Dicumarol is a potent reversible inhibitor of gap junctional intercellular communication

Dicumarol [3,3'-methylene-bis(4-hydroxycoumarin)] is a potent inhibitor of NAD(P)H:quinone oxidoreductase-1. Exposure of rat liver epithelial cells or of human skin fibroblasts to dicumarol resulted in a rapid and complete inhibition of connexin-43-dependent gap junctional intercellular communication (GJC). GJC was restored within 60min following removal of dicumarol. The concentration of dicumarol required for half maximal inhibition of GJC was 3muM, making dicumarol about 10-fold more effective in blocking GJC than 1-octanol and flufenamic acid, known inhibitors of GJC. Warfarin, a related coumarin derivative, also attenuated GJC, yet very high concentrations of 5-10mM were required. Dicumarol-induced downregulation of GJC was found not to be due to an interference with pathways enhancing the phosphorylation of connexin-43, such as epidermal growth factor receptor and extracellular signal-regulated kinase pathways. Rather, inhibition of GJC by dicumarol was paralleled by a reversible loss of a phosphorylated form ("P2") of connexin-43.     

α-Tocopheryl succinate pre-treatment attenuates quinone toxicity in prostate cancer PC3 cells

α-Tocopheryl succinate is one of the most effective analogues of vitamin E for inhibiting cell proliferation and inducing cell death in a variety of cancerous cell lines while sparing normal cells or tissues. αTocopheryl succinate inhibits oxidative phosphorylation at the level of mitochondrial complexes I and II, thus enhancing reactive oxygen species generation which, in turn, induces the expression of Nrf2-driven antioxidant/detoxifying genes. The cytoprotective role of Nrf2 downstream genes/proteins prompted us to investigate whether and how α-tocopheryl succinate increases resistance of PC3 prostate cancer cells to pro-oxidant damage. A 4 h α-tocopheryl succinate pre-treatment increases glutathione intracellular content, indicating that the vitamin E derivative is capable of training the cells to react to an oxidative insult. We found that α-tocopheryl succinate pre-treatment does not enhance paraquat-/hydroquinone-induced cytotoxicity whereas it exhibits an additional/synergistic effect on H₂O₂-/docetaxel-induced cytotoxicity.     

Inhibition of cell growth by overexpression of manganese superoxide dismutase (MnSOD) in human pancreatic carcinoma

Manganese superoxide dismutase (MnSOD) levels have been found to be low in human pancreatic cancer [Pancreas26, (2003), 23] and human pancreatic cancer cell lines [Cancer Res.63, (2003), 1297] when compared to normal human pancreas. We hypothesized that stable overexpression of pancreatic cancer cells with MnSOD cDNA would alter the malignant phenotype. MIA PaCa-2 cells were stably transfected with a pcDNA3 plasmid containing sense human MnSOD cDNA or containing no MnSOD insert by using the lipofectAMINE method. G418-resistant colonies were isolated, grown and maintained. Overexpression of MnSOD was confirmed in two selected clones with a 2-4-fold increase in MnSOD immunoreactive protein. Compared with the parental and neo control cells, the MnSOD-overexpressing clones had decreased growth rates, growth in soft agar and plating efficiency in vitro, while in vivo, the MnSOD-overexpressing clones had slower growth in nude mice. These results suggest that MnSOD may be a tumor suppressor gene in human pancreatic cancer.     

Inhibition of the SAPK/JNK pathway blocks the stimulatory effects of glutamine on fluid secretion by the Malpighian tubules of Rhodnius prolixus

Physiological levels of amino acids such as glutamine, glutamate, aspartate and proline increase the rates of fluid secretion and ion transport by serotonin-stimulated Malpighian tubules (MTs) of Rhodnius prolixus. Here, we examine the proposal that the effects of glutamine are mediated through activation of specific kinases to produce the observed increases in fluid secretion. The glutamine-dependent increase in MT fluid secretion rate was blocked by two chemically unrelated inhibitors of the stress activated protein kinase (SAPK) pathway, SP600125 and dicumarol. Inhibitors of phosphatidyl inositol-3 kinase, p38 mitogen activated protein kinase (MAPK), extracellular-signal regulated kinases and MAPK kinase did not block glutamine's effects on fluid secretion rate when applied at commonly used concentrations. Inhibitors of protein kinase A or C reduced fluid secretion rates of serotonin-stimulated MTs, but did not block the response to glutamine. The glutamine-dependent increase in fluid secretion was also insensitive to cytoskeletal disrupting agents and protein synthesis inhibitors. Results of this study are the first to suggest a role for the SAPK pathway in the control of fluid secretion rates by insect MTs.     

