Mapping of individual sensory nerve axons from digits to spinal cord with the transparent embedding solvent system.

Achieving uniform optical resolution for a large tissue sample is a major challenge for deep imaging. For conventional tissue clearing methods, loss of resolution and quality in deep regions is inevitable due to limited transparency. Here we describe the Transparent Embedding Solvent System (TESOS) method, which combines tissue clearing, transparent embedding, sectioning and block-face imaging. We used TESOS to acquire volumetric images of uniform resolution for an adult mouse whole-body sample. The TESOS method is highly versatile and can be combined with different microscopy systems to achieve uniformly high resolution. With a light sheet microscope, we imaged the whole body of an adult mouse, including skin, at a uniform 0.8 × 0.8 × 3.5 µm3 voxel resolution within 120 h. With a confocal microscope and a 40×/1.3 numerical aperture objective, we achieved a uniform sub-micron resolution in the whole sample to reveal a complete projection of individual nerve axons within the central or peripheral nervous system. Furthermore, TESOS allowed the first mesoscale connectome mapping of individual sensory neuron axons spanning 5 cm from adult mouse digits to the spinal cord at a uniform sub-micron resolution.

PDGF-BB accelerates TSCC via fibroblast lactates limiting miR-26a-5p and boosting mitophagy.

The tumor microenvironment and cancer-associated fibroblasts (CAFs) play crucial roles in tumor development, and their metabolic coupling remains unclear. Clinical data showed a positive correlation between PDGF-BB, CAFs, and glycolysis in the tumor microenvironment of oral tongue squamous cell carcinoma patients. In vitro, CAFs are derived from hOMF cells treated with PDGF-BB, which induces their formation and promotes aerobic glycolysis. Mitophagy increased the PDGF-BB-induced formation of CAF phenotypes and aerobic glycolysis, while autophagy inhibition blocked PDGF-BB-induced effects. Downregulation of miR-26a-5p was observed in CAFs; upregulation of miR-26a-5p inhibited the expression of mitophagy-related proteins ULKI, Parkin, PINK1, and LC3 and aerobic glycolysis in PDGF-BB-induced CAFs. PDGF-BB-induced CAFs promoted tumor cell proliferation, invasion, metastasis, NF-κB signaling pathway activation, and PDGF-BB secretion. Thus, PDGF-BB is associated with lactate-induced CAF formation and glucose metabolism reprogramming. These findings indicate potential therapeutic targets in oral tongue squamous cell carcinoma.

DTL promotes head and neck squamous cell carcinoma progression by mediating the degradation of ARGLU1 to regulate the Notch signaling pathway.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, with a high incidence in squamous epithelium. The E3 ubiquitin ligase DTL is a component of the CRL4A complex and is widely involved in tumor progression. We aimed to analyze the role of DTL in HNSCC and to explore its mechanism of action. Through clinical analysis, we found that DTL is upregulated in HNSCC tissues and is associated with the tumor microenvironment and poor survival in patients. Through gain-of-function and loss-of-function assays, we showed that DTL promotes cell proliferation and migration in vitro and tumor growth in vivo. Mass spectrometry analysis and immunoprecipitation assays showed that DTL interacts with ARGLU1 to promote K11-linked ubiquitination-mediated degradation of ARGLU1, thereby promoting the activation of the CSL-dependent Notch signaling pathway. Furthermore, siARGLU1 blocks the inhibitory effects of DTL knockdown on HNSCC cells. In this study, we showed that DTL promotes HNSCC progression through K11-linked ubiquitination of ARGLU1 to activate the CSL-dependent Notch pathway. These findings identify a promising therapeutic target for HNSCC.

MiR-3529-3p from PDGF-BB-induced cancer-associated fibroblast-derived exosomes promotes the malignancy of oral squamous cell carcinoma.

