Estramustine and its Derivatives in Potentiating Radiotherapy of Prostate Cancer

The purpose of this study was to deepen our knowledge of the combined use of estramustine and radiotherapy in the treatment of prostate cancer. Prostate cancer is a common disease, with a high variability between subjects in its malignant potential. In many cases, the disease is an incidental finding with little or no clinical significance. In other cases, however, prostate cancer may be an aggressive malignant disease, which, if the initial treatment fails, lacks an effective cure and may lead to severe symptoms, metastasis, and death despite all treatment. In many cases, the methods of treatment available at the moment provide cure or significant regression of symptoms, but often at the cost of considerable side effects. Estramustine, a cytostatic drug used for treating advanced cancer of the prostate, has been shown to inhibit prostate cancer progression and also to increase the sensitivity of cancer cells to radiotherapy. The goals of this study were, first, to find out whether it is possible to use either estramustine or an antibody against estramustine binding protein as carrier molecules for bringing therapeutic radioisotopes into prostate cancer cells, and, secondly, to gain more understanding of the mechanisms behind the known radiosensitising effect of estramustine. Estramustine and estramustine binding protein antibody were labelled with iodine-125 to study the biodistribution of these substances in mice. In the first experiment, both of the substances accumulated in the prostate, but radioiodinated estramustine also showed affinity to the liver and the lungs. Since the radiolabelled antibody was found out to accumulate more selectively to the prostate, we studied its biodistribution in nude mice with DU-145 human prostate cancer implants. In this experiment, the prostate and the tumour accumulated more radioactivity than other organs, but we concluded that the difference in the dose of radiation compared to other organs was not sufficient for the radioiodinated antibody to be advocated as a carrier molecule for treating prostate cancer. Mice with similar DU-145 prostate cancer implants were then treated with estramustine and external beam irradiation, with and without neoadjuvant estramustine treatment. The tumours responded to the treatment as expected, showing the radiation potentiating effect of estramustine. In the third experiment, this effect was found without an increase in the amount of apoptosis in the tumour cells, despite previous suggestions to the contrary. In the fourth experiment, we gave a similar treatment to the mice with DU-145 tumours. A reduction in proliferation was found in the groups treated with radiotherapy, and an increased amount of tumour hypoxia and tumour necrosis in the group treated with both neoadjuvant estramustine and radiation. This finding is contradictory to the suggestion that the radiation sensitising effect of estramustine could be attributed to its angiogenic activity.

Prostate cancer cell-specific VEGF siRNA delivery system using cell targeting peptide conjugated polyplexes

A polymeric gene carrier was developed to deliver vascular endothelial growth factor (VEGF) small interfering RNA (siRNA) for prostate cancer cells in a target-specific manner. Prostate cancer-binding peptide (PCP) was conjugated with polyethylenimine (PEI) via a poly(ethylene glycol) (PEG) linker (PEI-PEG-PCP). The PEI-PEG-PCP conjugate could effectively condense siRNA to form stable polyelectrolyte complexes (polyplexes) with an average diameter of approximately 150 nm in an aqueous solution. VEGF siRNA/PEI-PEG-PCP polyplexes exhibited significantly higher VEGF inhibition efficiency than PCP-unmodified polycationic carriers (PEI-PEG or PEI) in human prostate carcinoma cells (PC-3 cells). The enhanced gene silencing activity of VEGF siRNA/PEI-PEG-PCP was maintained even under serum conditions, owing to the steric stabilization of the polyplexes with hydrophilic PEG grafts. Confocal microscopic studies revealed that the siRNA/PEI-PEG-PCP polyplexes were delivered into PC-3 cells in a PCP ligand-specific manner.

MYC activity mitigates response to rapamycin in prostate cancer through eukaryotic initiation factor 4E-binding protein 1-mediated inhibition of autophagy.

