Department of Molecular Immunology and Toxicology (MITO) –

Prostate cancer diagnosis canada, Hőmérséklet és prosztatagyulladás - A prosztatitis hőmérsékletének súlyosbítása

Seladin-1 belongs to a subgroup of androgen-dependent genes associated with anti-proliferative, pro-differentiation, and pro-apoptotic functions and plays a protective role against oncogenic stress. The present study aims to investigate the localization and expression of Seladin-1 protein in normal and tumoral human prostatic tissues as well as to explore its role in proliferation and steroid secretion in androgen-dependent LnCaP and androgen-independent DU cell lines and in human prostate primary cell culture.

Proliferation Ki67 fluorescence labeling and cell counts and steroid secretion ELISA were assessed in cell lines and primary epithelial cell cultures. In human prostatic tissue and cells, Seladin-1 was mostly localized within epithelial and rarely within stromal cells and primarily present in secretory luminal cells of normal and tumoral prostate cells.

Its expression was increased in low-risk prostate cancer but reduced in advanced prostate cancers when compared to normal tissues. Seladin-1 inhibition by treatment with its specific inhibitor, UA 75 nMincreased proliferation in LnCaP and primary cell culture, as well as testosterone and dihydrotestosterone levels in both LnCaP and DU cell lines.

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Robert Sabbagh and Nicole Gallo-Payet are co-senior authors. Each author certifies that all conflicts of interest, including specific financial interests and relationships and affiliations relevant to the subject matter or materials discussed in the manuscript e.

E-mail: nicole.

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Seladin-1 involvement in proliferation and secretion suggests that its downregulation may be a major mechanism causing prostate cancer evolution. Seladin-1 may thus potentially decrease cell growth and steroid dependency in low-grade prostate cancer. Prostate —, In the normal prostate, androgen maintains equilibrium between proliferation, survival, and apoptosis [3,4].

However, during prostatic tumorigenesis, this equilibrium is altered, thus leading to uncontrolled proliferation by cancer cells [5]. Evidence suggests that this disequilibrium may, in part, result from cholesterol defects, since elevated cholesterol levels and abnormal cholesterol metabolism may alter signal transduction events in a manner that promotes tumor cell growth and inhibits apoptotic signals [6,7].

The Seladin-1 protein for SELective Alzheimer Disease Indicator 1; also known as DHCR24 for dehydrocholesterol reductase is involved in the late steps of cholesterol biosynthesis as a reductase for the conversion of desmosterol to cholesterol [8].

Seladin-1 is also involved in many events pertaining to prostate cancer progression. Recent studies have shown that Seladin-1 mRNA is overexpressed in the early stages of prostate cancer, but repressed in the more homok prosztatitis stages and in metastasis [9]. In human fasciculata cells in culture, we have recently shown that Seladin-1 is involved in androgen secretion following hormonal stimulation [13].

Based on these findings, our working hypothesis is that, in the early stages of prostate cancer, Seladin-1 expression is increased in order to slow down anarchic proliferation and decrease androgen secretion, while in more aggressive stages of the cancer, Seladin-1 expression is rather decreased, explaining the increase in cell proliferation and androgen activity.

Therefore, the aims of the present study were firstly to compare the The Prostate expression and localization of Seladin-1 protein in normal versus different tumoral Gleason scores prostate tissues as well as in two models of well-established androgen-dependent LnCaP and -independent prostate cancer diagnosis canada lines DU Louis, MO.

London, ON, Canada. All other chemicals were of A-grade purity. All prostate tissues were obtained either after transrectal ultrasound-guided TRUS biopsies or from radical prostatectomies.

Inhibition of DHCR24/Seladin-1 impairs cellular homeostasis in prostate cancer

Specimens were classified as either normal, benign prostatic hyperplasia BPH or tumoral tissues according to the Gleason system [14]. Gleason score is the addition of the two most prevalent Gleason grades present in the tissue.

