IGF-I and MAP kinase involvement in the stimulatory effects of LNCaP prostate cancer cell conditioned media on cell proliferation and protein synthesis in MC3T3-E1 osteoblastic cells

Bone metastases from prostate cancer cause abnormal new bone formation, however, the factors involved and the pathways leading to the response are incompletely defined. We investigated the mechanisms of osteoblast stimulatory effects of LNCaP prostate carcinoma cell conditioned media (CM). MC3T3-E1 osteoblastic cells were cultured with CM from confluent LNCaP cells. LNCaP CM stimulated MAP kinase, cell proliferation (3H-thymidine incorporation), and protein synthesis (14C-proline incorporation) in the MC3T3-E1 cells. The increases in cell proliferation and protein synthesis were prevented by inhibition of the MAP kinase pathway. IGF-I mimicked the effects of the CM on the MC3T3-E1 cells and inhibition of IGF-I action decreased the LNCaP CM stimulation of 3H-thymidine and 14C-proline incorporation and MAP kinase activity. The findings indicate that IGF-I is an important factor for the stimulatory effects of LNCaP cell CM on cell proliferation and protein synthesis in osteoblastic cells, and that MAP kinase is a component of the signaling pathway for these effects.     

Nuclear exclusion of the androgen receptor by melatonin

Androgen receptors (AR) play a crucial role in androgen-mediated processes and prostate cancer progression. The pineal hormone melatonin attenuates the androgen-dependent growth of benign and cancer prostate epithelial cells in vitro and may reverse clinical resistance to androgen ablation therapy in patients progressing on gonadotropin releasing hormone (GnRH) analogue. Where along the AR cascade does melatonin act remains to be determined. The effects of melatonin on AR localization, level and activity were assessed using androgen-insensitive prostate carcinoma PC3 cells stably transfected with a wild-type AR-expressing vector (PC3-AR).AR was localized to the PC3-AR cell nucleus in the absence of dihydrotestosterone (DHT). Melatonin caused a robust exclusion of the AR from the cell nucleus to the cytoplasm. The nuclear export inhibitor, leptomycin B prevented this process. The exclusion was selective since melatonin had no such effect on the nuclear localization of estrogen receptors alpha (ERalpha) in these cells.Melatonin also caused nuclear exclusion of the AR in the presence of DHT. In addition, it attenuated androgen induced reporter gene activity in PC3 cells co-transfected with the human AR and AR reporter plasmids. Elevated androgen concentrations counteracted melatonin's effects. Melatonin did not decrease AR level or androgen binding in the cells.The nuclear localization of the AR is a hallmark of its cellular activity. These data point to AR nuclear exclusion as a possible mechanism to attenuate androgen responses in target tissues.     

Synchronized prostate cancer cells for studying androgen regulated events in cell cycle progression from G1 into S phase

Androgen-ablation is a most commonly prescribed treatment for metastatic prostate cancer but it is not curative. Development of new strategies for treatment of prostate cancer is limited partly by a lack of full understanding of the mechanism by which androgen regulates prostate cancer cell proliferation. This is due, mainly, to the limitations in currently available experimental models to distinguish androgen/androgen receptor (AR)-induced events specific to proliferation from those that are required for cell viability. We have, therefore, developed an experimental model system in which both androgen-sensitive (LNCaP) and androgen-independent (DU145) prostate cancer cells can be reversibly blocked in G(0)/G(1) phase of cell cycle by isoleucine deprivation without affecting their viability. Pulse-labeling studies with (3)H-thymidine indicated that isoleucine-deprivation caused LNCaP and DU145 cells to arrest at a point in G(1) phase which is 12-15 and 6-8 h, respectively, before the start of S phase and that their progression into S phase was dependent on serum factors. Furthermore, LNCaP, but not DU145, cells required AR activity for progression from G(1) into S phase. Western blot analysis of the cell extracts prepared at regular intervals following release from isoleucine-block revealed remarkable differences in the expression of cyclin E, p21(Cip1), p27(Kip1), and Rb at the protein level between LNCaP and DU145 cells during progression from G(1) into S phase. However, in both cell types Cdk-2 activity associated with cyclin E and cyclin A showed an increase only when the cells transited from G(1) into S phase. These observations were further corroborated by studies using exponentially growing cells that were enriched in specific phases of the cell cycle by centrifugal elutriation. These studies demonstrate usefulness of the isoleucine-deprivation method for synchronization of androgen-sensitive and androgen-independent prostate cancer cells, and for examining the role of androgen and AR in progression of androgen-sensitive prostate cancer cells from G(1) into S phase.     

