• Molecules and Cells
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• v.37 no.8
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• pp.605-612
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• 2014

The p73 gene contains an extrinsic P1 promoter and an intrinsic P2 promoter, controlling the transcription of the pro-apoptotic TAp73 isoform and the anti-apoptotic ${\Delta}Np73$ isoform, respectively. The DNA methylation status of both promoters act equally in the epigenetic transcriptional regulation of their relevant isoforms. The aim of this study was to analyze the different effects of these p73 isoforms in 5-aza-2'-deoxycytidine (5-aza-dC)-induced apoptosis in breast cancer cells. We investigated the effects of the DNA demethylation agent, 5-aza-dC, on the T-47D breast cancer cell line, and evaluated the methylation status of the p73 promoters and expression of TAp73 and ${\Delta}Np73$. Furthermore, we assessed the expression of p53 and p73 isoforms in 5-aza-dC-treated T-47D cells and p53 knockout cells. 5-aza-dC induced significant anti-tumor effects in T-47D cells, including inhibition of cell viability, G1 phase arrest and apoptosis. This was associated with p73 promoter demethylation and a concomitant increase in TAp73 mRNA and protein expression. In contrast, the methylation status of promoter P2 was not associated with ${\Delta}Np73$ mRNA or protein levels. Furthermore, demethylation of P2 failed to inhibit the expression of ${\Delta}Np73$ with 5-aza-dC in the p53 knockdown cell model. Our study suggests that demethylation of the P1 and P2 promoters has opposite effects on the expression of p73 isoforms, namely up-regulation of TAp73 and down-regulation of ${\Delta}Np73$. We also demonstrate that p53 likely contributes to 5-aza-dC-induced ${\Delta}Np73$ transcriptional inactivation in breast cancer cells.

• Animal cells and systems
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• v.6 no.2
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• pp.171-180
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• 2002

Nitric oxide has high affinity for iron, and thus it can cause intracellular iron loss. We tested the idea that intracellular iron can be the primary target of NO toxicity by comparing the signaling mechanisms involved in cell death caused by iron depletion and that caused by NO. Treatment of HL-60 cells with a NO donor, S-nitroso-N-acetyl-DL-penicillamine (SNAP), decreased the intracellular iron level rapidly as that observed with the iron chelator deferoxamine (DFO). Iron chelators such as DFO and mimosine could induce death of human leukemic HL-60 cells by a mechanism requiring activation of p38 kinase, c-Jun N-terminal kinase, caspase-3 and caspase-8. DFO and SNAP also caused release of cytochrome c from mitochondria. Inhibition of p38 kinase by a selective inhibitor, SB203580, abolished the NO and DFO-induced cell death, release of cytochrome c, and activation of caspase-3 and caspase-8, thus indicating that p38 kinase lies upstream in the cell death processes. In a parallel situation, the cells that are sensitive to NO showed similar sensitivity to DFO. Moreover, simultaneous addition of ferric citrate, an iron-containing compound, inhibited the SNAP and DFO-induced activation of caspases and also blocked the NO-mediated cell cycle arrest at $G_1$ phase. Collectively, our data implicate that the NO-induced cell death of tumor cells including HL-60 cells is mediated by depletion of iron and further suggest that activation of p38 kinase lies upstream of cytochrome c release and caspase activation involved in this apoptotic process.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.23
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• pp.10407-10412
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• 2015

Background: ${\beta}$-elemene, extracted from herb medicine Curcuma wenyujin has potent anti-tumor effects in various cancer cell lines. However, the activity of ${\beta}$-elemene against glioma cells remains unclear. In the present study, we assessed effects of ${\beta}$-elemene on human glioma cells and explored the underlying mechanism. Materials and Methods: Human glioma U87 cells were used. Cell proliferation was determined with MTT assay and colony formation assay to detect the effect of ${\beta}$-elemene at different doses and times. Fluorescence microscopy was used to observe cell apoptosis with Hoechst 33258 staining and change of glioma apoptosis and cell cycling were analyzed by flow cytometry. Real-time quantitative PCR and Western-blotting assay were performed to investigated the influence of ${\beta}$-elemene on expression levels of Fas/FasL, caspase-3, Bcl-2 and Bax. The experiment was divided into two groups: the blank control group and ${\beta}$-elemne treatment group. Results: With increase in the concentration of ${\beta}$-elemene, cytotoxic effects were enhanced in the glioma cell line and the concentration of inhibited cell viability ($IC_{50}$) was $48.5{\mu}g/mL$ for 24h. ${\beta}$-elemene could induce cell cycle arrest in the G0/G1 phase. With Hoechst 33258 staining, apoptotic nuclear morphological changes were observed. Activation of caspase-3,-8 and -9 was increased and the pro-apoptotic factors Fas/FasL and Bax were upregulated, while the anti-apoptotic Bcl-2 was downregulated after treatment with ${\beta}$-elemene at both mRNA and protein levels. Furthermore, proliferation and colony formation by U87 cells were inhibited by ${\beta}$-elemene in a time and does-dependent manner. Conclusions: Our results indicate that ${\beta}$-elemene inhibits growth and induces apoptosis of human glioma cells in vitro. The induction of apoptosis appears to be related with the upregulation of Fas/FasL and Bax, activation of caspase-3,-8 and -9 and downregulation of Bcl-2, which then trigger major apoptotic cascades.