Cudraxanthone D Regulates Epithelial-Mesenchymal Transition by Autophagy Inhibition in Oral Squamous Cell Carcinoma Cell Lines.

Cudraxanthone D (CD), derived from the root bark of Cudrania tricuspidata, is a natural xanthone compound. However, the biological activity of CD in terms of human metabolism has been barely reported to date. Autophagy is known as a self-degradation process related to cancer cell viability and metastasis. Herein, we investigated the effects of CD on human oral squamous cell carcinoma (OSCC) metastatic related cell phenotype. We confirmed that CD effectively decreased proliferation and viability in a time- and dose-dependent manner in human OSCC cells. In addition, OSCC cell migration, invasion, and EMT were inhibited by CD. To further determine the underlying mechanism of CD's inhibition of cell metastatic potential, we established the relationship between EMT and autophagy in OSCC cells. Thus, our findings indicated that CD inhibited the potential metastatic abilities of OSCC cells by attenuating autophagy.

A Case of Seizure Revealing Fahr's Syndrome with Primary Hypoparathyroidism.

BACKGROUND Idiopathic basal ganglia calcification, also known as Fahr's disease or Fahr's syndrome, is a rare neurological disorder characterized by abnormal calcified deposits in the basal ganglia. Here, we report a case of Fahr's syndrome with calcification of the basal ganglia due to hypoparathyroidism in a patient with seizures. CASE REPORT A 52-year-old male patient visited our clinic with seizures. Brain computed tomography (CT) showed bilateral symmetrical calcifications in cerebellar white matter, the corpus striatum, the posterior thalami, and the centrum semiovale of both cerebral hemispheres. He had symptoms of hypocalcemia and low parathyroid hormone levels. The patient was diagnosed with Fahr's syndrome due to primary hypoparathyroidism. He underwent calcium supplementation and calcifediol treatment. His symptoms improved, and he was discharged from the hospital. CONCLUSIONS In patients with hypocalcemia accompanied by parathyroid dysfunction, neurological examination and CT should be performed to confirm abnormal intracranial calcification.

Cordycepin Accelerates Osteoblast Mineralization and Attenuates Osteoclast Differentiation In Vitro.

Bone homeostasis destruction is triggered by the uncontrolled activity of osteoblasts and osteoclasts. Targeting both the regulation of bone formation and resorption is a promising strategy for treating bone disorders. Cordycepin is a major component of Chinese caterpillar fungus Cordyceps militaris. It exerts a variety of biological actions in various cells and animal models. However, its function on bone metabolism remains unclear. In the present study, we discovered a dual-action function of cordycepin in murine MC3T3-E1 and RAW264.7 cells. MC3T3-E1 cells were cultured in an osteogenic medium in the presence of 1 µM cordycepin for up two weeks. Cordycepin was used for effects of osteoblast and osteoclast differentiation. Cell viability was measured using the MTT assay. Osteoblast differentiation was confirmed by alizarin red staining, ALP activity, western blot, and real-time PCR. Osteoclast differentiation and autophagic activity were confirmed via TRAP staining, pit formation assay, confocal microscopy, western blot, and real-time PCR. Cordycepin promoted osteoblast differentiation, matrix mineralization, and induction of osteoblast markers via BMP2/Runx2/Osterix pathway. On the other hand, RAW264.7 cells were differentiated into osteoclast by RANKL treatment for 72 h. 1 µM cordycepin significantly inhibited RANKL-induced osteoclast formation and resorption activity through disturbing the actin ring-formatted sealing zone and activating cathepsin K and MMP9. These findings indicate that cordycepin might be an innovative dual-action therapeutic agent for bone disease caused by an imbalance of osteoblasts and osteoclasts.

Induction of Apoptosis and Inhibition of Epithelial Mesenchymal Transition by α-Mangostin in MG-63 Cell Lines.

