In vitro toxicology evaluation of pharmaceuticals using Raman micro-spectroscopy

Raman micro-spectroscopy combined with multivariate analysis was employed to monitor real-time biochemical changes induced in living cells in vitro following exposure to a pharmaceutical. The cancer drug etoposide (topoisomerase II inhibitor) was used to induce double-strand DNA breaks in human type II pneumocyte-like cells (A549 cell-line). Raman spectra of A549 cells exposed to 100 microM etoposide were collected and classical least squares (CLS) analysis used to determine the relative concentrations of the main cellular components. It was found that the concentrations of DNA and RNA significantly (P < 0.05) decreased, whilst the concentration of lipids significantly (P < 0.05) increased with increasing etoposide exposure time as compared to control untreated A549 cells. The concentration of DNA decreased by 27.5 and 87.0% after 24 and 48 h exposure to etoposide respectively. Principal components analysis (PCA) successfully discriminated between treated and untreated cells, with the main variance between treatment groups attributed to changes in DNA and lipid. DNA fragmentation was confirmed by Western blot analysis of apoptosis regulator protein p53 and cell metabolic activity determined by MTT assay. The over-expression of p53 protein in the etoposide treated cells indicated a significant level of DNA fragmentation and apoptosis. MTT tests confirmed that cellular metabolic activity decreased following exposure to etoposide by 29.4 and 61.2% after 24 and 48 h, respectively. Raman micro-spectroscopy may find applications in the toxicology screening of other drugs, chemicals and new biomaterials, with a range of cell types.     

转录因子Snail的作用机制及其生理功能

Snail为起负调节作用的锌指转录因子,其序列和功能在不同种属动物中十分保守。Snail超家族成员在胚胎着床、胚胎发生、肿瘤发生、细胞命运决定、细胞周期调控、左右不对称发育及创伤愈合等生理或病理过程发挥重要作用。对Snail的进一步研究,不仅可以阐明Snail超家族的作用机制,而且可以为探究Snail相关的肿瘤治疗策略提供重要的理论基础。     

Cellular search migrations in normal development and carcinogenesis

This review describes the large group of morphogenetic processes designated as search migrations. Search migrations typically include two stages: i) search, when a group of cells or of the cytoplasmic processes migrate over the cell-free spaces, and ii) choice, the stage when migrating cells reach specific loci where they stop and undergo specific differentiations induced by local factors such as cell-cell contacts and humoral agents. Migrating cells that do not meet their targets usually undergo apoptosis. Numerous examples of search migrations range from gastrulation to formation of axon-muscle connections. Critical stages of carcinogenesis such as acquisition of cell ability for invasion may be regarded as the genetic aberration of normal search migration: cancer cells perform an endless search but cannot make final choice.     

Studying fatty aldehyde metabolism in living cells with pyrene-labeled compounds

The lack of fatty aldehyde dehydrogenase function in Sjögren Larsson Syndrome (SLS) patient cells not only impairs the conversion of fatty aldehydes into their corresponding fatty acid but also has an effect on connected pathways. Alteration of the lipid profile in these cells is thought to be responsible for severe symptoms such as ichtyosis, mental retardation, and spasticity. Here we present a novel approach to examine fatty aldehyde metabolism in a time-dependent manner by measuring pyrene-labeled fatty aldehyde, fatty alcohol, fatty acid, and alkylglycerol in the culture medium of living cells using HPLC separation and fluorescence detection. Our results show that in fibroblasts from SLS patients, fatty aldehyde is not accumulating but is converted readily into fatty alcohol. In control cells, in contrast, exclusively the corresponding fatty acid is formed. SLS patient cells did not display a hypersensitivity toward hexadecanal or hexadecanol, but 3-fold lower concentrations of the fatty alcohol than the corresponding fatty aldehyde were needed to induce toxicity in SLS patient and in control cells.     

