EMT-related transcription factors and protein stabilization mechanisms involvement in cadherin switch of head and neck squamous cell carcinoma.

Epithelial to mesenchymal transition (EMT) describes a process where epithelial tumor cells acquire mesenchymal characteristics. EMT often correlates with invasion and an increased cell migration potential by losing cellular polarity and cell-cell junctions. It is mainly induced by tumor-microenvironment factors, such as TGF-beta 1 and IL-6, which activate the increased expression of the EMT-transcription factor (TF) Slug. We previously reported the Slug/Krüppel-like factor 4 (KLF4) switch in EMT in HNSCC, and found, that in human papilloma virus (HPV)-negative HNSCC Slug gene expression was significant higher represented, than in HPV-positive HNSCC. The purpose of this study was to investigate the impact of KLF4 and Slug on the regulation of the cadherin switch and on the EMT phenotype. Gene expression of KLF4 positive correlated with E-cadherin in 71 head and neck squamous cell carcinoma (HNSCC) patient tissue samples, which we also confirmed by the investigation of the Cancer Genome Atlas database (TCGA). HPV-transcripts contributed to stabilization of KLF4 at protein level, and simultaneously upregulated E-cadherin. Furthermore, ectopic KLF4 overexpression was associated with epithelial gene expression by induction of E-cadherin, ß-catenin and 70-kDa heat shock protein (HSP-70). The presence of HSP-70 ensures the membranous localization of E-cadherin, therefore, the ability of cells to form cadherin/catenin complexes and cellular linkages. In conclusion, KLF4 is a major regulator of the epithelial cadherin-adhesion in HNSCC.

New Polymethoxyflavones from Hottonia palustris Evoke DNA Biosynthesis-Inhibitory Activity in An Oral Squamous Carcinoma (SCC-25) Cell Line.

Four new compounds, 5-hydroxy-2',6'-dimethoxyflavone (4), 5-hydroxy-2',3',6'-trimethoxyflavone (5), 5-dihydroxy-6-methoxyflavone (6), and 5,6'-dihydroxy-2',3'-dimethoxyflavone (7), and three known compounds, 1,3-diphenylpropane-1,3-dione (1), 5-hydroxyflavone (2), and 5-hydroxy-2'-methoxyflavone (3), were isolated from the aerial parts of Hottonia palustris. Their chemical structures were determined through the use of spectral, spectroscopic and crystallographic methods. The quantitative analysis of the compounds (1-7) and the zapotin (ZAP) in methanol (HP1), petroleum (HP6), and two chloroform extracts (HP7 and HP8) were also determined using HPLC-PDA. The biological activity of these compounds and extracts on the oral squamous carcinoma cell (SCC-25) line was investigated by considering their cytotoxic effects using the MTT assay. Subsequently, the most active compounds and extracts were assessed for their effect on DNA biosynthesis. It was found that all tested samples during 48 h treatment of SCC-25 cells induced the DNA biosynthesis-inhibitory activity: compound 1 (IC50, 29.10 ± 1.45 µM), compound 7 (IC50, 40.60 ± 1.65 µM) and extracts ZAP (IC50, 20.33 ± 1.01 µM), HP6 (IC50, 14.90 ± 0.74 µg), HP7 (IC50, 16.70 ± 0.83 µg), and HP1 (IC50, 30.30 ± 1.15 µg). The data suggest that the novel polymethoxyflavones isolated from Hottonia palustris evoke potent DNA biosynthesis inhibitory activity that may be considered in further studies on experimental pharmacotherapy of oral squamous cell carcinoma.

Electrochemical Assessment of Anticancer Compounds on the Human Tongue Squamous Carcinoma Cells.

