Search

Objective Salidroside is an important plant-derived aromatic compound with diverse biological properties. The main objective of this study was to synthesize salidroside from tyrosol using UDP-glucosyltransferase (UGT) with in situ regeneration of UDP-glucose (UDPG). Results The UDP-glucosyltransferase 85A1 (UGT85A1) from Arabidopsis thaliana,which showed high activity and regioselectivity towards tyrosol, was selected for the production of salidroside. Then, an in vitro cascade reaction for in situ regeneration of UDPG was constructed by coupling UGT85A1 to sucrose synthase from Glycine max (GmSuSy). The optimal UGT85A1-GmSuSy activity ratio of 1:2 was determined to balance the efficiency of salidroside production and UDP-glucose regeneration. Different cascade reaction conditions for salidroside production were also determined. Under the optimized condition, salidroside was produced at a titer of 6.0 g/L with a corresponding molar conversion of 99.6% and a specific productivity of 199.1 mg/L/h in a continuous feeding reactor. Conclusion This is the highest salidroside titer ever reported so far using biocatalytic approach.

The applications of flow chemistry (continuous flow reactions) in the synthesis of pharmaceuticals and fine chemicals require more advanced optimization algorithms to guide laboratory-scale and industry-scale optimization. Although several Bayesian Optimization (BO) frameworks have been developed, they are rarely equipped with state-of-the-art noise-handling acquisition functions and have not been benchmarked by multiple real-world continuous flow kinetic models. In this study, we developed FlowBO for flow chemistry, equipped with the recently-developed MOO algorithm qNEHVI that can better handle experimental noise and make parallel recommendations. Also, five kinetic models built from experimental results, including four series reactions, were used as benchmarks for FlowBO and two other recognized BO frameworks. The results show that FlowBO outperforms in all four series reaction cases with optimization results >99.9% for conversion and selectivity. At the same time, FlowBO offers a range of optimum advantages with a wide choice of temperature, residence time, and reactant concentration, facilitating process optimization for subsequent steps (i.e. separation).

Here we developed a continuous flow microreactor system to synthesis N-benzoylpiperazine, which is an important intermediate of novel nootropic Sunifiram. And the kinetics of the condensation amidation of piperazine and benzoic acid using HATU as coupling reagent was revealed in microreactor. A kinetic model was established, all the reactions kinetics parameters, including the reaction order of each reactant, rate constants, pre-exponential factors and activation energies were acquired. With this model the selectivity vs. reaction temperature/molar flow ratio were obtained to help understand this kind of reaction and further optimize operating conditions. Under the condition of using equivalent piperazine, 95.6% of selectivity and 91.2% of yield were obtained in microreactor with 50 s. To further expand the scope of carboxylic acid, cyclopropanecarboxylic acid was tried as acylating agent, and 92.8% of selectivity and 86.1% of yield were obtained in microreactor.

Reaction coupling separation systems using calcium fumarate as a substrate can break the reaction equilibrium and promote the production of l-malate. However, the low reusability and stability of fumarase limit its further application. In this study, partially purified fumarase of Thermus thermophilus (87.0 U mg−1) was immobilized within konjac-κ-carrageenan beads. An amalgamation of konjac and carrageenan gum (2%) was used to form the beads, and polyethylene polyamine (0.2%) and glutaraldehyde (0.1%) were used as the cross-linking agents. The pH and temperature profiles of free and immobilized fumarases were remarkably similar. The diffusion limit of immobilized fumarase caused a decline in the maximal velocity (Vmax), whereas the kinetic constant (Km) value increased. Optimization of the parameters for biotransformation by immobilized fumarase showed that 88.3% conversion of 200 mM calcium fumarate could be achieved at 55 °C within 8 h. The beads were stored for 30 days at 4 °C with minimal loss in activity and were reusable for up to 20 cycles with 78.1% relative activity. By recycling the reaction supernatant, a total amount of 3.98 M calcium fumarate was obtained with a conversion of 99.5%, which is the highest value ever reported.

