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【摘要】
Oxidative and nitrosative stress have been implicated in prostate carcinogenesis, but the cause(s) of redox imbalance in the gland remains poorly defined. We and others have reported that administration of testosterone plus 17ß-estradiol to Noble rats for 16 weeks induces dysplasia and stromal inflammation of the lateral prostate (LP) but not the ventral prostate. Here, using laser capture microdissected specimens, we found that the combined hormone regimen increased the expression of mRNA of specific members of NAD(P)H oxidase (NOX-1, NOX-2, and NOX4), nitric-oxide synthase , and cyclooxygenase (COX-2) in the LP epithelium and/or its adjacent inflammatory stroma. Accompanying these changes was the accumulation of 8-hydroxy-2'-deoxyguanosine, 4-hydroxynonenal protein adducts, and nitrotyrosine, primarily in the LP epithelium, suggesting that NOX, NOS, and COX may mediate hormone-induced oxidative/nitrosative stress in epithelium. We concluded that the oxidative/nitrosative damage resulting from the testosterone-plus-17ß-estradiol treatment is not solely derived from stromal inflammatory lesions but likely also originates from the epithelium per se. In this context, the up-regulation of COX-2 from epithelium represents a potential mechanism by which the hormone-initiated epithelium might induce inflammatory responses. Thus, we link alterations in the hormonal milieu with oxidative/nitrosative/inflammatory damage to the prostate epithelium that promotes carcinogenesis.
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Oxidative stress (OS) and nitrosative stress (NS) refer to a state of redox imbalance leading to excessive production of reactive oxygen species (ROS) and reactive nitrogen species (RNS), respectively, that overwhelms the antioxidant defenses of a cell. Such imbalances have recently been implicated in the genesis of prostate cancer (PCa). Thus, biomarkers of OS and NS have been reported in human prostatic intraepithelial neoplasia, also termed dysplasia purported PCa precursor, and in cancer of the prostate.1,2
Reactive oxygen species are normally generated as byproducts of aerobic respiration in the mitochondria, as well as a variety of inflammatory cells, producing large quantities of ROS and RNS.3 In addition to these two sources, superoxide-generating homologues of phagocytic NAD(P)H oxidase catalytic subunit gp91phox (NOX-1 and NOX-3, NOX-4, and NOX-5) and nitric-oxide synthase (NOS) family members, including inducible NOS (iNOS), endothelial NOS (eNOS), and neuronal NOS (nNOS), are primary O2 sources of superoxide (O2C?
【关键词】 hormones epithelial inflammation-mediated oxidative/nitrosative prostatic carcinogenesis
Materials and Methods
Animals and Hormone Treatments
Protocols of animal usage were approved by the University of Massachusetts Medical School Animal Care and Usage Committee. NBL rats were purchased from Charles River Laboratories (Kingston, NY), kept under standard conditions, and treated as previously reported.23,24 In brief, control rats (n = 8) were implanted with empty capsules. Hormone-treated rats (n = 8) were implanted with 2-cm lengths of Silastic tubing (1.0-mm inner diameter x 2.2-mm outer diameter; Dow Corning, Midland, MI) that were tightly packed with 14.4 ?? 2.1 mg of T (Sigma, St. Louis, MO), and 1-cm lengths of the same tubing were packed with 14.8 ?? 2.6 mg of E2 (Sigma). At the end of a 16-week treatment period, animals were sacrificed with an overdose of isoflurane, and VPs and LPs were excised. One half of each lobe was processed for histological examination, and the other half was snap frozen for RNA extraction or laser capture microdissection (LCM).
