Regulation of monoamine levels by typical and atypical antipsychotics in Caenorhabditis elegans mutant for nuclear distribution element genes.

Mammalian nuclear distribution genes encode proteins with essential roles in neuronal migration and brain formation during embryogenesis. The implication of human nuclear distribution genes, namely nudC and NDE1 (Nuclear Distribution Element 1)/NDEL1 (Nuclear Distribution Element-Like 1), in psychiatric disorders including schizophrenia and bipolar disorder, has been recently described. The partial loss of NDEL1 expression results in neuronal migration defects, while ndel1 null knockout (KO) leads to early embryonic lethality in mice. On the other hand, loss-of-function of the orthologs of nuclear distribution element genes (nud) in Caenorhabditis elegans renders viable worms and influences behavioral endophenotypes associated with dopaminergic and serotoninergic pathways. In the present work, we evaluated the role of nud genes in monoamine levels at baseline and after the treatment with typical or atypical antipsychotics. Dopamine, serotonin and octopamine levels were significantly lower in homozygous loss-of-function mutant worms KO for nud genes compared with wild-type (WT) C. elegans at baseline. While treatment with antipsychotics determined significant differences in monoamine levels in WT, the nud KO mutant worms appear to respond differently to the treatment. According to the best of our knowledge, we are the first to report the influence of nud genes in the monoamine levels changes in response to antipsychotic drugs, ultimately placing the nuclear distribution genes family at the cornerstone of pathways involved in the modulation of monoamines in response to different classes of antipsychotic drugs.

The preventive effect of liraglutide on the lipotoxic liver injury via increasing autophagy.

INTRODUCTION AND OBJECTIVES: The incidence of non-alcoholic fatty liver disease (NAFLD) is increasing. Previous studies indicated that Liraglutide, glucagon-like peptide-1 analogue, could regulate glucose homeostasis as a valuable treatment for Type 2 Diabetes. However, the precise effect of Liraglutide on NAFLD model in rats and the mechanism remains unknown. In this study, we investigated the molecular mechanism by which Liraglutide ameliorates hepatic steatosis in a high-fat diet (HFD)-induced rat model of NAFLD in vivo and in vitro. MATERIALS AND METHODS: NALFD rat models and hepatocyte steatosis in HepG2 cells were induced by HFD and palmitate fatty acid treatment, respectively. AMPK inhibitor, Compound C was added in HepG2 cells. Autophagy-related proteins LC3, Beclin1 and Atg7, and AMPK pathway-associated proteins were evaluated by Western blot and RT-PCR. RESULTS: Liraglutide enhanced autophagy as showed by the increased expression of the autophagy markers LC3, Beclin1 and Atg7 in HFD rats and HepG2 cells treated with palmitate fatty acid. In vitro, The AMPK inhibitor exhibited an inhibitory effect on Liraglutide-induced autophagy enhancement with the deceased expression of LC3, Beclin1 and Atg7. Additionally, Liraglutide treatment elevated AMPK levels and TSC1, decreased p-mTOR expression. CONCLUSIONS: Liraglutide could upregulate autophagy to decrease lipid over-accumulation via the AMPK/mTOR pathway.

Characterization of cell death events induced by anti-neoplastic drugs cisplatin, paclitaxel and 5-fluorouracil on human hepatoma cell lines: Possible mechanisms of cell resistance.

