Ju Dong Song

Diphenyleneiodonium induces ROS-independent p53 expression and apoptosis in human RPE cells

The diphenyleneiodonium (DPI) is widely used as an inhibitor of flavoenzymes, particularly NADPH oxidase. In this study, we investigated the effect of DPI on the apoptosis of human RPE cells. DPI treatment in ARPE‐19 cells evoked a dose‐ and time‐dependent growth inhibition, and also induced DNA fragmentation and protein content of the proapoptotic factor Bax. In addition, DPI significantly induced the expression and phosphorylation of p53, which induces proapoptotic genes in response to DNA damage or irreparable cell cycle arrest. ROS have been implicated as a key factor in the activation of p53 by many chemotherapeutic drugs. Recent data on the regulation of intracellular ROS by DPI are controversial. Therefore, we analyzed whether DPI could contribute to the generation of intracellular ROS. Although there was increase in ROS level from cells treated for 24 h with DPI, it was not detectable at early time points, required to induce p53 expression. And DPI‐induced p53 expression was not affected by the ROS scavenger NAC. We conclude that DPI induces the expression of p53 by ROS‐independent mechanism in ARPE‐19 cells, and renders cells sensitive to drug‐induced apoptosis by induction of p53 expression.

Redox factor-1 mediates NF-κB nuclear translocation for LPS-induced iNOS expression in murine macrophage cell line RAW 264.7

Redox‐sensitive transcriptional regulator redox factor‐1 (Ref‐1) is induced by oxidative stress and protects cells against it. However, the function of Ref‐1 in regulating nitric oxide (NO) synthesis in macrophages has not been defined. We investigated the role of Ref‐1 related to the regulation of NO synthesis in lipopolysaccharide (LPS)‐stimulated macrophage RAW 264.7 cells. LPS stimulates the up‐regulation and nuclear translocation of Ref‐1 in macrophages. Importantly, Ref‐1‐deficient macrophages using a small interfering RNA did not stimulate inducible NO synthase (iNOS) expression as well as nuclear factor‐κB nuclear translocation by stimulation with LPS. When the cells were pretreated with diphenyleneiodonium or p47phox small interfering RNA for inhibition of NADPH oxidase activity, LPS did not stimulate the nuclear translocation of Ref‐1. We next asked whether reactive oxygen species are sufficient for the nuclear translocation of Ref‐1 in macrophages. The direct use of H2O2 stimulated the translocation to the nucleus of nuclear factor‐κB, but not Ref‐1 and antioxidant N‐acetyl cysteine did not inhibit the LPS‐stimulated nuclear translocation of Ref‐1. These data suggest that Ref‐1 nuclear translocation in LPS‐stimulated macrophages requires the activation of other signalling molecules aside from reactive oxygen species followed by the activation of NADPH oxidase.

Cobalt protoporphyrin induces differentiation of monocytic THP-1 cells through regulation of cytoplasmic Ref-1-related NADPH oxidase activity

Cobalt protoporphyrin (CoPP) is a potent and effective metalloporphyrin inducer of heme oxygenase-1 (HO-1) activity in many tissues. Here, we report that CoPP induces differentiation of monocytic THP-1 cells into macrophage-like cells. CoPP induced a marked growth inhibition with a slight reduction in viability, and increased adhesion and spreading of THP-1 cells. However, other protoporphyrins did not. CoPP also resulted in expression of CD11b, MMP9, MSR1, CD14 and ICAM-1, which are differentiation markers for macrophages. Interestingly, we observed a decrease of cytoplasmic redox factor-1 (Ref-1) levels in the process of CoPP-induced differentiation of THP-1 cells. In addition, knockdown of Ref-1 by siRNA enhanced cell adhesion induced by CoPP. Furthermore, an inhibitor of NADPH oxidase, diphenyleneiodonium (DPI), completely abolished CoPP-induced adhesion of Ref-1-deficient cells using an siRNA. A cytosolic factor for NADPH oxidase activity, p47phox, was significantly increased in THP-1 cells by CoPP treatment. Κnockdown of Ref-1 increased CoPP-induced p47phox expression in THP-1 cells. Taken together, these results suggest that CoPP induces differentiation of monocytic THP-1 cells, and that the CoPP-induced differentiation is associated with cytoplasmic Ref-1-related NADPH oxidase activity.

Involvement of Peroxynitrite in NO Donor-Induced HO-1 Expression in Rat Articular Chondrocytes

Nitric oxide (NO) donors are a potent inducer of heme oxygenase-1 (HO-1). However, it is unclear whether or not HO-1 expression induced by NO donors is a direct consequence of NO released by NO donors. Here, we investigated the effects of NO donors on the expression of HO-1 in primary rat articular chondrocytes. NO donors (SIN-1, SNAP, and SNP) significantly induced the accumulation of HO-1 protein accompanied by an increase in HO-1 mRNA. NO donor-induced HO-1 expression exerted cytoprotection against NO and/or superoxide-induced cell death. Guanylate cyclase signaling was not associated with Nrf2 and HO-1 expression in NO donor-treated chondrocytes. Interestingly, NO scavenger carboxy-PTIO and SOD mimetic TEMPOL markedly inhibited NO donor-induced HO-1 expression in chondrocytes. In addition, NO donor-induced HO-1 expression was completely abrogated by the peroxynitrite scavenger MnTBAP. Since peroxynitrite can be physiologcally formed in the cell through reaction of NO with superoxide, we analyzed whether or not peroxynitrite could directly induce HO-1 expression in chondrocytes. Peroxynitrite treatment in chondrocytes evoked dose-and time-dependent Nrf2 and HO-1 expression. These results indicate that HO-1 expression induced by NO donors in rat articular chondrocytes is due to NO-mediated peroxynitrite rather than NO.

