Title page for etd-0804111-200936


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URN etd-0804111-200936
Author Yi-chien Fang
Author's Email Address f5812582@yahoo.com.tw
Statistics This thesis had been viewed 5572 times. Download 981 times.
Department Biological Sciences
Year 2010
Semester 2
Degree Ph.D.
Type of Document
Language English
Title The effect of m-3m3FBS and paroxetine on calcium homeostasis and viability in OC2 human oral cancer cells and canine MDCK renal tubular cells
Date of Defense 2011-05-24
Page Count 147
Keyword
  • Necrosis
  • tBHP
  • H2O2
  • caspase
  • paroxetine
  • Apoptosis
  • OC2 cells
  • MDCK cells
  • m-3M3FBS
  • Ca2+
  • BAPTA
  • Abstract The effect of 2,4,6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)- benzenesulfonamide (m-3M3FBS), a presumed phospholipase C activator, on cytosolic free Ca2+ concentrations ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells and OC2 human oral cancer cells was unclear. This study explored whether m-3M3FBS changed basal [Ca2+]i levels in suspended MDCK and OC2 cells by using fura-2 as a Ca2+-sensitive fluorescent dye. m-3M3FBS at concentrations between 0.1-20 μM increased [Ca2+]i in a concentration-dependent manner in MDCK cells, however in OC2 cells, m-3M3FBS at concentrations between 10-60 μM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signals were reduced partly by removing extracellular Ca2+ in the two cell types. m-3M3FBS-induced Ca2+ influx was inhibited by the store-operated Ca2+ channel blockers nifedipine, econazole and SK&F96365, and by the phospholipase A2 inhibitor aristolochic acid. In Ca2+-free medium, m-3M3FBS pretreatment abolished the [Ca2+]i rise induced by the endoplasmic reticulum Ca2+ pump inhibitors thapsigargin, cyclopiazonic acid or 2,5-di-tert-butylhydroquinone (BHQ). Conversely, pretreatment with thapsigargin, cyclopiazonic acid or BHQ partly reduced m-3M3FBS-induced [Ca2+]i rise. Inhibition of phospholipase C with U73122 did not alter m-3M3FBS-induced [Ca2+]i rise. Collectively, in MDCK and OC2 cells, m-3M3FBS induced [Ca2+]i rises by causing phospholipase C-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via store-operated Ca2+ channels and other unidentified Ca2+ channels. Additionally, 5-100 μM of m-3M3FBS killed cells in a concentration-dependent manner in OC2 cells. The cytotoxic effect of m-3M3FBS was not reversed by prechelating cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane- N,N,N',N'-tetraacetic acid (BAPTA). Propidium iodide staining data suggest that m-3M3FBS (20 or 50 μM) induced apoptosis in a Ca2+-independent manner.
    We were also interested in knowing whether BAPTA suppressed cell death during oxidative stress in MDCK cells. BAPTA loading altered tBHP (tert-butyl hydroperoxide) and H2O2-induced cell death in a concentration-dependent manner. This suggests that the cell death induced by tBHP and H2O2 appears to be Ca2+-dependent in MDCK cells. The tBHP and H2O2-induced cell death was not suppressed by 2 μM U73122 (PLC inhibitor), 50 μM zVAD-fmk (caspase inhibitor), 2 μM cyclosporin A (a potent inhibitor of the MPTP), 20 μM PD98059 (ERK inhibitor) or 2 μM SP600125 (JNK inhibitor). This suggests that the tBHP and H2O2-induced MDCK cells death was not via the PLC, MPTP, caspase, ERK or JNK pathways. Propidium iodide staining, caspase-3 activity assay and cell morphology data suggest that tBHP and H2O2-induced cell death was necrosis, not via apoptosis, and the cell death appears to be caspase-independent and Ca2+-dependent.
    The effect of the antidepressant paroxetine on [Ca2+]i in OC2 human oral cancer cells is unclear. This study also explored whether paroxetine changed basal [Ca2+]i levels in suspended OC2 cells by using fura-2 as a Ca2+-sensitive fluorescent dye. Paroxetine at concentrations between 100-1000 μM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced by 50% by removing extracellular Ca2+. Paroxetine-induced Ca2+ influx was inhibited by the store-operated Ca2+ channel blockers nifedipine, econazole and SK&F96365, the phospholipase A2 inhibitor aristolochic acid, and protein kinase C modulators. In Ca2+-free medium, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin abolished paroxetine–induced [Ca2+]i rise. Inhibition of PLC with U73122 did not alter paroxetine-induced [Ca2+]i rise. Paroxetine at 10-50 μM induced cell death in a concentration-dependent manner. The death was not reversed when cytosolic Ca2+ was chelated with BAPTA. Propidium iodide staining suggests that apoptosis played a role in the death. Collectively, in OC2 cells, paroxetine induced [Ca2+]i rise by causing PLC-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via store-operated Ca2+ channels in a manner regulated by protein kinase C and phospholipase A2. Paroxetine also induced cell death in a Ca2+-independent manner.
    Advisory Committee
  • Chen-Chih Kao - chair
  • Hsueh-Wen Chang - co-chair
  • Ko-Long Lin - co-chair
  • Chung-Ren Jan - advisor
  • Chen-Fu Shaw - advisor
  • Files
  • etd-0804111-200936.pdf
  • Indicate in-campus at 0 year and off-campus access at 1 year.
    Date of Submission 2011-08-04

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