Selective antitumor and apoptosis-inducing effects of the Src inhibitor PP1 in human tongue squamous cell carcinoma cells

Abstract

Src phosphorylation (activation) is associated with the proliferation and survival of numerous human cancer cells. The role of Src phosphorylation and expression, as well as its pharmacological inhibition by PP1, a Src inhibitor, in the growth of oral squamous cell carcinoma (OSCC), remain unclear. The present study explored whether Src is expressed and phosphorylated in HSC-3 human oral cancer cells and whether PP1 treatment affects the proliferation of these cells. Src was found to be highly expressed and phosphorylated in HSC-3 human oral cancer cells. Notably, treatment with PP1 at 10 µM significantly reduced cell proliferation and induced apoptosis, evidenced by DNA fragmentation, caspase-9 and −8 activation, and poly(ADP-ribose) polymerase cleavage. Mechanistically, PP1 not only inhibited Src phosphorylation but also disrupted a broad network of oncogenic pathways, including EGFR, JAK2, STAT-3, PKB and ERK-1/2 in HSC-3 cells. Furthermore, PP1 induced markers of ER stress and inhibited protein translation, as shown by increased eIF-2α phosphorylation and decreased S6 phosphorylation. The critical role of Src was confirmed by pharmacological inhibition and further validated when small interfering RNA-mediated knockdown mimicked the anti-proliferative effects of PP1. Importantly, these potent anticancer effects were conserved in another OSCC cell line (YD-10B) and, were validated in vivo, where PP1 suppressed tumor growth in a zebrafish xenograft model. Collectively, these findings suggest that PP1 exerts strong anticancer effects on human oral cancer by simultaneously inhibiting Src activity and disrupting a network of associated oncogenic pathways (EGFR, STAT-3, PKB and ERK-1/2).

Introduction

Oral squamous cell carcinoma (OSCC) is the predominant type of cancer in the upper aerodigestive tract, mainly affecting the head and neck area (1–3), with over 377,000 new cases and 177,000 deaths reported annually, making it one of the most common malignancies worldwide (4). It is known for its high metastatic potential within the oral cavity and ranks as one of the top 10 most prevalent cancers worldwide (5,6). This aggressive cancer not only has a high mortality rate but can also lead to significant facial disfigurement, severely impacting patients' quality of life (1,5,6). Emerging biomarkers such as salivary non-coding RNAs, including microRNAs and lncRNAs, offer promising non-invasive diagnostic and prognostic tools (4). Standard treatments for OSCC primarily involve surgery and radiotherapy, often supplemented by chemotherapy such as cisplatin, 5-fluorouracil and doxorubicin, and more recently, immunotherapy, including immune checkpoint inhibitors such as PD-1/PD-L1 blockers, targeting the tumor immune microenvironment (7). Additionally, molecular profiling has paved the way for personalized treatment approaches that aim to enhance clinical efficacy while reducing systemic toxicity (8). Despite extensive research efforts, the global incidence of OSCC continues to rise (9). Consequently, there remains a critical need for ongoing discovery of novel and effective therapeutic drugs and molecular targets to enhance treatment options for OSCC (10).

The proto-oncogene Src encodes a non-receptor tyrosine kinase, Src, which plays a critical role in cancer cell proliferation, survival and migration by contributing to malignant transformation and oncogenesis (11–13). Studies have shown that Src is highly activated through phosphorylation in various human solid cancers and cancer cells, including prostate, breast, colon and hematologic malignancies (14–16). Inhibiting Src using pharmacological inhibitors or gene silencing has demonstrated effectiveness in reducing tumor size and inhibiting cancer cell proliferation (17–19). This suggests that targeting Src activation and inhibition could be essential for treating cancers where Src hyperactivation drives oncogenic processes. Various growth factors, such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF) and hepatocyte growth factor (HGF), can trigger Src phosphorylation and activation, leading to the regulation of downstream targets such as phosphatidylinositide-3 kinase (PI3K), protein kinase B (PKB/Akt), signal transducer and activator of transcription-3 (STAT-3), and extracellular signal-regulated kinase-1/2 (ERK-1/2), which control cell proliferation, cycle and migration in colorectal and other cancers (20–23). Previous evidence has shown that Src is overexpressed in OSCC, and its overexpression is closely associated with OSCC development (24). Despite these findings, the precise role of Src activation in driving OSCC cell proliferation remains to be fully elucidated.

Given the critical role of Src, targeting its activity with selective inhibitors presents a promising therapeutic strategy. One such inhibitor is 4-Amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo-d-3,4-pyrimidine (PP1), a selective inhibitor of Src that has exhibited potent anticancer effects in hematological and solid tumors, including Ras-associated cancers, rat basophilic leukemia cells, and renal cancer cells (25–29). Nonetheless, the regulatory impact and mechanism of PP1 on human oral cancer cell proliferation remain insufficiently explored. Therefore, in the present study, it was aimed to investigate the expression and phosphorylation status of Src in HSC-3 human oral cancer cells and it was explored how treatment with PP1 influences the growth of these cells.

