Negative Prostate Cancer Cells Research Articles

Modification of the Adiponectin:Leptin Ratio May Underlie Obesity Dependent Prostate Cancer Progression

Obesity and prostate cancer exhibit a statistical link, which may be due to the paracrine/endocrine cell cycle effects of the adipocyte‐derived peptides leptin (LEP) and adiponectin (ADIPO). ADIPO induced AMPK–dependent phosphorylation of p27 on T198 (T198‐P) and increased total p27 protein and adiponectin receptor (ADIPOR1) protein levels in androgen‐receptor (AR) negative PC3 prostate cancer cells. Conversely, LEP induced the opposite effects on AMPK, T198‐P p27, total p27 and ADIPOR1 protein levels. LEP and ADIPO also had opposing effects in AR‐positive LNCaP cells. As observed in breast cancer cells, LEP treatment overcame the effects of ADIPO on all proteins examined in a concentration‐dependent manner. Furthermore, ADIPO administration was able to counteract the effects of LEP on all proteins measured. Our data suggest that ADIPO and LEP exert antagonistic effects on prostate epithelial cell cycle regulation, and modification of the ADIPO:LEP ratio may be one of the important factors that underlie the molecular link between obesity and prostate cancer progression.

Multiple defects in negative regulation of the PKB/Akt pathway sensitise human cancer cells to the antiproliferative effect of non-steroidal anti-inflammatory drugs

Antitumorigenic effects of non-steroidal anti-inflammatory drugs (NSAIDs) are well established in several types of cancer disease. However, the mechanisms driving these processes are not understood in all details. In our study, we observed significant differences in sensitivity of cancer epithelial cell lines to COX-independent antiproliferative effects of NSAIDs. The prostate cancer cell line LNCaP, lacking both critical enzymes in the negative control of PKB/Akt activation, PTEN and SHIP2, was the most sensitive to these effects, as assessed by analysing the cell cycle profile and expression of cell cycle regulating proteins. We found that p53 protein and its signalling pathway is not involved in early antiproliferative action of the selected NSAID—indomethacin. RNAi provided evidence for the involvement of p21 Cip1/Waf1, but not GDF-15, in antiproliferative effects of indomethacin in LNCaP cells. Interestingly, we also found that indomethacin activated PKB/Akt and induced nuclear localisation of p21 Cip1/Waf1 and Akt2 isoform. Our results are in agreement with other studies and suggest that maintaining of the p21 Cip1/Waf1 level and its intracellular localisation might be influenced by Akt2. Knock-down of SHIP2 by RNAi in PTEN negative prostate and colon cancer cell lines resulted in higher sensitivity to antiproliferative effects of indomethacin. Our data suggest novel mechanisms of NSAIDs antiproliferative action in cancer epithelial cells, which depends on the status of negative regulation of the PKB/Akt pathway and the isoform-specific action of Akt2. Thus, unexpectedly, multiple defects in negative regulation of the PKB/Akt pathway may contribute to increased sensitivity to chemopreventive effects of these widely used drugs.

3,3′-Diindolylmethane induces a G1 arrest in human prostate cancer cells irrespective of androgen receptor and p53 status

3,3′-Diindolylmethane (DIM) is a potential chemopreventive phytochemical derived from Brassica vegetables. In this study we characterized the effect of DIM on cell cycle regulation in both androgen-dependent LNCaP and androgen receptor negative p53 mutant DU145 human prostate cancer cells. DIM had an anti-proliferative effect on both LNCaP and DU145 cells, as it significantly inhibited [3H]-thymidine incorporation. FACS analysis revealed a DIM-mediated G1 cell cycle arrest. DIM strongly inhibited the expression of cdk2 and cdk4 protein and increased the expression of the cell cycle inhibitor p27Kip1 protein in LNCaP and DU145 cells. Promoter deletion studies with p27Kip1 reporter gene constructs showed that this DIM-mediated increase in p27Kip1 was dependent on the Sp1 transcription factor. Moreover, using a dominant negative inhibitor of p38 MAPK, we showed that the induction of p27Kip1 and subsequent G1 arrest by DIM involve activation of the p38 MAPK pathway in the DU145 cells. Taken together, our results indicate that DIM is able to stop the cell cycle progression of human prostate cancer cells regardless of their androgen-dependence and p53 status, by differentially modulating cell cycle regulatory pathways. The Sp1 and p38 MAPK pathways mediate the DIM cell cycle regulatory effect in DU145 cells.

