The neddylation inhibitor MLN4924 inhibits proliferation and triggers apoptosis of oral cancer cells but not for normal cells.

Increased neddylation benefits the survival of several types of cancer cells. The inhibition of neddylation has the potential to exert anticancer effects but is rarely assessed in oral cancer cells. This study aimed to investigate the antiproliferation potential of a neddylation inhibitor MLN4924 (pevonedistat) for oral cancer cells. MLN4924 inhibited the cell viability of oral cancer cells more than that of normal oral cells (HGF-1) with 100% viability, that is, IC50 values of oral cancer cells (CAL 27, OC-2, and Ca9-22) are 1.8, 1.4, and 1.9 µM. MLN4924 caused apoptotic changes such as the subG1 accumulation, activation of annexin V, pancaspase, and caspases 3/8/9 of oral cancer cells at a greater rate than in normal oral cells. MLN4924 induced greater oxidative stress in oral cancer cells compared to normal cells by upregulating reactive oxygen species and mitochondrial superoxide and depleting the mitochondrial membrane potential and glutathione. In oral cancer cells, preferential inductions also occurred for DNA damage (γH2AX and 8-oxo-2'-deoxyguanosine). Therefore, this investigation demonstrates that MLN4924 is a potential anti-oral-cancer agent showing preferential inhibition of apoptosis and promotion of DNA damage with fewer cytotoxic effects on normal cells.

Protein phosphatase 2A modulation and connection with miRNAs and natural products.

Protein phosphatase 2A (PP2A), a heterotrimeric holoenzyme (scaffolding, catalytic, and regulatory subunits), regulates dephosphorylation for more than half of serine/threonine phosphosites and exhibits diverse cellular functions. Although several studies on natural products and miRNAs have emphasized their impacts on PP2A regulation, their connections lack systemic organization. Moreover, only part of the PP2A family has been investigated. This review focuses on the PP2A-modulating effects of natural products and miRNAs' interactions with potential PP2A targets in cancer and non-cancer cells. PP2A-modulating natural products and miRNAs were retrieved through a literature search. Utilizing the miRDB database, potential PP2A targets of these PP2A-modulating miRNAs for the whole set (17 members) of the PP2A family were retrieved. Finally, PP2A-modulating natural products and miRNAs were linked via a literature search. This review provides systemic directions for assessing natural products and miRNAs relating to the PP2A-modulating functions in cancer and disease treatments.

Demethoxymurrapanine, an indole-naphthoquinone alkaloid, inhibits the proliferation of oral cancer cells without major side effects on normal cells.

Antioral cancer drugs need a greater antiproliferative impact on cancer than on normal cells. Demethoxymurrapanine (DEMU) inhibits proliferation in several cancer cells, but an in-depth investigation was necessary. This study evaluated the proliferation-modulating effects of DEMU, focusing on oral cancer and normal cells. DEMU (0, 2, 3, and 4 µg/mL) at 48 h treatments inhibited the proliferation of oral cancer cells (the cell viability (%) for Ca9-22 cells was 100.0 ± 2.2, 75.4 ± 5.6, 26.0 ± 3.8, and 15.4 ± 1.4, and for CAL 27 cells was 100.0 ± 9.4, 77.2 ± 5.9, 57.4 ± 10.7, and 27.1 ± 1.1) more strongly than that of normal cells (the cell viability (%) for S-G cells was 100.0 ± 6.6, 91.0 ± 4.6, 95.0 ± 2.6, and 95.8 ± 5.5), although this was blocked by the antioxidant N-acetylcysteine. The presence of oxidative stress was evidenced by the increase of reactive oxygen species and mitochondrial superoxide and the downregulation of the cellular antioxidant glutathione in oral cancer cells, but these changes were minor in normal cells. DEMU also caused greater induction of the subG1 phase, extrinsic and intrinsic apoptosis (annexin V and caspases 3, 8, and 9), and DNA damage (γH2AX and 8-hydroxy-2-deoxyguanosine) in oral cancer than in normal cells. N-acetylcysteine attenuated all these DEMU-induced changes. Together, these data demonstrate the preferential antiproliferative function of DEMU in oral cancer cells, with the preferential induction of oxidative stress, apoptosis, and DNA damage in these cancer cells, and low cytotoxicity toward normal cells.

