Effects of Reduced Extracellular Sodium Concentrations on Cisplatin Treatment in Human Tumor Cells: The Role of Autophagy.

Hyponatremia is the prevalent electrolyte imbalance in cancer patients, and it is associated with a worse outcome. Notably, emerging clinical evidence suggests that hyponatremia adversely influences the response to anticancer treatments. Therefore, this study aims to investigate how reduced extracellular [Na+] affects the responsiveness of different cancer cell lines (from human colon adenocarcinoma, neuroblastoma, and small cell lung cancer) to cisplatin and the underlying potential mechanisms. Cisplatin dose-response curves revealed higher IC50 in low [Na+] than normal [Na+]. Accordingly, cisplatin treatment was less effective in counteracting the proliferation and migration of tumor cells when cultured in low [Na+], as demonstrated by colony formation and invasion assays. In addition, the expression analysis of proteins involved in autophagosome-lysosome formation and the visualization of lysosomal areas by electron microscopy revealed that one of the main mechanisms involved in chemoresistance to cisplatin is the promotion of autophagy. In conclusion, our data first demonstrate that the antitumoral effect of cisplatin is markedly reduced in low [Na+] and that autophagy is an important mechanism of drug escape. This study indicates the role of hyponatremia in cisplatin chemoresistance and reinforces the recommendation to correct this electrolyte alteration in cancer patients.

The 3D in vitro Adrenoid cell model recapitulates the complexity of the adrenal gland.

The crosstalk between the chromaffin and adrenocortical cells is essential for the endocrine activity of the adrenal glands. This interaction is also likely important for tumorigenesis and progression of adrenocortical cancer and pheochromocytoma. We developed a unique in vitro 3D model of the whole adrenal gland called Adrenoid consisting in adrenocortical carcinoma H295R and pheochromocytoma MTT cell lines. Adrenoids showed a round compact morphology with a growth rate significantly higher compared to MTT-spheroids. Confocal analysis of differential fluorescence staining of H295R and MTT cells demonstrated that H295R organized into small clusters inside Adrenoids dispersed in a core of MTT cells. Transmission electron microscopy confirmed the strict cell-cell interaction occurring between H295R and MTT cells in Adrenoids, which displayed ultrastructural features of more functional cells compared to the single cell type monolayer cultures. Adrenoid maintenance of the dual endocrine activity was demonstrated by the expression not only of cortical and chromaffin markers (steroidogenic factor 1, and chromogranin) but also by protein detection of the main enzymes involved in steroidogenesis (steroidogenic acute regulatory protein, and CYP11B1) and in catecholamine production (tyrosine hydroxylase and phenylethanolamine N-methyltransferase). Mass spectrometry detection of steroid hormones and liquid chromatography measurement of catecholamines confirmed Adrenoid functional activity. In conclusion, Adrenoids represent an innovative in vitro 3D-model that mimics the spatial and functional complexity of the adrenal gland, thus being a useful tool to investigate the crosstalk between the two endocrine components in the pathophysiology of this endocrine organ.

ß3-adrenergic receptor on tumor-infiltrating lymphocytes sustains IFN-γ-dependent PD-L1 expression and impairs anti-tumor immunity in neuroblastoma.

Neuroblastoma (NB) is a heterogeneous extracranial tumor occurring in childhood. A distinctive feature of NB tumors is their neuroendocrine ability to secrete catecholamines, which in turn, via ß-adrenergic receptors ligation, may affect different signaling pathways in tumor microenvironment (TME). It was previously demonstrated that specific antagonism of ß3-adrenergic receptor (ß3-AR) on NB tumor cells affected tumor growth and progression. Here, in a murine syngeneic model of NB, we aimed to investigate whether the ß3-AR modulation influenced the host immune system response against tumor. Results demonstrated that ß3-AR antagonism lead to an immune response reactivation, partially dependent on the PD-1/PD-L1 signaling axis involvement. Indeed, ß3-AR blockade on tumor-infiltrating lymphocytes (TILs) dampened their ability to secrete IFN-γ, which in turn reduced the PD-L1 expression, caused by TILs infiltration, on NB tumor cells. Further investigations, through a genomic analysis on NB patients, showed that high ADRB3 gene expression correlates with worse clinical outcome compared to the low expression group, and that ADRB3 gene expression affects different immune-related pathways. Overall, results indicate that ß3-AR in NB TME is able to modulate the interaction between tumor and host immune system, and that its antagonism hits multiple pro-tumoral signaling pathways.

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.

Infantile hemangiomas ß3-adrenoceptor overexpression is associated with nonresponse to propranolol.