The most common oral cancer is squamous cell carcinoma (SCC) and its highest occurrence is in the tongue. Almost 30% of patients with one primary head and neck tumor will have a second primary malignancy. In recent studies, two novel plant extracts, andrographolide and cannabidiol (CBD), have been exploited for their anticancer effects. Here, we investigated the cytotoxic effects of these two compounds on SCC-25 cells, a human tongue squamous carcinoma cell line, and compared the outcomes with two chemotherapeutic drugs, cisplatin and fluorouracil. Electric cell substrate impedance sensing (ECIS) system was applied to measure frequency- and time-dependent impedance of SCC-25 cell-covered electrodes and to further assess subtle changes in cell morphology and micromotion in response to different concentrations (0, 10, 30, 100, and 300 µM) of these compounds. AlamarBlue and Annexin V/7-AAD binding assays were used to measure the concentration dependent changes in viability and apoptosis of SCC-25 cells. Our results demonstrate that 24 hours after exposure to 30 µM CBD can significantly decrease the micromotion rate, damage the integrity of cell morphology, reduce cell viability, and induce higher apoptosis in treated SCC-25 cells, while the other three drugs attain similar effects at the concentration of 100 µM or higher. The apoptosis-induced changes in cell morphology and micromotion monitored by ECIS correlate well with biochemical assays. Thus, both frequency- and time-dependent impedance measurements using ECIS can be used to real-time follow cancer cell activities in response to anticancer drugs with different temporal cytotoxicity profiles.

MG53 Inhibits the Progression of Tongue Cancer Cells through Regulating PI3K-AKT Signaling Pathway: Evidence from 3D Cell Culture and Animal Model.

MG53 is transcriptionally activated by the IRS-1/PI3K/AKT signal pathway, which is closely related with oncogenesis of several tumors. Here, the role of MG53 in the tumorigenesis of tongue cancer is analyzed in vitro and in vivo. The stable MG53 overexpression/knockdown SCC9 and SCC25 cells are constructed through retrovirus infection. Then a PLGA cylinder is used to provide a 3D culture environment for cell growth. Cell counting results suggest that overexpression of MG53 inhibits the cell proliferation and colony formation of SCC9 and SCC25 cells. While knockdown of MG53 has the opposite effect. Furthermore, knockdown of MG53 significantly promotes the invasion of SCC9 and SCC25 cells. Western blotting data confirm that MG53 affects the expression of the AKT signaling pathway. In a xenograft assay, knockdown of MG53 promotes the growth of xenograft which is induced by SCC25 cells in nude mice. The findings demonstrate that MG53 affects the biological behavior of human tongue cancer SCC9 and SCC25 cells.

Biological and molecular effects of bromodomain and extra-terminal (BET) inhibitors JQ1, IBET-151, and IBET-762 in OSCC cells.

BACKGROUND: Despite improvements in oral squamous cell carcinoma (OSCC) management, survival rates remain relatively low and novel anti-neoplastic agents are needed. Bromodomain and extra-terminal (BET) inhibitors proved to be promising agents for cancer treatment. We investigated the effects of three BET inhibitors (JQ1, IBET-151, IBET-762) on SCC-25 cell line and primary oral cancer cell culture. METHODS: Cell viability was evaluated by MTT. Protein levels of MCM5 and cleaved-PARP were estimated by Western blot. Clonogenic and migratory abilities were determined by colony forming and scratch assays. BET inhibitors effects on mRNA levels of E-Cadherin, Vimentin, SNAI1, SNAI2, CLU, SERPINI1, MCM5, c-Myc, E2F, IL7R, and PPARg were analyzed by qPCR. RESULTS: BET inhibitors significantly reduced oral cancer cell viability. JQ1 showed the greatest effect reducing cell viability to 10%, both in SCC-25 and primary OSCC cultures (P < 0.001), compared to control cells. Cells treated with BET inhibitors displayed a reduction to 50% in colony forming capacity compared to control cells (P < 0.0001) and the colonies were smaller; they also had a 50%-60% reduction in migratory capacity (P < 0.05) compared to untreated cells. BET inhibitors had a significant impact on genes related to epithelial to mesenchymal transition and other cancer cell markers, notably on MCM5, a gene related to cell cycle control. CONCLUSIONS: BET inhibitors induce both OSCC cell death and reduction of tumor aggressiveness. Molecular mechanisms of BET inhibition involve among others, MCM5 downregulation. Importantly, this study demonstrates for the first time the anti-tumoral effect of IBET-151 and IBET-762 in oral cancer.

