Enhancing the Efficiency of Mild-Temperature Photothermal Therapy for Cancer Assisting with Various Strategies.

Conventional photothermal therapy (PTT) irradiates the tumor tissues by elevating the temperature above 48 °C to exert thermal ablation, killing tumor cells. However, thermal ablation during PTT harmfully damages the surrounding normal tissues, post-treatment inflammatory responses, rapid metastasis due to the short-term mass release of tumor-cellular contents, or other side effects. To circumvent this limitation, mild-temperature photothermal therapy (MTPTT) was introduced to replace PTT as it exerts its activity at a therapeutic temperature of 42-45 °C. However, the significantly low therapeutic effect comes due to the thermoresistance of cancer cells as MTPTT figures out some of the side-effects issues. Herein, our current review suggested the mechanism and various strategies for improving the efficacy of MTPTT. Especially, heat shock proteins (HSPs) are molecular chaperones overexpressed in tumor cells and implicated in several cellular heat shock responses. Therefore, we introduced some methods to inhibit activity, reduce expression levels, and hinder the function of HSPs during MTPTT treatment. Moreover, other strategies also were emphasized, including nucleus damage, energy inhibition, and autophagy mediation. In addition, some therapies, like radiotherapy, chemotherapy, photodynamic therapy, and immunotherapy, exhibited a significant synergistic effect to assist MTPTT. Our current review provides a basis for further studies and a new approach for the clinical application of MTPTT.

Dual roles of TGF-ß signaling in the regulation of dental epithelial cell proliferation.

The purpose of this study is to investigate the molecular mechanisms and biological function of TGF-ß-activated Smad1/5 in dental epithelium. Immunohistochemistry was used to detect the expressions of TGF-ß signaling-related gene in mice molar germ. Primary dental epithelial cells were cultured and treated with TGF-ß1 at a concentration of 0.5 or 5 ng/mL. Small molecular inhibitors, SB431542 and ML347, was used to inhibite ALK5 and ALK1/2, respectively. Small interfering RNA was used to knock down Smad1/5 or Smad2/3. The proliferation rate of cells was evaluated by EdU assay. In the basal layer of dental epithelial bud TGF-ß1 and p-Smad1/5 were highly expressed, and in the interior of the epithelial bud TGF-ß1 was lowly expressed, whereas p-Smad2/3 was highly expressed. In primary cultured dental epithelial cells, low concentration of TGF-ß1 activated Smad2/3 but not Smad1/5, while high concentration of TGF-ß1 was able to activate both Smad2/3 and Smad1/5. SB431542 but not ML347 was able to block the phosphorylation of Smad2/3 by TGF-ß1. Either SB431542 or ML347 was able to block the phosphorylation of Smad1/5 by TGF-ß1. EdU staining showed that high concentration of TGF-ß1 promoted dental epithelial cell proliferation, which was reversed by silencing Smad1/5, whereas low concentration of TGF-ß1 inhibited cell proliferation, which was reversed by silencing Smad2/3. In conclusions, TGF-ß exhibits dual roles in the regulation of dental epithelial cell proliferation through two pathways. On the one hand, TGF-ß activates canonical Smad2/3 signaling through ALK5, inhibiting the proliferation of internal dental epithelial cells. On the other hand, TGF-ß activates noncanonical Smad1/5 signaling through ALK1/2-ALK5, promoting the proliferation of basal cells in the dental epithelial bud.

HDAC6 Regulates the Fusion of Autophagosome and Lysosome to Involve in Odontoblast Differentiation.

Odontoblast differentiation is an important process during tooth development in which pre-odontoblasts undergo elongation, polarization, and finally become mature secretory odontoblasts. Many factors have been found to regulate the process, and our previous studies demonstrated that autophagy plays an important role in tooth development and promotes odontoblastic differentiation in an inflammatory environment. However, it remains unclear how autophagy is modulated during odontoblast differentiation. In this study, we found that HDAC6 was involved in odontoblast differentiation. The odontoblastic differentiation capacity of human dental papilla cells was impaired upon HDAC6 inhibition. Moreover, we found that HDAC6 and autophagy exhibited similar expression patterns during odontoblast differentiation both in vivo and in vitro; the expression of HDAC6 and the autophagy related proteins ATG5 and LC3 increased as differentiation progressed. Upon knockdown of HDAC6, LC3 puncta were increased in cytoplasm and the autophagy substrate P62 was also increased, suggesting that autophagic flux was affected in human dental papilla cells. Next, we determined the mechanism during odontoblastic differentiation and found that the HDAC6 substrate acetylated-Tubulin was up-regulated when HDAC6 was knocked down, and LAMP2, LC3, and P62 protein levels were increased; however, the levels of ATG5 and Beclin1 showed no obvious change. Autophagosomes accumulated while the number of autolysosomes was decreased as determined by mRFP-GFP-LC3 plasmid labeling. This suggested that the fusion between autophagosomes and lysosomes was blocked, thus affecting the autophagic process during odontoblast differentiation. In conclusion, HDAC6 regulates the fusion of autophagosomes and lysosomes during odontoblast differentiation. When HDAC6 is inhibited, autophagosomes can't fuse with lysosomes, autophagy activity is decreased, and it leads to down-regulation of odontoblastic differentiation capacity. This provides a new perspective on the role of autophagy in odontoblast differentiation.

