3D-Printed Alginate/Pectin-Based Patches Loaded with Olive Leaf Extracts for Wound Healing Applications: Development, Characterization and In Vitro Evaluation of Biological Properties.

Traditional wound dressings may lack suitability for diverse wound types and individual patient requirements. In this context, this study aimed to innovate wound care by developing a 3D-printed patch using alginate and pectin and incorporating Olive Leaf Extract (OLE) as an active ingredient. Different polymer-to-plasticizer ratios were systematically examined to formulate a printable ink with optimal viscosity. The resultant film, enriched with OLE, exhibited a substantial polyphenolic content of 13.15 ± 0.41 mg CAE/g, showcasing significant antioxidant and anti-inflammatory properties. Notably, the film demonstrated potent scavenging abilities against DPPH, ABTS, and NO radicals, with IC50 values of 0.66 ± 0.07, 0.47 ± 0.04, and 2.02 ± 0.14 mg/mL, respectively. In vitro release and diffusion studies were carried out and the release profiles revealed an almost complete release of polyphenols from the patch within 48 h. Additionally, the fabricated film exhibited the capacity to enhance cell motility and accelerate wound healing, evidenced by increased collagen I expression in BJ fibroblast cells. Structural assessments affirmed the ability of the patch to absorb exudates and maintain the optimal moisture balance, while biocompatibility studies underscored its suitability for biomedical applications. These compelling findings endorse the potential application of the developed film in advanced wound care, with the prospect of tailoring patches to individual patient needs.

Synthesis and Characterization of a Biopolymer Pectin/Ethanolic Extract from Olive Mill Wastewater: In Vitro Safety and Efficacy Tests on Skin Wound Healing.

Wound-healing delay is one of the major problems of type 2 diabetes, representing also a clinical emergency in non-healing chronic wounds. Natural antioxidants show interesting wound-healing properties, including those extracted from waste derived from olive oil production. Olive mill wastewater is one of the main by-products of the olive oil-making process, and it is rich in high-value secondary metabolites, mainly hydroxytyrosol. We proposed an eco-friendly extraction method, employing both ultrasound-assisted and Soxhlet techniques and ethanol as a solvent, to recover valuable molecules from Roggianella cv (Olea europea L.) olive mill wastewater, which was further entrapped in a pectin polymer via an enzymatic reaction using porcine pancreatic lipase. Pectin, in combination with other substances, promoted and accelerated wound healing and demonstrated good potential to produce a biomedical conjugate for wound treatment. The antioxidant activity of the extracts and conjugate were evaluated against lipophilic (IC50 equal to 0.152 mg mL-1) and hydrophilic (IC50 equal to 0.0371 mg mL-1) radical species as well as the in vitro cytotoxicity via NRU, h-CLAT, and a wound-healing scratch assay and assessment. The pectin conjugate did not exert hemolytic effects on the peripheral blood, demonstrating interesting wound-healing properties due to its ability to stimulate cell proliferation in a dose-dependent manner.

ERα/LKB1 complex upregulates E-cadherin expression and stimulates breast cancer growth and progression upon adiponectin exposure.

Adiponectin is the major adipocytes-secreted protein involved in obesity-related breast cancer growth and progression. We proved that adiponectin promotes proliferation in ERα-positive breast cancer cells, through ERα transactivation and the recruitment of LKB1 as ERα-coactivator. Here, we showed that adiponectin-mediated ERα transactivation enhances E-cadherin expression. Thus, we investigated the molecular mechanism through which ERα/LKB1 complex may modulate the expression of E-cadherin, influencing tumor growth, progression and distant metastasis. We demonstrated that adiponectin increases E-cadherin expression in ERα-positive 2D and higher extent in 3D cultures. This occurs through a direct activation of E-cadherin gene promoter by ERα/LKB1-complex. The impact of E-cadherin on ERα-positive breast cancer cell proliferation comes from the evidence that in the presence of E-cadherin siRNA the proliferative effects of adiponectin is no longer noticeable. Since E-cadherin connects cell polarity and growth, we investigated if the adiponectin-enhanced E-cadherin expression could influence the localization of proteins cooperating in cell polarity, such as LKB1 and Cdc42. Surprisingly, immunofluorescence showed that, in adiponectin-treated MCF-7 cells, LKB1 and Cdc42 mostly colocalize in the nucleus, impairing their cytosolic cooperation in maintaining cell polarity. The orthotopic implantation of MCF-7 cells revealed an enhanced E-cadherin-mediated breast cancer growth induced by adiponectin. Moreover, tail vein injection of MCF-7 cells showed a higher metastatic burden in the lungs of mice receiving adiponectin-treated cells compared to control. From these findings it emerges that adiponectin treatment enhances E-cadherin expression, alters cell polarity and stimulates ERα-positive breast cancer cell growth in vitro and in vivo, sustaining higher distant metastatic burden.

