Mario Rende

Nerve growth factor (NGF) influences differentiation and proliferation of myogenic cells in vitro via TrKA

Classic studies have established that muscle cells exert trophic actions on neurons of the developing peripheral nervous system through the production of neurotrophins. For this reason neurotrophins are also known as ‘target‐derived factors’. During differentiation, muscle cells also express some neurotrophin receptors, such as the low‐affinity p75 neurotrophin receptor, which binds all neurotrophins, and the high affinity tyrosine kinase receptor TrKA, nerve growth factor (NGF) transducing receptor. The functional roles of these receptors in muscle cells are still unclear and only fragmentary and controversial data are available regarding the responsiveness of muscle cells to NGF. The aim of the present study is to investigate the effects of NGF on cells of myogenic lineage. The rat myogenic cell line L6, primary cultures of adult human myoblasts, and the human rhabdomyosarcoma cell line TE‐671 were used in this study. As expected, all the three cell types expressed NGF, p75 and TrKA. NGF was expressed by L6 and primary myoblasts following differentiation, but it was constitutively expressed at high levels in the TE‐671 rhabdomyosarcoma cells. In L6 myoblasts, p75 receptor was expressed in myoblasts but not in myotubes early after plating; while some primary human myoblasts expressed it at all the time‐points tested. Some fusiform cells of the TE‐671 rhabdomyosarcoma cell line also expressed p75. TrKA was constitutively immunodetected in all the three cell lines, suggesting that these cells may respond to NGF. Addition of exogenous NGF increased the fusion rate of both primary and L6 myoblasts; as well as the proliferation of the slowly dividing primary myoblasts. Consistently, blocking the action of endogenously produced NGF with a specific neutralizing antibody decreased the percentage of fusion in both primary and L6 myoblasts. On the contrary, blocking the binding of NGF to p75 did not affect the percentage of fusion. Furthermore, neither exogenous NGF nor NGF‐ or p75‐neutralizing antibodies appeared to affect the rhabdomyosarcoma cells, which have a high proliferation rate and do not fuse. Pharmacological inhibition of TrKA signal transduction with K252a (in the nM range) and tyrphostin AG879 (in the low μM range) resulted in a dramatic dose‐dependent decrease in proliferation of all of the myogenic cell lines tested. Interestingly, this was especially evident in the rapidly dividing rhabdomyosarcoma cell line. The TrKA inhibitors also blocked fusion of L6 and primary myoblasts and induced morphological changes characterized by the flattening of the cells and a ‘spider‐like’ rearrangement of the intermediate filaments in all three cell lines with some minor differences. A transfection study showed that p75‐overexpressing L6 cells do not fuse and present changes in their morphology similar to the TrKA‐inhibitors treated L6 cells. These data support the notion that NGF expression in skeletal muscle is not only associated with a classical target‐derived neurotrophic function for peripheral nervous system neurons, but also with an autocrine action which affects the proliferation, fusion into myotubes, and cell morphology of developing myoblasts. The present data also suggest that these effects of NGF are mediated by TrKA receptors and that a sustained presence of NGF is needed for increase fusion into myotubes. Lastly, the dramatic anti‐proliferative effect of TrKA inhibitors on myogenic cells, and especially on the TE‐671 rhabdomyosarcoma cell line, suggests that pharmacological interference with NGF signal transduction could be effective in the control of these malignancies.

Disruption of plasmepsin-4 and merozoites surface protein-7 genes in Plasmodium berghei induces combined virulence-attenuated phenotype

Blood stage malaria parasites causing a mild and self limited infection in mice have been obtained with either radiation or chemical mutagenesis showing the possibility of developing an attenuated malaria vaccine. Targeted disruption of plasmepsin-4 (pm4) or the merozoite surface protein-7 (msp7) genes also induces a virulence-attenuated phenotype in terms of absence of experimental cerebral malaria (ECM), delayed increase of parasitemia and reduced mortality rate. The decrease in virulence in parasites lacking either pm4 or msp7 is however incomplete and dependent on the parasite and mouse strain combination. The sequential disruption of both genes induced remarkable virulence-attenuated blood-stage parasites characterized by a self-resolving infection with low levels of parasitemia and no ECM. Furthermore, convalescent mice were protected against the challenge with P. berghei or P. yoelii parasites for several months. These observations provide a proof-of-concept step for the development of human malaria vaccines based on genetically attenuated blood-stage parasites.

