Yonghui Dong

The Fascinating Effects of Baicalein on Cancer: A Review

Cancer is one of the leading causes of death worldwide and a major global health problem. In recent decades, the rates of both mortality and morbidity of cancer have rapidly increased for a variety of reasons. Despite treatment options, there are serious side effects associated with chemotherapy drugs and multiple forms of drug resistance that significantly reduce their effects. There is an accumulating amount of evidence on the pharmacological activities of baicalein (e.g., anti-inflammatory, antioxidant, antiviral, and antitumor effects). Furthermore, there has been great progress in elucidating the target mechanisms and signaling pathways of baicalein’s anti-cancer potential. The anti-tumor functions of baicalein are mainly due to its capacities to inhibit complexes of cyclins to regulate the cell cycle, to scavenge oxidative radicals, to attenuate mitogen activated protein kinase (MAPK), protein kinase B (Akt) or mammalian target of rapamycin (mTOR) activities, to induce apoptosis by activating caspase-9/-3 and to inhibit tumorinvasion and metastasis by reducing the expression of matrix metalloproteinase-2/-9 (MMP-2/-9). In this review, we focused on the relevant biological mechanisms of baicalein involved in inhibiting various cancers, such as bladder cancer, breast cancer, and ovarian cancer. Moreover, we also summarized the specific mechanisms by which baicalein inhibited the growth of various tumors in vivo. Taken together, baicalein may be developed as a potential, novel anticancer drug to treat tumors.

Inhibitory effects of high glucose/insulin environment on osteoclast formation and resorption in vitro

Patients with type 2 diabetes mellitus (T2DM) exhibit hyperglycemia and hyperinsulinemia and increased risk of fracture at early stage, but they were found to have normal or even enhanced bone mineral density (BMD). This study was aimed to examine the molecular mechanisms governing changes in bone structure and integrity under both hyperglycemic and hyperinsulinemic conditions. Monocytes were isolated from the bone marrow of the C57BL/6 mice, induced to differentiate into osteoclasts by receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) and exposed to high glucose (33.6 mmol/L), high insulin (1 μmol/L), or a combination of high glucose/high insulin (33.6 mmol/L glucose and 1 μmol/L insulin). Cells cultured in α-MEM alone served as control. After four days of incubation, the cells were harvested and stained for tartrate resistant acid phosphatase (TRAP). Osteoclast-related genes including RANK, cathepsin K and TRAP were determined by using real-time PCR. The resorptive activity of osteoclasts was measured by using a pit formation assay. Osteoclasts that were derived from monocytes were of multinucleated nature and positive for TRAP, a characteristic marker of osteoclasts. Cell counting showed that the number of osteoclasts was much less in high glucose and high glucose/high insulin groups than in normal glucose and high insulin groups. The expression levels of RANK and cathepsin K were significantly decreased in high glucose, high insulin and high glucose/high insulin groups as compared with normal glucose group, and the TRAP activity was substantially inhibited in high glucose environment. The pit formation assay revealed that the resorptive activity of osteoclasts was obviously decreased in high glucose group and high glucose/high insulin group as compared with normal group. It was concluded that osteoclastogenesis is suppressed under hyperglycemic and hyperinsulinemic conditions, suggesting a disruption of the bone metabolism in diabetic patients.SummaryPatients with type 2 diabetes mellitus (T2DM) exhibit hyperglycemia and hyperinsulinemia and increased risk of fracture at early stage, but they were found to have normal or even enhanced bone mineral density (BMD). This study was aimed to examine the molecular mechanisms governing changes in bone structure and integrity under both hyperglycemic and hyperinsulinemic conditions. Monocytes were isolated from the bone marrow of the C57BL/6 mice, induced to differentiate into osteoclasts by receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) and exposed to high glucose (33.6 mmol/L), high insulin (1 μmol/L), or a combination of high glucose/high insulin (33.6 mmol/L glucose and 1 μmol/L insulin). Cells cultured in α-MEM alone served as control. After four days of incubation, the cells were harvested and stained for tartrate resistant acid phosphatase (TRAP). Osteoclast-related genes including RANK, cathepsin K and TRAP were determined by using real-time PCR. The resorptive activity of osteoclasts was measured by using a pit formation assay. Osteoclasts that were derived from monocytes were of multinucleated nature and positive for TRAP, a characteristic marker of osteoclasts. Cell counting showed that the number of osteoclasts was much less in high glucose and high glucose/high insulin groups than in normal glucose and high insulin groups. The expression levels of RANK and cathepsin K were significantly decreased in high glucose, high insulin and high glucose/high insulin groups as compared with normal glucose group, and the TRAP activity was substantially inhibited in high glucose environment. The pit formation assay revealed that the resorptive activity of osteoclasts was obviously decreased in high glucose group and high glucose/high insulin group as compared with normal group. It was concluded that osteoclastogenesis is suppressed under hyperglycemic and hyperinsulinemic conditions, suggesting a disruption of the bone metabolism in diabetic patients.

