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. 2026 Feb 13;20:557101. doi: 10.2147/DDDT.S557101

Lycopene Enhances the Sensitivity of Oral Squamous Cell Carcinoma to Cisplatin Through Inhibition of the PI3K/Akt Signaling Pathway and Reversal of Epithelial-Mesenchymal Transition

Ran Wang 1,2,*, Xinxing Lu 3,*, Riyue Yu 2, Zhaochen Shan 1,✉
PMCID: PMC12912039  PMID: 41710578

Abstract

Background

Cisplatin-based chemotherapy for oral squamous cell carcinoma (OSCC) is limited by intrinsic inefficacy and toxicity. Lycopene, a natural carotenoid, may enhance cisplatin’s therapeutic potential.

Objective

To explore lycopene’s chemo-sensitizing effects on cisplatin and its mechanisms in OSCC.

Methods

In vitro, CAL-27/SCC-9 cells were treated with lycopene and/or cisplatin; cell viability, colony formation, migration, invasion, and apoptosis were detected, and protein expression of MRP-1, PI3K/Akt/mTOR pathway components, and EMT markers was analyzed by Western blot. In vivo, a nude mouse xenograft model was used to verify the combination’s effect on tumor growth.

Results

Compared with cisplatin alone, the lycopene-cisplatin combination significantly inhibited OSCC cell proliferation, colony formation, migration, and invasion, while promoting apoptosis. Mechanistically, lycopene reversed cisplatin-induced upregulation of MRP-1 and activation of the PI3K/Akt/mTOR pathway, and restored cisplatin-suppressed E-cadherin while reducing N-cadherin and EpCAM. In vivo, the combination reduced tumor growth vs cisplatin alone without increasing toxicity.

Conclusion

This is the first report that lycopene enhances cisplatin sensitivity in OSCC by coupling inhibition of MRP-1-mediated drug efflux with suppression of PI3K/Akt/mTOR and EMT/stemness—filling the gap of lycopene’s synergistic mechanism with cisplatin, offering a strategy to improve therapeutic efficacy without exacerbating toxicity.

Keywords: oral squamous cell carcinoma, lycopene, cisplatin, chemoresistance, PI3K/Akt pathway

Introduction

Oral squamous cell carcinoma, the most prevalent subtype of head and neck malignancies, represents a substantial global health challenge attributable to its rapid invasive growth, high rates of recurrence, and limited efficacy of current therapeutic interventions.1,2 Despite advances in multimodal therapies, cisplatin—a platinum-based chemotherapeutic agent—remains a cornerstone for OSCC treatment.3 However, its clinical utility is severely constrained by drug resistance, as well as dose-dependent systemic toxicity, which lead to therapeutic failure and poor patient survival.4

The mechanisms underlying cisplatin resistance in OSCC are multifactorial, involving dysregulated survival signaling pathways and adaptive cellular plasticity.5 The PI3K/Akt/mTOR signaling pathway, a critical regulator of cell proliferation and apoptosis evasion, is frequently hyperactivated in resistant tumors, promoting chemoresistance and metastasis.6 Concurrently, epithelial-mesenchymal transition (EMT), a process marked by loss of epithelial marker (E-cadherin) and gain of mesenchymal markers (N-cadherin), facilitates tumor invasion, stemness acquisition, and therapeutic escape.7 These pathways not only drive resistance but also create a permissive microenvironment for tumor recurrence. Current strategies to overcome resistance, such as kinase inhibitors or EMT-targeting agents, face challenges in specificity and toxicity, underscoring the need for safer and multi-target approaches.8,9

Lycopene, a natural carotenoid found abundantly in tomatoes and watermelons, has garnered attention for its potential chemo-preventive and chemotherapeutic properties.10 Extensive researches have demonstrated lycopene’s potent antioxidant and antitumor activities, suggesting it may enhance the efficacy of conventional chemotherapeutic agents.11 Our previous research has demonstrated that lycopene is capable of suppressing PI3K/AKT pathway and EMT process in oral cancer,12 leading us to hypothesize that lycopene may have the potential to enhance the sensitivity of cisplatin, offering a promising strategy to overcome resistance and improve treatment outcomes.

In this context, our study aims to explore the chemo-sensitizing effects of lycopene on cisplatin and its underlying mechanisms in OSCC. Through comprehensive in vitro and in vivo experiments, we seek to provide evidence for the clinical application of lycopene as a cisplatin adjuvant, ultimately laying the groundwork for the advancement of more potent and well-tolerated treatment protocols to benefit OSCC patients in clinical practice.

Materials and Methods

Ethics Statement

All animal experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Capital Medical University (approval No. KYD-2024-0002-002, approval date: 2024–10-22). All procedures were performed in strict compliance with the Guide for the Care and Use of Laboratory Animals13 to ensure the welfare of laboratory animals. Additionally, the study adheres to the ARRIVE guidelines to guarantee the transparency and completeness of in vivo experiment reporting.14

Antibodies and Reagents

Cisplatin (DDP), lycopene (LYC), dimethyl sulfoxide (DMSO), and corn oil were purchased from Sigma. The primary antibodies used in this study included anti-MRP1, anti-β-actin, anti-p-Akt, anti-Akt, anti-p-mTOR, anti-mTOR, anti-Bax, anti-Bcl-2, anti-EpCAM, anti-N-cadherin, and anti-E-cadherin, all diluted at 1:1000. Additionally, HRP-labeled goat anti-mouse IgGs were used as secondary antibodies at a dilution of 1:2000. All antibodies were sourced from Abcam.