AIMS: This study aims to explore the role of exosomes from cancer-associated fibroblasts (CAFs) induced by PDGF-BB in promoting the malignancy of oral squamous cell carcinoma (OSCC) and provide new insight into the mechanism of OSCC progression and its treatment. MAIN METHODS: Exosomes were extracted from human oral mucosa fibroblasts (hOMFs) and CAFs. Differentially expressed miRNAs of exosomes between hOMFs and CAFs were analysed using high-throughput sequencing and self-programmed R software. Cal-27, a human tongue squamous carcinoma cell line, was treated with exosomes. Differentially expressed miRNAs between clinical cancer tissues and adjacent tissues and between hOMF and CAF exosomes were verified by qRT‒PCR. The effect of miR-3529-3p on Cal-27 cells was clarified by overexpressing or knocking down miR-3529-3p in Cal-27 cells. Sample expression and differentially expressed miRNA expression were compared between cancer and paracarcinoma tissues. KEY FINDINGS: We found that exosomes from CAFs (CAF-Exos) were internalized by tongue squamous carcinoma cells and promoted their proliferation, migration, invasion, and antiapoptotic effects. MiR-3529-3p was a significant differentially expressed miRNA between CAF-Exos and exosomes from hOMFs (hOMF-Exos). The overexpression of miR-3529-3p promoted proliferation, migration, and invasion and inhibited apoptosis of Cal-27 cells. SIGNIFICANCE: This study explores the role of PDGF-BB-induced CAFs in promoting malignancy in OSCC. This study will provide new insight into the mechanism of OSCC progression and its treatment.

miR-10a induces inflammatory responses in epileptic hippocampal neurons of rats via PI3K/Akt/mTOR signaling pathway.

Epilepsy is a common chronic neurological disorder worldwide. MicroRNAs (miRNAs) play an important role in the pathogenesis of epilepsy. However, the mechanism of the regulatory effect of miR-10a on epilepsy is unclear. In this study, we investigated the effect of miR-10a expression on the PI3K/Akt/mTOR signaling pathway and inflammatory cytokines in epileptic hippocampal neurons of rats. The miRNA differential expression profile of rat epileptic brain was analyzed using bioinformatic approaches. Neonatal Sprague-Dawley rat hippocampal neurons were prepared as epileptic neuron models in vitro by replacing culture medium with magnesium-free extracellular solution. The hippocampal neurons were transfected with miR-10a mimics, and transcript levels of miR-10a, PI3K, Akt and mTOR were detected by quantitative reverse transcription-PCR, and PI3K, mTOR, Akt, TNF-α, IL-1ß, IL-6 protein expression levels were detected by Western blot. Cytokines secretory levels were detected by ELISA. Sixty up-regulated miRNAs were identified in the hippocampal tissue of epileptic rats and might affect the PI3K-Akt signaling pathway. In the epileptic hippocampal neurons model, the expression levels of miR-10a were significantly increased, with decreasing levels of PI3K, Akt and mTOR, and increasing levels of TNF-α, IL-1ß and IL-6. The miR-10a mimics promoted the expression of TNF-α, IL-1ß and IL-6. Meanwhile, miR-10a inhibitor activated PI3K/Akt/mTOR pathway and inhibited cytokines secretion. Finally, cytokine secretion was increased by treated with PI3K inhibitor and miR-10a inhibitor. The miR-10a may promote inflammatory responses in rat hippocampal neurons by inhibiting the PI3K/Akt/mTOR pathway, suggesting that miR-10a may be one of the target therapeutic molecules for epilepsy treatment.

Analysis of mRNA-miRNA interaction network reveals the role of CAFs-derived exosomes in the immune regulation of oral squamous cell carcinoma.

BACKGROUND: Cancer-associated fibroblasts (CAFs) have significant tumor regulatory functions, and CAFs-derived exosomes (CAFs-Exo) released from CAFs play an important role in the progression of oral squamous cell carcinoma (OSCC). However, a lack of comprehensive molecular biological analysis leaves the regulatory mechanisms of CAFs-Exo in OSCC unclear. METHODS: We used platelet derived growth factor-BB (PDGF-BB) to induce the transformation of human oral mucosa fibroblast (hOMF) into CAFs, and extracted exosomes from the supernatant of CAFs and hOMF. We validated the effect of CAFs-Exo on tumor progression by exosomes co-culture with Cal-27 and tumor-forming in nude mice. The cellular and exosomal transcriptomes were sequenced, and immune regulatory genes were screened and validated using mRNA-miRNA interaction network analysis in combination with publicly available databases. RESULTS: The results showed that CAFs-Exo had a stronger ability to promote OSCC proliferation and was associated with immunosuppression. We discovered that the presence of immune-related genes in CAFs-Exo may regulate the expression of PIGR, CD81, UACA, and PTTG1IP in Cal-27 by analyzing CAFs-Exo sequencing data and publicly available TCGA data. This may account for the ability of CAFs-Exo to exert immunomodulation and promote OSCC proliferation. CONCLUSIONS: CAFs-Exo was found to be involved in tumor immune regulation through hsa-miR-139-5p, ACTR2 and EIF6, while PIGR, CD81, UACA and PTTG1IP may be potentially effective targets for the treatment of OSCC in the future.