Cdk4 Regulates Glioblastoma Cell Invasion and Stemness and Is Target of a Notch Inhibitor Plus Resveratrol Combined Treatment.

Glioblastoma multiforme (GBM) is one of the most aggressive types of cancer characterized by poor patient outcomes. To date, it is believed that the major cause of its recurrence and chemoresistance is represented by the enrichment of GBM stem cells (GSCs) sustained by the abnormal activation of a number of signaling pathways. In this study, we found that in GBM cells, treatment with low toxicity doses of the γ-secretase inhibitor RO4929097 (GSI), blocking the Notch pathway activity, in combination with resveratrol (RSV) was able to reverse the basal mesenchymal phenotype to an epithelial-like phenotype, affecting invasion and stemness interplay. The mechanism was dependent on cyclin D1 and cyclin-dependent kinase (CDK4), leading to a reduction of paxillin (Pxn) phosphorylation. Consequently, we discovered the reduced interaction of Pxn with vinculin (Vcl), which, during cell migration, transmits the intracellular forces to the extracellular matrix. The exogenous expression of a constitutively active Cdk4 mutant prevented the RSV + GSI inhibitory effects in GBM cell motility/invasion and augmented the expression of stemness-specific markers, as well as the neurosphere sizes/forming abilities in untreated cells. In conclusion, we propose that Cdk4 is an important regulator of GBM stem-like phenotypes and invasive capacity, highlighting how the combined treatment of Notch inhibitors and RSV could be prospectively implemented in the novel therapeutic strategies to target Cdk4 for these aggressive brain tumors.

Solid Lipid Nanoparticles Hydroquinone-Based for the Treatment of Melanoma: Efficacy and Safety Studies.

Classical melanoma therapy has several side effects that are responsible for a decrease in the final therapeutic efficacy. It is possible that the drug is degraded before reaching the target site and is metabolized by the body itself, resulting in repeated doses being administered throughout the day and a decrease in patient compliance. Drug delivery systems avoid degradation of the active ingredient, improve release kinetics, prevent the drug from being metabolized before reaching the site of action, and improve the safety and efficacy profiles of adjuvant cancer therapy. The solid lipid nanoparticles (SLNs) based on hydroquinone esterified with stearic acid realized in this work represent a chemotherapeutic drug delivery system that is useful in the treatment of melanoma. The starting materials were characterized by FT-IR and 1H-NMR, while the SLNs were characterized by dynamic light scattering. In efficacy studies, their ability to influence anchorage-dependent cell proliferation was tested on COLO-38 human melanoma cells. Furthermore, the expression levels of proteins belonging to apoptotic mechanisms were determined by analyzing the role of SLNs in modulating the expression of p53 and p21WAF1/Cip1. Safety tests were conducted to determine not only the pro-sensitizing potential but also the cytotoxicity of SLNs, and studies were conducted to assess the antioxidant and anti-inflammatory activity of these drug delivery.

Oleuropein Counteracts Both the Proliferation and Migration of Intra- and Extragonadal Seminoma Cells.

