Qunzhou Zhang

EGF induces epithelial-mesenchymal transition and cancer stem-like cell properties in human oral cancer cells via promoting Warburg effect.

“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.

(−)-Epigallocatechin-3-gallate inhibits human papillomavirus (HPV)-16 oncoprotein-induced angiogenesis in non-small cell lung cancer cells by targeting HIF-1α

PurposeTo investigate the effects of (−)-epigallocatechin-3-gallate (EGCG) on human papillomavirus (HPV)-16 oncoprotein-induced angiogenesis in non-small cell lung cancer (NSCLC) cells and the underlying mechanisms.MethodsNSCLC cells (A549 and NCI-H460) transfected with EGFP plasmids containing HPV-16 E6 or E7 oncogene were treated with different concentrations of EGCG for 16xa0h. The effects of EGCG on angiogenesis in vitro and in vivo were observed. The expression of HIF-1α, p-Akt, and p-ERK1/2 proteins in NSCLC cells was analyzed by Western blot. The levels of HIF-1α mRNA in NSCLC cells were detected by real-time RT-PCR. The concentration of VEGF and IL-8 in the conditioned media was determined by ELISA. HIF-1α, VEGF, and CD31 expression in A549 xenografted tumors of nude mice was analyzed by immunohistochemistry.ResultsHPV-16 E6 and E7 oncoproteins HIF-1α-dependently promoted angiogenesis in vitro and in vivo, which was inhibited by EGCG. Mechanistically, EGCG inhibited HPV-16 oncoprotein-induced HIF-1α protein expression but had no effect on HIF-1α mRNA expression in NSCLC cells. Additionally, 50 and 100xa0μmol/L of EGCG significantly reduced the secretion of VEGF and IL-8 proteins induced by HPV-16 E7 oncoprotein in NSCLC A549 cells. Meanwhile, HPV-16 E6 and E7 oncoproteins HIF-1α-dependently enhanced Akt activation in A549 cells, which was suppressed by EGCG. Furthermore, EGCG inhibited HPV-16 oncoprotein-induced HIF-1α and HIF-1α-dependent VEGF and CD31 expression in A549 xenografted tumors.ConclusionsEGCG inhibited HPV-16 oncoprotein-induced angiogenesis conferred by NSCLC through the inhibition of HIF-1α protein expression and HIF-1α-dependent expression of VEGF, IL-8, and CD31 as well as activation of Akt, suggesting that HIF-1α may be a potential target of EGCG against HPV-related NSCLC angiogenesis.

Neural Progenitor‐Like Cells Induced from Human Gingiva‐Derived Mesenchymal Stem Cells Regulate Myelination of Schwann Cells in Rat Sciatic Nerve Regeneration

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. Stem Cells Translational Medicine 2017;6:458–470

Bisphosphonate Induces Osteonecrosis of the Jaw in Diabetic Mice via NLRP3/Caspase-1-Dependent IL-1β Mechanism

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 (pu2009<u20090.001). Systemic treatment of mice with zoledronate (Zol), a nitrogen‐containing bisphosphonate, resulted in a 1.86‐ and 1.63‐fold increase in NLRP3/caspase‐1‐dependent IL‐1β secretion by db/+ and db/db BMDMs, respectively, compared with BMDMs derived from nontreated mice (pu2009<u20090.001). Importantly, systemic administration of pharmacological inhibitors of NLRP3 activation improved oral wound healing and suppressed BRONJ formation in db/db mice. Mechanistically, we showed that supplementation with intermediate metabolites of the mevalonate pathway, inhibitors of caspase‐1 and NLRP3 activation, an antagonist for P2X7R, or a scavenger of reactive oxygen species (ROS), robustly abolished Zol‐enhanced IL‐1β release from macrophages in response to NLRP3 activation (pu2009<u20090.001). Our findings suggest that diabetes‐associated chronic inflammatory response may have contributed to impaired socket wound healing and rendered oral wound susceptible to the development of BRONJ via NLRP3 activation in macrophages.

Induction of Salivary Gland–Like Cells from Dental Follicle Epithelial Cells:

The dental follicle (DF), most often associated with unerupted teeth, is a condensation of ectomesenchymal cells that surrounds the tooth germ in early stages of tooth development. In the present study, we aim to isolate epithelial stem-like cells from the human DF and explore their potential differentiation into salivary gland (SG) cells. We demonstrated the expression of stem cell–related genes in the epithelial components of human DF tissues, and these epithelial progenitor cells could be isolated and ex vivo expanded in a reproducible manner. The human DF-derived epithelial cells possessed clonogenic and sphere-forming capabilities, as well as expressed a panel of epithelial stem cell–related genes, thus conferring stem cell properties (hDF-EpiSCs). When cultured under in vitro 3-dimensional induction conditions, hDF-EpiSCs were capable to differentiate into SG acinar and duct cells. Furthermore, transplantation of hDF-EpiSC–loaded native de-cellularized rat parotid gland scaffolds into the renal capsule of nude mice led to the differentiation of transplanted hDF-EpiSCs into salivary gland–like cells. These findings suggest that hDF-EpiSCs might be a promising source of epithelial stem cells for the development of stem cell–based therapy or bioengineering SG tissues to repair/regenerate SG dysfunction.