Identifying M1-like macrophage related genes for prognosis prediction in lung adenocarcinoma based on a gene co-expression network.

Macrophages are one of the most important players in the tumor microenvironment. But the contribution of macrophages to lung adenocarcinoma (LUAD) is still controversial. The current study aimed to display an immune landscape to clarify the function of macrophages and detect prognostic hub genes in LUAD. The transcriptome data were adopted to screen differently expressed genes (DEGs) in The Cancer Genome Atlas database (TCGA). The cell type identification by estimating relative subsets of RNA transcripts (CIBERSORT) algorithm was used to reveal the immune landscape. Weighted gene co-expression network analysis (WGCNA) analysis was performed to identify the hub module associated with macrophages. Function Enrichment analysis was conducted on hub module genes. Moreover, univariate and multivariate Cox regression analyses were performed to identify prognostic hub genes. Kaplan-Meier (KM) and Time-dependent receiver operating characteristic (ROC) curves were plotted to assess the prognostic capacity of the four prognostic hub genes. The GES1196959 dataset from the Gene Expression Omnibus (GEO) database was downloaded to verify the differential expression of the 4 prognostic hub genes.

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

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

SAMHD1 as a prognostic and predictive biomarker in stage II colorectal cancer: A multicenter cohort study.

Background: The identification of high-risk population patients is key to the personalized treatment options for the stage II colorectal cancers. The use of proteomics in the prognosis of patients with stage II colorectal cancer remains unclear. Methods: Using quantitative proteomics, we analyzed proteins that are differentially expressed in the tumor and adjacent normal tissues of 11 paired colorectal cancer patients with and without recurrence selected by a nested case-control design. Of the 21 identified proteins, we selected one candidate protein. The association of the corresponding gene of the selected protein with overall survival (OS) and adjuvant chemotherapy was analyzed using two independent cohorts of patients with stages II colorectal cancer. Results: Sterile α motif and histidine-aspartate domain-containing protein 1 (SAMHD1) was selected as the candidate biomarker. A group of 124 patients (12.5%) were stratified into SAMHD1-high subgroup. The 5-year OS rate of SAMHD1-high patients was lower than that of SAMHD1-low patients with stage II colorectal cancer (discovery cohort: hazard ratio [HR] = 2.89, 95% confidence interval [CI], 1.17-7.18, P = 0.016; validation cohort: HR = 2.25, 95% CI, 1.17-4.34, P = 0.013). The Cox multivariate analysis yielded similar results. In a pooled database, the 5-year OS rate was significantly different between patients with and without adjuvant chemotherapy among stage II SAMHD1-low tumors than in patients with stage II SAMHD1-high tumors (88% vs. 77%, P = 0.032). Conclusions: SAMHD1-high expression could help in identifying patients with stage II colorectal cancer with poor prognosis and less benefit from adjuvant chemotherapy.

[Pilose antler polypeptide CNT14 promoted proliferation and migration of human oral mucosa fibroblast cells].

