Inhibition of proliferation and metastasis of nasopharyngeal carcinoma by kaempferol via down-regulation of c-Jun

Introduction

Nasopharyngeal carcinoma (NPC) is a primary head and neck malignancy originating from the nasopharyngeal mucosal epithelium. The standard therapeutic strategies for NPC include radiotherapy, chemotherapy, and molecular-targeted therapy (1). However, owing to its highly malignant, aggressive invasiveness and strong propensity for distant metastasis, patients with NPC often experience poor therapeutic outcomes accompanied by significant clinical challenges, such as locoregional recurrence, acquired radio-chemoresistance, and treatment-related toxicities (2). These factors collectively contribute to suboptimal survival and compromised quality of life in patients with NPC, underscoring the urgent need for more effective and safer therapeutic strategies (3). Angiogenesis is widely recognized as a critical driver of NPC progression and has a profound impact on patient prognosis (4). This highlights the substantial clinical significance and promising therapeutic potential of anti-angiogenic strategies in NPC management.

In recent years, substantial research has been devoted to evaluating the efficacy of natural compounds in oncology. Phytochemicals with phenolic structures, such as flavonoids, have exhibited antiproliferative effects in cancer cells while maintaining a favorable safety profile (5). Among these, Kaempferol (Kae) has emerged as one of the most extensively studied flavonoids, owing to its broad spectrum of potential health benefits (6). Preclinical studies have consistently demonstrated that Kae possesses potent anti-tumor, anti-inflammatory, cardioprotective, and neuroprotective properties (7,8). Established studies have revealed that Kae exerts its inhibitory effect on the proliferation of various tumor cells by inducing apoptosis (9,10). Moreover, Kae can modulate diverse cellular signaling pathways that disrupt the critical survival and regulatory mechanisms in cancer cells (11). For instance, in prostate cancer, Kae has been shown to suppress androgen receptor signaling and downregulate Ki67 expression, thereby inhibiting tumor proliferation (12). Moreover, Kae modulates the PI3K/Akt pathway to reduce oxidative stress and facilitate apoptosis and cell cycle arrest, thereby inhibiting mammary tumorigenesis (13). Similarly, Kae interferes with the Wnt/β-catenin axis and JMJD2C-mediated signaling to restrain colorectal cancer progression (14). Importantly, beyond these direct anti-tumor effects, Kae also demonstrates anti-angiogenic potential by targeting vascular endothelial growth factor (VEGF) receptor-2 and downregulating the PI3K/AKT, MEK, and ERK pathways in VEGF-stimulated human umbilical vein endothelial cells (15). These findings provide a mechanistic rationale for exploring the potential anti-angiogenic and anti-tumor roles of Kae in NPC, suggesting that Kae may hold promise as a therapeutic agent for this malignancy. Nevertheless, the precise mechanism underlying its anti-cancer activity in NPC remains incompletely understood and warrants further investigation.

c-Jun, the most active transcription factor in the activator protein-1 (AP-1) complex (16), plays a regulatory role in regulating cell growth and metabolic function (17). As a vital oncogenic transcription factor, elevated c-Jun expression is strongly associated with the initiation, progression, and prognosis of numerous malignancies (18). Sun et al. reported that c-Jun expression was markedly higher in NPC tissues compared to normal nasopharyngeal mucosa (NNM) tissues, with c-Jun overexpression identified as an independent risk factor of poor clinical prognosis in NPC patients (19). Moreover, silencing c-Jun expression has been shown to suppress NPC cell growth (20), implicating c-Jun as a crucial driver in NPC pathogenesis. VEGF is a principal pro-angiogenic factor that promotes the formation of new blood vessels from the pre-existing vascular network, playing an essential role in the growth and metastasis of tumors (21). Overexpression of VEGF has been frequently observed in NPC patients (22), and c-Jun signaling exacerbates cancer angiogenesis by upregulating VEGF expression (23). Wu et al. indicated that Kae exerts anti-metastatic effects in oral cancer cells by inhibiting c-Jun activity (24). Based on these findings, we hypothesize that Kae may regulate angiogenesis in NPC via the c-Jun/VEGF axis, which may contribute to the suppression of tumor growth and metastasis and ultimately improve treatment outcomes in NPC.