Cell Lines and Culture

Human OSCC cell lines CAL-27 and SCC-9 were obtained from the Cell Bank of Peking Union Medical College. Cells were maintained in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C in a humidified 5% CO2 incubator. Cells were routinely tested for mycoplasma contamination and used within 20 passages.

Cell Proliferation Assay

Cell viability was assessed using the CCK-8 kit (BioTECH) per the manufacturer’s protocol. Briefly, 5 × 103 cells were seeded in 96-well plates overnight. Cells were treated with indicated concentrations of LYC (0–8 μM), indicated concentrations of cisplatin (0–100 μM), or their combination for 24–48 h. Subsequently, 10 μL CCK-8 reagent was added to each well, and absorbance was measured at 450 nm using a microplate reader. Each experiment was independently repeated 5 times.

Colony Formation Assay

Cells were exposed to vehicle control, 0.5 μM lycopene (LYC), 5 μM cisplatin (DDP), or the combination of LYC and DDP for 48 hours. Following trypsinization, 200 cells per well were seeded into 6-well plates. After a 14-day incubation period, formed colonies were fixed with 4% paraformaldehyde, stained using Giemsa solution, and visualized under a dissecting microscope (Nikon, Tokyo, Japan) for counting. Colonies consisting of more than 50 cells were defined as viable for quantitative analysis. Each experiment was independently repeated 5 times.

Cell Migration Assay

Cells were cultured in 6-well plates until 90% confluency. Subsequently, cells were treated with vehicle control, 0.5 μM lycopene (LYC), 5 μM cisplatin (DDP), or the combination of LYC and DDP for 24 hours. A sterile pipette tip was used to create a linear scratch in the cell monolayer. Wound closure dynamics were tracked and photographed at 0 and 24 hours under an inverted microscope, while the degree of wound healing was quantified using Image J software (Scion). Each independent experiment was replicated five times to ensure result reliability.

Cell Invasion Assay

Cell invasion capacity was assessed using 24-well Biocoat cell culture inserts (BD Biosciences) equipped with 8-μm pore membranes pre-coated with Matrigel (1 mg/mL; BD Biosciences). The Matrigel-coated membranes were incubated at 37°C for 6 hours to allow gel formation. Cells were pre-treated with vehicle control, 0.5 μM lycopene (LYC), 5 μM cisplatin (DDP), or the combination of LYC and DDP for 48 hours, then seeded into the upper chamber of the inserts in serum-free medium. The lower chamber was filled with medium containing 10% fetal bovine serum (FBS) to act as a chemoattractant. After incubating for 24 hours, non-invading cells adhering to the upper membrane surface were carefully wiped off. Invading cells that had migrated to the lower surface were washed with PBS, fixed with paraformaldehyde, stained with Giemsa, and photographed for quantification. Each experiment was independently repeated 5 times.

Apoptosis Analysis by TUNEL Assay

Apoptotic cell death was assessed using the TUNEL Apoptosis Detection Kit (KeyGEN BioTECH, Nanjing, China) following the manufacturer’s protocol. CAL-27 and SCC-9 cells were seeded in 12-well plates at 1×105 cells and allowed to adhere overnight. Cells were then treated with vehicle control, 0.5 μM lycopene (LYC), 5 μM cisplatin (DDP), or LYC+DDP combination for 48 h.

Post-treatment, cells were fixed with 4% paraformaldehyde (30 min, room temperature), permeabilized with 0.1% Triton X-100 (10 min), and incubated with TUNEL reaction mixture (enzyme + label solution) for 1 h at 37°C in the dark. Nuclei were counterstained with DAPI (Sigma-Aldrich) for 5 min. Apoptotic cells (TUNEL-positive, red fluorescence) and total nuclei (DAPI, blue fluorescence) were visualized via fluorescence microscopy (Nikon Eclipse Ti, Tokyo, Japan). Five random fields per well were captured, and apoptosis rate was calculated as the ratio of TUNEL-positive cells to total nuclei. Each experiment was independently repeated five times.

Western Blot Analysis

Total protein was extracted using RIPA lysis buffer (Beyotime, Shanghai, China) containing protease and phosphatase inhibitors. Protein concentrations were measured by BCA assay. Equal amounts of protein were separated by SDS-PAGE, transferred to PVDF membranes, and probed with primary antibodies overnight at 4°C. Blots were developed using an ECL detection system and quantified using ImageJ software (NIH, Bethesda). β-actin served as the loading control. Each independent experiment was replicated five times to ensure result reliability.