PURPOSE: To explore the effect of pilose antler polypeptides CNT14 on proliferation and migration of human oral mucosa fibroblast (hOMF) cells and the related molecular mechanism. METHODS: The biosafety of pilose antler polypeptides CNT14 on hOMF cells was verified by live-dead cell staining kit.CCK-8 assay was used to detect the effect of pilose antler polypeptides CNT14 on hOMF cell proliferation. The effect of pilose antler polypeptides CNT14 on hOMF cell migration was detected by scratch test. Western blot was used to detect the expression of α-SMA, TGF-ß1, Smad2 and p-Smad2 proteins in hOMF cells stimulated by pilose antler polypeptides CNT14. The effect of Smad2 inhibitors on fibroblast activation induced by pilose antler polypeptides CNT14 was evaluated.The model of keratinized gingival defect was established in New Zealand white rabbits, and the regenerated gingival tissue was stained with H-E. The expression levels of α-SMA, TGF-ß1, Smad2 and p-Smad2 proteins in the gingival tissues of regenerated New Zealand white rabbits were detected by immunohistochemistry, and the ability of pilose antler polypeptides CNT14 to promote regeneration of oral gingival tissues was verified. Statistical analysis was performed with SPSS 20.0 software package. RESULTS: The survival rate of hOMF cells was above 95% after treated with pilose antler polypeptides CNT14. After stimulation of hOMF cells with pilose antler polypeptides CNT14, the proliferation and migration rates of hOMF cells were increased compared with the control group (P＜0.05). The expression of α-SMA, TGF-ß1, Smad2 and p-Smad2 proteins in hOMF cells stimulated by pilose antler peptide CNT14 was increased, and the difference was statistically significant(P＜0.05). The expression of α-SMA in fibroblasts induced by Smad2 inhibitor was decreased. In animal experiments, H-E staining showed that the inflammatory response of oral mucosal wounds of New Zealand white rabbits treated with CNT14 was less than that of the control group. Immunohistochemical staining results showed that the expressions of α-SMA, TGF-ß1, Smad2 and p-Smad2 in the regenerated gingival tissues of New Zealand white rabbits treated with CNT14 were significantly increased compared with those in the control group on the 9th and 11th days within the gingival wounds(P＜0.05). CONCLUSIONS: Pilose antler polypeptides CNT14 has good biosafety and can promote the proliferation and migration of human oral mucosa fibroblast cells, and the expression levels of α-SMA, TGF-ß1, Smad2 and p-Smad2 were increased, promoting the regeneration of gingival tissues.

PDGF-BB regulates the transformation of fibroblasts into cancer-associated fibroblasts via the lncRNA LURAP1L-AS1/LURAP1L/IKK/IκB/NF-κB signaling pathway.

The most abundant cells in the tumor microenvironment are cancer-associated fibroblasts (CAFs). They play an important role in oral squamous cell carcinoma (OSCC) angiogenesis, invasion and metastasis. Platelet-derived growth factor (PDGF)-BB has an obvious regulating effect on the formation of CAFs through binding to PDGF receptor (PDGFR)-ß, but the role of long non-coding (lnc)RNA in PDGF-BB-induced transformation of fibroblasts into CAFs remains poorly understood. Using an lncRNA ChIP, 370 lncRNA transcripts were identified to be significantly and differentially expressed between fibroblasts and PDGF-BB-induced fibroblasts, including 240 upregulated lncRNAs and 130 downregulated lncRNAs, indicating that lncRNAs are involved in the regulation of the transformation of CAFs. Previous studies have shown that the nuclear factor (NF)-κB signaling pathway plays an important role in the activation of CAFs. Dual-luciferase reporter assay and co-immunoprecipitation were conducted to confirm that the leucine-rich adaptor protein 1-like (LURAP1L), which is the target of lncRNA LURAP1L antisense RNA 1 (LURAP1L-AS1) had a positive regulatory effect on I-κB kinase (IKK)/NF-κB signaling. Therefore, LURAP1L-AS1 was selected and PDGF-BB was demonstrated to upregulate the expression of LURAP1L-AS1 and LURAP1L, which was reversed by a PDGFR-ß inhibitor. Subsequently, knocking down LURAP1L-AS1 suppressed the expression of PDGF-BB-induced fibroblast activation marker protein α-smooth muscle actin, fibroblast activation protein-α, PDGFR-ß and phosphorylated (p)-PDGFR-ß. IKKα, p-IĸB and p-NF-κB were downregulated by the knockdown of LURAP1L-AS1 and upregulated by overexpression of LURAP1L-AS1. The present study indicates that LURAP1L-AS1/LURAP1L/IKK/IĸB/NF-κB plays an important regulatory role in PDGF-BB-induced fibroblast activation and may become a potential target for the treatment of OSCC.

[Corrigendum] Epithelial mesenchymal transition induced by the CXCL9/CXCR3 axis through AKT activation promotes invasion and metastasis in tongue squamous cell carcinoma.

