5-5--6-6--tetrachloro-1-1--3-3--tetraethylbenzimidazolocarbocyanine and Breast-Neoplasms

The lichen compound usnic acid is used for its antimicrobial activities in cosmetic products and is also a component of slimming agents. Its effect against cancer cells was first noted over 30 years ago. In this study possible mechanisms of this effect were investigated using two human cell lines, the breast cancer cell line T-47D and the pancreatic cancer cell line Capan-2. Pure (+)-usnic acid from CLADONIA ARBUSCULA and (-)-usnic acid from ALECTORIA OCHROLEUCA were shown to be equally effective inhibitors of DNA synthesis, with IC (50) 4.2 microg/mL and 4.0 microg/mL for (+) and (-)-usnic acid against T-47D, and 5.3 microg/mL and 5.0 microg/mL against Capan-2, respectively. Flow cytometric analysis confirmed the inhibited entry into the S-phase and showed reduction in cell size. Classical apoptosis, as assessed by TUNEL staining, was not observed. Necrosis, measured by LDH release, was seen only in Capan-2 after exposure for 48 hours. Staining with the mitochondrial dye JC-1 demonstrated dose-dependent loss of mitochondrial membrane potential following treatment with usnic acid in both cell lines. In conclusion, usnic acid had a marked inhibitory effect on growth and proliferation of two different human cancer cell lines and led to loss of mitochondrial membrane potential. Cell survival was little affected; late necrosis was seen in one of the cell lines. No difference was noted between the two enantiomers.

Topics: Antineoplastic Agents, Phytogenic; Benzimidazoles; Benzofurans; Breast Neoplasms; Carbocyanines; Cell Cycle; Cell Line, Tumor; Cell Proliferation; DNA; Dose-Response Relationship, Drug; Female; Flow Cytometry; Humans; Inhibitory Concentration 50; Lichens; Membrane Potential, Mitochondrial; Necrosis; Pancreatic Neoplasms; Phytotherapy; Plant Extracts

Recent studies have shown that only breast cancer epithelial cells with intact p53 can induce metallothionein (MT) synthesis after exposure to metals. In this study, the potential role of p53 on regulation of MT was investigated. Results demonstrate that zinc and copper increased metal response elements (MREs) activity and MTF-1 expression in p53 positive MN1 and parental MCF7 cells. However, inactivation of p53 by treatment with pifithrin-alpha or the presence of inactive p53 inhibited MRE-dependent reporter gene expression in response to metals. MTF-1 levels remained unchanged after treatment with zinc in cells with nonfunctional p53. The introduction of wild-type p53 in MDD2 cells, containing nonfunctional p53, enhanced the ability of zinc to increase MRE-dependent reporter gene expression. The cellular level of p21Cip1/WAF1 was increased in MDD2 cells after p53 transfection, confirming the presence of active p53. The treatment of MN1 and parental MCF7 with trichostatin A led to a sixfold increase in the MRE activity in response to zinc. On the contrary, MRE activity remained unaltered in MDD2 cells with inactive p53. The above results demonstrate that activation of p53 is an important factor in metal regulation of MT.

Topics: Benzimidazoles; Breast Neoplasms; Carbocyanines; Cations, Divalent; Cell Line, Tumor; Copper; DNA-Binding Proteins; Female; Fluorescent Dyes; Gene Expression Regulation, Neoplastic; Genes, Reporter; Humans; Luciferases; Membrane Potentials; Metallothionein; Metals, Heavy; Mitochondria; Plasmids; Response Elements; Time Factors; Transcription Factor MTF-1; Transcription Factors; Transcription, Genetic; Transfection; Tumor Suppressor Protein p53; Zinc

Various thermotherapies are based on the induction of lethal heat in target tissues. Spatial and temporal instabilities of elevated temperatures induced in therapy targets require optimized treatment protocols and reliable temperature control methods during thermotherapies. Heat-stress induced effects on mitochondrial transmembrane potentials were analyzed in breast cancer cells, species MX1, using the potential sensor JC-1 (Molecular Probes, Invitrogen, Germany). Potential dependant labeling of heat-stressed cells was imaged and evaluated by fluorescence microscopy and compared with control cells. JC-1 stains mitochondria in cells with high mitochondrial potentials by forming orange-red fluorescent J-aggregates while in cells with depolarized or damaged mitochondria the sensor dye exists as green fluorescent monomers. In MX1 cells orange-red and green fluorescence intensities were correlated with each other after various heat-stress treatments and states of mitochondrial membrane potentials were deduced from the image data. With increasing stress temperatures the intensity of red fluorescent J-aggregates decreased while the green fluorescence intensity of JC-1 monomers increased. This heat-stress response happened in a nonlinear manner with increasing temperatures resulting in a nonlinear increase of red/green fluorescence ratios. These data indicated that mitochondria in MX1 cells were increasingly depolarized in response to increasing ambient temperatures.

Topics: Benzimidazoles; Breast Neoplasms; Carbocyanines; Cell Line, Tumor; Female; Fluorescent Dyes; Hot Temperature; Humans; Hyperthermia, Induced; Membrane Potential, Mitochondrial; Microscopy, Fluorescence

The estrogenic flavanone rac-8-prenylnaringenin (8-PN) and 3 derivatives (rac-7-(O-prenyl)naringenin-4'-acetate (7-O-PN), rac-5-(O-prenyl)naringenin-4',7-diacetate (5-O-PN), and rac-6-(1,1-dimethylallyl)naringenin (6-DMAN) were prepared by chemical synthesis and analyzed with respect to their toxicity and possible cell cycle effects in human acute myeloid leukemia (HL-60) cells. With the exception of 5-O-PN, all the other naringenins showed only weak toxic effects at concentrations below 50 micromol/l. A cell cycle analysis over several cell generations up to 4 days was carried out using the fluorescent dye carboxyfluorescein diacetate N-succinimidyl ester (CFSE) followed by propidium iodide (PI) staining at the end of the experiment. The well-studied flavonol quercetin was included in the analysis as a reference substance. All flavonoids affected cell proliferation, but the extent and the resulting changes in the proliferation pattern were specific for each substance. In contrast to the radical scavenging activity of quercetin, the tested flavanones showed no anti-oxidative properties using several different test systems. Similarly, the mitochondrial membrane potential (DeltaPsim) was hardly effected by these compounds, while both menadione and quercetin strongly reduced the potential after 1 h of treatment. The reported chemical modification of interesting lead substances (like the strongly estrogenic 8-PN) presents a promising approach to modulate the properties of a relevant substance in a pharmacologically desirable way. The low toxicity and weak cytostatic properties of the tested naringenin derivatives is encouraging for further studies on known naringenin target molecules.

Topics: Benzimidazoles; Breast Neoplasms; Carbocyanines; Cell Division; Cell Line, Tumor; Cell Membrane; Dose-Response Relationship, Drug; Flavanones; Fluorescent Dyes; Free Radical Scavengers; Germany, East; HL-60 Cells; Humans; Membrane Potentials; Mitochondria; Quercetin; Reactive Oxygen Species; Time Factors; Vitamin K 3