Tongue Squamous Carcinoma Research Articles

Golgi integral membrane protein 4 manipulates cellular proliferation, apoptosis, and cell cycle in human head and neck cancer.

The incidence of global head and neck cancer has increased markedly in the last 10 years, and its prognosis is poor, which seriously endangers people’s life and health. At present, there are few studies on its pathogenesis. Golgi integral membrane protein 4 (GOLIM4) is a major member of the Golgi apparatus transporter complex, and its role in tumor is unclear. The present study found that GOLIM4 was the key target protein downstream of stromal interaction molecule 1 (STIM1), which can inhibit the proliferation of head and neck cancer cells FaDu (human pharyngeal squamous carcinoma cell) and Tca-8113 (human tongue squamous carcinoma cell) with knockdown of GOLIM4 by lentivirus. And the decreased expression of GOLIM4 induced cellular apoptosis. Further experiments revealed that FaDu cell cycle progression was changed after GOLIM4 silence, G1 phase arrest and the number of G2/M cells decreased significantly. It was also found that the cells in S-phase decreased markedly after GOLIM4 was knocked down compared with the control group by 5-bromo-2′-deoxyuridine (BrdU) incorporation experiment. In conclusion, we found that GOLIM4, as the target gene downstream of STIM1, inhibited the proliferation of head and neck cancer, promoted apoptosis, and regulated cell cycle progression, and GOLIM4 is a novel oncogene in head and neck cancer and might help in developing promising targetted therapies for head and neck cancer patients.

Abstract 5912: Sentinel lymph node targeted therapy by oncolytic sendai virus suppresses micrometastasis of head and neck squamous cell carcinoma in an orthotopic nude mouse model

Abstract Background: Lymph node metastasis is one of the most important prognostic factors in head and neck squamous cell carcinoma (HNSCC). In clinical N0 cases, however, it is difficult to decide whether we should select elective neck dissection or watchful waiting even at present. We recently reported the successful oncolytic virotherapy against head and neck cancer using recombinant Sendai virus vector (rSeV). We herein focus on sentinel lymph node (SLN)-targeted therapy by oncolytic Sendai virus as a novel less invasive therapy. The objectives of this study are to investigate rSeV migration into SLN, and to elucidate anti-tumor effect on SLN micrometastasis of HNSCC in an orthotopic nude mouse model. Experimental design: We established SLN micrometastasis of HNSCC in an orthotopic nude mouse model. We inoculated HSC-3-M3, human highly metastatic tongue squamous carcinoma cells, into the left edge of the tongue, and two weeks after tumor inoculation we intratumorally injected a type of rSeV-GFP that selectively shows urokinase type plasminogen activator (uPA)-specific cell killing activity via cell-cell fusion, which is named “BioKnife”. We investigated rSeV migration into SLN by using immunostaining, RT-PCR, and in vivo imaging system. We also examined anti-tumor effect and its mechanisms through serial section examination of SLN by using anti-SeV antibody and the ApopTag peroxidase in situ apoptosis detection kit. Results: rSeV migration into SLN was clearly visible by fluorescent signals of rSeV in SLN using immunostaining and in vivo imaging system, and clarified by significant increases of relative mRNA expressions of GFP compared to control group in both tongue and SLN using RT-PCR (p < 0.05 respectively). Serial section examination of SLN showed that BioKnife selectively infected cancer cells and exhibited significant anti-tumor effect against SLN micrometastases compared to control groups (p < 0.05). We also detected apoptosis in SLN micrometastasis infected by BioKnife. Conclusions: This study demonstrated that BioKnife can migrate into metastatic SLN after intratumoral injection into primary tumor, and effectively suppresses SLN micrometastases in an orthotopic nude mouse model, suggesting that SLN targeted therapy of oncolytic rSeV has a great potential to provide a novel and promising alternative to elective neck dissection for clinical N0 patients with HNSCC. Citation Format: Yuya Tanaka, Koji Araki, Shingo Tanaka, Yoshihiro Miyagawa, Hiroshi Suzuki, Daisuke Kamide, Masayuki Tomifuji, Kosuke Uno, Eiko Harada, Taku Yamashita, Yasuji Ueda, Makoto Inoue, Akihiro Shiotani. Sentinel lymph node targeted therapy by oncolytic sendai virus suppresses micrometastasis of head and neck squamous cell carcinoma in an orthotopic nude mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5912.

