Plumbagin targets the GLUT1/MMP-2 axis to inhibit oral squamous cell carcinoma progression

Abstract

Aim

This study aimed to investigate the clinicopathological characteristics and prognostic value of GLUT1 in oral squamous cell carcinoma (OSCC) and the effect of plumbagin (PLB) on inhibiting OSCC invasion and metastasis through the GLUT1/Matrix Metalloproteinase 2 (MMP2) axis pathway.

Materials and methods

One hundred and twenty human OSCC specimens were collected. Immunohistochemistry was performed to analyze the expression, clinicopathological characteristics, and prognostic value of GLUT1 in these specimens. Cal27 and SCC9 cell lines were used to investigate the role of PLB in cell proliferation, migration, invasion, and metastasis in vitro and in vivo.

Results

Immunohistochemistry showed significant associations between GLUT1 expression and MMP2, tumor recurrence, lymphatic metastasis, and TNM stage. In vitro and in vivo experiments demonstrated that PLB inhibited OSCC cell proliferation, migration, and invasion by downregulating GLUT1 and MMP2. WZB117, a GLUT1 inhibitor, also reduced cell colony formation, migration, and invasion. However, univariate and multivariate analyses indicated that GLUT1 expression was not an independent prognostic marker for OSCC overall and disease-free survival.

Conclusions

The findings demonstrated a novel anti-cancer mechanism of plumbagin, inhibiting OSCC invasion and migration by suppressing the GLUT1/MMP2 axis pathway, providing a theoretical basis for the future clinical use of plumbagin in the treatment of OSCC.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12935-025-03915-7.

Background

Oral squamous cell carcinoma (OSCC) is the most common malignant tumor of the oral cavity and the eighth-most prevalent type of cancer worldwide [1, 2]. According to surveys, 34,864 new cases were expected worldwide in 2018, increasing to 377,713 in 2020, which is more than double the 185,976 cases observed in 1990. These data indicate a rapid increase in the prevalence of oral cancer, with OSCC accounting for 90% of these cases [3, 4]. OSCC exhibits rapid growth, local invasiveness, and early regional lymph node metastasis, resulting in high recurrence and mortality rates [3, 4]. Cervical lymph node metastasis is the most crucial prognostic factor for OSCC patients [5]. Approximately 30% of tongue squamous cell carcinomas (TSCC) with T1/T2 stage metastasize to the cervical lymph nodes [5]. Unfortunately, despite advancements in cancer diagnosis and treatment, the 5-year survival rate for OSCC remains one of the lowest among malignancies and has been stagnant at 50% for the last three decades [3, 6]. Additionally, the tongue, gingiva, buccal mucosa, and other anatomical structures in the oral cavity, when affected by OSCC, can lead to chewing and swallowing difficulties, as well as aesthetic and speech disorders. These dysfunctions severely impact patients’ quality of life [7].

The hallmarks of cancer are pivotal to accelerating tumor progression and include sustaining proliferative signaling, deregulating cellular energetics, and activating invasion and metastasis [6, 8]. Glucose transporter 1 (GLUT1) is a membrane protein in the GLUT transporter family and is ubiquitously expressed in the human body [9]. It plays a crucial role in maintaining glucose homeostasis by transporting glucose from the extracellular to the intracellular environment [10]. However, one of the hallmarks of cancer, aerobic glycolysis, results in increased glucose consumption by cancer cells. This phenomenon is known as the “Warburg effect“ [11].

GLUT1 is significantly overexpressed in various types of malignancies, as shown in the TIMER database (Figure S1), and it plays a regulatory role in tumor glucose metabolism [9]. Elevated GLUT1 expression in tumors leads to higher rates of glycolysis, which further increases tumor growth and the risk of metastasis [12]. Our studies, along with others, have also demonstrated that GLUT1 is correlated with lymph node metastasis and tumor stage in TSCC [13–15].

In nuclear medicine, fluorodeoxyglucose positron emission tomography (FDG-PET) plays a diagnostic role by measuring increased glucose uptake in tumors. It has been successfully applied in differentiating benign from malignant tumors, diagnosing tumor metastasis or recurrence, detecting unknown primary cancers, and in cancer screening [16, 17].

Given the function of GLUT1 in cancers, there is growing research focused on developing GLUT1 inhibitors for cancer treatment. These include natural inhibitors such as plumbagin, resveratrol, and phloretin, synthetic inhibitors like BAY-876, WZB117, and STF-31, as well as noncoding RNA inhibitors such as miRNA-340 and miRNA-233-3p [13, 18–20].

Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone, PLB) is a naturally active naphthoquinone compound isolated from the root of Plumbago zeylanica L. It possesses high pharmacological efficiency and minimal side effects in anticancer, antibacterial, antifungal, antiosteoporotic, antiatherosclerotic, antidiabetic, and antioxidant activities [21–25]. In terms of its anticancer properties, PLB exhibits anti-proliferative and anti-metastatic effects in various cancers [26, 27]. Additionally, our previous study demonstrated that PLB inhibits tumor proliferation in tongue squamous cell carcinoma (TSCC) via the PI3K/Akt/GLUT1 signaling pathway [13]. However, the role of GLUT1 in OSCC invasion and metastasis, and whether it functions through downstream regulators such as MMP-2, remains unexplored [28–30]. However, no studies have yet explored the role of PLB-mediated GLUT1 in OSCC invasion and metastasis. Therefore, the purpose of the present study is to investigate the clinicopathological characteristics and prognostic value of GLUT1 in OSCC, and the effect of PLB-mediated GLUT1 on OSCC invasion and metastasis.

Materials and methods

Clinical specimens

A total of 120 human specimens from 120 patients, each pathologically diagnosed with oral squamous cell carcinoma, were obtained from the Department of Pathology at the Stomatology Hospital of Xi’an Jiaotong University in 2022. The specimens were selected based on specific inclusion criteria: patients diagnosed with OSCC who underwent radical surgery and neck dissection as their initial treatment, had completed clinical information, and had no other malignant tumors. While clinicopathological analysis was performed on all 120 specimens, the survival analysis was conducted on 68 of these specimens [31]. All patients agreed and signed the informed consent form. This study was approved by the Medical Ethics Committee of Xi’an Jiaotong University’s Stomatological Hospital (2022-XJKQIEC-009-003).

Immunohistochemistry

Two pathologists using HE staining to verify the diagnosis further examined all pathological paraffin blocks. The pathologists then marked the effective areas. To create tissue microarray (TMA) paraffin blocks, a tissue core with a diameter of 1.00 mm was punched from the marked area and inserted into a recipient block measuring approximately 2 cm x 2 cm. An automatic immunostaining device, the Leica Bond MAX (Leica Microsystems, Wetzlar, DE), was used to conduct immunohistochemistry (IHC) staining.

Heat-induced epitope retrieval was performed at pH 9.0 for 20 min after tissue samples were deparaffinized and rehydrated with ethanol. Automated IHC staining was processed using Primary Antibodies against GLUT1 (ab40084, Abcam, Cambridge, UK, 1:200) and MMP-2 (ab37150, Abcam, Cambridge, UK, 1:500). The Bond Polymer Refine-HRP Detection (DS9800, Leica, Newcastle, UK) was used, which includes the incubation of the secondary antibody, development with DAB, and counterstaining with hematoxylin. The images were quantitatively analyzed and automatically scored using the IHC profiler of Image J [32]. The software quantifies the score as negative, low positive, positive, or high positive.

Cell culture

Two OSCC cell lines (Cal27 and SCC9) were obtained from the Institute of Oral and Maxillofacial Surgery of Nanchang University (Nanchang - China). The cells were cultured in a DMEM culture medium with 10% fetal bovine serum (FBS) (Gibco, Gaithersburg, USA). PLB (p7262, Sigma, Taufkirchen, DE) was dissolved in DMSO and then diluted to a stock concentration of 100 mM. Before use, the working concentration of PLB was diluted with DMEM, ensuring that DMSO constituted less than 0.05% of the PLB solution. For the control group, only an equal volume of DMEM was added to the cells.

Colony formation assay

Cal27 and SCC9 cells were seeded in a 6-well plate at a density of 500 cells per well. After 24 h, the cells were treated with PLB at concentrations of 0, 1, 5, and 10 µM for 24 h or with WZB117 (GLUT1 inhibitor) at concentrations of 0, 10, and 25 µM for 24 h. Following treatment, all mediums were aspirated, and the cells were washed twice with PBS. Fresh medium was then added to the 6-well plate, allowing the cells to proliferate freely for 14 days. Subsequently, the cells were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet. Finally, the cell colonies were counted.

Wound-healing assay

Cal27 and SCC9 cells were inoculated in 6-well plates at a density of 5 × 10^5 cells per well and allowed to reach 90% confluency. A wound was then created using a plastic pipette, and the cells were washed twice to remove any floating cells. The Cal27 and SCC9 cells were treated with PLB at concentrations of 0, 1, 5, and 10 µM for 24 h or with WZB117 at concentrations of 0, 10, and 25 µM for 24 h. Cell migration into the wound areas was observed and photographed using an inverted microscope (Olympus FSX100) and analyzed with Image-Pro Plus software. Each experiment was conducted independently three times.