m-Difluorobenzene is an important structural in many pharmaceutical agents and nature products. However, the conventional method of preparing m-difluorobenzene, which was adopts double diazotization from m-phenylenediamine, is very tricky with potential hazards associated with the strongly exothermic and thermal instability of the diazonium intermediate, and has severe coupled side reactions. Here, we report the continuous-flow methodology, which effectively addresses this series of process challenges by an extra inert solvent flow. The protocol rapidly synthesize m-Difluorobenzene with 85% total yield (the traditional method has 65% total yield). The molar ratio of hydrochloric acid used in the process deduce to 3.6 equivalent (traditional method requires 12 equivalent). Furthermore, the reaction time can be decreased from hours to within 40 s. Above all, the method has potential industrial application value. Graphical abstract

The rational design of enzymes with enhanced thermostability is efficient. Solvent-tolerant metalloprotease from Pseudomonas aeruginosa PT121 presents high Z-aspartame (Z-APM) synthesis activity, but insufficient thermostability. In this study, we enhanced enzyme thermostability using a rational strategy. Molecular dynamics (MD) simulation was applied to rapidly identify that the D28 and D116 mutations are likely to exhibit increased thermostability, and experimentation verified that the D28N and D116N mutants were more stable than the wild-type (WT) enzyme. In particular, the T

Acute liver failure (ALF) is a rapidly progressive disease with high morbidity and mortality rates. Liver transplantation and artificial liver (AL) support systems, such as ALs and bioartificial livers (BALs), are the two major therapies for ALF. Compared to ALs, BALs are composed of functional hepatocytes that provide essential liver functions, including detoxification, metabolite synthesis, and biotransformation. Furthermore, BALs can potentially provide effective support as a form of bridging therapy to liver transplantation or spontaneous recovery for patients with ALF. In this review, we systematically discussed the currently available state-of-the-art designs and manufacturing processes for BAL support systems. Specifically, we classified the cell sources and bioreactors that are applied in BALs, highlighted the advanced technologies of hepatocyte culturing and bioreactor fabrication, and discussed the current challenges and future trends in developing next-generation BALs for large-scale clinical applications.

It remains impossible to accurately assess the prognosis after thermal ablation in patients with hepatocellular carcinoma (HCC). Our aim was to build a nomogram to predict the survival rate of HCC patients after thermal ablation. We developed and validated a nomogram using data of 959 HCC patients after thermal ablation from two centers. Harrell’s concordance index (C-index), calibration plot and Decision curve analysis (DCA) were used to measure the performance of the nomogram, and we compared it with the Barcelona Clinic Liver Cancer (BCLC) staging system and a previous nomogram. Six variables including age, serum albumin, operation method, risk area, tumor number and early recurrence were selected to construct the nomogram. In the training cohort, internal validation cohort, and external validation cohort, the nomogram all had a higher C-index to predict survival rate than both the BCLC staging system and the previous nomogram (0.736, 0.558 and 0.698, respectively; 0.763, 0.621 and 0.740, respectively; and 0.825, 0.551 and 0.737, respectively). Calibration plots showed a high degree of consistency between prediction and actual observation. Decision curve analysis (DCA) presented that compared with BCLC system and the previous nomogram, our nomogram had the highest net benefit. In all three cohorts, the nomogram could accurately divide patients into three subgroups according to predicted survival risk. A nomogram was developed and validated to predict survival of HCC patients who underwent thermal ablation, which is helpful for prognostic prediction and individual surveillance in clinical practice.

The chlorination of aromatic carboxylic acids with SOCl2 has been effectively performed by reacting with a Brønsted acid as the catalyst. Based on this discovery, an efficient catalytic method that is cheaper than traditional catalytic methods was developed. 20 substrates were chlorinated offering excellent yields in a short reaction time. And the SOCl2/Brønsted acid system has been used in a larger scale preparative reaction. A dual activation mechanism was proposed to prove the irreplaceable system of SOCl2/Brønsted acid.

Non-genetic induction of somatic cells into neural crest stem-like cells (NCSCs) is promising for potential cell-based therapies for post-traumatic peripheral nerve regeneration. Here, we report that human gingiva-derived mesenchymal stem cells (GMSCs) could be reproducibly and readily induced into NCSCs via non-genetic approaches. Compared to parental GMSCs, induced NCSC population had increased expression in NCSC-related genes and displayed robust differentiation into neuronal and Schwann-like cells. Knockdown of the expression of Yes-associated protein 1 (YAP1), a critical mechanosensor and mechanotransducer, attenuated the expression of NCSC-related genes; specific blocking of RhoA/ROCK activity and non-muscle myosin II (NM II)-dependent contraction suppressed YAP1 and NCSC-related genes and concurrently abolished neural spheroid formation in NCSCs. Using a rat model of facial nerve defect, implantation of NCSC-laden nerve conduits promoted functional regeneration of the injured nerve. These promising findings demonstrate that induced NCSCs derived from GMSCs represent an easily accessible and promising source of neural stem-like cells for peripheral nerve regeneration.