BACKGROUND: Propranolol (antagonist of ß1-/ß2-AR but minimally active against ß3-AR) is currently the first-line treatment for infantile hemangiomas (IH). Its efficacy is attributed to the blockade of ß2-AR. However, its success rate is ~60%. Considering the growing interest in the angiogenic role of ß3-ARs, we evaluated a possible relationship between ß3-AR expression and response to propranolol. METHODS: Fifteen samples of surgical biopsies were collected from patients with IH. Three were taken precociously from infants and then successfully treated with propranolol (responder group). Twelve were taken later, from residual lesions noncompletely responsive to propranolol (nonresponder group). A morphometrical analysis of the percentage of ß1-, ß2-, and ß3-ARs positively stained area was compared between the two groups. RESULTS: While no difference was found in both ß1- and ß2-AR expression level, a statistically significant increase of ß3-AR positively stained area was observed in the nonresponder group. CONCLUSIONS: Although the number of biopsies is insufficient to draw definitive conclusions, and the different ß-AR pattern may be theoretically explained by the different timing of samplings, this study suggests a possible correlation between ß3-AR expression and the reduced responsiveness to propranolol treatment. This study could pave the way for new therapeutic perspectives to manage IH. IMPACT: Propranolol (unselective antagonist of ß1 and ß2-ARs) is currently the first-line treatment for IHs, with a success rate of ~60%. Its effectiveness has been attributed to its ability to block ß2-ARs. However, ß3-ARs (on which propranolol is minimally active) were significantly more expressed in hemangioma biopsies taken from patients nonresponsive to propranolol. This study suggests a possible role of ß3-ARs in hemangioma pathogenesis and a possible new therapeutic target.

Hydrochlorothiazide Use and Risk of Nonmelanoma Skin Cancers: A Biological Plausibility Study.

Recent studies reported the association between increased risk of nonmelanoma skin cancers (NMSCs) and the use of hydrochlorothiazide (HCTZ), one of the most commonly prescribed diuretic, antihypertensive drug, over the world. Although HCTZ is known to be photosensitizing, the mechanisms involved in its potential prophotocarcinogenic effects remain unclear. Under acute exposure, therapeutically relevant concentrations of HCTZ (70, 140, and 370 ng/mL) amplified UVA-induced double-strand breaks, oxidative DNA, and protein damage in HaCaT human keratinocytes, and this effect was associated to a defective activity of the DNA repair enzyme, OGG1. Oxidative damage to DNA, but not that to proteins, was reversible within few hours. After chronic, combined exposure to HCTZ (70 ng/mL) and UVA (10 J/cm2), for 9 weeks, keratinocytes acquired a dysplastic-like phenotype characterized by a multilayered morphology and alterations in cell size, shape, and contacts. At the ultrastructural level, several atypical and enlarged nuclei and evident nucleoli were also observed. These transformed keratinocytes were apoptosis resistant, exhibited enhanced clonogenicity capacity, increased DNA damage and inflammation, defective DNA repair ability, and increased expression of the oncogene ΔNp63α and intranuclear ß-catenin accumulation (a hallmark of Wnt pathway activation), compared to those treated with UVA alone. None of these molecular, morphological, or functional effects were observed in cells treated with HCTZ alone. All these features resemble in part those of preneoplastic lesions and NMSCs and provide evidence of a biological plausibility for the association among exposure to UVA, use of HCTZ, and increased risk of NMSCs. These results are of translational relevance since we used environmentally relevant UVA doses and tested HCTZ at concentrations that reflect the plasma levels of doses used in clinical practice. This study also highlights that drug safety data should be followed by experimental evaluations to clarify the mechanistic aspects of adverse events.

Effect of Beta 3 Adrenoreceptor Modulation on Patency of the Ductus Arteriosus.

ß3-adrenoreceptor (ß3-AR), a G-protein coupled receptor, has peculiar regulatory properties in response to oxygen and widespread localization. ß3-AR is expressed in the most frequent neoplasms, also occurring in pregnant women, and its blockade reduces tumor growth, indicating ß3-AR-blockers as a promising alternative to antineoplastic drugs during pregnancy. However, ß3-AR involvement in prenatal morphogenesis and the consequences of its blockade for the fetus remain unknown. In this study, after the demonstrated expression of ß3-AR in endothelial and smooth muscle cells of ductus arteriosus (DA), C57BL/6 pregnant mice were acutely treated at 18.5 of gestational day (GD) with indomethacin or with the selective ß3-AR antagonist SR59230A, or chronically exposed to SR59230A from 15.5 to 18.5 GD. Six hours after the last treatment, fetuses were collected. Furthermore, newborn mice were treated straight after birth with BRL37344, a ß3-AR agonist, and sacrificed after 7 h. SR59230A, at the doses demonstrated effective in reducing cancer progression (10 and 20 mg/kg) in acute and chronic mode, did not induce fetal DA constriction and did not impair the DA ability to close after birth, whereas at the highest dose (40 mg/kg), it was shown to cause DA constriction and preterm-delivery. BRL37344 administered immediately after birth did not alter the physiological DA closure.

