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Niflumic acid, a drug used for joint and muscular pain, affected Ca²⁺ signaling in different models. However, the effect of niflumic acid on Ca²⁺ homeostasis and Ca²⁺-related physiology in human osteosarcoma cells is unknown. This study examined the effect of niflumic acid on cytosolic free Ca²⁺ concentrations ([Ca²⁺]i) in MG63 human osteosarcoma cells. Intracellular Ca²⁺ concentrations in suspended cells were monitored by using the fluorescent Ca²⁺-sensitive dye fura- 2. Cell viability was examined by using 4-[3-[4-lodophenyl]-2-4(4-nitrophenyl)-2H-5-tetrazolio- 1,3-benzene disulfonate] water soluble tetrazolium-1 (WST-1). In MG63 cells, niflumic acid at concentrations of 250-750 μM evoked [Ca²⁺]i rises concentration-dependently. Niflumic acid-evoked Ca²⁺ entry was confirmed by Mn²⁺-induced quenching of fura-2 fluorescence. This entry was inhibited by nifedipine, econazole, SKF96365, the protein kinase C (PKC) activator phorbol 12-myristate 13 acetate (PMA), but was not affected by the PKC inhibitor GF109203X. In Ca²⁺- free medium, treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin (TG) inhibited niflumic acid-evoked [Ca²⁺]i rises. Conversely, treatment with niflumic acid abolished TG-evoked [Ca²⁺]i rises. Inhibition of phospholipase C (PLC) with U73122 also partly reduced niflumic acid-evoked [Ca²⁺]i rises. Niflumic acid killed cells at 200-500 μM in a concentration-dependent fashion. Chelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid/ AM (BAPTA/AM) did not reverse niflumic acid-induced cytotoxicity. Collectively, our data suggest that in MG63 cells, niflumic acid induced [Ca²⁺]i rises by evoking PLC-dependent Ca²⁺ release from the endoplasmic reticulum, and Ca²⁺ entry via PKC-sensitive store-operated Ca²⁺ entry. Niflumic acid also induced Ca²⁺-independent cell death., Competing Interests: The authors declared no conflicts of interest.

The effect of the natural product 3,3'-diindolylmethane (DIM) on cytosolic Ca(2+) concentrations ([Ca(2+)](i)) and viability in MG63 human osteosarcoma cells was explored. The Ca(2+)-sensitive fluorescent dye fura-2 was applied to measure [Ca(2+)](i). DIM at concentrations of 40-80 μM induced a [Ca(2+)](i) rise in a concentration-dependent manner. The response was reduced partly by removing Ca(2+). DIM-evoked Ca(2+) entry was suppressed by nifedipine, econazole, SK&F96365 and protein kinase C modulators. In the absence of extracellular Ca(2+), incubation with the endoplasmic reticulum Ca(2+) pump inhibitors thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) inhibited or abolished DIM-induced [Ca(2+)](i) rise. Incubation with DIM also inhibited thapsigargin or BHQ-induced [Ca(2+)](i) rise. Inhibition of phospholipase C with U73122 abolished DIM-induced [Ca(2+)](i) rise. At concentrations of 10-50 μM, DIM killed cells in a concentration-dependent manner. This cytotoxic effect was not altered by chelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Annexin V/propidium iodide staining data implicate that DIM (20 and 40 μM) induced apoptosis in a concentration-dependent manner. In sum, in MG63 cells, DIM induced a [Ca(2+)](i) rise by evoking phospholipase C-dependent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry via protein kinase C-sensitive store-operated Ca(2+) channels. DIM caused cell death that may involve apoptosis., (© 2011 The Authors. Basic & Clinical Pharmacology & Toxicology © 2011 Nordic Pharmacological Society.)

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 > or = 5 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 60 microM. 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 1 microM 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 200 microM, AM404 killed cells in a concentration-dependent manner presumably by inducing apoptotic cell death. The cytotoxic effect of 50 microM 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.

The effect of the natural product 3,3′-diindolylmethane (DIM) on cytosolic Ca2+ concentrations ([Ca2+]i) and viability in MG63 human osteosarcoma cells was explored. The Ca2+-sensitive fluorescent dye fura-2 was applied to measure [Ca2+]i. DIM at concentrations of 40-80 μM induced a [Ca2+]i rise in a concentration-dependent manner. The response was reduced partly by removing Ca2+. DIM-evoked Ca2+ entry was suppressed by nifedipine, econazole, SK&F96365 and protein kinase C modulators. In the absence of extracellular Ca2+, incubation with the endoplasmic reticulum Ca2+ pump inhibitors thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) inhibited or abolished DIM-induced [Ca2+]i rise. Incubation with DIM also inhibited thapsigargin or BHQ-induced [Ca2+]i rise. Inhibition of phospholipase C with U73122 abolished DIM-induced [Ca2+]i rise. At concentrations of 10-50 μM, DIM killed cells in a concentration-dependent manner. This cytotoxic effect was not altered by chelating cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). Annexin V/propidium iodide staining data implicate that DIM (20 and 40 μM) induced apoptosis in a concentration-dependent manner. In sum, in MG63 cells, DIM induced a [Ca2+]i rise by evoking phospholipase C-dependent Ca2+ release from the endoplasmic reticulum and Ca2+ entry via protein kinase C-sensitive store-operated Ca2+ channels. DIM caused cell death that may involve apoptosis. [ABSTRACT FROM AUTHOR]

Extraosseous Ewing sarcoma is now regarded as a member of the Ewing sarcoma/primitive neuroectodermal tumor (PNET) family. It typically involves the soft tissues of the chest wall, pelvis, paravertebral region, abdominal wall, retroperitoneal region and extremities of children, adolescents and young adults, but it seldom occurs in the female genital tract. We report an extremely rare case of retrospective diagnosis of vaginal extraosseous Ewing sarcoma/PNET which metastasized to the right frontoparietal scalp, skull, and dura. Surgical resection, followed by adjuvant radiotherapy and chemotherapy resulted in a favourable clinical outcome. Both the vaginal and head tumors had similar light microscopic features supporting the diagnosis. [Copyright &y& Elsevier]

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]