E3 ubiquitin ligase, RNF139, inhibits the progression of tongue cancer.

BACKGROUND: Tongue cancer is still one of the leading causes of mortality around the world. Recently, the ubiquitin system has been established as a critical modulator of tumors. In order to find the oral cancer related E3 ubiquitin ligases, we screened the human E3 ubiquitin ligase library and found that RING finger protein 139 (RNF139) regulated the biological behavior of tongue cancer cells. METHODS: MTT assay was used to analyze the cell viability changes of tongue cancer SCC9 and SCC25 cells caused by RNF139. The invasion ability of SCC9 and SCC25 cells with or without the knockdown of RNF139 was evaluated through transwell assay. The immunoblotting was recruited to determine the expression level of RNF139 in human tongue cancer tissues and para-carcinoma tissues. The effect of RNF139 on tumorigenicity of tongue cancer cells was analyzed by xenograft model on immunodeficient Balb/c nude mice. RESULTS: Overexpression of RNF139 inhibits the viability of tongue cancer cells since day 2. The colony formation ability of SCC9 and SCC25 cells was also decreased with the overexpression of RNF139. Knockdown of RNF139 significantly promoted the invasion ability of SCC9 and SCC25 cells. Furthermore, knockdown of RNF139 also induced the activation of AKT signaling pathway. While human tongue cancer tissues had low expression of RNF139. In nude mice, knockdown of RNF139 promoted the tumorigenicity of the SCC25 cells. CONCLUSIONS: Our data establish a role for RNF139 in regulating the progression of tongue cancer.

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.

Sphingosine kinase 1 mediates head & neck squamous cell carcinoma invasion through sphingosine 1-phosphate receptor 1.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is characterized by aggressive loco-regional invasion. Sphingosine kinase1 (SphK1), an enzyme in sphingolipid metabolism, is emerging as a key player in HNSCC pathology. The observation that SphK1 is overexpressed in all HNSCC stages and is associated with depth of tumor invasion, metastasis and clinical failure underscores the importance of SphK1 in HNSCC pathology. Still, the mechanisms underlying SphK1 regulation of invasion have not been delineated. Therefore, we sought to mechanistically describe how SphK1 regulates invasion in HNSCC. METHODS: Invasion assays were used to measure invasive ability of SphK1 overexpressing human tongue squamous cell carcinoma (SCC-25 cells). Western blotting, quantitative qPCR, ELISA and zymography were used to measure the effect of SphK1 and sphingosine 1-phoshate receptor 1 (S1P1) on invasion measures, MMP-2/9, E-cadherin, EGFR, IL-6/STAT3, in SCC-25 cells. RESULTS: SphK1 expression is elevated in cells with an invasive phenotype as compared to non-invasive phenotype. We show SphK1 overexpression increased EGF-induced EGFR/ERK and AKT activity, increased matrix metalloproteinase (MMP)-2/9 mRNA and reduced E-cadherin. SphK1 overexpression also increased IL-6 concentration and EGF-induced STAT3 phosphorylation, exemplifying that SphK1 modulates IL-6/STAT3 signaling. Notably, we show that S1P1 knockdown reduced IL-6/STAT3 signaling, representing another pathway by which SphK1/S1P regulates invasion. CONCLUSIONS: Taken together, our data suggest that SphK1 sits at the hub of multiple key signaling cascades, all which have been implicated in the regulation of invasiveness, making SphK1 an attractive target for the development of HNSCC therapies.

Interplay between Partial EMT and Cisplatin Resistance as the Drivers for Recurrence in HNSCC.

