CAL27 (CRL-2095) and SCC9 (CRL-1629) cells (human OSCC cell lines) were obtained from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Grand Island, New York, USA) and DMEM/F12 (Hyclone, Logan, Utah, USA) at 37°C with 5% CO₂, respectively. The two media contained 10% fetal bovine serum (FBS) (Procell Life Science&Technology Co., Ltd., Wuhan, Hubei, China), 1% penicillin and streptomycin (Sigma-Aldrich, Saint Louis, Missouri, USA), and 0.1% plasmocin prophylactic (In vivogen, San Diego, California, USA).

CAL27 and SCC9 cells at the logarithmic growth phase were plated into 96-well plates at a density of 8 × 10³ cells per well and cultured for 24 h. Then, the cells were incubated with 0, 5, 10, 20, 30, 40, 60, or 80 μM CA (B21175, Shanghai Yuanye Biological Technology Co., Ltd., Shanghai, China), which had been dissolved in dimethyl sulfoxide (DMSO) (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China). The final concentration of DMSO in the culture was ≤ 0.1%. After 24 h of CA treatment, the cells in each well were incubated with 10 μL of 5 mg/mL 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) (S19063, Source Leaf Biological Technology Co., Ltd., Shanghai, China) for 4 h. Afterward, the culture supernatant was discarded, and 150 μL DMSO was used to dissolve the formazan crystals in each well. Absorbance of the samples was measured at 490 nm using an Enzyme-labeled Instrument (HBS-1096A, NanJing DeTie Laboratory Equipment Co., Ltd., Nanjing, Jiangsu, China).

CAL27 and SCC9 cells at the logarithmic growth phase were plated into 6-well plates at a density of 3 × 10⁵ cells per well. When the cells reached > 90% confluence, they were scraped using a syringe needle and then cultured with CA (30 and 15 μM for CAL27 and SCC29 cells, respectively) for 0, 12 and 24 h. Fluorescence microscope (Eclipse TE 2000-S, Nikon Corp., Tokyo, Japan) and quantifiable ImageJ software were used to analyze the influence of CA on OSCC cells migration.

CAL27 and SCC9 cells at the logarithmic growth phase were plated into 6-well plates at a density of 2.5 × 10⁵ cells per well and incubated in an incubator at 37°C with 5% CO₂ for 24 h. Next, the CAL27 and SCC9 cells were incubated with 30 and 15 μM CA for 12 h, respectively. Afterward, the cells were processed using the eBioscietm Annexin V-FITC Apop Kit (BMS500FI-100, Invitrogen, Carlsbad, California, USA) according to the instructions of the manufacturer and analyzed using a CytoFLEX Flow Cytometer (C02945, Beckman Coulter, Inc. Brea, Carlsbad, California, USA).

CAL27 and SCC9 cells at the logarithmic growth phase were plated into 6-well plates at a density of 2.5 × 10⁵ cells per well and cultured for 24 h. Subsequently, the CAL27 and SCC9 cells were incubated with 30 and 15 μM CA for 12 h, respectively. Afterward, the cells were washed with phosphate-buffered saline (PBS) three times and then incubated with 5 mg/mL 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1) (C2006, Beyotime Biotechnology, Shanghai, China) for 20 min. Fluorescence intensity of the samples was measured using a fluorescence microscope (Eclipse TE 2000-S, Nikon Corp., Tokyo, Japan), and the related data were quantitatively analyzed using ImageJ software.

CAL27 and SCC9 cells at the logarithmic growth phase were plated into 6-well plates at a density of 2.5 × 10⁵ cells per well and then incubated for 24 h. Afterward, the CAL27 and SCC9 cells were exposed to 30 and 15 μM CA for 12 h, respectively. The cells were incubated with 10 μM DFCH-DA for 30 min, and the intracellular ROS levels were measured using the Reactive Oxygen Species (ROS) Assay Kit (S0033S, Beyotime Biotechnology, Shanghai, China) according to the instructions of the manufacturer.

For Ca²⁺ detection, the cells were incubated with 1 μM Fluo-4AM (S1060, Beyotime Biotechnology, Shanghai, China) for 40 min. Afterward, the culture supernatant was discarded, and the cells were washed three times with PBS. Fluorescence intensity of the samples was measured using a fluorescence microscope (Eclipse TE 2000-S, Nikon Corp., Tokyo, Japan), and the related data were quantitatively analyzed using ImageJ software.

