Effects and Mechanisms of Fucoxanthin from Hizikia fusiforme on Inhibiting Tongue Squamous Cell Carcinoma Proliferation via AKT/mTOR-Mediated Glycolysis

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Introduction
Oral cancer is a threat to global public health and accounts for 1%-2% of all cancers [1][2][3].It is closely related to factors such as frequent consumption of alcohol and hot foods, smoking, and a history of oral diseases.Tongue squamous cell carcinoma (TSCC) is the most prevalent oral cancer.It can be transferred to organs such as the lungs in late-stage TSCC, and lymph node metastasis is common [4].Currently, tongue cancer is treated mainly via comprehensive surgical sequences [5].Although treatment methods and technologies have improved, the mortality rate has not decreased [6].
Many Chinese monomers inhibit tumor growth [7,8].Drug components in natural plants cause few adverse reactions owing to their single components and thus allow for a wide range of treatments.Hence, they can be used as auxiliary or alternative methods for targeted treatment of tongue cancer.Terefore, fnding high-efciency traditional Chinese medicine monomers to treat tongue cancer has become a research focus.
Aberrant modifcations in energy metabolism are a prominent characteristic of tumor cells, pushing them towards aerobic glycolysis as a primary means of energy generation [9].Research has demonstrated that inhibiting the tumor glycolysis pathway efectively impedes tumor cell proliferation and induces tumor cell death.Additionally, studies have confrmed that the protein kinase B (AKT)/ mammalian target of the rapamycin (mTOR) pathway facilitates the crucial expression of enzymes involved in the glycolysis pathway, resulting in enhanced glucose uptake by tumor cells and dysregulated glycolytic activity [10][11][12].Tis phenomenon contributes to increased energy supply, tumor growth promotion, and uncontrolled tumor cell proliferation.Tese studies indicate that the AKT/mTOR signaling pathway is signifcantly associated with glycolytic metabolism.Hence, a potential therapeutic strategy for treating TSCC may involve targeting the glycolytic pathway, controlled via the AKT/mTOR signaling pathway, as well as attenuating tumor cell energy supply.
Hizikia fusiforme is a plant of the Sargassum family of the phylum Ochrophyta, which has been used as a drug to treat diseases for thousands of years in China and Japan [13].Research has demonstrated that Hizikia extract exhibits diverse efects, including antitumor, antifatigue, antiradiation, antiviral, antiaging, and immunomodulatory properties [14][15][16].Fucoxanthin is the main active ingredient in hijiki.Numerous studies have highlighted the ability of fucoxanthin to impede tumor cell growth by promoting apoptosis, inhibiting DNA synthesis, and arresting the cell cycle.Notably, fucoxanthin signifcantly inhibits various cancers, including cervical, breast, gastric, liver, and lung cancers.Bae et al. found that fucoxanthin partially attenuated alterations in gene expression associated with glycolysis and mitochondrial respiration involving hexokinase, peroxisome proliferator-activated receptor c coactivator 1β, and pyruvate dehydrogenase kinase 3 [17].Nevertheless, insufcient data exist on the regulatory efects of fucoxanthin, specifcally on TSCC.Our fndings will contribute to theoretical and experimental foundations for potential applications of fucoxanthin isolated from hijiki (Figure 1) for preventing and treating TSCC.

Efect of Fucoxanthin on CAL-27 Cell
Viability.CAL-27 cells in the logarithmic growth phase were aseptically cultured by seeding into 96-well plates at 5000 cells/well.Te cells were divided into the drug, control (no drug), and blank (blank medium only) groups.After a 24-hour incubation, the drug group was treated with various concentrations of fucoxanthin, and the control group received an equivalent volume of untreated medium.Six replicate wells were prepared per dose.After further incubation for 0, 6, 12, 18, and 24 hours, each well was supplemented with 10 μL of MTT solution (Solaribo, Beijing, China).Te cells were then incubated for 4 hours, and the absorbance (A) at 570 nm was estimated using a microplate reader.