Recent and growing literature has reported that oleuropein (OLE), the main polyphenol in olive leaf extract, inhibits tumor cell proliferation and reduces the invasiveness properties of cancer cells; therefore, OLE may play a significant role in the development of new drugs for cancer treatment. These antineoplastic properties have been reported in many experimental cancer models, but the effect of OLE on seminoma cells is yet to be evaluated. In the present study, we demonstrate, for the first time, that OLE reduces cell viability in both intra- and extragonadal TCAM-2 and SEM-1 seminoma cells, respectively, in a dose-dependent manner. As shown by Western-blot analysis, OLE exposure reduced cyclin-D1 expression and upregulated p21Cip/WAF1, concomitantly affecting the upstream pathway of NF-κB, leading to the reduction of its nuclear content, thereby suggesting that OLE could modulate cell-cycle regulators by inhibiting NF-κB. Moreover, Annexin V staining revealed that OLE induced apoptosis in cancer cells and upregulated the pro-apoptotic factor BAX. Through wound-healing scratch and transmigration assays, we also demonstrated that OLE significantly reduced the migration and motility of TCAM-2 and SEM-1 cells, and downregulated the expression of TGFß-1, which is known to be the main pro-fibrotic factor involved in the acquisition of the migratory and invasive properties of cancer cells. Collectively, our results indicate that OLE reduces seminoma cell proliferation, promotes apoptosis, and counteracts cell migration and motility. Further studies are needed to explore the molecular mechanisms underlying these observed effects.

Synthesis and evaluation of wound healing properties of hydro-diab hydrogel loaded with green-synthetized AGNPS: in vitro and in ex vivo studies.

In diabetic patients, the presence of neuropathy, peripheral vascular diseases and ischemia, leads to the formation of foot ulcerations with a higher risk of infection because the normal response to bacterial infection is missing. In the aim to control and treat diabetic foot ulcerations (DFUs), wound dressings that are able to absorb exudate, to prevent infections, and to promote wound healing are needed. For this reason, the aim of the present research was to synthetize a biocompatible hydrogel (called HyDrO-DiAb) composed of carboxymethylcellulose loaded with silver nanoparticles (AgNPs) for the treatment of diabetic foot ulcers. In this study, AgNPs were obtained by a green synthesis and, then, were dissolved in a CMC hydrogel that, after a freeze drying process, becomes a flexible and porous structure. The in vitro and in ex vivo wound healing activity of the obtained HyDrO-DiAb hydrogel was evaluated.

Barrier effect and wound healing activity of the medical device REF-FTP78 in the treatment of gastroesophageal reflux disease.

REF-FTP78 is a class IIb medical device present on the market with different trade names and developed for the treatment of gastroesophageal reflux disease (GERD). This medical device is based on polysaccharides from Aloe Barbadensis and fucoidans from brown seaweeds, such as Undaria pinnatifida and Fucus vesiculosus, and aims to exert a protective effect on the esophageal mucosa against the noxious components of refluxate. The present study reports on the efficacy of REF-FTP78 devoting a particular attention to the barrier effect and wound healing properties, combined with antioxidant and anti-inflammatory activities. Film-forming properties and barrier effect were investigated on in vitro reconstructed human esophageal epithelium, through TEER measurement and evaluation of caffeine and Lucifer yellow permeability, and in an ex vivo swine model of esophageal mucosa damage. Antioxidant and anti-inflammatory properties were evaluated in terms of scavenging activity towards DPPH, ABTS and NO radicals and a wound healing assay was carried out to study the influence of the product on cell migration. The obtained results highlighted a significant barrier effect, with a reduction in caffeine penetration equal to 65.3%, the ability to both repair and prevent the damage caused by an acid insult, confirmed by a good transepithelial resistance for the tissue treated with the tested item, and the capacity to promote wound healing. Furthermore, the tested product showed good antioxidant and anti-inflammatory properties in the performed radical scavenging assays. These findings support the use of REF-FTP78 in the treatment of GERD.

The Antioxidant Selenoprotein T Mimetic, PSELT, Induces Preconditioning-like Myocardial Protection by Relieving Endoplasmic-Reticulum Stress.