Osteosarcoma is the most common bone primary malignant tumor and nearly 30% of patients still die from osteosarcoma due to metastasis or recurrence. Thus, it is necessary to develop effective new chemotherapeutic agents for osteosarcoma treatment. α-Mangostin is a xanthone derivative shown to have antioxidant and anticarcinogen properties. However, the molecular mechanisms underlying the antimetastatic effects of osteosarcoma remain unclear. In metastasis progression, epithelial mesenchymal transition (EMT) is a process that plays important roles in development, cell polarity, and increased invasion and migration. This study focused on the induction of apoptosis and inhibition of EMT process by α-mangostin in human osteosarcoma cell line MG63. α-Mangostin treatments on MG63 cells not only showed the several lines of evidence of apoptotic cell death but also inhibited cell migration, invasion, and EMT-inducing transcription factor. In conclusion, we demonstrate that the α-mangostin induces apoptosis via mitochondrial pathway and suppresses metastasis of osteosarcoma cells by inhibiting EMT.

The Effects of Kaempferol-Inhibited Autophagy on Osteoclast Formation.

Kaempferol, a flavonoid compound, is derived from the rhizome of Kaempferia galanga L., which is used in traditional medicine in Asia. Autophagy has pleiotropic functions that are involved in cell growth, survival, nutrient supply under starvation, defense against pathogens, and antigen presentation. There are many studies dealing with the inhibitory effects of natural flavonoids in bone resorption. However, no studies have explained the relationship between the autophagic and inhibitory processes of osteoclastogenesis by natural flavonoids. The present study was undertaken to investigate the inhibitory effects of osteoclastogenesis through the autophagy inhibition process stimulated by kaempferol in murin macrophage (RAW 264.7) cells. The cytotoxic effect of Kaempferol was investigated by MTT assay. The osteoclast differentiation and autophagic process were confirmed via tartrate-resistant acid phosphatase (TRAP) staining, pit formation assay, western blot, and real-time PCR. Kaempferol controlled the expression of autophagy-related factors and in particular, it strongly inhibited the expression of p62/SQSTM1. In the western blot and real time-PCR analysis, when autophagy was suppressed with the application of 3-Methyladenine (3-MA) only, osteoclast and apoptosis related factors were not significantly affected. However, we found that after cells were treated with kaempferol, these factors inhibited autophagy and activated apoptosis. Therefore, we presume that kaempferol-inhibited autophagy activated apoptosis by degradation of p62/SQSTM1. Further study of the p62/SQSTM1 gene as a target in the autophagy mechanism, may help to delineate the potential role of kaempferol in the treatment of bone metabolism disorders.

A novel combination treatment to stimulate bone healing and regeneration under hypoxic conditions: photobiomodulation and melatonin.

Melatonin has anabolic effects on the bone, even under hypoxia, and laser irradiation has been shown to improve osteoblastic differentiation. The aim of this study was to investigate whether laser irradiation and melatonin would have synergistic effects on osteoblastic differentiation and mineralization under hypoxic conditions. MC3T3-E1 cells were exposed to 1% oxygen tension for the hypoxia condition. The cells were divided into four groups: G1-osteoblast differentiation medium only (as the hypoxic condition), G2-treatment with 50 µM melatonin only, G3-laser irradiation (808 nm, 80 mW, GaAlAs diode) only, and G4-treatment with 50 µM melatonin and laser irradiation (808 nm, 80 mW, GaAlAs diode). Immunoblotting showed that osterix expression was markedly increased in the melatonin-treated and laser-irradiated cells at 48 and 72 h. In addition, alkaline phosphatase activity significantly increased and continued to rise throughout the experiment. Alizarin Red staining showed markedly increased mineralized nodules as compared with only melatonin-treated or laser-irradiated cells at day 7, which significantly increased by day 14. Moreover, when melatonin-treated cells were laser-irradiated, the differentiation and mineralization of cells were found to involve p38 MAPK and PRKD1 signaling mechanisms. However, the enhanced effects of laser irradiation with melatonin were markedly inhibited when the cells were treated with luzindole, a selective melatonin receptor antagonist. Therefore, we concluded that laser irradiation could promote the effect of melatonin on the differentiation and mineralization of MC3T3-E1 cells under hypoxic conditions, and that this process is mediated through melatonin 1/2 receptors and PKRD/p38 signaling pathways.

The role of kaempferol-induced autophagy on differentiation and mineralization of osteoblastic MC3T3-E1 cells.

