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Targeted inhibition of the PTEN/PI3K/AKT pathway by YSV induces cell cycle arrest and apoptosis in oral squamous cell carcinoma

Journal of Translational Medicine volume 23, Article number: 145 (2025) Cite this article

Abstract

Background

Tyroservatide (YSV), a bioactive tripeptide, holds potential as an anti-tumor agent. However, its specific effects on oral squamous cell carcinoma (OSCC) have not been elucidated. This study aims to investigate the inhibitory effects of YSV on OSCC and explore the underlying molecular mechanisms.

Methods

A series of in vitro experiments were conducted to assess the impact of YSV on OSCC cell viability, colony formation, cell cycle, and apoptosis. RNA sequencing (RNA-seq), molecular docking, and western blotting were employed to investigate the molecular mechanisms. Additionally, a subcutaneous tumor model was established to validate the in vitro findings. Furthermore, PI3K inhibitors LY294002 and PI3K-IN-1, were used to confirm the role of the PTEN/PI3K/AKT pathway in YSV-mediated OSCC inhibition. Cell cycle and apoptosis were analyzed to assess the combined effect of YSV and LY294002.

Results

YSV significantly inhibited OSCC proliferation by inducing cell cycle arrest and apoptosis. RNA-seq and molecular docking revealed that YSV regulated the PTEN/PI3K/AKT signaling pathway. Western blotting confirmed the modulation of this pathway both in vitro and in vivo. The use of PI3K inhibitors, LY294002 and PI3K-IN-1, further validated the involvement of the PTEN/PI3K/AKT pathway in YSV-induced anti-tumor effects. Notably, the combination of YSV and LY294002 synergistically enhanced cell cycle arrest and apoptosis, demonstrating effective anti-tumor activity. In vivo experiments also supported these findings.

Conclusion

YSV inhibited the progression of OSCC by promoting cell cycle arrest and apoptosis through the regulation of the PTEN/PI3K/AKT signaling pathway. The combination of YSV and PI3K inhibitors, such as LY294002, exhibited enhanced anti-tumor activity, suggesting potential therapeutic strategies for OSCC treatment.

Graphical abstract

Introduction

Head and neck cancer (HNC) ranks as the 6th most prevalent malignancy worldwide, including oral, laryngeal and pharynx cancers [1, 2]. In 2022, there were over 700,000 global diagnoses and over 300,000 new deaths of HNC [3]. Among HNC, oral cancer is the most prevalent type, with oral squamous cell carcinoma (OSCC) comprising the largest proportion, constituting over 90% of cases [3]. With the deepening of research, significant progress has been made in the comprehensive sequential therapy for OSCC, including surgery followed by chemoradiotherapy and immunotherapy. Despite the multimodal treatment, the 5-year survival rate of OSCC patients remains very low [4, 5]. Meanwhile, the severe adverse reaction caused by chemoradiotherapy and immunotherapy needs to be considered for administration [6]. To this end, it’s still urgent to disclose more effective and less side effects drug treatment and eventually improve the overall survival rate of OSCC patients.

Recently, bioactive peptides with inherent biocompatibility have attracted significant attention in the fields of drug discovery and formulation design [7]. Tyroservatide (YSV) is a bioactive tripeptide composed of tyrosyl-seryl-valine, which exhibits excellent anti-tumor effect in various of tumors [8,9,10,11]. For example, YSV exerted anti-tumor activity by selectively upregulating p21 expression and inhibiting the activity of histone deacetylase (HDAC), thus inducing G0/G1 cell cycle arrest and inhibiting lung carcinoma cell proliferation [12]. YSV also showed the tumor inhibitory effect in hepatocarcinoma by altering the ultrastructure of tumor cells and inducing apoptosis [13]. YSV could effectively interfere with the tumor cells proliferation, adhesion, and invasion by downregulating the expression of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) and ultimately inhibiting abdominal cavity metastasis of ovarian carcinoma and liver metastasis of colon carcinoma [11]. Nevertheless, the cumulative information on the role of YSV in tumor suppression in relation to OSCC is limited, there are no studies available on the role and molecular mechanism; thus, further study is needed.