Loss of PTEN and activation of phosphoinositide 3-kinase are commonly observed in advanced prostate cancer. Inhibition of mammalian target of rapamycin (mTOR), a downstream target of phosphoinositide 3-kinase signaling, results in cell cycle arrest and apoptosis in multiple in vitro and in vivo models of prostate cancer. However, single-agent use of mTOR inhibition has limited clinical success, and the identification of molecular events mitigating tumor response to mTOR inhibition remains a critical question. Here, using genetically engineered human prostate epithelial cells (PrEC), we show that MYC, a frequent target of genetic gain in prostate cancers, abrogates sensitivity to rapamycin by decreasing rapamycin-induced cytostasis and autophagy. Analysis of MYC and the mTOR pathway in human prostate tumors and PrEC showed selective increased expression of eukaryotic initiation factor 4E-binding protein 1 (4EBP1) with gain in MYC copy number or forced MYC expression, respectively. We have also found that MYC binds to regulatory regions of the 4EBP1 gene. Suppression of 4EBP1 expression resulted in resensitization of MYC-expressing PrEC to rapamycin and increased autophagy. Taken together, our findings suggest that MYC expression abrogates sensitivity to rapamycin through increased expression of 4EBP1 and reduced autophagy.

The methyl-CpG-binding protein MECP2 is required for prostate cancer cell growth.

The incidence of prostate cancer is increasing in western countries because of population aging. Prostate cancer begins as an androgen-dependent disease, but it can become androgen independent at a later stage or in tumors recurring after an antihormonal treatment. Although many genetic events have been described to be involved in androgen-dependent and/or -independent prostate cancer growth, little is known about the contribution of epigenetic events. Here we have examined the possibility that the methyl-CpG-binding protein MECP2 might play a role in controlling the growth of prostate cancer cells. Inhibition of MECP2 expression by stable short hairpin RNA stopped the growth of both normal and cancer human prostate cells. In addition, ectopic expression of the MECP2 conferred a growth advantage to human prostate cancer cells. More importantly, this expression allowed androgen-dependent cells to grow independently of androgen stimulation and to retain tumorigenic properties in androgen-depleted conditions. Analysis of signaling pathways showed that this effect is independent of androgen receptor signaling. Instead, MECP2 appears to act by maintaining a constant c-myc level during antihormonal treatment. We further show that MECP2-expressing cells possess a functional p53 pathway and are still responsive to chemotherapeutic drugs.

Nonapical and Cytoplasmic Expression of Interleukin-8, CXCR1, and CXCR2 Correlates with Cell Proliferation and Microvessel Density in Prostate Cancer

Purpose: We characterized interleukin-8 (IL-8) and IL-8 receptor expression (CXCR1 and CXCR2) in prostate cancer to address their significance to this disease. Experimental Design: Immunohistochemistry was conducted on 40 cases of human prostate biopsy containing histologically normal and neoplastic tissue, excised from patients with locally confined or invasive androgen-dependent prostate cancer, and 10 cases of transurethral resection of the prostate material from patients with androgen-independent disease. Results: Weak to moderate IL-8 expression was strictly localized to the apical membrane of normal prostate epithelium. In contrast, membranous expression of IL-8, CXCR1, and CXCR2 was nonapical in cancer cells of Gleason pattern 3 and 4, whereas circumferential expression was present in Gleason pattern 5 and androgen-independent prostate cancer. Each of IL-8, CXCR1, and CXCR2 were also increasingly localized to the cytoplasm of cancer cells in correlation with advancing stage of disease. Cytoplasmic expression (but not apical membrane expression) of IL-8 in Gleason pattern 3 and 4 cancer correlated with Ki-67 expression (R = 0.79; P <0.001), cyclin D1 expression (R = 0.79; P <0.001), and microvessel density (R = 0.81; P <0.001). In vitro studies on androgen-independent PC3 cells confirmed the mitogenic activity of IL-8, increasing the rate of cell proliferation through activation of both CXCR1 and CXCR2 receptors. Conclusions: We propose that the concurrent increase in IL-8 and IL-8 receptor expression in human prostate cancer induces autocrine signaling that may be functionally significant in initiating and promoting the progression of prostate cancer by underpinning cell proliferation and angiogenesis.

HIF-1 and NF-kappaB-mediated upregulation of CXCR1 and CXCR2 expression promotes cell survival in hypoxic prostate cancer cells

