Antiproliferative actions of the synthetic androgen, mibolerone, in breast cancer cells are mediated by both androgen and progesterone receptors

Androgen signaling, mediated by the androgen receptor (AR), is a critical factor influencing growth of normal and malignant breast cells. Given the increasing use of exogenous androgens in women, a better understanding of androgen action in the breast is essential. This study compared the effects of 5alpha-dihydrotestosterone (DHT) and a synthetic androgen, mibolerone, on estradiol (E(2))-induced proliferation of breast cancer cells. DHT modestly inhibited E(2)-induced proliferation and mibolerone significantly inhibited proliferation in T-47D cells. The effects of both androgens could be reversed by an AR antagonist, suggesting that their actions were mediated, in part, by AR. Whereas high physiological doses (10-100nM) of DHT reduced E(2)-mediated induction of the estrogen-regulated gene progesterone receptor (PR) to basal levels, mibolerone at lower doses (1nM) eliminated PR expression, suggesting that mibolerone may also act via the PR. In the AR positive, PR-negative MCF-7 cells, mibolerone had modest effects on E(2)-induced proliferation, but was a potent inhibitor of proliferation in the AR positive, PR positive MCF-7M11 PRA cells. The effects of mibolerone in breast cancer cells were similar to those of the progestin, medroxyprogesterone acetate. Our results demonstrate that mibolerone can have both androgenic and progestagenic actions in breast cancer cells.     

Secretion of endogenous kallikreins 2 and 3 by androgen receptor-transfected PC-3 prostate cancer cells

Androgen independent PC-3 cells lack androgen receptor (AR) expression and do not produce kallikrein 2 (hK2) or 3 (prostate-specific antigen, PSA). In this paper, we examined the ability of androgens to stimulate PSA and hK2 production in AR transfected PC-3 cells (PC-3(AR)) and compared this to LNCaP cells. PSA and hK2 were measured in the culture medium and cell lysates using an ELISA-based immunofluorometric assay. Only androgens were able to induce PSA and hK2 secretion in PC-3(AR) cells in a dose- and time-dependent manner depending on the level of AR present. The level of androgen-induced PSA and hK2 secretion in PC-3(AR) cells was approximately 1.5 and 0.9% that induced in LNCaP cells, respectively. Insulin-like growth factor-I (IGF-I), which has been shown to activate AR in the absence of ligand, did not activate PSA secretion in the absence of androgen, but further increased the dihydrotestosterone-induced PSA secretion in PC-3(AR) cells. The lack of PSA and hK2 production in parental PC-3 cells is thus a result of their lack of AR expression. PSA and/or hK2 production in PC-3(AR) cells can thus serve as an endogenous reporter system to investigate AR action or to screen putative endocrine disrupters.     

Testosterone increases osteoprotegerin mRNA expression in mouse osteoblast cells.

The role that androgens play in the regulation of bone metabolism has been substantiated in animals and humans. We previously demonstrated that testosterone inhibits osteoclast differentiation stimulated by parathyroid hormone through the androgen receptor in mouse bone-cell cultures. However, the details of this mechanism are still unknown. The present study was aimed at examining whether testosterone would affect the mRNA levels of osteoprotegerin (OPG) and receptor activator of Nf kappa B ligand (RANKL) in mouse bone-cell cultures as well as mouse osteoblastic cell-line, MC3T3-E1 cells by employing semi-quantitative RT-PCR. Testosterone increased OPG mRNA expression in both mouse bone-cell cultures and MC3T3-E1 cells. 10-8 M PTH-(1-34) as well as 10-8M 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] inhibited OPG mRNA expression in mouse bone cells. 10-8 M testosterone antagonized OPG mRNA expression inhibited by 10-8 M PTH-(1-34), but failed to affect OPG mRNA expression inhibited by 10-8 M 1,25(OH)2D3. 10-8 M alpha-dehydrotestosterone, a non-aromatizable androgen, increased OPG mRNA expression. On the other hand, testosterone did not affect RANKL mRNA expression in MC3T3-E1 or mouse bone cells. In conclusion, the present study demonstrated that testosterone increased OPG mRNA expression in mouse bone-cell cultures and the osteoblastic cell line. These effects are likely to take place through the androgen receptor.     

