Uncoupling oxidative phosphorylation with 2,4-dinitrophenol promotes development of the adhesion phenotype.

OBJECTIVE: To determine the effect of uncoupling oxidative phosphorylation with 2,4-dinitrophenol (DNP) on adhesion phenotype development. DESIGN: Prospective experimental study. SETTING: Academic medical center. PATIENT(S): Women undergoing laparotomy for pelvic pain from whom normal peritoneum and adhesions were excised to create primary cultures of normal peritoneal and adhesion fibroblasts. INTERVENTION(S): Treatment of normal peritoneal and adhesion fibroblasts isolated from the same patient(s) with or without 0.2 mM DNP for 24 hours. MAIN OUTCOME MEASURE(S): Evaluation of adhesion phenotype markers type I collagen, vascular endothelial growth factor (VEGF), and hypoxia-inducible factor (HIF)-1α. RESULT(S): In agreement with prior findings, adhesion fibroblasts exhibited significantly higher basal levels of type I collagen, VEGF, and HIF-1α compared with normal peritoneal fibroblasts. Treatment of normal peritoneal fibroblasts with DNP resulted in significant increases in type I collagen (10.2 ± 1.4 vs. 18.4 ± 1.9 fg/µg RNA) and VEGF (8.2 ± 1.1 vs. 13.7 ± 0.4 fg/µg RNA) over baseline. HIF-1α levels did not increase when normal peritoneal fibroblasts were treated with DNP. CONCLUSION(S): The adhesion phenotype, which is normally expressed in response to hypoxia, is reproduced in a normoxic environment by uncoupling oxidative phosphorylation with DNP, as evidenced by an increase in type I collagen and VEGF. Acquisition of the adhesion phenotype was via a mechanism distinct from up-regulation of HIF-1α. These observations are consistent with the hypothesis that the adhesion phenotype represents a state of intracellular metabolic depletion.

Macrophages induce the adhesion phenotype in normal peritoneal fibroblasts.

OBJECTIVE: To determine whether macrophages, exposed to hypoxia, stimulate primary cultures of fibroblasts to acquire the adhesion phenotype. The adhesion phenotype has been previously characterized, in part, by increased fibroblast expression of transforming growth factor (TGF) ß1, vascular endothelial growth factor (VEGF), and type I collagen. DESIGN: Media collected from human macrophages cultured under hypoxic conditions (2% O(2)) were used to treat human peritoneal fibroblasts. Additionally, human peritoneal fibroblasts were treated with varying concentrations of TGF-ß1. Real-time reverse-transcription polymerase chain reaction and Western blot analysis were used to measure mRNA and protein levels, respectively, for select adhesion markers: TGF-ß1, VEGF, and, type I collagen. We hypothesized that macrophage secretion, under hypoxic conditions, is responsible for inducing the adhesion phenotype in human peritoneal fibroblasts. SETTING: University research laboratory. PATIENT(S): Human macrophages and peritoneal fibroblasts. INTERVENTION(S): Macrophage-fibroblast interaction. MAIN OUTCOME MEASURE(S): Ability of macrophages to induce the adhesion phenotype in human peritoneal fibroblasts. RESULT(S): Hypoxia treatment resulted in a significant increase in TGF-ß1 expression in human macrophages. Additionally, hypoxia treatment resulted in a significant increase in TGF-ß1, VEGF, and type I collagen mRNA and protein levels in normal peritoneal fibroblasts compared with normoxic conditions. Similarly, normal peritoneal fibroblasts treated with media collected from macrophages cultured under hypoxic conditions resulted in a significant increase in TGF-ß1, VEGF, and type I collagen mRNA and protein levels compared with normal peritoneal fibroblasts treated with media collected from macrophages cultured under normoxic conditions. Additionally, human peritoneal fibroblasts exposed to varying concentrations of TGF-ß1 exhibited a dose-dependent response in the expression of TGF-ß1, VEGF, and type I collagen. At a low TGF-ß1 concentration (12.5 ng TGF-ß1/mL medium), TGF-ß1, VEGF, and type I collagen were significantly increased. In contrast, at higher TGF-ß1 concentrations (25 and 50 ng TGF-ß1/mL media), TGF-ß1, VEGF, and type I collagen mRNA levels were significantly reduced compared with 12.5 ng TGF-ß1/mL medium. CONCLUSION(S): Human macrophages, cultured under hypoxic conditions, release factors that induce the expression of the adhesion phenotype in normal peritoneal fibroblasts. Particularly, TGF-ß1 reproduces this response by regulating the expression of TGF-ß1, VEGF, and type I collagen in a dose-dependent manner. Therefore, these findings highlight an important role for human macrophages in peritoneal wound healing.