Cell invasion assay

The instructions provided by the manufacturer (BD Biosciences, USA) were followed to construct Matrigel chambers. Briefly, Cal27 and SCC9 cells, resuspended in serum-free DMEM medium, were seeded into the upper chambers (50µL), while the lower chambers were filled with 500µL of DMEM medium containing 1% FBS. PLB at concentrations of 0, 1, 5, and 10 µM, or WZB117 at concentrations of 0, 10, and 25 µM, were separately added to the cell suspension in the upper chambers. After a 24-hour incubation period, the cells that had migrated to the bottom surface of the membrane were fixed with paraformaldehyde for 15 min and stained with 0.1% crystal violet. Finally, four randomly selected fields were photographed using an inverted microscope (Olympus FSX100), and these images were analyzed using Image J software. The experiments were conducted at least three times.

Western blot

After incubation with 5 µM PLB for 0, 12, 24, and 48 h, or PLB at 0, 1, 5, and 10 µM for 24 h, or only medium for the control group, 5 µM PLB for the treatment group, and 10 µM WZB117 for the inhibitor group, Cal27 and SCC9 cells were collected and lysed with RIPA lysis solution (ZHHC, Xi’an, China). Protein concentrations were analyzed using the BCA protein analysis kit (BOSTER, Wuhan, China). A PAGE gel (color gel) rapid matching kit (ZHHC, Xi’an, China) was then used to prepare the gel.

20 µg of total protein were denatured in a water bath, isolated by SDS electrophoresis, transferred to PVDF membranes, and then blocked in TBST with 5% skim milk for two hours. The primary antibodies, GLUT1 (ab40084, Abcam, Cambridge, UK, 1:500), MMP-2 (ab37150, Abcam, Cambridge, UK, 1:500), and GAPDH (BM3874, BOSTER, Wuhan, China, 1:2000), were incubated separately overnight at 4 °C.

The next day, the secondary antibody (BA1054, BOSTER, Wuhan, China, 1:5000) was incubated at room temperature for two hours. Finally, 200 µL of chemiluminescence solution (AR1197, BOSTER, Wuhan, China) was loaded onto the machine (Bio-Rad ChemiDoc XRS system), and the expression of the proteins was analyzed quantitatively with Image Lab software. The relative protein expression levels of all target proteins, including GLUT1, MMP-2, and any others analyzed by Western blot, were quantified using Image Lab software (Bio-Rad). The band intensities were measured by densitometric analysis and normalized to the internal control GAPDH to account for variations in protein loading. All values are expressed as the ratio of the target protein intensity to the corresponding GAPDH intensity. Each experiment was independently repeated at least three times to ensure reproducibility.

In vivo xenografts and metastasis models

The plan for animal experiments was reviewed and approved by the Institutional Animal Care and Use Committee of Xi’an Jiaotong University Health Science Center. Immunodeficient mice (six weeks old) were obtained from the Laboratory Animal Center, Xi’an Jiaotong University Health Science Center, and randomly assigned into a control group and a treatment group, with five mice in each group. Mice were randomly divided into control and treatment groups using a random number table. Investigators performing tumor measurement, histological examination, and data analysis were blinded to the treatment allocation to reduce bias. Cal27 cells (2 × 10^5 cells/100 µL of PBS) were inoculated subcutaneously into the right back. When the tumor volume reached 25 mm^3 to 50 mm^3, PLB dissolved in 25% polyethylene glycol (PEG) was injected intraperitoneally at a concentration of 1 mg/kg/day for three weeks in the treatment group, while the control group received an equivalent volume of 25% PEG. The formula V = 1/2a × b^2 (where a is the longest diameter of the tumor and b is the shortest diameter of the tumor) was used to measure and calculate the Cal27 tumor volume.

After three weeks of treatment, Cal27-derived tumors were excised from euthanized mice, and the expression levels of GLUT1 and MMP2 were further examined in vivo.

For the metastasis model, 1 × 10^6 Cal27 cells were injected into the lateral tail veins of anesthetized immunodeficient mice. PLB dissolved in 25% polyethylene glycol (PEG) was injected intraperitoneally at a concentration of 1 mg/kg/day for six weeks in the treatment group, while the control group received an equivalent volume of 25% PEG. The livers were collected from euthanized mice and stained with H&E. The number of liver tumor metastasis lesions in five randomized regions was quantitatively analyzed using Image Pro Plus software.

