Tongue Squamous Carcinoma Cell Line Research Articles

Cholesterol depletion affects caveolin-1 expression, migration and invasion of oral tongue squamous cell carcinoma cell lines

IntroductionCholesterol is a key lipid molecule within cell membranes. This is especially true in cavelolas, invaginated membrane nanodomains, which present the protein caveolin-1 (CAV-1). It is important to note that this structure is involved in many cell signalling pathways. Additionally, high cholesterol is seen in different tumor types but little is known in regards to oral tongue squamous cell carcinoma (OTSCC). The aim of this study was to evaluate the influence of cholesterol depletion on primary (SCC-25) and metastatic (HSC-3) OTSCC cell lines. Materials and methodsCell membrane fluidity, cell viability, gene and protein expression of CAV-1 and of epithelial-mesenchymal transition (EMT) markers, cell migration in Myogel and invasion-myoma assay were evaluated after cholesterol depletion with methyl-β-cyclodextrin (MβCD - 7.5, 10 or 15 mM) ResultsDecreased cell viability and increased membrane fluidity of SCC-25 cells was seen with cholesterol depletion but cell viability was less affected and there was no effect on membrane fluidity in HSC-3. Cholesterol depletion also decreased CAV-1 at 6 h but increased it after 24 h.; both epithelial and mesenchymal EMT genes were upregulated after 6 h, followed by downregulation at 24 h in SCC-25. In HSC-3, CAV-1 was downregulated, and E-cadherin gene (ECAD) was upregulated at 6 h. Only the protein β-catenin in SCC-25 was affected, and cell migration of both cell lines was decreased, affecting SCC-25 more intensely. The invasive capacity within human myoma organotypic model was increased in SCC-25 and decreased in HSC-3. ConclusionCholesterol depletion affects CAV-1 and ECAD inversely. This affect also depends on cell type since the invasive capacity was augmented in primary cells while decreased in metastatic cells.

Evaluation of the cytotoxic, anti-proliferative, anti-metastatic and pro-apoptotic effect of aqueous leaf extract of Annona muricata on oral tongue squamous cell carcinoma cell line (SCC-15): An in vitro study.

Oral cancer still represents the leading cause of mortality in India. Due to the drawbacks of current treatment options, a safe, low-cost therapy is the need of the hour. Recently, novel plant extracts with anti-cancer properties have gained greater attention. One among them is Annona muricata and its leaf extract, which has been studied for its anti-cancer effect against various cancers. However, studies on oral cancer cells are very much limited and hence the study. To evaluate the cytotoxic, anti-proliferative, anti-metastatic and pro-apoptotic effect of aqueous leaf extract of Annona muricata (ALEAM) against SCC-15 cell lines through in vitro assays. In vitro assays such as MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide], colony formation and wound healing assays were performed. Furthermore, to evaluate the underlying mechanism, gene and protein expression analysis of apoptotic/anti-apoptotic marker genes Bax, P53 and Bcl2, were done using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis. Student's t-test has been performed for analysis of experimental data. The results showed that ALEAM exhibited significant cytotoxic activity in a dose-dependent manner as well as inhibited colony formation and cell migration. The pro-apoptotic properties were affirmed by a highly significant drop in Bcl-2 gene expression and a highly significant rise in P53 and Bax genes in the study group compared to the control (P < 0.05). The current study provides evidence that ALEAM has the potential to be developed as a novel anti-cancer drug for the treatment of SCC after further clinical studies.

A Novel Tongue Squamous Cell Carcinoma Cell Line Escapes from Immune Recognition due to Genetic Alterations in HLA Class I Complex.