Animal Study

Male Balb/c nude mice (weighing 20–22 g, aged 6–8 weeks) were procured from the Experimental Animal Center of Capital Medical University and allowed to acclimate to the laboratory environment for one week prior to experimentation. CAL-27 cells (5 × 106) suspended in a 1:1 mixture of Matrigel and PBS were subcutaneously injected into the right flank. When tumors reached ~100 mm3, mice were randomized into four groups (n = 6/group):

  1. Vehicle control (corn oil, daily oral gavage)

  2. Lycopene (LYC) (LYC 6 mg/kg/day, intragastrically)

  3. Cisplatin (DDP) (DDP 3 mg/kg/week, intraperitoneally)

  4. Combination (LYC 6 mg/kg/day, intragastrically + DDP 3 mg/kg/week, intraperitoneally).

Tumor volume was measured every 3 days using calipers and calculated as (length × width2)/2.15 Mice were euthanized after 28 days, and tumors were excised for further analysis.

Immunofluorescence Staining

Xenograft tumor tissues were fixed in 10% phosphate-buffered formalin and embedded in paraffin for subsequent immunofluorescence analysis. Five-micrometer-thick paraffin sections were first deparaffinized and rehydrated, then treated with 3% H2O2 to inhibit endogenous peroxidase activity. Sections were incubated overnight at 4°C with primary antibodies against PCNA or E-cadherin (1:100 dilution, Sigma-Aldrich), followed by secondary antibodies (Invitrogen). After staining, sections were mounted with DAPI for nuclear counterstaining and analyzed via fluorescence microscope. For quantification, five random fields per tumor section were captured. The positive rate (percentage of positive cells or fluorescence intensity) from these fields was averaged to generate a single value per tumor (biological replicate). These values (n=6 mice per group) were then used for statistical comparisons between treatment groups. All experiments were independently repeated five times.

Statistical Analysis

All experimental assays were independently conducted in five biological replicates, with each replicate executed as an entirely separate experiment to ensure result reliability. Statistical computations were carried out using SPSS software (version 22.0).

For data distribution assessment: Normality was verified via the Shapiro–Wilk test and Q–Q plots; homogeneity of variances was evaluated using Levene’s test.

For statistical description: Data conforming to normal distribution were presented as mean ± standard deviation (SD); data with skewed distribution were reported as median (interquartile range, IQR).

For group comparisons: One-way analysis of variance (ANOVA) was applied to normally distributed data with equal variance, followed by Tukey’s post-hoc test for pairwise comparisons. For normally distributed data with unequal variance (as indicated by Levene’s test), Welch’s ANOVA was used instead, with Games–Howell post-hoc test for pairwise comparisons. For skewed data, the non-parametric Kruskal–Wallis H-test was applied, followed by Dunn’s post-hoc test for pairwise comparisons. A p-value < 0.05 was considered statistically significant.

Results

Lycopene Enhances Cisplatin-Mediated Cytotoxicity in OSCC Cells

To investigate the combinatorial effects of lycopene (LYC) and cisplatin (DDP) on the proliferation of OSCC cells, we conducted CCK-8 and colony formation assays. The CCK-8 assay revealed that lycopene (LYC) or cisplatin (DDP) alone inhibited the proliferation of CAL-27 and SCC-9 cells in a dose-dependent manner (Figure 1A–D). The combination treatment (0.5 μM lycopene + 5 μM cisplatin) resulted in a more pronounced reduction in cell proliferation compared to either agent alone at both 24 and 48 hours post-treatment (Figure 1E and F; ANOVA, p<0.001; Tukey, 0.5LYC + 5DDP vs 5DDP, p<0.001). Remarkably, the growth inhibitory effect generated by the 0.5 μM lycopene + 5 μM cisplatin (0.5 LYC + 5 DDP) regimen was comparable to that elicited by the 10 μM cisplatin (10 DDP) treatment (Figure 1E and F; 0.5LYC + 5DDP vs 5DDP: CAL-27 24H, ANOVA, p<0.001; Tukey, p=0.166; CAL-27 48H, ANOVA, p<0.001; Tukey, p=0.025; SCC-9 24H, ANOVA, p<0.001; Tukey, p=0.568; SCC-9 48H, ANOVA, p<0.001; Tukey, p=0.203).

Figure 1.

Figure 1

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Lycopene (LYC) enhances cisplatin (DDP) sensitivity to suppress OSCC cell proliferation in a dose- and time-dependent manner. (A–D) CCK-8 assay: indicated cells were treated with different concentrations of LYC or DDP as indicated. Cell viability was determined in 24h. (E and F) CCK-8 assay: indicated cells treated with vehicle, 0.5 μM LYC, 5 μM DDP, 0.5 μM LYC + 5 μM DDP, or 10 μM DDP for 24/48 h. Statistical comparisons (Tukey’s test) for (E and F) CAL-27 (24h): LYC+DDP vs DDP, p=0.166; LYC+DDP vs Control, p < 0.001. CAL-27 (48h): LYC+DDP vs DDP, p=0.025; LYC+DDP vs Control, p < 0.001. SCC-9 (24h): LYC+DDP vs DDP, p=0.568; LYC+DDP vs Control, p < 0.001. SCC-9 (48h): LYC+DDP vs DDP, p=0.203; LYC+DDP vs Control, p < 0.001. Each experiment was independently repeated 5 times.

Colony formation assays further confirmed that lycopene sensitized OSCC to cisplatin. The combination treatment (0.5 μM lycopene + 5 μM cisplatin) resulted in a more pronounced reduction in cell colonies compared to either agent alone (Figure 2A–D; CAL-27: ANOVA, p<0.001; Tukey, LYC + DDP vs DDP, p=0.002, LYC + DDP vs LYC, p=0.003; SCC-9: ANOVA, p<0.001; Tukey, LYC + DDP vs DDP, p=0.008, LYC + DDP vs LYC, p=0.007), demonstrating that lycopene could enhance the chemotherapeutic efficacy of cisplatin.