Pancreatic polypeptide is secreted from and controls differentiation through its specific receptors in osteoblastic MC3T3-E1 cells

Although the neuropeptide Y (NPY) family has been demonstrated to control bone metabolism, the role of pancreatic polypeptide (PP), which has structural homology with NPY and peptide YY (PYY) to share the NPY family receptors, in peripheral bone tissues has remained unknown. In the present study, we studied the regulatory roles of PP and its Y receptors using MC3T3-E1 cells, a murine transformed osteoblastic cell line, as a model for osteoblastic differentiation. We found that (1) PP mRNA was detected and increased during cell-contact-induced differentiation in MC3T3-E1 cells; (2) the immunoreactivity of PP was detected by radioimmunoassay and increased in culture medium during differentiation; (3) all the types of NPY family receptor mRNAs (Y1, Y2, Y4, Y5, and y6) were found to increase during differentiation; (4) PP stimulated differentiation in MC3T3-E1 cells in terms of ALP mRNA and BMP-2 mRNA. These findings suggested that MC3T3-E1 cells produce and secrete PP, which may in turn stimulate the differentiation of MC3T3-E1 through its specific receptors in an autocrine manner.     

DU-145 and PC-3 human prostate cancer cell lines express androgen receptor: implications for the androgen receptor functions and regulation

The majority of human prostate cancer cell lines, including the two "classical" cell lines DU-145 and PC-3, are reported to be androgen receptor (AR)-negative. However, other studies have provided evidence that the DU-145 and PC-3 cell lines express AR mRNA. These contradictory observations prompted us to investigate whether DU-145 and PC-3 cell lines express the androgen receptor. Using antipeptide antibodies directed against three distinct regions of the human AR protein and an improved method to detect AR protein in immunoblotting, we report that DU-145 and PC-3 cell lines express AR protein. We found that the relative levels of the AR mRNA and protein that were detected in DU-145 and PC-3 cell lines were lower than the LNCaP, an AR-positive cell line. Moreover, the antibody directed against the non-variant region (amino acids 299-315), but not the variant N- or C-terminal region (amino acids 1-20 and 900-919, respectively) of the human AR protein, detected the expression of AR in all prostate cancer cell lines. Notably, treatment of these cell lines with dihydrotestosterone (DHT) resulted in measurable increases in the AR protein levels and considerable nuclear accumulation. Although, treatment of DU-145 and PC-3 cells with DHT did not result in stimulation of the activity of an AR-responsive reporter, knockdown of AR expression in PC-3 cells resulted in decreases in p21(CIP1) protein levels, and a measurable decrease in the activity of the p21-luc-reporter. Our observations demonstrate the expression of AR protein in DU-145 and PC-3 prostate cancer cell lines.     

Chemotactic responses of osteoblastic MC3T3-E1 cells toward zinc chloride

Although zinc (Zn) is known to participate in bone formation, its exact role in the remodeling of this tissue has not been fully clarified. The present study was designed to investigate whether Zn has a role at the resorptive sites in vitro. We investigated the migration of osteoblastic MC3T3-E1 cells in response to Zn using a Boyden chamber assay. Exposure of MC3T3-E1 cells to Zn stimulated the migration of MC3T3-E1 cells. Checkerboard analysis revealed that the migration of MC3T3-E1 cells toward Zn was a directional (chemotaxis) rather than a random (chemokinesis) motion. Pretreatment of MC3T3-E1 cells with pertussis toxin completely blocked the chemotactic response of cells to Zn, indicating that it is mediated by G-protein-coupled receptors. Because the bone is one of the major Zn storage sites, we suggest that Zn released from bone-resorptive sites plays an important role in the recruitment of osteoblasts and bone renewal.     

Smad3 differently affects osteoblast differentiation depending upon its differentiation stage.