Subsequently to the publication of this paper, an interested reader drew to the authors' attention that two pairs of data panels containing strikingly similar data were featured in Fig. 4A and B. The authors have re­examined their data and realized that Fig. 4 was assembled incorrectly. The revised version of Fig. 4, containing the correct data for Fig. 4A and B, is shown on the next page. The authors regret the errors that were made in the preparation of the published figure, and confirm that these errors did not seriously affect the conclusions reported in the paper. The authors are grateful to the editor of Oncology Reports for allowing them the opportunity to publish a Corrigendum, and all the authors agree to this Corrigendum. Furthermore, they apologise to the readership for any inconvenience caused. [the original article was published in Oncology Reports 39: 1356-1368, 2018; DOI: 10.3892/or.2017.6169].

Longitudinal characteristics of lymphocyte responses and cytokine profiles in the peripheral blood of SARS-CoV-2 infected patients.

BACKGROUND: The dynamic changes of lymphocyte subsets and cytokines profiles of patients with novel coronavirus disease (COVID-19) and their correlation with the disease severity remain unclear. METHODS: Peripheral blood samples were longitudinally collected from 40 confirmed COVID-19 patients and examined for lymphocyte subsets by flow cytometry and cytokine profiles by specific immunoassays. FINDINGS: Of the 40 COVID-19 patients enrolled, 13 severe cases showed significant and sustained decreases in lymphocyte counts [0·6 (0·6-0·8)] but increases in neutrophil counts [4·7 (3·6-5·8)] than 27 mild cases [1.1 (0·8-1·4); 2·0 (1·5-2·9)]. Further analysis demonstrated significant decreases in the counts of T cells, especially CD8+ T cells, as well as increases in IL-6, IL-10, IL-2 and IFN-γ levels in the peripheral blood in the severe cases compared to those in the mild cases. T cell counts and cytokine levels in severe COVID-19 patients who survived the disease gradually recovered at later time points to levels that were comparable to those of the mild cases. Moreover, the neutrophil-to-lymphocyte ratio (NLR) (AUC=0·93) and neutrophil-to-CD8+ T cell ratio (N8R) (AUC =0·94) were identified as powerful prognostic factors affecting the prognosis for severe COVID-19. INTERPRETATION: The degree of lymphopenia and a proinflammatory cytokine storm is higher in severe COVID-19 patients than in mild cases, and is associated with the disease severity. N8R and NLR may serve as a useful prognostic factor for early identification of severe COVID-19 cases. FUNDING: The National Natural Science Foundation of China, the National Science and Technology Major Project, the Health Commission of Hubei Province, Huazhong University of Science and Technology, and the Medical Faculty of the University of Duisburg-Essen and Stiftung Universitaetsmedizin, Hospital Essen, Germany.

Comprehensive immunogenomic landscape analysis of prognosis-related genes in head and neck cancer.

Head and neck cancer is the sixth most common malignancy around the world, and 90% of cases are squamous cell carcinomas. In this study, we performed a systematic investigation of the immunogenomic landscape to identify prognostic biomarkers for head and neck squamous cell carcinoma (HNSCC). We analyzed the expression profiles of immune-related genes (IRGs) and clinical characteristics by interrogating RNA-seq data from 527 HNSCC patients in the cancer genome atlas (TCGA) dataset, including 41 HPV+ and 486 HPV- samples. We found that differentially expressed immune genes were closely associated with patient prognosis in HNSCC by comparing the differences in gene expression between cancer and normal samples and performing survival analysis. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to annotate the biological functions of the differentially expressed immunogenomic prognosis-related genes. Two additional cohorts from the Oncomine database were used for validation. 65, 56 differentially expressed IRGs was associated with clinical prognosis in total and HPV- samples, respectively. Furthermore, we extracted 10, 11 prognosis-related IRGs from 65, 56 differentially expressed IRGs, respectively. They were significantly correlated with clinical prognosis and used to construct the prognosis prediction models. The multivariable ROC curves (specifically, the AUC) were used to measure the accuracy of the prognostic models. These genes were mainly enriched in several gene ontology (GO) terms related to immunocyte migration and receptor and ligand activity. KEGG pathway analysis revealed enrichment of pathways related to cytokine-cytokine receptor interactions, which are primarily involved in biological processes. In addition, we identified 63 differentially expressed transcription factors (TFs) from 4784 differentially expressed genes, and 16 edges involving 18 nodes were formed in the regulatory network between differentially expressed TFs and the high-risk survival-associated IRGs. B cell and CD4 T cell infiltration levels were significantly negatively correlated with the expression of prognosis-related immune genes regardless of HPV status. In conclusion, this comprehensive analysis identified the prognostic IRGs as potential biomarkers, and the model generated in this study may enable an accurate prediction of survival.

