Mangiferin blocks proliferation and induces apoptosis of breast cancer cells via suppression of the mevalonate pathway and by proteasome inhibition.

Mangiferin is a natural xanthone glycoside with therapeutic potential. Herein, its cytotoxic properties were explored in a human cell model of breast adenocarcinoma. The results supported the multi-target nature of mangiferin action, as the inhibition of three enzymatic systems, namely HMG-CoA reductase, the proteasome and plasmin, respectively in charge of regulating cholesterol homeostasis, protein turnover and cell adhesion, was documented for the first time. Globally, mangiferin was able to selectively block breast cancer cell growth by inducing apoptosis and by arresting cell proliferation through a combined action on cholesterol and proteasome pathways, as well as to inhibit plasmin-mediated mechanisms of cell migration.

Pazopanib and sunitinib trigger autophagic and non-autophagic death of bladder tumour cells.

BACKGROUND: Tyrosine kinase inhibitors (TKI) such as sunitinib and pazopanib display their efficacy in a variety of solid tumours. However, their use in therapy is limited by the lack of evidence about the ability to induce cell death in cancer cells. Our aim was to evaluate cytotoxic effects induced by sunitinib and pazopanib in 5637 and J82 bladder cancer cell lines. METHODS: Cell viability was tested by MTT assay. Autophagy was evaluated by western blot using anti-LC3 and anti-p62 antibodies, acridine orange staining and FACS analysis. Oxygen radical generation and necrosis were determined by FACS analysis using DCFDA and PI staining. Cathepsin B activation was evaluated by western blot and fluorogenic Z-Arg-Arg-AMC peptide. Finally, gene expression was performed using RT-PCR Profiler array. RESULTS: We found that sunitinib treatment for 24 h triggers incomplete autophagy, impairs cathepsin B activation and stimulates a lysosomal-dependent necrosis. By contrast, treatment for 48 h with pazopanib induces cathepsin B activation and autophagic cell death, markedly reversed by CA074-Me and 3-MA, cathepsin B and autophagic inhibitors, respectively. Finally, pazopanib upregulates the α-glucosidase and downregulates the TP73 mRNA expression. CONCLUSION: Our results showing distinct cell death mechanisms activated by different TKIs, provide the biological basis for novel molecularly targeted approaches.

Cloning of sole proopiomelanocortin (POMC) cDNA and the effects of stocking density on POMC mRNA and growth rate in sole, Solea solea.

Proopiomelanocortin (POMC) is an important gene implicated in different functions, such as the stress response of the hypothalamus-pituitary-adrenal axis. The aim of the present study was to determine whether farming conditions, such as stocking density, can be considered a powerful stressor influencing in turn the growth rate in juvenile fish. Thus, POMC cDNA expression was investigated during adaptation to farming conditions in sole (Solea solea), as a model for studying the effects of rearing densities on stress response; different stocking densities (50, 100, and 250 animals/m(2)) were applied and, after 7 and 21 days, the fishes were examined for body weight and plasma cortisol levels as indicators of stress. In addition, proopiomelanocortin was cloned and sequenced from the brain of sole, allowing semi-quantitative RT-PCR to be performed to evaluate POMC mRNA expression in brain tissue. There was a significant increase in cortisol levels in fish reared at high stocking densities of 250/m(2) compared to fish reared at control densities of 100 and 50/m(2), in both experimental times, i.e., 7 and 21 days. The high stocking densities were also found to decrease the specific growth rate of fish. Moreover, it was demonstrated that the highest stocking density induced a significant decrease in sole POMC mRNA expression. It is concluded that POMC and cortisol are both involved in the stress response due to high rearing densities, during which cortisol may serve as a negative regulator of POMC.

Capsaicin-induced apoptosis of glioma cells is mediated by TRPV1 vanilloid receptor and requires p38 MAPK activation.

We provide evidence on the expression of the transient receptor potential vanilloid type-1 (TRPV1) by glioma cells, and its involvement in capsaicin (CPS)-induced apoptosis. TRPV1 mRNA was identified by quantitative RT-PCR in U373, U87, FC1 and FLS glioma cells, with U373 cells showing higher, and U87, FC1 and FLS cells lower TRPV1 expression as compared with normal human astrocytes. By flow cytometry we found that a substantial portion of both normal human astrocytes, and U87 and U373 glioma cells express TRPV1 protein. Moreover, we analyzed the expression of TRPV1 at mRNA and protein levels of glioma tissues with different grades. We found that TRPV1 gene and protein expression inversely correlated with glioma grading, with marked loss of TRPV1 expression in the majority of grade IV glioblastoma multiforme. We also described that CPS trigger apoptosis of U373, but not U87 cells. CPS-induced apoptosis involved Ca(2+) influx, p38 but not extracellular signal-regulated mitogen-activated protein kinase activation, phosphatidylserine exposure, mitochondrial permeability transmembrane pore opening and mitochondrial transmembrane potential dissipation, caspase 3 activation and oligonucleosomal DNA fragmentation. TRPV1 was functionally implicated in these events as they were markedly inhibited by the TRPV1 antagonist, capsazepine. Finally, p38 but not extracellular signal-regulated protein kinase activation was required for TRPV1-mediated CPS-induced apoptosis of glioma cells.

