ABSTRACT
In the present study, we investigated the anti-cancer effect of various potencies of Ruta graveolens (Ruta) on COLO-205 cell line, as evidenced by cytotoxicity, migration, clonogenecity, morphological and biochemical changes and modification in the levels of genes associated with apoptosis and cell cycle. On treatment of COLO-205 cells maximal effects were seen with mother tincture (MT) and 30C potencies, wherein decrease in cell viability along with reduced clonogenecity and migration capabilities were noted. In addition morphological and biochemical alterations such as nuclear changes (fragmented nuclei with condensed chromatin) and DNA ladder-like pattern (increased amount of fragmented DNA) in COLO-205 cells indicating apoptotic related cell death were seen. The expression of apoptosis and cell-cycle related regulatory genes assessed by reverse transcriptase-PCR revealed an up-regulation of caspase 9, caspase-3, Bax, p21 and p27 expression and down-regulation of Bcl-2 expression in treated cells. The mode of cell death was suggestive of intrinsic apoptotic pathway along with cell cycle arrest at the G2/M of the cell cycle. Our findings indicate that phytochemicals present in Ruta showed potential for natural therapeutic product development for colon carcinoma.
Subject(s)
Apoptosis/drug effects , Cell Cycle Checkpoints/drug effects , Colonic Neoplasms/drug therapy , DNA Fragmentation/drug effects , Ruta , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Colonic Neoplasms/pathology , Flow Cytometry , HumansABSTRACT
BACKGROUND: Impaired fracture healing is a recurring interdisciplinary medical challenge. Alternative treatment concepts, apart from conventional medicine, are popular, but scientific evidence on their effects is still lacking. Plant-derived substances are widely assumed to support bone homeostasis. To clarify the effects on bone healing mechanisms, a commercially available, homeopathic-spagyric remedy, containing inter alia two herbal substances with assumed osteogenic potential, equisetum arvense and bellis perennis, was analyzed. METHODS: Human fetal osteoblastic (hFOB) 1.19 cells were incubated with the test substance in serial dilutions from 10 to 0.00001%. Cell viability has been evaluated through ATP level (CTG assay) and MTT tetrazolium reduction. Cell proliferation was analyzed by BrdU incorporation and cell migration by wound healing assay (WHA) via image analysis. Additionally, determination of the expression of key genes via real-time PCR and proteins via proteome array for inflammation, cell proliferation, and angiogenesis were performed. RESULTS: An incubation of hFOB 1.19 cells with the test substance for 24/72 h showed no reduction in cell number, viability, or proliferation. Cell migration was unimpaired. The test substance induced inflammatory genes and growth factors along with genes of osseous regeneration (ALP, Col1, IL-1α, IL-6, IL-8, IL-10, Osteocalcin, Osteonectin, RUMX2, TGF, VEGFA). Increased protein expression was found in multiple cytokines, chemokines, and acute phase proteins. CONCLUSION: The test substance did not impair cell vitality parameters (MTT, CTG, BrdU, and WHA). A tendency to activate growth factors, bone regeneration genes, and proteins was shown for osteoblasts, indicating a possible positive effect on osteogenic processes.
Subject(s)
Cell Proliferation , Cell Survival , Osteoblasts , Plant Extracts , Humans , Osteoblasts/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Plant Extracts/pharmacology , Cell Movement/drug effects , Cell Line , Osteogenesis/drug effects , PhytotherapyABSTRACT
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
CONTEXT: For 2000 years, traditional Chinese medicine has been used as a remedy for general health improvement, including the fight against aging. Pearl powder has recently been used as a health food that has antioxidant, antiaging, antiradioactive, and tonic activities for cells; it is also applied to cure aphthous ulcer, gastric ulcer, and duodenal ulcer on clinical therapy. In addition, the mother of pearl, nacre, could enhance the cell adhesion and tissue regeneration of skin fibroblasts. OBJECTIVE: Fibroblast is regarded as indispensable in the processes of wound healing. Therefore, the effect of pearl extract (PL) on fibroblasts is investigated in this study. MATERIALS AND METHODS: PL is produced by a room temperature super extraction system (Taiwan patent no. I271 220). DMEM medium containing PL (300 µg/mL) was used to examine the effect of migration-promoting potential on human fibroblast cell line or human primary fibroblast cells in a wound healing model in vitro. RESULTS: Medium containing PL (300 µg/mL) demonstrated that the migratory cell numbers of fibroblasts were three times more than that without PL, and mRNA expression of collagen type III was higher than in collagen type I in fibroblasts. It revealed a migration-promoting potential of human fibroblasts in a wound healing model in vitro. DISCUSSION AND CONCLUSION: The present study found that the migration-promoting effect in PL, which could be a supplement in cell culture. These data suggest PL could be useful for enhancing the wound healing of fibroblasts.