Dichloroacetate induces apoptosis of epithelial ovarian cancer cells through a mechanism involving modulation of oxidative stress.

Epithelial ovarian cancer (EOC) cells are under intrinsic oxidative stress, which alters metabolic activity and reduces apoptosis. Key oxidative stress enzymes, including myeloperoxidase (MPO) and inducible nitric oxide synthase (iNOS), are upregulated and colocalized in EOC cells. Oxidative stress is also regulated, in part, by superoxide dismutase (SOD) and hypoxia-inducible factor (HIF) 1a. Dichloroacetate (DCA) converts anaerobic to aerobic metabolism and thus was utilized to determine the effects on apoptosis, iNOS, MPO, extracellular SOD (SOD-3), and HIF-1a, in EOC cells. Protein and messenger RNA (mRNA) levels of iNOS, MPO, SOD-3, and HIF-1a were evaluated by immunoprecipitation/Western blot and real-time reverse transcriptase-polymerase chain reaction (RT-PCR), respectively, utilizing SKOV-3 and MDAH-2774 treated with DCA. Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and caspase 3 assays. Dichloroacetate induced apoptosis, reduced MPO, iNOS, and HIF-1a, whereas increased SOD, in both EOC cell lines. In conclusion, reduction of enhanced oxidative stress-induced apoptosis of EOC cells, which may serve as future therapeutic intervention for ovarian cancer.

Reduction of hypoxia-induced angiogenesis in ovarian cancer cells by inhibition of HIF-1 alpha gene expression.

PURPOSE: The goal of this study was to investigate the effects of silencing HIF-1 alpha gene expression with specific small interfering RNA (siRNA) on VEGF production and angiogenesis in epithelial ovarian cancer (EOC) cells. METHODS: Two EOC cell lines, MDAH-2774 and SKOV-3, were cultured under normoxic (20% O(2)) and hypoxic (2% O(2)) conditions using standard techniques. After EOC cells were transfected with siRNA, HIF-1 alpha and VEGF mRNA levels were measured by real-time RT-PCR. Angiogenesis was evaluated utilizing an in vitro assay model consisting of human umbilical vein endothelial cells (HUVEC) and polymerized ECM Matrix. RESULTS: Both EOC cell lines evaluated constitutively expressed HIF-1 alpha and VEGF mRNA. HIF-1 alpha and VEGF mRNA levels were significantly increased in response to hypoxia (P < 0.05). Under hypoxic conditions, inhibition of HIF-1 alpha gene expression by a specific siRNA resulted in a significant reduction in HIF-1 alpha and VEGF mRNA levels (P < 0.05). In the in vitro angiogenesis model, supernatant from the hypoxic EOC cells induced the HUVEC to form a complex tubular network, a hallmark of angiogenesis. Semi-quantitative analysis of the angiogenesis assay revealed a significant reduction in tube formation when supernatant from HIF-1 alpha siRNA-treated hypoxic EOC cell was used (P < 0.05). CONCLUSION: Inhibition of HIF-1 alpha expression by specific siRNA resulted in a significant decrease in VEGF production and angiogenesis. Further investigation of HIF-1 alpha inhibition for anti-tumor activity is warranted and may potentially prove HIF-1 alpha as a therapeutic target in the management ovarian cancer.