Inhibition of SDF-1α/CXCR4 Signalling in Subchondral Bone Attenuates Post-Traumatic Osteoarthritis

Previous studies showed that SDF-1α is a catabolic factor that can infiltrate cartilage, decrease proteoglycan content, and increase MMP-13 activity. Inhibiting the SDF-1α/CXCR4 signalling pathway can attenuate the pathogenesis of osteoarthritis (OA). Recent studies have also shown that SDF-1α enhances chondrocyte proliferation and maturation. These results appear to be contradictory. In the current study, we used a destabilisation OA animal model to investigate the effects of SDF-1α/CXCR4 signalling in the tibial subchondral bone and the OA pathological process. Post-traumatic osteoarthritis (PTOA) mice models were prepared by transecting the anterior cruciate ligament (ACLT), or a sham surgery was performed, in a total of 30 mice. Mice were treated with phosphate buffer saline (PBS) or AMD3100 (an inhibitor of CXCR4) and sacrificed at 30 days post ACLT or sham surgery. Tibial subchondral bone status was quantified by micro-computed tomography (μCT). Knee-joint histology was analysed to examine the articular cartilage and joint degeneration. The levels of SDF-1α and collagen type I c-telopeptidefragments (CTX-I) were quantified by ELISA. Bone marrow mononuclear cells (BMMCs) were used to clarify the effects of SDF-1α on osteoclast formation and activity in vivo. μCT analysis revealed significant loss of trabecular bone from tibial subchondral bone post-ACLT, which was effectively prevented by AMD3100. AMD3100 could partially prevent bone loss and articular cartilage degeneration. Serum biomarkers revealed an increase in SDF-1α and bone resorption, which were also reduced by AMD3100. SDF-1α can promote osteoclast formation and the expression oftartrate resistant acid phosphatase (TRAP), cathepsin K (CK), and matrix metalloproteinase (MMP)-9 in osteoclasts by activating the MAPK pathway, including ERK and p38, but not JNK. In conclusion, inhibition of SDF-1α/CXCR4signalling was able to prevent trabecular bone loss and attenuated cartilage degeneration in PTOA mice.

Dose-dependent inhibitory effects of zoledronic acid on osteoblast viability and function in vitro

Zoledronic acid (ZA), which is one of the most potent and efficacious bisphosphonates, has been commonly used in clinical practice for the treatment of various bone disorders. The extensive use of ZA has been associated with increasing occurrence of jaw complications, now known as bisphosphonate-associated osteonecrosis of the jaw (BRONJ). However, the mechanism underlying BRONJ remains to be fully elucidated. The aim of the present study was to investigate the effects of different concentrations of ZA on the MC3T3-E1 murine preosteoblast cell line cells and examine the possible pathogenesis of BRONJ. In the present study, the effect of ZA on the viability, apoptosis, differentiation and maturation of MC3T3-E1 cells, as well as its relevant molecular mechanism, were examined The results of a Cell Counting Kit 8 assay, a flow cytometric Annexin-V/propidium iodide assay and western blot analysis demonstrated that ZA exhibited a significant inhibition of cell viability and induction of apoptosis at concentrations >10 µM. Subsequently, the effect of ZA on cell differentiation at concentrations <1 µM were investigated. In this condition, ZA inhibited bone nodule formation and decreased the activity of alkaline phosphatase. The results of reverse transcription-quantitative polymerase chain reaction and western blot analyses indicated that ZA downregulated the expression levels of the marker genes and proteins associated with osteogenic differentiation. Further investigation revealed that the suppression of differentiation by ZA was associated with decreased expression of bone morphogenetic protein-2 (BMP-2) and downregulation of the phosphorylation levels in the downstream extracellular signal-regulated kinase 1/2 and p38 pathways. These adverse effects of ZA were observed to be concentration-dependent. The results from the present study suggested that ZA at higher concentrations induces cytotoxicity towards osteoblasts, and ZA at lower concentrations suppresses osteoblast differentiation by downregulation of BMP-2. These results assist in further understanding the mechanisms of BRONJ.

