Patrizia Limonta

miR-205 Exerts Tumor-Suppressive Functions in Human Prostate through Down-regulation of Protein Kinase Cε

Limited information is available concerning the expression and role of microRNAs in prostate cancer. In this study, we investigated the involvement of miR-205 in prostate carcinogenesis. Significantly lower miR-205 expression levels were found in cancer than in normal prostate cell lines as well as in tumor compared with matched normal prostate tissues, with a particularly pronounced reduction in carcinomas from patients with local-regionally disseminated disease. Restoring the expression of miR-205 in prostate cancer cells resulted in cell rearrangements consistent with a mesenchymal-to-epithelial transition, such as up-regulation of E-cadherin and reduction of cell locomotion and invasion, and in the down-regulation of several oncogenes known to be involved in disease progression (i.e., interleukin 6, caveolin-1, EZH2). Our evidence suggests that these events are driven by the concurrent repression of specific predicted miR-205 targets, namely N-chimaerin, ErbB3, E2F1, E2F5, ZEB2, and protein kinase Cepsilon. Strikingly, the latter seemed to play a direct role in regulating epithelial-to-mesenchymal transition. In fact, its down-regulation led to a cell phenotype largely reminiscent of that of cells ectopically expressing miR-205. Overall, we showed for the first time that miR-205 exerts a tumor-suppressive effect in human prostate by counteracting epithelial-to-mesenchymal transition and reducing cell migration/invasion, at least in part through the down-regulation of protein kinase Cepsilon.

The luteinizing hormone-releasing hormone receptor in human prostate cancer cells: messenger ribonucleic acid expression, molecular size, and signal transduction pathway.

Evidence has accumulated indicating that LHRH might behave as an autocrine/paracrine growth inhibitory factor in some peripheral tumors. However, LHRH receptors in tumor cells have not been fully characterized, so far. The present experiments were performed to analyze: 1) the messenger RNA expression; 2) the molecular size; and 3) the signal transduction pathway of LHRH receptors in prostate cancer. For these studies, the human androgen-dependent LNCaP and androgen-independent DU 145 prostate cancer cell lines were used. 1) By RT-PCR, a complementary DNA product, which hybridized with a 32 P-labeled oligonucleotide probe specific for the pituitary LHRH receptor complementary DNA, was found both in LNCaP and in DU 145 cells. 2) Western blot analysis, using a monoclonal antibody raised against the human pituitary LHRH receptor, revealed the presence of a protein band of approximately 64 kDa (corresponding to the molecular mass of the pituitary receptor) in both cell lines. 3) In LNCaP and DU 145 cells, pertussis toxin completely abrogated the antiproliferative action of a LHRH agonist (LHRH-A). Moreover, LHRH-A substantially antagonized the pertussis toxin-catalyzed ADP-ribosylation of a Gai protein. Finally, LHRH-A significantly counteracted the forskolin-induced increase of intracellular cAMP levels in both cell lines. These data demonstrate that the LHRH receptor, which is present in prostate cancer cells, independently of whether they are androgen-dependent or not, corresponds to the pituitary receptor, in terms of messenger RNA expression and protein molecular size. However, at variance with the receptor of the gonadotrophs, prostate cancer LHRH receptor seems to be coupled to the Gai protein-cAMP signal transduction pathway, rather than to the Gaq/11-phospholipase C signaling system. This might be responsible for the different actions of LHRH in anterior pituitary and in prostate cancer. (Endocrinology 140: 5250 ‐5256, 1999)

LHRH analogues as anticancer agents: pituitary and extrapituitary sites of action

Two classes of luteinising hormone-releasing hormone (LHRH) analogues have been developed so far to be used for oncological therapies: LHRH agonists and antagonists. LHRH agonists are widely and successfully used for the management of steroid-dependent malignancies. Chronic administrations of these compounds result in downregulation and desensitisation of pituitary LHRH receptors and, therefore, in a complete suppression of gonadal function. LHRH agonist administration is effective, safe and reversible, suffering only from the ‘flare-up’ phenomenon at the beginning of treatment. LHRH antagonists suppress the pituitary-gonadal function by competing with native LHRH for binding to its pituitary receptor but without giving rise to the intracellular cascade of events evoked by the natural hormone or LHRH agonists. Synthetic peptides belonging to the last generations of LHRH antagonists have already been successful in clinical trials. They are completely devoid of the ‘flare-up’ phenomenon and seem to be free of side effects, such as histamine release. Recently, the expression of LHRH and LHRH receptors has been reported in a number of hormone-responsive tumours. In constrast with the pituitary LHRH receptor which is coupled to the Gq/11-PLC intracellular system of events, stimulation of the tumour LHRH receptor by LHRH is followed by the activation of a Gi protein and a decrease in cAMP levels. This intracellular pathway mediates the inhibitory action of the autocrine/paracrine LHRH system on tumour cell proliferation. The activation of LHRH receptors at tumour level may then represent an additional and more direct mechanism of action for the antitumoural activity of LHRH agonists. Surprisingly, LHRH antagonists also exert a marked antimitogenic activity on a number of hormone-responsive cancer cell lines, indicating that these compounds might behave as antagonists at pituitary level and as agonists at the level of the tumour. The observation that the inhibitory LHRH autocrine system is also present in some steroid-unresponsive cancer cell lines might suggest a possible clinical utility of LHRH analogues also for those tumours that have escaped the initial phase of hormone dependency.

