Liriopesides B inhibits proliferation and metastasis and induces apoptosis of oral squamous cell carcinoma via the PI3K/Akt/mTOR signaling pathway

Introduction

Oral cancer, a malignant neoplasm originating from oral epithelial tissues, is the most commonly represented by oral squamous cell carcinoma (OSCC), accounting for roughly 90% of cases. OSCC is characterized by aggressive behavior, high recurrence rates, and significant metastatic potential (1). Its development is closely linked to various risk factors, including tobacco use, alcohol consumption, mechanical trauma, viral infections, Candida infections, and genetic predisposition (2). Despite advancements in clinical diagnosis, treatment techniques, and multidisciplinary care, the five-year survival rate for individuals with OSCC remains relatively stagnant (3). Current therapeutic strategies primarily involve surgical resection, radiotherapy, chemotherapy, or an integration of these modalities (4). Nevertheless, these approaches frequently struggle to achieve optimal outcomes and are associated with challenges such as postoperative infections, immune suppression, and an increased risk of secondary malignancies. Chemotherapy, typically utilizing agents like 5-fluorouracil, paclitaxel, and cisplatin, aims to target rapidly proliferating cells and limit cancer spread (5). Although these chemotherapeutic agents can slow tumor progression, they also induce cytotoxicity in normal tissues, resulting in adverse reactions encompassing bone marrow suppression, nausea, vomiting, and organ damage, which markedly impair patients’ quality of life (6). Consequently, an urgent demand exists for innovative therapeutic compounds that can successfully suppress OSCC advancement while enhancing survival outcomes and elevating the general well-being of affected individuals.

Liriopesides B (LPB), a compound documented in the 2015 edition of the Chinese Pharmacopoeia (7), is derived from Ophiopogon vulgaris, a member of the Liliaceae family. Ophiopogon vulgaris possesses various therapeutic characteristics, encompassing immune-enhancing, anti-inflammatory, antibacterial, and cardiovascular protective effects (8). The plant contains abundant steroidal saponins, flavonoids, organic acids, and polysaccharides, with 72 distinct steroidal saponins having been isolated and identified (9). Many of these steroidal saponins have demonstrated significant anti-cancer activity in a range of malignancies (10). LPB has demonstrated anti-tumor activity in various cancers. Studies indicate that LPB inhibits proliferation and induces cell differentiation in ovarian cancer A2780 cells by reducing cancer antigen 125 (CA-125) levels and alkaline phosphatase activity (11). Furthermore, it significantly increases E-cadherin expression while decreasing Bcl-2 expression levels, thereby suppressing A2780 cell migration and inducing apoptosis (12). Additionally, LPB exerts anti-tumor effects in non-small cell lung cancer H460 and H1975 cells by inducing G1/S phase cell cycle arrest. This is achieved through downregulation of cyclin D1, cyclin D3, and CDK6 expression, alongside upregulation of the negative cell cycle regulator P21 (13). Given that ovarian cancer, non-small cell lung cancer, and OSCC all originate from epithelial tissues, it is hypothesized that LPB may exert anti-cancer effects on OSCC as well. However, the specific anti-OSCC effects and mechanisms of LPB still require comprehensive investigation.

In this investigation, human OSCC cell lines (SAS and Cal-27) were employed as in vitro models to investigate the impact of LPB on cell multiplication, motility, invasion, and apoptosis. High-throughput sequencing and bioinformatics analysis were performed to identify potential molecular mechanisms underlying LPB’s effects on OSCC. Differential gene enrichment and signaling pathways were confirmed through quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting (WB). Additionally, a subcutaneous xenograft model of OSCC was developed in nude mice to evaluate the in vivo efficacy of LPB. Xenograft tumor volume and weight were assessed, and histological analysis through Hematoxylin and Eosin (H&E) staining, quantitative reverse transcription polymerase chain reaction (qRT-PCR), and immunohistochemistry (IHC) was conducted to further explore LPB’s inhibitory effects on tumor growth and the underlying mechanisms. This investigation reveals a crucial understanding of the molecular pathways by which LPB suppresses OSCC, establishing a foundation for creating innovative therapeutic compounds in OSCC management. We present this article in accordance with the MDAR and ARRIVE reporting checklists (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-613/rc).

