In vitro antimetastatic potential of pseudolaric acid B in HSC-3 human tongue squamous carcinoma cell line.

OBJECTIVE: Pseudolaric acid B (PAB) is a novel diterpenoid derived from the traditional Chinese medicinal herb Cortex pseudolaricis that exerts anticancer, anti-inflammatory, and immunomodulatory properties. While the anticancer potential of PAB has been studied, its effects on metastasis have not been well-studied. This study aims to determine the inhibitory effects of PAB on HSC-3 human tongue squamous cell carcinoma (TSCC) cell line. DESIGN: Cell viability and soft agar colony formation assays were conducted to assess cellular proliferation and in vitro tumorigenic capacity of TSCC cells, respectively. Additionally, wound healing, transwell migration, and invasion assays were conducted to monitor the aggressive behavior of TSCC cells. Furthermore, Western blotting analysis was conducted to reveal the signaling pathways involved in the modulation of epithelial-mesenchymal transition (EMT). RESULTS: The migratory and invasive capacities of HSC-3 cells were suppressed by PAB irrespective of their proliferation states. PAB's effects on EMT involved upregulation of E-cadherin expression and downregulation of Twist; these were concomitantly accompanied by downregulated phosphorylation of epidermal growth factor receptor (EGFR). CONCLUSIONS: PAB suppresses human TSCC in vitro by regulating Twist/E-cadherin through the EGFR signaling pathway. PAB may have potential as a candidate antimetastatic drug for TSCC treatment.

Cancer chemo-preventive role of grape seed oil and cisplatin as a combination adjuvant therapy in the treatment of tongue squamous cell carcinoma: A biological in-vitro study.

OBJECTIVES: Grape seed oil (GSO) has recently gained popularity due to its anticancer properties. This study aimed to investigate the efficacy of combining cisplatin (CP) and GSO in tongue squamous cell carcinoma (TSCC) treatment. METHODS: In this study, human tongue carcinoma cell line (HNO-97) was treated with CP and GSO alone or in combination. The effects of CP and GSO on cytotoxicity and cell cycle arrest were studied using the MTT assay and flowcytometry, respectively. The apoptotic markers, including p53 and caspase 8, were assessed using reverse-transcription polymerase chain reaction (RT-PCR), caspase 3 using immunohistochemistry, and the angiogenic marker vascular endothelial growth factor (VEGF) using enzyme-linked immunosorbent assay (ELISA). RESULTS: The IC50 drug concentrations were found to be 16.4 ug/mL of GSO and 2.18 ug/mL of CP. When compared to the untreated control group, the percentage of S phase cells and apoptotic cells was significantly higher in the GSO, CP, and GSO/CP combination therapy groups. Furthermore, p53, caspase 8, caspase 3 expression were significantly upregulated in the GSO-and CP-treated groups, with evident upregulation with GSO/CP combination therapy. However, VEGF levels were significantly lower in the GSO-, CP-, and combined GSO/CP-treated groups. CONCLUSIONS: GSO has both an apoptotic and antiangiogenic effect in the treatment of TSCC, suggesting a new strategy for phytochemical-based combination therapy.

Preoperative Prediction of the Aggressiveness of Oral Tongue Squamous Cell Carcinoma with Quantitative Parameters from Dual-Energy Computed Tomography.

