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Suppression of DHT-Induced Paracrine Stimulation of Endothelial Cell Growth by Estrogens Via Prostate Cancer Cells
Abstract
Background:
Androgen modulation of angiogenesis in prostate cancer may be not directly mediated by androgen receptor (AR) as AR is not detected in the prostatic endothelial cells.
Methods:
We examined the paracrine stimulation of cell proliferation by prostate tumor cells and its modulation by androgen and estrogens in a murine endothelial cell line (MEC) that does not express AR.
Results:
Tumor cell conditioned media (TCM) collected from LAPC-4 or LNCaP prostatic tumor cells produced a time- and concentration-dependent induction of cell growth in MECs, which was parallel to the VEGF concentration in the TCM. This TCM-induced cell growth in MECs was enhanced by the treatment of prostatic tumor cells with dihydrotestosterone (DHT). Both the TCM-stimulation and DHT-enhancement effects in MECs were completely blocked by SU5416, a specific VEGF receptor antagonist. Co-administration of 17α-estradiol or 17β-estradiol with DHT in prostatic tumor cells completely inhibited the DHT-enhancement effect while treatment with DHT, 17α-estradiol or 17β-estradiol did not produce any significant direct effect in MECs. Moreover, administration of 17α-estradiol or 17β-estradiol in xenograft animals with LAPC-4 or LNCaP prostate tumor significantly decreased the microvessel number in the tumor tissues.
Conclusions:
Our study indicated that prostate tumor cells regulate endothelial cell growth through a paracrine mechanism, which is mainly mediated by VEGF; and DHT is able to modulate endothelial cell growth via tumor cells, which is inhibited by 17α-estradiol and 17β-estradiol. Thus, both17α-estradiol and 17β-estradiol are potential agents for anti-angiogenesis therapy in androgen-responsive prostate cancer.
... As cancer cells lost their ability to divide in a controlled manner, they require a dedicated blood supply to provide oxygen and nutrients for growth. Cancer cells can produce various growth factors including VEGF [260], to induce angiogenesis [261]. ...
... On the other hand, PCA3 lncRNA is able to promote PCa angiogenesis by potentiating androgen-induced production of growth factors, including VEGF, in PCa cells. These growth factors then function in a paracrine fashion to stimulate endothelial cell growth and vessel formation [219,244,260]. ...
... Knockdown of MALAT1 in BCa cells results in the attenuation of endothelial cell proliferation, cell migration, and tube formation in a co-culturing human umbilical vein endothelial cell and BCa cell assay. This effect appears to be mediate by miR-145 sponging, which leads to an increased expression of VEGF, an angiogenic factor acting in a paracrine manner [286,260]. ...
Long noncoding RNAs (lncRNAs) are defined as noncoding RNA transcripts with a length greater than 200 nucleotides. Research over the last decade has made great strides in our understanding of lncRNAs, especially in the biology of their role in cancer. In this article, we will briefly discuss the biogenesis and characteristics of lncRNAs, then review their molecular and cellular functions in cancer by using prostate and breast cancer as examples. LncRNAs are abundant, diverse, and evolutionarily, less conserved than protein-coding genes. They are often expressed in a tumor and cell-specific manner. As a key epigenetic factor, lncRNAs can use a wide variety of molecular mechanisms to regulate gene expression at each step of the genetic information flow pathway. LncRNAs display widespread effects on cell behavior, tumor growth, and metastasis. They act intracellularly and extracellularly in an autocrine, paracrine and endocrine fashion. Increased understanding of lncRNA's role in cancer has facilitated the development of novel biomarkers for cancer diagnosis, led to greater understanding of cancer prognosis, enabled better prediction of therapeutic responses, and promoted identification of potential targets for cancer therapy.
... [67] It has been shown that AR is not expressed in the ECs from microvessels of some organs such as the prostate gland and skin, and androgen is therefore unable to stimulate EC growth directly via AR. [68][69][70] However, androgen is able to affect EC proliferation indirectly through a paracrine fashion (Figure 2). By using prostate cancer cells and ECs from mouse skin, we have demonstrated that androgen was able to upregulate VEGF expression in prostate cancer cells, which was secreted from these cancer cells and stimulated EC proliferation. ...
... By using prostate cancer cells and ECs from mouse skin, we have demonstrated that androgen was able to upregulate VEGF expression in prostate cancer cells, which was secreted from these cancer cells and stimulated EC proliferation. [70,71] This paracrine action may be an important mechanism in synchronizing angiogenesis and tumor growth in tumor progression. ...
... It is worthwhile to note that both direct and indirect androgen actions on EC proliferation (see Figure 2) may be modulated by other hormones such as estrogens. [70,71] We have shown that estrogens via ERs produce a ligand, receptor-isoform and cell specific modulation of androgen actions in multiple systems. [72][73][74][75] In ECs that express both AR and ERs, estrogens produce an ER-dependent modification of androgen actions on gene expression and cell proliferation in an ER-ligand specific manner. ...
The roles of androgens on cardiovascular physiology and pathophysiology are controversial as both beneficial and detrimental effects have been reported. Although the reasons for this discrepancy are unclear, multiple factors such as genetic and epigenetic variation, sex-specificity, hormone interactions, drug preparation and route of administration may contribute. Recently, growing evidence suggests that androgens exhibit beneficial effects on cardiovascular function though the mechanism remains to be elucidated. Endothelial cells (ECs) which line the interior surface of blood vessels are distributed throughout the circulatory system, and play a crucial role in cardiovascular function. Endothelial progenitor cells (EPCs) are considered an indispensable element for the reconstitution and maintenance of an intact endothelial layer. Endothelial dysfunction is regarded as an initiating step in development of atherosclerosis and cardiovascular diseases. The modulation of endothelial functions by androgens through either genomic or nongenomic signal pathways is one possible mechanism by which androgens act on the cardiovascular system. Obtaining insight into the mechanisms by which androgens affect EC and EPC functions will allow us to determine whether androgens possess beneficial effects on the cardiovascular system. This in turn may be critical in the prevention and therapy of cardiovascular diseases. This article seeks to review recent progress in androgen regulation of endothelial function, the sex-specificity of androgen actions, and its clinical applications in the cardiovascular system.
... Angiogenic factors, such as vascular endothelial growth factor (VEGF), are highly expressed in tissues of BPH and play a significant role in development and progression (12)(13)(14)(15)(16). Several studies have shown that androgen, a key hormone regulating BPH, is a positive regulator for VEGF expression (17,18). However, Wen et al. (19,20) reported that DHT, a most potent androgen responsible for BPH, failed to alter VEGF expression in prostate cancer cells. Thus, the exact mechanism for regulating VEGF in prostate tissues is controversial and unclear. ...
... The level of VEGF immunostaining is higher in BPH epithelial cells compared with that in stromal cells, even in prostate cancer tissues (24,36,37). Although there is some controversy (20), the androgen including DHT is regarded as an inducing factor for VEGF expression (17,18,38). In this study, we detected expression of VEGFA in DHT-stimulated prostatic epithelial cells but not in vascular endothelial cells (Fig. 2A). ...
Benign prostatic hyperplasia (BPH), a common disease in elderly males, is accompanied by non-malignant growth of prostate tissues, subsequently causing hypoxia and angiogenesis. Although VEGF-related angiogenesis is one of the therapeutic targets of prostate cancer, there is no previous study targeting angiogenesis for treatment of BPH. Dihydrotestosterone (DHT)-induced expressions of vascular endothelial growth factor (VEGF) in prostate epithelial RWPE-1 cells and human umbilical vascular endothelial cells (HUVECs). Conditioned media (CM) from DHT-treated RWPE-1 cells were transferred to HUVECs. Then, 6SL inhibited proliferation, VEGFR-2 activation, and tube formation of HUVECs transferred with CM from DHT-treated RWPE-1 cells. In the rat BPH model, 6SL reduced prostate weight, size, and thickness of the prostate tissue. Formation of vessels in prostatic tissues were also reduced with 6SL treatment. We found that 6SL has an ameliorative effect on in vitro and in vivo the BPH model via inhibition of VEGFR-2 activation and subsequent angiogenesis. These results suggest that 6SL might be a candidate for development of novel BPH drugs.
... A recent study showed that DHT could induce VEGF production by prostate cancer cells that in turn activated proliferation of ECs. 43 This effect could be defeated by estrogens. 44 The mechanism of VEGF induction in cancer cells may be important for the formation of tumor neovessels and further tumor propagation. ...
... For instance, estrogens are able to influence the effects of androgens on cell growth in ECs that do not express AR in a paracrine manner. 43 However, other androgen/estrogen-dependent vasoactive agents that explain the complexity of vasodilatory/vasoconstrictor effects of sex hormones on vascular endothelium, may exist. 39 ...
Cardiovascular effects of android hormones in normal and pathological conditions can lead to either positive or negative effects. The reason for this variation is unknown, but may be influenced by gender-specific effects of androids, heterogeneity of the vascular endothelium, differential expression of the androgen receptor in endothelial cells (ECs) and route of androgen administration. Generally, androgenic hormones are beneficial for ECs because these hormones induce nitric oxide production, proliferation, motility, and growth of ECs and inhibit inflammatory activation and induction of procoagulant, and adhesive properties in ECs. This indeed prevents endothelial dysfunction, an essential initial step in the development of vascular pathologies, including atherosclerosis. However, androgens can also activate endothelial production of some vasoconstrictors, which can have detrimental effects on the vascular endothelium. Androgens also activate proliferation, migration, and recruitment of endothelial progenitor cells (EPCs), thereby contributing to vascular repair and restoration of the endothelial layer. In this paper, we consider effects of androgen hormones on EC and EPC function in physiological and pathological conditions.
... Regarding the possible interaction with DHT and paracrine factors for vascular cells, a recent study suggests that DHT can induce endothelial cell growth through a paracrine mechanism that is mainly mediated by VEGF in prostate cancer cells [39]. In addition to such a paracrine system, the autocrine and lumicrine manner of DHT and secreted factors may be essential regulator for the maintenance of reproductive organs [40]. ...