The incubation and CA treatment protocols of CAL27 and SCC9 cells were the same as 2.6 Quantitation of intracellular ROS and Ca²⁺ .The collected cells were fixed at 4°C for 4 h, embedded in 1% agarose solution, and then fixed in 1% osmium tetroxide for the secondary fixation. After dehydration with ethanol, the cells were embedded in the embedding plate and polymerized for 48 h. The ultra-microtome (Leica UC7, Leica, Weztlar, Germany) was used to cut the resin blocks into 60-80 nm and loaded the slices on a 150-mesh cuprum grids. After the sections were stained with uranyl acetate and lead citrate, images were captured using a transmission electron microscope (H-7650, HITACHI, Japan).

All the animal experiments were conducted under the guidance of the Animal Ethics and Welfare Committee of Jilin University (NO. SY202101004). Male BALB/c nude mice (5 weeks old) (Wei-tongli-hua Laboratory Animal Technology Company, Beijing, China) were provided with sufficient food and water and maintained at 23 ± 1°C under 12 h/12 h light/dark cycle.

CAL27 and SCC9 cells (1 × 10⁷) were subcutaneously inoculated into the right dorsum of BALB/c nude mice. When the tumor volume reached 100 mm³, the mice in each group were randomly divided into two sub-groups (n = 6 per sub-group) and intraperitoneally injected every other day for 14 d with the physiological saline containing < 0.1% DMSO (control mice) or 20 mg/kg of CA dissolved in DMSO with a final concentration of < 0.1% (CA-treated mice). The volumes of the tumors and the body weights of the mice were measured before each administration. Tumor volume was calculated according to the following formula: length (mm) × [width (mm)]² × 0.5. After the last treatment, the mice were euthanized via carbon-dioxide asphyxiation. The tumors were removed and homogenized for western blot analysis. Peripheral-blood samples were collected from the mice for blood analyses using an automatic blood analyzer. The tumor, heart, liver, spleen, and kidney of the mice were fixed in 4% polyformaldehyde (POM) (AR0211, Dingguo Changsheng Biotechnology Co., Ltd., Beijing, China) for 48 h for histopathological examination.

Following the POM fixation, the samples were embedded in paraffin and then sectioned at 5 μm thickness by using a microtome (Leica, Wetzlar, Germany). The sections were stained with hematoxylin and eosin by using a standard protocol. After the slices were dehydrated and cleared with ethanol and xylene, images were captured using an Eclipse TE 2000-S fluorescence microscope (Nikon Corp., Tokyo, Japan).

The paraffin sections prepared from tumor tissues were deparaffinized and repaired with proteinase K. After breaking the membrane with 0.1% triton, terminal deoxynucleotidyl transferase (Tdt) enzyme and dUTP were added according to the instructions of TUNEL assay kit (G1205, Servicebio technology Co., Ltd., Wuhan, China), and incubated at 37°C for 2 h. The nuclei were stained with 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) and incubated in the dark for 10 minutes. Fluorescent images were captured using an Eclipse TE 2000-S fluorescence microscope (Nikon Corp., Tokyo, Japan).

CAL27 and SCC9 cells at the logarithmic growth phase were plated into 6-well plates at a density of 2.5 × 10⁵ cells per well and cultured for 24 h. Afterward, the CAL27 and SCC9 cells were treated with 30 and 15 μM of CA for 24 h, respectively. The treated cells and the tumor tissues obtained from the BALB/c nude mice were homogenized using the RIPA Lysis Buffer (PC101, EpiZyme, Shanghai, China). The total-protein concentrations of the samples were measured using a Standard BCA Protein Assay Kit (23225, Thermo Fisher Scientific, Waltham, Massachusetts, USA) following the manufacturer’s instructions. Lysates with 30–40 μg total protein were electrophoresed using the One-Step PAGE Gel Fast Preparation Kit (PG213, EpiZyme, Shanghai, China) at 90–120 V and then transferred onto a PVDF membranes (Merck Millipore, Billerica, MA, USA) at 100 V for 2 h. The membranes were blocked with the NcmBlot blocking buffer (P30500, NCM Biotech, Suzhou, Jiangsu, China) and then incubated overnight at 4 °C with antibodies against Cleaved poly (ADP-Ribose) polymerase (PARP1) (A19612), PARP1 (A19596), Cleaved Caspase-3 (A2156), Caspase-3 (A2156), Caspase-9 (A2636), Bad (A19595), Bax (A19684) (all from ABclonal Technology Co., Ltd., Wuhan, Hubei, China); Cleaved Caspase-9 (Asp315, Cell Signaling Technology, Boston, Massachusetts, USA); B-cell lymphoma-2 (Bcl-2) (BSM-33047M, Beijing Biosynthesis Biotechnology Co., Ltd., Beijing, China); and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (SY0102, Elabscience Biotechnology Co., Ltd., Wuhan, Hubei, China). After washing the membranes with TBST, they were incubated at 4°C for 4 h with the corresponding goat anti-rabbit IgG (H+L) (peroxidase/HRP-conjugated) (E-AB-1003) or anti-mouse IgG (H+L) (peroxidase/HRP-conjugated) (E-AB-1001) (Elabscience Biotechnology Co., Ltd., Wuhan, Hubei, China). The protein signals were detected using electrochemiluminescence (ECL) detection kits (Merck Millipore, Billerica, MA, USA), and the signal intensities were quantified using ImageJ software.