The present study sought to delineate the anticancer effect of Kae on NPC both in vitro and in vivo, and to unravel the underlying molecular mechanism mediating its therapeutic action. The findings underscore the potential of Kae as a promising candidate in the treatment of NPC, offering valuable insights into its mechanistic role in inhibiting tumor progression. We present this article in accordance with the MDAR and ARRIVE reporting checklists (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1889/rc).

Methods

Cell culture

Human NPC cell lines C666-1 (iCell-h378) cells were obtained from iCell Bioscience Inc. (Shanghai, China). The cells were free of mycoplasma infection, as identified by STR. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin, and maintained in a humidified environment with 5% CO₂ at 37 ℃.

Cell Counting Kit-8 (CCK-8)

C666-1 cells were seeded into a 96-well plate at a density of 1×10⁴ cells/well and incubated for 24 hours to ensure complete adherence. Cell viability was evaluated using the CCK-8 assay kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions, with absorbance at 450 nm measured via a microplate reader.

Colony formation assay

C666-1 cells were plated in a 6-well plate at a density of 500 cells per well and subjected to the designated treatments, followed by incubation for 14 days under 5% CO₂ at 37 ℃ to allow for colony formation. A colony was defined as a cluster of ≥50 cells. Subsequently, cells were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 10 min, and stained with 0.1% crystal violet for 10 min. For quantification, images of each well were captured, and colonies were counted manually. To ensure objectivity and reproducibility, counting was performed independently by two investigators blinded to the treatment groups, and the colony numbers were quantified from at least three independent wells per group. The colony formation rate was calculated as (number of colonies counted / number of cells initially seeded) × 100%.

Flow cytometry for apoptosis analysis

C666-1 cells were treated with 0, 10, 20, and 40 µM Kae for 48 hours. Apoptotic rates were assessed using the Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (BD Biosciences, San Jose, CA, USA; 556547). Then, the cells were harvested, washed twice with PBS, and incubated with Annexin V and propidium iodide (PI). Flow cytometric analysis was performed via a flow cytometer (BD Biosciences).

Wound healing assay

C666-1 cells were cultured in 6-well plates until reaching 70–80% confluency. A uniform linear scratch was then carefully generated in each well using a 200-µL sterile pipette tip, and detached cells were gently removed by washing with PBS. Cells were subsequently maintained in a serum-free medium to exclude the influence of cell proliferation. Wound closure was monitored at 0, 24, and 48 hours after scratching, and images were captured at identical positions using an inverted microscope. The migration ability was quantified by measuring the scratch width at each time point using ImageJ software, and the wound closure rate was calculated as [(initial wound width − wound width at indicated time) / initial wound width] × 100%. Each experiment was performed in triplicate.

Transwell

Cell invasion was assessed using Transwell chambers with 8-µm pore polycarbonate membranes (Corning, New York, NY, USA). The upper surface of the membranes was pre-coated with 50 µL of Matrigel diluted at 1:8 in serum-free medium and incubated at 37 ℃ for 30 min to allow gelation. C666-1 cells were starved in serum-free Ham’s F-12K medium for 24 h, then harvested, washed twice with PBS, and resuspended in serum-free medium at a final density of 2´10⁵ cells/mL. A total of 200 µL of the cell suspension was seeded into the upper chamber, while 600 µL of complete medium supplemented with 10% fetal bovine serum (FBS) was added to the lower chamber as a chemoattractant. After incubation for 48 hours at 37 ℃ with 5% CO₂, non-invading cells on the upper surface of the membrane were gently removed with cotton swabs. The cells that had invaded through the Matrigel and membrane to the lower surface were fixed with 4% formaldehyde for 10 minutes and stained with 0.1% crystal violet for 30 minutes at room temperature. After air-drying, the membranes were carefully excised and mounted on glass slides. After air-drying, the membranes were carefully excised and mounted on glass slides. The number of invaded cells was counted under a light microscope (Olympus, Tokyo, Japan) at ×100 magnification in three randomly selected fields. Each experiment was independently repeated three times.