Androgen receptor signaling is required for androgen-sensitive human prostate cancer cell proliferation and survival

Background  

Androgens and androgen receptors (AR) regulate normal prostate development and growth. They also are involved in pathological development of prostatic diseases, including benign prostatic hyperplasia (BPH) and prostate cancer (PCa). Antiandrogen therapy for PCa, in conjunction with chemical or surgical castration, offers initial positive responses and leads to massive prostate cell death. However, cancer cells later appear as androgen-independent PCa. To investigate the role of AR in prostate cell proliferation and survival, we introduced a vector-based small interfering RNA (siRNA). This siRNA targeted 5'-untranslated region of AR mRNA for extended suppression of AR expression in androgen-sensitive human prostate LNCaP cells.     

Inhibition of macrophage migration inhibitory factor or its receptor (CD74) attenuates growth and invasion of DU-145 prostate cancer cells

Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, is overexpressed in prostate cancer, but the mechanism by which MIF exerts effects on tumor cells remains undetermined. MIF interacts with its identified membrane receptor, CD74, in association with CD44, resulting in ERK 1/2 activation. Therefore, we hypothesized that increased expression or surface localization of CD74 and MIF overexpression by prostate cancer cells regulated tumor cell viability. Prostate cancer cell lines (LNCaP and DU-145) had increased MIF gene expression and protein levels compared with normal human prostate or benign prostate epithelial cells (p < 0.01). Although MIF, CD74, and CD44 variant 9 expression were increased in both androgen-dependent (LNCaP) and androgen-independent (DU-145) prostate cancer cells, cell surface of CD74 was only detected in androgen-independent (DU-145) prostate cancer cells. Therefore, treatments aimed at blocking CD74 and/or MIF (e.g., inhibition of MIF or CD74 expression by RNA interference or treatment with anti-MIF- or anti-CD74- neutralizing Abs or MIF-specific inhibitor, ISO-1) were only effective in androgen-independent prostate cancer cells (DU-145), resulting in decreased cell proliferation, MIF protein secretion, and invasion. In DU-145 xenografts, ISO-1 significantly decreased tumor volume and tumor angiogenesis. Our results showed greater cell surface CD74 in DU-145 prostate cancer cells that bind to MIF and, thus, mediate MIF-activated signal transduction. DU-145 prostate cancer cell growth and invasion required MIF activated signal transduction pathways that were not necessary for growth or viability of androgen-dependent prostate cells. Thus, blocking MIF either at the ligand (MIF) or receptor (CD74) may provide new, targeted specific therapies for androgen-independent prostate cancer.     

The Hippo Pathway Effector YAP Regulates Motility,Invasion, and Castration-Resistant Growth of Prostate Cancer Cells