The development of embryonic external genitalia (eExG) into characteristic male structures, such as urethra and penile erectile tissues, depends on 5α-dihydrotestosterone (DHT). Although the corpus cavernosum (CC) is well known as essential for erectile function in adults, its developmental process and its dependency on DHT have been unknown. To reveal the dimorphic formation of the murine CC from the embryonic stage, we first analyzed the production of the protein vascular endothelial growth factor receptor-2 (FLK1) via its expression (hereinafter referred as "expression of FLK1") and the expression of alpha-smooth muscle actin (ACTA2) and collagen type 1 (COL1A1) in developing external genitalia. The 5-α reductase type 2 encoded by the SRD5A2 gene has been suggested to be a crucial enzyme for male sexual differentiation, as it converts testosterone (T) into DHT in the local urogenital organs. In fact, SRD5A2 mutation results in decreased synthesis of DHT, which leads to various degrees of masculinized human external genitalia (ExG). We further investigated the expression profile of SRD5A2 during the formation of the murine CC. We observed that SRD5A2 was expressed in smooth muscle of the CC. To determine the role of SRD5A2 in CC formation, we analyzed the formation of erectile tissue in the male Srd5a2 KO mice and measured the levels of androgens in the ExG by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Intriguingly, there were no obvious defects in the CCs of male Srd5a2 KO mice, possibly due to increased T levels. The current study suggests possible redundant functions of androgens in CC development.
... However, in the cardiac setting T has been shown a detrimental effect on overall RV function in the context of increased afterload [16], the key role of androgen on modulating PA remodeling still lacks of evidence. In our previous studies, we demonstrated that DHT, which avoids the estrogen action of T participated in vascular angiogenesis [17], and we further explored whether endogenous androgen and exogenous DHT (5 mg/kg/d) repletion has regulatory effect on the progression of PAH. ...
Pulmonary arterial hypertension (PAH) is more popular among females than males. However, female patients exhibit better prognosis than men, sex hormones may partly explain such sex paradox. Estrogens are disease modifiers in PAH, androgen effects on PAH are yet incompletely characterized. In this study, we sought to determine the effect of androgen depletion and dihydrotestoterone (DHT) repletion on a rat model of monocrotaline-induced PAH (MCT-PH) and to further clarify the possible mechanisms. MCT-PH was induced in male Sprague-Dawley (SD) rats as well as castrated rats with or without concomitant DHT repletion. Our research showed that rats with PAH exhibited cardiopulmonary alterations including induction of right ventricular systolic pressure (RVSP), pulmonary vascular remodeling, right ventricular hypertrophy (RVH) and fibrosis. Moreover, MCT upregulated expression of vascular cell proliferative proteins (PCNA and Ki67), matrix metalloproteinase-2 (MMP-2) and apoptotic proteins (Bax and Bcl-2) in pulmonary artery, and promoted pro-inflammatory cytokines expression (IL-6, TNF-α and IL-1β) and oxidative stress level (SOD activity and MDA level) in perivascular lung tissue. The magnitude of these PAH-induced changes was significantly partly inhibited by castration. DHT replacement reversed castration-action on MCT-related cardiopulmonary alteration. We studied the detrimental effect of endogenous androgen and exogenous DHT in MCT-PH rats, which may be through stimulation of vascular cell proliferation, gelatinolytic activity, apoptosis, perivascular inflammation and oxidative stress.
... Our results show that particu-larly genistein and daidzein have different biological activities in prostate cancer cells when they depend on androgen signaling (49). Thus, the effect of these flavonoids in prostate cancer cells might be related to their estrogenic activity (50,51). Phloretin and apigenin are flavonoids that change their activity to a lesser extent between LNCaP vs LNCaP-R and PC-3 vs PC-3-AR cells, and this could be explained by their weaker estrogenic activity (52). ...
... For siRNA transfection, DU145 cells were seeded in a 96-well plate, or 12-well plate or 60-mm dishes in phenol red-free DMEM containing 5% stripped FBS without antibiotics. Twenty-four hours later, the cells were transfected with various concentrations of siRNA (10, 50 and 100 nM) using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen) in OPTI-MEM medium as previously described (24,25). Five hours after transfection, transfection reagents were replaced with DMEM medium and cells were treated with or without various dose NSC for different durations as indicated in each experiment. ...
Castration-resistant prostate cancer (CRPC) is a major cause of prostate cancer (Pca) death. Chemotherapy is able to improve the survival of CRPC patients. We previously found that NSC606985 (NSC), a highly water-soluble camptothecin analog, induced cell death in Pca cells via interaction with topoisomerase 1 and activation of the mitochondrial apoptotic pathway. To further elucidate the role of NSC, we studied the effect of NSC on ER-stress and its association with NSC-induced cell death in Pca cells. NSC produced a time- and dose-dependent induction of GRP78, CHOP and XBP1s mRNA, and CHOP protein expression in Pca cells including DU145, indicating an activation of ER-stress. However, unlike conventional ER-stress in which GRP78 protein is increased, NSC produced a time- and dose-dependent U-shape change in GRP78 protein in DU145 cells. The NSC-induced decrease in GRP78 protein was blocked by protease inhibitors, N-acetyl-L-leucyl-L-leucylnorleucinal (ALLN), a lysosomal protease inhibitor, and epoxomicin (EPO), a ubiquitin-protease inhibitor. ALLN, but not EPO, also partially inhibited NSC-induced cell death. However, both 4-PBA and TUDCA, two chemical chaperons that effectively reduced tunicamycin-induced ER-stress, failed to attenuate NSC-induced GRP78, CHOP and XBP1s mRNA expression and cell death. Moreover, knockdown of NSC induction of CHOP expression using a specific siRNA had no effect on NSC-induced cytochrome c release and NSC-induced cell death. These results suggest that NSC produced an atypical ER-stress that is dissociated from NSC-induced activation of the mitochondrial apoptotic pathway and NSC-induced cell death in DU145 prostate cancer cells.
... Cell proliferation assay. Cell proliferation assays were performed using HUVEC essentially as described (26)(27)(28). HUVECs were resuspended to a density of 2x10 4 /ml. ...
A recent discovery revealed that microRNAs (miRNAs) have an essential effect in the development and progression of gastric cancer (GC). It has already been shown that miR‑125a may inhibit tumor development by targeting human epidermal growth factor receptor-2 (Her-2) in GC; however, the other roles of miR‑125a in gastric cancer remained to be explored. Our study confirmed that miR‑125a was indeed capable of modulating the expression of VEGF-A in gastric cancer cells. In vitro, low expression of miR‑125a was able to maintain the secretion of VEGF-A, while the latter increased Akt phosphorylation level in endothelial cells and thereby promoted the proliferation, migration and angiogenesis of human umbilical vein endothelial cells (HUVECs). Our investigation showed that miR‑125a expression decreased significantly in gastric cancer comparing with normal gastric tissue and was negatively correlated with the expression of VEGF-A (P<0.05). In vivo, the expression of miR‑125a was inversely proportional to microvessel density (MVD) (r=-0.5382, P<0.001). The results of this study suggested that low expression of miR‑125a predict a worse survival in gastric cancer patients. Collectively, our results indicated that miR‑125a regulated the paracrine of VEGF-A in gastric cancer and thereby controlled the angiogenesis of the tumor.
... Supporting this hypothesis, withdrawal of androgenic signaling using AR antagonists (e.g., flutamide and bicalutamide) or inhibitors of steroid metabolism (e.g., finasteride and dutasteride) reduced hematuria during prostate surgery or in patients with benign prostatic hyperplasia (BPH) and in combination therapy with bicalutamide-goserelin (a gonadotropin-releasing hormone agonist) and dutasteride (an inhibitor of 5a-reductase isoenzymes types 1 and 2) induced profound vascular collapse and reduced prostatic tissue vascularity in human CaP patients (Godoy et al. 2013). Results from other studies also indicated that prostate tumor cells regulated EC growth through a paracrine mechanism, which was mainly mediated by VEGF, and demonstrated that DHT was able to modulate EC growth via tumor cells (Wen et al. 2013) and that the induction of VEGF was mediated by binding of the transcription factor AR and SP1 to the core promoter region of VEGF (Eisermann et al. 2013). ...
Androgen receptor (AR) is a ligand-inducible transcription factor, and a member of the steroid-thyroid-retinoid receptor superfamily, that mediates the biological effects of androgens in a wide range of physiological and pathological processes. Androgen receptor (AR) expression was identified in vascular cells nearly 20 years ago, and recent research showed that AR mediates a variety of actions of androgens in endothelial and vascular smooth muscle cells. In this mini-review, we review evidence indicating the importance of AR in human endothelial cell homeostatic and pathogenic processes. While a role for AR in modulation of human endothelial cell biology is evident, the molecular mechanisms by which AR regulates human endothelial cell homeostasis and disease processes are not fully understood. Understanding these mechanisms could provide critical insights into the pathogenesis of disease processes ranging from cardiovascular disease to cancer that are major causes of human morbidity and mortality.
... Our results show that particu-larly genistein and daidzein have different biological activities in prostate cancer cells when they depend on androgen signaling (49). Thus, the effect of these flavonoids in prostate cancer cells might be related to their estrogenic activity (50,51). Phloretin and apigenin are flavonoids that change their activity to a lesser extent between LNCaP vs LNCaP-R and PC-3 vs PC-3-AR cells, and this could be explained by their weaker estrogenic activity (52). ...
Cancer cells show different metabolic requirements than normal cells. In prostate cancer, particularly, glycolytic metabolism differs in androgen-responsive and nonresponsive cells. In addition, some natural compounds with antiproliferative activities are able to modify glucose entry into cells by either modulating GLUT transporters expression or by altering glucose binding. The aim of this work was to study the regulation of some glucose transporters -GLUT1 and GLUT4- in both androgen-sensitive (LNCaP) and insensitive (PC-3) prostate cancer cells by four structurally different flavonoids (i.e. genistein, phloretin, apigenin and daidzein). Glucose uptake was measured using nonradiolabeled 2-deoxyglucose. The evaluation of protein levels as well as subcellular distribution of GLUT1/4 were analyzed by western blot and immunocytochemistry respectively. Androgen-insensitive LNCaP-R and androgen-sensitive PC-3-AR cells were used to study the effect of androgen signaling. Additionally a docking simulation was employed to compare interactions between flavonoids and XylE, a bacterial homologue of GLUT 1-4. Results show for the first time the presence of functionally relevant GLUT4 transporter in prostate cancer cells. Furthermore differences in GLUT1 and GLUT4 levels and glucose uptake were found, without differences on subcellular distribution, after incubation with flavonoids. Docking simulation showed that all compounds interact with the same location of transporters. More importantly, differences between androgen sensitive and insensitive prostate cancer cells were found in both glucose transporter protein levels and glucose uptake. Thus, phenotypic characteristics of prostate cancer cells are responsible for the different effect of these flavonoids in glucose uptake and in GLUTs expression rather than their structural differences, being the most effective in reducing cell growth the highest in modifying glucose uptake and GLUT transporters levels.
Background
Androgens, the known drivers of prostate cancer (PCa), have been indicated as important metabolic regulators with a relevant role in stimulating lipid metabolism. Also, the relationship between obesity and the aggressiveness of PCa has been established. However, it is unknown if the androgenic hormonal environment may alter the response of PCa cells to lipid availability.
