Detection of prostatic-inhibin-like peptide in the cytoplasm of LNCaP cells,a human prostatic adenocarcinoma cell line

Prostatic inhibin peptide (PIP) is a 94-amino acid peptide involved in various cellular functions. The concentration of this peptide changes with prostatic pathophysiology suggesting a role in various disease conditions; present study was undertaken to investigate the presence of this peptide in two human prostatic cell lines: LNCaP and PC3 cells. The LNCaP cells showed an intense intracellular fluorescence pattern after staining with rabbit-anti-PIP antiserum and FITC conjugated goat antirabbit-IgG, while the PC3 cells did not exhibit any fluorescence. There was no alteration in the concentration of PIP in LNCaP cells with or without supplementation of steroids in culture medium. Immunoblot analysis indicates similarities between PIP from LNCaP cells and that from the human seminal plasma. Thus, present study demonstrates the presence of PIP in a human prostatic cell line, i.e., LNCaP cells. Its intracellular concentration is androgen independent, and has a close similarity with PIP isolated from the human seminal plasma. © 1994 Wiley-Liss, Inc.     

IGF-I secretion by prostate carcinoma cells does not alter tumor-bone cell interactions in vitro or in vivo

BACKGROUND: IGF-I is an important growth and differentiative factor for osteoblasts and may have a role in defining prostate cancer risk and skeletal metastases. METHODS: Conditioned media (CM) from human prostate cancer (PC), C4-2 and C4-2B, which produce osteoblastic lesions, and PC-3, which causes osteolysis, was added to MC3T3-E1 bone cultures. SCID mice were injected intratibially with these engineered cells. Tumor bearing tibiae were analyzed by microCT and pQCT. RESULTS: CM from PC cells increased osteoblast proliferation and differentiation and was unaltered by the type of PC cell, IGF-I antibodies, or exogenous IGF-I and IGFBP2. Study of intratibial PC tumors in SCID mice showed that C4-2 cells grew slowly preserving bone structure, while PC-3 tumors caused rapid osteolysis. Overexpression of IGF-I did not change either tumor progression or skeletal response. CONCLUSIONS: IGF-I is neither necessary nor sufficient for the osteoblastic response to PC metastases.     

Induction of cell cycle arrest and apoptosis in human prostate carcinoma cells by a recombinant adenovirus expressing p27(Kip1)

BACKGROUND: Adp27(Kip1), a recombinant adenovirus, was evaluated for expression of p27, a cyclin-dependent kinase inhibitor (CDKI) and tumor suppressor protein, in human prostate carcinoma cells. Effects of p27(Kip1) on cell cycle and apoptosis were analyzed. METHODS: We evaluated the effects of overexpression of p27(Kip1) in the human prostate carcinoma cell lines LNCaP, DU-145, and PC-3 in vitro and in vivo. Growth curve studies, cell cycle analysis, terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL), and annexin V-fluorescein isothiocyanate apoptosis analyses were conducted to determine effects of p27(Kip1) on cell cycle. CDKI activity assays and Western blots were conducted to determine presence/activities of CDKIs. RESULTS: Adp27(Kip1)-induced protein levels increased in a dose-dependent manner; p27(Kip1) protein was detected within 6 hr of infection with Adp27(Kip1) and remained stable for at least 48 hr. The activities of Cdk2, Cdk4, and Cdc2 kinases were inhibited 24 hr after infection with Adp27(Kip1). Bromodeoxyuridine incorporation demonstrated a dose-dependent decrease in S-phase cells 24 hr postinfection. TUNEL analysis revealed an induction of apoptosis (10 pfu/cell) within 48 hr of infection in all cell lines. Growth curve analyses demonstrated that Adp27(Kip1) infection inhibited proliferation of all cell lines tested and decreased cell numbers for Adp27(Kip1)-infected LNCaP and PC-3 cells by 96 hr. Cell cycle analysis of DNA content demonstrated an accumulation of cells in G0/G1-phase 24-120 hr after Adp27(Kip1)-infection. In vivo studies demonstrated a reduction in LNCaP xenograft tumor growth rates in mice injected with Adp27(Kip1). CONCLUSION: Exogenous p27(Kip1) overexpression results in cell cycle regulation in the human prostate carcinoma cell lines tested, representing the first use of this vector on prostate cancer cell lines in vitro and in vivo. Moreover, p27(Kip1) expression is associated with an increase in early apoptosis, which represents a recently discovered function for this protein. It also represents the first time this association has been observed in prostate carcinoma cell lines. This study provides support for the further development of Adp27(Kip1) as a potential therapeutic vector in the treatment of adenocarcinoma of the prostate.     