Efects of Fucoxanthin on CAL-27 Cell Ability to Form
Colonies.CAL-27 cells in the logarithmic growth phase were aseptically cultured by seeding into 6-well plates at 5000 cells/well and then incubated for 24 hours.Te experiment consisted of a control group (untreated) and low-, medium-, and high-dose administration groups (3, 6, and 9 μg/mL, respectively), with three replicate wells per group.After incubating for 24 hours, the cell culture media were replenished every other day, and the cells were cultured for 14 days until visible colonies formed.After removing the cell culture medium, the wells were washed three times with phosphate-bufered saline (PBS).Subsequently, the cells were fxed in a 4% paraformaldehyde solution for 15 minutes and then stained with a 0.5% crystal violet solution for 15 minutes.After discarding the staining solution, the wells were rinsed with distilled water, and the excess water was drained.Photographs of the colonies in each well were captured using a camera and counted under a microscope.

Efect of Fucoxanthin on CAL-27
Apoptosis.CAL-27 cells in the logarithmic growth phase were aseptically cultured by seeding into 12-well plates at 5 × 10 4 cells/mL and then incubated for 24 hours.After a 24-hour incubation with 0, 3, 6, or 9 μg/mL fucoxanthin, the cells were collected and suspended in 100 μL double-staining incubation solution.Next, 5 μL of annexin V-FITC staining solution and 5 μL of propidium iodide (PI) staining solution (BD, Tokyo, Japan) were added to the cell suspension.Subsequently, the cells were incubated in the dark at room temperature for 15 minutes before detection.Apoptosis was assessed using fow cytometry (BD FACSVerse, Franklin Lakes, NJ, USA), and the proportions of early-and late-stage apoptotic cells were quantifed.

Efect of Fucoxanthin on CAL-27 Cell
Cycle.CAL-27 cells in the logarithmic growth phase were aseptically cultured by seeding into 6-well culture plates at 5 × 10 4 cells/mL and then incubated for 24 hours.After incubation with 0, 3, 6, or 9 μg/mL fucoxanthin for 24 hours, the cells were rinsed twice with cold PBS.Subsequently, cells were detached and fxed by adding 1 mL of 70% ethanol at 4 °C for at least 18 hours.After two washes with precooled PBS, 0.5 mL of RNAseA/PI dye solution (BD, Tokyo, Japan) was applied and left for 20 minutes at room temperature.Te cell cycle distribution in each group was investigated using fow cytometry.After exposure to fucoxanthin, the cells were harvested, and a small amount of cell suspension was used to determine the cell count using a cell counter.Glucose consumption, ATP production, and lactic acid production (Solaribo, Beijing, China) were assessed by measuring the absorbance at specifc wavelengths.Absorbance readings were obtained at 555 nm for glucose, 570 nm for lactic acid, and 340 nm for ATP, adhering to the manufacturer's guidelines and using a microplate reader.Te glucose consumption, ATP production, and lactic acid production were calculated from the obtained absorbance values.Measurement of intracellular ROS was performed using 2, 7-dichlorofuorescin-diacetate (Solaribo, Beijing, China).For fow cytometry, cells were collected for analysis.

Efect of Fucoxanthin on Hexokinase and PK Activity in
CAL-27 Cells.CAL-27 cells in the logarithmic growth phase were aseptically cultured by seeding into 6-well culture plates at 5 × 10 4 cells/mL and then incubated for 24 hours.After treatment with fucoxanthin, the cells were harvested, and a small amount of cell suspension was used to count the cells using a cell counter.Te enzymatic activities of hexokinase and PK (Solaibo, Beijing, China) were evaluated by quantifying the absorbance at 340 nm using a microplate reader, adhering to the manufacturer's guidelines.Te hexokinase and PK activities were calculated from the obtained absorbance values.