Purpose
The present study evaluated the effect of 5α-dihydrotestosterone (DHT) in regulating lipid metabolism, and the interplay between this hormone and low-density lipoprotein (LDL)-cholesterol in modulating PCa cells fate.
Methods
Non-neoplastic and neoplastic PCa cells were treated with 10 nM DHT, and the expression of fatty acids transporter, fatty acid synthase (FASN), and carnitine palmitoyltransferase 1 A (CPT1A) evaluated. PCa cells were also exposed to LDL (100 μg/ml) in the presence or absence of DHT.
Results
Treatment with DHT upregulated the expression of FASN and CPT1A in androgen-sensitive PCa cells. In contrast, LDL supplementation suppressed FASN expression regardless of the presence of DHT, whereas augmenting CPT1A levels. Our results also showed that LDL-cholesterol increased PCa cells viability, proliferation, and migration dependently on the presence of DHT. Moreover, LDL and DHT synergistically enhanced the accumulation of lipid droplets in PCa cells.
Conclusions
The obtained results show that androgens deregulate lipid metabolism and enhance the effects of LDL increasing PCa cells viability, proliferation and migration. The present findings support clinical data linking obesity with PCa and first implicate androgens in this relationship. Also, they sustain the application of pharmacological approaches targeting cholesterol availability and androgens signaling simultaneously.
Sex steroids such as estrogen and testosterone are key mediators of angiogenesis. They are implicated in both physiological and pathological angiogenesis such as during the menstrual cycle, wound healing and cancer growth and progression. Sex steroids regulate many aspects of angiogenesis through both classic genomic transcription modulation and rapid non-genomic signaling pathways. In this capacity, sex steroids modulate endothelial and progenitor cell functions such as proliferation, migration and attachment, which are all essential components involved in neovascularization. Since sex steroids are known to augment angiogenesis which is vital to tumor progression and growth, common treatment of hormone responsive tumors is through sex steroid receptor antagonists or hormone deprivation. Due to the involvement of sex steroids in necessary physiological functions as well as the potential to promote pathological angiogenesis, it is fundamental that the mechanisms behind sex steroid-mediated neovascularization are understood.
Objective: This study investigated the anti-prostate cancer activity of sika deer (Cervus nippon) velvet antler (DVA) extract on the expression of Prostate-Specific Antigen (PSA) and migration-related genes in LNCaP human prostate cancer cells. Methodology: The DVA was divided into three sections: Upper DVA (U-DVA), middle DVA (M-DVA) and bottom DVA (B-DVA) and an aqueous extract was prepared. The U-DVA was non-toxic up to 1,000 μg mL⁻¹ to RWPE-1 transformed human prostate cells. The U-DVA (125-1,000 μg mL⁻¹) which exhibited the most potent ORAC activity, attenuated the migration of LNCaP cells. Results: In addition, U-DVA inhibited the expression of PSA and migration-related genes such as matrix metalloproteinase-9 and vascular endothelial growth factor. In contrast, U-DVA stimulated the expression of tissue inhibitor of metalloproteinase TIMP-1 and TIMP-2. Conclusion: This is the first report of DVA having an effect on a human-specific cancer. These results indicate that U-DVA possesses anti-prostate cancer potential via modulation of several genes. Further investigations to explore individual bioactive compounds in U-DVA that are responsible for these in vivo effects as well as clinical trials in humans are needed.
It remains unclear whether estrogen is produced in prostate cancer (PCa) and how it functions in PCa.
To examine the production of estrogen in PCa cells, the concentration of estrogen in the medium in which LNCaP cells and PCa-derived stromal cells (PCaSC) were co-cultured, was measured by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), while aromatase (CYP19) mRNA expression was confirmed by real-time polymerase chain reaction (RT-PCR) methods. To verify whether estrogen is synthesized from testosterone in PCaSC functions, PCaSC were co-cultured with breast cancer MCF-7-E10 cells, which were stably-transfected with ERE-GFP, in the presence of testosterone. GFP expression was detected when PCaSCs could synthesize estrogen. The proliferation of PC-3 cells in the presence of PCaSC was determined by cell count.
PCaSC metabolized excessive testosterone to estrogen, which activated estrogen receptor in breast cancer cells. Moreover, estrogen synthesized from testosterone in PCaSC regulated the proliferation of PC-3 cell via repression of some unknown growth factors that were secreted from PCaSC.
A chimeric co-culture method between breast cancer cells and PCaSC revealed the production of active estrogen in PCaSC. High-dose testosterone therapy might introduce a new potential strategy to treat CRPC.
Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.
Vascular endothelial growth factor (VEGF) is one of the most important inducers of angiogenesis, therefore blocking angiogenesis has led to great promise in the treatment of various cancers and inflammatory diseases. VEGF, expressed in response to soluble mediators such as cytokines and growth factors, is important in the physiological development of blood vessels as well as development of vessels in tumors. In cancer patients VEGF levels are increased, and the expression of VEGF is associated with poor prognosis in diseases. VEGF is a mediator of angiogenesis and inflammation which are closely integrated processes in a number of physiological and pathological conditions including obesity, psoriasis, autoimmune diseases and tumor. Mast cells can be activated by anti-IgE to release potent mediators of inflammation and can also respond to bacterial or viral antigens, cytokines, growth factors and hormones, leading to differential release of distinct mediators without degranulation. Substance P strongly induces VEGF in mast cells, and IL-33 contributes to the stimulation and release of VEGF in human mast cells in a dose-dependent manner and acts synergistically in combination with Substance P. Here we report a strong link between VEGF and mast cells and we depict their role in inflammation and immunity.
A recent explosion in newly discovered vascular growth factors has coincided
with exploitation of powerful new genetic approaches for studying vascular
development. An emerging rule is that all of these factors must be used in
perfect harmony to form functional vessels. These new findings also demand
re-evaluation of therapeutic efforts aimed at regulating blood vessel growth
in ischaemia, cancer and other pathological settings.
Growing evidences support that androgen displays beneficial effects on cardiovascular functions although the mechanism of androgen actions remains to be elucidated. Modulation of endothelial cell growth and function is a potential mechanism of androgen actions. We demonstrated in the present study that androgens [dihydrotestosterone (DHT) and testosterone], but not 17β-estradiol, produced a time- and dose-dependent induction of cell proliferation in primary human aortic endothelial cells (HAECs) as evident by increases in viable cell number and DNA biosynthesis. Real-time qRT-PCR analysis showed that DHT induced androgen receptor (AR), cyclin A, cyclin D1, and vascular endothelial growth factor (VEGF) gene expression in a dose- and time-dependent manner. The addition of casodex, a specific AR antagonist, or transfection of a specific AR siRNA blocked DHT-induced cell proliferation and target gene expression, indicating that the DHT effects are mediated via AR. Moreover, coadministration of SU5416 to block VEGF receptors, or transfection of a specific VEGF-A siRNA to knockdown VEGF expression, produced a dose-dependent blockade of DHT induction of cell proliferation and cyclin A gene expression. Interestingly, roscovitine, a selective cyclin-dependent kinase inhibitor, also blocked the DHT stimulation of cell proliferation with a selective inhibition of DHT-induced VEGF-A expression. These results indicate that androgens acting on AR stimulate cell proliferation through upregulation of VEGF-A, cyclin A, and cyclin D1 in HAECs, which may be beneficial to cardiovascular functions since endothelial cell proliferation could assist the repair of endothelial injury/damage in cardiovascular system.
Clinical data and models of human disease indicate that androgen receptor (AR) activity is essential for prostate cancer development, growth, and progression. The dependence of prostatic adenocarcinoma on AR signaling at all stages of disease has thereby been exploited in the treatment of disseminated tumors, for which ablation of AR function is the goal of first-line therapy. Although these strategies are initially effective, recurrent tumors arise with restored AR activity, and no durable treatment has yet been identified to combat this stage of disease. New insights into AR regulation and the mechanisms underlying resurgent AR activity have provided fertile ground for the development of novel strategies to more effectively inhibit receptor activity and prolong the transition to therapeutic failure.
Prostate cancer remains the most common non-cutaneous malignancy among American men. Since the advent of PSA testing, most men are diagnosed with localized disease, but a proportion of men will be diagnosed with metastatic disease, many will eventually receive chemotherapy with docetaxel and prednisone. However, responses are not durable and all men will ultimately progress on this treatment. As such, continued efforts are geared towards the discovery of new agents and mechanisms of targeting prostate cancer. Angiogenesis has been shown to play an important role in tumorigenesis, proliferation and metastasis in prostate cancer. Here we discuss the major angiogenic signaling pathway involving VEGF in prostate cancer progression and the role of various promising agents that targets this pathway. This includes bevacizumab, thalidomide and its analogues, tyrosine kinase inhibitors sorafenib and AZD2171, and other inhibitors of angiogenic signaling pathways. Results of key clinical trials associated with the use of these agents and future directions are discussed herein.
To better understand direct and indirect androgen action on rat prostatic growth and function, the various cell populations within the intact adult ventral, dorsal, and lateral prostate lobes were characterized for the presence or absence of androgen receptor (AR). Polyclonal rabbit antibodies raised against amino acids 1-21 of the rat AR (PG-21) were used in combination with a library of monoclonal antibodies directed against cell-specific antigens for positive cellular identification. Luminal epithelial cells were strongly AR positive, with an order of ventral greater than lateral greater than or equal to dorsal. In the lateral lobe, staining intensity was strongest in the peripheral regions, whereas a similar gradient was not apparent in the ventral and dorsal prostate. Basal epithelial cells were AR negative in all regions of the three lobes. Periacinar smooth muscle was strongly positive for AR, and this staining did not vary with the thickness of the muscle layer. Endothelial cells of the vasculature were AR negative, while the perivascular smooth muscle cells were AR positive. The majority of stromal fibroblasts were AR negative, although a number of AR-positive fibroblastic-appearing cells were observed within the ventral and dorsal lobes. Staining with ED2, a specific marker for tissue macrophages, revealed that fixed macrophages were present in significant quantities in the stroma of intact rat prostate lobes. Since these were frequently identified as AR positive, macrophages may partially account for the appearance of AR-positive stromal cells. Thus, the present findings indicate a complex pattern of AR expression among different cell types of the three prostate lobes. Cells types that express AR can potentially be considered as direct targets of androgen action, whereas those lacking AR should be considered as indirect targets or androgen-insensitive cells.
