Malignant Prostate Cell Lines Research Articles

Abstract 4079: Control of survivin expression by IGF-I in prostate epithelial cells.

Abstract Survivin, a unique member of the inhibitor of apoptosis (IAP) proteins, is overexpressed in numerous cancers through poorly defined mechanisms. We show that insulin-like growth factor-I (IGF-I), which is implicated in the etiology of prostate cancer, induces the expression of Survivin in a variety of pre-neoplastic and malignant prostate cell lines. Using the pre-neoplastic NRP-152 rat prostate cell line as a model, we showed that silencing Survivin by lentiviral-mediated shRNA represses IGF-I-stimulated cell growth, suggesting that the induction of Survivin is critical to this growth response. Moreover, we show that IGF-I induces the expression of Survivin through a transcriptional mechanism that is mediated in part by the PI3K/Akt/mTORC1 pathway. The CDE and CHR response elements in the proximal region of the Survivin promoter are decisive for this IGF-I response. TGF-β signaling antagonists similarly induced this promoter activity and rendered cells refractory to further promoter activation by IGF-I. Suppression of TGF-β signaling, either by TGF-β receptor kinase inhibitors or by silencing Smads 2 and 3, induced Survivin expression and promoted cell growth similar to that induced by IGF-I. TGF-β receptor antagonists also rescued cells from down-regulation of Survivin expression and growth suppression by pharmacological inhibitors of PI3K, Akt, MEK and mTORC1. Sh-RNA gene silencing studies suggest that mTORC1 induces while mTORC2 represses the expression of Survivin. We conclude that IGF-I signaling through a PI3K/Akt/mTORC1-dependent mechanism elevates expression of Survivin and promotes growth of prostate epithelial cells by suppressing Smad-dependent autocrine TGF-β signaling. Citation Format: David Danielpour, Kyung Song, Eswar Shankar, Jiayi Yang, Kara L. Bane. Control of survivin expression by IGF-I in prostate epithelial cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4079. doi:10.1158/1538-7445.AM2013-4079

Expression of tissue factor and tissue factor pathway inhibitor in microparticles and subcellular fractions of normal and malignant prostate cell lines

The association between cancer and thrombogenesis has been recognized since 1865, and tissue factor (TF) is important at various stages in the natural history of the disease. It is involved in cancer angiogenesis, growth and metastasis. TF pathway inhibitor (TFPI), being the major physiological regulator of the TF-dependent coagulation pathway, is also important in establishing net procoagulant potential. In this study, we determine TF and TFPI levels in three prostate epithelial cell lines, one of normal and two of malignant origin. Cells were grown in standard maintenance conditions and harvested at more than 90% confluence. These were fractionated into cytosol, membrane and nuclei for analysis. Microparticles secreted into the culture medium were also analysed. TF and TFPI levels were determined using an ELISA. TF expression in these cells was also visualized using immunocytochemistry. There was absence of TF and TFPI in nuclei of all cell lines. TF expression was higher in subcellular fractions and microparticles of normal prostate cells than cancer cells. In contrast, levels of TFPI (structurally resembling a secreted, rather than transmembrane protein) in microparticles of normal prostate cells were much lower than tumour cells. In conclusion, the activity of prostate cancer cells themselves is unlikely to be the source of hypercoagulability in patients, but might precipitate chains of events that would produce such an effect.

Distribution and Possible Function of Cannabinoid Receptor Subtype 1 in the Human Prostate

Background: Cannabinoid receptor subtype 1 (CB1) has a relationship to the proliferation of various cells including malignant tumoral cells. We investigated and compared the expression of CB1 in benign and malignant human prostate tissues and in benign and malignant human prostate cell lines, as well as its function for the proliferation of human prostate cancer cells. Methods: Real-time quantitative PCR was performed to compare its expressions in human prostate tissues (normal, benign hyperplasia, and cancer) and prostate cell lines (3 normal and 3 malignant). For localization of CB1, immunofluorescent staining with rabbit anti-CB1 polyclonal antibodies and tetramethyl isothiocyanate (TRITC)-labeled swine anti-rabbit immunoglobulin (DAKO) were used under fluorescence microscope. To further analyze whether cell death was induced by anandamide (non-selective agonist for CB1/CB2) via a receptor dependent mechanism, the viability of DU145 cells, which is known as androgen-insensitive prostate cancer cell, was measured using MTT assay. Results: CB1mRNA was found to be expressed in the all 3 human prostate tissues, however, CB1 protein was expressed in BPH and low grade malignant PC tissues, but not in high grade malignant PC tissues. CB1 as for cell lines, the expression of CB1 was low in malignant cell lines except for DU145. Anandamide elicited cell death, which was significantly inhibited by AM251 (selective antagonist for CB1), indicating that cell death induced by anandamide in DU145 cells was mediated by CB1. Anandamide time-dependently elicits up-regulation of CB1 in DU145 cells. Conclusions: CB1 may be an inhibitory regulator of androgen-insensitive human prostate cancer epithelial cell growth.