Methods

Reagents and antibodies

LPB, SC79 and dimethyl sulfoxide (DMSO) were sourced from MCE (Shanghai, China). Dulbecco’s Modified Eagle’s Medium (DMEM), DMEM-F12, and fetal bovine serum (FBS) were obtained from Gibco (Carlsbad, CA, USA). Antibiotic-Antimycotic solution was procured from Sigma-Aldrich (St. Louis, MO, USA). Cell Counting Kit-8 (CCK-8), TRIzol, reverse transcription kits, and SYBR Green Master Mix were supplied by Invitrogen (Shanghai, China). Matrigel was acquired from Corning Co. Ltd. (Corning, NY, USA). The Mir-X™ miRNA First-Strand Synthesis Kit was sourced from Takara (Kusatsu, Shiga, Japan). BCA and ECL assay kits were procured from Beyotime (Shanghai, China). The Apoptosis Detection Kit was procured from 4A Biotech (Suzhou, China). The Dual-Luciferase Reporter Gene Detection Kit was obtained from Promega (Madison, WI, USA). Anti-PI3K (1:1,000), anti-mTOR (1:2,000), anti-p-mTOR (1:1,000), anti-Akt (1:2,000), anti-p-Akt (1:1,000), and anti-GAPDH (1:1,000) antibodies were procured from Boster Biological Technology (Wuhan, China). Anti-S6 (1:1,000), anti-p-S6 (1:1,000), anti-4EBP1 (1:2,000), and anti-p-4EBP1 (1:1,000) antibodies were sourced from Bioworld (Nanjing, China).

Cell lines and culture

Human OSCC cell lines, CAL-27, SAS, and SCC-9, were provided by Kebai Biotechnology Co., Ltd. (Nanjing, China). SAS and SCC-9 cells were cultured in the DMEM-F12 medium, whereas CAL-27 cells were kept in the DMEM medium. The complete culture medium consisted of 89% medium, 10% FBS, and 1% Antibiotic-Antimycotic solution. Cells were kept at 37 °C in a 5% CO₂ atmosphere. For cell experiments, each group was set up with at least three replicates, and each experiment was independently repeated at least three times.

Cell proliferation assay and colony forming analysis

CAL-27, SAS, and SCC-9 cells (70–80% confluence) were placed into 96-well plates at 5×10³ cells/well. Following 24 hours of incubation, researchers substituted the initial growth medium with a fresh solution containing either DMSO or specified amounts of LPB according to test parameters. Subsequently, after 48 hours, each well received 10 µL of CCK-8 reagent and optical density readings were obtained at 450 nm utilizing a plate analyzer (Infinite MFlex, TECAN, Männedorf, Switzerland) following 2 hours of cultivation at 37 °C with 5% CO₂. Regarding colony formation assays, researchers placed 500 cells in 6-well plates, and after 24 hours, exchanged the growth medium with fresh solution containing various LPB concentrations. Following 12 days of cultivation, cell fixation was achieved using 1 mL methanol solution at ambient conditions for 30 minutes, followed by crystal violet (0.1%) staining for an additional 30 minutes.

Migration and invasion assay

The wound healing assay was performed to assess the migratory capacity of the cells. A sum of 5×10⁴ cells per well was placed into 6-well plates. Once the cells reached near-confluence, the original medium was discarded. A scratch wound was generated utilizing a 200 µL pipette tip, and the wound was immediately visualized under a microscope. DMSO or LPB was added according to the experimental cohorts. The healing progress was monitored and recorded at 24 and 48 hours after treatment using a microscope. The Transwell assay (8.0 µm, Corning, NY, USA) was utilized to evaluate cell migration and invasion capabilities following LPB administration. For the migration assay, 500 µL of complete culture medium was introduced to the lower chamber, and cells in optimal condition were resuspended in a serum-free medium. A 200 µL aliquot of cell suspension (5×10⁴ cells per chamber) was placed in the upper chamber. For the invasion assay, Matrigel was thawed overnight at 4 °C, diluted to 0.2 µg/mL in serum-free medium, and allowed to solidify by incubation at 37 °C for 45 minutes. After 24 hours of incubation (48 hours for invasion), non-migratory cells were eliminated from the membrane utilizing a cotton swab. Cells that traversed to the lower chamber underwent methanol fixation and crystal violet (0.1%) staining.

