A Role of the Heme Degradation Pathway in Shaping Prostate Inflammatory Responses and Lipid Metabolism
2020; Elsevier BV; Volume: 190; Issue: 4 Linguagem: Inglês
10.1016/j.ajpath.2019.12.008
ISSN1525-2191
AutoresLisa Vikström Lilljebjörn, Eva Csizmadia, Andreas Hedblom, Giacomo Canesin, Alireza Kalbasi, Mailin Li, Farah Kramer, Karin Bornfeldt, Barbara Wegiel,
Tópico(s)Neonatal Health and Biochemistry
ResumoThe molecular mechanisms of prostate inflammation are unclear. We hypothesized that heme oxygenase 1 (HMOX1; HO-1), an enzyme responsible for degradation of heme to carbon monoxide, bilirubin, and iron, is an important regulator of inflammation and epithelial responses in the prostate. Injection of non-uropathogenic Escherichia coli (MG1655 strain) or phosphate-buffered saline into the urethra of mice led to increased numbers of CD45+ leukocytes and mitotic markers (phosphorylated histone H3 and phosphorylated ERK1/2) in the prostate glands. Leukocyte infiltration was elevated in the prostates harvested from mice lacking HO-1 in myeloid compartment. Conversely, exogenous carbon monoxide (250 ppm) increased IL-1β levels and suppressed cell proliferation in the prostates. Carbon monoxide did not affect the number of infiltrating CD45+ cells in the prostates of E. coli– or phosphate-buffered saline–treated mice. Interestingly, immunomodulatory effects of HO-1 and/or carbon monoxide correlated with early induction of the long-chain acyl-CoA synthetase 1 (ACSL1). ACSL1 levels were elevated in response to E. coli treatment, and macrophage-expressed ACSL1 was in part required for controlling of IL-1β expression and prostate cancer cell colony growth in soft agar. These results suggest that HO-1 and/or carbon monoxide might play a distinctive role in modulating prostate inflammation, cell proliferation, and IL-1β levels in part via an ACSL1-mediated pathway. The molecular mechanisms of prostate inflammation are unclear. We hypothesized that heme oxygenase 1 (HMOX1; HO-1), an enzyme responsible for degradation of heme to carbon monoxide, bilirubin, and iron, is an important regulator of inflammation and epithelial responses in the prostate. Injection of non-uropathogenic Escherichia coli (MG1655 strain) or phosphate-buffered saline into the urethra of mice led to increased numbers of CD45+ leukocytes and mitotic markers (phosphorylated histone H3 and phosphorylated ERK1/2) in the prostate glands. Leukocyte infiltration was elevated in the prostates harvested from mice lacking HO-1 in myeloid compartment. Conversely, exogenous carbon monoxide (250 ppm) increased IL-1β levels and suppressed cell proliferation in the prostates. Carbon monoxide did not affect the number of infiltrating CD45+ cells in the prostates of E. coli– or phosphate-buffered saline–treated mice. Interestingly, immunomodulatory effects of HO-1 and/or carbon monoxide correlated with early induction of the long-chain acyl-CoA synthetase 1 (ACSL1). ACSL1 levels were elevated in response to E. coli treatment, and macrophage-expressed ACSL1 was in part required for controlling of IL-1β expression and prostate cancer cell colony growth in soft agar. These results suggest that HO-1 and/or carbon monoxide might play a distinctive role in modulating prostate inflammation, cell proliferation, and IL-1β levels in part via an ACSL1-mediated pathway. Inflammation in the prostate is a common urogenital tract condition in men that leads to pain.1Schatteman P.H. Hoekx L. Wyndaele J.J. Jeuris W. Van Marck E. Inflammation in prostate biopsies of men without prostatic malignancy or clinical prostatitis: correlation with total serum PSA and PSA density.Eur Urol. 2000; 37: 404-412Crossref PubMed Scopus (136) Google Scholar,2Delongchamps N.B. de la Roza G. Chandan V. Jones R. Sunheimer R. Threatte G. Jumbelic M. Haas G.P. Evaluation of prostatitis in autopsied prostates--is chronic inflammation more associated with benign prostatic hyperplasia or cancer?.J Urol. 2008; 179: 1736-1740Crossref PubMed Scopus (104) Google Scholar It has been postulated that prostate inflammation also leads to an increased risk of prostate cancer; however, studies have reached conflicting results.3Roberts R.O. Bergstralh E.J. Bass S.E. Lieber M.M. Jacobsen S.J. Prostatitis as a risk factor for prostate cancer.Epidemiology. 