tretinoin and lactacystin

Topics: Acetylcysteine; Animals; Annexin A2; Antineoplastic Agents; Cell Line, Tumor; HEK293 Cells; HL-60 Cells; Humans; Leukemia, Promyelocytic, Acute; MCF-7 Cells; Mice; Mice, Inbred C57BL; Mice, Knockout; S100 Proteins; Tretinoin; U937 Cells; Ubiquitination

The proteasome inhibition and mitochondrial dysfunction are involved in pathomechanism of Parkinson's disease. The main aim of this study was to assess how particular culture conditions of human dopaminergic neuroblastoma SH-SY5Y cells could affect the extent of neurodegeneration induced by proteasome inhibitor-lactacystin (LC) and mitochondrial toxin-rotenone (Rot). This study revealed that induction of neuronal differentiation of SH-SY5Y cells with retinoic acid (RA-SH-SY5Y) caused a higher resistance of these cells to LC-evoked cell death when compared to undifferentiated cells (UN-SH-SY5Y). In contrast, RA-SH-SY5Y cells were more vulnerable than the UN-SH-SY5Y to Rot-induced cell damage. Furthermore, we found that a prolonged incubation of the cells under low serum condition (PLSC) significantly increased the LC toxicity in both differentiated and undifferentiated cells. Next, the effects of combined treatment with LC and Rot on cell viability were studied in RA-SH-SY5Y cells under PLSC and normal low serum condition (NLSC). At a low concentration, Rot (0.001-1 μM) attenuated the LC-evoked cell death in RA-SH-SY5Y cells exposed to NLSC. In contrast, under PLSC low concentrations of Rot lacked neuroprotective action while its higher levels (10 μM) enhanced the LC toxicity. Further, we showed that low concentrations of celastrol (Cel; 0.001 μM), a putative neuroprotective agent with antioxidant and anti-inflammatory properties, were able to partially attenuate the Rot-evoked toxicity under both PLSC and NLSC. On the other hand, Cel (0.001 and 0.01 μM) attenuated the LC-induced cell damage only under PLSC. Interestingly, higher concentrations of Cel (>1 μM) reduced cell viability in both UN- and RA-SH-SY5Y but only in UN-SH-SY5Y cells the effect was enhanced under PLSC. The obtained data indicate that toxicity of LC and Rot in SH-SY5Y cell line depends on the stage of cell differentiation and is enhanced in cells cultured for a longer time in low serum medium. Moreover, the neuroprotective properties of Rot and Cel against the LC-induced cell damage can be observed only under particular low serum conditions.

Topics: Acetylcysteine; Cell Death; Cell Differentiation; Cell Survival; Cells, Cultured; Culture Media, Serum-Free; Dose-Response Relationship, Drug; Drug Interactions; Humans; Nerve Degeneration; Neuroprotective Agents; Parkinson Disease; Pentacyclic Triterpenes; Rotenone; Time Factors; Tretinoin; Triterpenes

It has been shown that store-operated Ca(2+) influx (SOC) plays critical roles in the activation of endothelial nitric oxide (NO) synthase (eNOS) and generation of NO in endothelial cells. Recent studies indicate stromal interaction molecule 1 (STIM1) is the molecule responsible for SOC activation following Ca(2+) depletion in the ER. Retinoic acids (RA) have beneficial effects in the treatment of renal diseases. The mechanism of the RA action is still largely unknown. In the current study, we used primary cultured rat mesangial cells to examine the effect of RA on SOC and STIM1. In these cells, BK caused concentration-dependent [Ca(2+)](i) mobilization. Treatment of the cells with RA, while it had no effect on the initial peak, reduced the plateau phase of BK-mediated [Ca(2+)](i) response, indicating the inhibition of SOC by RA. The level of STIM1 protein but not mRNA in RA-treated cells was significantly reduced. RA treatment did not affect TGF-beta-mediated gradual Ca(2+) influx which occurred by superoxide anion-mediated mechanism, indicating RA treatment specifically inhibited SOC in mesangial cells. RT-PCR and Western blot analysis demonstrated that eNOS was expressed in rat mesangial cells grown in media containing 11 and 30 but not 5.5 mM glucose. Downregulation of STIM1 protein and BK-induced SOC by RA treatment or STIM1 dsRNA were associated with abolished NO production. The 26S proteasome inhibitor lactacystin blocked the RA-mediated downregulation of BK-induced SOC, suggesting that ubiquitin-proteasome pathway may be involved in RA-mediated STIM1 protein downregulation in rat mesangial cells. Our data suggest that glucose-induced eNOS expression and NO production in mesangial cells may contribute to hyperfiltration in diabetes and RA may exert beneficial effects by downregulation of STIM1 and SOC in mesangial cells.

