Na+/K+-ATPase as a target for cardiotonic steroids and cisplatin

The sodium (Na+)/potassium (K+)-ATPase is an ion pump located on the surface of all animal cells. It pumps three sodium ions out of the cell while pumping two potassium ions into the cell, hydrolyzing one ATP molecule as the driving force for the reaction. Na+/K+-ATPase forms and maintains the electrochemical gradient in cells, which provides the basis for the excitability of nerve and muscle tissues and contributes to the osmotic regulation of cell volume. In addition, the electrochemical Na+ gradient is the driving force for the secondary transport of nutrients such as amino acids, sugars, and drugs. Recently, the Na+/K+-ATPase has been studied as an important target for cancer treatment, as it has been implicated in the development and progression of many cancers. Na+/K+-ATPase forms a phosphoenzyme intermediate (EP) during ATP hydrolysis.　Cardiotonic steroids have been used to treat congestive heart failure and arrhythmias, and recently their anti-cancer activities have been reported. Ouabain, a specific inhibitor of Na+/K+-ATPase, is a cardiotonic steroid that binds to EP, inhibiting its dephosphorylation and the release of inorganic phosphate. Cisplatin is one of the most potent anti-tumor agents. Many studies have examined the relationship between cisplatin and Na+/K+-ATPase from the viewpoint of cisplatin accumulation and the prevention of nephrotoxicity. It has been suggested that the transport of cisplatin into cells is mediated by the Na+/K+-ATPase and that Na+/K+-ATPase activity is inhibited by cisplatin, although the underlying mechanism remains unclear.　In this review, we evaluate the mechanisms underlying inhibition of Na+/K+-ATPase by cisplatin. We also summarize the structure, function, and enzymatic reaction of Na+/K+-ATPase, as well as the potential for the pump to serve as a target for ouabain and cisplatin. Finally, we will describe experiments conducted by our group showing the mechanism of Na+/K+-ATPase inhibition by cisplatin, and the combined effects of ouabain and cisplatin on cancer cell viability

Suppression of osteoclastogenesis by lactoferrin

Recent research has shown that lactoferrin indirectly suppresses osteoclastogenesis by affecting osteoblasts and periodontal ligament fibroblasts. However, the mechanism by which lactoferrin directly affects osteoclastogenesis is yet to be reported. Therefore, this study examined the direct effects of lactoferrin on RANKL-induced osteoclast differentiation of murine osteoclastic RAW 264.7 cells. The number of osteoclasts was determined by counting the number of cells positive for tartrate-resistant acid phosphatase staining. The effect of lactoferrin on the number of osteoclasts was measured, and the effect on the mRNA expression of osteoclast differentiation markers was assayed using real-time PCR. Lactoferrin decreased the number of osteoclasts (_2 nuclei) and large osteoclasts (_8 nuclei) in a dose-dependent manner without affecting the viability of RAW 264.7 cells. Additionally, it only mediated these effects within 48 h of culturing the RAW 264.7 cells with RANKL. Lactoferrin also significantly inhibited RANKL-induced mRNA expressions of osteoclastic differentiation genes, such as NFATc1, RANK, DC-STAMP, and OC-STAMP. Thus, these findings suggest that lactoferrin directly suppresses osteoclastogenesis within 48 h of culturing the RAW 264.7 cells with RANKL. Therefore, lactoferrin may be a novel and innovative therapy for treating bone diseases

How much medium do you use for cell culture? Medium volume influences mineralization and osteoclastogenesis in vitro

Bone is maintained by a balance between bone formation and resorption. This remodeling is controlled by a wide variety of systemic and local factors including hormones, cytokines and mechanical stresses. The present in vitro study examined the impact of medium volume, using 0.4, 0.6, 0.8, 1.0, 1.5 and 2.0 ml/well in a 24-well plate, on the differentiation of osteoblasts and osteoclasts. There were no differences in the alkaline phosphatase activity of osteoblasts amongst the groups; however, the area of mineral deposition was decreased in a media volume-dependent manner. A co-culture of osteoblastic cells with bone marrow cells revealed a reduction in the total number of osteoclastic tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells (>= 2 nuclei), whereas the formation of large osteoclastic TRAP-positive multinuclear cells (>= 8 nuclei) was increased, in a media volume-dependent manner. There were also no differences in receptor activator of nuclear factor-kappa B ligand mRNA and total osteoprotegerin (OPG) protein expression levels amongst the groups, however the concentration of OPG decreased in a media volume-dependent manner. In conclusion, the present study demonstrated that the suppression of mineralization in osteoblastic cells and the stimulation of osteoclast fusion are dependent on the medium volume, indicating that media volume is an important factor in in vitro cell culture systems

