Periprostatic Adipose Tissue Microenvironment: Metabolic and Hormonal Pathways During Prostate Cancer Progression.

The periprostatic adipose tissue (PPAT) is a site of invasion of prostate cancer (PCa) and is part of the microenvironment. It was shown that PPAT secretes factors and fatty acids (FAs) that alter the microenvironment of the PCa. The PPAT secretome of patients with PCa-T3 stage (PPAT-T3) has a metabolic profile enriched in several pathways related to energy production, indicating a greater energy requirement by the tumor, when compared to that of patients in the PCa-T2 stage (PPAT-T2). PPAT-T3 also shows enrichment in pathways related to hormone response, polyamine synthesis, and control of protein synthesis, through amino acid, RNA, and nucleotide metabolism. PPAT-T2 and PPAT-BPH secretomes have less complex metabolic profile, both related with energy balance, while PPAT-BPH has hormone response through insulin pathway. Undoubtedly, a deeper characterization of the human PPAT will lead to a better understanding of the disease and possibly allow new stratification factors and the design of a specific therapy that targets crucial components of the tumor microenvironment as another way to treat or control the disease.

Lupane Triterpenoids and New Derivatives as Antiproliferative Agents Against Prostate Cancer Cells.

BACKGROUND/AIM: This study examined the potential role of natural triterpenoids lupeol, calenduladiol and heliantriol B2, and a set of 19 derivatives, as antiproliferative and antimetastatic agents against prostate cancer cells. MATERIALS AND METHODS: Natural triterpenoids were isolated from Chuqiraga erinaceae. Analogs were obtained by transformations of lupeol and calenduladiol. The effects of compounds on PC-3 and LNCaP cells were determined using the MTT assay. Compounds with half-maximal inhibitory concentration <70 µM were evaluated as antimetastatic agents by a wound-healing assay. RESULTS: Lupeol-3ß-sulfate, a new semisynthetic lupane, was the most active compound. In general, sulfated derivatives displayed higher activity than the lead against both cell lines. A new analog, calenduladiol-3ß-monosulfate, inhibited the migration of PC-3 cells; heliantriol B2 and 3ß-aminolupane inhibited the migration of LNCaP cells in a concentration-dependent manner. CONCLUSION: Our study provides novel agents with cytotoxic effects on prostate cancer cells, which may represent a potential new therapeutic approach for prostate cancer.

Human Periprostatic Adipose Tissue: Secretome from Patients With Prostate Cancer or Benign Prostate Hyperplasia.

BACKGROUND/AIM: Periprostatic adipose tissue (PPAT) directs tumour behaviour. Microenvironment secretome provides information related to its biology. This study was performed to identify secreted proteins by PPAT, from both prostate cancer and benign prostate hyperplasia (BPH) patients. PATIENTS AND METHODS: Liquid chromatography-mass spectrometry-based proteomic analysis was performed in PPAT-conditioned media (CM) from patients with prostate cancer (CMs-T) (stage T3: CM-T3, stage T2: CM-T2) or benign disease (CM-BPH). RESULTS: The highest number and diversity of proteins was identified in CM-T3. Locomotion was the biological process mainly associated to CMs-T and reproduction to CM-T3. Immune responses were enriched in CMs-T. Extracellular matrix and structural proteins were associated to CMs-T. CM-T3 was enriched in proteins with catalytic activity and CM-T2 in proteins with defense/immunity activity. Metabolism and energy pathways were enriched in CM-T3 and those with immune system functions in CMs-T. Transport proteins were enriched in CM-T2 and CM-BPH. CONCLUSION: Proteins and pathways reported in this study could be useful to distinguish stages of disease and may become targets for novel therapies.

Comparative proteomics of soluble factors secreted by human breast adipose tissue from tumor and normal breast.