2004; 15: 93-99Crossref PubMed Scopus (103) Google Scholar,4Dennis L.K. Lynch C.F. Torner J.C. Epidemiologic association between prostatitis and prostate cancer.Urology. 2002; 60: 78-83Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar Ninety-five percent of prostatitis cases involve chronic prostatitis or chronic pelvic pain syndrome in which bacteria cannot be detected but inflammatory cells are present. In some cases, no inflammatory component can be detected. Five percent of men with chronic bacterial prostatitis present with no detectable bacteria in the prostate, and 50% lack leukocytosis in the prostate.5Hua V.N. Williams D.H. Schaeffer A.J. Role of bacteria in chronic prostatitis/chronic pelvic pain syndrome.Curr Urol Rep. 2005; 6: 300-306Crossref PubMed Scopus (18) Google Scholar Fecal Escherichia coli, bacteria causing sexually transmitted diseases or uropathogenic bacteria, are among the pathogens found to be associated with urogenital tract infection and linked to prostatitis. E. coli was previously detected in prostate tissues6Boehm B.J. Colopy S.A. Jerde T.J. Loftus C.J. Bushman W. Acute bacterial inflammation of the mouse prostate.Prostate. 2012; 72: 307-317Crossref PubMed Scopus (57) Google Scholar,7Funahashi Y. Wang Z. O'Malley K.J. Tyagi P. DeFranco D.B. Gingrich J.R. Takahashi R. Majima T. Gotoh M. Yoshimura N. Influence of E. coli-induced prostatic inflammation on expression of androgen-responsive genes and transforming growth factor beta 1 cascade genes in rats.Prostate. 2015; 75: 381-389Crossref PubMed Scopus (22) Google Scholar; however, these studies are limited by the number of patients without comprehensive data confirming this observation. Inflammation in the prostate can be driven by activation of residential macrophages and/or infiltration of leukocytes in response to pathogen-associated molecular patterns or danger-associated molecular patterns released from bacteria or damaged cells, respectively.8Liu Y.-C. Zou X.-B. Chai Y.-F. Yao Y.-M. Macrophage polarization in inflammatory diseases.Int J Biol Sci. 2014; 10: 520-529Crossref PubMed Scopus (589) Google Scholar Chronic inflammation is propagated by continuous release of reactive oxygen species and proinflammatory mediators from immune cells and may lead to damage of the prostate tissue.9Coussens L.M. Werb Z. Inflammation and cancer.Nature. 2002; 420: 860-867Crossref PubMed Scopus (11233) Google Scholar Further, unresolved inflammatory response is linked to gastric, colon, and prostate cancer.10Mantovani A. Allavena P. Sica A. Balkwill F. Cancer-related inflammation.Nature. 2008; 454: 436-444Crossref PubMed Scopus (7906) Google Scholar Elkahwaji et al11Elkahwaji J.E. Hauke R.J. Brawner C.M. Chronic bacterial inflammation induces prostatic intraepithelial neoplasia in mouse prostate.Br J Cancer. 2009; 101: 1740-1748Crossref PubMed Scopus (51) Google Scholar found that chronic bacterial infection of the mouse prostate led to the development of prostatic intraepithelial neoplasia lesions after induction of prostatitis by intraurethral injection of E. coli into C3H/HeOuJ mice. In this model, infiltration of leukocytes into the prostate stroma was associated with increased epithelial DNA damage and proliferation and lower expression of the tumor suppressors, PTEN and p27Kip1.11Elkahwaji J.E. Hauke R.J. Brawner C.M. Chronic bacterial inflammation induces prostatic intraepithelial neoplasia in mouse prostate.Br J Cancer. 