Effects of carvacrol and 2,6-diisopropylphenol (propofol) on reactive oxygen species (ROS)-, calcium (Ca2+)- and caspase-3-associated apoptosis in human normal cells and non-normal cells

Autor: Wei-Zhe Liang, 梁維哲
Rok vydání: 2012
Druh dokumentu: 學位論文 ; thesis
Popis: 101
The effect of the natural essential oil carvacrol or the anesthetic propofol on cell viability, cell cycle distribution, reactive oxygen species (ROS), intracellular free Ca2+ concentrations ([Ca2+]i) and caspase-3-associated apoptosis in human normal cells or non-normal cells is unclear. Human gingival fibroblasts (HGF), human oral cancer cell line (OC2) and human glioblastoma cell line (DRTBG-05MG, HGB) were used in this study. Cell viability was measured by detecting reagent water soluble tetrazolium salt-1 (WST-1). Apoptosis was detected by Annexin V/propidium iodide (PI) staining, cell cycle distribution was detected by PI staining, and ROS was detected by membrane-permeable probe dichlorofluorescein diacetate (DCFH-DA) or hydroethidine (HE) staining. Apoptosis, cell cycle distribution and ROS were analyzed by flow cytometry. The Ca2+-sensitive fluorescent dye fura-2 was applied to measure [Ca2+]i. Caspase-3 expression was detected by western blotting. Carvacrol at 200-800 μM decreased the cell viability of OC2 or HGB cells in a concentration-dependent manner and 1,000 μM carvacrol almost killed all OC2 or HGB cells, but in HGF cells, 200-800 μM carvacrol did not significantly kill cells and 1,000 μM carvacrol decreased only about 63% of cell viability. Similarly, propofol at concentrations between 300 and 600 μM decreased the cell viability of OC2 or HGB cells in a concentration-dependent manner and 700 μM propofol almost killed all OC2 or HGB cells, but in HGF cells, 300-600 μM propofol did not significantly kill cells and 700 μM propofol decreased about 62% of cell viability. In OC2 or HGB cells, carvacrol (200 μM, 400 μM or 600 μM) or propofol (300 μM, 400 μM or 500 μM) induced apoptosis, increased ROS production, evoked cell cycle arrest and activated caspase-3. The caspase-3 inhibitor (DEVD-CHO) partially decreased the apoptotic effect induced by carvacrol or propofol. On the other hand, in OC2 or HGB cells, carvacrol at concentrations between 400 μM and 1,000 μM induced a [Ca2+]i rise in a concentration-dependent manner and the signal was reduced by removal of extracellular Ca2+. In HGF cells, carvacrol at 1000 μM did not induce immediate [Ca2+]i rises in Ca2+-containing or Ca2+-free medium. Similarly, propofol at concentrations between 400 μM and 1,000 μM induced a [Ca2+]i rise in a concentration-dependent manner in OC2 or HGB cells, but not in HGF cells. This cytotoxic effect was not reversed in carvacrol-treated groups, but was partially reversed in propofol-treated groups when cytosolic Ca2+ was chelated with 1,2-bis(2-aminophenoxy)ethane-N,N,N'',N''-tetraacetic acid/acetoxy methyl (BAPTA-AM) in OC2 or HGB cells. The apoptotic effect of propofol was also partially decreased by BAPTA-AM treatment in OC2 and HGB cells. In OC2 and HGB cells, carvacrol- or propofol-induced Ca2+ signal was not altered by L-type voltage-gated Ca2+ channel blocker nifedipine, store-operated Ca2+ channel blocker econazole or SK&;F96365) and protein kinase C (PKC) activator phorbol myristate acetate (PMA), but was inhibited by PKC inhibitor GF109203X. When extracellular Ca2+ was removed, incubation with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin (TG) or 2,5-di-tert-butylhydroquinone (BHQ) abolished carvacrol- or propofol-induced [Ca2+]i rises. Incubation with carvacrol or propofol also abolished TG or BHQ-induced [Ca2+]i rises. Inhibition of phospholipase C (PLC) with U73122 abolished carvacrol- or propofol-induced [Ca2+]i rises. Together, first, in HGF cells, carvacrol (200-800 μM) or propofol (300-600 μM) did not induce [Ca2+]i rises and cell death. Second, in OC2 or HGB cells, carvacrol induced [Ca2+]i rises and cell death that might involve ROS- and caspase-3-associated apoptosis. Third, in OC2 or HGB cells, propofol induced [Ca2+]i rises and cell death that might involve ROS-, Ca2+- and caspase-3-associated apoptosis. Lastly, in OC2 or HGB cells, carvacrol or propofol induced [Ca2+]i rises by inducing PLC-dependent Ca2+ release from the endoplasmic reticulum and Ca2+ entry via PKC-sensitive, non store-operated Ca2+ channels.
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