Tumor progression depends on the tumor-stroma interaction. In the breast, adipose tissue is the predominant stromal type. We have previously demonstrated that conditioned media (CMs) from explants of human adipose tissue of tumor breasts (hATT) increase proliferation and migration of breast cancer epithelial cells when compared to human adipose tissue from normal breasts (hATN). In this work, we aim to identify specific proteins and molecular/biological pathways associated with the secretion profile of hATT and hATN explants. hATT-CMs and hATN-CMs were separated by SDS-PAGE and analyzed by means of two-dimensional nano-liquid chromatography-mass spectrometry. The data was analyzed using ProteoIQ and FunRich software. In addition, 42 cytokines from hATT-CMs and hATN-CMs were assayed by a protein antibody assay. Compared to hATN-CMs, hATT-CMs showed greater protein diversity. We found that hATT-CMs presented a greater amount of proteins related to complement system activity, metabolism and immune system, as well as proteins involved in a variety of biological processes such as signal transduction and cell communication. Specifically, apolipoprotein AI and AII, complement component 3, and vimentin and desmin were significantly increased in hATT-CMs versus hATN-CMs. Moreover, a multivariate discriminant analysis of the cytokines detected by the array showed that IL-6, MCP-2 and GRO cytokines were sufficient and necessary to differentiate hATT-CMs from hATN-CMs. This analysis also showed that the levels of these three cytokines, taken together, correlated with stage and histological grade of the tumor in the hATT-CMs group, and with body mass index in the hATN-CMs group.

Phytochemical Study of the Genus Salpichroa (Solanaceae), Chemotaxonomic Considerations, and Biological Evaluation in Prostate and Breast Cancer Cells.

Twelve Salpichroa taxa have been phytochemically analyzed. From the aerial parts of S. scandens, four known salpichrolides A, C, I, S, and an unreported withanolide named salpichrolide V (1), were isolated. In S. dependens, S. gayi, S. glandulosa subsp. glandulosa, S. glandulosa subps. weddellii, S. leucantha, S. micrantha, S. microloba, S. proboscidea, S. ramosissima, S. tristis var. tristis, and S. weberbauerii, no withanolides were found. The chemical content of ca. 85% of the Salpichroa taxa is in agreement with molecular studies, which suggest that Salpichroa and Jaborosa, a genus considered morphologically close to Salpichroa, are distant in the systematic of the Solanoideae subfamily. Moreover, the in vitro cytotoxic activity of a set of natural salpichrolides and derivatives was examined against two prostate carcinoma cell lines (PC3 and LNCaP) and two human breast cancer cell lines (MCF-7 and T47D). Several compounds showed moderate activity (IC50  = 64.91 - 29.97 µm).

Role of 20-Hydroxyeicosatetraenoic Acid (20-HETE) in Androgen-Mediated Cell Viability in Prostate Cancer Cells.

20-Hydroxyeicosatetraenoic acid (20-HETE) is generated intracellularly through the ω-hydroxylation of arachidonic acid by the cytochrome P450 (in humans, CYP4A11 and CYP4F2). 20-HETE induces mitogenic responses in different cancer cells. The aim of this study was to analyze how 20-HETE impacts cell survival, proliferation, and apoptosis in prostate cancer cells. Incubation of the human androgen-sensitive cells (LNCaP) with 1-10 µM HET0016 (a selective inhibitor of 20-HETE synthesis) reduced cell viability by 49*-64%* (*p < 0.05 vs. control). This was explained by a reduction in cell proliferation (vehicle, 46 ± 3%; 1 µM, 23 ± 3%*; 10 µM, 28 ± 3%*) and by an increase in apoptosis (vehicle, 2.1 ± 0%; 1 µM, 16 ± 4%*; 10 µM, 31 ± 3%*). Furthermore, the increase in LNCaP cell viability induced by dihydrotestosterone (DHT, 0.1 nM) was abrogated by 30*-42%* by 1-10 µM HET0016. Incubation with 20-HETE (5-1000 nM) increased LNCaP cell viability up to 50%*, together with a 70%* reduction in apoptosis. PC-3 (androgen-insensitive) cell viability was not affected by either HET0016 or 20-HETE. In LNCaP cells, HET0016 (10 µM) diminished the expression of androgen receptors (AR): messenger RNA (mRNA) (40%*) and protein (50%*). DHT (10 nM) augmented CYP4F2 protein expression (1.9-fold*) and 20-HETE levels (50%*). Oppositely, enzalutamide (AR antagonist) reduced CYP4F2 mRNA and protein expressions by 30 and 25%, respectively. Thus, intracellular availability of 20-HETE is necessary to sustain LNCaP cell viability. 20-HETE may act as a signaling molecule in the pathways involved in LNCaP cell viability upon stimulation of the AR. This effect may be partially attributed to its role on securing normal AR expression levels that in turn contribute to maintain intracellular levels of 20-HETE.