Statistical analysis

All statistical analyses were conducted using SPSS Statistics 24.0 (IBM, Armonk, NY, United States). The χ² test was performed to determine the association between GLUT1 and clinicopathological characteristics as well as MMP2. Student’s t-test was applied for statistical analysis. Additionally, univariate and multivariate regression analyses, along with Kaplan-Meier survival analysis, were performed to estimate the prognostic value of GLUT1 in OSCC patients. Data from this study are expressed as the mean ± standard deviation of at least three independent experiments. Statistical significance is indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001.

Results

The clinicopathological characteristics and prognostic value of GLUT1 in OSCC

Immunohistochemical staining showed high expression of GLUT1 in 60 cases (50%) and low expression of GLUT1 in 60 cases (50%) (Table 1). Furthermore, abnormally activated GLUT1 in OSCC was significantly correlated with tumor recurrence (χ² = 4.10, P = 0.04), lymphatic metastasis (χ² = 6.60, P = 0.01), and clinic stage (χ² = 4.48, P = 0.03), but uncorrelated with gender (χ² = 1.32, P = 0.25), age (χ2 = 0.14, P = 0.71) and tumor differentiation (χ2 = 0.35, P = 0.84) and subsites (χ² = 5.65, P = 0.34) (Table 1). The data suggested that GLUT1 may contribute to tumor invasion and metastasis in OSCC. IHC staining revealed that GLUT1 was predominantly localized on the plasma membrane of OSCC tumor cells, which is consistent with its function as a glucose transporter. In some cases, faint cytoplasmic staining was also observed, possibly due to high expression or protein turnover. Additionally, MMP2 was stained by IHC and primarily expressed in the cytoplasm of the tumor margin (Fig. 1). Interestingly, MMP2 staining was also strong in most OSCC specimens with high-expression GLUT1, and GLUT1 was statistically correlated with MMP2 (χ² = 30, P = 0.00) (Fig. 1; Table 1).

Table 1.

The clinicopathological characteristics of GLUT1 in human oral squamous cell carcinoma (OSCC)

Clinicopathological characteristics of OSCC patients——IHC profiler
Characteristics	Patients		GLUT1
	n	%	High	Low	Χ²	p-value
No. of patients	120	100.00	60	60
Gender
Male	78	65.00	36	42	1.32	0.25
Female	42	35.00	24	18
Age
>60 years	72	60.00	37	35	0.14	0.71
≤ 60 years	48	40.00	23	25
Tumor recurrence
Yes	34	28.30	22	12	4.10	0.04
No	86	71.70	38	48
Lymphatic metastasis
Yes	66	55.00	40	26	6.60	0.01
No	54	45.00	20	34
Differentiation
Well	85	70.80	42	43	0.35	0.84
Moderate	30	25.00	16	14
Poorly	5	4.20	2	3
TNM stage
I/II	41	34.20	15	26	4.48	0.03
III/IV	79	65.80	45	34
MMP2
High	60	50.00	45	15	30.00	0.00
Low	60	50.00	15	45
Subsites
Mucosal Lip	24	20.0	17	9
Buccal Mucosa	10	8.3	4	7
Floor of the Mouth	9	7.5	4	6	5.65	0.34
Hard Palate	5	4.2	2	3
Oral Tongue	39	32.5	23	19
Gum	25	20.8	10	16

Fig. 1.

Immunohistochemical analysis of GLUT1 and MMP-2 expression in human oral squamous cell carcinoma (OSCC); Representative images are shown for strong or weak expression of GLUT1 and MMP-2 in well or poorly differentiated OSCC (original magnification ×40 and ×200)

Kaplan-Meier survival analysis indicated that overall survival (OS) (P = 0.0258) and disease-free survival (DFS) (P = 0.0006) were significantly shorter in patients with OSCC with high GLUT1 expression (Fig. 2). For OS, univariate Cox regression analysis showed that GLUT1 (HR, 4.05; 95% CI, 1.07–15.34; p = 0.04) was a risk factor for OS, and multivariate Cox regression analysis showed that GLUT1 was not an independent prognostic factor for OS in OSCC patients (Tables 2 and 3). For DFS, univariate Cos regression analysis showed that Tumor recurrence (HR, 26.68; 95% CI, 8.97–79.36; p = 0.00), Lymphatic metastasis (HR, 4.66; 95% CI, 2.01–10.79; p = 0.00), TNM stage (HR, 5.58; 95% CI, 2.09–14.93; p = 0.00), and GLUT1 (HR, 3.57; 95% CI, 1.64–7.74, p = 0.00) were risk factors for DFS in OSCC patients. Multivariate Cox regression analysis showed that Tumor recurrence (HR, 36.73; 95% CI, 9.98-135.23, p = 0.00) was an independent risk factor for patient prognosis (Tables 2 and 3).