Figure 2.

Figure 2

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Lycopene (LYC) synergizes with cisplatin (DDP) to suppress colony formation in oral squamous cell carcinoma (OSCC) cells. (A and B) Colony formation assay of CAL-27 cells treated with vehicle (Control), indicated LYC alone, indicated DDP alone, or LYC+DDP combination for 48h. (C and D) Colony formation assay of SCC-9 cells treated with vehicle (Control), indicated LYC alone, indicated DDP alone, or LYC+DDP combination for 48h. Statistical comparisons (Tukey’s test): CAL-27: LYC+DDP vs DDP, p=0.002; LYC+DDP vs LYC, p=0.003. SCC-9: LYC+DDP vs DDP, p=0.008; LYC+DDP vs LYC, p=0.007. Each experiment was independently repeated 5 times.

Lycopene Enhances Cisplatin’s Inhibition of Migration and Invasion in OSCC Cells

The wound healing assay assessed the impact of lycopene (LYC), cisplatin (DDP), and their combination (LYC+DDP) on CAL-27 and SCC-9 cell migration. The results showed that 5 μM cisplatin alone exerted a moderate inhibitory effect on migration, whereas the combination treatment (0.5 μM lycopene + 5 μM cisplatin) resulted in the greatest reduction in gap closure, thereby indicating that lycopene enhances cisplatin-mediated inhibition of cell migration (Figure 3A–D; CAL-27: ANOVA, p<0.001; Tukey, LYC + DDP vs DDP, p<0.001, LYC + DDP vs LYC, p<0.001; SCC-9: ANOVA, p<0.001; Tukey, LYC + DDP vs DDP, p=0.034, LYC + DDP vs LYC, p<0.001).

Figure 3.

Figure 3

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Lycopene (LYC) enhances cisplatin (DDP)-mediated inhibition of OSCC cell migration. Green dotted lines represent the initial scratch boundaries at 0 h and the reference boundaries for assessing scratch closure at 24 h in the wound healing assay, serving to quantify the gap closure rate. (A and B) Wound healing assay of CAL-27 cells treated with vehicle control, 0.5 μM LYC, 5 μM DDP, or 0.5 μM LYC + 5 μM DDP for 24 h. Images were captured at 0 h and 24 h to quantify gap closure. (C and D) Wound healing assay of SCC-9 cells under the same treatment conditions as above. Statistical comparisons (Tukey’s test): CAL-27: LYC+DDP vs DDP, p < 0.001; LYC+DDP vs LYC, p < 0.001. SCC-9: LYC+DDP vs DDP, p=0.034; LYC+DDP vs LYC, p < 0.001. Each experiment was independently repeated 5 times.

Similarly, the transwell assay revealed that the combination treatment (0.5 μM lycopene + 5 μM cisplatin) resulted in the fewest invasive cells, indicating lycopene enhances cisplatin’s anti-invasive effect, consistent with the migration assay results (Figure 4A–D; CAL-27: ANOVA, p<0.001; Tukey, LYC + DDP vs DDP, p=0.036, LYC + DDP vs LYC, p=0.032; SCC-9: ANOVA, p<0.001; Tukey, LYC + DDP vs DDP, p=0.036, LYC + DDP vs LYC, p=0.032). Collectively, these results indicate that lycopene significantly enhanced cisplatin’s inhibitory effect on cell migration and invasion.

Figure 4.

Figure 4

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Lycopene (LYC) enhances cisplatin (DDP)-mediated inhibition of OSCC cell invasion. (A and B) Transwell invasion assay of CAL-27 cells treated with vehicle control, LYC alone, DDP alone, or LYC+DDP combination. Invaded cells were stained and counted to evaluate invasive capacity. (C and D) Transwell invasion assay of SCC-9 cells under the same treatment conditions as above. Statistical comparisons (Tukey’s test): CAL-27: LYC+DDP vs DDP, p=0.036; LYC+DDP vs LYC, p=0.032. SCC-9: LYC+DDP vs DDP, p=0.036; LYC+DDP vs LYC, p=0.032. Each experiment was independently repeated 5 times.

Lycopene Potentiates Cisplatin-Induced Apoptosis in OSCC Cells

Apoptosis effect was evaluated using TUNEL assays. The results indicated that cisplatin alone caused a slight increase in apoptosis compared to the vehicle control group, while the combination treatment led to a markedly higher apoptosis rate compared to cisplatin alone (Figure 5A–D; CAL-27: ANOVA, p<0.001; Tukey, LYC + DDP vs DDP, p=0.002, LYC + DDP vs LYC, p=0.001; SCC-9: ANOVA, p<0.001; Tukey, LYC + DDP vs DDP, p<0.001, LYC + DDP vs LYC, p=0.001), demonstrating lycopene’s ability to sensitize OSCC cells to cisplatin-induced apoptosis.

Figure 5.