Yes-associated protein (YAP) is an effector of the Hippo tumor suppressor pathway. The functional significance of YAP in prostate cancer has remained elusive. In this study, we first show that enhanced expression of YAP is able to transform immortalized prostate epithelial cells and promote migration and invasion in both immortalized and cancerous prostate cells. We found that YAP mRNA was upregulated in androgen-insensitive prostate cancer cells (LNCaP-C81 and LNCaP-C4-2 cells) compared to the level in androgen-sensitive LNCaP cells. Importantly, ectopic expression of YAP activated androgen receptor signaling and was sufficient to promote LNCaP cells from an androgen-sensitive state to an androgen-insensitive state in vitro, and YAP conferred castration resistance in vivo. Accordingly, YAP knockdown greatly reduced the rates of migration and invasion of LNCaP-C4-2 cells and under androgen deprivation conditions largely blocked cell division in LNCaP-C4-2 cells. Mechanistically, we found that extracellular signal-regulated kinase–ribosomal s6 kinase signaling was downstream of YAP for cell survival, migration, and invasion in androgen-insensitive cells. Finally, immunohistochemistry showed significant upregulation and hyperactivation of YAP in castration-resistant prostate tumors compared to their levels in hormone-responsive prostate tumors. Together, our results identify YAP to be a novel regulator in prostate cancer cell motility, invasion, and castration-resistant growth and as a potential therapeutic target for metastatic castration-resistant prostate cancer (CRPC).     

Flavonoid ampelopsin inhibits the growth and metastasis of prostate cancer in vitro and in mice

The objective of this study was to evaluate the chemopreventive effect of a novel flavonoid, ampelopsin (AMP) on the growth and metastasis of prostate cancer cells. AMP showed the more potent activity in inhibiting the proliferation of androgen-sensitive LNCaP and, to less extent, androgen-independent PC-3 human prostate cancer cell lines in vitro, primarily by induction of apoptosis associated with down-regulation of bcl-2. On the other hand, AMP showed much less activity in inhibiting the proliferation of normal prostate epithelial cells than that of prostate cancer cell lines. AMP also inhibited the migration and invasion of PC-3 cells in vitro associated with down-regulation of CXCR4 expression. In the animal study using an orthotopic prostate tumor model, AMP (150 and 300 mg/kg body weight) inhibited the growth of PC-3 tumors and lymph node and lung metastases in a dose-dependent manner. Compared to the control mice, mice treated with AMP at 300 mg/kg BW had reduced final tumor weight by 49.2% (P<0.05), lymph node metastases by 54.5% (P?=?0.3) and lung metastases by 93% (P<0.05), but had no apparent alteration on food intake or body weight. The in vivo anti-growth and anti-metastasis activities of AMP were associated with induction of apoptosis and inhibition of proliferation of prostate cancer cells, reduction of prostate tumor angiogenesis, and reduction of CXCR4 expression. Our results provide supporting evidence to warrant further investigation to develop AMP as a novel efficacious and safe candidate agent against progression and metastasis of prostate cancer.     

UDP-glucuronosyltransferase 2B15 (UGT2B15) and UGT2B17 enzymes are major determinants of the androgen response in prostate cancer LNCaP cells

Uridine diphosphate-glucuronosyltransferase 2 (UGT2)B15 and B17 enzymes conjugate dihydrotestosterone (DHT) and its metabolites androstane-3alpha, 17beta-diol (3alpha-DIOL) and androsterone (ADT). The presence of UGT2B15/B17 in the epithelial cells of the human prostate has been clearly demonstrated, and significant 3alpha-DIOL glucuronide and ADT-glucuronide concentrations have been detected in this tissue. The human androgen-dependent cancer cell line, LNCaP, expresses UGT2B15 and -B17 and is also capable of conjugating androgens. To assess the impact of these two genes in the inactivation of androgens in LNCaP cells, their expression was inhibited using RNA interference. The efficient inhibitory effects of a UGT2B15/B17 small interfering RNA (siRNA) probe was established by the 70% reduction of these UGT mRNA levels, which was further confirmed at the protein levels. The glucuronidation of dihydrotestosterone (DHT), 3alpha-DIOL, and ADT by LNCaP cell homogenates was reduced by more than 75% in UGT2B15/B17 siRNA-transfected LNCaP cells when compared with cells transfected with a non-target probe. In UGT2B15/B17-deficient LNCaP cells, we observe a stronger response to DHT than in control cells, as determined by cell proliferation and expression of eight known androgen-sensitive genes. As expected, the amounts of DHT in cell culture media from control cells were significantly lower than that from UGT2B15/B17 siRNA-treated cells, which was caused by a higher conversion to its corresponding glucuronide derivative. Taken together these data support the idea that UGT2B15 and -B17 are critical enzymes for the local inactivation of androgens and that glucuronidation is a major determinant of androgen action in prostate cells.     

