OSCC cell lines (HSC-3 and HN12) were obtained from the Japanese Collection of Research Bioresources (JCRB) Cell Bank. The cells were routinely cultured in high glucose DMEM supplemented with 10% fetal bovine serum (Invitrogen Life Technologies, Carlsbad, CA, United States) and 1% antibiotics at 37°C in a 5% CO₂ incubator. The primary antibodies anti-PERK, anti-p-PERK, anti-eIF2α, anti-p-eIF2α were purchased from Cell Signaling Technology (1:1000, United States). The primary antibodies anti-ATF4, anti-caspase-3, anti-cleaved-caspase-3, anti-Bcl2, anti-Bax were obtained from Affinity (1:500, United States). Antibodies α-tubulin, anti-HSP60, anti-IF1, anti-VDAC, and anti-CHOP were obtained from Abcam (1:1000, Cambridge, MA). The OXPHOS complexes were obtained from Thermo Fisher Scientific (1:1000, United States). Antibody p-Ser was obtained from Abcam (1:1000, AP0932, China). Goat anti-rabbit/mouse secondary antibody was purchased from ZSGB-BIO (Beijing, China). Nebivolol was purchased from Selleck (Shanghai, China). 4-Phenylbutyrate (4-PBA) was purchased from Sigma-Aldrich (St. Louis, MO, United States). MitoTracker Red was obtained from Solarbio (Beijing, China).

Briefly, cells were seeded in 96-well plates at a density of 2 × 104 cells per well and treated with different concentrations of nebivolol (0, 1, 5, 10, 15, 20, and 50 μM) for 24 h. The cell proliferation rates were determined by CCK-8 solution and incubated at 37°C for 1 h. Absorbance was measured at a wavelength of 450 nm using a plate reader.

HSC-3 and HN12 cells were cultured in 6-well plates at a density of 500 cells per well. After being treated with different concentrations of nebivolol (0, 5, 10, and 15 μM) for 24 h, cells were incubated in a humidified atmosphere of 5% CO₂ at 37°C for 14 days until colony formation was observed. Then, cells were fixed in 4% paraformaldehyde for 30 min and stained with 1% crystal violet for 30 min. The stained cells were captured by stereoscopic microscope.

Flow cytometry was used to measure cell apoptosis and the cell cycle distribution. Cell apoptosis assay was evaluated by Annexin V-FITC apoptosis detection kit (Nanjing KeyGen Biotech Co. Ltd., Nanjing, China) according to the manufacturer’s instructions. The cell cycle distributions were evaluated by a cell cycle detection kit (Nanjing KeyGen Biotech Co. Ltd., Nanjing, China) according to the manufacturer’s instructions.

The apoptotic cells were evaluated by using DeadEnd^TM Fluorometric TUNEL system (Promega) according to the manufacturer’s instructions. The percentage of apoptotic and nonapoptotic cells was observed by fluorescent microscope.

HSC-3 and HN12 were cultured in a 10 cm dish and incubated with vehicle or 10 μM nebivolol for 24 h. The cells were harvested and fixed in a 3% glutaraldehyde solution. Then, cells were prefixed in Karnovsky’s solution and washed with cacodylate buffer. After dehydration with alcohol, the cells were embedded in Poly/Bed 812 resins and polymerized and observed under an electron microscope (EM 902A, Zeiss, Oberkochen, Germany).

OSCC cells were cultured in a 20 mm glass plate and treated with or without nebivolol for 24 h. Later, the cells were stained with 1 nM MitoTracker Red and 10 nM Hoechst at 37°C for 15 min. The images were captured by Confocal Olympus fluorescence microscope.

Total RNA was gathered by TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. The concentration of total RNA was measured by NanoDrop One (Thermo Fisher Scientific). cDNA was prepared from 2 μg RNA, which utilized PrimeScript RT Reagent kit with gDNA Eraser (Takara). qRT‐PCR was performed using SYBR Mix on a Bio‐Rad CFX96 Real‐Time System. The relative mRNA expressions were normalized by the GAPDH gene and calculated by using the 2^−∆∆Ct method when compared to the control groups. The relative primers’ sequences were listed in Supplementary Table 1.