Flow cytometry assay

Cells were harvested when confluence reached 70–80% and placed at 1×10⁵ cells per well in 6-well plates. After a 24-hour incubation, the culture medium was substituted with 2 mL of complete medium containing varying concentrations of LPB. Following an additional 48-hour incubation, the supernatant was collected into 15 mL centrifuge tubes. Cells were then trypsinized (without ethylenediaminetetraacetic acid) and collected into the same centrifuge tubes containing the supernatant. The solution underwent centrifugation at 1,000 rpm for 5 minutes under ambient conditions. After a single wash with pre-cooled PBS (4 °C), cells were resuspended in 1× binding buffer. Annexin V Alexa Fluor 647 and PI were introduced for apoptosis detection via flow cytometry.

qRT-PCR test

RNA from cell and tissue samples was procured utilizing the TRIzol reagent. A reverse transcription kit facilitated messenger RNA (mRNA) generation, whereas miRNA production employed the Mir-X™ miRNA First-Strand Synthesis Kit. Measurement of mRNA and miRNA levels was accomplished through SYBR Green Master Mix with a QuantStudio-5 platform (Applied Biosystems, Foster City, CA, USA). The specific primers for gene analysis are depicted in Tables S1,S2. For normalization purposes, GAPDH served as the mRNA reference, while U6 functioned as the miRNA control. Expression values were ascertained via the 2^-ΔΔCt formula.

RNA sequencing and bioinformatics analysis

Cells at 70–80% confluency were collected and split into a blank control cohort and an experimental cohort (5×10⁵ cells per well). Total RNA was procured from each cohort, and transcriptome analysis was conducted. The initial dataset underwent filtration to secure premium-quality information, followed by transcriptome quantitative analysis using Top Hat2 (Version: 2.1.1) (http://ccb.jhu.edu/software/tophat), Cufflinks software (Version: 2.1.1) (http://cole-trapnell-lab.github.io/cufflinks/). Differential analysis of biological replicates was performed using DESeq2 (https://www.r-project.org/), with P<0.05 as the default threshold. For normalization, the TMM method was applied, followed by differential analysis with DEGseq (https://www.bioconductor.org/packages/release/bioc/html/DEGseq.html), using the thresholds: multiple testing correction q<0.01; |log₂Fold Change| ≥1. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of differentially expressed mRNAs were executed utilizing R software (Version 4.2.1).

Western blotting

Following a 24-hour exposure to LPB or DMSO, cells underwent lysis in RIPA buffer comprising protease inhibitors. The lysed samples were subjected to centrifugation at 12,000 g for 15 minutes while maintained at 4 °C, and supernatant protein content was ascertained via BCA analysis. The specimens were combined with equivalent amounts of 5× protein loading buffer and subjected to thermal treatment at 100 °C for 5 minutes. The protein components were resolved via SDS-PAGE, transferred onto PVDF membranes (Millipore, MA, USA), and blocked utilizing 5% skim milk solution in TBST for 1 hour at ambient conditions. The membranes were incubated with primary antibodies overnight at 4 °C, then exposed to matching secondary antibodies at ambient conditions the following day. Protein visualization was accomplished utilizing the ECL reagent and the BIO-RAD imaging platform.

The xenograft tumor model

A protocol was prepared before the study without registration. Nude male mice (4–6 weeks old, weighing 16–18 g) were procured from the Guangxi Medical University Experimental Animal Center. SAS cells, with a fusion rate of 70–80%, were resuspended to a level of 1×10⁷ cells/mL. A sum of 6×10⁶ cells was subcutaneously injected into the left axilla of each mouse. The treated mice were housed in individually ventilated cages (IVCs within specific pathogen free (SPF) animal rooms. Upon tumors attaining roughly 200 mm³, the mice were arbitrarily split into three cohorts (4 mice per cohort). Mice were weighed, and 100 µL of the drug was administered per 10 g of body weight daily for 2 consecutive weeks. After this period, the mice were euthanized, and the xenografted tumors were harvested for subsequent examination. The animal experiments were approved by the Ethics Committee of Guangxi Medical University (202112003), in compliance with institutional guidelines for the care and use of animals.

Histology and IHC analysis

Specimen preparation involved fixation in 4% paraformaldehyde at ambient conditions, sequential ethanol dehydration, paraffin block preparation, and microtome sectioning (RM2255, Leica) to achieve 4 µm thickness. Following rehydration, specimens underwent H&E staining to examine tumor morphology via inverted microscopy. For immunochemical analysis, heat-mediated antigen recovery utilized sodium citrate solution (0.01 mol/L, pH 6.0) at 95 °C for 20 minutes, followed by endogenous peroxidase neutralization (#PV-6000, ZSGB-Bio, China) for 15 minutes. The specimens were exposed to primary antibodies overnight at 4 °C, succeeded by secondary antibody application at ambient conditions for 30 minutes. Protein visualization employed DAB detection and hematoxylin counterstaining to evaluate PI3K/Akt/mTOR pathway component expression through inverted microscopy.