A fully continuous-flow diazotization–hydrolysis protocol has been developed for the preparation of p-cresol. This process started from the diazotization of p-toluidine to form diazonium intermediate. The reaction was then quenched by urea and subsequently followed by a hydrolysis to give the final product p-cresol. Three types of byproducts were initially found in this reaction sequence. After an optimization of reaction conditions (based on impurity analysis), side reactions were eminently inhibited, and a total yield up to 91% were ultimately obtained with a productivity of 388 g/h. The continuous-flow methodology was used to avoid accumulation of the highly energetic and potentially explosive diazonium salt to realize the safe preparation for p-cresol.

// Qilin Xu 1 , Qunzhou Zhang 1 , Yasutaka Ishida 1, 2 , Souren Hajjar 1 , Xudong Tang 1, 3 , Haoran Shi 1 , Chi V. Dang 4 , Anh D. Le 1, 5 1 Department of Oral and Maxillofacial Surgery and Pharmacology, University of Pennsylvania School of Dental Medicine, Philadelphia, Pennsylvania, USA 2 Department of Molecular Oral Medicine and Maxillofacial Surgery, Graduate School of Biomedical Sciences, Hiroshima University, Japan 3 Institute of Biochemistry and Molecular Biology, Guangdong Medical University, Zhanjiang, Guangdong, China 4 Abramson Cancer Center, Perelman School of Medicine at The University of Pennsylvania, Philadelphia, Pennsylvania, USA 5 Department of Oral & Maxillofacial Surgery, Penn Medicine Hospital of The University of Pennsylvania, Philadelphia, Pennsylvania, USA Correspondence to: Anh D. Le, email: Anh.Le@uphs.upenn.edu Keywords: EGF, EMT, cancer stem cells, Warburg effect, oral cancer Received: June 30, 2016 Accepted: November 21, 2016 Published: December 01, 2016 ABSTRACT “Warburg effect”, the enhanced glycolysis or aerobic glycolysis, confers cancer cells the ability to survive and proliferate even under stressed conditions. In this study, we explored the role of epidermal growth factor (EGF) in orchestrating Warburg effect, the epithelial-mesenchymal transition (EMT) process, and the acquisition of cancer stem-like cell properties in human oral squamous cell carcinoma (OSCC) cells. Our results showed that EGF induces EMT process in OSCC cells, which correlates with the acquisition of cancer stem-like properties, including the enrichment of CD44 + /CD24 − population of cancer cells and an increased expression of CSC-related genes, aldehyde dehydrogenase-1 (ALDH1) and Bmi-1. We also showed that EGF concomitantly enhanced L-lactate production, while blocking glycolysis by 2-deoxy-D-glucose (2-DG) robustly reversed EGF-induced EMT process and CSC-like properties in OSCC cells. Mechanistically, we demonstrated that EGF promoted EMT process and CSC generation through EGFR/PI3K/HIF-1α axis-orchestrated glycolysis. Using an orthotopic tumor model of human OSCC (UM-SCC1) injected in the tongue of BALB/c nude mice, we showed that treatment with 2-DG in vivo significantly inhibited the metastasis of tumor cells to the regional cervical lymph nodes and reduced the expression of ALDH1 and vimentin in both in situ tumors and tumor cell-invaded regional lymph nodes. Taken together, these findings have unveiled a new mechanism that EGF drives OSCC metastasis through induction of EMT process and CSC generation, which is driven by an enhanced glycolytic metabolic program in OSCC cells.

