Quercetin-induced apoptosis in colorectal tumor cells: possible role of EGF receptor signaling.

Flavonoids are among the best candidates for mediating the protective effect of diets rich in fruits and vegetables with respect to colorectal cancer. To gain additional information about their growth effects on colorectal tumors and their cellular mechanisms of action, a series of related flavonoids was added to cultures of colonic tumor cells. Most compounds induced growth inhibition and cell loss at concentrations of 1-100 microM, relative effectivity being quercetin > apigenin > fisetin > robinetin and kaempferol. Myricetin was only slightly effective. Quercetin was the strongest inducer of apoptosis in a process that was reversible until 10 hours by flavonoid removal and until 24 hours by fetal calf serum. Cells were preferentially retained in the S phase. On the cellular level, quercetin sensitivity was correlated with epidermal growth factor (EGF) receptor levels, rapid growth, and poor differentiation, indicating the possibility of targeting those cells most harmful for the organism. The flavonoid transiently inhibited EGF receptor phosphorylation but had only little effect on other signaling molecules. Even after recovery of receptor phosphorylation, cells remained resistant to EGF stimulation. In summary, the data indicate that inhibition of EGF receptor kinase is an integral part of quercetin-induced growth inhibition, but additional mechanisms also contribute to the overall effect.

Antagonistic effects of combination photosensitization by hypericin, meso-tetrahydroxyphenylchlorin (mTHPC) and photofrin II on Staphylococcus aureus.

Photodynamic therapy involves the application of a photosensitizer activated by visible light to generate cytotoxic reactive oxygen. In addition to clinical investigations, in vitro studies concerning photodynamic potency of sensitizers as well as quantification of illumination procedures are necessary. In our investigation, the objective was to evaluate not only the effects of photosensitizer and light on Gram-positive Staphylococcus aureus, but also to investigate possible synergistic or antagonistic effects of these sensitizers. Therefore, we used hypericin, Photofrin II, porfimer sodium and meso-tetrahydroxyphenylchlorin (mTHPC) alone, as well as in combination. Log-phase cells of S. aureus exhibited a marked sensitivity to white thermal light irradiation in the presence of Photofrin II and mTHPC. However, hypericin caused a rather stimulated growth expressed in increased optical density (OD) and increase of total cell count (TCC) of the culture. Combination sensitization of S. aureus by Photofrin II and mTHPC with hypericin likewise caused a stimulation of bacterial growth. No synergistic effects were obtained by combination of Photofrin II and mTHPC; photoresponse of S. aureus was rather decreased by using combined porphyrins. In comparison, TCC and colony-forming units (CFU) were suppressed in the presence of mTHPC after an illumination procedure as well as in dark reactions. These effects were also obtained in the combination photosensitization by mTHPC and Photofrin II. In the presence the of hypericin, photodynamic effects of mTHPC and Photofrin II were inhibited. It was finally concluded that hypericin in our model is not a proper sensitizer for combination photo-sensitization due to antagonistic effects on photodynamic activity of mTHPC and Photofrin II.

Inactivation of viruses by beta-propiolactone in human cryo poor plasma and IgG concentrates.

Virus inactivation by cold treatment with beta-propiolactone (BPL) was investigated in human cryo poor plasma and purified IgG concentrates spiked with relevant human viruses or appropriate animal model viruses. The samples were treated with 0.1 or 0.25% BPL for 300 or 480 min, respectively. Residual infectivity was determined by standard microtitration assays on tissue culture cells. The inactivation of all viruses tested was more effective in IgG than in plasma. IgG: R1=4-5.5 log10 for vesicular stomatitis virus (VSV). Semliki Forest virus (SFV), bovine virus diarrhoea virus (BVDV), murine encephalomyelitis virus (MEV), feline calicivirus (FVC), suid parvovirus (PPV), simian virus 40 (SV40); R1=2-4 log10 for suid herpesvirus type 1 (SHV-1), bovine herpesvirus type 1 (BHV-1), human immunodeficiency virus type 2 (HIV-2), simian immunodeficiency virus (SIVagm3). Plasma: R1=3-5 log10 for VSV, SFV, BVDV, SHV-1, MEV:R1=0-3 log10 for HIV-1, SIVagm3 BHV-1, FCV, PPV, SV40. After addition of SIVagm3, HIV-2, and PPV to plasma or IgG, spontaneous inactivation without further addition of BPL was observed. These results demonstrate that treatment with BPL has a limited capacity to inactivate viruses. Different inactivation kinetics were observed in plasma and IgG concentrates. Therefore, virus inactivation by BPL must be tested for individual blood products independently and should not be extrapolated from other model systems.

