Anna Maria Stabile

Novel Localization of Low Affinity NGF Receptor (p75) in the Stroma of Prostate Cancer and Possible Implication in Neoplastic Invasion: An Immunohistochemical and Ultracytochemical Study

The localization of low affinity nerve growth factor receptor (p75) in prostate carcinogenesis is still unclear. Our aim was to reinvestigate the localization of p75 in normal and pathological prostate and to check a possible correlation to neoplastic grading.

The tricyclic antidepressant amitriptyline is cytotoxic to HTB114 human leiomyosarcoma and induces p75(NTR)-dependent apoptosis.

Nerve growth factor (NGF) receptors, TrKA and p75NTR, are being investigated in cancer therapy. Our previous data show that, in HTB114 uterine leiomyosarcoma cells, p75NTR-dependent apoptosis is inducible by cytotoxic drugs and can suppress nerve growth factor-dependent growth. Although amitriptyline can kill cancer cells and bind TrKA/B, its effects on p75NTR-dependent apoptosis are unknown. The aim of this paper was to evaluate the antineoplastic potential of amitriptyline, and the role of p75NTR-dependent apoptosis in the chemoresistant uterine HTB114 leiomyosarcoma. Using proliferation assays and fluorescence-activated cell sorting analysis, we found that amitriptyline caused a marked reduction in HTB114 cell viability, associated with the parallel upregulation of p75NTR expression. This converted the TrKA+-proliferating cells into TrKA+/p75NTR+, leading to downregulation of TrKA-prosurvival signaling (AKT) and activation of p75NTR-dependent apoptosis (through caspase-3). Overall, we provide novel evidence that HTB114 uterine leiomyosarcoma cells are highly sensitive to amitriptyline, supporting the role of p75NTR-dependent apoptosis as a novel cytotoxic mechanism of this drug and of p75NTR as an inducible stress receptor and a novel target in clinical oncology.

Anti-proliferative effects of GW441756, a novel inhibitor of NGFreceptor tyrosine kinase a (TRKA), in human sarcoma

Several experimental data have shown a relevant involvement of Nerve Growth Factor (NGF) in neoplastic proliferation and survival. NGF acts through two receptors and two distinct transduction pathways: high affinity TrKA receptor connected to proliferation via AKT, and low-affinity p75 receptor connected to apoptosis via caspases. Our previous studies have shown that TrKA inhibitors have a relevant anti-proliferative effects in neoplasia. GW441756 is a novel TrKA-inhibitor (IC50 = 2nM) that displays an higher selectivity on TrKA respect to previous inhibitors and a 100-fold selectivity over a range of other kinases. However, its biological effects in cancer cells is still unknown. The aim of our study was to investigate in vitro the biological effects of this new inhibitor in human muscle sarcoma cancer cell line HTB114, at basal condition and following GW441756 administration. As revealed by thymidine incorporation, GW441756 induced a dose-dependent decrease in neoplastic proliferation. Contemporaneously, Annexin V assay demonstrated a dramatic dose-dependent increase in apoptosis. To further characterize these results, we studied AKT and caspases activation by cytofluorimetric analysis. In agreement to its anti-proliferative-pro-apoptotic effects, GW441756 did not induce AKT activation, but produced a relevant increase of caspase-3 that, in turn, leads to apoptosis. In conclusion, GW441756 has a comparable anti-neoplastic effect respect to previous TrKA inhibitors, but with a lower effective dosage (10 μM vs 50 μM of other TrKAinhibitors). Our data have a preclinical relevance to achieve anti-proliferative effects in muscular sarcomas by TrKA-inhibitors administration.

Antitumor activity and expression profiles of genes induced by sulforaphane in human melanoma cells

