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Background/purpose: Far-infrared (FIR) therapy is a safe and noninvasive source for medical applications. Animal study has shown the effects of FIR in promoting nerve repair. However, the cellular mechanism is not well known. Nerve growth factor (NGF) treated neuron-like PC12 cells for neurite outgrowth have been widely employed as the in vitro model for neural regeneration.Methods: In this study, we tried to evaluate the potential of FIR in promoting neurite outgrowth and related mechanism by using NGF-treated neuron-like PC12 cells as a cellular model. We found that FIR could promote neurites outgrowth of neuron-like PC12 cells at earlier culture period.Results: The neurite outgrowth-enhancing effect of FIR irradiation was more obvious when lower NGF concentration (1 ng/ml and 10 ng/ml) was added into the medium. We also found that FIR had no thermal effects on culture medium. The effects of FIR in promoting neurite outgrowth were dose dependent, and higher power density of FIR provided more effects for improving neurite outgrowth. The mechanism of FIR in promoting neurite outgrowth was through AKT1 pathway.Conclusion: The effects of FIR irradiation on promoting neurite outgrowth and neural regeneration of NGF-treated neuron-like PC12 cells are dose dependent and through activation of AKT1 phosphorylation. This study provided important information for understanding the cellular mechanism of FIR in promoting neurite outgrowth and possible neural regeneration for further clinical applications. [ABSTRACT FROM AUTHOR]

Objective: To investigate the efficacy, safety and the duration of treatment effectiveness of intra-articular hyaluronic acid (Artz, Japan) in patients with ankle osteoarthritis (OA).Method: As a prospective clinical trial, 93 patients with unilateral ankle pain for at least 6 months and radiographically classified as Kellgren-Lawrence grade I or II ankle OA were included. After five weekly intra-articular Artz injections, the Ankle Osteoarthritis Scale (AOS), the American Orthopaedic Foot and Ankle Society (AOFAS) ankle/hindfoot score, ankle sagittal range of motion (ROM), patients' global satisfaction, local adverse events and consumption of rescue analgesics were analyzed.Results: Seventy-five patients completed the study. Significant improvement in AOS and AOFAS ankle/hindfoot scores was noted at 1 week, 1 month, 3 months and 6 months post the fifth injection (P < 0.001 compared with baseline). The mean reduction of AOS score was 1.9, 2.6, 2.5 and 2.6 at each following visit (P < 0.001). The mean AOFAS ankle/hindfoot score improved from 64 points at baseline to 75, 78, 78, and 78 points at 1 week, 1 month, 3 months and 6 months, respectively, post the fifth injection (P < 0.001). Ankle sagittal ROM did not improve significantly (P > 0.05). The majority of patients reported satisfaction at 1 week (100%), 1 month (100%), 3 months (90.7%) and 6 months (86.7%) follow-up. Local adverse events occurred in 6.7% of patients. Acetaminophen consumption dropped significantly following treatment (P < 0.001).Conclusion: Five weekly intra-articular injections of Artz provide pain relief and functional improvements in patients with Kellgren-Lawrence grades I and II ankle OA. The clinical effect was rapid at 1 week and may last for 6 months or more. [ABSTRACT FROM AUTHOR]

Objective: To investigate the effects of intraarticular hyaluronic acid (HA) (Artzal, Seikagaku Corp., Japan) in geriatric participants with unilateral knee osteoarthritis (OA).Method: This was a prospective, observer-blind study with 6 months follow-up done in the setting of an outpatient rehabilitation department in a university-affiliated tertiary care medical center. Sixty-eight patients, aged 65 years or above, with symptoms and radiographic evidence of unilateral knee OA for at least 6 months were recruited. Patients received five weekly intraarticular injections of Artzal into symptomatic knees. Fifty-six participants completed the study. Fifty age-, body mass- and gender-matched healthy individuals were selected as control. Visual analog scale (VAS), Lequesne index and four balance tests including single-leg stance test (SLS), function reach test (FRT), timed "Up-and-Go" test (TUG) and Berg balance scale (BBS) were assessed before injection and at each follow-up visit in the OA group. Four balance tests were obtained on healthy participants for data comparison.Results: Before Artzal injections, the OA group showed significantly worse VAS, Lequesne index and four balance tests scores than did the control group (P < 0.001). Significant improvement in all outcome measures were noted at 1 week, 1, 3 and 6 months post the fifth injection compared with baseline before injection. Local adverse events were reported in four patients (7.1%).Conclusion: Significant improvement in pain, physical function and balance tests was demonstrated after five weekly Artzal injections in geriatric patients with knee OA. The effect had rapid onset at 1 week and may last for 6 months. [ABSTRACT FROM AUTHOR]