Materials and methods

Chemicals and antibodies

PP1 was purchased from Calbiochem. Media for culturing cells and reagents, including Roswell Park Memorial Institute (RPMI)-1640 medium, Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin, and phosphate-buffered saline (PBS) were purchased from Welgene, Inc. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) reagent was acquired from Promega Corporation. Bradford's reagent was purchased from Bio-Rad Laboratories, Inc. A protease inhibitor cocktail (100X) was obtained from Calbiochem. Control siRNA (cat. no. sc-37007) and Src siRNA (cat. no. sc-29228) were obtained from Santa Cruz Biotechnology, Inc. LY294002 and PD98059 were procured from Enzo Life Sciences. Cell culture plastic wares were purchased from SPL Life Sciences. The antibodies used are provided in detail in Table I.

Table I.

Antibodies used for western blot analysis.

Antibody	Dilution	Supplier	Cat. no.
p-Src (T416)	1:2,000	Cell Signaling Technology, Inc.	2101
Src	1:2,000	Cell Signaling Technology, Inc.	2108
p-EGFR (Y1068)	1:2,000	Cell Signaling Technology, Inc.	2234
EGFR	1:2,000	Cell Signaling Technology, Inc.	2646
p-JAK2 (Y1007/1008)	1:2,000	Cell Signaling Technology, Inc.	sc-3776
JAK2	1:2,000	Santa Cruz Biotechnology, Inc.	sc-278
p-STAT-3 (Y705)	1:2,000	Santa Cruz Biotechnology, Inc.	sc-8059
STAT-3	1:2,000	Santa Cruz Biotechnology, Inc.	sc-8019
p-PKB (S473)	1:2,000	Cell Signaling Technology, Inc.	9271
PKB	1:2,000	Cell Signaling Technology, Inc.	9272
p-ERK-1/2 (T202/Y204)	1:2,000	Cell Signaling Technology, Inc.	9101
ERK-1/2	1:2,000	Cell Signaling Technology, Inc.	9102
Procaspase-9	1:2,000	Enzo Life Sciences	ADI-AAM-139
Procaspase-8	1:2,000	Cell Signaling Technology, Inc.	9746
PARP	1:2,000	Santa Cruz Biotechnology, Inc.	sc-53643
p-eIF-2α (S51)	1:2,000	Abcam	ab32157
eIF-2α	1:2,000	Cell Signaling Technology, Inc.	9722
p-S6 (S235/S236)	1:2,000	Cell Signaling Technology, Inc.	2211
S6	1:2,000	Cell Signaling Technology, Inc.	2317
ATF4	1:2,000	Santa Cruz Biotechnology, Inc.	sc-390063
β-Actin	1:10,000	MilliporeSigma	A5441
Goat anti-rabbit IgG-HRP	1:5,000	Jackson Immuno Research Laboratories, Inc.	111-035-045
Goat anti-mouse IgG-HRP	1:5,000	Jackson Immuno Research Laboratories, Inc.	115-035-062

p-, phosphorylated; eIF-2α, eukaryotic initiation factor-2α; ATF4, activating transcription factor 4; S6, ribosomal protein S6; PARP, poly(ADP-ribose) polymerase.

Cell culture

Human cancer cell lines (HSC-3, YD-10B and A549) were obtained from the Japanese Cancer Research Resources Bank. Human gingival fibroblasts (HGFs) were purchased from the American Type Culture Collection. HSC-3 and YD-10B cells, as well as HGFs, were cultured in high-glucose DMEM (MilliporeSigma), while A549 cells were cultured in RPMI-1640 medium, both supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin, at 37°C in a humidified atmosphere with 5% CO₂.

Cell count assay

HSC-3 and YD-10B cells were seeded at a density of 2×10⁴ cells/500 µl/well, while HGFs were seeded at a density of 2.5×10⁴ cells/500 µl/well in a 24-well plate and incubated overnight. Subsequently, the cells were treated with either vehicle control (0.1% DMSO) or varying concentrations of PP1 (0.1, 0.5, 1, 5 and 10 µM) for 24 h. The cells were then washed twice with PBS, and the number of viable cells, indicated by the absence of trypan blue staining, was determined using a phase-contrast microscope. The cell count assay was conducted in triplicate, and the data presented represent the mean ± SE of three independent experiments.

DNA fragmentation assay

The method for analyzing DNA fragmentation was previously outlined in detail (27). Intact or fragmented DNA was extracted and subjected to electrophoresis at 50 V on a 1.8% agarose gel containing GelRed Nucleic Acid Stain (cat. no. 41003; Biotium, Inc.) for 40 min. Subsequently, the intact or fragmented DNA was visualized and captured under UV illumination post-staining with ethidium bromide (0.1 µg/ml) using a Gel documentation system (Gel Doc-XR; Bio-Rad Laboratories, Inc.).

Preparation of whole-cell lysates

HSC-3 and YD-10B cells were seeded at a density of 2×10⁵ cells/ml/well in a 6-well plate overnight. Following this, the cells were treated with the vehicle control (0.1% DMSO), PP1, and other reagents for the specified durations. At designated time points, the conditioned HSC-3 cells were washed, harvested, and lysed in a radioimmunoprecipitation assay (RIPA) buffer (MilliporeSigma) supplemented with a protease inhibitor cocktail (1X). The whole-cell lysates underwent centrifugation at 12,000 × g for 20 min at 4°C, and the resulting supernatant was preserved for protein concentration determination using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Inc.).

