Involvement of prostate specific antigen in the stimulation of LNCaP cell growth

Since its identification in 1979, prostatic specific antigen (PSA) has been used extensively as a serum marker for diagnosis and prognosis of prostate cancer. In addition, PSA is an immunohistochemical marker for the identification of prostatic tissues and cells in histological specimens. PSA is found in normal prostate, benign prostatic hypertrophy (BPH) tissue, in cancer of the prostate, and its metastases as well as in other hormone dependent cancers, such as breast and ovarian carcinoma. However, the importance of PSA as a regulator of cell growth generally has not been appreciated. The role of PSA in the development of prostate or other hormone-dependent cancers has remained unclear. We therefore examined the role of PSA in the control of cell growth using both the PSA positive cell line, LNCaP cells and the PSA negative cell line PC-3 and DU145. LNCaP cell growth was stimulated by the conditioned medium (CM) from LNCaP cells, but not by CM from PC-3 or DU145 cells. No such stimulation was observed when PC-3 or DU145 cells were exposed to CM from LNCaP cells nor from CM produced by their own lines. The stimulation of LNCaP cell growth by its own CM could not be attributed to the high level of insulin-like growth factor binding protein-2 (IGFBP-2) present in the CM since even higher level of IGFBP-2 was also found to be present in CM from both PC-3 and DU145 CMs. High level of PSA and 66 kDa epidermal growth factor (EGF) were present in LNCaP CM as measured by Western blotting. The stimulation of LNCaP cell growth by its own CM was eliminated partially by PSA or EGF antibody. Stimulation of DNA biosynthesis in LNCaP cells by LNCaP CM or pure PSA was also observed. These data indicate that PSA and EGF are involved in the growth regulation of PSA positive LNCaP cell line.     

Resveratrol pretreatment desensitizes AHTO-7 human osteoblasts to growth stimulation in response to carcinoma cell supernatants.

Resveratrol, a natural phytoestrogen, has been reported to promote differentiation of murine MC3T3-E1 osteoblasts and to inhibit proliferation of prostate cancer cell lines. In the present study we tested the effects of resveratrol on the increased proliferation of human AHTO-7 osteoblastic cell line induced by conditioned media (CM) from a panel of carcinoma cell lines. This compound was found to modulate AHTO-7 proliferation in a tamoxifen-sensitive mechanism at lower concentrations, but failed to induce the osteoblast differentiation marker alkaline phosphatase (ALP) in contrast to vitamin D3. The proliferative response of AHTO-7 cells to conditioned media from carcinoma cell lines was diminished (30-71.4% inhibition) upon pretreatment with 0.5 microM resveratrol. Highest inhibition was demonstrated for pancreas (BxPC3, Panc-1), breast (ZR75-1) and renal (ACHN) carcinoma cell line supernatants whereas the effect on colon carcinoma (SW620, Colo320DM) cell CM and prostate cancer (PC3, DU145 and LNCaP) CM was less pronounced. Direct addition of resveratrol affected only supernatants of cell lines (<25% inhibition) exhibiting growth stimulatory activity for normal WI-38 lung fibroblasts. Resveratrol inhibited proliferation of DU145 and LNCaP cells in concentrations exceeding 5 microM, altered cell cycle distribution of all prostate cancer cell lines in concentrations as low as 0.5 microM, but did not inhibit the production of osteoblastic factors by these lines. In conclusion, resveratrol failed to induce ALP activity as marker of osteoblast differentiation in human osteoblastic AHTO-7 cells, however, inhibited their response to osteoblastic carcinoma-derived growth factors in concentrations significantly lower than those to reduce growth of cancer cells, thus effectively modulating tumor - osteoblast interaction.     