Recruitment of HDAC4 by transcription factor YY1 represses HOXB13 to affect cell growth in AR-negative prostate cancers

HOXB13 is a homeodomain protein implicated to play a role in growth arrest in AR (androgen receptor)-negative prostate cancer cells. Expression of HOXB13 is restricted to the AR-expressing prostate cells. In this report, we demonstrate that the HDAC inhibitor NaB (sodium butyrate) was able to induce cell growth arrest and to increase HOXB13 expression in AR-negative prostate cancer cells. We also show that both HDAC4 and YY1 participated in the repression of HOXB13 expression through an epigenetic mechanism involving histone acetylation modification. Specifically, co-immunoprecipitation assays revealed that HDAC4 and YY1 formed a complex. The chromatin immunoprecipitation (ChIP) assays verified that HDAC4 was recruited to HOXB13 promoter by YY1. Moreover, promoter truncation and point mutation studies determined that the two proximal YY1 binding sites on the HOXB13 promoter were essential for the recruitments of YY1 and HDAC4. Data presented in this report suggest that YY1 and HDAC4 affected cell growth by repressing transcriptional regulation of HOXB13 through an epigenetic modification of histones.

Androgen receptor-negative human prostate cancer cells induce osteogenesis in mice through FGF9-mediated mechanisms.

In prostate cancer, androgen blockade strategies are commonly used to treat osteoblastic bone metastases. However, responses to these therapies are typically brief, and the mechanism underlying androgen-independent progression is not clear. Here, we established what we believe to be the first human androgen receptor-negative prostate cancer xenografts whose cells induced an osteoblastic reaction in bone and in the subcutis of immunodeficient mice. Accordingly, these cells grew in castrated as well as intact male mice. We identified FGF9 as being overexpressed in the xenografts relative to other bone-derived prostate cancer cells and discovered that FGF9 induced osteoblast proliferation and new bone formation in a bone organ assay. Mice treated with FGF9-neutralizing antibody developed smaller bone tumors and reduced bone formation. Finally, we found positive FGF9 immunostaining in prostate cancer cells in 24 of 56 primary tumors derived from human organ-confined prostate cancer and in 25 of 25 bone metastasis cases studied. Collectively, these results suggest that FGF9 contributes to prostate cancer-induced new bone formation and may participate in the osteoblastic progression of prostate cancer in bone. Androgen receptor-null cells may contribute to the castration-resistant osteoblastic progression of prostate cancer cells in bone and provide a preclinical model for studying therapies that target these cells.

Androgen receptor mediates the expression of UDP‐glucuronosyltransferase 2 B15 and B17 genes

Enhanced androgen receptor (AR) activity by increased testosterone availability may play important roles in prostate cancer progressing to castration resistant state. Comparison of expression profiles in androgen dependent and independent prostate tumors demonstrated a marked increase of the expression of UDP-glucuronosyltransferase 2B15 (UGT2B15), an androgen catabolic enzyme. We investigated mechanisms controlling the differential expression of UGT2B15 and B17 in response to androgen treatments. Gene expression was determined by RT-PCR. The association of AR with UGT2B15/B17 genes was determined by Chromatin immuno-precipitation (CHIP). RNA interference was used to knock-down gene expression. UGT2B15 and B17 genes were not expressed in AR negative prostate cancer cell lines, PC3 and DU145, while they were expressed in AR positive cell lines, LNCaP, LNCaP-abl (an androgen independent LNCaP sub-line), and VCaP. The expression levels of UGT2B15/B17 were up-regulated in LNCaP-abl comparing to those in LNCaP. These results suggest the requirement of AR for the expression of UGT2B15/B17. Treatment with DHT down-regulated the expression of UGT2B15/B17 in LNCaP in a time and dose dependent manner and this down-regulation was competitively antagonized by flutamide and bicalutimide, suggesting a pathway mediated by AR. Further CHIP experiments demonstrated the direct interaction of AR with the promoter regions of UGT2B15/B17 genes. Knocking down AR expression in LNCaP significantly reduced the expression of UGT2B15/B17 and completely inhibited the DHT-induced down-regulation of UGT2B15/B17 genes. We demonstrated that UGT2B15 and B17 are primary androgen-regulated genes and AR is required for both their basal expression and their androgen-regulated expression.

