dicumarol and Pancreatic-Neoplasms

Base excision repair (BER) is an essential pathway for pancreatic ductal adenocarcinoma (PDA) survival. Attempts to target this repair pathway have failed due to lack of tumor-selectivity and very limited efficacy. The. Quinone Oxidoreductase 1 (NQO1) bioactivatable drug, ß-lapachone (ARQ761 in clinical form), can provide tumor-selective and enhanced synergy with BER inhibition. ß-Lapachone undergoes NQO1-dependent futile redox cycling, generating massive intracellular hydrogen peroxide levels and oxidative DNA lesions that stimulate poly(ADP-ribose) polymerase 1 (PARP1) hyperactivation. Rapid NAD(+)/ATP depletion and programmed necrosis results. To identify BER modulators essential for repair of ß-lapachone-induced DNA base damage, a focused synthetic lethal RNAi screen demonstrated that silencing the BER scaffolding protein, XRCC1, sensitized PDA cells. In contrast, depleting OGG1 N-glycosylase spared cells from ß-lap-induced lethality and blunted PARP1 hyperactivation. Combining ß-lapachone with XRCC1 knockdown or methoxyamine (MeOX), an apyrimidinic/apurinic (AP)-modifying agent, led to NQO1-dependent synergistic killing in PDA, NSCLC, breast and head and neck cancers. OGG1 knockdown, dicoumarol-treatment or NQO1- cancer cells were spared. MeOX + ß-lapachone exposure resulted in elevated DNA double-strand breaks, PARP1 hyperactivation and TUNEL+ programmed necrosis. Combination treatment caused dramatic antitumor activity, enhanced PARP1-hyperactivation in tumor tissue, and improved survival of mice bearing MiaPaca2-derived xenografts, with 33% apparent cures.. Targeting base excision repair (BER) alone has limited therapeutic potential for pancreatic or other cancers due to a general lack of tumor-selectivity. Here, we present a treatment strategy that makes BER inhibition tumor-selective and NQO1-dependent for therapy of most solid neoplasms, particularly for pancreatic cancer.

Topics: Animals; Autophagy; Catalase; Cell Line, Tumor; Cell Survival; Dicumarol; DNA Breaks, Double-Stranded; DNA Glycosylases; DNA Repair; DNA-Binding Proteins; Female; Humans; Hydroxylamines; Mice; Mice, Nude; NAD(P)H Dehydrogenase (Quinone); Naphthoquinones; Pancreatic Neoplasms; Poly(ADP-ribose) Polymerases; Reactive Oxygen Species; Transplantation, Heterologous; X-ray Repair Cross Complementing Protein 1

The enzyme human NAD(P)H quinone oxidoreductase-1 (NQO1), which is overexpressed in several types of tumor cell, is considered a design target for cancer therapeutics. We identify new coumarin-based competitive inhibitors of NQO1, one of which is nanomolar. Using computational docking and molecular dynamics, we obtain insights into the structural basis of inhibition. Selected inhibitors were then assessed for off-target effects associated with dicoumarol and were found to have differing effects on superoxide formation and mitochondrial respiration. A comparison of NQO1 inhibition and off-target effects for dicoumarol and its derivatives suggests that the ability of dicoumarol to kill cancer cells is independent of NQO1 inhibition, that cellular superoxide production by dicoumarol does not seem linked to NQO1 inhibition but may be related to mitochondrial decoupling, and that superoxide does not appear to be a major determinant of cytotoxicity. Implications are discussed for NQO1 inhibition as an anticancer drug design target and superoxide generation as the dicoumarol-mediated mechanism of cytotoxicity.