Detection of Smell Print Differences Between Nonmalignant and Malignant Prostate Cells with An Electronic Nose

To determine whether an electronic nose can differentiate cultured nonmalignant and malignant prostatic cells from each other and whether the smell print is secreted to the surrounding medium. Prostatic nonmalignant (EP-156T and controls) and malignant (LNCaP) cell lines, as well as conditioned and unconditioned media, were collected. The smell prints of the samples were analyzed by a ChemPro(®) 100 electronic nose device. The data were normalized and dimension reduction was conducted. The samples were classified and misclassification rates were calculated. The electronic nose differentiated the nonmalignant and malignant cell lines from each other, achieving misclassification rates of 2.9-3.6%. Cells did not differ from the conditioned medium but differed from the unconditioned medium (misclassification rates: 0.0-25.6%). Malignant and nonmalignant prostatic cell lines have distinct smell prints. Prostatic cancer cells seem to modify the smell print of their medium.

Abstract 2176: Deletion of 3p14 is linked to ERG-fusion positive prostate cancers and harbors the FOXP1 tumor suppressor gene

Abstract Genetic rearrangements involving 3p14 are frequent alterations in prostate cancer, affecting approximately 15-20% of tumors. Both the clinical significance and the tumor suppressor at 3p14 locus are currently unknown. We performed SNP array analysis of 72 primary prostate cancers and show a commonly deleted region at 3p14 in 14 (19%) of the tumors. Our data indicate the existence of a small, recurrent deletion in a significant fraction of prostate cancers harboring 8 genes only. Ten of 14 tumors with 3p14 deletions detected in our study included the FOXP1 locus. To estimate the prevalence and clinical significance of FOXP1 deletions, we analyzed a tissue microarray containing 4,699 prostate cancers with histopathological and clinical follow-up data. Heterozygous FOXP1 deletions were found in 15% of tumors, and were strongly associated with ERG-fusion positive cancers (p<0.0001). No homozygous deletions were detected. FOXP1 deletions were unrelated to tumor stage or Gleason grade. FOXP1 deletions were linked to early PSA recurrence in ERG-positive cancers (p=0.0576) but not in ERG negative tumors (p=0.5425). To better understand the association between ERG fusion and FOXP1 expression, we performed FOXP1 immunohistochemistry on our TMA. FOXP1 expression was found in virtually all (95%) prostate cancers, and was particularly strong in ERG-fusion positive tumors. This finding argues for an important role of FOXP1 in prostate cancer in general and for ERG fusion positive tumors in particular. Since FOXP1 is a transcriptional repressor, we hypothesized that upregulation might be a consequence of ERG activation in order to control transcription of ERG target genes. However, neither expression of ERG in LNCaP cells, or depletion of ERG in VCaP cells had an effect on FOXP1 mRNA levels, strongly arguing against a direct link between ERG and FOXP1 at least in the tested cell lines. In addition neither the transcription nor the steady state levels of FOXP1 protein were modulated by activation of AR signaling. Finally, colony formation assays in both benign and malignant prostate cell lines revealed that FOXP1 may act as a tumor suppressor. In summary, our study demonstrates that FOXP1 functions as one of the tumor suppressor genes in prostate cancer that is targeted by genomic deletions at 3p14. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2176. doi:1538-7445.AM2012-2176

Abstract B62: Protein inhibitor of activated STAT1 (PIAS1) is expressed in prostate cancer and stimulates cellular proliferation through inhibition of p21