Western blot analysis

Proteins (40 µg) were separated via 10 or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane (MilliporeSigma). The membranes were blocked with 5% (w/v) skim milk in Tris-buffered saline (TBS) (10 mM Tris, 150 mM NaCl) containing 0.05% (v/v) Tween 20 (TBST) for 2 h, followed by incubation with the respective primary antibodies against the target proteins listed in Table I at 4°C. After three washes with TBST, the membranes were incubated with the appropriate secondary antibodies of horseradish peroxidase (HRP)-conjugated anti-goat immunoglobulin (IgG) or anti-mouse IgG or anti-rabbit IgG for 2 h at room temperature. Subsequently, the membranes were washed thrice with TBST and visualized using ECL reagents (Advansta,), and the chemiluminescent signals were detected using an imaging system (NFEC-2025-08-307766). Equal protein actin levels were used as a loading control. The band intensity was quantified using ImageJ (version 1.53; National Institutes of Health).

Small interfering RNA (siRNA) transfection

Transfection of HSC-3 cells was carried out using 100 pM of control siRNA (sc-37007) or Src siRNA (sc-29228) in combination with Lipofectamine RNAiMAX (Invitrogen; Thermo Fisher Scientific, Inc.). The specific sequences of these siRNAs were not provided by the manufacturer as they are proprietary pooled products and scrambled sequences. The siRNA-lipid complexes were allowed to form for 15 min at room temperature and then added to the cells. Cells were incubated with the transfection complexes at 37°C in a humidified atmosphere containing 5% CO₂ for 48 h. After transfection, whole-cell lysates were collected and subjected to western blot analysis for Src and actin expression levels.

Cancer cell preparation for zebrafish injection

Before injection, HSC-3 cells were labeled in vitro with 20 µg/ml of 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI; Invitrogen; Thermo Fisher Scientific, Inc.) for 30 min. A total of 5 nl of tumor cell solution containing 50 DiI-labeled HSC-3 cells was injected into the perivitelline cavity (yolk sac) of each embryo using a microinjector.

Zebrafish tumor xenograft model

Fertilized zebrafish (Danio rerio) eggs from the transgenic Tg (fli1:EGFP) strain, which expresses enhanced green fluorescent protein (EGFP) under the fli1 promoter, were incubated at 28°C in sea salt water and maintained under standard laboratory conditions. At 48 h post-fertilization (hpf), embryos were dechorionated using sharp-tipped forceps and anesthetized with 0.04 mg/ml tricaine (cat. no. MS-222; MilliporeSigma). The anesthetized embryos were then transferred onto a 2% agarose gel for microinjection.

DiI-labeled HSC-3 cells were resuspended in complete DMEM medium (Welgene, Inc.), and 5 nl of the cell suspension, containing ~50 cells, was injected into the yolk sac of each embryo using a microinjector (World Precision Instruments). Following injection, embryos were immediately transferred into system water. Successful cell implantation was confirmed by fluorescence microscopy. Subsequently, embryos were immersed in system water containing vehicle control (0.1% DMSO) or varying concentrations of the PP1 compound and maintained at 28°C. Tumor growth and invasion were monitored at 24 and 48 h post-injection using fluorescence microscopy.

At the experimental endpoint, zebrafish embryos were euthanized by immersion in 0.3 mg/ml tricaine for 25 min after cessation of opercular movement to ensure irreversible euthanasia and prevent recovery (30,31). All animal (zebrafish) experiments were conducted in compliance with the Institutional Animal Care and Use Committee (IACUC) guidelines and were approved (approval no. KM2025-014) by the Ethics Committee of Keimyung University (Dalseo, Korea).

Statistical analysis

Data are presented as the mean ± standard error (SE) of three independent experiments. Prior to analysis, data distribution was assessed for normality. Since the data followed a normal distribution, statistical significance among groups was evaluated using one-way ANOVA, followed by Dunnett's post hoc test, to compare multiple treatment groups against the control. All analyses were performed using SPSS software, version 20 (IBM Corp.). P<0.05 was considered to indicate a statistically significant difference.

Results

Src exhibits significant expression and phosphorylation levels in HSC-3 human oral cancer cells

Initially, Western blot analysis was employed to assess the basal total (T-) expression and phosphorylation (p-) status of Src in HSC-3 human oral cancer cells, A549 human lung cancer cells, and normal HGFs cultured in 10% FBS-containing media for 8 h. In Fig. 1A, substantial levels of T-Src in both normal and cancerous cells (lower panel) are illustrated. Notably, HSC-3 cells exhibited markedly elevated p-Src levels compared with A549 cells and HGFs. The triplicate experimental results (Fig. 1B and C) further confirmed the significant difference in the p-Src/T-Src ratio between HSC-3 and A549 cells. As previously observed, the Src phosphorylation in cancer cells is stimulated by growth factors. Given that the FBS present in the culture medium for HSC-3 cells contains various growth factors and mitogens (32), an investigation was conducted to determine if the observed hyperphosphorylation of Src in HSC-3 cells is influenced by FBS and its constituents. To this end, HSC-3 cells were cultured in media devoid of FBS or with 10% FBS for different durations. At each time interval, levels of p-Src and T-Src in HSC-3 cells grown with or without 10% FBS were measured. Minimal levels of p-Src were detected in HSC-3 cells cultured without FBS, whereas cells incubated with 10% FBS exhibited elevated p-Src levels at the time points examined (Fig. 1D). These results suggest that Src hyperphosphorylation in HSC-3 cells is dependent on the presence of FBS.