The castration-induced regression and testosterone stimulated regrowth of the vasculature in the rat ventral prostate lobe were studied using stereological techniques. Seven days after castration, the endothelial cell proliferation rate (bromodeoxyuridine labeling index); the total weights of blood vessel walls, blood vessel lumina, endothelial cells, glandular epithelial cells; and total organ weight were all decreased. Within 2 days after sc treatment with testosterone, the total weights of blood vessel walls, endothelial cells, and vascular lumina, as well as the endothelial cell proliferation rate, were all normalized. In contrast to the rapid response of the vasculature, the total weight of glandular epithelium and total organ weight were not normalized during the 4 days of testosterone treatment. Growth of the vasculature apparently precedes growth of the glandular epithelium. The testosterone- dependent factors stimulating the vasculature are unknown, but factors derived from epithelial cells, mast cells (which accumulate in the prostate during the first day of testosterone treatment), and tissue macrophages could all be involved. Castration-induced regression and testosterone-stimulated regrowth of the prostatic vasculature can be used as an experimental model to study factors regulating angiogenesis and organ growth in the prostate.
The sequence of events that leads to tumor vessel regression and the functional characteristics of these vessels during hormone-ablation therapy are not known. This is because of the lack of an appropriate animal model and monitoring technology. By using in vivo microscopy and in situ molecular analysis of the androgen-dependent Shionogi carcinoma grown in severe combined immunodeficient mice, we show that castration of these mice leads to tumor regression and a concomitant decrease in vascular endothelial growth factor (VEGF) expression. Androgen withdrawal is known to induce apoptosis in Shionogi tumor cells. Surprisingly, tumor endothelial cells begin to undergo apoptosis before neoplastic cells, and rarefaction of tumor vessels precedes the decrease in tumor size. The regressing vessels begin to exhibit normal phenotype, i.e., lower diameter, tortuosity, vascular permeability, and leukocyte adhesion. Two weeks after castration, a second wave of angiogenesis and tumor growth begins with a concomitant increase in VEGF expression. Because human tumors often relapse following hormone-ablation therapy, our data suggest that these patients may benefit from combined anti-VEGF therapy.
Estrogens influence the differentiation and maintenance of reproductive tissues and affect lipid metabolism and bone remodeling. Two estrogen receptors (ERs) have been identified to date, ERalpha and ERbeta. We previously generated and studied knockout mice lacking estrogen receptor alpha and reported severe reproductive and behavioral phenotypes including complete infertility of both male and female mice and absence of breast tissue development. Here we describe the generation of mice lacking estrogen receptor beta (ERbeta -/-) by insertion of a neomycin resistance gene into exon 3 of the coding gene by using homologous recombination in embryonic stem cells. Mice lacking this receptor develop normally and are indistinguishable grossly and histologically as young adults from their littermates. RNA analysis and immunocytochemistry show that tissues from ERbeta -/- mice lack normal ERbeta RNA and protein. Breeding experiments with young, sexually mature females show that they are fertile and exhibit normal sexual behavior, but have fewer and smaller litters than wild-type mice. Superovulation experiments indicate that this reduction in fertility is the result of reduced ovarian efficiency. The mutant females have normal breast development and lactate normally. Young, sexually mature male mice show no overt abnormalities and reproduce normally. Older mutant males display signs of prostate and bladder hyperplasia. Our results indicate that ERbeta is essential for normal ovulation efficiency but is not essential for female or male sexual differentiation, fertility, or lactation. Future experiments are required to determine the role of ERbeta in bone and cardiovascular homeostasis.
Recent studies have found that blood flow to the rat ventral prostate gland is drastically reduced at an early time after castration. These observations caused us to reevaluate the effects of castration on the various cell populations of the ventral prostate, especially those in the prostatic vascular system. Sections of ventral prostate glands obtained at different times after castration were analyzed using the TUNEL (terminal deoxynucleotide transferase-mediated dUTP nick END labeling) staining method to quantify apoptosis in different cell types. The results of this analysis showed a significant increase in TUNEL staining of prostate endothelial and (nonendothelial) stromal cells as early as 12 h postcastration that continued to 24 h after castration. In contrast, TUNEL labeling of prostate epithelial cells was not significantly increased compared with control values until 72 h after castration. The use of dual immunohistochemical staining procedures (anti-CD31 for endothelial cells or antismooth muscle actin for smooth muscle cells combined with TUNEL labeling) allowed us to confirm that the TUNEL-positive vascular cells at these early times after castration were endothelial in nature, whereas smooth muscle cells surrounding the prostate glands or portions of the afferent vascular endothelium were rarely TUNEL labeled. Electron microscopic evaluation of ventral prostate tissues at 48 h after castration provided further morphological evidence for the occurrence of apoptosis in prostate endothelial cells. Finally, the Lendrum-Fraser histochemical procedure used to identify fibrin leakage in tissues with vascular damage was applied to sections of the ventral prostate gland. This stain revealed diffuse fibrin accumulation in periglandular areas outside the capillaries and blood vessels in prostates from 24-h castrated rats, but not in prostates of sham-operated rats. Our results confirm an early effect of castration on the vascular system of the rat ventral prostate identified by increased apoptosis of endothelial cells and vascular leakiness. As these changes temporally precede the loss of epithelial cells, we propose that they may be causal rather than incidental to regression of the rat ventral prostate after castration.
In normal rats and mice, immunostaining with specific antibodies revealed that nuclei of most prostatic epithelial cells harbor estrogen receptor beta (ERbeta). In rat ventral prostate, 530- and 549-aa isoforms of the receptor were identified. These sediment in the 4S region of low-salt sucrose gradients, indicating that prostatic ERbeta does not contain the same protein chaperones that are associated with ERalpha. Estradiol (E(2)) binding and ERbeta immunoreactivity coincide on the gradient, with no indication of ERalpha. In prostates from mice in which the ERbeta gene has been inactivated (BERKO), androgen receptor (AR) levels are elevated, and the tissue contains multiple hyperplastic foci. Most epithelial cells express the proliferation antigen Ki-67. In contrast, prostatic epithelium from wild-type littermates is single layered with no hyperplasia, and very few cells express Ki-67. Rat ventral prostate contains an estrogenic component, which comigrates on HPLC with the testosterone metabolite 5alpha-androstane-3beta,17beta-diol (3betaAdiol). This compound, which competes with E(2) for binding to ERbeta and elicits an estrogenic response in the aorta but not in the pituitary, decreases the AR content in prostates of wild-type mice but does not affect the elevated levels seen in ERbeta knockout (BERKO) mice. Thus ERbeta, probably as a complex with 3betaAdiol, is involved in regulating the AR content of the rodent prostate and in restraining epithelial growth. These findings suggest that ligands specific for ERbeta may be useful in the prevention and/or clinical management of prostatic hyperplasia and neoplasia.
Normal adult prostate epithelium of both human and rat origin was transplanted with Matrigel into intact or androgen-ablated (i.e., castrated) nude mice. Within these transplants, an influx of mouse mesenchymal cells was one of the earliest events to occur resulting in the development of a collar of smooth muscle cells and fibroblasts surrounding the transplanted epithelium. A subset of these surrounding stromal cells express androgen receptor (AR). The surrounded transplanted epithelium initially expresses high molecular weight cytokeratins characteristic of prostatic basal cells and AR. In both intact and androgen-ablated hosts, this epithelium subsequently develops a patent lumen producing a rudimentary glandular acini. Only in the nonablated hosts, however, do these rudimentary acini undergo a further proliferative growth phase, as determined by Ki67 immunocytochemical stainings and the development of a low molecular weight cytokeratin positive layer of luminal (i.e., secretory) epithelial cells. Because AR is expressed in both the donor epithelium and host (i.e., mouse) stromal cells, this androgen-stimulated growth response could involve either autocrine pathways initiated within donor normal adult epithelial cells themselves or paracrine pathways initiated within the AR-expressing subset of mouse stromal cells. To resolve this issue, mice carrying the testicular feminized mutation in the X-linked AR gene were cross-bred to AR-wt nude mice to produce AR-null nude male mice. None of the cells in these AR-null nude male mice express functional AR protein. Therefore, these animals can be used to prevent any possibility of host stromal cell paracrine involvement in initiating an androgen-stimulated growth response when normal adult or malignant prostatic epithelial cells are transplanted into these null hosts. In these AR-null nude male mice, the androgen-stimulated growth of normal adult prostatic epithelial cells did not occur (i.e., androgen-induced growth response of normal prostatic epithelial cells requires stromal cell paracrine involvement). In contrast, using four different prostatic cancer models (i.e., human PC-82, human LNCaP, human LAPC-4, and rat R3327G), the androgen-stimulated growth of prostatic cancer cells occurred identically in both AR-null and AR-wt nude male mice (i.e., a direct autocrine mechanism is responsible for androgen-stimulated growth of malignant prostatic epithelial cells). In summary, a fundamental change in the mechanism for androgen-stimulated growth occurs during the transformation from normal to malignant prostatic epithelial cells.
Often those diseases most evasive to therapeutic intervention usurp the human body's own cellular machinery or deregulate normal physiological processes for propagation. Tumor-induced angiogenesis is a pathological condition that results from aberrant deployment of normal angiogenesis, an essential process in which the vascular tree is remodeled by the growth of new capillaries from preexisting vessels. Normal angiogenesis ensures that developing or healing tissues receive an adequate supply of nutrients. Within the confines of a tumor, the availability of nutrients is limited by competition among actively proliferating cells, and diffusion of metabolites is impeded by high interstitial pressure (Jain RK. Cancer Res 47: 3039-3051, 1987). As a result, tumor cells induce the formation of a new blood supply from the preexisting vasculature, and this affords tumor cells the ability to survive and propagate in a hostile environment. Because both normal and tumor-induced neovascularization fulfill the essential role of satisfying the metabolic demands of a tissue, the mechanisms by which cancer cells stimulate pathological neovascularization mimic those utilized by normal cells to foster physiological angiogenesis. This review investigates mechanisms of tumor-induced angiogenesis. The strategies used by cancer cells to develop their own blood supply are discussed in relation to those employed by normal cells during physiological angiogenesis. With an understanding of blood vessel growth in both normal and abnormal settings, we are better suited to design effective therapeutics for cancer.
Although pre-menopausal women enjoy relative cardiovascular protection, hormone (oestrogen+/-progestin)-replacement therapy has not shown cardiovascular benefits in post-menopausal women, suggesting that the effects of oestrogens on the cardiovascular system are much more complex than previously expected. Endothelial cells, smooth muscle cells, cardiac myocytes and fibroblasts, the cellular components of blood vessels and the heart, play important roles in cardiovascular health and disease. During the development and progression of cardiovascular disease, changes occur both in the structure and function of these cells, resulting in a wide range of abnormalities, which affect growth, death and physiological function. These cells contain functional oestrogen receptors and are targets for oestrogen action. This review focuses on recent studies on the effects of oestrogen on cardiovascular cell function. Oestrogens, particularly 17beta-oestradiol, exert multiple effects on cardiovascular cells, and these effects may contribute to the gender-associated protection against cardiovascular diseases.
