Health-Promoting Properties of Natural Flavonol Glycosides Isolated from Staphylea pinnata L.

Staphylea, also called bladdernuts, is a genus of plants belonging to the family Staphyleaceae, widespread in tropical or temperate climates of America, Europe, and the Far East. Staphylea spp. produce bioactive metabolites with antioxidant properties, including polyphenols which have not been completely investigated for their phytotherapeutic potential, even though they have a long history of use for food. Here, we report the isolation of six flavonol glycosides from the hydroalcoholic extract of aerial parts of Staphylea pinnata L., collected in Italy, using a solid-phase extraction technique. They were identified using spectroscopic, spectrometric, and optical methods as three quercetin and three isorhamnetin glycosides. Among the flavonol glycosides isolated, isoquercetin and quercetin malonyl glucoside showed powerful antioxidant, antimicrobial, and wound healing promoting activity and thus are valuable as antiaging ingredients for cosmeceutical applications and for therapeutic applications in skin wound repair.

Comparative Proteomic Profiling of Secreted Extracellular Vesicles from Breast Fibroadenoma and Malignant Lesions: A Pilot Study.

Extracellular vesicles (EVs) shuttle proteins, RNA, DNA, and lipids crucial for cell-to-cell communication. Recent findings have highlighted that EVs, by virtue of their cargo, may also contribute to breast cancer (BC) growth and metastatic dissemination. Indeed, EVs are gaining great interest as non-invasive cancer biomarkers. However, little is known about the biological and physical properties of EVs from malignant BC lesions, and even less is understood about EVs from non-malignant lesions, such as breast fibroadenoma (FAD), which are clinically managed using conservative approaches. Thus, for this pilot study, we attempted to purify and explore the proteomic profiles of EVs from benign breast lesions, HER2+ BCs, triple-negative BCs (TNBCs), and continuous BC cell lines (i.e., BT-549, MCF-10A, and MDA-MB-231), combining experimental and semi-quantitative approaches. Of note, proteome-wide analyses showed 49 common proteins across EVs harvested from FAD, HER2+ BCs, TNBCs, and model BC lines. This is the first feasibility study evaluating the physicochemical composition and proteome of EVs from benign breast cells and primary and immortalized BC cells. Our preliminary results hold promise for possible implications in precision medicine for BC.

miR-216a Acts as a Negative Regulator of Breast Cancer by Modulating Stemness Properties and Tumor Microenvironment.

Breast cancer is the most frequent malignancy in females in terms of both incidence and mortality. Underlying the high mortality rate is the presence of cancer stem cells, which divide indefinitely and are resistant to conventional chemotherapies, so causing tumor relapse. In the present study, we identify miR-216a-5p as a downregulated microRNA in breast cancer stem cells vs. the differentiated counterpart. We demonstrate that overexpression of miR-216a-5p impairs stemness markers, mammosphere formation, ALDH activity, and the level of Toll-like receptor 4 (TLR4), which plays a significant role in breast cancer progression and metastasis by leading to the release of pro-inflammatory molecules, such as interleukin 6 (IL-6). Indeed, miR-216a regulates the crosstalk between cancer cells and the cells of the microenvironment, in particular cancer-associated fibroblasts (CAFs), through regulation of the TLR4/IL6 pathway. Thus, miR-216a has an important role in the regulation of stem phenotype, decreasing stem-like properties and affecting the cross-talk between cancer cells and the tumor microenvironment.

The Role of Exo-miRNAs in Cancer: A Focus on Therapeutic and Diagnostic Applications.

