Unveiling the Role of Tryptophan 2,3-Dioxygenase in the Angiogenic Process.

BACKGROUND: Indoleamine 2,3-dioxygenase (IDO1) and tryptophan-2,3-dioxygenase (TDO) are the two principals enzymes involved in the catabolization of tryptophan (Trp) into kynurenine (Kyn). Despite their well-established role in the immune escape, their involvement in angiogenesis remains uncertain. We aimed to characterize TDO and IDO1 in human umbilical venular endothelial cells (HUVECs) and human endothelial colony-forming cells (ECFCs). METHODS: qRT-PCR and immunofluorescence were used for TDO and IDO1 expression while their activity was measured using ELISA assays. Cell proliferation was examined via MTT tests and in in vitro angiogenesis by capillary morphogenesis. RESULTS: HUVECs and ECFCs expressed TDO and IDO1. Treatment with the selective TDO inhibitor 680C91 significantly impaired HUVEC proliferation and 3D-tube formation in response to VEGF-A, while IDO1 inhibition showed no effect. VEGF-induced mTor phosphorylation and Kyn production were hindered by 680C91. ECFC morphogenesis was also inhibited by 680C91. Co-culturing HUVECs with A375 induced TDO up-regulation in both cell types, whose inhibition reduced MMP9 activity and prevented c-Myc and E2f1 upregulation. CONCLUSIONS: HUVECs and ECFCs express the key enzymes of the kynurenine pathway. Significantly, TDO emerges as a pivotal player in in vitro proliferation and capillary morphogenesis, suggesting a potential pathophysiological role in angiogenesis beyond its well-known immunomodulatory effects.

Iron Oxide Nanoparticles: Selectively Targeting Melanoma Cells In Vitro by Inducing DNA Damage via H2AX Phosphorylation and Hindering Proliferation through ERK Dephosphorylation.

This study investigates the distinctive characteristics of iron oxide magnetic nanoparticles (mNPs) and their potential application in cancer therapy, focusing on melanoma. Three types of mNPs, pre-validated for safety, underwent molecular analysis to uncover the activated signaling pathways in melanoma cells. Using the Western blot technique, the study revealed that mNPs induce cytotoxicity, hinder proliferation through ERK1/2 dephosphorylation, and prompt proapoptotic effects, including DNA damage by inducing H2AX phosphorylation. Additionally, in vitro magnetic hyperthermia notably enhanced cellular damage in melanoma cells. Moreover, the quantification of intracellular iron levels through Inductively Coupled Plasma Mass Spectrometry (ICP-MS) analysis unveils the precise dosage required to induce cellular damage effectively. These compelling findings not only shed light on the therapeutic potential of mNPs in melanoma treatment but also open exciting avenues for future research, heralding a new era in the development of targeted and effective cancer therapies. Indeed, by discerning the effective dose, our approach becomes instrumental in optimizing the therapeutic utilization of iron oxide magnetic nanoparticles, enabling the induction of precisely targeted and controlled cellular responses.

Proton Therapy, Magnetic Nanoparticles and Hyperthermia as Combined Treatment for Pancreatic BxPC3 Tumor Cells.

We present an investigation of the effects on BxPC3 pancreatic cancer cells of proton therapy combined with hyperthermia, assisted by magnetic fluid hyperthermia performed with the use of magnetic nanoparticles. The cells' response to the combined treatment has been evaluated by means of the clonogenic survival assay and the estimation of DNA Double Strand Breaks (DSBs). The Reactive Oxygen Species (ROS) production, the tumor cell invasion and the cell cycle variations have also been studied. The experimental results have shown that the combination of proton therapy, MNPs administration and hyperthermia gives a clonogenic survival that is much smaller than the single irradiation treatment at all doses, thus suggesting a new effective combined therapy for the pancreatic tumor. Importantly, the effect of the therapies used here is synergistic. Moreover, after proton irradiation, the hyperthermia treatment was able to increase the number of DSBs, even though just at 6 h after the treatment. Noticeably, the magnetic nanoparticles' presence induces radiosensitization effects, and hyperthermia increases the production of ROS, which contributes to cytotoxic cellular effects and to a wide variety of lesions including DNA damage. The present study indicates a new way for clinical translation of combined therapies, also in the vision of an increasing number of hospitals that will use the proton therapy technique in the near future for different kinds of radio-resistant cancers.