The castration-induced regression and testosterone stimulated regrowth of the vasculature in the rat ventral prostate lobe were studied using stereological techniques. Seven days after castration, the endothelial cell proliferation rate (bromodeoxyuridine labeling index); the total weights of blood vessel walls, blood vessel lumina, endothelial cells, glandular epithelial cells; and total organ weight were all decreased. Within 2 days after sc treatment with testosterone, the total weights of blood vessel walls, endothelial cells, and vascular lumina, as well as the endothelial cell proliferation rate, were all normalized. In contrast to the rapid response of the vasculature, the total weight of glandular epithelium and total organ weight were not normalized during the 4 days of testosterone treatment. Growth of the vasculature apparently precedes growth of the glandular epithelium. The testosterone- dependent factors stimulating the vasculature are unknown, but factors derived from epithelial cells, mast cells (which accumulate in the prostate during the first day of testosterone treatment), and tissue macrophages could all be involved. Castration-induced regression and testosterone-stimulated regrowth of the prostatic vasculature can be used as an experimental model to study factors regulating angiogenesis and organ growth in the prostate.
The vasculature plays an important role in the normal and malignant prostate. Under basal conditions both glandular epithelial and stromal prostate cells produce an abundance of blood flow and angiogenesis regulating substances and the expression of these is generally increased in prostate tumors. The proportion of proliferating endothelial cells is high in the normal prostate compared to other tissues in the body. After castration effects on the vasculature, such as decreased blood flow and vascular regression, precede effects on the glandular compartment. Correspondingly, hormone induced prostate growth is characterized by early effects on the vasculature such as increased blood flow and endothelial cell proliferation, thus indicating that the vasculature may be involved in the androgenic regulation of the prostate. Prostatic intraepithelial neoplasia (PIN) and prostate cancer are associated with increased vascular density and in experimental models prostate cancer growth is apparently angiogenesis-depe...
Recent studies have found that blood flow to the rat ventral prostate gland is drastically reduced at an early time after castration. These observations caused us to reevaluate the effects of castration on the various cell populations of the ventral prostate, especially those in the prostatic vascular system. Sections of ventral prostate glands obtained at different times after castration were analyzed using the TUNEL (terminal deoxynucleotide transferase-mediated dUTP nick END labeling) staining method to quantify apoptosis in different cell types. The results of this analysis showed a significant increase in TUNEL staining of prostate endothelial and (nonendothelial) stromal cells as early as 12 h postcastration that continued to 24 h after castration. In contrast, TUNEL labeling of prostate epithelial cells was not significantly increased compared with control values until 72 h after castration. The use of dual immunohistochemical staining procedures (anti-CD31 for endothelial cells or antismooth muscle actin for smooth muscle cells combined with TUNEL labeling) allowed us to confirm that the TUNEL-positive vascular cells at these early times after castration were endothelial in nature, whereas smooth muscle cells surrounding the prostate glands or portions of the afferent vascular endothelium were rarely TUNEL labeled. Electron microscopic evaluation of ventral prostate tissues at 48 h after castration provided further morphological evidence for the occurrence of apoptosis in prostate endothelial cells. Finally, the Lendrum-Fraser histochemical procedure used to identify fibrin leakage in tissues with vascular damage was applied to sections of the ventral prostate gland. This stain revealed diffuse fibrin accumulation in periglandular areas outside the capillaries and blood vessels in prostates from 24-h castrated rats, but not in prostates of sham-operated rats. Our results confirm an early effect of castration on the vascular system of the rat ventral prostate identified by increased apoptosis of endothelial cells and vascular leakiness. As these changes temporally precede the loss of epithelial cells, we propose that they may be causal rather than incidental to regression of the rat ventral prostate after castration.
Androgen independent prostate cancer growth and metastasis are a major cause of prostate cancer death. Aberrant androgen receptor activation due to androgen receptor mutation is an important mechanism of androgen independence. We determined the effectiveness and mechanism of 17α-estradiol (Sigma®) in blocking aberrant androgen receptor activation due to androgen receptor mutation.
We used LNCaP and MDA Pca-2b prostatic tumor cells (ATCC®) containing a mutated androgen receptor and WT estrogen receptor β to test 17α-estradiol inhibition of aberrant androgen receptor activation of prostate specific antigen gene expression and cell growth. Cotransfection analysis was used to further elucidate the mechanism of 17α-estradiol action. Xenograft animals with an LNCaP prostate tumor were prepared to study the in vivo effect of 17α-estradiol on tumor growth inhibition.
In LNCaP cells 17α-estradiol produced a dose dependent inhibition of cyproterone acetate (Sigma) or dihydrotestosterone induced prostate specific antigen gene expression. In MDA Pca-2b cells 17α-estradiol inhibited cortisol (Sigma) induced prostate specific antigen expression and blocked dihydrotestosterone and cortisol induced cell proliferation in LNCaP and MDA Pca-2b cells, respectively. Cotransfection analysis showed that 17α-estradiol inhibition of aberrant androgen receptor activation of prostate specific antigen gene expression was medicated via estrogen receptors. In xenograft mice with LNCaP prostate cancer 17α-estradiol but not 17β-estradiol (Sigma) significantly inhibited tumor growth, although each estrogen tended to decrease tumor growth.
Results suggest that 17α-estradiol with less classic estrogenic activity is a potential therapeutic agent for androgen independent prostate cancer due to androgen receptor mutation.
Solid tumors can be thought of as multicellular 'organs' that consist of a variety of cells as well as a scaffold of noncellular matrix. Stromal-epithelial crosstalk is integral to prostate cancer progression and metastasis, and androgen signaling is an important component of this crosstalk at both the primary and metastatic sites. Intratumoral production of androgen is an important mechanism of castration resistance and has been the focus of novel therapeutic approaches with promising results. Various other pathways are important for stromal-epithelial crosstalk and represent attractive candidate therapeutic targets. Hedgehog signaling has been associated with tumor progression, growth and survival, while Src family kinases have been implicated in tumor progression and in regulation of cancer cell migration. Fibroblast growth factors and transforming growth factor beta signaling regulate cell proliferation, apoptosis and angiogenesis in the prostate cancer microenvironment. Integrins mediate communication between the cell and the extracellular matrix, enhancing growth, migration, invasion and metastasis of cancer cells. The contribution of stromal-epithelial crosstalk to prostate cancer initiation and progression provides the impetus for combinatorial microenvironment-targeting strategies.
It is becoming increasingly clear that angiogenesis plays a crucial role in prostate cancer (CaP) survival, progression, and metastasis. Tumor angiogenesis is a hallmark of advanced cancers and an attractive treatment target in multiple solid tumors. By understanding the molecular basis of resistance to androgen withdrawal and chemotherapy in CaP, the rational design of targeted therapeutics is possible. This review summarizes the recent advancements that have improved our understanding of the role of angiogenesis in CaP metastasis and the potential therapeutic efficacy of inhibiting angiogenesis in this disease. Current therapeutic options for patients with metastatic hormone-refractory CaP are very limited. Targeting vasculature is a developing area, which shows promise for the control of late stage and recurrent CaP disease and for overcoming drug resistance. We discuss angiogenesis and its postulated mechanisms and focus on the regulation of angiogenesis in CaP progression and the therapeutic beneficial effects associated with targeting of the CaP vasculature to overcome the resistance to current treatments and CaP recurrence.
Androgens acting via the androgen receptor play critical roles in prostate development, growth, and pathogenesis. There are two potent androgens, testosterone and dihydrotestosterone (DHT), in humans and mammals. DHT is converted from testosterone by 5alpha-reductase isozymes. Two 5alpha-reductase isozymes have been identified. Although both isozymes are expressed, 5alpha-reductase-2 is the predominant isozyme in the human prostate. Mutations in 5alpha-reductase-2 gene cause the 5alpha-reductase-2 deficiency syndrome. Affected 46, XY individuals have a small, nonpalpable, and rudimentary prostate in adulthood. Neither benign prostate hyperplasia (BPH) nor prostate cancer has been reported in these patients. The prostate is small in animals with 5alpha-reductase-2 gene knockout or treated with specific 5alpha-reductase inhibitors. 5alpha-reductase isozymes are molecular targets for the prevention and treatment of BPH and prostate cancer. Moreover, androgen actions on prostate gene expression and cell growth are directly modulated by estrogen receptor ligands via protein-protein interactions. The studies of 5alpha-reductases and androgen actions highlight the importance of 5alpha-reductase isozymes in male sexual differentiation and prostate physiology and pathophysiology.
The critical role played by stroma-epithelium crosstalk in carcinogenesis and progression of prostate cancer has been increasingly recognized. These interactions are mediated by a variety of paracrine factors secreted by cancer cells and/or stromal cells. In human prostate cancer, reactive stroma is characterized by an increase in myofibroblasts and a corresponding amplification of extracellular matrix production and angiogenesis. Permanent genetic mutations have been reported in stromal cells as well as in tumour cells. Transforming growth factor-beta, vascular endothelial growth factor, platelet-derived growth factor and fibroblast growth factor signalling pathways are involved in the process of angiogenesis, whereas hepatocyte growth factor, insulin-like growth factor-1, epidermal growth factor, CXC12 and Interleukin-6 play active roles in the progression, androgen-independent conversion and distal metastasis of prostate cancer. Some soluble factors have reciprocal interactions with androgens and the androgen receptor (AR), and can even activate AR in the absence of the androgen ligand. In this article, we review the complex interactions between cancer cells and the surrounding microenvironment, and discuss the potential therapeutic targets in the stromal compartment of prostate cancer.
Our purpose in this paper is to present and summarize the available evidence pertaining to the association of noncontraceptive estrogens and cardiovascular disease. A particular focus will be the association of cardiovascular disease with the estrogens used for menopausal therapy. The association of cardiovascular disease and estrogens used for lactation suppression, pregnancy maintenance, and therapy for prostatic cancer and cardiovascular disease prophylaxis will also be examined. This review will not address the relationship between cardiovascular disease and estrogens used for contraceptive purposes because 1) comprehensive reviews of this topic already exist and 2) although estrogens and oral contraceptives are often used interchangeably in the medical literature, oral contraceptives are unlike noncontraceptive estrogens used for menopausal therapy.
The effect of the middle T oncogene of polyoma virus was studied in vivo using a replication-defective selectable retrovirus. Injection of virus into newborn and adult mice resulted in the rapid appearance of cavernous hemangiomas. Infection of embryos did not yield transgenic mice; therefore, embryonal stem (ES) cells were used as an alternative system. Several infected ES cell clones were established that constitutively expressed middle T and its associated tyrosine kinase activity. Chimeric embryos obtained by blastocyst injection of individual ES cell clones were specifically arrested at midgestation, when multiple hemangiomas disrupted blood vessel formation. From these tumors endothelial cell lines were established that retained expression of von Willebrand factor yet were tumorigenic in vivo. These results suggest that middle T acts in endothelial cells as a single-step oncogene and that ES cells provide a valuable system for the study of growth control during embryogenesis.
