Biomedical subjects

Lorena González

Publications and source records attributed to Lorena González.

9 recordsLinked to original sources

PubMed2006

Role of c-Src in human MCF7 breast cancer cell tumorigenesis.

To study the role of c-Src in breast cancer tumorigenesis, we generated a cell line derived from MCF7 carrying an inducible dominant negative c-Src (c-SrcDN: K295M/Y527F) under tetracycline control (Tet-On system). c-SrcDN expression caused phenotypic changes, relocation of c-Src, Fak, and paxillin, and loss of correct actin fiber assembly. These alterations were coupled to increased Fak-Tyr(397) autophosphorylation and to inhibition of Fak-Tyr(925), p130(CAS), and paxillin phosphorylation. An increased association of total Src with Fak and a decreased interaction of p130(CAS) and p85-PI3K with Fak were also observed. SrcDN inhibited cell attachment, spreading, and migration. Serum and EGF-induced stimulation of cell proliferation and Akt phosphorylation were also significantly reduced by SrcDN, whereas p27(Kip1) expression was increased. Consistently, silencing c-Src expression by siRNA in MCF7 cells significantly reduced cell migration, attachment, spreading and proliferation. Inoculation of MCF7 cells carrying inducible SrcDN to nude mice generated tumors. However, doxycycline administration to mice significantly reduced tumorigenesis, and when doxycycline treatment was installed after tumor development, a significant tumor regression was observed. In both situations, inhibition of tumorigenesis was associated with decreased Ki67 staining and increased apoptosis in tumors. These data undoubtedly demonstrate the relevance of the Src/Fak complex in breast cancer tumorigenesis.

Animals↗

PubMed2006

Inhibition of proliferation and induction of apoptosis in human breast cancer cells by lauryl gallate.

Lauryl gallate is an antioxidant food additive showing low toxicity to normal cells. Here, its antiproliferative effect has been studied on three human breast cancer cell lines: estrogen-dependent, wild-type p53, MCF7; estrogen-independent, non-functional p53, MDA-MB-231 and MCF7 ADR, which overexpresses P-glycoprotein (P-gp) and displays a multidrug-resistant phenotype. Lauryl gallate inhibited proliferation and induced cell cycle alterations in all three cell lines without altering P-gp functionality in the drug-resistant cells. A stable arrest in G(1) phase was observed in MCF7, while a slow-down of cell cycle progression was induced in the other two cell lines. Lauryl gallate increased p53 expression only in MCF7, and upregulated p21(Cip1) and reduced cyclin D1 levels in all three cell lines. The induction of apoptosis, demonstrated by annexin V-FITC labeling, PARP cleavage and mitochondrial membrane depolarization and morphological alterations, were clearly detected in MCF7 ADR and MDA-MB-231 and to a minor extent in MCF7. Overexpression of Bcl-2 in MCF7 ADR cells demonstrated its protective role against morphological alterations and apoptosis. Lauryl gallate induction of p21(Cip1) and apoptosis observed in all three cell lines was regulated by Erk1/2 activation. These findings suggest a potential use of lauryl gallate against tumors harboring p53 mutations and drug-resistant phenotypes.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2005

The genome project of Taenia solium.

We have constituted a consortium of key laboratories at the National Autonomous University of Mexico to carry out a genomic project for Taenia solium. This project will provide powerful resources for the study of taeniasis/cysticercosis, and, in conjunction with the Echinococcus granulosus and Echinococcus multilocularis genome project of expressed sequence tags (ESTs), will mark the advent of genomics for cestode parasites. Our project is planned in two consecutive stages. The first stage is being carried out to determine some basic parameters of the T. solium genome. Afterwards, we will evaluate the best strategy for the second stage, a full blown genome project. We have estimated the T. solium genome size by two different approaches: cytofluorometry on isolated cyton nuclei, as well as a probabilistic calculation based on approximately 2000 sequenced genomic clones, approximately 3000 ESTs, resulting in size estimates of 270 and 251 Mb, respectively. In terms of sequencing, our goal for the first stage is to characterize several thousand EST's (from adult worm and cysticerci cDNA libraries) and genomic clones. Results obtained so far from about 16,000 sequenced ESTs from the adult stage, show that only about 40% of the T. solium coding sequences have a previously sequenced homologue. Many of the best hits are found with mammalian genes, especially with humans. However, 1.5% of the hits lack homologues in humans, making these genes immediate candidates for investigation on pharmaco-therapy, diagnostics and vaccination. Most T. solium ESTs are related to gene regulation, and signal transduction. Other important functions are housekeeping, metabolism, cell division, cytoskeleton, proteases, vacuolar transport, hormone response, and extracellular matrix activities. Preliminary results also suggest that the genome of T. solium is not highly repetitive.