Statistical analysis

Statistical analysis was executed utilizing SPSS 20.0 software. Data are denoted as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was utilized to assess variations between multiple cohorts. Pairwise comparisons were made using Tukey’s or Dunnett’s method. Statistical significance was set at P<0.05.

Results

LPB changes the morphology and impedes the proliferation of OSCC cells

The chemical structure of LPB, procured from the National Center for Biotechnology Information, is presented in Figure 1A. The CCK-8 assay was utilized to assess LPB’s toxic impact on CAL-27, SAS, and SCC-9 cells, revealing a dose-dependent suppression of OSCC cell growth (Figure 1B). Based on the logit model, the half-maximal inhibitory concentration (IC₅₀) values for CAL-27, SAS, and SCC-9 cells were calculated to be 11.81±0.51, 13.81±0.72, and 8.10±0.32 µmol/L, respectively (Figure 1B). Therefore, a concentration of 12 µmol/L was selected as the IC₅₀, and CAL-27 and SAS cells were chosen for further experiments. In the cell morphology observation experiment, treatment with 6 and 12 µmol/L LPB for 24 hours weakened the adhesion ability of the three OSCC cell lines. The cells exhibited altered morphology, with enlarged intercellular spaces. LPB-treated cells were in poor condition, with a noticeable reduction in cell number and an increase in floating cells (Figure 1C). Similar results were observed in the colony formation assay, where LPB treatment markedly inhibited colony formation (Figure 1D). These observations collectively indicate that LPB suppresses the growth of OSCC cells.

Figure 1 LPB alters morphology and inhibits the proliferation of OSCC cells. (A) Chemical structure of LPB. (B) The inhibitory effect of LPB on the proliferation of CAL-27, SAS, and SCC-9 cells. (C) The effect of LPB on the morphology of CAL-27 and SAS cells (×100). (D) LPB suppressed the clonal formation abilities of CAL-27 and SAS cells (crystal violet staining). **, P<0.01; ***, P<0.001. LPB, liriopesides B; OSCC, oral squamous cell carcinoma.

LPB suppresses the invasion and migration of OSCC cells

To evaluate LPB’s influence on cell migration and invasion, transwell migration and invasion assays, as well as a wound healing assay, were conducted. In the transwell migration and invasion assays, the number of SAS and CAL-27 cells exposed to LPB was notably diminished (Figure 2A,2B). Correspondingly, the wound healing rates of SAS and CAL-27 cells exposed to LPB (6 and 12 µmol/L) were markedly decreased in a dose-dependent manner (Figure 2C,2D). The MMP-2 and MMP-9 gene levels were markedly reduced in SAS and CAL-27 cells, while the E-cadherin gene level was notably elevated, further confirming that LPB inhibits cell migration in these cell lines (Figure 2E).

Figure 2 LPB inhibits migration and invasion of OSCC cells in vitro. (A) Transwell migration assay of SAS and CAL-27 cells (crystal violet staining, ×200). (B) LPB inhibits the invasion ability of SAS and CAL-27 cells (crystal violet staining, ×200). (C,D) Wound healing assay of SAS and CAL-27 cells. (E) LPB downregulated the relative expression of MMP-2 and MMP-9 while upregulating the expression of E-cadherin in SAS and CAL-27 cells. *, P<0.05; **, P<0.01; ***, P<0.001. LPB, liriopesides B; OSCC, oral squamous cell carcinoma.

LPB induces the apoptosis of OSCC cells

Cell death analysis utilizing flow cytometry was conducted to assess apoptotic levels in SAS and CAL-27 cell lines. After 24 hours of LPB treatment, the apoptosis rates increased markedly in a dose-dependent manner (Figure 3A). The apoptosis rates for SAS and CAL-27 cells exposed to 12 µmol/L LPB were 25.6% and 25%, respectively, while those treated with 24 µmol/L LPB reached 40% and 49.6%, respectively. Additionally, examination revealed heightened expression of the death-promoting genes Bax and Bad, while levels of the survival-associated gene Bcl-2 showed a notable decrease (Figure 3B). These results demonstrate that LPB induces apoptosis in OSCC cells in vitro.

Figure 3 LPB induces apoptosis in OSCC cells. (A) LPB induces apoptosis in SAS and CAL-27 cells. (B) LPB upregulated the relative expression of Bax and Bad and downregulated the relative expression of Bcl-2 in SAS and CAL-27 cells. *, P<0.05; **, P<0.01; ***, P<0.001. LPB, liriopesides B; OSCC, oral squamous cell carcinoma.