2009; 101: 1740-1748Crossref PubMed Scopus (51) Google Scholar Furthermore, intraurethral injection of bacterial lipopolysaccharide led to hyperplasia, inflammation, and bleeding in the prostate.12Dos Santos Gomes F.O. Oliveira A.C. Ribeiro E.L. da Silva B.S. Dos Santos L.A.M. de Lima I.T. Silva A. da Rocha Araujo S.M. Goncalves T. de Melo-Junior M.R. Peixoto C.A. Intraurethral injection with LPS: an effective experimental model of prostatic inflammation.Inflamm Res. 2018; 67: 43-55Crossref PubMed Scopus (13) Google Scholar Degradation of heme is catalyzed by heme oxygenase 1 (HO-1) and generates biliverdin, ferrous iron, and carbon monoxide.13Tenhunen R. Marver H.S. Schmid R. The enzymatic conversion of heme to bilirubin by microsomal heme oxygenase.Proc Natl Acad Sci U S A. 1968; 61: 748-755Crossref PubMed Scopus (1510) Google Scholar,14Tenhunen R. Marver H.S. Schmid R. Microsomal heme oxygenase: characterization of the enzyme.J Biol Chem. 1969; 244: 6388-6394Abstract Full Text PDF PubMed Google Scholar HO-1 and CO have major immunomodulatory properties.15Naito Y. Takagi T. Higashimura Y. Heme oxygenase-1 and anti-inflammatory M2 macrophages.Arch Biochem Biophys. 2014; 564: 83-88Crossref PubMed Scopus (240) Google Scholar, 16Otterbein L.E. Soares M.P. Yamashita K. Bach F.H. Heme oxygenase-1: unleashing the protective properties of heme.Trends Immunol. 2003; 24: 449-455Abstract Full Text Full Text PDF PubMed Scopus (1014) Google Scholar, 17Wegiel B. Hanto D.W. Otterbein L.E. The social network of carbon monoxide in medicine.Trends Mol Med. 2013; 19: 3-11Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar Recently, we found that carbon monoxide treatment after infection potentiates bacterial clearance18Otterbein L.E. May A. Chin B.Y. Carbon monoxide increases macrophage bacterial clearance through Toll-like receptor (TLR)4 expression.Cell Mol Biol (Noisy-le-grand). 2005; 51: 433-440PubMed Google Scholar and amplifies proinflammatory responses in macrophages in a mouse model of sepsis in part via Nalp3 inflammasome–dependent release of IL-1β.19Wegiel B. Larsen R. Gallo D. Chin B.Y. Harris C. Mannam P. Kaczmarek E. Lee P.J. Zuckerbraun B.S. Flavell R. Soares M.P. Otterbein L.E. Macrophages sense and kill bacteria through carbon monoxide-dependent inflammasome activation.J Clin Invest. 2014; 124: 4926-4940Crossref PubMed Scopus (126) Google Scholar This mechanism involves a direct effect of carbon monoxide on bacteria facilitating active release of ATP from bacteria. Extracellular ATP, acting as a danger-associated molecular pattern, binds to the macrophage-expressed P2X7 receptor,19Wegiel B. Larsen R. Gallo D. Chin B.Y. Harris C. Mannam P. Kaczmarek E. Lee P.J. Zuckerbraun B.S. Flavell R. Soares M.P. Otterbein L.E. Macrophages sense and kill bacteria through carbon monoxide-dependent inflammasome activation.J Clin Invest. 2014; 124: 4926-4940Crossref PubMed Scopus (126) Google Scholar activating the Nalp3 inflammasome via changes in intracellular potassium levels to drive IL-1β expression.20Ayna G. Krysko D.V. Kaczmarek A. Petrovski G. Vandenabeele P. Fesus L. ATP release from dying autophagic cells and their phagocytosis are crucial for inflammasome activation in macrophages.PLoS One. 2012; 7: e40069Crossref PubMed Scopus (101) Google Scholar, 21Bours M.J. Dagnelie P.C. Giuliani A.L. Wesselius A. Di Virgilio F. P2 receptors and extracellular ATP: a novel homeostatic pathway in inflammation.Front Biosci (Schol Ed). 2011; 3: 1443-1456Crossref PubMed Google Scholar, 22Jalilian I. Peranec M. Curtis B.L. Seavers A. Spildrejorde M. Sluyter V. Sluyter R. Activation of the damage-associated molecular pattern receptor P2X7 induces interleukin-1beta release from canine monocytes.Vet Immunol Immunopathol. 