This study aims to investigate the role of partial epithelial to mesenchymal transition (pEMT)-related proteins in modulating Cisplatin resistance in head and neck squamous cell carcinoma (HNSCC). SCC-25 cells were pre-treated with TGF-beta1 followed by transient Krüppel-like Factor 4 (KLF4)-overexpression and Cisplatin treatment. Cell growth, cell morphological changes and cell migration were assessed using Juli BR live cell video-microscopy. In addition, Ki-67 and Slug immunostaining and follow-up image cytometric analysis of primary and recurrent HNSCC tumors were performed to evaluate the proliferation index (PI) and the EMT-like phenotype. We observed that proliferating and Slug-positive tumor cells expand after therapy in HNSCC. Subsequently, protein analysis revealed the stabilization of Slug, upregulation of Vimentin and phospho-p38 (p-p38) in Cisplatin-resistant SCC-25 cells. Moreover, KLF4-overexpression contributed to Cisplatin sensitivity by reduction of Slug at the protein level. This work strongly suggests that an pEMT-like pathway is activated in recurrent and Cisplatin-resistant HNSCC. Finally, stable KLF4-overexpression might sensitize HNSCC tumor cells for Cisplatin treatment.

KLF4, Slug and EMT in Head and Neck Squamous Cell Carcinoma.

Epithelial to mesenchymal transition (EMT) is clinically relevant in head and neck squamous cell carcinoma (HNSCC). We hypothesized that EMT-transcription factors (EMT-TFs) and an anti-EMT factor, Krüppel-like-factor-4 (KLF4) regulate EMT in HNSCC. Ten control mucosa and 37 HNSCC tissue samples and three HNSCC cell lines were included for investigation of EMT-TFs, KLF4 and vimentin at mRNA and protein levels. Slug gene expression was significantly higher, whereas, KLF4 gene expression was significantly lower in HNSCC than in normal mucosa. In the majority of HNSCC samples, there was a significant negative correlation between KLF4 and Slug gene expression. Slug gene expression was significantly higher in human papilloma virus (HPV) negative HNSCC, and in tumor samples with irregular p53 gene sequence. Transforming-growth-factor-beta-1 (TGF- ß1) contributed to downregulation of KLF4 and upregulation of Slug. Two possible regulatory pathways could be suggested: (1) EMT-factors induced pathway, where TGF-ß1 induced Slug together with vimentin, and KLF4 was down regulated at the same time; (2) p53 mutations contributed to upregulation and stabilization of Slug, where also KLF4 could co-exist with EMT-TFs.

Anticancer effects induced by artichoke extract in oral squamous carcinoma cell lines.

BACKGROUND: Oral squamous cell carcinoma is occupying the eighth position of all malignant neoplasia worldwide. Nowadays, natural compounds found in vegetables and fruits are important resources of many anticancer drugs especially those with high levels of phytochemicals representing an efficient strategy for cancer prevention and treatment. Artichoke (Cynara cardunculus L.) is a kind of antioxidant-rich vegetables demonstrated a potential anticancer activity on various types of cancer cells related to its content of phenolic compounds. Anticarcinogenic effects of polyphenolic extracts were reported to cause a reduction in cell viability, inhibition of cell growth, and initiation of apoptotic mechanisms. The present study aimed to investigate the cell cycle arrest, cytotoxic, and apoptotic effects of artichoke extract against the invasive oral squamous cell carcinoma. RESULTS: A pure extract from the edible part and leaves of fresh artichoke was added to oral squamous carcinoma cell lines and to control group to evaluate the expression of caspase-9, Bcl-2, and Bax genes. Artichoke extract demonstrated the highest cytotoxic effect against cancer cell lines which increased in a time-dependent manner. No apparent effects were observed in the normal control group. Expression of Bax and caspase-9 genes revealed a highly significant increase in cancer cell lines (p = 0.0001) when compared to the control group. In addition to a highly significant decrease (p = 0.005) in Bcl-2 of cancer cells. It was demonstrated that artichoke extract induced cell growth arrest at G2/M phase which revealed a significant increase (p < 0.05) in comparison to the untreated control group. CONCLUSION: Artichoke exerts potent cell cycle arrest, cytotoxic, and apoptotic effects on oral squamous carcinoma cell lines.

Effects of Resveratrol, Berberine and Their Combinations on Reactive Oxygen Species, Survival and Apoptosis in Human Squamous Carcinoma (SCC-25) Cells.