Proopiomelanocortin gene expression and beta-endorphin localization in the pituitary, testis, and epididymis of stallion.

Proopiomelanocortin (POMC) is a precursor protein that contains the sequences of several bioactive peptides including adrenocorticotropin (ACTH), beta-endorphin (beta-EP), and melanocyte-stimulating-hormone (MSH). POMC is synthesized in the pituitary gland, brain, and many peripheral tissues. Immunoreactive POMC-derived peptides as well as POMC-like mRNA have been evidenced in several nonpituitary tissues, thus suggesting that POMC is actively synthesized by these tissues. The present study was aimed at evaluating if also in the case of stallion POMC-derived peptide, beta-EP, is produced locally in the testis, thus playing effects in a paracrine/autocrine fashion. To investigate this hypothesis the POMC gene expression was analyzed using 3' RACE-PCR and Northern Blot approaches in the testis and epididimys of stallion; moreover, immunocytochemical localization for beta-EP was also performed through confocal laser microscopy. The immunofluorescence results showed a positive beta-EP reaction not only in cellular nest of pituitary but also in the testis and genital tract of stallion, which function could be related with sperm mobility. Such role seem not to be no dependent on the peptide synthesized locally, because the molecular biology approach demonstrated the presence of POMC transcript in the pituitary only. In fact the Northern Blot analysis showed the presence of a single POMC transcript in the pituitary while no signal was detected in the testis and epididimys. The same results were obtained by applied 3' RACE-PCR analysis. In conclusion, opioid-derived peptide beta-EP is present in the genital tract of stallion, but is not locally produced as in other mammalian, and nonmammalian models; its possible biological function at testicular level could be linked to a long-loop feed-back mechanisms.

Expression of proopiomelanocortin and its cleavage enzyme genes in Rana esculenta and Xenopus laevis gonads.

Proopiomelanocortin (POMC) is the precursor protein of different hormones and neuropeptides, and the POMC-derived peptides are produced through proteolytic cleavage. Prohormone convertase PC1 and PC2 are enzymes responsible for the cleavage of the POMC prohormone. The coexpression of POMC, PC1, and PC2 genes was previously described in the brain and the pituitary gland of Rana esculenta and Xenopus laevis, but no data are available for the gonad. The present work demonstrates a gonadal POMC convertase gene expression in Rana esculenta and Xenopus laevis.

Placental expression of substance P and vasoactive intestinal peptide: evidence for a local effect on hormone release.

The present study evaluated vasoactive intestinal peptide (VIP) and substance P (SP) mRNA expressions and the localization of both peptides in first- and third-trimester human placentas. VIP and SP mRNAs were detected by slot blot analysis in first- and third-trimester placental tissues. By immunohistochemistry both neuropeptides were localized in the trophoblast (syncytium and cytotrophoblastic cells) of the chorionic villi. Because little information is available on the role of VIP and/or SP on the secretion of placental hormones, we investigated the effect of these neuropeptides on human chorionic gonadotropin (hCG) and progesterone release from primary cultured human trophoblastic and JEG-3 cells. The addition of increasing doses of VIP resulted in a dose-dependent stimulation of hCG release from cultured human trophoblast and JEG-3 cells. Increasing doses of VIP also dose-dependently stimulated progesterone secretion from primary cultured trophoblastic cells at all time points evaluated and from JEG-3 cells only after 3 h. SP did not affect hCG and progesterone secretion either in cultured human trophoblast or in JEG-3 cells. In conclusion, the present study demonstrates that VIP and SP are mainly expressed in human trophoblasts, and that VIP modulates the in vitro secretion of hCG and progesterone, suggesting a different role in trophoblastic function of the two peptides.

Hypericum perforatum methanolic extract inhibits growth of human prostatic carcinoma cell line orthotopically implanted in nude mice.

The antiproliferative effect of serotonin-reuptake inhibitors (SSRI) and serotonin antagonists has been demonstrated in prostate tumors. Since Hypericum perforatum components act as serotonin-reuptake inhibitors and exert cytotoxic effects on several human cancer cell lines, in this work we analyzed the effect of a treatment with Hypericum perforatum extract (HPE) on the growth of human prostate cancer cells in vitro and in vivo. This study highlighted a significant reduction of tumor growth and number of metastasis suggesting that this natural compound may be useful in the treatment of prostate cancer.

Evaluation of ovarian POMC mRNA through quantitative RT-PCR analysis in Rana esculenta.