Animals↗

PubMed2004

Prolactin induces c-Myc expression and cell survival through activation of Src/Akt pathway in lymphoid cells.

Stimulation of resting W53 cells (lymphoid murine cells expressing prolactin (PRL) receptor) by PRL induced expression of growth-related immediate-early genes (IEG), and proliferation through activation of the Src kinases. Since IEG are essential for cell cycle progression, we have studied how PRL controls expression of c-Myc mRNA and c-Fos. Stimulation of W53 cell proliferation by PRL required activation of MAPK, as the Mek1/2 inhibitor PD184352 eliminated Erk1/2 stimulation, cell proliferation, and expression of c-Fos mRNA. In contrast, PD184352 did not alter PRL activation of c-Myc mRNA expression or stimulation of p70S6K, Akt, and the Jak2/Stat5 pathway. Activation of the PI3K by PRL was necessary for the expression of c-MycmRNA and W53 cell proliferation, as the PI3K inhibitor LY294002 abolished them. However, it did not modify PRL stimulation of c-Fos mRNA expression or activation of Erk1/2 and Stat5. Furthermore, rapamycin, an inhibitor of mTOR and consequently of p70S6K, did not alter PRL stimulation of c-Myc and c-Fos mRNA expression and it had a very minor inhibitory effect on PRL stimulation of W53 cell proliferation. In addition, rapamycin did not affect PRL stimulation of Akt or Stat5. However, it reinforced PRL activation of Erk1/2. Overexpression of a constitutively activated Akt (myristoylated Akt) in W53 cells overcame the inhibitory effect of LY294002 on c-Myc expression, as well as cell death upon PRL deprivation. Consistently, inducible expression of Akt-CAAX Box in W53 cells caused inhibition of c-Myc expression. PRL stimulation of W53 cells resulted in Akt translocation to the nucleus, phosphorylation of FKHRL1 transcription factor, and its nuclear exclusion. In contrast, induced expression of Akt-CAAX Box caused inhibition of FKHRL1 phosphorylation. Furthermore, transient expression of nonphosphorylatable FKHRL1-A3 mutant impaired PRL-induced activation of the c-Myc promoter. Akt activation also resulted in phosphorylation and inhibition of glycogen synthetase kinase 3 (GSK3), which in turn promoted c-Myc stability. Consistently, treatment of W53 with selective inhibitors of GSK3 such as SB415286 and lithium salts resulted in increased levels of c-Myc. Also, overexpression of c-Myc in W53 cells overcame the decrease in cell proliferation induced by LY294002. These findings defined a PRL-signalling cascade in W53 cells, involving Src kinases/PI3K/Akt/FKHRL1-GSK3, that mediates stimulation of c-Myc expression.

Cell Cycle↗

PubMed2003

Src mediates prolactin-dependent proliferation of T47D and MCF7 cells via the activation of focal adhesion kinase/Erk1/2 and phosphatidylinositol 3-kinase pathways.

Prolactin (PRL) stimulates breast cancer cell proliferation; however, the involvement of PRL-activated signaling molecules in cell proliferation is not fully established. Here we studied the role of c-Src on PRL-stimulated proliferation of T47D and MCF7 breast cancer cells. We initially observed that PRL-dependent activation of focal adhesion kinase (Fak), Erk1/2, and cell proliferation was mediated by c-Src in T47D cells, because expression of a dominant-negative form of c-Src (SrcDM, K295A/Y527F) blocked the PRL-dependent effects. The Src inhibitor PP1 abrogated PRL-dependent in vivo activation of Fak, Erk1/2, p70S6K, and Akt and the proliferation of T47D and MCF7 cells; Janus kinase 2 (Jak2) activation was not affected. However, in vitro, Fak and Jak2 kinases were not directly inhibited by PP1, demonstrating the effect of PP1 on c-Src kinase as an upstream activator of Fak. Expression of Fak mutant Y397F abrogated PRL-dependent activation of Fak, Erk1/2, and thymidine incorporation, but had no effect on p70S6K and Akt kinases. MAPK kinase 1/2 (Mek1/2) inhibitor PD184352 blocked PRL-induced stimulation of Erk1/2 and cell proliferation; however, p70S6K and Akt activation were unaffected. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 abolished cell proliferation and activation of p70S6K and Akt; however, PRL-dependent activation of Erk1/2 was not modified. Moreover, we show that both c-Src/PI3K and c-Src/Fak/Erk1/2 pathways are involved in the up-regulation of c-myc and cyclin d1 expression mediated by PRL. The previous findings suggest the existence of two PRL-dependent signaling cascades, initiated by the c-Src-mediated activation of Fak/Erk1/2 and PI3K pathways that, subsequently, control the expression of c-Myc and cyclin D1 and the proliferation of T47D and MCF7 breast cancer cells.

