Laikui Liu

Snail overexpression induces an epithelial to mesenchymal transition and cancer stem cell-like properties in SCC9 cells.

Local invasiveness and distant metastasis are critical factors that contribute to oral squamous cell carcinoma-related deaths. Increasing evidence has shown that the epithelial to mesenchymal transition (EMT) is involved in cancer progression and is associated with the ‘stemness’ of cancer cells. Snail is a transcriptional factor that can induce EMT and preserve stem-cell function, which may induce resistance to radio- and chemotherapies in the cells. In the present study, SCC9 cells were transfected with an empty vector or a vector encoding human Snail (SCC9-S). Overexpression of Snail induced SCC9 cells to undergo EMT, in which the cells presented a fibroblast-like appearance, downregulated the epithelial markers E-cadherin and β-catenin, upregulated the mesenchymal marker vimentin, and associated with highly invasive and metastatic properties. Furthermore, the induction of EMT promoted cancer stem cell (CSC)-like characteristics in the SCC9-S cells, such as low proliferation, self-renewal, and CSC-like markers expression. These results indicate that overexpression of Snail induces EMT and promotes CSC-like traits in the SCC9 cells. Further understanding the role of Snail in cancer progression may reveal new targets for the prevention or therapy of oral cancers.

The Hippo transducer TAZ promotes epithelial to mesenchymal transition and cancer stem cell maintenance in oral cancer

The Hippo pathway has emerged as a fundamental regulator in tissue growth, organ size and stem cell functions, and tumorigenesis when deregulated. However, its roles and associated molecular mechanisms underlying oral squamous cell carcinoma (OSCC) initiation and progression remain largely unknown. Here, we identified TAZ, the downstream effector of Hippo signaling, as a novel bona fide oncogene by promoting cell proliferation, migration/invasion and chemoresistance in OSCC. TAZ promoted epithelial‐to‐mesenchymal transition (EMT) and also was involved in TGF‐β1‐induced EMT in oral cancer cells. Furthermore, enriched TAZ sustained self‐renewal, maintenance, tumor‐seeding potential of oral cancer stem cells (CSCs). Remarkably, enforced TAZ overexpression conferred CSCs‐like properties on differentiated non‐CSCs and fueled phenotypic transition from non‐CSCs to CSCs‐like cells. Mechanistically, TAZ‐TEADs binding and subsequent transcriptional activation of EMT mediators and pluripotency factors are presumably responsible for TAZ‐mediated EMT and non‐CSCs‐to‐CSCs conversion. Importantly, aberrant TAZ overexpression was found to be associated with tumor size, pathological grade and cervical lymph node metastasis, as well as unfavorable prognosis. Pharmacological repression of TAZ by simvastatin resulted in potent anti‐cancer effects against OSCC. Taken together, our findings have revealed critical links between TAZ, EMT and CSCs in OSCC initiation and progression, and also established TAZ as a novel cancer biomarker and viable druggable target for OSCC therapeutics.

Membrane Type 1 Matrix Metalloproteinase induces an epithelial to mesenchymal transition and cancer stem cell-like properties in SCC9 cells

BackgroundTissue invasion and metastasis are acquired abilities of cancer and related to the death in oral squamous cell carcinoma (OSCC). Emerging observations indicate that the epithelial-to-mesenchymal transition (EMT) is associated with tumor progression and the generation of cells with cancer stem cells (CSCs) properties. Membrane Type 1 Matrix Metalloproteinase (MT1-MMP) is a cell surface proteinase, which is involved in degrading extracellular matrix components that can promote tumor invasion and cell migration.MethodsIn the current study, we utilized SCC9 cells stably transfected with an empty vector (SCC9-N) or a vector encoding human MT1-MMP (SCC9-M) to study the role of MT1-MMP in EMT development.ResultsUpon up-regulation of MT1-MMP, SCC9-M cells underwent EMT, in which they presented a fibroblast-like phenotype and had a decreased expression of epithelial markers (E-cadherin, cytokeratin18 and β-catenin) and an increased expression of mesenchymal markers (vimentin and fibronectin). We further demonstrated that MT1-MMP-induced morphologic changes increased the level of Twist and ZEB, and were dependent on repressing the transcription of E-cadherin. These activities resulted in low adhesive, high invasive abilities of the SCC9-M cells. Furthermore, MT1-MMP-induced transformed cells exhibited cancer stem cell (CSC)-like characteristics, such as low proliferation, self-renewal ability, resistance to chemotherapeutic drugs and apoptosis, and expression of CSCs surface markers.ConclusionsIn conclusion, our study indicates that overexpression of MT1-MMP induces EMT and results in the acquisition of CSC-like properties in SCC9 cells. Our growing understanding of the mechanism regulating EMT may provide new targets against invasion and metastasis in OSCC.