Inhibition of MMPs supports amoeboid angiogenesis hampering VEGF-targeted therapies via MLC and ERK 1/2 signaling.

BACKGROUND: In the past decades studies on anti-tumoral drugs inhibiting matrix metalloproteinase (MMPs) were disappointing. Recently, we demonstrated that mature endothelial cells (ECs) and endothelial colony forming cells (ECFCs) can switch between invasion modes to cope with challenging environments, performing the "amoeboid angiogenesis" in the absence of proteases activity. METHODS: We first set out to investigate by ELISA if the inhibitors of the main protease family involved in angiogenesis were differently expressed during breast cancer progression. We used Marimastat, a broad-spectrum MMP inhibitor, as a means of inducing amoeboid characteristics and studied VEGF role in amoeboid angiogenesis. Thus, we performed invasion and capillary morphogenesis assay, morphological, cell signaling and in vivo mouse studies. RESULTS: Our data showed that TIMP1, TIMP2, alpha2-antiplasmin, PAI-1 and cystatin increase in breast cancer serum of patients with primary cancer and lymph node positive compared to healthy women. In vitro results revealed that the most high-powered protease inhibitors able to induce amoeboid invasion of ECFCs were TIMP1, 2 and 3. Surprisingly, Marimastat promotes ECFC invasion and tubular formation in vitro and in vivo, inducing amoeboid characteristics. We observed that the combination of Marimastat plus VEGF doesn't boost neither cell invasion nor vessel formation capacity. Moreover, inhibition of VEGF activity with Bevacizumab in the presence of Marimastat confirmed that amoeboid angiogenesis is independent from the stimulus of the main vascular growth factor, VEGF. CONCLUSIONS: We underline the importance to consider the amoeboid mechanism of endothelial and cancer cell invasion, probably responsible for the failure of synthetic metalloproteinase inhibitors as cancer therapy and tumor resistance to VEGF-targeted therapies, to set-up new drugs to be used in cancer therapy.

miR-214-Enriched Extracellular Vesicles Released by Acid-Adapted Melanoma Cells Promote Inflammatory Macrophage-Dependent Tumor Trans-Endothelial Migration.

The understanding of the molecular mechanisms leading to melanoma dissemination is urgently needed in view of the identification of new targets and the development of innovative strategies to improve patients' outcomes. Within the complexity of tumor intercellular communications leading to metastatic dissemination, extracellular vesicles (EV) released by tumor cells are central players. Indeed, the ability to travel through the circulatory system conveying oncogenic bioactive molecules even at distant sites makes EV capable of modulating recipient cells to facilitate metastatic dissemination. The dynamic remodeling of the tumor microenvironment might influence, along with a number of other events, tumoral EV release. We observed that, in melanoma, extracellular acidosis increases the release of EV enriched in miR-214, an onco-miRNA involved in melanoma metastasis. Then, miR-214-enriched EV were found to induce a state of macrophage activation, leading to an overproduction of proinflammatory cytokines and nitric oxide. Such an inflammatory microenvironment was able to alter the endothelial cell permeability, thereby facilitating the trans-endothelial migration of melanoma cells, a crucial step in the metastatic cascade. The use of synthetic miR-214 inhibitors and miR-214 overexpression allowed us to demonstrate the key role of miR-214 in the EV-dependent induction of macrophage activation. Overall, our in vitro study reveals that the release of tumor miR-214-enriched EV, potentiated by adapting tumor cells to extracellular acidosis, drives a macrophage-dependent trans-endothelial migration of melanoma cells. This finding points to miR-214 as a potential new therapeutic target to prevent melanoma intravasation.

Star-Shaped Magnetic-Plasmonic Au@Fe3O4 Nano-Heterostructures for Photothermal Therapy.