Regeneration of peripheral nerve injury remains a major clinical challenge. Recently, mesenchymal stem cells (MSCs) have been considered as potential candidates for peripheral nerve regeneration; however, the underlying mechanisms remain elusive. Here, we show that human gingiva-derived MSCs (GMSCs) could be directly induced into multipotent NPCs (iNPCs) under minimally manipulated conditions without the introduction of exogenous genes. Using a crush-injury model of rat sciatic nerve, we demonstrate that GMSCs transplanted to the injury site could differentiate into neuronal cells, whereas iNPCs could differentiate into both neuronal and Schwann cells. After crush injury, iNPCs, compared with GMSCs, displayed superior therapeutic effects on axonal regeneration at both the injury site and the distal segment of the injured sciatic nerve. Mechanistically, transplantation of GMSCs, especially iNPCs, significantly attenuated injury-triggered increase in the expression of c-Jun, a transcription factor that functions as a major negative regulator of myelination and plays a central role in dedifferentiation/reprogramming of Schwann cells into a progenitor-like state. Meanwhile, our results also demonstrate that transplantation of GMSCs and iNPCs consistently increased the expression of Krox-20/EGR2, a transcription factor that governs the expression of myelin proteins and facilitates myelination. Altogether, our findings suggest that transplantation of GMSCs and iNPCs promotes peripheral nerve repair/regeneration, possibly by promoting remyelination of Schwann cells mediated via the regulation of the antagonistic myelination regulators, c-Jun and Krox-20/EGR2.

Odontogenic tumors are lesions derived from the epithelial and/or mesenchymal elements of the tooth-forming apparatus and are therefore found within the jaw bone or soft tissue overlying teeth. These tooth development-associated tumors are generally benign, but several odontogenic tumors are capable of aggressive growth and locally invasive behavior with a high rate of recurrence. Benign odontogenic tumors can pose diagnostic challenges because of overlapping histology, a lack of distinct phenotypic and molecular features, and an unclear pathogenesis of the tumors. In this article, we will review the possible origins, immunophenotypic features, and genetic alterations of the common benign odontogenic tumors.

Phase relations in the Zn–V–Sb ternary system have been studied experimentally for the whole composition range for two temperatures, 600°C and 800°C, using scanning electron microscopy coupled with energy-dispersive spectrometry and X-ray diffraction. The ternary compound VZnSb, mentioned in the literature, still exists at 600°C, but disappears at 800°C. Moreover, the experimental results indicate that eight tri-phase regions could be confirmed in the system at 600°C, and four tri-phase regions exist in the 800°C isothermal section. The maximum solubility of Zn in V3Sb2 and V3Sb is up to 14.3 at.% and 3.6 at.% at 600°C, and 6.2 at.% and 2.7 at.% at 800°C, respectively.

Monoacylated symmetrical diamines are building blocks or intermediates of several well-established drugs. However, for the symmetrical diamines, the two amine groups located in a similar chemical environment make the selective monoacylation rather difficult and no kinetics study has ever been reported before. To comprehend this kind of reaction more deeply, a continuous flow microfluidic system was developed to evaluate the reaction kinetics of m-phenylenediamine and benzoic anhydride. At first, the reaction order of each reactant, pre-exponential factors and activation energies were determined in turn. The possible reaction mechanism was postulated from the reaction order. Then, a series of validation experiments were designed to show the accuracy and the applied range (concentration and temperature) of the kinetic model. Next, to obtain a controlled selectivity, the effects of initial concentration, reaction temperature and molar ratio on the selectivity were investigated through the model simulation. In the end, under the conditions optimized by the kinetic model, 96.9% selectivity and 92.5% yield were obtained in the microreactor.

Diabetes mellitus is an established risk factor associated with bisphosphonate-related osteonecrosis of the jaw (BRONJ). Sustained activation of Nod-like receptor (NLR) family, pyrin domain-containing protein 3 (NLRP3) inflammasome contributes to the persistent inflammation and impaired cutaneous wound healing in diabetic mice and human. We have recently demonstrated a compelling linkage between M1 macrophages and BRONJ conditions in both murine and human diseases. The aim of this study was to determine whether NLRP3 inflammasome activation is involved in BRONJ development in diabetic mice. We showed an increased incidence of delayed oral wound healing and bone necrosis of extraction sockets in db/db mice compared with those in nondiabetic db/+ controls, which correlated with an elevated expression of NLRP3, caspase-1, and IL-1β in macrophages residing at local wounds. Constitutively, bone marrow-derived macrophages from db/db mice (db/db BMDMs) secrete a relatively higher level of IL-1β than those from db/+ mice (db/+ BMDMs). Upon stimulation by NLRP3 activators, the secretion of IL-1β by db/db BMDMs was 1.77-fold higher than that by db/+ BMDMs (p