PurposeHuman melanoma is a highly aggressive incurable cancer due to intrinsic cellular resistance to apoptosis, reprogramming, proliferation and survival during tumour progression. Sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables, plays a role in carcinogenesis in many cancer types. However, the cytotoxic molecular mechanisms and gene expression profiles promoted by SFN in human melanoma remain unknown.MethodsThree different cell lines were used: two human melanoma A375 and 501MEL and human epidermal melanocytes (HEMa). Cell viability and proliferation, cell cycle analysis, cell migration and invasion and protein expression and phosphorylation status of Akt and p53 upon SFN treatment were determined. RNA-seq of A375 was performed at different time points after SFN treatment.ResultsWe demonstrated that SFN strongly decreased cell viability and proliferation, induced G2/M cell cycle arrest, promoted apoptosis through the activation of caspases 3, 8, 9 and hampered migration and invasion abilities in the melanoma cell lines. Remarkably, HEMa cells were not affected by SFN treatment. Transcriptomic analysis revealed regulation of genes involved in response to stress, apoptosis/cell death and metabolic processes. SFN upregulated the expression of pro-apoptotic genes, such as p53, BAX, PUMA, FAS and MDM2; promoted cell cycle inhibition and growth arrest by upregulating EGR1, GADD45B, ATF3 and CDKN1A; and simultaneously acted as a potent inhibitor of genotoxicity by launching the stress-inducible protein network (HMOX1, HSPA1A, HSPA6, SOD1).ConclusionOverall, the data show that SFN cytotoxicity in melanoma derives from complex and concurrent mechanisms during carcinogenesis, which makes it a promising cancer prevention agent.

A role for NGF and its receptors TrKA and p75NTR in the progression of COPD.

Abstract Nerve growth factor and its receptors, TrkA and p75NTR, are involved in inflammation and airways diseases, but their role in chronic obstructive pulmonary disease is still unclear and not well investigated. our data indicate the stage dependent variation of nerve growth factor and its receptors in chronic obstructive pulmonary disease progression. In fact, for the first time, this study evaluates the presence of nerve growth factor and its receptors in serum and in peripheral blood mononuclear cells of patients with different stages of chronic obstructive pulmonary disease compared to healthy subjects, non-smoker and current smoker. Serum monocyte chemoattractant protein-1, tumor necrosis factor-α, interleukin-10 and forced expiratory volume in 1 s were also analyzed. Compared to healthy subjects, chronic obstructive pulmonary disease patients presented a staging-dependent increase in serum nerve growth factor, negatively correlated to forced expiratory volume in 1 s and positively to monocyte chemoattractant Protein-1. The percentage of p75NTR+ peripheral blood mononuclear cells increased in early stages of chronic obstructive pulmonary disease (I–II), while TrKA+ peripheral blood mononuclear cells increased in late stages (III–IV). Our data demonstrate the involvement and modulation of nerve growth factor and its receptors in chronic obstructive pulmonary disease and in its staging.

β-NGF and β-NGF receptor upregulation in blood and synovial fluid in osteoarthritis

Abstract Osteoarthritis (OA) of the knee is the most common form of non-traumatic joint disease. Previous studies have shown the involvement of β-NGF and its receptors TrKA and p75NTR in OA-related pain, but their role in its pathogenesis is still unclear. The aim of our study was to investigate the amount of β-NGF and the expression levels of its receptors on cells isolated from synovial fluid and blood from OA patients who had undergone total knee arthroplasty, in order to check any possible correlation with the disease staging. Our results show a progressive stage-related increase of β-NGF and its receptors both in serum and synovial fluid. Furthermore, with respect to control subjects, OA patients show an increased amount of inflammatory monocytes along with an increased expression of β-NGF, TrKA and p75NTR. In conclusion, our study suggests a stage-related modulation of β-NGF and its receptors in the inflammatory process of OA.

LY294002 induces in vitro apoptosis and overexpression of p75NTR in human uterine leiomyosarcoma HTB 114 cells

Abstract Uterine leiomyosarcoma is a severe neoplasia resistant to conventional therapies. In previous studies, we have shown that human SK-UT-1 (ATCC HTB114) uterine leiomyosarcoma cell line secretes nerve growth factor (NGF) and expresses its receptors tyrosine kinase A receptor (TrKA) and low affinity nerve growth factor receptor (p75NTR). Furthermore, we have demonstrated that direct chemical inhibition or IgG neutralization of TrKA receptor induce apoptosis through p75NTR. In the present study, HTB114 cells were exposed to the phosphoinositide 3-kinase (PI3K) inhibitor LY294002 with and without β-NGF: apoptosis, cell cycle, activation of caspase-3 and protein kinase B (AKT) and TrKA/p75NTR phenotypic expression were evaluated. According to the type of exposure, LY294002 not only induced a relevant increase in apoptosis, but also produced a novel and unexpected phenotypic modulation of the NGF receptors with a downregulation of TrKA and an upregulation of p75NTR. This latter increase enhanced HTB114 apoptosis. Our study confirms that the interference on NGF transduction is a promising therapeutical approach in uterine leiomyosarcoma.