Immune checkpoint inhibitors (ICI) have made progress in the field of anticancer treatment, but a certain number of PD-L1 negative OSCC patients still have limited benefits from ICI immuno-therapy because of primary immune evasion due to immunodeficiency. However, in existing human OSCC cell lines, cell models that can be used to study immunodeficiency have not been reported. The objective of this study was to establish a PD-L1 negative OSCC cell line, profile whether the presence of mutated genes is associated with immune deficiency, and explore its influence on the immune recognition of CD8+ T cells in vitro. Here, we established a novel tongue SCC cell line (WU-TSC-1), which escapes from immune recognition by antigen presentation defects. This cell line was from a female patient who lacked typical causative factors. The expression of PD-L1 was negative in the WU-TSC-1 primary tumor, transplanted tumor, cultured cells and lipopolysaccharide stimulation. Whole exome sequencing (WES) revealed that WU-TSC-1 harbored missense mutations, loss of copy number and structural variations in human leukocyte antigen (HLA) class I/II genes. The tumor mutation burden (TMB) score was high at 292.28. In addition, loss of heterozygosity at beta-2-microglobulin (B2M)-a component of all HLA class I complex allotypes-was detected. Compared with the commonly used OSCC cell lines, genetic alterations in HLA class I and B2M impeded the proteins' translation and inhibited the activation and killing effect of CD8+ T cells. In all, the WU-TSC-1 cell line is characterized by genetic variations and functional defects of the HLA class I complex, leading to escape from recognition by CD8+ T cells.

Activated fibroblasts induce immune escape of TSCC through CCL25/AKT pathway.

Accumulating evidence suggests that activated fibroblasts are the key cells in the T-cell response to tumor immunosuppression. We attempted to investigate the effect of activated fibroblasts on PD-L1 expression and the related immune escape mechanism in tongue squamous cell carcinoma. Western blotting, qPCR, and other techniques were used to study the expression of PD-L1 in tongue squamous cell carcinoma cells and the nude mouse model of transplanted tumors in vivo; clinical tissue samples were verified. In addition, we established a direct coculture model of T cells and tongue squamous cell carcinoma cells explore the mechanisms of immune escape. We found that PDGF-BB induces fibroblast activation by facilitating the oversecretion of chemokine CCL25. Further analysis showed that CCL25 derived from activated fibroblasts activated the Akt signaling pathway to promote PD-L1 expression. The activated fibroblasts inhibited T-cell IFN-γ secretion through the CCL25/Akt/PD-L1 pathway, which indirectly inhibited T-cell proliferation. Activated fibroblasts can induce the high expression of PD-L1 in the oral and tongue squamous cell carcinoma cell line Cal-27 via the CCL25/CCR9/p-Akt axis, to significantly inhibit the proliferation and IFN-γ secretion of T cells and promote the immune escape of tongue squamous cell carcinoma cells.

Evaluation of Apoptotic Effect of Betanin Nanoparticles Against Squamous Cell Carcinoma Cell Line Compared to Doxorubicin

Aim: to evaluate the anticancer effect of nano sized betanine particles (betanine NP) on squamous cell carcinoma cell line compared to doxorubicin (DOX) by measuring apoptosis through caspase 3. Material and Methods: Three groups of the human tongue squamous cell carcinoma cell line (SCC 25) were created, and two of them were treated with DOX and betanine NP. The third group served as a negative control and was not given any treatment. To evaluate the concentration of caspase 3 using the ElISA technology, different quantities of betanin NP and DOX were administered to SCC25 at 48- and 72-hour intervals. Results: Caspase 3 levels in the current investigation were 501.69.93, 543.86.71 pg/ml for DOX and 336.913.1, 405.53.5, and respectively for betanine NP in 48h and 72h intervals. Additionally, morphological analysis was performed to demonstrate the cells’ apoptotic alterations. There was a highly statistically significant difference between samples of various materials, as revealed by the ANOVA test for Comparison between groups using the ELISA technique for caspase 3 detection at 48h and 72h (p0.001). These findings suggest that DOX has a stronger apoptotic effect on SCC25 cells than betanin NP. Conclusion: Our findings explained that DOX and betanine NP can induce cancer cell death against SCC 25 cell lines by increasing the concentration of caspase 3. DOX has higher apoptotic effect on SCC25 cells than betanin NP according to ELISA technique.

Proanthocyanidins-loaded complex coacervates-based drug delivery attenuates oral squamous cell carcinoma cells metastatic potential through down-regulating the Akt signaling pathway.