Oxidative stress and endoplasmic reticulum stress (ERS) are strictly involved in myocardial ischemia/reperfusion (MI/R). Selenoprotein T (SELENOT), a vital thioredoxin-like selenoprotein, is crucial for ER homeostasis and cardiomyocyte differentiation and protection, likely acting as a redox-sensing protein during MI/R. Here, we designed a small peptide (PSELT), encompassing the redox site of SELENOT, and investigated whether its pre-conditioning cardioprotective effect resulted from modulating ERS during I/R. The Langendorff rat heart model was employed for hemodynamic analysis, while mechanistic studies were performed in perfused hearts and H9c2 cardiomyoblasts. PSELT improved the post-ischemic contractile recovery, reducing infarct size and LDH release with and without the ERS inducer tunicamycin (TM). Mechanistically, I/R and TM upregulated SELENOT expression, which was further enhanced by PSELT. PSELT also prevented the expression of the ERS markers CHOP and ATF6, reduced cardiac lipid peroxidation and protein oxidation, and increased SOD and catalase activities. An inert PSELT (I-PSELT) lacking selenocysteine was ineffective. In H9c2 cells, H2O2 decreased cell viability and SELENOT expression, while PSELT rescued protein levels protecting against cell death. In SELENOT-deficient H9c2 cells, H2O2 exacerbated cell death, that was partially mitigated by PSELT. Microscopy analysis revealed that a fluorescent form of PSELT was internalized into cardiomyocytes with a perinuclear distribution. Conclusions: The cell-permeable PSELT is able to induce pharmacological preconditioning cardioprotection by mitigating ERS and oxidative stress, and by regulating endogenous SELENOT.

A Tara Gum/Olive Mill Wastewaters Phytochemicals Conjugate as a New Ingredient for the Formulation of an Antioxidant-Enriched Pudding.

Olive mill wastewater, a high polyphenols agro-food by-product, was successfully exploited in an eco-friendly radical process to synthesize an antioxidant macromolecule, usefully engaged as a functional ingredient to prepare functional puddings. The chemical composition of lyophilized olive mill wastewaters (LOMW) was investigated by HPLC-MS/MS and 1H-NMR analyses, while antioxidant profile was in vitro evaluated by colorimetric assays. Oleuropein aglycone (5.8 µg mL-1) appeared as the main compound, although relevant amounts of an isomer of the 3-hydroxytyrosol glucoside (4.3 µg mL-1) and quinic acid (4.1 µg mL-1) were also detected. LOMW was able to greatly inhibit ABTS radical (IC50 equal to 0.019 mg mL-1), displaying, in the aqueous medium, an increase in its scavenger properties by almost one order of magnitude compared to the organic one. LOMW reactive species and tara gum chains were involved in an eco-friendly grafting reaction to synthesize a polymeric conjugate that was characterized by spectroscopic, calorimetric and toxicity studies. In vitro acute oral toxicity was tested against 3T3 fibroblasts and Caco-2 cells, confirming that the polymers do not have any effect on cell viability at the dietary use concentrations. Antioxidant properties of the polymeric conjugate were also evaluated, suggesting its employment as a thickening agent, in the preparation of pear puree-based pudding. High performance of consistency and relevant antioxidants features over time (28 days) were detected in the milk-based foodstuff, in comparison with its non-functional counterparts, confirming LOWM as an attractive source to achieve high performing functional foods.

Novel Insights into the Antagonistic Effects of Losartan against Angiotensin II/AGTR1 Signaling in Glioblastoma Cells.

New avenues for glioblastoma therapy are required due to the limited mortality benefit of the current treatments. The renin-angiotensin system (RAS) exhibits local actions and works as a paracrine system in different tissues and tumors, including glioma. The glioblastoma cell lines U-87 MG and T98G overexpresses Angiotensin II (Ang II)/Angiotensin II type I receptor (AGTR1) signaling, which enhances in vitro and in vivo local estrogen production through a direct up-regulation of the aromatase gene promoters p I.f and p I.4. In addition, Ang II/AGTR1 signaling transactivates estrogen receptor-α in a ligand-independent manner through mitogen-activated protein kinase (MAPK) activation. The higher aromatase mRNA expression in patients with glioblastoma was associated with the worst survival prognostic, according to The Cancer Genome Atlas (TCGA). An intrinsic immunosuppressive glioblastoma tumor milieu has been previously documented. We demonstrate how Ang II treatment in glioblastoma cells increases programmed death-ligand 1 (PD-L1) expression reversed by combined exposure to Losartan (LOS) in vitro and in vivo. Our findings highlight how LOS, in addition, antagonizes the previously documented neoangiogenetic, profibrotic, and immunosuppressive effects of Ang II and drastically inhibits its stimulatory effects on local estrogen production, sustaining glioblastoma cell growth. Thus, Losartan may represent an adjuvant pharmacological tool to be repurposed prospectively for glioblastoma treatment.