Exosomes are extracellular vesicles released into biological fluids where they act as carriers of various molecules, including proteins, lipids, and RNAs, between cells, modulating or perturbing specific physiological processes. Recently, it has been suggested that tumoral cells release excessive amounts of exosomes that, through their cargo, promote tumor progression, stimulating growth, angiogenesis, metastasis, insensitivity to chemotherapy, and immune evasion. Increasing evidence highlights exosomal microRNAs (exo-miRNAs) as important players in tumorigenesis. MicroRNA (miRNA) are a class of small non-coding RNA able to regulate gene expression, targeting multiple mRNAs and inducing translational repression and/or mRNA degradation. Exo-miRNAs are highly stable and easily detectable in biological fluids, and for these reasons, miRNAs are potential cancer biomarkers useful diagnostically and prognostically. Furthermore, since exosomes are natural delivery systems between cells, they can be appropriately modified to carry therapeutic miRNAs to specific recipient cells. Here we summarize the main functions of exo-miRNAs and their possible role for diagnostic and therapeutic applications.

Ultrasmall Carbon Nanodots as Theranostic Nanoheaters for Precision Breast Cancer Phototherapy: Establishing the Translational Potential in Tumor-in-a-Dish Models.

This study investigates the remarkable attributes of sulfur-doped carbon nanodots (CDs) synthesized in high yield and a narrow size distribution (4.8 nm). These CDs exhibit notable features, including potential bioelimination through renal clearance and efficient photothermal conversion in the near-infrared region with multicolor photoluminescence across the visible spectrum. Our research demonstrates high biocompatibility and effective near-infrared (NIR)-triggered photothermal toxicity when targeting mammospheres and patient-derived tumor organoids. Moreover, the study delves into the intricate cellular responses induced by CD-mediated hyperthermia. This involves efficient tumor mass death, activation of the p38-mitogen-activated protein kinase (MAPK) pathway, and upregulation of genes associated with apoptosis, hypoxia, and autophagy. The interaction of CDs with mammospheres reveals their ability to penetrate the complex microenvironment, impeded at 4 °C, indicating an energy-dependent endocytosis mechanism. This observation underscores the CDs' potential for targeted drug delivery, particularly in anticancer therapeutics. This investigation contributes to understanding the multifunctional properties of sulfur-doped CDs and highlights their promising applications in cancer therapeutics. Utilizing 3-D tumor-in-a-dish patients' organoids enhances translational potential, providing a clinically relevant platform for assessing therapeutic efficacy in a context mirroring the physiological conditions of cancerous tissues.

MCT4-driven CAF-mediated metabolic reprogramming in breast cancer microenvironment is a vulnerability targetable by miR-425-5p.

Multiple oncogenic alterations contribute to breast cancer development. Metabolic reprogramming, deeply contributing to tumor microenvironment (TME) education, is now widely recognized as a hallmark of cancer. The reverse Warburg effect induces cancer-associated fibroblasts (CAFs) to produce and secrete L-lactate, enhancing malignant characteristics such as neoangiogenesis, metastatic dissemination, and treatment resistance. Monocarboxylate transporter (MCT) 4 is involved in lactate efflux from CAFs into stromal and epithelial cells. Here, we first assess the expression of miR-425-5p and its target MCT4 in breast cancer CAFs and normal fibroblasts. We analyzed the metabolic changes induced by miR-425-5p in CAFs and its role in the education of breast cancer epithelial cells. We show that miR-425-5p-induced MCT4 knockdown decreased lactate extrusion from CAFs and its availability in the TME. miR-425-5p overexpression induced profound metabolic transformation in CAFs, ultimately influencing breast cancer metabolism. Furthermore, miR-425-5p impaired the capacity of CAFs to sustain vessel formation and breast cancer cell migration, viability, and proliferation. These findings emphasize the key role of miR-425-5p in breast cancer metabolism and aggressiveness, and its possible importance for breast cancer therapy and monitoring.