Biomimetic mineralization of dentin induced by agarose gel loaded with calcium phosphate

A novel biomimetic mineralization system was designed to induce a layer of hydroxyapatite on a demineralized dentin surface. This system was constructed as follows. A layer of 0.5% agarose gel containing 0.26M Na(2) HPO(4) was used to cover acid-etched dentin slices, followed by a layer of agarose gel without phosphate ions. Then a neutral 0.13M CaCl(2) solution was added onto the ion-free gel surface. The mineralization system (dentin-agarose gel containing phosphate ions-CaCl(2) solution) was kept in a water bath at 37°C, and the gel and CaCl(2) solution were replaced at various intervals. The results showed that the deposited hydroxyapatite crystals densely packed to each other, completely covered the dentin surface, and occluded the dentinal tubules after 10 days of biomimetic mineralization in vitro. Therefore, this method may provide the experimental basis for dentin remineralization and for a new method to treat dentin hypersensitivity and dental caries.

Comparison of MTT assay, flow cytometry, and RT-PCR in the evaluation of cytotoxicity of five prosthodontic materials

In the present study, the cytotoxic effects of five prosthodontic materials on the L929 cell line were assessed by flow cytometry (FCM), reverse transcription PCR (RT-PCR), and MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazoli-umbromide) assay. The cells were treated with eluates resin (RE), pressable ceramics (PC), Co-Cr alloy-porcelain (CC), Ni-Cr alloy-porcelain (NC), and diatomite ceramics (DC). The cytotoxicity of all the materials tested by the MTT assay was grade 1. By FCM analysis, apoptosis rates of DC and PC were low, with no significant difference from the control (p > 0.05). The rest of the groups induced much higher apoptosis rates (p < 0.05), with the highest in the RE group. The necrotic cell levels of RE was also significantly increased (p < 0.05). Bcl-2 and Bax mRNA expression were determined by RT-PCR, and the Bax/Bcl-2 ratio in the DC and PC groups were not significantly different from the control (p > 0.05), whereas CC, NC, and RE groups showed significant differences (p < 0.05). Taken together, the results suggest that FCM and RT-PCR analyses can supplement the traditional MTT assay in evaluating the cytotoxicity of prosthodontic materials for selecting highly biocompatible materials.

Tumor-associated macrophages correlate with the clinicopathological features and poor outcomes via inducing epithelial to mesenchymal transition in oral squamous cell carcinoma

BackgroundBoth tumor-associated macrophages (TAMs) and the epithelial to mesenchymal transition (EMT) of cancer cells play key roles in promoting tumor progression. However, whether TAMs could induce EMT in the progression of oral squamous cell carcinoma (OSCC) remains undefined.ResultsHere we detected the expression of macrophages markers CD68 and CD163, epithelial marker E-cadherin and mesenchymal marker vimentin in 127 OSCC patients by using semi-quantitative immunohistochemistry. CD68 and CD163 expression was not confined to the infiltrating TAMs, but also detected in cancer cells. The high number of CD68-positive macrophages was correlated with poor overall survival. Meanwhile, the expression of CD163 both in macrophages and in cancer cells was associated with poor overall survival and had a significant prognostic impact in OSCC. Importantly, the expression of CD163 in cancer cells had a significant relationship with E-cadherin and vimentin. Furthermore, the incubation of TAMs conditioned medium resulted in a fibroblast-like appearance of cancer cells (HN4, HN6 and SCC9) together with the decreased/increased expression of E-cadherin/ vimentin, which were correlated with the enhanced ability of migration and invasion.ConclusionsOur results indicate that TAMs could promote the EMT of cancer cells, thereby leading to the progression of oral cancer.

The histone demethylase LSD1 is a novel oncogene and therapeutic target in oral cancer

The histone demethylase LSD1 functions as a key pro-oncogene and attractive therapeutic target in human cancer. Here we sought to interrogate the oncogenic roles of LSD1 in OSCC tumorigenesis and therapeutic intervention by integrating chemical-induced OSCC model, genetic and pharmacological loss-of-function approaches. Our data revealed that aberrant LSD1 overexpression in OSCC was significantly associated with tumor aggressiveness and shorter overall survival. Increased abundance of LSD1 was detected along with disease progression in DMBA- or 4NQO-induced OSCC animal models. LSD1 depletion via siRNA-mediated knockdown in OSCC cells resulted in impaired cell proliferation, migration/invasion, tumorsphere formation and reduced xenograft growth while inducing cell apoptosis and enhancing chemosensitivity to 5-FU. Moreover, treatments of LSD1 chemical inhibitors (pargyline and tranylcypromine) induced its protein reduction probably via enhanced protein degradation and produced similar phenotypic changes resembling LSD1 silencing in OSCC cells. Pharmacological inhibition of LSD1 by intraperitoneal delivery of these inhibitors resulted in impaired xenograft overgrowth. Taken together, our data reveal the tumorigenic roles of LSD1 and identified LSD1 as a novel biomarker with diagnostic and prognostic significance, and also establish that targeting LSD1 by chemical inhibitors is a viable therapeutic strategy against OSCC.