Inhibition of PRMT5 suppresses osteoclast differentiation and partially protects against ovariectomy-induced bone loss through downregulation of CXCL10 and RSAD2

Protein arginine methyltransferase 5 (PRMT5) is an arginine methylation methyltransferase that regulates various physiological processes. Abnormal PRMT5 activity has been reported in inflammation and various types of cancers. Because osteoclast differentiation is characterized by the activation of inflammation-related pathways, we speculated that PRMT5 may play a role in this process. In the present study, we found that PRMT5 was upregulated during osteoclast differentiation. Knockdown of PRMT5 with siRNA in bone marrow mononuclear cells (BMMs) resulted in inhibition of receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation. Consistent with the PRMT5 knockdown results, the PRMT5 inhibitor EPZ015666 (EPZ) suppressed osteoclast differentiation and bone resorption. Intraperitoneal administration of EPZ prevented ovariectomy-induced bone loss. Moreover, RANKL-induced NF-κB and MAPK activation was inhibited by EPZ. Expression microarrays showed that the expression of several osteoclast formation-related genes was altered by EPZ treatment, including chemokine C-X-C motif ligand 10 (CXCL10). Administration of recombinant CXCL10 partially reversed the osteoclastogenesis inhibition effect of the PRMT5 inhibitor. Intriguingly, RSAD2, which is a reported antiviral protein, was apparently suppressed when PRMT5 was inhibited. Knockdown of RSAD2 with siRNA in BMMs led to inhibition of osteoclast differentiation. Subsequent ChIP-qPCR identified that both PRMT5 inhibition and knockdown resulted in decreased H3R8 or/and H4R3 methylation at CXCL10 and RSAD2 promotors. In conclusion, our study found that PRMT5 is an activator of osteoclast differentiation and inhibition of PRMT5 partially suppressed osteoclastogenesis through downregulation of CXCL10 and RSAD2.

Decreased osteoclastogenesis, osteoblastogenesis and low bone mass in a mouse model of type 2 diabetes

The effect of type 2 diabetes mellitus (T2DM) on bone is controversial. Therefore, the present study investigated whether T2DM causes osteoporosis and explored the underlying mechanisms involved in this process. The effects of T2DM on bone physiology were analyzed in a mouse model of T2DM; KK/Upj‑Ay/J (KK‑Ay) mice develop diabetes after 8 weeks and exhibit stable diabetes symptoms and signs after 10 weeks when fed a KK‑Ay mouse maintenance fodder. Diabetic mice exhibited hyperglycemia, hyperinsulinemia and increased body and fat pad weight in comparison with C57BL/6 non-diabetic mice. Furthermore, diabetic mice demonstrated low bone weight and bone mineral density in the femur, tibia and fifth lumbar vertebra. Using von Kossa and tartrate-resistant acid phosphatase (TRAP) staining, alkaline phosphatase and TRAP activity analyses and gene profiling it was demonstrated that osteoblastogenesis and osteoclastogenesis were impaired in diabetic mice. To evaluate the bone biomechanics, the ultimate load of the bone was analyzed. It was found that the ultimate load of the tibia in diabetic mice was lower than that in the controls. The results from the present study suggest that bone metabolism is impaired in T2DM, resulting in decreased osteoblastogenesis, osteoclastogenesis and bone mass.

Flavonoids Active Against Osteosarcoma: A Review of the Molecular Mechanisms Involved

Osteosarcoma is the most frequent primitive malignant bone tumor affecting adolescents and young adults worldwide. The tumor exhibits aggressive growth in the primary site and readily metastasizes to other organs. There has been no significant improvement in the 5-year survival rate since the 1970s and the figure remains at 60-70%. In addition, the side effects of chemotherapeutic drugs and resistance to chemotherapy compromise the effects of treatment for osteosarcoma. In recent years, the development of flavonoids drugs inhibiting carcinogenesis is attracting great interest in the scientific community. Flavonoids are one kind of polyphenolic compounds widely found in vegetables and fruits. Moreover, flavonoids have become popular compounds, exhibiting comprehensive antitumor activities, while being safe and inexpensive. Here, the literature on the benefits afforded by flavonoids in terms of osteosarcoma treatment is reviewed and certain flavonoids and their effects on osteosarcoma are discussed. These compounds can perturb the cell cycle, induce apoptosis, inhibit tumor cell invasion and metastasis, potentiate the actions of chemotherapeutic agents, trigger autophagy, and stimulate antitumor activity in vivo. In summary, we highlight the currently well-accepted flavonoid compounds and detail the molecular mechanisms by which flavonoids may treat osteosarcoma, and thus the flavonoids exhibit great promise as anti-osteosarcoma agents.