Fig. 2.

Potential prognostic value of GLUT1 in patients with human oral squamous cell carcinoma (OSCC); (A) Over survival curves (P = 0.0258) and (B) Disease-free survival (P = 0.0258)

Table 2.

Univariate and multivariate analysis of various prognostic parameters in patients with OSCC for OS

	Univariate analysis				Multivariate analysis
	P-value	Hazard Ratio (HR)	95% confidence interval		P-value	Hazard Ratio (HR)	95% confidence interval
Gender	0.44	0.63	0.19–2.05
Age	0.47	0.65	0.20–2.14
Tumor recurrence	0.16	2.34	0.71–7.70		0.90	1.09	0.30–3.93
Lymphatic metastasis	0.06	3.51	0.93–13.3		0.17	2.68	0.66–10.94
Differentiation	0.23	1.77	0.69–4.55
TNM stage	0.20	2.40	0.63–9.10
MMP2	0.20	2.26	0.65–7.81		0.65	1.39	0.34–5.68
GLUT1	0.04	4.05	1.07–15.34		0.23	2.61	0.54–12.68
Subsites	0.34	1.16	0.85–1.58

Table 3.

Univariate and multivariate analysis of various prognostic parameters in patients with OSCC for DFS

	Univariate analysis				Multivariate analysis
	P-value	Hazard Ratio (HR)	95% confidence interval		P-value	Hazard Ratio (HR)	95% confidence interval
Gender	0.56	0.80	0.37–1.71
Age	0.81	1.10	0.50–2.39
Tumor recurrence	0.00	26.68	8.97–79.36		0.00	36.73	9.98-135.23
Lymphatic metastasis	0.00	4.66	2.01–10.79		0.52	1.54	0.41–5.75
Differentiation	0.97	1.02	0.51–2.01
TNM stage	0.00	5.58	2.09–14.93		0.07	4.71	0.87–25.46
MMP2	0.06	2.043	0.959–4.353		0.60	1.32	0.46–3.79
GLUT1	0.00	3.57	1.64–7.74		0.35	0.61	0.22–1.71
Subsites	0.19	1.15	0.93–1.41

Anticancer effects of plumbagin in OSCC in vitro

In the colony formation assay, clonal colonies of Cal27 and SCC9 cells significantly decrease with increasing PLB concentrations (Cal27, P < 0.0001 and SCC9, P < 0.0001) (Fig. 3A). Next, the effect of PLB on cell migration and invasion was investigated by wound-healing and Matrigel-coated transwell experiments. After incubation for 24 h., the number of cells migrating to the wounded area was significantly reduced in a PLB concentration-dependent manner (Cal27, P < 0.0001 and SCC9, P < 0.0001) (Fig. 3B). Similarly, the results of the cell invasion assay indicated that the invasiveness of Cal27 and SCC9 cells was distinctly decreased when pre-treated with PLB at 0, 1, 5, and 10 µM (Cal27, P < 0.0001 and SCC9, P < 0.0001) (Fig. 3C).

Fig. 3.

Anticancer effects of Plumbagin (PLB) in human oral squamous cell carcinoma (OSCC). (a) Colony formation assay was performed to assess the cell proliferation capacity of PLB treatment at 0µM, 1µM, 5µM, and 10µM; (b) Migration ability of Cal27 and SCC9 cells was examined by wound-healing assay after treatment with PLB; (c) The effect of PLB at various dose on OSCC invasion capacity was examined using Matrigel-coated transwell; (d) Western blot analysis of GLUT1 and MMP-2 (dose-dependent); and (e) Western blot analysis of GLUT1 and MMP-2 (time-dependent). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Western blot results demonstrated that the treatment with PLB remarkably downregulated the expression level of GLUT1 (Cal27, 1µM, 5µM, and 10µM PLB, P < 0.0001; PLB for 12 h, 24 h, and 48 h, P < 0.0001; SCC9, 1µM, 5µM, and 10µM PLB, P < 0.0001; PLB for 12 h, 24 h and 48 h, P < 0.0001) and MMP-2 (Cal27, 1µM, 5µM and 10µM PLB, P < 0.0001; PLB for 12 h, 24 h, and 48 h, P < 0.0001; SCC9, 1µM, 5µM and 10µM PLB, P < 0.0001; PLB for 12 h, 24 h, and 48 h, P < 0.0001) in a dose- and time-dependent manner (Fig. 3D, E). Overall, these findings indicate that PLB significantly reduces cell proliferation, migration, and invasion in OSCC in a concentration-dependent manner by inhibiting the expression of GLUT1 and MMP-2.