The mitochondrial mass was evaluated by using acridine orange 10-nonyl bromide (NAO, HY-D0993). Cells were treated with or without 10 µM nebivolol for 24 h. Later, the treated cells were gathered and incubated with 5 µM NAO for 20 min and analyzed by using the flow cytometer (Beckman FC500, Brea, CA, United States).

The OSCC cells were reseeded in 24-well microplates which were treated with and without 10 µM nebivolol for 2 h. The corresponding treated cells’ oxygen consumption rate (OCR) was measured by Seahorse XFp Extracellular Flux Analyzer (Seahorse Bioscience). The sequential final concentrations of injected substances were 2.5 μM oligomycin, 1 μM FCCP, 1 μM rotenone, and antimycin. Each group was determined five times and normalized according to the number of cells.

The cells were incubated with or without 10 μM nebivolol for 24 h and then harvested and resuspended in PBS. The cells were incubated with 5 μM H2DCFDA for 30 min in the dark. Finally, the fluorescence intensity of the 2,7-dichlorofluorescein dye was quantified by using a plate reader.

Cells were treated with or without 10 µM nebivolol for 24 h. Then, cells were gathered to measure mitochondrial membrane potential by using Mitochondrial Membrane Potential Assay Kit (C2006, Beyotime, Shanghai, China) according to the manufacturer’s instructions. The treated cells were gathered and analyzed by using the flow cytometer (Beckman FC500, Brea, CA, United States).

The cells were stimulated with different concentrations of nebivolol. The proteins were gathered in each group. The instructions of western blot analysis were described elsewhere. The primary antibodies were described above. The results were visualized by using an enhanced chemiluminescence Western-blotting detection reagent (Millipore, Billerica, MA, United States).

The small interfering RNAs (siRNAs) were designed and obtained by Ribobio (Guangzhou, China) and were transfected with Lipofectamine RNAiMAX reagent (Invitrogen) according to the manufacturer’s protocol. The sequences of siRNA were as follows: si iNOS1: GTG​CTG​TAT​TTC​CTT​ACG​A and: si iNOS2: GAA​GCG​TAT​CAC​GAA​GAT.

After transfection for almost 48 h, the groups were treated with or without nebivolol for 3 h or 12 h, and the corresponding gene and protein expressions were evaluated by quantitative real-time PCR (qPCR) and Western blot.

For gene knockdown, lentiviruses knockdown ATF4 or negative vectors were designed and purchased from JiKai (Shanghai, China). The cells were transfected with lentiviruses for 72 h and selected with 2 μg/ml puromycin (Sigma). The stable expression cell lines were treated with nebivolol for 12 h or 24 h. The corresponding gene expressions were evaluated by qPCR, and the cell viability was detected by CCK-8 assay.

The OSCC patients were diagnosed by two experienced pathologists. OSCC tissue samples were obtained from the Hospital of Stomatology, Sichuan University, with informed consent from patients. The tissues were collected and used in accordance with a protocol approved by the Human Research Ethics Committee of the West China Hospital of Stomatology, Sichuan University.

Twenty female C57BL/6 mice, weighing 16–20 g, 4–6 weeks old, were used to induce oral carcinomas. Protocols for animal experiments were approved by the Animal Care and Use Committee, State Key Laboratory of Oral Diseases, Sichuan University, in compliance with the guidelines approved by the National Institute of Health (NIH) in the United States. The mice were housed in the specific pathogen-free (SPF) unit of the animal facility in accordance with institutional guidelines. 4NQO was used to establish the oral carcinomas model. The mice were fed up with 100 μg/ml 4NQO chemistry medicine to induce tongue tumor model. Meanwhile, animals were randomly allocated into different groups and were treated with a single daily intraperitoneal injection of 25 mg/kg nebivolol or 125 mg/kg 6OHDA every two weeks. After 24 weeks, the mice were euthanized, and the mice tongues were gathered to prepare for immunohistochemistry analysis.