Nanog down-regulates the Wnt signaling pathway via β-catenin phosphorylation during epidermal stem cell proliferation and differentiation

BackgroundSkin tissue homeostasis is maintained by a balance between the proliferation and differentiation of epidermal stem cells (EpSCs). EpSC proliferation and differentiation are complex processes regulated by many factors and signaling pathways. This study aimed to explore the connection between the Nanog and the Wnt/β-catenin pathway in the proliferation and differentiation of EpSCs.ResultsOur results demonstrated that during the study period, EpSC underwent differentiation when incubated in the presence neuropeptide substance P (SP), there was an opposing expression trend of Nanog and β-catenin after SP treatment, which could be antagonized by the Wnt antagonist, Dkk-1. The transduced EpSCs had a greater proliferative ability than the SP treatment group and they did not undergo differentiation upon SP treatment. More important, β-catenin expression was down-regulated but phosphorylated β-catenin expression and phosphorylated GSK-3β expression was up-regulated upon Nanog overexpression.ConclusionsThese results strongly suggest that Nanog plays an important role in maintaining the proliferation and differentiation homeostasis of EpSCs by promoting β-catenin phosphorylation via GSK-3β to inhibit the activity of the Wnt/β-catenin signaling pathway. This is important for precise regulation of proliferation and differentiation of EpSC in the application of tissue engineering.

REV-ERB agonism suppresses osteoclastogenesis and prevents ovariectomy-induced bone loss partially via FABP4 upregulation

REV‐ERBs (REV‐ERBα and REV‐ERBβ) are transcription repressors and circadian regulators. Previous investigations have shown that REV‐ERBs repress the expression of target genes, including MMP9 and CX3CR1, in macrophages. Because MMP9 and CX3CR1 reportedly participate in receptor activator of nuclear factor‐κB ligand (RANKL)‐induced osteoclastogenesis, we inferred that REV‐ERBs might play a role in osteoclastogenesis. In the present study, we found that the REV‐ERBα level decreased significantly during RANKL‐induced osteoclast differentiation from primary bone marrow–derived macrophages (BMMs). REV‐ERBα knockdown by small interfering RNA in BMMs resulted in the enhanced formation of osteoclasts, whereas REV‐ERBβ knockdown showed no effect on osteoclast differentiation. Moreover, the REV‐ERB agonist SR9009 inhibited osteoclast differentiation and bone resorption. Intraperitoneal SR9009 administration prevented ovariectomy‐induced bone loss; this effect was accompanied by decreased serum RANKL and C‐terminal telopeptide of type I collagen levels and increased osteoprotegerin levels. Further investigation revealed that NF‐κB and MAPK activation and nuclear factor of activated T cells, cytoplasmic 1, and c‐fos expression were suppressed by SR9009. The level of reactive oxygen species was also decreased by SR9009, with NADPH oxidase subunits also being down‐regulated. In addition, an expression microarray showed that FABP4, an intracellular lipid‐binding protein, was up‐regulated by REV‐ERB agonism. BMS309403, an inhibitor of FABP4, partially prevented the suppression of osteoclastogenesis by SR9009 through stabilizing phosphorylation of p65. To summarize, our results proved that the REV‐ERB agonism inhibited osteoclastogenesis partially via FABP4 up‐regulation.—Song, C., Tan, P., Zhang, Z., Wu, W., Dong, Y., Zhao, L., Liu, H., Guan, H., Li, F. REV‐ERB agonism suppresses osteoclastogenesis and prevents ovariectomy‐induced bone loss partially via FABP4 upregulation. FASEB J. 32, 3215–3228 (2018). www.fasebj.org

Role of IFT88 in icariin‑regulated maintenance of the chondrocyte phenotype

Maintenance of the chondrocyte phenotype is crucial for cartilage repair during tissue engineering. Intraflagellar transport protein 88 (IFT88) is an essential component of primary cilia, shuttling signals along the axoneme. The hypothesis of the present study was that IFT88 could exert an important role in icariin-regulated maintenance of the chondrocyte phenotype. To this end, the effects of icariin on proliferation and differentiation of the chondrogenic cell line, ATDC5, were explored. Icariin-treated ATDC5 cells and primary chondrocytes expressed IFT88. Icariin has been demonstrated to aid in the maintenance of the articular cartilage phenotype in a rat model of post-traumatic osteoarthritis (PTOA). Icariin promoted chondrocyte proliferation and expression of the chondrogenesis marker genes, COL II and SOX9, increased ciliary assembly, and upregulated IFT88 expression in a concentration- and time-dependent manner. Icariin-treated PTOA rats secreted more cartilage matrix compared with the controls. Knockdown of IFT88 expression with siRNA reduced extracellular signal-regulated kinase (ERK) phosphorylation, and icariin upregulated IFT88 expression by promoting ERK phosphorylation. Thus, IFT88 serves a major role in icariin-mediated maintenance of the chondrocyte phenotype, promoting ciliogenesis and IFT88 expression by increasing ERK phosphorylation. Icariin may therefore be useful for maintenance of the cartilage phenotype during tissue engineering.