Efect of Fucoxanthin on the AKT/mTOR Signaling Pathway and Glycolytic-Related Gene Expression in CAL-27
Cells.CAL-27 cells in the logarithmic growth phase were aseptically cultured by seeding into 6-well culture plates at 5 × 10 4 cells/mL and then incubated for 24 hours.After exposure to fucoxanthin, total cellular RNA was extracted with TRIzol.Subsequently, the RNA was solubilized in 25 μL of DEPC water, and the RNA concentration was measured using a NanoDrop spectrophotometer.Next, cDNA was synthesized per the guidelines of the reverse transcription kit (Tokyo, Japan).Te reverse transcription system consisted of PrimeScript Bufer RT Enzyme Mix (1.0 μL), RT Primer Mix (1.0 μL), 5× PrimeScript Bufer 2 (for Real-time) (4.0 μL), and RNAse-free dH 2 O (4.0 μL).Te reaction was conducted at 37 °C and 85 °C for 5 seconds and incubated at 95 °C for 30 seconds and then subjected to 40 denaturation cycles at 95 °C for 5 seconds and annealing/extension at 60 °C for 30 seconds.Table 1 lists the primer amplifcation sequences used in the reverse transcription quantitative polymerase chain reaction (RT-qPCR).

Efect of Fucoxanthin on the AKT/mTOR Signaling Pathway and Glycolytic-Related Protein Expression in CAL-27
Cells.CAL-27 cells in the logarithmic growth phase were aseptically cultured by seeding into 6-well culture plates at 5 × 10 4 cells/mL and then incubated for 24 hours.Following exposure to fucoxanthin, 150 μL of RIPA lysate supplemented with phosphatase and protease inhibitors was added to each well to extract cellular proteins.After centrifugation and supernatant collection, protein concentrations were assessed using the Bradford method and subsequently normalized.Next, protein samples totaling 30 μg were separated through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).After separation, the proteins were transferred onto NC membranes (Millipore Co., Billerica, MA, USA), and the membranes were blocked with 5% skim milk for 2 hours.Te membranes were incubated overnight and then exposed to specifc antibodies: Cyclin CDK4 (ABclonal Technology, Wuhan, China), Cyclin D1 (ABclonal Technology), p21 (ABclonal Technology), AKT (ABclonal Technology), mTOR (ABclonal Technology), RPS6 (ABclonal Technology), p-AKT (ABclonal Technology), p-mTOR (ABclonal Technology), p-RPS6 (ABclonal Technology), HK (ABclonal Technology), and PKM (ABclonal Technology) diluted at 1 : 2500 and GAPDH (Cell Signaling Technology, Beverly, MA, USA) diluted at 1 : 4000.After three washes with TBST bufer solution for 10 minutes each, the membranes were incubated with suitable secondary antibodies (1 : 4000, ABclonal Technology) at room temperature for 60 minutes.After three Journal of Food Biochemistry additional washes with TBST bufer solution, the protein immunoblots were constructed using ECL chemiluminescence reagent.Te protein expression levels were analyzed using a Tanon imaging system (Shanghai, China).ImageJ (MD, USA) was used for the image analysis, and the relative gray values representing the target protein expression were normalized to GAPDH, which served as an internal reference.

Statistical Analysis.
Acquired data were analyzed with GraphPad Prism 7.0.Measurement data are presented as means ± standard deviation.Te means of the two groups were statistically compared using t-tests.One-way analysis of variance was conducted to compare the means of several groups.

Efects of Fucoxanthin on Apoptosis and Mitochondrial
Membrane Potential of CAL-27 Cells.Te apoptotic rate of CAL-27 cells in the control group was 10.92% (Figure 3(a)).At 3 μg/mL, fucoxanthin increased the apoptosis rate of CAL-27 cells to 14.69%.At 6 μg/mL fucoxanthin, the CAL-27 cell apoptosis rate increased signifcantly to 25.20% and continued to increase with the fucoxanthin concentration, reaching 39.90% at 9 μg/mL.In CAL-27 cells, early and late apoptosis rates increased when the fucoxanthin concentration increased.Tus, fucoxanthin efectively promoted CAL-27 cell apoptosis, supporting MTT assay fndings on the inhibition of cell proliferation.Flow cytometry was used to determine alterations in MMP in CAL-27 cells treated with various fucoxanthin concentrations.As the fucoxanthin concentration increased, the proportion of red fuorescence in the CAL-27 cells decreased, and the proportion of green fuorescence increased (Figure 3(b)).JC-1 fuorescence results in both the FITC and PE channels in the control cells showed that 98.90% of the CAL-27 cells fuoresced red, and 0.70% of the CAL-27 cells fuoresced green.Increasing the fucoxanthin concentrations to 3, 6, and 9 μg/mL decreased the proportions of CAL-27 showing red fuorescence to 92.50%, 83.70%, and 67.40%, respectively, and increased the proportion of CAL-27 cells showing green fuorescence to 6.89%, 15.50%, and 32.20%, respectively.Tus, fucoxanthin reduced the MMP in CAL-27 cells.