Figure 1.

Elevated Src expression and phosphorylation in HSC-3 human oral cancer cells. (A) HSC-3 cells, A549 cells and human gingival fibroblasts (HGFs) were cultured in medium containing 10% FBS for 8 h, followed by preparation of whole-cell lysates for Western blot analysis. (B) HSC-3 cells and A549 cells were cultured in medium containing 10% FBS for 8 h, followed by the preparation of whole-cell lysates for Western blot analysis. (C) Densitometric analysis of the data in (B) showing phosphorylation levels of Src normalized to total Src expression in HSC-3 cells and A549 cells. Data are presented as the mean ± SE from three independent experiments. *P<0.05 compared with A549 cells. (D) HSC-3 cells were cultured in medium containing either 0 or 10% FBS for the indicated times. Whole-cell lysates were prepared at each time point and subjected to Western blot analysis.

Exposure to PP1 at 5 or 10 µM specifically results in a notable decrease in the proliferation of HSC-3 cells

As previously mentioned, PP1 (depicted in Fig. 2A) is a specific inhibitor of Src (25). To assess the potential of PP1 as a novel candidate drug for anti-oral cancer treatment and evaluate Src as a potential molecular target in oral cancer cells, HSC-3 cells were treated with various concentrations of PP1 (0.1, 0.5, 1, 5, or 10 µM) for 24 h, and cell survival was assessed using a cell count assay. Normal HGFs were included in the study for comparison and specificity. Notably, as shown in Fig. 2B, treatment with PP1, particularly at 5 and 10 µM for 24 h, significantly decreased the survival of HSC-3 cells. By contrast, the survival of HGFs was unaffected by the tested concentrations. Microscopic examination, depicted in Fig. 2C, further demonstrated that treatment with PP1 at 5 and 10 µM for 24 h markedly inhibited the proliferation of HSC-3 cells (upper panels), while there was no significant impact on the proliferation of HGFs following exposure to PP1 at the administered doses (lower panels). These findings highlight the selectivity of PP1 in suppressing the proliferation of HSC-3 cells, underscoring its potential as a targeted therapy.

Figure 2.

Effects of PP1 on the proliferation of HSC-3 human oral cancer cells and normal HGFs. (A) Chemical structure of PP1. (B) HSC-3 cells and HGFs were treated with vehicle control (0.1% DMSO) or the indicated concentrations of PP1 for 24 h, followed by cell counting to determine cell survival. (C) Representative phase-contrast images of cells treated as described in (B), captured at ×100 magnification (scale bar, 100 µm). Images are representative of three independent experiments. Data are presented as the mean ± SE from three independent experiments. *P<0.05 compared with control values.

Exposure to PP1 at 10 µM triggers apoptosis in HSC-3 cells, accompanied by the activation of caspase-9 and −8 and the cleavage of poly(ADP-ribose) polymerase (PARP)

Inhibition of cancer cell growth often involves the initiation of apoptosis, a form of programmed cell death (3). One characteristic hallmark of apoptosis is the presence of fragmented nuclear DNA. Therefore, it was investigated whether treatment with PP1 could induce apoptosis in HSC-3 cells through a DNA fragmentation assay. Notably, as illustrated in Fig. 3A, treatment with PP1 at concentrations of 1, 5, or 10 µM for 24 h resulted in genomic DNA fragmentation in HSC-3 cells. The activation of caspase-9 and caspase-8 is a crucial step in programmed cell death induced by various apoptotic stimuli in human cancer cells (33). Caspases are typically expressed as inactive precursor forms with high molecular weights in cells (34). Upon exposure to apoptotic triggers, caspases undergo processing into active, low molecular weight forms (35). Therefore, it was investigated whether treatment with PP1 impacts the expression and activation of (pro)caspase-9 and (pro)caspase-8 in HSC-3 cells. As depicted in Fig. 3B, results from triplicate experiments revealed that treatment with PP1, particularly at 10 µM for 2 h, significantly reduced the levels of procaspase-9 and procaspase-8 while increasing the levels of cleaved (active) caspase-9 and caspase-8 in HSC-3 cells. Active caspase-9 and caspase-8 are involved in cleaving various intracellular proteins, including PARP, which is crucial for cell proliferation (36). Intriguingly, treatment with PP1 at concentrations of 1 or 10 µM for 2 h also led to a substantial accumulation of cleaved PARP in HSC-3 cells. The densitometric analysis for levels of procaspase-9, procaspase-8 and cleaved PARP, normalized to actin, is demonstrated in Fig. 3C.

Figure 3.

Effects of PP1 on apoptosis and the expression of apoptosis-related markers in HSC-3 human oral cancer cells. (A) HSC-3 cells were exposed to vehicle control (0.1% DMSO) or PP1 (1, 5, and 10 µM) for 24 h. Genomic DNA was extracted and analyzed on a 1.8% agarose gel. (B) HSC-3 cells were treated with vehicle control or PP1 (1 or 10 µM) for 2 h, and whole-cell lysates were prepared and subjected to Western blot analysis. Blots are representative of three independent experiments. (C) Densitometric analysis of the data in (B) showing expression levels of procaspase-9, procaspase-8, and cleaved PARP normalized to control actin protein in HSC-3 cells. Data are presented as the mean ± SE from three independent experiments. *P<0.05 compared with control values.