Oral cancer, constituted up to 90% by squamous cell carcinomas, is a significant health burden globally. Grape seed proanthocyanidins (PA) have been suggested as a potential chemopreventive agent for oral cancer. However, their efficacy can be restricted due to the low bioavailability and bioaccessibility. Inspired by sandcastle worm adhesive, we adapted the concept of complex coacervation to generate a new type of drug delivery platform. Complex coacervates are a dense liquid phase formed by the associative separation of a mixture of oppositely charged polyelectrolytes, can serve as a drug delivery platform to protect labile cargo. In this study, we developed a complex coacervates-based delivery of PA. The release kinetics was measured, and anticancer effects were determined in two human tongue squamous cell carcinoma cell lines. The results showed that complex coacervate successfully formed and able to encapsulate PA. Additionally, PA were steadily released from the system in a pH-dependent manner. The drug delivery system could significantly inhibit the cell proliferation, migration, and invasion of cancer cells. Moreover, it could markedly reduce the expression of certain matrix metalloproteinases (MMP-2, 9, and 13) crucial to metastatic processes. We also found that suppression of protein kinase B (Akt) pathway might be the underlying mechanism for these anticancer activities. Taken together, complex coacervates-based delivery of PA can act as an effective anticancer approach for oral cancer therapy.

G-Quadruplex Aptamer-Ligand Characterization.

In this work we explore the structure of a G-rich DNA aptamer termed AT11-L2 (TGGTGGTGGTTGTTGTTGGTGGTGGTGGT; derivative of AT11) by evaluating the formation and stability of G-quadruplex (G4) conformation under different experimental conditions such as KCl concentration, temperature, and upon binding with a variety of G4 ligands (360A, BRACO-19, PDS, PhenDC3, TMPyP4). We also determined whether nucleolin (NCL) can be a target of AT11-L2 G4. Firstly, we assessed by circular dichroism, UV and NMR spectroscopies the formation of G4 by AT11-L2. We observed that, for KCl concentrations of 65 mM or less, AT11-L2 adopts hybrid or multiple topologies. In contrast, a parallel topology predominates for buffer containing 100 mM of KCl. The Tm of AT11-L2 in 100 mM of KCl is 38.9 °C, proving the weak stability of this sequence. We also found that upon titration with two molar equivalents of 360A, BRACO-19 and PhenDC3, the G4 is strongly stabilized and its topology is maintained, while the addition of 3.5 molar equivalents of TMPyP4 promotes the disruption of G4. The KD values between AT11-L2 G4, ligands and NCL were obtained by fluorescence titrations and are in the range of µM for ligand complexes and nM when adding NCL. In silico studies suggest that four ligands bind to the AT11-L2 G4 structure by stacking interactions, while the RBD1,2 domains of NCL interact preferentially with the thymines of AT11-L2 G4. Finally, AT11-L2 G4 co-localized with NCL in NCL-positive tongue squamous cell carcinoma cell line.

Geniposide inhibits cell proliferation and migration in human oral squamous carcinoma cells via AMPK and JNK signaling pathways

Iridoids are a special class of cyclopentanoid monoterpenes, which exhibit a wide range of biological effects. The present study aimed to investigate the potential effects of three iridoids genipin, geniposide and geniposidic acid on three human oral squamous cell carcinoma (OSCC) cell lines HSC-2, SCC-9 and A253 in addition to studying the possible underlying mechanisms. Cell viability assay revealed that geniposide treatment significantly suppressed the proliferation of all three cancer cell lines. In addition, geniposide induced SCC-9 cell cycle arrest at the G2/M phase (flow cytometry) through downregulation of cyclin-dependent kinase 2 and Cyclin A2 expression (western blot analysis), whilst also inducing cell apoptosis (flow cytometry and acridine orange/ethidium bromide staining) by dissipating the mitochondrial membrane potential (flow cytometry), and upregulating the expression of cleaved caspase-3 and cleaved poly-ADP ribose polymerase (western blot analysis). A wound-healing assay indicated that geniposide impaired SCC-9 cell migration by increasing the expression of E-cadherin (western blot analysis), whilst suppressing the expression of MMP-2 (western blot analysis). Western blot analysis also demonstrated that geniposide induced autophagy in SCC-9 cells by upregulating the expression of Beclin-1 and light chain 3-II. Mechanistically, geniposide activated the 5'-AMP-activated protein kinase signaling pathway and inhibited the JNK signaling pathway in SCC-9 cells (western blot analysis). The present results indicated that geniposide is able to inhibit the proliferation and migration of the tongue squamous carcinoma cell line SCC-9, suggesting a potential strategy for OSCC treatment.

