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The Effect of Z-Ligustilide on the Mobility of Human Glioblastoma T98G Cells.

ABSTRACT: Z-ligustilide (LIG), an essential oil extract from Radix Angelica sinensis, has broad pharmaceutical applications in treating cardio-vascular diseases and ischemic brain injury. Recently, LIG has been connected to Glioblastoma multiforme (GBM) because of its structural similarity to 3-n-alkyphthalide (NBP), which is specifically cytotoxic to GBM cells. Hence, we investigated LIG's effect on GBM T98G cells. The study shows that LIG can significantly reduce T98G cells' migration in a dose-dependent manner. Furthermore, the attenuation of cellular mobility can be linked to the activity of the Rho GTPases (RhoA, Rac1 and Cdc42), the three critical molecular switches governing cytoskeleton remodeling; thus, regulating cell migration. LIG significantly reduces the expression of RhoA and affects in a milder manner the expression of Cdc42 and Rac1.

SUBMITTER: Yin J

PROVIDER: S-EPMC3689746 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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The Effect of Z-Ligustilide on the Mobility of Human Glioblastoma T98G Cells.

Yin Jun J Wang Chengyuan C Mody Aaron A Bao Lin L Hung Shen-Hsiu SH Svoronos Spyros A SA Tseng Yiider Y

PloS one 20130621 6

Z-ligustilide (LIG), an essential oil extract from Radix Angelica sinensis, has broad pharmaceutical applications in treating cardio-vascular diseases and ischemic brain injury. Recently, LIG has been connected to Glioblastoma multiforme (GBM) because of its structural similarity to 3-n-alkyphthalide (NBP), which is specifically cytotoxic to GBM cells. Hence, we investigated LIG's effect on GBM T98G cells. The study shows that LIG can significantly reduce T98G cells' migration in a dose-dependen ...[more]

PMID: 23805245

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Project description:BackgroundObesity-related central nervous system (CNS) pathologies like neuroinflammation and reactive gliosis are associated with high-fat diet (HFD) related elevation of saturated fatty acids like palmitic acid (PA) in neurons and astrocytes of the brain.MethodsHuman neuroblastoma cells SH-SY5Y (as a neuronal model) and human glioblastoma cells T98G (as an astrocytic model), were treated with 100-500 µM PA, oleic acid (OA) or lauric acid (LA) for 24 h or 48 h, and their cell viability was assessed by 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effects of stable overexpression of γ-synuclein (γ-syn), a neuronal protein recently recognized as a novel regulator of lipid handling in adipocytes, and transient overexpression of Parkinson's disease (PD) α-synuclein [α-syn; wild-type (wt) and its pathogenic mutants A53T, A30P and E46K] in SH-SY5Y and T98G cells, were also evaluated. The effects of co-treatment of PA with paraquat (PQ), a Parkinsonian pesticide, and leptin, a hormone involved in the brain-adipose axis, were also assessed. Cell death mode and cell cycle were analyzed by Annexin V/PI flow cytometry. Reactive oxygen species (ROS) level was determined using 2',7'-dichlorofluorescien diacetate (DCFH-DA) assay and lipid peroxidation level was determined using thiobarbituric acid reactive substances (TBARS) assay.ResultsMTT assay revealed dose- and time-dependent PA cytotoxicity on SH-SY5Y and T98G cells, but not OA and LA. The cytotoxicity was significantly lower in SH-SY5Y-γ-syn cells, while transient overexpression of wt α-syn or its PD mutants (A30P and E46K, but not A53T) modestly (but still significantly) rescued the cytotoxicity of PA in SH-SY5Y and T98G cells. Co-treatment of increasing concentrations of PQ exacerbated PA's neurotoxicity. Pre-treatment of leptin, an anti-apoptotic adipokine, did not successfully rescue SH-SY5Y cells from PA-induced cytotoxicity-suggesting a mechanism of PA-induced leptin resistance. Annexin V/PI flow cytometry analysis revealed PA-induced increase in percentages of cells in annexin V-positive/PI-negative quadrant (early apoptosis) and subG0-G1 fraction, accompanied by a decrease in G2-M phase cells. The PA-induced ROS production and lipid peroxidation was at greater extent in T98G as compared to that in SH-SY5Y.DiscussionIn conclusion, PA induces apoptosis by increasing oxidative stress in neurons and astrocytes. Taken together, the results suggest that HFD may cause neuronal and astrocytic damage, which indirectly proposes that CNS pathologies involving neuroinflammation and reactive gliosis could be prevented via the diet regimen.

