Photodynamic therapy reduces cell viability, migration and triggers necroptosis in prostate tumor cells.

Prostate cancer is the most common cancer in American men, aside from skin cancer. As an alternative cancer treatment, photodynamic laser therapy (PDT) can be used to induce cell death. We evaluated the PDT effect, using methylene blue as a photosensitizer, in human prostate tumor cells (PC3). PC3 were subjected to four different conditions: DMEM (control); laser treatment (L-660 nm, 100 mW, 100 J.cm-2); methylene blue treatment (MB-25 µM, 30 min), and MB treatment followed by low-level red laser irradiation (MB-PDT). Groups were evaluated after 24 h. MB-PDT treatment reduced cell viability and migration. However, because MB-PDT did not significantly increase the levels of active caspase-3 and BCL-2, apoptosis was not the primary mode of cell death. MB-PDT, on the other hand, increased the acid compartment by 100% and the LC3 immunofluorescence (an autophagy marker) by 254%. Active MLKL level, a necroptosis marker, was higher in PC3 cells after MB-PDT treatment. Furthermore, MB-PDT resulted in oxidative stress due to a decrease in total antioxidant potential, catalase levels, and increased lipid peroxidation. According to these findings, MB-PDT therapy is effective at inducing oxidative stress and reducing PC3 cell viability. In such therapy, necroptosis is also an important mechanism of cell death triggered by autophagy.

High-fat diet during sexual maturation induces hyperplastic differentiation of rat prostate and higher expression of AR45 isoform and ERα.

We examined the consequences of high-fat diet (HFD) on prostate histophysiology in two periods along sexual maturation of rats and the impact on the gland in adulthood. After weaning, male Wistar rats were fed a balanced diet (4 % fat-C3, C6, C9) or a HFD (20 % fat- HF3, HF6, HF9) for 3, 6 or 9 weeks. Fat deposit weights, blood glucose and levels of serum testosterone and estrogen were measured. Prostate was evaluated for histology, proliferative and apoptotic cell index, and for the expression of androgen (AR), estrogen receptors type α (ERα) and aromatase. HFD did not affect estrogen levels and elevated serum testosterone only in HF9. HFD reduced prostate weight in HF6 and increased it in adulthood (HF9) but relative prostate weight was unchanged among groups. Cell proliferation, height and density were higher in epithelium of all HFD-groups, compared to controls, featuring the epithelial hyperplasia. Epithelial apoptosis was lower in HF9. HF3 and HF9 exhibited higher expressions of ERα, indicating that HFD triggers a new activation of ERα expression in the acinar epithelium. The content of prostatic aromatase was also elevated in HF9. Increased numbers of AR-positive cells were observed in all HFD groups, and western blotting analysis showed an increase in the truncated form of 45 kDa (AR45) and a reduction in the expression of 110 kDa-AR for HF3 and HF9. In conclusion, excessive dietary fats during sexual maturation of rats led to developmental programming of the prostate, inducing a hyperplastic status with perturbations in AR isoforms expression and reactivation of ERα in adulthood, whose implications for posterior prostatic health could be detrimental.

Wound healing activity of the hydroethanolic extract of the leaves of Maytenus ilicifolia Mart. Ex Reis.

BACKGROUND AND AIM: Maytenus ilicifolia has analgesic, healing, antioxidant and anti-inflammatory properties. This study evaluated effect of the hydroalcoholic extract of M. ilicifolia leaves on skin wound repair. EXPERIMENTAL PROCEDURE: Wounds were induced on mice and treated with the extract. The treatment was performed daily, until day 7 after wound induction. Wound closure was measured and the features of the repaired tissue were investigated, including mast cell quantification, neutrophil and macrophage activities, collagen deposition, angiogenesis, and pro-metalloproteases and metalloproteases 2 and 9 activity (pro-MMPs and MMPs). RESULTS AND CONCLUSION: The M. ilicifolia extract accelerated the closure of wounds. The extract at a concentration of 4% was found to be effective, presenting anti-inflammatory effects and hemoglobin increased, along with increased soluble, total and type III collagens in the wound. In addition, there was an increase in pro-MMP9 and MMP9 activity after day 7th of treatment. The phenolic compounds and tannins present in this plant could be associated with the anti-inflammatory and healing activities observed in this study. Therefore, the ability to modulate essential parameters for accelerated and adequate healing as shown here suggests that the use of standardised extracts of M. ilicifolia and its fractions enriched in polyphenols may represent a therapeutic strategy for the treatment of wounds.

