Papers by Antonio Filippini
Journal of Immunology, Jun 15, 2010
CXCL13-CXCR5 interactions support prostate cancer cell migration and invasion in a PI3K p110-, SR... more CXCL13-CXCR5 interactions support prostate cancer cell migration and invasion in a PI3K p110-, SRC-and FAK-dependent fashion (98.5)
Cancers
The use of a higher dose per fraction to overcome the high radioresistance of prostate cancer cel... more The use of a higher dose per fraction to overcome the high radioresistance of prostate cancer cells has been unsuccessfully proposed. Herein, we present PC3 and DU-145, castration-resistant prostate cancer cell lines that survived a clinically used ultra-higher dose per fraction, namely, radioresistant PC3 and DU-145 cells (PC3RR and DU-145RR). Compared to PC3, PC3RR showed a higher level of aggressive behaviour, with enhanced clonogenic potential, DNA damage repair, migration ability and cancer stem cell features. Furthermore, compared to PC3, PC3RR more efficiently survived further radiation by increasing proliferation and down-regulating pro-apoptotic proteins. No significant changes of the above parameters were described in DU-145RR, suggesting that different prostate cancer cell lines that survive ultra-higher dose per fraction do not display the same grade of aggressive phenotype. Furthermore, both PC3RR and DU-145RR increased antioxidant enzymes and mesenchymal markers. Our d...
Thanks to the presence of high levels of monounsaturated fatty acids, extra virgin olive oil is b... more Thanks to the presence of high levels of monounsaturated fatty acids, extra virgin olive oil is beneficial for health as it is associated with protection against liver steatosis, some types of cancer and cardiovascular diseases. Here we study different responses between hepatocarcinoma cell lines (Hep3B and Huh7.5) vs a healthy hepatocyte control (THLE-2) following the treatment with oleic acid (OA), in particular we have observed a dose-dependent response (50-300 M). First of all, we observed a different neutral lipid accumulation between the hepatocarcinoma cells and the healthy control. Moreover, we have found a differential viability rate; a reduction of hepatocarcinoma cell lines at 300 M OA dosage but unchanged vitality in healthy hepatocytes through Alamar Blue staining. Furthermore, we evaluated the apoptotic rate by western blot, we have found that the levels of cleaved PARP appear differently modulated in the hepatocarcinoma cell lines following the treatment with OA. We...
Cancers
The endothelium is the innermost layer of all blood and lymphatic vessels composed of a monolayer... more The endothelium is the innermost layer of all blood and lymphatic vessels composed of a monolayer of specialized endothelial cells (ECs). It is regarded as a dynamic and multifunctional endocrine organ that takes part in essential processes, such as the control of blood fluidity, the modulation of vascular tone, the regulation of immune response and leukocyte trafficking into perivascular tissues, and angiogenesis. The inability of ECs to perform their normal biological functions, known as endothelial dysfunction, is multi-factorial; for instance, it implicates the failure of ECs to support the normal antithrombotic and anti-inflammatory status, resulting in the onset of unfavorable cardiovascular conditions such as atherosclerosis, coronary artery disease, hypertension, heart problems, and other vascular pathologies. Notably, it is emerging that the ability of ECs to adapt their metabolic status to persistent changes of the tissue microenvironment could be vital for the maintenance...
Exploration of Targeted Anti-tumor Therapy, 2022
Aim: B-cell lymphoma-2 (Bcl-2)-like protein-10 (Bcl2L10) is the less studied member of Bcl-2 fami... more Aim: B-cell lymphoma-2 (Bcl-2)-like protein-10 (Bcl2L10) is the less studied member of Bcl-2 family proteins, with the controversial role in different cancer histotypes. Very recently, Bcl2L10 expression in melanoma tumor specimens and its role in melanoma response to therapy have been demonstrated. Here, the involvement of Bcl2L10 on the in vitro and in vivo properties associated with melanoma aggressive features has been investigated. Methods: Endogenous Bcl2L10 protein expression was detected by western blotting analysis in a panel of patient-derived and commercially available human melanoma cells. In vitro assays to evaluate clonogenicity, cell proliferation, cell migration, cell invasion, and in vitro capillary-like structure formation [vasculogenic mimicry (VM)] have been performed by using human melanoma cells stably overexpressing Bcl2L10 or transiently transfected for loss/gain function of Bcl2L10, grown under two- or three-dimensional (3D) conditions Xenograft melanoma mod...