Figure 5

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Lycopene (LYC) promotes cisplatin (DDP)-induced apoptosis in OSCC cells. (A and B) TUNEL assay of CAL-27 cells treated with vehicle control, LYC alone, DDP alone, or LYC+DDP combination. Apoptotic cells (stained brown) were visualized and counted to assess apoptosis levels. (C and D) TUNEL assay of SCC-9 cells under the same treatment conditions as above. Statistical comparisons (Tukey’s test): CAL-27: LYC+DDP vs DDP, p=0.002; LYC+DDP vs LYC, p=0.001. SCC-9: LYC+DDP vs DDP, p < 0.001; LYC+DDP vs LYC, p=0.001. Each experiment was independently repeated 5 times.

We then analyzed apoptosis-related proteins via Western blotting. The results showed significantly upregulated Bax (Figure 6A–D, CAL-27: ANOVA, p<0.001; Tukey, LYC + DDP vs DDP, p<0.001; SCC-9: ANOVA, p<0.001; Tukey, LYC + DDP vs DDP, p<0.001) and downregulated Bcl-2 (Figure 6A–D; CAL-27: ANOVA, p<0.001; Tukey, LYC + DDP vs DDP, p<0.001; SCC-9: ANOVA, p<0.001; Tukey, LYC + DDP vs DDP, p=0.041) in the combination group compared to the cisplatin monotherapy group, indicating that lycopene enhances cisplatin-induced apoptosis.

Figure 6.

Figure 6

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Lycopene (LYC) synergizes with cisplatin (DDP) to modulate key signaling pathways and molecular markers in OSCC cells. (A) Western blot analysis of proteins related to drug efflux (MRP-1), PI3K/Akt/mTOR signaling (p-AKT, AKT, p-m-TOR, m-TOR), epithelial-mesenchymal transition (E-cadherin, N-cadherin, EpCam), and apoptosis (Bax, bcl-2) in CAL-27 and SCC-9 cells under different treatment conditions (Control, LYC alone, DDP alone, LYC + DDP). β-actin served as a loading control. B–E: Quantitative analysis of relative protein band densities normalized to the control group for CAL-27 (B and C) and SCC-9 (D and E) cells. Key statistical comparisons (Tukey’s test, see main text for full details): Bax (CAL-27 and SCC-9): LYC+DDP vs DDP, p < 0.001; Bcl-2 (SCC-9): LYC+DDP vs DDP, p=0.041; pAKT/AKT (CAL-27): DDP vs LYC+DDP, p=0.007; (SCC-9): p=0.009; p-mTOR/mTOR (CAL-27): DDP vs LYC+DDP, p < 0.001; (SCC-9): p=0.021; MRP-1 (CAL-27): Control vs DDP, p < 0.001, Control vs LYC, p < 0.001, Control vs LYC+DDP, p=0.083; (SCC-9): Control vs DDP, p < 0.001, Control vs LYC, p < 0.001, Control vs LYC+DDP, p=0.121; E-cadherin (SCC-9): LYC+DDP vs DDP, p < 0.001; N-cadherin (SCC-9): LYC+DDP vs DDP, p=0.006. Each experiment was independently repeated 5 times.

Lycopene Enhances Cisplatin Sensitivity in OSCC Cells via Modulation of MRP-1 Expression, EMT Process and PI3K/AKT/mTOR Signaling Pathway

To explore the mechanism of lycopene’s action on chemo-sensitivity, we examined the expression of MRP-1, which was a key protein involved in drug efflux and cisplatin chemoresistance.16,17 Western blot analysis revealed distinct patterns of MRP-1 expression across treatment groups. Compared to the vehicle control, cisplatin (DDP) monotherapy significantly upregulated MRP-1 expression in both CAL-27 (Figure 6A, C and E; ANOVA, p<0.001; Tukey, Control vs DDP, p<0.001) and SCC-9 (Figure 6A, C and E; ANOVA, p<0.001; Tukey, Control vs DDP, p<0.001) cells, consistent with its role in promoting drug efflux and chemoresistance. In contrast, lycopene (LYC) alone markedly downregulated MRP-1 expression (Figure 6A, C and E; CAL-27: ANOVA, p<0.001; Tukey, Control vs LYC, p<0.001; SCC-9: ANOVA, p<0.001; Tukey, Control vs LYC, p<0.001), demonstrating its intrinsic ability to suppress this resistance-associated transporter. Notably, the combination of LYC and DDP restored MRP-1 levels to near-baseline (control group) values (Figure 6A, C and E; CAL-27: ANOVA, p<0.001; Tukey, Control vs LYC+DDP, p=0.083; SCC-9: ANOVA, p<0.001; Tukey, Control vs LYC+DDP, p=0.121), effectively counteracting cisplatin-induced MRP-1 overexpression. This normalization of MRP-1 expression in the combination group correlated with enhanced cisplatin sensitivity, as evidenced by reduced cell viability and colony formation compared to DDP monotherapy (Figure 1E and F, 2A–D).