Swimming reduces fatty acids-associated hypothalamic damage in mice.

The arcuate and the paraventricular and lateral hypothalamic nuclei, related to hunger and satiety control, are generally compromised by excess fatty acids. In this situation, fatty acids cause inflammation via TLR4 (toll like receptor 4) and the nuclei become less responsive to the hormones leptin and insulin, contributing to the development of obesity. In this work, these nuclei were analyzed in animals fed with high-fat diet and submitted to swimming without and with load for two months. For this, frontal sections of the hypothalamus were immunolabelled with GFAP (glial fibrillary acidic protein), synaptophysin, IL-6 (interleukin 6) and TLR4. Also, proteins extracted from the hypothalamus were analyzed using Western blotting (GFAP and synaptophysin), fluorometric analysis for caspases 3 and 7, and CBA (cytometric bead array) for Th1, Th2, and Th17 profiles. The high-fat diet significantly caused overweight and, in the hypothalamus, decreased synapses and increased astrocytic reactivity. The swimming with load, especially 80 % of the maximum load, reduced those consequences. The high-fat diet increased TLR4 in the arcuate nucleus and the swimming exercise with 80 % of the maximum load showed a tendency of reducing this expression. Swimming did not significantly influence the inflammatory or anti-inflammatory cytokines in the hypothalamus or in plasma. The high-fat diet in sedentary animals increased the expression of caspases 3 and 7 and swimming practice reduced this increment to levels compatible with animals fed on a normal diet. The set of results conclude that the impact of swimming on the damage caused in the hypothalamus by a high-fat diet is positive. The different aspects analyzed in here point to better cellular viability and conservation of the synapses in the hypothalamic nuclei of overweight animals that practiced swimming with a load.

Effect of glucose and palmitate environment on proliferation and migration of PC3-prostate cancer cells.

Recent studies have been trying to find out how diet and metabolic changes such as dyslipidaemia, hyperglycaemia, and hyperinsulinaemia can stimulate cancer progression. This investigation aimed to evaluate the effect of high concentrations of fatty acids and/or glucose in tumour prostate cells, focusing on the proliferation/migration profile and oxidative stress. PC3 cells were treated with high concentration of saturated fatty acid (palmitate, 100 µM), glucose (220 mg/dL), or both for 24 or 48 h. Results demonstrated that PC3 cells showed a significant increase in proliferation after 48 h of treatment with glucose and palmitate+glucose. Cell proliferation was associated with reduced levels of AMPK phosphorylation in glucose group at 24 and 48 h of treatment, while palmitate group presented this result only after 48 h of treatment. Also, there was a significant increase in cell migration between time 0 and 48 h after all treatments, except in the control. Catalase activity was increased by palmitate in the beginning of treatment, while glucose presented a later effect. Also, nitrite production was increased by glucose only after 48 h, and the total antioxidant activity was enhanced by palmitate in the initial hours. Thus, we conclude that the high concentration of the saturated fatty acid palmitate and glucose in vitro influences PC3 cells and stimulates cellular activities related to carcinogenesis such as cell proliferation, migration, and oxidative stress in different ways. Palmitate presents a rapid and initial effect, while a glucose environment stimulates cells later on, maintaining high levels of cell proliferation.

Pathological lesions and global DNA methylation in rat prostate under streptozotocin-induced diabetes and melatonin supplementation.