Growth of human prostate cancer cells is significantly suppressed in vitro with sodium butyrate through apoptosis

Histone deacetylase inhibitors (HDACis) have shown significant antiproliferative and apoptotic properties in various types of cancer cells, including prostate cancer cells, and are therefore being evaluated as a treatment modality. However, the mechanism by which sodium butyrate (SB) induces apoptosis is not completely understood. We focused on SB which exists in the intestine and is therefore expected to have less adverse effects. In this study, three prostate cancer cell lines (LNCaP, DU145 and PC-3) were treated in vitro with different concentrations of SB. Cell proliferation was studied by the XTT assay; cell cycle analysis and induction of apoptosis were studied by laser scanning cytometry. Western blot analysis was used to study p21, p27, CDK2, CDK4, CDK6, caspase-3, caspase-7, Fas, FADD, TRADD, Bcl-2 and Bax protein expression. SB inhibited cell growth and induced apoptosis in a concentration-dependent manner in human prostate cancer cells (LNCaP, DU145 and PC-3). Western blot analysis showed dose-dependent increases of p21 levels in DU145 and PC-3 cells, and dose-dependent decreases of CDK2, CDK4, CDK6 and procaspase-3 protein levels in all three prostate cancer cell lines. Bcl-xL was significantly down-regulated in DU145 cells, and Bcl-2 was significantly down-regulated in PC-3 and LNCaP cells. No significant changes were observed in procaspase-7, TRADD and Bax expression, although slight decreases in Fas and FADD expression were seen in all three prostate cancer cell lines. Analysis of cell morphology using laser scanning microscopy detected condensed and fragmented nuclei. In conclusion, SB induces G1 and G2 arrest by increasing p21 expression resulting in CDK2, CDK4 and CDK6 down-regulation. SB potently induced apoptosis, which was accompanied by DNA fragmentation, down-regulated Bcl-2 in LNCaP and PC-3 cells, Bcl-xL in DU145 cells, and down-regulated procaspase-3, but not procaspase-7, in these human prostate cancer cell lines. These results suggest that SB may serve as a new modality for the treatment of hormone refractory prostate cancer.     

Antiproliferative effects of AVN944, a novel inosine 5-monophosphate dehydrogenase inhibitor, in prostate cancer cells

Inosine 5-monophosphate dehydrogenase II, a key enzyme in the de novo synthesis of purine nucleotides, is expressed in prostate tumors and prostate cancer cells. AVN944 is a new, specific, noncompetitive IMPDH inhibitor. In this study, we investigated the effects of IMPDH inhibitor AVN944 on LNCaP, CWR22Rv1, DU145 and PC-3 human prostate cancer cells. AVN944 inhibited proliferation of these 4 prostate cancer cell lines and was associated with cell cycle G1 arrest of LNCaP cells and S-phase block of androgen-independent CWR22Rv1, DU145 and PC-3 cells. AVN944 induced caspase-dependentand caspase-independent cell death in LNCaP, CWR22Rv1, and DU145 cells. AVN944 induced expression of p53-target proteins Bok, Bax and Noxa in androgen-responsive cell lines and suppressed expression of survivin in prostate cancer cells regardless of their androgen sensitivity. AVN944 also induced differentiation of androgen-independent prostate cancer cells as indicated by morphological changes and increased expression of genes coding for prostasomal proteins, keratins and other proteins, including tumor suppressor genes MIG-6 and NDRG1. AVN944-differentiated androgen-independent DU145 and PC-3 cells are sensitized to TRAIL-induced apoptosis as demonstrated by induction of caspases and PARP cleavage. In summary, AVN944 inhibited the growth of human prostate cancer cells by inducing cell cycle arrest, cell death as well as differentiation. AVN944 is a novel, promising therapeutic agent that might be combined with other agents for treatment of human prostate cancer.     

RANKL-dependent and RANKL-independent mechanisms of macrophage-osteoclast differentiation in breast cancer

