (B) The expression pattern and quantification results of CD11c+/CD83+/MHCII+ DCs in mice spleen and TDLN. (C) The expression pattern and quantification results of CD8+/IFN-γ+/IL-2+ CTLs in mice TIL, TDLN and spleen. (D) The IHC staining images and quantification results of CD8 and CD86 in tumor tissues. (Scale bar=100 μm)

Moving beyond innate immunity, we explored magnolol’s regulation of adaptive immunity. We examined whether DCs can be activated by magnolol, thus triggering T-cell-mediated immunoregulation. In Figure 3B, the activation of DCs (CD11c+/CD83+/MHCII+) in spleenocytes (SP) and TDLN was observed in the magnolol-treated group. Subsequently, we investigated whether cytotoxic T cells (CTLs) might be affected after DCs activation. As indicated in Figure 3C, the levels of CTLs in tumor-infiltrating lymphocytes (TIL), TDLN, and SP all showed an increase in the magnolol-treated group. CD8+/IFN-γ+/IL-2+ CTLs were all elevated by magnolol. Finally, we investigated whether tumor-associated macrophages were affected by magnolol. In Figure 3D, the M1-type population of macrophages increased with magnolol treatment in BM and SP. Concurrently, the protein expression levels of CD8 and CD86 increased in the tumor tissue with magnolol treatment (Figure 3E). In summary, magnolol may systematically and locally trigger positive immunoregulation involving innate and adaptive immunity.

Magnolol effectively suppressed the accumulation of immunosuppressive cells and the expression of related factors in OSCC. We then validated whether immunosuppressive factors and cells were decreased by magnolol in the MOC1-bearing model. As indicated in Figure 4A, the expression of myeloid-derived suppressor cells (MDSCs, CD11b+/Gr-1+) in spleenocytes (SP) and bone marrow (BM) was reduced by magnolol. Additionally, tumor-associated macrophage (CD11b+/CD206+) was decreased by magnolol in SP and TIL as well (Figure 4B). Furthermore, regulatory T cells (Tregs) identified as CD4+/CD25+/FOXP3+ with negative immuno-regulation capacity, were also decreased by magnolol in SP (Figure 4C). Moreover, the protein expression of CD206 and FOXP3, which represent TAM (M2) and Tregs in tumor tissue, was diminished by magnolol (Figure 4D). As shown in Figure 4E, magnolol also suppressed immunosuppressive factors, such as VEGF and IDO, which regulate immune cell recruitment and T cell inactivation (26, 27). Taken together, immuno-suppressive factors and cells were suppressed by magnolol, thereby diminishing negative immunoregulation in the tumor microenvironment.

Figure 4.
Figure 4.
Figure 4.

Inhibiting negative immunoregulation by magnolol on MOC1-bearing mice. (A) The expression pattern and quantification results of CD11b+/Gr-1+ expressed MDSCs in mice BM and SP. (B) The expression pattern and quantification results of CD11b+/CD206+ M2 in mice spleen and TIL. (C) The expression pattern and quantification results of CD4+/CD25+/FOXP3+ Tregs in mice SP. (D) The IHC staining images and quantification results of CD206 and FOXP3 in tumor tissues. (E) The IHC staining images and quantification results of VEGF and IDO in tumor tissues. (Scale bar=100 μm)

Discussion

Magnolol has been reported to effectively induce apoptosis and abolish stemness properties in OSCC cells (17, 18). Moreover, magnolol has demonstrated anti-tumor effects in various cancers, including non-small cell lung cancer and breast cancer, as reported by our group (28, 29). However, its effects on OSCC require further exploration. To investigate the inhibitory effects of magnolol on OSCC growth in vivo, we evaluated its effectiveness using an orthotopic MOC1 model. Administering 40 mg/kg and 60 mg/kg of magnolol significantly curtailed tumor growth without elevating AST, ALT, CREA, and γGT levels in serum (Figure 2B-C). These findings suggest that the doses of magnolol utilized are both efficacious in inhibiting OSCC growth and are well-tolerated.

M1 macrophages, CD8+ T cells, and NK cells, which serve as key antitumor effector cells, are pivotal in combating tumors (30-32). DCs bolster the anti-tumor capabilities of specific T cells through antigen presentation (33). Immunotherapy can heighten the expression and activation of these immune cells, thereby facilitating tumor suppression (34, 35). Moreover, both immunologic and non-immunologic agents might enhance the effectiveness of immunotherapy by augmenting the abundance of these immune cells (19, 23, 36). The increased presence of tumor-infiltrating CD8+ T cells corresponds with extended survival among OSCC patients (37). Our results show that treatment with magnolol effectively increased the percentage of activated CD8+ T cells, DCs, NK cells, and polarization of M1 macrophages in spleen and TDLN (Figure 3A-D). Additionally, it significantly promoted the infiltration of these immune cells into the tumor tissues (Figure 3E).

M2 macrophages and Tregs are immunosuppressive cells that reverse the activation of antitumor effector cells, leading to tumor immune evasion (19, 23). In addition to suppressing antitumor effector cells, M2 macrophages contribute to tumor growth, metastasis, and angiogenesis (22, 38). In OSCC patients, higher Treg counts in groups are associated with shorter survival times than those with lower Treg counts (39). Targeting these immunosuppressive cells may boost the effectiveness of immunotherapy against cancer (40, 41). Our results showed that magnolol markedly reduced the population of Tregs and the polarization of M2 macrophages, while also decreased the tumor infiltration of both Tregs and M2 macrophages Figure 3-4.

In conclusion, magnolol was shown to promote anti-tumor immunity by inducing anti-tumor effector cells and suppressing immunosuppressive cells in the orthotopic MOC1 model. Our results suggest that magnolol demonstrates potential as a therapeutic agent for OSCC, offering antitumor efficacy and immunomodulatory benefits, which indicate promising clinical applications.

Acknowledgements

Experiments and data analysis were partially conducted at the Medical Research Core Facilities Center, Office of Research & Development, China Medical University, Taichung, Taiwan, ROC.

Footnotes

  • Authors’ Contributions

    CFT, TLL, and FTH conducted all experiments, carried out statistical analyses, and compiled the data. CJYM, WTC, and HMC drafted the initial version of the manuscript. MCH, JHC and SHK conceptualized the presented idea, supervised the findings of this study, conducted the literature review, and finalized the manuscript.

  • Funding

    The study received support from the Chang Bing Show Chwan Memorial Hospital, Changhua, Taiwan (ID: BRD-111053), and Shu-Zen Junior College of Medicine and Management, Kaohsiung, Taiwan (ID: KAFGH-ZY_A_111018).

  • Conflicts of Interest

    The Authors affirm that they have no financial interests that might be construed as conflicting with the findings or conclusions presented in this study.

  • Received May 8, 2024.
  • Revision received June 6, 2024.
  • Accepted June 20, 2024.

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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Magnolol’s Therapeutic Efficacy and Immunomodulatory Effects in Oral Squamous Cell Carcinoma
CHIEN-FU TSENG, HSIN-MING CHEN, TSAI-LAN LIAO, FEI-TING HSU, CHI-JUNG YEH, WEI-TING CHEN, SANG-HENG KOK
In Vivo Sep 2024, 38 (5) 2152-2164; DOI: 10.21873/invivo.13678
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