Chronic hyperglycemia increases production of reactive oxygen species, which favors carcinogenesis. The association between diabetes and prostate cancer is controversial. Melatonin has antioxidant, anti-inflammatory, and antiproliferative properties. We investigated whether low doses of melatonin prevent the tissue alterations caused by diabetes and alter prostate histology of healthy rats. We also investigated whether experimental diabetes promoted the development of pathological lesions in the ventral prostate of rats. Melatonin was provided in drinking water (10 µg/kg/day) from age 5 weeks until the end of experiment. Diabetes was induced at 13 weeks by administration of streptozotocin (40 mg/kg, ip). Rats were euthanized at 14 or 21 weeks. Histological and stereological analyses were carried out and the incidence and density of malignant and pre-malignant lesions were assessed. Immunohistochemical assays of α-actin, cell proliferation (PCNA), Bcl-2, glutathione S-transferase (GSTPI), and DNA methylation (5-methylcytidine) were performed. Melatonin did not elicit conspicuous changes in the prostate of healthy animals; in diabetic animals there was a higher incidence of atrophy (93%), microinvasive carcinoma (10%), proliferative inflammatory atrophy, PIA (13%), prostatitis (26%), and prostate intraepithelial neoplasia, PIN (20%) associated with an increase of 40% in global DNA methylation. Melatonin attenuated epithelial and smooth muscle cell (smc) atrophy, especially at short-term diabetes-and normalized incidence of PIN (11%), inflammatory cells infiltrates, prostatitis (0%) and PIA (0%) at long-term diabetes. MLT was effective in preventing inflammatory disorders and PIN under diabetic condition. Although MLT has antioxidant action, it did not influence DNA methylation and not avoid carcinogenesis at low doses.

Role of the TNF-α receptor type 1 on prostate carcinogenesis in knockout mice.

BACKGROUND: TNF-α is a key cytokine involved in prostate carcinogenesis and is mediated by the TNF-α receptor type 1 (TNFR-1). This receptor triggers two opposite pathways: cell death or cell survival and presents a protective or stimulator role in cancer. Thus, the purpose of this study was to evaluate the role of TNF signaling in chemically induced prostate carcinogenesis in mice. METHODS: C57bl/6 wild type (WT) and p55 TNFR-1 knockout mice (KO) were treated with mineral oil (control) or N-methyl N-nitrosurea (MNU) in association with testosterone (MNU+T, single injection of 40 mg/kg and weekly injection 2 mg/kg, respectively) over the course of 6 months. After this induction period, prostate samples were processed for histological and biochemical analysis. RESULTS: MNU+T treatment led to the development of prostate intraepithelial neoplasia (PIN) and adenocarcinoma (PCa) in both WT and KO animals; however, the incidence of PCa was lower in KO group than in WT. Cell proliferation analysis showed that PCNA levels were significantly lower in the KO group, even after carcinogenesis induction. Furthermore, the prostate of KO animals had lower levels of p65 and p-mTOR after treatment with MNU+T than WT. There was also a decrease in prostate androgen receptor levels after induction of carcinogenesis in both KO and WT mice. Regarding the extracellular matrix in the prostate, KO mice had higher levels of fibronectin and lower levels of matrix metalloproteinase 2 (MMP2) after carcinogenesis. Finally, there was a similar increase in apoptosis in both groups after carcinogenesis, indicating that the TNAFr1 pathway in prostate carcinogenesis presented proliferative, and not apoptotic, stimuli. CONCLUSIONS: TNF-α, through its receptor TNFR-1, promoted cell proliferation and cell survival in prostate by activation of the AKT/mTOR and NFKB pathway, which stimulated prostate carcinogenesis in chemically induced mice. Prostate 76: 917-926, 2016. © 2016 Wiley Periodicals, Inc.

Differential ontogenetic exposure to obesogenic environment induces hyperproliferative status and nuclear receptors imbalance in the rat prostate at adulthood.