Hypoxic cancer cells are resistant to treatment, leading to the selection of cells with a more malignant phenotype. The expression of interleukin-8 (IL-8) plays an important role in the tumorigenesis and metastasis of solid tumors including prostate cancer. Recently, we detected elevated expression of IL-8 and IL-8 receptors in human prostate cancer tissue. The objective of the current study was to determine whether hypoxia increases IL-8 and IL-8 receptor expression in prostate cancer cells and whether this contributes to a survival advantage in hypoxic cells. IL-8, CXCR1 and CXCR2 messenger RNA (mRNA) expression in PC3 cells was upregulated in response to hypoxia in a time-dependent manner. Elevated IL-8 secretion following hypoxia was detected by enzyme-linked immunosorbent assay, while immunoblotting confirmed elevated receptor expression. Attenuation of hypoxia-inducible factor (HIF-1) and nuclear factor-kappaB (NF-kappaB) transcriptional activity using small interfering RNA (siRNA), a HIF-1 dominant-negative and pharmacological inhibitors, abrogated hypoxia-induced transcription of CXCR1 and CXCR2 in PC3 cells. Furthermore, chromatin-IP analysis demonstrated binding of HIF-1 and NF-kappaB to CXCR1. Finally, inhibition of IL-8 signaling potentiated etoposide-induced cell death in hypoxic PC3 cells. These results suggest that IL-8 signaling confers a survival advantage to hypoxic prostate cancer cells, and therefore, strategies to inhibit IL-8 signaling may sensitize hypoxic tumor cells to conventional treatments.

Novel microtubule-targeting agents, pyrrolo-1,5-benzoxazepines, induce cell cycle arrest and apoptosis in prostate cancer cells

Advanced hormone-refractory prostate cancer is associated with poor prognosis and limited treatment options. Members of the pyrrolo-1,5-benzoxazepine (PBOX) family of compounds exhibit anti-cancer properties in cancer cell lines (including multi-drug resistant cells), ex vivo patient samples and in vivo mouse tumour models with minimal toxicity to normal cells. Recently, they have also been found to possess anti-angiogenic properties in vitro. However, both the apoptotic pathways and the overall extent of the apoptotic response induced by PBOX compounds tend to be cell-type specific. Since the effect of the PBOX compounds on prostate cancer has not yet been elucidated, the purpose of this study was to investigate if PBOX compounds induce anti-proliferative effects on hormone-refractory prostate cancer cells. We examined the effect of two representative PBOX compounds, PBOX-6 and PBOX-15, on the androgen-independent human prostate adenocarcinoma cell line, PC3. PBOX-6 and -15 displayed anti-proliferative effects on PC3 cells, mediated initially through a sustained G2/M arrest. G2/M arrest, illustrated as DNA tetraploidy, was accompanied by microtubule depolymerisation and phosphorylation of anti-apoptotic proteins Bcl-2 and Bcl-xL and the mitotic spindle checkpoint protein BubR1. Phosphorylation of BubR1 is indicative of an active mitotic checkpoint and results in maintenance of cell cycle arrest. G2/M arrest was followed by apoptosis illustrated by DNA hypoploidy and PARP cleavage and was accompanied by degradation of BubR1, Bcl-2 and Bcl-xL. Furthermore, sequential treatment with the CDK1-inhibitor, flavopiridol, synergistically enhanced PBOX-induced apoptosis. In summary, this in vitro study indicates that PBOX compounds may be useful alone or in combination with other agents in the treatment of hormone-refractory prostate cancer.

HES6 drives a critical AR transcriptional programme to induce castration-resistant prostate cancer through activation of an E2F1-mediated cell cycle network

Castrate-resistant prostate cancer (CRPC) is poorly characterized and heterogeneous and while the androgen receptor (AR) is of singular importance, other factors such as c-Myc and the E2F family also play a role in later stage disease. HES6 is a transcription co-factor associated with stem cell characteristics in neural tissue. Here we show that HES6 is up-regulated in aggressive human prostate cancer and drives castration-resistant tumour growth in the absence of ligand binding by enhancing the transcriptional activity of the AR, which is preferentially directed to a regulatory network enriched for transcription factors such as E2F1. In the clinical setting, we have uncovered a HES6-associated signature that predicts poor outcome in prostate cancer, which can be pharmacologically targeted by inhibition of PLK1 with restoration of sensitivity to castration. We have therefore shown for the first time the critical role of HES6 in the development of CRPC and identified its potential in patient-specific therapeutic strategies.

The androgen receptor induces a distinct transcriptional program in castration-resistant prostate cancer in man

The androgen receptor (AR) regulates prostate cell growth in man, and prostate cancer is the commonest cancer in men in the UK. We present a comprehensive analysis of AR binding sites in human prostate cancer tissues, including castrate-resistant prostate cancer (CRPC). We identified thousands of AR binding sites in CRPC tissue, most of which were not identified in PC cell lines. Many adjacent genes showed AR regulation in xenografts but not in cultured LNCaPs, demonstrating an in-vivo-restricted set of AR-regulated genes. Functional studies support a model of altered signaling in vivo that directs AR binding. We identified a 16 gene signature that outperformed a larger in-vitro-derived signature in clinical data sets, showing the importance of persistent AR signaling in CRPC.

