HSC-3 Cells Research Articles

Pengaruh ekstrak biji melinjo terhadap viabilitas dan apoptosis sel hsc-3 (Laporan Penelitian)

Background: Squamous cell carcinoma (SCC) is the most common form of oral cancer. SCC treatment generally uses surgical procedures, chemotherapy, etc. Currently there are more than 60% of anticancer compounds obtained from natural ingredients, one of which is Gnetum gnemon L. (G. gnemon L.) or commonly called melinjo. Objective: To determine whether the G. gnemon L. seed ethanolic extract with concentrations of 1, 10, and 100 µg/mL could reduce viability and induce apoptosis in the HSC-3 cell line. Methods: Laboratory experimental research (in vitro) was conducted using the cell line HSC-3. Cells were treated with G. gnemon L. seed ethanolic extract with concentrations of 1, 10, and 100 µg/mL for 24 hours. Ethanol 70% was used as solvent and negative control. Doxorubicin was used as a positive control. The treated cells were analyzed using MTT assay and flow cytometry to examine changes in cell viability and apoptosis that occurred. Results: G. gnemon L. seed ethanolic extract with concentrations of 1, 10, and 100 µg/mL could significantly reduce viability and induce apoptosis in the HSC-3 cell line (p<0.05). The ability to reduce viability increased with the increase in extract concentration as many as 8,169 cells, 5,789 cells, and 3,068 cells and also induces apoptosis by 8.33 ± 0.93%, 16.55 ± 1.51%, and 28.73 ± 0.89% of cells tested sequentially. Conclusion: G. gnemon L. seed ethanolic extract with concentrations of 1, 10, and 100 µg/mL for 24 hours was able to reduce viability and induce apoptosis in the HSC-3 cell line.

Cytotoxicity differences from roots and leaves ethanol extracts of annona muricata linn. on HSC-3

Objective: The aim of this study is to examine cytotoxicity differences between ethanol extract of A. muricata roots and leaves against HSC-3 cell line.
 Material and Methods: HSC-3 cell lines were treated with ethanol extracts of A. muricata roots and leaves with 100, 30, 10, and 1 μg/mL concentrations for 24 hours. The percentage of cell viability was measured using MTT assay.
 Results: The results showed significant differences (p <0.001) of treatments group compared to negative control. Ethanol extract of A. muricata roots showed the highest cytotoxicity against HSC-3 cell lines.
 Conclusion: There is difference in cytotoxicity between ethanol extracts of A. muricata roots and leaves against HSC-3 cell lines where root extracts showed higher cytotoxicity.

Arsenic Trioxide Triggers Apoptosis of Metastatic Oral Squamous Cells Carcinoma with Concomitant Downregulation of GLI1 in Hedgehog Signaling.

Given the lack of advances in Oral Squamous Cell Carcinoma (OSCC) therapy in recent years, pharmacological strategies to block OSCC-related signaling pathways have gained prominence. The present study aimed to evaluate the therapeutic potential of Arsenic Trioxide (ATO) concerning its antitumoral effects and the inhibition of the Hedgehog (HH) pathway in OSCC. Initially, ATO cytotoxicity was assessed in a panel of cell lines. Cell viability, cell cycle, death patterns, and cell morphology were analyzed, as well as the effect of ATO on the expression of HH pathway components. After the cytotoxic assay, HSC3 cells were chosen for all in vitro assays. ATO increased apoptotic cell death and nuclear fragmentation in the sub-G1 cell cycle phase and promoted changes in cell morphology. In addition, the reduced expression of GLI1 indicated that ATO inhibits HH activity. The present study provides evidence of ATO as an effective cytotoxic drug for oral cancer treatment in vitro.