Phosphatidylinositol-3-kinase (PI3K), also known as a lipid kinase, produces PIP3 and acts as a second messenger to regulate AKT translocation to plasma membrane [14, 15]. Then, AKT is activated through phosphorylation modification on threonine 308 and serine 473 [16]. PI3K/AKT signaling pathway, involved in various cellular biological processes, including proliferation, cell cycle, apoptosis, metastasis and metabolism, controls hallmarks of human cancers [17, 18]. Researchers have reported that the PI3K/AKT signaling pathway is aberrantly activated in over 90% of OSCC patients [19]. In addition, upregulated PI3K/AKT signaling could enhance radiotherapy and cytostatic drug resistances in the therapy of OSCC [20, 21]. The above evidence indicates that PI3K/AKT is a reasonable therapeutic target for OSCC patients. It was reported that YSV could increase the mRNA and protein expression of phosphatase and tensin homolog (PTEN), p21 and p27 and inhibit the mRNA and protein expression of AKT in hepatocarcinoma [22]. PTEN is a tumor-suppressor operator that inhibits PI3K/AKT in diminishing tumor growth, metastasis and drug resistance [23]. p21 and p27 are cell cycle regulators, which can inhibit the binding of cyclins in G1 and S phases to cyclin dependent kinase (CDK) and ultimately inhibit cell proliferation. p21 and p27 was also considered as the downstream target of the PTEN/PI3K/AKT signaling pathway [24]. Taken all aspects above, we suppose that YSV maybe exert OSCC progression inhibitory effect via targeting PTEN/PI3K/AKT signaling pathway.

In the present study, we first assessed the effect of YSV against OSCC cells. The key phenotypes of the impact of YSV against OSCC were investigated, including cell cycle arrest and apoptosis. To reveal the potential underlying molecular mechanism of these effects, transcriptomic analysis was conducted to identify the signaling pathways that YSV may modulate systematically. Moreover, molecular docking was employed to evaluate potential target sites. These results were further confirmed through the use of a signaling pathway inhibitor. Furthermore, in vivo experiments were conducted to validate the safety and efficacy of YSV via nude mouse xenograft models. Collectively, we proved that YSV could exert an excellent anti-tumor effect through regulating the PTEN/PI3K/AKT signaling pathway, which provides a theoretic basis for the clinical application, however, the detailed molecular mechanism still needs further exploration for the clinical drug delivery.

Materials and methods

Peptide synthesis and characterization

YSV was prepared by solid phase peptide synthesis (SPPS) using N-terminal Fmoc protected amino acids with side chains properly protected by protecting groups and 2-chlorotrityl chloride resin. Fmoc-amino acids were obtained from GL Biochem (Shanghai, China). High performance liquid chromatography (HPLC) system and mass spectrometry (MS, Waters ZQ2000, England) were used for purification and characterization of the synthesized YSV.

Cell culture

Two human OSCC cell lines SCC-15 and CAL-27 cells were purchased from the American Type Culture Collection. Both cells were cultured in DMEM (Gibco, USA) with 10% FBS (Gibco, USA), 1% penicillin/streptomycin solution (Solarbio, China) and maintained in 5% CO2 and 37 °C.

Cell counting Kit-8 assay

The cell viability was measured using CCK-8 (Beyotime, China) according to the manufacturer’s protocol. Cells were seeded into 96-well plates at a density of 4000 cells/well. After 24, 48, and 72 hours, the supernatant was replaced by empty DMEM and CCK-8 solution (10:1) and incubated for 2 hours at 37 °C. The optical density (OD) at 450 nm was measured by the automatic microplate reader (BioTek ELX808 American).

Cell viability was calculated by the formula: cell viability = [(experimental wells’ OD - blank wells’ OD)/(control wells’ OD - blank wells’ OD)]  × 100%)

Colony formation assay

Cells were plated into 6-well plates at 400 cells/well and cultured for 7 days. The culture medium was changed every two days. At day 7, cell clonies were washed with phosphate-buffered saline (PBS), fixed with formaldehyde for 15 minutes and stained with 0.5% crystal violet for 20 minutes.

Flow cytometric analysis

Cells were seeded into 6-well plates at 3 × 105 cells/well and cultured for 48 hours. For analysis of cell cycle, cells were fixed in 70% ethanol overnight at 4 °C, rinsed twice with PBS and stained with PI, buffer and RNase A at 37 °C for 30 minutes using the Cell Cycle and Apoptosis Analysis Kit (Beyotime, China). For analysis of apoptosis, cells were stained with Annexin V-FITC and propidium iodide (PI) for 15 minutes using the Annexin V-FITC Apoptosis Detection Kit (Solarbio, China).

Western blotting analysis

The western blotting protocol was based on our earlier publication [25]. Briefly, the total proteins of cells and OSCC tumor tissues were extracted by RIPA (Solarbio, China). The concentration of proteins was measured by a BCA Protein Assay Kit (Beyotime, China) according to the manufacturer’s protocol. Equal amounts of proteins (20 µg/lane) were separated by 4–20% SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% BSA for 1.5 hours at room temperature and then incubated with primary antibodies overnight at 4 ̊C. The antibodies including: CDK1 (1:1000, Sino Biological Inc, China), Cyclin B1 (1:1000, Beyotime, China), Bcl-2 (1:1000, Beyotime, China), Bax (1:1000, proteintech, China), Cleaved caspase-3 (1:1000, CST, USA), PTEN (1:1000, proteintech, China), PI3K (1:1000, Wanleibio, China), p-PI3K (1:1000, Wanleibio, China), AKT (1:1000, Wanleibio, China), p-AKT (1:1000, Wanleibio, China), GAPDH (1:10000, proteintech, China), HRP-labeled goat anti-rabbit IgG (1:1000, Beyotime, China). The HRP-conjugated secondary antibodies were incubated for 1 hour at room temperature. Target proteins were visualized with a Super-Enhanced chemiluminescence detection kit (Beyotime, China).