HSC-3 cells were gathered, washed, and resuspended in DMEM, which were subcutaneously injected (2 x 10⁵ cells (200 μl)) into mucosal tissue of the tongue area of athymic BALB/c nude mice. After 2 weeks of implantation, mice were anesthetized and the tumor was measured. Meanwhile, animals were randomly allocated into different groups and were treated with an intraperitoneal injection of 20 mg/kg nebivolol or the same dose of 0.9% NaCl buffer every three days. Then, the engrafted tumors were fixed in 4% paraformaldehyde overnight, which were prepared for the following histological analyses and morphologic analyses.

The distribution of β-Ⅲ-tubulin was evaluated by IHC in the 4NQO‐induced mice OSCC model and tumor xenograft model. IHC was used to determine tumor-related markers in tumor xenograft tissues and the 4NQO‐induced mice OSCC model. The standard protocol for IHC was similar to the previous one. The images were scanned by Aperio Digital Pathology Systems (ScanScope GL; Aperio Technologies).

HSC-3 and HN12 cells were treated with or without nebivolol for 12 h. Briefly, the cells were fixed with 4% paraformaldehyde. Then, 0.1% Triton-100 punched the cells for 2 min, which were blocked by 10% FBS for 30 min. Subsequently, cells were incubated overnight with anti-ATF4 (1:100) at 4°C. The next day, Alexa Fluor 488-conjugated goat anti-rabbit (1:750, Invitrogen) was used as a fluorescent second antibody. The cell nuclei were stained with 4,6-diamino-2-phenylindole (DAPI). The images were captured by confocal laser-scanning microscopy (CLSM, Tokyo, Japan and Olympus, FV3000, Japan).

To identify the nebivolol effect on tumor angiogenesis, CD31 antibody was used to stain specimens in mice oral tumor model. Alexa Fluor 488-conjugated goat anti-rabbit (1:750, Invitrogen) was used as a fluorescent second antibody. The cell nuclei were stained with DAPI. The images were captured by an Olympus fluorescence microscope.

HSC-3 and HN12 cells were treated with or without nebivolol for 12 h. Then, cells were gathered to measure the activities of complexes I by using Mitochondrial Respiratory Chain Complex Ⅰ Activity Assay Kit (D799471, Sangong, Shanghai, China) according to the manufacturer’s instructions. Absorbance was measured at A₃₄₀ by using spectrophotometric (Agilent Cary 100). The concentrations of protein were relatively quantified by BCA kit (Thermo Fisher Scientific).

All experiments were carried out independently at least three times. The results are calculated as the means ± standard deviation (SD) using GraphPad Prism software (version 7.2; GraphPad Software, Inc., La Jolla, CA, United States). The differences between the experimental groups and controls were assessed by single-factor analysis of variance (ANOVA) and Student’s t-test. A p-value less than 0.05 (p < 0.05) was regarded as a statistically significant result.

In order to determine the distribution of nerve fibers in OSCC tissues, the neuromarker β-Ⅲ-tubulin was used as the target protein. IHC experiments were performed on the pathological tissue sections of the patients who had been clinically diagnosed with OSCC or OLK. Representative images of β-Ⅲ-tubulin staining of OLK and OSCC samples are shown in Figure 1A. Nerves existed in OSCC tissue. In addition, the tumor tissue wrapped the nerve bundles like a sleeve, suggesting that the nerve is a component of the TME.

The 4NQO-induced OSCC model was established. 6OHDA was injected periodically in the intraperitoneal cavity (Figure 1B). After 24 weeks, the tissues of the tongue lesions and important organs were gathered for histological examination. As shown in Figure 1C, when compared to the control groups, the tumor sizes in the 6OHDA groups were significantly reduced. Besides, the representative images of tongue tumors were exhibited in HE staining (Figure 1D). In addition, the lesions were analyzed in degrades. The percentage of dysplasia in the 6OHDA administration groups was significantly increased when compared to the 4NQO-induced groups. However, the percentage of tumor lesions was apparently reversed when compared to the percentage of dysplasia (Figure 1E). Collectively, these results demonstrated that using 6OHDA to destroy the sympathetic nervous system could delay the progression of OSCC. Although 6OHDA halted cancer growth in tongue mice, the organs suffered from toxicity in different degrees when compared to the control groups.