Smad3, a critical component of the TGF-beta signaling pathways, plays an important role in the regulation of bone formation. However, how Smad3 affects osteoblast at the different differentiation stage remains still unknown. In the present study, we examined the effects of Smad3 on osteoblast phenotype by employing mouse bone marrow ST-2 cells and mouse osteoblastic MC3T3-E1 cells at the different differentiation stage. Smad3 overexpression significantly inhibited bone morphogenetic protein-2 (BMP-2)-induced ALP activity in ST-2 cells, indicating that Smad3 suppresses the commitment of pluripotent mesenchymal cells into osteoblastic cells. Smad3 increased the levels of COLI and ALP mRNA at 7 day cultures in MC3T3-E1 cells, and its effects on COL1 were decreased as the culture periods progress, although its effects on ALP were sustained during 21 day cultures. Smad3 overexpression enhanced the level of Runx2 and OCN mRNA at 14 day and 21 day cultures. Smad3 increased the levels of MGP and NPP-1 mRNA, although the extent of increase in MGP and NPP-1 was reduced and enhanced during the progression of culture period, respectively. Smad3 did not affect the level of ANK mRNA. On the other hand, Smad3 enhanced the level of MEPE mRNA at 14 and 21 day cultures, although Smad3 decreased it at 7 day cultures. In conclusion, Smad3 inhibits the osteoblastic commitment of ST-2 cells, while promotes the early stage of differentiation and maturation of osteoblastic committed MC3T3-E1 cells. Also, Smad3 enhanced the expression of mineralization-related genes at the maturation phase of MC3T3-E1 cells.     

The effects of BIG-3 on osteoblast differentiation are not dependent upon endogenously produced BMPs

BMPs play an important role in both intramembranous and endochondral ossification. BIG-3, BMP-2-induced gene 3 kb, encodes a WD-40 repeat protein that accelerates the program of osteoblastic differentiation in vitro. To examine the potential interactions between BIG-3 and the BMP-2 pathway during osteoblastic differentiation, MC3T3-E1 cells stably transfected with BIG-3 (MC3T3E1-BIG-3), or with the empty vector (MC3T3E1-EV), were treated with noggin. Noggin treatment of pooled MC3T3E1-EV clones inhibited the differentiation-dependent increase in AP activity observed in the untreated MC3T3E1-EV clones but did not affect the increase in AP activity in the MC3T3E1-BIG-3 clones. Noggin treatment decreased the expression of Runx2 and type I collagen mRNAs and impaired mineralized matrix formation in MC3T3E1-EV clones but not in MC3T3E1-BIG-3 clones. To determine whether the actions of BIG-3 on osteoblast differentiation converged upon the BMP pathway or involved an alternate signaling pathway, Smad1 phosphorylation was examined. Basal phosphorylation of Smad1 was not altered in the MC3T3E1-BIG-3 clones. However, these clones did not exhibit the noggin-dependent decrease in phosphoSmad1 observed in the MC3T3E1-EV clones, nor did it decrease nuclear localization of phosphoSmad1. These observations suggest that BIG-3 accelerates osteoblast differentiation in MC3T3-E1 cells by inducing phosphorylation and nuclear translocation of Smad1 independently of endogenously produced BMPs.     

CD73-generated adenosine promotes osteoblast differentiation

CD731 is a GPI-anchored cell surface protein with ecto-5'-nucleotidase enzyme activity that plays a crucial role in adenosine production. While the roles of adenosine receptors (AR) on osteoblasts and osteoclasts have been unveiled to some extent, the roles of CD73 and CD73-generated adenosine in bone tissue are largely unknown. To address this issue, we first analyzed the bone phenotype of CD73-deficient (cd73(-/-)) mice. The mutant male mice showed osteopenia, with significant decreases of osteoblastic markers. Levels of osteoclastic markers were, however, comparable to those of wild-type mice. A series of in vitro studies revealed that CD73 deficiency resulted in impairment in osteoblast differentiation but not in the number of osteoblast progenitors. In addition, over expression of CD73 on MC3T3-E1 cells resulted in enhanced osteoblastic differentiation. Moreover, MC3T3-E1 cells expressed adenosine A(2A) receptors (A(2A)AR) and A(2B) receptors (A(2B)AR) and expression of these receptors increased with osteoblastic differentiation. Enhanced expression of osteocalcin (OC) and bone sialoprotein (BSP) observed in MC3T3-E1 cells over expressing CD73 were suppressed by treatment with an A(2B)AR antagonist but not with an A(2A) AR antagonist. Collectively, our results indicate that CD73 generated adenosine positively regulates osteoblast differentiation via A(2B)AR signaling.     