A Comparison of Lysosomal Enzymes Expression Levels in Peripheral Blood of Mild- and Severe-Alzheimer’s Disease and MCI Patients: Implications for Regenerative Medicine Approaches

The association of lysosomal dysfunction and neurodegeneration has been documented in several neurodegenerative diseases, including Alzheimer’s Disease (AD). Herein, we investigate the association of lysosomal enzymes with AD at different stages of progression of the disease (mild and severe) or with mild cognitive impairment (MCI). We conducted a screening of two classes of lysosomal enzymes: glycohydrolases (β-Hexosaminidase, β-Galctosidase, β-Galactosylcerebrosidase, β-Glucuronidase) and proteases (Cathepsins S, D, B, L) in peripheral blood samples (blood plasma and PBMCs) from mild AD, severe AD, MCI and healthy control subjects. We confirmed the lysosomal dysfunction in severe AD patients and added new findings enhancing the association of abnormal levels of specific lysosomal enzymes with the mild AD or severe AD, and highlighting the difference of AD from MCI. Herein, we showed for the first time the specific alteration of β-Galctosidase (Gal), β-Galactosylcerebrosidase (GALC) in MCI patients. It is notable that in above peripheral biological samples the lysosomes are more sensitive to AD cellular metabolic alteration when compared to levels of Aβ-peptide or Tau proteins, similar in both AD groups analyzed. Collectively, our findings support the role of lysosomal enzymes as potential peripheral molecules that vary with the progression of AD, and make them useful for monitoring regenerative medicine approaches for AD.

Anticarcinogenic activities of sulforaphane are influenced by Nerve Growth Factor in human melanoma A375 cells

Melanoma is a severe form of cancer, resistant to conventional therapies. According to in vitro studies, sulforaphane, a dietary component, has been considered a promising antineoplastic candidate. The present study analyzes the in vitro biological effects of sulforaphane in A375 melanoma cell line with or without the addition of Nerve Growth Factor. For the first time, our results show that a supplementation of Nerve Growth Factor partially reverses the sulforaphane-induced: i) inhibition of cell migration, ii) pro apoptotic changes in cell cycle and iii) modulation of active caspase-3. Furthermore, we report the sulforaphane-induced modulation in the expression of Nerve Growth Factor receptors TrKA and p75NTR, shifting their ratio from pro survival to pro apoptotic. In conclusion, the present study evidences that in vivo the antineoplastic effects of sulforaphane may be reduced by the contemporaneous presence of other biological elements such as Nerve Growth Factor and it contributes to a better definition of the real in vivo potentiality of sulforaphane as antineoplastic candidate.

Nicotine induces overexpression of low affinity p75 NGF receptor in monocytes

In inflammatory pulmonary diseases, cigarette smoking is a major risk factor influencing the phenotype of immune cells and their functions (Arnson et al., 2010). Nicotine is a relevant constituent of cigarette smoking and, on monocytes, it binds nicotinic acetylcholine receptors inducing a quantitative increase in the levels of CD14 and a decrease in TNF-a. Altogether, these data supports an anti-inflammatory effect of nicotine on these cells.Our previous studies have shown that NGF and its receptors TrKA and p75 are involved in inflammatory pulmonary diseases. In particular, we have shown that smokers present an increase in p75 expression on monocytes, but the cause that triggers this increase is actually unknown. In addition, cigarette smoking seems not to vary the TrKA expression on these cells. The aim of our study was to investigate in vitro the biological effects of nicotine on the percentage of TrKA- and p75-positive monocytes from human healthy non smoker donors. Cytofluorimetric investigation confirms that in monocytes nicotine treatment does not influence the percentage of TrKA, but induces a relevant dose-dependent increase in the percentage of p75 that, in turn, could be an element in the general anti-inflammatory effects of nicotine. Consequently, in order to investigate the involvement of p75 in inflammatory/non-inflammatory mechanisms, we added in vitro the inflammatory stimulus MCP-1 on monocytes. Cytofluorimetric investigation have shown that, in this latest inflammatory condition, the percentage of p75-positive monocytes was greatly reduced. In conclusion, our data support the hypothesis that nicotine-induced p75 overexpression could be involved in its anti-inflammatory effect.