In addition, IF staining was performed to analyze the expression of CD31 after being treated with nebivolol in the mice oral tumor model, which aims to analyze the nebivolol effect on tumor angiogenesis. As shown in Supplementary Figure 1, the expression of CD31 (green) was apparently reduced in nebivolol-treated mice when compared to the control group. This indicated that nebivolol triggered significant inhibition of tumor angiogenesis (Supplementary Figure 1A).

In order to discover a drug that could be alternated to 6OHDA, nebivolol was used, which could not only halt cancer growth but also show inapparent toxicity to organs. As shown in Figure 2A, the orthotopic tongue tumor mice were established and nebivolol was administrated at appointed times. The growing trend of tumor sizes was observed between the two groups in the tumor xenograft model. The growth of tumors in the nebivolol treatment group was significantly slower than that in the control group (Figure 2B). The representative images of tongue tumors were exhibited (Figure 2C). Moreover, the same trend of tumor growth was observed in the 4NQO‐induced mice OSCC model (Figure 1C). Besides, the representative images of tongue tumors were exhibited in HE staining (Figure 2D). In addition, the lesions were analyzed and histopathological grades evaluation were performed. The percentage of dysplasia in the 6OHDA administration groups was significantly increased when compared to the 4NQO-induced groups. However, the percentage of tumor lesions was apparently reversed when compared to the percentage of dysplasia (Figure 1E). These resulted illustrated that nebivolol halted cancer growth in the orthotopic tongue tumor mice model and the 4NQO-induced tongue mice model without toxicity in the organs (Figure 2E).

We used RT-qPCR to detect the expression levels of each adrenergic receptor subtype gene (ADRβ1, ADRβ2, and ADRβ3) in OSCC cells. The cDNAs of normal human keratinocyte (HOK) and OSCC cell lines (Cal27, HSC-3, HSC-4, HN4, and HN12) were collected, and the expression levels and differences of the corresponding receptor genes in each cell line were detected by RT-qPCR. Experimental results showed that the expression levels of neurotransmitter receptor genes in various OSCC cell lines are significantly different from those of normal keratinocytes. Compared with normal human keratinocytes HOK, the β1 receptor mRNA of HN12, HSC-3, and HSC-4 in OSCC cells is highly expressed. However, there was no significant difference among the expression of ADRβ2 and ADRβ3 receptors in OSCC cells and HOK cells (Supplementary Figure 1B, C, D).

To examine the effect of nebivolol on the growth of OSCC cells, nebivolol was applied on HSC-3 and HN12 cells. As shown in Figure 3A, the cell viability significantly decreased with the increasing concentration of nebivolol in OSCC cells at 24 h. Nebivolol induced a dose-dependent cytotoxic effect in the OSCC cells. Similarly, the number of HSC-3 and HN12 cell colonies was significantly reduced after being treated with an increasing concentration of nebivolol. The clonogenicity of OSCC cells was significantly inhibited in a dose-dependent manner (Figures 3B,C).

In order to illustrate the association of apoptosis and antitumor activities induced by nebivolol, the apoptotic ratio was evaluated. As shown in Figures 3D,E, there were a significantly elevated percentage of apoptotic cells when treated with an increased concentration of nebivolol for 24 h. Consistently, after being treated with nebivolol for 24 h, flow cytometric double staining by Annexin V-FITC apoptosis detection showed an obvious effect on apoptosis induction in OSCC cells. With the increasing concentrations of nebivolol treatment, the proportion of live cells was significantly decreased. Adversely, the proportion of apoptotic cells was significantly increased in OSCC cells when compared to the control groups (Figure 3F). Furthermore, after nebivolol treatment, the ratio of cleaved-caspase 3 and procaspase 3 as well as Bcl-2 and Bax was significantly increasing (Figure 3G). To sum up, these data illustrated that nebivolol inhibited the growth of OSCC cells by inducing apoptosis.

