NMR Metabolomics of Arctium lappa L., Taraxacum officinale and Melissa officinalis: A Comparison of Spontaneous and Organic Ecotypes.

Officinal plants are a source of metabolites whose chemical composition depends on pedoclimatic conditions. In this study, the NMR-based approach was applied to investigate the impacts of different altitudes and agronomical practices (Land, Mountain Spontaneous, and Organically Grown Ecotypes, namely LSE, MSE, and OE, respectively) on the metabolite profiles of Burdock root, Dandelion root and aerial part, and Lemon balm aerial part. Sugars, amino acids, organic acids, polyphenols, fatty acids, and other metabolites were identified and quantified in all samples. Some metabolites turned out to be tissue-specific markers. Arginine was found in roots, whereas myo-inositol, galactose, glyceroyldigalactose moiety, pheophytin, and chlorophyll were identified in aerial parts. Caftaric and chicoric acids, 3,5 di-caffeoylquinic acid, and chlorogenic and rosmarinic acids were detected in Dandelion, Burdock and Lemon balm, respectively. The metabolite amount changed significantly according to crop, tissue type, and ecotype. All ecotypes of Burdock had the highest contents of amino acids and the lowest contents of organic acids, whereas an opposite trend was observed in Lemon balm. Dandelion parts contained high levels of carbohydrates, except for the MSE aerial part, which showed the highest content of organic acids. The results provided insights into the chemistry of officinal plants, thus supporting nutraceutical-phytopharmaceutical research.

Comprehensive metabolite and biological profile of "Sulmona Red Garlic" ecotype's aerial bulbils.

"Sulmona Red Garlic" is a well-known Italian traditional product. Bulbs, used for culinary purposes, have been largely investigated for their medicinal properties whereas aerial bulbils are usually removed as waste material. Here, for the first time, chemical composition and biological properties of the hydroalcoholic extract from aerial bulbils were investigated. Complementary information on metabolite composition were obtained using both NMR based untargeted and HPLC-DAD targeted methodologies. The NMR analysis revealed the presence of sugars, organic acids, amino acids, organosulphur compounds (methiin, alliin, allicin and cycloalliin), and other secondary metabolites. In particular, methiin and alliin were identified for the first time in the NMR spectra of aerial bulbil garlic extracts. Polyphenol content was determined by HPLC-DAD analysis: catechin, chlorogenic acid, and gallic acid turned out to be the most abundant phenolics. Hydroalcoholic extract blocked cell proliferation of colon cancer cell line HCT116 with an IC50 of 352.07 µg/mL, while it was non-toxic to myoblast cell line C2C12. In addition, it caused seedling germination reduction of two edible and herbaceous dicotyledon species, namely Cichorium intybus and C. endivia. Moreover, the same extract reduced the gene expression of TNF-α (tumor necrosis factor), HIF1-α (hypoxia-inducible factor), VEGFA (vascular endothelial growth factor), and transient receptor potential (TRP) M8 (TRPM8) indicating the ability to contrast cancer development through the angiogenic pathway. Final, in silico experiments were also carried out supporting the biological effects of organosulphur compounds, particularly alliin, which may directly interact with TRPM8. The results here reported suggest the potential use of garlic aerial bulbils often considered a waste product as a source in phytotherapeutic remedies.

Characterization of the Chemopreventive Properties of Cannabis sativa L. Inflorescences from Monoecious Cultivars Grown in Central Italy.