Abstract: The effect of the environmental toxicant nonylphenol on cytosolic free Ca2+ concentration ([Ca2+] i ) and proliferation has not been explored in human osteoblast-like cells. This study examined whether nonylphenol alters Ca2+ levels and causes cell death in MG63 human osteosarcoma cells. [Ca2+] i and cell death were measured using the fluorescent dyes fura-2 and WST-1 respectively. Nonylphenol at concentrations above 3μM increased [Ca2+] i in a concentration-dependent manner. The Ca2+ signal was reduced by 90% by removing extracellular Ca2+. The nonylphenol-induced Ca2+ influx was insensitive to blockade of L-type Ca2+ channel blockers. After pretreatment with 10μM nonylphenol, 1μM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) failed to induce [Ca2+] i rises. Inhibition of phospholipase C with 2μM U73122 did not change nonylphenol-induced [Ca2+] i rises. The nonylphenol-induced [Ca2+] i rises were enhanced or inhibited by phorbol myristate acetate or GF 109203X, respectively. At concentrations of 10 and 20μM nonylphenol killed 55% and 100% cells, respectively. The cytotoxic effect of 10μM nonylphenol was unaltered by pre-chelating cytosolic Ca2+ with BAPTA. Collectively, in MG63 cells, nonylphenol induced [Ca2+] i rises by causing Ca2+ release from intracellular stores and Ca2+ influx from extracellular space. Furthermore, nonylphenol can cause Ca2+-unrelated cytotoxicity in a concentration-dependent manner. [Copyright &y& Elsevier]

Objective Magnolol, a polyphenol compound from herbal medicines, was shown to alter physiology in various cell models. However, the effect of magnolol on Ca 2+ homeostasis and its related physiology in oral cancer cells is unclear. This study examined whether magnolol altered Ca 2+ signaling and cell viability in OC2 human oral cancer cells. Methods Cytosolic Ca 2+ concentrations ([Ca 2+ ] i ) in suspended cells were measured by using the fluorescent Ca 2+ -sensitive dye fura-2. Cell viability was examined by 4-[3-[4-lodophenyl]-2-4(4-nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate] water soluble tetrazolium-1 (WST-1) assay. Results Magnolol at concentrations of 20–100 μM induced [Ca 2+ ] i rises. Ca 2+ removal reduced the signal by approximately 50%. Magnolol (100 μM) induced Mn 2+ influx suggesting of Ca 2+ entry. Magnolol-induced Ca 2+ entry was partially suppressed by protein kinase C (PKC) regulators, and inhibitors of store-operated Ca 2+ channels. In Ca 2+ -free medium, treatment with the endoplasmic reticulum Ca 2+ pump inhibitor 2,5-di- tert -butylhydroquinone (BHQ) abolished magnolol-evoked [Ca 2+ ] i rises. Conversely, treatment with magnolol abolished BHQ-evoked [Ca 2+ ] i rises. Inhibition of phospholipase C (PLC) with U73122 partially inhibited magnolol-induced [Ca 2+ ] i rises. Magnolol at 20–100 μM decreased cell viability, which was not reversed by pretreatment with the Ca 2+ chelator 1,2-bis(2-aminophenoxy)ethane- N,N,N',N' -tetraacetic acid-acetoxymethyl ester (BAPTA/AM). Conclusions Together, in OC2 cells, magnolol induced [Ca 2+ ] i rises by evoking partially PLC-dependent Ca 2+ release from the endoplasmic reticulum and Ca 2+ entry via PKC-sensitive store-operated Ca 2+ entry. Magnolol also caused Ca 2+ -independent cell death. Therefore, magnolol-induced cytotoxicity may not be involved in activation mechanisms associated with intracellular Ca 2+ mobilization in oral cancer cells. [ABSTRACT FROM AUTHOR]

Puerarin is a natural compound and has been used as herb medication in a number of countries, especially in Asia. The effect of puerarin on Ca 2+ signaling is unknown in renal cells. This study examined whether puerarin affected Ca 2+ physiology in MDCK renal tubular cells. Cytosolic free Ca 2+ levels ([Ca 2+ ] i ) were measured using the fluorescent dye fura-2. Cell viability was examined by using WST-1 assay. Puerarin induced [Ca 2+ ] i rises and the response was reduced by removing extracellular Ca 2+ . Puerarin-induced Ca 2+ entry was not altered by protein kinase C (PKC) activity, but was inhibited by nifedipine. In Ca 2+ -free medium, treatment with the endoplasmic reticulum Ca 2+ pump inhibitor 2,5-di-tert-butylhydroquinone (BHQ) or thapsigargin partly inhibited puerarin-evoked [Ca 2+ ] i rises. Inhibition of phospholipase C (PLC) with U73122 did not change puerarin-induced [Ca 2+ ] i rises. Puerarin at 25–50 μM caused cytotoxicity, which was not reversed by pretreatment with the Ca 2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM). Collectively, in MDCK cells, puerarin induced [Ca 2+ ] i rises by evoking PLC-independent Ca 2+ release from the endoplasmic reticulum and other unknown stores, and Ca 2+ entry via nifedipine-sensitive, PKC-insensitive Ca 2+ entry pathways. Puerarin also caused Ca 2+ -independent cell death. [ABSTRACT FROM AUTHOR]