Measurement of inotropic effects by a newly developed guinea pig papillary muscle bioassay.

In order to measure inotropic influences of physiologically occurring substances and drugs we used a newly developed guinea pig papillary muscle (GPPM) bioassay. GPPM were suspended in air and surface coated with buffer (Krebs-Henseleit solution). The muscles were stimulated (pulsating direct current, 1.5 V; 0.5 Hz, 20 ms duration) which led to contraction. This method enables measurements of inotropic effects up to 5 days, contrary to previous studies (1 day), in which immersions of GPPM in buffer were performed. In order to investigate the comparability of the new method we measured the effect of metabolites (citric acid cycle), lactic acid, lactate, and extracellular pH on muscle contractility. The H(+)-dependent decrease of the contractile force of the GPPM can be compensated by an increased Ca(2+)-concentration. Further, the influence of catecholamines (isoproterenol) on the contractility was investigated. As a result, isoproterenol caused arrhythmias and extrasystoles as it was observed in clinical studies. Several pharmaceutical substances were tested to show the reproducibility and repeatability of the bioassay.

Natural products derived from plants as potential drugs for the photodynamic destruction of tumor cells.

Chlorophyll and some of its synthetically produced derivatives are important sensitizers in photodynamic cancer therapy. Other natural products from plants with light dependent activity include quinones like hypericin and fagopyrin. These compounds have extended pi-electron systems which upon photoexcitation with visible light are responsible for singlet oxygen production. Other plant constituents which show UV light induced photosensitizing activity are furanocumarins like psoralen and angelicin, aflatoxins and alkaloids, as well as thiophene derivatives, terthienyl and several polyacetylenes. The photodynamic properties of a chelidonium alkaloid derivative and data for its potential clinical applicability are given.

Wavelength-dependent photoresponse of biological and aqueous model systems using the photodynamic plant pigment hypericin.

Photodynamic eradication of tumour cells in vivo depends on the presence of a photosensitizer, light delivery to the cells, and an oxygen supply. Hypericin, a polycyclic quinone with absorption maxima in the ultraviolet and visible ranges, was prepared for clinical use as a photosensitizer. Due to antitumoral and antineoplastic activities as well as the generation of singlet oxygen after photoexcitation, hypericin was applied in clinical oncology and photodynamic therapy. Hypericin was administered subcutaneously (20 micrograms hypericin in 200 microliters Nacl/pyridine solution) into the ante brachium (forearm) of two volunteers. After the diffusion and equilibration of 120 min phototesting was carried out using outdoor light exposure, halogen lamp, laser 514 nm (argon), laser 632 nm (argon dye) and laser 670 nm (diode laser), from 60 to 120 J cm-2. Positive phototests to outdoor light exposure, halogen lamp and laser 514 nm were characterized by rubescence, oozing, vesiculation and darting pain. Phototests with laser 632 nm and 670 nm showed no effects after irradiation. When hypericin was administered topically on skin, erythema and flaring could not be induced by any irradiation. These results suggest that hypericin is a potent photosensitizer only within the UV and green light ranges. This characteristic photoresponse could also be obtained in guinea pig papillary muscle (GPPM) bioassay, which may be established as a model for photosensitizer testing. Irradiation of hypericin-incubated GPPM with 514 nm (20 J cm-2) led to a decrease of the contractile force of about 31%. However, excitation with 632 nm and 670 nm did not cause inotropic effects on GPPM. In addition, hypericin and Photosan 3 were shown to be capable of sensitizing the photo-oxidation of sodium linoleate. This assay should be established for testing interactions between photosensitizers and light sources in vitro.

Hypericin in phototherapy.