Hemp bioproducts hold great promise as valuable materials for nutraceutical and pharmaceutical applications due to their diverse bioactive compounds and potential health benefits. In line with this interest and in an attempt to valorize the Lazio Region crops, this present study investigated chemically characterized hydroalcoholic and organic extracts, obtained from the inflorescences of locally cultivated Felina 32, USO 31, Ferimon and Fedora 17 hemp varieties. In order to highlight the possible chemopreventive power of the tested samples, a bioactivity screening was performed, which included studying the antimutagenic activity, radical scavenging power, cytotoxicity in human hepatoma HepG2 cells, leakage of lactate dehydrogenase (LDH) and modulation of the oxidative stress parameters and glucose-6-phosphate dehydrogenase (G6PDH) involved in the regulation of the cell transformation and cancer proliferation. Tolerability studies in noncancerous H69 cholangiocytes were performed, too. The organic extracts showed moderate to strong antimutagenic activities and a marked cytotoxicity in the HepG2 cells, associated with an increased oxidative stress and LDH release, and to a G6PDH modulation. The hydroalcoholic extracts mainly exhibited radical scavenging properties with weak or null activities in the other assays. The extracts were usually well-tolerated in H69 cells, except for the highest concentrations which impaired cell viability, likely due to an increased oxidative stress. The obtained results suggest a possibility in the inflorescences from the Felina 32, USO 31, Ferimon and Fedora 17 hemp varieties as source of bioactive compounds endowed with genoprotective and chemopreventive properties that could be harnessed as preventive or adjuvant healing strategies.

Byproducts of Globe Artichoke and Cauliflower Production as a New Source of Bioactive Compounds in the Green Economy Perspective: An NMR Study.

The recovery of bioactive compounds from crop byproducts leads to a new perspective way of waste reutilization as a part of the circular economy. The present study aimed at an exhaustive metabolite profile characterization of globe artichoke and cauliflower byproducts (leaves, stalks, and florets for cauliflower only) as a prerequisite for their valorization and future implementations. The metabolite profile of aqueous and organic extracts of byproducts was analyzed using the NMR-based metabolomics approach. Free amino acids, organic acids, sugars, polyols, polyphenols, amines, glucosinolates, fatty acids, phospho- and galactolipids, sterols, and sesquiterpene lactones were identified and quantified. In particular, globe artichoke byproducts are a source of health-beneficial compounds including chiro-inositol (up to 10.1 mg/g), scyllo-inositol (up to 1.8 mg/g), sesquiterpene lactones (cynaropicrin, grosheimin, dehydrocynaropicrin, up to 45.5 mg/g in total), inulins, and chlorogenic acid (up to 7.5 mg/g), whereas cauliflower byproducts enclose bioactive sulfur-containing compounds S-methyl-L-cysteine S-oxide (methiin, up to 20.7 mg/g) and glucosinolates. A variable content of all metabolites was observed depending on the crop type (globe artichoke vs. cauliflower) and the plant part (leaves vs. stalks). The results here reported can be potentially used in different ways, including the formulation of new plant biostimulants and food supplements.

Modulatory Properties of Food and Nutraceutical Components Targeting NLRP3 Inflammasome Activation.

Inflammasomes are key intracellular multimeric proteins able to initiate the cellular inflammatory signaling pathway. NLRP3 inflammasome represents one of the main protein complexes involved in the development of inflammatory events, and its activity has been largely demonstrated to be connected with inflammatory or autoinflammatory disorders, including diabetes, gouty arthritis, liver fibrosis, Alzheimer's disease, respiratory syndromes, atherosclerosis, and cancer initiation. In recent years, it has been demonstrated how dietary intake and nutritional status represent important environmental elements that can modulate metabolic inflammation, since food matrices are an important source of several bioactive compounds. In this review, an updated status of knowledge regarding food bioactive compounds as NLRP3 inflammasome modulators is discussed. Several chemical classes, namely polyphenols, organosulfurs, terpenes, fatty acids, proteins, amino acids, saponins, sterols, polysaccharides, carotenoids, vitamins, and probiotics, have been shown to possess NLRP3 inflammasome-modulating activity through in vitro and in vivo assays, mainly demonstrating an anti-NLRP3 inflammasome activity. Plant foods are particularly rich in important bioactive compounds, each of them can have different effects on the pathway of inflammatory response, confirming the importance of the nutritional pattern (food model) as a whole rather than any single nutrient or functional compound.