Radiation modulates expression and related activities of c-Met protein in oral tongue squamous cell carcinoma cell lines

Objectivesc-Met, a receptor tyrosine kinase, is involved in the growth, invasion and metastasis of a variety of cancers. In a set of cell lines from several solid tumors, a five-fold increase in c-Met expression after irradiation has been reported. This study aimed to assess if c-Met is likewise abundantly expressed in oral tongue squamous cell carcinoma (OTSCC) upon exposure to irradiation, followed by a Met-induced biological response.Materials and methodsSix OTSCC cell lines were exposed to gamma radiation doses of 2, 4, and 6 Gray. The changes in c-Met protein levels were assessed by western blot and flow cytometry. c-Met gene expression, cell migration, proliferation and cell cycle assays were performed as phenotypic readouts.ResultsIrradiation resulted in upregulation of c.Met in all cell lines with different time kinetics. On average the cells displayed minimal c-Met expression on their surface ranging from 5 to 30% of total protein. Abrupt downregulation of c-Met surface expression occurred one hour after radiation but recovered 48 h post-radiation. Intracellularly, the highest level of expression was found on day 5 after radiation exposure. Irradiation induced aggressive invasive potential of the cells as determined in cell migration assays, particularly in cell lines with the highest c-Met expression.ConclusionsThese results provide novel insights into both intracellular and extracellular dynamics of c-Met expression profiles upon irradiation of OTSCC cells in vitro. It might also suggest that radiation enhances cell migration, indicative of invasiveness, through c-Met up-regulation, at least for certain types of OTSCC cells.

Dracaena cinnabari Methanolic Extract impairing Migration and inhibiting Growth of Nasopharyngeal Carcinoma Cells through Induction of Intrinsic Apoptotic Pathway In Vitro

The resin of Dracaena cinnabari Balf. f. has demonstrated antiviral, antibacterial, antioxidative, analgesic and anti-inflammatory properties, which presents some proof for its effectiveness in traditional use. Despite the broad health benefits of Dracaena cinnabari, few research has been conducted on its anti-tumour effect. We have previously shown that Dracaena cinnabari Balf. f methanolic extract (DCBME) induces apoptotic cell death in human tongue squamous cell carcinoma cell line and significantly reduces its migration capacity. The anti-tumour potential and mechanism of action of DCBME in Nasopharyngeal Carcinoma (NPC) have not yet been elucidated. In our recent paper, we showed the disruption of mitochondrial membrane potential and the induction of the intrinsic apoptotic pathway through caspase 3/7 activation. In the present study, we aim to investigate the effect of DCBME on proliferation, morphological changes, cell death, cell cycle arrest and cell migration of NPC cells. NPC cells (C666-1 and HK-1) were treated with DCBME and the mechanism underlying its anti-tumour effect was explored by examining cell proliferation, induction of apoptosis and cell migration using MTT assay, phase-contrast and fluorescence microscopy, propidium iodide staining, DNA fragmentation and wound healing assays. DCBME significantly inhibited NPC cell proliferation in a time and dose-dependent manner. It induced marked apoptotic morphological changes, apoptotic DNA laddering and S and/or G2/M-phase cell cycle arrest. DCBME also inhibited the migration of NPC cells. Together with our previously reported anti-tumour effect of DCBME, the findings suggest that DCBME deserves further investigation as a promising anti-tumour agent for human Nasopharyngeal Carcinoma cells therapy.

Roles of IGFBP-3 in cell migration and growth in an endophytic tongue squamous cell carcinoma cell line