The effect of androgen depletion/repletion on the cell types of the rat ventral prostate has been studied using light microscope autoradiography and morphometric analysis. All prostatic cells were assigned to one of the following groups: epithelial, endothelial, stromal (periacinar), or stromal (interacinar). With the exception of the latter group, the numbers of all other cell types were significantly reduced by androgen depletion (castration), and increased by repletion thereafter. During the repletion period, the labeling index of all cell types rose in parallel and peaked 72 hours postinitiation of androgen replacement. The size of the proliferating pool just prior to the peak day was determined. Forty-five percent of the total prostatic cell population was found to be labeled. The epithelial cell group contained the greatest proportion of labeled cells, but significant percentages occurred in the other types. These results precisely define the temporal effects of androgen replacement on prostatic cell population dynamics in the castrate rat.
This article has no abstract; the first 100 words appear below.
THE growth of solid neoplasms is always accompanied by neovascularization. This new capillary growth is even more vigorous and continuous than a similar outgrowth of capillary sprouts observed in fresh wounds or in inflammation.¹ Many workers have described the association between growing solid malignant tumors and new vessel growth.²³⁴⁵⁶ However, it has not been appreciated until the past few years that the population of tumor cells and the population of capillary endothelial cells within a neoplasm may constitute a highly integrated ecosystem. In this ecosystem the mitotic index of the two cell populations may depend upon each other. Tumor cells . . .
Supported by a grant (5 RO1 CA08185–06) from the National Cancer Institute, a grant from the American Cancer Society, National Chapter (IC-28), and gifts from the Merck Company and the Alza Corporation.
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From the Department of Surgery, Children's Hospital Medical Center and Harvard Medical School, Boston, Massachusetts 02115.
Angiogenesis is a critical event in wound healing, tumor growth, and the inflammatory vasculitides. Since women have a higher incidence of many vasculitic diseases, we examined the effects of female sex steroids, particularly estradiol, on human umbilical vein endothelial cell (HUVEC) behavior in vitro and on angiogenesis in vivo.
HUVECs were grown in estrogen-free medium before each assay. Exogenous 17 beta-estradiol (1 to 5 nmol/L) increased cell attachment to laminin, types I and IV collagen, and fibronectin, as well as to tissue culture plastic. After a confluent monolayer of cells was "wounded" by scraping, estradiol-treated (10(-8) mol/L) cells migrated into the wound three times faster than untreated cells. Cell proliferation on plastic and on laminin increased threefold to fivefold, respectively, in the presence of estradiol. Estradiol also enhanced the ability of HUVECs to organize into tubular networks when plated on a reconstituted basement membrane, Matrigel. Estradiol effects on both the "wounding" assay and tube formation were blocked by the specific estrogen receptor antagonist ICI 182,780. Ovariectomy markedly decreased in vivo vascularization of Matrigel plugs coinjected with basic fibroblast growth factor in mice. With estrogen replacement, angiogenesis was increased to the levels observed in nonovariectomized mice.
These studies demonstrate that, in vitro and in vivo, estradiol enhances endothelial cell activities important in neovascularization and suggest a promoting influence of estrogens on angiogenesis.
Linomide is a p.o. active antiangiogenic agent that has been demonstrated to be effective in suppressing the in vivo growth of rat and human prostatic cancer xenografts. The present studies were conducted to determine whether the angiogenic molecules, vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) and basic fibroblast growth factor (bFGF) are expressed in vitro by DU-145, PC-3, TSU-PR1, and LnCaP human prostate cancer cell lines and whether Linomide inhibits the secretion of these angiogenic molecules. Additionally, two different androgen-responsive human prostatic cancer xenograft models (i.e., PC-82 and A-2) were used to determine whether androgen ablation-induced reduction in tumor growth is associated with a reduction in tumor VEGF and/or bFGF levels. These studies demonstrated that both VEGF and bFGF proteins are expressed to different degrees in the human prostatic cancer cell lines. The secretion of VEGF but not bFGF is up-regulated by hypoxia. Linomide is unable to inhibit either basal or hypoxia-induced secretion of VEGF. Linomide also has no effect on secreted bFGF levels. Castration inhibited tumor VEGF but had no effect on bFGF levels in both the androgen-responsive PC-82 and A-2 human prostatic cancers when grown in severe combined immunodeficient mice. When given in combination, castration potentiated the inhibition of tumor growth induced by Linomide alone. This potentiation is not due to a further inhibition in tumor VEGF levels induced by castration. Although both castration and Linomide inhibit angiogenesis, the former accomplishes it by inhibiting VEGF secretion, whereas the latter has multiple effects at several steps in the angiogenic process other than VEGF secretion. Based on their different but complementary mechanisms of action, simultaneous combination of androgen ablation with Linomide enhances the anti-prostatic cancer efficacy compared to either monotherapies alone and warrants testing in humans.
Background:
Androgenic steroids regulate the development and size of the mammalian prostate gland. The mechanism(s) for this growth control might involve a direct effect on prostate cell proliferation and survival as well as more complex effects on the tissue environment supporting nourishment and oxygenation. In this study, we evaluated an animal model of androgen action on the prostate, the rat ventral prostate gland, to determine whether acute androgen withdrawal, by means of castration, might alter the primary blood flow to the prostate gland and for the effects of castration on prostatic endothelial cell viability.
Methods:
Groups of rats studied included intact control males, males that had been surgically castrated, or males that received a sham-surgical castration. Relative blood flow (RBF) to the rat ventral prostate glands and rat bladders were measured at 18 and 24 hr after castration or sham castration using a fluorescent microsphere infusion technique. Thin sections from fixed and embedded rat ventral prostate glands obtained from unoperated or 12-hr castrated rats were analyzed by the TUNEL immunostaining technique to microscopically identify and quantify apoptotic epithelial, stromal, and endothelial cells.
Results:
RBF to the rat ventral prostate was reduced by 38%, at 18 hr after castration when compared with intact or sham-operated rats and by 45% at 24 hr after castration (P=0.038 unoperated/0.025 sham operated). In contrast, RBF to the bladder was not significantly different between any of the groups in the 24-hr castrate experiment. TUNEL staining analysis of ventral prostate tissues obtained from 12-hr castrated rats showed only rare TUNEL-positive epithelial cells similar to the control tissue but significantly increased TUNEL labeling for endothelial and other ventral prostate stromal cells.
Conclusions:
Castration resulted in a rapid and significant reduction of blood flow to the mature rat ventral prostate gland that was not seen in the bladder. This reduction precedes the appearance of apoptosis in the epithelial cells of the tissue but more coincided with the appearance of TUNEL-positive prostate vascular endothelial and stromal cells, suggesting that androgens support the survival of cells in the vascular and stromal compartment of the rat prostate as well as in the prostatic epithelium. These preliminary data support the concept that androgen action on the prostate might involve primary regulation of prostate blood flow and prostate vascular cell vitality.
In previous studies, we have demonstrated that androgen ablation-induced growth inhibition of androgen-responsive PC-82 and A-2 human prostate cancer xenografts involves not only direct activation of programmed (apoptotic) death of these cells but also indirect activation of this death process via a decrease in tumor angiogenesis secondary to a reduction in tumor vascular endothelial growth factor (VEGF) levels. To determine whether androgens consistently regulate angiogenesis via control of VEGF levels, an additional human (i.e., LnCaP) and two rodent (i.e., Dunning G and H) androgen-sensitive prostate cancer sublines were tested. Androgen ablation causes a decrease in the subsequent growth rate of each of these three additional prostate cancer sublines, and this growth inhibition is consistently associated with a >60% reduction in tumor VEGF levels. To examine whether androgens regulate VEGF levels not only in malignant but also in normal prostatic tissue, male rats were castrated, and the temporal changes in the VEGF content of ventral prostate tissue were determined. One week after castration, VEGF content decreased to <20% within the ventral prostate. Subsequent replacement with exogenous androgen to long-term castrated rats stimulated an 8-fold rise in ventral prostate VEGF content within 1 week. To evaluate whether androgen regulation of VEGF is due to a direct effect of androgen on prostatic cells, the dose-response ability of androgens to increase VEGF levels in media of LnCaP cells grown in vitro was tested. These studies demonstrate that androgens directly stimulate VEGF secretion in these cells. The presence of 4-5-fold higher levels of VEGF in prostatic fluid versus seminal vesicle fluid obtained from benign prostatic hyperplasia and clinically localized prostate cancer patients suggests that elevated levels of VEGF may contribute to the progression of these prostatic conditions by promoting angiogenesis. In summary, one of the mechanisms for androgen sensitivity for the control of the growth of both normal and malignant prostatic tissue is via its stimulation of VEGF levels.
Vascular endothelial growth factor (VEGF), an important regulator of endothelial cell physiology, was identified some 10 years ago and has, since then, been recognised as the major growth factor relatively specific for endothelial cells (reviewed in Ferrara and Davis-Smyth 1997). VEGF is a dimeric glycoprotein, closely related to placenta growth factor (PIGF). Both VEGF and PIGF are distantly related in structure to the platelet-derived growth factors A and B (PDGF A and PDGF B) (Heldin et al. 1993). Three novel growth factors belonging to the family of VEGF, PIGF and the two PDGFs were recently discovered. These growth factors, termed vascular endothelial growth factor B/VEGF-related factor (VEGF-B/VRF) (Grimmond et al. 1996; Olofsson et al. 1996a), vascular endothelial growth factor C/VEGF-related protein (VEGF-C/VRP) (Joukov et al. 1996; Lee et al. 1996)] and c-fos-induced growth factor (F1GF) (Orlandini et al. 1996) share structural features typical of the VEGF/PDGF growth factor family. The prominent structural similarities between VEGF-related growth factors, several of which target endothelial cells, and FIGF suggest the possibility that F1GF also targets endothelial cells, despite its identification as a fibroblast growth factor. Based on these criteria, we propose that the name FIGF should be changed to VEGF-D to indicate its structural and functional relatedness to other VEGFs.
We examined the changes in tumor Doppler flow signals as well as prostatic volume in 11 cases of prostatic cancer, before and after castration, by using power Doppler imaging, and compared the changes in vascular resistance (RI) and prostatic volume (PV) in 11 cases. RI, except for one case, and PV decreased after castration in all cases without exception. In both, the manner of decrease showed an exponential curve. However, the reduction time of RI fell in an extremely narrow range compared with that of PV. Tumor vascular flow was influenced by castration, resulting in a decrease of RI. The dynamic change of vascular flow occurred earlier than that of PV. Analysis of changes in tumor blood flow signals could offer valuable information on early therapeutic effects in patients with prostatic cancer; however, it might not be a useful parameter for the prediction of prognosis.
