Human Multi-Lineage Liver Organoid Model Reveals Impairment of CYP3A4 Expression upon Repeated Exposure to Graphene Oxide.

Three-dimensional hepatic cell cultures can provide an important advancement in the toxicity assessment of nanomaterials with respect to 2D models. Here, we describe liver organoids (LOs) obtained by assembling multiple cell lineages in a fixed ratio 1:1:0.2. These are upcyte® human hepatocytes, UHHs, upcyte® liver sinusoidal endothelial cells, LSECs, and human bone marrow-derived mesenchymal stromal cells, hbmMSCs. The structural and functional analyses indicated that LOs reached size stability upon ca. 10 days of cultivation (organoid maturation), showing a surface area of approximately 10 mm2 and the hepatic cellular lineages, UHHs and LSECs, arranged to form both primitive biliary networks and sinusoid structures, alike in vivo. LOs did not show signs of cellular apoptosis, senescence, or alteration of hepatocellular functions (e.g., dis-regulation of CYP3A4 or aberrant production of Albumin) for the entire culture period (19 days since organoid maturation). After that, LOs were repeatedly exposed for 19 days to a single or repeated dose of graphene oxide (GO: 2-40 µg/mL). We observed that the treatment did not induce any macroscopic signs of tissue damage, apoptosis activation, and alteration of cell viability. However, in the repeated dose regimen, we observed a down-regulation of CYP3A4 gene expression. Notably, these findings are in line with recent in vivo data, which report a similar impact on CYP3A4 when mice were repeatedly exposed to GO. Taken together, these findings warn of the potential detrimental effects of GO in real-life exposure (e.g., occupational scenario), where its progressive accumulation is likely expected. More in general, this study highlights that LOs formed by many cell lineages can enable repeated exposure regimens (suitable to mimic accumulation); thus, they can be suitably considered alternative or complementary in vitro systems to animal models.

Small molecule approaches to targeting RNA.

The development of innovative methodologies to identify RNA binders has attracted enormous attention in chemical biology and drug discovery. Although antibiotics targeting bacterial ribosomal RNA have been on the market for decades, the renewed interest in RNA targeting reflects the need to better understand complex intracellular processes involving RNA. In this context, small molecules are privileged tools used to explore the biological functions of RNA and to validate RNAs as therapeutic targets, and they eventually are to become new drugs. Despite recent progress, the rational design of specific RNA binders requires a better understanding of the interactions which occur with the RNA target to reach the desired biological response. In this Review, we discuss the challenges to approaching this underexplored chemical space, together with recent strategies to bind, interact and affect biologically relevant RNAs.

Identification of a Dual Autophagy and REV-ERB Inhibitor with in Vivo Anticancer Efficacy.

The autophagy process appears as a promising target for anticancer interventions. Chloroquine (CQ) and its derivative hydroxychloroquine (HCQ) are the only FDA-approved autophagy flux inhibitors. Although diverse anticancer clinical trials are providing encouraging results, several limitations associated with the need of high dosage and long-term administration of these autophagy inhibitors are also emerging. We showed that the inhibition of REV-ERB, a nuclear receptor regulating circadian rhythm and metabolism, enhances CQ-mediated cancer cell death and identified a class of dual inhibitors of autophagy and REV-ERB displaying an in vitro anticancer activity against diverse tumor cells greatly higher than CQ. Herein, we describe our lead optimization strategy that led to the identification of compound 24 as a dual autophagy and REV-ERB inhibitor, showing improved potency in blocking autophagy, enhanced toxicity against cancer cells, optimal drug-like properties, and efficacy in a mouse xenograft model of melanoma as a single anticancer agent.

Autophagy and cancer drug resistance in dialogue: Pre-clinical and clinical evidence.

The emergence of drug resistance is a major challenge for oncologists. Resistance can be categorized as acquired or intrinsic; the alteration of several biological mechanisms contributes to both intrinsic and acquired resistance. Macroautophagy/autophagy is the primary process in eukaryotes for the degradation of macromolecules and organelles. This process is critical in maintaining cellular homeostasis. Given its function as either a pro-survival or a pro-death phenomenon, autophagy has a complex physio-pathological role. In some circumstances, autophagy can confer chemoresistance and promote cell survival, whereas in others it can promote chemosensitivity and contribute to cell death. The role of autophagy in the modulation of cancer drug resistance reflects its impact on apoptosis and metastasis. The regulation of autophagy in cancer is mediated by various factors including AMP-activated protein kinase (AMPK), MAPK, phosphoinositide 3-kinase (PI3K)-AKT, BECN1 and ATG proteins. Non-coding RNAs are among the main regulators of autophagy, e.g., via the modulation of chemoresistance pathways. Due to the significant contribution of autophagy in cancer drug resistance, small molecule modulators and natural compounds targeting autophagy have been introduced to alter the response of cancer cells to chemotherapy. Furthermore, nanotherapeutic approaches based on autophagy regulation have been introduced in pre-clinical cancer therapy. In this review we consider the potential for using autophagy regulators for the clinical treatment of malignancies.