Quantitative reverse transcription polymerase chain reaction (qRT-PCR)

Total RNA was isolated using TRIzol reagent, and first-strand cDNA synthesis was conducted utilizing the Hifair^®II 1st Strand cDNA Synthesis SuperMix for qRT-PCR. qRT-PCR was carried out on an ABI 7300 real-time PCR system with Hieff^® qPCR SYBR Green Master Mix. The primer sequences utilized in the analysis were detailed below: c-Jun-F: 5'-GTGCCGAAAAAGGAAGCTGG-3'; c-Jun-R: 5'-CTGCGTTAGCATGAGTTGGC-3'; VEGF-F: 5'-ATCCAATCGA GACCCTGGTG-3'; VEGF-R: 5'-ATCTCTCCTATGTGCTGGCC-3'; GAPDH-F: 5'-TCAAGAAGGTGGTGAAGCAGG-3'; GAPDH-R: 5'-TCAAAGGTGGAGGAGTGGGT-3'.

Western blotting

C666-1 cells and tumor tissues were lysed using radioimmunoprecipitation assay (RIPA) buffer (Beyotime), and the extracted protein was separated on a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel before being transferred onto nitrocellulose membranes. Membranes were blocked with 5% non-fat milk and incubated with primary and secondary antibodies (Beyotime), and the protein detection was carried out using an ECL kit (Pierce, Rockford, IL, USA). The primary antibodies utilized in this study included c-Jun (CST, Danvers, MA, USA; 9165T), VEGF (Abclonal, Wuhan, China; A0280), and GAPDH (Proteintech, Rosemont, IL, USA; 60004-1-Ig).

Cell transfection

To knock down c-Jun expression, three predesigned siRNAs targeting c-Jun (c-Jun siRNA-11, -755, and -915) were synthesized by GENCEFE Biotech (Wuxi, China), with a negative control siRNA (NC siRNA) serving as the transfection control. The sequences were as follows: c-Jun siRNA-11, sense: 5'-AGAUGGAAACGACCUUCUATT-3', Antisense: 5'-UAGAAGGUCGUUUCCAUCUTT-3'; c-Jun siRNA-755, Sense: 5'-GGAUCAAGGCGGAGAGGAATT-3', Antisense: 5'-UUCCUCUCCGCCUUGAUCCTT-3'; c-Jun siRNA-915, sense: 5'-GGCACAGCUUAAACAGAAATT-3', Antisense: 5'-UUUCUGUUUAAGCUGUGCCTT-3'; NC siRNA, sense: 5'-UUCUCCGAACGUGUCACGUTT-3', antisense: 5'-ACGUGACACGUUCGGAGAATT-3'.

C666-1 cells were seeded in 6-well plates at a density of 3.5×10⁵ cells/well and incubated overnight at 37 ℃ in a humidified incubator with 5% CO₂. Transfection was performed using jetPRIME^® transfection reagent (Polyplus, Illkirch, France; 101000046) according to the manufacturer’s instructions. Briefly, 15 nM of each siRNA was diluted in 200 µL jetPRIME^® buffer, mixed with 4 µL jetPRIME^® reagent, vortexed gently, and incubated at room temperature for 10 minutes. The transfection mixture was then added to each well containing cells in serum-containing medium. After 4–6 hours of incubation at 37 ℃, the medium was replaced with fresh complete medium. Cells were harvested 48 hours post-transfection for qRT-PCR and Western blot analysis to assess the transfection efficiency.

Animals and xenograft experiments

Four-week-old BALB/c nude mice were procured from Zhuhai BesTest Bio-Tech Co. and acclimatized for one week under standardized laboratory conditions. The animals were housed in a specific pathogen-free (SPF) environment with a relative humidity of 60–65% and a temperature of 23±2 ℃, maintained on a 12-hour light/dark cycle, with free access to standard chow and water. Experiments were performed under a project license (No. 202510245) granted by the Ethics Review Institutional Animal Care and Use Committee (IACUC), Hubei Provincial Center for Disease Control and Prevention, in compliance with internationally recognized national and institutional guidelines for the care and use of animals. A protocol was prepared before the study without registration.