Additional file 2: Figure S2. Representative examples of asexual parasites. ER: Early rings; LR: ... more Additional file 2: Figure S2. Representative examples of asexual parasites. ER: Early rings; LR: Late rings; ET: Early trophozoites; LT: Late trophozoites; ES: Early schizonts; LS: Late schizonts. Scale bar: 5 μm.
Cell Calcium, 2022
In comparison with normal cells, cancer cells are equipped with a higher number of lysosomes, inv... more In comparison with normal cells, cancer cells are equipped with a higher number of lysosomes, involved in degradative and non-degradative roles. In particular, the lysosome is a Ca 2+ signalling hub, and the enhancement of this interconnected machinery in cancer cells has recently prompted investigations into the role that lysosomal ion channels play in oncology. The present review reports findings about the emerging role of lysosomal Ca 2+ channels: Two-Pore Channels (TPCs), Transient Receptor Potential Cation Channels (TRPMLs; mucolipins), and Purinergic X Receptor 4 (P2×4R), in a variety of cancer models, highlighting their impact on crucial functions such as the regulation of autophagy and the composition of the tumour microenvironment, including the secretion-mediated interplay with immune and endothelial cells. Notably, recent evidence indicates that, by regulating tumour secretome, lysosomal Ca 2+ signalling can affect the composition of the tumour-infiltrating immune cell repertoire. Intriguingly, the data so far available show that the protumoral/antitumoral role of lysosomal Ca 2+ channels can differ according to the specific genetic context, types of cancer and the malignancy stage, and signals from the microenvironment.
Italian journal of anatomy and embryology, 2017
Two Pore Channels (TPCs) are an emerging family of intracellular channels, expressed on acidic co... more Two Pore Channels (TPCs) are an emerging family of intracellular channels, expressed on acidic compartments, which mediate calcium signaling evoked by NAADP. In particular, we demonstrated that TPC2 isoform has a main role in angiogenesis (Favia et al. PNAS 2014 Nov 4;111(44):E4706-15). TPC2 inhibition is emerging as a key therapeutic step in a range of important pathological conditions including the progression and metastatic potential of cancer, Parkinson’s disease, and Ebola virus infection. We introduce naringenin, a natural flavonoid, as a novel TPC2 inhibitor as shown by electrophysiological evidence in a heterologous system, i.e. Arabidopsis vacuoles lacking endogenous TPCs. In view of the control exerted by TPC2 on intracellular calcium signaling and angiogenesis, we demonstrate that naringenin dampens intracellular calcium responses of human endothelial cells stimulated with VEGF, histamine or NAADP-AM, but not with ATP or Angiopoietin-1. The ability of naringenin to impair...
Cancers, 2020
Melanoma is one of the most aggressive and treatment-resistant human cancers. The two-pore channe... more Melanoma is one of the most aggressive and treatment-resistant human cancers. The two-pore channel 2 (TPC2) is located on late endosomes, lysosomes and melanosomes. Here, we characterized how TPC2 knockout (KO) affected human melanoma cells derived from a metastatic site. TPC2 KO increased these cells’ ability to invade the extracelullar matrix and was associated with the increased expression of mesenchymal markers ZEB-1, Vimentin and N-Cadherin, and the enhanced secretion of MMP9. TPC2 KO also activated genes regulated by YAP/TAZ, which are key regulators of tumourigenesis and metastasis. Expression levels of ORAI1, a component of store-operated Ca2+ entry (SOCE), and PKC-βII, part of the HIPPO pathway that negatively regulates YAP/TAZ activity, were reduced by TPC2 KO and RNA interference knockdown. We propose a cellular mechanism mediated by ORAI1/Ca2+/PKC-βII to explain these findings. Highlighting their potential clinical significance, patients with metastatic tumours showed a ...