Here, we synthesize a Au@Fe3O4 core@shell system with a highly uniform unprecedented star-like shell morphology with combined plasmonic and magnetic properties. An advanced electron microscopy characterization allows assessing the multifaceted nature of the Au core and its role in the growth of the peculiar epitaxial star-like shell with excellent crystallinity and homogeneity. Magnetometry and magneto-optical spectroscopy revealed a pure magnetite shell, with a superior saturation magnetization compared to similar Au@Fe3O4 heterostructures reported in the literature, which is ascribed to the star-like morphology, as well as to the large thickness of the shell. Of note, Au@Fe3O4 nanostar-loaded cancer cells displayed magneto-mechanical stress under a low frequency external alternating magnetic field (few tens of Hz). On the other hand, such a uniform, homogeneous, and thick magnetite shell enables the shift of the plasmonic resonance of the Au core to 640 nm, which is the largest red shift achievable in Au@Fe3O4 homogeneous core@shell systems, prompting application in photothermal therapy and optical imaging in the first biologically transparent window. Preliminary experiments performing irradiation of a stable water suspension of the nanostar and Au@Fe3O4-loaded cancer cell culture suspension at 658 nm confirmed their optical response and their suitability for photothermal therapy. The outstanding features of the prepared system can be thus potentially exploited as a multifunctional platform for magnetic-plasmonic applications.

The "End Life" of the Grape Pomace Waste Become the New Beginning: The Development of a Virtuous Cycle for the Green Synthesis of Gold Nanoparticles and Removal of Emerging Contaminants from Water.

During the last decades, the demand for processes developed according to the Circular Economy Principles has increased, searching for an alternative life for wastes. For this purpose, a one-pot green approach is exploited during this work to synthesize gold nanoparticles (AuNPs) by using grape pomace waste from Vitis vinifera. A raw aqueous extract of grape seeds, skin, and stems is used for AuNPs synthesis. UV-Vis, XPS, SEM, and ATR-FTIR spectroscopies demonstrate the main role of the extract's polyphenolic components in stabilizing nanoparticles. XRD, DLS, and Zeta Potential analyses were used to characterize AuNPs. Moreover, the ionic strength, pH, and temperature role was investigated through the Surface Plasmon Resonance (SPR) band observation to assess AuNPs' stability and photostability. For foreseeing the as-synthesized AuNPs' potential use in cosmetic and biomedical fields as multifunctional platforms, their antioxidant, and skin-lightening properties were tested, together with their sunscreen ability. A preliminary in-vitro evaluation is reported about the AuNPs' cytoprotective effects against H2O2 oxidative stress-induced in normal human dermal fibroblasts. Briefly, the possibility of reusing the grape pomace waste after the AuNPs synthesis as an adsorbent for the efficient removal of emergent contaminants is preliminarily discussed in the paper, further valorizing the use of waste according to a bio circular approach.

Olive phenols preserve lamin B1 expression reducing cGAS/STING/NFκB-mediated SASP in ionizing radiation-induced senescence.

Senescence occurs upon critical telomere shortening, or following DNA damage, oncogenic activation, hypoxia and oxidative stress, overall referred to stress-induced premature senescence (SIPS). In response to DNA damage, senescent cells release cytoplasmic chromatin fragments (CCFs), and express an altered secretome, the senescence-associated secretory phenotype (SASP), which contributes to generate a pro-inflammatory and pro-tumoral extracellular milieu. Polyphenols have gained significant attention owing to their anti-inflammatory and anti-tumour activities. Here, we studied the effect of oleuropein aglycone (OLE) and hydroxytyrosol (HT) on DNA damage, CCF appearance and SASP in a model of irradiation-induced senescence. Neonatal human dermal fibroblasts (NHDFs) were γ-irradiated and incubated with OLE, 5 µM and HT, 1 µM. Cell growth and senescence-associated (SA)-ß-Gal-staining were used as senescence markers. DNA damage was evaluated by Comet assay, lamin B1 expression, release of CCFs, cyclic GMP-AMP Synthase (cGAS) activation. IL-6, IL-8, MCP-1 and RANTES were measured by ELISA assay. Our results showed that OLE and HT exerted a protective effect on 8 Gy irradiation-induced senescence, preserving lamin B1 expression and reducing cGAS/STING/NFκB-mediated SASP. The ability of OLE and HT to mitigate DNA damage, senescence status and the related SASP in normal cells can be exploited to improve the efficacy and safety of cancer radiotherapy.

Smart Magnetic Nanocarriers for Multi-Stimuli On-Demand Drug Delivery.