Subsequently, we examined the expression profiles of key components in the PI3K/AKT/mTOR signaling pathway using Western blotting analysis (Figure 6A). The results demonstrated that the phosphorylation levels of AKT and mTOR, which are critical for cell survival and proliferation,18 were markedly decreased in the LYC+DDP group compared to the DDP alone group (Figure 6A, B and D; CAL-27 pAKT/AKT: ANOVA, p<0.001, Tukey, DDP vs LYC+DDP, p=0.007; SCC-9 pAKT/AKT: ANOVA, p<0.001; Tukey, DDP vs LYC+DDP, p=0.009; CAL-27 p-mTOR/mTOR: ANOVA, p<0.001; Tukey, DDP vs LYC+DDP, p<0.001; SCC-9 mTOR/mTOR: ANOVA, p<0.001; Tukey, DDP vs LYC+DDP, p=0.021). This indicates that lycopene potentiates cisplatin’s inhibitory effect by deactivating the PI3K/AKT/mTOR signaling pathway.

Then we explored the expression of EMT markers19 (E-cadherin, N-cadherin) and cell stemness marker20 (EpCAM). Compared to the vehicle control, cisplatin monotherapy significantly downregulated E-cadherin (Figure 6A, C and E; CAL-27: ANOVA, p<0.001; Tukey, Control vs DDP, p=0.006; SCC-9: ANOVA, p<0.001; Tukey, Control vs DDP, p=0.031) while upregulating N-cadherin (Figure 6A, C and E; CAL-27: ANOVA, p<0.001; Tukey, Control vs DDP, p<0.001; SCC-9: ANOVA, p<0.001; Tukey, Control vs DDP, p<0.001) and EpCAM (Figure 6A, C and E; CAL-27: ANOVA, p<0.001; Tukey, Control vs DDP, p<0.001; SCC-9: ANOVA, p<0.001; Tukey, Control vs DDP, p<0.001) in both CAL-27 and SCC-9 cells, indicating cisplatin-induced EMT progression and stemness enhancement. In contrast, lycopene alone upregulated E-cadherin (Figure 6A, C and E; CAL-27: ANOVA, p<0.001; Tukey, Control vs LYC, p<0.001; SCC-9: ANOVA, p<0.001; Tukey, Control vs LYC, p<0.001) and suppressed N-cadherin (Figure 6A, C and E; CAL-27: ANOVA, p<0.001; Tukey, Control vs LYC, p<0.001; SCC-9: ANOVA, p<0.001; Tukey, Control vs LYC, p=0.037) and EpCAM (Figure 6A, C and E; CAL-27: ANOVA, p<0.001; Tukey, Control vs LYC, p=0.008; SCC-9: ANOVA, p<0.001; Tukey, Control vs LYC, p<0.001) expression. Notably, in the LYC+DDP group, LYC not only counteracted cisplatin-driven EMT but also attenuates stem cell characteristics in both cell lines: E-cadherin was upregulated (Figure 6A, C and E; CAL-27: ANOVA, p<0.001; Tukey, LYC+DDP vs DDP, p=0.042; SCC-9: ANOVA, p<0.001; Tukey, LYC+DDP vs DDP, p<0.001) and N-cadherin was downregulated (Figure 6A, C and E; CAL-27: ANOVA, p<0.001; Tukey, LYC+DDP vs DDP, p<0.001; SCC-9: ANOVA, p<0.001; Tukey, LYC+DDP vs DDP, p=0.006).

These results demonstrate that lycopene (LYC) effectively reverses the cisplatin (DDP)-induced epithelial-mesenchymal transition (EMT) process and attenuates stemness phenotypes in OSCC cells.

Lycopene Enhances Sensitivity of Cisplatin in a Nude Mouse Xenograft Model

To further validate the chemo-sensitizing effect of lycopene (LYC) on cisplatin (DDP) in oral squamous cell carcinoma (OSCC), we conducted in vivo experiments using a nude mouse xenograft model. The results demonstrated that lycopene significantly enhanced the antitumor efficacy of cisplatin. Specifically, the combination treatment group (LYC+DDP) exhibited the most substantial reduction in tumor volume and weight compared to the DDP group (Figure 7A–C; Tumor volume: ANOVA, p<0.001; Tukey, LYC+DDP vs DDP, p<0.001; Tumor weight: ANOVA, p<0.001; Tukey, LYC+DDP vs DDP, p<0.001), indicating that lycopene potentiates the chemo-therapeutic effect of cisplatin. In contrast, the DDP group showed limited tumor growth inhibition, while the LYC group showed moderate tumor reduction. The vehicle control group exhibited rapid tumor growth and the heaviest tumor weights. Notably, lycopene was well tolerated at the administered dose, with no significant differences in body weight or signs of toxicity observed among the groups (Figure 7D).

Figure 7.

Figure 7

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Lycopene (LYC) synergizes with cisplatin (DDP) to suppress tumor growth in OSCC xenograft models. (A) Representative images of tumors excised from nude mice bearing CAL-27 xenografts in each treatment group. (B) Growth curves of CAL-27 xenograft tumors over time, showing tumor volume (mm3) measured at indicated time points (Days 1–28). (C) Final tumor weights (mg) of excised CAL-27 xenografts in each treatment group. (D) Body weight changes of nude mice during the treatment period, indicating that the treatment did not cause significant systemic toxicity compared to control. Statistical comparisons (Tukey’s test): Tumor volume and weight: LYC+DDP vs DDP, p < 0.001. Sample size: n = 6 nude mice per treatment group.

