Melanin extract from Gallus gallus domesticus promotes proliferation and differentiation of osteoblastic MG-63 cells via bone morphogenetic protein-2 signaling

BACKGROUND/OBJECTIVES: Gallus gallus domesticus (GD) is a natural mutant breed of chicken in Korea with an atypical characterization of melanin in its tissue. This study investigated the effects of melanin extracts of GD on osteoblast differentiation and inhibition of osteoclast formation. MATERIALS/METHODS: The effects of the melanin extract of GD on human osteoblast MG-63 cell differentiation were examined by evaluating cell viability, osteoblast differentiation, and expression of osteoblast-specific transcription factors such as bone morphogenetic protein 2 (BMP-2), small mothers against decapentaplegic homologs 5 (SMAD5), runt-related transcription factor 2 (RUNX2), osteocalcin and type 1 collagen (COL-1) by reverse transcription-polymerase chain reaction and western blotting analysis. We investigated the inhibitory effect of melanin on the osteoclasts formation through tartrate-resistant acid phosphatase (TRAP) activity and TRAP stains in Raw 264.7 cell. RESULTS: The melanin extract of GD was not cytotoxic to MG-63 cells at concentrations of 50-250 µg/mL. Alkaline phosphatase (ALP) activity and bone mineralization of melanin extract-treated cells increased in a dose-dependent manner from 50 to 250 µg/mL and were 149% and 129% at 250 µg/mL concentration, respectively (P < 0.05). The levels of BMP-2, osteocalcin, and COL-1 gene expression were significantly upregulated by 1.72-, 4.44-, and 2.12-fold in melanin-treated cells than in the control cells (P < 0.05). The levels of RUNX2 and SMAD5 proteins were higher in melanin-treated cells than in control vehicle-treated cells. The melanin extract attenuated the formation of receptor activator of nuclear factor kappa-B ligand-induced TRAP-positive multinucleated RAW 264.7 cells by 22%, and was 77% cytotoxic to RAW 264.7 macrophages at a concentration of 500 µg/mL. CONCLUSIONS: This study provides evidence that the melanin extract promoted osteoblast differentiation by activating BMP/SMADs/RUNX2 signaling and regulating transcription of osteogenic genes such as ALP, type I collagen, and osteocalcin. These results suggest that the effective osteoblastic differentiation induced by melanin extract from GD makes it potentially useful in maintaining bone health.

Migration of human neural stem cells toward an intracranial glioma

Many in vivo and in vitro studies have demonstrated the targeted migration of neural stem cells (NSC) to infiltrating brain tumors, including malignant glioma, highlighting a potential therapeutic approach. However, there is not enough information to apply this approach to clinical therapy. The most important things in stem cell therapy for brain tumors involve selecting the appropriate neural progenitor type and optimizing the efficiency of the cell engraftment. By histological analysis using two different live-dyes, human NSCs were shown to migrate away from the transplanted site in the direction of the expanding C6 glioma and to intermix with the tumor bed, especially with the tumor core. This intermixing occurred within 7 days when NSCs were implanted into glioma model. The time course of migratory HB1.F5 with the greatest mobility of three NSC lines was as follows. As early as 3 days after transplantation, several NSCs were found leaving the implant site, primarily approaching microsatellites and frontier cells located near the site of NSC implantation. Through 7 days post-transplantation, massive numbers of NSCs continued to be attracted to and interspersed with C6 glioma, and were finally distributed extensively throughout the whole tumor bed, including the core and penumbra of the tumor mass. However, NSCs appeared to penetrate into the tumor mass very well, whereas normal fibroblast cells could not migrate. These findings strengthen the potential for human NSCs as attractive vehicles to improve therapeutic gene delivery to cancer or glioma if they are optimized to selectively kill neoplastic cells.

Protein Expression Profiles in a Rat Cirrhotic Model Induced by Thioacetamide / 대한간학회지

BACKGROUND/AIMS: The reactive oxygen species from thioacetamide (TAA) induces rat liver cirrhosis that resembles the human disease, and it can serve as a suitable animal model for studying human liver cirrhosis. The aim of this study was to identify the molecular protein signatures via a proteomics approach with using a rat model with TAA-induced liver cirrhosis. METHODS: Male Wistar rats were treated with 0.3 g/L TAA in their drinking water. The animals were then sacrificed at 9 and 30 weeks after TAA administration. The development of liver cirrhosis was observed with histological study. The livers were processed for proteins extraction and the proteins were analyzed by 2-dimensional electrophoresis. The proteins were identified by matrix-assisted laser desorption ionizing time-of-flight mass spectrometry and this was validated by immunohistochemical staining. RESULTS: On the proteomics analysis of the liver tissues, a total of 88 proteins showed significant change in their expression between the controls and the cirrhotic rats. When the proteins were categorized by their function, they included ECM/cellular skeleton, cell proliferation/death signal, metabolism, DNA damage/stress and immune response related proteins. The level of expression gradually increased up to 30 weeks for interleukin-6 (IL-6) precursor, transforming growth factor-beta (TGF-beta) induced protein, TIMP-1 and MMP-9. Cytochrome P450 2B, which is required for the metabolic activation of TAA, also showed the same increasing pattern. In contrast, the expression level of the proteins did not show a significant change at 9 weeks, but this increased to 3-fold at 30 weeks for carbonic anhydrase VII, ras related protein Rab 6, Annexin A2, neurofibromatosis type 2 and aldehyde dehydrogenase. CONCLUSIONS: This study showed that there is a repertoire of proteins during the development of liver cirrhosis via TAA. In this model, IL-6, TGF-beta, MMP-9 and TIMP-1 were reconfirmed as the molecular signatures during the development of TAA-induced liver cirrhosis.

Proteomics of Protein Expression Profiling in Tissues with Different Radiosensitivity / 대한방사선종양학회지

PURPOSE: The purpose of this study was to identify Radiosensitivity of proteins in tissues with different radiosensitivity. MATERIALS AND METHODS: C3H/HeJ mice were exposed to 10 Gy. The mice were sacrifiud 8 hrs after radiation. Their spleen and liver tissues were collected and analyzed histologicaly for apoptosis. The expressions of radiosusceptibility protein were analyzed by 2-dimensional electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. RESULTS: The peak of apoptosis levels were 35.3+/-1.7% in spleen and 0.6+/-0.2% in liver at 8 hrs after radiation. Liver, radioresistant tissues, showed that the levels of ROS metabolism related to proteins such as cytochromm c, glutathione S transferase, NADH dehydrogenase, riken cDNA and peroxiredoxin VI increased after radiation. The expression of cytochrome c increased significantly in spleen and liver tissues after radiation. In spleen, radiosensitivity tissue, the identified proteins showed a significantly quantitative alteration following radiation. It was categorized as signal transduction, apoptosis, cytokine, Ca signal related protein, stress-related protein, cytoskeletal regulation, ROS metabolism, and others. CONCLUSION: Differences of radiation-induced apoptosis by tissues specifted were coupled with the induction of related radiosensitivity and radioresistant proteins. The result suggests that apoptosis relate protein and redox proteins play important roles in this radiosusceptibility.