Xenograft study models

The animal experiment was approved by the Animal Care and Use Committee of the Peking University Health Science Center (No. LA2022229), using the approved protocol of the Animal Ethical and Welfare Committee, and following the Committee of Peking University Health Science Center’s Animals Usage Guideline. Healthy male BALB/c-nu nude mice (5 weeks) were randomly divided into eight groups, and different OSCC cells were injected according to the groups’ name. A volume of 100 µL of SCC-15 or CAL-27 cells suspension (5 × 106 cells/100 µL) was subcutaneously injected into the armpit of nude mice. Then the mice were given intra-peritoneal injection of 0 mg/kg (control group) or 50 mg/kg YSV or 50 mg/kg YSV combination usage of 50 mg/kg LY294002 twice per week when the tumor volume was about 50 mm3. And the size of tumor was measured every 3 days and estimate it using the formula: V = length × width2/2. About 4 weeks later, euthanasia was performed on the mice and the tumors were removed completely for HE and western blotting.

Hematoxylin–eosin (HE) staining

The corresponding tissues were fixed with 4% polyformalin for 24 hours, embedded in paraffin, and sliced into 4 μm thick sections. Then the slides were deparaffinized, rehydrated and stained with hematoxylin and eosin. Finally, the slides were sealed with neutral gum and observed under an optical microscope (Nikon Eclipse Ci-L).

RNA isolation and real-time PCR analysis

Total RNA from OSCC cells was extracted by Trizol (Invitrogen, USA). Subsequently, an equivalent quantity of total RNA from each group was transcribed into cDNA using the Reverse Transcription Kit (TaKaRa, Japan). A real-time PCR reaction was formulated by mixing 1µL of cDNA, 1µL of forward and reverse primers (both at a concentration of 10µM), 5µL of universal SYBR Green Fast qPCR Mix (ABclonal, China), and 3µL of RNase-free water. This prepared mixture then underwent real-time PCR analysis on the ABI 7500 Sequence Detection System (Invitrogen, USA). GAPDH served as the internal control gene. All real-time PCR data were analyzed using the comparative CT method (ΔΔCT). The primers sequences are listed in Table 1.

Table 1 Primers for real-time PCR
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RNA sequencing

To further explore the underlying mechanisms of YSV against OSCC, RNA sequencing (RNA-seq) of OSCC cells exposured to YSV was conducted. YSV was applied to SCC-15 cells at doses of 1 mg/mL for 48 hours. Then total RNA was extracted by Trizol (Takara, Japan), followed by library preparation according to Illumina standard instruction (VAHTS Universal V6 RNA-seq Library Prep Kit for Illumina®). The protocol of high-throughput sequencing was according to the manufacturer’s instructions (Illumina). Differentially expressed genes (DEGs) were identify as those with a False Discovery Rate (FDR) value above the threshold (Q < 0.05) and|log2fold change|>1 by edgeR software.

Bioinformatics analysis

Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of DEGs was analysis by Database for Annotation Visualization and Integrated Discovery (DAVID, https://david.ncifcrf.gov/).

Molecular docking

The structure of YSV was downloaded from PubChem database (https://pubchem.ncbi.nlm.nih.gov/) and was shown in Fig. 1a, b. The proteins structures of PTEN, PI3K and AKT were obtained from PDB database (http://www.rcsb.org/). PyMOL software was used to remove water and small molecule ligands from proteins. Then AutoDockTools 1.5.7 was used for hydrogenation of protein molecules to obtain PDBQT files and determine active pockets and Vina 1.1.2 program was employed to calculate the binding energy. Finally, PyMOL was used to visualize the optimal combination model. The smaller binding energy between YSV and the target proteins indicated the stronger binding force. The binding energy ≤ -5.0 kcal/mol indicated that they could combine, and the binding energy ≤ -7.0 kcal/mol indicated the excellent binding strength.