Phase diagram of the Zn-V-Sb ternary system has been studied experimentally by means of scanning electron microscopy coupled with energy-dispersive spectrometric and x-ray diffraction. Twelve different tri-phase regions could be confirmed in this system at 450 °C. Moreover, a new ternary compound named VZnSb, whose composition range spanned from 30.4-32.4 at.% V, 30.5-34.0 at.% Zn and 34.7-37.1 at.% Sb, is positively identified for the first time, and the crystal structure of VZnSb is not resolved. The VZnSb phase could be equilibrated with V3Sb, SbZn, Sb2Zn3, Sb3Zn4 and η-Zn. Experimental results indicated that the solubility of V in SbZn, Sb3Zn4, Sb2Zn3 and α-Sb is very limited. On the other hand, the maximum solubility of Zn in VSb2, VSb and V3Sb is up to 1.8, 9.1 and 3.7 at.%, respectively. The solubility of Sb in VZn3 is up to 0.4 at.%, and η-Zn dissolves up to 1.3 at.% V and 2.0 at.% Sb.

In the oral cavity, the tongue is the anatomic subsite most commonly involved by invasive squamous cell carcinoma. Current treatment protocols often require significant tissue resection to achieve adequate negative margins and optimal local tumor control. Reconstruction of the tongue while preserving and/or restoring its critical vocal, chewing, and swallowing functions remains one of the major challenges in head and neck oncologic surgery. We investigated the in vitro feasibility of fabricating a novel combinatorial construct using porcine small intestinal submucosa extracellular matrix (SIS-ECM) and human gingiva-derived mesenchymal stem cells (GMSCs) as a GMSC/SIS-ECM tissue graft for the tongue reconstruction. We developed a rat model of critical-sized myomucosal defect of the tongue that allowed the testing of therapeutic effects of an acellular SIS-ECM construct versus a GMSC/SIS-ECM construct on repair and regeneration of the tongue defect. We showed that the GMSC/SIS-ECM construct engrafted at the host recipient site, promoted soft tissue healing, and regenerated the muscular layer, compared to the SIS-ECM alone or nontreated defect controls. Furthermore, our results revealed that transplantation of the GMSC/SIS-ECM construct significantly increased the expression of several myogenic transcriptional factors and simultaneously suppressed the expression of type I collagen at the wounded area of the tongue. These compelling findings suggest that, unlike the tongue contracture and fibrosis of the nontreated defect group, transplantation of the combinatorial GMSC/SIS-ECM constructs accelerates wound healing and muscle regeneration and maintains the overall tongue shape, possibly by both enhancing the function of endogenous skeletal progenitor cells and suppressing fibrosis. Together, our findings indicate that GMSC/SIS-ECM potentially served as a myomucosal graft for tongue reconstruction postsurgery of head and neck cancer.

Pancreatic progenitor (PP) cells are tissue-committed cells, which can differentiate into all kinds of pancreatic cells. They are potential candidates for regeneration of pancreatic tissue. However, it is unfeasible to acquire PP cells from pancreatic tissues and expand them in vitro. Generation of PP cells from adipose tissue-derived mesenchymal stem cells (AD-MSCs) would provide an unlimited source of PP cells. Here we developed a 2-step stepwise protocol, which induced AD-MSCs to generate FOXA2- or SOX17-positive definitive endoderm (DE) (5 days) and pancreatic and duodenal homeobox gene 1 (PDX1)-positive PP cells (4-6 days). By mimicking the developmental progress in embryonic development, we optimized the timing and combination of cytokines to activate the key signaling pathways during pancreatic development. We found that activating the Nodal/Activin signal with Activin A could induce differentiation of AD-MSCs toward DE, which could be further promoted by the Wnt signaling pathway activator Wnt3a. Besides, transient T (BRACHYURY)(+) mesendodermal cells were observed during formation of DE from AD-MSCs. Subsequently, the Wnt signaling pathway inhibitor Dkk1 along with retinoic acid/FGF2 (60 ng/mL) further induced AD-MSC-derived DE cells to differentiate into PDX1-positive PP cells. The derived PP cells were capable to form pancreatic endocrine or exocrine cells. In conclusion, we established a stepwise protocol that could derive DE and PP cells from AD-MSCs. It might provide an unlimited source of autologous PP cells for pancreatic diseases.