Hypothalamic Opiatergic Tone During Pregnancy, Parturition and Lactation in the Rat

The concentrations of beta-endorphin have been shown to change in the rat brain during pregnancy and lactation. This study has been performed in order to analyze whether also brain opioid receptors might undergo significant modifications during these two physiological situations. The maximal binding capacity (Bmax) and the constant of affinity (Ka) of the mu-subpopulation of opioid receptors have been evaluated in the hypothalami of female rats at different stages of pregnancy (7, 15 and 22 days), on the day of parturition (12-18 h after delivery) and 6-8 days postpartum (both in lactating and in nonlactating animals). Female rats killed on the day of estrus served as controls. The receptor binding assay has been performed utilizing [3H]-dihydromorphine [( 3H]-DHM) as the ligand for the mu-opioid receptors. Hypothalamic concentrations of beta-endorphin as well as serum levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and prolactin have also been evaluated by radioimmunoassay. The results showed that the concentration of hypothalamic mu-opioid receptors increased during pregnancy, being significantly higher than in the controls at days 15 and 22 of gestation. After delivery, the concentration of these receptors returned towards control values, regardless on whether the animals were lactating or not. The Ka values of [3H]-DHM for the mu-receptors did not change significantly in the different groups of experimental animals. Hypothalamic beta-endorphin content showed a modest though not significant increase at the end of gestation (day 22) and returned to control values 12-18 h after delivery.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulatory and Inhibitory Effects of the Opioids on Gonadotropin Secretion

In order to gain additional information on the role played by the opioids in the control of the secretion of anterior pituitary gonadotropins, morphine (an opioid agonist) and naloxone (an opioid antagonist) have been injected intraventricularly (i.v.t.) into normal or castrated male rats. The animals were killed by decapitation at different time intervals after treatment and serum luteinizing hormone (LH), follicle-stimulating hormone (FSH) and prolactin were measured by radioimmunoassay. Animals injected i.v.t. with 0.9% saline solution and sacrificed at the same time intervals served as controls. When morphine (at the dose of 200 and 400 micrograms/rat) and naloxone (at the dose of 7.5 and 15 micrograms/rat) were injected i.v.t. into normal male rats, a significant increase of serum levels of LH was observed 10 and 20 min after injection. There was no effect at 5, 40 and 60 min. Lower doses of morphine (6.25, 12.5, 25, 50 and 100 micrograms/rat) given i.v.t. were ineffective. When morphine (200 micrograms/rat) and naloxone (either in the dose of 7.5 micrograms/rat or of 15 micrograms/rat) were given simultaneously, serum LH was significantly higher than in the saline-treated controls both at 10 and 20 min. However, the increases of serum LH levels induced by the combined treatment were in both instances lower than those produced by the administration of either drug alone. Morphine (200 micrograms/rat) when administered i.v.t. to normal male rats significantly enhanced prolactin release at 10 and 20 min, and this effect of morphine was blunted by the concomitant i.v.t. administration of naloxone (7.5 and 15 micrograms/rat).(ABSTRACT TRUNCATED AT 250 WORDS)

Luteinizing hormone-releasing hormone agonists interfere with the mitogenic activity of the insulin-like growth factor system in androgen-independent prostate cancer cells

We have previously shown that LHRH agonists exert a direct and specific inhibitory action on the proliferation of the androgen-independent DU 145 prostate cancer cell line; however, the cellular mechanisms mediating this antiproliferative action are not well defined. It is well known that the insulin-like growth factor (IGF) system plays a crucial role in the local regulation of the growth of androgen-independent prostate cancer. The present experiments were performed to evaluate whether LHRH agonists might exert their antimitogenic effect by interfering with the activity of the locally expressed IGF system. To this purpose, the effects of the LHRH agonist Zoladex (LHRH-A) on 1) the mitogenic action of IGF-I, 2) the tyrosine phosphorylation of type 1 IGF-I receptor (IGF-IR), 3) the concentration of IGF-IR, and 4) the secretion of IGF-binding protein-3 were studied. The results obtained show that in DU 145 cells, LHRH-A 1) counteracts the mitogenic action of IGF-I in a dose-dependent manner, 2) prevents th...

Growth-Inhibitory Activity of Melatonin on Human Androgen-Independent DU 145 Prostate Cancer Cells

The pineal hormone melatonin has been shown to exert a direct oncostatic activity on neoplastic cells, particularly from breast cancer. In the present study, we evaluated the effects of melatonin on the proliferation and on the cell cycle distribution of human androgen‐independent DU 145 prostate cancer cells. Experiments were also performed to gain insights into the possible mechanism of action of the hormone.