N-acetylcysteine attenuates PGE(2) and ROS production stimulated by 4-META/MMA-based resin in murine osteoblastic cells

This study examined the effects of N-acetylcysteine (NAC) on the inflammatory reactions of murine osteoblastic cells cultured on the 4-methacryloxyethyl trimellitate anhydride/methyl methacrylate (4-META/MMA)-based resin. Superbond C&B (SB) was used as the 4-META/MMA-based resin and placed in a 48-well cell culture plate. The cells were cultured in alpha MEM (control) as well as on SB and SB in alpha MEM with NAC (SB+NAC). They were examined using the WST-1 proliferation assay, real-time PCR, enzyme-linked immunosorbent assay (ELISA), intracellular reactive oxygen species (ROS) measurements, and cellular glutathione (GSH) detection. COX-2 and IL-6 gene expressions were upregulated in SB; however, they were suppressed by NAC. Furthermore, PGE2 production in the culture medium was increased in SB, whereas NAC decreased the PGE(2) production. NAC lowered the ROS level in the culture medium and significantly increased the intracellular GSH level. The present in vitro study demonstrated that NAC might be effective for dental material detoxification

Intramedullary Insetting of Silicone Implant for Lateral Stability in Distal Interphalangeal Joint Arthroplasty

Summary:. Silicone implant arthroplasty is an alternative surgical intervention for painful and deformed osteoarthritis of the distal interphalangeal (DIP) joints. DIP joint stability is essential for hand function; however, it carries a potential risk of postoperative joint instability. To address this concern, an intramedullary implant insetting method was used to maintain joint stability by minimum resection of the head of the middle phalanx and preserving the collateral ligament. In the new method, the length of the bone excision was limited to maintain the lateral cortical bone with the insertion of the collateral ligament, and the medullary cavity of the middle phalanx was partially removed to intentionally set the hinge part of the silicone implant in the medullary canal. Between 20 digits of the conventional approach and 23 digits of the intramedullary insetting method, there were no significant differences in patient demographics (ie, age, affected hand, and finger), and clinical characteristics (ie, active DIP joint arc, DIP joint extension loss, grip strength, visual analog scale, and Quick Disabilities of the Arms, Shoulder and Hand questionnaire score) before and over 6 months after surgery. However, postoperative joint instability was significantly lower with the intramedullary insetting method, with a significantly shorter length of bone excision of the middle phalanx. This new approach is more beneficial than the conventional approach for preventing postoperative joint instability

Release from optimal compressive force suppresses osteoclast differentiation

Bone remodeling is an important factor in orthodontic tooth movement. During orthodontic treatment, osteoclasts are subjected to various mechanical stimuli, and this promotes or inhibits osteoclast differentiation and fusion. It has been previously reported that the release from tensile force induces osteoclast differentiation. However, little is known about how release from compressive force affects osteoclasts. The present study investigated the effects of release from compressive force on osteoclasts. The number of tartrate-resistant acid phosphatase (TRAP) -positive multinucleated osteoclasts derived from RAW264.7 cells was counted, and gene expression associated with osteoclast differentiation and fusion in response to release from compressive force was evaluated by reverse transcription-quantitative polymerase chain reaction. Osteoclast number was increased by optimal compressive force application. On release from this force, osteoclast differentiation and fusion were suppressed. mRNA expression of NFATc1 was inhibited for 6 h subsequent to release from compressive force. mRNA expression of the other osteoclast-specific genes, TRAP, RANK, matrix metalloproteinase-9, cathepsin-K, chloride channel 7, ATPase H+ transporting vacuolar proton pump member I, dendritic cell-specific transmembrane protein and osteoclast stimulatory transmembrane protein (OC-STAMP) was significantly inhibited at 3 h following release from compressive force compared with control cells. These findings suggest that release from optimal compressive force suppresses osteoclast differentiation and fusion, which may be important for developing orthodontic treatments