BACKGROUND: Experimental data indicate that high-fat diet (HFD) may alter proliferative activity and prostate health. However, the consequences of HFD exposure during different periods of ontogenetic development on prostate histophysiology remain to be elucidated. Herein, we compare the influence of obesogenic environment (OE) due to maternal obesity and HFD at different periods of life on proliferative activity and nuclear receptors frequency in the rat ventral prostate and a possible relationship with metabolic and hormonal alterations. METHODS: Male Wistar rats (19 weeks old), treated with balanced chow (Control group-C; 3% high-fat, 3.5 Kcal/g), were compared with those exposed to HFD (20% high-fat, 4.9 kcal/g) during gestation (G-maternal obesity), gestation and lactation (GL), from post-weaning to adulthood (WA), from lactation to adulthood (LA) and from gestation to adulthood (GA). After the experimental period, the ventral prostate lobes were removed and analyzed with different methods. RESULTS: Metabolic data indicated that G and GL rats became insulin resistant and WA, LA, and GA became insulin resistant and obese. There was a strong inverse correlation between serum testosterone (∼133% lower) and leptin levels (∼467% higher) in WA, LA, and GA groups. Estrogen serum levels increased in GA, and insulin levels increased in all groups, especially in WA (64.8×). OE-groups exhibited prostatic hypertrophy, since prostate weight increased ∼40% in G, GL, LA, and GA and 31% in WA. As indicated by immunohistochemistry, all HFD-groups except G exhibited an increase in epithelial cell proliferation (PCNA-positive) and a decrease in frequency of AR- and ERß-positive epithelial cells; there was also an increment of ERα-positive stromal cells in comparison with control. Cells containing PPARγ increased in both epithelium and stroma of all OE groups and those expressing LXRα decreased, particularly in groups OE-exposed during gestation (G, GL and GA). CONCLUSIONS: OE leads to prostate hypertrophy regardless of the period of development and, except when restricted to gestation, leads to a hyperproliferative status which was correlated to downregulation of AR and LXRα and upregulation of ERα and PPARγ signaling.

Prostate hyperplasia caused by long-term obesity is characterized by high deposition of extracellular matrix and increased content of MMP-9 and VEGF.

Recent studies have shown a positive association of cancer and obesity, but the morphological and molecular mechanisms involved in this relationship are still unknown. This study analysed the impact of long-term obesity on rat prostate, focusing on stromal changes. Male adult Wistar rats were treated with high-fat diet to induce obesity, while the control group received a balanced diet. After 30 weeks of feeding, the ventral prostate was analysed by immunohistochemistry for cell proliferation, smooth muscle α-actin, vimentin, chondroitin sulphate and metalloproteinases (MMP-2 and 9). The content of androgen receptor (AR), oestrogen receptors (ERs) and vascular endothelial growth factor (VEGF) was measured by Western blotting, and activity of catalase and Glutathione-S-Transferase (GST) were quantified by enzymatic assay. Long-term obesity decreased testosterone plasma levels by 70% and resulted in stromal prostate hyperplasia, as evidenced by increased collagen fibres. Such stromal hyperplasia was associated with increased number of blood vessels and raised VEGF content, and increased expression of chondroitin sulphate, vimentin, α-actin and MMP-9. In spite of the high cell density in prostate, the proliferative activity was lower in the prostates of obese rats, indicating that hyperplasia was established during the early phases in this obesity model. AR levels increased significantly, whereas the ERα decreased in this group. Moreover, the levels of catalase and GST were changed considerably. These findings indicate that long-term obesity, besides disturbing the antioxidant control, causes intense stromal remodelling and release of factors that create an environment that can promote proliferative disorders in the gland, culminating with diffuse hyperplasia.

High-fat diet obesity associated with insulin resistance increases cell proliferation, estrogen receptor, and PI3K proteins in rat ventral prostate.