DPSCs from Inflamed Pulp Modulate Macrophage Function via the TNF-α/IDO Axis

Human dental pulp stem cells (DPSCs) can be isolated from inflamed pulp derived from carious teeth with symptomatic irreversible pulpitis (I-DPSCs), which possess stemness and multidifferentiation potentials similar to DPSCs from healthy pulp. Since macrophages—essential cell players of the pulpal innate immunity—can regulate pulpal inflammation and repair, the authors investigated the immunomodulatory effects of DPSCs/I-DPSCs on macrophage functions and their underlying mechanisms. Similar to DPSCs, I-DPSCs were capable of colony-forming efficiency and adipogenic and osteo/dentinogenic differentiation under in vitro induction conditions. I-DPSCs also expressed a similar phenotypic profile of mesenchymal stem cell markers, except a relatively higher level of CD146 as compared with DPSCs. Coculture of DPSCs or I-DPSCs with differentiated THP-1 cells, the human monocyte cell line, markedly suppressed tumor necrosis factor α (TNF-α) secretion in response to stimulation with lipopolysaccharides (LPS) and/or nigericin. However, unlike TNF-α, the secreted level of interleukin 1β was not affected by coculture with DPSCs or I-DPSCs. Furthermore, DPSC/I-DPSC-mediated inhibition of TNF-α secretion by macrophages was abolished by pretreatment with 1-methyl-D-tryptophan, a specific inhibitor of indoleamine-pyrrole 2,3-dioxygenase (IDO), but not by NSC-398, a specific inhibitor of COX-2, suggesting IDO as a mediator. Interestingly, IDO expression was significantly augmented in macrophages and mesenchymal stromal cells in inflamed human pulp tissues. Collectively, these findings show that I-DPSCs, similar to DPSCs, possess stem cell properties and suppress macrophage functions via the TNF-α/IDO axis, thereby providing a physiologically relevant context for their innate immunomodulatory activity in the dental pulp and their capability for pulp repair.

Loss of Notch3 Signaling Enhances Osteogenesis of Mesenchymal Stem Cells from Mandibular Torus

Mandibular torus (MT) is a common intraoral osseous outgrowth located on the lingual surface of the mandible. Histologic features include hyperplastic bone consisting of mature cortical and trabecular bone. Some theories on the etiology of MT have been postulated, such as genetic factors, masticatory hyperfunction, trauma, and continued growth, but the underlying mechanism remains largely unknown. In this study, we investigated the potential role of mesenchymal stem cells (MSCs) derived from human MT in the pathogenesis of bone outgrowth. We demonstrated that MT harbored a distinct subpopulation of MSCs, with enhanced osteogenic and decreased adipogenic differentiation capacities, as compared with their counterparts from normal jaw bone. The increased osteogenic differentiation of mandibular torus MSCs was associated with the suppression of Notch3 signaling and its downstream target genes, Jag1 and Hey1, and a reciprocal increase in the transcriptional activation of ATF4 and NFATc1 genes. Targeted knockdown of Notch3 expression by transient siRNA transfection promoted the expression of osteogenic transcription factors in normal jaw bone MSCs. Our data suggest that the loss of Notch3 signaling may contribute partly to bone outgrowth in MT, as mediated by enhanced MSC-driven osteogenic differentiation in the jaw bone.

3D bio-printed scaffold-free nerve constructs with human gingiva-derived mesenchymal stem cells promote rat facial nerve regeneration

Despite the promising neuro-regenerative capacities of stem cells, there is currently no licensed stem cell-based product in the repair and regeneration of peripheral nerve injuries. Here, we explored the potential use of human gingiva-derived mesenchymal stem cells (GMSCs) as the only cellular component in 3D bio-printed scaffold-free neural constructs that were transplantable to bridge facial nerve defects in rats. We showed that GMSCs have the propensity to aggregate into compact 3D-spheroids that could produce their own matrix. When cultured under either 2D- or 3D-collagen scaffolds, GMSC spheroids were found to be more capable of differentiating into both neuronal and Schwann-like cells than their adherent counterparts. Using a scaffold-free 3D bio-printer system, nerve constructs were printed from GMSC spheroids in the absence of exogenous scaffolds and allowed to mature in a bioreactor. In vivo transplantation of the GMSC-laden nerve constructs promoted regeneration and functional recovery when used to bridge segmental defects in rat facial nerves. Our findings suggest that GMSCs represent an easily accessible source of MSCs for 3D bio-printing of scaffold-free nervous tissue constructs with promising potential application for repair and regeneration of peripheral nerve defects.

Neural Crest Stem-Like Cells Non-genetically Induced from Human Gingiva-Derived Mesenchymal Stem Cells Promote Facial Nerve Regeneration in Rats

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.

Cellular Plasticity–Targeted Therapy in Head and Neck Cancers:

Head and neck cancer is one of the most frequent human malignancies worldwide, with a high rate of recurrence and metastasis. Head and neck squamous cell carcinoma (HNSCC) is cellularly and molecularly heterogeneous, with subsets of undifferentiated cancer cells exhibiting stem cell-like properties, called cancer stem cells (CSCs). Epithelial-mesenchymal transition, gene mutation, and epigenetic modification are associated with the formation of cellular plasticity of tumor cells in HNSCC, contributing to the acquisition of invasive, recurrent, and metastatic properties and therapeutic resistance. Tumor microenvironment (TME) plays a supportive role in the initiation, progression, and metastasis of head and neck cancer. Stromal fibroblasts, vasculature, immune cells, cytokines, and hypoxia constitute the main components of TME in HNSCC, which contributes not only to the acquisition of CSC properties but also to the recurrence and therapeutic resistance of the malignancies. In this review, we discuss the potential mechanisms underlying the development of cellular plasticity, especially the emergence of CSCs, in HNSCC. We also highlight recent studies implicating the complex interplays among TME components, plastic CSCs, tumorigenesis, recurrence, and therapeutic resistance of HNSCC. Finally, we summarize the treatment modalities of HNSCC and reinforce the novel concept of therapeutic targeting CSCs in HNSCC.