Objectives: To determine whether quantitative parameters derived from dual-energy computed tomography (DECT) were predictive of the aggressiveness of oral tongue squamous cell carcinoma (OTSCC) including the pathologic stages, histologic differentiation, lymph node status, and perineural invasion (PNI). Methods: Between August 2019 and March 2021, 93 patients (mean age, 54.6 ± 13.8 years; 66 men) with pathologically diagnosed OTSCC were enrolled in this prospective study. Preoperative DECT was performed and quantitative parameters (e.g., slope of the spectral Hounsfield unit curve [λHu], normalized iodine concentration [nIC], normalized effective atomic number [nZeff], and normalized electron density [nRho]) were measured on arterial phase (AP) and venous phase (VP) DECT imaging. Quantitative parameters from DECT were compared between patients with different pathologic stages, histologic differentiation, lymph node statuses, and perineural invasion statuses. Logistic regression analysis was utilized to assess independent parameters and the diagnostic performance was analyzed by the receiver operating characteristic curves (ROC). Results: λHu and nIC in AP and λHu, nZeff, and nIC in VP were significantly lower in stage III-IV lesions than in stage I-II lesions (p < 0.001 to 0.024). λHu in VP was an independent predictor of tumor stage with an odds ratio (OR) of 0.29, and area under the curve (AUC) of 0.80. λHu and nIC were higher in well-differentiated lesions than in poorly differentiated lesions (p < 0.001 to 0.021). The nIC in VP was an independent predictor of histologic differentiation with OR of 0.31, and AUC of 0.78. λHu and nIC in VP were lower in OTSCCs with lymph node metastasis than those without metastasis (p < 0.001 to 0.005). λHu in VP was the independent predictor of lymph node status with OR of 0.42, and AUC of 0.74. No significant difference was found between OTSCCs without PNI and those with PNI in terms of the quantitative DECT parameters. Conclusion: DECT can be a complementary means for the preoperative prediction of the aggressiveness of OTSCC.

Tongue squamous cell carcinoma resists hyperthermia treatment by promoting Id-1 expression mediated EMT.

Epithelial-mesenchymal transition (EMT) is a key initial step in the recurrence and metastasis of tongue squamous cell carcinoma (TSCC). Hyperthermia (HT) may reduce the rate of postoperative recurrence and distant metastasis by reversing the process of EMT of tumor cells, but the molecular mechanism is unclear. This study aims to investigate the role of inhibitor of differentiation/DNA binding-1 (Id-1) in HT mediated reversal of EMT of TSCC cells, and to provide a new approach for the treatment of TSCC using therapeutic gene targeting. After the combination of RNA interference with Id-1 and HT, the morphology of TSCC cells changed from spindle-like to pebble-like, and the arrangement of cells changed from loose and disorderly to compact and orderly. The silencing of Id-1 gene enhances the efficacy of HT by affecting the expression of EMT markers in TSCC cells. This study suggests that the Id-1 gene in TSCC cells can regulate transforming growth factor-beta 1, thereby affecting the expression of EMT markers, to achieve the effect of reducing HT.

Quercetin induces tongue squamous cell carcinoma cell apoptosis via the JNK activation-regulated ERK/GSK-3α/ß-mediated mitochondria-dependent apoptotic signaling pathway.

Tongue squamous cell carcinoma (SCC) is a most common type of oral cancer. Due to its highly invasive nature and poor survival rate, the development of effective pharmacological therapeutic agents is urgently required. Quercetin (3,3',4',5,7-pentahydroxyflavone) is a polyphenolic flavonoid found in plants and is an active component of Chinese herbal medicine. The present study investigated the pharmacological effects and possible mechanisms of quercetin on apoptosis of the tongue SCC-derived SAS cell line. Following treatment with quercetin, cell viability was assessed via the MTT assay. Apoptotic and necrotic cells, mitochondrial transmembrane potential and caspase-3/7 activity were analyzed via flow cytometric analyses. A caspase-3 activity assay kit was used to detect the expression of caspase-3 activity. Western blot analysis was performed to examine the expression levels of proteins associated with the MAPKs, AMPKα, GSK3-α/ß and caspase-related signaling pathways. The results revealed that quercetin induced morphological alterations and decreased the viability of SAS cells. Quercetin also increased apoptosis-related Annexin V-FITC fluorescence and caspase-3 activity, and induced mitochondria-dependent apoptotic signals, including a decrease in mitochondrial transmembrane potential and Bcl-2 protein expression, and an increase in cytosolic cytochrome c, Bax, Bak, cleaved caspase-3, cleaved caspase-7 and cleaved poly (ADP-ribose) polymerase protein expression. Furthermore, quercetin significantly increased the protein expression levels of phosphorylated (p)-ERK, p-JNK1/2 and p-GSK3-α/ß, but not p-p38 or p-AMPKα in SAS cells. Pretreatment with the pharmacological JNK inhibitor SP600125 effectively reduced the quercetin-induced apoptosis-related signals, as well as p-ERK1/2 and p-GSK3-α/ß protein expression. Both ERK1/2 and GSK3-α/ß inhibitors, PD98059 and LiCl, respectively, could significantly prevent the quercetin-induced phosphorylation of ERK1/2 and GSK3-α/ß, but not JNK activation. Taken together, these results suggested that quercetin may induce tongue SCC cell apoptosis via the JNK-activation-regulated ERK1/2 and GSK3-α/ß-mediated mitochondria-dependent apoptotic signaling pathway.