Two different hepatoma cell lines were incubated for 48h with chemotherapeutic drugs cisplatin, paclitaxel and 5-FU to determine their ability to induce cytotoxicity and DNA fragmentation as well as to modify the expression of some cell death-related genes that could be involved in the resistance to therapy. We observed that cisplatin and paclitaxel induced cytotoxicity, but significant differences between both cell lines, were found only in the case of paclitaxel. At 48h, apoptosis was clearly present in Hep3B cells treated with cisplatin and HepG2 cells treated with paclitaxel. 5-FU induced cytotoxicity in both cell lines but only at higher concentrations than the other two drugs, triggering apoptosis and necrosis in HepG2 cells and only necrosis in Hep3B. When a time course was performed for the first 8h of treatment to elucidate the initial mechanism of cell death responsible for DNA fragmentation, we observed that 5-FU in Hep3B, and cisplatin in both cell lines, induces primary necrosis, whereas at the concentration tested here, paclitaxel clearly triggers apoptosis in both cell lines. HepG2 cells were weakly sensitive to 5-FU in the first 8h of treatment, so the primary mechanism of cell death was not clear, but results seem to indicate that it could be apoptosis. At 48h, Bax was not up-regulated with any of the treatments, whereas cisplatin was able to induce Bcl-xL down-regulation in both cell lines. Treatment with 5-FU also down-regulated Bcl-xL in HepG2 cells. We also measured variations in the expression of survivin, an inhibitor of apoptosis that has also been involved in mitototic catastrophe. Hep3B cells seem to show an increase in protein levels with all treatments. Exposure to paclitaxel resulted in the highest effect. In the case of HepG2 cells, there was a decrease in survivin expression when cells were treated with 5FU and paclitaxel, both treatments showing complete loss of the protein. Using an antibody that recognizes unprocessed caspase-3, we observed that the enzyme was assumingly activated in HepG2 cells treated with 5FU and paclitaxel, but only weakly after treatment with cisplatin. Hep3B cells did not show activation since the levels of the pro-enzyme remained the same as that in the control. In conclusion, the three drugs tested in this study could induce cell death, with paclitaxel being more effective inducing apoptosis. 5FU was only effective at high doses and its mechanism seems to be primarily related to necrosis in Hep3B and probably apoptosis in HepG2. Cisplatin mechanism of cell death is probably mediated by the decrease in anti-apoptotic protein Bcl-xL whereas paclitaxel and 5FU are decreasing the apoptosis inhibitor survivin. According to pro-enzyme levels, caspase-3 was only activated in HepG2 cells, whereas in the case of Hep3B cells the mechanisms of toxicity appear to be caspase-3-independent at the time and concentrations tested in this study. The resistance of Hep3B cells to death induced by chemotherapy could be related to an increase in the expression of IAP survivin, which can decrease cell response to the treatment or even switch the type of death from apoptosis to another kind, making therapy less efficient.

Apoptotic events induced by naturally occurring retinoids ATRA and 13-cis retinoic acid on human hepatoma cell lines Hep3B and HepG2.

Two hepatoma cell lines were incubated for 72 h with ATRA and its analog 13cisRA and according to MTT assay, Hep3B cells were highly susceptible whereas HepG2 cells were more resistant to the treatment. At the high concentration of 166 microM, retinoids were able to induce apoptosis in both cell lines and the highest effect was observed in HepG2 cells treated with ATRA. TUNEL-based photometric ELISA showed that at the same retinoid concentration tested by flow cytometry, both cell lines showed apoptosis whereas plasma membranes were not significantly disrupted. Inhibitors of apoptosis Bcl-xL and survivin were downregulated in Hep3B cells by treatment with both retinoids. Bax, a pro-apoptotic protein, was not significantly upregulated in Hep3B cells, but was slightly increased in HepG2 cells treated with 13cisRA. Both procaspase-3 and procaspase-8 were cleaved in Hep3B cells, suggesting apoptosis could be triggered through the extrinsic pathway. In the case of HepG2 cells, lack of caspase activation suggests a mechanism dependent on other kind of proteases.

Changes in the expression of cytoskeletal proteins in the CNS of transferrin transgenic mice.

Apotransferrin injected intracranially into young rats has been shown in our laboratories to induce an early differentiation of oligodendroglial cells and an increased deposition of myelin. The expression of some myelin-specific proteins such as myelin basic protein (MBP) and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) and of their mRNAs were significantly increased in these animals. Also, in the cytoskeleton obtained from isolated myelin, it was found that several microtubule associated proteins (MAPs), particularly the stable tubule only peptide (STOP) and MAP 1B, as well as actin and tubulin were markedly increased. In the present paper, we compare the changes in expression of brain and myelin cytoskeletal proteins in a newly generated transferrin transgenic mouse (Tg), overexpressing the human transferrin gene, with the results obtained in aTf-injected rats. In the myelin cytoskeletal fraction of Tg mice there was a significant increase in the expression of MBP, tubulin, tau and STOP, similarly to what was previously found in the aTf-injected rats. Immunohistochemical studies showed that a variance with what occurs in the aTf-injected model, in which the above mentioned changes were limited to the corpus callosum, in the Tg mice the changes in expression of cytoskeletal proteins were observed in the various anatomical areas studied such as cerebral cortex, brain stem and cerebellum. There was also an increased expression of neurofilaments in the Tg animals, in contrast with results obtained in aTf-injected rats, suggesting that in the Tg mice, the continuous overexpression of Tf might also induce some neuronal changes. Changes in tau, total and acetylated tubulin and MAP 1B were observed in both neurons and OLGc. The increase in STOP was more significant in OLGc while the changes in MAP2 were exclusively found in neurons.