Topics: Antineoplastic Agents; Binding Sites; Cell Death; Cell Line, Tumor; Coumarins; Dicumarol; Drug Screening Assays, Antitumor; Humans; Models, Molecular; NAD(P)H Dehydrogenase (Quinone); Oxygen Consumption; Pancreatic Neoplasms; Protein Binding; Structure-Activity Relationship; Superoxides

Dicumarol is a naturally occurring anticoagulant derived from coumarin that induces cytotoxicity and oxidative stress in human pancreatic cancer cells (Cullen, J. J., Hinkhouse, M. M., Grady, M., Gaut, A. W., Liu, J., Zhang, Y., Weydert, C. J. D., Domann, F. E., and Oberley, L. W. (2003) Cancer Res. 63, 5513-5520). Although dicumarol has been used as an inhibitor of the two-electron reductase NAD(P)H:quinone oxidoreductase (NQO1), dicumarol is also thought to affect quinone-mediated electron transfer reactions in the mitochondria, leading to the production of superoxide (O2*-) and hydrogen peroxide (H(2)O(2)). We hypothesized that mitochondrial production of reactive oxygen species mediates the increased susceptibility of pancreatic cancer cells to dicumarol-induced metabolic oxidative stress. Dicumarol decreased clonogenic survival equally in both MDA-MB-468 NQO1(-) and MDA-MB-468 NQO1+ breast cancer cells. Dicumarol decreased clonogenic survival in the transformed fibroblast cell line IMRSV-90 compared with the IMR-90 cell line. Dicumarol, with the addition of mitochondrial electron transport chain blockers, decreased clonogenic cell survival in human pancreatic cancer cells and increased superoxide levels. Dicumarol with the mitochondrial electron transport chain blocker antimycin A decreased clonogenic survival and increased superoxide levels in cells with functional mitochondria but had little effect on cancer cells without functional mitochondria. Overexpression of manganese superoxide dismutase and mitochondrial-targeted catalase with adenoviral vectors reversed the dicumarol-induced cytotoxicity and reversed fluorescence of the oxidation-sensitive probe. We conclude mitochondrial production of reactive oxygen species mediates the increased susceptibility of cancer cells to dicumarol-induced cytotoxicity.

Topics: Antineoplastic Agents; Breast Neoplasms; Catalase; Cell Line; Cell Line, Transformed; Cell Line, Tumor; Cell Survival; Dicumarol; Electron Transport; Female; Humans; Hydrogen Peroxide; Kinetics; Mitochondria; NAD(P)H Dehydrogenase (Quinone); Oxidative Stress; Pancreatic Neoplasms; Reactive Oxygen Species; Recombinant Proteins; Superoxide Dismutase; Tumor Stem Cell Assay; Uncoupling Agents

quinone oxidoreductase (NQO(1)) catalyzes the two-electron reduction of quinones to hydroquinones. This reaction is believed to prevent the one-electron reduction of quinones that would result in redox cycling with generation of superoxide (O(2)(.-)). We have recently demonstrated that inhibition of NQO(1) with dicumarol increases intracellular O(2)(.-) production and inhibits the in vitro malignant phenotype of pancreatic cancer cells (J. Cullen et al., Cancer Res., 63: 5513-5520, 2003). We hypothesized that inhibition of NQO(1) would increase cell killing, induce oxidative stress, and inhibit in vivo tumor growth.. In the human pancreatic cancer cell line MIA PaCa-2, dicumarol decreased cell viability, as measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and decreased clonogenic survival. Dicumarol increased the percentage of apoptotic cells in a time-dependent and dose-dependent manner as measured by 3,3'-diaminobenzidine staining and flow cytometry, which was associated with cytochrome c release and poly(ADP-ribose) polymerase cleavage. Dicumarol also induced oxidative stress as evidenced by increased total glutathione and oxidized glutathione, as well as sensitizing to cell killing mediated by menadione. In established orthotopic pancreatic tumors in nude mice, intratumoral injections of dicumarol slowed tumor growth and extended survival.. Inhibition of NQO(1) with dicumarol induces cell killing and oxidative stress in pancreatic cancer cells and speculate that dicumarol may prove to be useful in pancreatic cancer therapeutics.