NMR, RP-HPLC-PDA-ESI-MSn, and RP-HPLC-FD Characterization of Green and Oolong Teas (Camellia sinensis L.).

Untargeted (NMR) and targeted (RP-HPLC-PDA-ESI-MSn, RP-HPLC-FD) analytical methodologies were used to determine the bioactive components of 19 tea samples, characterized by different production processes (common tea and GABA tea), degrees of fermentation (green and oolong teas), and harvesting season (autumn and spring). The combination of NMR data and a multivariate statistical approach led to a statistical model able to discriminate between GABA and non-GABA teas and green and oolong teas. Targeted analyses showed that green and GABA green teas had similar polyphenol and caffeine contents, but the GABA level was higher in GABA green teas than in regular green tea samples. GABA oolong teas showed lower contents of polyphenols, caffeine, and amino acids, and a higher content of GABA, in comparison with non-GABA oolong teas. In conclusion, the results of this study suggest that the healthy properties of teas, especially GABA teas, have to be evaluated via comprehensive metabolic profiling rather than only the GABA content.

Metabolomic Profiling of Fresh Goji (Lycium barbarum L.) Berries from Two Cultivars Grown in Central Italy: A Multi-Methodological Approach.

The metabolite profile of fresh Goji berries from two cultivars, namely Big Lifeberry (BL) and Sweet Lifeberry (SL), grown in the Lazio region (Central Italy) and harvested at two different periods, August and October, corresponding at the beginning and the end of the maturation, was characterized by means of nuclear magnetic resonance (NMR) and electrospray ionization Fourier transform ion cyclotron resonance (ESI FT-ICR MS) methodologies. Several classes of compounds such as sugars, amino acids, organic acids, fatty acids, polyphenols, and terpenes were identified and quantified in hydroalcoholic and organic Bligh-Dyer extracts. Sweet Lifeberry extracts were characterized by a higher content of sucrose with respect to the Big Lifeberry ones and high levels of amino acids (glycine, betaine, proline) were observed in SL berries harvested in October. Spectrophotometric analysis of chlorophylls and total carotenoids was also carried out, showing a decrease of carotenoids during the time. These results can be useful not only to valorize local products but also to suggest the best harvesting period to obtain a product with a chemical composition suitable for specific industrial use. Finally, preliminary studies regarding both the chemical characterization of Goji leaves generally considered a waste product, and the biological activity of Big Lifeberry berries extracts was also investigated. Goji leaves showed a chemical profile rich in healthy compounds (polyphenols, flavonoids, etc.) confirming their promising use in the supplements/nutraceutical/cosmetic field. MG63 cells treated with Big Lifeberry berries extracts showed a decrease of iNOS, COX-2, IL-6, and IL-8 expression indicating their significant biological activity.

A Multimethodological Characterization of Cannabis sativa L. Inflorescences from Seven Dioecious Cultivars Grown in Italy: The Effect of Different Harvesting Stages.

The chemical profile of the female inflorescence extracts from seven Cannabis sativa L. dioecious cultivars (Carmagnola, Fibranova, Eletta Campana, Antal, Tiborszallasi, Kompolti, and Tisza) was monitored at three harvesting stages (4, 14, and 30 September), reaching from the beginning of flowering to end of flowering/beginning of seed formation, using untargeted nuclear magnetic resonance (NMR) and targeted (ultra-high-performance liquid chromatography (UHPLC) and spectrophotometry) analyses. The tetrahydrocannabinol content was always below the legal limits (<0.6%) in all the analyzed samples. The NMR metabolite profile (sugars, organic acids, amino acids, and minor compounds) subjected to principal components analysis (PCA) showed a strong variability according to the harvesting stages: samples harvested in stage I were characterized by a high content of sucrose and myo-inositol, whereas the ones harvested in stage II showed high levels of succinic acid, alanine, valine, isoleucine, phenylalanine, and threonine. Samples harvested in stage III were characterized by high levels of glucose, fructose, choline, trigonelline, malic acid, formic acid, and some amino acids. The ratio between chlorophylls and carotenoids content indicated that all plants grew up exposed to the sun, the Eletta Campana cultivar having the highest pigment amount. Tiborszallasi cultivar showed the highest polyphenol content. The highest antioxidant activity was generally observed in stage II. All these results suggested that the Cannabis sativa L. inflorescences of each analyzed dioecious hemp cultivar presented a peculiar chemical profile affected by the harvesting stage. This information could be useful for producers and industries to harvest inflorescences in the appropriate stage to obtain samples with a peculiar chemical profile suitable for proper applications.

