ABSTRACT
Triple-negative breast cancer (TNBC), representing the most aggressive form of breast cancer with currently no targeted therapy available, is characterized by an inflammatory and hypoxic tumor microenvironment. To date, a broad spectrum of anti-tumor activities has been reported for phenanthroindolizidine alkaloids (PAs), however, their mode of action in TNBC remains elusive. Thus, we investigated six naturally occurring PAs extracted from the plant Tylophora ovata: O-methyltylophorinidine (1) and its five derivatives tylophorinidine (2), tylophoridicine E (3), 2-demethoxytylophorine (4), tylophoridicine D (5), and anhydrodehydrotylophorinidine (6). In comparison to natural (1) and for more-in depth studies, we also utilized a sample of synthetic O-methyltylophorinidine (1s). Our results indicate a remarkably effective blockade of nuclear factor kappa B (NFκB) within 2 h for compounds (1) and (1s) (IC50 = 17.1 ± 2.0 nM and 3.3 ± 0.2 nM) that is different from its effect on cell viability within 24 h (IC50 = 13.6 ± 0.4 nM and 4.2 ± 1 nM). Furthermore, NFκB inhibition data for the additional five analogues indicate a structure-activity relationship (SAR). Mechanistically, NFκB is significantly blocked through the stabilization of its inhibitor protein kappa B alpha (IκBα) under normoxic as well as hypoxic conditions. To better mimic the TNBC microenvironment in vitro, we established a 3D co-culture by combining the human TNBC cell line MDA-MB-231 with primary murine cancer-associated fibroblasts (CAF) and type I collagen. Compound (1) demonstrates superiority against the therapeutic gold standard paclitaxel by diminishing spheroid growth by 40% at 100 nM. The anti-proliferative effect of (1s) is distinct from paclitaxel in that it arrests the cell cycle at the G0/G1 state, thereby mediating a time-dependent delay in cell cycle progression. Furthermore, (1s) inhibited invasion of TNBC monoculture spheroids into a matrigel®-based environment at 10 nM. In conclusion, PAs serve as promising agents with presumably multiple target sites to combat inflammatory and hypoxia-driven cancer, such as TNBC, with a different mode of action than the currently applied chemotherapeutic drugs.
Subject(s)
Alkaloids , Triple Negative Breast Neoplasms , Alkaloids/pharmacology , Alkaloids/therapeutic use , Animals , Cell Line, Tumor , Cell Proliferation , Collagen Type I , Humans , Indole Alkaloids , Indolizines , Inflammation , Mice , NF-KappaB Inhibitor alpha , NF-kappa B/pharmacology , Paclitaxel/pharmacology , Phenanthrenes , Phenanthrolines , Triple Negative Breast Neoplasms/pathology , Tumor Microenvironment , TylophoraABSTRACT
Fermentation of the marine-derived fungus Aspergillus falconensis, isolated from sediment collected from the Red Sea, Egypt on solid rice medium containing 3.5% NaCl yielded a new dibenzoxepin derivative (1) and a new natural isocoumarin (2) along with six known compounds (3-8). Changes in the metabolic profile of the fungus were induced by replacing NaCl with 3.5% (NH4)2SO4 that resulted in the accumulation of three further known compounds (9-11), which were not detected when the fungus was cultivated in the presence of NaCl. The structures of the new compounds were elucidated by HRESIMS and 1D/2D NMR as well as by comparison with the literature. Molecular docking was conducted for all isolated compounds on crucial enzymes involved in the formation, progression and metastasis of cancer which included human cyclin-dependent kinase 2 (CDK-2), human DNA topoisomerase II (TOP-2) and matrix metalloproteinase 13 (MMP-13). Diorcinol (7), sulochrin (9) and monochlorosulochrin (10) displayed notable stability within the active pocket of CDK-2 with free binding energy (ΔG) equals to -25.72, -25.03 and -25.37 Kcal/mol, respectively whereas sulochrin (9) exerted the highest fitting score within MMP-13 active center (ΔG = -33.83 Kcal/mol). In vitro cytotoxic assessment using MTT assay showed that sulochrin (9) exhibited cytotoxic activity versus L5178Y mouse lymphoma cells with an IC50 value of 5.1 µM and inhibition of migration of MDA-MB 231 breast cancer cells at a concentration of 70 µM.