EGCG inhibits growth of tumoral lesions on lip and tongue of K-Ras transgenic mice through the Notch pathway.

Epigallocatechin-3-gallate (EGCG), the main active ingredient of green tea, exhibits low toxic side effect and versatile bioactivities, and its anti-cancer effect has been extensively studied. Most of the studies used cancer cell lines and xenograft models. However, whether EGCG can prevent tumor onset after cancer-associated mutations occur is still controversial. In the present study, Krt14-cre/ERT-Kras transgenic mice were developed and the expression of K-RasG12D was induced by tamoxifen. Two weeks after induction, the K-Ras mutant mice developed exophytic tumoral lesions on the lips and tongues, with significant activation of Notch signaling pathway. Administration of EGCG effectively delayed the time of appearance, decreased the size and weight of tumoral lesions, relieved heterotypic hyperplasia of tumoral lesions, and prolonged the life of the mice. The Notch signaling pathway was significantly inhibited by EGCG in the tumoral lesions. Furthermore, EGCG significantly induced cell apoptosis and inhibited the proliferation of tongue cancer cells by blocking the activation of Notch signaling pathway. Taken together, these results indicate EGCG as an effective chemotherapeutic agent for tongue cancer by targeting Notch pathway.

Cytotoxicity mechanisms of plumbagin in drug-resistant tongue squamous cell carcinoma.

OBJECTIVES: To evaluate the inhibitory effect and mechanism of plumbagin (PLB) against drug-resistant tongue squamous cell carcinoma (TSCC), and whether its antitumour effect is not affected by tumour drug resistance. METHODS: TSCC sensitive CAL27 cells and drug-resistant CAL27/RE cells were used to study the cytotoxicity and mechanism of PLB in vitro, including CCK-8 analysis, colony formation, DAPI staining, flow cytometry assay, transmission electron microscopy, western blotting assay, autophagy, apoptosis and ROS fluorescent probes. BALB/c nude mice xenograft models were used to study the growth inhibitory effect of PLB in vivo. KEY FINDINGS: The results showed that the cell viability and proliferation inhibition and apoptosis induction abilities of PLB on drug-resistant cells were more obvious than that on sensitive cells. And PLB induced protective autophagy in TSCC cells. Mechanistically, PLB induced apoptosis and autophagy by generating reactive oxygen species to mediate JNK and AKT/mTOR pathways. Finally, the growth inhibitory effect of PLB against drug-resistant TSCC was also confirmed in vivo. CONCLUSIONS: PLB will be a promising anticancer agent to overcome drug-resistant TSCC without being affected by its drug resistance properties.

YY1-mediated PTEN dephosphorylation antagonizes IR-induced DNA repair contributing to tongue squamous cell carcinoma radiosensitization.