Chemico-Biological Characterization of Torpedino Di Fondi® Tomato Fruits: A Comparison with San Marzano Cultivar at Two Ripeness Stages.

Torpedino di Fondi (TF) is a hybrid tomato landrace developed in Sicily and recently introduced in the south Lazio area along with the classical San Marzano (SM) cultivar. The present study aimed at characterizing TF tomatoes at both pink and red ripening stages, and at comparing them with traditional SM tomatoes. A multidisciplinary approach consisting of morphological, chemical (FT-ICR MS, NMR, HPLC, and spectrophotometric methods), and biological (antioxidant and antifungal in vitro activity) analyses was applied. Morphological analysis confirmed the mini-San Marzano nature and the peculiar crunchy and solid consistency of TF fruits. Pink TF tomatoes displayed the highest content of hydrophilic antioxidants, like total polyphenols (0.192 mg/g), tannins (0.013 mg/g), flavonoids (0.204 mg/g), and chlorophylls a (0.344 mg/g) and b (0.161 mg/g), whereas red TF fruits were characterized by the highest levels of fructose (3000 mg/100 g), glucose (2000 mg/100 g), tryptophan (2.7 mg/100 g), phenylalanine (13 mg/100 g), alanine (25 mg/100 g), and total tri-unsaturated fatty acids (13% mol). Red SM fruits revealed the greatest content of lipophilic antioxidants, with 1234 mg/g of total carotenoids. In agreement with phenolics content, TF cultivar showed the greatest antioxidant activity. Lastly, red TF inhibited Candida species (albicans, glabrata and krusei) growth.

1H-NMR metabolomics reveals the Glabrescione B exacerbation of glycolytic metabolism beside the cell growth inhibitory effect in glioma.

BACKGROUND: Glioma is the most common and primary brain tumors in adults. Despite the available multimodal therapies, glioma patients appear to have a poor prognosis. The Hedgehog (Hh) signaling is involved in tumorigenesis and emerged as a promising target for brain tumors. Glabrescione B (GlaB) has been recently identified as the first direct inhibitor of Gli1, the downstream effector of the pathway. METHODS: We established the overexpression of Gli1 in murine glioma cells (GL261) and GlaB effect on cell viability. We used 1H-nuclear magnetic resonance (NMR) metabolomic approach to obtain informative metabolic snapshots of GL261 cells acquired at different time points during GlaB treatment. The activation of AMP activated protein Kinase (AMPK) induced by GlaB was established by western blot. After the orthotopic GL261 cells injection in the right striatum of C57BL6 mice and the intranasal (IN) GlaB/mPEG5kDa-Cholane treatment, the tumor growth was evaluated. The High Performance Liquid Chromatography (HPLC) combined with Mass Spectrometry (MS) was used to quantify GlaB in brain extracts of treated mice. RESULTS: We found that GlaB affected the growth of murine glioma cells both in vitro and in vivo animal model. Using an untargeted 1H-NMR metabolomic approach, we found that GlaB stimulated the glycolytic metabolism in glioma, increasing lactate production. The high glycolytic rate could in part support the cytotoxic effects of GlaB, since the simultaneous blockade of lactate efflux with α-cyano-4-hydroxycinnamic acid (ACCA) affected glioma cell growth. According to the metabolomic data, we found that GlaB increased the phosphorylation of AMPK, a cellular energy sensor involved in the anabolic-to-catabolic transition. CONCLUSIONS: Our results indicate that GlaB inhibits glioma cell growth and exacerbates Warburg effect, increasing lactate production. In addition, the simultaneous blockade of Gli1 and lactate efflux amplifies the anti-tumor effect in vivo, providing new potential therapeutic strategy for this brain tumor.