To establish the tumor xenograft model, C666-1 cells (2×10⁶ per mouse) were subcutaneously injected into the right axillary region of BALB/c nude mice, and the mice were randomly allocated into two groups (n=5 per group): the NC group and the Kae treatment group. When the inoculated tumor volume reached approximately 100–200 mm³, mice in the treatment group received Kae via oral gavage at a dose of 20 mg/kg once daily for 24 days, while control mice were administered an equivalent volume of physiological saline. Tumor size was measured every three days using vernier calipers, and volumes were calculated accordingly. During treatment, animal health and behavior (including activity, coat condition, posture, signs of paralysis or respiratory distress) were monitored daily. All animals were observed to be in normal condition throughout the experiment. The total experimental duration was 38 days. Ten mice were used in total, this sample size and grouping design were determined based on two key considerations: first, to ensure that each group had a sufficient number of biological replicates to reduce the impact of individual differences between mice on experimental results, which is essential for obtaining statistically reliable data; second, the 10 mice (5 per group) are sufficient to support the subsequent experimental analyses planned in this study, with no animal found dead during the experiment. At the experimental endpoint, all 10 mice were humanely euthanized using CO₂ inhalation, in accordance with institutional ethical guidelines. The humane endpoint was defined as a tumor volume exceeding 2,000 mm³, or signs of severe distress or illness, none of which were reached prior to planned euthanasia. At the endpoint, the attending veterinarian confirmed the death of the mice based on the disappearance of heartbeat and breathing, and the tumors were excised, rinsed with saline, weighed, and fixed in 4% paraformaldehyde for subsequent analyses.

TUNEL staining

Tumor tissues were fixed in formalin, dehydrated, embedded in paraffin, and sectioned into 4 µm slices. Apoptotic cells were detected using the One-Step TUNEL Apoptosis Assay Kit (Beyotime) according to the manufacturer’s protocol. Sections were incubated with TUNEL reaction mixture at 37 ℃ for 2 hours, counterstained with PI, and subsequently examined under a fluorescence microscope (Axioskop 2, Zeiss) at 200× magnification.

Hematoxylin-eosin (H&E) staining

H&E staining was performed to evaluate the pathological features of the tumor tissues, which were first subjected to paraffin embedding, cut into 4 µm-sections, and then underwent routine H&E staining. The images were captured under a microscope.

Statistical analysis

All data are presented as means ± standard deviation (SD). Statistical analyses were performed utilizing GraphPad Prism 8.4.2 software. Comparisons between two groups were made using an unpaired Student’s t-test. For comparisons among multiple groups, one-way analysis of variance (ANOVA) was performed followed by Tukey’s multiple comparisons test, with P value <0.05 deemed statistically significant.

Results

Kae suppresses the proliferation of C666-1 cells in a concentration-dependent and time-dependent manner

In the present study, C666-1 cells were exposed to increasing concentrations of Kae for 48 h (Figure 1A). The results revealed that Kae treatment (≥5 µM) notably reduced cell viability (P=0.02, P<0.001), with a half-maximal inhibitory concentration (IC50) value of 48.33 µM (Figure 1B). Consequently, Kae concentrations of 10, 20, and 40 µM were selected for subsequent experiments, as concentrations exceeding the IC50 threshold may induce nonspecific cytotoxic effects. To accurately assess the biological effects of Kae on C666-1 cells, experimental concentrations were maintained below the IC50, in alignment with the most common drug concentration selection criteria (25). Further investigation into the time-dependent effects of Kae on C666-1 cell viability was conducted using the CCK-8 assay at 0, 24, 48, and 72 h (Figure 1C-1F). The findings indicated that at 24 hours, a significant reduction in cell viability was observed only at a Kae concentration of 40 µM (P=0.003). However, at 48 and 72 hours, even 10 µM Kae treatment significantly reduced cell viability (P<0.001), with progressively greater inhibition observed at higher concentrations. These results suggest that Kae exerts a concentration- and time-dependent inhibitory effect on the viability of C666-1 cells. Consistently, the colony formation assay further demonstrated that Kae significantly impaired the clonogenic ability of C666-1 cells in a concentration-dependent manner (P<0.001; Figure 1G), underscoring its potent anti-proliferative activity in C666-1 cells.

Figure 1 Kae suppresses the proliferation of C666-1 cells. (A) CCK-8 assay of C666-1 cells treated with increasing concentrations of Kae for 48 h. (B) Calculation of IC50 values of C666-1 cells obtained by fitting Kae. (C-F) CCK-8 assay of C666-1 cells treated with Kae (10, 20, and 40 µM) for 0, 24, 48, and 72 h. (G) The clone formation rates of C666-1 cells treated with Kae were examined by colony formation assay (crystal violet staining). *, P<0.05; **, P<0.01; ***, P<0.001 vs. Blank. CCK-8, Cell Counting Kit-8; IC50, half-maximal inhibitory concentration; Kae, Kaempferol.