2012; 149: 86-91Crossref PubMed Scopus (22) Google Scholar, 23Petrovski G. Ayna G. Majai G. Hodrea J. Benko S. Madi A. Fesus L. Phagocytosis of cells dying through autophagy induces inflammasome activation and IL-1beta release in human macrophages.Autophagy. 2011; 7: 321-330Crossref PubMed Scopus (54) Google Scholar Monocyte-derived IL-1β has antiproliferative properties in prostate cancer cells and blocks androgen receptor–dependent expression of–prostate-specific antigen.24Culig Z. Hobisch A. Herold M. Hittmair A. Thurnher M. Eder I.E. Cronauer M.V. Rieser C. Ramoner R. Bartsch G. Klocker H. Konwalinka G. Interleukin 1beta mediates the modulatory effects of monocytes on LNCaP human prostate cancer cells.Br J Cancer. 1998; 78: 1004-1011Crossref PubMed Scopus (43) Google Scholar In contrast, others found that IL-1β promotes bone marrow metastases and neuroendocrine phenotype of prostate cancer cells.25Liu Q. Russell M.R. Shahriari K. Jernigan D.L. Lioni M.I. Garcia F.U. Fatatis A. Interleukin-1beta promotes skeletal colonization and progression of metastatic prostate cancer cells with neuroendocrine features.Cancer Res. 2013; 73: 3297-3305Crossref PubMed Scopus (74) Google Scholar IL-1 receptor 1 knockout mice have low expansion of c-kit+ progenitors in the inflamed prostate linking abnormal proliferation with IL-1 signaling.26Wang L. Zoetemelk M. Chitteti B.R. Ratliff T.L. Myers J.D. Srour E.F. Broxmeyer H. Jerde T.J. Expansion of prostate epithelial progenitor cells after inflammation of the mouse prostate.Am J Physiol Renal Physiol. 2015; 308: F1421-F1430Crossref PubMed Scopus (11) Google Scholar Previous data from our group indicate a strong induction of HO-1 in macrophages after infection with E. coli or Enterococcus faecalis and the importance of HO-1 in the generation of IL-1β.19Wegiel B. Larsen R. Gallo D. Chin B.Y. Harris C. Mannam P. Kaczmarek E. Lee P.J. Zuckerbraun B.S. Flavell R. Soares M.P. Otterbein L.E. Macrophages sense and kill bacteria through carbon monoxide-dependent inflammasome activation.J Clin Invest. 2014; 124: 4926-4940Crossref PubMed Scopus (126) Google Scholar Long-chain acyl-CoA synthetase 1 (ACSL1), one of five isoforms of an enzyme family that activate fatty acids by coupling them to coenzyme A, has been previously reported to be upregulated in tumors27Wu X. Daniels G. Lee P. Monaco M.E. Lipid metabolism in prostate cancer.Am J Clin Exp Urol. 2014; 2: 111-120PubMed Google Scholar and is induced during bacterial infection via a toll-like receptor 4–dependent mechanism. ACSL1 mediates phospholipid turnover in lipopolysaccharide-stimulated macrophages28Rubinow K.B. Wall V.Z. Nelson J. Mar D. Bomsztyk K. Askari B. Lai M.A. Smith K.D. Han M.S. Vivekanandan-Giri A. Pennathur S. Albert C.J. Ford D.A. Davis R.J. Bornfeldt K.E. Acyl-CoA synthetase 1 is induced by Gram-negative bacteria and lipopolysaccharide and is required for phospholipid turnover in stimulated macrophages.J Biol Chem. 2013; 288: 9957-9970Crossref PubMed Scopus (47) Google Scholar among other effects.29Kanter J.E. Kramer F. Barnhart S. Averill M.M. Vivekanandan-Giri A. Vickery T. Li L.O. Becker L. Yuan W. Chait A. Braun K.R. Potter-Perigo S. Sanda S. Wight T.N. Pennathur S. Serhan C.N. Heinecke J.W. Coleman R.A. Bornfeldt K.E. Diabetes promotes an inflammatory macrophage phenotype and atherosclerosis through acyl-CoA synthetase 1.Proc Natl Acad Sci U S A. 2012; 109: E715-E724Crossref PubMed Scopus (189) Google Scholar ACSL1 converts free long-chain fatty acids into fatty acyl-CoA esters, thereby playing a key role in lipid biosynthesis and β-oxidation. Conditional deletion of ACSL1 in myeloid cells prevents macrophage