A novel anticancer agent,retigeric acid B,displays proliferation inhibition,S phase arrest and apoptosis activation in human prostate cancer cells

Retigeric acid B (RB), a naturally occurring pentacyclic triterpenic acid, has been noted for its antifungal properties in vitro. Here, we observed that RB inhibited prostate cancer cell proliferation and induced cell death in a dose-dependent manner, but exerted very little inhibitory effect on noncancerous prostate epithelial cell viability. Treatment of androgen-independent PC-3 cells with RB caused a moderate increase in p21^Cip1, and enforced the cell cycle arrest in the S phase. A block of S phase was accompanied with decreases in cyclin B, and increases in cyclin E and cyclin A proteins and phosphorylated retinoblastoma protein (pRb), whereas the expression of cdk2 remained almost unchanged in PC-3 cells exposed to RB. Moreover, RB significantly inhibited DNA synthesis with a dose-dependent reduction in the incorporation of BrdU into DNA, and enhanced apoptosis of PC-3 cells with induction of a higher ratio of Bax/Bcl-2 proteins, and activation of caspase-3 which, in turn, promoted the cleavage of poly (ADP-ribose) polymerase (PARP). However, pretreatment with the pan-caspase inhibitor z-VAD-fmk only partially alleviated RB-triggered apoptosis in PC-3 cells, suggesting the involvement of both caspase-dependent and caspase-independent pathways. Additionally, treatment of androgen-sensitive LNCaP cells with RB led to a reduction in the expression of androgen receptor (AR), and subsequently decreased the transactivity of AR. These observations help to support the search for promising candidates to treat prostate cancer.     

Effect of steroid hormones on vaginal epithelial cells: Anin vitro model for steroid hormone action

Conditions have been standardized to maintain rat vaginal epithelial cellsin vitro with more than 95% viability. Cultured epithelial cells were used to study the effects of normal fetal calf scrum, estradiol and progesterone on the incorporation of [³H]-uridine in RNA and incorporation of [¹⁴C]-aminoacids in proteins. While fetal calf serum and estradiol stimulate the incorporation of both uridine and afno acids, progesterone did not show any effect. Estradiol treated vaginal cells show typical fcroridges (indicative of keratinization of cells) in contrast to estradiol deprived cells, which show microvilli on cell surface when examined in scanning electron microscope.     

Stimulatory effects of antiandrogens on LNCaP human prostate tumor cell growth, EGF-receptor level and acid phosphatase secretion

LNCaP cells (derived from a lymph node carcinoma of the human prostate) show androgen responsive growth. Progestagens, estradiol and antiandrogens competed with androgens for binding to the androgen receptor in the cells to a higher extent than in other androgen-sensitive systems. Optimal growth (3–4 fold increase in DNA content of 6 day cell cultures vs controls) was observed after addition of the synthetic androgen R1881 (0.1 nM). Both steroidal and non-steroidal antiandrogens did not suppress the androgen responsive growth. At a concentration of 10 nM cyproterone acetate or 100 nM RU 23908, growth was even stimulated to an extent comparable to that observed after addition of androgen. Cyproterone acetate and RU 23908 also increased the number of epidermal growth factor receptors expressed at the cell surface to a comparable level as did the androgen. Like androgens, cyproterone acetate, RU 23908 or estradiol stimulated the secretion per cell of prostate specific acid phosphatase in the culture fluid. In conclusion, antiandrogens can exert striking stimulatory effects on the proliferation of LNCaP cells probably due to a defective androgen receptor system. It is discussed that comparable changes in the specificity of the androgen receptor in prostate cancer cells may give these cells an advantage in growth rate and may contribute to development of tumors characterized as hormone independent.     