Effect of Overexpressed Ref-1 on AKT Phosphorylation for NO Production in Mouse Aortic Endothelial Cell Line

Redox factor-1 (Ref-1) is essential for repair of oxidatively damaged DNA and also govern the reductive activation of many transcription factors. In this study, we examined the effect of overexpressed Ref-1 on AKT activation for nitric oxide (NO) production in mouse aortic endothelial (MAE) cells. Adenoviral-mediated overexpression of Ref-1 enhanced NO production in unstimulated- as well as bradykinin-stimulated MAE cells. Importantly, forced overexpression of Ref-1 induced direct phosphorylation of AKT in cells. And, a PI3K inhibitor wortmannin completely abolished the increase in AKT phosphorylation by stimulation of bradykinin and/or overexpressed Ref-1. In addition, inhibition of AKT activity with HA-tagged activation-deficient AKT suppressed Ref-1-induced endothelial NO synthase (eNOS) phosphorylation and resulted in a corresponding inhibition of unstimulated- and bradykinin-stimulated NO production. These results suggest that Ref-1 stimulates direct phosphorylation of AKT for eNOS enzyme activity in murine endothelial cells.

Anti-apoptotic Activity of Heme Oxygenase-1 Up-regulated by Etoposide in Human Retinal Pigment Epithelial Cells

The topoisomerase II inhibitor etoposide causes an accumulation of DNA double strand breaks within the nuclei of cells. In this study, we investigated the effect of etoposide on the cell growth and apoptosis of human RPE cells. Etoposide evoked a significant inhibition of cell growth, and also induced DNA fragmentation in ARPE-19 cells. In addition, etoposide significantly up-regulated the expression of heme oxygenase-1 (HO-1), which is a stress-responsive protein and is known to play a protective role against the oxidative injury. And, etoposide-induced HO-1 expression was affected by the ROS scavenger N-acetyl cysteine. We also used oligonucleotides interfering with HO-1 mRNA (siRNA) for the inhibition of HO-1 expression. Interestingly, knock-down of the HO-1 gene significantly increased the level of DNA fragmentation in etoposide-treated ARPE-19 cells. In conclusion, these results suggest that up-regulated HO-1 plays as an anti-apoptotic factor in the process of apoptosis of ARPE-19 cells stimulated by etoposide.

Adenoviral-Mediated Ref-1 Overexpression Potentiates NO Production in Bradykinin-Stimulated Endothelial Cells

The dual-function protein redox factor-1 (Ref-1) is essential for base excision repair of oxidatively damaged DNA and also governs the activation of many redox-sensitive transcription factors. We examined the role of Ref-1 in regulation of nitric oxide (NO) synthesis employing adenoviral-mediated overexpression of Ref-1 in bradykinin-stimulated endothelial cells. Intracellular NO was detected with the NO-sensitive fluorophore DAF-2. Overexpression of Ref-1 potentiates bradykinin-stimulated NO production in endothelial cells. And, cells infected with AdRef-1 showed higher fluorescence intensity compared with uninfected or AdDl312-infected cells. In parallel with this, overexpression of Ref-1 also stimulated endothelial NO synthase (eNOS) enzyme activity, compared with uninfected or AdDl312-infected cells, in bradykinin-stimulated cells as well as in unstimulated cells. These results suggest that Ref-1 implicates in endothelium-dependent vasorelaxation resulting from NO production in vascular system.

NADPH oxidase inhibitor diphenyleneiodonium induces p53 expression and cell cycle arrest in several cancer cell lines

The Diphenyleneiodonium (DPI) is widely used as an inhibitor of flavoenzymes, particularly NADPH oxidase. In this study, we investigated the effect of DPI on the cell growth progression of human colon cancer cells HCT-116 (wild-type p53), HT-29 (p53 mutant) and human breast cancer cells MCF-7 (wild-type p53). DPI treatment in cancer cells evoked a dose- and time-dependent growth inhibition, and also induced the cell cycle arrest in G2/M phase. The peak of cell population arrested in G2/M phase was observed at 12 hr after treatment of DPI. In addition, DPI significantly induced the expression of p53, which induces proapoptotic genes in response to DNA damage or irreparable cell cycle arrest, at 6 hr in DPI-stimulated cells. However, a catechol apocynin, which inhibits the assembly of NADPH oxidase, did not induce p53 expression. This suggest that p53 expression induced by DPI is not associated with the inhibition of NADPH oxidase. In conclusion, we suggest that DPI induces the expression of wild-type p53 by ROS-independent mechanism in several cancer cells, and upregulated p53 may be involved in regulatory mechanisms for growth inhibition and cell cycle arrest at G2/M phase in DPI-stimulated cells.