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 PCRRNA 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

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

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.