Chromatographic Analyses, In Vitro Biological Activities, and Cytotoxicity of Cannabis sativa L. Essential Oil: A Multidisciplinary Study.

Due to renewed interest in the cultivation and production of Italian Cannabis sativa L., we proposed a multi-methodological approach to explore chemically and biologically both the essential oil and the aromatic water of this plant. We reported the chemical composition in terms of cannabinoid content, volatile component, phenolic and flavonoid pattern, and color characteristics. Then, we demonstrated the ethnopharmacological relevance of this plant cultivated in Italy as a source of antioxidant compounds toward a large panel of enzymes (pancreatic lipase, α-amylase, α-glucosidase, and cholinesterases) and selected clinically relevant, multidrug-sensible, and multidrug-resistant microbial strains (Staphylococcus aureus, Helicobacter pylori, Candida, and Malassezia spp.), evaluating the cytotoxic effects against normal and malignant cell lines. Preliminary in vivo cytotoxicity was also performed on Galleria mellonella larvae. The results corroborate the use of this natural product as a rich source of important biologically active molecules with particular emphasis on the role exerted by naringenin, one of the most important secondary metabolites.

Synergistic inhibition of the Hedgehog pathway by newly designed Smo and Gli antagonists bearing the isoflavone scaffold.

Aberrant activation of the Hedgehog (Hh) pathway is responsible for the onset and progression of several malignancies. Small molecules able to block the pathway at the upstream receptor Smoothened (Smo) or the downstream effector Gli1 have thus emerged recently as valuable anticancer agents. Here, we have designed, synthesized, and tested new Hh inhibitors taking advantage by the highly versatile and privileged isoflavone scaffold. The introduction of specific substitutions on the isoflavone's ring B allowed the identification of molecules targeting preferentially Smo or Gli1. Biological assays coupled with molecular modeling corroborated the design strategy, and provided new insights into the mechanism of action of these molecules. The combined administration of two different isoflavones behaving as Smo and Gli antagonists, respectively, in primary medulloblastoma (MB) cells highlighted the synergistic effects of these agents, thus paving the way to further and innovative strategies for the pharmacological inhibition of Hh signaling.

A multi-methodological approach in the study of Italian PDO "Cornetto di Pontecorvo" red sweet pepper.

A multi-methodological approach was applied to study red sweet peppers (Capsicum annuum L.) ecotype "Cornetto di Pontecorvo" grown in a greenhouse or in open field. This approach includes morphological analysis, chemical composition determination, and biological activity evaluation of different extracts from pepper fruits. Untargeted analyses, namely NMR spectroscopy and mass spectrometry, allowed the comprehensive pepper metabolite profile of pepper pulp, peel and seeds hydroalcoholic and organic extracts to be determined, showing the presence of sugars, organic acids, amino acids and other secondary metabolites. Targeted analyses, such as HPLC-PDA, HPLC-TLC and spectrophotometric analyses allowed polyphenols, tannins, flavonoids and pigments content to be determined. Samples quality and freshness were verified by the low content of biogenic amines and mycotoxins, as determined using HPLC-FLD and HPLC-MS, respectively. Preliminary biological results demonstrated the ability of the organic extracts to inhibit α-amylase, a key enzyme in the control of glucose metabolism.

A promising natural product, pristimerin, results in cytotoxicity against breast cancer stem cells in vitro and xenografts in vivo through apoptosis and an incomplete autopaghy in breast cancer.