Animals↗

PubMed2003

Multivariate optimisation of a cyclodextrin-assisted-capillary zone electrophoretic method for the separation of torasemide and its metabolites.

In this work, a rapid cyclodextrin-assisted capillary electrophoretic method is developed for the separation of the diuretic torasemide and three of its metabolites. Both fractional factorial and central composite designs were employed to optimise the separation method. The factors studied were pH, concentration of methyl-beta-cyclodextrin, concentration of the background electrolyte and percentage of acetonitrile as organic modifier. Monitored response was a composite quality response (Q*) which balanced conflicting normalized responses, such as resolution and migration time. Optimal separation of the four studied compounds was achieved in less than 6.5 min, using an electrolyte of 60 mM borate buffer with no organic modifier and 25 mM methyl-beta-cyclodextrin concentration adjusted to pH 8.0 at a potential of 30 kV. Detection wavelength and temperature were 197 nm and 20 degrees C respectively. This work means a significant improvement with regard to a previous separation method for these compounds developed in our laboratory.

Cyclodextrins↗

PubMed2002

Application of capillary zone electrophoresis to the screening of some angiotensin II receptor antagonists.

A capillary zone electrophoretic (CZE) method was optimized for the separation of five angiotensin II receptor antagonists (Losartan, Irbesartan, Valsartan, Telmisartan and Eprosartan) and two of their metabolites (EXP 3174 and Candesartan M1) by means of experimental design methodologies. The aim of this study was to define rapidly experimental conditions under which the analytes can be resolved for quantitation. The effects of the buffer (pH, concentration and composition), the organic modifier and voltage were studied. Critical factors were identified in a screening design (fractional factorial design) and sequentially an optimization design (central composite design) was used to choose optimal conditions for separation. The most favorable electrophoretic conditions were found by setting the resolution at a threshold value (Rs < or = 1.5) and minimizing, if possible, analysis time. Successful results were obtained with a 50 mM potassium dihydrogen phosphate:boric acid (25:75 v/v) buffer at pH 5.5 in the presence of 5% methanol and application of a 25 kV voltage. Analysis time was 8 min in a conventional fused-silica capillary (50 cm effective length) in a normal cationic mode (anode at the inlet and cathode at the outlet) after hydrostatical sample injection for 30 s.

Acrylates↗

PubMed2002

Separation of the high-ceiling diuretic Torasemide and its metabolites by capillary zone electrophoresis with diode-array detection.

A capillary zone electrophoretic method was developed for the separation of the high-ceiling loop diuretic Torasemide and three of its metabolites (M1, M3 and M5) using an experimental design approach. Two different experimental designs were employed to optimize the developed method: (i) a fractional factorial design examining six factors at two levels (2(6-2)) and (ii) a central composite design examining two factors at two levels (2(2)+2x2+p). The factors studied were: pH, buffer concentration, proportion of boric acid in the mixed boric acid:potassium dihydrogen phosphate background electrolyte, temperature, applied voltage and percentage of organic modifier. Resolution between peaks was established as response. Separation of the four studied compounds was achieved in less than 8 min, using an electrolyte of 20 mM boric acid:potassium dihydrogen phosphate (75:25 v/v) with 15% MeOH adjusted to pH 9.7 with KOH, at a potential of 28 kV. Detection wavelength and temperature were 206 nm and 35 degrees C, respectively.

Diuretics↗

PubMedDate not supplied

Isolation and molecular characterization of a mouse renal microvascular endothelial cell line.

Murine endothelial cells (ECs) have proven difficult to obtain and maintain in culture. Long-term maintenance of normal ECs remains a difficult task. In this article we report the establishment of the first cellular line of renal microvascular endothelium obtained from normal tissue. Cells were isolated, cloned, and maintained by serial passages for longer than 24 mo, using endothelial cell growth supplement (ECGS) and gelatin-coated plates. Their morphology and ultrastructure, expression of von Willebrand factor, presence of smooth muscle alpha-actin, vimentin, cytokeratin filaments, capillary structures formed on Matrigel, and some typical ECs surface molecules were the criteria used to characterize cultured ECs. When examined for responsiveness to Shiga toxin-1, 13-20% of cytotoxicity was observed when coincubated with lipopolysaccharides. This cytotoxicity was not observed for normal lung ECs (1G11). Consequently, REC-A4 line retains characteristics of resting microvascular ECs and represents a useful in vitro model to study biological and physiopathological properties of renal endothelium.

Actins↗