miR-124通过靶向调控PKM2基因的表达抑制前列腺癌PC3细胞的增殖

目的:探讨miR-124抑制前列腺癌PC3细胞增殖活性的机制。方法:利用荧光素酶报告基因验证miR-124能否靶向作用于丙酮酸激酶M2型(PKM2)3'端非编码区(UTR)。将miR-124 mimic转染至PC3细胞后,采用实时荧光定量PCR和Western印迹检测前列腺癌PC3细胞PKM2 mRNA和蛋白的表达水平,MTT法检测miR-124 mimic与PKM2 siRNA对PC3细胞生长活性的影响。结果:与人前列腺上皮细胞RWPE-1比较,PC3细胞中PKM2 mRNA和蛋白水平表达分别上调(5.12±0.35)倍和(4.05±0.20)倍(P0.05)。荧光素酶报告基因结果证实,PKM2是miR-124调控的靶基因,miR-124可特异性地结合于PKM2 mRNA的3'-UTR。转染miR-124 mimic 24 h后,PKM2蛋白水平下调至阴性对照组(0.16±0.04)倍(P0.05),但对PKM2 mRNA表达无显著影响(P0.05)。MTT结果显示,转染miR-124 mimic和PKM2 siRNA都能显著抑制PC3细胞的增殖,但miR-124 mimic对PC3细胞生长活性的抑制能力较PKM2 siRNA强。转染miR-124 mimic和PKM2 siRNA 24 h和48 h后,PC3细胞的增殖率分别为(66.20±5.10)%和(82.10±6.35)%(P0.05)、(49.34±2.37)%和(70.10±5.80)%(P0.05)。结论:miR-124可通过靶向调控PKM2基因的表达而抑制前列腺癌PC3细胞的增殖。     

Transfer of functional prostasomal CD59 of metastatic prostatic cancer cell origin protects cells against complement attack

BACKGROUND: Prostasomes are secretory granules produced, stored, and released, by the glandular epithelial cells of the prostate. They express the glycosylphosphatidylinositol (GPI)-anchored complement regulatory protein CD59, which has been shown to be transferred to spermatozoa and erythrocytes. METHODS: The CD59 content of prostasomes isolated from seminal fluid and malignant prostate cells (PC-3, DU145, and LNCaP) and the transfer of prostasomal CD59 to rabbit erythrocytes (RE) and to PIPLC-treated and unmanipulated cancer cells were investigated using FACS. All prostasomes were also incubated with RE and tested in a hemolytic assay. RESULTS: Prostasomes from cancer cells had higher expression of CD59 than those of normal cells. Prostasomal CD59 of different origin could be transferred to RE, malignant cell lines stripped of CD59 by PIPLC, or unmanipulated LNCaP cells. Malignant cell prostasomes had an increased ability to inhibit complement-mediated lysis compared to those from non-malignant cells. CONCLUSIONS: These results point to a novel mechanism by which prostasomes can protect prostatic malignant cells from complement attack.     

Osteopontin enhances the cell proliferation induced by the epidermal growth factor in human prostate cancer cells

BACKGROUND: Susceptibility to extracellular matrix and growth factors has been demonstrated to play a critical role in the development of prostate cancer (PCa) metastases. The aim of this study was to elucidate some mechanisms by which stroma controls tumor progression. METHODS: In our study we tested the growth ability of the LNCaP human prostatic cell line in steroid-free culture conditions in response to osteopontin (OPN), a non-collageneous matrix protein, localized in large amounts in the bone. RESULTS: In the LNCaP cell model, OPN stimulates cell proliferation in serum-free medium and colony growth at high dilution but this effect is visible only in presence of epidermal growth factor (EGF). Proliferation induced by OPN is accompanied by a sustained activation of EGF receptor (EGFR) whose phosphorylation is detectable up to 12 hr after treatment in association with EGF. The colocalization of integrin beta1, a ligand of OPN, and of EGFR on the cellular membrane, suggests that the association of these cell surface receptors may be the principal mechanism involved in the long-term activation of the EGFR. CONCLUSIONS: Our data describe a new possible mechanism involved in the establishment of bone metastases which may also account for the formation of androgen-independent cellular clones, frequently responsible of the clinical progression of PCa.     

Regulation of prostatic carcinoma cell proliferation and secretory activity by extracellular matrix and stromal secretions.