Oriented Tailoring of Plastic Antibodies for Prostate Specific Antigen and Application of the Imprinted Material as Ionophore in Potentiometric Detection

Prostate Specific Antigen (PSA) is the biomarker of choice for screening prostate cancer throughout the population, with PSA values above 10 ng/mL pointing out a high probability of associated cancer1. According to the most recent World Health Organization (WHO) data, prostate cancer is the commonest form of cancer in men in Europe2. Early detection of prostate cancer is thus very important and is currently made by screening PSA in men over 45 years old, combined with other alterations in serum and urine parameters. PSA is a glycoprotein with a molecular mass of approximately 32 kDa consisting of one polypeptide chain, which is produced by the secretory epithelium of human prostate. Currently, the standard methods available for PSA screening are immunoassays like Enzyme-Linked Immunoabsorbent Assay (ELISA). These methods are highly sensitive and specific for the detection of PSA, but they require expensive laboratory facilities and high qualify personal resources. Other highly sensitive and specific methods for the detection of PSA have also become available and are in its majority immunobiosensors1,3-5, relying on antibodies. Less expensive methods producing quicker responses are thus needed, which may be achieved by synthesizing artificial antibodies by means of molecular imprinting techniques. These should also be coupled to simple and low cost devices, such as those of the potentiometric kind, one approach that has been proven successful6. Potentiometric sensors offer the advantage of selectivity and portability for use in point-of-care and have been widely recognized as potential analytical tools in this field. The inherent method is simple, precise, accurate and inexpensive regarding reagent consumption and equipment involved. Thus, this work proposes a new plastic antibody for PSA, designed over the surface of graphene layers extracted from graphite. Charged monomers were used to enable an oriented tailoring of the PSA rebinding sites. Uncharged monomers were used as control. These materials were used as ionophores in conventional solid-contact graphite electrodes. The obtained results showed that the imprinted materials displayed a selective response to PSA. The electrodes with charged monomers showed a more stable and sensitive response, with an average slope of -44.2 mV/decade and a detection limit of 5.8X10-11 mol/L (2 ng/mL). The corresponding non-imprinted sensors showed smaller sensitivity, with average slopes of -24.8 mV/decade. The best sensors were successfully applied to the analysis of serum samples, with percentage recoveries of 106.5% and relatives errors of 6.5%.

Improving the early diagnostic of prostate cancer by multiple biomarker detection with new biosensing devices

Prostate cancer (PCa) is the most common form of cancer in men, in Europe (World Health Organization data). The most recent statistics, in Portuguese territory, confirm this scenario, which states that about 50% of Portuguese men may suffer from prostate cancer and 15% of these will die from this condition. Its early detection is therefore fundamental. This is currently being done by Prostate Specific Antigen (PSA) screening in urine but false positive and negative results are quite often obtained and many patients are sent to unnecessary biopsy procedures. This early detection protocol may be improved, by the development of point-of-care cancer detection devices, not only to PSA but also to other biomarkers recently identified. Thus, the present work aims to screen several biomarkers in cultured human prostate cell lines, serum and urine samples, developing low cost sensors based on new synthetic biomaterials. Biomarkers considered in this study are the following: prostate specific antigen (PSA), annexin A3 (ANXA3), microseminoprotein-beta (MSMB) and sarcosine (SAR). The biomarker recognition may occurs by means of molecularly imprinted polymers (MIP), which are a kind of plastic antibodies, and enzymatic approaches. The growth of a rigid polymer, chemically stable, using the biomarker as a template allows the synthesis of the plastic antibody. MIPs show high sensitivity/selectivity and present much longer stability and much lower price than natural antibodies. This nanostructured material was prepared on a carbon solid. The interaction between the biomarker and the sensing-material produces electrical signals generating quantitative or semi-quantitative data. These devices allow inexpensive and portable detection in point-of-care testing.

T Cells Bearing a Chimeric Antigen Receptor against Prostate-Specific Membrane Antigen Mediate Vascular Disruption and Result in Tumor Regression.