Endoplasmic reticulum stress,autophagic and apoptotic cell death,and immune activation by a natural triterpenoid in human prostate cancer cells

Though the current therapies are effective at clearing an early stage prostate cancer, they often fail to treat late-stage metastatic disease. We aimed to investigate the molecular mechanisms underlying the anticancer effects of a natural triterpenoid, ganoderic acid DM (GA-DM), on two human prostate cancer cell lines: the androgen-independent prostate carcinoma (PC-3), and androgen-sensitive prostate adenocarcinoma (LNCaP). Cell viability assay showed that GA-DM was relatively more toxic to LNCaP cells than to PC-3 cells (IC₅₀s ranged 45-55 µM for PC-3, and 20-25 µM for LNCaP), which may have occurred due to differential expression of p53. Hoechst DNA staining confirmed detectable nuclear fragmentation in both cell lines irrespective of the p53 status. GA-DM treatment decreased Bcl-2 proteins while it upregulated apoptotic Bax and autophagic Beclin-1, Atg5, and LC-3 molecules, and caused an induction of both early and late events of apoptotic cell death. Biochemical analyses of GA-DM-treated prostate cancer cells demonstrated that caspase-3 cleavage was notable in GA-DM-treated PC-3 cells. Interestingly, GA-DM treatment altered cell cycle progression in the S phase with a significant growth arrest in the G2 checkpoint and enhanced CD4 ⁺ T cell recognition of prostate tumor cells. Mechanistic study of GA-DM-treated prostate cancer cells further demonstrated that calpain activation and endoplasmic reticulum stress contributed to cell death. These findings suggest that GA-DM is a candidate for future drug design for prostate cancer as it activates multiple pathways of cell death and immune recognition.     

Juglone,isolated from Juglans mandshurica Maxim,induces apoptosis via down-regulation of AR expression in human prostate cancer LNCaP cells

Juglone is a natural compound which has been isolated from Juglans mandshurica Maxim. Recent studies have shown that juglone had various pharmacological effects such as anti-viral, anti-bacterial and anti-cancer. However, its anti-cancer activity on human prostate cancer LNCaP cell has not been examined. Thus, the current study was designed to elucidate the molecular mechanism of apoptosis induced by juglone in androgen-sensitive prostate cancer LNCaP cells. MTT assay was performed to examine the anti-proliferative effect of juglone. Occurrence of apoptosis was detected by Hoechst 33342 staining and flow cytometry in LNCaP cells treated with juglone for 24 h. The result shown that juglone inhibited the growth of LNCaP cells in a dose-dependent manner. Morphological changes of apoptotic body formation after juglone treatment were observed by Hoechst 33342 staining. This apoptotic induction was associated with loss of mitochondrial membrane potential, and caspase-3, -9 activation. Moreover, we found that juglone significantly inhibited the expression levels of androgen receptor (AR) and prostate-specific antigen (PSA) in a dose-dependent manner, as well as abrogated up-regulation of AR and PSA genes with and/or without dihydrotestosterone (DHT). Take together, our results demonstrated that juglone might induce the apoptosis in LNCaP cell via down-regulation of AR expression. Therefore, our results indicated that juglone may be a potential candidate of drug for androgen-sensitive prostate cancer.     