Ferroptosis-Regulated Natural Products and miRNAs and Their Potential Targeting to Ferroptosis and Exosome Biogenesis.

Ferroptosis, which comprises iron-dependent cell death, is crucial in cancer and non-cancer treatments. Exosomes, the extracellular vesicles, may deliver biomolecules to regulate disease progression. The interplay between ferroptosis and exosomes may modulate cancer development but is rarely investigated in natural product treatments and their modulating miRNAs. This review focuses on the ferroptosis-modulating effects of natural products and miRNAs concerning their participation in ferroptosis and exosome biogenesis (secretion and assembly)-related targets in cancer and non-cancer cells. Natural products and miRNAs with ferroptosis-modulating effects were retrieved and organized. Next, a literature search established the connection of a panel of ferroptosis-modulating genes to these ferroptosis-associated natural products. Moreover, ferroptosis-associated miRNAs were inputted into the miRNA database (miRDB) to bioinformatically search the potential targets for the modulation of ferroptosis and exosome biogenesis. Finally, the literature search provided a connection between ferroptosis-modulating miRNAs and natural products. Consequently, the connections from ferroptosis-miRNA-exosome biogenesis to natural product-based anticancer treatments are well-organized. This review sheds light on the research directions for integrating miRNAs and exosome biogenesis into the ferroptosis-modulating therapeutic effects of natural products on cancer and non-cancer diseases.

Modulation of AKT Pathway-Targeting miRNAs for Cancer Cell Treatment with Natural Products.

Many miRNAs are known to target the AKT serine-threonine kinase (AKT) pathway, which is critical for the regulation of several cell functions in cancer cell development. Many natural products exhibiting anticancer effects have been reported, but their connections to the AKT pathway (AKT and its effectors) and miRNAs have rarely been investigated. This review aimed to demarcate the relationship between miRNAs and the AKT pathway during the regulation of cancer cell functions by natural products. Identifying the connections between miRNAs and the AKT pathway and between miRNAs and natural products made it possible to establish an miRNA/AKT/natural product axis to facilitate a better understanding of their anticancer mechanisms. Moreover, the miRNA database (miRDB) was used to retrieve more AKT pathway-related target candidates for miRNAs. By evaluating the reported facts, the cell functions of these database-generated candidates were connected to natural products. Therefore, this review provides a comprehensive overview of the natural product/miRNA/AKT pathway in the modulation of cancer cell development.

Oxidative-Stress-Mediated ER Stress Is Involved in Regulating Manoalide-Induced Antiproliferation in Oral Cancer Cells.

Manoalide provides preferential antiproliferation of oral cancer but is non-cytotoxic to normal cells by modulating reactive oxygen species (ROS) and apoptosis. Although ROS interplays with endoplasmic reticulum (ER) stress and apoptosis, the influence of ER stress on manoalide-triggered apoptosis has not been reported. The role of ER stress in manoalide-induced preferential antiproliferation and apoptosis was assessed in this study. Manoalide induces a higher ER expansion and aggresome accumulation of oral cancer than normal cells. Generally, manoalide differentially influences higher mRNA and protein expressions of ER-stress-associated genes (PERK, IRE1α, ATF6, and BIP) in oral cancer cells than in normal cells. Subsequently, the contribution of ER stress on manoalide-treated oral cancer cells was further examined. ER stress inducer, thapsigargin, enhances the manoalide-induced antiproliferation, caspase 3/7 activation, and autophagy of oral cancer cells rather than normal cells. Moreover, N-acetylcysteine, an ROS inhibitor, reverses the responses of ER stress, aggresome formation, and the antiproliferation of oral cancer cells. Consequently, the preferential ER stress of manoalide-treated oral cancer cells is crucial for its antiproliferative effect.