GnRH Receptors in Cancer: From Cell Biology to Novel Targeted Therapeutic Strategies

The crucial role of pituitary GnRH receptors (GnRH-R) in the control of reproductive functions is well established. These receptors are the target of GnRH agonists (through receptor desensitization) and antagonists (through receptor blockade) for the treatment of steroid-dependent pathologies, including hormone-dependent tumors. It has also become increasingly clear that GnRH-R are expressed in cancer tissues, either related (i.e. prostate, breast, endometrial, and ovarian cancers) or unrelated (i.e. melanoma, glioblastoma, lung, and pancreatic cancers) to the reproductive system. In hormone-related tumors, GnRH-R appear to be expressed even when the tumor has escaped steroid dependence (such as castration-resistant prostate cancer). These receptors are coupled to a G(αi)-mediated intracellular signaling pathway. Activation of tumor GnRH-R by means of GnRH agonists elicits a strong antiproliferative, antimetastatic, and antiangiogenic (more recently demonstrated) activity. Interestingly, GnRH antagonists have also been shown to elicit a direct antitumor effect; thus, these compounds behave as antagonists of GnRH-R at the pituitary level and as agonists of the same receptors expressed in tumors. According to the ligand-induced selective-signaling theory, GnRH-R might assume various conformations, endowed with different activities for GnRH analogs and with different intracellular signaling pathways, according to the cell context. Based on these consistent experimental observations, tumor GnRH-R are now considered a very interesting candidate for novel molecular, GnRH analog-based, targeted strategies for the treatment of tumors expressing these receptors. These agents include GnRH agonists and antagonists, GnRH analog-based cytotoxic (i.e. doxorubicin) or nutraceutic (i.e. curcumin) hybrids, and GnRH-R-targeted nanoparticles delivering anticancer compounds.

Growth‐inhibitory effects of luteinizing hormone‐releasing hormone (LHRH) agonists on xenografts of the DU 145 human androgen‐independent prostate cancer cell line in nude mice

Experiments have been performed to clarify whether LHRH agonists might decrease growth of hormone‐unresponsive prostate cancer in vivo. Male nude mice were injected s.c. with the human androgen‐independent prostate tumor DU 145 cells; osmotic minipumps releasing the LHRH agonist Zoladex (LHRH‐A) for 14 days were simultaneously implanted under the skin. Treatment with LHRH‐A induced a significant decrease in tumor growth up to the end of the treatment. In subsequent experiment, minipumps releasing LHRH‐A were implanted in nude mice either 7 or 14 days after cell inoculation. When the treatment was started 7 days after inoculation of the cells, tumor growth was significantly decreased up to 28 days; thereafter, tumor volume remained lower than in controls, although not significantly. When LHRH‐A was administered beginning 14 days after cell inoculation, tumor growth was not significantly affected at any time interval considered. LHRH‐A did not appear to induce apoptosis in DU 145 cells, at least on the basis of the apoptotic index and immunohistochemical staining of the p53 protein. On the other hand, treatment with LHRH‐A was accompanied by a significant decrease of the concentration of epidermal growth factor receptors in DU 145 prostate cancer specimens. Our results show that the LHRH agonist used significantly inhibits the growth of DU 145 androgen‐independent prostate tumor xenografts in nude mice. Int. J. Cancer 76:506–511, 1998.© 1998 Wiley‐Liss, Inc.

Modulation by sex steroids of brain opioid receptors: Implications for the control of gonadotropins and prolactin secretion☆

Several experiments have been performed in order to analyze whether physiological or experimental changes of the endocrine environment might modify the binding characteristics of brain mu and kappa opioid receptors in the brain of the female and male rat. (a) In a first series of experiments, it has been observed that in the whole brain of regularly cycling female rats the number of mu receptors shows variation during the different phases of the estrous cycle. In particular a significant increase of the number of mu receptors has been observed in the morning of proestrus and in the afternoon of estrus. (b) In a second series of experiments, it has been shown that the administration of estrogens brings about a significant increase in the number of mu receptors in the hippocampus and in the thalamus of ovariectomized rats, while the administration of a regime including estrogen and progesterone induces a significant decrease of the number of mu receptors in the hypothalamus and in the corpus striatum. These data seem to indicate that hypothalamic mu receptors may be involved in the positive but not in the negative feedback control of LH secretion. (c) In a third series of experiments, it has been found that the number of mu receptors in the whole brain of 15- and 22-month-old male rats and in the hypothalamus of 22-month-old male rats is significantly lower than in the same tissues of young animals; moreover, the administration to old animals of testosterone does not modify the number of hypothalamic mu opioid receptors, indicating that the decline of brain mu receptors in old animals is not the consequence of the physiological decline of testosterone secretion but probably represents an autonomous phenomenon. (d) In a fourth series of experiments, it was shown that, in young male rats, the concentration of kappa receptors is extremely variable in different regions of the brain. The highest concentrations have been found in the hypothalamus and in the striatum; also in the mesencephalon and in the amygdala kappa receptors are present in rather elevated quantities; lower concentrations have been found in the thalamus, the frontal poles, the hippocampus and in the anterior and posterior cerebral cortex. These experiments have shown in addition that the process of aging induces an increase of the number of kappa receptors in the amygdala and in the thalamus; no age-linked modifications were observed in the other structures examined.(ABSTRACT TRUNCATED AT 400 WORDS)