PP1 decreases levels of p-Src and downstream oncogenic signaling pathways in HSC-3 cells

To understand the molecular signaling events leading to PP1-induced apoptosis, its effects on major oncogenic pathways known to be associated with Src were next investigated. It was demonstrated that treatment with PP1 at 1 or 10 µM resulted in a dose- and time-dependent reduction in p-Src levels, while levels of T-Src remained unaffected, indicating the drug's effectiveness in inhibiting Src activity (Fig. 4A). PP1 (10 µM) also decreased levels of p-PKB and p-ERK-1/2 at the examined time points (2, 8 and 24 h), without altering their total protein expression levels. Interestingly, the effect on STAT-3 and EGFR was delayed. While treatment for 2 or 8 h had no impact, a 24-h exposure to PP1 caused a dose-dependent decrease in both the phosphorylation and total expression levels of these proteins. As illustrated in Fig. 4B, results from triplicate experiments further validated the ability of PP1 treatment at 10 µM for 8 h to significantly reduce levels of p-Src without affecting its total expression in HSC-3 cells. The densitometric data for p-Src levels normalized by T-Src levels in HSC-3 cells is provided in Fig. 4C.

Figure 4.

Effects of PP1 on the phosphorylation and expression of Src and other signaling proteins in HSC-3 human oral cancer cells. (A) HSC-3 cells were exposed to vehicle control (0.1% DMSO) or PP1 (1 or 10 µM) for the indicated times. At each time point, whole-cell lysates were prepared and subjected to Western blot analysis. (B) HSC-3 cells were treated with vehicle control or PP1 (10 µM) for 8 h in triplicate Whole-cell lysates were prepared and analyzed using Western blotting. (C) Densitometric analysis of the data in (B) showing phosphorylation levels of Src normalized to total Src expression in HSC-3 cells. Data are presented as the mean ± SE from three independent experiments. *P<0.05 compared with control at the indicated time.

Inhibition of downstream signaling pathways reduces HSC-3 cell proliferation

As aforementioned, it was shown that PP1 inhibits a network of key signaling proteins in HSC-3 cells, including EGFR, Janus kinase 2 (JAK-2), STAT-3, PKB and ERK-1/2 (Fig. 4A). To determine whether the inhibition of these individual pathways contributes to the overall proliferation-inhibitory effect of PP1, the effects of erlotinib (an EGFR inhibitor), AG490 (a JAKs/STATs inhibitor), LY294002 (a PI3K/PKB inhibitor) and PD98059 (an ERK-1/2 inhibitor) on the proliferation of HSC-3 cells were assessed. As depicted in Fig. 5A-D, treatment with erlotinib, AG490, LY294002 and PD98059 resulted in a dose-dependent reduction in the proliferation of HSC-3 cells. Microscopic examination further verified that each pharmacological inhibitor could diminish the proliferation of HSC-3 cells in a concentration-dependent manner (Fig. 5E).

Figure 5.

Effects of erlotinib, AG490, LY294002, or PD98059 on the proliferation of HSC-3 human oral cancer cells. (A-D) HSC-3 cells were treated for 24 h with the indicated concentrations of (A) erlotinib, (B) AG490, (C) LY294002, or (D) PD98059. Vehicle control (0.1% DMSO) was used for comparison. Cell survival was determined by cell count analysis. Data represent the mean ± SE of three independent experiments. *P<0.05 compared with vehicle control. (E) Representative phase-contrast images of treated cells from (A-D), captured at ×100 magnification (scale bar, 100 µm). Images are representative of three independent experiments.

PP1 induces endoplasmic reticulum (ER) stress and inhibits protein translation markers in HSC-3 cells

ER stress and the regulation of protein translation are critical pathways that can influence cancer cell proliferation and apoptosis (37–39). Key markers in these pathways include the eukaryotic initiation factor-2α (eIF-2α), activating transcription factor 4 (ATF4), and ribosomal protein S6 (rpS6), which is a component of the 40S ribosomal subunit (37–39). This led us to investigate the effects of PP1 on the phosphorylation and expression levels of eIF-2α, ATF4, and S6 in HSC-3 cells. The results revealed that treatment with PP1 at 1 or 10 µM for 8 or 24 h resulted in a slight, time- and dose-dependent increase in levels of p-eIF-2α without affecting its total expression in HSC-3 cells (Fig. 6). Moreover, treatment with PP1, particularly at 10 µM for 8 or 24 h, increased the expression levels of ATF4 in HSC-3 cells. The effect on S6 phosphorylation was observed only at a higher dose and later time points. Specifically, while shorter treatments (2 h) or lower-dose treatments (1 µM at 8 h) had no effect, PP1 at 10 µM significantly decreased S6 phosphorylation at 8 and 24 h. Throughout these experiments, control actin protein expression levels remained consistent.

Figure 6.