In this study, we report the realization of drug-loaded smart magnetic nanocarriers constituted by superparamagnetic iron oxide nanoparticles encapsulated in a dual pH- and temperature-responsive poly (N-vinylcaprolactam-co-acrylic acid) copolymer to achieve highly controlled drug release and localized magnetic hyperthermia. The magnetic core was constituted by flower-like magnetite nanoparticles with a size of 16.4 nm prepared by the polyol approach, with good saturation magnetization and a high specific absorption rate. The core was encapsulated in poly (N-vinylcaprolactam-co-acrylic acid) obtaining magnetic nanocarriers that revealed reversible hydration/dehydration transition at the acidic condition and/or at temperatures above physiological body temperature, which can be triggered by magnetic hyperthermia. The efficacy of the system was proved by loading doxorubicin with very high encapsulation efficiency (>96.0%) at neutral pH. The double pH- and temperature-responsive nature of the magnetic nanocarriers facilitated a burst, almost complete release of the drug at acidic pH under hyperthermia conditions, while a negligible amount of doxorubicin was released at physiological body temperature at neutral pH, confirming that in addition to pH variation, drug release can be improved by hyperthermia treatment. These results suggest this multi-stimuli-sensitive nanoplatform is a promising candidate for remote-controlled drug release in combination with magnetic hyperthermia for cancer treatment.

uPAR Controls Vasculogenic Mimicry Ability Expressed by Drug-Resistant Melanoma Cells.

Malignant melanoma is a highly aggressive skin cancer characterized by an elevated grade of tumor cell plasticity. Such plasticity allows adaptation of melanoma cells to different hostile conditions and guarantees tumor survival and disease progression, including aggressive features such as drug resistance. Indeed, almost 50% of melanoma rapidly develop resistance to the BRAFV600E inhibitor vemurafenib, with fast tumor dissemination, a devastating consequence for patients outcomes. Vasculogenic mimicry (VM), the ability of cancer cells to organize themselves in perfused vascular-like channels, might sustain tumor spread by providing vemurafenib-resistant cancer cells with supplementary ways to enter into circulation and disseminate. Thus, this research aims to determine if vemurafenib resistance goes with the acquisition of VM ability by aggressive melanoma cells, and identify a driving molecule for both vemurafenib resistance and VM. We used two independent experimental models of drug-resistant melanoma cells, the first one represented by a chronic adaptation of melanoma cells to extracellular acidosis, known to drive a particularly aggressive and vemurafenib-resistant phenotype, the second one generated with chronic vemurafenib exposure. By performing in vitro tube formation assay and evaluating the expression levels of the VM markers EphA2 and VE-cadherin by Western blotting and flow cytometer analyses, we demonstrated that vemurafenib-resistant cells obtained by both models are characterized by an increased ability to perform VM. Moreover, by exploiting the CRISPR-Cas9 technique and using the urokinase plasminogen activator receptor (uPAR) inhibitor M25, we identified uPAR as a driver of VM expressed by vemurafenib-resistant melanoma cells. Thus, uPAR targeting may be successfully leveraged as a new complementary therapy to inhibit VM in drug-resistant melanoma patients, to counteract the rapid progression and dissemination of the disease.

A Possible Role for PAI-1 Blockade in Melanoma Immunotherapy.

In their new article in the Journal of Investigative Dermatology, Tseng et al. (2021) confirm that the sensitivity of melanoma cells to anti‒PD-L1 checkpoint inhibitor therapy is correlated with high PD-L1 surface expression. By blocking PD-L1 membrane clearing, controlled by LRP1 and PAI-1, the expression of high-cell-surface levels of PD-L1 was maintained.

CRISPR/Cas9 uPAR Gene Knockout Results in Tumor Growth Inhibition, EGFR Downregulation and Induction of Stemness Markers in Melanoma and Colon Carcinoma Cell Lines.