Identification of cancer hallmark-associated gene and lncRNA cooperative regulation pairs and dictate lncRNA roles in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is the most common malignant tumour in the oral and maxillofacial region. Numerous cancers share ten common traits ("hallmarks") that govern the transformation of normal cells into cancer cells. Long non-coding RNAs (lncRNAs) are important factors that contribute to tumorigenesis. However, very little is known about the cooperative relationships between lncRNAs and cancer hallmark-associated genes in OSCC. Through integrative analysis of cancer hallmarks, somatic mutations, copy number variants (CNVs) and expression, some OSCC-specific cancer hallmark-associated genes and lncRNAs are identified. A computational framework to identify gene and lncRNA cooperative regulation pairs (GLCRPs) associated with different cancer hallmarks is developed based on the co-expression and co-occurrence of mutations. The distinct and common features of ten cancer hallmarks based on GLCRPs are characterized in OSCC. Cancer hallmark insensitivity to antigrowth signals and self-sufficiency in growth signals are shared by most GLCRPs in OSCC. Some key GLCRPs participate in many cancer hallmarks in OSCC. Cancer hallmark-associated GLCRP networks have complex patterns and specific functions in OSCC. Specially, some key GLCRPs are associated with the prognosis of OSCC patients. In summary, we generate a comprehensive landscape of cancer hallmark-associated GLCRPs that can act as a starting point for future functional explorations, the identification of biomarkers and lncRNA-based targeted therapy in OSCC.

The HSP90/Akt pathway may mediate artemether-induced apoptosis of Cal27 cells.

Tongue squamous cell carcinoma is the most common malignant tumor in oral and maxillofacial regions. Recent research has found that artemether can inhibit growth and induce apoptosis of cancer cells, although the mechanism is not clear. The present study aimed to explore the correlation between the HSP90/Akt pathway and artemether-induced apoptosis of Cal27 cells. A cell counting kit-8 and flow cytometry were used to detect the proliferation and apoptosis of Cal27 cells, respectively, mRNA expression was examined by quantitative RT-PCR, and protein expression was detected by western blotting. Our data revealed that artemether can inhibit growth and induce apoptosis of Cal27 cells. As the artemether concentration was increased, we observed downregulation of the expression of HSP90, p-Akt and p-mTOR in Cal27 cells, whereas the expression of Akt was not significantly changed. We also observed a time-dependent decrease in the expression of HSP90, p-Akt and p-mTOR during exposure to 0.1 mg·mL-1 artemether. In conclusion, the HSP90/Akt pathway may be involved in artemether-induced apoptosis of Cal27 cells.

Epithelial mesenchymal transition induced by the CXCL9/CXCR3 axis through AKT activation promotes invasion and metastasis in tongue squamous cell carcinoma.

The present study aimed to assess the induction of epithelial-mesenchymal transition (EMT), invasion, and metastasis by the chemokine CXCL9/receptor CXCR3 axis in tongue squamous cell carcinoma (TSCC), unveiling the underlying mechanisms and providing new insights into the prevention and treatment of oral cancer metastasis. The expression levels of CXCL9 and CXCR3 in TSCC tissue specimens were determined by immunohistochemistry, assessing differences between samples with cervical lymph node metastasis and those without. Moreover, protein expression or activity in the TSCC Cal-27 cell line was controlled by neutralizing antibodies, gene transfection, or knock-out. Then, alterations of cell proliferation, migration, invasion, and the cytoskeleton were analyzed by CCK-8, cell scratch, Transwell, and cyto-skeleton staining assays, respectively. Alterations of EMT markers (E-cadherin and vimentin) in Cal-27 cells were detected by immunofluorescence and western blotting. In addition, western blotting was utilized to detect protein expression levels of Akt2, p-Akt2, eIF4E and p-eIF4E, and to explore the regulatory roles and mechanisms of the CXCL9/CXCR3 axis in invasion and metastasis. Significantly increased expression levels of CXCL9 and CXCR3 were detected in tissue specimens with lymph node metastasis compared with those without (P<0.01). Overexpression of CXCL9/CXCR3 in Cal-27 cells resulted in cytoskeleton alterations, decreased E-cadherin expression, increased vimentin levels, enhanced migration and invasion (P<0.05), and increased phosphorylated Akt2 and eIF4E levels (P<0.05). These results revealed that in TSCC, the CXCL9/CXCR3 axis could activate the Akt signaling pathway, with EMT and cytoskeleton rearrangement, promoting invasion and metastasis.