Immunofluorescence staining revealed that the combination treatment significantly reduced PCNA-positive cells (Figure 8A and B, ANOVA, p<0.001; Tukey, LYC+DDP vs DDP, p=0.001) and increased E-cadherin-positive cells (an epithelial marker, Figure 8C and D, ANOVA, p<0.001; Tukey, LYC+DDP vs DDP, p=0.008), suggesting that lycopene reverses epithelial-mesenchymal transition (EMT) and enhances chemotherapeutic sensitivity.

Figure 8.

Figure 8

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Lycopene (LYC) synergizes with cisplatin (DDP) to modulate proliferation and epithelial - mesenchymal transition (EMT) - related markers in OSCC xenograft tumors. (A and B) Immunofluorescence staining of PCNA in tumor sections from different treatment groups. DAPI (blue) was used for nuclear staining. The merged images show the co-localization, and the positive rate of PCNA was quantified in (B). (C and D) Immunofluorescence staining of E-cadherin in tumor sections from different treatment groups. DAPI (blue) was used for nuclear staining. The merged images show the co-localization, and the positive rate of E-cadherin was quantified in (D). Statistical comparisons (Tukey’s test): PCNA: LYC+DDP vs DDP, p=0.001. E-cadherin: LYC+DDP vs DDP, p=0.008. Each experiment was independently repeated 5 times.

These findings suggest that lycopene enhances the sensitivity of OSCC cells to cisplatin in vivo, providing a promising strategy for improving chemotherapeutic outcomes in OSCC patients.

Discussion

Oral squamous cell carcinoma (OSCC), as a dominant subtype of head and neck malignancies, presents a severe challenge to global public health due to its aggressive biological behavior and limited therapeutic options.21 Despite advancements in multimodal therapies, cisplatin remains a cornerstone in its treatment.22 However, cisplatin is severely hampered by drug resistance and toxicity, which greatly limit its clinical utility. Thus, the exploration of novel therapeutic strategies is urgently needed.

Lycopene, a natural carotenoid, has demonstrated potential chemo-preventive and chemotherapeutic properties in numerous studies. Its antioxidant and antitumor activities have attracted extensive attention.10

Based on previous studies, it was supposed that lycopene could synergize with cisplatin to overcome chemoresistance in OSCC. In the present study, functional assays collectively demonstrated the synergistic efficacy of lycopene and cisplatin in OSCC. CCK-8 proliferation assays revealed that the combination of 0.5 μM lycopene and 5 μM cisplatin significantly reduced cell viability compared to either agent alone at 24 and 48 hours. Notably, the growth inhibitory efficacy of the 0.5 μM lycopene + 5 μM cisplatin regimen was comparable to that of the 10 μM cisplatin treatment (Figure 1E and F). This may indicate that lycopene can enhance the cytotoxicity of cisplatin, allowing for a potential reduction in the effective dose of cisplatin while maintaining or even improving therapeutic outcomes, which may help mitigate the dose dependent toxicity associated with high-dose cisplatin monotherapy. Colony formation assays further corroborated this effect, showing a drastic reduction in viable colonies under combination treatment (Figure 2A–D), indicative of long-term growth suppression. Wound healing and Transwell invasion assays highlighted lycopene’s ability to enhance cisplatin’s anti-metastatic effects, with combination-treated cells exhibiting minimal gap closure (Figure 3A–D) and reduced invasive capacity (Figure 4A–D). Apoptosis analysis via TUNEL staining and Western blotting revealed that the combination therapy markedly increased apoptotic rates (Figure 5A–D), accompanied by upregulated Bax and downregulated bcl-2 expression (Figure 6A and D). These results collectively underscore lycopene’s role in sensitizing OSCC cells to cisplatin by targeting proliferation, migration, invasion and apoptotic resistance.

A critical contributor to cisplatin resistance in OSCC is the overexpression of multidrug resistance-associated protein 1 (MRP-1), a drug efflux pump that reduces intracellular cisplatin accumulation.16,17 The results showed that cisplatin monotherapy significantly elevated MRP-1 expression in OSCC cells, aligning with prior reports that platinum-based therapies paradoxically activate drug efflux pumps to evade cytotoxicity.23 This upregulation likely contributes to the suboptimal clinical efficacy of cisplatin by reducing intracellular drug accumulation. Strikingly, lycopene alone suppressed MRP-1 expression below control levels, while the LYC+DDP combination restored MRP-1 to baseline, effectively neutralizing cisplatin’s unintended induction of this resistance marker. By normalizing MRP-1 levels, lycopene ensures greater intracellular cisplatin retention, thereby amplifying its cytotoxic effects. These findings are consistent with studies demonstrating that natural compounds, such as curcumin and quercetin, reverse chemoresistance by modulating MRP-1 and other ATP-binding cassette transporters.4,24 This suggests that lycopene may enhance cisplatin sensitivity by reducing drug efflux via downregulating MRP-1.

Beyond drug efflux, hyperactivation of the PI3K/Akt/mTOR pathway is a hallmark of cisplatin-resistant tumors, promoting cell survival and apoptosis evasion.25,26 Western blotting demonstrated that lycopene synergized with cisplatin to reduce phosphorylation levels of Akt and mTOR in OSCC cells. This pathway inactivation was functionally linked to increased apoptosis,27 as shown by elevated Bax/bcl-2 ratios and TUNEL-positive cells. These observations corroborate reports that PI3K/Akt/mTOR inhibition destabilizes mitochondrial integrity and activates caspase cascades, rendering cancer cells susceptible to cisplatin-induced apoptosis.6 Notably, the combination therapy achieved greater suppression of PI3K/Akt/mTOR signaling than either agent alone, highlighting lycopene’s role in augmenting cisplatin’s pro-apoptotic effects.