ID1‐, ID2‐, and ID3‐regulated gene expression in E2A positive or negative prostate cancer cells

The inhibitor of differentiation (Id) proteins are expressed in prostate cancer (PCA). However, there is a general lack of Id isoform-specific downstream effectors. Id1, Id2, or Id3 were silenced in PCA cell lines LNCaP, DU145, and PC3 using gene-specific small interfering RNA (siRNA). The effect of Id gene silencing on representative genes involved in apoptosis (p53, SNAIL2), proliferation (p21, p16), and tumor invasion (E-cadherin and MMP9) was investigated by real-time PCR. Expression of E-proteins, the primary Id interaction partners was also evaluated to understand the molecular mechanism of action. The Id proteins regulated the expression of CDKNIs p16 and p21 even in the absence of E-proteins. Loss of Id1 and Id3 up- or downregulated E-cadherin expression in E-protein negative or positive PCA cell lines, respectively. The effect of Id genes on cell proliferation was also independent of CDKNIs in p16 and p21 null PC3 cells. The p53-independent anti-apoptotic effect of Id2 was mediated in part by transcriptional repressor SNAI2. MMP9 seems to be the common target of all three Id genes (Id1, Id2, and Id3). The overall effect of Id proteins on proliferation and apoptosis is independent of E-proteins. E-proteins can however determine the magnitude of response or in some cases even reverse the Id-mediated target gene expression. Evaluating E-protein expression in conjunction with Id proteins will allow better understanding of the molecular mechanism of action of Id proteins and increase their prognostic significance in PCA.

Pristimerin induces apoptosis by targeting the proteasome in prostate cancer cells

Pristimerin is a natural product derived from the Celastraceae and Hippocrateaceae families that were used as folk medicines for anti inflammation in ancient times. Although it has been shown that pristimerin induces apoptosis in breast cancer cells, the involved mechanism of action is unknown. The purpose of the current study is to investigate the primary target of pristimerin in human cancer cells, using prostate cancer cells as a working model. Nucleophilic susceptibility and in silico docking studies show that C6 of pristimerin is highly susceptible towards a nucleophilic attack by the hydroxyl group of N-terminal threonine of the proteasomal chymotrypsin subunit. Consistently, pristimerin potently inhibits the chymotrypsin-like activity of a purified rabbit 20S proteasome (IC50 2.2 micromol/L) and human prostate cancer 26S proteasome (IC50 3.0 micromol/L). The accumulation of ubiquitinated proteins and three proteasome target proteins, Bax, p27 and I kappa B-alpha, in androgen receptor (AR)-negative PC-3 prostate cancer cells supports the conclusion that proteasome inhibition by pristimerin is physiologically functional. This observed proteasome inhibition subsequently led to the induction of apoptotic cell death in a dose- and kinetic-dependent manner. Furthermore, in AR-positive, androgen-dependent LNCaP and AR-positive, androgen-independent C4-2B prostate cancer cells, proteasome inhibition by pristimerin results in suppression of AR protein prior to apoptosis. Our data demonstrate, for the first time, that the proteasome is a primary target of pristimerin in prostate cancer cells and inhibition of the proteasomal chymotrypsin-like activity by pristimerin is responsible for its cancer cell death-inducing property.