Design and development of plastic antibodies against SARS-CoV-2 RBD based on molecularly imprinted polymers that inhibit in vitro virus infection.

The present research study reports the development of plastic antibodies based on Molecularly Imprinted Polymers (MIPs) capable of selectively binding a portion of the novel coronavirus SARS-CoV-2 spike protein. Indeed, molecular imprinting represents a very promising and attractive technology for the synthesis of MIPs characterized by specific recognition abilities for a target molecule. Given these characteristics, MIPs can be considered tailor-made synthetic antibodies obtained by a templating process. After in silico analysis, imprinted nanoparticles were synthesized by inverse microemulsion polymerization and their ability to prevent the interaction between ACE2 and the receptor-binding domain of SARS-CoV-2 was investigated. Of relevance, the developed synthetic antibodies are capable of significantly inhibiting virus replication in Vero cell culture, suggesting their potential application in the treatment, prevention and diagnosis of SARS-CoV-2 infection.

Adipocyte-derived extracellular vesicles promote breast cancer cell malignancy through HIF-1α activity.

Extracellular vesicles (EVs) are emerging key protagonists in intercellular communication between adipocytes and breast cancer (BC) cells. Here, we described a new mechanism by which EVs released by mature adipocytes promoted breast cancer cell malignancy "in vitro" and "in vivo". We found that adipocyte-derived EVs enhanced growth, motility and invasion, stem cell-like properties, as well as specific traits of epithelial-to-mesenchymal transition in both estrogen receptor positive and triple negative BC cells. Of note, adipocyte-derived EVs aid breast tumor cells in lung metastatic colonization after tail-vein injection in mice. These EV-mediated effects occur via the induction of HIF-1α activity, since they were abrogated by the use of the HIF-1α inhibitor KC7F2 or in cells silenced for HIF-1α expression. Moreover, using an "ex vivo" model of obese adipocytes we found that the depletion of EVs counteracted the ability of obese adipocytes to sustain pro-invasive phenotype in BC cells. Interestingly, EVs released by undifferentiated adipocytes failed to induce aggressiveness and HIF-1α expression. These findings shed new light on the role of adipocyte-derived EVs in breast cancer progression, suggesting the possibility to target HIF-1α activity to block the harmful adipocyte-tumor cell dialogue, especially in obese settings.

Overexpression of p75NTR in Human Seminoma: A New Biomarker?

Several studies have demonstrated that the p75NTR low-affinity receptor of Nerve Growth Factor (NGF), is produced in abnormally large amounts in several human cancer types. However, the role of p75NTR varies substantially depending on cell context, so that a dual role of this receptor protein in tumor cell survival and invasion, as well as cell death, has been supported in recent studies. Herein we explored for the first time the expression of p75NTR in human specimens (nr = 40) from testicular germ cell tumors (TGCTs), mostly seminomas. Nuclear overexpression of p75NTR was detected by immunohistochemistry in seminoma tissue as compared to normal tissue, whereas neither NGF nor its high-affinity TrkA receptor was detected. An increased nuclear staining of phospho-JNK, belonging to the p75NTR signaling pathway and its pro-apoptotic target gene, p53, was concomitantly observed. Interestingly, our analysis revealed that decreased expression frequency of p75NTR, p-JNK and p53 was related to staging progression, thus suggesting that p75NTR may represent a specific marker for seminoma and staging in TGCTs.

FSH-R Human Early Male Genital Tract, Testicular Tumors and Sperm: Its Involvement in Testicular Disorders.

The follicle-stimulating hormone receptor (FSH-R) expression was always considered human gonad-specific. The receptor has also been newly detected in extragonadal tissues. In this finding, we evaluated FSH-R expression in the human male early genital tract, in testicular tumors, and in sperm from healthy and varicocele patients. In sperm, we also studied the mechanism of FSH-R action. Immunohystochemistry and Western blot analysis showed FSH-R presence in the first pathways of the human genital tract, in embryonal carcinoma, and in sperm, but it was absent in seminoma and in lower varicocele. In sperm, FSH/FSH-R activity is mediated by G proteins activating the PKA pathway, as we observed by using the H89. It emerged that increasing FSH treatments induced motility, survival, capacitation, and acrosome reaction in both sperm samples. The different FSH-R expression in tumor testicular tissues may be discriminate by tumor histological type. In spermatozoa, FSH-R indicates a direct action of FSH in these cells, which could be beneficial during semen preparation for in vitro fertilization procedures. For instance, FSH positive effects could be relevant in idiopathic infertility and in the clinic surgery of varicocele. In conclusion, FSH-R expression may be considered a molecular marker of testicular disorders.