Exposure to polychlorinated biphenyls enhances lipid peroxidation in human normal peritoneal and adhesion fibroblasts: a potential role for myeloperoxidase.

Nitric oxide, superoxide, and lipid peroxidation (LPO) produced under oxidative stress may contribute to the development of postoperative adhesions. The objective of this study was to determine the effects of polychlorinated biphenyls (PCBs) on LPO, superoxide dismutase, myeloperoxidase (MPO), and nitrite/nitrate in human normal peritoneal and adhesion fibroblasts. PCB treatment reduced inducible nitric oxide synthase (iNOS) expression as well as levels of nitrite/nitrate in both cell lines. Although there was no difference in iNOS expression between the two cell lines, adhesion fibroblasts manifested lower basal levels of MPO compared to normal peritoneal fibroblasts. There was a reduction in MPO expression and its activity in response to PCB treatment in normal peritoneal fibroblasts; however, this effect was minimal in adhesion fibroblasts. Moreover, adhesion fibroblasts manifested higher levels of LPO compared to normal peritoneal fibroblasts, whereas PCB treatment increased LPO levels in both cell types. We conclude that PCBs promote the development of the adhesion phenotype by generating an oxidative stress environment. This is evident by lower iNOS, MPO, and nitrite/nitrate and a simultaneous increase in LPO. Loss of MPO activity, possibly through a mechanism involving MPO heme depletion and free iron release, is yet another source of oxidative stress.

Myeloperoxidase serves as a redox switch that regulates apoptosis in epithelial ovarian cancer.

OBJECTIVES: Resistance to apoptosis is a key feature of cancer cells and is believed to be regulated by nitrosonium ion (NO(+))-induced S-nitrosylation of key enzymes. Nitric oxide (NO), produced by inducible nitric oxide synthase (iNOS), is utilized by MPO to generated NO(+). We sought to investigate the expression of myeloperoxidase (MPO) and iNOS in epithelial ovarian cancer (EOC) and determine their effect on S-nitrosylation of caspase-3 and its activity as well as apoptosis. METHODS: MPO and iNOS expression were determined using immunofluorescence in SKOV-3 and MDAH-2774 and EOC tissue sections. S-nitrosylation of caspase-3 and its activity, levels of MPO and iNOS, as well as apoptosis, were evaluated in the EOC cells before and after silencing MPO or iNOS genes with specific siRNA probes utilizing real-time RT-PCR, ELISA, and TUNEL assays. RESULTS: MPO and iNOS are expressed in EOC cell lines and in over 60% of invasive EOC cases with no expression in normal ovarian epithelium. Indeed, silencing of MPO or iNOS gene expression resulted in decreased S-nitrosylation of caspase-3, increased caspase-3 activity, and increased apoptosis but with a more significant effect when silencing MPO. CONCLUSION: MPO and iNOS are colocalized to the same cells in EOC but not in the normal ovarian epithelium. Silencing of either MPO or iNOS significantly induced apoptosis, highlighting their role as a redox switch that regulates apoptosis in EOC. Understanding the mechanisms by which MPO functions as a redox switch in regulating apoptosis in EOC may lead to future diagnostic tools and therapeutic interventions.

S-nitrosylation of caspase-3 is the mechanism by which adhesion fibroblasts manifest lower apoptosis.