The mechanism of plumbagin in suppressing OSCC invasion and metastasis

WZB117, a GLUT1 inhibitor, was used to mimic the effects of PLB inhibition of GLUT1 on the biological behaviors of OSCC cells, such as proliferation, invasion, and metastasis. Interestingly, WZB117 exhibited similar effects to PLB in terms of the biological functions of OSCC cell lines. The downregulation of GLUT1 significantly inhibited cell proliferation (Cal27, 10µM WZB117 and 25µM WZB117, P < 0.0001; SCC9, 10µM WZB117 and 25µM WZB117, P < 0.0001), migration (Cal27 and SCC9, P < 0.0001) and invasive (Cal27 and SCC9, P < 0.0001) ability in a concentration-dependent manner, which GLUT1 was involved in the regulation of OSCC invasion and metastasis (Fig. 4A-C). Additionally, the downregulation of GLUT1 by both PLB and WZB117 further inhibited MMP-2 expression (Cal27 and SCC9, P < 0.0001) (Fig. 4D). To sum up, our results evidenced that PLB suppressed OSCC invasion and metastasis by blocking the GLUT1/MMP-2 axis.

Fig. 4.

Anticancer effect of GLUT1/MMP-2 axis in human oral squamous cell carcinoma (OSCC)

(a) Cell proliferation under WZB117 treatment; (b) Cell migration under WZB117 treatment; (c) Cell invasion under WZB117 treatment; and (d) Inhibition of GLUT1 and MMP-2 by PLB and WZB117. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Anti-growth and anti-metastasis effects of plumbagin in OSCC in vivo

To further investigate the inhibitory role of PLB in OSCC growth and metastasis, xenografts and metastasis models were established. After 21 days, Cal27-derived tumors from both the control group and treatment group were harvested and compared. The final tumor volume in the treatment group was reduced to 23.61% tumor volume in the control group (P < 0.001) (Fig. 5A & C). Additionally, there was a significant difference in tumor volume between the control group and the treatment group within 21 days (P < 0.05) (Fig. 5B). IHC assessed the expression of GLUT1, MMP-2, and Ki67 in vivo.

Fig. 5.

Effect of Plumbagin (PLB) on the growth and metastasis of Cal27 cell tumor in vivo. (A) Tumor volume comparison (photograph); (B) Tumor growth curve; (C) Final tumor volume comparison; (D–G) IHC staining of GLUT1, MMP-2, and Ki67; and (H–I) Liver metastasis images (H&E staining). *P < 0.05, **P < 0.01, ***P < 0.001

The results showed that the expression of GLUT1, MMP-2, and Ki67 was inhibited after 21 days of PLB treatment. Specifically, GLUT1 included 1 -/+ and 4 ++/+++ in the control group, and 4 -/+ and 1 ++/+++ in the treatment group; MMP-2 included 1 -/+ and 4 ++/+++ in the control group and 3 -/+ and 2 ++/+++ in the treatment group; Ki67 positive cells 155 in the control group and 74.92 in the treatment group. An experiment on OSCC metastasis showed that there were more OSCC metastatic nodes on the surface of the liver and significantly larger liver metastatic lesions in the control group than in the treatment group, as presented by the HE staining section (Fig. 5H-I).

Discussion

Oral squamous cell carcinoma (OSCC) is the most frequently occurring head and neck malignancy, with incidence and mortality rates rising worldwide each year [2, 33]. OSCC is notorious for its local invasive growth and early regional lymphatic metastasis, resulting in a poor prognosis for OSCC patients [3, 4]. The high level of tumor invasion and metastasis is mainly responsible for the poor prognosis of OSCC [34]. Thus, it is imperative to elucidate the pathogenesis and mechanisms associated with OSCC to develop effective therapeutic medicine.