Abstract Introduction: PIAS (Protein Inhibitors of Activated STAT) proteins have originally been identified as inhibitors of the JAK/STAT pathway but they have a wide role in the regulation of transcription. Besides their DNA binding ability they also act as SUMO-E3 ligases, thus interacting with and modulating the activity of various proteins. Whilst PIAS proteins have previously been reported to regulate androgen receptor activity in prostate cancer, studies that systematically address PIAS protein expression patterns and their role in the regulation of cell growth and cell death in cancer have not been performed yet. The aim of this study is to investigate both in vivo and in vitro expression patterns of PIAS1 in prostate specimens and cell lines and to elucidate the functional role of PIAS1 in the regulation of proliferation, cell cycle progression, and apoptosis. Experimental procedures: In vivo expression of PIAS1 was determined by immunohistochemical analysis of a tissue microarray of benign and malignant human prostate cancer samples (n=90). For in vitro studies, the expression levels of mRNA and protein were determined by Western Blot and Real Time PCR. Localization studies were performed by immunofluorescence and cell fractionation. For down-regulation of PIAS1, cells were transfected with 25nM of siRNA. For overexpression of PIAS1, cells were transfected with 3μg of pEGFP-PIAS1. Proliferation was measured using [3H] thymidine incorporation assay. Apoptosis and cell cycle analysis were determined by FACS after propidium iodide staining. Clonogenicity was determined by a 2-D colony formation assay. Results: Tissue microarray data reveal that PIAS1 expression is significantly higher in malignant areas in comparison to benign tissue in clinical prostate cancer specimens. Furthermore, the staining pattern of PIAS1 correlates with the staining patterns of the proliferation markers PCNA and Ki67, thus suggesting a pro-proliferative role for PIAS1. Most benign and malignant prostate cell lines express PIAS1 mRNA and protein. PIAS1 predominantly shows nuclear localisation. Downregulation of PIAS1 leads to a ∼50% decrease in cell proliferation and clonogenicity after 96 h. At the same time, PIAS1 depletion does not significantly influence apoptosis. Decreased proliferation correlates with an increased nuclear expression of the cell-cycle regulator p21 at mRNA and protein level (up to 6-fold). Furthermore, PIAS1 down-regulation leads to a significant increase of the percentage of cells in G1-phase and a decrease of cells in S-phase of the cell cycle. Overexpression of PIAS1 decreases expression of p21, leading to a significant increase of cells in S-phase, and subsequently to an accelerated proliferation rate. Conclusion: Our data indicate that PIAS1 has pro-proliferative effects in prostate cancer cell lines and tissue. We demonstrate that PIAS1 influences cell cycle progression via the regulation of p21 expression and thereby affects cell proliferation. Our results clearly show that PIAS1 may be a new target for improved prostate cancer therapy. Citation Format: Julia Hoefer, Martin Puhr, Georg Schaefer, Helmut Klocker, Holger Erb, Ian G. Mills, Ludger Hengst, Zoran Culig. Protein inhibitor of activated STAT1 (PIAS1) is expressed in prostate cancer and stimulates cellular proliferation through inhibition of p21 [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr B62.

HOXC8 Inhibits Androgen Receptor Signaling in Human Prostate Cancer Cells by Inhibiting SRC-3 Recruitment to Direct Androgen Target Genes

HOX (homeobox) genes encode homeodomain-containing transcription factors critical to development, differentiation, and homeostasis. Their dysregulation has been implicated in a variety of cancers. Previously, we showed that a subset of genes of the HOXC cluster is upregulated in primary prostate tumors, lymph node metastases, and malignant prostate cell lines. In the present study, we show that HOXC8 inhibits androgen receptor (AR)-mediated gene induction in LNCaP prostate cancer cells and HPr-1 AR, a nontumorigenic prostate epithelial cell line. Mechanistically, HOXC8 blocks the AR-dependent recruitment of the steroid receptor coactivators steroid receptor coactivator-3 (SRC-3), and CREB binding protein to the androgen-regulated prostate-specific antigen gene enhancer and inhibits histone acetylation of androgen-regulated genes. Inhibition of androgen induction by HOXC8 is reversed upon expression of SRC-3, a member of the SRC/p160 steroid receptor cofactor family. Coimmunoprecipitation studies show that HOXC8 expression inhibits the hormone-dependent interaction of AR and SRC-3. Finally, HOXC8 expression increases invasion in HPr-1 AR nontumorigenic cells. These data suggest a complex role for HOXC8 in prostate cancer, promoting invasiveness while inhibiting AR-mediated gene induction at androgen response element-regulated genes associated with differentiated function of the prostate. A greater understanding of HOXC8 actions in the prostate and its interactions with androgen signaling pathways may elucidate mechanisms driving the onset and progression of prostate cancer.

Vitamin D pathway gene variants and prostate cancer prognosis

Observational studies linking vitamin D deficiency with increased prostate cancer (PCa) mortality and the pleiotropic anticancer effects of vitamin D in malignant prostate cell lines have initiated trials examining potential therapeutic benefits of vitamin D metabolites. There have been some successes but efforts have been hindered by risk of inducing hypercalcemia. A limited number of studies have investigated associations between variants in vitamin D pathway genes with aggressive forms of PCa. Increased understanding of relevant germline genetic variation with disease outcome could aid in the development of vitamin-D-based therapies. We undertook a comprehensive analysis of 48 tagging single-nucleotide polymorphisms (tagSNPs) in genes encoding for vitamin D receptor (VDR), vitamin D activating enzyme 1-alpha-hydroxylase (CYP27B1), and deactivating enzyme 24-hydroxylase (CYP24A1) in a cohort of 1,294 Caucasian cases with an average of 8 years of follow-up. Disease recurrence/progression and PCa-specific mortality risks were estimated using adjusted Cox proportional hazards regression. There were 139 cases with recurrence/progression events and 57 cases who died of PCa. Significantly altered risks of recurrence/progression were observed in relation to genotype for two VDR tagSNPs (rs6823 and rs2071358) and two CYP24A1 tagSNPs (rs927650 and rs2762939). Three VDR tagSNPs (rs3782905, rs7299460, and rs11168314), one CYP27B1 tagSNP (rs3782130), and five CYP24A1 tagSNPs (rs3787557, rs4809960, rs2296241, rs2585428, and rs6022999) significantly altered risks of PCa death. Genetic variations in vitamin D pathway genes were found to alter both risk of recurrence/progression and PCa-specific mortality.