A Coregulatory Role for the Mediator Complex in Prostate Cancer Cell Proliferation and Gene Expression

Androgen receptor (AR) signaling pathways are important for the survival and proliferation of prostate cancer cells. Because AR activity is facilitated by distinct coregulatory factors and complexes, it is conceivable that some of these proteins might also play a role in promoting prostate oncogenesis. The multisubunit Mediator complex is an important coactivator for a broad range of regulatory transcriptional factors including AR, yet its role in prostate cancer is unclear. Here, we used RNA interference to knock down the expression of two integral Mediator components, MED1/TRAP220 and MED17, in prostate cancer cells. MED1/TRAP220 plays a particularly important role in androgen signaling in that it serves as a direct binding target for AR. We found that the knockdown of either subunit markedly decreases transcription from transiently transfected androgen-responsive reporter genes, as well as inhibits androgen-dependent expression of endogenous AR target genes. We show for the first time that loss of either MED1/TRAP220 or MED17 in prostate cancer cells significantly decreases both androgen-dependent and -independent cellular proliferation, inhibits cell cycle progression, and increases apoptosis. Furthermore, we show that MED1/TRAP220 is overexpressed in both AR-positive and -negative prostate cancer cells lines, as well as in 50% (10 of 20) of the clinically localized human prostate cancers we examined, thus suggesting that MED1/TRAP220 hyperactivity may have implications in prostate oncogenesis. In sum, our data suggest that Mediator plays an important coregulatory role in prostate cancer cell proliferation and survival, and therefore, may represent a new target for therapeutic intervention.

Reactive oxygen species mediate androgen receptor‐ and serum starvation‐elicited downstream signaling of ADAM9 expression in human prostate cancer cells

The A Disintegrin And Metalloprotease (ADAM) family is a group of transmembrane proteins containing cell adhesive and proteolytic functional domains. ADAM9 expression was shown to be mediated by androgen receptor (AR) and stress conditions. This study determined a common mediator responsible for ADAM9 protein regulation which supports human prostate cancer (PCa) cell growth and survival. ADAM9 protein expression was measured under androgen, anti-androgen, hydrogen peroxide, and/or serum starvation conditions in PCa cells. The roles of reactive oxygen species (ROS) were assessed in the presence or absence of recombinant catalase, or in cells stably transfected with either catalase- or a control neo-cDNA expression vector. ROS was assayed by dihydroethidium (DHE) followed by FACS analysis. ADAM9 protein expression was upregulated by androgen in AR-positive but not in AR-negative PCa cells. The anti-androgen bicalutamide effectively blocked this induction. While serum starvation enhanced ADAM9 expression in AR-positive PCa cells, this stress condition did not alter ADAM9 expression in AR-negative PCa cells. Parallel results also showed that androgen treatment or serum starvation enhanced ROS only in AR-positive but not in AR-negative PCa cells. ROS appears to be a common downstream mediator of androgen- or serum starvation-induced ADAM9 expression since addition of hydrogen peroxide or introduction of catalase, either enhanced or abolished respectively ADAM9 protein expression by both AR-positive and -negative PCa cells. These findings suggest that ROS is: a common mediator responsible for ADAM9 protein induction in human PCa cells, downstream from AR, and stress response signaling.

Steroid Receptor Coactivator-3 and Activator Protein-1 Coordinately Regulate the Transcription of Components of the Insulin-Like Growth Factor/AKT Signaling Pathway

Steroid receptor coactivator (SRC)-3, also called amplified in breast cancer 1, is a member of the p160 nuclear receptor coactivator family involved in transcriptional regulation of target genes. SRC-3 is frequently amplified and/or overexpressed in hormone-sensitive and hormone-insensitive tumors. We reported previously that SRC-3 stimulated prostate cell growth in a hormone-independent manner through activation of AKT signaling pathway. However, the underlying mechanism remains undefined. Here, we exploited the mifepristone-induced SRC-3 LNCaP prostate cancer cell line generated in our laboratory to identify SRC-3-regulated genes by oligonucleotide microarray analysis. We found that SRC-3 up-regulates the expression of multiple genes in the insulin-like growth factor (IGF)/AKT signaling pathway that are involved in cell proliferation and survival. In contrast, knockdown of SRC-3 in PC3 (androgen receptor negative) prostate cancer cells and MCF-7 breast cancer cells reduces their expression. Similarly, in prostate glands of SRC-3 null mice, expressions of these components in the IGF/AKT signal pathway are also reduced. Chromatin immunoprecipitation assay revealed that SRC-3 was directly recruited to the promoters of these genes, indicating that they are direct targets of SRC-3. Interestingly, we showed that recruitment of SRC-3 to two target promoters, IRS-2 and IGF-I, requires transcription factor activator protein-1 (AP-1). Taken together, our results clearly show that SRC-3 and AP-1 can coordinately regulate the transcription of multiple components in the IGF/AKT pathway to ensure ligand-independent cell proliferation and survival of cancer cells.