In this study, we evaluated the effects of obesity and insulin resistance induced by a high-fat diet on prostate morphophysiology, focusing on cell proliferation, expression of androgen (AR) and estrogen receptors (ER) and proteins of the insulin signaling pathway. Adult male Wistar rats were fed a high-fat diet (20% fat) for 15 weeks, whereas control animals received a balanced diet (4% fat). Both groups were then divided and treated for 2 weeks with 1 mg/kg body weight/day of the aromatase inhibitor letrozole or vehicle only. The ventral prostate was analyzed with immunohistochemical, histopathological, stereological, and Western blotting methods. Obese rats showed insulin resistance, hyperinsulinemia, and reduced plasma testosterone levels. The incidence of prostatic intraepithelial neoplasia (PIN) was 2.7 times higher in obese rats and affected 0.4% of the gland compared with 0.1% PIN areas found in control rats. Obesity doubled cell proliferation in both prostate epithelium and stroma. AR content decreased in the prostate of obese rats and estrogen receptor beta (ERß) increased in this group. Protein levels of insulin receptor substrate 1 and protein kinase B diminished in the obese group, whereas phosphatidylinositol 3-kinase (PI3K) increased significantly. Most structural changes observed in the prostate of obese rats normalized after letrozole treatment, except for increased stromal cell proliferation and ERß expression, which might be associated with insulin resistance. This experimental model of obesity and insulin resistance induced by a high-fat diet increases cell proliferation in rat prostate. Such alterations are associated with decreased levels of AR and increased ERß and PI3K proteins. This change can facilitate the establishment of proliferative lesions in rat prostate.

Proliferation and apoptotic rates and increased frequency of p63-positive cells in the prostate acinar epithelium of alloxan-induced diabetic rats.

The effects of experimental type 1 diabetes were investigated in the acinar epithelium of rat ventral prostate, focusing on the rates of cell proliferation and the frequency of apoptosis and p63-positive cells. Type 1 diabetes was induced in adult male Wistar rats by a single alloxan administration (42 mg/kg b.w.) and its effects were analysed for 1 week and 3 months after the establishment of the disease. A group of diabetic rats was treated daily with 5 IU of insulin during 1 week after diabetes had been diagnosed. Immunocytochemical methods for the localization of cell proliferation antigen (PCNA), androgen receptor (AR) and p63 protein were carried out, and apoptotic cells were identified by TUNEL essay. In diabetic rats, testosterone levels reduced drastically after 1 week and in a lower degree after 3 months. In short-term diabetic rats, cell proliferation decreased, and in medium-term, epithelial apoptotic rates increased. In both periods after the onset of diabetes, the frequency of p63-positive cells doubled. Insulin treatment was effective in preventing testosterone decrease, p63-positive cell increase and apoptotic rates, but did not interfere in cell proliferation. This investigation shows that, soon after diabetes onset, there are important modifications in cell proliferation within the acinar prostatic epithelium, and in longer term, there is a marked impact on kinetics of differentiation and cell death, which may initially be attributable to an androgenic fall, but is probably also because of other factors related to diabetes, as changes are considerably different from those resulting from castration.

Diabetes induces stromal remodelling and increase in chondroitin sulphate proteoglycans of the rat ventral prostate.

Extracellular matrix (ECM) remodelling is an important process involved in prostate cancer progression. Alterations in ECM caused by diabetes in different tissues such as kidney is well described; however, it is poorly investigated in prostate. The aim of this study was to evaluate changes in ECM of rat prostate showing gland atrophy caused by diabetes and their implications in development of malignant lesions. Diabetes was induced in Wistar rats using alloxan (45 mg/kg bw). After 90 days of diabetes onset, animals were killed and ventral prostate was removed and prepared for light microscopy following immunoreaction for fibronectin, chondroitin sulphate and Picrossirius staining for collagen fibres. Proteoglycans (PG) were identified at transmission electron microscopy after fixation with Cuprolinic Blue. Diabetes led to a thickening of 25% in the acinar basement membrane accompanied by increase and disorganization of its proteoglycans (P1). Three additional populations of prostatic stromal PGs were identified: collagen fibril linked (P2) and interstitial (P3) and (P4) PGs. Diabetes increased P3 and mainly P4 which had higher dimension and accumulated around the smooth muscle cells. In addition, an increase in chondrotin sulphate (33%, mainly in sites where P4 were noted) and collagen (44%) was noted in diabetic rats, whereas fibronectin did not change. Atrophic changes observed in rat ventral prostate after diabetes are accompanied by stromal remodelation related to increase in collagen and chondroitin sulphate proteoglycans. Thus, diabetes can promote a stromal microenvironment rich in elements that could favour cell migration, proliferation and pathological process.

