Melissa officinalis extract induces apoptosis and inhibits proliferation in colon cancer cells through formation of reactive oxygen species.

PURPOSE: Efficient strategies for the prevention of colon cancer are extensively being explored, including dietary intervention and the development of novel phytopharmaceuticals. Safe extracts of edible plants contain structurally diverse molecules that can effectively interfere with multi-factorial diseases such as colon cancer. In this study, we describe the antiproliferative and proapoptotic effects of ethanolic lemon balm (Melissa officinalis) leaves extract in human colon carcinoma cells. We further investigated the role of extra- and intracellular reactive oxygen species (ROS). METHODS: Antitumor effects of lemon balm extract (LBE) were investigated in HT-29 and T84 human colon carcinoma cells. Inhibition of proliferation was analyzed by DNA quantification. The causal cell cycle arrest was determined by flow cytometry of propidium iodide-stained cells and by immunoblotting of cell cycle regulator proteins. To investigate apoptosis, cleavage of caspases 3 and 7 was detected by immunoblotting and fluorescence microscopy. Phosphatidylserine externalization was measured by Annexin V assays. Mechanistic insights were gained by measurement of ROS using the indicator dyes CM-H2DCFDA and Cell ROX Green. RESULTS: After 3 and 4 days of treatment, LBE inhibited the proliferation of HT-29 and T84 colon carcinoma cells with an inhibitory concentration (IC50) of 346 and 120 µg/ml, respectively. Antiproliferative effects were associated with a G2/M cell cycle arrest and reduced protein expression of cyclin dependent kinases (CDK) 2, 4, 6, cyclin D3, and induced expression of cyclin-dependent kinase inhibitor 2C (p18) and 1A (p21). LBE (600 µg/ml) induced cleavage of caspases 3 and 7 and phosphatidylserine externalization. LBE-induced apoptosis was further associated with formation of ROS, whereas quenching of ROS by antioxidants completely rescued the colon carcinoma cells from LBE-induced apoptosis. CONCLUSIONS: Lemon balm (Melissa officinalis) extract inhibits the proliferation of colon carcinoma cells and induces apoptosis through formation of ROS. Taken together, LBE or subfractions thereof could be used for the prevention of colon cancer.

Which nerve fibers mediate the axon reflex flare in human skin?

Axon reflex vasodilatation due to transcutaneous electrical stimulation in human skin was measured by laser Doppler imaging. Constant current pulses of 10 mA, 0.2 ms, delivered at 1 or 10 Hz for 2 min through a probe of 30 mm2 surface area did not induce a significant flare response, though this stimulus previously has been found supra-maximal for cutaneous polymodal (mechano-heat responsive) C-nociceptors in microneurography experiments. Pulses of the same strength from a pointed probe yielding a higher current density induced an extended and persistent flare. This type of stimulus previously has been proven to recruit mechano-insensitive C-units in microneurography experiments, in contrast to stimuli from the 30 mm2 probe. It is concluded that mechano-insensitive C-nociceptors and not polymodal C-units mediate the axon reflex flare in human skin.

Low-dose lidocaine reduces secondary hyperalgesia by a central mode of action.

Sodium channel blockers are approved for intravenous administration in the treatment of neuropathic pain states. Preclinical studies have suggested antihyperalgesic effects on the peripheral as well as the central nervous system. The objective of this study was to determine mechanisms of action of low-dose lidocaine in experimental induced, secondary hyperalgesia. In a first experimental trial, participants (n=12) received lidocaine systemically (a bolus injection of 2 mg/kg in 10 min followed by an intravenous infusion of 2 mg kg(-1)h(-1) for another 50 min). In a second trial, a modified intravenous regional anesthesia (IVRA) was administered to exclude possible central analgesic effects. In one arm, patients received an infusion of 40 ml lidocaine, 0.05%; in the other arm 40 ml NaCl, 0.9%, served as a control. In both trials capsaicin, 20 microgram, was injected intradermally and time course of capsaicin-induced pain, allodynia and hyperalgesia as well as axon reflex flare was determined. The capsaicin-induced pain was slightly reduced after systemic and regional application of the anesthetic. The area of pin-prick hyperalgesia was significantly reduced by systemic lidocaine, whereas the inhibition of hyperalgesia was absent during regional administration of lidocaine. In contrast, capsaicin-induced flare was significantly decreased after both treatments. We conclude that systemic lidocaine reduces pin-prick hyperalgesia by a central mode of action, which could involve blockade of terminal branches of nociceptors. A possible role for tetrodotoxin resistant sodium channels in the antihyperalgesic effect of low-dose lidocaine is discussed.

Interspecific variation in the chelonian primary visual system.

The primary visual system of 21 species of turtles, distributed among nine of the existing 12 families, were studied by autoradiography. In all species, contralateral visual projections exist to 15 targets: two hypothalamic structures (nucleus suprachiasmaticus and n. periventricularis), three major thalamic visual centres (nucleus ovalis, n. geniculatus lateralis ventralis and n. geniculatus lateralis dorsalis) and two minor thalamic targets (nucleus dorsolateralis anterior and n. ventrolateralis), five pretectal sites (nucleus geniculatus pretectalis, n. opticus pretectalis ventrolateralis, n. lentiformis mesencephali, n. posterodorsalis and n. griseus tectalis), two strata of the optic tectum (stratum opticum and s. fibrosum et griseum superficiale), and a single tegmental target (nucleus opticus tegmenti). In contrast to the stability of contralateral visual projections, their ipsilateral counterparts varied considerably between species, being limited to the hypothalamus in some species, and involving the majority of the primary visual centres in others. This variation is not readily explainable in terms of taxonomic position or of differences in mode of life.