DLX3 Inhibits the Proliferation of Human Dental Pulp Cells Through Inactivation of Canonical Wnt/ß-Catenin Signaling Pathway.

Homeodomain gene Distal-less-3 (Dlx3) plays an important role during tooth development. Our previous studies indicate that DLX3 inhibits proliferation of human dental pulp cells (hDPCs). However, the mechanism of DLX3 regulating proliferation of hDPCs and maintaining the quiescence of the cells remain unknown. Given the importance of canonical Wnt signaling in the proliferation of dental pulp cell and tooth development, we hypothesized that DLX3 inhibited proliferation of hDPCs through inactivation of canonical Wnt signaling. With overexpression or knock-down of DLX3 in primary hDPCs, we found DLX3 down regulated canonical Wnt signaling and its downstream target genes. And when the DLX3 overexpressed-cells were treated with lithium chloride, the proliferation inhibition by DLX3 was reversed. We also found that DLX3 enhanced the expression of DKK1 and the reduced proliferation of hDPCs by DLX3 was reversed with knock-down of DKK1. Furthermore, luciferase reporter assay and chromatin immunoprecipitation assay showed DLX3 was able to bind to Dkk1 promoter region from nucleotides (nt) -1656 to -1245, and stimulated Dkk1 promoter activity. Mutagenesis studies further revealed two DLX3 responsive elements in Dkk1 promoter. Taken together, our data indicate that DLX3 inhibits proliferation of hDPCs via inactivation of Wnt/ß-catenin signaling pathway by directly binding to Dkk1 promoter and increasing its expression.

TGF-ß signaling inhibits canonical BMP signaling pathway during palate development.

During early palate development, gene expression and regulation exhibit heterogeneity along the anterior-posterior axis. Transforming growth factor-ß (TGF-ß) and bone morphogenetic protein (BMP) signaling pathways play essential roles in secondary palatal formation but the exact relationship between the TGF-ß and BMP pathways in palate development remains unknown. Here, we demonstrate that, during early secondary palate development, phospho-(p)Smad1/5/8 is highly expressed in the anterior palate but relatively lowly expressed in the posterior palate. Conversely, pSmad2/3 has a lower expression level in the anterior palate than in the posterior palate. With the BRE-Gal reporter, we found that the canonical BMP signaling pathway was not activated in the anterior palate but exhibited a moderate level in the posterior palate. Co-immunoprecipitation revealed that Smad4 bound to pSmad1/5/8 only in the posterior palate and not in the anterior palate. Knocking-out of Tgfbr2 (Wnt1-Cre;Tgfbr2 f/f;BRE) in the palatal mesenchyme enhanced canonical BMP activity in the posterior palate but not in the anterior palate, because of decreased pSmad2/3. pSmad1/5/8-Smad4 complexes were found to be dramatically increased in posterior palatal mesenchymal cells at embryonic day 13.5 cultured in the presence of SB525334. Proximity ligation assay also showed pSmad1/5/8-Smad4 complexes were increased in the posterior palate of Wnt1-Cre;Tgfbr2 f/f mice. Therefore, the reduction of pSmad2/3 level in the palatal mesenchyme of Wnt1-Cre;Tgfbr2 f/f;BRE mice contributes primarily to the increase of pSmad1/5/8-Smad4 complexes leading to enhanced canonical BMP activity in the posterior palate. Moreover, during early development, canonical BMP signaling operates in the posterior palate but is completely absent in the anterior palate. Canonical TGF-ß signaling suppresses canonical BMP signaling activity in the posterior palate by competing limited Smad4.