Ionizing radiation (IR) confers a survival advantage in tongue squamous cell carcinoma (TSCC), however, IR resistance limits its efficacy. Although Yin Yang 1 (YY1) has been reported to play a role in genotoxic drug resistance by accelerating DNA repair, its role in TSCC radioresistance remains unclear. In this study, we examined YY1 mRNA and protein expression in human tongue cancer samples using qRT-PCR and western blotting, respectively. DNA array data identified YY1 mRNA expression in IR sensitivity or resistance cell lines and tissues. Tongue carcinoma primary cells and CAL27 cells with YY1 stably overexpressed or knocked-down were exposed to IR and evaluated for cell proliferation and apoptosis by CCK8-assay and caspase-3 assay, respectively. We also examined DNA damage- or repair-related indicators, such as YY1, p-H2AX, nuclear PTEN, p-PTEN, and Rad51 through Western blot analysis. Additionally, we explored the mechanism of IR-induced PTEN nuclear translocation by introducing a series of PTEN phosphorylation site mutations and co-IP assay. We observed that YY1 mRNA and protein are highly expressed in TSCC tissues, which was correlated with worse overall survival. Moreover, higher expression of YY1 and Rad51 was observed in radioresistant cells and tissues, overexpression of YY1 led to IR resistance in TSCC cells, whereas YY1 knockdown sensitized TSCC cells to IR. The underlying mechanism showed that the overexpression of YY1 upregulated nuclear PTEN and Rad51 expression, which is essential for DNA repair. IR upregulated YY1, nuclear PTEN, and Rad51; thus, knockdown of YY1 completely blocked IR-induced upregulation of nuclear PTEN/Rad51. IR upregulated PTEN phosphorylation, and mutation of the phosphorylation site of Ser380 nearly completely blocked IR-induced PTEN nuclear translocation. Furthermore, the phosphatase PP2A negatively regulated pS380-PTEN, and knockdown of YY1 completely blocked IR-induced pS380-PTEN through PP2A. In conclusion, knockdown of YY1 enhanced TSCC radiosensitivity through PP2A-mediated dephosphorylation of PTEN Ser380; thus, antagonizing the IR-induced nuclear PTEN/Rad51 axis and targeting YY1 may reverse IR resistance in TSCC.

The in vitro and in vivo antitumor effects of Dracaena cinnabari resin extract on oral cancer.

OBJECTIVE: To study the potential for apoptosis induction of Dracaena cinnabari Balf. f methanolic extract (DCBME) on tongue squamous cell carcinoma cell line, H103. We evaluated the chemopreventive activity of DCBME against 4-nitroquinolone-1-oxide (4NQO)-induced tongue carcinogenesis in rat. DESIGN: Phase contrast microscope, acridine orange/propidium iodide (AO/PI) analysis of cells under fluorescence microscope, annexin-V flow-cytometry, DNA fragmentation, mitochondrial membrane potential, and caspase 3/7, 8 and 9 assays were performed. In vivo study, the rats were given 4NQO in their drinking water. The tongue was subjected to histopathological study to evaluate the incidence of squamous cell carcinoma (SCC). RESULTS: DCBME showed cytotoxic effect on H103 cells in a dose- and time-dependent manner. Furthermore, DCBME showed low cytotoxic effect on a normal cell line. In H103 cells, it caused cell morphology changes, S and G2/M-phase cell cycle arrest, significant reduction of cell migration and induced apoptosis through the intrinsic (mitochondrial) pathway. The incidence of SCC was 85.7% in the induced cancer and vehicle groups while in rats treated with DCBME at 100, 500 and 1000 mg/kg was 57.1%, 28.6% and 14.3%, respectively. CONCLUSIONS: (DCBME)-apoptosis induction reported in this work can be exploited as a potential antitumor agent with applications in medicinal treatments of tongue SCC.

SDF-1-loaded PLGA nanoparticles for the targeted photoacoustic imaging and photothermal therapy of metastatic lymph nodes in tongue squamous cell carcinoma.

The combination of photothermal therapy and targeted chemotherapy can produce much greater cytotoxicity than chemotherapy. Herein, we developed multifunctional targeted polymeric nanoparticles (NPs) loaded with indocyanine green (ICG) and doxorubicin (DOX) for the targeted photoacoustic imaging and photothermal ablation of oral cancer cells. The chemokine SDF-1, a specific antibody, was conjugated to NPs by the carbodiimide method. The NPs were automatically targeted to tumour tissue in vitro and in vivo through CXCR4-SDF-1 interactions. The results of in vivo and in vitro photoacoustic imaging and photothermal therapy experiments showed that the multifunctional NPs had excellent photoacoustic imaging characteristics and photothermal therapy capabilities. The photothermal material heated rapidly after laser irradiation, and the resulting heat increased cell metabolism and membrane permeability, which increased cellular NP uptake. The encapsulated drug (DOX) was released immediately after the liquid core was transformed into a gas via laser effects, which killed tumour cells while producing strong photoacoustic signals in vitro and in vivo. Thus, we concluded that the chemokine SDF-1 can be applied for the targeted chemotherapy of metastatic lymph nodes of oral squamous cell carcinoma (OSCC) and is more effective for treating oral cancer when combined with photothermal therapy than when used alone.

Outcomes after up-front surgery and metronomic neoadjuvant chemotherapy with S-1 or UFT for early tongue squamous cell carcinoma.

BACKGROUND: Our aim was to investigate the disease-free survival in patients with tongue squamous cell carcinoma receiving metronomic neoadjuvant chemotherapy with 5-fluorouracil prodrugs (UFT or S-1) plus bleomycin compared with those who had up-front surgery retrospectively. METHODS: In this retrospective study, 108 patients with stages I to II tongue squamous cell carcinoma who had undergone surgery were divided into the "surgery group" or "neoadjuvant chemotherapy group." RESULTS: A total of 41 patients received up-front surgery; 67 received metronomic neoadjuvant chemotherapy with UFT plus bleomycin (39) or S-1 plus bleomycin (28). The rate of disease-free survival was the primary outcome measure. Neoadjuvant 5-fluorouracil prodrugs did not correlate higher with improved disease-free survival than up-front surgery (72 and 54%, respectively; hazard ratio for recurrence or death, 0.54; 95% confidence interval [CI], 0.28 to 1.03; P = 0.06). Patients who received S-1 were more likely than those who received UFT to have pathological complete response (46% vs. 15%; P = 0.007). Neoadjuvant S-1 significantly improved disease-free survival as compared with up-front surgery (79% vs. 54%; hazard ratio, 0.41; 95% CI, 0.15 to 0.98; P = 0.04). However, neoadjuvant UFT did not improve disease-free survival as compared with up-front surgery (67% vs. 54%, respectively; hazard ratio, 0.66; 95% CI, 0.31 to 1.33; P = 0.24). CONCLUSIONS: Neoadjuvant S-1 chemotherapy, as compared with up-front surgery, significantly improved disease-free survival among patients with tongue squamous cell carcinoma. CLINICAL RELEVANCE: A choice of drugs before neoadjuvant metronomic chemotherapy is needed.

Safflower polysaccharide inhibits the development of tongue squamous cell carcinoma.

BACKGROUND: Safflower polysaccharide (SPS) is one of the most important active components of safflower (Carthamus tinctorius L.), which has been confirmed to have the immune-regulatory function and antitumor effect. This study aimed to explore the effects of safflower polysaccharide (SPS) on tongue squamous cell carcinoma (TSCC). METHODS: HN-6 cells were treated with 5 µg/mL cisplatin and various concentrations of SPS (0, 0.02, 0.04, 0.08, 0.16, 0.32, 0.64, and 1.28 mg/mL), and cell proliferation was measured. After treatment with 5 µg/mL cisplatin and 0.64 mg/mL SPS, the induction of apoptosis and the protein and mRNA expression of Bax, Bcl-2, COX-2, and cleaved caspase-3 in HN-6 cells were quantified. In addition, HN-6 cells were implanted into mice to establish an in vivo tumor xenograft model. Animals were randomly assigned to three groups: SPS treatment, cisplatin treatment, and the model group (no treatment). The body weight, tumor volume, and tumor weight were measured, and the expression of the above molecules was determined. RESULTS: SPS treatment (0.02-0.64 mg/mL) for 24-72 h inhibited HN-6 cell proliferation. In addition, 0.64 mg/mL SFP markedly induced apoptosis in HN-6 cells and arrested the cell cycle at the G0/G1 phase. Compared with the control group, the expression of Bcl-2 and COX-2 was markedly reduced by SPS treatment, whereas the expression of Bax and cleaved caspase-3 was increased. Moreover, SPS significantly inhibited the growth of the tumor xenograft, with similar changes in the expression of Bcl-2, COX-2, Bax, and cleaved caspase-3 in the tumor xenograft to the in vitro analysis. CONCLUSIONS: Our results indicated that SPS may inhibit TSCC development through regulation of Bcl-2, COX-2, Bax, and cleaved caspase-3 expression.