As Gleason grade is scaled from 1 being normal to 5 being undifferentiated ; in prostatitis szarvasmarha of tumorigenicity, the Gleason score ranges from 6 low-risk cancer to 10 high-risk cancer. For patients undergoing TRUS biopsies, two additional prostate biopsies were taken for this study, one in the apical zone of the prostate, the area most susceptible to cancer [15], and the other at the prostate base.

A prosztatitis hőmérsékletének súlyosbítása.

Negative biopsies and normal areas of positive core biopsies served as negative controls. Biopsies used for immunofluorescence were either scored as normal no sign of pathologyBPH, or scored under Gleason grade ranging from 3 to 5 as our goal was to evaluate Seladin-1 expression at the cellular level and for this purpose, only one area of tissue was analyzed and evaluated sequentially.

For patients who underwent radical prostatectomy for localized prostate cancer, once the prostate was removed, it was immediately sent to the uropathologist who studied it in detail after which a 1 cm3 of fresh normal adjacent tissue and tumoral tissue were given for this project.

Prostatectomies used for Western blot analysis and primary cell culture were scored under the Gleason score and subcategorized as low-risk cancer Gleason prostate calcifications psaintermediate risk Gleason 7and high-risk cancer Gleason 8— Since whole tissue was submitted to total protein extraction, no distinction could gyógyított prosztatit éhség made regarding Gleason grade.

Tissue Processing and Immunofluorescence Studies Prostate biopsy samples were fixed in 3.

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Specificity of the antibody was confirmed and characterized in both rat and humans [10,16,17]. The Prostate Human tissue sections, normal human primary epithelial cell, and LnCaP or DU cell lines were incubated with the primary anti-Seladin-1 antiserum or with the pre-immune sera bothovernight at 48C followed by incubation with a secondary conjugated anti-rabbit antibody coupled with Alexa-Fluor nm ; green for 1 hr at room temperature.

To confirm cell proliferation indices, LnCaP and DU cells lines were also incubated with the monoclonal mouse anti-human Ki67 antigen, clone MIB-1 followed by incubation with a secondary conjugated anti-mouse antibody coupled with Prostate cancer diagnosis canada nm ; red for 1 hr at room temperature.

Nuclei were visualized by DAPI staining ,; blue.

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Slides were mounted with Vectashield prostate cancer diagnosis canada medium and examined under a fluorescence Nikon Eclipse inverted microscope Nikon, Mississauga, ON, Canada equipped for epi-illumination. In all instances, no specific staining was observed with pre-immune substitution of antibody or with omission of primary antiserum for same dilution and exposure time. All images were acquired using identical camera settings for prostate cancer diagnosis canada and brightness unless indicated in the figure.

Prosztatarák metasztázisok experiment was performed in duplicate for each cell line and condition. Number of red-stained Ki67 cells superposed to the nucleus was measured and expressed as percentage of total cell count.

The Ki67 epitope is strictly exposed during active phase of the cell cycle and detected within the nucleus [18]. The patients had no prior treatment. Each marked blocks for benign and cancer sites was sampled Battista et al.

The number of patients in this TMA was 84 with a total of cores. The TMA paraffin block was sectioned into 0.

Dvostruki navodi Male or female, age 18 years or older 2. A prior diagnosis of multiple myeloma with documented disease progression in need of treatment at time of screening. Received prior lines of therapy Appendix Dincluding lenalidomide and a proteasome inhibitor PIeither sequential or in the same line, and is refractory prostate cancer diagnosis canada and refractory or refractory to lenalidomide in the last line.

Slides were counterstained with hematoxylin and staining bluing reagent. Non-specific reactivity was assessed by omission of the primary antibody and comparison to intrinsic stromal immunoreactivity. Following prostatectomy, retrieved normal prostatic tissues 1 cm3 were gently cut into smaller entities using a scalpel and kept moist in MEM medium.

The supernatant was removed. At the end of each dissociation period, cells were disrupted by gentle aspiration, centrifuged 10 min at g prostate cancer diagnosis canada cell pellets resuspended in MEM medium and maintained at C. Culture medium was changed 24 hr after initial addition of the medium. Cells were washed four times with serum-free complete CDM. On the fourth day of culture, epithelial cells were stimulated for 24 hr without control or with the Seladin-1 inhibitor UA 75 nM.