Exosomal microRNAs synergistically trigger stromal fibroblasts in breast cancer.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype. TNBC progression is sustained by recruitment of a strong tumor microenvironment (TME) mainly composed of cancer-associated fibroblasts (CAFs) able to endorse tumor hallmarks. Increasing evidences demonstrate that exosomes mediate the crosstalk between cancer cells and the TME. We examined TNBC-derived exosomes and their microRNA (miRNA) cargo in activation of normal fibroblasts (NFs) toward CAFs. We demonstrated that TNBC cell-derived exosomes increased NF collagen contraction and migration alongside CAF molecular markers. Exosome-activated fibroblasts promoted the invasion potential of normal breast epithelial cells, as assessed by an organotypic co-culture assay that resembled the in vivo context. We also investigated TNBC cell-derived exosome cargo in activating NFs to CAFs by performing small RNA sequencing. We found that the synergistic action of miR-185-5p, miR-652-5p, and miR-1246 boosted fibroblast migration and contraction, promoting specific CAF subspecialization toward a pro-migratory functional state. These data highlight the role of breast cancer cells in re-education of the TME and their contribution to tumor evolution.

Selection of RNA aptamers targeting hypoxia in cancer.

Hypoxia plays a crucial role in tumorigenesis and drug resistance, and it is recognised as a major factor affecting patient clinical outcome. Therefore, the detection of hypoxic areas within the tumour micro-environment represents a useful way to monitor tumour growth and patients' responses to treatments, properly guiding the choice of the most suitable therapy. To date, non-invasive hypoxia imaging probes have been identified, but their applicability in vivo is strongly limited due to an inadequate resistance to the low oxygen concentration and the acidic pH of the tumour micro-environment. In this regard, nucleic acid aptamers represent very powerful tools thanks to their peculiar features, including high stability to harsh conditions and a small size, resulting in easy and efficient tumour penetration. Here, we describe a modified cell-SELEX (Systematic Evolution of Ligands by EXponential enrichment) approach that allows the isolation of specific RNA aptamers for the detection of the hypoxic phenotype in breast cancer (BC) cells. We demonstrated the effectiveness of the proposed method in isolating highly stable aptamers with an improved and specific binding to hypoxic cells. To our knowledge, this is the first example of a cell-SELEX approach properly designed and modified to select RNA aptamers against hypoxia-related epitopes expressed on tumour cell surfaces. The selected aptamers may provide new effective tools for targeting hypoxic areas within the tumour with great clinical potential.

Erratum: Exosomal microRNAs synergistically trigger stromal fibroblasts in breast cancer.

[This corrects the article DOI: 10.1016/j.omtn.2022.02.013.].

A dominant mutation etiologic for human tricho-dento-osseous syndrome impairs the ability of DLX3 to downregulate ΔNp63α.

The homeodomain transcription factors play crucial roles in many developmental processes ranging from organization of the body plan to differentiation of individual tissues. The homeodomain protein Distal-less-3 (DLX3) has an essential role in epidermal stratification and development of ectodermal appendages, placenta and bones. A four-nucleotide deletion in the human DLX3 gene is etiologic for the human hereditary tricho-dento-osseous (TDO) ectodermal dysplasia, a dominant syndrome characterized by abnormalities in hair, nails, teeth, and bones. We have previously demonstrated that DLX3 gene expression induces degradation of ΔNp63α, a specific product of the TP63 gene, a master regulator of multi-layered epithelia. Here we show that the DLX3(TDO) mutant protein is unable to promote ΔNp63α protein degradation and impairs the expression of cell cycle regulatory proteins and skin differentiation markers. However, we found that in cell expressing equal amounts of mutant and wild-type DLX3, ΔNp63α protein level is efficiently regulated implying that genetic heterozygosity at the DLX3 locus protects TDO patients from developing severe p63-associated skin defects.

Identification of a novel RNA aptamer that selectively targets breast cancer exosomes.

Breast cancer is a leading cause of cancer mortality in women. Despite advances in its management, the identification of new options for early-stage diagnosis and therapy of this tumor still represents a crucial challenge. Increasing evidence indicates that extracellular vesicles called exosomes may have great potential as early diagnostic biomarkers and regulators of many cancers, including breast cancer. Therefore, exploiting molecules able to selectively recognize them is of great interest. Here, we developed a novel differential SELEX strategy, called Exo-SELEX, to isolate nucleic acid aptamers against intact exosomes derived from primary breast cancer cells. Among the obtained sequences, we optimized a high-affinity aptamer (ex-50.T) able to specifically recognize exosomes from breast cancer cells or patient serum samples. Furthermore, we demonstrated that the ex.50.T is a functional inhibitor of exosome cellular uptake and antagonizes cancer exosome-induced cell migration in vitro. This molecule provides an innovative tool for the specific exosome detection and the development of new therapeutic approaches for breast cancer.