Human breast adipose tissue: characterization of factors that change during tumor progression in human breast cancer.

BACKGROUND: Adipose microenvironment is involved in signaling pathways that influence breast cancer. We aim to characterize factors that are modified: 1) in tumor and non tumor human breast epithelial cell lines when incubated with conditioned media (CMs) from human breast cancer adipose tissue explants (hATT) or normal breast adipose tissue explants (hATN); 2) in hATN-CMs vs hATT-CMs; 3) in the tumor associated adipocytes vs. non tumor associated adipocytes. METHODS: We used hATN or hATT- CMs on tumor and non-tumor breast cancer cell lines. We evaluated changes in versican, CD44, ADAMTS1 and Adipo R1 expression on cell lines or in the different CMs. In addition we evaluated changes in the morphology and expression of these factors in slices of the different adipose tissues. The statistical significance between different experimental conditions was evaluated by one-way ANOVA. Tukey's post-hoc tests were performed within each individual treatment. RESULTS: hATT-CMs increase versican, CD44, ADAMTS1 and Adipo R1 expression in breast cancer epithelial cells. Furthermore, hATT-CMs present higher levels of versican expression compared to hATN-CMs. In addition, we observed a loss of effect in cellular migration when we pre-incubated hATT-CMs with chondroitinase ABC, which cleaves GAGs chains bound to the versican core protein, thus losing the ability to bind to CD44. Adipocytes associated with the invasive front are reduced in size compared to adipocytes that are farther away. Also, hATT adipocytes express significantly higher amounts of versican, CD44 and Adipo R1, and significantly lower amounts of adiponectin and perilipin, unlike hATN adipocytes. CONCLUSIONS: We conclude that hATT secrete a different set of proteins compared to hATN. Furthermore, versican, a proteoglycan that is overexpressed in hATT-CMs compared to hATN-CMs, might be involved in the tumorogenic behavior observed in both cell lines employed. In addition, we may conclude that adipocytes from the tumor microenvironment show a less differentiated state than adipocytes from normal microenvironment. This would indicate a loss of normal functions in mature adipocytes (such as energy storage), in support of others that might favor tumor growth.

Synthesis and cytotoxic evaluation of four new 6E-hydroximinosteroids.

Four new 6E-hydroximinosteroids (1, 2a, 3 and 4) have been synthesized from the corresponding ketones, 2ß,3ß-dihydroxy-5α-cholestan-6-one (5), 2α,3α-dihydroxy-5α-cholestan-6-one (6), 2ß,3α-dihydroxy-5α-cholestan-6-one (7) and 2ß,3α-dihydroxy-5α-cholestan-6-one-disulfate (8). The cytotoxic activity of the steroidal oximes was evaluated against two prostate carcinoma cell lines (PC-3 and LNCaP) and compared with that of five polyhydroxylated sulfated analogs (8-12). Oxime 3 and trisulfated analog 11 were the most active compounds with IC50 values of 10.8µM (PC-3) and 7.9µM (LNCaP), respectively.

Human adipose tissue from normal and tumoral breast regulates the behavior of mammary epithelial cells.

INTRODUCTION: Stromal-epithelial interactions mediate both breast development and breast cancer progression. In the present work, we evaluated the effects of conditioned media (CMs) of human adipose tissue explants from normal (hATN) and tumor (hATT) breast on proliferation, adhesion, migration and metalloproteases activity on tumor (MCF-7 and IBH-7) and non-tumor (MCF-10A) human breast epithelial cell lines. MATERIALS AND METHODS: Human adipose tissues were obtained from patients and the conditioned medium from hATN and hATT collected after 24 h of incubation. MCF-10A, MCF-7 and IBH-7 cells were grown and incubated with CMs and proliferation and adhesion, as well as migration ability and metalloprotease activity, of epithelial cells after exposing cell cultures to hATN- or hATT-CMs were quantified. The statistical significance between different experimental conditions was evaluated by one-way ANOVA. Tukey's post hoc tests were performed. RESULTS: Tumor and non-tumor breast epithelial cells significantly increased their proliferation activity after 24 h of treatment with hATT-CMs compared to control-CMs. Furthermore, cellular adhesion of these two tumor cell lines was significantly lower with hATT-CMs than with hATN-CMs. Therefore, hATT-CMs seem to induce significantly lower expression or less activity of the components involved in cellular adhesion than hATN-CMs. In addition, hATT-CMs induced pro-MMP-9 and MMP-9 activity and increased the migration of MCF-7 and IBH-7 cells compared to hATN-CMs. CONCLUSIONS: We conclude that the microenvironment of the tumor interacts in a dynamic way with the mutated epithelium. This evidence leads to the possibility to modify the tumor behavior/phenotype through the regulation or modification of its microenvironment. We developed a model in which we obtained CMs from adipose tissue explants completely, either from normal or tumor breast. In this way, we studied the contribution of soluble factors independently of the possible effects of direct cell contact.