KRAS-regulated glutamine metabolism requires UCP2-mediated aspartate transport to support pancreatic cancer growth.

The oncogenic KRAS mutation has a critical role in the initiation of human pancreatic ductal adenocarcinoma (PDAC) since it rewires glutamine metabolism to increase reduced nicotinamide adenine dinucleotide phosphate (NADPH) production, balancing cellular redox homeostasis with macromolecular synthesis1,2. Mitochondrial glutamine-derived aspartate must be transported into the cytosol to generate metabolic precursors for NADPH production2. The mitochondrial transporter responsible for this aspartate efflux has remained elusive. Here, we show that mitochondrial uncoupling protein 2 (UCP2) catalyses this transport and promotes tumour growth. UCP2-silenced KRASmut cell lines display decreased glutaminolysis, lower NADPH/NADP+ and glutathione/glutathione disulfide ratios and higher reactive oxygen species levels compared to wild-type counterparts. UCP2 silencing reduces glutaminolysis also in KRASWT PDAC cells but does not affect their redox homeostasis or proliferation rates. In vitro and in vivo, UCP2 silencing strongly suppresses KRASmut PDAC cell growth. Collectively, these results demonstrate that UCP2 plays a vital role in PDAC, since its aspartate transport activity connects the mitochondrial and cytosolic reactions necessary for KRASmut rewired glutamine metabolism2, and thus it should be considered a key metabolic target for the treatment of this refractory tumour.

Knockdown of Leptin Receptor Affects Macrophage Phenotype in the Tumor Microenvironment Inhibiting Breast Cancer Growth and Progression.

Aberrant leptin (Ob) signaling, a hallmark of obesity, has been recognized to influence breast cancer (BC) biology within the tumor microenvironment (TME). Here, we evaluated the impact of leptin receptor (ObR) knockdown in affecting BC phenotype and in mediating the interaction between tumor cells and macrophages, the most abundant immune cells within the TME. The stable knockdown of ObR (ObR sh) in ERα-positive and ERα-negative BC cells turned the tumor phenotype into a less aggressive one, as evidenced by in vitro and in vivo models. In xenograft tumors and in co-culture experiments between circulating monocytes and BC cells, the absence of ObR reduced the recruitment of macrophages, and also affected their cytokine mRNA expression profile. This was associated with a decreased expression and secretion of monocyte chemoattractant protein-1 in ObR sh clones. The loss of Ob/ObR signaling modulated the immunosuppressive TME, as shown by a reduced expression of programmed death ligand 1/programmed cell death protein 1/arginase 1. In addition, we observed increased phagocytic activity of macrophages compared to control Sh clones in the presence of ObR sh-derived conditioned medium. Our findings, addressing an innovative role of ObR in modulating immune TME, may open new avenues to improve BC patient health care.

Controlled Release of 5-FU from Chi-DHA Nanoparticles Synthetized with Ionic Gelation Technique: Evaluation of Release Profile Kinetics and Cytotoxicity Effect.