Kae induces apoptosis and inhibits the migration and invasion of C666-1 cells

The role of Kae in inducing apoptosis in C666-1 cells was examined through flow cytometric assay, and the findings revealed that exposure to 10, 20, and 40 µM Kae for 48 h markedly elevated the proportion of apoptotic cells, exhibiting a concentration-dependent elevation in apoptotic cell rates (Figure 2A; P<0.001). Moreover, the impact of Kae on the migratory and invasive capabilities of C666-1 cells was evaluated. Wound healing assays were employed to assess cell migratory capacity, with relative migration rates quantified according to the distance traversed by the cells. The data demonstrated that all concentrations of Kae considerably diminished the migration rate of C666-1 cells at both 24 and 48 h (Figure 2B; P<0.001). In addition, Kae treatment markedly decreased the number of cells invading through the Matrigel, as detected by Transwell assays (Figure 2C; P<0.001). These results collectively demonstrate that Kae significantly induced apoptosis and suppressed cell migration and invasion in a concentration-dependent manner.

Figure 2 Kae induces apoptosis and inhibits the migration and invasion of C666-1 cells. (A) Flow cytometric analysis of apoptosis in C666-1 cells exposed to Kae at different concentrations for 48 h. (B) Cell migration was assessed via the Wound healing assay (´100). (C) Cell invasion was detected through the Transwell assay (crystal violet staining, ´200). ***, P<0.001 vs. Blank. Kae, Kaempferol.

Kae inhibits the mRNA and protein of c-Jun and VEGF in C666-1 cells

To delve deeper into the antitumor mechanisms of Kae in C666-1 cells, the expression levels of c-Jun and VEGF were analyzed using qRT-PCR and Western blot. As illustrated in Figure 3A-3C, Kae treatment led to a pronounced downregulation of both mRNA (c-Jun: P=0.004, P<0.001; VEGF: P=0.03, P<0.001) and protein (c-Jun: P=0.02, P<0.001; VEGF: P=0.004, P<0.001) expression levels of c-Jun and VEGF. Furthermore, with increasing concentrations of Kae, the mRNA and protein expression of c-Jun and VEGF showed a further significant decline (P<0.001), indicating a dose-dependent inhibitory effect. As a proto-oncogene, c-Jun is overexpressed across various malignancies, where it plays a crucial role in promoting tumor angiogenesis by transcriptionally regulating VEGF expression (23), suggesting that Kae may exert its antitumor effects by modulating c-Jun, thereby impeding VEGF-mediated signaling and disrupting angiogenesis, ultimately suppressing the proliferation and metastatic potential of NPC cells.

Figure 3 Kae inhibits the mRNA and protein of c-Jun and VEGF in C666-1 cells. (A,B) The mRNA levels of c-Jun and VEGF were tested by qRT-PCR after treatment with increasing concentrations of Kae (10, 20, and 40 μM). (C) Protein expression of c-Jun and VEGF were assessed by Western blot after treatment with increasing concentrations of Kae (10, 20, and 40 μM). Densitometric quantification of protein bands was performed using ImageJ software, and relative protein expression was calculated by normalizing c-Jun and VEGF intensity to loading control GAPDH. *, P<0.05; **, P<0.01; ***, P<0.001 vs. Blank. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Kae, kaempferol; qRT-PCR, quantitative reverse transcription polymerase chain reaction; VEGF, vascular endothelial growth factor.

Silencing of c-Jun attenuates the inhibitory effect of Kae on the proliferation of C666-1 cells

To validate our hypothesis, c-Jun siRNA was designed and transfected into C666-1 cells to determine the role of c-Jun in mediating the effects of Kae on NPC cell survival. The silencing efficiency of three siRNA sequences was assessed using qRT-PCR (Figure 4A) and Western blot (Figure 4B), revealing that all siRNA groups exhibited varying degrees of inhibition on c-Jun expression (P<0.01). Among them, siRNA-1 demonstrated the strongest inhibitory effect on both mRNA and protein expression of c-Jun and was selected as the optimal siRNA for subsequent experiments.