Pharmacological Research, 2021
Naringenin is a powerful inhibitor of SARS-CoV-2 infection in vitro Recently, an interesting revi... more Naringenin is a powerful inhibitor of SARS-CoV-2 infection in vitro Recently, an interesting review appeared in Pharmacological Research presented a list of candidate drugs against SARS-CoV-2 and COVID-19 [1]. In the present insight, we highlight novel experimental evidence that the flavanone Naringenin, targeting the endo-lysosomal Two-Pore Channels (TPCs), could be added to the list of potential weapons against SARS-CoV-2 infection and COVID-19 disease. Coronaviruses (CoV) are a large family of enveloped positive-sense single-stranded RNA viruses including the highly pathogenic SARS-CoV-1 and-2, MERS-CoV, and other four human CoV of less recent zoonotic origin (229E, OC43, HKU1, NL63) known to cause illnesses ranging from the common cold to acute respiratory tract infection. The current COVID-19 pandemic is one of the largest challenges in medicine and public health and the management of COVID-19 patients is burdened by the lack of antivirals hampering SARS-CoV-2 replication without side effects. Possible pharmacological strategies for the management of COVID-19 patients include drug repurposing such as in the case of hydroxychloroquine, an anti-malarial drug, which has proved to be very controversial [2]. In this scenario, the availability of well tolerated drugs actively impairing virus replication at the early stages of infection represents a promising option and an urgent medical need. Intriguingly, it has been shown that CoV infection depends on trafficking of virions to lysosomal compartments and processing of the S protein by lysosomal proteases is required for productive entry to occur. In this context, the role played by endo-lysosomal TPCs on CoV biology and the feasibility of blocking the intracellular pathway of the virus by inhibiting these channels was preliminarily inferred by our group [3-5] and confirmed by other authors [6-8]. Genetic ablation of TPCs or TPC blockers have been previously shown to reduce infectivity affecting trafficking through the endo-lysosomal system of Ebola virus and MERS-CoV [9,10]. Finally, it has recently been demonstrated that a two-pore channel 2 (TPC2) is critically important for SARS-CoV-2 entry into cells and tetrandrine, a TPC inhibitor, previously described to block Ebola virus trafficking in a TPC-dependent manner [9], was found to impede the entry process of SARS-CoV-2 pseudoviruses [11]. During the infection process SARS-CoV replication takes place in specific membrane-enclosed cellular compartments, hidden from innate immunity, and membrane fusion events are of pivotal importance for the replication of the virus. Cathepsins B and L, belonging to the papain subfamily of cysteine proteases and located predominantly in endo-lysosomal vesicles, have been described to be fundamental for cleavage of viral S protein allowing the release of the fusogenic peptide S2. Of note, tetrandrine treatment suppressed the replication of common cold related human coronaviruses (HCoV) [12]. These findings support the potential role of TPC-dependent membrane trafficking and translocation of endosomes containing CoVs particles. Moreover, TPCs are known to promote endo-lysosomal trafficking and evidence is emerging of their contributions to trafficking dysfunction [13,14]. Therefore, inhibition of TPCs should both impair the fusogenic potential of the endo-lysosomal system and alter the normal trafficking, limiting viral replication. Noteworthy, our recent evidence has shown that the activity of human TPC channels can be inhibited by the natural flavonoid compound Naringenin (Nar) [4], one of the main flavonoids present in the human diet. In the present study, we focus on Nar and show data pointing to a novel anti-CoV pharmacological strategy, highly promising for efficient and safe prophylaxis and therapy.
Cells, 2021
The flavonoid naringenin (Nar), present in citrus fruits and tomatoes, has been identified as a b... more The flavonoid naringenin (Nar), present in citrus fruits and tomatoes, has been identified as a blocker of an emerging class of human intracellular channels, namely the two-pore channel (TPC) family, whose role has been established in several diseases. Indeed, Nar was shown to be effective against neoangiogenesis, a process essential for solid tumor progression, by specifically impairing TPC activity. The goal of the present review is to illustrate the rationale that links TPC channels to the mechanism of coronavirus infection, and how their inhibition by Nar could be an efficient pharmacological strategy to fight the current pandemic plague COVID-19.