The ionic gelation technique allows us to obtain nanoparticles able to function as carriers for hydrophobic anticancer drugs, such as 5-fluoruracil (5-FU). In this study, reticulated chitosan- docosahexaenoic acid (Chi-DHAr) nanoparticles were synthesized by using a chemical reaction between amine groups of chitosan (Chi) and carboxylic acids of docosahexaenoic acid (DHA) and the presence of a link between Chi and DHA was confirmed by FT-IR, while the size and morphology of the obtained Chi-DHAr nanoparticles was evaluated with dynamic light scattering (DLS) and scanning electron microscopy (SEM), respectively. Drug-loading content (DLC) and drug-loading efficiency (DLE) of 5-FU in Chi-DHAr nanoparticles were 33.74 ± 0.19% and 7.9 ± 0.26%, respectively, while in the non-functionalized nanoparticles (Chir + 5FU), DLC, and DLE were in the ranges of 23.73 ± 0.14%, 5.62%, and 0.23%, respectively. The in vitro release profile, performed in phosphate buffer saline (PBS, pH 7.4) at 37 °C, indicated that the synthetized Chi-DHAr nanoparticles provided a sustained release of 5-FU. Based on the obtained regression coefficient value (R2), the first order kinetic model provided the best fit for both Chir and Chi-DHAr nanoparticles. Finally, cytotoxicity studies of chitosan, 5-FU, Chir, Chir + 5-FU, Chi-DHAr, and Chi-DHAr + 5-FU nanoparticles were conducted. Overall, Chi-DHAr nanoparticles proved to be much more biocompatible than Chir nanoparticles while retaining the ability to release the drug with high efficiency, especially towards specific types of cancerous cells.

Leptin and Notch Signaling Cooperate in Sustaining Glioblastoma Multiforme Progression.

Glioblastoma multiforme (GBM) is the most malignant form of glioma, which represents one of the commonly occurring tumors of the central nervous system. Despite the continuous development of new clinical therapies against this malignancy, it still remains a deadly disease with very poor prognosis. Here, we demonstrated the existence of a biologically active interaction between leptin and Notch signaling pathways that sustains GBM development and progression. We found that the expression of leptin and its receptors was significantly higher in human glioblastoma cells, U-87 MG and T98G, than in a normal human glial cell line, SVG p12, and that activation of leptin signaling induced growth and motility in GBM cells. Interestingly, flow cytometry and real-time RT-PCR assays revealed that GBM cells, grown as neurospheres, displayed stem cell-like properties (CD133+) along with an enhanced expression of leptin receptors. Leptin treatment significantly increased the neurosphere forming efficiency, self-renewal capacity, and mRNA expression levels of the stemness markers CD133, Nestin, SOX2, and GFAP. Mechanistically, we evidenced a leptin-mediated upregulation of Notch 1 receptor and the activation of its downstream effectors and target molecules. Leptin-induced effects on U-87 MG and T98G cells were abrogated by the selective leptin antagonist, the peptide LDFI (Leu-Asp-Phe-Ile), as well as by the specific Notch signaling inhibitor, GSI (Gamma Secretase Inhibitor) and in the presence of a dominant-negative of mastermind-like-1. Overall, these findings demonstrate, for the first time, a functional interaction between leptin and Notch signaling in GBM, highlighting leptin/Notch crosstalk as a potential novel therapeutic target for GBM treatment.

Statins Reduce Intratumor Cholesterol Affecting Adrenocortical Cancer Growth.

Mitotane causes hypercholesterolemia in patients with adrenocortical carcinoma (ACC). We suppose that cholesterol increases within the tumor and can be used to activate proliferative pathways. In this study, we used statins to decrease intratumor cholesterol and investigated the effects on ACC growth related to estrogen receptor α (ERα) action at the nuclear and mitochondrial levels. We first used microarray to investigate mitotane effect on genes involved in cholesterol homeostasis and evaluated their relationship with patients' survival in ACC TCGA. We then blocked cholesterol synthesis with simvastatin and determined the effects on H295R cell proliferation, estradiol production, and ERα activity in vitro and in xenograft tumors. We found that mitotane increases intratumor cholesterol content and expression of genes involved in cholesterol homeostasis, among them INSIG, whose expression affects patients' survival. Treatment of H295R cells with simvastatin to block cholesterol synthesis decreased cellular cholesterol content, and this affected cell viability. Simvastatin reduced estradiol production and decreased nuclear and mitochondrial ERα function. A mitochondrial target of ERα, the respiratory complex IV (COXIV), was reduced after simvastatin treatment, which profoundly affected mitochondrial respiration activating apoptosis. Additionally, simvastatin reduced tumor volume and weight of grafted H295R cells, intratumor cholesterol content, Ki-67 and ERα, COXIV expression and activity and increase terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells. Collectively, these data demonstrate that a reduction in intratumor cholesterol content prevents estradiol production and inhibits mitochondrial respiratory chain-inducing apoptosis in ACC cells. Inhibition of mitochondrial respiration by simvastatin represents a novel strategy to counteract ACC growth.