Connection between Radiation-Regulating Functions of Natural Products and miRNAs Targeting Radiomodulation and Exosome Biogenesis.

Exosomes are cell-derived membranous structures primarily involved in the delivery of the payload to the recipient cells, and they play central roles in carcinogenesis and metastasis. Radiotherapy is a common cancer treatment that occasionally generates exosomal miRNA-associated modulation to regulate the therapeutic anticancer function and side effects. Combining radiotherapy and natural products may modulate the radioprotective and radiosensitizing responses of non-cancer and cancer cells, but there is a knowledge gap regarding the connection of this combined treatment with exosomal miRNAs and their downstream targets for radiation and exosome biogenesis. This review focuses on radioprotective natural products in terms of their impacts on exosomal miRNAs to target radiation-modulating and exosome biogenesis (secretion and assembly) genes. Several natural products have individually demonstrated radioprotective and miRNA-modulating effects. However, the impact of natural-product-modulated miRNAs on radiation response and exosome biogenesis remains unclear. In this review, by searching through PubMed/Google Scholar, available reports on potential functions that show radioprotection for non-cancer tissues and radiosensitization for cancer among these natural-product-modulated miRNAs were assessed. Next, by accessing the miRNA database (miRDB), the predicted targets of the radiation- and exosome biogenesis-modulating genes from the Gene Ontology database (MGI) were retrieved bioinformatically based on these miRNAs. Moreover, the target-centric analysis showed that several natural products share the same miRNAs and targets to regulate radiation response and exosome biogenesis. As a result, the miRNA-radiomodulation (radioprotection and radiosensitization)-exosome biogenesis axis in regard to natural-product-mediated radiotherapeutic effects is well organized. This review focuses on natural products and their regulating effects on miRNAs to assess the potential impacts of radiomodulation and exosome biogenesis for both the radiosensitization of cancer cells and the radioprotection of non-cancer cells.

Physapruin A Enhances DNA Damage and Inhibits DNA Repair to Suppress Oral Cancer Cell Proliferation.

The selective antiproliferation to oral cancer cells of Physalis peruviana-derived physapruin A (PHA) is rarely reported. Either drug-induced apoptosis and DNA damage or DNA repair suppression may effectively inhibit cancer cell proliferation. This study examined the selective antiproliferation ability of PHA and explored detailed mechanisms of apoptosis, DNA damage, and repair. During an ATP assay, PHA provided high cytotoxicity to two oral cancer cell lines (CAL 27 and Ca9-22) but no cytotoxicity to two non-malignant oral cells (HGF-1 and SG). This selective antiproliferation of PHA was associated with the selective generation of reactive oxygen species (ROS) in oral cancer cells rather than in non-malignant oral cells, as detected by flow cytometry. Moreover, PHA induced other oxidative stresses in oral cancer cells, such as mitochondrial superoxide generation and mitochondrial membrane potential depletion. PHA also demonstrated selective apoptosis in oral cancer cells rather than non-malignant cells in annexin V/7-aminoactinmycin D and caspase 3/7 activity assays. In flow cytometry and immunofluorescence assays, PHA induced γH2AX expressions and increased the γH2AX foci number of DNA damages in oral cancer cells. In contrast, the mRNA expressions for DNA repair signaling, including homologous recombination (HR) and non-homologous end joining (NHEJ)-associated genes, were inhibited by PHA in oral cancer cells. Moreover, the PHA-induced changes were alleviated by the oxidative stress inhibitor N-acetylcysteine. Therefore, PHA generates selective antiproliferation, oxidative stress, and apoptosis associated with DNA damage induction and DNA repair suppression in oral cancer cells.