LPB attenuates OSCC cells via the PI3K/Akt/mTOR signal cascade

To investigate how LPB affects OSCC, RNA-seq was conducted on SAS cells treated with LPB. The analysis revealed 5,864 distinctly modulated mRNAs, comprising 2,676 enhanced and 3,188 diminished genes, as illustrated in Figure 4A and the supplementary table (https://cdn.amegroups.cn/static/public/tcr-2025-613-1.xls). GO and KEGG enrichment analyses were performed to elucidate the potential mechanisms by which LPB exerts its inhibitory effects on OSCC. GO enrichment analysis suggested that LPB may influence the metabolic processes of OSCC cells (Figure 4B). The KEGG examination demonstrated substantial enrichment of differentially regulated genes in metabolic pathways, PI3K-Akt cascade, mTOR signaling, and tight junction networks (Figure 4C). Considering that mTOR signaling operates downstream of PI3K-Akt, which critically influences OSCC cell movement and metabolism, we hypothesized that LPB functions via the PI3K/Akt/mTOR axis (14-16). To validate this proposition, we employed WB techniques to examine crucial protein expression within the PI3K/Akt/mTOR cascade in OSCC cells (Figure 4D). Our findings showed unchanged levels of 4E-BP1 and p-4EBP1 in cohorts exposed to LPB (12, 24 µmol/L). Nevertheless, we observed marked reductions in PI3K, Akt, p-Akt, mTOR, p-mTOR, S6, and p-S6 expression, confirming that LPB suppresses protein expression within the PI3K/Akt/mTOR signaling network. Additionally, in LPB cells treated with the Akt activator SC79, cell viability showed a significant increase (P<0.05) (Figure 5A). The relative expression levels of migration and invasion-related factors MMP-2 and MMP-9 were markedly elevated across two OSCC cell lines, while the relative expression of pro-apoptotic genes Bax and Bad decreased, with upregulation of the anti-apoptotic gene Bcl-2 (Figures 5B,5C). Our rescue experiments further demonstrate the critical role of the PI3K signaling pathway in LPB’s anti-OSCC effects.

Figure 4 LPB inhibits OSCC progression via the PI3K/Akt/mTOR signaling pathway in vitro. (A) Differential gene expression between SAS cells treated with LPB and untreated SAS cells. (B) GO analysis of differentially expressed genes. (C) KEGG analysis of differentially expressed genes. (D) The effect of LPB on the protein expression of PI3K, Akt, p-Akt, mTOR, p-mTOR, S6, p-S6, 4EBP1, and p-4EBP1 in SAS cells. *, P<0.05; **, P<0.01. BP, biological process; CC, cell component; D6, SAS cells treated with LPB; GO, Gene Ontology; HC, SAS cells untreated with LPB; KEGG, Kyoto Encyclopedia of Genes and Genomes; LPB, liriopesides B; MF, molecular function; OSCC, oral squamous cell carcinoma.

Figure 5 Rescue assay further demonstrated LPB inhibited OSCC progression via the PI3K/Akt/mTOR signaling pathway. (A) SC79 reverses the LPB-induced CAL-27 and SAS cells viability inhibition. (B) The effect of LPB on the relative expression of MMP-2 and MMP-9 were rescued by SC79 in CAL-27 and SAS cells. (C) The effect of LPB on the relative expression of Bax, Bad and Bcl-2 were rescued by SC79 in CAL-27 and SAS. *, P<0.05; **, P<0.01; ***, P<0.001. LPB, liriopesides B; OSCC, oral squamous cell carcinoma.

LPB regulates the growth, migration and apoptosis of OSCC through the PI3K/Akt/mTOR signaling cascade in vivo

Tumor weight and volume measurements were used to evaluate the impact of LPB on the growth of xenografted tumors. In contrast to the control cohort, tumors in the LPB-treated cohort exhibited slower growth with prolonged administration. On day 6 of treatment, the tumor volume in the LPB-treated cohort was notably diminished (P<0.01). By day 14, the tumor volumes in the low-dose LPB cohort (5 mg/kg) and high-dose LPB cohort (10 mg/kg) were 878.19±198.55 and 734.62±181.54 mm³, respectively, compared to 1,722.00±200.18 mm³ in the control cohort, with significant differences observed (P<0.001) (Figure 6A). Tumor weights in the control cohort, LPB low-dose cohort (5 mg/kg), and LPB high-dose cohort (10 mg/kg) were 1.00±0.10, 0.75±0.15, and 0.54±0.12 g, respectively. The average tumor inhibition rates in the high-dose cohort were 25.69% and 46.63%, respectively. The tumor weight in the LPB treatment cohorts was notably diminished (P<0.05) (Figure 6B).