Biomolecules, 2020
Caveolae are flask-shaped invaginations of the plasma membrane found in numerous cell types and a... more Caveolae are flask-shaped invaginations of the plasma membrane found in numerous cell types and are particularly abundant in endothelial cells and adipocytes. The lipid composition of caveolae largely matches that of lipid rafts microdomains that are particularly enriched in cholesterol, sphingomyelin, glycosphingolipids, and saturated fatty acids. Unlike lipid rafts, whose existence remains quite elusive in living cells, caveolae can be clearly distinguished by electron microscope. Despite their similar composition and the sharing of some functions, lipid rafts appear more heterogeneous in terms of size and are more dynamic than caveolae. Following the discovery of caveolin-1, the first molecular marker as well as the unique scaffolding protein of caveolae, we have witnessed a remarkable increase in studies aimed at investigating the role of these organelles in cell functions and human disease. The goal of this review is to discuss the most recent studies related to the role of cav...
International Journal of Radiation Biology, 2020
Purpose: Radiation therapy (RT), by using ionizing radiation (IR), destroys cancer cells inducing... more Purpose: Radiation therapy (RT), by using ionizing radiation (IR), destroys cancer cells inducing DNA damage. Despite several studies are continuously performed to identify the best curative dose of IR, the role of dose-rate, IR delivered per unit of time, on tumor control is still largely unknown. Materials and methods: Rhabdomyosarcoma (RMS) and prostate cancer (PCa) cell lines were irradiated with 2 or 10 Gy delivered at dose-rates of 1.5, 2.5, 5.5 and 10.1 Gy/min. Cell-survival rate and cell cycle distribution were evaluated by clonogenic assays and flow cytometry, respectively. The production of reactive oxygen species (ROS) was detected by cytometry. Quantitative polymerase chain reaction assessed the expression of anti-oxidant-related factors including NRF2, SODs, CAT and GPx4 and miRNAs (miR-22,-126,-210,-375,-146a,-34a). Annexin V and caspase-8,-9 and-3 activity were assessed to characterize cell death. Senescence was determined by assessing b-galactosidase (SA-b-gal) activity. Immunoblotting was performed to assess the expression/ activation of: i) phosphorylated H2AX (c-H2AX), markers of DNA double strand breaks (DSBs); ii) p19 Kip1/Cip1 , p21 Waf1/Cip1 and p27 Kip1/Cip1 , senescence-related-markers; iii) p62, LC3-I and LC3-II, regulators of autophagy; iv) ATM, RAD51, DNA-PKcs, Ku70 and Ku80, mediators of DSBs repair. Results: Low dose-rate (LDR) more efficiently induced apoptosis and senescence in RMS while high dose-rate (HDR) necrosis in PCa. This paralleled with a lower ability of LDR-RMS and HDR-PCa irradiated cells to activate DSBs repair. Modulating the dose rate did not differently affect the antioxidant ability of cancer cells. Conclusion: The present results indicate that a stronger cytotoxic effect was induced by modulating the dose-rate in a cancer cell-dependent manner, this suggesting that choose the dose-rate based on the individual patient's tumor characteristics could be strategic for effective RT exposures.
Frontiers in Immunology, 2020
Atherosclerosis is a progressive vascular disease representing the primary cause of morbidity and... more Atherosclerosis is a progressive vascular disease representing the primary cause of morbidity and mortality in developed countries. Formerly, atherosclerosis was considered as a mere passive accumulation of lipids in blood vessels. However, it is now clear that atherosclerosis is a complex and multifactorial disease, in which the involvement of immune cells and inflammation play a key role. A variety of studies have shown that autophagy-a cellular catalytic mechanism able to remove injured cytoplasmic components in response to cellular stress-may be proatherogenic. So far, in this context, its role has been investigated in smooth muscle cells, macrophages, and endothelial cells, while the function of this catabolic protective process in lymphocyte functionality has been overlooked. The few studies carried out so far, however, suggested that autophagy modulation in lymphocyte subsets may be functionally related to plaque formation and development. Therefore, in this research, we aimed at better clarifying the role of lymphocyte subsets, mainly regulatory T cells (Tregs), in human atherosclerotic plaques and in animal models of atherosclerosis investigating the contribution of autophagy on immune cell homeostasis. Here, we investigate basal autophagy in a mouse model of atherosclerosis, apolipoprotein E (ApoE)-knockout (KO) mice, and we analyze the role of autophagy in driving Tregs polarization. We observed defective maturation of Tregs from ApoE-KO mice in response to tumor growth factor-β (TGFβ). TGFβ is a well-known autophagy inducer, and Tregs maturation defects in ApoE-KO mice seem to be related to autophagy impairment. In this work, we propose that autophagy underlies Tregs maturation, advocating that the study of this process in atherosclerosis may open new therapeutic strategies.