accumulation in the artery wall in diabetic mice, indicating a role for ACSL1 in inflammatory responses.29Kanter J.E. Kramer F. Barnhart S. Averill M.M. Vivekanandan-Giri A. Vickery T. Li L.O. Becker L. Yuan W. Chait A. Braun K.R. Potter-Perigo S. Sanda S. Wight T.N. Pennathur S. Serhan C.N. Heinecke J.W. Coleman R.A. Bornfeldt K.E. Diabetes promotes an inflammatory macrophage phenotype and atherosclerosis through acyl-CoA synthetase 1.Proc Natl Acad Sci U S A. 2012; 109: E715-E724Crossref PubMed Scopus (189) Google Scholar Because ACSL1 and HO-1 regulate metabolic stress and immune responses, we hypothesized that HO-1 and/or carbon monoxide crosstalks with ACSL1 signaling in macrophages during inflammatory responses in the prostate. We report here that ACSL1 is a potential target of the heme degradation pathway in macrophages. Nonuropathogenic Escherichia coli MG1655, originally acquired from the Keio library, was provided by Bernard Strauss (Massachusetts Institute of Technology, Cambridge, MA). This E. coli strain has been described previously.19Wegiel B. Larsen R. Gallo D. Chin B.Y. Harris C. Mannam P. Kaczmarek E. Lee P.J. Zuckerbraun B.S. Flavell R. Soares M.P. Otterbein L.E. Macrophages sense and kill bacteria through carbon monoxide-dependent inflammasome activation.J Clin Invest. 2014; 124: 4926-4940Crossref PubMed Scopus (126) Google Scholar This strain of bacteria was used in bacterial infection experiments in vitro and in vivo. Bacteria were inoculated in Luria broth medium from a frozen aliquot obtained from a single colony. Fresh Luria broth was inoculated with a 1 mL aliquot of the overnight culture and subcultured at 37°C until bacteria suspensions reached an OD measured at 600 nm of 0.45. Cells were pelleted at 3220 × g (5 minutes) and resuspended in sterile phosphate-buffered saline (PBS). Serial dilutions of stock suspension (1:1000 to 1:109) were plated in duplicates on Luria broth agar and incubated at 37°C in the o/n culture. Colony counts were averaged and used to estimate the concentration of bacteria in the prepared stock with OD measured at 600 nm of 0.45. Appropriate volumes of the bacteria dilutions were used to prepare injections for infection in vivo or for in vitro treatments. C57BL/6 male mice (Jackson Laboratories, Bar Harbor, ME) 7 to 10 weeks of age weighing 20 to 25 g were used (after at least 3 days of acclimatization) for in vivo prostatitis model, and both sexes of mice were used for bone marrow isolation. Acsl1flfl:LysM-Cre and Acsl1wt/wt-Cre control mice, also with the C57BL/6 background, were previously described.29Kanter J.E. Kramer F. Barnhart S. Averill M.M. Vivekanandan-Giri A. Vickery T. Li L.O. Becker L. Yuan W. Chait A. Braun K.R. Potter-Perigo S. Sanda S. Wight T.N. Pennathur S. Serhan C.N. Heinecke J.W. Coleman R.A. Bornfeldt K.E. Diabetes promotes an inflammatory macrophage phenotype and atherosclerosis through acyl-CoA synthetase 1.Proc Natl Acad Sci U S A. 2012; 109: E715-E724Crossref PubMed Scopus (189) Google Scholar To establish models of pathogen-induced and sterile prostatitis, intraurethral instillation of E. coli MG1655 (2 × 108 CFU per animal) or PBS (n = 3 to 6 per group) was performed. Injections were performed using a 30-gauge syringe connected to lubricated polyvinyl tubing (0.8 mm across) inserted into the urinary bladder via the urethra. The procedures were performed with the mice under 3% isoflurane-induced anesthesia. After the procedure, the mice were monitored for changes in behavior associated with pain or distress, and body weight records were kept. The animals were kept in ventilated cages (up to five mice per cage) in a 12-hour light-dark cycle and were provided water and food ad libitum at all times. The procedures were approved by the Institutional Animal