Topics: Acetylcysteine; Animals; Blotting, Western; Bradykinin; Calcium; Calcium Signaling; Cells, Cultured; Cysteine Proteinase Inhibitors; Gene Expression; Glucose; Membrane Glycoproteins; Mesangial Cells; Nitric Oxide; Nitric Oxide Synthase Type III; Proteasome Inhibitors; Rats; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Stromal Interaction Molecule 1; Transforming Growth Factor beta; Tretinoin

Parkinson's disease (PD) is a slowly progressing neurodegenerative disorder with no clear etiology. Pathological hallmarks of the disease include the loss of dopaminergic neurons from the substantia nigra (SN) and the presence of Lewy bodies (LBs) (alpha-synuclein and ubiquitin-positive, eosinophilic, cytoplasmic inclusions) in many of the surviving neurons. Experimental modeling of PD neurodegeneration using the neurotoxins 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenyl-pyridinium (MPP(+)) has identified changes in gene expression of different endoplasmic reticulum (ER) stress proteins associated with MPTP- and PD-related neurodegeneration. We show that the protein disulfide isomerase (PDI) family member pancreatic protein disulfide isomerase (PDIp), previously considered exclusively expressed in pancreatic tissue, is uniquely upregulated among PDI family members within 24 h following exposure of retinoic acid (RA)-differentiated SH-SY5Y human neuroblastoma cells to either 1 mM MPP(+) or 10 microM of the highly specific proteasome inhibitor lactacystin. RT-PCR confirms PDIp expression in brain of post-mortem human PD subjects and immunohistochemical studies demonstrate PDIp immunoreactivity in LBs. Collectively, these findings suggest that increased PDIp expression in dopaminergic (DA) neurons might contribute to LB formation and neurodegeneration, and that this increased PDIp expression may be the result of proteasome impairment.

Topics: 1-Methyl-4-phenylpyridinium; Acetylcysteine; Aged; Aged, 80 and over; Analysis of Variance; Animals; Blotting, Northern; Blotting, Western; Cell Differentiation; Cell Line, Tumor; Dopamine; Female; Gene Expression Regulation; Humans; Immunohistochemistry; Lewy Bodies; Male; Middle Aged; Neuroblastoma; Nuclear Proteins; Parkinsonian Disorders; Postmortem Changes; Rats; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors; Tretinoin

p11 is a member of the S100 family of proteins, is the cellular ligand of annexin II, and interacts with the carboxyl region of 85-kDa cytosolic phospholipase A(2) (cPLA(2)), inhibiting cPLA(2) activity and arachidonic acid (AA) release. We studied the effect of retinoic acid (RA) on PLA(2) activity in human bronchial epithelial cells and whether p11 contributes to these effects. The addition of 10(-6) M RA resulted in reduced p11 protein levels at 4 days, with the greatest effect observed on days 6 and 7. This effect was dose related (10(-6) to 10(-9) M). RA treatment (10(-6) M) had no effect on cPLA(2) protein levels. p11 mRNA levels were unchanged at 6 and 8 days of treatment (correlating with maximum p11 protein reduction). Treatment with RA reduced p11 levels in control cells and in cells transfected with a p11 expression vector, suggesting a posttranslational mechanism. Lactacystin (10(-6) M), an inhibitor of the human 26S proteasome, blocked the decrease in p11 observed with RA treatment. Compared with control cells (n = 3), RA-treated cells (n = 3) had significantly increased AA release after treatment with the calcium ionophore A-23187 (P = 0.006). Therefore, RA reduces p11 protein expression and increases PLA(2) activity and AA release.