Fig. 1
figure 1

YSV inhibited the proliferation of OSCC cells. (a) Two-dimensional chemical structure of YSV; (b) Three-dimensional chemical structure of YSV; (c) The molecular weight of YSV was identified by MS; (d) The proliferation curve of SCC-15 cells (left panel) and CAL-27 cells (right panel) after cells were treated with 0, 0.5, 1 and 2 mg/mL YSV; (e) The colony formation of SCC-15 and CAL-27 cells after cells were treated with 0, 0.5, 1 and 2 mg/mL YSV for 7 days; (f) The quantitative analysis results of (e). * P < 0.05; ** P < 0.01; *** P < 0.001; n = 3. Asterisks represent the differences between cells treated with 0.5, 1 and 2 mg/mL YSV and cells treated with 0 mg/mL YSV

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Statistical analysis

Statistical evaluations were conducted by GraphPad Prism software 8.0 (San Diego, USA). To ensure the validity of the data, experiments were repeated at least three times. All Error bars were represented as mean ± standard deviation (SD, n = 3). Homogeneity tests were performed on two or more groups of data, followed by one-way analysis of variance. P < 0.05 was considered significant.

Results

Effective inhibition of cell proliferation in OSCC cell lines by YSV

YSV was synthesized by SPPS and identified by MS and HPCL in this study. The result showed that the purity of YSV was 96.84% (Fig. 1c). Initially, we detected the inhibitory effects of YSV on OSCC cells’ proliferation. The CCK-8 assay results showed that after treatment with various concentrations of YSV (0 mg/mL, 0.5 mg/mL, 1 mg/mL, 2 mg/mL), the ability of cell proliferation was diminished with increasing YSV concentrations (Fig. 1d). Furthermore, the cell colony formation ability was also decreased as the concentration of YSV increased (Fig. 1e, f). Accordingly, these data collectively suggested that the concentration-dependent inhibitory effect of YSV on the proliferation of OSCC cells.

Effective arresting of cell cycle and inducing of cell apoptosis in OSCC cell lines by YSV

We have confirmed the inhibitory activity of YSV on the proliferation of OSCC cells, however, the underlying molecular mechanism is still unclear. We then sought to investigate whether YSV plays a functional role in the cell cycle. The results of flow cytometry analysis showed in comparison with that in the control group, cells in the experimental group treated with YSV exhibited a significant increase in the proportion of G2/M phases (Fig. 2a, b). In addition, the mRNA and protein expression levels of G2/M phase-related proteins CDK1 and Cyclin B1 was downregulated in a YSV dose-dependently manner (Fig. 2c-e).

Fig. 2
figure 2

YSV induced cell cycle arrest of OSCC cells in vitro. (a, b) Distribution (a) and statistical analysis (b) of cell cycles of SCC-15 cells (up) and CAL-27 cells (bottom), respectively, treated with 0, 0.5, 1 and 2 mg/mL YSV for 48 hours; (c) The protein expression levels of CDK1 and Cyclin B1 in SCC-15 cells (left) and CAL-27 cells (right), treated with 0, 0.5, 1 and 2 mg/mL YSV for 48 hours; (d) The quantitative analysis results of (c). (e) The mRNA expression levels of CDK1 and Cyclin B1 in SCC-15 cells (left) and CAL-27 cells (right), treated with 0, 0.5, 1 and 2 mg/mL YSV for 48 hours. *, #, & P < 0.05; **, ##, && P < 0.01; *** P < 0.001; n = 3. Asterisks represent the differences between G1 phase or the protein and mRNA levels of cells treated with 0.5, 1 and 2 mg/mL YSV (YSV-treated groups) and cells treated with 0 mg/mL YSV (control groups), pounds represent the differences between S phase in YSV-treated groups and control group; ampersands represent the differences between G2/M phase in YSV-treated groups and control group

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Then to investigate whether YSV could induce OSCC cell apoptosis, we detected apoptotic rates via flow cytometry after cells were treated with various concentrations of YSV. The results showed that the apoptosis rate of cells increased in line with the ascending YSV concentration for 24 hours (Fig. 3a, b). The aforementioned conclusion was reinforced via the detection of mRNA and protein expression levels of cell apoptosis-related proteins, where YSV dose-dependently upregulated expression of Bax and Cleaved caspase-3 while downregulating expression of Bcl-2 (Fig. 3c-f). These results strongly suggested that YSV effectively arrested cell cycle and promoted cell apoptosis.