PTK787/ZK 222584 (1-[4-chloroanilino]-4-[4-pyridylmethyl] phthalazine succinate) is a potent inhibitor of vascular endothelial growth factor (VEGF) receptor tyrosine kinases, active in the submicromolar range. It also inhibits other class III kinases, such as the platelet-derived growth factor (PDGF) receptor beta tyrosine kinase, c-Kit, and c-Fms, but at higher concentrations. It is not active against kinases from other receptor families, such as epidermal growth factor receptor, fibroblast growth factor receptor-1, c-Met, and Tie-2, or intracellular kinases such as c-Src, c-Abl, and protein kinase C-alpha. PTK787/ZK 222584 inhibits VEGF-induced autophosphorylation of kinase insert domain-containing receptor (KDR), endothelial cell proliferation, migration, and survival in the nanomolar range in cell-based assays. In concentrations up to 1 microM, PTK787/ZK 222584 does not have any cytotoxic or antiproliferative effect on cells that do not express VEGF receptors. After oral dosing (50 mg/kg) to mice, plasma concentrations of PTK787/ZK 222584 remain above 1 microM for more than 8 h. PTK787/ZK 222584 induces dose-dependent inhibition of VEGF and PDGF-induced angiogenesis in a growth factor implant model, as well as a tumor cell-driven angiogenesis model after once-daily oral dosing (25-100 mg/kg). In the same dose range, it also inhibits the growth of several human carcinomas, grown s.c. in nude mice, as well as a murine renal carcinoma and its metastases in a syngeneic, orthotopic model. Histological examination of tumors revealed inhibition of microvessel formation in the interior of the tumor. PTK787/ZK 222584 is very well tolerated and does not impair wound healing. It also does not have any significant effects on circulating blood cells or bone marrow leukocytes as a single agent or impair hematopoetic recovery after concomitant cytotoxic anti-cancer agent challenge. This novel compound has therapeutic potential for the treatment of solid tumors and other diseases where angiogenesis plays an important role.
The hormonally regulated growth of some human carcinomas represents an important therapeutic target. We report that androgens modulate the angiogenic activity of hormone responsive human prostate cancer.
To define further the critical mechanisms underlying hormone responsiveness we examined the angiogenic mediator, vascular endothelial growth factor messenger (m) RNA and protein in response to androgens in vitro as well as the angiogenic response of xenografts of human prostate cancer after androgen withdrawal in vivo.
In vitro androgen deprivation of LnCaP prostate cancer cells led to decreased vascular endothelial growth factor mRNA and protein expression as well as a 5-fold destabilization in vascular endothelial growth factor mRNA transcripts. In addition, androgen withdrawal inhibited the hypoxic induction of vascular endothelial growth factor mRNA. In mice bearing LnCaP tumors castration resulted in a rapid decrease in mRNA expression and markedly reduced tumor neovascularization.
These findings implicate sex steroids as an important stimulus for vascular endothelial growth factor regulation in hormone sensitive tumors and demonstrate the reversal of neovascularization after hormone withdrawal as an early event in the tumor response to therapy.
The vasculature of the prostate responds to androgens. Androgens most likely affect the vasculature indirectly by modulating the expression of angiogenic factors in the cells of the prostate. Most studies to date have examined the production of angiogenic factors by the prostate luminal epithelium. Here we examine the effects of androgen on production of three angiogenic factors, vascular endothelial growth factor (VEGF), angiopoietin-1, and angiopoietin-2, by the three major cell types in the prostate.
The ability of androgen to modulate VEGF, angiopoietin-1, and angiopoietin-2 production in cultured mouse prostate luminal epithelial, basal epithelial, and smooth muscle cells (SMCs) was assessed by Western blot and RT-PCR.
The production of VEGF was modulated by androgens in both luminal epithelial and prostate SMCs but not in basal epithelial cells. However, in prostate luminal epithelial cell cultures, VEGF was predominately secreted apically, suggesting that in vivo most of the epithelium-derived VEGF is unavailable to the underlying blood vessels. In addition, prostate luminal epithelial cells produced angiopoietin-2, an angiogenesis inhibitor. In contrast, prostate SMCs produced angiopoietin-1, a positive modulator of angiogenesis. Synthesis of the angiopoietins did not respond to androgen treatment.
Prostate smooth muscle may play an important role in regulating vascular responses to androgen.
The vasculature plays an important role in the normal and malignant prostate. Under basal conditions both glandular epithelial and stromal prostate cells produce an abundance of blood flow and angiogenesis regulating substances and the expression of these is generally increased in prostate tumors. The proportion of proliferating endothelial cells is high in the normal prostate compared to other tissues in the body. After castration effects on the vasculature, such as decreased blood flow and vascular regression, precede effects on the glandular compartment. Correspondingly, hormone induced prostate growth is characterized by early effects on the vasculature such as increased blood flow and endothelial cell proliferation, thus indicating that the vasculature may be involved in the androgenic regulation of the prostate. Prostatic intraepithelial neoplasia (PIN) and prostate cancer are associated with increased vascular density and in experimental models prostate cancer growth is apparently angiogenesis-dependent since tumor growth and progression can be inhibited by antiangiogenic treatment. Moreover, vascular density has been related to prognosis in prostate cancer patients. A better understanding of the pathways regulating angiogenesis in the normal prostate and how these pathways change during malignant transformation can hopefully lead to better prognostic markers and therapies for the large group of patients with prostate cancer. The purpose of this review is therefore to summarize the current knowledge on the role and regulation of the vasculature in the prostate and its potential clinical applications.
The interaction between cancer cells and their microenvironment is a promising area for the development of novel therapeutic anti-cancer modalities. The formation of new blood vessels, angiogenesis, is an important step in cancer progression. Angiogenesis is a complex multistep process involving close orchestration of endothelial cells, extracellular matrix, and soluble factors. Essentially every step has been found to be regulated by inducers and inhibitors. Prostate cancer has the ability to produce angiogenic factors such as metalloproteinases, vascular endothelial growth factor, fibroblast growth factor 2, transforming growth factor-beta and cyclooxygenase-2. In several studies in prostate cancer an increased microvessel density is associated with poorer prognosis. On the other hand several endogenous inhibitors of angiogenesis have been described in prostate cancer e.g., angiostatin, endostatin, prostate specific antigen (PSA), thrombospondin-1, interleukin 10, interferons and retinoids. The expanding insight in the process of angiogenesis has resulted in a large number of pharmaceutical agents that have been tested in preclinical studies and are currently tested in clinical trials. These agents inhibit endothelial cell proliferation or migration and induce apoptosis. This ultimately will affect the formation of new vessels thereby inducing tumor dormancy. Because antiangiogenic treatment is cytostatic rather than cytotoxic, patients will need long-term therapy to prevent regrowth of the tumor. Prostate cancer is an ideal tumor for antiangiogenic studies because of the availability of a reliable tumor marker, PSA, the indolent clinical course of this cancer and the low rate of proliferation even in metastatic sites. Furthermore, clinical studies showed limited side effects, which is advantageous in this elderly patient group. Whether the ultimate antiangiogenic treatment is effective as a single agent or in combination with radiation therapy, chemotherapy or immunotherapy remains to be determined.
Excerpt
Mammalian organisms depend on their vasculature todeliver nutrients and oxygen to all of their tissues, totransport products (such as hormones and antibodies)from certain cells to distant parts of the body, and to carryaway waste products. The development of a functioningvasculature, as well as its proper integration into the tissues it serves, depends on myriad interactions and communications between the many cell types involved. Although a large number of signals are involved inmediating these intercellular communications, a greatdeal of focus has been directed to growth factors that aremembers of either the vascular endothelial growth factor(VEGF) family or the angiopoietin family. Why the focuson these two families of growth factors? First of all, thesetwo families of growth factors are unique in that they actvia receptors that are largely restricted to the vasculatureendothelium—this very restricted distribution of their receptors indicates that these two families of growth factorsevolved to play very particular roles specifically involving the vasculature. Moreover, genetic approaches—involving gene knockouts and transgenic overexpression inmice—have spectacularly confirmed the very critical andvery specific roles played by members of these twogrowth factor families during vascular development.Thus, the focus on the VEGFs and angiopoietins seemswell-placed based on their action via vascular-specific receptors and the confirmation of their critical and specificvascular roles based on genetic studies in mice. Since theVEGFs have been extensively dealt with in a number ofexcellent reviews (Eriksson and Alitalo 1999; Ferrara1999; Yancopoulos et al. 2000; Carmeliet et al. 2001),this review highlights work from our laboratory regarding the angiopoietins, although much of this work is presented in the context of the complementary and reciprocal actions of the angiopoietins as compared to theVEGFs...
Prostate cancer is a leading cause of cancer death in American males. Androgens play an essential role in prostate development, growth and pathogenesis of benign prostate hyperplasia, and prostate cancer. Although testosterone is the main androgen secreted from the testes, dihydrotestosterone (DHT), a more potent androgen converted from testosterone by 5alpha-reductase isozymes, type 1 and 2, is the major androgen in the prostate cells. Thus, 5alpha-reductase(s) are critical in determining androgen activity in the prostate. However, it is unclear in prostate tumor cells whether 1 or 2 5alpha-reductase isozymes are expressed and whether they are functionally important. In the present report, we studied the importance of 5alpha-reductase isozymes in the androgen induction of prostate-specific antigen (PSA) gene expression in LNCaP prostatic tumor cells. Treatment with either testosterone or DHT in LNCaP cells produced dose- and time-dependent increases in PSA levels in the cell media and in PSA messenger RNA (mRNA) levels in the cells. However, testosterone-induced but not DHT-induced PSA gene expression was significantly inhibited by finasteride, a 5alpha-reductase inhibitor, in a dose-dependent manner. Furthermore, we demonstrated for the first time that both 5alpha-reductase-1 and 5alpha-reductase-2 mRNAs were expressed in LNCaP cells using reverse transcriptase-polymerase chain reaction (RT-PCR) and RT-PCR Southern blot analysis. These results suggest that both 5alpha-reductase isozymes are present and functionally important in prostatic tumor LNCaP cells and that DHT is a major mediator of androgen induction of PSA gene expression in these cells.
Recent studies show that testosterone-stimulated growth of the glandular tissue in the ventral prostate in adult castrated rats is preceded by increased epithelial VEGF synthesis, endothelial cell proliferation, vascular growth, and increased blood flow. These observations suggest that testosterone-stimulated prostate growth could be angiogenesis dependent, and that VEGF could play a central role in this process.