Figure 6 LPB inhibits growth, migration, and apoptosis of OSCC in vivo. (A) The effect of LPB on tumor volume in SAS xenograft nude mice. (B) The effect of LPB on tumor weight in SAS xenograft nude mice. (C) Histological analysis of tumor tissues in SAS xenograft nude mice (H&E staining, ×100). (D) The effect of LPB on the expression of proteins related to the PI3K/Akt/mTOR pathway (DAB staining, magnification). (E) The effect of LPB on the gene expression of MMP-2, MMP-9, Bax, Bad, and Bcl-2 in SAS xenograft tumors. *, P<0.05; **, P<0.01; ***, P<0.001. LPB, liriopesides B; OSCC, oral squamous cell carcinoma.

Histopathological evaluation of tumor tissue sections via H&E staining revealed cellular atypia, nuclear pleomorphism, and mitotic figures in the tumors of all cohorts. However, in contrast to the control cohort, tumors in the LPB treatment cohorts (5, 10 mg/kg) exhibited extensive areas of necrosis with uniform pink staining, inflammatory cell infiltration, vacuolation-like changes, and fibrous tissue hyperplasia, particularly in the high-dose LPB cohort (Figure 6C). IHC was performed to analyze the expression of key proteins associated with the PI3K/Akt/mTOR signaling cascade in tumor tissues. The PI3K, p-mTOR, and p-S6 levels were markedly diminished in the LPB treatment cohorts (5, 10 mg/kg). Additionally, the expression of Akt and S6 was notably decreased in the high-dose LPB cohort. These findings indicate that LPB may inhibit tumor growth in vivo by regulating the PI3K/Akt/mTOR signaling cascade (Figure 6D).

The relative expression levels of MMP-2 in the control cohort, LPB low-dose cohort (5 mg/kg), and LPB high-dose cohort (10 mg/kg) were 0.78±0.24, 0.38±0.09, and 0.33±0.15, respectively, exhibiting notable statistical variations (P<0.001). The relative expression levels of MMP-9 in the three cohorts were 0.82±0.22, 0.48±0.10, and 0.50±0.20, exhibiting notable statistical variations (P<0.01). The relative expression levels of Bax were 1.20±0.25, 1.96±0.55, and 1.78±0.39, respectively, with significant differences (P<0.05). The measured Bax expression quantities were documented at 1.37±0.39, 2.76±0.49, and 3.15±0.28, with significant differences (P<0.001). No statistical difference was detected in the relative expression of Bcl-2 among the cohorts (P>0.05). These findings indicate that LPB diminishes the quantities of migration and invasion-associated factors MMP-2 and MMP-9 within nude mice tumor specimens while enhancing the quantities of apoptosis-promoting genes Bax and Bad, confirming the in vitro observations (Figure 6E).

LPB shows preliminary biological safety

Throughout the administration period, no significant variations were detected in the developmental patterns of nude mice across the control, low-dose, and high-dose cohorts. No signs of drug toxicity were noted, and the mice’s activity, diet, and overall condition remained normal. No mortality occurred, and the mental state of the mice was stable. Body weights did not differ markedly between the control cohort (18.43±2.04 g), the low-dose cohort (18.85±2.22 g), and the high-dose cohort (18.65±1.03 g) (P>0.05) (Figure 7A). H&E staining of tissue sections from the heart, liver, and kidneys displayed no abnormalities in these organs (Figure 7B). To further assess the effects of LPB on organ function, serum biochemical markers were analyzed. No significant differences were found in ALT, AST, Cr, LDH, CK, and CK-MB levels between the low-dose and high-dose cohorts (P>0.05), indicating that LPB does not adversely affect heart, liver, or kidney function in nude mice (Figure 7C).

Figure 7 Preliminary in vivo safety evaluation of LPB. (A) The effect of LPB on body weight in SAS xenograft nude mice. (B) Histological analysis of heart, kidney, and liver tissues in SAS xenograft nude mice (H&E staining, ×200). (C) The effect of LPB on serum biochemical indices in SAS xenograft nude mice. ALT, alanine aminotransferase; AST, aspartate aminotransferase; CK, creatine kinase; Cr, creatinine; LDH, lactate dehydrogenase; LPB, liriopesides B.