A unique galectin signature in human prostate cancer progression suggests galectin-1 as a key target for treatment of advanced disease.

Galectins, a family of glycan-binding proteins, influence tumor progression by modulating interactions between tumor, endothelial, stromal, and immune cells. Despite considerable progress in identifying the roles of individual galectins in tumor biology, an integrated portrait of the galectin network in different tumor microenvironments is still missing. We undertook this study to analyze the "galectin signature" of the human prostate cancer microenvironment with the overarching goal of selecting novel-molecular targets for prognostic and therapeutic purposes. In examining androgen-responsive and castration-resistant prostate cancer cells and primary tumors representing different stages of the disease, we found that galectin-1 (Gal-1) was the most abundantly expressed galectin in prostate cancer tissue and was markedly upregulated during disease progression. In contrast, all other galectins were expressed at lower levels: Gal-3, -4, -9, and -12 were downregulated during disease evolution, whereas expression of Gal-8 was unchanged. Given the prominent regulation of Gal-1 during prostate cancer progression and its predominant localization at the tumor-vascular interface, we analyzed the potential role of this endogenous lectin in prostate cancer angiogenesis. In human prostate cancer tissue arrays, Gal-1 expression correlated with the presence of blood vessels, particularly in advanced stages of the disease. Silencing Gal-1 in prostate cancer cells reduced tumor vascularization without altering expression of other angiogenesis-related genes. Collectively, our findings identify a dynamically regulated "galectin-specific signature" that accompanies disease evolution in prostate cancer, and they highlight a major role for Gal-1 as a tractable target for antiangiogenic therapy in advanced stages of the disease.

Human periprostatic adipose tissue: its influence on prostate cancer cells.

BACKGROUND/AIMS: Adipose microenvironment is involved in signaling pathways that influence prostate cancer (PCa) progression. However, the role of human periprostatic adipose tissue (PPAT) from patients with benign prostatic hyperplasia (BPH) has not been studied and compared to that of PPAT from PCa patients. The aim of this paper was to investigate the influence of factors derived from both PPATs on the behavior of androgen-dependent and castration resistant PCa cells. METHODS: PPAT conditioned media (CM) were obtained from tissue samples from patients with clinically primary PCa (TPPAT) or BPH (BPPAT). Cell adhesion, proliferation, migration and metalloproteinase expression were evaluated following exposure of LNCaP (androgen dependent) and PC3 (androgen independent) prostate cancer cell lines to BPPAT or TPPAT CM. RESULTS: Proliferation or motility of LNCaP or PC3 cells were not significantly affected by TPPAT or BPPAT CM. The number of LNCaP but not PC3 cells attached to components of TPPAT CM significantly decreased compared to cells attached to BPPAT CM. PPAT produced and released pro-MMP-9. Zymograms demonstrated that TPPAT CM induced a significant increase in pro-MMP-9 activity compared to BPPAT CM in LNCaP cells but not in PC3 cells. CONCLUSIONS: We conclude that TPPAT released factors, such as pro-MMP-9, could induce the invasive capacity of LNCaP cells and speculate that PPAT derived factors could, in the early stages of prostate cancer, modulate disease progression.

Adipocyte differentiation influences the proliferation and migration of normal and tumoral breast epithelial cells.