BACKGROUND: Kaempferol, a kind of flavonol, has been reported to possess various osteogenic biological activities, such as inhibiting bone resorption of osteoclasts and promoting the differentiation and mineralization of preosteoblasts. However, the precise cellular mechanism of action of kaempferol in osteogenesis is elusive. Autophagy is a major intracellular degradation system, which plays an important role in cell growth, survival, differentiation and homeostasis in mammals. Recent studies showed that autophagy appeared to be involved in the degradation of osteoclasts, osteoblasts and osteocytes, potentially pointing to a new pathogenic mechanism of bone homeostasis and bone marrow disease. The potential correlation between autophagy, osteogenesis and flavonoids is unclear. METHODS: The present study verified that kaempferol promoted osteogenic differentiation and mineralization and that it elevated osteogenic gene expression based on alkaline phosphatase (ALP) activity, alizarin red staining and quantitative PCR. And then we found that kaempferol induced autophagy by acridine orange (AO) and monodansylcadaverine (MDC) staining and autophagy-related protein expression. The correlation between kaempferol-induced autophagy and the osteogenic process was confirmed by the autophagy inhibitor 3-methyladenine (3-MA). RESULTS: Kaempferol promoted the proliferation, differentiation and mineralization of osteoblasts at a concentration of 10 µM. Kaempferol showed cytotoxic properties at concentrations above 50 µM. Concentrations above 10 µM decreased ALP activity, whereas those up to 10 µM increased ALP activity. Kaempferol at concentrations up to 10 µM also increased the expression of the osteoblast- activated factors RUNX-2, osterix, BMP-2 and collagen I according to RT-PCR analyses. 10 µM or less, the higher of the concentration and over time, kaempferol promoted the activity of osteoblasts. Kaempferol induced autophagy. It also increased the expression of the autophagy-related factors beclin-1, SQSTM1/p62 and the conversion of LC3-II from LC3-I. The application of 3-MA decreased the activity of ALP and the autophagy induced by kaempferol. In the RT-PCR analysis, the expression of RUNX-2, osterix, BMP-2 and collagen I was decreased. CONCLUSION: The present study showed that kaempferol stimulated the osteogenic differentiation of cultured osteoblasts by inducing autophagy.

α-Mangostin Induces Apoptosis and Cell Cycle Arrest in Oral Squamous Cell Carcinoma Cell.

Mangosteen has long been used as a traditional medicine and is known to have antibacterial, antioxidant, and anticancer effects. Although the effects of α-mangostin, a natural compound extracted from the pericarp of mangosteen, have been investigated in many studies, there is limited data on the effects of the compound in human oral squamous cell carcinoma (OSCC). In this study, α-mangostin was assessed as a potential anticancer agent against human OSCC cells. α-Mangostin inhibited cell proliferation and induced cell death in OSCC cells in a dose- and time-dependent manner with little to no effect on normal human PDLF cells. α-Mangostin treatment clearly showed apoptotic evidences such as nuclear fragmentation and accumulation of annexin V and PI-positive cells on OSCC cells. α-Mangostin treatment also caused the collapse of mitochondrial membrane potential and the translocation of cytochrome c from the mitochondria into the cytosol. The expressions of the mitochondria-related proteins were activated by α-mangostin. Treatment with α-mangostin also induced G1 phase arrest and downregulated cell cycle-related proteins (CDK/cyclin). Hence, α-mangostin specifically induces cell death and inhibits proliferation in OSCC cells via the intrinsic apoptosis pathway and cell cycle arrest at the G1 phase, suggesting that α-mangostin may be an effective agent for the treatment of OSCC.

Autophagic Cell Death by Poncirus trifoliata Rafin., a Traditional Oriental Medicine, in Human Oral Cancer HSC-4 Cells.

Poncirus trifoliata Rafin. has long been used as anti-inflammatory and antiallergic agent to treat gastrointestinal disorders and pulmonary diseases such as indigestion, constipation, chest fullness, chest pain, bronchitis, and sputum in Korea. P. trifoliata extract has recently been reported to possess anticancer properties; however, its mechanisms of action remain unclear. In this study, its antiproliferative effects and possible mechanisms were investigated in HSC-4 cells. The methanol extract of P. trifoliata (MEPT) significantly decreased the proliferation of HSC-4 cells (inhibitory concentration (IC)50 = 142.7 µg/mL) in a dose-dependent manner. While there were no significant changes observed upon cell cycle analysis and ANNEXIN V and 7-AAD double staining in the MEPT-treated groups, the intensity of acidic vesicular organelle (AVO) staining and microtubule-associated protein 1 light chain (LC) 3-II protein expression increased in response to MEPT treatment. Furthermore, 3-methyladenine (3-MA, autophagy inhibitor) effectively blocked the MEPT-induced cytotoxicity of HSC-4 cells and triggered the activation of p38 and extracellular signal-regulated kinases (ERK) proteins. Taken together, our results indicate that MEPT is a potent autophagy agonist in oral cancer cells with antitumor therapeutic potential that acts through the mitogen-activated protein kinase (MAPK) pathway.