Tribulus terrestris fruit extract inhibits autophagic flux to diminish cell proliferation and metastatic characteristics of oral cancer cells.

Elevated autophagy is highly associated with cancer development and progression. Fruit extracts of several plants inhibit activity of autophagy-related protease ATG4B and autophagy activity in colorectal cancer cells. However, the effects of these plant extracts in oral cancer cells remain unclear. In this study, we found that the extracted Tribulus terrestris fruit (TT-(fr)) and Xanthium strumarium fruit had inhibitory effects on autophagy inhibition in both SAS and TW2.6 oral cancer cells. Moreover, the fruit extracts had differential effects on cell proliferation of oral cancer cells. In addition, the fruit extracts hampered cell migration and invasion of oral cancer cells, particularly in TT-(fr) extracts. Our results indicated that TT-(fr) extracts consistently inhibited autophagic flux, cell growth and metastatic characteristics of oral cancer cells, suggesting TT-(fr) might contain function ingredient to suppress oral cancer cells.

Correction: Ou-Yang, F. et al. Antiproliferation for Breast Cancer Cells by Ethyl Acetate Extract of Nepenthes thorellii x (ventricosa x maxima). Int. J. Mol. Sci. 2019, 20, 3238.

The authors would like to make corrections to their published paper [...].

Antiproliferation for Breast Cancer Cells by Ethyl Acetate Extract of Nepenthes thorellii x (ventricosa x maxima).

Extracts from the Nepenthes plant have anti-microorganism and anti-inflammation effects. However, the anticancer effect of the Nepenthes plant is rarely reported, especially for breast cancer cells. Here, we evaluate the antitumor effects of the ethyl acetate extract of Nepenthes thorellii x (ventricosa x maxima) (EANT) against breast cancer cells. Cell viability and flow cytometric analyses were used to analyze apoptosis, oxidative stress, and DNA damage. EANT exhibits a higher antiproliferation ability to two breast cancer cell lines (MCF7 and SKBR3) as compared to normal breast cells (M10). A mechanistic study demonstrates that EANT induces apoptosis in breast cancer cells with evidence of subG1 accumulation and annexin V increment. EANT also induces glutathione (GSH) depletion, resulting in dramatic accumulations of reactive oxygen species (ROS) and mitochondrial superoxide (MitoSOX), as well as the depletion of mitochondrial membrane potential (MMP). These oxidative stresses attack DNA, respectively leading to DNA double strand breaks and oxidative DNA damage in γH2AX and 8-oxo-2'deoxyguanosine (8-oxodG) assays. Overall these findings clearly revealed that EANT induced changes were suppressed by the ROS inhibitor. In conclusion, our results have shown that the ROS-modulating natural product (EANT) has antiproliferation activity against breast cancer cells through apoptosis, oxidative stress, and DNA damage.

Clathrin-mediated endocytosis is required for ANE 30-100K-induced autophagy.

BACKGROUND: We identified an autophagy-inducing areca nut (AN) ingredient (AIAI) in the 30-100 kDa fraction of AN extract (ANE 30-100K). This study was to analyze the role of endocytosis in ANE 30-100K-induced autophagy. METHODS: We used benzyl alcohol, dynasore, and shRNA of clathrin and dynamin to assess whether ANE 30-100K-induced cytotoxicity and accumulation of microtubule-associated protein 1 light chain 3 (LC3)-II were affected in oral (OECM-1) and esophageal (CE81T/VGH) carcinoma cells. RESULTS: Both benzyl alcohol and dynasore effectively reduced ANE 30-100K-induced cytotoxicity and LC3-II accumulation in OECM-1 and CE81T/VGH cells. Downregulated protein expression of both clathrin and dynamin by their shRNA also significantly attenuated ANE 30-100K-induced elevation of LC3-II levels in CE81T/VGH cells. CONCLUSIONS: These results indicate that AIAI may be engulfed by cells through clathrin-mediated endocytosis, which promotes the execution of the following autophagy program.

iPARTS2: an improved tool for pairwise alignment of RNA tertiary structures, version 2.