Previous studies from our laboratory have shown that reconstituted basement membrane and stromal secretory products are important regulators of benign prostatic epithelial cell growth and differentiation. In the present study we evaluated the impact of extracellular matrix (ECM) and soluble stromal secretory products on the proliferation and secretory activity of the androgen-responsive prostatic carcinoma cell line LNCaP. In these studies, dihydrotestosterone (DHT) was a potent mitogen for LNCaP cells cultured on plastic or on type I collagen. The growth response to DHT was greatly attenuated when LNCaP cells were grown on prostatic stromal ECM. Cells grown on stromal ECM also exhibited clustered morphology compared to the monolayer growth observed on plastic and secreted elevated levels of prostate specific antigen (PSA) and prostatic acid phosphatase (PAP). These findings indicate that cultivation of LNCaP on stromal ECM will promote the expression of differentiated functions. In additional studies, stromal cell conditioned medium (SCM) significantly increased PSA/PAP secretion by LNCaP cells in the presence of 10 nM DHT. The enhancement of DHT-induced PSA/PAP secretion by SCM was most pronounced when LNCaP cells were grown on stromal ECM. SCM did not significantly alter LNCaP proliferation. These studies indicate that prostatic stromal ECM and soluble secretory products will promote differentiated function in cultured LNCaP cells. In addition, we show that DHT can act as either a growth or differentiation-promoting stimulus depending on the presence of stromal factors.     

Lithium suppresses cell proliferation by interrupting E2F-DNA interaction and subsequently reducing S-phase gene expression in prostate cancer

BACKGROUND: Lithium is an existing drug for bipolar disorder and its uptake was recently linked to reduced tumor incidence compared to the general population. The major target of lithium action is glycogen synthase kinase 3 (GSK-3). Since GSK-3 expression and activation are associated with prostate cancer progression, the anti-cancer potential of lithium on prostate cancer was investigated in this study. METHODS: Multiple prostate cancer cell lines were treated with lithium chloride (LiCl). Cell proliferation and cell cycle distribution were analysed. DNA replication was determined using BrdU labeling assay. Genome-wide screening of gene expression was performed using cDNA microarray assay. GSK-3beta gene-specific silencing was conducted using small interferencing RNA (siRNA) transfection. E2 factor (E2F) transactivation was evaluated using reporter gene assay and E2F-DNA interaction was determined with chromatin-immunoprecipitation assay (ChIP). RESULTS: LiCl significantly inhibited cell proliferation, which was associated with reduced DNA replication and S-phase cell cycle arrest. LiCl significantly decreased the expression of multiple DNA replication-related genes, including cell division cycle 6 (cdc6), cyclin A, cyclin E, and cdc25C, which are regulated by E2F factor during cell cycle. A novel GSK-3-specific inhibitor TDZD-8 and GSK-3beta siRNA also suppressed the expression of these E2F target genes, indicating that LiCl-induced anti-cancer effect was associated with GSK-3beta inhibition. Furthermore, LiCl suppressed E2F transactivation by interrupting the interaction of E2F1 factor with its target gene promoter. CONCLUSIONS: These data indicated that LiCl suppresses cancer cell proliferation by disrupting E2F-DNA interaction and subsequent E2F-mediated gene expression in prostate cancer.     

Independent role of phosphoinositol-3-kinase (PI3K) and casein kinase II (CK-2) in EGFR and Her-2-mediated constitutive NF-kappaB activation in prostate cancer cells

BACKGROUND: Recent research has highlighted the potential role of EGFR and Her-2 in the constitutive activation of NF-kappaB (NF-kappaB) in prostate cancer cells, although the mechanism by which these receptors activate NF-kappaB in these cells remains unclear. METHODS AND RESULTS: Using pharmacological and genetic approaches we show that in PC-3 cells, EGFR and Her-2 are involved in the constitutive activation of NF-kappaB through two different mechanisms. EGFR activates NF-kappaB through the PI3K/Akt pathway that leads to the phosphorylation of IkappaBalpha on serines 32 and 36, thereby promoting the nuclear translocation of the p65 subunit. In contrast, Her-2 activates NF-kappaB through Casein Kinase II (CK-2) activation independently of IkappaBalpha phosphorylation on serines 32 and 36. CONCLUSIONS: Our study not only directly clarifies the signaling pathways involved in NF-kappaB activation in prostate cancer cell lines and but also provides a framework for further studies in the clinical characterization and management of prostate cancer.     