Introducing a novel chemotherapeutic supplement prepared by silver nanoparticles green-formulated by Salvia officinalis leaf aqueous extract to treat the human oral squamous cell carcinoma

• A clear peak at the wavelength of 448 nm confirms the nanoparticles formation. • The peaks related to the aliphatic O-H and C-H stretching, C=C, C=O and Ag-O are at the wavelengths of 3507, 2913, 1369, 1665 and 462 cm -1 . • The average size of 50-90 nm was recorded. This research displays the synthesis of Silver nanoparticles (Ag NPs) by Salvia officinalis . The structural features of the synthesized nanocomposite were investigated by FT-IR, FE-SEM, UV-Vis studies. MTT assay was used on common human oral squamous cell carcinoma cell lines i.e., KB, HSC-4, HSC-3, HSC-2, and Ca9-22 to survey the cytotoxicity and anti-human oral squamous cell carcinoma effects of the synthesized nanocomposite. The synthesized nanocomposite had very low cell viability and high anti-human oral squamous cell carcinoma activities dose-dependently against KB, HSC-4, HSC-3, HSC-2, and Ca9-22 cell lines without cytotoxicity on the normal cell line (HUVEC). To determine the antioxidant properties of the synthesized nanocomposite, the DPPH test was used in the presence of butylated hydroxytoluene as the positive control. The synthesized nanocomposite inhibited half of the DPPH molecules in the concentration of 181 µg/mL. Maybe significant anti-human oral squamous cell carcinoma potentials of the synthesized nanocomposite against common human oral squamous cell carcinoma cell lines are linked to their antioxidant activities.

TEAD1 Silencing Regulates Cell Proliferation and Resistance to 5-Fluorouracil in Cutaneous Squamous Cell Carcinoma.

Cutaneous squamous cell carcinoma (cSCC) is a skin malignant tumor account for approximately one-third of all nonmelanoma skin cancers. Studies have shown that TEA domain transcription factor 1 (TEAD1) is discovered to be involved in the pathogenesis of some human cancers, but to our knowledge its role in cSCC has not been reported. Samples from 16 cSCC patients and 27 healthy individuals were obtained for immunohistochemical staining of TEAD1. The expressions of TEAD1 in SCL-1, HSC-1 cells compared with the primary neonatal human epithelial keratinocytes were detected by Western blot and RT-qPCR. Proliferation and cell cycle of TEAD1 knockdown in cSCC cell lines were examined by MTT and flow cytometry analysis. Annexin V/PI and JC-1 staining were used to determine the cell apoptosis. The expression of TEAD1 decreased significantly in cSCC compared to its expression in normal skin tissues and cell lines. Down-regulation of TEAD1 in cSCC cell lines promoted cell growth via regulation of the G2/M progression. Additionally, silence of TEAD1 also protected cells against 5-Fluorouracil-induced apoptosis and decreased the expression of apoptosis-related protein (p53). Our results suggested that TEAD1 expression is down-regulated and functioned as a tumor suppressor in cSCC and that it may serve as a biomarker or therapeutic target of cSCC.

New Perspectives on the Sustainable Employment of Chestnut Shells as Active Ingredient against Oral Mucositis: A First Screening

Oral mucositis (OM), a common side effect of oncological treatment, is an oral mucosal disorder characterized by painful ulcerations and increased risk of infection. The use of natural antioxidants to suppress the redox imbalance responsible for the OM condition has emerged as an interesting approach to prevent/treat OM. This study aims to explore the chestnut (Castana sativa) shells as potential active ingredient against OM. Therefore, chestnut shells were extracted at different temperatures (110-180 °C) by Subcritical Water Extraction (SWE), aiming to recover antioxidants. The extracts were also evaluated against microorganisms present in the oral cavity as well as on human oral cell lines (TR146 and HSC3). The highest phenolic content was obtained with the extraction temperature of 110 °C, exhibiting the best antioxidant/antiradical activities and scavenging efficiencies against HOCl (IC50 = 4.47 μg/mL) and ROO• (0.73 μmol TE/mg DW). High concentrations of phenolic acids (e.g., gallic and protocatechuic acids) and flavanoids (catechin, epicatechin and rutin) characterized the phenolic profile. The antimicrobial activity against several oral microorganisms present in the oral cavity during OM, such as Streptococcus, Staphylococcus, Enterococcus, and Escherichia, was demonstrated. Finally, the effects on HSC3 and TR146 cell lines revealed that the extract prepared at 110 °C had the lowest IC50 (1325.03 and 468.15 µg/mL, respectively). This study highlights the potential effects of chestnut shells on OM.