Influence of luteolin on the invasion and migration of an human tongue squamous carcinoma cell line

Influence of luteolin on the invasion and migration of an human tongue squamous carcinoma cell line

RETRACTED ARTICLE: MicroRNA-488 inhibits tongue squamous carcinoma cell invasion and EMT by directly targeting ATF3

BackgroundIt has been reported that the expression of activating transcription factor 3 (ATF3) is closely associated with both microRNA (miRNA) processing and the progress of many cancers. Our study aimed to explore the interaction between ATF3 and miR-488 in tongue squamous cell carcinoma (TSCC).MethodsQuantitative real-time PCR was performed to detect the levels of ATF3 and miR-488 in TSCC tissues and cell lines. Cell invasion and epithelial–mesenchymal transition (EMT) were assessed to determine the biological functions of miR-488 and ATF3 in TSCC cells. The mRNA and protein levels of ATF3 were measured using quantitative RT-PCR and western blotting. Luciferase assays were performed to validate ATF3 as an miR-488 target in TSCC cells.ResultsWe found that the level of miR-488 significantly decreased and the expression of ATF3 significantly increased in TSCC tissues and cell lines. A low level of miR-488 was closely associated with increased expression of ATF3 in TSCC tissues. Introducing miR-488 significantly inhibited the invasion and EMT of TSCC cells, and knockdown of miR-488 promoted both processes. The bioinformatics analysis predicted that ATF3 is a potential target gene of miR-488. The luciferase reporter assay showed that miR-488 could directly target ATF3. ATF3 silencing had similar effects to miR-488 overexpression on TSCC cells. Overexpression of ATF3 in TSCC cells partially reversed the inhibitory effects of the miR-488 mimic.ConclusionmiR-488 inhibited cell invasion and EMT of TSCC cells by directly downregulating ATF3 expression.

Long non-coding RNA CASC15 promotes tongue squamous carcinoma progression through targeting miR-33a-5p.

Long non-coding RNAs (lncRNAs) have gained a lot of attention because they participate in several human disorders, including tumors. This study determined the role of LncRNA CASC15 (cancer susceptibility candidate 15) in the development of tongue squamous cell carcinoma (TSCC). Here, we identified that CASC15 expression was upregulated in TSCC samples and cell lines. We showed that overexpression of CASC15 promoted cell proliferation, cycle, and migration in TSCC. In addition, we revealed that miR-33a-5p expression was downregulated in TSCC tissues and cell lines. Moreover, we showed that the expression of CASC15 was negatively related with miR-33a-5p expression in TSCC tissues. Ectopic expression of miR-33a-5p suppressed cell proliferation, cycle, and migration in TSCC. Elevated expression of CASC15 suppressed miR-33a-5p expression and promoted ZEB1 expression in SCC4 cell. Ectopic expression of CASC15 promoted TSCC cell proliferation, cycle, and migration through targeting miR-33a-5p. These results suggested that lncRNA CASC15 and miR-33a-5p might be exploited as new markers of TSCC and were potential treatment targets for TSCC patients.

Biochemical and functional characterization of the human tissue kallikrein 9.

Human tissue kallikrein 9 (KLK9) is a member of the kallikrein-related family of proteases. Despite its known expression profile, much less is known about the functional roles of this protease and its implications in normal physiology and disease. We present here the first data on the biochemical characterization of KLK9, investigate parameters that affect its enzymatic activity (such as inhibitors) and provide preliminary insights into its putative substrates. We show that mature KLK9 is a glycosylated chymotrypsin-like enzyme with strong preference for tyrosine over phenylalanine at the P1 cleavage position. The enzyme activity is enhanced by Mg2+ and Ca2+, but is reversibly attenuated by Zn2+ KLK9 is inhibited in vitro by many naturally occurring or synthetic protease inhibitors. Using a combination of degradomic and substrate specificity assays, we identified candidate KLK9 substrates in two different epithelial cell lines [the non-tumorigenic human keratinocyte cells (HaCaT) and the tumorigenic tongue squamous carcinoma cells (SCC9)]. Two potential KLK9 substrates [KLK10 and midkine (MDK)] were subjected to further validation. Taken together, our data delineate some functional and biochemical properties of KLK9 for future elucidation of the role of this enzyme in health and disease.