uPAR is a globular protein, tethered to the cell membrane by a GPI-anchor involved in several cancer-related properties and its overexpression commonly correlates with poor prognosis and metastasis. We investigated the consequences of uPAR irreversible loss in human melanoma and colon cancer cell lines, knocking out its expression by CRISPR/Cas9. We analyzed through flow cytometry, western blotting and qPCR, the modulation of the most known cancer stem cells-associated genes and the EGFR while we observed the proliferation rate exploiting 2D and 3D cellular models. We also generated uPAR "rescue" expression cell lines as well as we promoted the expression of only its 3'UTR to demonstrate the involvement of uPAR mRNA in tumor progression. Knocking out PLAUR, uPAR-encoding gene, we observed an inhibited growth ratio unexpectedly coupled with a significant percentage of cells acquiring a stem-like phenotype. In vivo experiments demonstrated that uPAR loss completely abrogates tumorigenesis despite the gained stem-like profile. Nonetheless, we proved that the reintroduction of the 3'UTR of PLAUR gene was sufficient to restore the wild-type status validating the hypothesis that such a region may act as a "molecular sponge". In particular miR146a, by binding PLAUR 3' UTR region might be responsible for uPAR-dependent inhibition of EGFR expression.

Enhanced Antitumoral Activity and Photoacoustic Imaging Properties of AuNP-Enriched Endothelial Colony Forming Cells on Melanoma.

Near infrared (NIR)-resonant gold nanoparticles (AuNPs) hold great promise in cancer diagnostics and treatment. However, translating the theranostic potential of AuNPs into clinical applications still remains a challenge due to the difficulty to improve the efficiency and specificity of tumor delivery in vivo as well as the clearance from liver and spleen to avoid off target toxicity. In this study, endothelial colony forming cells (ECFCs) are exploited as vehicles to deliver AuNPs to tumors. It is first demonstrated that ECFCs display a great capability to intake AuNPs without losing viability, and exert antitumor activity per se. Using a human melanoma xenograft mouse model, it is next demonstrated that AuNP-loaded ECFCs retain their capacity to migrate to tumor sites in vivo 1 day after injection and stay in the tumor mass for more than 1 week. In addition, it is demonstrated that ECFC-loaded AuNPs are efficiently cleared by the liver over time and do not elicit any sign of damage to healthy tissue.

Synthesis and characterization of modified magnetic nanoparticles as theranostic agents: in vitro safety assessment in healthy cells.

Over the past few decades nanotechnology has paved its way into cancer treatment procedures with the use of nanoparticles (NPs) for contrast media and therapeutic agents. Iron based NPs are the most investigated since they can be used for drug delivery, imaging and when magnetically activate employed as local heat sources in cancer hyperthermia. In this work, was performed synthesis, characterization and biological evaluation of different types of iron oxide nanoparticles (mNPs'), as promising material for tumor hyperthermia. The surface of mNPs' has modified with inorganic stabilizing agents to particularly improve characteristics such as their magnetic properties, colloidal stability and biocompatibility. The successful coating of mNPs' was confirmed by morphological and structural characterization by transmission electron microscopy (TEM) and Fourier-Transform Infra-Red spectroscopy (FT-IR), while their hydrodynamic diameter was studied by using Dynamic light scattering (DLS). X-ray Diffraction (XRD) proved that the crystallite phase of mNPs' is the same with the pattern of magnetite. Superparamagnetic behavior and mNPs' response under the application of alternating magnetic field (AMF) were also thoroughly investigated and showed good heating efficiency in magnetic hyperthermia experiments. The contrast ability in magnetic resonance imaging (MRI) is also discussed indicating that mNPs are negative MRI contrast types. Nonetheless the effects of mNPs on cell viability was performed by MTT on human keratinocytes, human embryonic kidney cells, endothelial cells and by hemolytic assay on erythrocytes. In healthy keratinocytes wound healing assay in different time intervals was performed, assessing both the cell migration and wound closure. Endothelial cells have also been studied in functional activity performing capillary morphogenesis. In vitro studies showed that mNPs are safely taken by the healthy cells and do not interfere with the biological processes such as cell migration and motility.

uPAR-expressing melanoma exosomes promote angiogenesis by VE-Cadherin, EGFR and uPAR overexpression and rise of ERK1,2 signaling in endothelial cells.