Abstract 5758: Deletions of olfactomedin 4 gene is associated with progression of prostate cancer

Abstract The human olfactomedin 4 gene (OLFM4) encodes an olfactomedin-related glycoprotein, which our group first cloned and characterized in myeloid cells and mapped to chromosome 13q14.3. In a subsequent study, we have found that OLFM4 was expressed in normal prostate tissue, but its expression was lost in 10.9% (6/55) of well-differentiated prostate caner (Gleason score 4-6), 22.2% (8/36) of intermediately differentiated prostate cancer (Gleason score 7) and 58.8% (40/68) of advanced prostate cancer tissues (Gleason score 8-10). The loss of expression of OLFM4 was significantly associated with a higher Gleason score for prostate cancer (p<0.001). To study the mechanisms of loss of expression of OLMF4 gene in prostate cancer, we investigated alterations in the genetics and epigenetics of the OLFM4 gene in human prostate cancer patients. We looked for deletions and mutations of the OLFM4 gene and the status of methylation of the OLFM4 promoter region in benign and malignant prostate cell lines and in the paired normal and cancerous prostate epithelial cells that were obtained from whole mount prostate tissues by laser capture microdissection (LCM). The results showed that deletions and mutations of the OLFM4 gene is the primary cause of loss of expression of OLFM4 gene in human prostate cancer. We found that 28% (4/14) of primary prostate cancer cells had loss of heterozygosity (LOH) at rs 2298231 of the OLFM4 gene and 14% (2/14) of primary prostate cancer cells revealed deletion in exon 5 of the OLFM4 gene. We also found deletions in the promoter region in tumor cell lines, LNCaP and DU145, as well as exon 5 deletions in DU145 cells. There were no significant differences in the methylation status compared to adjacent normal (n=8), Prostatic Intraepithelial Neoplasia (PIN, n=3), low grade prostate cancer cells (Gleason score 4-7) and high grade prostate cancer cells (Gleason score 8-10, n=7) from the LCM samples studied. To further explore the function of the OLMF4 gene in prostate cancer, we have developed and studied the pathological changes in an OLFM4 knockout mouse model. Histopathological analysis of prostate tissue from OLMF4 wild type mice revealed the following: 5 out of 5 mice were normal at ages of 10 to 12 months, and 1 out of 5 showed prostate dysplastic changes at 18-20 month of age. The prostate from OLFM4 homozygous knock-out (KO) mice revealed that 7 out of 9 mice developed prostatic epithelial hyperplasia, and lower grade prostatic intraepithelial neoplasia (low-grade PIN) in mice at age of 10 to 12 months. By the age of 18 to 20 months, we found that 12 out of 18 KO mice developed high grade prostate intraepithelial neoplasia (high-grade PIN) in which 4 out of 18 developed into prostate cancer and 1 out of 18 showed evidence of lung metastasis. Our results suggest that OLFM4 gene is a candidate for the putative prostate cancer likely tumor suppressor gene located on chromosome 13q and is involved in the progression of prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5758.

Abstract 64: Differences in lipogenesis between cancer cells in culture and solid tumors

Abstract Elevated lipogenesis is a characteristic feature of cancer. Fatty acid synthase (FASN) overexpression has been found in many human cancers. Tumor cells in culture and solid tumor models are essential tools to study cancer biology. A comparison of the models can reveal tumor microenvironmental influences on lipogenesis. Here we have characterized differences in lipid components such as total fatty acid (tFA) and phosphatidylcholine (PtCho) levels between tumor cells in culture and solid tumors, with 1H MR spectroscopy (MRS) of lipid-soluble cell or tumor extracts derived from breast and prostate cancer cell lines. MCF-12A, nonmalignant human mammalian epithelial cells, MDA-MB-231, an estrogen receptor (ER)/progesterone receptor (PR)-negative metastatic human breast cancer cell line and MCF-7, an ER/PR-positive poorly metastatic human breast cancer cell line, as well as PC-3 and DU-145, which are both androgen independent malignant human prostate cell lines were used in this study. Fully relaxed 1H MR spectra of lipid-soluble extracts from cells in culture and tumors were acquired on a Bruker Avance 500 MR spectrometer using tetramethylsilane (TMS) as an internal concentration standard. Total FA and PtCho levels were quantified as A.U./cell or A.U./g using integrals of the 1.3 ppm and 3.2 ppm signals in the 1H NMR spectra, respectively, normalized to the cell number or tumor weight (g). Patterns of PtCho and tFA levels were similar in cells, but different in tumors. Both PtCho and tFA levels were significantly higher in MCF-7 cells compared to MCF-12A and MDA-MB-231 cells. Interestingly, the levels of PtCho and tFA in nonmalignant MCF-12A cells and malignant MDA-MB-231 cells were comparable. However, the level of tFA in MDA-MB-231 tumors was significantly higher than in MCF-7 tumors. It has been known that hypoxia and low pH conditions activate FASN activity. FASN or/and other lipogenic enzymes in MDA-MB-231 tumors may be more sensitive to those microenvironmental factors. The tFA level of DU-145 cells was significantly higher compared with PC-3 cells. However, PtCho and tFA levels in PC-3 and DU-145 tumors were not significantly different. FASN protein levels in MCF-12A and MCF-7 cells were not significantly different. Since MCF-12A medium contains epidermal growth factor, which activates FASN, we need to further investigate the involvement of FASN without its activators and with only de novo fatty acid. Our results revealed that tFA levels of cells and tumors do not show similar patterns. The differences between cells and tumors can arise from environmental factors found in solid tumors such as depletion of nutrients and oxygen, changes in pH, as well as cancer cell and stromal/endothelial cell interactions. Additional studies are necessary to unravel the tumor microenvironmental factors responsible for the differences in lipid metabolism observed between cells grown in culture as compared to the same cells grown as solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 64.