Fucoxanthin Arrested the Cell Cycle of CAL-27 Cells in G1
Phase. Figure 4(a) presents the fow cytometry results of the intracellular cycle distribution in CAL-27 cells treated with diferent fucoxanthin concentrations.As the fucoxanthin concentration increased, the number of CAL-27 cells in the G1 phase increased signifcantly (p < 0.05), and the ratio of cells in the S to G2/M phase decreased signifcantly.At 9 μg/mL fucoxanthin, the number of CAL-27 cells in the G2/M phase was 0, and the S phase cell percentage was 4.49%.Tese fndings indicate that fucoxanthin induced cell cycle arrest at the G1 phase in CAL-27 cells.Subsequently, the expression levels of G1 phase-specifc Cyclin CDK4, Cyclin D1, and p21 were detected.Compared with the control group, the expression levels of CDK4 and Cyclin D1 in fucoxanthin group were decreased, and the expression level of p21 was increased (Figure 4(b)).

Fucoxanthin Inhibited Glycolysis in CAL-27 Cells.
Figure 5 illustrates fucoxanthin's regulatory efects on the glycolytic pathway in CAL-27 cells.Glucose uptake, ATP generation, and lactate production measurements revealed that fucoxanthin signifcantly and dose-dependently inhibited these metabolic parameters in CAL-27 cells compared with those of the control group (p < 0.05; Figures 5(a)-5(c)).Compared with the control group, the     * p < 0.05 and * * p < 0.01 compared with the control group. 6 Journal of Food Biochemistry fucoxanthin, the relative expression of the glycolysis-related protein hexokinase decreased signifcantly (p < 0.05; Figure 7(h)).Compared with the model group, 24 hours of treatment with diferent fucoxanthin concentrations signifcantly reduced PKM protein expression levels (Figure 7(i)).Fucoxanthin at 3 μg/mL signifcantly decreased the relative expression levels of the glycolysis-related protein, PKM (p < 0.05), indicating that fucoxanthin controlled the glycolysis pathway of CAL-27 cells via the AKT/mTOR signaling cascade (Figure 8).