Histopathology and LCM
Formalin-fixed samples were processed for light microscopy. Twelve step-sections sampled serially through an entire half-LP/ventral prostate (VP), in a single-blinded manner, were subjected to histological examination for inflammatory, dysplastic, and malignant lesions by I.L.22 The other half of the LP was embedded in Tissue-Tek O.C.T. compound (Sakura Finetek USA, Torrance, CA) and then snap frozen in liquid nitrogen. Serial frozen sections were cryocut from each specimen. The first section of a replicate series was fixed, stained with hematoxylin, and used as a guide for the LCM (Arcturus, Mountain View, CA) conducted on the next serial sections, as previously described.25
Radioimmunoassay (RIA) of Serum T and E2 Levels
Serum total T and E2 levels from at least five animals in the control and in the T+E2-treated groups were measured by RIA, which was conducted in the ILAT Steroid RIA Laboratory, University of Massachusetts Medical School (Worcester, MA). The RIA kits for T and E2 assays were provided by Diagnostic Products (Los Angeles, CA) and Diagnostic Systems Laboratories (Webster, TX), respectively. The sensitivities of the T and E2 assays are 0.04 ng/ml and 2.2 pg/ml, respectively.
RNA Isolation, cDNA Synthesis, and Real-Time Quantitative Polymerase Chain Reaction
Total RNA was isolated and reverse-transcribed into cDNA as previously described.18 Real-time quantitative polymerase chain reaction (PCR) was performed with the iCycler IQ Real-Time PCR detection system (Bio-Rad Laboratories, Hercules, CA) as reported.24 Intron-spanning, gene-specific primers for nNOS (sense: 5'-CCCTGGCCAATGTGAGGTTC-3', antisense: 5'-TCCTCTCCCCTCCCAGTTCC-3'), iNOS (sense: 5'-CACCTTGGAGTTCACCCAGT-3', antisense: 5'-ACCACTCGTACTTGGGATGC-3'), eNOS (sense: 5'-CCAGCCAGGGGACCACATAG-3', antisense: 5'-CAGTTGTTCCACGGCCACAG-3'), COX-1 (sense: 5'-GCCTCGACCACTACCAATGT-3', antisense: 5'-AGGTGGCATTCACAAA CTCC-3'), and COX-2 (sense: 5'-GCTTTTCAACCAGCAGTTCC-3', antisense: 5'-CTGCTTGTACAGCGATTGGA-3') were designed with PRIMER 3 software, purchased from MWG Biotech (High Point, NC), and optimized for real-time PCR. Primers and PCR conditions for NOX-1, NOX-2, NOX-4, superoxide dismutase 1 (SOD-1), superoxide dismutase 2 (SOD-2), and ribosomal protein L19 (RPL19) have been documented previously.18
PCR products generated by each primer pair were column-purified by the Wizard SV gel and PCR clean-up system (Promega, Madison, WI) and quantified spectrophotometrically at 260 nm. The molecular mass of target cDNA was calculated on the following basis (Real-time PCR Instruction Manual; Applied Biosystems, Foster City, CA): mass of double-stranded DNA = number of bp x 1.096 x 10C21 g. Stock solutions of 300,000 copies of each standard cDNA/5 µl were prepared and serially diluted (1:10) with nuclease-free water. The target-gene transcript copy numbers in tissues/LCM samples were determined with a standard curve generated by plotting cycles at threshold against the logarithmic values of the standard copy number. The loading control was normalized by using the RPL19 level.
Immunohistochemical Analysis of OS/NS Biomarkers
Immunohistochemical staining for 8-hydroxy-2'-deoxy-guanosine (8-OHdG), 4-hydroxynonenal (4-HNE), and nitrotyrosine was conducted on paraffin slides as previously described.18,26 For controls, the primary antibodies were preincubated with either excess antigens (Sigma) or 10 mmol/L nitrotyrosine (Calbiochem, San Diego, CA) or were replaced with the corresponding normal isotype serum (Zymed, South San Francisco, CA).
Statistical Analyses
The statistical significance of the difference in expression levels between treatment groups was determined with Systat software (Student version 6.0.1) (SPSS, Chicago, IL) for one-way analysis of variance, followed by Tukey??s post hoc analyses or Student??s t-test. A P value of 0.05 was taken as a statistically significant difference between two groups.