The Use of Ultrasonography to Study Teratogenicity in Ruminants: Evaluation of Ipomoea carnea in Goats

Background: Ipomoea carnea (I. carnea) is a poisonous plant found in Brazil and other tropical countries that often poison livestock. The plant contains the alkaloids calystegines and mainly swainsonine, which inhibit cellular enzymes and cause systematic cell death. The objective of this study was to evaluate the perinatal effects of I. carnea in goats. Methods: Forty‐seven pregnant goats were randomly allocated into 5 treatment groups and given the following doses (g/kg BW) of I. carnea: 0 (IC0), 1.0 (IC1), 3.0 (IC3), 5.0 (IC5) and 7.5 (IC7). The treatment animals were given fresh I. carnea from day 27 of gestation to parturition. Weight gains and serum biochemistry were evaluated. Fetuses were evaluated using ultrasonographic measurements. Results: Goats from the IC7 group showed clinical signs of poisoning. Ultrasound examination revealed that I. carnea feeding in all treatment groups reduced fetal movement compared to the controls. There was an increase in the total number of birth defects (retrognathia and arthrogyposis) in the IC7 and IC5 groups compared to the controls. Conclusion: The results show that I. carnea has teratogenic potential in goats. In addition, ultrasounds were useful in evaluating fetotoxicity and teratogenicity. Birth Defects Res (Part B) 00:1–7, 2012. © 2012 Wiley Periodicals, Inc.     

Cardiolipin and electron transport chain abnormalities in mouse brain tumor mitochondria: lipidomic evidence supporting the Warburg theory of cancer

Otto Warburg first proposed that cancer originated from irreversible injury to mitochondrial respiration, but the structural basis for this injury has remained elusive. Cardiolipin (CL) is a complex phospholipid found almost exclusively in the inner mitochondrial membrane and is intimately involved in maintaining mitochondrial functionality and membrane integrity. Abnormalities in CL can impair mitochondrial function and bioenergetics. We used shotgun lipidomics to analyze CL content and composition in highly purified brain mitochondria from the C57BL/6J (B6) and VM/Dk (VM) inbred strains and from subcutaneously grown brain tumors derived from these strains to include an astrocytoma and ependymoblastoma (B6 tumors), a stem cell tumor, and two microgliomas (VM tumors). Major abnormalities in CL content or composition were found in all tumors. The compositional abnormalities involved an abundance of immature molecular species and deficiencies of mature molecular species, suggesting major defects in CL synthesis and remodeling. The tumor CL abnormalities were also associated with significant reductions in both individual and linked electron transport chain activities. A mathematical model was developed to facilitate data interpretation. The implications of our findings to the Warburg cancer theory are discussed.     

Recognition and lysis by natural killers of tumor cells with participation of laminin

According to the data obtained in the present work, the receptor complex of mouse natural killers (NK) includes laminin, an antibody that blocks NK activity (NKA) regardless of the presence of a complement. Preincubation of mouse splenocytes with antilaminin serum led to a decrease in their NKA towards tumor cell-stargets (CT) with the NKA activity decreasing by a factor of 2 with respect to cultivated cells of rat hepatoma HTC and by a factor of 10 with respect to cultivated cells of human erythroblastosis K562. Pretreatment of splenocytes with normal nonimmune serum did not lead to a change of NKA. The pattern after tumor-cell preincubation with antilaminin serum was quite different; pretreatment of CT K562 led to a twofold decrease in the sensitivity of these cells to NK lysis, whereas the pretreatment of CT K562, on the contrary, made them twice as sensitive to NK lysis. Electrophoretic separation of proteins of CT plasma membranes with subsequent immunoblotting with antilaminin immune serum revealed the presence of laminin on HTC cell plasma membrane, which was identified as laminin 8/9 by the mass-spectrometry method, while no laminin was detected on K562 cells. Preincubation of splenocytes with laminin did not affect NKA with respect to CT K562 and HTC. Pretreatment of CT K562 and HTC with laminin decreased the NKA to zero. The obtained data allow for the suggestion of the doubtless participation of laminin and its receptors in CT cytolysis by NK.     

Parallels and Antagonisms of Embryogenesis and Carcinogenesis

The main similarities of embryonic and tumor cells, as well as the mechanisms preventing the malignant transformation of embryonic cells, are presented in this review. Special attention is paid to the role of specific polypeptide growth factors in reciprocally excluding processes: embryogenesis and carcinogenesis. Based on the presented analysis, new potential targets for antitumor drugs are considered.