After being treated with nebivolol in OSCC cells, there were abundant cytoplasmic vacuoles observed apparently when compared to the control groups (Figure 4A). TEM was employed to detect the origination and cell ultrastructure of the observed vacuole. Compared to the control groups, there were numerous vacuoles detected after being treated with nebivolol. Furthermore, the vacuoles were derived from swollen portions of the ER and megamitochondria (Figure 4A). These results demonstrated that nebivolol-induced cytoplasmic vacuoles were derived from swollen ER and mitochondria, which might be associated with nebivolol-induced apoptosis in OSCC cells.

Much evidence has approved that ER stress was closely related to apoptosis (Hetz and Papa, 2018). In order to reveal the activated signal pathway of nebivolol-induced apoptosis in OSCC cells. The proteins expressions of ER stress markers were detected. As shown in Figure 4B, the results revealed that the proportion of phosphorylated protein level, p-PERK, and p-eIF2α significantly gradually elevated after increased concentrations treatment of nebivolol when compared to the corresponding protein level in the control groups. While the protein expression of PERK and eIF2α was still maintained at the same level, the protein level ratio of phosphorylated proteins compared to corresponding total proteins significantly increased after treatment with an increased dose of nebivolol. Subsequently, the significantly increased expression of ATF4 and CHOP was also observed after being treated with an increased dose of nebivolol when compared to the control groups (Figure 4B). In addition, the gene expression of the ER stress downstream pathway was also supported by the activated signal pathway (Figures 4C,D). These data revealed that the PERK-eIF2α-ATF4-CHOP axis signal pathway was activated when treated with nebivolol in OSCC cells. To ascertain that nebivolol-induced ER stress led to apoptosis, cells were treated with the ER stress inhibitor 4-PBA and nebivolol. 4-PBA significantly decreased nebivolol-induced overexpression of the ER stress markers (Figure 4E). Meanwhile, the cell viability was significantly enhanced after the combined treatment with nebivolol and 4-PBA when compared to the nebivolol groups (Figures 4F,G). These results demonstrated that nebivolol activated apoptosis via ER stress signaling pathway in OSCC cells.

The integrated stress response (ISR) is a countermeasure that is initiated when the cell is damaged from outside or inside. To investigate whether nebivolol induces ISR gene expression, the qPCR was applied. As shown in Figure 5A, the ISR genes were significantly increased in the presence of nebivolol for 12 h. As the typical transcription factor of ISR response, ATF4 could start a series of gene expressions that respond to stress (Pakos-Zebrucka et al., 2016). After being treated with nebivolol, the expressions of ATF4 significantly increased and transferred from cytoplasm to cell nucleus when compared to the control groups (Figure 5B).

To identify the explicit effect of ATF4 activation on ISR gene expression in nebivolol-treated OSCC cells, the knockdown ATF4 lentivirus was used in OSCC cells. The ISR genes were detected. As shown in Figure 5C, as compared to the nebivolol groups, ATF4 knockdown cells suppressed ISR genes expression when treated with nebivolol for 12 h. Meanwhile, the cell viability was significantly enhanced in ATF4 knockdown cell lines treated with nebivolol for 12 h when compared to the nebivolol-induced groups (Figure 5D). These data demonstrated that translation of ER and mitochondrial regulated nebivolol-mediated ISR gene expression through ATF4 activation.

To further study the upstream signaling pathway of nebivolol-induced ER stress, the NO-associated synthases were analyzed. The marked increased iNOS expressions were observed after being treated with nebivolol for 3 h, which indicated that nebivolol triggered the expression of iNOS to induce ER stress in OSCC cells (Figure 5E). In order to elucidate the role of iNOS in nebivolol-induced ER stress, the cells were then treated with nebivolol in the presence or absence transfected with si iNOS. The gene expression of iNOS was significantly inhibited in the nebivolol-induced si iNOS groups (Figure 5F). Furthermore, the downstream ER stress markers were also detected. The protein expressions of ATF4 and CHOP significantly decreased in the nebivolol-induced si iNOS groups when compared to the nebivolol-treated groups. The proportion of p-eIF2α protein expression significantly decreased in the nebivolol-induced si iNOS2 groups when compared to the nebivolol-treated groups (Figure 5G). Overall, these data revealed that nebivolol triggered the expression of iNOS to activate ER stress signal pathway in OSCC cells.