Discussion
Because of the lack of adequate therapy, cancer currently ranks as the second most prevalent cause of mortality [18].
Severe adverse efects and chemoresistance are the main   Journal of Food Biochemistry problems restricting current chemotherapy [19].Fewer adverse efects and greater pharmacology make natural goods a valuable resource for the pharmaceutical industry [20,21].Increasing evidence suggests that fucoxanthin isolated from S. fusiforme is a novel anticancer drug that can inhibit tumor development through various pathways.Calabrone et al. discovered that fucoxanthin suppressed prostate cancer cell growth and impeded vascular network formation in endothelial cells.qPCR and membrane antibody microarray showed that fucoxanthin downregulated the expression of various genes, including angiopoietin-2, CXCL5, TGFβ, IL6, STAT3, MMP1, TIMP1, and TIMP2 in prostate and endothelial cells [22].Wang et al. reported that fucoxanthin signifcantly inhibits cell adhesion molecule (CAM) expression triggered by infammatory factors, leading to a reduction in MCF-7 and endothelial cell adhesion [23].Suppression of the NF-κB signaling pathway inhibited CAM in endothelial cells, as evidenced by decreased phosphorylation levels of IκB-α, and NF-κB p65.A previous study revealed that fucoxanthin inhibited Te current study showed that fucoxanthin time-and dose-dependently inhibited CAL-27 cell activity.Moreover, fucoxanthin efectively inhibited the formation of CAL-27 cell colonies.Flow cytometry results further revealed that fucoxanthin induced CAL-27 cell apoptosis and reduced MMP.Tese fndings demonstrate the signifcant antiproliferative efects of fucoxanthin on CAL-27 cells.Tumor cells exhibit heightened glucose uptake and utilization of aerobic glycolysis as the primary feature of glucose metabolism.Although aerobic glycolysis is less efcient in net ATP production than in oxidative phosphorylation, it provides energy quickly and can meet the energy demands of rapidly proliferating tumor cells.Lung carcinoma is a highly aggressive cancer and exhibits the foremost etiology of mortality associated with cancer.Metabolomic investigations revealed an accumulation in the biosynthesis of nucleotides, amino acids, glycolysis pathway, tricarboxylic acid cycle, and glutathione metabolism [26].Inhibiting energy metabolism in cancer cells is a potential approach to cancer treatment, yet it remains a signifcant challenge to overcome.A previous study revealed that electrostimulation resulted in mitochondrial dysfunction, thereby inhibiting the electron transport chain and glycolysis pathways.Tis ultimately led to a severe energy supply crisis, causing cancer cell death [27].Additionally, neoplastic cells stimulate the glycolytic pathway by elevating glucose uptake.Te high glycolysis levels provide energy for unrestricted proliferation of neoplastic cells, and the generated intermediate products promote tumor growth [28].
A primary contributing factor of abnormal activity in the glycolytic pathway in tumor cells is the upregulation of key enzymes involved in the glycolytic pathway [29].Hexokinase is a crucial rate-limiting factor in the glycolytic pathway.Its primary function is to catalyze the conversion of glucose molecules entering the cell into glucose-6-phosphate.In several cancers, hexokinase is expressed abnormally.PK is the ultimate enzyme restricting the glycolysis rate and converts phosphoenolpyruvate and adenosine diphosphate (ADP) to pyruvate and ATP in the cytoplasm, thereby facilitating tumor cell proliferation.Tis enzyme has a pivotal function in advancing various tumors.Li et al. discovered that microRNA-let-7b-5p (let-7b-5p) represses the expression of hexokinase-2 through its interaction with the 3′untranslated region of hexokinase-2 mRNA.A previous study demonstrated the efcacy of let-7b-5p in restricting breast tumor growth and metastasis by inhibiting hexokinase-2-mediated aerobic glycolysis both in vitro and in vivo [30].Andrographolide, a diterpenoid lactone, is a natural anticancer agent because it suppresses cancer cell proliferation.Consequently, andrographolide signifcantly impeded human lung cancer cell viability and inhibited aerobic glycolysis through reduced lactate generation [31].
Te current study showed that fucoxanthin signifcantly increased the content of ROS and reduced glucose uptake, ATP generation, lactate production, and the enzyme activities of hexokinase and PK.Intervention with diferent concentrations of fucoxanthin signifcantly decreased the expression levels of glycolytic-related proteins, specifcally hexokinase and PK, in CAL-27 cells.Flow cytometry results showed that fucoxanthin arrested the CAL-27 cell cycle at the G1 phase.Western blot results showed that the expression levels of Cyclin CDK4, Cyclin D1, and p21 proteins in G1 phase were signifcantly changed.Tese results suggested that fucoxanthin can regulate cell glycolysis and effectively impede the rapid proliferation of CAL-27 cells, blocking tumor cell energy supply and inducing cell cycle arrest.
Studies have indicated that the AKT/mTOR signaling pathway is a crucial factor in regulating the glycolytic process [32,33].AKT is a critical driving factor of the tumor glycolytic phenotype by upregulating the expression and membrane translocation of glucose transporters and phosphorylating essential glycolytic enzymes, thereby regulating glycolysis [34].Additionally, AKT can be activated via phosphorylation by phosphatidylinositol-3-kinase (PI3K).Once activated, AKT activates the downstream efector molecule, mTORC, and binds to Raptor, forming mTOR complex 1. RPS6, which facilitates protein synthesis and tumor cell proliferation, can be activated via phosphorylation of p70 ribosomal protein S6 kinase by mTORC1 [35].Additionally, activation of mTORC1 can modulate the expression levels of glycolytic enzymes by controlling downstream transcription factors [36].Treatment with triptolide signifcantly inhibited intrahepatic cholangiocarcinoma (ICC) cell proliferation and glycolysis dose-and time-dependently.Subsequent analysis revealed that the inhibitory efect of triptolide on glycolysis in ICC cells was achieved by targeting the AKT/mTOR signaling pathway.Furthermore, triptolide inhibits tumor cell proliferation and glycolysis in mice with AKT/YapS127A mutations [37].Tese results indicate that fucoxanthin can decrease the expression levels of AKT/mTOR pathwayrelated proteins, including p-AKT, p-mTOR, and p-RPS6, in CAL-27 cells.Tis suggests that fucoxanthin can modulate the glycolytic pathway in CAL-27 cells via the AKT/mTOR signaling pathway.