Exosomes (Exos) have been reported to promote pre-metastatic niche formation, proliferation, angiogenesis and metastasis. We have investigated the role of uPAR in melanoma cell lines-derived Exos and their pro-angiogenic effects on human microvascular endothelial cells (HMVECs) and endothelial colony-forming cells (ECFCs). Melanoma Exos were isolated from conditioned media of A375 and M6 cells by differential centrifugation and filtration. Tunable Resistive Pulse Sensing (TRPS) and Nanoparticle tracking analysis were performed to analyze dimension and concentration of Exos. The CRISPR-Cas 9 technology was exploited to obtain a robust uPAR knockout. uPAR is expressed in melanoma Exos that are internalized by HMVECs and ECFCs, enhancing VE-Cadherin, EGFR and uPAR expression in endothelial cells that undergo a complete angiogenic program, including proliferation, migration and tube formation. uPAR loss reduced the pro-angiogenic effects of melanoma Exos in vitro and in vivo by inhibition of VE-Cadherin, EGFR and uPAR expression and of ERK1,2 signaling in endothelial cells. A similar effect was obtained with a peptide that inhibits uPAR-EGFR interaction and with the EGFR inhibitor Gefitinib, which also inhibited melanoma Exos-dependent EGFR phosphorylation. This study suggests that uPAR is required for the pro-angiogenic activity of melanoma Exos. We propose the identification of uPAR-expressing Exos as a potentially useful biomarker for assessing pro-angiogenic propensity and eventually monitoring the response to treatment in metastatic melanoma patients.

Cell-Mediated Release of Nanoparticles as a Preferential Option for Future Treatment of Melanoma.

Targeted and immune therapies have unquestionably improved the prognosis of melanoma patients. However the treatment of this neoplasm still requires approaches with a higher therapeutic index, in order to reduce shortcomings related to toxic effects and aspecific targeting. This means developing therapeutic tools derived with high affinity molecules for tumor components differentially expressed in melanoma cells with respect to their normal counterpart. Nanomedicine has sought to address this problem owing to the high modulability of nanoparticles. This approach exploits not only the enhanced permeability and retention effect typical of the tumor microenvironment (passive targeting), but also the use of specific "molecular antennas" that recognize some tumor-overexpressed molecules (active targeting). This line of research has given rise to the so-called "smart nanoparticles," some of which have already passed the preclinical phase and are under clinical trials in melanoma patients. To further improve nanoparticles partition within tumors, for some years now a line of thought is exploiting the molecular systems that regulate the innate tumor-homing activity of platelets, granulocytes, monocytes/macrophages, stem cells, endothelial-colony-forming cells, and red blood cells loaded with nanoparticles. This new vision springs from the results obtained with some of these cells in regenerative medicine, an approach called "cell therapy." This review takes into consideration the advantages of cell therapy as the only one capable of overcoming the limits of targeting imposed by the increased interstitial pressure of tumors.

Parvovirus B19 activates in vitro normal human dermal fibroblasts: a possible implication in skin fibrosis and systemic sclerosis.

OBJECTIVE: Fibrosis is the most characteristic pathological hallmark of SSc, a connective tissue disease characterized by vascular and immunological abnormalities, inflammation and enhanced extracellular matrix production, leading to progressive fibrosis of skin and internal organs. We previously demonstrated that parvovirus B19 (B19V) can infect normal human dermal fibroblasts (NHDFs) and that B19V persists in SSc fibroblasts. In this study, we investigated whether parvovirus B19V is able to activate in vitro NHDFs and to induce in these cells some phenotypic features similar to that observed in the SSc fibroblasts. METHODS: We preliminarily analysed the time course of B19V infection in cultured NHDFs, then we investigated the ability of B19V to induce cell migration, invasive phenotype and mRNA expression of some profibrotic and/or proinflammatory genes. RESULTS: We confirmed our previous findings that B19V infects NHDFs, but the infection is not productive. After incubation with B19V, NHDFs showed a significant increase of both migration and invasiveness, along with mRNA expression of different profibrotic genes (α-SMA, EDN-1, IL-6, TGF-ß1 receptors 1 and 2, Col1α2), some genes associated with inflammasome platform (AIM2, IFI16, IL-1ß, CASP-1) and genes for metalloprotease (MMP 2, 9 and 12). CONCLUSION: These data suggest that B19V can activate dermal fibroblasts and may have a role in the pathogenesis of fibrosis. B19V-induced fibroblast migration and invasiveness could be due to the B19V-associated MMP9 overexpression and activation. Moreover, the up-regulation of MMP12, typical of SSc, could link the B19V infection of fibroblasts to the anti-angiogenic process.

uPAR Knockout Results in a Deep Glycolytic and OXPHOS Reprogramming in Melanoma and Colon Carcinoma Cell Lines.