Genome-wide analysis of DHEA- and DHT-induced gene expression in mouse hypothalamus and hippocampus

Dehydroepiandrosterone (DHEA) is the most abundant steroid in humans and a multi-functional neuroactive steroid that has been implicated in a variety of biological effects in both the periphery and central nervous system. Mechanistic studies of DHEA in the periphery have emphasized its role as a prohormone and those in the brain have focused on effects exerted at cell surface receptors. Recent results demonstrated that DHEA is intrinsically androgenic. It competes with DHT for binding to androgen receptor (AR), induces AR-regulated reporter gene expression in vitro, and exogenous DHEA administration regulates gene expression in peripheral androgen-dependent tissues and LnCAP prostate cancer cells, indicating genomic effects and adding a level of complexity to functional models. The absence of information about the effect of DHEA on gene expression in the CNS is a significant gap in light of continuing clinical interest in the compound as a hormone replacement therapy in older individuals, patients with adrenal insufficiency, and as a treatment that improves sense of well-being, increases libido, relieves depressive symptoms, and serves as a neuroprotective agent. In the present study, ovariectomized CF-1 female mice, an established model for assessing CNS effects of androgens, were treated with DHEA (1 mg/day), dihydrotestosterone (DHT, a potent androgen used as a positive control; 0.1 mg/day) or vehicle (negative control) for 7 days. The effects of DHEA on gene expression were assessed in two regions of the CNS that are enriched in AR, hypothalamus and hippocampus, using DNA microarray, real-time RT-PCR, and immunohistochemistry. RIA of serum samples assessed treatment effects on circulating levels of major steroids. In hypothalamus, DHEA and DHT significantly up-regulated the gene expression of hypocretin (Hcrt; also called orexin), pro-melanin-concentrating hormone (Pmch), and protein kinase C delta (Prkcd), and down-regulated the expression of deleted in bladder cancer chromosome region candidate 1 (Dbccr1) and chitinase 3-like 3 (Chi3l3). Two-step real-time RT-PCR confirmed changes in the expression of three genes (Pmch, Hcrt and Prkcd) using the same RNA sample employed in the microarray experiment. Immunohistochemistry showed augmentation of prepro-hypocretin (pHcrt) neuropeptide protein expression by DHEA and DHT in hypothalamus, consistent with the localization of orexin neurons. In hippocampus, DHT down-regulated the expression of Prkcd, while DHEA did not have significant effects. RIA results supported the view that DHEA-induced effects were mediated through AR. The current study identified neurogenomic effects of DHEA treatment on a subset of genes directly implicated in the regulation of appetite, energy utilization, alertness, apoptosis, and cell survival. These changes in gene expression in the CNS represent a constellation of effects that may help explain the diverse benefits attributed to replacement therapy with DHEA. The data also provide a new level of detail regarding the genomic mechanism of action of DHEA in the CNS and strongly support a central role for the androgen receptor in the production of these effects. More broadly, the results may be clinically significant because they provide new insights into processes that appear to mediate the diverse CNS effects attributed to DHEA.     

Apelin stimulates proliferation and suppresses apoptosis of mouse osteoblastic cell line MC3T3-E1 via JNK and PI3-K/Akt signaling pathways

The aim of this study was to investigate the effects of apelin on proliferation and apoptosis of mouse osteoblastic MC3T3-E1 cells. APJ was expressed in MC3T3-E1 cells. Apelin did not affect Runx2 expression, alkaline phosphatase (ALP) activity, osteocalcin and type I collagen secretion, suggesting that it has no effect on osteoblastic differentiation of MC3T3-E1 cells. However, apelin stimulated MC3T3-E1 cell proliferation and inhibited cell apoptosis induced by serum deprivation. Our study also shows that apelin decreased cytochrome c release and caspase-3, capase-8 and caspase-9 activation in serum-deprived MC3T3-E1 cells. Apelin activated c-Jun N-terminal kinase (JNK) and Akt (phosphatidylinositol 3-kinase downstream effector), and the JNK inhibitor SP600125, the phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002 or the Akt inhibitor 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (HIMO) inhibited its effects on proliferation and serum deprivation-induced apoptosis. Furthermore, apelin protected against apoptosis induced by the glucocorticoid dexamethasone or TNF-alpha. Apelin stimulates proliferation and suppresses serum deprivation-induced apoptosis of MC3T3-E1 cells and these actions are mediated via JNK and PI3-K/Akt signaling pathways.     