Effects of PP1 on the expression and phosphorylation of ER stress and translation-related markers in HSC-3 human oral cancer cells. HSC-3 cells were exposed to vehicle control (0.1% DMSO) or PP1 (1 and 10 µM) for the indicated times. Whole-cell lysates were collected at each time point and analyzed using Western blotting.

Src Knockdown significantly inhibits the growth of HSC-3 cells. To genetically validate that Src is the critical target responsible for the proliferation of HSC-3 cells, siRNA transfection experiments were performed. HSC-3 cells were transfected with either control or Src siRNA at a concentration of 100 pM for 48 h. Subsequently, Src expression levels were measured, and any changes in cell proliferation were monitored. Western blotting results depicted in Fig. 7A revealed a significant decrease in Src protein expression in HSC-3 cells transfected with Src siRNA compared with those transfected with control siRNA, confirming the efficacy of Src siRNA transfection. Notably, cell count analysis (Fig. 7B) and microscopic observations (Fig. 7C) demonstrated that Src knockdown significantly suppressed the proliferation of HSC-3 cells compared with cells transfected with control siRNA, providing strong evidence for the crucial role of Src expression and phosphorylation in the proliferation of HSC-3 cells.

Figure 7.

Effects of Src knockdown on the proliferation of HSC-3 human oral cancer cells. (A) HSC-3 cells were transfected with 100 pM of control siRNA (siCON) or Src siRNA (siSrc) for 48 h. Whole-cell lysates were collected and subjected to Western blot analysis. (B) Cell viability of transfected cells was determined by cell count assay in triplicate. Data represent the mean ± SE of three independent experiments. *P<0.05 compared with control. (C) Representative phase-contrast images of HSC-3 cells transfected with siCON or siSrc, captured at ×100 magnification (scale bar, 100 µm). Images are representative of three independent experiments.

PP1 also exhibits proliferation-inhibitory and pro-apoptotic effects on tumorigenic YD-10B cells

To determine whether PP1′s proliferation-inhibitory and pro-apoptotic effects are confined to metastatic tongue squamous cell carcinoma HSC-3 cells, its impact on another OSCC cell line, YD-10B, was further examined. Consistent with observations in HSC-3 cells, a 24-h treatment with PP1 resulted in a concentration-dependent decrease of YD-10B cell survival (Fig. 8A and B). In addition, PP1 treatment induced the accumulation of fragmented DNA (Fig. 8C) and enhanced the cleavage of caspase-9, caspase-8, and PARP (Fig. 8D). The effect on intracellular signaling pathways was then assessed (Fig. 8E). Similar to the effects in HSC-3 cells, PP1 treatment reduced the phosphorylation levels of Src, JAK-2, PKB, and ERK-1/2. However, unlike in HSC-3 cells, levels of p-STAT-3 remained unchanged. Furthermore, PP1 treatment dose-dependently reduced levels of p-S6 while inducing a modest increase in levels of p-eIF-2α.

Figure 8.

Effects of PP1 on survival, apoptosis and the expression and phosphorylation of cellular proteins in YD-10B cells. (A) YD-10B cells were exposed to vehicle control (0.1% DMSO) or PP1 (1, 5, or 10 µM) for 24 h, followed by cell counting to assess cell survival. Data are presented as the mean ± SE from three independent experiments. *P<0.05 compared with control. (B) Representative phase-contrast images of treated cells from (A), captured at ×100 magnification (scale bar, 100 µm). Images are representative of three independent experiments. (C) YD-10B cells were exposed to vehicle control (0.1% DMSO) or PP1 (1, 5, and 10 µM) for 24 h. Genomic DNA was extracted and analyzed on a 1.8% agarose gel. (D and E) YD-10B cells were treated with vehicle control or PP1 (1, 5, and 10 µM) for 24 h. Whole-cell lysates were prepared and subjected to Western blot analysis.

PP1 demonstrates antitumor activity in zebrafish xenograft model. To assess the in vivo antitumor effects of PP1 on oral cancer growth, a zebrafish cancer model was established using Tg (fli1:EGFP) embryos implanted with DiI-labeled HSC-3 cells (Fig. 9A). Treatment with PP1 resulted in a dose-dependent decrease in fluorescence intensity at both 24 and 48 h post-implantation (hpi) (Fig. 9B). Specifically, 2.5 µM of PP1 demonstrated significant differences at both time points, whereas 1 µM of PP1 showed significant differences only at 48 hpi (Fig. 9C). Furthermore, treatment with 1 and 2.5 µM PP1 significantly reduced the mean area at both 24 and 48 hpi (Fig. 9D).

Figure 9.

In vivo evaluation of the antitumor effects of PP1 in a zebrafish xenograft model. (A) Representative images of DiI-labeled HSC-3 cells used for implantation. (B) DiI-labeled HSC-3 cells were implanted into Tg (fli1:EGFP) zebrafish embryos and treated with vehicle control (0.1% DMSO) or the indicated concentrations of PP1. Representative fluorescence images show tumor xenografts at 0, 24, and 48 h post-implantation (hpi). Imagea are representative of three independent experiments. (C and D) Quantification of (C) fluorescence intensity and (D) mean tumor area from the images in (B) at the indicated time points. Data are presented as the mean ± SE from three independent experiments. *P<0.05 compared with control.