Care and Use Committees at Beth Israel Deaconess Medical Center. Prostates (lateral or ventral lobes) and blood samples were harvested from the different mouse groups at 2 hours, 6 hours, 24 hours, and 3 weeks after injection. The same time points as indicated above were chosen for investigation of the role of carbon monoxide in this model. Animals injected with E. coli or PBS as well as negative controls inhaled carbon monoxide (250 ppm) for 1 hour, starting 1 hour after injection. The role of myeloid-derived HO-1 in bacteria-induced or sterile prostatitis was investigated by using conditional knockout mice deficient in HO-1 in myeloid cells. These animals were generated by crossing mice with the Hmox1 gene flanked with LoxP sites (Hmoxfl/fl) as previously described,30Wegiel B. Hedblom A. Li M. Gallo D. Csizmadia E. Harris C. Nemeth Z. Zuckerbraun B.S. Soares M. Persson J.L. Otterbein L.E. Heme oxygenase-1 derived carbon monoxide permits maturation of myeloid cells.Cell Death Dis. 2014; 5: e1139Crossref PubMed Scopus (42) Google Scholar with transgenic mice expressing Cre recombinase under control of the myeloid specific LysM promoter (LysM-Cre) (Jackson Laboratories). LysM-Cre:Hmox1fl/fl and Hmox1fl/fl homozygotes with the C57BL/6 background were verified by PCR-based genotyping.31Mamiya T. Katsuoka F. Hirayama A. Nakajima O. Kobayashi A. Maher J.M. Matsui H. Hyodo I. Yamamoto M. Hosoya T. Hepatocyte-specific deletion of heme oxygenase-1 disrupts redox homeostasis in basal and oxidative environments.Tohoku J Exp Med. 2008; 216: 331-339Crossref PubMed Scopus (26) Google Scholar Bone marrow was isolated and prepared as previously described.19Wegiel B. Larsen R. Gallo D. Chin B.Y. Harris C. Mannam P. Kaczmarek E. Lee P.J. Zuckerbraun B.S. Flavell R. Soares M.P. Otterbein L.E. Macrophages sense and kill bacteria through carbon monoxide-dependent inflammasome activation.J Clin Invest. 2014; 124: 4926-4940Crossref PubMed Scopus (126) Google Scholar Bone marrow–derived macrophages (BMDMs) differentiated in the presence of macrophage–colony-stimulating factor (Prospec, Rehovot, Israel) (20 ng/mL) for 5 days were scraped off the culture dishes with cell lifters and suspended in fresh RPMI 1640 medium, 10% fetal bovine serum (FBS), and 1% penicillin-streptomycin medium (Life Technologies, Carlsbad, CA). The cells were counted and replated in 100-mm dishes (3 × 106 cells per plate). Bacteria (E. coli MG1655) were prepared as described above. Macrophages were washed twice in PBS to remove residual antibiotics and were then stimulated with bacteria (10 multiplicity of infection) for 1 to 2 hours in antibiotic-free culture media. Sterile PBS (pH 7.4) was added to control plates. Macrophages from stimulated and control groups were treated with carbon monoxide (250 ppm) for 1 hour. Bacteria were removed after 2 hours of stimulation, and medium with antibiotics (1 mL medium per 170,000 cells) was added to the plates. Supernatants from the BMDMs were harvested 24 hours after stimulation, filtered, and stored at −20°C. For soft agar colony assay, a base layer of 0.5% sterile agar RPMI 1640 medium, 10% FBS, and 1% penicillin and streptomycin (Life Technologies) was casted into six-well plates, 1 mL per well, and left to solidify. PC3 and VCaP prostate cancer cells, previously validated and/or authenticated in Dr. Balk's laboratory,32Yu Z. Cai C. Gao S. Simon N.I. Shen H.C. Balk S.P. Galeterone prevents androgen receptor binding to chromatin and enhances degradation of mutant androgen receptor.Clin Cancer Res. 2014; 20: 4075-4085Crossref PubMed Scopus (75) Google Scholar were used for the study. These cells were grown to 60% to 80% confluence in RPMI 1640 medium with 10% FBS, detached by trypsinization, and diluted to the final concentration of 10,000 cells/mL