GLUT1 is a critical regulatory factor in the glucose metabolism of cancer. Elevated levels of GLUT1 increase the flow of glucose into the cell, thereby facilitating a higher rate of anaerobic glycolysis, which results in rapid cancer cell proliferation and greater risks of metastasis [12, 35]. In our study, GLUT1 was expressed in all 120 OSCC specimens, and the staining intensity of GLUT1 was related to tumor recurrence, lymphatic metastasis, and clinic stage in OSCC. Additionally, survival analyses of OS and DFS revealed that GLUT1 is correlated with poor prognosis in patients with OSCC. Although high GLUT1 expression was associated with worse OS and DFS in univariate analysis, multivariate Cox regression analysis revealed that GLUT1 was not an independent prognostic factor for OSCC patients. This suggests that the prognostic significance of GLUT1 is likely influenced by its correlation with other clinicopathological parameters, such as tumor recurrence and lymphatic metastasis. Therefore, while GLUT1 may reflect tumor aggressiveness, its clinical application as a standalone prognostic biomarker should be interpreted with caution. Consistent with our previous study, the highest GLUT1 expression was found around tumor islands, followed by the tumor margin, and the lowest expression was in the tumor center (Fig. 1) [13]. A meta-analysis found that high GLUT1 expression in OSCC was associated with lymph node metastasis, clinical stage, tissue differentiation, and overall survival [15]. However, our study also found a correlation between GLUT1 overexpression and tumor recurrence, as well as DFS, which its high invasive capacity might cause.

In the development of anti-cancer drugs, natural herbal extracts are favored by researchers due to their fewer adverse effects and low costs [36]. As a type of natural herbal compound, PLB exhibits remarkable anticancer properties with low side effects [21]. In this study, we found that PLB had an inhibitory impact on OSCC proliferation, migration, and invasion in vitro, suppressed OSCC tumor growth, and reduced liver metastatic lesions in vivo. It is known that GLUT1, an enhancer factor of cancers, promotes cancer progression by increasing glucose metabolism [18]. Sinha first found that PBL effectively inhibited GLUT1 expression to reduce angiogenesis in ovarian cancer cells [37].

Our previous study also found that PLB inhibited GLUT1 expression and translocation through the regulation of PI3K/Akt signaling, which in turn effectively induced apoptosis in TSCC [13]. A liposomal formulation containing plumbagin and genistein can synergistically suppress prostate cancer cell growth by targeting GLUT1 and Akt3 [38]. Additionally, GLUT1 plays a key role in cancer invasion and metastasis [34]. MMP-2 can degrade the extracellular matrix to promote cancer cell migration and metastasis and also drives lymphangiogenesis, and provide a route for cancer cell metastasis [39, 40].

It was reported that the upregulation of MMP-2 through activation of PI3K/AKT signaling promoted the migration and invasion of bladder cancer cells [41]. In lung cancer, upregulated GLUT1 increases the expression of MMP-2 [42]. Our study demonstrated that PLB suppressed cell proliferation, migration, and invasion in OSCC in a dose-dependent manner and decreased the expression level of GLUT1 and MMP-2 in a dose- and time-dependent manner. Combined with the molecular experimental results of PLB anti-tumor performance in this study (Fig. 3D, E), we speculate that PLB may exert its anti-tumor effect by inhibiting the expression of GLUT1. WZB117 is a small molecule compound inhibitor of GLUT 1. Studies have shown that it can downregulate glycolysis, induce cell cycle arrest, and inhibit cancer cell growth in vitro and in vivo, and is expected to be further developed as a prototype compound for new anticancer therapeutics as a GLUT1 and glucose transport inhibitor [43]. To verify this hypothesis, we used the GLUT1 inhibitor WZB117 to mimic the effects of PLB. The results showed that WZB117 and PLB showed similar effects on the biological behavior of OSCC cell lines, both inhibiting cell proliferation, migration, and invasion in a concentration-dependent manner by downregulating GLUT1 expression (Fig. 4A, B, C). Importantly, WZB117 downregulated the expression of MMP2 by inhibiting GLUT1, thereby suppressing the migration and invasion behaviors of OSCC cells (Fig. 4D). Although we demonstrated that GLUT1 downregulation by WZB117 significantly inhibited OSCC cell proliferation, migration, and invasion, a limitation of our study is the absence of gain-of-function assays (e.g., GLUT1 overexpression) to confirm the pro-metastatic role of GLUT1. Future studies will include both overexpression and knockdown models to fully elucidate the causal relationship between GLUT1 and OSCC metastasis. Taken together, this suggests that GLUT1 is involved in downregulating MMP-2 expression under PLB treatment of OSCC. As reported in the literature, the upregulation of GLUT1-mediated glycolysis promotes the invasive-metastatic potential of OSCC [30].