The cellular and humoral mechanisms accounting for tumour osteolysis in metastatic breast cancer are uncertain. Osteoclasts, the specialised multinucleated cells responsible for tumour osteolysis, are derived from monocyte/macrophage precursors. Breast cancer-derived tumour-associated macrophages (TAMs) are capable of osteoclast differentiation but the cellular and humoral mechanisms controlling this activity are uncertain. In this study, TAMs were isolated from primary breast cancers and cultured in the presence and absence of cytokines/growth factors influencing osteoclastogenesis. Extensive TAM-osteoclast differentiation occurred only in the presence of RANKL and M-CSF; this process was inhibited by OPG and RANK:Fc, decoy receptors for RANKL. Breast cancer-derived fibroblasts and human bone stromal cells expressed mRNA for RANKL, OPG and TRAIL, and co-culture of these fibroblasts with human monocytes stimulated osteoclast formation by a RANKL-dependent mechanism. Osteoclast formation and lacunar resorption also occurred by a RANKL-independent mechanism when the conditioned medium from breast cancer cells, MDA-MB-231 and MCF-7, was added (with M-CSF) to monocyte cultures. Our findings indicate that TAMs in breast cancer are capable of osteoclast differentiation and that breast cancer-derived fibroblasts and breast cancer cells contribute to this process by producing soluble factors that influence osteoclast formation by RANKL-dependent and RANKL-independent mechanisms respectively.     

矮壮素对小鼠前成骨细胞系MC3T3-E1骨骼发育相关基因及蛋白的影响及其机制

目的：探讨矮壮素（chlorocholine chloride,CCC）对小鼠前成骨细胞系MC3T3-E1细胞骨骼发育相关基因及蛋白表达情况的影响,评价矮壮素对骨骼发育的影响,并探究其可能的作用机制。方法：选取处于对数生长期的MC3T3-E1细胞接种于96孔板中,以8、40、200、1 000 μg/mL矮壮素进行染毒。MTT法检测矮壮素染毒后不同时间（24、48、72 h）MC3T3-E1细胞的相对存活率。实时荧光定量PCR法检测矮壮素染毒48 h后MC3T3-E1细胞中骨骼发育相关基因mRNA的表达情况,包括成骨相关基因碱性磷酸酶（ALP）、骨钙素（OCN）、胰岛素样生长因子1（IGF-1）、生长激素受体（GHR）;破骨相关基因核因子κB受体活化因子配体（RANKL）、骨保护素（OPG）、巨噬细胞集落刺激因子（M-CSF）。使用Western blot测定矮壮素染毒48 h后MC3T3-E1细胞中骨骼发育相关功能蛋白及MAPK信号通路蛋白的表达情况,包括碱性磷酸酶（ALP）、生长激素受体（GHR）、骨形成蛋白2（BMP2）、转录因子Runt相关蛋白2（Runx2）。结果：不同浓度矮壮素对MC3T3-E1细胞染毒不同时间均无明显细胞毒性;矮壮素染毒24 h,1 000 μg/mL矮壮素染毒组细胞中ALP、OCN、RANKL mRNA表达明显增高（P < 0.05）,且细胞中RANKL与OPG的mRNA表达量比值明显上升（P < 0.05）,GHR、IGF-1、OPG、M-CSF mRNA的表达无明显变化（P > 0.05）。1 000 μg/mL组MC3T3-E1细胞GHR和BMP2蛋白表达明显降低,磷酸化ERK蛋白表达量降低和磷酸化JNK蛋白表达量升高。结论：矮壮素对MC3T3-E1细胞无明显细胞毒性,但可能会抑制MC3T3-E1细胞向成熟成骨细胞的分化,且有可能促进破骨细胞分化;其作用机制可能与MAPK通路有关,磷酸化ERK降低和磷酸化JNK升高可能会抑制小鼠前成骨细胞骨骼发育相关蛋白的表达。     

Differential expression of cell surface molecules in prostate cancer cells

The expression of 119 cell surface molecules was catalogued for three prostate cancer cell lines, LNCaP, PC3, and DU145, all of which were established from metastases. Many of these molecules are common to all three cell lines, whereas some are differentially expressed. More prostate basal epithelial cell-specific than luminal epithelial cell-specific molecules are detected, especially in DU145 and PC3 cells. The cancer cells also express molecules that are not normally associated with prostate epithelial cells. As a population, expression of these molecules appears to be heterogeneous. This heterogeneity may be an inherent property of the population.     

Role of fibroblast growth factor receptor signaling in prostate cancer cell survival.