in RPMI 1640 medium, 20% FBS, and 2% penicillin and streptomycin. These cell lines were not authenticated in our laboratory but were previously described and characterized.32Yu Z. Cai C. Gao S. Simon N.I. Shen H.C. Balk S.P. Galeterone prevents androgen receptor binding to chromatin and enhances degradation of mutant androgen receptor.Clin Cancer Res. 2014; 20: 4075-4085Crossref PubMed Scopus (75) Google Scholar Each suspension was mixed 1:1 with sterile soft agar 0.7% (37 to 39°C) at a final concentration of 0.35% agar and 10% FBS, applied to each well on top of the base layer, and left to incubate in the o/n culture. The cells in the soft agar were treated for 2 to 3 weeks with supernatants from differentiated BMDMs treated with bacteria at 10 multiplicity of infection and carbon monoxide (Bacteria Stimulation and Carbon Monoxide Treatment of BMDMs). Medium was changed every third day (0.5 mL per well per time). The RPMI 1640 medium with 10% FBS was used as the negative control. PNT1A, VCaP, DU145, and LNCaP cell lines were previously described.33Wegiel B. Gallo D. Csizmadia E. Harris C. Belcher J. Vercellotti G.M. Penacho N. Seth P. Sukhatme V. Ahmed A. Pandolfi P.P. Helczynski L. Bjartell A. Persson J.L. Otterbein L.E. Carbon monoxide expedites metabolic exhaustion to inhibit tumor growth.Cancer Res. 2013; 73: 7009-7021Crossref PubMed Scopus (244) Google Scholar One thousand cells were seeded in 96-well plate and treated with supernatants from BMDM treated with E. coli as described above. Viability and proliferation were assessed on day 2 of the culture by staining with crystal violet. Stained and dry cells were dissolved in 10% acetic acid and processed for measurement of absorbance at 562 nm. Prostate tissue was sonicated in lysis buffer (50 mmol/L Tris chloride, pH 7.0, 150 mmol/L sodium chloride, 0.5% NP-40, 2 mmol/L EDTA, 25 mmol/L sodium fluoride, 0.1% SDS, 1 tablet per 10-mL buffer Complete MiniProtease Inhibitor Cocktail; Roche, Indianapolis, IN). The lysates were centrifuged (Microfuge 18 Centrifuge, Beckman Coulter, Bromma, Sweden) for 20 minutes at 18,000 × g, and supernatants were transferred to clean Eppendorf tubes. The centrifugation was repeated once more, and clear supernatants were transferred to new Eppendorf tubes. The protein concentrations in the samples were measured using the Pierce BCA Protein Assay (Thermo Scientific, Waltham, MA), according to the manufacturer's instructions. The samples were adjusted with distilled water to equal concentration. Samples were prepared for electrophoresis by adding 4× sample buffer (Life Technologies) that contained 10% β-mercaptoethanol and boiled for 5 minutes. Samples were loaded (20 to 25 μg per well) in NuPAGE Novex 4% to 12% Bis-Tris Protein Gels (Life Technologies) using Precision Plus Protein Kaleidoscope (Bio Rad, Hercules, CA) as the size marker. Electrophoresis was performed in 2-N-morpholino ethanesulfonic acid SDS running buffer (Life Technologies) at 90 to 120 V. Separated proteins were transferred to polyvinylidene difluoride membranes (Amersham, Piscataway, NJ; Bio Rad) in transfer buffer (33 mmol/L Tris, 0.19 mol/L glycine, and 20% methanol) at 80 V for 1.5 hours. Membranes were blocked in 5% fat-free milk in Tris-buffered saline (TBS) (50 mmol/L Tris hydrochloride, pH 7.0, 150 mmol/L sodium chloride) for 1 hour in the o/n culture at +4°C followed by incubation with the following primary antibodies: HO-1 1:1000 (ab13248, Abcam, Cambridge, MA), ACSL1 1:1000 (D2H5) (catalog number 9189; Cell Signaling, Beverly, MA), IL-1β 1:500 (ab1413, Millipore, Billerica, MA), phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) 1:1000 (catalog number 9101; Cell Signaling), TLR4 1:1000 (ab13556; Abcam), and β-actin (catalog number A5316; Sigma-Aldrich, St. Louis, MO). After