Urinary Dickkopf-3 and Contrast-Associated Kidney Damage.

BACKGROUND: Administration of iodinated contrast medium (CM) during invasive cardiovascular procedures may be associated with impairment of kidney function. OBJECTIVES: Urinary dickkopf-3 (DKK3), a stress-induced renal tubular epithelium-derived glycoprotein, has been identified as a biomarker predicting both acute kidney injury (AKI) and persistent kidney dysfunction. METHODS: Urinary DKK3/creatinine ratio (uDKK3/uCr), urine and serum neutrophil gelatinase-associated lipocalin (uNGAL, sNGAL) and serum cystatin C (sCyC) were assessed in 458 patients with chronic kidney disease scheduled for invasive cardiovascular procedures requiring CM administration with universal adoption of nephroprotective interventions. Contrast-associated AKI (CA-AKI) was defined as serum creatinine increase ≥0.3 mg/dl at 48 h after CM administration. Persistent kidney dysfunction was defined as persistent estimated glomerular filtration rate reduction ≥25% at 1 month compared with baseline. RESULTS: CA-AKI occurred in 64 or the 458 patients (14%), and baseline uDKK3/uCr ≥491 pg/mg was the best threshold for its prediction. Net reclassification improvement (NRI) was significantly increased by adding baseline uDKK3/uCr to the Mehran, Gurm, and National Cardiovascular Data Registry (NCDR) scores (all p < 0.05), and the same applied to integrated discrimination improvement (IDI) when adding uDKK3/uCr to the Gurm and NCDR scores (p < 0.001). Persistent kidney dysfunction occurred in 57 of the 458 patients (12%) and baseline uDKK3/uCr ≥322 pg/mg appeared as the best threshold for its prediction. Adding baseline uDKK3/uCr to the Mehran, Gurm, and NCDR scores significantly increased IDI and NRI (all p < 0.001). CONCLUSIONS: Baseline uDKK3/uCr seems to be a reliable marker for improving the identification of patients with chronic kidney disease undergoing invasive coronary and peripheral procedures at risk for AKI and persistent kidney dysfunction.

miR-34c-3p targets CDK1 a synthetic lethality partner of KRAS in non-small cell lung cancer.

Lung cancer is still the leading cause of death by cancer worldwide despite advances both in its detection and therapy. Multiple oncogenic driver alterations have been discovered, opening the prospective for new potential therapeutic targets. Among them, KRAS mutations represent the most frequent oncogene aberrations in non-small cell lung cancer (NSCLC) patients with a negative prognostic impact, but effective therapies targeting KRAS are not well characterized yet. Here, we demonstrate that the microRNA miR-34c-3p is a positive prognostic factor in KRAS-mutated NSCLC patients. Firstly, looking at the TGCA dataset, we found that high miR-34c-3p expression correlated with longer survival of KRAS-mutated NSCLC patients. In vitro assays on immortalized and patient-derived primary NSCLC cells revealed that miR-34c-3p overexpression increased apoptosis and lowered proliferation rate in KRASmut cells. Computational analysis and in vitro assays identified CDK1, one of the most promising lethal targets for KRAS-mutant cancer, as a target of miR-34c-3p. Moreover, the combination of CDK1 inhibition (mediated by RO3306) and miR-34c-3p overexpression resulted in an additive effect on the viability of KRASmut-expressing cells. Altogether, our findings demonstrate that miR-34c-3p is a novel biomarker that may allow tailored treatment for KRAS-mutated NSCLC patients.

Modulating the Crosstalk between the Tumor and the Microenvironment Using SiRNA: A Flexible Strategy for Breast Cancer Treatment.