We have previously found that adhesion fibroblasts exhibit lower apoptosis and higher protein nitration as compared with normal peritoneal fibroblasts. In this study, we sought to determine whether the decreased apoptosis observed in adhesion fibroblasts is caused by lower caspase-3 activity due to an increase in caspase-3 S-nitrosylation. For this study, we have utilized primary cultures of fibroblasts obtained from normal peritoneum and adhesion tissues of the same patient(s). Cells were treated with increasing concentrations of peroxynitrite and cell lysates were immunoprecipitated with anti-caspase-3 polyclonal antibody. The biotinylated proteins were detected using a nitrosylation detection kit. Caspase-3 activity and apoptosis were measured by colorimetric and TUNEL assays, respectively. Our results showed that caspase-3 S-nitrosylation is significantly higher in adhesion fibroblasts as compared with normal peritoneal fibroblasts. This increase in S-nitrosylation resulted in a 30% decrease in caspase-3 activity in adhesion fibroblasts. Peroxynitrite treatment resulted in a dose response increase in caspase-3 S-nitrosylation, leading to a decrease in caspase-3 activity and apoptosis in normal peritoneal fibroblasts. We conclude that S-nitrosylation of caspase-3 is the reason for its decreased activity and subsequent decrease in apoptosis of adhesion fibroblasts. The mechanism by which caspase-3 S-nitrosylation occurs is not fully understood. However, the role of hypoxia in the formation of peroxynitrite via superoxide production may suggest a possible mechanism.

Hypoxia regulates iNOS expression in human normal peritoneal and adhesion fibroblasts through nuclear factor kappa B activation mechanism.

OBJECTIVE: To determine the mechanism by which hypoxia increases expression of iNOS in human normal peritoneal and adhesion fibroblasts. DESIGN: Prospective experimental study. SETTING: University medical center. PATIENT(S): Primary cultures of fibroblasts from normal peritoneum and adhesion tissues. INTERVENTION(S): Hypoxia-treated cells. MAIN OUTCOME MEASURE(S): We used real-time reverse transcription-polymerase chain reaction to quantify mRNA levels of iNOS and nuclear factor kappa B (NF-kappaB). Western blots were used to determine iNOS, NF-kappaB, IkappaB-alpha, and phospho-IkappaB expression levels in normal peritoneal and adhesion fibroblasts in response to hypoxia. RESULT(S): Hypoxia resulted in a significant increase in iNOS and NF-kappaB expression in normal and adhesion fibroblasts. Furthermore, both cell types manifested lower levels of NF-kappaB, cytoplasmic phospho-IkappaB-alpha, and iNOS proteins. In contrast, they manifested higher levels of cytoplasmic IkappaB-alpha and IkappaB-alpha/NF-kappaB ratios as well as a phosphorylated-IkappaB-alpha/NF-kappaB ratio. Under hypoxic conditions, both cell types exhibited significantly decreased cytoplasmic NF-kappaB, IkappaB-alpha levels, and significantly increased cytoplasmic phospho-IkappaB-alpha, iNOS, and NF-kappaB protein levels. CONCLUSION(S): Hypoxia increases iNOS expression by a mechanism involving activation of NF-kappaB. The ratio of IkappaB-alpha/NF-kappaB or IkappaB-alpha/p-IkappaB-alpha can be used to monitor activation.

Hypoxia-generated superoxide induces the development of the adhesion phenotype.

Adhesion fibroblasts exhibit higher TGF-beta1 and type I collagen expression as compared to normal peritoneal fibroblasts. Furthermore, exposure of normal peritoneal fibroblasts to hypoxia results in an irreversible increase in TGF-beta1 and type I collagen. We postulated that the mechanism by which hypoxia induced the adhesion phenotype is through the production of superoxide either directly or through the formation of peroxynitrite. To test this hypothesis, normal peritoneal and adhesion fibroblasts were treated with superoxide dismutase (SOD), a superoxide scavenger, and xanthine/xanthine oxidase, a superoxide-generating system, under normoxic and hypoxic conditions. Also, cells were treated with peroxynitrite. TGF-beta1 and type I collagen expression was determined before and after all treatments using real-time RT/PCR. Hypoxia treatment resulted in a time-dependent increase in TGF-beta1 and type I collagen mRNA levels in both normal peritoneal and adhesion fibroblasts. Similarly, treatment with xanthine oxidase, to endogenously generate superoxide, resulted in higher mRNA levels of TGF-beta1 and type I collagen in both normal peritoneal and adhesion fibroblasts. In contrast, treatment with SOD, to scavenge endogenous superoxide, resulted in a decrease in TGF-beta1 and type I collagen expression in adhesion fibroblasts to levels seen in normal peritoneal fibroblasts; no effect on the expression of these molecules was seen in normal peritoneal fibroblasts. Exposure to hypoxia in the presence of SOD had no effect on mRNA levels of TGF-beta1 and type I collagen in either normal peritoneal or adhesion fibroblasts. Peroxynitrite treatment alone significantly induced both adhesion phenotype markers. In conclusion, hypoxia, through the production of superoxide, causes normal peritoneal fibroblasts to acquire the adhesion phenotype. Scavenging superoxide, even in the presence of hypoxia, prevented the development of the adhesion phenotype. These findings further support the central role of free radicals in the development of adhesions.