UA, in prostate cancer diagnosis canada concentrations, is an important inhibitor of cholesterol biosynthetic activity of Seladin-1 [20] and has been shown in our recent studies to decrease adrenocorticotropin-induced steroid secretion without having any effect on cell morphology or behavior [16,17]. Moreover, in the nanomolar concentration, UA has previously been shown to be a specific inhibitor of the Dreductase activity of Seladin-1 and having no impact on cholesterol membrane trafficking [20—22].

Finally, UA nM concentration has also been previously used in cancerous cell types such as glia and neuroblastoma [21]. On the fifth day of culture, cells were examined under a phase-contrast microscope Leica Corp. This cell culture method offers a very high percentage prostate cancer diagnosis canada separation and isolation between epithelial and stromal prostatic cells resulting in a mostly pure epithelial culture.

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To clearly characterize the purity, nature, and functionality of our epithelial primary cell culture, immunofluorescence for cytokeratin 8 and PSA was performed. Cytokeratin 8 allows for discrimination prostate cancer diagnosis canada epithelial and stromal cells as it is only expressed in epithelial cells, while PSA is secreted by active epithelial prostatic cells [19,22].

Cell Line Culture and Steroid Measurements LnCaP and DU cells lines were specifically chosen in the course of this study because of prostate cancer diagnosis canada differences relative to androgen sensitivity prostate cancer diagnosis canada proliferative behavior, hence appropriate to address our specific aims. LnCaP cells are androgen-dependent and exhibit a moderate rate of proliferation while DU cells are androgen-independent and have a high proliferating index.

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Cells were stimulated once daily for two consecutive days, without control or with the Seladin-1 inhibitor UA 75 nM. On the third day of culture, cells were examined under a phase-contrast Leica Corp. Results obtained are within the limit range of detection.

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Total testosterone and DHT sensitivities were, respectively, set at 6. Total Protein Extraction and Western Blotting Total protein extraction was performed on frozen human prostate tissues, obtained from prostatectomies, as well as on LnCaP and DU cell lines. Western blotting of Seladin-1 was performed immediately following protein extraction as previously described [16,17]. For human prostate tissue samples, quantification of blots was normalized to actin.

Following Seladin-1 incubation, membranes were stripped for 2 hr with glycine 0. Results are expressed as the fold increase of Seladin-1 to actin ratio for tumoral over normal adjacent tissue. In cell lines, however, for same amount of protein loaded on the prostate cancer diagnosis canada gel, actin or total MAPK were not equivalent between each cell line. Immunoreactive bands were scanned by laser densitometry and expressed in arbitrary units. Cells were scrapped with a rubber policeman and 25 ml of the cell suspension was counted under a phase-contrast microscope.

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For each experiment, four independent cell count measurements were performed, with total counts measured in duplicate and twice per duplicate. In LnCaP and DU cell lines, trypan blue, the most common prostate cancer diagnosis canada dye to distinguish between viable from non-viable cells [23], was added to the cell suspension for 1 min 0.

Trypan blue positive cells dead cells were reported as the percentage of total cell count. Statistical analyses of the data were performed using one-way ANOVA, followed by a test of simple effects when appropriate. Comparison between BPH and tumoral tissue did not reach statistical significance, due to prostate cancer diagnosis canada sample size and large inter-individual variations. In control tissue Fig. Upon initiation of cancer, Seladin-1 protein expression in epithelial cells increased as shown in Gleason grade 3 tissues Fig.

Immunofluorescence images taken at identical pszichés merevedèsi zavar kezelése times showed that Seladin-1 was present in the luminal side of glands Fig. Images from Gleason grade 4 biopsies Fig.

In Gleason grade 5 Fig. No specific staining was detected when the primary antibody was omitted Fig. These in situ observations were also supported by Western blotting performed on tissues obtained from human prostatectomies Fig.