Insulin-like growth factor binding protein-3 (IGFBP-3) is a member of the IGFBP family that has high affinity for IGFs and functions as either an oncogene or tumor suppressor in various types of cancer. We previously found that IGFBP3 mRNA levels are higher in endophytic-type human tongue squamous cell carcinoma (TSCC) that is more invasive and more prone to metastasis than exophytic and superficial types. This finding prompted us to investigate the roles of IGFBP-3 in TSCC using SAS cells, which were originally derived from endophytic-type TSCC. Specifically, we used SAS cells that express a fluorescent ubiquitination-based cell-cycle indicator (Fucci). RNA-sequencing analysis indicated that IGFBP-3 is associated with cell migration and cell growth. In fact, IGFBP-3 knockdown downregulates cell migration and causes cells to arrest in G1. This migratory potential appears to be cell cycle–independent. IGFBP-3 knockdown also reduced levels of secreted IGFBP-3; however, decreased migratory potential was not rescued by exogenous recombinant human IGFBP-3. Furthermore, ERK activity was downregulated by IGFBP-3 depletion, which suggests that MEK/ERK signaling may be involved in IGFBP-3-mediated cell migration. We therefore conclude that intracellular IGFBP-3 enhances cell migration independently of the cell cycle in TSCC with a higher metastatic potential.

An Ex vivo Apoptotic and Cytotoxic Effects of Frankincense on Oral Squamous Cell Carcinoma Cell Line

BACKGROUND: Oral squamous cell carcinoma is accounting for almost 90% of oropharyngeal cancer diagnoses. Natural herbal medicine can use as an alternative, or complementary, or adjunctive for cancer treatment. Frankincense and its combination with myrrh have anticancer effects on different cancer types. METHODS: In this research, aqueous and methanolic extracts of frankincense and the combination of aqueous extract of frankincense and myrrh were applied on tongue squamous cell carcinoma cell line to study their cytotoxic and apoptotic effect by the assessment of cell viability and cytotoxicity, caspase 3 and 8 activation, reactive oxygen species activity, mitochondrial membrane potential, morphological changes, and nuclear area factor measurements. RESULTS: The result showed that aqueous and methanolic extracts of frankincense have cytotoxic and apoptotic effects in a concentration-dependent manner with an IC50 value of 21.05 ± 1.27 μM for aqueous extract, 36.72 ± 2.07 μM for methanolic extract, and IC50 value of 1.31 ± 0.04 μM for combination of aqueous extract of frankincense and myrrh extract after 24 h. CONCLUSION: Different extracts of frankincense and the combination of aqueous extract of frankincense and myrrh extract exhibited cytotoxic and apoptotic effects by reducing the cell viability and activating caspases 3 and 8 causing intrinsic- and extrinsic-mediated apoptosis pathways activation with the involvement of oxidative stress that was conceivable with cytonuclear morphological alterations results.

Stabilization of a DNA aptamer by ligand binding

G-rich aptamers such as AS1411 are small oligonucleotides that present several benefits comparatively to monoclonal antibodies, since they are easier to manufacture and store, have small size and do not stimulate an immune response. We analyzed AT11-B1, a modified sequence of AT11 (itself a modified version of AS1411), in which one thymine was removed from the bulge region. We studied G-quadruplex (G4) formation/stabilization using PhenDC3, PDS, BRACO-19, TMPyP4 and 360A ligands by different biophysical techniques, namely circular dichroism (CD), Förster resonance energy transfer (FRET-melting) and nuclear magnetic resonance (NMR). The CD spectra showed that AT11-B1 adopts a predominant G4 of parallel topology when the buffer contains KCl or when ligands are added. PhenDC3 induced a ΔTm of 30 °C or more of the G4 structure as shown by CD- and FRET-melting experiments. The ligands demonstrate high affinity for AT11-B1 G4 and the NMR studies revealed that the AT11-B1 G4 involves four G-tetrad layers. The in silico studies suggest that all ligands bind AT11-B1 G4, namely, by stacking interactions, with the possible exception of PDS that may bind to the loop/groove interface. In addition, molecular dynamics simulations revealed that nucleolin (NCL) interacts with the AT11-B1 G4 structure through the RNA binding domain (RBD) 2 and the 12-residue linker between RBD1,2. Moreover, AT11-B1 G4 was internalized into a NCL-positive tongue squamous cell carcinoma cell line. In a nutshell, this study may help the identification of the ligands scaffolds to bind and stabilize AT11-B1, improving the targeting towards NCL that is overexpressed in cancer cells.

IDO1 Inhibition Reduces Immune Cell Exclusion Through Inducing Cell Migration While PD-1 Blockage Increases IL-6 and -8 Secretion From T Cells in Head and Neck Cancer.