Ellagic acid, a natural phenol compound found in numerous fruits and vegetables, causes various physiological effects in different cell models. However, the effect of this compound on Ca 2+ homeostasis in liver cells is unknown. This study examined the effect of ellagic acid on intracellular Ca 2+ concentration ([Ca 2+ ] i ) and established the relationship between Ca 2+ signaling and cytotoxicity in liver cells. The data show that ellagic acid induced concentration-dependent [Ca 2+ ] i rises in HepG2 human hepatoma cells, but not in HA22T, HA59T human hepatoma cells or AML12 mouse hepatocytes. In HepG2 cells, this Ca 2+ signal response was reduced by removing extracellular Ca 2+ and was inhibited by store-operated Ca 2+ channel blockers (2-APB, econazole or SKF96365) and the protein kinase C (PKC) inhibitor GF109203X. In Ca 2+ -free medium, pretreatment with the endoplasmic reticulum Ca 2+ pump inhibitor thapsigargin abolished ellagic acid-induced [Ca 2+ ] i rises. Conversely, incubation with ellagic acid abolished thapsigargin-induced [Ca 2+ ] i rises. Inhibition of phospholipase C (PLC) with U73122 also abolished ellagic acid-induced [Ca 2+ ] i rises. Ellagic acid (25–100 μM) concentration-dependently caused cytotoxicity in HepG2, HA22T or HA59T cells, but not in AML12 cells. Furthermore, this cytotoxic effect was partially prevented by prechelating cytosolic Ca 2+ with BAPTA-AM only in HepG2 cells. Together, in HepG2 cells, ellagic acid induced [Ca 2+ ] i rises by inducing PLC-dependent Ca 2+ release from the endoplasmic reticulum and Ca 2+ entry via PKC-sensitive store-operated Ca 2+ channels. Moreover, ellagic acid induced Ca 2+ -associated cytotoxicity. [ABSTRACT FROM AUTHOR]

Abstract: The effect of N-(4-hydroxyphenyl) arachidonoyl-ethanolamide (AM404), a drug commonly used to inhibit the anandamide transporter, on intracellular free Ca2+ levels ([Ca2+]i) and viability was studied in human MG63 osteosarcoma cells using the fluorescent dyes fura-2 and WST-1, respectively. AM404 at concentrations ≥5μM increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 60μM. The Ca2+ signal was reduced partly by removing extracellular Ca2+. AM404 induced Mn2+ quench of fura-2 fluorescence implicating Ca2+ influx. The Ca2+ influx was sensitive to La3+, Ni2+, nifedipine and verapamil. In Ca2+-free medium, after pretreatment with 1μM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), AM404-induced [Ca2+]i rise was abolished; and conversely, AM404 pretreatment totally inhibited thapsigargin-induced [Ca2+]i rise. Inhibition of phospholipase C with U73122 did not change AM404-induced [Ca2+]i rise. At concentrations between 10 and 200μM, AM404 killed cells in a concentration-dependent manner presumably by inducing apoptotic cell death. The cytotoxic effect of 50μM AM404 was partly reversed by prechelating cytosolic Ca2+ with BAPTA/AM. Collectively, in MG63 cells, AM404 induced [Ca2+]i rise by causing Ca2+ release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca2+ influx via L-type Ca2+ channels. AM404 caused cytotoxicity which was possibly mediated by apoptosis. [Copyright &y& Elsevier]

Abstract: The effect of the antidepressant desipramine on intracellular Ca2+ movement and viability in prostate cancer cells has not been explored previously. The present study examined whether desipramine could alter Ca2+ handling and viability in human prostate PC3 cancer cells. Cytosolic free Ca2+ levels ([Ca2+] i ) in populations of cells were measured using fura-2 as a probe. Desipramine at concentrations above 10μM increased [Ca2+] i in a concentration-dependent manner. The responses saturated at 300μM desipramine. The Ca2+ signal was reduced by half by removing extracellular Ca2+, but was unaffected by nifedipine, nicardipine, nimodipine, diltiazem or verapamil. In Ca2+-free medium, after treatment with 300μM desipramine, 1μM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) failed to release Ca2+ from endoplasmic reticulum. Conversely, desipramine failed to release more Ca2+ after thapsigargin treatment. Inhibition of phospholipase C with U73122 did not affect desipramine-induced Ca2+ release. Overnight incubation with 10–800μM desipramine decreased viability in a concentration-dependent manner. Chelation of cytosolic Ca2+ with BAPTA did not reverse the decreased cell viability. Collectively, the data suggest that in PC3 cells, desipramine induced a [Ca2+] i increase by causing Ca2+ release from endoplasmic reticulum in a phospholipase C-independent fashion and by inducing Ca2+ influx. Desipramine decreased cell viability in a concentration-dependent, Ca2+-independent manner. [Copyright &y& Elsevier]