Discussion

Previous studies have shown that Src is highly phosphorylated and active in various human cancers, including tongue, ductal carcinoma, and breast cancer, with its hyperactivation being associated with tumor progression (3,40,41). Although PP1 is a well-known Src inhibitor, the present study is the first to specifically elucidate its potent antitumor effects in the oral cancer HSC-3 cell model through disruption of multiple oncogenic signaling pathways and induction of ER stress. The present study demonstrated that Src is highly expressed and phosphorylated (activated) in an FBS-dependent manner in HSC-3 cells. Furthermore, inhibition of Src using PP1, a pharmacological Src inhibitor, or siRNA-mediated gene silencing significantly suppressed proliferation and induced apoptosis in HSC-3 cells. Importantly, similar anti-proliferative and pro-apoptotic effects of PP1 were observed in YD-10B oral cancer cells. In addition, in vivo experiments using a zebrafish xenograft model further confirmed that PP1 reduces tumor growth and invasion. Collectively, these findings suggest that Src is a crucial mediator of oral cancer cell survival and that PP1 exerts its therapeutic effects by dismantling a broad network of oncogenic signaling pathways.

In initial experiments, total Src was expressed in multiple cell lines; however, HSC-3 oral cancer cells exhibited a uniquely high ratio of phosphorylated Src (p-Src) to total Src. These results indicate that HSC-3 cells rely heavily on Src signaling for survival and proliferation, highlighting Src as a promising therapeutic target. Accordingly, the effects of the Src inhibitor PP1, which has demonstrated antitumor efficacy in other cancers, including glioblastoma and leukemia, were examined (28,42,43). The present study confirmed its therapeutic potential, showing that treatment with PP1 at sub-micromolar concentrations significantly and selectively inhibited the proliferation of HSC-3 cells without affecting normal HGFs. Mechanistically, PP1 acted on its primary target, resulting in a dose-dependent inhibition of Src phosphorylation. Notably, PP1 exerted broader effects on beyond Src, substantially suppressing the phosphorylation of key downstream signaling molecules, including EGFR, PKB/Akt, and ERK-1/2. This observation is consistent with previous studies describing extensive crosstalk between Src and these pathways in other cancers, such as breast cancer and melanoma (44–47). Collectively, these data suggest that PP1 exerts its potent antitumor effects by inhibiting its primary target, Src, while simultaneously disrupting the broader oncogenic network that drives HSC-3 cell proliferation. To further confirm that Src is the critical target of PP1, siRNA-mediated knockdown of Src was performed. Src silencing mimicked the anti-proliferative effects of PP1, providing further evidence that Src is a crucial survival factor for HSC-3 cells.

The present investigation revealed that PP1 disrupts oncogenic signaling in HSC-3 cells through a multi-pronged mechanism. It was found that PP1 effectively suppresses the JAK-2/STAT-3 signaling axis, a key driver of proliferation and stemness in OSCC cells (48,49). Given that STAT-3 is a promising therapeutic target in OSCC due to its role in regulating cell survival genes (50–53), the effect of PP1 on this pathway was investigated. It was revealed that PP1 effectively suppresses the phosphorylation of both JAK-2 and STAT-3 in HSC-3 cells. However, regarding the uninhibited STAT-3 phosphorylation in YD-10B cells (Fig. 8E), this finding suggests PP1′s antitumor effects may exhibit cell line specificity or context dependence, underscoring the heterogeneity of oral cancer and providing insights for future personalized therapies. Since direct inhibition of this pathway by AG490 similarly suppressed cell proliferation, it is suggested that PP1′s anti-proliferative effect is mediated, at least in part, through the inhibition of the JAK-2/STAT-3 signaling axis. The comprehensive signaling inhibition by PP1 ultimately triggers the induction of apoptosis (54–56). The findings of the present study clearly indicate the hallmarks of programmed cell death, including DNA fragmentation, PARP cleavage, and the activation of both the intrinsic (caspase-9) and extrinsic (caspase-8) apoptotic pathways. This demonstrates that inhibiting the master regulator Src, upon which HSC-3 cells are highly dependent, triggers a catastrophic collapse of downstream proliferation-inhibitory pathways like JAK-2/STAT-3.

In addition to triggering these canonical apoptotic pathways, the present findings suggest that PP1 also induces significant ER stress, a process known to trigger apoptosis in OSCC (57). Specifically, a clear increase was observed in phosphorylation of the ER stress marker eIF-2α and an upregulation of the transcription factor ATF4. This is significant as the phosphorylation of eIF-2α halts general protein synthesis and concurrently upregulates the transcription factor ATF4, which together orchestrate the cellular response to ER stress (58–60). Concurrently, PP1 suppressed the phosphorylation of the translational activator S6, further confirming the inhibition of global translation (39). Taken together, these findings suggest that PP1 exerts its potent growth-suppressive effects by activating apoptotic pathways while simultaneously inducing ER stress and inhibiting global protein translation. This indicates that disruption of Src signaling by PP1 triggers downstream events including cellular stress and apoptosis.