Response Surface Methodology (RSM) Powered Formulation Development, Optimization and Evaluation of Thiolated Based Mucoadhesive Nanocrystals for Local Delivery of Simvastatin.

In oral administration systems, mucoadhesive polymers are crucial for drug localization and target-specific activities. The current work focuses on the application of thiolated xanthan gum (TXG) to develop and characterize a novel mucoadhesive nanocrystal (NC) system of simvastatin (SIM). Preparation of SIM-NC was optimized using response surface methodology (RSM) coupled with statistical applications. The concentration of Pluronic F-127 and vacuum pressure were optimized by central composite design. Based on this desirable approach, the prerequisites of the optimum formulation can be achieved by a formulation having 92.568 mg of F-127 and 77.85 mbar vacuum pressure to result in EE of 88.8747% and PS of 0.137.835 nm. An optimized formulation was prepared with the above conditions along with xanthan gum (XG) and TXG and various parameters were evaluated. A formulation containing TXG showed 98.25% of SIM at the end of 96 h. Regarding the mucoadhesion potential evaluated by measuring zeta potential, TXG-SIM-NC shoed the maximum zeta potential of 16,455.8 ± 869 mV at the end of 6 h. The cell viability percentage of TXG-SIM-NC (52.54 ± 3.4% with concentration of 50 µg/mL) was less than the plain SIM, with XG-SIM-NC showing the highest cytotoxicity on HSC-3 cells. In vivo pharmacokinetic studies confirm the enhanced bioavailability of formulated mucoadhesive systems of SIM-NC, with TXG-SIM-NC exhibiting the maximum.

An Innovative PTD-IVT-mRNA Delivery Platform for CAR Immunotherapy of ErbB(+) Solid Tumor Neoplastic Cells.

Chimeric antigen receptor (CAR) immunotherapy includes the genetic modification of immune cells to carry such a receptor and, thus, recognize cancer cell surface antigens. Viral transfection is currently the preferred method, but it carries the risk of off-target mutagenicity. Other transfection platforms have thus been proposed, such the in vitro transcribed (IVT)-mRNAs. In this study, we exploited our innovative, patented delivery platform to produce protein transduction domain (PTD)-IVT-mRNAs for the expression of CAR on NK-92 cells. CAR T1E-engineered NK-92 cells, harboring the sequence of T1E single-chain fragment variant (scFv) to recognize the ErbB receptor, bearing either CD28 or 4-1BB as co-stimulatory signaling domains, were prepared and assessed for their effectiveness in two different ErbB(+) cancer cell lines. Our results showed that the PTD-IVT-mRNA of CAR was safely transduced and expressed into NK-92 cells. CAR T1E-engineered NK-92 cells provoked high levels of cell death (25-33%) as effector cells against both HSC-3 (oral squamous carcinoma) and MCF-7 (breast metastatic adenocarcinoma) human cells in the co-incubation assays. In conclusion, the application of our novel PTD-IVT-mRNA delivery platform to NK-92 cells gave promising results towards future CAR immunotherapy approaches.

CLEFMA Induces the Apoptosis of Oral Squamous Carcinoma Cells through the Regulation of the P38/HO-1 Signalling Pathway.