The role of NLRP3 inflammasome in 5-fluorouracil resistance of oral squamous cell carcinoma

Background5-Fluorouracil (5-FU) is a widely used drug for the therapy of cancer. However, the chemoresistance of tumor cells to 5-FU usually limits its clinical effectiveness. In this study, we explored the role of NLRP3 inflammasome in 5-FU resistance of oral squamous cell carcinoma (OSCC).MethodsThe mRNA and protein expression levels of NLRP3, Caspase1 and IL-1β in resected OSCC specimens or cell lines were measured respectively by quantitative real time-PCR (qRT-PCR) and western blot. NLRP3 and Ki-67 expression in paraffin-embedded OSCC tissues was determined by immunohistochemistry. The correlation between 5-FU treatment and the expression and activation of NLRP3 inflammasome was further examined by evaluating NLRP3 and IL-1β expression in OSCC cell lines without or with NLRP3 knocked down. Cell viabilities of OSCC cells were determined by the MTT assay. Apoptosis and intracellular reactive oxygen species (ROS) of OSCC cells induced by 5-FU were measured by the flow cytometer. The carcinogen-induced tongue squamous carcinoma mice model was established by continuous oral administration of 4-nitroquinoline 1-oxide in wild-type BALB/c, Nlrp3−/− and Caspase1−/− mice. Tumor incidence were observed and tumor area were evaluated.ResultsIn the clinical analysis, expression and activation of NLRP3 inflammasome was clearly increased in OSCC tissues of patients who received 5-FU-based chemotherapy. Multivariate Cox regression analysis revealed that this high expression was significantly correlated with tumor stage and differentiation, and was associated with poor prognosis. Moreover, 5-FU treatment increased expression and activation of NLRP3 inflammasome in OSCC cells in a cell culture system and xenograft mouse model. Silencing of NLRP3 expression significantly inhibited OSCC cell proliferation and enhanced 5-FU-induced apoptosis of OSCC cells. Further investigation showed that intracellular ROS induced by 5-FU promoted the expression and activation of NLRP3 inflammasome and increased the production of interleukin (IL)-1β, which then mediated the chemoresistance. With the carcinogen-induced OSCC model, we found less and later tumor incidence in Nlrp3−/− and Caspase1−/− mice than wild-type mice. And greater decrease of tumor area was observed in the gene deficient mice treated with 5-FU.ConclusionsOur findings suggest that NLRP3 inflammasome promoted 5-FU resistance of OSCC both in vitro and in vivo, and targeting the ROS/NLRP3 inflammasome/IL-1β signaling pathway may help 5-FU-based adjuvant chemotherapy of OSCC.

Generation and characterization of a human oral squamous carcinoma cell line SCC-9 with CRISPR/Cas9-mediated deletion of the p75 neurotrophin receptor

ObjectiveTo investigate the importance of the p75 neurotrophin receptor (p75NTR) in human tongue squamous carcinoma cells, we exploited the CRISPR/Cas9 technology to establish a p75NTR-knockout SCC-9 cell line and to explore the effect on biological functions. Materials and methodsThe clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease (Cas9) system was used to generate genomic deletion mutants of p75NTR in the tongue squamous carcinoma cell lines SCC-9. Single-guide RNA (sgRNA) sequences were designed to target the p75NTR genomic sequence and were cloned into plasmid pGK1.1. The linearized vector was electroporated into SCC-9 cells and p75NTR deletion was confirmed using Cruiser™ enzyme digestion and PCR amplification. SCC-9 clones with successful deletion of p75NTR were identified and verified by sequencing and selected for functional testing in cell proliferation, invasion, migration, and colony-forming assays. ResultsCompared with control cells, p75NTR-knockout SCC-9 cells showed significantly diminished abilities to proliferate, invade, migrate, and form colonies, indicating a reduction in pro-tumorigenic behavior. ConclusionThese data demonstrate, first, that the CRISPR/Cas9 system is a simplified method for generating p75NTR knockouts with relatively high efficiency, and second, that deletion of p75NTR suppresses several tumor-promoting properties of SCC-9 cells, suggesting that p75NTR is a potential target for the development of novel therapies for tongue cancer.

Nicotine may promote tongue squamous cell carcinoma progression by activating the Wnt/β-catenin and Wnt/PCP signaling pathways.