We describe the first local use of hypericin as photosensitizer for photodynamic therapy in a patient with recurrent malignant mesothelioma. Hypericin is a polycyclic quinone, which has been shown to possess in vivo and in vitro antiretroviral and photosensitizing activity; moreover, it is used in depressive disorders. The semiquinone radical, singlet oxygen, and superoxide anion radical are reported to be the toxic agents in hypericin phototherapy. Our first experience with locally applied hypericin in a superficial tumor-plate was performed 8 weeks after the systemic administration of hematoporphyrin derivatives. For tumor light illumination we used an argon pumped dye laser tuned to 632 nm. Owing to satisfactory results, we repeated the same therapy 4 weeks later- and no therapeutic effect was noted. Following this, we proved the interstitial application of HPD and the combination of interstitial HDP and superficially applied hypericin. The subsequent light illumination 6 hours later had no efficacy in the HDP-photosensitized area but there was tumor destruction in the field with both administered photosensitizers. Our first experience suggests a potentiation of two photosensitizers: hematoporphyrin derivatives and hypericin.

Determination of Photosan III in human plasma.

In order to develop an accurate and quick method for the determination of Photosan III in human plasma, we used statistically planned experiments with an aim to identify the factors that can influence the analysis. Through a series of 20 experiments based on acid extraction of the porphyrin from the plasma and subsequent fluorescence analysis a calibration was obtained between 0 and 3.3 x 10(-5) M. The deviation of the parameters around the regression line is 3.02%, the coefficient of variation 3.55%.

A quantitative determination of singlet oxygen with horseradish peroxidase.

A singlet oxygen determination method based on the formation of dimethyl-(2,5)-2-methoxy-5-hydroperoxydihydrofuran (DMFO2) followed by acid hydrolysis with 0.1 N H2SO4 is described. Hydrogen peroxide is formed thereby as a product of hydrolysis which is then determined by the horseradish peroxidase-catalyzed formation of the homovanillic acid dimer. Since DMFO2 is a crystalline compound with sufficient stability it can be used as a primary standard for the determination. The peroxidase-catalyzed hydrogen peroxide determination allows detection of singlet oxygen concentrations as low as 10(-7) M.

tBOOH acts as a suicide substrate for catalase.

The effects of t-butyl hydroperoxide (tBOOH) on bovine liver catalase were investigated. tBOOH is accepted as a substrate of catalase and in the absence of hydrogen donors leads to a destruction of the enzyme via compound II formation. During the decomposition of this enzyme-substrate complex catalase serves as internal hydrogen donor which results in destruction of the enzyme. Evidence for this destruction is given by: a decrease of the Soret band in the uv/vis spectrum, iron release from the enzyme, decrease of the catalatic activity of the enzyme measured by oxygen release from hydrogen peroxide. Hydrogen donors like NADH and o-dianisidine have been found to protect the enzyme from destruction by tBOOH but lead to a structural alteration of the enzyme, shown by alteration of the electrophoretic mobility. In the presence of the hydrogen donor tBOOH is completely reduced to t-butanol, which is thought to proceed in a peroxidase-like reaction.

Serum urate level and cancer.

Histograms of the serum uric acid level of cancer patients (n = 501) and healthy controls (n = 15150) are compared. The statistical evaluation of the data yields significantly lower uric acid levels for cancer patients than for healthy persons (P less than 0.0001 for males, P less than 0.001 for females). These results support the theory that reactive species of oxygen (singlet oxygen and oxygen radicals) play an important role during carcinogenesis.

A nonenzymatic method for determination of hydrogen peroxide and organic peroxides.

Reduction of hydrogen peroxide and organic peroxides (t-butyl hydroperoxide and linoleic acid hydroperoxide) was achieved with homovanillic acid as hydrogen donor in the presence of the triethylenetetramine-Fe3+ complex. By the catalytic action of this complex, homovanillic acid is oxidized to its fluorescent dimer. Based on this reaction a fluorometric method for the measurement of the hydroperoxides mentioned above is described. The method can be extended to the determination of substrate-enzyme systems that produce hydrogen peroxide, e.g., glucose-glucose oxidase. The method allows the determination of substances such as hydrogen peroxide and t-butyl hydroperoxide with an accuracy and precision of less than 3%. Glucose can be determined with similar precision and an accuracy of 4.7%.

[Electrophoretic studies on the effect of different types of radiation (60Co, electrons, x-ray, photons, UV) on low-molecular ribonucleic acids]. / Elektrophoretische Untersuchungen des Einflusses von verschiedenen Strahlungsarten (60Co, Elektronen, Röntgen, Photonen und UV) auf niedermolekulare Ribonukleinsäuren.

