Effect of verbascoside on apoptosis and metastasis in human oral squamous cell carcinoma.

Despite significant advances in therapy, the 5-year survival rates for patients with advanced stage oral cancers still remains poor as an appropriate treatment has not been found yet, due to side effects of chemo/radiotherapy. Verbascoside (VB), a major bioactive constituent of the Tsoong herb, displays pharmacological properties by exhibiting anti-oxidative, anti-inflammatory and anti-cancer activities. However, the underlining function and mechanism of VB in human oral squamous cell carcinoma (OSCC) remains unclear. In this study, we show that VB significantly decreased the viability and metastasis of HN4 and HN6 tumor cells, while promoting apoptosis. A xenograft OSCC mouse model further showed that intraperitoneal injection of VB strongly inhibited growth and lung metastasis of implanted tumor cells. Immunoblot analysis confirmed that VB effectively suppressed nuclear factor (NF)-κB activation and downstream Bcl-2/Bcl-XL expression, resulting in increased OSCC cell apoptosis. In addition, VB suppressed mRNA and protein expression of matrix metalloproteinase-9 via suppression of NF-κB activation, thereby inhibiting tumor cell metastasis. Inspiringly, compared to cisplatin-treated group, VB is a biocompatible agent without signficant side effects in vivo. Collectively, our results demonstrate that VB effectively inhibits OSCC tumor cell growth and metastasis via suppression of IκB kinase complex (IKK)/NF-κB-related signaling activation, suggesting that VB has potential use as a potent anticancer agent in OSCC therapeutic strategies.

Depletion of abundant human serum proteins by per se imprinted cryogels based on sample heterogeneity.

Macroporous cryogels were prepared and used to deplete abundant proteins. It was accomplished based on the sample heterogeneity rather than any exogenous assistance. Human serum was added in monomer solutions to synthesize molecularly imprinted polymers; therein some abundant proteins were imprinted in the polyacrylamide cryogels. Meanwhile the rare components remained aqueous. Chromatography and electrophoresis showed that albumin, serotransferrin, and most globulins were depleted by columns packed with the molecularly imprinted polymers. After the depletion, lower abundance proteins were revealed by SDS-PAGE, peptide fingerprint analysis, and identified by MALDI-TOF-MS. This is an example that a "per se imprint" protocol enables to gradually dimidiate proteomes, simplify sample complexities, and facilitate further proteome profiling or biomarker discovery.

The effects of altered BMP4 signaling in first branchial-arch-derived murine embryonic orofacial tissues.

The first branchial arch (BA1), which is derived from cranial neural crest (CNC) cells, gives rise to various orofacial tissues. Cre mice are widely used for the determination of CNC and exploration of gene functions in orofacial development. However, there is a lack of Cre mice specifically marked BA1's cells. Pax2-Cre allele was previously generated and has been widely used in the field of inner ear development. Here, by compounding Pax2-Cre and R26R-mTmG mice, we found a specific expression pattern of Pax2+ cells that marked BA1's mesenchymal cells and the BA1-derivatives. Compared to Pax2-Cre; R26R-mTmG allele, GFP+ cells were abundantly found both in BA1 and second branchial arch in Wnt1-Cre;R26R-mTmG mice. As BMP4 signaling is required for orofacial development, we over-activated Bmp4 by using Pax2-Cre; pMes-BMP4 strain. Interestingly, our results showed bilateral hyperplasia between the upper and lower teeth. We also compare the phenotypes of Wnt1-Cre; pMes-BMP4 and Pax2-Cre; pMes-BMP4 strains and found severe deformation of molar buds, palate, and maxilla-mandibular bony structures in Wnt1-Cre; pMes-BMP4 mice; however, the morphology of these orofacial organs were comparable between controls and Pax2-Cre; pMes-BMP4 mice except for bilateral hyperplastic tissues. We further explore the properties of the hyperplastic tissue and found it is not derived from Runx2+ cells but expresses Msx1, and probably caused by abnormal cell proliferation and altered expression pattern of p-Smad1/5/8. In sum, our findings suggest altering BMP4 signaling in BA1-specific cell lineage may lead to unique phenotypes in orofacial regions, further hinting that Pax2-Cre mice could be a new model for genetic manipulation of BA1-derived organogenesis in the orofacial region.

Chromatin Accessibility Predetermines Odontoblast Terminal Differentiation.

Embryonic development and stem cell differentiation are orchestrated by changes in sequential binding of regulatory transcriptional factors to their motifs. These processes are invariably accompanied by the alternations in chromatin accessibility, conformation, and histone modification. Odontoblast lineage originates from cranial neural crest cells and is crucial in dentinogenesis. Our previous work revealed several transcription factors (TFs) that promote odontoblast differentiation. However, it remains elusive as to whether chromatin accessibility affects odontoblast terminal differentiation. Herein, integration of single-cell RNA-seq and bulk RNA-seq revealed that in vitro odontoblast differentiation using dental papilla cells at E18.5 was comparable to the crown odontoblast differentiation trajectory of OC (osteocalcin)-positive odontogenic lineage. Before in vitro odontoblast differentiation, ATAC-seq and H3K27Ac CUT and Tag experiments demonstrated high accessibility of chromatin regions adjacent to genes associated with odontogenic potential. However, following odontoblastic induction, regions near mineralization-related genes became accessible. Integration of RNA-seq and ATAC-seq results further revealed that the expression levels of these genes were correlated with the accessibility of nearby chromatin. Time-course ATAC-seq experiments further demonstrated that odontoblast terminal differentiation was correlated with the occupation of the basic region/leucine zipper motif (bZIP) TF family, whereby we validated the positive role of ATF5 in vitro. Collectively, this study reports a global mapping of open chromatin regulatory elements during dentinogenesis and illustrates how these regions are regulated via dynamic binding of different TF families, resulting in odontoblast terminal differentiation. The findings also shed light on understanding the genetic regulation of dentin regeneration using dental mesenchymal stem cells.