The evaluation of changes in the expression of specific genes requires accurate measurement of the corresponding mRNA concentration, especially when the gene is expressed at a very low level. We previously showed that the proopiomelanocortin (POMC) gene is expressed in the ovary of the frog Rana esculenta, and, to evaluate its mRNA content in frog ovary, we have now developed a sensitive quantitative RT-PCR method. This study provides evidence for the validation of this method and for the effects of captivity and hypophysectomy on POMC gene expression in the ovary of this anuran. Our data indicate that ovarian POMC gene is involved in short-term captivity stress response and seems not influenced by pituitary. These results are discussed taking into account the knowledge of the role played by opioids in stress response; moreover, a local control of POMC gene expression is also suggested.

Differential splicing of three gonadotropin-releasing hormone transcripts in the ovary of seabream (Sparus aurata).

Previous studies demonstrated the presence of high-affinity GnRH binding sites and compounds with GnRH-like activity in the ovary of seabream, Sparus aurata, providing evidence for the role of GnRH as a paracrine/autocrine regulator of ovarian function in this species. In the present study, the expression of three forms of GnRH (salmon, chicken-II, and seabream) genes in this marine teleost species was demonstrated for the first time. Moreover, there is evidence for differential splicing and intronic expression of cGnRH-II and sbGnRH. Treatment of seabream follicle-enclosed oocytes with salmon GnRH stimulated reinitiation of oocyte meiosis, whereas chicken GnRH-II treatment was without effect. Novel information was also provided about organization of cGnRH-II and seabream GnRH transcripts, confirming that GnRH gene organization is maintained through evolution, despite changes in the size and sequence of exons and introns.

Gilthead seabream (Sparus aurata) vitellogenin: purification, partial characterization, and validation of an enzyme-linked immunosorbent assay (ELISA).

VTG was purified from seabream Sparus aurata plasma by ion exchange chromatography on a DEAE-Sepharose column. The vitellogenin was characterized and its properties were determined. The molecular mass of the native form, obtained by Sephadex G-200 column, was around 450 kDa, whereas an apparent molecular mass of 180 kDa was detected by electrophoresis under denaturing and reducing conditions, suggesting a dimeric form for the native protein. The presence of carbohydrates was determined using concanavalin A, while the presence of phosphate groups was detected by Stains-all, a cationic stain. These data together with the sex specificity, the estrogen inducibility, and the cross-reactivity of the abVTG against the major yolk proteins identifies this protein as vitellogenin. The validated ELISA was used for a rapid and reliable measurement of plasma VTG changes related with those of estradiol-17beta in female broodstock.

Presence and activity of compounds with GnRH-like activity in the ovary of seabream Sparus aurata.

The binding and activity of gonadotropin-releasing hormone (GnRH) were characterized in the mature gilthead seabream (Sparus aurata) ovary by use of an analogue of salmon GnRH([D-Arg6,Trp7,Leu8,Pro9-N(Et)]GnRH, sGnRH-A) as labeled ligand. The binding of 125I-sGnRH-A to the seabream ovarian membrane preparation was saturable, displaceable, reversible, and dependent on time, temperature and tissue concentration. Addition of unlabeled s-GnRH-A displaced the radio-ligand in a dose-related manner, indicating the presence of one class of high-affinity binding sites with an equilibrium dissociation constant of 45.5 +/- 6.2 nM. Addition of other GnRH peptides, including salmon GnRH ([Trp7,Leu8]GnRH) and chicken GnRH-II ([His5,Trp7,Tyr8]GnRH), also displaced 125I-sGnRH-A; all these peptides bound with lower affinities than sGnRH-A to the seabream ovarian binding site. In this study, we also demonstrated the presence of compounds with GnRH-like activity in the ovary of seabream. Seabream ovarian extract stimulated pituitary gonadotropin release from the goldfish pituitary and displaced 125I-sGnRH-A binding in the seabream ovary. Furthermore, addition of sGnRH-A to cultured seabream oocytes directly stimulated reinitiation of oocyte meiosis, as indicated by germinal vesicle breakdown. Overall, the present study characterizes GnRH-binding sites in the seabream ovary and supports the hypothesis that GnRH or compounds with GnRH-like activity play an autocrine/paracrine role in the regulation of ovarian function in the seabream ovary.

Seasonal changes in plasma growth hormone and prolactin concentrations of the frog Rana esculenta.

Plasma growth hormone (GH), prolactin (PRL), and vitellogenin (VTG) concentrations were determined during the annual reproductive cycle of the frog Rana esculenta. Plasma GH and PRL were measured using a RIA that employed purified bullfrog (Rana catesbeiana) GH and PRL as standards and radioligand, and their respective antibodies. Using ELISA, plasma VTG titers were related to ovarian weight. GH, PRL, and VTG displayed different trends related to season and sex. In male frogs the GH and PRL trends have been found parallel, showing the highest concentrations (35 and 85 ng/ml, respectively) during the winter months. In the female frogs, the GH trend behaved differently from that in males; in fact, plasma GH changed during the annual reproductive cycle, showing two main peaks occurring during the reproductive period and the autumn ovarian recrudescence that are well correlated with the vitellogenesis as well as with the main changes in ovarian weight.