The purpose of this research was to evaluate the impact and the underlying molecular mechanism of CLEFMA-induced cell death in human OSCC. The anti-tumour properties of CLEFMA in oral cancer were explored using colony formation, flow cytometry, human apoptosis array, Western blot, and immunohistochemistry assays. The in vivo anti-tumour effect of CLEFMA administered by oral gavage was evaluated using SCC-9-derived xenograft-bearing nude mouse models. CLEFMA significantly suppressed colony formation and elicited cellular apoptosis in oral cancer cells. CLEFMA treatment remarkably increased phosphorylated p38 and HO-1 along with cleavage of poly ADP-ribose polymerase and activation of caspase-8, -9, and -3 in HSC-3 and SCC-9 cells. Administration of HO-1 small interfering RNA significantly protected the cells from CLEFMA-induced caspase-3, -8, and -9 activation. Attenuation of p38 activity by the pharmacologic inhibitor SB203580 dramatically reduced CLEFMA-induced caspase-3, -8, and -9 activation and HO-1 expression in OSCC. The subcutaneous murine xenograft models showed that CLEFMA in vivo suppressed tumour growth in implanted SCC-9 cells. All of these findings indicated that CLEFMA induced apoptosis through the p38-dependent rise in HO-1 signal transduction cascades in OSCC.

TWIST1 regulates proliferation, migration, and invasion and is a prognostic marker for oral tongue squamous cell carcinoma.

Epithelial-mesenchymal transition is one of the main mechanisms for tumor progression and metastasis. Transcription factors such as TWIST1 are key regulators of the epithelial-mesenchymal transition and are regarded as potential therapeutic targets for the treatment of cancer. The purpose of this study was to examine TWIST1 as a possible epithelial-mesenchymal transition-related prognostic biomarker in oral epithelial dysplasia and oral tongue squamous cell carcinomas, as well as the biological behavior of TWIST1-silencing in oral tongue squamous cell carcinomas cell lines. Immunohistochemical analysis of TWIST1, E-cadherin, and N-cadherin was carried out in 47 samples representing oral epithelial dysplasia and 41 oral tongue squamous cell carcinomas. The suppression of TWIST1 expression was performed using shRNA-expression vectors in HSC-3 and SCC-9 cells to investigate in vitro the impact of TWIST1 on proliferation, apoptosis, viability, migration, and invasion of SCC-9 and HSC-3 cells. The expression of nuclear TWIST1 was significantly higher in oral tongue squamous cell carcinomas than in oral epithelial dysplasis (p < 0.0001), whereas TWIST1 in the cytoplasm was more expressed in oral epithelial dysplasis (p=0.012). The high cytoplasmic expression of TWIST1 was significantly associated with shortened overall survival (p < 0.05), and increased nuclear TWIST1 expression was significantly related to high risk of recurrence (p=0.03). Knockdown of TWIST1 in oral tongue squamous cell carcinomas cells induced the expression of E-cadherin and inhibited N-cadherin, which were followed by decreased proliferation, migration, and invasion. Our research suggests that TWIST1 is linked to the development of oral tongue carcinogenesis and may be used as a prognostic indicator and therapeutic target for oral tongue squamous cell carcinomas patients.

Cigarette smoking induces the activation of RIP2/caspase-12/NF-κB axis in oral squamous cell carcinoma.

Cigarette smoking is one of the major risk factors for the occurrence and progression of oral squamous cell carcinoma (OSCC). Receptor-interacting protein 2 (RIP2) has been involved in mucosal immunity and homeostasis via a positive regulation of nuclear factor κB (NF-κB) transcription factor activity. Caspase-12 can bind to RIP2 and dampen mucosal immunity. However, the roles of RIP2/NF-κB and caspase-12 in OSCC induced by cigarette smoking remain unknown. Herein, we investigated the effects of cigarette smoking on the RIP2/NF-κB and caspase-12 in human OSCC tissues and OSCC cell lines (HSC-3). We first observed that RIP2 mediated NF-κB activation and caspase-12 upregulation in OSCC patients with cigarette smoking and cigarette smoke extract (CSE)-treated HSC-3 cells, respectively. Moreover, we confirmed that the downregulation of RIP2 by siRNA resulted in the reduction of caspase-12 expression and NF-κB activity in the presence of CSE treatment in vitro. In summary, our results indicated that cigarette smoking induced the activation of the RIP2/caspase-12/NF-κB axis and it played an important role in the development of OSCC. The RIP2/caspase-12/NF-κB axis could be a target for OSCC prevention and treatment in the future.

1-[4-(2-Dimethylaminoethoxy)phenylcarbonyl]-3,5-Bis(3,4,5-Trimethoxybenzylidene)- 4-Piperidone Hydrochloride and Related Compounds: Potent Cytotoxins Demonstrate Greater Toxicity to Neoplasms than Non- Malignant Cells.

The incidence of cancer has been increasing worldwide. Unfortunately, the drugs used in cancer chemotherapy are toxic to both neoplasms and normal tissues, while many available medications have low potencies. Conjugated α,β-unsaturated ketones differ structurally from contemporary anticancer medications , some of which have noteworthy antineoplastic properties. This study aimed to design and synthesize highly potent cytotoxins with far greater toxicity to neoplasms than to non-malignant cells. A series of N-acyl-3,5-bis(benzylidene)-4-piperidone hydrochlorides 4a-n were prepared and evaluated against Ca9-22, HSC-2, HSC-3, and HSC-4 squamous cell carcinomas as well as against HGF, HPLF, and HPC non-malignant cells. QSAR and western blot analyses were performed. The majority of compounds display submicromolar CC50 values towards the neoplasms; the figures for some of the compounds are below 10-7 M. In general, 4a-n have much lower CC50 values than those of melphalan, 5-fluorouracil, and methotrexate, while some compounds are equitoxic with doxorubicin. The compounds are far less toxic to the non-malignant cells, giving rise to substantial selectivity index (SI) figures. A QSAR study revealed that both potency and the SI data were controlled to a large extent by the electronic properties of the substituents in the arylidene aryl rings. Two representative compounds, 4f and 4g, caused apoptosis in HSC-2 cells. The compounds in series 4 are potent cytotoxins displaying tumor-selective toxicity. In particular, 4g with an average CC50 value of 0.04 μM towards four malignant cell lines and a selectivity index of 46.3 is clearly a lead molecule that should be further evaluated.

Pd nanoparticles supported over agar-agar functionalized Fe3O4 as a biodegradable composite nanomedicine for the in-vitro anti-human oral cancer application

In recent years, magnetic bio-nanocomposites have been evolved as potential new generation materials. Consequently, we demonstrate herein a novel Pd NPs adorned agar-agar modified magnetic Fe3O4 nanoparticles as a novel anticancer agent (Fe3O4@agar-agar/Pd). The post-synthetically modified material was meticulously characterized through several analytical techniques, such as Fourier Transformed Infra Red (FT-IR) spectroscopy, scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), energy dispersive X-ray analysis (EDX), elemental mapping, transmission electron microscopy (TEM), vibrating-sample magnetometer (VSM), X-ray diffraction analysis (XRD), and inductively coupled plasma-atomic emission spectrometry (ICP-AES) analysis. The as synthesized Fe3O4@agar-agar/Pd bio-nanocomposite was investigated for antioxidant and human anti-oral cancer assays. In such studies a number of cell lines, viz., HSC-3, HSC-2, and Ca9-22 were used in determining the cytotoxicity. Notably, Fe3O4@agar-agar/Pd bio-nanocomposite exhibit significant anti-oral cancer properties against HSC-3, HSC-2, and Ca9-22 cancer cell lines following time and dose-dependent manner. The corresponding IC50 values were determined as 195, 187, and 239 μg/mL respectively. DPPH radical scavenging method was used to determine the antioxidant activity of Fe3O4@agar-agar/Pd bio-nanocomposite. The significant IC50 value suggested the material having very good antioxidant potential. The anti-human oral cancer effect of our material is believed to be due to its antioxidant effects.

Peptide-DNA origami as a cryoprotectant for cell preservation.