Subject(s)
Cell Movement/drug effects , Materia Medica/pharmacology , Medicine, Chinese Traditional , Skin/drug effects , Up-Regulation/drug effects , Wound Healing/drug effects , Animals , Cell Line , Cells, Cultured , Collagen Type III/genetics , Collagen Type III/metabolism , Dermatologic Agents/isolation & purification , Dermatologic Agents/pharmacology , Foreskin/cytology , Humans , Male , Materia Medica/isolation & purification , RNA, Messenger/metabolism , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Skin/cytology , Skin/metabolism , Unionidae/metabolismABSTRACT
BACKGROUND: Drugs of plant origin such as Arnica montana, Calendula officinalis or Hypericum perforatum have been frequently used to promote wound healing. While their effect on wound healing using preparations at pharmacological concentrations was supported by several in vitro and clinical studies, investigations of herbal homeopathic remedies on wound healing process are rare. The objective of this study was to investigate the effect of a commercial low potency homeopathic remedy Similasan® Arnica plus Spray on wound closure in a controlled, blind trial in vitro. METHODS: We investigated the effect of an ethanolic preparation composed of equal parts of Arnica montana 4x, Calendula officinalis 4x, Hypericum perforatum 4x and Symphytum officinale 6x (0712-2), its succussed hydroalcoholic solvent (0712-1) and unsuccussed solvent (0712-3) on NIH 3T3 fibroblasts. Cell viability was determined by WST-1 assay, cell growth using BrdU uptake, cell migration by chemotaxis assay and wound closure by CytoSelect ™Wound Healing Assay Kit which generated a defined "wound field". All assays were performed in three independent controlled experiments. RESULTS: None of the three substances affected cell viability and none showed a stimulating effect on cell proliferation. Preparation (0712-2) exerted a stimulating effect on fibroblast migration (31.9%) vs 14.7% with succussed solvent (0712-1) at 1:100 dilutions (p < 0.001). Unsuccussed solvent (0712-3) had no influence on cell migration (6.3%; p > 0.05). Preparation (0712-2) at a dilution of 1:100 promoted in vitro wound closure by 59.5% and differed significantly (p < 0.001) from succussed solvent (0712-1), which caused 22.1% wound closure. CONCLUSION: Results of this study showed that the low potency homeopathic remedy (0712-2) exerted in vitro wound closure potential in NIH 3T3 fibroblasts. This effect resulted from stimulation of fibroblasts motility rather than of their mitosis.
Subject(s)
Arnica/chemistry , Calendula/chemistry , Comfrey/chemistry , Fibroblasts/drug effects , Hypericum/chemistry , Materia Medica/pharmacology , Plant Extracts/pharmacology , Wound Healing/drug effects , Animals , Cell Movement/drug effects , Cell Proliferation/drug effects , Fibroblasts/physiology , Mice , NIH 3T3 CellsABSTRACT
Kangfuxin (KFX) is the ethanol extract of Periplaneta Americana L., which has been widely used in Traditional Chinese Medicine for the treatment of injury in clinic with a long history. However, the biological influence of KFX in the different wound stages was not investigated comprehensively yet. This study aims to investigate the influence of KFX in the various wound healing activities with cellular and animal models, including the influence of KFX in 1) proliferation and cells cycle of kerationcytes and fibroblasts; 2) migration and chemotaxis of these skin cells; 3) secretion of EGF and VEGF; 4) the healing rate; 5) synthesis and deposition of different types of collagen; 6) as well as the pro-angiogenesis effect. KFX was shown to/for 1) promote the kerationcytes proliferation and regulate the cells cycle of skin fibroblasts significantly; 2) obviously stimulate the migration of kerationcytes and chemotaxis of fibroblasts; 3) the trend to promote EGF and VEGF secretion both in vitro & in vivo; 4) accelerate the wound closure, collagen synthesis and angiogenesis. KFX was demonstrated to accelerate wound healing and improve the healing quality by multiple regulation. Results of this study provide the comprehensive evidence for the application of KFX as a novel therapeutics for wound treatment.