Elucidation of the molecular mechanisms governing human adipose-derived mesenchymal stem cells (hASCs) osteogenic differentiation is of great importance for improving the treatment of bone-related diseases. In this study, we examined the role of microRNA (miR)-100 on the osteogenesis of hASCs. Overexpression of miR-100 inhibited osteogenic differentiation of hASCs in vitro, whereas downregulation of miR-100 enhanced the process. Target prediction analysis and dual luciferase report assay confirmed that bone morphogenetic protein receptor type II (BMPR2) was a direct target of miR-100. Furthermore, knockdown of BMPR2 by RNA interference inhibited osteogenic differentiation of hASCs, similar as the effect of upregulation miR-100. Taken together, our findings imply that miR-100 plays a negative role in osteogenic differentiation and might act through targeting BMPR2.

Diabetes mellitus is an established risk factor associated with bisphosphonate-related osteonecrosis of the jaw (BRONJ). Sustained activation of Nod-like receptor (NLR) family, pyrin domain-containing protein 3 (NLRP3) inflammasome contributes to the persistent inflammation and impaired cutaneous wound healing in diabetic mice and human. We have recently demonstrated a compelling linkage between M1 macrophages and BRONJ conditions in both murine and human diseases. The aim of this study was to determine whether NLRP3 inflammasome activation is involved in BRONJ development in diabetic mice. We showed an increased incidence of delayed oral wound healing and bone necrosis of extraction sockets in db/db mice as compared to those in non-diabetic db/+ controls, which correlated with an elevated expression of NLRP3, caspase-1 and IL-1β in macrophages residing at local wounds. Constitutively, bone marrow-derived macrophages from db/db mice (db/db BMDMs) secrete a relatively higher level of IL-1β than those from db/+ mice (db/+ BMDMs). Upon stimulation by NLRP3 activators, the secretion of IL-1β by db/db BMDMs was 1.77-fold higher than that by db/+ BMDMs (p

The radiation characteristics of the omnidirectional periodic circular dielectric rod leaky-wave antenna with arbitrary grating profile are investigated by the network method combining with staircase approximation. In the analysis, the arbitrary grating profiles are transformed to a multilayer rectangular periodic structure, which is represented by a radial transmission line with a series current source. As a result, the calculations are greatly simplified and the effects of the grating profile on the performance of the grating antenna are systematically studied in a very efficient way. Some useful guidelines for the design and fabrication of the omnidirectional dielectric grating antenna are thereby suggested.

Although the epithelial-mesenchymal transition (EMT) is a normal process that occurs during development, it is thought to be associated with cancer progression and metastasis. Emerging evidence links mesenchymal stem cells (MSCs) in the tumor microenvironment with the occurrence of EMT in cancer progression. In this study, the human breast cancer cell line MCF7 was co-cultured with human adipose-derived MSCs (hAD-MSCs) in a transwell system. Co-cultured cells were analyzed for changes in cellular morphology, EMT markers, protein expression and tumor characteristics. We found that co-cultured MCF7 cells underwent EMT and established a stable mesenchymal phenotype after prolonged co-culturing. Here, we demonstrate that paracrine transforming growth factor-β1 (TGF-β1) secreted by hAD-MSCs regulated the establishment of EMT in MCF7 cells by targeting the ZEB/miR-200 regulatory loop. The downregulation of paracrine TGF-β1 levels can inhibit and reverse the EMT progress by downregulating ZEB1/2 and upregulating miR-200b and miR-200c. The maintenance of a stable mesenchymal state by MCF7 cells required the establishment of autocrine TGF-β signaling to drive and sustain ZEB expression, which had been initiated by the prolonged co-culturing with hAD-MSCs. These results suggest that MSCs may promote breast cancer metastasis by stimulating and facilitating the EMT process.