Fig. 3
figure 3

YSV induced apoptosis of OSCC cells in vitro. (a, b) Representative flow cytometry plots (a) and statistical analysis (b) of SCC-15 cells (up) and CAL-27 cells (bottom), respectively, treated with 0, 0.5, 1 and 2 mg/mL YSV for 48 hours; (c) The protein expression levels of Bcl-2, Bax and Cleaved caspase-3 in SCC-15 cells (left) and CAL-27 cells (right), treated with 0, 0.5, 1 and 2 mg/mL YSV for 48 hours; (d) The quantitative analysis results of (c); (e) The mRNA expression levels of Bcl-2 and Bax in SCC-15 cells (left) and CAL-27 cells (right), treated with 0, 0.5, 1 and 2 mg/mL YSV for 48 hours. * P < 0.05; ** P < 0.01; *** P < 0.01; n = 3. Asterisks represent the differences between the protein and mRNA levels of cells treated with 0.5, 1 and 2 mg/mL YSV and cells treated with 0 mg/mL YSV

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RNA-seq and molecular docking indicate that YSV may target the PTEN/PI3K/AKT signaling pathway

To further illustrate the molecular mechanism underlying the inhibitory effect of YSV on OSCC cells, RNA-seq was performed on cells from the experimental group (treated with 1 mg/mL YSV) and the control group (treated with equal volume of PBS). Correlation heatmap result demonstrated a strong correlation among the samples (Fig. 4a). Principal component analysis (PCA) indicated the significant differences of transcriptomes between the two groups (Fig. 4b). Subsequently, heatmaps and volcano maps were employed to depict the differentially expressed genes (DEGs) between the two groups (Fig. 4c). The comparison between the treatment group and the control group identified 829 DEGs, consisting of 280 upregulated and 549 downregulated (Fig. 4d). KEGG categorized of DEGs including the Cancers: specific, Cancer: overview, signal transduction and cell growth and death (Fig. 4e). The result of KEGG enrichment of DEGs showed that PI3K/AKT signaling pathway had the highest representation (Fig. 5a). The different expression genes in PI3K/AKT signaling pathway were marker in Fig. 5b. It is worth noting that PI3K/AKT signaling pathway closely related to cell proliferation, cell cycle arrest and cell apoptosis. Consequently, it could be inferred that YSV may exert the inhibitory effects on OSCC cells through PI3K/AKT signaling pathway. Considering that PI3K and AKT serve as pivotal targets in the PI3K/AKT signaling pathway and PTEN is a key regulator of this pathway, molecular docking was conducted to investigate the binding interactions between YSV and PTEN, PI3K and AKT. The docking results provide a visual representation of the overall three-dimensional structures of the two complexes (Fig. 5c). The binding energies of YSV with PTEN, PI3K and AKT were calculated to be − 7.0, − 6.4 and − 7.1 kcal/mol, respectively (Fig. 5d), indicating the stable binding of YSV to PTEN, PI3K and AKT.

Fig. 4
figure 4

RNA-seq and molecular docking analysis revealed that PI3K/AKT was the core signaling pathway of YSV against OSCC. (a) Correlation heatmap among the different samples. (b) PCA analysis; (c) The heatmap of DEGs; (d) Volcano plot depicting DEGs. The cell cycle related genes CDK1 and Cyclin B1 and cell apoptosis related genes BCL2 and BAX have been marked; (e) KEGG classification analysis

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Fig. 5
figure 5

RNA-seq and molecular docking analysis revealed that PTEN/PI3K/AKT was the core signaling pathway of YSV against HNSCC. (a) KEGG pathway analysis; (b) A visualized PI3K/AKT pathway heatmap with related gene expression levels; (c) Molecular docking between YSV and PTEN, PI3K and AKT; (d) Binding affinities of YSV with the proteins in (c)

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We then detected the protein expression levels of PTEN, PI3K, p-PI3K, AKT and p-AKT in the two OSCC cell lines to validate the regulatory effect of YSV on the PTEN/PI3K/AKT signaling pathway. As shown in Fig. 6a, b, the protein expression levels of PTEN gradually increased and that of p-PI3K and p-AKT gradually decreased along with the increase of YSV concentration in comparison to the control group, indicating that YSV could suppress this signaling pathway via activating PTEN and inhibiting the phosphorylation of PI3K and AKT in a dose-dependent manner.

Fig. 6
figure 6

YSV regulated the PTEN/PI3K/AKT signaling pathway. (a) The protein expression levels of PTEN, PI3K, p-PI3K, AKT and p-AKT in SCC-15 cells (left) and CAL-27 cells (right), treated with 0, 0.5, 1 and 2 mg/mL YSV for 48 hours; (b) The quantitative analysis results of (a). * P < 0.05; ** P < 0.01; *** P < 0.001; n = 3. Asterisks represent the differences between the protein levels of cells treated with 0.5, 1 and 2 mg/mL YSV and cells treated with 0 mg/mL YSV

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YSV inhibits tumor growth in vivo

To evaluate the anti-tumor effect of YSV in vivo, a xenograft tumor model was established via subcutaneous implantation of SCC-15 cells and CAL-27 cells in nude mice (Fig. 7a). The YSV treatment group showed a significant reduction in tumor volume (Fig. 7b-d) as well as a marked decrease in the tumor volume growth rate (Fig. 7c, d) compared to the control group, indicating that YSV effectively inhibited tumor growth in vivo.