Figure 4 Silencing of c-Jun attenuates the inhibitory effect of Kae on the proliferation of C666-1 cells. (A,B) The silencing efficiency of siRNAs on c-Jun mRNA and protein levels were assessed through qRT-PCR and Western blot analysis. (C-F) CCK-8 assay of C666-1 cells treated with Kae, c-Jun siRNA, and c-Jun siRNA + Kae for 0, 24, 48, and 72 h. (G) The clone formation rates of C666-1 cells treated with Kae, c-Jun siRNA, and c-Jun siRNA + Kae were examined by colony formation assay (crystal violet staining). **, P<0.01 and ***, P<0.001 vs. NC siRNA or Blank. Kae, kaempferol; NC, negative control; qRT-PCR, quantitative reverse transcription polymerase chain reaction.

Next, we explored the time-dependent inhibitory effect of Kae on cell viability under c-Jun gene silencing conditions (Figure 4C-4F). Treatment with c-Jun siRNA alone led to a significant decline in C666-1 cell viability after 24 hours (P<0.01), indicating that c-Jun silencing markedly attenuates C666-1 cell viability. However, upon co-treatment with Kae, c-Jun-deficient C666-1 cells displayed a partial restoration of viability compared to cells treated with Kae alone, suggesting that c-Jun gene silencing partially rescued the cell growth inhibition induced by Kae. Similarly, colony formation assays further corroborated that c-Jun silencing attenuated the inhibitory effect of Kae on the clonogenic ability of C666-1 cells (Figure 4G). These results indicate that c-Jun plays a crucial role in the mechanism of Kae’s antitumor activity against NPC. Specifically, Kae appears to primarily regulate cell proliferation and survival through the c-Jun signaling pathway.

Silencing of c-Jun inhibits the pro-apoptotic and anti-migratory/invasive effects of Kae on C666-1 cells

As illustrated in Figure 5A, flow cytometric analysis revealed a significant increase in the apoptosis rate of C666-1 cells following Kae treatment (P<0.001), while c-Jun silencing also exhibited a pro-apoptotic effect. However, the apoptosis rate in the c-Jun siRNA + Kae group was markedly decreased compared to the Kae treatment group (P<0.001) and was nearly equivalent to that in the c-Jun siRNA group. This finding suggested that the pro-apoptotic effect of Kae was attenuated upon c-Jun silencing. Furthermore, wound healing assay results further substantiated that the anti-migratory effect of Kae on C666-1 cell migration was attenuated following c-Jun silencing, with a significant difference observed at 48 hours (Figure 5B; P<0.001). Likewise, Transwell assays demonstrated that both Kae treatment and c-Jun knockdown significantly suppressed the invasive capacity of C666-1 cells (P<0.01). Nevertheless, in the presence of c-Jun gene silencing, the inhibitory effect of Kae on C666-1 cell invasion was also diminished, rendering it comparable to that of the c-Jun siRNA group (Figure 5C). These findings underscore the critical role of c-Jun as a molecular target underlying the anti-migratory and anti-invasive effects of Kae, indicating that the suppression of NPC cell migration and invasion by Kae is dependent on c-Jun expression.

Figure 5 Silencing of c-Jun inhibits the pro-apoptotic and anti-migratory/invasive effects of Kae on C666-1 cells. (A) Flow cytometric analysis of C666-1 cells apoptosis. (B) Cell migration was assessed via the Wound healing assay (´100). (C) Cell invasion was detected through the Transwell assay (crystal violet staining, ´200). **, P<0.01; ***, P<0.001 vs. Blank. ^###, P<0.01 vs. Kae. FITC, fluorescein isothiocyanate; Kae, Kaempferol.

Kae impedes tumor growth and c-Jun/VEGF expression in vivo

To further validate the in vitro findings, a xenograft model was established in BALB/c nude mice. As shown in Figure 6A-6C, Kae treatment significantly reduced tumor volume compared to the NC group, while no significant difference in body weight was observed between the two groups, indicating robust antitumor efficacy with low toxicity in vivo. TUNEL staining revealed a marked increase in apoptotic cells in the Kae-treated tumors (P=0.049; Figure 6D), suggesting that Kae effectively induces apoptosis in NPC tissues. Moreover, H&E staining showed that tumors in the NC group exhibited densely packed tumor cells with large, hyperchromatic nuclei, and no apparent necrosis or inflammatory infiltration. In contrast, the Kae-treated group presented with focal hemorrhage and extensive infiltration of inflammatory cells within the tumor tissue (Figure 6E), indicating histological damage to the tumors.