BackgroundImmune checkpoint inhibitors (ICIs), primarily anti-PD-1, are currently used to treat patients with recurrent or metastatic head and neck squamous cell carcinoma (HNSCC). However, only a minority of patients benefit from these costly therapies. Therefore, there is an unmet need to better understand the effect of ICIs on immune effector cells. This study aimed to investigate the effect of a PD-1 antibody and an IDO1 inhibitor on different lymphocyte populations (NK, CD4+, and CD8+ T cells) in term of migration, cytotoxicity, and cytokine release in the presence of HNSCC cells.MethodsUsing a microfluidic chip, we injected HSC-3 cells (an oral tongue squamous cell carcinoma cell line) embedded in a human tumor-derived matrix “myogel/fibrin” together with NK, CD4+, and CD8+ T cells in separate channels. The two channels were connected with microchannels. The PD-1 antibody nivolumab and IDO1 inhibitor epacadostat were added to the microfluidic chips. Lymphocyte migration and cytotoxicity were examined under fluorescent microscopy and cytokine release was measured using a FirePlex Human Discovery Cytokines Immunoassay.ResultsEpacadostat significantly increased the migration and infiltration of NK and CD4+ T cells, but not CD8+ T cells, towards the cancer cells. Nivolumab did not exhibit a similar effect. While CD8+ T cells alone showed near to no migration, adding CD4+ T cells enhanced migration towards the cancer cells. There was a mild nonsignificant increase in apoptosis of HSC-3 cells after adding epacadostat to lymphocytes. In contrast, HSC-3 proliferation was not affected by lymphocytes regardless of ICIs. Nivolumab significantly increased release of MIP1-α, IL-6, and IL-8 from NK, CD4+, and CD8+ T cells, respectively.ConclusionsThis study revealed that each subpopulation of lymphocytes respond differently to ICIs. We also revealed the subpopulation of lymphocytes responsible for the increases in specific serum cytokines after ICI treatment.

The Tongue Squamous Carcinoma Cell Line Cal27 Primarily Employs Integrin α6β4-Containing Type II Hemidesmosomes for Adhesion Which Contribute to Anticancer Drug Sensitivity.

Integrins are heterodimeric cell surface glycoproteins used by cells to bind to the extracellular matrix (ECM) and regulate tumor cell proliferation, migration and survival. A causative relationship between integrin expression and resistance to anticancer drugs has been demonstrated in different tumors, including head and neck squamous cell carcinoma. Using a Cal27 tongue squamous cell carcinoma model, we have previously demonstrated that de novo expression of integrin αVβ3 confers resistance to several anticancer drugs (cisplatin, mitomycin C and doxorubicin) through a mechanism involving downregulation of active Src, increased cell migration and invasion. In the integrin αVβ3 expressing Cal27-derived cell clone 2B1, αVβ5 expression was also increased, but unrelated to drug resistance. To identify the integrin adhesion complex (IAC) components that contribute to the changes in Cal27 and 2B1 cell adhesion and anticancer drug resistance, we isolated IACs from both cell lines. Mass spectrometry (MS)-based proteomics analysis indicated that both cell lines preferentially, but not exclusively, use integrin α6β4, which is classically found in hemidesmosomes. The anticancer drug resistant cell clone 2B1 demonstrated an increased level of α6β4 accompanied with increased deposition of a laminin-332-containing ECM. Immunofluorescence and electron microscopy demonstrated the formation of type II hemidesmosomes by both cell types. Furthermore, suppression of α6β4 expression in both lines conferred resistance to anticancer drugs through a mechanism independent of αVβ3, which implies that the cell clone 2B1 would have been even more resistant had the upregulation of α6β4 not occurred. Taken together, our results identify a key role for α6β4-containing type II hemidesmosomes in regulating anticancer drug sensitivity.