GLP-2 Prevents Neuronal and Glial Changes in the Distal Colon of Mice Chronically Treated with Cisplatin.

Cisplatin is a chemotherapeutic agent widely used for the treatment of solid cancers. Its administration is commonly associated with acute and chronic gastrointestinal dysfunctions, likely related to mucosal and enteric nervous system (ENS) injuries, respectively. Glucagon-like peptide-2 (GLP-2) is a pleiotropic hormone exerting trophic/reparative activities on the intestine, via antiapoptotic and pro-proliferating pathways, to guarantee mucosal integrity, energy absorption and motility. Further, it possesses anti-inflammatory properties. Presently, cisplatin acute and chronic damages and GLP-2 protective effects were investigated in the mouse distal colon using histological, immunohistochemical and biochemical techniques. The mice received cisplatin and the degradation-resistant GLP-2 analog ([Gly2]GLP-2) for 4 weeks. Cisplatin-treated mice showed mucosal damage, inflammation, IL-1ß and IL-10 increase; decreased number of total neurons, ChAT- and nNOS-immunoreactive (IR) neurons; loss of SOX-10-IR cells and reduced expression of GFAP- and S100ß-glial markers in the myenteric plexus. [Gly2]GLP-2 co-treatment partially prevented mucosal damage and counteracted the increase in cytokines and the loss of nNOS-IR and SOX-10-IR cells but not that of ChAT-IR neurons. Our data demonstrate that cisplatin causes mucosal injuries, neuropathy and gliopathy and that [Gly2]GLP-2 prevents these injuries, partially reducing mucosal inflammation and inducing ENS remodeling. Hence, this analog could represent an effective strategy to overcome colonic injures induced by cisplatin.

ß3-Adrenoceptors as Putative Regulator of Immune Tolerance in Cancer and Pregnancy.

Understanding the mechanisms of immune tolerance is currently one of the most important challenges of scientific research. Pregnancy affects the immune system balance, leading the host to tolerate embryo alloantigens. Previous reports demonstrated that ß-adrenergic receptor (ß-AR) signaling promotes immune tolerance by modulation of NK and Treg, mainly through the activation of ß2-ARs, but recently we have demonstrated that also ß3-ARs induce an immune-tolerant phenotype in mice bearing melanoma. In this report, we demonstrate that ß3-ARs support host immune tolerance in the maternal microenvironment by modulating the same immune cells populations as recently demonstrated in cancer. Considering that ß3-ARs are modulated by oxygen levels, we hypothesize that hypoxia, through the upregulation of ß3-AR, promotes the biological shift toward a tolerant immunophenotype and that this is the same trick that embryo and cancer use to create an aura of immune-tolerance in a competent immune environment. This study confirms the analogies between fetal development and tumor progression and suggests that the expression of ß3-ARs represents one of the strategies to induce fetal and tumor immune tolerance.

Archetype JC polyomavirus DNA associated with extracellular vesicles circulates in human plasma samples.

BACKGROUND: JC polyomavirus (JCPyV) establishes a stable and successful interaction with the host, causing progressive multifocal leukoencephalopathy (PML) in immunocompromised subjects. Recently, it has been reported that JCPyV, like other viruses, may exploit extracellular vesicles (EV) in cell cultures. OBJECTIVE: To investigate the presence of JCPyV-DNA in EV circulating in human plasma obtained from patients at risk for PML. STUDY DESIGN: JCPyV-DNA status was studied in EV obtained from 170 plasma samples collected from 120 HIV positive patients and 50 healthy donors. EV were extracted from plasma and characterized by Nanoparticle tracking analysis, by western blot for presence of tetraspanin CD63, CD81, annexin II, cythocrome C protein and, finally, by immunoelectron microscopy (IEM). Presence and quantitation of JCPyV-DNA were assessed with Multiplex real-time TaqMan PCR assay. RESULTS: The JCPyV-DNA plasma prevalence in 120 HIV positive patients and 50 healthy donors was 28% and 4%, respectively. The investigation performed on well-characterized plasma EV reported JCPyV-DNA detection in 15 out of 36 (42%) of the viremic samples (14 were from HIV patients and 1 from healthy people) at a mean level of 23.5 copies/mL. The examination of EV selected samples reported the percentage of JCPyV-DNA in EV of 5.4% of the total viral load. Moreover, IEM reported the presence of JCPyV Vp1 antigen in plasma-derived EV. CONCLUSION: The potential role of EV-associated JCPyV-DNA open new avenues and mechanistic insights into the molecular strategies adopted by this polyomavirus to persist in the host and spread to the central nervous system.