Recognition of O6MeG Lesions by MGMT and Mismatch Repair Proficiency may be a Prerequisite for Low-Dose Radiation Hypersensitivity

Low-dose hyper-radiosensitivity (HRS) is the phenomenon whereby cells exposed to radiation doses of less than approximately 0.5 Gy exhibit increased cell killing relative to that predicted from back-extrapolating high-dose survival data using a linear-quadratic model. While the exact mechanism remains to be elucidated, the involvement of several molecular repair pathways has been documented. These processes in turn are also associated with the response of cells to O6-methylguanine (O6MeG) lesions. We propose a model in which the level of low-dose cell killing is determined by the efficiency of both pre-replicative repair by the DNA repair enzyme O6-methylguanine methyltransferase (MGMT) and post-replicative repair by the DNA mismatch repair (MMR) system. We therefore hypothesized that the response of cells to low doses of radiation is dependent on the expression status of MGMT and MMR proteins. MMR (MSH2, MSH6, MLH1, PMS1, PMS2) and MGMT protein expression signatures were determined in a panel of normal (PWR1E, RWPE1) and malignant (22RV1, DU145, PC3) prostate cell lines and correlated with clonogenic survival and cell cycle analysis. PC3 and RWPE1 cells (HRS positive) were associated with MGMT and MMR proficiency, whereas HRS negative cell lines lacked expression of at least one (MGMT or MMR) protein. MGMT inactivation had no significant effect on cell survival. These results indicate a possible role for MMR-dependent processing of damage produced by low doses of radiation.

Differential Expression of Secretory Phospholipases A2 in Normal and Malignant Prostate Cell Lines: Regulation by Cytokines, Cell Signaling Pathways, and Epigenetic Mechanisms

Upregulation of group IIA phospholipase A2 (sPLA2-IIA) correlates with prostate tumor progression suggesting prooncogenic properties of this protein. Here, we report data on expression of three different sPLA2 isozymes (groups IIA, V, and X) in normal (PrEC) and malignant (DU-145, PC-3, and LNCaP) human prostate cell lines. All studied cell lines constitutively expressed sPLA2-X, whereas sPLA2-V transcripts were identified only in malignant cells. In contrast, no expression of sPLA2-IIA was found in PrEC and DU-145 cells, but it was constitutively expressed by IFN-γ in LNCaP and PC-3 cells. Expression of sPLA2-IIA is upregulated in PC-3 and in PrEC cell in a signal transducer and activator of transcription-1-dependent manner, but not in LNCaP cell. Additional signaling pathways regulating sPLA2-IIA expression include cAMP/protein kinase A, p38 mitogen-activated protein kinase, protein kinase C, Rho-kinase, and mitogen-activated/extracellular response protein kinase / extracellular signal-regulated kinase. No deletions were revealed in the sPLA2-IIA gene from DU-145 cells lacking the expression of sPLA2-IIA. Reexpression of sPLA2-IIA was induced by 5-aza-2′-deoxycytidine demonstrating that DNA methylation is implicated in the regulation of sPLA2-II. Together, these data suggest that sPLA2-IIA and sPLA2-V, but not sPLA2-X, are differentially expressed in normal and malignant prostate cells under the control of proinflammatory cytokines; epigenetic mechanisms appear involved in the regulation of sPLA2-IIA expression, at least in DU-145 cells.