Synergistic Cytotoxic Effect of Honey Bee Venom and Cisplatin on Tongue Squamous Cell Carcinoma Cell Line

BACKGROUND: Tongue cancer is one of the most common head and neck cancers in the world. Nowadays, natural compounds are important resources of many anti-cancer drugs. Venom from honey bees possesses potent anti-cancer activities. Cisplatin is a chemotherapeutic drug that has been used for decades to treat cancer cells. Recently, combination therapy has been a popular treatment choice for cancer patients. AIM: This study was conducted to evaluate the synergistic cytotoxic effect of honey bee venom (BV) and cisplatin on tongue squamous cell carcinoma 25 (SCC-25) cell lines. METHODS: The cytotoxic effect was determined using methyl thiazol tetrazolium assay, microscopic examination, real-time polymerase chain reaction (RT-PCR), and statistical analysis. RESULTS: The findings revealed that the cytotoxic potential of the tested drugs on SCC-25 cells was dose-dependent. Microscopic examination showed that BV and cisplatin alone and in combination mainly produced apoptotic cell death. Regarding RT-PCR results, P53 and caspase-3 expression levels were significantly increased in SCC-25-treated cells (p = 0.0001). CONCLUSION: The combined use of BV and cisplatin induced a marked synergistic cytotoxic effect on SCC-25 cell line.

5-Azacitidine and Trichostatin A induce DNA damage and apoptotic responses in tongue squamous cell carcinoma: An in vitro study

ObjectiveThe present in vitro study aims to investigate the potential use of epigenetic inhibitors as treatment modalities in tongue squamous cell carcinoma. DesignThe human tongue squamous cell carcinoma cell line (CAL-27) was cultured and exposed to varying concentrations of 5-Azacitidine (5-Aza) or Trichostatin A (TSA) in the culture medium. The cell apoptosis was evaluated using Annexin V/PI by flow cytometry. To evaluate DNA damage response, γH2AX foci analysis was performed using immunofluorescence. Single cell gel electrophoresis (SCGE) was applied to measure DNA strand breaks. Gene expression was assessed by quantitative real-time PCR. ResultsThe results showed that 5-Aza and TSA had apoptotic effects on the SCC cell line at concentrations of 50–200 µM and 0.5–5 µM, respectively. Immunofluorescence analysis showed increased expression of γH2AX, the marker of DNA damage response after treatment of 5-Aza and TSA that was associated with increased DNA strand breaks. The expressions of urokinase plasminogen activator, its receptor and matrix metalloproteinase-2, were significantly reduced in TSA- and 5-Aza-treated cells. ConclusionsOur results showed that 5-Aza and TSA increase apoptotic and DNA damage response in squamous cell carcinoma cell line while reducing the expression of tumor invasion genes that further indicating the potential therapeutic value of two epigenetic modifiers in squamous cell carcinoma.

Melatonin Suppresses Oral Squamous Cell Carcinomas Migration and Invasion through Blocking FGF19/FGFR 4 Signaling Pathway.

Oral squamous cell carcinomas (OSCCs) are one of the most prevalent malignancies, with a low five-year survival rate, thus warranting more effective drugs or therapy to improve treatment outcomes. Melatonin has been demonstrated to exhibit oncostatic effects. In this study, we explored the anti-cancer effects of melatonin on OSCCs and the underlying mechanisms. A human tongue squamous cell carcinoma cell line (SCC-15) was treated with 2 mM melatonin, followed by transwell migration and invasion assays. Relative expression levels of Fibroblast Growth Factor 19 (FGF19) was identified by Cytokine Array and further verified by qPCR and Western blot. Overexpression and downregulation of FGF19 were obtained by adding exogenous hFGF19 and FGF19 shRNA lentivirus, respectively. Invasion and migration abilities of SCC-15 cells were suppressed by melatonin, in parallel with the decreased FGF19/FGFR4 expression level. Exogenous hFGF19 eliminated the inhibitory effects of melatonin on SCC-15 cells invasion and migration, while FGF19 knocking-down showed similar inhibitory activities with melatonin. This study proves that melatonin suppresses SCC-15 cells invasion and migration through blocking the FGF19/FGFR4 pathway, which enriches our knowledge on the anticancer effects of melatonin. Blocking the FGF19/FGFR4 pathway by melatonin could be a promising alternative for OSCCs prevention and management, which would facilitate further development of novel strategies to combat OSCCs.