Abstract: The effect of anandamide on human osteoblasts is unclear. This study examined the effect of anandamide on viability, apoptosis, mitogen-activated protein kinases (MAPKs) and Ca2+ levels in MG63 osteosarcoma cells. Anandamide at 50–200μM decreased cell viability via apoptosis as demonstrated by propidium iodide staining and activation of caspase-3. Immunoblotting suggested that anandamide induced expression of ERK, JNK and p38 MAPK. Anandamide-induced cell death and apoptosis were reversed by SB203580, but not by PD98059 and SP600125, suggesting that anandamide''s action was via p38 MAPK, but not via ERK and JNK. Anandamide at 1–100μM induced [Ca2+] i increases. Removal of extracellular Ca2+ decreased the anandamide response, indicating that anandamide induced Ca2+ influx and Ca2+ release. Chelation of intracellular Ca2+ with BAPTA reversed anandamide-induced cell death and p38 MAPK phosphorylation. Collectively, in MG63 cells, anandamide induced [Ca2+] i increases which evoked p38 MAPK phosphorylation. This p38 MAPK phosphorylation subsequently activated caspase-3 leading to apoptosis. [Copyright &y& Elsevier]

Abstract: Melittin, a peptide from bee venom, is thought to be a phospholipase A2 activator and Ca2+ influx inducer that can evoke cell death in different cell types. However, the effect of melittin on cytosolic free Ca2+ concentration ([Ca2+]i) and viability has not been explored in human osteoblast-like cells. This study examined whether melittin altered [Ca2+]i and killed cells in MG63 human osteosarcoma cells. [Ca2+]i changes and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. Melittin at concentrations above 0.075 μM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was abolished by removing extracellular Ca2+. Melittin-induced Ca2+ entry was confirmed by Mn2+ quenching of fura-2 fluorescence at 360 nm excitation wavelength which was Ca2+-insensitive. The melittin-induced Ca2+ influx was unchanged by modulation of protein kinase-C activity with phorbol 12-myristate 13-acetate (PMA) and GF 109203X, or inhibition of phospholipase A2 with AACOCF3 and aristolochic acid; but was substantially inhibited by blocking L-type Ca2+ channels. At concentrations of 0.5 μM and 1 μM, melittin killed 33% and 45% of cells, respectively, via inducing apoptosis. Lower concentrations of melittin failed to kill cells. The cytotoxic effect of 1 μM melittin was completely reversed by pre-chelating cytosolic Ca2+ with BAPTA. Taken together, these data showed that in MG63 cells, melittin induced a [Ca2+]i increase by causing Ca2+ entry through L-type Ca2+ channels in a manner independent of protein kinase-C and phospholipase A2 activity; and this [Ca2+]i increase subsequently caused apoptosis. [Copyright &y& Elsevier]

Abstract: The effect of miconazole, an anti-fungal drug, on cytoplasmic free Ca2+ concentrations ([Ca2+]i) in human osteosarcoma cells (MG63) was explored by using the Ca2+-sensitive dye fura-2. Miconazole acted in a concentration-dependent manner with an EC50 of 75 μM. The Ca2+ signal comprised a gradual rise and a sustained elevation. Removal of extracellular Ca2+ reduced 50% of the signal. In Ca2+-free medium, the [Ca2+]i rise induced by 1 μM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) was completely inhibited by pretreatment with 20 μM miconazole. Pretreatment with thapsigargin partly inhibited miconazole-induced Ca2+ release. The miconazole-induced Ca2+ release was not changed by inhibition of phospholipase C with 2 μM U73122. By using tetrazolium as a fluorescent probe, it was shown that 10–100 μM miconazole decreased cell proliferation rate in a concentration-dependent manner. Collectively, this study shows that miconazole induces [Ca2+]i rises in human osteosarcoma cells via releasing Ca2+ mainly from the endoplasmic reticulum in a manner independent of phospholipase C activity, and by causing Ca2+ influx. Furthermore, miconazole may be cytotoxic to the cells at higher concentrations. [Copyright &y& Elsevier]