ABSTRACT
Gonolobus condurango plant extract is used as an anticancer drug in some traditional systems of medicine including homeopathy, but it apparently lacks any scientific validation. Further, no detailed study is available to suggest whether condurango-glycoside-A (CGA), a major ingredient of condurango serves as a potent anticancer compound. Therefore, we investigated apoptosis-inducing ability of CGA against cervix carcinoma cells (HeLa). ß-galactosidase-activity and DNA damage were critically studied at different time points; while induced DNA-damage was observed at 9-12th hours, senescence of cells appeared at a later stage (18th hour after CGA treatment), implicating thereby a possible role of DNA damage in inducing pre-mature cell senescence. Concurrently, the number of cells undergoing apoptosis increased along with increase in reactive oxygen species (ROS) generation. Expression of p53 was also up-regulated, indicating that apoptosis could have been mediated through p53 pathway. DCHFDA (4',6-Diamidino-2-phenylindole dihydrochloride) assay, acridine orange/ethidium bromide staining and annexin V/PI assay results collectively confirmed that apoptosis was induced by increased ROS generation. Reduction in proliferation of cells was further evidenced by the cell cycle arrest at G0/G1 stage. Expression profiles of certain relevant genes and proteins like p53, Akt, Bcl-2, Bax, cytochrome c and caspase 3 also provided evidence of ROS mediated p53 up-regulation and further boost in Bax expression and followed by cytochrome c release and activation of caspase 3. Overall results suggest that CGA initiates ROS generation, promoting up-regulation of p53 expression, thus resulting in apoptosis and pre-mature senescence associated with DNA damage.
Subject(s)
Apoptosis/drug effects , Cellular Senescence/drug effects , DNA Damage , Marsdenia/chemistry , Pregnanes/pharmacology , Reactive Oxygen Species/metabolism , Tumor Suppressor Protein p53/metabolism , Apoptosis/genetics , Cell Cycle Checkpoints/drug effects , Cell Cycle Checkpoints/genetics , Cell Movement/drug effects , Cell Movement/genetics , Cell Proliferation/drug effects , Cell Survival/drug effects , Cell Survival/genetics , Cellular Senescence/genetics , G1 Phase/drug effects , G1 Phase/genetics , Gene Expression Regulation, Neoplastic/drug effects , HeLa Cells , Humans , Mass Spectrometry , Pregnanes/chemistry , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Resting Phase, Cell Cycle/drug effects , Resting Phase, Cell Cycle/genetics , Signal Transduction/drug effects , Signal Transduction/geneticsABSTRACT
The chemical structure of the main fluorescenting compound in the ethanolic extract (mother tincture) of the American yellow jasmine, Gelsemium sempervirens, was determined by employing (1)H nuclear magnetic resonance (NMR), (13)C NMR, mass spectroscopy, high-performance liquid chromatography (HPLC), correlation spectroscopy (COSY), and Fourier transform infrared (FTIR) spectroscopy analyses. Spectrofluorometric analysis has been made of the mother tincture and its agitated serial dilutions (up to 12th potency) prepared according to a homeopathic procedure in which serial, agitated dilutions were made separately in glass and polypropylene containers. The succussions were made by employing three different modes: hand jerk, sonication, and vortexing. The chemical formula of scopoletin, the main fluorescent compound, was determined to be C(10)H(8)O(4) having a molecular weight of 192.17. Significant differences were noted between the remedies prepared in the two types of containers. Further, a comparison between any two methods of agitation revealed significant differences in fluorometric data of remedies at certain potency levels. The biological (anticancer) action of the crude extract, the alkaloid scopoletin, and 2C potency of Gelsemium sp were tested in vitro on the HeLa cell line through fluorescence microscopy, the 3(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, and fluorescent activated cell sorting (FACS). The role of nanoparticles presumably derived from the containers, their orientation, and their interaction with the starting substance during the dynamization process initiated by different modes of agitation could possibly be attributed to the differences noted in the fluorometric data of potencies prepared in the two types of containers and among the three different means of succussion tested.
Subject(s)
Alkaloids/chemistry , Antineoplastic Agents/pharmacology , Gelsemium/chemistry , Plant Extracts/pharmacology , Alkaloids/analysis , Alkaloids/isolation & purification , Apoptosis/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Formazans/metabolism , G1 Phase/drug effects , HeLa Cells , Humans , Molecular Structure , Molecular Weight , Tetrazolium Salts/metabolismABSTRACT
OBJECTIVE: To investigate the proliferation inhibition and the differentiation effects of realgar (As4S4) nano-particles on human acute myeloid leukemia cell line HL-60. METHOD: Cell viability was determined by MTT and PI-stained cell cycle assays. The realgar induced morphological changes on cells were examined after Wright-Giemsa staining. The cell differentiation was evaluated with NBT and specific cell surface antigen (CD11b and CD14) expression assays. RESULT: HL-60 cells exhibited obvious morphological features of differentiation after the realgar treatment. A 24 h incubation of the cells with 0.25-1.0 micromol x L(-1) realgar caused a great increase in NBT reduction ability. The expressions of CD11b and CD14 were augmented in cells treated with 0.50 micromol x L(-1) realgar for 48 h, and cell cycles were arrested in G1 phase. CONCLUSION: Low dose realgar induces differentiation in human acute myeloid leukemia cell line HL-60.