Topics: Animals; Apoptosis; Blotting, Western; Cell Line, Tumor; Cell Survival; Cytochromes c; Dicumarol; Dose-Response Relationship, Drug; Electrons; Enzyme Inhibitors; Flow Cytometry; Glutathione; Humans; Hydroquinones; Mice; Mice, Nude; NAD(P)H Dehydrogenase (Quinone); Oxidative Stress; Oxygen; Pancreatic Neoplasms; Phenotype; Poly(ADP-ribose) Polymerases; Quinone Reductases; Time Factors; Uncoupling Agents; Vitamin K 3

Manganese superoxide dismutase (MnSOD) levels have been found to be low in human pancreatic cancer [Pancreas 26, (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.

Topics: Agar; Aged; Animals; Antioxidants; Cell Proliferation; Cells, Cultured; Dicumarol; DNA, Complementary; Gene Expression; Humans; Male; Mice; Mice, Nude; Neoplasm Transplantation; Pancreatic Neoplasms; Superoxide Dismutase; Superoxides; Transfection

NADPH:quinone oxidoreductase (NQO(1)), a homodimeric, ubiquitous, flavoprotein, catalyzes the two-electron reduction of quinones to hydroquinones. This reaction prevents the one-electron reduction of quinones by cytochrome P450 reductase and other flavoproteins that would result in oxidative cycling with generation of superoxide (O(2)(.-)). NQO(1) gene regulation may be up-regulated in some tumors to accommodate the needs of rapidly metabolizing cells to regenerate NAD(+). We hypothesized that pancreatic cancer cells would exhibit high levels of this enzyme, and inhibiting it would suppress the malignant phenotype. Reverse transcription-PCR, Western blots, and activity assays demonstrated that NQO(1) was up-regulated in the pancreatic cancer cell lines tested but present in very low amounts in the normal human pancreas. To determine whether inhibition of NQO(1) would alter the malignant phenotype, MIA PaCa-2 pancreatic cancer cells were treated with a selective inhibitor of NQO(1), dicumarol. Dicumarol increased intracellular production of O(2)(.-), as measured by hydroethidine staining, and inhibited cell growth. Both of these effects were blunted with infection of an adenoviral vector containing the cDNA for manganese superoxide dismutase. Dicumarol also inhibited cell growth, plating efficiency, and growth in soft agar. We conclude that inhibition of NQO(1) increases intracellular O(2)(.-) production and inhibits the in vitro malignant phenotype of pancreatic cancer. These mechanisms suggest that altering the intracellular redox environment of pancreatic cancer cells may inhibit growth and delineate a potential strategy directed against pancreatic cancer.

Topics: Adenocarcinoma; Blotting, Western; Cell Division; Dicumarol; Enzyme Inhibitors; Humans; NAD(P)H Dehydrogenase (Quinone); Pancreatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Superoxide Dismutase; Superoxides; Up-Regulation

Using 4 human cancer cell lines, the relevance of NAD(P)H: quinone oxidoreductase (DT-diaphorase) activity to mitomycin C (MMC)-induced cytotoxicity was investigated. KB cells (oral epidermoid carcinoma) had more than 4 times higher DT-diaphorase activity than PH101 (pancreatic cancer), SH 101 (gastric cancer), or K562 (myelogenous leukemia) cells. The sensitivity to MMC was greatest in KB cells. Concentrations causing 50% inhibition of cell growth (IC50 value: microgram/ml) by 30 min treatment with MMC were 0.4 in KB, 1.1 in PH101, 1.6 in SH 101, and 1.9 in K 562. Treatment with 1.5 micrograms/ml of MMC induced DNA total cross links, and the indices were 0.18 in KB, 0.10 in SH101, 0.09 in SH101, and 0.06 in K 562. When DT-diaphorase activity was inhibited by non-toxic dicoumarol (50 microM), DNA damage and cytotoxic activity induced by MMC were decreased in all cells examined. Especially in KB cells, it was remarkable. Since it was shown that the level of cellular DT-diaphorase activities were correlated with the responses to MMC, we suggest that bioreduction by DT-diaphorase may activate MMC.

Topics: Carcinoma, Squamous Cell; Cell Division; Dicumarol; DNA Damage; Humans; Leukemia, Myeloid; Mitomycin; Mouth Neoplasms; NAD(P)H Dehydrogenase (Quinone); Pancreatic Neoplasms; Stomach Neoplasms; Tumor Cells, Cultured