Malignant lesions in the ventral prostate of alloxan-induced diabetic rats.

The aim of this study was to evaluate the changes caused by chronic diabetes in the rat ventral prostate and to establish a correlation between diabetes and the development of prostatic lesions. Male rats received alloxan (42 mg/kg b.w.) to induce diabetes. Ninety days after diabetes diagnosis, animals were sacrificed and the ventral prostate was removed and prepared for general and immunohistochemical analyses. The total area showing different types of lesions was estimated. Diabetes led to a decrease in the body and prostatic weights, as well as in testosterone levels. The prostate morphology and stereology showed high variation in the diabetic group. Some animals had light changes; the great majority had an intense epithelial atrophy; and other rats showed premalignant and malignant lesions in the prostate. Such epithelial atrophy was, in some samples, combined with chronic inflammation, similar to proliferative inflammatory atrophy (PIA). The diabetic group also presented high incidence of prostatitis, adenocarcinoma and prostatic intra-epithelial neoplasia (PIN). Samples with adenocarcinoma had poorly differentiated acini with high levels of cellular proliferation and nuclear atypia. These lesions exhibited an invasive feature showing Bcl-2-positive cells and interruptions in the basement membrane. An association of PIA, PIN and adenocarcinoma was detected in one sample. Reduced androgen levels have a synergic effect to insulin dysfunction promoting negative effects in the rat prostate. Diabetic individuals had a high incidence of prostatitis, and this inflammation could stimulate the incidence of other forms of prostatic pathology.

Increased pancreatic islet mass is accompanied by activation of the insulin receptor substrate-2/serine-threonine kinase pathway and augmented cyclin D2 protein levels in insulin-resistant rats.

It is well known that glucocorticoids induce peripheral insulin resistance in rodents and humans. Here, we investigated the structural and ultrastructural modifications, as well as the proteins involved in beta-cell function and proliferation, in islets from insulin-resistant rats. Adult male Wistar rats were made insulin resistant by daily administration of dexamethasone (DEX; 1mg/kg, i.p.) for five consecutive days, whilst control (CTL) rats received saline alone. Structure analyses showed a marked hypertrophy of DEX islets with an increase of 1.7-fold in islet mass and of 1.6-fold in islet density compared with CTL islets (P < 0.05). Ultrastructural evaluation of islets revealed an increased amount of secreting organelles, such as endoplasmic reticulum and Golgi apparatus in DEX islets. Mitotic figures were observed in DEX islets at structural and ultrastructural levels. Beta-cell proliferation, evaluated at the immunohistochemical level using anti-PCNA (proliferating cell nuclear antigen), showed an increase in pancreatic beta-cell proliferation of 6.4-fold in DEX islets compared with CTL islets (P < 0.0001). Increases in insulin receptor substrate-2 (IRS-2), phosphorylated-serine-threonine kinase AKT (p-AKT), cyclin D(2) and a decrease in retinoblastoma protein (pRb) levels were observed in DEX islets compared with CTL islets (P < 0.05). Therefore, during the development of insulin resistance, the endocrine pancreas adapts itself increasing beta-cell mass and proliferation, resulting in an amelioration of the functions. The potential mechanisms that underlie these events involve the activation of the IRS-2/AKT pathway and activation of the cell cycle, mediated by cyclin D(2). These adaptations permit the maintenance of glycaemia at near-physiological ranges.