Subject(s)
Biological Products/pharmacology , Periplaneta/chemistry , Regeneration/drug effects , Wound Healing/drug effects , Animals , Cell Cycle/drug effects , Cell Movement/drug effects , Cell Proliferation/drug effects , Collagen/metabolism , Epidermal Growth Factor/metabolism , Fibroblasts/drug effects , Fibroblasts/metabolism , Humans , Keratinocytes/drug effects , Keratinocytes/metabolism , Materia Medica/pharmacology , Skin/drug effects , Skin/metabolism , Vascular Endothelial Growth Factor A/metabolismABSTRACT
A systematic study was undertaken of luminescent aqueous solutions of homeopathic preparation of sodium chloride at a dilution from D1 to D30, produced by "Weleda" company (Moscow) was carried out. It was shown that intensity of luminescence versus the degree of dilution is a non-monotonous function with several maxima, the main maximum corresponds to 13-14 decimal dilution. The dynamics of spectra was registered for several weeks. A systematic study of water samples (D1-D30) exposed to a similar procedure of potentization but without salt addition was also performed. The difference in the luminescence spectra of water of different stages of potentization was shown. The motility of infusoria Spirostoma ambiquum in solutions being examined was studied. A significant negative correlation between the infusoria motility and luminescence intensity was registered.
Subject(s)
Cell Movement/drug effects , Ciliophora/physiology , Sodium Chloride/pharmacology , Animals , Homeopathy , Luminescent Measurements , Materia Medica/pharmacology , Sodium Chloride/chemistry , SolutionsABSTRACT
BACKGROUND: Several homeopathic remedies are applied in the treatment of periodontal inflammation. However, little is known about their basic active principles. Therefore, we aimed at investigating the effects of homeopathic drugs in periodontal inflammation by observing lymphocyte migration activity in vitro. MATERIAL AND METHODS: Lymphocytes from blood samples of 3 periodontitis patients and 3 matched healthy volunteers were extracted and embedded in collagen matrix migration assays together with highly diluted (D12 and C200) aqueous extracts from Mercurius solubilis, Silicea, Sulphur, Tuberculinum, or placebo. Lymphocyte migration and lymphocyte speed were observed in a 60-min time frame. Statistical analysis was performed using univariate statistics and SiZer time series analysis. RESULTS: While C-dilutions did not reveal clear differences between placebo and substances, strong effects were observed in D-dilutions compared to placebo. The strongest effects were achieved in lymphocytes exposed to Sulfur D12. While most specific effects were observed in Sulphur D12 showing an activating effect on periodontitis patient lymphocytes (mean activity: 11,1% (placebo) vs. 23,8% (verum)), there was no effect in healthy volunteers (25,8% (placebo) vs. 25,6% (verum)). SiZer analysis confirmed this effect to be significant. CONCLUSION: The basic active principles of highly diluted substances are still a matter of controversial debate. Although conclusions are limited due to low sample size, results from our pilot study might encourage further investigations on the role of highly diluted Sulphur in the treatment of periodontitis. Apart from a reproduction study with Sulphur, other immunological experiments, i.e. the investigation of cell limes via flow cytometry, should be performed to underpin these results.
Subject(s)
Lymphocytes/drug effects , Materia Medica/pharmacology , Cell Movement/drug effects , Cells, Cultured , Humans , In Vitro Techniques , Periodontal Diseases/therapy , Pilot ProjectsABSTRACT
The effect on in vitro migration of leucocytes and lymphocytes of various drugs used in anaesthesia have been determined in the concentration range 10(-2) to 10(-6) M. The drugs included, thiopentone, bupivacaine, lignocaine, adrenaline, noradrenaline, hydrocortisone, morphine (with and without preservative), lorazepam, suxamethonium, pancuronium and atropine. Toxicity and effect on random mobility after incubation for 1 and 18 h were also determined. Thiopentone depressed leucocyte function at a concentration of 10(-5) M which is comparable to clinical plasma concentrations. Increasing the duration of exposure of the cells to the drugs significantly lowered the concentrations at which depression of function was observed. At concentrations used during local infiltration in clinical practice, bupivacaine and lignocaine were toxic to both leucocytes and lymphocytes. Adrenaline, whilst having no direct effect on cell function, potentiated the effect of lignocaine. Morphine showed no effect at 10(-4) M, a level 1,000 times greater than the reported toxic plasma levels. However, this level falls within the range reported for drug addicts. No effects were found for the other drugs.