As shown by phase-contrast examination, LnCaP cells exhibited a fibroblast-like appearance Fig. Detection of Seladin-1in human prostatic tissues. Specific labeling of Seladin-1was detected on single biopsied tissue by immunofluorescence A or on Gleason graded tissue microarray TMA by immunohistochemistry B.

Panels Ae andf arerepresentative ofnonspecific labeling of Prostate cancer diagnosis canada revealedby omission of primary antibody Ae orpre-immune serum Af.

Seladin-1 protein expression in human prostatic tissues retrieved following prostatectomies.

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A: Representative Western blotting of Seladin-1 in prostatic tissues of varying Gleason scores. Seladin-1 72 kDa quantification 15 mg loading krónikusan prosztatitis oka protein, normalized to actin, 42 kDa. B: Densitometric quantification of Seladin-1 human prostatic tissues, performed as kevés vizelet in the Materials and Methods Section.

Results are expressed as the fold increase of Seladin-1to actin ratio for tumoral over normal adjacent tissue and BPH was setat arelativevalue of1. Lysateproteinswere subjectedto Westernblottingusing specific antibodiesagainstSeladin-1Inset, expressed as box and whiskers plot, is representative of the foldincreaseinSeladin-1expressionof tumoralovernormaladjacent tissue for each patientincludedin the study.

Immunofluorescence labeling revealed that Seladin-1 in LnCaP cells was highly present within the cytoplasm and the nucleus, whereas in DU cells, the expression level was lower in both compartments Fig.

In fact, exposure time had to be increased by 4. Pre-immune serum substitution of the Seladin-1 antibody showed no specific staining data not shown. Such treatment had no effect on prostate cancer diagnosis canada Seladin-1 subcellular localization Fig. Proliferation assays were next performed in both cell lines. Trypan blue assay Fig. As shown in Figure 4E,F, a 2-day treatment with UA significantly increased testosterone secretion by approximately 1.

Finally, to further correlate proliferation data obtained in cell lines with more physiological conditions, normal human prostatic epithelial cells were prepared for primary culture. This epithelial cell culture was pure and functional, since the vast majority of cells expressed cytokeratin 8 and PSA Fig.

These human prostatic epithelial cells were intensely prostate cancer diagnosis canada for Seladin-1 which was localized in both the cytoplasm and nucleus, hence corroborating observations in whole prostatic tissues and in cell lines Fig. Moreover, we demonstrate that in LnCaP and human primary cultures of normal epithelial cells, Seladin-1 inhibition accelerates cell proliferation and increases androgen secretion in these cells.

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All of these results indicate a role for Seladin-1 against prostate cancer progression. Relationship Between Seladin-1Bimodal Expression and Proliferation Defects in Seladin-1 expression have been shown in several types of cancers, mainly in those related to Battista et al.

Seladin-1 protein expression in the human prostatic cell lines LnCaP androgen-dependent and DU androgen-independent. Scale bars, mm. After formaldehyde fixation and permeabilization with 0. To represent similar photomicrographs, LnCaP cell images were acquired using a 0.

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Images are representative of three different experiments. Scale bars, 40 mm. Sel-1, Seladin-1; C, control. Cells were cultured for 3 days and treatedfor two consecutivedays 24 hr afterplating withoutor withUA UA,75 nM induplicateinmmNuncPetridishesasdescribed in the Materials and Methods Section. Cell lysate proteins were subjected to Western blotting using specific antibodies against Seladin-1Results are expressed as densitometric values obtained for 15 mg loading of protein normalized to cells, due to variation in cell number between the two cell lines.

Impact of the Seladin-1inhibitor,UA, on proliferation and androgen secretion in human prostatic cell lines, LnCaP androgendependent and DU androgen-independent.

A,B: Proliferation assays using immunofluorescence of the cell-cycle proliferation antigen, Ki The resulting pinkish color indicates colocalization of Ki67 with the nucleus proliferative cell. C: Cell counts as measured with a hemocytometer counting chamber. Each cell count was performedin duplicate. D: Cell death assaysperformedusingTrypanblue exclusion.