Cryopreservation of cells is essential for the conservation and cold chain of bioproducts and cell-based medicines. Here, we demonstrate that self-assembled DNA origami nanostructures have a substantial ability to protect cells undergoing freeze-thaw cycles; thereby, they can be used as cryoprotectant agents, because their nanoscale morphology and ice-philicity are tailored. In particular, a single-layered DNA origami nanopatch functionalized with antifreezing threonine peptides enabled the viability of HSC-3 cells to reach 56% after 1 month of cryopreservation, surpassing dimethyl sulfoxide, which produced 38% viability. It also exhibited minimal dependence on the cryopreservation period and freezing conditions. We attribute this outcome to the fact that the peptide-functionalized DNA nanopatches exert multisite actions for the retardation of ice growth in both intra- and extracellular regions and the protection of cell membranes during cryopreservation. This discovery is expected to deepen our fundamental understanding of cell survival under freezing environment and affect current cryopreservation technologies.

ABCG2, CD44 and SOX9 are increased with the acquisition of drug resistance and involved in cancer stem cell activities in head and neck squamous cell carcinoma cells.

Cancer stem cells are a sub-population of cancer cells with self-renewal activity that play key roles in tumor resistance to chemotherapy and radiation. Several cancer stem cell markers have been identified to correlate with clinical prognosis. However, which marker is associated with which cancer stem cell characteristic is unclear. The present study aimed to clarify the relationship between cancer stem cell markers associated with drug resistance acquisition and the characteristics of cancer stem cells. We generated cisplatin-resistant head and neck squamous cell carcinoma cells by culturing cells in increasing concentrations of cisplatin. The cisplatin-resistant head and neck squamous cell carcinoma cells also acquired multidrug resistance and were named resistant HSC-3 (R HSC-3) cells. R HSC-3 showed no differences in cell proliferation or cell cycle distributions compared with parental cells. R HSC-3 cells showed increased drug excretion ability and elevated expression of ATP-binding cassette subfamily G member 2 (ABCG2), a drug excretion pump. R HSC-3 cells also highly expressed CD44, a cancer stem cell marker, and exhibited enhanced cell invasion and spheroid formation abilities. Furthermore, the stem cell-related factor SRY-box transcription factor 9 (SOX9) was identified as increased in R HSC-3 cells by microarray analysis. Knockdown experiments showed that SOX9 and ABCG2 were involved in the drug excretion ability of R HSC3 cells and ABCG2 was involved in the spheroid formation ability of R HSC-3 cells. These results indicate that CD44, SOX9 and ABCG2 expression levels were enhanced in head and neck squamous cell carcinoma cells that acquired multidrug resistance and that these molecules are important for maintaining cancer stem cell characteristics. Overall, regulating CD44, SOX9 and ABCG2 may be a strategy to inhibit cancer stem cells.

Bis(chloroacetamidino)-Derived Heteroarene-Fused Anthraquinones Bind to and Cause Proteasomal Degradation of tNOX, Leading to c-Flip Downregulation and Apoptosis in Oral Cancer Cells.