Melatonin and prostate cancer cell proliferation: interplay with castration,epidermal growth factor,and androgen sensitivity

BACKGROUND: Potential modulatory effects of melatonin on the proliferation of androgen-sensitive LNCaP and androgen-insensitive PC-3 and DU 145 prostate cancer cells were reported recently. In this study, we investigated the effects of combined melatonin and castration on LNCaP tumor growth in vivo, the interactions between melatonin and epidermal growth factor (EGF) on LNCaP cell proliferation, and melatonin actions on the proliferation of PC-3 and DU 145 cells. METHODS: Tumor development and growth in castrated nude mice inoculated with LNCaP cells or in intact animals inoculated with DU 145 cells, with or without daily melatonin treatment, were monitored by observation and caliper measurement. MT(1) receptor expression in native or transfected prostate cancer cell lines was examined by immunocytochemistry or 2-[(125)I]iodomelatonin binding. Cyclin D1 expression in LNCaP cells was assessed by Western blotting, and cell proliferation was measured by thymidine incorporation and/or cell count. RESULTS: Melatonin treatment was associated with further decreases in LNCaP tumor incidence and growth rate in castrated nude mice. Melatonin and 2-iodomelatonin (a melatonin receptor agonist) attenuated EGF-stimulated increases in LNCaP cell proliferation and cyclin D1 levels. Melatonin had no effect on the proliferation or growth of MT(1) receptor-expressing DU 145 cells, and of PC-3 cells in which MT(1) receptor protein was undetectable. The proliferation of transfected PC-3 cells expressing MT(1) receptor was unaffected by 2-iodomelatonin. CONCLUSION: Together with previous data, the present results indicate synergistic action of melatonin and castration in inhibiting the growth of androgen-sensitive LNCaP tumor. Androgen-sensitive prostate cancer cell proliferation may be modulated by opposite changes in cyclin D1 levels induced by activated MT(1) and EGF receptors. In androgen-insensitive prostate cancer cells, MT(1) receptor-mediated signal transduction may become defective not only through changes in membrane receptor protein expression and/or functions, but also by means of alterations in downstream postreceptor signaling events.     

Inhibition of prostatic epithelial cell proliferation by a factor secreted specifically by prostatic stromal cells

Stromal cells from the prostate were recently shown to inhibit clonal growth of the prostatic carcinoma cell lines PC-3 (hormone-independent) and LNCaP (hormone-sensitive) in coculture. Our study revealed that stromal cell-conditioned medium strongly inhibited proliferation of PC-3 and LNCaP cells when grown in monolayer culture. Antiproliferative activity was found to be reversible, and was produced specifically by prostatic stromal cells and not by stromal cells derived from skin, foreskin, uterus, kidney, and Wilms' tumor. Inhibition was not species-specific, since the cell lines AT-2.1 and MATLyLu, derived from the Dunning rat prostate tumor, were also sensitive. No inhibition, however, occurred on breast and renal carcinoma cell lines, suggesting a prostate-specific action. The putative inhibiting factor(s) could be concentrated and partially purified by ammonium sulfate precipitation. The possible role in stromal control of epithelial cell proliferation is discussed.     

Effect of increasing ratio of estrogen: androgen on proliferation of normal human prostate stromal and epithelial cells, and the malignant cell line LNCaP

BACKGROUND: Changes in steroid ratios seen in the aging male are thought to promote prostate disease. The aims of this study were to compare the effects of varied ratios of steroids on growth of normal stromal and epithelial cell isolates, and the prostate cancer cell line, LNCaP. METHODS: The effect of altered steroid ratios on cell proliferation of normal stromal (PrSC) and epithelial (PrEC) prostate cells, and the malignant cell line, LNCaP, were assessed. RESULTS: Increasing the ratios of both estrogen:dihydrotestosterone (DHT) and DHT:estrogen, stimulated PrSC proliferation, with increasing estrogen:DHT having the greatest effect. LNCaP proliferation was increased significantly by both steroids, but altered ratios had no additional effect. PrEC proliferation was unaffected when cells were grown alone, despite presence of androgen receptors (AR) and estrogen receptors (ER). When grown in co-culture PrEC cell proliferation was significantly increased by treatments. CONCLUSIONS: PrSC proliferation is stimulated by an increasing ratio of estrogen:androgen. Proliferation of normal epithelial cells is stimulated as a result of an indirect action of steroids mediated by stromal cells. Malignant prostate cancer cells have an altered response in comparison.