Tumorigenesis is a complex and multistep process in which sequential mutations in oncogenes and tumor-suppressor genes result in enhanced proliferation and apoptosis escape. Over the past decades, several studies have provided evidence that tumors are more than merely a mass of malignant cancer cells, with the tumor microenvironment (TME) also contributing to cancer progression. For this reason, the focus of cancer research in recent years has shifted from the malignant cancer cell itself to the TME and its interactions. Since the TME actively participates in tumor progression, therapeutic strategies targeting it have created great interest. In this context, much attention has been paid to the potential application of small interfering RNA (siRNA), a class of non-coding RNA that has the ability to downregulate the expression of target genes in a sequence-specific way. This is paving the way for a novel therapeutic approach for the treatment of several diseases, including cancer. In this review, we describe recent efforts in developing siRNA therapeutics for the treatment of breast cancer, with particular emphasis on TME regulation. We focus on studies that adapt siRNA design to reprogram/re-educate the TME and eradicate the interplay between cancer cells and TME.

Targeting Ephrin Receptor Tyrosine Kinase A2 with a Selective Aptamer for Glioblastoma Stem Cells.

Despite the benefits associated with radiotherapy and chemotherapy for glioblastoma (GBM) treatment, most patients experience a relapse following initial therapy. Recurrent or progressive GBM usually does not respond anymore to standard therapy, and this is associated with poor patient outcome. GBM stem cells (GSCs) are a subset of cells resistant to radiotherapy and chemotherapy and play a role in tumor recurrence. The targeting of GSCs and the identification of novel markers are crucial issues in the development of innovative strategies for GBM eradication. By differential cell SELEX (systematic evolution of ligands by exponential enrichment), we have recently described two RNA aptamers, that is, the 40L sequence and its truncated form A40s, able to bind the cell surface of human GSCs. Both aptamers were selective for stem-like growing GBM cells and are rapidly internalized into target cells. In this study, we demonstrate that their binding to cells is mediated by direct recognition of the ephrin type-A receptor 2 (EphA2). Functionally, the two aptamers were able to inhibit cell growth, stemness, and migration of GSCs. Furthermore, A40s was able to cross the blood-brain barrier (BBB) and was stable in serum in in vitro experiments. These results suggest that 40L and A40s represent innovative potential therapeutic tools for GBM.

Potential and Challenges of Aptamers as Specific Carriers of Therapeutic Oligonucleotides for Precision Medicine in Cancer.

Due to the progress made in the area of precision and personalized medicine in the field of cancer therapy, strategies to selectively and specifically identify target molecules causative of the diseases are urgently needed. Efforts are being made by a number of different laboratories, companies, and researchers to develop therapeutic molecules that selectively recognize the tissues and the cells of interest, exhibit few or no off-target and side effects, are non-immunogenic, and have a strong action. Aptamers, artificially selected single-stranded DNA or RNA oligonucleotides, are promising molecules satisfying many of the requirements needed for diagnosis and precision medicine. Aptamers can also couple to their native mechanism of action the delivery of additional molecules (oligonucleotides, siRNAs, miRNAs) to target cells. In this review, we summarize recent progress in the aptamer-mediated strategy for the specific delivery of therapeutic oligonucleotides.

Highly Homogeneous Biotinylated Carbon Nanodots: Red-Emitting Nanoheaters as Theranostic Agents toward Precision Cancer Medicine.