Capsaicin, a Component of Red Peppers, Inhibits the Growth of Androgen-Independent, p53 Mutant Prostate Cancer Cells

Capsaicin is the major pungent ingredient in red peppers. Here, we report that it has a profound antiproliferative effect on prostate cancer cells, inducing the apoptosis of both androgen receptor (AR)-positive (LNCaP) and -negative (PC-3, DU-145) prostate cancer cell lines associated with an increase of p53, p21, and Bax. Capsaicin down-regulated the expression of not only prostate-specific antigen (PSA) but also AR. Promoter assays showed that capsaicin inhibited the ability of dihydrotestosterone to activate the PSA promoter/enhancer even in the presence of exogenous AR in LNCaP cells, suggesting that capsaicin inhibited the transcription of PSA not only via down-regulation of expression of AR, but also by a direct inhibitory effect on PSA transcription. Capsaicin inhibited NF-kappa activation by preventing its nuclear migration. In further studies, capsaicin inhibited tumor necrosis factor-alpha-stimulated degradation of IkappaBalpha in PC-3 cells, which was associated with the inhibition of proteasome activity. Taken together, capsaicin inhibits proteasome activity which suppressed the degradation of IkappaBalpha, preventing the activation of NF-kappaB. Capsaicin, when given orally, significantly slowed the growth of PC-3 prostate cancer xenografts as measured by size [75 +/- 35 versus 336 +/- 123 mm(3) (+/-SD); P = 0.017] and weight [203 +/- 41 versus 373 +/- 52 mg (+/-SD); P = 0.0006; capsaicin-treated versus vehicle-treated mice, respectively]. In summary, our data suggests that capsaicin, or a related analogue, may have a role in the management of prostate cancer.

Role of SRC-1 in the Promotion of Prostate Cancer Cell Growth and Tumor Progression

Prostate cancer is initially androgen dependent and there is evidence that androgen receptor continues to play a role in androgen-independent prostate cancer. Androgen receptor activity depends both on the level of androgens and on the level of coactivators that interact with androgen receptor. Our goal was to evaluate the role of the androgen receptor coactivator SRC-1 in prostate cancer progression. Using tissue arrays to measure SRC-1 protein levels, we found that increased SRC-1 expression in clinically localized, androgen-dependent cancer is associated with clinical and pathologic variables of increased tumor aggressiveness. Interestingly, there was variable expression of SRC-1 in normal prostate tissue which correlated with the staining intensity of the corresponding cancer tissue. To test the contribution of SRC-1, we examined its role in androgen-dependent LNCaP and androgen-independent C4-2 prostate cancer cell lines. Using small interfering RNA to reduce expression of androgen receptor, we found that androgen receptor was required both for cell growth and for basal expression of prostate-specific antigen in the androgen-independent C4-2 cell line. Thus, although the cells can grow in an androgen-depleted medium, they remained androgen receptor dependent. Reduction of SRC-1 expression significantly reduced growth and altered androgen receptor target gene regulation in both LNCaP and C4-2 cell lines whereas it had no effect on the growth of the androgen receptor-negative PC-3 and DU145 prostate cancer cell lines. Although the requirement for androgens and androgen receptor in the development of prostate cancer is well established, our study implicates enhanced androgen receptor activity through elevated expression of SRC-1 in the development of more aggressive disease in men with prostate cancer.