Simple SummaryNew-generation anthraquinone derivatives attached with different heterocycles and bearing chloroacetamidines in the side chains have been synthesized to reduce side effects and drug resistance. In this study, we identified the cellular target of the studied compounds through ligand binding assays and in silico simulations. Our results illustrate that the studied compounds bound to and targeted the tumor-associated NADH oxidase (tNOX) in oral cancer cells. tNOX is a growth-related protein and is found to be expressed in cancer cells but not in non-transformed cells, and its knockdown by RNA interference in tumor cells overturns cancer phenotypes, supporting its role in cellular growth. We also identified that tNOX bound to the studied compounds and underwent degradation, which was correlated with apoptosis induction in oral cancer cells.Anthraquinone-based intercalating compounds, namely doxorubicin and mitoxantrone, have been used clinically based on their capacity to bind DNA and induce DNA damage. However, their applications have been limited by side effects and drug resistance. New-generation anthraquinone derivatives fused with different heterocycles have been chemically synthesized and screened for higher anticancer potency. Among the compounds reported in our previous study, 4,11-bis(2-(2-chloroacetamidine)ethylamino)anthra[2,3-b]thiophene-5,10-dione dihydrochloride (designated 2c) was found to be apoptotic, but the direct cellular target responsible for the cytotoxicity remained unknown. Here, we report the synthesis and anticancer properties of two other derivatives, 4,11-bis(2-(2-chloroacetamidine)ethylamino)naphtho[2,3-f]indole-5,10-dione dihydrochloride (2a) and 4,11-bis(2-(2-chloroacetamidine)ethylamino)-2-methylanthra[2,3-b]furan-5,10-dione dihydrochloride (2b). We sought to identify and validate the protein target(s) of these derivatives in oral cancer cells, using molecular docking simulations and cellular thermal shift assays (CETSA). Our CETSA results illustrate that these derivatives targeted the tumor-associated NADH oxidase (tNOX, ENOX2), and their direct binding downregulated tNOX in p53-functional SAS and p53-mutated HSC-3 cells. Interestingly, the compounds targeted and downregulated tNOX to reduce SIRT1 deacetylase activity and increase Ku70 acetylation, which triggers c-Flip ubiquitination and induces apoptosis in oral cancer cells. Together, our data highlight the potential value of these heteroarene-fused anthraquinones in managing cancer by targeting tNOX and augmenting apoptosis.

FUT6 inhibits the proliferation, migration, invasion, and EGF-induced EMT of head and neck squamous cell carcinoma (HNSCC) by regulating EGFR/ERK/STAT signaling pathway.

Glycosylation change is one of the landmark events of tumor occurrence and development, and tumor cells may be inhibited by regulating the aberrant expression of glycosyltransferases. Currently, fucosyltransferase VI (FUT6), which is involved in the synthesis of α-1, 3 fucosyl bond, has been detected to be closely associated with multiple tumors, but its function and mechanism in head and neck squamous cell carcinoma (HNSCC) still need further research. In this study, FUT6 knockdown and overexpression strategies were used to investigate the effects of FUT6 on cell proliferation, migration, and invasion, as well as the growth and metastasis of HNSCC in a xenografts mouse model. The protein expression levels of epidermal growth factor receptor (EGFR), extracellular signal-regulated kinase (ERK), Signal Transducer and Activator of Transcription (STAT), protein kinase B (AKT), c-Myc, and epithelial-mesenchymal transition (EMT) markers were determined by western blot analysis. Our research found that the mRNA expression of FUT6 was lower in HNSCC tissues than in normal mucosal epithelial tissues. In Cal-27 and FaDu cells, FUT6 overexpression inhibited cell proliferation, migration and invasion, causing upregulation of ZO-1 and E-cadherin, downregulation of N-cadherin and Vimentin, and finally decreased the phosphorylation levels of EGFR, ERK, STAT, and c-Myc. In HSC-3 cells, knockdown of FUT6 promoted cell proliferation, migration and invasion, downregulating ZO-1 and E-cadherin, upregulating N-cadherin and Vimentin, and increased the phosphorylation levels of EGFR, ERK, STAT, and c-Myc. In the HNSCC xenografts mouse, FUT6 overexpression inhibited tumor growth and metastasis. In summary, FUT6 controls the proliferation, migration, invasion, and EGF-induced EMT of HNSCC by regulating EGFR/ERK/STAT signaling pathway, indicating its potential future therapeutic application for HNSCC.

Proof-of-Principle Study Suggesting Potential Anti-Inflammatory Activity of Butyrate and Propionate in Periodontal Cells.