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.

Regulation of the p75 Neurotrophin Receptor in a Rat Myogenic Cell Line (L6)

Neurotrophins are expressed in muscle cells both during development and postnatally. Furthermore, during development muscle cells express high levels of the common p75 neurotrophin receptor, which binds all neurotrophins. Only fragmentary and controversial data are available regarding the responsiveness of muscle cells to neurotrophins and the importance of low-affinity p75 receptor in muscle development. The present study investigates in vitro the immunocytochemical expression of p75 in a rat myogenic cell line (L6) at various time points and in response to different coating substrates as a first step in elucidating the regulation of p75 in muscle. We found that in L6 myoblasts, p75 is expressed only at very early stages of maturation and its levels of expression are regulated by the nature of the coating substrates. p75 expression decreases in cells growing on substrates more suitable for myoblast fusion into myotubes. Time course analysis indicates a reverse correlation between myoblast fusion into myotubes and the levels of p75 expression. Myotubes were always p75 negative. Substrates not suitable for the fusion process induced a prolonged presence of p75 in myoblasts with an increase of their apoptosis. We conclude that expression of p75, at least in this in vitro condition, is regulated by the stages of myoblast differentiation and the nature of the coating substrates. According to the observed time- and substrate-related evidences, future studies should investigate in vivo both the regulation of p75 in the myoblast fusion and the effects and the importance of neurotrophins binding during myoblast differentiation.

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.

Prosaposin is immunolocalized to muscle and prosaptides promote myoblast fusion and attenuate loss of muscle mass after nerve injury

Prosaposin is the precursor of the saposins and has both neurotrophic and myelinotrophic activity in vitro and in vivo. Using an antibody specific for the holoprotein, an immunocytochemical survey demonstrated intense staining of adult rat skeletal, cardiac, and smooth muscle cells. Prosaposin immunoreactivity in muscle appears dependent on innervation, as denervated adult rat skeletal muscles showed decreased immunostaining that returned to normal levels after reinnervation. TX14(A), a peptide derived from the neurotrophic sequence of prosaposin, attenuated the decline in muscle mass loss following nerve injury induced by a constricting ligature. In vitro, both L6 myoblasts and primary chick‐embryo myoblasts showed similar prosaposin immunopositivity, mainly in myotubes. TX14(A) induced a threefold increase in L6 myoblast fusion during early stages of differentiation without affecting cell proliferation. The fusion process was decreased in vitro in a dose‐dependent fashion by addition of a neutralizing anti‐prosaposin antibody. These data suggest that, in addition to neurotrophic and myelinotrophic activities, prosaposin has myotrophic properties.

Modulation of serotonin 5-HT3 receptor expression in injured adult rat spinal cord motoneurons

The effects of sciatic nerve lesions on the expression of serotonin 5-HT3 receptor (5-HT3R) alpha subunit in motoneurons of the spinal cord was investigated by semi-quantitative immunohistochemistry. Following sciatic nerve crush, a significant reduction in density of staining in motoneurons was observed in longitudinal sections of the ventral horn at 3 and 15 days on the lesioned side when compared to the contralateral side (p<0.01). At 30 days after crush, after completion of sciatic nerve regeneration and reinnervation of peripheral targets, intensity of staining had returned to normal. Conversely, after sciatic nerve cut, a lesion that does not allow for target reinnervation, highly significant reductions were observed at 3, 15, 30 and 45 days. These results suggest a role for functional contacts with muscular targets in the maintenance of 5-HT3R expression in spinal motoneurons.

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.