Mitochondrial aconitase and citrate metabolism in malignant and nonmalignant human prostate tissues

BackgroundIn prostate cancer, normal citrate-producing glandular secretory epithelial cells undergo a metabolic transformation to malignant citrate-oxidizing cells. m-Aconitase is the critical step involved in this altered citrate metabolism that is essential to prostate malignancy. The limiting m-aconitase activity in prostate epithelial cells could be the result of a decreased level of m-aconitase enzyme and/or the inhibition of existing m-aconitase. Earlier studies identified zinc as an inhibitor of m-aconitase activity in prostate cells; and that the depletion of zinc in malignant cells is an important factor in this metabolic transformation. However, a possibility remains that an altered expression and level of m-aconitase enzyme might also be involved in this metabolic transformation. To address this issue, the in situ level of m-aconitase enzyme was determined by immunohistochemical analysis of prostate cancer tissue sections and malignant prostate cell lines.ResultsThe immunocytochemical procedure successfully identified the presence of m-aconitase localized in the mitochondrial compartment in PC-3, LNCaP, and DU-145 malignant prostate cell lines. The examination of prostate tissue sections from prostate cancer subjects demonstrated that m-aconitase enzyme is present in the glandular epithelium of normal glands, hyperplastic glands, adenocrcinomatous glands, and prostatic intraepithelial neoplastic foci. Quantitative analysis of the relative level of m-aconitase in the glandular epithelium of citrate-producing adenomatous glands versus the citrate-oxidizing adenocarcinomatous glands revealed no significant difference in m-aconitase enzyme levels. This is in contrast to the down-regulation of ZIP1 zinc transporter in the malignant glands versus hyperplastic glands that exists in the same tissue samples.ConclusionThe results demonstrate the existence of m-aconitase enzyme in the citrate-producing glandular epithelial cells; so that deficient m-aconitase enzyme is not associated with the limiting m-aconitase activity that prevents citrate oxidation in these cells. The level of m-aconitase is maintained in the malignant cells; so that an altered enzyme level is not associated with the increased m-aconitase activity. Consequently, the elevated zinc level that inhibits m-aconitase enzyme is responsible for the impaired citrate oxidation in normal and hyperplastic prostate glandular epithelial cells. Moreover, the down-regulation of ZIP1 zinc transporter and corresponding depletion of zinc results in the increase in the activity of the existing m-aconitase activity in the malignant prostate cells. The studies now define the mechanism for the metabolic transformation that characterizes the essential transition of normal citrate-producing epithelial cells to malignant citrate-oxidizing cells.

Ribosomal Protein L19 Is a Prognostic Marker for Human Prostate Cancer

Microquantity differential display analysis of gene expression profiles between benign (PNT2) and malignant (PC3M) human prostate cell lines identified the gene encoding ribosomal protein L19 (RPL19) to be overexpressed in the malignant cells. Northern blot hybridization analysis done on a wide range of human cell lines and tissues confirmed the level of RPL19 mRNA to be 5-fold higher in malignant cell lines and 8-fold higher in malignant tissues, when compared with their benign counterparts. Analysis of RPL19 mRNA expression by in situ hybridization revealed a significant increase of RPL19 expression in a substantial number of prostate cancers. All of the eight normal prostatic tissues were unstained (100%). Of 32 benign prostatic hyperplasia (BPH) tissues, 15 (46.9%) were unstained, 9 (28.1%) stained weakly, and 8 (25%) stained moderately. Among 87 carcinomas, only 7 (8.1%) were unstained, whereas 22 (25.2%) stained weakly, 21 (24.1%) stained moderately, and 37 (42.61%) stained strongly. The intensity of staining of the malignant specimens was significantly higher than that of normal and BPH specimens (chi(2): n = 127, P < 0.001). Gleason scores of the carcinomas correlated with RPL19 expression (chi(2): n = 87, P < 0.001). Kaplan-Meier survival analysis confirmed increased RPL19 expression to be highly predictive of shorter patient survival (P < 0.05), revealing RPL19 to be a sensitive predictor of prostate cancer progression. Expression of this protein could be a valuable marker in prostate cancer diagnosis and patient management.

EAAC1 is expressed in rat and human prostate epithelial cells; functions as a high-affinity L-aspartate transporter; and is regulated by prolactin and testosterone

BackgroundProstate epithelial cells accumulate a high level of aspartate that is utilized as a substrate for their unique function of production and secretion of enormously high levels of citrate. In most mammalian cells aspartate is synthesized; and, therefore is a non-essential amino acid. In contrast, in citrate-producing prostate cells, aspartate is an essential amino acid that must be derived from circulation. The prostate intracellular/extracellular conditions present a 40:1 concentration gradient. Therefore, these cells must possess a plasma membrane-associated aspartate uptake transport process to achieve their functional activity. In earlier kinetic studies we identified the existence of a unique Na+-dependent high-affinity L-aspartate transport process in rat prostate secretory epithelial cells. The present report is concerned with the identification of this putative L-aspartate transporter in rat and human prostate cells.ResultsThe studies show for the first time that EAAC1 is expressed in normal rat prostate epithelial cells, in normal and hyperplastic human prostate glands, and in human malignant prostate cell lines. EAAC1 expression and high-affinity L-aspartate transport are correspondingly down-regulated by EAAC1 siRNA knock down. Exposure of prostate cells to physiological levels of prolactin or testosterone results in an up-regulation of EAAC1 expression and a corresponding increase in the high-affinity transport of L-aspartate into the cells.ConclusionThis study shows that EAAC1 functions as the high-affinity L-aspartate transporter that is responsible for the uptake and accumulation of aspartate in prostate cells. In other cells (predominantly excitable tissue cells), EAAC1 has been reported to function as a glutamate transporter rather than as an aspartate transporter. The regulation of EAAC1 expression and L-aspartate transport by testosterone and prolactin is consistent with their regulation of citrate production in prostate cells. The identification of EAAC1 as the high-affinity L-aspartate transporter now permits studies to elucidate the mechanism of hormonal regulation of EAAC1 gene expression, and to investigate the mechanism by which the cellular environment effects the functioning of EAAC1 as an aspartate transporter or as a glutamate transporter.