Visual system degeneration in the glaucomatous albino quail.

The peripheral (eye, retina, optic nerve) and central (primary optic tracti and centers, centrifugal visual tractus and nucleus) visual system of an imperfect albino quail mutant with a sex linked recessive gene was examined in 32 specimens ages 1 week - 16 months-hatch using various histological techniques. During the first weeks the visual system was normal and comparable in its overall organization to that found in the pigmented quail. However, the ipsilateral retinal projections were observed to be weaker in the young mutant, then completely disappeared two months after birth. Initial signs of the bupthalmos, a form of spontaneous glaucoma, appeared between the 3rd and 5th months. This was characterized by a distention of the eye linked to an increase in intraocular pressure. The pathological process was progressive and at 16 months the eye was very prominent, the anterior chamber deep and a large and globular cornea was noted. The glaucoma progressively induced different histopathological changes in the visual system including: cupping of the optic disc, degeneration of optic axons and their parent ganglion and centrifugal cells and cavernous degeneration. All of these phenomena were identifiable at about the 10th post-natal month and progressed in a relatively constant and orderly manner. The retinal projections to the nucleus ectomamillaris, ventral and lateral optic tectum and ventral pretectum were the first to degenerate. The degeneration of optic fibers attaining the dorsal pretectum and dorsal thalamus occurred later. Furthermore the retrograde degeneration in the centrifugal isthmo-optic nucleus progressed from the external to the internal pole. The mechanisms involved in the selective degeneration of centrifugal and centripetal optic fibers is discussed.

[Demonstration of a retino-thalamo-hippocampal pathway in the rat]. / Mise en évidence d'une voie rétino-thalamo-hippocampique chez le rat.

Investigations using double labeling by axonal transport of tracers have shown that in the rat, four nuclei of the anterior thalamus (anterior dorsal, anterior ventral, lateral anterior, bed nucleus of the stria terminalis) that project to the CA1 region of the hippocampus also receive a discrete input from the contralateral eye. The significance of this telencephalic visual pathway is discussed in a phylogenetic context.

[Anatomical study of the afferent pathways of the rostral telencephalon in the Gallus domesticus chick]. / Etude anatomique des afférences du télencéphale rostral chez le poussin de Gallus domesticus L.

The neuroanatomical method involving retrograde axonal transport of horseradish peroxidase (HRP) was employed in the chick Gallus domesticus to investigate ascending projection systems to the rostral telencephalon. Following either extensive or restricted injections of the enzyme into different regions of the latter, differential bilateral or unilateral projections onto the Wulst (hyperstriatum accessorium, hyperstriatum intercalatus superior, hyperstriatum dorsale) were demonstrated from the dorsal thalami complex (nucleus dorsolateralis anterior thalami, pars lateralis). Other nuclei of the dorsal thalamus shown to project to the rostral telencephalon were nuclei, dorsolateralis medialis, dorsolateralis posterior, dorsomedialis anterior, parvocellularis and rotundus. At the level of the mesencephalon, rostral telencephalic projections were found to originate in the substantia grisea centralis, the strata cellulare externum and internum, the lateral reticular formation and the area ventralis. More caudally peroxidase label was localized in the nuclei locus, ceruleus, subceruleus dorsale, subceruleus ventrale and within the principal Vth nucleus. The results obtained using restricted injections of HRP made it possible to more precisely identify the telencephalic projections sites of several of the above areas and nuclei. The data is compared to previous findings regarding ascending projection system in different avian species and some methodological problems in the use of HRP technique are discussed.

[Primary visual centers in the frog (Rana esculenta L.) and the problem of ipsilateral projections. Radioautographic study]. / Les centres visuels primaires chez la grenouille (Rana esculenta L.) et le problème des projections ipsilatérales. Etude radioautographique.

The topographic organization of the frog's primary visual system has been reexamined in Rana esculenta using autoradiographic technique. 10 to 24 hrs following an intraocular injection of tritiated proline, an intense labelling was observed contralaterally in the various retinal projection centres (preoptic area); neuropils of Bellonci; corpus geniculatum of the thalamus; posterior thalamus; posterodorsal preoptic area; magnocellular pretectal nucleus and basal optic nucleus; tectal laminae A to G of Potter), as already described. Furthermore, the presence of a ipsilateral projection within the hypothalamus (preoptic area), the thalamus (neuropils of Bellonci and corpus geniculatum) and the pretectium (posterior thalami neuropil, pretectal optic area) is confirmed. On the other hand, the existence of ipsilateral retinotectal (to Potter's lamina F) and retino-tegmentomescencephalic projections (to the basal optic nucleus), which were thusfar questioned, has been demonstrated.

[Problems posed by the interpretation of peroxidase labelling of various neurons in Cyprinidae]. / Problèmes posés par l'interprétation du marquage peroxydasique de certains neurones chez les Cyprinidés

The origin of a centrifugal visual pathway in Cyprinids could not be demonstrated with the technique involving the labelling of cell bodies by retrograde transport of Horseradish Peroxidase. The hypothalamic labelling following intraocular injection of HRP is localized in neurosecretory structures which take up the enzyme that has passed into the circulatory system. Identical results were obtained following direct intracardiac injection. Thus extreme caution must be taken in attempting to interpret HRP results.