Effects of genistein on expression of bone markers during MC3T3-E1 osteoblastic cell differentiation

In this in vitro study, the hypothesis that the beneficial effects of dietary genistein on bone are through the modulation of the bone marker synthesis by osteoblastic MC3T3-E1 cells was tested, and the possible roles of estrogen receptors in the actions of genistein on osteoblastic cells were also examined. Interleukin-6 production was decreased 40% to 60% in osteoblastic cells treated with genistein from either day 8-16 or day 12-16, at dietarily achievable concentrations (10(-10) to 10(-8) M) (P<0.05). The mRNA expression of osteoprotegerin increased about 140% in cells treated from with genistein day 4-8 at a concentration of 10(-8) M (P<0.05). The ratio of estrogen receptor-alpha to beta expression increased 10-fold from day 0 to 12 of culture (P<0.05). Correlating with this time-dependent variation in estrogen receptor expression, treatments of 17beta-estradiol and genistein had opposite dose patterns on the ratio of estrogen receptor-alpha to beta expression following treatment from day 4 to 6 compared to from day 0 to 2. The addition of ICI-182,780, an estrogen receptor blocker, reduced the inhibitory effect of genistein on IL-6 production by 30-50%. In summary, these findings suggest that the beneficial skeletal effects of genistein, at dietarily achievable levels, appear to be mediated, at least in part, by interleukin-6 and osteoprotegerin, and estrogen receptors play important roles in the inhibition of interleukin-6 synthesis by genistein in osteoblastic MC3T3-E1 cells.     

Prostate cancer cells and bone stromal cells mutually interact with each other through bone morphogenetic protein-mediated signals

Functional interactions between cancer cells and the bone microenvironment contribute to the development of bone metastasis. Although the bone metastasis of prostate cancer is characterized by increased ossification, the molecular mechanisms involved in this process are not fully understood. Here, the roles of bone morphogenetic proteins (BMPs) in the interactions between prostate cancer cells and bone stromal cells were investigated. In human prostate cancer LNCaP cells, BMP-4 induced the production of Sonic hedgehog (SHH) through a Smad-dependent pathway. In mouse stromal MC3T3-E1 cells, SHH up-regulated the expression of activin receptor IIB (ActR-IIB) and Smad1, which in turn enhanced BMP-responsive reporter activities in these cells. The combined stimulation with BMP-4 and SHH of MC3T3-E1 cells cooperatively induced the expression of osteoblastic markers, including alkaline phosphatase, bone sialoprotein, collagen type II α1, and osteocalcin. When MC3T3-E1 cells and LNCaP cells were co-cultured, the osteoblastic differentiation of MC3T3-E1 cells, which was induced by BMP-4, was accelerated by SHH from LNCaP cells. Furthermore, LNCaP cells and BMP-4 cooperatively induced the production of growth factors, including fibroblast growth factor (FGF)-2 and epidermal growth factor (EGF) in MC3T3-E1 cells, and these may promote the proliferation of LNCaP cells. Taken together, our findings suggest that BMPs provide favorable circumstances for the survival of prostate cancer cells and the differentiation of bone stromal cells in the bone microenvironment, possibly leading to the osteoblastic metastasis of prostate cancer.     

Serial passage of MC3T3-E1 cells alters osteoblastic function and responsiveness to transforming growth factor-beta1 and bone morphogenetic protein-2.

The murine-derived clonal MC3T3-E1 cell is a well-studied osteoblast-like cell line. To understand the effects of serial passages on its cellular function, we examined changes in cell morphology, gap junctional intercellular communication (GJIC), proliferation, and osteoblastic function between early passage (<20) and late passage (>65) cells. MC3T3-E1 cells developed an elongated, spindle shape after multiple passages. Intercellular communication decreased significantly (33%) in late vs. early passage cells. Transforming growth factor-beta1 (TGF-beta1) stimulated cell proliferation in early passage cells and induced c-fos expression, while it inhibited proliferation in late passage cells. Using alkaline phosphatase (ALP) activity and osteocalcin (OC) secretion as markers for osteoblastic function and differentiation, we demonstrated that both markers were significantly reduced after multiple cell passages. Bone morphogenetic protein-2 (BMP-2) significantly enhanced ALP activity and OC secretion in early passage cells while TGF-beta1 exerted an opposite effect. Both BMP-2 and TGF-beta1 had minimal effects on late passage cells. We conclude that serial passage alters MC3T3-E1 cell morphology, and significantly diminishes GJIC, osteoblastic function, TGF-beta1-mediated cell proliferation, and responsiveness to TGF-beta1 and BMP-2. Cell passage numbers should be clearly defined in functional studies involving MC3T3-E1 cells.