Project description:Mammalian cells were grown as multicellular aggregates (spheroids) in an effort to determine the signaling events required for two cellular transformations states; primary foreskin fibroblasts (HFF-2) and glioblastoma cancer (T98G) cells, to survive at room temperature under oxygen and nutrient-deprived conditions for extended periods of time (2 weeks) and subsequently grown out from the arrested state as adherent monolayers. HFF-2 cells were cultured in DMEM supplemented with 15% fetal bovine serum and 5% carbon dioxide humidified air at 37 degrees C. T98G cells were cultured in EMEM with 10% FBS, 5% non-essential amino acids and 5% carbon dioxide humidified air at 37 degreesC. Monolayers were grown in T-185 flasks to 60% confluency then split into T-185 flasks coated with a 1% agarose mix in a 2:1 media/water ratio. Cells were suspended in 30 ml of supplemented media and grown for 4 days in order to form multicellular spheroids as described previously by our group (J. Cell. Physiol., 206 [2006] 526-536; see GSE1364 and GSE1455 for similar experiments with HEK293 cells). The suspension was removed from the flasks and centrifuged (1500 x g, 2 min) and the media removed. The pellet was returned to the flasks and then placed in vacuum bags (Dri-shield 2000 moisture barrier bag from Surmount Inc., USA; Cat. number 70068), which were sealed immediately under vacuum (Deni Magic Vac, Champion model; Keystone Manufacturing, USA). Vacuum-sealed flasks were stored for 2 weeks (in the dark) at room temperature. Recovery was initiated by removing the flask from the bag and resuspending the spheroids in supplemented media and placing the flasks in a 5% CO2/humidified air incubator maintained at 37 degreesC. Timepoints for transcriptional analysis were monolayer (control), 4 day growth spheroids, 2 week stored spheroids and 7 day growth back to monolayers. There were 67 HFF-2 and 50 T98G genes that exhibited at least a 10-fold change in expression during the course of arrest and recovery. Eleven of these genes were common to both cell lines. While the trends were the same between the two cell lines for genes that were in common, the timing of the responses were clearly different. The primary fibroblasts showed significant gene expression changes during spheroid formation and returned to their initial monolayer state after the two week arrest, whereas the glioblastoma cells exhibited major changes in gene expression during the recovery process. Furthermore, the T98G cells never returned to the monolayer expression levels during the time course of the recovery phase (7 days growth under monolayer conditions). Major differences in the transcriptional data were also found between the cell lines with regards to the putative cellular location of the encoded proteins. Of the genes with significant fold changes (+/- 10-fold) for which location information is available, the primary fibroblasts were significantly enriched in genes encoding extracellular (32%) and membrane-associated (26%) proteins. The glioblastoma cells, on the other hand, had only 17% extracellular and 13% membrane-associated. The expression level trends for all genes above the 10-fold cutoff were as observed for the "in common" genes; HFF-2 genes had returned to monolayer expression levels after 7 days of recovery whereas the T98G line was still exhibiting expression values much different from that of the monolayer control.

Dataset Information

The Effect of Z-Ligustilide on the Mobility of Human Glioblastoma T98G Cells.

Publications

The Effect of Z-Ligustilide on the Mobility of Human Glioblastoma T98G Cells.

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