To investigate the effects and the possible underlying mechanisms of nicotine stimulation on tongue squamous cell carcinoma (TSCC) progression, a TSCC cell line Cal27 and 34 samples of paraffin-embedded TSCC were examined. Immunofluorescence, western blot analysis, and TOP/FOP flash, CCK-8, wound healing and Transwell invasion assays were used to evaluate Cal27 in response to nicotine stimulation. We also investigated expression levels of related proteins of Wnt/β-catenin and Wnt/PCP pathways in paraffin-embedded TSCC samples with or without a history of smoking by immunohistochemistry. Nicotine stimulation can promote proliferation, migration, and invasion of TSCC cells in vitro, downregulate E-cadherin, and activate the Wnt/β-catenin and Wnt/PCP pathways, which could be antagonized by the α7 nicotine acetylcholine receptor (α7 nAChR) inhibitor α-BTX. Moreover, the expression levels of β-catenin, Wnt5a and Ror2 were higher in TSCC patients with a history of smoking than those without a history of smoking. Our results suggest nicotine may promote tongue squamous carcinoma cells progression by activating the Wnt/β-catenin and Wnt/PCP signaling pathways and may play a significant role in the progression and metastasis of smoking-related TSCC.

Stromal interaction molecule 1 regulates growth, cell cycle, and apoptosis of human tongue squamous carcinoma cells.

Oral tongue squamous cell carcinoma (OTSCC) is the most common type of oral carcinomas. However, the molecular mechanism by which OTSCC developed is not fully identified. Stromal interaction molecule 1 (STIM1) is a transmembrane protein, mainly located in the endoplasmic reticulum (ER). STIM1 is involved in several types of cancers. Here, we report that STIM1 contributes to the development of human OTSCC. We knocked down STIM1 in OTSCC cell line Tca-8113 with lentivirus-mediated shRNA and found that STIM1 knockdown repressed the proliferation of Tca-8113 cells. In addition, we also showed that STIM1 deficiency reduced colony number of Tca-8113 cells. Knockdown of STIM1 repressed cells to enter M phase of cell cycle and induced cellular apoptosis. Furthermore, we performed microarray and bioinformatics analysis and found that STIM1 was associated with p53 and MAPK pathways, which may contribute to the effects of STIM1 on cell growth, cell cycle, and apoptosis. Finally, we confirmed that STIM1 controlled the expression of MDM2, cyclin-dependent kinase 4 (CDK4), and growth arrest and DNA damage inducible α (GADD45A) in OTSCC cells. In conclusion, we provide evidence that STIM1 contributes to the development of OTSCC partially through regulating p53 and MAPK pathways to promote cell cycle and survival.

Antiproliferative and Apoptosis-inducing Effect of exo-Protoporphyrin IX based Sonodynamic Therapy on Human Oral Squamous Cell Carcinoma

Sonodynamic therapy (SDT) is an innovative modality for cancer treatment. But the biological effect of SDT on oral squamous cell carcinoma has not been studied. Our previous study has shown that endo-Protoporphyrin IX based SDT (ALA-SDT) could induce apoptosis in human tongue squamous carcinoma SAS cells through mitochondrial pathway. Herein, we investigated the effect of exo- Protoporphyrin based SDT (PpIX-SDT) on SAS cells in vitro and in vivo. We demonstrated that PpIX-SDT increased the ratio of cells in the G2/M phase and induced 3–4 times more cell apoptosis compared to sonocation alone. PpIX-SDT caused cell membrane damage prior to mitochondria damage and upregulated the expression of Fas and Fas L, while the effect was suppressed if cells were pre-treated with p53 inhibitor. Additionally, we examined the SDT-induced cell apoptosis in two cell lines with different p53 status. The increases of p53 expression and apoptosis rate in wild-type p53 SAS cells were found in the SDT group, while p53-mutated HSC-3 cells did not show such increase. Our data suggest that PpIX-SDT suppress the proliferation of SAS cells via arresting cell cycle at G2/M phase and activating the extrinsic Fas-mediated membrane receptor pathway to induce apoptosis, which is regulated by p53.

Synthetic Isoliquiritigenin Inhibits Human Tongue Squamous Carcinoma Cells through Its Antioxidant Mechanism.