Fig. 7
figure 7

YSV inhibited OSCC progression in vivo. (a) The diagram showing the establishment of Xenograft study models via injection of SCC-15 and CAL-27 cells; (b) Representative images of tumor samples; (c) Tumor volume (left) and tumor weight (right) of the mice injection of SCC-15 cells treated with 50 mg/kg YSV compared with the control group; (d) Tumor volume (left) and tumor weight (right) of the mice injection of CAL-27 cells treated with 50 mg/kg YSV compared with the control group; (e) HE staining images of tumor of the mice treated with 0 mg/kg and 50 mg/kg YSV. * P < 0.05, ** P < 0.01, *** P < 0.001, n = 3. Asterisks represent the differences between mice treated with 50 mg/kg YSV and 0 mg/kg YSV

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Furthermore, HE staining was performed on the tumor tissues of two groups of mice (Fig. 7e). The results indicated that tumor cells in the control group exhibited a larger cell volume and a tight arrangement with variable shapes, including circles, spindles, and polygons. There was a considerable number of cells, enriched cytoplasm, abundant megakaryocytes, obvious cell heterogeneity, frequent nuclear division, and mild focal necrosis in some areas. In contrast, the YSV treatment group showed are duction in tumor cell number, fewer instances of nuclear division and significant tumor cell necrosis were observed. Necrotic cells exhibited dissolution, shrinkage or fragmentation, leading to an enlarged necrotic area. Generally, in the absence of drug interference, tumor necrosis intensifies with increasing volume. However, compared to the control group, the tumors in the treatment group showed smaller volumes but more severe necrosis, suggesting that YSV treatment accelerated tumor necrosis. This pathological evidence further confirms the anti-tumor effect of YSV. Meanwhile, YSV treatment at a dose of 50 mg/kg did not cause significant toxicity in major organs (Fig. 10c).

YSV inhibits OSCC proliferation through the PTEN/PI3K/AKT signaling pathway

Next, PI3K inhibitor was employed to investigate the impact of YSV on cell cycle and cell apoptosis through regulating the PTEN/PI3K/AKT signaling pathway. We first detected the cell proliferation ability via CCK-8 assay after cells treated with two classic PI3K inhibitors PI3K-IN-1and LY294002. The results showed that PI3K-IN-1 and LY294002 both could decrease the cell proliferation ability of OSCC cells, meanwhile, the combination usage of LY294002 and YSV performed the optimal inhibitory effect in cell proliferation. (Fig. 8a, b). Therefore, LY294002 was used in the following investigations. Compared to treatment with YSV alone, co-treatment with LY294002 further arrested the cell cycle at the G2/M (Fig. 8c, d). At the same time, the cell apoptosis rate was markedly increased upon the co-treatment (Fig. 8e, f).

Fig. 8
figure 8

YSV induced cell cycle arrest and apoptosis of OSCC by regulating PTEN/PI3K/AKT signaling pathway in vitro. (a, b) The proliferation curve of SCC-15 cells (a) and CAL-27 cells (b) after cells were treated with YSV, PI3K-IN-1, LY294002 or the combination usage of YSV and PI3K-IN-1 or LY294002; (c, d) Distribution (c) and statistical analysis (d) of cell cycles of SCC-15 cells (up) and CAL-27 cells (bottom), respectively, treated with LY294002, YSV and the two combined usage; (e, f) Representative flow cytometry plots (e) and statistical analysis (f) of SCC-15 cells (up) and CAL-27 cells (bottom), respectively, treated with LY294002, YSV and the two combined usage; (g) The protein expression of CDK1, Cyclin B1, Bcl-2, Bax, Cleaved caspase-3, PTEN, PI3K, p-PI3K, AKT and p-AKT in SCC-15 cells (left) and CAL-27 cells (right), treated with LY294002, YSV and the two combined usage. *, #, & P < 0.05; **, ##, && P < 0.01; n = 3. Asterisks represent the differences between G1 phase or the apoptosis rete, pounds represent the differences between S phase; ampersands represent the differences between G2/M phase

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Moreover, when co-administered with LY294002, the protein expression levels of CDK1, Cyclin B1 and Bcl-2 was significantly diminished, while the expression levels of Bax and Cleaved-caspase 3 was further increased compared to YSV treatment alone (Fig. 8g). Accordingly, these results confirmed that YSV indeed induced cell cycle arrest and cell apoptosis in OSCC cells through the PTEN/PI3K/AKT signaling pathway, ultimately leading to tumor cell proliferation inhibitory.

Finally, the mechanism of YSV’s anti-tumor effect in vivo was further confirmed by western blotting. In comparison to the control group, there was a significant reduction in the expression of CDK1 and Cyclin B1 in tumor tissues of the treatment group (Fig. 9). Meanwhile, the expression of Bcl-2 was notably decreased while the expression of the Bax and Cleaved-caspase 3 were significantly increased in the mice treated with YSV compared to the control group. Additionally, the protein expression level of PTEN was increased and that of p-PI3K and p-AKT was reduced significantly. In summary, these findings collectively demonstrated that YSV could suppress the PTEN/PI3K/AKT signaling pathway in vivo, inducing cell cycle arrest and cell apoptosis, thereby inhibiting tumor proliferation.

Fig. 9
figure 9

YSV induced cell cycle arrest and apoptosis of OSCC by regulating PI3K/AKT signaling pathway in vivo. The protein expression levels of CDK1, Cyclin B1, Bcl2, Bax, Cleaved caspase-3, PTEN, PI3K, p-PI3K, AKT and p-AKT in the tumor tissues of the mice injection of SCC-15 cells (left) and CAL-27 cells (right)

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The combination usage of YSV and LY294002 inhibits tumor progression in vivo

To further investigate the effect of combination usage of YSV and LY294002 on OSCC, in vivo experiment of mice treated with YSV and LY294002 was also conducted. The combination of YSV and LY294002 resulted in the most significant inhibition of tumor growth (Fig. 10a, b). Meanwhile, the treatment of YSV and LY294002 did not cause significant toxicity in major organs (Fig. 10c). These results indicated that the combination usage of YSV and LY294002 performed the optimal inhibitory effect of OSCC.

Fig. 10
figure 10

The combination usage of YSV and LY294002 inhibited OSCC progression in vivo. (a) Representative images of tumor samples; (b) Tumor volume (left) and tumor weight (right) of the mice injection of SCC-15 cells treated with PBS, YSV, LY294002, or the combination of YSV and LY294002; (c) HE staining images of tumor, heart, liver, spleen, lung and kidney of the mice treated with PBS, YSV, LY294002, or the combination of YSV and LY294002. * P < 0.05, ** P < 0.01, *** P < 0.001, n = 3. Asterisks represent the differences between mice treated with YSV or LY294002 and PBS; pounds represent the differences between mice treated with the combination of YSV and LY294002 and LY294002; ampersands represent the differences between mice treated with combination of YSV and LY294002 and YSV

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Discussion

In this study, we explored the potential therapeutic effects of YSV in combating OSCC through a series of experiments both in vitro and in vivo. Initially, we evaluated the impact of YSV on OSCC cell proliferation, cell cycle, and cell apoptosis. Additionally, a xenograft tumor model of OSCC was also established to detect the anti-tumor efficacy of YSV in vivo. To gain deeper insights into the underlying molecular mechanism of the inhibitory activity of YSV against OSCC, we performed RNA-seq on YSV-treated OSCC cells. KEGG analysis of DEGs demonstrated that the PI3K/AKT signaling pathway was the potential key regulator signaling pathway of YSV against OSCC. Notably, aberrant activation of the PI3K/AKT signaling pathway is a well-established hallmark of the progression of numerous tumors, which was also frequently detected in abnormal activation in OSCC. Therefore, we supposed that YSV exerted its anti-tumor effect by targeting the PTEN/PI3K/AKT signaling pathway. Molecular docking is a vital method for predicting the binding between drugs and targets, which can provide a better reference for the application of clinical drugs. Thus, molecular docking was then conducted and the results indicated that YSV could stably bind with the PTEN, PI3K and AKT proteins. Our experimental results showed that YSV decreased the protein expression level of PI3K and AKT in a dose-dependent manner. Moreover, the PI3K inhibitor LY294002 was employed to explore whether YSV performed excellent anti-tumor effect by suppressing the PTEN/PI3K/AKT signaling pathway. The combination of YSV and LY294002 further enhanced YSV’s capacity to inhibit OSCC cell proliferation, cell cycle and cell apoptosis, thereby demonstrating that YSV inhibited OSCC cell proliferation by promoting G2/M phase arrest and apoptosis via the PTEN/PI3K/AKT signaling pathway.

Cell cycle checkpoints are crucial regulatory mechanisms to ensure the normal progression of the cell cycle in eukaryotic cells [26]. A hallmark of tumor cells is the lack of G1 checkpoint control, leading them to mainly rely on the S and G2/M checkpoints to prevent DNA damage and cell death [26]. When the prolonged arrest of tumor cells in the S and G2/M phases, accumulated DNA damage could consequently result in cell apoptosis. Therefore, S and G2/M phase arresting plays a crucial role in combating tumors. At present, many clinical chemotherapy drugs target the S or G2/M phase, such as cytarabine and paclitaxel [27]. Our results indicate that YSV significantly induces cell cycle arrest in the G2/M phase. The mechanism behind G2/M phase arrest relies on decreasing the expression of cyclin B and CDK1.

Cell apoptosis is one of the internal monitoring and balancing mechanisms of cells, which can promptly eliminate harmful and abnormal cells [28]. Inducing cell apoptosis is considered as an effective strategy for tumor prevention and treatment [29]. Our study suggested that YSV significantly enhances cell apoptosis in OSCC cells. Mechanistically, YSV significantly decreased the protein expression level of anti-apoptotic molecule Bcl-2 and increased the protein expression level of pro-apoptotic Bax. The reduced Bcl-2 then releases cytochrome C into the cytoplasm, promoting the activation of Cleaved caspase-3, which is also a hallmark of cell apoptosis [30]. Our results also indicated a significant increase in the expression level of Cleaved caspase-3 following YSV treatment. Accordingly, our results suggested that YSV could exhibit anti-tumor effect by promoting cell apoptosis.

The aberrant activation of the PTEN/PI3K/AKT signaling pathway is frequently observed in OSCC [31]. A series of cancer-related functions, including cell proliferation [32], adhesion [33] and apoptosis [34], is associated with the PI3K/AKT signaling pathway. Our results suggested that YSV could target the PTEN/PI3K/AKT signaling pathway in OSCC cells, facilitating cell cycle arrest and inducing cell apoptosis, thereby enhancing its anti-tumor activity. More interestingly, we observed that the combination usage of YSV and LY294002 exerted a stronger inhibitory effect on cell proliferation and cell apoptosis than using them alone. We predicted that YSV also has other oncogenic gene signaling pathways targeting OSCC, as our RNA-seq analysis results indicated that YSV also has the potential to regulate signaling pathways, such as MAPK [35] and JAK-STAT [36], to exert its anti-tumor effects, which may be account for the reason why combination therapy is more effective. Further studies are needed to identify this issue. In addition, the clinical trial in OSCC suggested that the tumor response in monotherapy targeting PI3K and AKT is 5.3% [37] and 2.5% [38], respectively, suggesting that the effect of PI3K and AKT inhibitors may not seem remarkable. Moreover, due to the activation of compensatory signaling pathways, tumors may develop drug resistance bypassing the inhibited signaling pathways [39]. Many promising singlepathway targeted drugs have produced disappointing clinical results [40]. In contrast, combination therapy and multi-pathway targeted drugs are more promising for tumor treatment [41, 42]. Therefore, we reasonably assumed that the combination usage of YSV and PTEN/PI3K/AKT inhibitor could produce a marked anti-tumor effect and consequently achieve better therapeutic effect of OSCC.

Conclusions

In summary, our study preliminarily revealed that YSV played a vital role in suppressing the proliferation of OSCC via regulating PTEN/PI3K/AKT signaling pathway, which provided a new perspective of YSV in the treatment of OSCC. Meanwhile, this work also provides a novel option for multi-pathway combination therapy for OSCC.

Data availability

The data supporting this study are available from the corresponding author according to reasonable requirements.

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Funding

This research was funded by National Natural Science Foundation of China, grant number 81072214 and 30371547.

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Author notes

  1. Lin Qiu and Qian Gao contributed equally to this work.

Authors and Affiliations

  1. Central Laboratory, Peking University School and Hospital of Stomatology & National Center for Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Research Center of Oral Biomaterials and Digital Medical Devices, Beijing, 100081, China

    Lin Qiu, Qian Gao, Yiheng Liao & Cuiying Li

  2. National Key Laboratory of Efficacy and Mechanism on Chinese Medicine for Metabolic Diseases, Beijing Research Institute of Chinese Medicine, Beijing University of Chinese Medicine, Beijing, 102488, China

    Xinxin Li

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Contributions

Lin Qiu: Conceptualization, Methodology, Software, Writing – review & editing, Investigation, Formal analysis. Qian Gao: Writing – original draft, Writing – review & editing, Visualization, Investigation. Yiheng Liao: Writing – review & editing, Methodology. Xinxin Li: Writing – review & editing, Visualization, Validation, Methodology, Investigation, Supervision. Cuiying Li: Writing – review & editing, Validation, Supervision, Project administration, Investigation, Funding acquisition, Data curation.

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Correspondence to Xinxin Li or Cuiying Li.

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The ethics approval statements of all animal experiments were approved by the Institutional Animal Care and Use Committee of the Peking University Health Science Center and followed the Committee of Peking University Health Science Center’s Animals Usage Guideline.

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Qiu, L., Gao, Q., Liao, Y. et al. Targeted inhibition of the PTEN/PI3K/AKT pathway by YSV induces cell cycle arrest and apoptosis in oral squamous cell carcinoma. J Transl Med 23, 145 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12967-025-06169-z

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Journal of Translational Medicine

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