Short-chain fatty acids (SCFAs) are potent immune modulators present in the gingival crevicular fluid. It is therefore likely that SCFAs exert a role in periodontal health and disease. To better understand how SCFAs can module inflammation, we screened acetic acid, propionic acid, and butyric acid for their potential ability to lower the inflammatory response of macrophages, gingival fibroblasts, and oral epithelial cells in vitro. To this end, RAW 264.7 and primary macrophages were exposed to LPSs from Porphyromonas gingivalis (P. gingivalis) with and without the SCFAs. Moreover, gingival fibroblasts and HSC2 oral epithelial cells were exposed to IL1β and TNFα with and without the SCFAs. We report here that butyrate was effective in reducing the lipopolysaccharide (LPS)-induced expression of IL6 and chemokine (C-X-C motif) ligand 2 (CXCL2) in the RAW 264.7 and primary macrophages. Butyrate also reduced the IL1β and TNFα-induced expression of IL8, chemokine (C-X-C motif) ligand 1 (CXCL1), and CXCL2 in gingival fibroblasts. Likewise, butyrate lowered the induced expression of CXCL1 and CXCL2, but not IL8, in HSC2 cells. Butyrate further caused a reduction of p65 nuclear translocation in RAW 264.7 macrophages, gingival fibroblasts, and HSC2 cells. Propionate and acetate partially lowered the inflammatory response in vitro but did not reach the level of significance. These findings suggest that not only macrophages, but also gingival fibroblasts and oral epithelial cells are susceptive to the anti-inflammatory activity of butyrate.

P53 status modifies cytotoxic activity of lactoferrin under hypoxic conditions.

Lactoferrin (LF) is an iron binding glycoprotein of the transferrin family with a wide spectrum of biological effects, including anti-cancer activity. However, the detailed molecular mechanisms of anti-cancer activity of LF have not been fully determined. In this study, we tried to clarify cytotoxic functions of LF on various cell lines under hypoxic conditions and elucidate those molecular mechanisms. Cytotoxic activity of LF on cell lines was found to have a range of sensitivities. Hypoxia decreased sensitivity to LF in KD (lip fibroblast) but increased that in HSC2 (oral squamous cell carcinoma). Expression analyses further revealed that LF treatments increased hypoxic HIF-1α, -2α and p53 proteins in KD but attenuated them in HSC2 cells, and decreased HIF-1 target gene, DEC2, in KD but increased it in HSC2, suggesting a possible relationship between LF-modified DEC2 expression and HIF-α protein. MTT assay strikingly demonstrated that cells expressing mutant-type p53 (MT5) were more sensitive to LF than control HepG2 (hepatoma), suggesting an important role of the p53 signal. Knock-down of TP53 (p53 gene) interestingly reduced sensitivity to LF in HepG2, suggesting that p53 may be a target of LF cytotoxic activity. Further analyses with a ferroptosis promoter or inhibitor demonstrated that LF increased ACSL4 in hypoxic MT5, suggesting LF-induced ferroptosis in cells expressing mutant-type p53. In conclusion, hypoxia was found to regulate cytotoxic activities of LF differently among various cell lines, possibly through the p53 signaling pathway. LF further appeared to regulate ferroptosis through a modification of ACSL4 expression.

The marine natural product mimic MPM-1 is cytolytic and induces DAMP release from human cancer cell lines

Bioprospecting contributes to the discovery of new molecules with anticancer properties. Compounds with cytolytic activity and the ability to induce immunogenic cell death can be administered as intratumoral injections with the aim to activate anti-tumor immune responses by causing the release of tumor antigens as well as damage-associated molecular patterns (DAMPs) from dying cancer cells. In the present study, we report the cytolytic and DAMP-releasing effects of a new natural product mimic termed MPM-1 that was inspired by the marine Eusynstyelamides. We found that MPM-1 rapidly killed cancer cells in vitro by inducing a necrosis-like death, which was accompanied by lysosomal swelling and perturbation of autophagy in HSC-3 (human oral squamous cell carcinoma) cells. MPM-1 also induced release of the DAMPs adenosine triphosphate (ATP) and high mobility group box 1 (HMGB1) from Ramos (B-cell lymphoma) and HSC-3 cells, as well as cell surface expression of calreticulin in HSC-3 cells. This indicates that MPM-1 has the ability to induce immunogenic cell death, further suggesting that it may have potential as a novel anticancer compound.