Combined high energy of extracorporeal shock wave and 5-FU effectively suppressed the proliferation and growth of tongue squamous cell carcinoma

BackgroundWe tested the hypothesis that extracorporeal shock wave (ECSW)-assisted 5-FU therapy effectively suppressed human tongue squamous carcinoma cell line SAS (i.e., SAS cells) proliferation and tumor growth. Methods and resultsIn vitro study showed that as compared with lower ECSW energy (≤0.12 mJ/mm2), higher ECSW energy (≥0.25–035 mJ/mm2) significantly suppressed the SAS cell proliferation and upregulated tumor cell apoptosis/DNA-damage/oxidative-stress, whereas combined higher ECSW energy (0.35 mJ/mm2) and 5-FU (20uM) further significantly altered the expressions of these parameters (all p < 0.001). Adult male nude mice (NM) (n = 36) were equally categorized into group 1 (2.0 × 105 SAS cells were implanted into NM back), group 2 [SAS in NM back + stepwise-increased ECSW energy (from 0.05/0.1/0.3/to 0.5 mJ/mm2)/500 impulses which applied to the tumor at days 9/12/15/21], group 3 (SAS in NM back + 5-FU/i.p./7 mg/kg/every 3-day) and group 4 (SAS in NM back + ECSW + 5-FU) and tumors were removed from each animal by day-28. The result showed that tumor volume and tumor weight were significantly progressively reduced from group 1 to group 4 (all p < 0.0001). The protein expressions of apoptotic (mitochondrial-Bax/cleaved-caspase3/cleaved-PARP/cyclophyllin-D), autophagic (ratio of LC3B-II/LC3B-I) and oxidative-stress (NOX-1/NOX-2) biomarkers displayed an opposite pattern of tumor mass among the groups, whereas the cell-stress signaling (p-PI3K/p-Akt/p-m-TOR, and ASK1/MKK4/MKK7/p38/p-JNK/p-c-JUN), MAP kinase family members (RAS/cRAF/KRAS/BRAF/p-ERK1/2), tumor protein (p53) and cellular levels of angiogenesis/DNA-damage (α-SMA+/VEGF+/γ-H2AX+) exhibited an identical pattern of tumor mass among the groups (all p < 0.0001). ConclusionCombined high-energy ECSW and 5-FU offers an additional benefit for suppressing the cancer cell proliferation and tumor growth.

Tongue Cancer Cell-Derived CCL20 Induced by Interaction With Macrophages Promotes CD163 Expression on Macrophages.

BackgroundCD163-positive macrophages contribute to the aggressiveness of oral squamous cell carcinoma. We showed in a previous report that CD163-positive macrophages infiltrated not only to the cancer nest but also to its surrounding epithelium, depending on the presence of stromal invasion in tongue carcinogenesis. However, the role of intraepithelial macrophages in tongue carcinogenesis remains unclear. In this study, we assessed the biological behavior of intraepithelial macrophages on their interaction with cancer cells.Materials and MethodsWe established the indirect coculture system (intraepithelial neoplasia model) and direct coculture system (invasive cancer model) of human monocytic leukemia cell line THP-1-derived CD163-positive macrophages with SCC25, a tongue squamous cell carcinoma (TSCC) cell line. Conditioned media (CM) harvested from these systems were analyzed using cytokine array and enzyme-linked immunosorbent assay and extracted a specific upregulated cytokine in CM from the direct coculture system (direct CM). The correlation of both this cytokine and its receptor with various clinicopathological factors were evaluated based on immunohistochemistry using clinical samples from 59 patients with TSCC. Moreover, the effect of this cytokine in direct CM on the phenotypic alterations of THP-1 was confirmed by real-time polymerase chain reaction, western blotting, immunofluorescence, and transwell migration assay.ResultsIt was shown that CCL20 was induced in the direct CM specifically. Interestingly, CCL20 was produced primarily in SCC25. The expression level of CCR6, which is a sole receptor of CCL20, was higher than the expression level of SCC25. Our immunohistochemical investigation showed that CCL20 and CCR6 expression was associated with lymphatic vessel invasion and the number of CD163-positive macrophages. Recombinant human CCL20 induced the CD163 expression and promoted migration of THP-1. We also confirmed that a neutralizing anti-CCL20 antibody blocked the induction of CD163 expression by direct CM in THP-1. Moreover, ERK1/2 phosphorylation was associated with the CCL20-driven induction of CD163 expression in THP-1.ConclusionsTongue cancer cell-derived CCL20 that was induced by interaction with macrophages promotes CD163 expression on macrophages.