Since its first release in 2010, iPARTS has become a valuable tool for globally or locally aligning two RNA 3D structures. It was implemented by a structural alphabet (SA)-based approach, which uses an SA of 23 letters to reduce RNA 3D structures into 1D sequences of SA letters and applies traditional sequence alignment to these SA-encoded sequences for determining their global or local similarity. In this version, we have re-implemented iPARTS into a new web server iPARTS2 by constructing a totally new SA, which consists of 92 elements with each carrying both information of base and backbone geometry for a representative nucleotide. This SA is significantly different from the one used in iPARTS, because the latter consists of only 23 elements with each carrying only the backbone geometry information of a representative nucleotide. Our experimental results have shown that iPARTS2 outperforms its previous version iPARTS and also achieves better accuracy than other popular tools, such as SARA, SETTER and RASS, in RNA alignment quality and function prediction. iPARTS2 takes as input two RNA 3D structures in the PDB format and outputs their global or local alignments with graphical display. iPARTS2 is now available online at http://genome.cs.nthu.edu.tw/iPARTS2/.

R3D-BLAST2: an improved search tool for similar RNA 3D substructures.

BACKGROUND: RNA molecules have been known to play a variety of significant roles in cells. In principle, the functions of RNAs are largely determined by their three-dimensional (3D) structures. As more and more RNA 3D structures are available in the Protein Data Bank (PDB), a bioinformatics tool, which is able to rapidly and accurately search the PDB database for similar RNA 3D structures or substructures, is helpful to understand the structural and functional relationships of RNAs. RESULTS: Since its first release in 2011, R3D-BLAST has become a useful tool for searching the PDB database for similar RNA 3D structures and substructures. It was implemented by a structural-alphabet (SA)-based method, which utilizes an SA with 23 structural letters to encode RNA 3D structures into one-dimensional (1D) structural sequences and applies BLAST to the resulting structural sequences for searching similar substructures of RNAs. In this study, we have upgraded R3D-BLAST to develop a new web server named R3D-BLAST2 based on a higher quality SA newly constructed from a representative and sufficiently non-redundant list of RNA 3D structures. In addition, we have modified the kernel program in R3D-BLAST2 so that it can accept an RNA structure in the mmCIF format as an input. The results of our experiments on a benchmark dataset have demonstrated that R3D-BLAST2 indeed performs very well in comparison to its earlier version R3D-BLAST and other similar tools RNA FRABASE, FASTR3D and RAG-3D by searching a larger number of RNA 3D substructures resembling those of the input RNA. CONCLUSIONS: R3D-BLAST2 is a valuable BLAST-like search tool that can more accurately scan the PDB database for similar RNA 3D substructures. It is publicly available at http://genome.cs.nthu.edu.tw/R3D-BLAST2/ .

Effect of Flat-Plane Splint Vertical Thickness on Disc Displacement Without Reduction: A Retrospective Matched-Cohort Study.

