Efecto de esfingolípidos y Agelosina B en la movilización de Ca2+ y la señalización celular en células de cáncer de mama (MCF-7) y su relación con la apoptosis / Effect of sphingolipids and Agelosin B on Ca2 + mobilization and cell signaling in breast cancer cells (MCF-7) and its relationship with apoptosis

Los esfingolípidos, como la ceramida (Cer), la ceramida-1-fosfato (C-1-P), la esfingosina (Sph) y la esfingosina-1-fosfato (S-1P) estan relacionados con la señalización intracelular en procesos como crecimiento celular, movilización intracelular de Ca+2 y apoptósis. En este trabajo se evaluó el efecto de estos esfingolípidos en la homeostasis de Ca+2 intracelular y en la apoptósis en células de cáncer de mama MCF-7. Se utilizaron fluoróforos específicos para el Ca+2 y microscopía confocal. Se demostró que en estas células, la Sph (20 uM), la Cer (10uM), la S-P (2uM) y la C--P (uM) aumentaron la concentración intracelular ce Ca+2, induciendo su liberación desde el retículo endoplasmático (RE). Además, se observo que la esfingosina abrioun canal de Ca2+ en la membrana plasmática. También se demostró que la Cer inhibe parcialmente la actividad de la Ca2+-ATPasa del RE (SERCA), de forma dosis dependiente, mientras que la ceramina, su análogo no hidrolisable la inhibe totalmente. La Sph también inhibe completamente la actividad de la SERCA, a la misma concentración que induce la liberación del Ca+2 del RE. Asimismo, se evaluó el efecto de estos esfingolípidos sobre la inducción de la apotósis en células MCF-7 evidenciando que el tratamiento con la Cer, la ceramida, la Sph inducen toxicidad. También se observo que mientras la ceramida activo la caspasa 7 y la caspasa 8, el esfingolipido natural, la Cer no tuvo ningún efecto. Por su parte, la Sph activa la caspasa 8 sin modificar la activdad de la caspasa 7. Tanto la Cer, como la ceramida y la Sph, disminuyeron la expresión de la proteína Bcl-2 amti-apoptótica, y también indujeron la fragmentación de ADN, visualizada mediante la técnica de TUNEL, demostrando que estos esfingolípidos inducen apoptósis en MCF-7. La agelasina B, toxina purificada a partir de la esponja marina Agelas clathrodes tiene un efecto citotóxico un orden de magnitud mayor en MCF-7, en comparación con fibroplastos humanos. La agelasina B induce la liberación del Ca+2 almacenado en el RE en celulas MCF-7, ademas de inhibir la actividad de la SERCA en un 100%. También se demostró que esta toxina induce apoptosis, ya que disminuye el potencial de membrana mitocondrial, activa la caspasa 8, disminuye la expresion de la proteina Bcl-2 e induce fragmentación del ADN de las células MCF-7. Este mecanismo es similar al efecto de la tapsigargina.

Human placental lipid induces mitogenesis and melanogenesis in B16F10 melanoma cells.

A hydroalcoholic extract of fresh term human placenta was found to be mitogenic as well as melanogenic on B16F10 mouse melanoma in an in vitro culture. The extract, a reservoir of a large number of bioactive molecules, was resolved to get the lipid fraction. Its activity was evaluated on B16F10 mouse melanoma by assessing the change in cellular morphology, growth and melanin induction. The lipid fraction, placental total lipid fraction (PTLF) tested in the study employed doses of 0 01 to 200 microg/ml; optimum growth and melanization accompanied by morphological changes were recorded at 10 and 100 microg/ml respectively. At intermediate doses growth and melanization were found to show a pattern of change over between growth and melanization and finally reached at an inverse relation at the respective optimal dose of response. Compared with defined sphingolipids, C(2) ceramide and sphingosine-1-phosphate, the results were mostly corroborative. The duality of biological response of sphingolipids as reported in numerous studies was comparable for the PTLF suggesting that its active component is a sphingolipid and showing its use for pigment recovery in vitiligo.

Ceramide glycanase from rat mammary tissues: inhibition by PPMP(D-/L-) and its probable role in signal transduction.

A ceramide glycanase (CGase activity has been characterized from lactating rat mammary tissue which cleaves the glycosidic bond between sphingosine and the glucose chain of a glycosphingolipid (GSL) thus liberating the intact oligosaccharide chain from a GSL. The majority (65%) of the hydrolase activity was detected in the supernatant fraction when the rat mammary tissue homogenate was centrifuged at 100,000 x g. Attempts to purify the enzyme indicated that the CGase protein is of hydrophobic nature as it binds to hydrophobic columns. The enzyme has been partially purified using hydrophobic columns in tandem. The partially purified protein was found to be immunoreactive to the antibody raised against the purified clam CGase. The immunostained band corresponded to a 64 kDa protein as also found with the clam enzyme. This immuno cross-reactivity indicated probable structural similarities between CGase proteins isolated from widely separated species in the evolutionary tree. The rat CGase was found to have a specific detergent requirement for optimal activity, and the pH optimum was found to be between 5 and 6. The enzyme activity is partially heat stable. It is not a divalent cation requiring enzyme; however, the activity is totally inhibited in the presence of mercury, indicative of a sulfhydryl group in the active site of the enzyme. The rat mammary CGase activity is inhibited in the presence of both D- and L-PPMP (1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol. HCl), homologs of PDMP (1-phenyl-2-decanoylamino-3-morpholino-1-propanol. HCl), a well-known inhibitor of GlcT-1 (Ceramide: UDP-Glc Glucosyltransferase), an enzyme in the glycolipid synthetic pathway. The inhibition seems to be of a competitive nature and the same type of inhibition is also observed with clam CGase. The CGase activity was found to be highest in lactating tissue compared to the activity found in either pregnant or post-lactating rat mammary tissues. Tissue survey indicated the presence of high levels of CGase in lactating rat liver, uterus, and ovary; moderate activity was detected in kidney and spleen. Both virgin and male rat mammary tissue also indicated a basic level of CGase activity. However, newborn spleen and mammary tissue showed a comparable level of activity to that found in lactating rat tissues. This report is mainly concerned with the characterization of CGase activity from a mammalian source and its importance in cellular processes.