Isoliquiritigenin (ISL), a natural antioxidant, has antitumor activity in different types of cancer cells. However the antitumor effect of ISL on human tongue squamous carcinoma cells (TSCC) is not clear. Here we aimed to investigate the effects of synthetic isoliquiritigenin (S-ISL) on TSCC and elucidate the underlying mechanisms. S-ISL was synthesized and elucidated from its nuclear magnetic resonance spectrum and examined using high performance liquid chromatography. The effects of S-ISL on TSCC cells (Tca8113) were evaluated in relation to cell proliferation, apoptosis and adhesion, migration, and invasion using sulforhodamine B assay, fluorescence microscopy technique, flow cytometry (FCM) analysis, and Boyden chamber assay. The associated regulatory mechanisms were examined using FCM and fluorescence microscopy for intracellular reactive oxygen species (ROS) generation, Gelatin zymography assay for matrix metalloproteinase (MMP) activities, and Western blot for apoptosis regulatory proteins (Bcl-2 and Bax). Our data indicated that S-ISL inhibited Tca8113 cell proliferation, adhesion, migration, and invasion while promoting the cell apoptosis. Such effects were accompanied by downregulation of Bcl-2 and upregulation of Bax, reduction of MMP-2 and MMP-9 activities, and decreased ROS production. We conclude that S-ISL is a promising agent targeting TSCC through multiple anticancer effects, regulated by its antioxidant mechanism.

MicroRNA-137 suppresses tongue squamous carcinoma cell proliferation, migration and invasion.

Tongue squamous cell carcinoma (TSCC) is the most frequent type of oral malignancy. Increasing evidence has shown that miRNAs play key roles in many biological processes such as cell development, invasion, proliferation, differentiation, metabolism, apoptosis and migration. qRT-PCR analysis was performed to measure miR-137 expression. CCK-8 analysis, cell colony formation, wound-healing analysis and invasion were performed to detect resultant cell functions. The direct target of miR-137 was labelled and measured by luciferase assay and Western blotting. We demonstrated that expression of miR-137 was downregulated in TSCC tissues compared to matched normal ones. miR-137 expression was downregulated in TSCC lines (SCC4, SCC1, UM1 and Cal27) compared to the immortalized NOK16B cell line and normal oral keratinocytes in culture (NHOK). In addition, we have shown that miR-137 expression was epigenetically regulated in TSCCs. Overexpression of miR-137 suppressed TSCC proliferation and colony formation. Ectopic expression of miR-137 promoted expression of the epithelial biomarker, E-cadherin, and inhibited the mesenchymal biomarker, N-cadherin, as well as vimentin and Snail expression, indicating that miR-137 suppressed TSCC epithelial-mesenchymal transition (EMT). We also showed that ectopic expression of miR-137 inhibited TSCC invasion and migration. In addition, we identified SP1 as a direct target gene of miR-137 in SCC1 cells. SP1 overexpression rescued inhibitory effects exerted by miR-137 on cell proliferation and EMT. These results indicate that miR-137 acted as a tumour suppressor in TSCC by targeting SP1.

PDGF-D/PDGFRβ promotes tongue squamous carcinoma cell (TSCC) progression via activating p38/AKT/ERK/EMT signal pathway

Platelet-derived growth factor D (PDGF-D) signaling plays significant roles during the development and progression of human malignancies via interacting with the receptor of PDGF-D (PDGFR). Meanwhile, the majority of human tumor metastasis is closely associated with epithelial-mesenchymal transition (EMT). However, the underlying mechanism between PDGF-D/PDGFR signaling and EMT which involved in tumor metastasis remain dismal. This study aimed to investigate the role of PDGF-D signaling during EMT process of tongue squamous cell carcinoma (TSCC). In our study, the expression of PDGF-D and PDGFR were examined in primary TSCC samples and the expression of PDGF-D was also determined in TSCC cell lines. In addition, the correlation between PDGF-D expression and TSCC aggressive histopathological features was analyzed. Our results implied that upregulation of PDGFRβ in UM1 cells induced with exogenous PDGF-D can remarkably promote tumor cells invasiveness; conversely, when using small interfering RNA (siRNA), the invasiveness can be severely prohibited. Furthermore, PDGF-D downstream signal molecules p38, AKT, ERK and EMT biomarkers (E-cadherin, N-cadherin, Vimentin and snail) were measured using Western blot. Our results showed that PDGF-D can induce p38, AKT and ERK phosphorylation; downregulate epithelial markers and upregulate mesenchymal markers. On the contrary, PDGFRβ siRNA significantly prohibited p38, AKT and ERK phosphorylation; inhibited EMT process. Function analysis revealed that PDGFRβ siRNA obviously interfered with UM1 cell migration and invasion, according to transwell and wound healing assay. In conclusion, this study suggested that EMT process can be triggered by the PDGF-D/PDGFRβ axis in TSCC, and then involved in the tumor cell invasion via activation of p38/AKT/ERK/EMT pathway.