MYC-Associated Factor MAX is a Regulator of the Circadian Clock.

The circadian transcriptional network is based on a competition between transcriptional activator and repressor complexes regulating the rhythmic expression of clock-controlled genes. We show here that the MYC-associated factor X, MAX, plays a repressive role in this network and operates through a MYC-independent binding to E-box-containing regulatory regions within the promoters of circadian BMAL1 targets. We further show that this "clock" function of MAX is required for maintaining a proper circadian rhythm and that MAX and BMAL1 contribute to two temporally alternating transcriptional complexes on clock-regulated promoters. We also identified MAX network transcriptional repressor, MNT, as a fundamental partner of MAX-mediated circadian regulation. Collectively, our data indicate that MAX regulates clock gene expression and contributes to keeping the balance between positive and negative elements of the molecular clock machinery.

Disulfide-Mediated Bioconjugation: Disulfide Formation and Restructuring on the Surface of Nanomanufactured (Microfluidics) Nanoparticles.

This study is about (1) nanomanufacturing (focusing on microfluidic-assisted nanoprecipitation), (2) advanced colloid characterization (focusing on field flow fractionation), and (3) the possible restructuring of surface disulfides. Disulfides are dynamic and exchangeable groups, and here we specifically focus, first, on their use to introduce biofunctional groups and, second, on their re-organization, which may lead to variable surface chemistries and uncontrolled cell interactions. The particles were obtained via microfluidic-assisted (flow-focused) nanoprecipitation of poly(ethylene glycol)-b-poly(ε-caprolactone) bearing or not a 2-pyridyl disulfide (PDS) terminal group, which quantitatively exchanges with thiols in solution. In this study, we have paid specific attention to size characterization, thereby also demonstrating the limitations of dynamic light scattering (DLS) as a stand-alone technique. By using asymmetric flow field flow fractionation coupled with DLS, static light scattering (SLS), and refractive index detectors, we show that relatively small amounts of >100 nm aggregates (cryogenic transmission electron microscopy and SLS/DLS comparison suggesting them to be wormlike micelles) dominated the stand-alone DLS results, whereas the "real" size distributions picked <50 nm. Our key result is that the kinetics of the conjugation based on PDS-thiol exchange was controlled by the thiol pKa, and this also determined the rate of the exchange between the resulting disulfides and glutathione (GSH). In particular, more acidic thiols (e.g., peptides, where a cysteine is flanked by cationic residues) react faster with PDS, but their disulfides hardly exchange with GSH; the reverse applies to thiols with a higher pKa. Disulfides that resist against restructuring via thiol-disulfide exchange allow for a stable bioconjugation, although they may be bad news for payload release under reducing conditions. However, experiments of both thiol release and nanoparticles uptake in cells (HCT116) show that also the disulfides formed from less-acidic and, therefore, less-reactive, and more exchangeable thiols were stable for at least a few hours even in a GSH-rich (10 mM) environment; this suggests a sufficiently long stability of surface groups to achieve, for example, a cell-targeting effect.

A broad-spectrum antibiotic, DCAP, reduces uropathogenic Escherichia coli infection and enhances vorinostat anticancer activity by modulating autophagy.

