ABSTRACT
Nature has provided us with a wide spectrum of disease healing phytochemicals like Artonin E, obtained from the root bark of Artocarpus elasticus. This molecule had been predicted to be drug-like, possessing unique medicinal properties. Despite strides made in chemotherapy, prognosis of the heterogenous aggressive triple negative breast cancer is still poor. This study was conducted to investigate the mechanism of inhibition of Artonin E, a prenylated flavonoid on MDA-MB 231 triple negative breast cancer cell, with a view of mitigating the hallmarks displayed by these tumors. The anti-proliferative effect, mode of cell death and the mechanism of apoptosis induction were investigated. Artonin E, was seen to effectively relinquish MDA-MB 231 breast cancer cells of their apoptosis evading capacity, causing a half-maximal growth inhibition at low concentrations (14.3, 13.9 and 9.8 µM) after the tested time points (24, 48 and 72 hours), respectively. The mode of cell death was observed to be apoptosis with defined characteristics. Artonin E was seen to induce the activation of both extrinsic and intrinsic caspases initiators of apoptosis. It also enhanced the release of total reactive oxygen species which polarized the mitochondrial membrane, compounding the release of cytochrome c. Gene expression studies revealed the upregulation of TNF-related apoptosis inducing ligand and proapoptotic genes with down regulation of anti-apoptotic genes and proteins. A G2/M cell cycle arrest was also observed and was attributed to the observed upregulation of p21 independent of the p53 status. Interestingly, livin, a new member of the inhibitors of apoptosis was confirmed to be significantly repressed. In all, Artonin E showed the potential as a promising candidate to combat the aggressive triple negative breast cancer.
Subject(s)
Antineoplastic Agents, Phytogenic/toxicity , Apoptosis/drug effects , Flavonoids/toxicity , Artocarpus/chemistry , Artocarpus/metabolism , Caspases/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Cyclin-Dependent Kinase Inhibitor p21/metabolism , DNA Fragmentation/drug effects , Female , G2 Phase Cell Cycle Checkpoints/drug effects , Humans , M Phase Cell Cycle Checkpoints/drug effects , Microscopy, Fluorescence , Plant Roots/chemistry , Plant Roots/metabolism , Reactive Oxygen Species/metabolism , Triple Negative Breast Neoplasms/metabolism , Triple Negative Breast Neoplasms/pathology , Tumor Suppressor Protein p53/metabolism , Up-Regulation/drug effectsABSTRACT
Artonin E is a prenylated flavonoid isolated from the stem bark of Artocarpus elasticus Reinw.(Moraceae). This study aimed to investigate the apoptotic mechanisms induced by artonin E in a metastatic human ovarian cancer cell line SKOV-3 in vitro. MTT assay, clonogenic assay, acridine orange and propidium iodide double staining, cell cycle and annexin V analyses were performed to explore the mode of artonin E-induced cell death at different time points. DNA laddering, activation of caspases-3, -8, and -9, multi-parametric cytotoxicity-3 analysis by high-content screening, measurement of reactive oxygen species generation, and Western blot were employed to study the pathways involved in the apoptosis. MTT results showed that artonin E inhibited the growth of SKOV-3 cells, with IC50 values of 6.5±0.5 µg/mL after 72 h treatment, and showed less toxicity toward a normal human ovarian cell line T1074, with IC50 value of 32.5±0.5 µg/mL. Results showed that artonin E induced apoptosis and cell cycle arrest at the S phase. This compound also promoted the activation of caspases-3, -8, and -9. Further investigation into the depletion of mitochondrial membrane potential and release of cytochrome c revealed that artonin E treatment induced apoptosis via regulation of the expression of pro-survival and pro-apoptotic Bcl-2 family members. The expression levels of survivin and HSP70 proteins were also down regulated in SKOV-3 cells treated with artonin E. We propose that artonin E induced an antiproliferative effect that led to S phase cell cycle arrest and apoptosis through dysregulation of mitochondrial pathways, particularly the pro- and anti-apoptosis signaling pathways.