Molecular characterization of an improved vector for evaluation of the tumor suppressor versus oncogene abilities of the androgen receptor

There is a growing body of evidence demonstrating that the function of the ligand-occupied androgen receptor (AR) within the nuclei of normal prostatic epithelial cells acts as a tumor suppressor gene. This is in contrast to the well-documented ability of the AR within prostate cancer cells to function as an oncogene. Thus, many groups are attempting to understand the biochemistry and signaling cascade differences involved in the switching of AR from a tumor suppressor to an oncogene. To do this, of plasmid vectors for transgenic expression of AR are very useful. AR negative PC-3 human prostate cancer cells were transfected with a plasmid containing the full length coding sequence of AR without its 5'- or 3'-untranslated regions (UTRs) (i.e., pSG5-AR). Transgenic expression of the AR protein results in profound growth inhibition which is not relieved by the addition of ligand. A new expression vector for the AR, pAR-IRES-EGFP, has been constructed that contains full-length 5'-UTR which includes the identified translation regulatory regions, the full length coding sequence and the partial 3'-UTR, which includes the identified post-transcriptional regulatory regions. When PC-3 cells were transfected with the pAR-IRES-EGFP vector, it was found that transgenic AR protein expression was not growth inhibitory until ligand was added. These pSG5-AR versus pSAR-IRES-EGFP clones are being studied to determine the molecular pathways explaining their different response to AR and ligand.

Nerve growth factor induces the re-expression of functional androgen receptors and p75(NGFR) in the androgen-insensitive prostate cancer cell line DU145.

One of the paracrine/autocrine factors regulating prostate growth and differentiation is nerve growth factor (NGF). The role of NGF and its receptors in the prostate, however, remains controversial. We have shown that NGF treatment of human prostate cancer cell lines reduced their tumorigenicity, both in vitro and in vivo. To investigate the involvement of NGF as a differentiation factor in prostate cancer cells. We exposed the androgen-independent/androgen receptor (AR)-negative prostate cancer cell line DU145 to NGF to study whether this neurotrophin could revert DU145 cells to a less malignant phenotype. DU145 cells were treated with NGF, then ARs and NGF receptor p75(NGFR) expression and telomerase activity were studied. Finally, we investigated whether re-expression of ARs could restore the androgen sensitivity in this cell line. NGF treatment induced a reversion of DU145 cells to a less malignant phenotype, characterized by the re-expression of ARs and p75(NGFR) NGF receptors. Re-expression of ARs restored the androgen sensitivity, as suggested by the fact that exposure to dihydrotestosterone stimulated the growth of NGF-treated DU145 cells. This effect was blocked by androgen antagonist drugs, such as hydroxyflutamide and cyproterone acetate, which also induced apoptotic death of NGF-treated cells. The hypothesis that a differentiation pathway is activated by exogenous NGF in DU145 cells is also supported by findings indicating that NGF-treated DU145 cells expressed a low telomerase activity, as a result of a decrease in human telomerase reverse transcriptase transcription.

Mechanisms of constitutive NF‐κB activation in human prostate cancer cells

AbstractBACKGROUNDActivation of the NF‐κB transcription factor has been previously demonstrated in two androgen receptor negative prostate cancer cell lines. We wished to extend this work to additional prostate cancer cells and to characterize the mechanisms responsible for constitutive NF‐κB activation.METHODSElectrophoretic mobility shift assays were performed to measure NF‐κB DNA‐binding activity in prostate cancer cell lines, and immunohistochemistry was performed to detect nuclear localization of NF‐κB in prostate cancer tissues. Western blot analysis was used to study the status of IκBα. Transient transfection assays were employed to characterize the contributions of IκB kinase (IKK), MAPK kinase kinases (MAPKKKs), androgen receptor (AR), and tyrosine phosphorylation to the constitutive activation of NF‐κB in the prostate cancer cell lines.RESULTSConstitutive NF‐κB activity was observed in AR‐negative cell lines as well as in the prostate cancer patient samples, but was not present in AR positive cells. A “super‐repressor” IκBα, as well as dominant negative forms of IKKβ and NF‐κB‐inducing kinase (NIK), and tyrosine kinase inhibition were able to suppress NF‐κB activity in the cells with constitutive activation.CONCLUSIONSThe constitutive activation of NF‐κB observed in prostate cancer cells is likely due to a signal transduction pathway involving tyrosine kinases, NIK, and IKK activation. Prostate 52:183–200, 2002. © 2002 Wiley‐Liss, Inc.