Subject(s)
Antineoplastic Agents/pharmacology , Aspergillus/chemistry , Polyketides/pharmacology , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/isolation & purification , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Drug Screening Assays, Antitumor , Humans , Mice , Molecular Docking Simulation , Molecular Structure , Optical Imaging , Polyketides/chemistry , Polyketides/isolation & purification , Structure-Activity RelationshipABSTRACT
Current therapies for treating pancreatic ductal adenocarcinoma (PDAC) are largely ineffective, with the desmoplastic environment established within these tumors being considered a central issue. We established a 3D spheroid co-culture in vitro model using a PDAC cell line (either PANC-1 or Capan-2), combined with stellate cells freshly isolated from pancreatic tumors (PSC) or hepatic lesions (HSC), and human type I collagen to analyze the efficiency of the chemotherapeutic gemcitabine (GEM) as well as two novel drug candidates derived from natural products: pseudopterosin (PsA-D) and O-methyltylophorinidine (TYLO). Traditional 2D in vitro testing of these agents for cytotoxicity on PANC-1 demonstrated IC50 values of 4.6 (±0.47) nM, 34.02 (±1.35) µM, and 1.99 (± 0.13) µM for Tylo, PsA-D, and GEM, respectively; these values were comparable for Capan-2: 5.58 (±1.74) nM, 33.94 (±1.02) µM, and 0.41 (±0.06) µM for Tylo, PsA-D, and GEM, respectively. Importantly, by assessing the extent of viable cells within 3D co-culture spheroids of PANC-1 with PSC or HSC, we could demonstrate a significant lack of efficacy for GEM, while TYLO remained active and PsA-D showed slightly reduced efficacy: GEM in PANC-1/PSC (IC50 = >100 µM) or PANC-1/HSC (IC50 = >100 µM) spheroids, TYLO in PANC-1/PSC (IC50 = 3.57 ± 1.30 nM) or PANC-1/HSC (IC50 = 6.39 ± 2.28 nM) spheroids, and to PsA-D in PANC-1/PSC (IC50 = 54.42 ± 12.79 µM) or PANC-1/HSC (IC50 = 51.75 ± 0.60 µM). Microscopic 3D rendering supported these cytotoxicity outcomes, showing little or no morphological spheroid structure change during this period of rapid cell death. Our results support the use of this 3D spheroid co-culture in vitro model having a desmoplastic microenvironment for the identification of possible novel chemotherapeutic drug candidates for PDAC, such as TYLO and PsA-D.
ABSTRACT
The marine-derived fungus Aspergillus falconensis, isolated from sediment collected from the Canyon at Dahab, Red Sea, yielded two new chlorinated azaphilones, falconensins O and P (1 and 2) in addition to four known azaphilone derivatives (3-6) following fermentation of the fungus on solid rice medium containing 3.5% NaCl. Replacing NaCl with 3.5% NaBr induced accumulation of three additional new azaphilones, falconensins Q-S (7-9) including two brominated derivatives (7 and 8) together with three known analogues (10-12). The structures of the new compounds were elucidated by 1D and 2D NMR spectroscopy and HRESIMS data as well as by comparison with the literature. The absolute configuration of the azaphilone derivatives was established based on single-crystal X-ray diffraction analysis of 5, comparison of NMR data and optical rotations as well as on biogenetic considerations. Compounds 1, 3-9, and 11 showed NF-κB inhibitory activity against the triple negative breast cancer cell line MDA-MB-231 with IC50 values ranging from 11.9 to 72.0 µM.
Subject(s)
Antineoplastic Agents/chemistry , Aquatic Organisms/chemistry , Aspergillus/chemistry , Benzopyrans/chemistry , Geologic Sediments/microbiology , Pigments, Biological/chemistry , Animals , Antineoplastic Agents/pharmacology , Aquatic Organisms/isolation & purification , Aspergillus/isolation & purification , Benzopyrans/pharmacology , Cell Line, Tumor/drug effects , Indian Ocean , Inhibitory Concentration 50 , Pigments, Biological/pharmacologyABSTRACT
Didymellanosine (1), the first analogue of the decahydrofluorene-class of natural products bearing a 13-membered macrocyclic alkaloid conjugated with adenosine, and a new benzolactone derivative, ascolactone C (4) along with eight known compounds (2, 3, 5-10), were isolated from a solid rice fermentation of the endophytic fungus Didymella sp. IEA-3B.1 derived from the host plant Terminalia catappa. In addition, ascochitamine (11) was obtained when (NH4)2SO4 was added to rice medium and is reported here for the first time as a natural product. Didymellanosine (1) displayed strong activity against the murine lymphoma cell line L5178Y, Burkitt's lymphoma B cells (Ramos) and adult lymphoblastic leukemia T cells (Jurkat J16), with IC50 values of 2.0, 3.3 and 4.4 µM, respectively. When subjected to a NFκB inhibition assay, didymellanosine (1) moderately blocked NFκB activation in the triple-negative breast cancer cell line MDA-MB 231. In an antimicrobial assay, ascomylactam C (3) was the most active compound when tested against a panel of Gram-positive bacteria including drug-resistant strains with MICs of 3.1-6.3 µM, while 1 revealed weaker activity. Interestingly, both compounds were also found active against Gram-negative Acinetobacter baumannii with MICs of 3.1 µM, in the presence of a sublethal concentration (0.1 µM) of colistin.