Overexpression of ecto-protein kinases in prostasomes of metastatic cell origin

Prostasomes are secretory granules produced, stored, and released by the glandular epithelial cells of the prostate. They express numerous enzymes whose physiological roles have so far not been fully evaluated. In this study, we investigated the expression and function of prostasomal protein kinases and ATPase. The protein kinase activities of prostasomes isolated from seminal fluid and malignant prostate cell lines (PC-3, DU145, and LNCaP) were investigated using the model phosphorylation substrates histone and casein, as well as the plasma proteins C3 and fibrinogen, in combination with specific protein kinase inhibitors. The prostasomal ATPase activity was also evaluated. The expression of protein kinases and ATPase on prostasomes was verified by flow cytometry. Prostasomes (intact or solubilized with octylglucoside or saponin) from prostate cancer cells had higher expression of protein kinases A, C, and casein kinase II compared to prostasomes isolated from seminal plasma, resulting in higher phosphorylation of both exogenous and endogenous substrates. Using intact prostasomes, it was found that prostasomes of metastatic origin had lower ATPase activity, resulting in higher residual ATP available for the phosphorylation reaction. Finally, complement component C3 and fibrinogen (two proteins whose activities are modulated by phosphorylation) were identified as physiologically relevant phosphorylation substrates. These results indicate that prostasomes are capable of modifying proteins possibly involved in the innate response by extracellular phosphorylation mediated by ecto-kinases. This is a novel mechanism by which prostatic malignant cells may interact with their environment.

HZIP1 zinc uptake transporter down regulation and zinc depletion in prostate cancer

BackgroundThe genetic and molecular mechanisms responsible for and associated with the development and progression of prostate malignancy are largely unidentified. The peripheral zone is the major region of the human prostate gland where malignancy develops. The normal peripheral zone glandular epithelium has the unique function of accumulating high levels of zinc. In contrast, the ability to accumulate zinc is lost in the malignant cells. The lost ability of the neoplastic epithelial cells to accumulate zinc is a consistent factor in their development of malignancy. Recent studies identified ZIP1 (SLC39A1) as an important zinc transporter involved in zinc accumulation in prostate cells. Therefore, we investigated the possibility that down-regulation of hZIP1 gene expression might be involved in the inability of malignant prostate cells to accumulate zinc. To address this issue, the expression of hZIP1 and the depletion of zinc in malignant versus non-malignant prostate glands of prostate cancer tissue sections were analyzed. hZIP1 expression was also determined in malignant prostate cell lines.ResultshZIP1 gene expression, ZIP1 transporter protein, and cellular zinc were prominent in normal peripheral zone glandular epithelium and in benign hyperplastic glands (also zinc accumulating glands). In contrast, hZIP1 gene expression and transporter protein were markedly down-regulated and zinc was depleted in adenocarcinomatous glands and in prostate intra-epithelial neoplastic foci (PIN). These changes occur early in malignancy and are sustained during its progression in the peripheral zone. hZIP1 is also expressed in the malignant cell lines LNCaP, PC-3, DU-145; and in the nonmalignant cell lines HPr-1 and BPH-1.ConclusionThe studies clearly establish that hZIP1 gene expression is down regulated and zinc is depleted in adenocarcinomatous glands. The fact that all the malignant cell lines express hZIP1 indicates that the down-regulation in adenocarcinomatous glands is likely due to in situ gene silencing. These observations, coupled with the numerous and consistent reports of loss of zinc accumulation in malignant cells in prostate cancer, lead to the plausible proposal that down regulation of hZIP1 is a critical early event in the development prostate cancer.