Subject(s)
Apoptosis/drug effects , Cell Proliferation/drug effects , Flavonoids/pharmacology , Mitochondria/drug effects , Apoptosis/genetics , Artocarpus/chemistry , Blotting, Western , Caspase 3/metabolism , Caspase 8/metabolism , Caspase 9/metabolism , Cell Line, Tumor , Cell Proliferation/genetics , Cell Survival/drug effects , Cell Survival/genetics , Cytochromes c/metabolism , DNA Fragmentation/drug effects , Enzyme Activation/drug effects , Female , Flavonoids/chemistry , Flow Cytometry , Humans , Inhibitory Concentration 50 , Mitochondria/metabolism , Molecular Structure , Ovarian Neoplasms/genetics , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Proto-Oncogene Proteins c-bcl-2/metabolism , Reactive Oxygen Species/metabolism , S Phase Cell Cycle Checkpoints/drug effects , S Phase Cell Cycle Checkpoints/geneticsABSTRACT
Detailed phytochemical investigation has been carried out on the bark of Artocarpus elasticus Reinw. ex Blume, which led to the isolation of artonin E (1), a new dihydrobenzoxanthone derivative named elastixanthone (2), cycloartobiloxanthone (3) and artobiloxanthone (4). Structures of these compounds were elucidated on the basis of various spectroscopic (UV, IR, ID-NMR and 2D-NMR) and MS data. Compounds 1-3 displayed outstanding scavenging activity for 1,1-diphenylpicrylhydrazyl (DPPH) with IC5o values of 11.5, 21.6 and 40.0 µg/mL, respectively. In addition, compounds 1-3 displayed broad spectrum antimicrobial activities against thirteen different bacterial strains when tested using the disc diffusion assay. Cytotoxic screening revealed that artonin E (1). constantly exhibited strong cytotoxic activity against human estrogen receptor (ER+) positive breast cancer (MCF-7) and human estrogen receptor (ER-) negative (MDA-MB 231) cells in comparison with the other two, with IC50 values of 2.6 and 13.5 µg/mL, respectively, without being toxic towards the WRL68 (human normal liver) cell line (IC50 value more than 30 [µg/mL). However, the compound was inactive against HepG2 (human liver carcinoma) cancer cells.
Subject(s)
Antineoplastic Agents, Phytogenic/chemistry , Antineoplastic Agents, Phytogenic/pharmacology , Artocarpus/chemistry , Phytochemicals/pharmacology , Molecular Structure , Phytochemicals/chemistryABSTRACT
Staphylococci are facultative anaerobes, perfectly spherical un-encapsulated cocci, with a diameter not exceeding 1 micrometer in diameter. Staphylococcus aureus are generally harmless and remain confined to the skin unless they burrow deep into the body, causing life-threatening infections in bones, joints, bloodstream, heart valves and lungs. Among the 20 medically important staphylococci species, Staphylococcus aureus is one of the emerging human pathogens. Streptomycin had its highest potency against Staphylococcus infections despite the likelihood of getting a resistant type of staphylococcus strains. Methicillin-resistant S. aureus (MRSA) is the persister type of Staphylococcus aureus and was evolved after decades of antibiotic misuse. Inadequate penetration of the antibiotic is one of the principal factors related to success/failure of the therapy. The active drug needs to reach the bacteria at concentrations necessary to kill or suppress the pathogen's growth. In turn the effectiveness of the treatment relied on the physical properties of Staphylococcus aureus. Thus understanding the cell integrity, shape and roughness is crucial to the overall influence of the therapeutic agent on S. aureus of different origins. Hence our experiments were designed to clarify ultrastructural changes of S. aureus treated with streptomycin (synthetic compound) in comparison to artonin E (natural compound). In addition to the standard in vitro microbial techniques, we used transmission electron microscopy to study the disrupted cell architecture under antibacterial regimen and we correlate this with scanning electron microscopy (SEM) to compare results of both techniques.