Figure 6 Kae impedes tumor growth and c-Jun/VEGF expression in vivo. (A) The gross features of the tumor in the NC and Kae-treated groups (five samples in each group). (B) Tumor volume curve. (C) Body weight curve of mice in each group. (D) Representative fluorescence images of TUNEL assay (green: TUNEL-positive cells; blue: DAPI) and quantification of apoptotic rate (´20). (E) H&E staining of tumor tissues (´20). (F) Western blot analysis of c-Jun and VEGF protein levels in xenograft tumor tissues. Densitometric quantification of protein bands was performed using ImageJ software, and relative protein expression was calculated by normalizing c-Jun and VEGF intensity to loading control GAPDH. *, P<0.05; **, P<0.01, ***, P<0.001 vs. NC. DAPI, 4’,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Kae, Kaempferol; NC, negative control; VEGF, vascular endothelial growth factor.

Consistent with the in vitro results, Western blot analysis demonstrated a significant reduction in the protein expression levels of c-Jun and VEGF in the Kae-treated group compared to controls (P=0.002, P<0.0001; Figure 6F), supporting the hypothesis that Kae exerts its antitumor activity through suppression of the c-Jun/VEGF signaling pathway.

Discussion

NPC is a prevalent malignancy of the head and neck, frequently posing clinical challenges due to its concealed location and nonspecific symptoms during early stages (26). The present study demonstrates that Kae exerts a profound inhibitory effect on the proliferation of C666-1 cells in a concentration- and time-dependent manner, while simultaneously promoting apoptosis and attenuating both migratory and invasive behaviors. Mechanistic investigations reveal that Kae may suppress NPC cell growth and metastasis by downregulating c-Jun expression and consequently disrupting VEGF-mediated signaling, with the above results further validated in an in vivo xenograft mouse model. These findings not only deepen our understanding of the potential therapeutic role of Kae in NPC treatment but also position the c-Jun/VEGF axis as a promising therapeutic target for NPC. Moreover, this study provides new insights for future anticancer research and the development of novel treatment approaches.

Consistent with previous reports that underscore the growth-inhibitory effects of Kae in various cancer cell lines (12,27,28), our in vitro and in vivo results reveal that Kae substantially diminishes C666-1 cell viability and colony-forming ability, while significantly impeding tumor growth and inducing histological damage in a mouse xenograft model. Notably, even at relatively low concentrations (10 µM), Kae obviously impeded cell viability following 48 h of treatment, highlighting its potent cytotoxicity against NPC cells. Apoptosis induction is the well-established mechanism through which numerous anticancer agents exert their effects, and our flow cytometric analysis coupled with TUNEL staining confirmed that Kae markedly increased apoptotic cell populations, corroborating prior findings from previous studies in head and neck cancer (29) and cervical cancer (30). Furthermore, it’s pronounced to suppress migration and invasion, as evidenced by wound healing and Transwell assays, suggesting its potential to mitigate metastatic dissemination in NPC. Of particular interest, Kae treatment elicited a substantial downregulation of VEGF expression, which may underlie its anti-migratory and anti-invasive properties. Considering that VEGF functions as a pivotal regulator in tumor angiogenesis and metastasis (31) and its overexpression is closely linked to advanced clinical stage, lymph node metastasis, and poor prognosis in NPC patients (32), the inhibitory effect of Kae on VEGF provides further compelling evidence of its therapeutic promise in NPC management while underscoring the clinical relevance of targeting VEGF-related signaling pathways in this malignancy.