Hypoxia down-regulates DNA double strand break repair gene expression in prostate cancer cells

Background and purpose Intratumoral hypoxia has been correlated with poor clinical outcome in prostate cancer. Prostate cancer cells can be genetically unstable and have altered DNA repair. We, therefore, hypothesized that the expression of DNA double-strand break (DNA-dsb) repair genes in normal and malignant prostate cultures can be altered under hypoxic conditions. Methods and materials The expression of homologous recombination (HR) and non-homologous recombination (NHEJ) genes following gas hypoxia (0.2%) or exposure to HIF1α-inducing agent, CoCl 2 (100 μM), was determined for normal diploid fibroblasts (GM05757) and the pre-malignant and malignant prostate cell lines, BPH-1, 22RV-1, DU145 and PC3. RNA and protein levels were determined using RT-PCR and Western blotting. Additionally, p53 genotype and function, the level of hypoxia-induced apoptosis, and cell cycle distribution, were determined to correlate to changes in DNA-dsb gene expression. Results Induction of hypoxia was confirmed using HIF1α and VEGF expression in gas- and CoCl 2-treated cultures. Hypoxia (48–72 h of 0.2% O 2) decreased RNA expression of a number of HR-related genes (e.g. Rad51, Rad52, Rad54, BRCA1, BRCA2) in both normal and malignant cultures. Similar decreases in RNA pertaining to the NHEJ-related genes (e.g. Ku70, DNA-PKcs, DNA Ligase IV, Xrcc4) were observed. In selected cases, hypoxia-mediated decreases in RNA expression led to decreased DNA-dsb protein expression. CoCl 2-treated cultures did not show decreased DNA-dsb protein expression. The ability of hypoxia to down-regulate Rad51 and other HR-associated genes under hypoxia was not correlated to c-Abl or c-Myc gene expression, p53 genotype or function, propensity for hypoxia-mediated apoptosis, or specific changes in cell cycle distribution. Conclusions Hypoxia can down-regulate expression of DNA-dsb repair genes in both normal and cancer cells. If associated with a functional decrease in DNA-dsb repair, this observation could provide a potential basis for the observed genetic instability within tumor cells exposed to hypoxia.

Influence of p53 and bcl-2 on chemosensitivity in benign and malignant prostatic cell lines

The administration of cancer chemotherapeutic agents results in an increase in the apoptotic cells in the tumor: therefore, it has been assumed that anticancer drugs exhibit their cytotoxic effects via apoptotic signaling pathways. Characteristics that confer sensitivity to drug-induced apoptosis are, a functional p53 protein and expression of the apoptosis-promoting protein, bax. The role of p53 and bax/bcl-2 in drug-induced apoptosis was assessed in six prostate cell lines, 1532T, 1535T, 1542T, 1542N, BPH-1 and LNCaP using TD 50 concentrations of etoposide, vinblastine and estramustine. Cell death was monitored morphologically by fluorescent microscopy, and by flow cytometry (Annexin-V assay). Apoptotic morphology was rather low and ranged from 0.1% to 12.1%, 3.0% to 6.0% and 0.1% to 8.5% for etoposide, estramustine and vinblastine, respectively. Annexin-V binding and flow cytometry indicated apoptotic propensities of 0% to 4%, 0% to 3% and 0% to 5%, respectively. The percentage of cells responding to drug-induced apoptosis was, on average, higher in the tumor cell lines than in the normal cell lines, but showed no correlation with p53 status. The percentage of cells showing necrosis, assessed by Annexin binding and Propidium Iodide permeability in aqueous medium, tended to be much higher, and was found to be at the level of 5% to 30%. Immunoblotting demonstrated that bax and bcl-2 proteins were expressed at a basal level in all cell lines, but did not increase after exposure to TD 50 doses of the three drugs. The ratio of bax and bcl-2, measured by laser scanning densitometry, was not altered by the drug-induced DNA damage. The results suggest that apoptosis is not a major mechanism of drug-induced cell death in prostate cell lines and appears to be independent of p53 status and bax/bcl-2 expression.

Expression of heat shock proteins and heat shock protein messenger ribonucleic acid in human prostate carcinoma in vitro and in tumors in vivo

Heat shock proteins (HSPs) are thought to play a role in the development of cancer and to modulate tumor response to cytotoxic therapy. In this study, we have examined the expression of hsf and HSP genes in normal human prostate epithelial cells and a range of prostate carcinoma cell lines derived from human tumors. We have observed elevated expressions of HSF1, HSP60, and HSP70 in the aggressively malignant cell lines PC-3, DU-145, and CA-HPV-10. Elevated HSP expression in cancer cell lines appeared to be regulated at the post-messenger ribonucleic acid (mRNA) levels, as indicated by gene chip microarray studies, which indicated little difference in heat shock factor (HSF) or HSP mRNA expression between the normal and malignant prostate cell lines. When we compared the expression patterns of constitutive HSP genes between PC-3 prostate carcinoma cells growing as monolayers in vitro and as tumor xenografts growing in nude mice in vivo, we found a marked reduction in expression of a wide spectrum of the HSPs in PC-3 tumors. This decreased HSP expression pattern in tumors may underlie the increased sensitivity to heat shock of PC-3 tumors. However, the induction by heat shock of HSP genes was not markedly altered by growth in the tumor microenvironment, and HSP40, HSP70, and HSP110 were expressed abundantly after stress in each growth condition. Our experiments indicate therefore that HSF and HSP levels are elevated in the more highly malignant prostate carcinoma cells and also show the dominant nature of the heat shock-induced gene expression, leading to abundant HSP induction in vitro or in vivo.