Discussion

OSCC is a highly prevalent malignancy characterized by a high rate of metastasis and poor prognosis (17). Chemotherapy remains a standard treatment for advanced OSCC (18). While current first-line chemotherapy agents can extend patient survival to some extent, they are often associated with limitations such as low specificity and significant toxic side effects (19). Recent studies have indicated that natural compounds derived from traditional Chinese medicine may offer a promising alternative for cancer treatment, with anti-cancer effects observed across various cancer types. Compared to conventional chemotherapy drugs, these natural products generally exhibit lower toxicity to normal cells, enabling the administration of higher doses with reduced adverse effects (20). LPB, a saponin active compound extracted from Ophiopogon japonicus, demonstrates anticancer efficacy against multiple malignancies, encompassing breast cancer (12) and non-small cell lung cancer (13). Research has also found that the combination of gemcitabine and LPB can significantly inhibit the proliferation, invasion, and migration of pancreatic cancer cells while promoting the apoptosis process (21). Nevertheless, the medicinal value of LPB against OSCC has yet to be investigated. This investigation represents the initial examination of LPB’s anti-OSCC capabilities and its associated molecular pathways utilizing both in vitro and in vivo models (Figure 8), as well as assessing its safety profile in vivo.

Figure 8 Anti-tumor mechanism of liriopesides B.

Uncontrolled proliferation and loss of contact inhibition are hallmark traits of cancer cells and fundamental drivers of tumor initiation and progression (22). The accumulation of genetic mutations, chromosomal instability, and epigenetic alterations disrupt the regulatory systems governing cancer cell proliferation and apoptosis. Several studies have demonstrated that numerous anti-cancer agents exert their therapeutic effects by inhibiting cancer cell proliferation. For instance, Fuling Granules have been shown to markedly suppress ovarian cancer cell proliferation via cell cycle arrest, apoptosis induction, and cellular senescence (23). Similarly, Sheng et al. reported that LPB reduced proliferation and colony formation in non-small cell lung cancer in a dose- and time-dependent manner (13), aligning with the findings presented in this study.

Metastasis, the spread of cancer from the primary site to distant organs, is closely linked to poor patient prognosis and survival (24). Preventing metastasis is critical for improving clinical outcomes in OSCC treatment. One key mechanism driving cancer cell metastasis is epithelial-mesenchymal transition (EMT), which promotes decreased cellular adhesion and morphological transformation to spindle-shaped cells, enabling cells to become motile and invasive. E-cadherin, a marker of epithelial cell-cell adhesion, serves as an indicator of the metastatic potential of cancer cells and is commonly used to evaluate cancer progression (25). During the early stages of tumor spread in the local area, matrix metalloproteinases (MMPs) are secreted to degrade and remodel extracellular matrix components such as collagen, elastin, gelatin, and casein, which helps tumor cells overcome tissue barriers and migrate to distant sites (26). MMPs are upregulated in various cancers and are associated with poor patient survival. Notably, MMP-2, MMP-9, MMP-11, and MMP-14 are particularly overexpressed in breast cancer, esophageal cancer, and head and neck cancers (27). As gelatinases in the MMP family, MMP-9 and MMP-2 play pivotal roles in the pathophysiology of tumors. Inhibition of MMP secretion represents a promising therapeutic strategy to impede cancer metastasis (28). The results from both in vitro and in vivo experiments in this investigation demonstrate that LPB markedly downregulates MMP-9, MMP-2, and E-cadherin levels in OSCC tumor tissues, suggesting that LPB effectively inhibits OSCC cell migration.

Abnormal regulation of programmed cell death markedly influences cancer development and advancement (29). Cell death programs operate through two main routes: the internal mitochondrial cascade and the external receptor-mediated pathway, each producing distinctive cellular changes leading to death (30). The internal pathway, alternatively called the mitochondrial route, responds to various toxic challenges, including oncogenic pressure, therapeutic agents, and metabolic alterations (31). These factors influence the equilibrium between death-promoting (e.g., Bax, Bad) and death-preventing (e.g., Bcl-2) proteins, modifying mitochondrial barrier function and enabling cytochrome c release into cellular space. This initiates apoptosome formation, triggering death-executing proteins including Caspase-9, -3, -6, and -7 (32). The Bcl-2 protein cohort crucially maintains mitochondrial integrity and regulates cellular death processes. Interventions targeting Bcl-2 proteins have emerged as vital strategies in cancer treatment development (33). For instance, compounds activating Bax, such as SMBA1 and BAM7, demonstrate cell death induction in cancer cells both in vitro and in vivo (34). The flow cytometry reveals LPB’s concentration-dependent effect on OSCC cell death. Additional gene expression studies confirm that LPB treatment enhances death-promoting genes Bax and Bad while reducing death-preventing Bcl-2 expression, aligning with experimental observations. These results suggest LPB promotes OSCC cell death via mitochondrial pathway regulation through Bcl-2 protein family modulation.