Head and neck cancer is one of the 10 most common cancers worldwide. Over-expression of EGFR has been demonstrated in approximately 90% of head and neck cancers, which is associated with progressive tumor growth, metastasis, recurrence, and resistance to chemo- and radiation therapies. Recurrent tumors are often therapy-resistant and may have their origin in resistant cancer stem cells (CSCs) and/or in tumor cells with an epithelial-mesenchymal transition (EMT) phenotype. Recently it is increasingly accepted that metabolic changes in cancer cells can drive tumor formation and progression. In our study, we used 20ng/ml EGF to treat human tongue cancer cell line, SCC-1 cells. Our results demonstrated that EGF induces EMT in oral SCC cells as characterized by a downregulation of E-cadherin (an epithelial marker), an upregulation of vimentin (a mesenchymal marker), increased invasive and metastatic capacities, and acquisition of stem-like properties such as the increased expression of ALDH1 and enrichment of CD44+/CD24− proportion of cancer cells. We also found that EGF treatment concomitantly enhanced L-lactate production and blocking glycolysis by 2-DG robustly reversed EGF-induced EMT and CSC-like phenotypes of OSCC cells. Then we further demonstrated that EGF regulates glycolysis/EMT/CSCs axis through downstream EGFR/PI3K signaling pathways in an EGFR/PI3K-dependent but AKT-independent manner. Importantly, we showed that in vivo treatment with 2-DG remarkably reduced the expression of ALDH1 and vimentin at both the in situ tumors and regional lymph nodes. Taken together, these findings have demonstrated that EGF drives tumor growth and metastasis through induction of EMT and CSC generation, which is orchestrated by an enhanced glycolytic metabolic program in OSCC cells. This work was supported by National Institute of Health Research Grant, R01DE 019932, Oral and Maxillofacial Surgery Foundation (OMSF) Research Grant, and the Schoenleber funding support Citation Format: Qilin Xu, Qunzhou Zhang, Ahn D. Le. EGF induces epithelial to mesenchymal transition and cancer stem-like cell properties in human head and neck cancer cells via promoting Warburg Effect. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1143. doi:10.1158/1538-7445.AM2015-1143

Head and neck cancer is one of the 10 most common cancers worldwide. Despite advances in therapeutic research in the last few decades, morbidity and mortality remain unchanged. Recurrent and metastatic oral squamous cell carcinoma (OSCC) of the head and neck may have correlated with epithelial-mesenchymal transition (EMT), a property acquired by cancer stem cells (CSCs). In our study, SCC-1 cells (a human tongue cancer cell line) treated with epidermal growth factor (EGF) underwent EMT as confirmed by morphological changes and expression of EMT-related markers using Western blot, immunocytostaining, and flow cytometric analyses. Our data showed that, in comparison with non-treated cells, EGF treatment induced SCC-1 cells to undergo EMT, characterized by spindle-shaped mesenchymal-like morphology, an altered suppression of E-cadherin and promotion of vimentin expression, and an enhanced invasive capacity. Our results also indicated that EGF significantly enriched the fraction of CD44+/CD24low/- CSCs in SCC-1 cells, from 7∼8% to 25∼29%, and simultaneously increased the expression of aldehyde dehydrogenase 1 (ALDH1), a common marker for stem cells. Moreover, the purified CD44+/CD24low/- CSC population displayed stem cell properties associated with EMT phenotypes and increased migration and invasion abilities in vitro. Using a murine orthotopic tongue cancer model transplanted with stably GFP-expressing SCC-1 cells (GFP-SCC-1), we confirmed that EGF can promote tumor metastasis to cervical lymph nodes, and simultaneously augmented CD44 and ALDH1 expression in these invasive tumor cells. Taken together, we have demonstrated that EGF-induced EMT process is closely associated with the acquisition of CSC properties in human OSCC, which may contribute to the increased distant metastasis and recurrence of head and neck cancer. Further elucidation of the underlying mechanisms will facilitate development of new generation of effective therapies that aim to specifically target CSCs and eliminate the “root” of cancer recurrence. This work was supported by National Institute of Health Research Grant, R01DE 019932, Oral and Maxillofacial Surgery Foundation Research Support, 002894, and the Schoenleber funding support. Citation Format: Qilin Xu, Qunzhou Zhang, Anh Le. EGF induces epithelial-mesenchymal transition and cancer stem cell properties in human oral squamous cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1059. doi:10.1158/1538-7445.AM2014-1059