Having established the mechanism in HSC-3 cells, we next examined if these findings could be extended to other oral cancer contexts before translating them into an in vivo system. PP1 at 10 µM similarly suppressed cell proliferation and induced apoptosis in another oral cancer cell line, YD-10B, further supporting its multi-targeted inhibitory effects on human oral cancer cells. To translate these in vitro findings into a living system, a zebrafish xenograft model was employed, which provides a rapid and visually accessible platform for evaluating the in vivo efficacy of potential anticancer agents (61). Consistent with the cell-based assays, PP1 treatment significantly suppressed tumor growth in vivo. Taken together, these results provide robust, multi-level evidence supporting the therapeutic potential of PP1 by confirming its mechanism of action, extending its efficacy across oral cancer models, and demonstrating its antitumor activity in a whole-organism context.

Mechanistically, PP1 is known to interfere with key signaling pathways, such as Src, JAK/STAT (62) and PI3K/PKB (63), that drive tumor cell proliferation and survival. The reduction in tumor burden observed in the present study may reflect the inhibition of these oncogenic signals by PP1 in vivo. Although PP1 is widely used as a Src inhibitor, it may have off-target effects. In the present experiments, we focused primarily on Src-dependent endpoints, including the phosphorylation of key Src substrates and downstream functional readouts such as cell proliferation. The selective modulation of these Src-associated pathways by PP1 suggests that the major biological effects observed are predominantly Src-mediated, making it unlikely that off-target effects account for the key findings. Future studies using more selective Src inhibitors could further validate these observations and strengthen the translational relevance of our results.

In this study, STAT-3 was examined as a representative transcription factor regulated downstream of Src signaling. Although PP1 inhibited STAT-3 phosphorylation in HSC-3 cells, this effect was not observed in YD-10B cells. This finding suggests that, while Src may function as a common upstream activator, the downstream signaling networks may vary among different oral cancer subtypes. The differential response of STAT-3 phosphorylation to PP1 treatment between HSC-3 and YD-10B cells further indicates that intrinsic molecular differences may influence PP1 sensitivity. Future studies will investigate potential differences in upstream receptor tyrosine kinase activation, baseline JAK/STAT signaling activity, and feedback regulatory mechanisms between these two cell lines. Identifying the genetic or signaling factors responsible for this divergence will help clarify the context-dependent effects of PP1 and provide deeper mechanistic insight into its therapeutic potential. Importantly, validation of these findings in patient-derived models and the assessment of Src expression and phosphorylation in clinical OSCC specimens will be critical steps in confirming the translational relevance of targeting Src. As a next step, murine xenograft experiments are planned to further evaluate the antitumor and pro-apoptotic effects of PP1 in a physiologically and immunologically more complex environment. These follow-up studies will help determine whether the therapeutic potential observed in zebrafish model can be extended to higher-order in vivo systems and will provide essential evidence for subsequent preclinical development.

Taken together, as summarized in the proposed model (Fig. 10), the present study identifies Src as a critical vulnerability in oral cancer cells. The findings demonstrate that the inhibitor PP1 effectively targets this vulnerability by inhibiting Src phosphorylation and disrupting a broad network of oncogenic signaling pathways, including EGFR, JAK-2, STAT-3, PKB and ERK-1/2. This coordinated inhibition ultimately triggers potent apoptotic responses, as evidenced by DNA fragmentation, PARP cleavage, and activation of caspase-9 and −8. These results provide a strong preclinical rationale for the further development of PP1 as a therapeutic agent for OSCC.

Figure 10.

Proposed model illustrating the anticancer effects of PP1. (A) Proposed mechanism of the anti-proliferative and apoptosis-inducing effects of PP1 in HSC-3 human oral cancer cells. (B) Schematic diagram of the zebrafish xenograft model.

In conclusion, the present study identifies Src as a critical survival factor that is hyperactivated in human oral cancer cells. The Src inhibitor PP1 exerts potent anti-proliferative and pro-apoptotic effects in vitro across multiple oral cancer cell lines and, importantly, suppresses tumor growth in vivo. Mechanistically, PP1 achieves this by inhibiting Src activity while simultaneously dismantling a network of associated oncogenic signaling pathways, including EGFR, JAK-2, STAT-3, PKB, and ERK-1/2. Collectively, these findings establish a strong preclinical rationale for targeting Src in oral cancer and highlight PP1 as a promising therapeutic agent for its treatment.

Funding Statement

The present study was supported by the Keimyung University Dongsan Medical Center in 2024 (grant no. DS2024-0001).

Availability of data and materials

The data generated in the present study may be requested from the corresponding author.

Authors' contributions

BCJ and YMS conceptualized the study. BCJ, YMS, SLK and SW developed methodology and conducted investigation. SLK, HK and SW performed software analysis. BCJ, YMS, HK, SLK and SW validated and visualized data, performed formal analysis, and wrote, reviewed and edited the manuscript. YMS and BCJ provided resources. SLK, HK and SW curated data. BCJ, YMS and SLK prepared the original draft of the manuscript. SW generated supplementary data. BCJ and YMS supervised the study, conducted project administration and acquired funding. BCJ and SW confirm the authenticity of all the raw data. All authors read and approved the final version of the manuscript.

Ethics approval and consent to participate

All animal (zebrafish) experiments were conducted in compliance with the Institutional Animal Care and Use Committee (IACUC) guidelines and were approved (approval no. KM2025-014) by the Ethics Committee of Keimyung University (Dalseo, Korea).

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.