The statistical significance of the differences between the control and CA-treatment groups was determined using one-way analysis of variance (ANOVA). Post-hoc multiple comparisons (Dunn’s test) were performed using DSS 25.0 software (IBM Corporation, Armonk, New York, USA). P < 0.05 was considered significant.

CA significantly inhibited the viability of the OSCC cell lines CAL27 and SCC9 (IC₅₀ 34.66 and 13.23 μM, respectively) (P < 0.05, Figure 1A ). CA increased the early/late apoptosis of CAL27 and SCC9 cells to 36.77% and 29.1%, respectively ( Figure 1B ). The migration of OSCC cells was time-dependent. Compared with the control cells, CA treatment significantly inhibited the migration ability of these OSCC cells (P < 0.05, Figure 1C ).

After CAL27 and SCC9 cells were incubated with CA for 12 h, increased green fluorescence intensity was observed, indicating that CA increased the ROS levels (P < 0.001, Figure 2A ) and Ca²⁺ influx (P < 0.001, Figure 2B ) in OSCC cells. Moreover, the measurement of mitochondrial membrane potential proved that after OSCC cells were exposed to CA, more green fluorescence from JC-1 monomer was observed, the intensity of the red fluorescence aggregated in the matrix of the mitochondria decreased, and the red/green fluorescence ratio was significantly reduced by > 60% in the CA-treated cells (P < 0.01, Figure 2C ), indicating that the level of mitochondrial depolarization increased, and the MMP decreased, and thus the cells were in the early apoptotic stage. The images obtained by TEM showed that the mitochondrial cristae of CAL27 and SCC9 cells were arranged neatly and densely ( Figure 2D ). After CA incubation, the mitochondria were severely swollen, the matrix was dissolved, and the mitochondrial cristae was missing and accompanied by vacuoles ( Figure 2D ). The administration of CA affected the structure and function of mitochondria.

In CA-exposed CAL27 and SCC9 cells, the ratios of the levels of Cleaved PARP1, Caspase-3, and Caspase-9 to total PARP1, Caspase-3, and Caspase-9 levels, respectively, were increased (P < 0.05, Figure 3 ). Additionally, Bcl-2 was downregulated, whereas Bax and Bad were upregulated (P < 0.01, Figure 3 ). These in vitro results preliminarily showed that CA induced apoptosis in OSCC cells by reducing the MMP.

In CAL27- and SCC9-xenotransplanted BALB/c nude mice, CA treatment for 14 d significantly inhibited the tumor growth (P < 0.01, Figures 4A – C and 5A – C ) without affecting the body weights or organ indices of the animals ( Figures 4D and 5D ; Table S1 ). Compared with CTRL mice, the green fluorescence in tumor tissues enhanced, in other words, the number of TUNEL positive cells increased after CA administration, indicating that CA administration promoted tumor tissue apoptosis ( Figures 4E and 5E ). The cardiomyocytes and hepatocytes of the animals were still neatly arranged, the spleen had no significant infiltration of inflammatory cells, and the glomeruli had no obvious lesions, indicating that CA did not impact the histological features of the mice ( Figures 4F and 5F ).

Results from the peripheral-blood analyses indicated increased monocyte number and decreased platelet distribution width (PDWcv) in the SCC9-xenotransplanted BALB/c nude mice (P < 0.05, Tables S2 ). No significant changes in other indicators were found in the mice transplanted with the OSCC cells ( Tables S2 ).

Consistent with the in vitro results, CA administration for 14 d increased the ratios of the levels of Cleaved PARP1, Caspase-3, and Caspase-9 to total PARP1, Caspase-3, and Caspase-9 levels in the xenografts, respectively (P < 0.01, Figure 6 ). Furthermore, Bax and Bad were upregulated, whereas Bcl-2 was downregulated (P < 0.05, Figure 6 ).