The central mechanistic insight from our study is the role of c-Jun in mediating the anticancer effects of Kae. As a proto-oncogene frequently upregulated in various malignancies, c-Jun is known for its ability to regulate VEGF transcription and promote tumor angiogenesis (23). Tsai et al. have reported that knockdown of c-Jun expression significantly suppressed VEGF expression (33). Consistent with this, our results revealed that Kae effectively downregulated the expression of c-Jun and VEGF in C666-1 cells as well as in an in vivo model, thereby disrupting angiogenesis and tumor progression. In the context of NPC, c-Jun acts as a key downstream effector of multiple oncogenic signaling pathways and transcriptionally activates a repertoire of target genes involved in cell cycle progression, apoptosis resistance, and epithelial-mesenchymal transition (EMT), thereby promoting tumor cell proliferation, survival, metastasis, and angiogenesis (34). Additionally, aberrant activation of the c-Jun/AP-1 signaling axis has been linked to radioresistance and enhanced metastatic potential in NPC, further underscoring its central regulatory role in driving NPC progression. To further elucidate the role of c-Jun in Kae’s antitumor activity, siRNA-mediated knockdown of c-Jun was performed in C666-1 cells. This intervention markedly inhibited cell viability, colony formation, migration, and invasion, underscoring that c-Jun is indeed a crucial regulator of NPC cell proliferation and metastasis. More importantly, the attenuation of Kae’s inhibitory effects on cell proliferation, migration, and invasion, accompanied by a partial reversal of its pro-apoptotic activity upon c-Jun knockdown, strongly suggests that Kae exerts its antitumor effects predominantly via the c-Jun/VEGF signaling axis. These findings align well with accumulating evidence from other tumor models indicating that Kae can suppress oncogenic transcriptional programs by targeting key signaling nodes upstream or downstream of c-Jun, thereby converging on pathways governing angiogenesis and metastatic dissemination (34,35). While prior research has highlighted the role of Kae in modulating PI3K/Akt/mTOR and JMJD2C/β-catenin pathways in cervical and colorectal cancers (14,30), our findings provide novel evidence that Kae primarily exerts its anti-NPC effects via the c-Jun/VEGF axis, which has remained largely unexplored in previous studies. These findings highlight the multifaceted regulatory potential of Kae and suggest the need for future broader mechanistic studies using a multi-omics approach to map the complete molecular picture of Kae activity in NPC.

Conclusions

Taken together, this study provides the first evidence that Kae exerts potent anti-tumor effects against NPC by suppressing cell proliferation, inducing apoptosis, and inhibiting migration and invasion via modulation of the c-Jun/VEGF axis. Importantly, this work identifies the c-Jun/VEGF pathway as a previously underexplored molecular target of Kae in NPC, thereby offering novel mechanistic insight into its anti-angiogenic and anti-metastatic potential. These findings not only expand the current understanding of Kae’s anticancer mechanisms but also highlight its promise as a low-toxicity therapeutic candidate for NPC.

Despite these promising findings, certain limitations must be acknowledged. First, our cellular experiments were conducted solely in the C666-1 cell line, and future studies should extend these observations to other NPC cell models, such as HK-1, to verify the generalizability of our results. Second, comprehensive pharmacokinetic/pharmacodynamic (PK/PD) evaluations are warranted to determine optimal dosing regimens and assess systemic safety profiles in vivo. In addition, prior evidence indicates that Kae enhances cisplatin-induced autophagy and reduces oxidative damage in renal models (36), while reversing 5-fluorouracil resistance in colorectal cancer cells (37); therefore, combinatorial strategies should be explored to evaluate Kae as an adjuvant to existing NPC therapies, including anti-angiogenic agents (e.g., bevacizumab), immune checkpoint inhibitors, and conventional chemoradiotherapy. Lastly, translational studies involving patient-derived organoids may help validate c-Jun and VEGF expression as predictive biomarkers of Kae sensitivity, thereby paving the way for biomarker-guided individualized therapy and further advancing Kae toward clinical application.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR and ARRIVE reporting checklists. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1889/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1889/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1889/prf

Funding: The study was supported by the Quanzhou Municipal Guiding Scientific and Technological Plan Projects in the Medical and Health Field (No. 2023N040S), Quanzhou Municipal Science and Technology Program Project (No.2023NS060), and Fujian Provincial Natural Science Foundation (No. 2023J01722).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1889/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Experiments were performed under a project license (No. 202510245) granted by the Ethics Review Institutional Animal Care and Use Committee (IACUC), Hubei Provincial Center for Disease Control and Prevention, in compliance with internationally recognized national or institutional guidelines for the care and use of animals. A protocol was prepared before the study without registration.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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