Effect of low-level laser therapy on oral keratinocytes exposed to bisphosphonate.

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a side effect of bisphosphonate therapy. However, its pathophysiology is not yet fully elucidated, and effective treatment of BRONJ remains unclear. The aim of this study is to investigate the effects of alendronate on oral keratinocytes and of low-level laser therapy (LLLT) on alendronate-treated keratinocytes, specifically by evaluating their viability, apoptosis, and wound healing function after irradiation. Oral keratinocyte cells (HaCaT) were exposed to 25 µM alendronate. Then, laser irradiation was performed with a low-level Ga-Al-As laser (λ = 808 ± 3 nm, 80 mW, and 80 mA; NDLux, Seoul, Korea) using 1.2 J/cm(2) energy dose. Viability was analyzed using MTT assay. Apoptosis was measured by Hoechst staining, caspase assay. Changes in secretion of IL-8, VEGF, and collagen type I were studied by ELISA and immunofluorescence microscopy. Scratch wound assays were also performed to measure cellular migration. Our results show that alendronate inhibits keratinocyte viability, expression of IL-8, VEGF, and collagen type I which are intimately related to healing events and cell migration while promoting apoptosis. Our results serve to demonstrate the utility of LLLT in partially overcoming the inhibitory effects of this bisphosphonate. From these results, the authors believe that the present study will provide an experimental basis for a fuller explanation of the clinical effects of LLLT as a BRONJ treatment modality.

Stanford type A aortic dissection secondary to infectious aortitis: a case report.

Nowadays, infectious aortitis has become a rare disease thanks to antibiotics, but remains life-threatening. We present a case of a patient with acupuncture-induced infectious aortitis leading to aortic dissection. Chest computed-tomogram scan revealed Stanford type A dissection with pericardial effusion. Under the impression of an impending rupture, emergent surgery was performed. During surgery, infectious aortitis was identified incidentally, so she underwent resection of the infected aorta including surrounding tissues. Then the ascending aorta and hemi-arch were replaced with a prosthetic graft as an in situ fashion. The resected tissue and blood cultures revealed Staphylococcus aureus, so prolonged antibiotherapy was prescribed.

Low-level laser therapy induces the expressions of BMP-2, osteocalcin, and TGF-ß1 in hypoxic-cultured human osteoblasts.

The aim of this study was to examine the effect of low-level laser therapy (LLLT) on the cell viability and the expression of hypoxia-inducible factor-1s (HIF-1s), bone morphogenic protein-2 (BMP-2), osteocalcin, type I collagen, transforming growth factor-ß1 (TGF-ß1), and Akt in hypoxic-cultured human osteoblasts. Human fetal osteoblast cells (cell line 1.19) were cultured under 1 % oxygen tension for 72 h. Cell cultures were divided into two groups. At the experimental side, low-level laser (808 nm, GaAlAs diode) was applied at 0, 24, and 48 h. After irradiation, each cell culture was incubated 24 h more under hypoxia. Total energy was 1.2, 2.4, and 3.6 J/cm(2), respectively. Non-irradiated cultures served as controls. Comparisons between the two groups were analyzed by t test; a p value <0.05 was considered statistically significant. Hypoxia resulted in a decrease in the expression of type I collagen, osteocalcin, and TGF-ß1 (p < 0.001, p < 0.001, and p < 0.01, respectively). Cell viability and BMP-2 expression were not decreased by hypoxic condition. On the other hand, LLLT on hypoxic-cultured osteoblast promoted the expression of BMP-2, osteocalcin, and TGF-ß1 (p < 0.05, p < 0.01, and p < 0.001, respectively). Cell proliferation was also increased time-dependently. However, hypoxia decreased in type I collagen expression (p < 0.001), and LLLT did not affect type I collagen expression in hypoxic-cultured osteoblasts. Furthermore, LLLT inhibited HIF-1 and Akt expression in hypoxic conditioned osteoblasts. We concluded that LLLT induces the expression of BMP-2, osteocalcin, and TGF- ß1 in 1 % hypoxic-cultured human osteoblasts.