Construction of epithelial membrane protein 1 eukaryotic expression vector and its influence on migration and invasion of human oral tongue squamous carcinoma cells

This study aimed to construct a eukaryotic expression vector pEGFP-N1-EMP1 of epithelial mem-brane protein 1 (EMP1) and investigate its influence on migration and invasion of human oral tongue squamous carcinoma cells. The human EMP1 gene was amplified by reverse transcription polymerase chain reaction and then ligated into the pEGFP-N1 vector by double restriction endonuclease digestion to construct pEGFP-N1-EMP1 recombinant plasmid. After sequencing identification, pEGFP-N1-EMP1 recombinant plasmid and pEGFP-N1 plasmid were transfected into human oral tongue squamous carcinoma Tb3.1 cell line. The expression of green fluorescent protein in cells was observed after transfection using an inverted fluorescence microscope. The overexpression of EMP1 mRNA was identified at 24, 48, and 72h after transfection by real-time fluorescence quantitative polymerase chain reaction. The effect of EMP1 overexpression on migration and invasion of Tb3.1 cells was detected by Transwell assay. The full-length EMP1 gene sequence was successfully obtained. Sequence analysis showed that the EMP1 gene was inserted into the pEGFP-N1 vector correctly. Green fluorescence was observed in the transfected cells under fluorescence microscopy. The results of real-time fluorescence quantitative polymerase chain reaction indicated that the expression of EMP1 at 24h after pEGFP-N1-EMP1 transfection was significantly higher than the other groups. Transwell assays indicated that overexpression of the EMP1 gene could significantly inhibit the migration and invasion ability of Tb3.1 cells. The eukaryotic expression vector of EMP1 was successfully constructed, and EMP1 overexpression was confirmed to inhibit the migration and inva-sion of oral tongue squamous carcinoma cells in vitro. This study laid a foundation for further investigation on the influence of the EMP1 gene on the metastasis of oral tongue squamous carcinoma and its molecular mechanism. .

Role of CD133+ cells in tongue squamous carcinomas: Characteristics of 'stemness' in vivo and in vitro.

The objective of the present study was to determine the ‘stemness’ characteristics of CD133+ cells (harvested from the squamous cell tongue carcinoma Tca-8113 cell line) in vitro and to observe the tumourigenicity of the CD133+ cells in the bodies of NOD/SCID mice. Single cells from the Tca-8113 cell line were observed for multiplication capacity in vitro. The suspending and pelletizing phenomena of Tca-8113 cells in vitro were also observed, and the expression of CD133 in squamous cell carcinoma of the tongue was measured. The CD133+ cells from the Tca-8113 cell line were purified, and their multiplication capacity and differentiation potency were observed. The NOD/SCID mouse model was established, and the tumourigenicity of the CD133+ cells was determined. The Tca-8113 cells were observed to emerge in the form of suspending tumour spheres in squamous cell carcinoma of the tongue. Monoplasts with sustainable multiplication capacity accounted for ~5.32% of the spheres, and 0.95% of the CD133+ cells were expressed in squamous cell carcinoma of the tongue, with stronger multiplication capacity and differentiation potency in vitro. Stronger tumourigenicity was also observed in the bodies of the NOD/SCID mice. CD133– cells exhibited a multiplication capacity to a certain extent. Overall, the CD133+ cells in squamous cell carcinoma of the tongue are characterised by relatively strong tumourigenicity capacity in vivo and in vitro. To a certain extent, these CD133+ cells demonstrate the characteristics of ‘stemness’.

Integrin αvβ3 expression in tongue squamous carcinoma cells Cal27 confers anticancer drug resistance through loss of pSrc(Y418)