Conclusions
Fucoxanthin can block cell glycolysis, limit tumor cell energy supply, and inhibit tongue cancer cell proliferation by suppressing the AKT/mTOR signaling pathway, thereby causing cell cycle arrest in the G1 phase.Additional in vivo studies are required to explore the mechanisms underlying fucoxanthin's efects.Tese fndings will establish a scientifc foundation for developing fucoxanthin as a therapeutic intervention for tongue cancer.

Figure 2 :
Figure 2: Efect of fucoxanthin on CAL-27 cell viability.(a) Inhibitory efect of fucoxanthin on the growth of tongue carcinoma CAL-27 cells compared with control cultures treated with DMEM.Data are presented as means ± standard deviation (SD; n � 5).(b) Fucoxanthin's efect on CAL-27 cell colony formation.Colony numbers were counted using ImageJ.* p < 0.05 and * * p < 0.01, compared with the control group.

Figure 3 :
Figure3: Fucoxanthin-triggered apoptosis and mitochondrial membrane potential in CAL-27 cells.Data are presented as means ± SD (n � 3).(a) Flow cytometry was used to evaluate apoptosis after 24 h of treatment with 0, 3, 6, and 9 μg/mL fucoxanthin.Flow cytometry analysis of CAL-27 cells divided the cell population into four quadrants based on FITC annexin V and PI staining.Q1 represented dead cells (negative for FITC annexin V and positive for PI); Q2 represented cells in the end stage of apoptosis (positive for both FITC annexin V and PI); Q3 represented cells undergoing apoptosis (positive for FITC annexin V and negative for PI); Q4 represented viable cells that did not undergo apoptosis (negative for both FITC annexin V and PI).(b) Mitochondrial membrane potential of fucoxanthin-treated CAL-27 cells.*p < 0.05 and * * p < 0.01 compared with the control group.

Figure 4 :Figure 5 :Figure 6 :
Figure 4: Cell cycle distribution of fucoxanthin-treated CAL-27 cells.Data are presented as means ± SD (n � 3).(a) Sub-G1 peak (apoptotic peak) after treatment with 0, 3, 6, and 9 μg/mL fucoxanthin for 24 h.Te DNA content of cells in the G2 and M phases was twice that of cells in the G0 and G1 phases.Cells in the S phase exhibited a DNA content within the range of the aforementioned extremes.(b) Efect of fucoxanthin on cell cycle-related protein expressions of CAL-27 cells.

Table 1 :
Primers used for the RT-qPCR.
progression of diverse types of cancers.We conducted multiple experiments measuring the expression of potential signaling molecules in response to fucoxanthin stimulation for AKT/mTOR involvement.Te mRNA levels of apoptosis-related genes in CAL-27 cells were assessed via RT-qPCR measurements (Figure6).Te antiapoptosis genes AKT, mTOR, and RPS6 showed no signifcant changes as the fucoxanthin concentration increased (p > 0.05; Figures6(a)-6(c)).Compared with those of the control group, the expression levels of the glycolysis-related enzyme genes, hexokinase and PKM, were signifcantly decreased in the fucoxanthin-treated group (p < 0.decreased signifcantly with 3 μg/mL fucoxanthin (p < 0.01; Figure7(b)).At 6 μg/mL fucoxanthin, the relative expression levels of p-mTOR and p-RPS6 decreased signifcantly (p < 0.01; Figures7(d) and 7(g)).Tese fndings demonstrated that fucoxanthin inhibited activation of the AKT/ mTOR signaling pathway in CAL-27 cells.At >6 μg/mL