The cellular recycling pathway of autophagy plays a fundamental role in adaptive responses to nutrient deprivation and other forms of stress under physiological and pathological conditions. However, autophagy can also be a double-edge sword during certain bacterial infections (such as urinary tract infections) and in cancer, where it can be hijacked by the pathogens and cancer cells, respectively, to promote their own survival. Thus, autophagy modulation can potentially have multiple effects in multiple contexts and this property can be leveraged to improve outcomes. In this report, we identify that a broad-spectrum antibiotic, 2-((3-(3, 6-dichloro-9H-carbazol-9-yl)-2-hydroxypropyl) amino)-2-(hydroxymethyl) propane-1, 3-diol (DCAP) modulates autophagy. We employed combined biochemical, fluorescence microscopy and correlative light electron microscopy approaches to demonstrate that DCAP treatment blocks autophagy at the late stages by preventing autophagolysosome maturation and interrupting the autophagic flux. We further show that, DCAP significantly reduces UPEC infection in urinary tract epithelial cells via inhibition of autophagy. Finally, we reveal that DCAP enhances the anticancer activity of the histone acetyltransferase (HDAC) inhibitor, vorinostat, which has been reported to increase susceptibility to bacterial infections as a common adverse effect. Collectively, our data support the concept that DCAP represents a valuable chemical scaffold for the development of an innovative class of bactericidal autophagy inhibitors for treatment of urinary tract infections and/or for adjuvant therapy in cancer treatment.

Autophagy is essential for maintaining the growth of a human (mini-)organ: Evidence from scalp hair follicle organ culture.

Autophagy plays a crucial role in health and disease, regulating central cellular processes such as adaptive stress responses, differentiation, tissue development, and homeostasis. However, the role of autophagy in human physiology is poorly understood, highlighting a need for a model human organ system to assess the efficacy and safety of strategies to therapeutically modulate autophagy. As a complete, cyclically remodelled (mini-)organ, the organ culture of human scalp hair follicles (HFs), which, after massive growth (anagen), spontaneously enter into an apoptosis-driven organ involution (catagen) process, may provide such a model. Here, we reveal that in anagen, hair matrix keratinocytes (MKs) of organ-cultured HFs exhibit an active autophagic flux, as documented by evaluation of endogenous lipidated Light Chain 3B (LC3B) and sequestosome 1 (SQSTM1/p62) proteins and the ultrastructural visualization of autophagosomes at all stages of the autophagy process. This autophagic flux is altered during catagen, and genetic inhibition of autophagy promotes catagen development. Conversely, an anti-hair loss product markedly enhances intrafollicular autophagy, leading to anagen prolongation. Collectively, our data reveal a novel role of autophagy in human hair growth. Moreover, we show that organ-cultured scalp HFs are an excellent preclinical research model for exploring the role of autophagy in human tissue physiology and for evaluating the efficacy and tissue toxicity of candidate autophagy-modulatory agents in a living human (mini-)organ.

Circadian clock: Time for novel anticancer strategies?

Disruption of the circadian clock is associated with a variety of human pathologies, including cancer. Rather than being a mere consequence of a global changes associated with the cancer cell transcriptome, the aberrant clock gene expression observed in many tumors may serve for cancer cell survival. This scenario suggests the provocative hypothesis that pharmacological modulation of clock-related proteins may be suitable as an effective anticancer strategy. In this review, we focus on the functions of the druggable circadian nuclear receptors, REV-ERBα and REV-ERBß, in cancer cell survival and describe the potential development of small molecule compounds that modulate REV-ERB activity as novel anticancer therapeutics. In addition, we debate the use of circadian rhythm-based synthetic lethal approaches to identify yet unexplored anticancer strategies.

Synthesis and in Vitro Anticancer Activity of the First Class of Dual Inhibitors of REV-ERBß and Autophagy.

Autophagy inhibition is emerging as a promising anticancer strategy. We recently reported that the circadian nuclear receptor REV-ERBß plays an unexpected role in sustaining cancer cell survival when the autophagy flux is compromised. We also identified 4-[[[1-(2-fluorophenyl)cyclopentyl]amino]methyl]-2-[(4-methylpiperazin-1-yl)methyl]phenol, 1 (ARN5187), as a novel dual inhibitor of REV-ERBß and autophagy. 1 had improved cytotoxicity against BT-474 breast cancer cells compared to chloroquine, a clinically relevant autophagy inhibitor. Here, we present the results of structure-activity studies, based around 1, that disclose the first class of dual inhibitors of REV-ERBß and autophagy. This study led to identification of 18 and 28, which were more effective REV-ERBß antagonists than 1 and were more cytotoxic to BT-474. The combination of optimal chemical and structural moieties of these analogs generated 30, which elicited 15-fold greater REV-ERBß inhibitory and cytotoxic activities compared to 1. Furthermore, 30 induced death in a panel of tumor cell lines at doses 5-50 times lower than an equitoxic amount of chloroquine but did not affect the viability of normal mammary epithelial cells.