Insulin-like Growth Factor (IGF)-binding Protein-3 Induces Apoptosis and Mediates the Effects of Transforming Growth Factor-β1 on Programmed Cell Death through a p53- and IGF-independent Mechanism

Insulin-like growth factor (IGF) binding protein-3 (IGFBP-3) is known to block IGF action and inhibit cell growth. IGFBP-3 is thought to act by sequestering free IGFs or, possibly, act via a novel IGF-independent mechanism. Supporting its role as a primary growth inhibitor, IGFBP-3 production has been shown to be increased by cell growth-inhibitory agents, such as transforming growth factor-beta (TGF-beta), and the tumor suppressor gene p53. In this paper, we demonstrate, for the first time, a novel function of IGFBP-3 as an apoptosis-inducing agent and show that this action is mediated through an IGF.IGF receptor-independent pathway. In the p53 negative prostate cancer cell line, PC-3, the addition of recombinant IGFBP-3 resulted in a dose-dependent induction of apoptosis. 125I-IGFBP-3 bound with high affinity to specific proteins in PC-3 cell lysates and plasma membrane preparations. These membrane-associated molecules may serve as receptors that mediate the direct effect of IGFBP-3 on apoptosis. In addition, in an IGF receptor-negative mouse fibroblast cell line, treatment with recombinant IGFBP-3 as well as transfection of the IGFBP-3 gene induced apoptosis, suggesting that neither IGFs nor IGF receptors are required for this action. Furthermore, treatment with TGF-beta1, a known apoptosis-inducing agent, resulted in the induction of IGFBP-3 expression 6-12 h before the onset of apoptosis. This effect of TGF-beta1 was prevented by co-treatment with IGFBP-3-neutralizing antibodies or IGFBP-3-specific antisense thiolated oligonucleotides. These findings suggest that IGFBP-3 induces apoptosis through a novel pathway independent of either p53 or the IGF.IGF receptor-mediated cell survival pathway and that IGFBP-3 mediates TGF-beta1 induced apoptosis in PC-3 cells.

Expression of c-erb B-2/neu proto-oncogene in human prostatic cancer tissues and cell lines.

Both amplification and overexpression of c-erb B-2/neu have been associated with the progression and possible prognosis of a number of human cancers. In this study, we demonstrated that c-erb B-2/neu may also play an important role in human prostate cancer. Our conclusion is based on the following observations: (1) A monoclonal antibody raised against a peptide sequence from the C-terminal domain of the human c-erb B-2/neu gene product reacted positively with 68.7% (11 of 16) of the human prostatic cancer tissue extracts analyzed by western blot procedure. These results were supported by the immunohistochemical staining of the prostatic cancer specimens; 80% (12 of 15) showed positive staining, primarily around the plasma membranes of the prostatic cancer cells. c-erb B-2/neu oncoprotein was not detectable in normal prostate tissues (five examined by immunohistochemical staining and three by western blotting) or in human benign prostatic hyperplasia (two examined by immunohistochemical staining and six by western blotting) and was expressed less abundantly with lower intensity in "normal" human prostate tissues adjacent to cancerous prostate tissue (5 of 12 examined by immunohistochemical staining). We observed no evidence of c-erb B-2/neu gene amplification in 10 fresh human prostatic cancer specimens examined by Southern blotting and in the cultured human prostatic cancer cell lines PC-3, DU-145, and LNCaP. (2) The c-erb B-2/neu protein was detected in both androgen-receptor-positive (LNCaP) and -negative (PC-3 and DU-145) human prostate cancer cell lines. Positive immunostaining of c-erb B-2/neu protein was found to be associated predominantly with the plasma membranes of PC-3 cells, but was also found to be widespread in the cytoplasmic region of the LNCaP cells and in the perinuclear region of the DU-145 cells. (3) Like prostate-specific antigen (PSA) expression, c-erb B-2/neu mRNA expression was also positively regulated by androgen in androgen-receptor-positive LNCaP cells in vitro and LNCaP tumors in vivo. When LNCaP tumors were grown in castrated male hosts, levels of c-erb B-2/neu and PSA mRNA expression decreased initially, but rebounded at 3 wk to levels comparable to those expressed by tumors maintained in intact adult male hosts.