Integrins play key roles in the regulation of tumor cell adhesion, migration, invasion and sensitivity to anticancer drugs. In the present study we investigate the mechanism of resistance of tongue squamous carcinoma cells Cal27 with de novo integrin αvβ3 expression to anticancer drugs. Cal27-derived cell clones, obtained by transfection of plasmid containing integrin subunit β3 cDNA, as compared to control cells demonstrate: expression of integrin αvβ3; increased expression of integrin αvβ5; increased adhesion to fibronectin and vitronectin; resistance to cisplatin, mitomycin C, doxorubicin and 5-fluorouracil; increased migration and invasion, increased amount of integrin-linked kinase (ILK) and decreased amounts of non-receptor tyrosine kinase (Src) and pSrc(Y418). Knockdown of ILK and integrin β5 in cells expressing integrin αvβ3 ruled out their involvement in drug resistance. Opposite, Src knockdown in Cal27 cells which led to a reduction in pSrc(Y418), as well as treatment with the pSrc(Y418) inhibitors dasatinib and PP2, conferred resistance to all four anticancer drugs, indicating that the loss of pSrc(Y418) is responsible for the observed effect. We identified differential integrin signaling between Cal27 and integrin αvβ3-expressing cells. In Cal27 cells integrin αv heterodimers signal through pSrc(Y418) while this is not the case in integrin αvβ3-expressing cells. Finally, we show that dasatinib counteracts the effect of cisplatin in two additional head and neck squamous cell carcinoma (HNSCC) cell lines Cal33 and Detroit562. Our results suggest that pSrc(Y418) inhibitors, potential drugs for cancer therapy, may reduce therapeutic efficacy if combined with chemotherapeutics, and might not be recommended for HNSCC treatment.

(286) Human tongue squamous carcinoma sensitizes tongue primary afferents in male and female mice

(286) Human tongue squamous carcinoma sensitizes tongue primary afferents in male and female mice

Silver Nanoparticles Exhibit the Dose-Dependent Anti-Proliferative Effect against Human Squamous Carcinoma Cells Attenuated in the Presence of Berberine.

The biological activity of nanosize silver particles towards oral epithelium-derived carcinoma seems to be still underinvestigated. We evaluated the influence of low doses of nanosize scale silver particles on the proliferation and viability of malignant oral epithelial keratinocytes in vitro, alone and in conjunction with the plant alkaloid berberine. Cells of human tongue squamous carcinoma SCC-25 (ATCC CRL-1628), cultivated with the mixture of Dulbecco's modified Eagle’s medium, were exposed to silver nanoparticles alone (AgNPs, concentrations from 0.31 to 10 μg/mL) and to a combination of AgNPs with berberine chloride (BER, 1/2 IC50 concentration) during 24 h and 48 h. The cytotoxic activity of AgNPs with diameters of 10 nm ± 4 nm was measured by 3-(4,5-dimethyl-2-thiazyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Cell cycle analysis was performed by treating cells with propidium iodide followed by flow-activated cell sorting. RT-QPCR reaction was used to assess expression of anti-apoptotic proteins Bcl-2 and pro-apoptotic protein Bcl-2-associated X protein Bax genes expression. Monodisperse silver nanoparticles at a concentration of 10 μg/mL arrested SCC-25 cells cycle after 48 h at the G0/G1 phase in a dose- and time-dependent manner through disruption G0/G1 checkpoint, with increase of Bax/Bcl-2 ratio gene expression. AgNPs exhibit cytotoxic effects on SCC-25 malignant oral epithelial keratinocytes, which is diminished when combined with BER. The AgNPs concentration required to inhibit the growth of carcinoma cells by 50% (IC50) after 48 h was estimated at 5.19 μg/mL. AgNPs combined with BER increased the expression of Bcl-2 while decreasing the ratio of Bax/Bcl-2 in SCC-25 cells. Silver particles at low doses therefore reduce the proliferation and viability of oral squamous cell carcinoma cells. SCC-25 cells are susceptible to damage from AgNPs-induced stress, which can be regulated by the natural alkaloid berberine, suggesting that nanoparticles may be potentially used in a chemoprevention/chemotherapy by augmentation of action of standard anti-cancer drugs.

Cellular Uptake and Photo-Cytotoxicity of a Gadolinium(III)-DOTA-Naphthalimide Complex "Clicked" to a Lipidated Tat Peptide.

A new bifunctional macrocyclic chelator featuring a conjugatable alkynyl-naphthalimide fluorophore pendant group has been prepared and its Gd(III) complex coupled to a cell-penetrating lipidated azido-Tat peptide derivative using Cu(I)-catalysed “click” chemistry. The resulting fluorescent conjugate is able to enter CAL-33 tongue squamous carcinoma cells, as revealed by confocal microscopy, producing a very modest anti-proliferative effect (IC50 = 93 µM). Due to the photo-reactivity of the naphthalimide moiety, however, the conjugate’s cytotoxicity is significantly enhanced (IC50 = 16 µM) upon brief low-power UV-A irradiation.