A paradoxical yet clinically significant finding of this study is that cisplatin monotherapy, while cytotoxic, inadvertently promotes EMT and stemness in OSCC cells—a phenomenon linked to chemoresistance and metastatic progression.7,12,28 Our data show that cisplatin downregulates E-cadherin and upregulates N-cadherin and EpCAM, suggesting that surviving cells adopt a mesenchymal and stem-like phenotype to evade therapy. This aligns with reports that platinum-based drugs activate pro-survival pathways to drive EMT and CSC (cancer stem cell) enrichment in residual tumors.5,6 Critically, lycopene not only reverses baseline EMT/stemness but also neutralizes cisplatin-induced adaptive changes. By restoring E-cadherin and suppressing N-cadherin/EpCAM, lycopene reinstates epithelial integrity and reduces CSC populations, thereby impairing metastatic potential and sensitizing cells to cisplatin.

The ability of lycopene to counteract cisplatin’s EMT/stemness-inducing effects may stem from its dual inhibition of PI3K/Akt/mTOR and NF-κB pathways, both of which are implicated in EMT regulation and CSC maintenance.12,28,29 For instance, PI3K/Akt activation by cisplatin can stabilize Snail, a transcriptional repressor of E-cadherin, while NF-κB signaling promotes EpCAM expression.7,19 Lycopene’s suppression of these pathways likely disrupts the transcriptional machinery driving EMT and stemness. This mechanistic synergy between lycopene and cisplatin highlights the importance of targeting adaptive resistance mechanisms to improve chemotherapeutic outcomes.

In vivo validation using a xenograft model reinforced these findings. Mice treated with the lycopene-cisplatin combination exhibited significantly smaller tumor volumes and weights compared to cisplatin monotherapy. Immunofluorescence analysis of tumor tissues revealed reduced PCNA (a proliferation marker) and increased E-cadherin expression in the combination group, confirming the dual anti-proliferative and EMT-reversing effects observed in vitro. Importantly, no significant toxicity or body weight loss was observed, underscoring lycopene’s favorable safety profile as an adjuvant.

Existing studies on lycopene in OSCC have focused primarily on its single-agent antitumor effects—for example, our prior work showed lycopene inhibits OSCC via PI3K/Akt/mTOR and EMT suppression,12 while others highlighted its isolated antioxidant or pro-apoptotic roles.11 None addressed the critical clinical need of overcoming cisplatin resistance, the main limitation of platinum-based OSCC therapy.5,22 Our study fills this gap by shifting focus from “lycopene monotherapy” to “lycopene-cisplatin synergy,” directly targeting clinical chemo-resistance.

Mechanistically, prior research linked lycopene to a single signaling node28 or resistance-related protein.4 In contrast, we identified a triple synergistic mechanism targeting interrelated cisplatin resistance drivers: (1) reversed MRP-1-mediated drug efflux, (2) inactivated PI3K/Akt/mTOR-driven survival, and (3) restored EMT/stemness-induced therapeutic escape, which providing a more comprehensive explanation for lycopene’s chemo-sensitizing effect.

While these findings highlight lycopene’s therapeutic promise, several limitations warrant consideration. First, the study focused on two OSCC cell lines; future work should validate these mechanisms in primary patient-derived cells or organoids to enhance translational relevance. Second, the interplay between lycopene and other resistance pathways, such as NF-κB or STAT3, remains unexplored. Additionally, pharmacokinetic studies are needed to optimize lycopene dosing and bioavailability, ensuring its efficacy in clinical settings. Combinatorial trials integrating lycopene with immune checkpoint inhibitors or targeted therapies could further enhance therapeutic outcomes by addressing both chemoresistance and immune evasion.

Conclusions

This study establishes lycopene as a potent chemosensitizer in OSCC, capable of reversing cisplatin resistance through suppression of MRP-1-mediated drug efflux, inactivation of PI3K/Akt/mTOR survival signaling, and reversal of EMT and stemness phenotypes. The synergistic efficacy of lycopene and cisplatin, coupled with minimal toxicity, positions this combination as a promising strategy to improve clinical outcomes in OSCC patients.

Acknowledgments

The authors thank the laboratory staff of the Outpatient Department of Oral and Maxillofacial Surgery (Capital Medical University) for technical support in cell culture and animal experiments.

Funding Statement

This work was supported by the Research Fund of Beijing Shijitan Hospital Affiliated to Capital Medical University (Grant No.: 2023-c09).

Abbreviations

OSCC, Oral Squamous Cell Carcinoma; LYC, Lycopene; DDP, Cisplatin; EMT, Epithelial mesenchymal transition; CSC, Cancer Stem Cell; mTOR, Mammalian target of rapamycin; PCNA, Proliferating Cell Nuclear Antigen; PI3K, Phosphatidylinositol 3 kinase.

Disclosure

The authors declare no conflicts of interest in this work.

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