Several natural products have been suggested as effective agents for the treatment of cancer. Given the important role of CSCs (Cancer Stem Cells) in cancer, which is a trendy hypothesis, it is worth investigating the effects of pristimerin on CSCs as well as on the other malignant cells (MCF-7 and MDA-MB-231) of breast cancer. The anti-growth activity of pristimerin against MCF-7 and MCF-7s (cancer stem cell enriched population) cells was investigated by real time viability monitorization (xCELLigence System®) and ATP assay, respectively. Mode of cell death was evaluated using electron and fluorescence microscopies, western blotting (autophagy, apoptosis and ER-stress related markers) and flow cytometry (annexin-V staining, caspase 3/7 activity, BCL-2 and PI3K expressions). Pristimerin showed an anti-growth effect on cancer cells and cancer stem cells with IC50 values ranging at 0.38-1.75µM. It inhibited sphere formation at relatively lower doses (<1.56µM). Apoptosis was induced in MCF-7 and MCF-7s cells. In addition, extensive cytoplasmic vacuolation was observed, implying an incompleted autophagy as evidenced by the increase of autophagy-related proteins (p62 and LC3-II) with an unfolded protein response (UPR). Pristimerin inhibited the growth of MCF-7 and MDA-MB-231-originated xenografts in NOD.CB17-Prkdcscid/J mice. In mice, apoptosis was further confirmed by cleavage of PARP, activation of caspase 3 and/or 7 and TUNEL staining. Taken together, pristimerin shows cytotoxic activity on breast cancer both in vitro and in vivo. It seems to represent a robust promising agent for the treatment of breast cancer. Pristimerin's itself or synthetic novel derivatives should be taken into consideration for novel potent anticancer agent(s).

Identification of a novel chalcone derivative that inhibits Notch signaling in T-cell acute lymphoblastic leukemia.

Notch signaling is considered a rational target in the therapy of several cancers, particularly those harbouring Notch gain of function mutations, including T-cell acute lymphoblastic leukemia (T-ALL). Although currently available Notch-blocking agents are showing anti-tumor activity in preclinical studies, they are not effective in all the patients and often cause severe side-effects, limiting their widespread therapeutic use. Here, by functional and biological analysis of the most representative molecules of an in house library of natural products, we have designed and synthetized the chalcone-derivative 8 possessing Notch inhibitory activity at low micro molar concentration in T-ALL cell lines. Structure-activity relationships were afforded for the chalcone scaffold. Short term treatments with compound 8 resulted in a dose-dependent decrease of Notch signaling activity, halted cell cycle progression and induced apoptosis, thus affecting leukemia cell growth. Taken together, our data indicate that 8 is a novel Notch inhibitor, candidate for further investigation and development as an additional therapeutic option against Notch-dependent cancers.

Polymeric glabrescione B nanocapsules for passive targeting of Hedgehog-dependent tumor therapy in vitro.

AIM: With the purpose of delivering high doses of glabrescione B (GlaB) to solid tumors after systemic administration, long-circulating GlaB-loaded oil-cored polymeric nanocapsules (NC-GlaB) were formulated. MATERIALS & METHODS: Synthesis of GlaB and its encapsulation in nanocapsules (NCs) was performed. Empty and GlaB-loaded NCs were assessed for their physico-chemical properties, in vitro cytotoxicity and in vivo biodistribution. RESULTS: GlaB was efficiently loaded into NCs (∽90%), which were small (∽160 nm), homogeneous and stable upon storage. Further, GlaB and NC-GlaB demonstrated specific activities against the cancer stem cells. Preliminary studies in tumor-bearing mice supported the ability of NC to accumulate in pancreatic tumors. CONCLUSION: This study provides early evidence that NC-GlaB has the potential to be utilized in a preclinical setting and justifies the need to perform therapeutic experiments in mice.