The effect of estradiol on the expression of estrogen, progesterone, androgen, and prolactin receptors in human peritoneal fibroblasts.

PURPOSE: To assess the ability of fibroblasts isolated from normal peritoneum and adhesion tissues to express various hormone receptors when cultured with exogenous estradiol. METHODS: Primary cultures of fibroblasts from normal human peritoneum and adhesion tissue were treated with zero (control), 10(-10), 10(-8), and 10(-6) M concentrations of 17beta-estradiol. We performed real time reverse transcriptase polymerase chain reaction to determine mRNA levels of estradiol-alpha receptor (ER-alpha) and estradiol-beta receptor (ER-beta), progesterone receptor (P-R), androgen receptor (A-R), and prolactin receptor (PRL-R) in the two types of fibroblast cultures. RESULTS: In the control groups, P-R and A-R were higher in normal than in adhesion fibroblasts. In adhesion cells, ER-alpha were higher at 10(-8) estradiol; ER-beta were higher at 10(-6) M estradiol; P-R remained constant; A-R showed a higher expression at 10(-10) and 10(-8) M estradiol; and PRL-R showed an exponential increase at 10(-10) M estradiol. CONCLUSIONS: The inflammatory-like changes manifested by adhesion fibroblasts enhance the anabolic hormones receptor expression (ER-alpha, ER-beta, PRL, and A-R), when exposed to estradiol.

Nitric oxide synthase isoforms expression in fibroblasts isolated from human normal peritoneum and adhesion tissues.

OBJECTIVE: To determine the expression of nitric oxide synthases (NOSs) and their modulation by hypoxia in human peritoneal (NF) and adhesion fibroblasts (ADF). DESIGN: Prospective experimental study. SETTING: University medical center. PATIENT(S): Fibroblasts from peritoneum and adhesion tissues. INTERVENTION(S): Hypoxia and silencing inducible NOS (iNOS) gene expression in fibroblasts. MAIN OUTCOME MEASURE(S): We used reverse-transcriptase polymerase chain reaction to quantify messenger RNA (mRNA) levels of NOS isoforms. Griess assay was used to measure NO levels. RESULT(S): The mRNA copies/mug RNA of neuronal NOS (nNOS) and endothelial NOS (eNOS) were 6.6 x 10(3) in NF, 5.7 x 10(3) in ADF and 7.0 x 10(3) in NF, 6.1 x 10(3) in ADF, respectively. The mRNA copies/mug RNA of iNOS were 31.3 x 10(3) in NF and 33.0 x 10(3) in ADF. Hypoxia increased iNOS mRNA copies/mug RNA from 31.3 x 10(3) to 61.3 x 10(3) in NF and from 33.0 x 10(3) to 63.9 x 10(3) in ADF, whereas there were no changes in mRNA levels of nNOS and eNOS in NF and ADF. Nitric oxide levels were lower in ADF (0.94 micromol/L) than NF (1.97 micromol/L). Silencing iNOS decreased NO levels in NF (from 1.97 micromol/L to 0.41 micromol/L) and in ADF (from 0.94 micromol/L to 0.27 micromol/L). CONCLUSION(S): Nitric oxide synthases are differentially expressed in NF and ADF, with iNOS being the most expressed and the main source of NO. Hypoxia was shown to alter the expression of NOSs and NO in NF and ADF.