In addition, the results showed that the proportion of HN12 cells was significantly increased in the S phase and reduced in the S and G2/M phase following treatment with 10 and 15 μM nebivolol when compared to the proportions in the corresponding phases of the control group cells. Thus, nebivolol significantly arrested cell cycle progression at G0/G1 phase (Figure 5H).

Although the inhibitor 4-PBA significantly rescued the cell viability when treated with nebivolol for 12 h, the inhibitor 4-PBA could not recover cell viability after being treated with nebivolol for 24 h. In order to reveal the underlying mechanism of nebivolol-induced apoptosis after ER stress signal pathway being activated in OSCC cells, we examined the possible involved ROS levels. As shown in Figure 6A, the ROS levels were apparently enhanced after being treated with nebivolol for 24 h when compared to the control groups. Thus, it was confirmed that nebivolol could induce the production of ROS through mitochondrial activation in OSCC cells. However, there was no significant difference in mitochondrial membrane potential in HSC-3 and HN12 cells (Supplementary Figure 2A). An experiment was also implemented by using NAC to detect the effects of inhibitors on nebivolol-induced apoptosis. It could be demonstrated that, in Figure 6B, the cell viability was significantly rescued by a combination of NAC and nebivolol. Herein, the results revealed that the accumulated ROS induced apoptosis after being treated with nebivolol.

In order to demonstrate the reason for ROS accumulation, the protein expression of OXPHOS complexes was measured. There was no significant difference in proteins expression of OXPHOS complexes and mitochondrial structural protein expression HSP60 in the presence of nebivolol for 12 h when compared to the control groups (Figure 6C). These data indicated that protein expression of OXPHOS complexes’ subunits was not affected after being stimulated with nebivolol. While the OCR were sequentially measured, in the presence of nebivolol, the OCR was significantly reduced which indicated the dysfunction of mitochondrial functions. Specifically, the basal respiration, ATP production, and maximal respiration were significantly decreased in the nebivolol-treated groups when compared with the control groups (Figures 6D,E). To determine the reason for the dysfunction of mitochondria, we observed the morphology of mitochondria. After being stimulated with nebivolol, the morphology of mitochondria could be depicted as fragmented and intermediate bodies which changed from tubular mitochondrial filaments by using MitoTracker Red. Mitochondria in the nebivolol-treated groups were more fragmented and intermediate bodies and less tubular than those in the matched control groups (Figure 6F). These results verified that the changed mitochondrial morphology led to mitochondrial dysfunction which may affect mitochondrial respiratory function. Besides, NAO was used to detect mitochondrial mass. Flow cytometric data demonstrated that nebivolol exhibited no significant effect in mitochondrial mass when compared to the control groups (Figure 6G). The reduced mitochondrial respiration capacities were not related to OXPHOS complexes and the mitochondrial mass. Thus, the activity of OXPHOS complexes was detected to observe its relationship with mitochondrial respiration. Nebivolol significantly decreased the activity of complex I (Figure 6H). These indicated that the activity of OXPHOS complex I diminished after being stimulated with nebivolol which could affect the capability of ETC resulting in mitochondrial dysfunction. It is known that the phosphorylation of serine residues regulates the activity of proteins of OXPHOS (García-Bermúdez et al., 2015). The protein level of p-Ser was significantly downregulated in response to nebivolol treatment when compared to the control groups. However, we observed no significant difference in the protein expression of IF1 in OSCC cells which may indicate that nebivolol affects the activity of mitochondria and does not affect respiratory complex V in OSCC cells (Supplementary Figure 2B). Together, our data suggested that nebivolol disturbed mitochondrial capacity and increased ROS level to induce apoptosis in OSCC cells.