The PI3K/Akt/mTOR cascade serves as a crucial mechanism in controlling essential cellular functions encompassing multiplication, migration, invasion, and apoptosis, which are central to tumor development and advancement (35). This signaling network’s hyperactivity has been observed across numerous cancer types, including OSCC (16,36). Prior investigations have revealed that modulating the PI3K/Akt/mTOR axis can markedly impact OSCC cell behavior. For instance, genipin was shown to induce autophagy and apoptosis in OSCC cells via the PI3K/Akt/mTOR cascade while concurrently inhibiting cell proliferation (37). Similarly, PX-866, a PI3K inhibitor, induced apoptosis, cell cycle arrest, and autophagy, with a concomitant reduction in OSCC invasiveness (38). These findings underscore the therapeutic potential of targeting the PI3K/Akt/mTOR cascade in cancer treatment. In this investigation, LPB was detected to modulate the PI3K/Akt/mTOR signaling pathway, as evidenced by differential gene expression analysis via high-throughput sequencing. Both WB analysis and in vivo data revealed a reduction in key protein levels within this pathway in OSCC cell lines and transplanted tumor tissues following LPB treatment. These observations indicate that LPB achieves its anti-OSCC impact through suppression of the PI3K/Akt/mTOR signaling network.

Despite these promising findings, LPB’s clinical translation faces numerous challenges. The foremost obstacle is the lack of systematic pharmacokinetic and toxicological studies. While our research preliminarily evaluated changes in body weight, pathological indicators and serum biochemical parameters in vivo, comprehensive safety assessment such as immunotoxicity tests or long-term toxicity experiments remain to be conducted. Critically, the low oral bioavailability of steroidal saponins, stemming from gastrointestinal instability, poor membrane permeability, significant first-pass effects, and susceptibility to gut microbiota-mediated hydrolysis, constitutes a fundamental bottleneck for clinical application (10). Overcoming this limitation necessitates focusing primarily on and artificial synthesis (8), targeted delivery systems such as liposomal, emulsion-based or nanoparticulate carriers (39), chemical structural modifications guided by quantitative structure-activity relationship (QSAR) studies to optimize pharmacokinetic profiles (40). However, neither approach has yet been applied to LPB. In combination therapies, saponins had promising anticancer effects when combined with other therapeutics (41). Though LPB synergizes with gemcitabine to suppress pancreatic cancer progression (21), its interactions with radiotherapy, immunotherapy, and other treatment modalities remain unexplored. Mechanistically, although our transcriptome sequencing revealed preliminary antitumor effects of OSCC, the precise biochemical processes governing LPB’s regulation of the PI3K/Akt/mTOR pathway and its specific target proteins warrant in-depth investigation. Given that most saponin-based drug research remains at the preclinical stage (8,10), advancing LPB into clinical trials after thorough safety validation represents not only an indispensable step but a critical pathway toward clinical translation.

Conclusions

In conclusion, this investigation reveals fresh perspectives on the anti-OSCC effects of LPB, demonstrating its capacity to regulate the PI3K/Akt/mTOR signaling cascade. The correlation between LPB’s anti-cancer activity and changes in MMP expression has also been established, while the safety of LPB in vivo was evaluated for the first time. These observations establish a robust basis for advancing LPB toward clinical applications as a potential anti-OSCC therapeutic agent.

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-2025-613/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-613/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-613/prf

Funding: This investigation was financially supported by the National Natural Science Foundation of China under Grant (grant number: 82360187), the Guangxi Science and Technology Base and Talents Special Project (grant number: 2021AC18031), the Guangxi Medical and Health Suitable Technology Development and Popularization Applications Project (grant number: S2021085), and the Nanning Qingxiu District Science and Technology Plan (grant number: 2021004).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-613/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. The animal experiments were approved by the Ethics Committee of Guangxi Medical University (202112003), in compliance with institutional guidelines for the care and use of animals.

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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