Results
T+E2 Regimen Maintained Physiological T Levels and Elevated Levels of E2
RIA analyses of serum T and E2 levels (Figure 1, A and B , respectively) revealed that the number and the size of Silastic capsules used maintained the serum T at physiological levels (untreated control rats = 1.17 ?? 0.28 ng/ml versus T+E2-treated rats = 1.66 ?? 0.49 ng/ml; P > 0.05) and increased E2 levels four- to fivefold (39.6 ?? 3.5 pg/ml, P < 0.05) when compared with the untreated control rats (8.0 ?? 1.14 pg/ml). The elevated E2 levels are within the physiological range of cyclic fluctuation of E2 (from 17 ?? 2 to 88 ?? 2 pg/ml) in female rats during their 4-day estrous cycle.27
Figure 1. T+E2 co-treatment results in changes in the sex hormone milieu (A and B) and a lobe-specific induction of stromal inflammatory response and focal epithelial dysplastic lesions in LPs (CCE), but not in VPs (F and G), of treated rats. After a 16-week implantation of Silastic capsules containing T and E2, serum levels of T and E2 were determined by radioimmunoassay. A: Serum T levels of treated rats were not significantly different from those of untreated control rats. B: Serum E2 levels of treated rats were significantly (*P < 0.05) increased by four- to fivefold compared with levels in untreated control rats. Data represent the mean ?? SEM of values for at least five animals. C: Normal control LP. Epithelial tubules are lined by a single layer of cuboidal secretory cells; the stroma comprises primarily a periacinar layer of smooth muscle cells and interstitial fibroblasts. Note the scarcity of inflammatory cells in the interstitial stroma. D: T+E2-treated LP. Dysplastic epithelium associated with stromal inflammation is shown. Infiltrating inflammatory cells accumulate in the stroma juxtaposed to the prostatic epithelium exhibiting a continuum of normal morphology to dysplasia, as evidenced by the presence of pleomorphic nuclei (red arrows), the earliest recognizable sign of preneoplasia. E: T+E2-treated LP. Epithelial dysplasia with no inflammatory cell infiltration in the adjacent stroma. The inset illustrates a dysplastic lesion characterized by deranged epithelial cells with pleomorphic nuclei and loss of cell polarity. F: Normal control VP. Glandular acini are composed of columnar epithelial cells surrounded by a thin layer of smooth muscles and fibroblasts. Inflammatory cells are rarely detected in the interstitial stroma. G: T+E2-treated VP. A representative region of the VP shows no induction of epithelial dysplasia and stromal inflammatory response.
T+E2 Co-Treatment Selectively Induced Inflammation and Premalignant Dysplasia in the LPs, but Not in the VPs, of Treated Rats
The T+E2-induced histopathological changes in the NBL rat model have been reported previously.22,24 In this article, we give only the morphological alterations directly relevant to the present study. The LPs and VPs of untreated rats were histologically normal (Figure 1, C and F , respectively). Dysplasia and inflammation developed specifically in the LPs (Figure 1D) , but not in the VPs (Figure 1G) , of all treated rats. A spectrum of focal dysplastic epithelial lesions associated with variable degrees of chronic inflammation in the stroma was observed in the LPs of treated rats, as has been reported previously.23 We found, however, that focal dysplastic lesions were sometimes evident in the LP region in the absence of inflammation (Figure 1, E and inset).
T+E2 Co-Treatment Selectively Increased the Expression of NOXs, NOSs, and COXs in the LPs, but Not in the VPs, of Treated Animals
In the VP, only the expression of eNOS mRNA was significantly increased following T+E2 treatment (Figure 2B) . In contrast, in the LP, T+E2 treatment significantly increased mRNA levels of all three NOX isoforms (Figure 2C , left: bulk tissue) compared with those of untreated controls. However, the mRNA levels of nNOS (Figure 2B , left: bulk tissue), SOD-1, and SOD-2 (data not shown) remained unchanged in the LPs and VPs following T+E2 exposure.
Figure 2. Aberrant up-regulation of (A) O2C?
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作者单位：From the Department of Environmental Health,* Division of Environmental Genetics and Molecular Toxicology, University of Cincinnati Medical Center, Cincinnati, Ohio; and the Department of Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts