Marilyn M. Hinkhouse

Redox Regulation of Pancreatic Cancer Cell Growth: Role of Glutathione Peroxidase in the Suppression of the Malignant Phenotype

Pancreatic cancer has low levels of antioxidant enzymes including manganese superoxide dismutase (MnSOD), which converts superoxide radical (O(2)(*-)) into hydrogen peroxide (H(2)O(2)), and glutathione peroxidase (GPx), which converts H(2)O(2) into water. Recent studies have demonstrated that overexpression of MnSOD has a tumor-suppressive effect in pancreatic cancer. However, GPx overexpression has been shown to reverse the tumor cell growth inhibition caused by MnSOD overexpression in other types of cancer. Our aims were to determine if overexpression of GPx alters in vitro pancreatic cancer cell behavior and if delivering the GPx gene directly to tumor xenografts alters growth and survival. In vitro, AdGPx slowed tumor growth by 39% and AdMnSOD slowed tumor growth by 35%. AdGPx also decreased plating efficiency and growth in soft agar. The combination of AdGPx and AdMnSOD had the greatest effect on tumor cell growth suppression with a 71% reduction in cell growth compared to controls. In vivo, either AdGPx or AdMnSOD alone slowed tumor growth by 51% and 54%, respectively, while the combination of AdGPx and AdMnSOD potentiated tumor growth suppression by 81% of controls and increased animal survival. GPx may be a tumor suppressor gene in pancreatic cancer. Delivery of the GPx gene alone or in combination with the MnSOD gene may prove beneficial for treatment of pancreatic cancer.

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.

Targeting NAD(P)H:Quinone oxidoreductase (NQO1) in pancreatic cancer

NAD(P)H:Quinone oxidoreductase (NQO1) functions as an important part of cellular antioxidant defense by detoxifying quinones, thus preventing the formation of reactive oxygen species. The aims of our study were to determine if NQO1 is elevated in pancreatic cancer specimens and pancreatic cancer cell lines and if so, would compounds previously demonstrated to redox cycle with NQO1 be effective in killing pancreatic cancer cells. Immunohistochemistry of resected pancreatic specimens demonstrated an increased immunoreactivity for NQO1 in pancreatic cancer and pancreatic intraepithelial neoplasia (PanIN) specimens versus normal human pancreas. Immunocytochemistry and Western immunoblots demonstrated inceased immunoreactivity in pancreatic cancer cells when compared to a near normal immortalized human pancreatic ductal epithelial cell line and a colonic epithelial cell line. Streptonigrin, a compound known to cause redox cycling in the presence of NQO1, decreased clonogenic survival and decreased anchorage‐independent growth in soft agar. Streptonigrin had little effect on cell lines with absent or reduced levels of NQO1. The effects of streptonigrin were reversed in pancreatic cancer cells pretreated with dicumarol, a known inhibitor of NQO1. NQO1 may be a therapeutic target in pancreatic cancer where survival is measured in months.

Effects of endotoxin on regulation of intestinal smooth muscle nitric oxide synthase and intestinal transit

BACKGROUND The disrupted intestinal transit during endotoxemia may be mediated by nitric oxide (NO). We hypothesized that the isoforms of nitric oxide synthase (NOS) are up-regulated in intestinal smooth muscle during endotoxemia and that the scavenging of NO will normalize transit. METHODS Rats were given Escherichia coli lipopolysaccharide (LPS) 10 mg/kg intravenously and were killed 4 hours later. To determine the activity of NOS isoforms in the jejunum and ileum, the conversion of tritiated L-arginine to tritiated L-citrulline was measured. Western immunoblots were performed by incubating the extracted protein with specific polyclonal antibodies. To determine intestinal transit, rats were divided into 4 groups: 0.9% sodium chloride 1 mL/h intravenously for 5 hours, LPS 10 mg/kg intravenous bolus plus 1 mL/h 0.9% sodium chloride intravenously, LPS plus oxyhemoglobin 0.5 g/kg/h intravenously, and oxyhemoglobin 0.5 g/kg/h intravenously. RESULTS LPS increased the constitutive and inducible NOS enzyme activities in the jejunum and ileum. Western blots demonstrated that LPS up-regulates both the NOS1 and NOS2 isoforms in jejunal and ileal smooth muscle. Oxyhemoglobin alone increased intestinal transit compared with controls, whereas endotoxemia increased intestinal transit, which was ameliorated with infusions of oxyhemoglobin. CONCLUSIONS NO may play a major role in mediating the rapid intestinal transit induced by endotoxemia.

Treatment of Pancreatic Cancer Cells with Dicumarol Induces Cytotoxicity and Oxidative Stress

Purpose: NAD(P)H:quinone oxidoreductase (NQO1) 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 (O2.-). We have recently demonstrated that inhibition of NQO1 with dicumarol increases intracellular O2.- 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 NQO1 would increase cell killing, induce oxidative stress, and inhibit in vivo tumor growth. Experimental Design and Results: 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. Conclusions: Inhibition of NQO1 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.

Pathophysiology of adynamic ileus

We hypothesized that the inhibitoryneurotransmitters nitric oxide (NO) and vasoactiveintestinal peptide (VIP) may play a role in thedisrupted gastrointestinal motility of endotoxemia.Strain gauge transducers on the stomach and small intestine of dogsdetermined interdigestive gastrointestinal motility.Tissue levels of NO synthase and VIP and serum levels ofnitrite/nitrate (NO2-/NO3-) and VIP weremeasured. Following completion of the baseline studies,dogs were given a single dose of E. colilipopolysaccharide, 200 μg/kg intravenously, and thestudies were repeated for the next three days. Following endotoxin bolus, the migrating motor complex(MMC) was delayed for two days while serum VIP wasincreased on postendotoxin day 1 and serumNO2-/NO3-was increased on postendotoxin day 2. There were nochanges in gut smooth muscle levels of NO synthase orVIP. We conclude that a single, sublethal dose ofendotoxin results in prolongation of the MMC withdistinct but independent increases in serum levels ofVIP and NO2-/NO3-

Effect of endotoxin on canine colonic motility and transit

Diarrhea is a common problem in patients who have episodes of sepsis and are beingfed enterally. Endotoxemia results in gastrointestinal motor dysfunction characterized by slowed gastric emptying and rapid intestinal transit; however, the effect of endotoxin on colonic motility is unknown. The aim of our study was to determine the effects of a single sublethal dose of endotoxin on colonic motility and transit. Seven dogs underwent construction of a 50 cm colonic Thiry-Vella fistula. Five manometry catheters were sewn into the colonic lumen at 8 cm intervals along the fistula. Following recovery, the fistula was perfused with an isotonic solution at 2.9 ml/min, and fasting and postprandial colonic motility was determined. Liquid transit was assessed by bolus ofa nonabsorbable marker instilled into the proximal end of the Thiry-Vella fistula. Recordings of gastrointestinal contractile activity were made digitally to determine contractile frequencies and motility indexes. Following completion of the baseline studies, each dog was given a single dose ofE. coli lipopolysaccharide, 200 γg/kg intravenously, and studies were repeated daily for the next 3 days. Endotoxin doubled the fasting colonic contractile frequency on postendotoxin day 1 and also increased motility indexes on that same day. Fasting motility indexes and contractile activity were decreased on postendotoxin days 2 and 3. The postprandial frequency of contractions and motility indexes were decreased on postendotoxin day 3. Fasting colonic liquid transit was rapid on postendotoxin day 1, whereas postprandial liquid transit was rapid on both postendotoxin days 1 and 2. Endotoxin temporarily speeds liquid transit and increases both the frequency and strength of colonic contractions. These effects may contribute to the diarrhea that occurs during episodes of sepsis.

Mechanisms of Impaired Gallbladder Contractile Response in Chronic Acalculous Cholecystitis

The mechanisms involved in the impaired gallbladder contractile response in chronic acalculous cholecystitis are unknown. To determine the mechanisms that may lead to impaired gallbladder emptying in chronic acalculous cholecystitis, gallbladder specimens removed during hepatic resection (controls) and after cholecystectomy for chronic acalculous cholecystitis were attached to force transducers and placed in tissue baths with oxygenated Krebs solution. Electrical field stimulation (EFS) (1 to 10 Hz, 0.1 msec, 70 V) or the contractile agonists, CCK-8 (10-9 to 10-5) or K+ (80 mmol/L), were placed separately in the tissue baths and changes in tension were determined. Patients with chronic acalculous cholecystitis had a mean gallbladder ejection fraction of 12% ± 4%. Pathologic examination of all gallbladders removed for chronic acalculous cholecystitis revealed chronic cholecystitis. Spontaneous contractile activity was present in gallbladder strips in 83% of control specimens but only 29% of gallbladder strips from patients with chronic acalculous cholecystitis (P < 0.05 vs. controls). CCK-8 contractions were decreased by 54% and EFS-stimulated contractions were decreased by 50% in the presence of chronic acalculous cholecystitis (P < 0.05 vs. controls). K+-induced contractions were similar between control and chronic acalculous cholecystitis gallbladder strips. The impaired gallbladder emptying in chronic acalculous cholecystitis appears to be due to diminished spontaneous contractile activity and decreased contractile responsiveness to both CCK and EFS.

DEHYDROEPIANDROSTERONE AND ESTRONE 17-KETOSTEROID REDUCTASES IN MCF-7 HUMAN BREAST CANCER CELLS

SummaryThe identification of several steroid-transforming enzymes within human breast cancers has led to speculation that the growth of some hormone-responsive tumors might be mediated in part by intracellularly derived estrogens. Reports that MCF-7 human breast cancer cells can transform both estrone (E1)1 to estradiol (E2) and dehydroepiandrosterone (DHEA) to the estrogenic steroid 5-androstenediol (AED), have prompted us to investigate the 17-ketosteroid reductase activities (17-KSRs) which mediate these potentially important reactions. Enzyme assays were performed by quantifying the amounts of [3H]AED or [3H]E2 former from [3H]DHEA or [3H]E1, respectively, by various subcellular preparations from MCF-7 cells under a variety of experimental conditions. DHEA 17-KSR was found to be localized exclusively within cytosol, whereas the E1 17-KSR activity appeared to be nearly equally divided between the soluble and particulate cytoplasmic subfractions. The particulate E1 17-KSR appeared capable of utilizing NADH or NADPH, whereas both the cytosolic form of this enzyme and the soluble DHEA 17-KSR activity showed a strict requirement for NADPH. Although both of the soluble 17-KSRs also showed similar pH optima, several other features suggested that they are different enzymes in MCF-7. E1 did not inhibit the conversion of DHEA to AED, and DHEA did not interfere with the transformation of E1 to E2, indicating that major differences in substrate specificity exist between the two cytosolic activities. Furthermore, DHEA 17-KSR activity within cytosol stored at −20°C deteriorated almost completely over twelve weeks of storage, whereas E1 17-KSR activity remained stable. Finally, although both enzymes were found to be subject to product inhibition, AED inhibited DHEA 17-KSR competitively, whereas cytosolic E1 17-KSR activity was inhibited by E2 in noncompetitive fashion. Studies of the oxidation of E2 to E1 by MCF-7 cells showed that this transformation is catalyzed by both soluble and particulate 17-hydroxysteroid oxidases which utilize either NAD or NADP as cofactor. Having previously reported the presence of a particulate NADP(H)-linked androstenedione (AE) 17-ketosteroid oxidoreductase in MCF-7, we now suggest that at least three different enzymes, one particulate and two soluble forms, participate in the conversion of 17-ketosteroids to their hormonally active 17-hydroxysteroid derivatives within this cell line. The restricted substrate requirements of each enzyme provide a rationale for developing selective enzyme inhibitors which could provide important investigational tools and potentially effective therapeutic agents.

Endotoxin temporarily impairs canine jejunal absorption of water, electrolytes, and glucose

Enteral feeding during and after episodes of sepsis may be beneficial. The aim of our study was to determine the effects of a single sublethal dose of endotoxin on canine jejunal absorption. Following a 240 kcal liquid meal, absorption studies were performed in eight dogs with 75 cm jejunal Thiry-Vella fistulas. These fistulas were perfused with an isotonic solution containing polyethylene glycol to calculate absorption. Each dog was then given a single dose ofEscherichia coli lipopolysaccharide, 200 μg/kg intravenously, and the studies were repeated for the next 3 days. Following endotoxin bolus infusion, net absorption of water, electrolytes and glucose was decreased for 2 days and returned to baseline values on postendotoxin day 3. A single sublethal dose of endotoxin temporarily impairs canine jejunal absorption. Although enteral feeding may be advantageous, jejunal absorption may be temporarily impaired following an episode of endotoxemia.