BACKGROUND: Expression of fibroblast growth factors (FGFs) is increased in a substantial fraction of human prostate cancers in vivo and in prostate cancer cell lines. Altered FGF signaling can potentially have a variety of effects, including stimulating cell proliferation and inhibiting cell death. To determine the biologic significance of altered FGF signaling in human prostate cancer, we disrupted signaling by expression of a dominant-negative (DN) FGF receptor in prostate cancer cell lines. METHODS: PC-3, LNCaP, and DU145 prostate cancer cells were stably transfected with DN FGFR constructs, and LNCaP and DU145 cells were infected with a recombinant adenovirus expressing DN FGFR-1. The effect of DN FGFR-1 expression was assessed by colony-formation assays, cell proliferation assays, flow cytometry, and cytogenetic analysis. Key regulators involved in the G(2)-to-M cell cycle transition were assessed by western blotting to examine cyclin B1 expression and by in vitro kinase assay to assess cdc2 kinase activity. RESULTS: Stable transfection of the DN FGFR-1 construct inhibited colony formation by more than 99% in all three cell lines. Infection of LNCaP and DU145 prostate cancer cells with adenovirus expressing DN FGFR-1 led to extensive cell death within 48 hours. Flow cytometry and cytogenetic analysis revealed that the DN FGFR-1 receptor led to arrest in the G(2) phase of the cell cycle before cell death. Cyclin B1 accumulated in DN FGFR-1-infected LNCaP cells, but cdc2 kinase activity was decreased. CONCLUSIONS: These findings reveal an unexpected dependence of prostate cancer cells on FGF receptor signal transduction to traverse the G(2)/M checkpoint. The mechanism for the G(2) arrest is not clear. Our results raise the possibility that FGF-signaling antagonists might enhance the cell death induced by other prostate cancer therapies.     

Stromal cells from human benign prostate hyperplasia produce a growth-inhibitory factor for LNCaP prostate cancer cells,identified as interleukin-6

To understand specific interactions between stromal cells and epithelial cells in benign prostatic hyperplasia (BPH) and prostatic adenocarcinoma, we developed stromal-cell cultures from normal human prostate (PNX) and BPH (BH101), composed of fibroblasts and myofibroblasts. Their role in epithelial-cell growth was studied using the established cancer cell lines LNCaP, PC3 and DU 145 and an SV40 large T-immortalized normal epithelial-cell line, PNTIA, in double-diffusion co-culture chambers. PNTIA was stimulated by PNX (x 1.6) and more strongly by BH101 stromal cells (x 2.7). Conversely, LNCaP growth decreased by 50% in the presence of BH101 stromal cells (stromal/epithelial ratio: 10). A BH101-conditioned medium (CM), obtained in serum-free conditions, induced 90% inhibition of [³H]thymidine incorporation of the LNCaP androgen-sensitive cell line. Two other androgen-independent prostate cancer cell lines were either insensitive to BH101 CM (PC3) or slightly inhibited (40% for DU145). BH101 produced large amounts of IL-Iβ, IL-6 and IL-8. HPLC gel filtration enabled separation of an inhibitory fraction which contained IL-6. IL-6 was demonstrated to be responsible for the strong inhibitory effect since an IL-6-neutralizing antibody abolished this inhibition, which was reproduced by human recombinant IL-6. Recombinant IL-6 growth inhibition was observed only on LNCaP prostate cancer androgen-sensitive cells. © 1996 Wiley-Liss, Inc.     

Altered response to thyroid hormones by prostate and breast cancer cells

Transferrin, an abundant bone marrow constituent, has been shown to be a potent mitogen in vitro in the prostate cancer cell line PC3. T4 (L-thyroxine) and T3 (3′,3,5-tri-iodo-L-thyronine) are regulators of cell metabolism. In this study, the effects of nonphysiological concentrations (about two orders of magnitude higher) of T4, T3, T2 (3,5-di-iodo-L-thyronine), RT3 (reverse T3, 3′,5′,3-tri-iodo-L-thyronine) and transferrin (about three orders of magnitude lower) were tested on the prostate cancer cell lines PC3, DU145 and LNCaP, and the breast cancer cell line MCF-7. In PC3 cells, increased proliferation by transferrin could be reversed by the addition of T3 or T4. T4 decreased proliferation in all cell lines tested, while transferrin increased proliferation in PC3 cells only. T3 decreased proliferation in PC3, LNCaP and MCF-7 cells but had no effect on DU145 cells. T4 and T3 gave two-state behavior in LNCaP cells. These results were combined to determine the essential iodines which produced the observed proliferative effects. Cell lines responded differently to T4, T3, T2, RT3 and transferrin suggesting a specific interaction among the compounds tested and the different cell lines. Finally, regulation of gene expression was demonstrated using DU145 cells. Upregulation of c-fos mRNA was observed in cultures at early time-points in the presence of T4, transferrin or both. Decreased expression was observed at later time-points with no expression at 4 h. An explanation for these results may be a change in thyroid hormone receptor/ligand affinity. Thus, the interactions between thyroid hormones and cancer cells may be different from those between thyroid hormones and normal cells. Received: 28 January 1999 / Accepted: 26 August 1999     