Inhibition of Hedgehog-dependent tumors and cancer stem cells by a newly identified naturally occurring chemotype.

Hedgehog (Hh) inhibitors have emerged as valid tools in the treatment of a wide range of cancers. Indeed, aberrant activation of the Hh pathway occurring either by ligand-dependent or -independent mechanisms is a key driver in tumorigenesis. The smoothened (Smo) receptor is one of the main upstream transducers of the Hh signaling and is a validated target for the development of anticancer compounds, as underlined by the FDA-approved Smo antagonist Vismodegib (GDC-0449/Erivedge) for the treatment of basal cell carcinoma. However, Smo mutations that confer constitutive activity and drug resistance have emerged during treatment with Vismodegib. For this reason, the development of new effective Hh inhibitors represents a major challenge for cancer therapy. Natural products have always represented a unique source of lead structures in drug discovery, and in recent years have been used to modulate the Hh pathway at multiple levels. Here, starting from an in house library of natural compounds and their derivatives, we discovered novel chemotypes of Hh inhibitors by mean of virtual screening against the crystallographic structure of Smo. Hh functional based assay identified the chalcone derivative 12 as the most effective Hh inhibitor within the test set. The chalcone 12 binds the Smo receptor and promotes the displacement of Bodipy-Cyclopamine in both Smo WT and drug-resistant Smo mutant. Our molecule stands as a promising Smo antagonist able to specifically impair the growth of Hh-dependent tumor cells in vitro and in vivo and medulloblastoma stem-like cells and potentially overcome the associated drug resistance.

The plant-derived triterpenoid tingenin B is a potent anticancer agent due to its cytotoxic activity on cancer stem cells of breast cancer in vitro.

Despite the rapid advances in chemotherapy regimens, the outcome of patients with breast cancer is not satisfactory. One of the reasons of this dissatisfaction is that subsets of cells in tumors which referred as cancer stem cells (CSCs) show and/or gain resistance to therapies. Thus, compounds that target CSCs are urgently needed. Since some are already used in the clinic, natural products have great potential for further development as anti cancer drugs. The aim of this study is to investigate the cytotoxic activity of tingenin b (or 22ß-hydroxytingenone) which is a quinone-methide triterpenoid structurally related to tingenone, against breast CSCs (stem-cell enriched population from MCF-7 cell line, MCF-7s). It has been found that tingenin b was cytotoxic against MCF-7s (IC50 value for 48 h was found to be 2.38 µM) by inducing apoptosis. It was evident by Annexin V staining positivity, decreased mitochondrial membrane potential and Bcl-2 dephosphorylation with a concomitant increase in Bax protein expression. In addition, endoplasmic reticulum stress was also found to be involved in tingenin b-induced cell death. In conclusion, the results warrant further studies aimed at elucidating and corroborating its possible use in the treatment of breast cancer.

Gli1/DNA interaction is a druggable target for Hedgehog-dependent tumors.

Hedgehog signaling is essential for tissue development and stemness, and its deregulation has been observed in many tumors. Aberrant activation of Hedgehog signaling is the result of genetic mutations of pathway components or other Smo-dependent or independent mechanisms, all triggering the downstream effector Gli1. For this reason, understanding the poorly elucidated mechanism of Gli1-mediated transcription allows to identify novel molecules blocking the pathway at a downstream level, representing a critical goal in tumor biology. Here, we clarify the structural requirements of the pathway effector Gli1 for binding to DNA and identify Glabrescione B as the first small molecule binding to Gli1 zinc finger and impairing Gli1 activity by interfering with its interaction with DNA. Remarkably, as a consequence of its robust inhibitory effect on Gli1 activity, Glabrescione B inhibited the growth of Hedgehog-dependent tumor cells in vitro and in vivo as well as the self-renewal ability and clonogenicity of tumor-derived stem cells. The identification of the structural requirements of Gli1/DNA interaction highlights their relevance for pharmacologic interference of Gli signaling.