Aberrant blood vessels enable tumor growth, provide a barrier to immune infiltration, and serve as a source of protumorigenic signals. Targeting tumor blood vessels for destruction, or tumor vascular disruption therapy, can therefore provide significant therapeutic benefit. Here, we describe the ability of chimeric antigen receptor (CAR)-bearing T cells to recognize human prostate-specific membrane antigen (hPSMA) on endothelial targets in vitro as well as in vivo. CAR T cells were generated using the anti-PSMA scFv, J591, and the intracellular signaling domains: CD3ζ, CD28, and/or CD137/4-1BB. We found that all anti-hPSMA CAR T cells recognized and eliminated PSMA(+) endothelial targets in vitro, regardless of the signaling domain. T cells bearing the third-generation anti-hPSMA CAR, P28BBζ, were able to recognize and kill primary human endothelial cells isolated from gynecologic cancers. In addition, the P28BBζ CAR T cells mediated regression of hPSMA-expressing vascular neoplasms in mice. Finally, in murine models of ovarian cancers populated by murine vessels expressing hPSMA, the P28BBζ CAR T cells were able to ablate PSMA(+) vessels, cause secondary depletion of tumor cells, and reduce tumor burden. Taken together, these results provide a strong rationale for the use of CAR T cells as agents of tumor vascular disruption, specifically those targeting PSMA. Cancer Immunol Res; 3(1); 68-84. ©2014 AACR.

Lipids and FA analysis of canine prostate tissue

It is widely reported that an association exists between dietary fat intake and the incidence of prostate cancer in humans. To study this association, there is a need for an animal model where prostate carcinogenesis occurs spontaneously. The canine prostate is considered a suitable experimental model for prostate cancer in humans since it is morphologically similar to the human prostate and both humans and dogs have a predisposition to benign and malignant prostate disease. In this study, the FA and lipids profiles of the normal canine prostate tissue from nine dogs were examined. The total lipid content of the canine prostate tissue was 1.7±0.5% (wet weight). The lipid composition analysis using TLC-FID showed that the two major lipid classes were phospholipids and TAG. Total FA, phospholipid, and TAG FA analysis showed that the major FA were palmitic acid (16∶0), stearic acid (18∶0), oleic acid (18∶1), linoleic acid (18∶2n−6), and arachidonic acid (20∶4n−6), The n−3 FA were present at <3% of total FA and included α-linolenic acid (18∶3n−3) (in total and TAG tissue FA), EPA (20∶5n−3) (not in TAG), and DHA (22∶6n−3) (not in TAG). The n−3/n−6 ratio was 1∶11, 1∶13, and 1∶8 in total, phospholipid, and TAG FA, respectively. This study shows the canine prostate has a low level of n−3 FA and a low n−3/n−6 ratio. This is perhaps due to low n−3 content of the diet of the dogs. FA analysis of dogfoods available in Australia showed that the n−3 content in both supermarket and premium bran dogfoods was <3% (wet weight), and the n−3/n−6 ratio was low.

Lobe Variation Effects of Experimental Diabetes and Insulin Replacement on Rat Prostate

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Age-related histopathological lesions in the Mongolian gerbil ventral prostate as a good model for studies of spontaneous hormone-related disorders

The Meriones unguiculatus (Mongolian) gerbil has demonstrated significant prostatic responses to hormonal treatments, and to drugs against human prostatic hyperplasia Spontaneous neoplasia develops in the older animals. Thirty gerbils (age 18 months) were divided into non-affected and prostatic lesion bearers and the prostate lesions were evaluated morphologically, immunohistochemically and quantitatively. The most frequent changes were in epithelial sites and, namely prostatic intraepithelial neoplasias, microinvasive carcinomas and adenocarcinomas. In the stromal compartment, cellular hyperplasia, when verified, was always associated with the sites of anomalous epithelium. Additionally, larger deposition of collagen fibrils, generating stromal fibrosis, was found in all the old gerbils analysed. The quantitative analysis showed that prostatic tissue proportions differed in altered areas, being specific for each lesion type. Isolated nuclear and nucleolar parameters were not effective in diagnosing the malign potential of lesions. However, the cellular proliferation and death indexes indicated larger cellular turnover in invasive lesions such as carcinomas. With these analyses, it could be verified that old gerbils present high propensity to develop spontaneous prostate changes and this may aid in a better understanding of the biological behaviour of human prostate cancer.