Electron irradiation (45 MeV) with the chosen doses modifies the molecule composition of isolated dry yeast RNA. High-energy irradiation will crack the RNA which then forms new chains of macromolecular nucleic acids. Other radiation types (60Co, photons, X-rays) do not modify dry RNA, but when irradiated in aqueous solution, these macromolecular bands will be built, too. After UV irradiation with 254 nm delivered over 24 hours these macromolecular bands disappear completely in the electrophoretic diagram. This wavelength corresponds to the absorption maximum of purines, thus stimulating them and reducing their stability.

[Fundamental model assays on chemical damages to ribonucleic acids caused by oxygen radicals in aqueous solution]. / Grundlegende Modellversuche über chemische Schädigungen von Ribonukleinsäuren durch Sauerstoffradikale in wässriger Lösung.

A partial decomposition of RNA molecules in Saccharomyces cerevisiae is caused by photochemically produced superoxide anion radical (O2-.). Hydroxyl radicals (OH.) lead to complete destruction of RNA. Such radicals as originating from peroxidase-hydrogen peroxide reaction cause a partial decomposition of RNA molecules. All RNA species are damaged to the same extent by radicals. Damages due to oxygen radicals can be prevented or reduced by strong radical captors (dimethyl sulfoxide, 1,4-diazabicyclo-(2,2,2)-octane or superoxide dismutase). All these reactions proceed in aqueous solution. Correlations with the use of radicals in radiotherapy are discussed.

Acute respiratory failure after cardiac surgery: clinical experience with the application of continuous arteriovenous hemofiltration.

Hemodynamic and oxygen measurements were obtained before and during 24 h of continuous arteriovenous hemofiltration (CAVH) in 36 postoperative cardiac surgery patients with severe acute pulmonary failure. During the first 6 h, the low mean arterial pressure averaged only 50 +/- 7 mm Hg; PaO2 was 90 torr on an inspired oxygen fraction of 0.86 +/- 0.03; and lactic acid was 10.5 +/- 6 mmol/L. Of the 34 patients recovering from shock within 12 h, only 24 (67%) were hospital survivors. Cardiac index, oxygen availability index, oxygen consumption, and PaO2 increased during CAVH. This treatment decreased serum levels of the myocardial depressant factor, thus allowing catecholamine support to be reduced. We conclude that CAVH eliminates cardiopulmonary toxic substances partly responsible for shock. Our patients' improved hemodynamic and respiratory function suggests that CAVH may be useful in postoperative cardiac surgery patients with respiratory and hemodynamic failure.

[Hemofiltration as therapy in acute pulmonary failure in cardiogenic shock]. / Hämofiltration als Therapie des akuten Lungenversagens im kardiogenen Schock.

In 29 cardiosurgical patients in cardiogenic shock after extracorporal circulation complicated by acute pulmonary failure, it was impossible to restore normal postoperative arterial oxygen tension, in-spite of optimal pharmacotherapy and ideal conditions with a conventional volume-controlled respirator. These patients were subject to continuous arteriovenous haemofiltration; in all of them the start of haemofiltration immediately led to a significant reduction of respiratory oxygen supply with an increase in arterial oxygen tension. Pulmonary shunt volume decreased. At the same time there was an increase in arteriovenous oxygen difference, arterial oxygen content and oxygen transport capacity. Pulmonary artery pressure as well as pulmonary vascular resistance decreased noticeably, whereas there was an increase in total peripheral vascular resistance. Starting haemofiltration with decreasing left ventricular filling pressure, accompanied by a rise in blood pressure and an increase in total peripheral resistance, led to an improvement of the haemodynamic situation as well as pulmonary oxygen diffusion, thereby ensuring oxygen perfusion of peripheral tissue. The results suggest a causal relation between the improvement of the clinical condition of the patient and the elimination of cardiopulmonary toxic agents like myocardial depressant factor (MDF) and shock mediators due to arteriovenous haemofiltration.

[Changes in serum protein patterns in cancer patients during radiotherapy]. / Veränderungen im Serumproteinmuster von Karzinompatientinnen bei Strahlentherapie.

The serum protein spectrum of a PAGE separation shows characteristic and permanent modifications in patients of premenopausal age submitted to a curative or postoperative irradiation by high dose rate afterloading method or percutaneous telecobalt therapy for carcinoma of the cervix. In older patients, the serum protein spectrum remains nearly unchanged during and after radiotherapy.