PURPOSE: The efficacy of occlusal splints for treating temporomandibular disorders (TMDs) remains controversial. This study aimed to evaluate and compare the effectiveness of flat-plane splints (FPSs) with a vertical thickness of 3 mm (VT3) and 5 mm (VT5) in treating disc displacement without reduction (DDWOR). MATERIALS AND METHODS: This retrospective matched-cohort study selected the study participants from 400 TMD patients treated in the Department of Oral and Maxillofacial Surgery of Tainan Sin-Lau Hospital, Tainan, Taiwan, between August 2013 and July 2015. The thickness of occlusal splints was the predictor variable. The outcome variables included joint crepitus sound, deviation of the mandible during opening, TMD-associated headache, myofascial pain with referral, temporomandibular joint (TMJ) arthralgia, and maximum assisted opening. The case and control groups were matched 1:1 by propensity scoring to ensure that there were no statistical differences in the categorical variables and continuous variables. The analysis used χ2 tests, t tests, and multiple regression analyses. RESULTS: We allocated 162 patients into 2 groups, with 81 patients each: VT3 group and VT5 group. Both VT3 FPSs and VT5 FPSs effectively improved the DDWOR. At 12 months after treatment, the VT5 group showed remarkable improvement in joint crepitus sound and TMJ arthralgia compared with the VT3 group. No statistical differences in other TMJ-associated symptoms such as deviation of the mandible during opening, TMD-associated headache, and myofascial pain with referral were observed between the 2 groups. CONCLUSIONS: Both VT3 and VT5 FPSs can effectively improve various clinical symptoms of DDWOR. We suggest that VT5 FPS treatment for at least 1 year is a suitable option for DDWOR patients with joint crepitus sound and TMJ arthralgia. For DDWOR patients without joint crepitus sound and TMJ arthralgia, there are no differences between the VT3 and VT5 FPSs.

Temporal relationship between dysthymia and temporomandibular disorder: a population-based matched case-control study in Taiwan.

BACKGROUND: Numerous studies have reported a relationship between depression and temporomandibular disorders (TMD), but the conclusions remain undefined. The aim of this article was to examine the temporal relationship between depression and TMD. METHODS: In this retrospective matched case-control study, we recruited all samples from a randomsample sub-dataset of one million insured individuals for the year 2005 (Longitudinal Health Insurance Database (LHID2005)). All beneficiaries were enrolled in the National Health Insurance (NHI) programme in Taiwan. We used propensity scoring and matched the case and control groups (1:1) by ten confounding factors to detect the effect of different types of depression on TMD. RESULTS: The positive correlative factors of TMD included the total number of times medical advice was sought for an unspecified anomaly of jaw size plus malocclusion (TTSMA-JS, p = 0.045), the total number of times medical advice was sought for an anxiety state (TTSMA-AS, p = 0.000), and the total number of times medical advice was sought for a panic disorder (TTSMA-P, p = 0.009). Dysthymia (synonymous with chronic depression) had an effect on TMD. The odds ratio (OR) of dysthymia for TMD measured by multiple logistic regression was 1.91 (p = 0.008) after adjusting for demographic factors, psychiatric comorbidities, and maxillofacial confounders. CONCLUSIONS: This study demonstrated the established temporal relationship between dysthymia and TMD. The inclusion of a psychiatrist on the TMD management team is appropriate.

Antiproliferation of Cryptocarya concinna-derived cryptocaryone against oral cancer cells involving apoptosis, oxidative stress, and DNA damage.

BACKGROUND: Cryptocarya-derived crude extracts and their compounds have been reported to have an antiproliferation effect on several types of cancers but their impact on oral cancer is less well understood. METHODS: We examined the cell proliferation effect and mechanism of C. concinna-derived cryptocaryone (CPC) on oral cancer cells in terms of cell viability, apoptosis, reactive oxygen species (ROS), mitochondrial depolarization, and DNA damage. RESULTS: We found that CPC dose-responsively reduced cell viability of two types of oral cancer cells (Ca9-22 and CAL 27) in MTS assay. The CPC-induced dose-responsive apoptosis effects on Ca9-22 cells were confirmed by flow cytometry-based sub-G1 accumulation, annexin V staining, and pancaspase analyses. For oral cancer Ca9-22 cells, CPC also induced oxidative stress responses in terms of ROS generation and mitochondrial depolarization. Moreover, γH2AX flow cytometry showed DNA damage in CPC-treated Ca9-22 cells. CPC-induced cell responses in terms of cell viability, apoptosis, oxidative stress, and DNA damage were rescued by N-acetylcysteine pretreatment, suggesting that oxidative stress plays an important role in CPC-induced death of oral cancer cells. CONCLUSIONS: CPC is a potential ROS-mediated natural product for anti-oral cancer therapy.

