Proliferation Inhibition, DNA Damage, and Cell-Cycle Arrest of Human Astrocytoma Cells after Acrylamide Exposure

Abstract

Acrylamide (ACR) has been recognized as a neurological and reproductive toxin in humans and laboratory animals. This study aimed to determine the effects of ACR-induced DNA damage on cell cycle regulation in human astrocytoma cell lines. Treatment of U-1240 MG cells with 2 mM ACR for 48 h resulted in a significant inhibition of cell proliferation as evaluated by Ki-67 protein expression and MTT assay. The analysis of DNA damage with the comet assay showed that treatment of the cells with 0.5, 1, and 2 mM ACR for 48 h caused significant increases in DNA damage by 3.5-, 4-, and 14-fold, respectively. Meanwhile, analysis of cell-cycle arrest with flow cytometry revealed that the ACR treatments resulted in significant increases in the G(0)/G(1)-arrested cells in a time- and dose-dependent manner. Expression of DNA damage-associated/checkpoint-related signaling molecules, including phosphorylated-p53 (pp53), p53, p21, p27, Cdk2, and cyclin D(1), in three human astrocytoma cell lines (U-1240 MG, U-251 MG, and U-87 MG) was also analyzed by immunoblotting. Treatment of the three cell lines with 2 mM ACR for 48 h caused marked increases in pp53 and Cdk2, as well as decreases in cyclin D(1) and p27. Moreover, increases in p53 and p21 were detected in both U-1240 and U-87 MG cells, whereas no marked change in p53 and a decrease in p21 were observed in U-251 MG cells. To address the involvement of ataxia telangiectasia mutated/ATM-Rad3-related (ATM/ATR) kinase in the signaling of ACR-induced G(0)/G(1) arrest, caffeine was used to block the ATM/ATR pathway in U-1240 MG cells. Caffeine significantly attenuated the ACR-induced G(0)/G(1) arrest as well as the expression of DNA damage-associated/checkpoint-related signaling molecules in a dose-dependent manner. This in vitro study clearly demonstrates the critical role of ATM/ATR in the signaling of ACR-induced cell-cycle arrest in astrocytoma cells.

Acrylamide inhibits cellular differentiation of human neuroblastoma and glioblastoma cells

“…Okuno et al (2006) reported that the ERK pathway causes phosphorylation of p53 at Ser15 and the cytotoxicity of ACR in SH-SY5Y cells without RA-induced differentiation. Our previous studies showed that ACR induced cytotoxicity in U-1240 MG cells through the phosphorylation of p53 at Ser15 during ATM/ATR signaling (Chen et al, 2010). The current study further demonstrated that the phosphorylation of ERK and JNK were decreased by ACR exposure in U-1240 MG cells without BA-induced differentiation.…”

“…The toxic effects of ACR on neurons have been investigated intensively, including the reduction of cell viability, the induction of apoptosis and p53 phosphorylation (Okuno et al, 2006;Sumizawa and Igisu, 2007), the formation of perikaryal inclusion bodies (Hartley et al, 1997), and the neurodegeneration of signal transduction (Yuzo et al, 2001). ACR-induced DNA breaks will increase p21 expression and G0/G1 cell-cycle arrest by inhibiting cyclin D1/Cdk4/6 kinase activity, which in turn causes cellular mitochondria collapse and apoptosis (Chen et al, 2010(Chen et al, , 2013. Our study further suggested that ACR impairs the differentiation efficiency of human neurons and glia cells and that the interactions of neurons and glia cells in the neuronal system may be affected by ACR-induced neurotoxicity.…”

“…Cell viability was analyzed with an MTT assay according to our previous protocols (Chen et al, 2010). In brief, cells were placed in 96 well microtiter plate at a density of 1000 cells per well in a final volume of 100 μL of culture medium for 1 day prior to the experiment.…”

“…Protein expressions were measured with Western blot analysis, which was modified from our previous protocol (Chen et al, 2009(Chen et al, , 2010. Cells were harvested in lysis buffer (Sigma, St Louis, MO) and left on ice for 20 min and then were centrifuged (8000 × g for 30 min at 4°C).…”

Flower extracts of Filipendula ulmaria (L.) Maxim inhibit the proliferation of the NCI-H460 tumour cell line

“…Nonetheless, considering the previously obtained results in cellular proliferation, an increase in the number of cells in the G1 phase of the cell cycle would make sense. In fact, cell cycle arrests cause decrease in cellular proliferation (Chen et al, 2010).…”