Stromal tissue regulates the development and differentiation of breast epithelial cells, with adipocytes being the main stromal cell type. The aim of the present study was to evaluate the effect of adipocyte differentiation on proliferation and migration, as well as to assess the activity of heparanase and metalloproteinase-9 (MMP-9), in normal (NMuMG) and tumoral (LM3) murine breast epithelial cells. NMuMG and LM3 cells were grown on irradiated 3T3-L1 cells (stromal support, SS) at various degrees of differentiation [preadipocytes (preA), poorly differentiated adipocytes (pDA) and mature adipocytes (MA)] and/or were incubated in the presence of conditioned medium (CM) derived from each of these three types of differentiated cells. Cells grown on a plastic support or in fresh medium served as the controls. Cell proliferation was measured with a commercial colorimetric kit, and the motility of the epithelial cells was evaluated by means of a wound-healing assay. Heparanase activity was assessed by quantifying heparin degradation, and the expression of MMP-9 was determined using Western blotting. The results indicate that cell proliferation was increased after 24 and 48 h in the NMuMG and LM3 cells grown on preA, pDA and MA SS. In the NMuMG cells cultured on SS in the presence of all three types of CM, proliferation was enhanced. LM3 cell migration was increased in the presence of all three types of CM and in cells grown on preA SS. Heparanase activity was increased in the NMuMG cells incubated with all three types of CM, and in the LM3 cells incubated with the CM from pDA and MA. Both the NMuMG and LM3 cell lines presented basal expression of MMP-9; however, a significant increase in MMP-9 expression was observed in the LM3 cells incubated with each of the three types of CM. In conclusion, adipocyte differentiation influences normal and tumoral breast epithelial cell proliferation and migration. Heparanase and MMP-9 appear to be involved in this regulation. The experimental model presented in this study is in keeping with the characteristics of the physiological environment of breast epithelial cells, in terms of both the soluble and insoluble factors present and the stromal structure per se.

Homeostatic response under carcinogen withdrawal, heme oxygenase 1 expression and cell cycle association.

BACKGROUND: Chronic injury deregulates cellular homeostasis and induces a number of alterations leading to disruption of cellular processes such as cell cycle checkpoints and apoptosis, driving to carcinogenesis. The stress protein heme oxygenase-1 (HO-1) catalyzes heme degradation producing biliverdin, iron and CO. Induction of HO-1 has been suggested to be essential for a controlled cell growth. The aim of this work was to analyze the in vivo homeostatic response (HR) triggered by the withdrawal of a potent carcinogen, p-dimethylaminoazobenzene (DAB), after preneoplastic lesions were observed. We analyzed HO-1 cellular localization and the expression of HO-1, Bcl-2 and cell cycle related proteins under these conditions comparing them to hepatocellular carcinoma (HC). METHODS: The intoxication protocol was designed based on previous studies demonstrating that preneoplastic lesions were evident after 89 days of chemical carcinogen administration. Male CF1 mice (n = 18) were used. HR group received DAB (0.5 % w/w) in the diet for 78 days followed by 11 days of carcinogen deprivation. The HC group received the carcinogen and control animals the standard diet during 89 days. The expression of cell cycle related proteins, of Bcl-2 and of HO-1 were analyzed by western blot. The cellular localization and expression of HO-1 were detected by immnunohistochemistry. RESULTS: Increased expression of cyclin E/CDK2 was observed in HR, thus implicating cyclin E/CDK2 in the liver regenerative process. p21cip1/waf1 and Bcl-2 induction in HC was restituted to basal levels in HR. A similar response profile was found for HO-1 expression levels, showing a lower oxidative status in the carcinogen-deprived liver. The immunohistochemical studies revealed the presence of macrophages surrounding foci of necrosis and nodular lesions in HR indicative of an inflammatory response. Furthermore, regenerative cells displayed changes in type, size and intensity of HO-1 immunostaining. CONCLUSION: These results demonstrate that the regenerative capacity of the liver is still observed in the pre-neoplastic tissue after carcinogen withdrawal suggesting that reversible mechanism/s to compensate necrosis and to restitute homeostasis are involved.

Synthesis of encoded modified oligonucleotide libraries. Part 1: orthogonal chemistry.

The basis for further development of combinatorial libraries of modified oligonucleotides tagged by a codifying sequence is discussed. The chemistry involved in the orthogonal synthesis of both strands and some representative examples of building blocks are presented.