Adult male mice were castrated and after 1 week treated with testosterone and vehicle, or with testosterone and a soluble chimeric VEGF-receptor flt(1-3)IgG protein.
Treatment with testosterone markedly increased endothelial cell proliferation, vascular volume, and organ weight in the ventral prostate lobe in the vehicle groups, but these responses were inhibited but not fully prevented by anti-VEGF treatment. The testosterone-stimulated increase in epithelial cell proliferation was unaffected by flt(1-3)IgG, but endothelial and epithelial cell apoptosis were increased in the anti-VEGF compared to the vehicle-treated groups.
This study suggests that testosterone stimulates vascular growth in the ventral prostate lobe indirectly by increasing epithelial VEGF synthesis and that this is a necessary component in testosterone-stimulated prostate growth.
Estrogens, including diethylstilbestrol (DES), were used as the primary medical treatment for metastatic prostate cancer for many years but have been superceded in the past two decades by luteinizing hormone-releasing hormone (LHRH) agonists, primarily because of the cardiovascular toxicity associated with oral estrogen therapy. Recently, a renewed interest in estrogen therapy for prostate cancer in the United States has developed as a result of 3 major issues. First, when measured by declines in prostate-specific antigen of > or = 50%, clinical trials have demonstrated activity of DES, DES-diphosphate, and the estrogenic herbal therapy PC-SPES in 21%-86% of patients treated in phase II trials of androgen-independent prostate cancer patients. Second, the recent description of estrogen receptor (ER)-b has led to a reevaluation of the role of estrogens in normal prostate development and cancer pathogenesis. In contrast to ER-a, ER-b is strongly expressed in normal prostate epithelium. Furthermore, loss of ER-b expression has been demonstrated in prostate cancers, suggesting a possible role for this pathway in the development of cancer. Finally, the issues of cost and safety of estrogens are being reassessed in the current environment of rising health care costs and improved cardiovascular care. In Europe, estrogen therapy is more accepted as a low-cost and effective alternative to LHRH agonists and antiandrogens. Toxicity of DES and other estrogens has also been attenuated by strategies that use lower doses and parenteral routes of administration, thereby avoiding hepatic first-pass metabolism and decreasing the risk of thromboembolism. Nonetheless, there remain many unanswered questions about the role of estrogen therapy in prostate cancer, including differences between specific drugs, optimal dose, timing, and patient selection. Further research is needed.
Sex steroid hormones play a central role in the development and progression of prostate and breast cancers. The biological functions of these and other steroid hormones are mediated by a family of closely related steroid hormone receptors (SHRs), with the androgen receptor (AR) mediating the effects of testosterone and related androgens, and the classical estrogen receptor (ERalpha) mediating the effects of estradiol. Recent studies have begun to elucidate the complex pathways through which SHRs regulate gene expression, and their interaction with other cellular pathways. These studies have also begun to reveal molecular mechanisms underlying the diverse spectrum of effects mediated by steroid hormone analogues in different tissues. A major advance has been the finding that certain drugs induce unique conformational changes in SHRs that alter their interactions with transcriptional coactivator and corepressor proteins, resulting in cell type specific responses. These unique conformational changes appear responsible for the tissue specific effects of the selective estrogen receptor modulators (SERMs) in breast cancer. SHRs are clearly well established therapeutic targets in cancer, and drug development has continued to focus on agents that either block steroid hormone production or bind to and modulate their receptors. The identification of multiple proteins and pathways that mediate the downstream functions of SHRs may eventually provide additional therapeutic targets. This review outlines the basic biology of SHR structure and function, with a focus on AR and ERalpha. Hormonal therapies in prostate and breast cancer that directly target AR and ERalpha, respectively, are then presented and possible novel drug targets in the SHR pathway are discussed.
The relative role of the two estrogen receptors, ERalpha and ERbeta, in mediating angiogenic responses in adult human endothelium is unknown. The aim of this study was to determine whether novel ERalpha-selective agonists, propyl pyrazole triol (PPT) and the tetrahydrochrysene (R,R-THC), up-regulate the expression of vascular endothelial growth factor receptor-2 (VEGFR-2), and promote VEGF-stimulated endothelial cell proliferation in primary cultures of adult female microvascular endothelial cells co-expressing endogenous ERalpha and ERbeta.
Confluent primary cultures of microvascular endothelial cells isolated from human myometrium were incubated with 17beta-estradiol (1 and 10 nM), PPT (10 nM to 3 microM), or R,R-THC (10 nM to 3 microM) for 18 hours and VEGFR-2 expression measured by biotin-VEGF165 binding and flow cytometry. Endothelial cell proliferation was assessed in microvascular endothelial cells after incubation with 17beta-estradiol (10 nM), PPT (100 nM), and R,R-THC (100 nM) for 6 days using a tetrazolium-based bioassay.
Both PPT and R,R-THC increased VEGFR-2 expression on myometrial microvascular endothelial cells in a dose-dependent manner, reaching a maximum at 1 microM. Approximately 40% of myometrial microvascular endothelial cell isolates only express ERbeta and do not express ERalpha, and in these neither PPT, R,R-THC, nor 17beta-estradiol increased VEGF binding. PPT- or R,R-THC-stimulated increase in VEGF binding was significantly different between ERalpha+ and ERalpha- microvascular endothelial cell samples (P < .001 and P < .05, respectively). PPT, R,R-THC, and 17beta-estradiol significantly augmented VEGF-stimulated microvascular endothelial cell proliferation in ERalpha+ (P < .05), but not in ERalpha- samples.
This angiogenic effect of 17beta-estradiol on adult female microvascular endothelial cells is mediated by ERalpha, rather than ERbeta.
Androgens via the androgen receptor (AR) play crucial roles in prostate physiology and pathophysiology. These androgen actions can be either inhibited or potentiated by estrogens. The mechanisms of these seemingly opposing estrogen effects are unclear. We studied the effects of estrogens on the modulation of androgen induction of prostate specific antigen (PSA) gene expression and prostate tumor cell growth. Cotransfection analyses in CV-1, DU-145, and PC-3 cells showed that dihydrotestosterone (DHT)-induced PSA transcription activity was inhibited by 17beta-estradiol, diethylstilbestrol, ICI182780, and 17alpha-estradiol, but not by tamoxifen via estrogen receptor alpha (ERalpha). In the presence of ERbeta, 17beta-estradiol and diethylstilbestrol had no significant effect, while 17alpha-estradiol inhibited and ICI182780 and tamoxifen potentiated DHT action. When both ERalpha and ERbeta were present, all ER-ligands except tamoxifen inhibited DHT action. The inhibition of DHT action by 17beta-estradiol via ERalpha was mainly dependent on the DNA binding domain, while the 17alpha-estradiol effect was mainly dependent on the ERalpha carboxyl terminus. Treatment with DHT in LAPC-4 prostate tumor cells that express a wild-type AR and both ERbeta and ERalpha greatly increased the PSA gene expression and cell growth. These DHT effects were significantly attenuated by the addition of 17alpha-estradiol, 17beta-estradiol, or cyproterone acetate in a dose-dependent manner. These results indicate that estrogens produce an ER-isoform- and ER-ligand-specific modulation of DHT induction of PSA gene expression and prostate tumor cell growth, providing a molecular basis for designing favorable agents for the prevention and control of prostate cancer.
Tumor growth and metastasis depend on neovascularization, the growth of new blood vessels. Recent findings have revealed that tumor neovascularization is regulated in part by monocytes, which are myeloid lineage cells from the bone marrow. Tumors exhibit significant monocyte infiltrates, which are actively recruited to the tumor microenvironment. Upon tumor infiltration, monocytes can participate in tumor neovascularization. Monocytes can either differentiate into macrophages, which express proangiogenic growth factors, or into endothelial-like cells, which may directly participate in neovascularization. Preliminary studies in animals suggest that modulation of bone marrow-derived cell trafficking into tumors will provide a useful new approach in cancer therapy.
The regulation of the prostate size by androgens may be partly the result of androgen effects on the prostatic vasculature. We examined the effect of changes in androgen levels on the expression of a variety of angiogenic factors in the mouse prostate and determined if vascular endothelial growth factor (VEGF)-A and the angiopoietins are involved in the vascular response to androgens.
Expression of angiogenic factors in prostate was quantitated using real-time PCR at different times after castration and after administration of testosterone to castrated mice. Angiopoietins were localized in prostate by immunohistochemistry and in situ hybridization. The roles of VEGF and the angiopoietins in regeneration of the prostate were examined in mice inoculated with cells expressing soluble VEGF receptor-2 or soluble Tie-2.
Castration resulted in a decrease in VEGF-A, VEGF-B, VEGF-C, placenta growth factor, FGF-2, and FGF-8 expression after 1 day. In contrast, VEGF-D mRNA levels increased. No changes in angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), hepatocyte growth factor, VEGF receptor-1, VEGF receptor-2, or tie-2 mRNA levels were observed. Administration of testosterone to castrated mice had the opposite effect on expression of these angiogenic factors. Ang-2 was expressed predominantly in prostate epithelial cells whereas Ang-1 was expressed in epithelium and smooth muscle. Inoculation of mice with cells expressing soluble VEGF receptor-2 or Tie-2 blocked the increase in vascular density normally observed after administration of testosterone to castrated mice. The soluble receptors also blocked the increase in prostate weight and proliferation of prostatic epithelial cells.
VEGF-A and angiopoietins are required for the vascular response to androgens and for the ability of the prostate to regenerate in response to androgens.
The proximal region of the prostatic ducts harbor the prostatic epithelial stem cells. As stem cell niches in other organs are highly vascularized, we determined if the proximal region is more highly vascularized than the remaining regions of the prostate. The effect of androgen on vascular density in the different prostatic regions was also examined.
Sections from prostates were immunostained with antibodies to CD31, and the vascular density in proximal, intermediate, and distal regions was calculated by image analysis software. Vascular density was compared in prostates from castrated mice that received daily inoculations of testosterone or vehicle alone for 3 days. To examine the role of angiogenic factors in the response to androgen, some animals were also treated with soluble VEGF receptor-2-Fc or Tie-2--Fc fusion proteins, which inhibit the activities of VEGF and angiopoietins, respectively. The endothelial proliferative response to androgen was determined by double staining sections with antibodies to CD31 and Ki-67.
In prostates from intact mice, vascular density was highest in the proximal region and lowest in the distal region. Administration of testosterone to castrated mice increased vascular density to the greatest extent in the distal and intermediate regions. The increase in vascular density required VEGF and the angiopoietins. Endothelial cell proliferation was less sensitive to androgen in the proximal region than the remainder of the prostate.
Vascular density is highest in the proximal region of the prostate, but the proximal vessels are less responsive to testosterone.