PLB achieves tumor invasion inhibition through intricate mechanisms. Studies have shown that p53, an essential tumor suppressor protein, can play an anti-tumor role by inhibiting GLUT1 [44]. Moreover, berberine (BBM), a traditional Chinese medicine for the treatment of leukopenia, has been shown to inhibit the proliferation, migration, and invasion of TNBC cells by regulating the PI3K/Akt/MDM2/p53 and PI3K/Akt/mTOR signaling pathways [45]. Overall, our results suggest that PLB targets the GLUT1/MMP2 axis and inhibits OSCC invasion and migration. However, its regulatory mechanism is complex and needs to be further studied. Based on relevant research findings, we speculate that PLB may play an anti-tumor invasion and migration role through the PI3K/Akt/p53/GLUT1/MMP2 axis.

Epithelial-mesenchymal transition(EMT) is a key process for tumor cells to acquire invasion and metastasis capabilities, which is related to tumor glucose metabolism. High concentrations of glucose promote the migration and invasion of breast cancer cells and promote the EMT process [46]. 3D cultured breast cancer cells undergoing EMT show enhanced aerobic glycolysis [47]. GLUT1 is a key protein that promotes glucose transport during glycolysis. Studies have shown that silencing of GLUT1 leads to inhibition of TGFβ1-induced cell proliferation and decreased expression of the EMT marker vimentin [48]. However, a limitation of the current study is the lack of direct quantification of EMT markers to confirm the involvement of the GLUT1/MMP-2 axis in EMT regulation. Future studies will incorporate Western blot or immunofluorescence analysis of canonical EMT markers such as E-cadherin, N-cadherin, and vimentin in OSCC cells following PLB treatment or GLUT1 inhibition. This will help to further validate the mechanistic role of GLUT1/MMP-2 signaling in OSCC invasion and metastasis via EMT modulation.

Additionally, the primary role of MMP-2 in cancer progression is to degrade the components of the basement membrane, enabling cancer cells to migrate out of the primary tumor to form metastases [49]. MMP2 plays an important role in regulating EMT. Reduced MMP2 expression in breast cancer inhibits the EMT process [50]. In addition, studies have shown that MMP2 mediates the EMT process of laryngeal squamous cell carcinoma through PI3K/Akt-NF-κB [51]. In our study, we found that by inhibiting GLUT1 expression, the expression level of MMP2 is reduced, which may suppress the biological behaviors of OSCC cells, such as migration and invasion, possibly through the inhibition of the EMT process. However, further experiments are needed to verify this hypothesis.

Notwithstanding the insights garnered from our study findings, a comprehensive understanding of the anticancer effects of PLB and the specific influence of GLUT1 on lymphatic metastasis necessitates further investigations. Future studies are anticipated to delve into the role of the PLB-mediated GLUT1/MMP-2 axis in OSCC lymphangiogenesis. Such endeavors are expected to expedite the development and enhance the clinical application of PLB as a promising anticancer drug for the treatment of OSCC. Additionally, future studies should investigate the M category and the expressions of GLUT1 and MMP-2. Despite its promising anticancer efficacy, the clinical translation of Plumbagin may be limited by potential adverse effects, such as cytotoxicity, hepatotoxicity, nephrotoxicity, immunosuppression, and oxidative stress. To overcome these limitations, several strategies are being explored, including the use of nanoformulations (e.g., liposomal PLB or PEGylated PLB), targeted drug delivery systems, and combination therapies with protective agents. Structural modifications and prodrug strategies may also enhance tumor selectivity and minimize systemic toxicity. Future studies should include detailed pharmacokinetic and toxicological profiling of PLB to evaluate its safety and guide clinical development.

Conclusions

Our findings have provided novel insights into the anti-cancer mechanism of Plumbagin, demonstrating its capacity to impede tumor growth, invasion, and metastasis in human oral squamous cell carcinoma by disrupting the GLUT1/MMP-2 signaling axis. These results highlight PLB as a promising therapeutic candidate for OSCC, warranting further exploration in clinical trials to evaluate its efficacy and safety in patients. Additionally, the development of PLB analogs with enhanced bioavailability and targeted delivery could optimize its therapeutic potential for future clinical applications.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Author contributions

F.H., W.W., S.A.E, and S.N. contributed to data collection, interpretation of data, designing the study, and writing the original manuscript. S.A.E has critically revised the manuscript. J.T, Y.H, and S.J contributed to the study conception and design and critically reviewed the manuscript. All authors have approved the final manuscript before its submission.

Funding

This work was supported by the National Natural Science Foundation of China (Grant No. 81860477).

Data availability

The datasets used and analyzed during the study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

The study was conducted by the Declaration of Helsinki and received approval from the Medical Ethics Committee of Xi’an Jiaotong University’s Stomatological Hospital (2022-XJKQIEC-009-003). Informed consent was obtained from all patients.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.