Androgens down-regulate myosin light chain kinase in human prostate cancer cells

Androgens play a major role in the growth and survival of primary prostate tumors. The molecular mechanisms involved in prostate cancer progression are not fully understood but genes that are regulated by androgens clearly influence this process. We searched for new androgen-regulated genes using the Affymetrix GeneChip Human Genome U95 Set in the androgen-sensitive LNCaP prostate cancer cell line. Analysis of gene expression profiles revealed that myosin light chain kinase (MLCK) mRNA levels were markedly down-regulated by the synthetic androgen R1881. The microarray data were confirmed by ribonuclease protection assays. RNA and protein analyses revealed that LNCaP cells express both long (non-muscle) and short (smooth muscle) isoforms, and that both isoforms are down-regulated by androgens. Taken together, these data identify MLCK as a novel downstream target of the androgen signalling pathway in prostate cells.     

Modulation of YY1 and p53 expression by transforming growth factor-β3 in prostate cell lines

Transforming growth factor-β (TGF-β) is the prototype of a family of secreted polypeptide growth factors. These cytokines play very important roles during development, as well as in normal physiological and disease processes, by regulating a wide array of cellular processes, such as cell growth, differentiation, migration, apoptosis, and extracellular matrix production. TGF-β utilizes a multitude of intracellular signalling pathways in addition to Smads with actions that are dependent on circumstances, including dose, target cell type, and context. The aims of this research were (i) to verify the effects of dose-dependent TGF-β3 treatment on YY1 and p53 expression, in BPH-1 cell line, human benign prostate hyperplasia, and two prostate cancer cell lines, LNCaP, which is androgen-sensitive, and DU-145, which is androgen-non responsive, (ii) establish a correlation between p53 and YY1 and (iii) determine the expression of a number of important intracellular signalling pathways in TGF-β3-treated prostate cell lines. The expression of YY1, p53, PI3K, AKT, pAKT, PTEN, Bcl-2, Bax, and iNOS was evaluated through Western blot analysis on BPH-1, LNCaP, and DU-145 cultures treated with 10 and 50 ng/ml of TGF-β3 for 24 h. The production of nitric oxide (NO) was determined by Griess reagent and cell viability through MTT assay. The results of this research demonstrated profound differences in the responses of the BPH-1, LNCaP, and DU-145 cell lines to TGF-β3 stimulation. We believe that the findings could be important because of the clinical relevance that they may assume and the therapeutic implications for TGF-β treatment of prostate cancer.     

Requirement of androgen-dependent activation of protein kinase Czeta for androgen-dependent cell proliferation in LNCaP Cells and its roles in transition to androgen-independent cells

A cell line that we designed, AILNCaP, proliferated in androgen-depleted medium after emerging from long-term androgen-depleted cultures of an androgen-sensitive prostate cancer cell line, LNCaP. Using this cell line as a model of progression to androgen independence, we demonstrated that the activity of the mammalian target of rapamycin/p70 S6 kinase transduction pathway is down-regulated after androgen depletion in LNCaP, whereas its activation is related to transition of this cell line to androgen-independent proliferation. Kinase activity of protein kinase Czeta is regulated by androgen stimulation in LNCaP cells, whereas it is activated constitutively in AILNCaP cells under androgen-depleted conditions. Treatment with a protein kinase Czeta pseudosubstrate inhibitor reduced p70 S6 kinase activity and cell proliferation in both cell lines. We identified that both protein kinase Czeta and p70 S6 kinase were associated in LNCaP cells and this association was enhanced by the androgen stimulation. We examined the expression of phospho-protein kinase Czeta and phospho-p70 S6 kinase in hormone-naive prostate cancer specimens and found that the expression of both kinases was correlated with each other in those specimens. Significant correlation was observed between the expression of both kinases and Ki67 expression. Most of the prostate cancer cells that survived after prior hormonal treatment also expressed both kinases. This is the first report that shows the significance of this pathway for both androgen-dependent and -independent cell proliferation in prostate cancer. Our data suggest that protein kinase Czeta/mammalian target of rapamycin/S6 kinase pathway plays an important role for the transition of androgen-dependent to androgen-independent prostate cancer cells.