International Journal of Molecular Sciences, 2019
Endothelial cells (ECs) constitute the innermost layer that lines all blood vessels from the larg... more Endothelial cells (ECs) constitute the innermost layer that lines all blood vessels from the larger arteries and veins to the smallest capillaries, including the lymphatic vessels. Despite the histological classification of endothelium of a simple epithelium and its homogeneous morphological appearance throughout the vascular system, ECs, instead, are extremely heterogeneous both structurally and functionally. The different arrangement of cell junctions between ECs and the local organization of the basal membrane generate different type of endothelium with different permeability features and functions. Continuous, fenestrated and discontinuous endothelia are distributed based on the specific function carried out by the organs. It is thought that a large number ECs functions and their responses to extracellular cues depend on changes in intracellular concentrations of calcium ion ([Ca2+]i). The extremely complex calcium machinery includes plasma membrane bound channels as well as int...
Malaria Journal, 2017
Background: Although malaria is a preventable and curable human disease, millions of people risk ... more Background: Although malaria is a preventable and curable human disease, millions of people risk to be infected by the Plasmodium parasites and to develop this illness. Therefore, there is an urgent need to identify new anti-malarial drugs. Ca 2+ signalling regulates different processes in the life cycle of Plasmodium falciparum, representing a suitable target for the development of new drugs. Results: This study investigated for the first time the effect of a highly specific inhibitor of nicotinic acid adenine dinucleotide phosphate (NAADP)-induced Ca 2+ release (Ned-19) on P. falciparum, revealing the inhibitory effect of this compound on the blood stage development of this parasite. Ned-19 inhibits both the transition of the parasite from the early to the late trophozoite stage and the ability of the late trophozoite to develop to the multinucleated schizont stage. In addition, Ned-19 affects spontaneous intracellular Ca 2+ oscillations in ring and trophozoite stage parasites, suggesting that the observed inhibitory effects may be associated to regulation of intracellular Ca 2+ levels. Conclusions: This study highlights the inhibitory effect of Ned-19 on progression of the asexual life cycle of P. falciparum. The observation that Ned-19 inhibits spontaneous Ca 2+ oscillations suggests a potential role of NAADP in regulating Ca 2+ signalling of P. falciparum.
Current hypertension reviews, Jan 4, 2017
Hypertension and aging are characterized by vascular remodelling and stiffness as well as endothe... more Hypertension and aging are characterized by vascular remodelling and stiffness as well as endothelial dysfunction. Endothelial function declines with age, since aging is associated with senescence of the endothelium due to increased rate of apoptosis and reduced regenerative capacity of the endothelium. Different phenotypes of hypertension have been described in younger and adult subjects with hypertension. In younger patients functional and structural alterations of resistance arteries occur as the earliest vascular alterations which have prognostic significance and may contribute to stiffness of large arteries through wave reflection. In individuals above age of 50 years as well as in subjects with long-lasting elevated blood pressure, vascular changes occur predominantly in conduit arteries which become stiffer. Activation of renin-angiotensin-aldosterone and endothelin systems plays a key role in endothelial dysfunction, vascular remodelling, and aging by inducing reactive oxyge...