Cell-based semiquantitative assay for sulfated glycosaminoglycans facilitating the identification of chondrogenesis.

Glycosaminoglycans (GAGs), in particular chondroitin sulfate, are an accepted marker of chondrogenic cells. In this study, a cell-based sulfated GAG assay for identifying the chondrogenesis of mesenchymal stem cells was developed. Based on fluorescent staining using safranin O and 4',6-diamidino-2-phenylindole (DAPI), this method was highly sensitive. The results were both qualitative and quantitative. The method is suitable for identifying the chondrogenic process and also for screening compounds. The method may be helpful for discovering novel bioactive compounds for cartilage regeneration.

Concentration effects of grape seed extracts in anti-oral cancer cells involving differential apoptosis, oxidative stress, and DNA damage.

BACKGROUND: Grape seeds extract (GSE) is a famous health food supplement for its antioxidant property. Different concentrations of GSE may have different impacts on cellular oxidative/reduction homeostasis. Antiproliferative effect of GSE has been reported in many cancers but rarely in oral cancer. METHODS: The aim of this study is to examine the antioral cancer effects of different concentrations of GSE in terms of cell viability, apoptosis, reactive oxygen species (ROS), mitochondrial function, and DNA damage. RESULTS: High concentrations (50-400 µg/ml) of GSE dose-responsively inhibited proliferation of oral cancer Ca9-22 cells but low concentrations (1-10 µg/ml) of GSE showed a mild effect in a MTS assay. For apoptosis analyses, subG1 population and annexin V intensity in high concentrations of GSE-treated Ca9-22 cells was increased but less so at low concentrations. ROS generation and mitochondrial depolarization increased dose-responsively at high concentrations but showed minor changes at low concentrations of GSE in Ca9-22 cells. Additionally, high concentrations of GSE dose-responsively induced more γH2AX-based DNA damage than low concentrations. CONCLUSIONS: Differential concentrations of GSE may have a differentially antiproliferative function against oral cancer cells via differential apoptosis, oxidative stress and DNA damage.

Tumor-associated macrophages promote oral cancer progression through activation of the Axl signaling pathway.

BACKGROUND: Recent studies suggest that tumor-associated macrophages (TAMs) promote tumor growth and metastasis. Our previous report demonstrated that Axl signaling promotes carcinogenesis and progression of oral squamous cell carcinoma (OSCC). This study aims to test the potential involvement of growth arrest-specific gene 6 (Gas6)/Axl signaling in the protumoral effect of TAMs. METHODS: Co-culture experiments by incubation of OSCC cells (YD38 and OE) and macrophages (THP-1) were performed. The expression of Gas6/Axl and epithelial-mesenchymal transition (EMT) genes were examined in YD38 and OE cells. The effect of Gas6/Axl signaling on co-cultured cancer cells was further investigated by knocking down Axl expression and neutralizing Gas6. Axl and TAM distribution were analyzed by immunohistochemistry in OSCC tissues. RESULTS: Activation of Axl signaling and increased expression of mesenchymal markers, along with increased invasion/migration ability of OSCC cells, was noted upon co-culture with THP-1. Neutralization of Gas6 in the co-culture system or knockdown of Axl in YD38 caused the co-culture effects to be diminished. Co-culture with THP-1 increased nuclear factor (NF)-κB nuclear translocation and transcription activity in YD38 cells. A significant association between the TAM count and expression of phosphorylated Axl (P = 0.004) was found in vivo cancer tissues. CONCLUSIONS: TAMs play a protumor role in OSCC and likely promote tumor progression through activation of the Gas6/Axl-NF-κB signaling pathway. Therefore, Gas6/Axl and NF-κB signaling in OSCC cells may be a putative target for therapeutic intervention.