BACKGROUND: Levels of cellular Reactive Oxygen Species (ROS) influence the oxidized/reduced states of cellular proteins, and create redox-signaling pathways that can activate transcription factors, kinases, and phosphatases. ROS levels can be increased radically by external factors, including ionizing and UV radiation or exposure to chemical compounds. These increased ROS levels can, in turn, lead to oxidative damage of DNA. Natural plant treatments against cancer can modulate these processes by inducing or decreasing ROS production. METHODS: Here we report new observations that squamous carcinoma (SCC-25) cells, exposed to 24 hours of combined resveratrol and berberine treatment, contain increased ROS levels. Using flow cytometry, for drug activity characteristics, an accumulation of ROS was observed. A combination of different dyes, CellROX Green (Life Technologies) and DCFH-DA (Sigma), allowed for flow cytometric estimation of levels of cellular ROS as well as cellular localization. RESULTS: Live staining and microscopic observations confirmed the accumulation of ROS in SCC-25 cells following a combination treatment at concentrations of 10µg/ml. Additionally, the cytotoxicity of the compounds was significantly improved after their combined application. Additive effects were observed for doses lower than the calculated IC50 of berberine [IC50=23µg/ml] and resveratrol [IC50=9µg/ml]. Viability (MTS) assays and analysis of isobolograms revealed a significant impact on cell viability upon combination treatment. CONCLUSION: These results suggest that administration of berberine, in the presence of resveratrol, could be decreased even to 50% (half the IC50 for berberine) for cancer treatment.

Interaction between MALAT-1, CCR7 and correlated genes in oral squamous cell carcinoma.

OBJECTIVE: This study focuses on the feasible molecular mechanism of the interaction between MALAT-1, CCR7 and related genes in oral squamous cell carcinoma, to find new target molecules that can block the lymph node metastasis. METHODS: The expression of MALAT-1, miRNA-320s, SRSF1, YB-1 and CCR7 were detected in T3/T4-phase OSCC tissues of two groups with or without lymph node metastasis using real-time qPCR. CO-IP and western blot to test the interaction of RNAs (MALAT-1, miRNA-320s) with SRSF1 protein or YB-1 were evaluated by CO-IP, Western blot and real-time qPCR. The expression change of chemokine receptor CCR7 were investigated using CO-IP, Western blot and real-time qPCR after silencing miRNA-320d (one of the miRNA-320s family members) by transfection of miRNA mimics to explore related signaling pathway. RESULTS: The expression levels of MALAT-1 SRSF1 and CCR7 in OSCC tissues with were differentially higher compared with those of samples without lymph node metastasis as well as para-carcinoma tissues, exclusive of miRNA-320d. Moreover, it is confirmed that the target RNA (MALAT-1, miRNA-320s) and SRSF1 protein can combine with each other, based on the statistically significant difference compared with negative control group (P<0.05). In addition, the expression of CCR7 was higher than the negative control group after silencing miRNA-320d. CONCLUSION: SRSF1 is likely to mediate the interactive relationship between MALAT-1 and miRNA-320d. CCR7 expression can be distinctly increased by silencing miRNA-320d. The effect of long-chain non-coding RNA MALAT-1 on chemokine receptor CCR7 and possibly further influence on lymph node metastasis of oral squamous cell carcinoma are revealed in molecular level to offer help for prevention and treatment of OSCC in future.

The E3 ubiquitin ligase RNF135 regulates the tumorigenesis activity of tongue cancer SCC25 cells.

Several E3 ubiquitin ligases have been confirmed that they are related to the tumorigenesis. This study aims to find the tongue cancer-related E3 ubiquitin ligase. The E3 ubiquitin ligase library was screened. The effect of candidate molecule on tongue cancer was validated through cell viability, cell proliferation, colony formation, invasive assay in vitro, and the xenograft model in vivo. The E3 ubiquitin ligase RNF135 significantly promoted the expression of PTEN and TP53 in SCC25 cells. The overexpression of RNF135 inhibited the viability, proliferation, and invasion of SCC25 cells. Knockdown of RNF135 had the opposite effects. Furthermore, RNF135 regulates the tumorigenesis activity of SCC25 cells in vivo. Our results demonstrated that RNF135 had the potential to affect the development of the tongue cancer in vitro. The further in vivo study is helpful to fully understand the role of it.