Urokinase Plasminogen Activator (uPA) Receptor (uPAR) is a well-known GPI-anchored three-domain membrane protein with pro-tumor roles largely shown in all the malignant tumors where it is over-expressed. Here we have exploited the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 gene knock out approach to investigate its role in the oxidative metabolism in human melanoma and colon cancer as the consequences of its irreversible loss. Knocking out PLAUR, a uPAR-encoding gene, in A375p, A375M6 and HCT116, which are two human melanoma and a colon carcinoma, respectively, we have observed an increased number of mitochondria in the two melanoma cell lines, while we evidenced an immature biogenesis of mitochondria in the colon carcinoma culture. Such biological diversity is, however, reflected in a significant enhancement of the mitochondrial spare respiratory capacity, fueled by an increased expression of GLS2, and in a decreased glycolysis paired with an increased secretion of lactate by all uPAR KO cells. We speculated that this discrepancy might be explained by an impaired ratio between LDHA and LDHB.

A potentiated cooperation of carbonic anhydrase IX and histone deacetylase inhibitors against cancer.

The emergence of tumour recurrence and resistance limits the survival rate for most tumour-bearing patients. Only, combination therapies targeting pathways involved in the induction and in the maintenance of cancer growth and progression might potentially result in an enhanced therapeutic efficacy. Herein, we provided a prospective combination treatment that includes suberoylanilide hydroxamic acid (SAHA), a well-known inhibitor of histone deacetylases (HDACs), and SLC-0111, a novel inhibitor of carbonic anhydrase (CA) IX. We proved that HDAC inhibition with SAHA in combination with SLC-0111 affects cell viability and colony forming capability to greater extent than either treatment alone of breast, colorectal and melanoma cancer cells. At the molecular level, this therapeutic regimen resulted in a synergistically increase of histone H4 and p53 acetylation in all tested cell lines. Overall, our findings showed that SAHA and SLC-0111 can be regarded as very attractive combination providing a potential therapeutic strategy against different cancer models.

Prep1 regulates angiogenesis through a PGC-1α-mediated mechanism.

Angiogenesis depends on a delicate balance between the different transcription factors, and their control should be considered necessary for preventing or treating diseases. Pre-B-cell leukemia transcription factor regulating protein 1 (Prep1) is a homeodomain transcription factor that plays a primary role in organogenesis and metabolism. Observations performed in a Prep1 hypomorphic mouse model, expressing 3-5% of the protein, show an increase of embryonic lethality due, in part, to defects in angiogenesis. In this study, we provide evidence that overexpression of Prep1 in mouse aortic endothelial cells (MAECs) stimulates migration, proliferation, and tube formation. These effects are paralleled by an increase of several proangiogenic factors and by a decrease of the antiangiogenic gene neurogenic locus notch homolog protein 1 (Notch1). Prep1-mediated angiogenesis involves the activation of the p160 Myb-binding protein (p160)/peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) pathway. Indeed, Prep1 overexpression increases its binding with p160 and induces a 4-fold increase of p160 and 70% reduction of PGC-1α compared with control cells. Incubation of MAECs with a synthetic Prep1(54-72) peptide, mimicking the Prep1 region involved in the interaction with p160, reverts the proangiogenic effects mediated by Prep1. In addition, Prep1 levels increase by 3.2-fold during the fibroblast growth factor ß (bFGF)-mediated endothelial colony-forming cells' activation, whereas Prep1(54-72) peptide reduces the capability of these cells to generate tubular-like structures in response to bFGF, suggesting a possible role of Prep1 both in angiogenesis from preexisting vessels and in postnatal vasculogenesis. Finally, Prep1 hypomorphic heterozygous mice, expressing low levels of Prep1, show attenuated placental angiogenesis and vessel formation within Matrigel plugs. All of these observations indicate that Prep1, complexing with p160, decreases PGC-1α and stimulates angiogenesis.-Cimmino, I., Margheri, F., Prisco, F., Perruolo, G., D'Esposito, V., Laurenzana, A., Fibbi, G., Paciello, O., Doti, N., Ruvo, M., Miele, C., Beguinot, F., Formisano, P., Oriente, F. Prep1 regulates angiogenesis through a PGC-1α-mediated mechanism.