Effect of dicumarol,a Nad(P)h: quinone acceptor oxidoreductase 1 (DT-diaphorase) inhibitor on ubiquinone redox cycling in cultured rat hepatocytes

Ubiquinol is considered to serve as an endogenous antioxidant. However, the mechanism by which the redox state of intracellular ubiquinone (UQ) is maintained is not well established. The effect of dicumarol, an inhibitor of NAD(P)H: quinone acceptor oxidoreductase 1 (NQO1=DT-diaphorase, EC 1.6.99.2), on the reduction of UQ in cultured rat hepatocytes was investigated in order to clarify whether or not NQO1 is involved in reducing intracellular UQ. A concentration of 5 μM dicumarol, which does not inhibit cytosolic NADPH-dependent UQ reductase in vitro , was observed to almost completely inhibit NQO1 and thereby to stimulate cytotoxicity of 2-methyl-1,4-naphthoquinone (menadione) in cultured rat hepatocytes. However, 5 μM dicumarol did not inhibit reduction of endogenous UQ-9, as well as exogenous UQ-10 added to the hepatocytes. In addition, it did not stimulate the formation of thiobarbituric acid reactive substances (TBARS) in the hepatocytes. These results suggested that NQO1 is not involved in maintaining UQ in the reduced state in the intact liver cells.     

Inhibition of 4-nitroquinoline 1-oxide induced unscheduled DNA synthesis in primary cultures of rat urothelial cells by dicumarol and pyrophosphate

Primary cultures of rat urothelial cells were exposed to hydroxyurea, [3H]thymidine, and 4-nitroquinoline 1-oxide (NQO) or N-hydroxy-4-aminoquinoline 1-oxide (HAQO) in a serum-free media for 2 h; unscheduled DNA synthesis (UDS) was measured by autoradiography. Both NQO and HAQO produced unscheduled DNA synthesis. Dicumarol, an inhibitor of NQO nitroreductase, inhibited the activity of NQO and, to a lesser extent, HAQO. Pyrophosphate, an inhibitor of seryl-AMP synthetase, inhibited the activity of both compounds. Neither dicumarol nor pyrophosphate, under similar experimental conditions, inhibited the activity of N-hydroxy-N-2-acetylaminofluorene (N-OH-AAF). These results support the idea that nitro-reductase and seryl-AMP synthetase may be involved in the activation of NQO.     

Effect of Dicumarol,a NAD(P)H: Quinone Acceptor Oxidoreductase 1 (DT-diaphorase) Inhibitor on Ubiquinone Redox Cycling in Cultured Rat Hepatocytes

Ubiquinol is considered to serve as an endogenous antioxidant. However, the mechanism by which the redox state of intracellular ubiquinone (UQ) is maintained is not well established. The effect of dicumarol, an inhibitor of NAD(P)H: quinone acceptor oxidoreductase 1 (NQO1=DT-diaphorase, EC 1.6.99.2), on the reduction of UQ in cultured rat hepatocytes was investigated in order to clarify whether or not NQO1 is involved in reducing intracellular UQ. A concentration of 5 &#119 M dicumarol, which does not inhibit cytosolic NADPH-dependent UQ reductase in vitro, was observed to almost completely inhibit NQO1 and thereby to stimulate cytotoxicity of 2-methyl-1,4-naphthoquinone (menadione) in cultured rat hepatocytes. However, 5 &#119 M dicumarol did not inhibit reduction of endogenous UQ-9, as well as exogenous UQ-10 added to the hepatocytes. In addition, it did not stimulate the formation of thiobarbituric acid reactive substances (TBARS) in the hepatocytes. These results suggested that NQO1 is not involved in maintaining UQ in the reduced state in the intact liver cells.     

Inhibition of Cell Growth by Overexpression of Manganese Superoxide Dismutase (MnSOD) in Human Pancreatic Carcinoma

Manganese superoxide dismutase (MnSOD) levels have been found to be low in human pancreatic cancer [Pancreas26, (2003), 23] and human pancreatic cancer cell lines [Cancer Res.63, (2003), 1297] when compared to normal human pancreas. We hypothesized that stable overexpression of pancreatic cancer cells with MnSOD cDNA would alter the malignant phenotype. MIA PaCa-2 cells were stably transfected with a pcDNA3 plasmid containing sense human MnSOD cDNA or containing no MnSOD insert by using the lipofectAMINE method. G418-resistant colonies were isolated, grown and maintained. Overexpression of MnSOD was confirmed in two selected clones with a 2-4-fold increase in MnSOD immunoreactive protein. Compared with the parental and neo control cells, the MnSOD-overexpressing clones had decreased growth rates, growth in soft agar and plating efficiency in vitro, while in vivo, the MnSOD-overexpressing clones had slower growth in nude mice. These results suggest that MnSOD may be a tumor suppressor gene in human pancreatic cancer.