Purified extract from Clematis mandshurica prevents adenoviral-TRAIL induced apoptosis on rat articular chondrocytes.

Since TNF-related apoptosis inducing ligand (TRAIL) is one of several apoptotic stimuli on articular chondrocytes, the modulation of the mechanism mediated by TRAIL could be considered as a novel strategy for the treatment of osteoarthritis (OA). Previous studies demonstrated that Clematis mandshurica prevents staurosporin-induced apoptosis in articular chondrocytes. This study was undertaken to examine whether Clematis mandshurica could prevent TRAIL-induced apoptosis in articular chondrocytes. Our data show that Clematis mandshurica prevents adenoviral TRAIL (Ad-TRAIL)-induced apoptosis in primary cultured articular chondrocytes. Clematis mandshurica prevents Ad-TRAIL-induced down-regulation of 14-3-3 and phosphorylated Akt. In addition, Clematis mandshurica treatment prevents the Ad-TRAIL-induced reduction of the interactions between 14-3-3 with phospho-ser112-Bad and phospho-ser136-Bad, and BcL-xL with phospho-ser155-Bad. A better understanding of the mechanism underlying inhibition of apoptosis in OA chondrocytes by Clematis mandshurica might lead to the development of a new therapeutic strategy for OA.

Inactivation of the UGPase1 gene causes genic male sterility and endosperm chalkiness in rice (Oryza sativa L.).

A rice genic male-sterility gene ms-h is recessive and has a pleiotropic effect on the chalky endosperm. After fine mapping, nucleotide sequencing analysis of the ms-h gene revealed a single nucleotide substitution at the 3'-splice junction of the 14th intron of the UDP-glucose pyrophosphorylase 1 (UGPase1; EC2.7.7.9) gene, which causes the expression of two mature transcripts with abnormal sizes caused by the aberrant splicing. An in vitro functional assay showed that both proteins encoded by the two abnormal transcripts have no UGPase activity. The suppression of UGPase by the introduction of a UGPase1-RNAi construct in wild-type plants nearly eliminated seed set because of the male defect, with developmental retardation similar to the ms-h mutant phenotype, whereas overexpression of UGPase1 in ms-h mutant plants restored male fertility and the transformants produced T(1) seeds that segregated into normal and chalky endosperms. In addition, both phenotypes were co-segregated with the UGPase1 transgene in segregating T(1) plants, which demonstrates that UGPase1 has functional roles in both male sterility and the development of a chalky endosperm. Our results suggest that UGPase1 plays a key role in pollen development as well as seed carbohydrate metabolism.

A purified extract from Clematis mandshurica prevents staurosporin-induced downregulation of 14-3-3 and subsequent apoptosis on rat chondrocytes.

OBJECTIVE: To dissect the mechanism of the protection of staurosporin-induced apoptosis on rat chondrocytes by a purified extract from Clematis mandshurica. DESIGN: Primary cultured rat articular chondrocytes as well as RCJ3.1C.18 cells were incubated with 1 microM staurosporin and 300 microg/ml purified extract from Clematis mandshurica. Western blot assay, silencing 14-3-3 gene and immunoprecipitation were conducted. RESULTS: Clematis mandshurica prevented staurosporin-induced downregulation of several antiapoptotic bcl-2 family proteins Bcl-xL and Bcl-2, and staurosporin-induced upregulation of an apoptotic bcl-2 family protein Bax. Clematis mandshurica also prevented staurosporin-induced downregulation of a premitochondrial antiapoptotic protein 14-3-3. It is noticeable that siRNA to 14-3-3 abolished the prevention of caspase-3 activation by Clematis mandshurica. Furthermore viability assay corroborated that silencing of 14-3-3 gene abolished this apoptosis protection efficacy by Clematis mandshurica. Immunoprecipitation assay elucidated that Clematis mandshurica prevented the staurosporin-induced reduction of the interactions between 14-3-3 with phospho-ser112-Bad and Bcl-xL to phospho-ser155-Bad. CONCLUSIONS: Clematis mandshurica prevents staurosporin-induced apoptosis of rat chondrocytes via 14-3-3.