overnight incubation, membranes were washed in 1× TBS for 2 × 10 minutes and then incubated with horseradish peroxidase–conjugated secondary antibodies [anti-mouse IgG, horseradish peroxidase–linked antibody (catalog number 7076; Cell Signaling) or anti-rabbit IgG, horseradish peroxidase–linked antibody (catalog number 7074; Cell Signaling) at 1:5000 dilution for 1 hour at room temperature on orbital shaker, followed by washing twice for 15 minutes in TBS. Proteins were detected on radiofilm after signal development with SuperSignal West Pico Chemiluminescent Substrate and SuperSignal West FemtoChemiluminescent Substrate (Thermo Scientific). Before reprobing with different antibodies, membranes were stripped in ReBlot Plus Strong Antibody Stripping Solution (MilliporeSigma, Burlington, MA) for 10 minutes. Membranes were washed in TBS for 20 minutes and stored until reprobed. Total RNA was isolated from prostate tissues according to the manufacturer's instructions using an RNeasy MiniRNA Isolation Kit (Qiagen, Germantown, MD). cDNA was synthesized with a iScript cDNA synthesis kit (Bio-Rad) using 1 μg isolated RNA following the manufacturer's protocol: 25°C for 10 minutes, 37°C for 2 hours, and 85°C for 5 minutes. Gene expression levels were measured by real-time PCR using SYBR-Green PCR Master Mix (Life Technologies and Applied Biosystems, Foster City, CA) using the following primers: IL-1β, forward: 5′-TGGGCCTCAAA-GGAAAGA-3′, reverse: 5′-GGTGCTGATGTACCAGTT-3′; IL-10, forward: 5′-CCAAGCCTTATCGGAAATGA-3′, reverse: 5′-TTTTCACAGGGGAGAAATCG-3′; ACSL1: forward: 5′-CTACTACGACCGATGTCAGAACCA-3′, reverse: 5′-GGAAATCCACTCATAGGGCTGG-3′; and β-actin, forward: 5′-TAGACTTCGAGCAGGAGATGGC-3′, reverse: 5′-CCACAGGATTCCATACCCAAGA-3′. The following program was applied: 95°C for 10 minutes, 94°C for 30 seconds, 58°C for 55 seconds, 72°C for 1 minute, 95°C for 1 minute, 55°C for 30 seconds, and 95°C for 30 seconds (step 2 to 4 for 40 cycles). Standard software of Stratagen MxPro 3005P version 4.10 (Agilent Technologies, Santa Clara, CA) was used to measure relative changes in mRNA levels and normalized to the β-actin levels. Prostate tissues were fixed in zinc fixative (0.1 mol/L Tris hydrochloride, 0.5 g/L of calcium acetate, 5 g/L of zinc chloride, and 5 g/L of zinc acetate dihydrate) for 48 hours, dehydrated in a series of alcohols with increasing percentages and treated with xylene, and then embedded in paraffin. The paraffin blocks were cut in 5-μm sections using microtome and mounted on microscope glass slides (Thermo Scientific). Deparaffinated and rehydrated sections were washed in distilled water three times for 2.5 minutes and placed in PBS (pH 7.4) for 5 minutes. Sections were fixed on the slide and antigen retrieved following the optimized protocol for each antibody as specified below [see CD45, phosphorylated histone H3 (p-HH3), and HO-1]. After antigen retrieval, sections were washed twice for 5 minutes in PBS and then incubated with 7% horse serum diluted in PBS for 30 minutes to block unspecific binding. Primary antibodies were applied on each section for overnight incubation at +4°C. The following day slides were washed three times in PBS followed by 10 minutes of blocking in 0.5% hydrogen peroxide solution. After an additional wash in PBS, biotinylated secondary antibodies (Vector Laboratories, Burlingame, CA) were diluted 1:300 in PBS and applied on each section and incubated at room temperature for 1 hour. Slides were washed twice for 5 minutes in 1× PBS. Avidin-biotin peroxidase complexes were prepared from an ABC kit (Vectastain, Vector Laboratories) (two drops of A and two drops of B in 10 mL of PBS) and was let to stabilize at room temperature for 30 minutes and then appli
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