Topics: Acetylcysteine; Annexin A2; Antineoplastic Agents; Arachidonic Acid; Bronchi; Calcimycin; Cell Line; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Cytosol; Epithelial Cells; Gene Expression; Humans; Ionophores; Multienzyme Complexes; Peptides; Phospholipases A; Proteasome Endopeptidase Complex; Protein Processing, Post-Translational; Respiratory Mucosa; RNA, Messenger; S100 Proteins; Transfection; Tretinoin; Ubiquitins

Membrane-permeable proteasome inhibitors, lactacystin (LC) and N-acetyl-Leu-Leu-norleucinal (ALLN), but not calpain inhibitor Z-Leu-leucinal (ZLL), prevented LFA-1/ICAM-1-dependent cellular adhesion of TPA-stimulated HL-60 cells. These proteasome inhibitors affected neither the induction of monocytic differentiation nor the accompanying protein-tyrosine phosphorylation. They suppressed the increase in the avidity of LFA-1 to ICAM-1 without changing the expression of these molecules. Immunoblotting using monoclonal antibody FK-1, which reacts specifically with polyubiquitinated proteins, demonstrated that the proteasome inhibitors caused the drastic accumulation of the polyubiquitinated proteins in the membrane fraction of TPA-treated HL-60 cells. This indicates that accompanying activation of LFA-1, TPA induces the polyubiquitination of the membrane proteins, which are rapidly degraded by proteasomes. These data taken together show that proteolysis mediated by the ubiquitin-proteasome system is a prerequisite for the induction of LFA-1-dependent adhesion of HL-60 cells.

Topics: Acetylcysteine; Adenosine Triphosphatases; Antibody Affinity; Cell Adhesion; Cell Differentiation; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; HL-60 Cells; Humans; Intercellular Adhesion Molecule-1; Leupeptins; Lymphocyte Function-Associated Antigen-1; Monocytes; Multienzyme Complexes; Proteasome Endopeptidase Complex; Tetradecanoylphorbol Acetate; Tretinoin; Ubiquitins

Acute promyelocytic leukemia (APL) is associated with a chromosomal translocation t(15;17) and successfully differentiated by all-trans-retinoic acid (ATRA) in vivo as well as in vitro. The PML-retinoic acid receptor alpha (RARA) oncoprotein, which is generated by the translocation, blocks the differentiation, and ATRA is thought to modulate the dominant negative function of PML-RARA. However, the molecular effect of ATRA on PML-RARA is unknown. In this study, we showed by means of immunoblotting that the expression of PML-RARA decreased within 12 h in APL cells treated with ATRA at concentrations greater than 0.1 microM. The decrease of PML-RARA was associated with restoration of the normal subcellular PML localization. PML-RARA transcripts were not down-regulated by ATRA. However, lactacystin, a specific inhibitor of the proteasome, almost completely inhibited the decrease of PML-RARA. These data indicate that the PML-RARA degradation is accelerated by pharmacological concentrations of ATRA, suggesting that ATRA allows APL cells to differentiate by relieving the differentiation block.

Topics: Acetylcysteine; Animals; Antibodies; Cell Differentiation; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Drug Stability; Humans; Immunoblotting; Leukemia, Promyelocytic, Acute; Neoplasm Proteins; Nuclear Proteins; Oncogene Proteins, Fusion; Promyelocytic Leukemia Protein; Rabbits; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Transcription Factors; Tretinoin; Tumor Cells, Cultured; Tumor Suppressor Proteins