Identification of proteins interacting with protein kinase C-δ in hyperthermia-induced apoptosis and thermotolerance of Tca8113 cells.

The purpose of the present study was to investigate the differential proteins that interact with protein kinase C­Î´ (PKC­Î´) in hyperthermia­induced apoptosis as well as thermotolerance in Tca8113 cells, and furthermore, to investigate the mechanisms of these processes in tumor cells. Activation of PKC­Î´ was previously indicated to be involved in the heat sensitivity and thermal resistance of tongue squamous carcinoma cells. Tca8113 cell apoptosis was induced by incubation at 43˚C for 80 min and the thermotolerant Tca8113 cells (TT­Tca8113) were generated through a gradient temperature­elevating method. The apoptotic rate of the cells was determined by flow cytometry, while cleavage and activation of PKC­Î´ were analyzed by western blot analysis. The proteins that interacted with PKC­Î´ in the Tca8113 and TT­Tca8113 cells were identified by co­immunoprecipitation coupled with mass spectrometry. Co­immunoprecipitation analysis followed by electrospray ionization mass spectrometric analysis were utilized to identify the pro­ and anti­apoptotic proteins that interacted with PKC­Î´. Significant cell apoptosis was observed in Tca8113 cells following hyperthermia, and the apoptotic rate was significantly higher than that in the control group (P<0.05). Marked PKC­Î´ cleavage fragmentation was also identified. By contrast, the apoptotic rate of the TT­Tca8113 cells was not significantly increased following hyperthermia and no PKC­Î´ cleavage fragmentation was observed. Among the proteins interacting with PKC­Î´, 39 were found to be involved in the promotion of apoptosis and 16 in the inhibition of apoptosis of Tca8113 cells; these proteins were known to be involved in the regulation of cell proliferation, apoptosis, transcription and intracellular protein transport. The results of the present study provided evidence that PKC­Î´ is a crucial factor in the heat sensitivity and thermal resistance of tongue squamous carcinoma cells and elucidated the underlying molecular basis, which may aid in the improvement of hyperthermic cancer treatments.

Hyperthermia inhibited the migration of tongue squamous cell carcinoma through TWIST2.

BACKGROUND: Hyperthermia has been shown promising in the treatment of head and neck squamous cell carcinoma (HNSCC); however, the mechanism underlying hyperthermia reducing tumor metastasis is poorly elucidated. TWIST2, an important transcription factor of epithelial-mesenchymal transition (EMT), plays a critical role in the tumor progression and metastasis. The role of TWIST2 in tongue squamous cell carcinoma (TSCC) and its association with hyperthermia still have not been reported. METHOD: The correlations between TWIST2 expression and the clinical-pathologic characteristics of 89 patients with TSCC were evaluated by immunohistochemical staining. TSCC cell lines transfected with siRNA against TWIST2 were heated for 40 min at 42.5°C, and the migration capability of cells was examined by migration assay. Xenograft tumors in nude mice were treated by hyperthermia, and TWIST2 expression was measured. RESULTS: Our data showed that TWIST2 expression was associated with the metastasis of human TSCC. In Tca8113 and Cal-27 cells, TWIST2-siRNA treatment can reduce cell migration ability and has no effect on the cell proliferation and apoptosis. Hyperthermia can decrease the level of TWIST2 in TSCC and inhibit the migration of cells. CONCLUSIONS: This demonstrated that hyperthermia might decrease the migration of Tca8113 and Cal-27 cells by reducing TWIST2 expression. Altogether, these findings suggest an as yet undescribed link between TWIST2 and hyperthermia in TSCC.