Faseb Journal, 2002
cADPR, a potent calcium-mobilizing intracellular messenger synthesized by ADP-ribosyl cyclases re... more cADPR, a potent calcium-mobilizing intracellular messenger synthesized by ADP-ribosyl cyclases regulates openings of ryanodine receptors (RyR). Here we report that in the rat testis, a functional cADPR Ca 2؉ release system is essential for the contractile response of peritubular smooth muscle cells (PSMC) to endothelin (ET). We previously showed that this potent smooth muscle agonist elicits intracellular Ca 2؉ release in PSMC and seminiferous tubule contraction via activation of ETA and ETB receptors. ETB-R induces the mobilization of a thapsigargin-sensitive but IP 3-independent intracellular Ca 2؉ pool. Stimulation of permeabilized PSMC with cADPR was found to elicit large Ca 2؉ releases blocked by either a selective antagonist of cADPR or a RyR blocker, but not by heparin. Western blotting and confocal fluorescence microscopy indicated the specific expression of type 2 RyR in perinuclear localization. ET was found to stimulate the activity of ADP-ribosyl cyclase. Microinjection of the selective cADPR antagonist 8NH 2-cADPR completely abolished subsequent stimulation of Ca 2؉ signaling via ETA and ETB receptors. cADPR therefore appears to have an obligatory role for ETA-R and ETB-R-mediated calcium signaling in PSMC. However, ETB-R seem to be coupled exclusively to cADPR whereas ETA-R activation may be linked to IP 3 and cADPR signaling pathways.
Toll-like receptors (TLRs) recognize microbial/viral-derived components that trigger innate immun... more Toll-like receptors (TLRs) recognize microbial/viral-derived components that trigger innate immune response and conflicting data implicate TLR agonists in cancer, either as protumor or antitumor agents. We previously demonstrated that TLR3 activation mediated by its agonist poly(I:C) induces antitumor signaling, leading to apoptosis of prostate cancer cells LNCaP and PC3 with much more efficiency in the former than in the second more aggressive line. The transcription factor hypoxia-inducible factor 1 (HIF-1) regulates several cellular processes, including apoptosis, in response to hypoxia and to other stimuli also in normoxic conditions. Here we describe a novel protumor machinery triggered by TLR3 activation in PC3 cells consisting of increased expression of the specific I.3 isoform of HIF-1α and nuclear accumulation of HIF-1 complex in normoxia, resulting in reduced apoptosis and in secretion of functional vascular endothelial growth factor (VEGF). Moreover, we report that, in the less aggressive LNCaP cells, TLR3 activation fails to induce nuclear accumulation of HIF-1α. However, the transfection of I.3 isoform of hif-1α in LNCaP cells allows poly(I:C)-induced HIF-1 activation, resulting in apoptosis protection and VEGF secretion. Altogether, our findings demonstrate that differences in the basal level of HIF-1α expression in different prostate cancer cell lines underlie their differential response to TLR3 activation, suggesting a correlation between different stages of malignancy, hypoxic gene expression, and beneficial responsiveness to TLR agonists.
Journal of Cellular and Molecular Medicine, 2014
Toll-like receptors (TLRs) are a family of highly conserved transmembrane proteins expressed in e... more Toll-like receptors (TLRs) are a family of highly conserved transmembrane proteins expressed in epithelial and immune cells that recognize pathogen associated molecular patterns. Besides their role in immune response against infections, numerous studies have shown an important role of different TLRs in cancer, indicating these receptors as potential targets for cancer therapy. We previously demonstrated that the activation of TLR3 by the synthetic double-stranded RNA analogue poly I:C induces apoptosis of androgen-sensitive prostate cancer (PCa) LNCaP cells and, much less efficiently, of the more aggressive PC3 cell line. Therefore, in this study we selected LNCaP cells to investigate the mechanism of TLR3-mediated apoptosis and the in vivo efficacy of poly I:C-based therapy. We show that interferon regulatory factor-3 (IRF-3) signalling plays an essential role in TLR3-mediated apoptosis in LNCaP cells through the activation of the intrinsic and extrinsic apoptotic pathways. Interestingly, hardly any apoptosis was induced by poly I:C in normal prostate epithelial cells RWPE-1. We also demonstrate for the first time the direct anticancer effect of poly I:C as a single therapeutic agent in a well-established human androgen-sensitive PCa xenograft model, by showing that tumour growth is highly impaired in poly I:C-treated immunodeficient mice. Immunohistochemical analysis of PCa xenografts highlights the antitumour role of poly I:C in vivo both on cancer cells and, indirectly, on endothelial cells. Notably, we show the presence of TLR3 and IRF-3 in both human normal and PCa clinical samples, potentially envisaging poly I:C-based therapy for PCa.
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Papers by Antonio Filippini