Biomimetic Mineralization and Bioactivity of Phosphorylated Chitosan

Phosphorylated chitosans were synthesized as templates to manipulate hydroxyapatite (HA) crystal nucleation, growth and microstructure. Two kinds of insoluble phosphorylated chitosan were soaked in saturated Ca(OH)2 solution for 4 d and in 1.5× SBF (simulated body fluid) solutions for 14 d at 37 °C for biomimetic mineralization. A lower [P]-content of phosphorylated chitosan promoted greater mineralization than higher [P]-content. Phosphorylated chitosan inhibited osteoblast proliferation and differentiation in vitro, while calcium phosphate phosphorylated chitosan composites did not.

Overexpression of miR-29b reduces collagen biosynthesis by inhibiting heat shock protein 47 during skin wound healing

Skin scar formation is characterized by excessive synthesis and aberrant deposition of collagens during wound healing. MicroRNAs are endogenous gene regulators critically involved in diverse biological events including skin scar formation and hold considerable promise as therapeutic targets. However, the detailed molecular mechanisms responsible for collagen production during skin wound repair and scar formation remain incompletely known. Here our data revealed that significant downregulation of miR-29b and upregulation of heat shock protein 47 (HSP47) were observed during wound healing in both excisional and burn wound models and also detected in facial skin scar as compared to adjacent healthy skin. HSP47, a specific chaperon for collagen production and secretion, was identified as a novel and direct post-transcriptional target of miR-29b in skin fibroblasts via bioinformatics prediction and experimental validation. Moreover, the regulatory functions of miR-29b in collagen biosynthesis are partially achieved through modulating HSP47 expression in skin fibroblasts. Furthermore, the profibrotic growth factor TGF-β1 inhibited miR-29b transcription by activating TGF-β/Smads signaling and in turn depressed HSP47 and enhanced collagen 1 production. In contrast, the proinflammatory cytokines IL-1β and TNF-α significantly induced miR-29b transcription via activating NF-κB signaling but had no significant effect on HSP47 and collagen production in skin fibroblasts. Importantly, local delivery of miR-29b lentiviral particles inhibited HSP47 expression and collagen biosynthesis as well as suppressed angiogenesis, thus reducing scar formation in an excisional wound splinting model. Collectively, our data reveal that miR-29b can reduce collagen biosynthesis during skin wound healing likely via post-transcriptional inhibition of HSP47 expression. These findings also suggest that therapeutic targeting of miR-29b/HSP47 might be a viable alternative strategy to prevent or reduce scar formation.

Hyaluronan synthase 2 expressed by cancer-associated fibroblasts promotes oral cancer invasion

BackgroundHyaluronan synthases (HAS) control the biosynthesis of hyaluronan (HA) and critically modulate the tumor microenviroment. Cancer-associated fibroblasts (CAFs) affect the progression of a tumor by remolding the matrix. However, little is known about the role of HAS from CAFs in this process. This study aimed to determine the role of hyaluronan synthase 2 (HAS2) from CAFs in the progression of oral squamous cell carcinoma (OSCC) invasion.MethodsHAS isoforms 1, 2, and 3 in paired sets of CAFs and normal fibroblasts (NFs) were examined by real-time PCR, and the expression of HAS2 and α-SMA in OSCC tissue sections was further evaluated using immunohistochemical staining. Furthermore, we used a conditioned culture medium model to evaluate the effects of HAS2 from CAFs on the invasion and epithelial-mesenchymal transition (EMT) of the oral cancer cells Cal27. Finally, we compared the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) between CAFs and NF, and between CAFs with or without HAS2 knockdown using an antibody array and western blotting.ResultsCAFs expressed higher levels of HAS2 than the paired NFs. HAS2 expression was consistent with α-SMA-positive myofibroblasts in the stroma of OSCC, and these were significantly correlated advanced clinical stages and cervical lymph node metastasis. Knocking down HAS2 with a specific siRNA or treatment with a HAS inhibitor markedly attenuated CAF-induced invasion and EMT of Cal27 cells. Higher MMP1 and lower TIMP1 levels were detected in the supernatants of CAFs relative to NFs. Knocking down HAS2 could decrease the expression of MMP1 and increase that of TIMP1 in CAFs.ConclusionsHAS2 is one of the key regulators responsible for CAF-mediated OSCC progression and acts by modulating the balance of MMP1 and TIMP1.