A recombinant adenovirus expressing wild-type Bax induces apoptosis in prostate cancer cells independently of their Bcl-2 status and androgen sensitivity

Using a binary co-transfection strategy of Ad/GT Bax and Ad/PGK-GV16, we have succeeded in inducing overexpression of Bax protein in three prostate cell lines (androgen-insensitive DU145 and PC3, and androgen-sensitive LNCaP). The expression of Bax protein by this system was sufficient to induce all three prostate lines to undergo apoptosis. The fact that DU145 cells which have a p53 mutation and are deficient in Bax, responded to this treatment, suggests that this effect is independent of these pathways. Initiation of the cleavage of Caspase-3 (CPP32/Yama/apopain) and PARP (poly (ADP-ribose) polymerase) by the introduction of Bax were confirmed by western blot analysis. Bcl-2 expression is relevant in the progression of prostate cancer and contributes to an androgen, apoptotic-resistant phenotype in the advanced stages. We examined stable Bcl-2 overexpressing DU145, PC3 and LNCaP cell lines as models of advanced prostate cancer. The adenoviral co-transfection system induced Bax protein expression and apoptosis even in these Bcl-2 transfected cell lines. Taken together, our results suggest that this Bax expression system might represent a useful gene therapy strategy when applied to the treatment of prostate cancer and its efficacy would be independent of the Bcl-2 status and androgen sensitivity of these cancers.     

Monocyte chemotactic protein-1 mediates prostate cancer-induced bone resorption

Prostate cancer preferentially metastasizes to bone, resulting in high mortality. Strategies to inhibit prostate cancer metastasis include targeting both tumor-induced osteoblastic lesions and underlying osteoclastic activities. We and others have previously shown that blocking receptor activator of nuclear factor-kappaB ligand (RANKL) partially blocks tumor establishment and progression in bone in murine models. However, levels of RANKL in the cell lines used in these studies were very low, suggesting that soluble factors other than RANKL may mediate the cancer-induced osteoclast activity. To identify these factors, a human cytokine antibody array was used to measure cytokine expression in conditioned medium collected from primary prostate epithelial cells (PrEC), prostate cancer LNCaP and its derivative C4-2B, and PC3 cells. All prostate cancer cells produced high amounts of monocyte chemotactic protein-1 (MCP-1) compared with PrEC cells. Furthermore, levels of interleukin (IL)-6, IL-8, GROalpha, ENA-78, and CXCL-16 were higher in PC3 than LNCaP. These results were confirmed by ELISA. Finally, human bone marrow mononuclear cells (HBMC) were cultured with PC3 conditioned medium. Although both recombinant human MCP-1 and IL-8 directly stimulated HBMC differentiation into osteoclast-like cells, IL-8, but not MCP-1, induced bone resorption on dentin slices with 21 days of culture in the absence of RANKL. However, the conditioned medium-induced bone resorption was inhibited by MCP-1 neutralizing antibody and was further synergistically inhibited with IL-8 antibody, indicating that MCP-1, in addition to IL-8, mediates tumor-induced osteoclastogenesis and bone resorption. MCP-1 may promote preosteoclast cell fusion, forming multinucleated tartrate-resistant acid phosphatase-positive osteoclast-like cells. This study may provide novel therapeutic targets for treatment of prostate cancer skeletal metastasis.