NSC 95397

Stimulation of Suicidal Erythrocyte Death by the CDC25 Inhibitor NSC-95397.[Pubmed:27889774]

Cell Physiol Biochem. 2016;40(3-4):597-607.

BACKGROUND/AIMS: The CDC25B inhibitor NSC-95397 triggers apoptosis of tumor cells and is thus considered for the treatment of malignancy. The substance is effective in part by modification of gene expression. Similar to apoptosis of nucleated cells erythrocytes may undergo eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Eryptosis may be triggered by increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide, as well as activation of protein kinases. The present study explored, whether NSC-95397 induces eryptosis and, if so, to shed some light on the mechanisms involved. METHODS: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ROS formation from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. RESULTS: A 48 hours exposure of human erythrocytes to NSC-95397 significantly increased the percentage of annexin-V-binding cells (>/= 1 microM), significantly decreased forward scatter (>/= 2.5 microM), and significantly increased Fluo3-fluorescence (>/= 1 microM), DCFDA fluorescence (5 microM) and ceramide abundance (>/= 5 microM). The effect of NSC-95397 (5 microM) on annexin-V-binding was slightly, but significantly blunted by removal of extracellular Ca2+ and by addition of the protein kinase C inhibitor staurosporine (1 microM). CONCLUSIONS: NSC-95397 triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part requiring entry of Ca2+ and activation of staurosporine sensitive kinase(s).

The cytotoxic agents NSC-95397, brefeldin A, bortezomib and sanguinarine induce apoptosis in neuroendocrine tumors in vitro.[Pubmed:20150630]

Anticancer Res. 2010 Jan;30(1):149-56.

UNLABELLED: The aim of this study was to investigate the apoptosis resulting from NSC 95397, brefeldin A, bortezomib and sanguinarine in neuroendocrine tumor cell lines. MATERIALS AND METHODS: A multiparametric high-content screening assay for measurement of apoptosis was used. The human pancreatic carcinoid cell line, BON-1, human typical bronchial carcinoid cell line NCI-H727 and the human atypical bronchial carcinoid cell line NCI-H720 were tested. After incubation with cytotoxic drugs, the DNA-binding dye Hoechst 33342, fluorescein-tagged probes that covalently bind active caspase-3 and chloromethyl-X-rosamine to detect mitochondrial membrane potential were added. Image acquisition and quantitative measurement of fluorescence was performed using automated image capture and analysis instrument ArrayScan. In addition, nuclear morphology was examined on microscopic slides stained with May-Grunewald-Giemsa. RESULTS: A time- and dose-dependent activation of caspase-3 and increase in nuclear fragmentation and condensation were observed for the drugs using a multiparametric apoptosis assay. These results were confirmed with nuclear morphological examination on microscopic slides. CONCLUSION: NSC 95397, brefeldin A, bortezomib and sanguinarine induced caspase-3 activation with modest changes in nuclear morphology.

Quinone-induced Cdc25A inhibition causes ERK-dependent connexin phosphorylation.[Pubmed:15652497]

Biochem Biophys Res Commun. 2005 Feb 25;327(4):1016-23.

Gap junctional intercellular communication (GJC) varies during progression of the cell cycle. We propose here that Cdc25A, a dual specificity phosphatase crucial for cell cycle progression, is linked to connexin (Cx) phosphorylation and the modulation of GJC. Inhibition of Cdc25 phosphatases in rat liver epithelial cells employing a 1,4-naphthoquinone-based inhibitor, NSC95397, induced cell cycle arrest, tyrosine phosphorylation of the epidermal growth factor receptor (EGFR), and activation of extracellular signal-regulated kinases ERK-1 and -2. ERK activation was blocked by specific inhibitors of MAPK/ERK kinases 1/2 or of the EGFR tyrosine kinase. An EGFR-dephosphorylation assay suggested that Cdc25A interacts with the EGFR, with inhibition by NSC95397 resulting in activation of the receptor. As a consequence of ERK activation, Cx43 was phosphorylated, resulting in a downregulation of GJC. Loss of GJC was prevented by inhibition of ERK activation. In summary, cell cycle and GJC are connected via Cdc25A and the EGFR-ERK pathway.

NAD(P)H:quinone oxidoreductase-1-dependent and -independent cytotoxicity of potent quinone Cdc25 phosphatase inhibitors.[Pubmed:14718602]

J Pharmacol Exp Ther. 2004 Apr;309(1):64-70.

Cdc25 dual-specificity phosphatases coordinate cell cycle progression and cellular signaling. Consequently, Cdc25 inhibitors represent potential anticancer agents. We evaluated >10,000 compounds for inhibition of human Cdc25 phosphatases and identified many potent and selective inhibitors, which all contained a quinone. Bioreductive enzymes frequently detoxify or activate quinones. Therefore, we evaluated the effect of NAD(P)H:quinone oxidoreductase-1 (NQO1) and reductase-rich microsomes on the activity of three quinone-containing Cdc25 inhibitors: 2-(2-hydroxyethylsulfanyl)-3-methyl-1,4-naphthoquinone (Cpd 5, compound 5; NSC 672121), 2,3-bis-(2-hydroxyethylsulfanyl)-1,4-naphthoquinone (NSC 95397), and 6-chloro-7-(2-morpholin-4-yl-ethylamino)quinoline-5,8-dione (NSC 663284). Each inhibitor was reduced by human NQO1 (K(m) of 0.3-0.5 microM) but none by microsomes. Compounds were evaluated with six cancer cell lines containing different amounts of NQO1: HT-29 (1056 nmol/mg/min), HCT116 (660 nmol/mg/min), sublines HCT116-R30A (28 nmol/mg/min) and HCT-116R30A/NQ5 (934 nmol/mg/min), MDA-MB-231/Q2 (null NQO1), and subline MDA-MB-231/Q6 (124 nmol/mg/min) but containing similar amounts of microsomal cytochrome P450 reductase and cytochrome b(5) reductase. Growth inhibition and G2/M arrest by Cpd 5 was proportional to NQO1 levels, requiring 4- to 5-fold more Cpd 5 to inhibit HCT-116 or HCT-116R30A/NQ5 compared with HCT-116R30A. In contrast, in all tested cell lines irrespective of NQO1 level, growth inhibition and G2/M arrest by NSC 95375 and NSC 663284 were similar (average IC(50) of 1.3 +/- 0.3 and 2.6 +/- 0.4 microM, respectively). NSC 95375 and NSC 663284 also caused similar Cdk1 hyperphosphorylation, indicating similar Cdc25 inhibition. However, lower Cpd 5 concentrations were needed to produce Cdk1 hyperphosphorylation in sublines with minimal NQO1. Thus, NQO1 detoxified Cpd 5, probably by reducing it to a less active hydroquinone, whereas NSC 95397- and NSC 663284-generated cytotoxicity was unaffected by NQO1.

NSC 95397 is a potent, selective Cdc25 dual specificity phosphatase inhibitor (Ki=32 nM (Cdc25A), 96 nM (Cdc25B), 40 nM (Cdc25C); IC50=22.3 nM (human Cdc25A), 56.9 nM (human Cdc25C), 125 nM (Cdc25B)). NSC 95397 inhibits mitogen-activated protein kinase phosphatase-1 (MKP-1) and suppresses proliferation and induces apoptosis in colon cancer cells through MKP-1 and ERK1/2 pathway.

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