Very recent red-emissive carbon nanodots (CDs) have shown potential as near-infrared converting tools to produce local heat useful in cancer theranostics. Besides, CDs seem very appealing for clinical applications combining hyperthermia, imaging, and drug delivery in a single platform capable of selectively targeting cancer cells. However, CDs still suffer from dramatic dot-to-dot variability issues such that a rational design of their structural, optical, and chemical characteristics for medical applications has been impossible so far. Herein, we report for the first time a simple and highly controllable layer-by-layer synthesis of biotin-decorated CDs with monodisperse size distribution, well established polymeric shell thickness, and degree of surface functionalization, endowed with strong red luminescence and the ability to convert NIR light into heat. These CDs, henceforth named CDs-PEG-BT, consist of a carbonaceous core passivated with biotin-terminated PEG2000 chains, which we demonstrate as active targeting groups to recognize cancer cells. The CDs-PEG-BT are designed to efficiently incorporate a high amount of anticancer drugs such as irinotecan (16-28%) and to act as NIR-activated nanoheaters capable of triggering local hyperthermia and massive drug release inside tumors, thus provoking sudden and efficient tumor death. The potential of the irinotecan-loaded CDs-PEG-BT (CDs-PEG-BT@IT) in fluorescence imaging was studied on 2D cultures and on complex 3D spheroids mimicking in vivo tumor architectures, showing their capability of selectively entering cancer cells through biotin receptors overexpressed in cell membranes. The efficient anticancer effect of these CDs was thoroughly assessed on multicellular 3D spheroids and patient organoids (tumor-on-a-dish preclinical models) to predict the drug response in humans in view of personalized medicine applications. CDs-PEG-BT@IT have a smart combination of properties, which pave the way to their real-world use as anticancer theranostic agents for image-guided photothermal applications.

The Discovery of RNA Aptamers that Selectively Bind Glioblastoma Stem Cells.

Glioblastoma (GBM) is the most aggressive primary brain tumor in adults. Despite progress in surgical and medical neuro-oncology, prognosis for GBM patients remains dismal, with a median survival of only 14-15 months. The modest benefit of conventional therapies is due to the presence of GBM stem cells (GSCs) that cause tumor relapse and chemoresistance and, therefore, that play a key role in GBM aggressiveness and recurrence. So far, strategies to identify and target GSCs have been unsuccessful. Thus, the development of an approach for GSC detection and targeting would be fundamental for improving the survival of GBM patients. Here, using the cell-systematic evolution of ligand by exponential (SELEX) methodology on human primary GSCs, we generated and characterized RNA aptamers that selectively bind GSCs versus undifferentiated GBM cells. We found that the shortened version of the aptamer 40L, which we have called A40s, costained with CD133-labeled cells in human GBM tissue, suggestive of an ability to specifically recognize GSCs in fixed human tissues. Of note, both 40L and A40s were rapidly internalized by cells, allowing for the delivery of the microRNA miR-34c and the anti-microRNA anti-miR-10b, demonstrating that these aptamers can serve as selective vehicles for therapeutics. In conclusion, the aptamers 40L and A40s can selectively target GSCs. Given the crucial role of GSCs in GBM recurrence and therapy resistance, these aptamers represent innovative drug delivery candidates with a great potential in the treatment of GBM.

Aptamer-miR-34c Conjugate Affects Cell Proliferation of Non-Small-Cell Lung Cancer Cells.

MicroRNAs (miRNAs) are key regulators of different human processes that represent a new promising class of cancer therapeutics or therapeutic targets. Indeed, in several tumor types, including non-small-cell lung carcinoma (NSCLC), the deregulated expression of specific miRNAs has been implicated in cell malignancy. As expression levels of the oncosuppressor miR-34c-3p are decreased in NSCLC compared to normal lung, we show that reintroduction of miR-34c-3p reduces NSCLC cell survival in vitro. Further, in order to deliver the miR-34c-based therapeutic selectively to tumor cells, we took advantage of a reported nucleic acid aptamer (GL21.T) that binds and inhibits the AXL transmembrane receptor and is rapidly internalized in the target cells. By applying methods successfully used in our laboratory, we conjugated miR-34c to the GL21.T aptamer as targeting moiety for the selective delivery to AXL-expressing NSCLC cells. We demonstrate that miR-34c-3p and the GL21.T/miR-34c chimera affect NSCLC cell proliferation and are able to overcome acquired RTK-inhibitor resistance by targeting AXL receptor. Thus, the GL21.T/miR-34c chimera exerts dual inhibition of AXL at functional and transcriptional levels and represents a novel therapeutic tool for the treatment of NSCLC.