rottlerin and Neuroblastoma

Neuroblastoma is one of the most common solid tumors in children younger than 1 year of age, with poor prognosis and survival rates. Therefore, novel molecular targets and therapeutic strategies are needed to prolong patient survival. For this purpose, we investigated the effects of rottlerin and genistein separately and in combination on neuroblastoma cells (SH-SY5Y, Kelly). First, the effects of rottlerin and genistein were investigated on cell proliferation. Different rottlerin (1-50 µM) and genistein (5-150 µM) doses were used as experimental groups compared to the control (DMSO/vehicle). The IC50 dose was found to be 5 µM for rottlerin and 30 µM for genistein (P < 0.0001). Other analyses, such as colony formation assays, annexin V/propidium iodide staining, matrigel invasion assays, and Western blot analysis, were performed with these doses and their combinations. To assess statistical significance, statistical analysis was conducted using the one-way ANOVA with the post hoc Tukey test. Our results showed that IC50 doses of rottlerin and genistein induced a significant reduction in cell proliferation, colony formation, and invasion in neuroblastoma cells (P < 0.0001). The combination of these doses increased the levels of inhibition of cell proliferation and invasion while decreasing the level of apoptosis (P 0.0001). Furthermore, these agents caused G1-cell cycle arrest in these cells. Our western blot data showed that rottlerin and genistein treatments markedly inhibit elongation factor 2 kinase (EF2K) and other pro-tumorigenic, metastatic proteins in neuroblastoma cells. These agents probably showed their anti-proliferative, anti-metastatic, and pro-apoptotic effects through EF2K downregulation. Our results suggested that rottlerin and genistein have inhibitory effects on cancer cell proliferation, invasion, and cell cycle and induce apoptosis in both cell lines. Combined treatment with rottlerin and genistein may be a viable approach and beneficial to neuroblastoma patients as the combined effect significantly suppresses the above-mentioned pathways.

Topics: Apoptosis; Cell Line, Tumor; Cell Proliferation; Cell Survival; Child; Elongation Factor 2 Kinase; G1 Phase Cell Cycle Checkpoints; Genistein; Humans; Neuroblastoma

Phosphodiesterases are promising targets for pharmacological intervention against various diseases. There are already inhibitors of PDE3, PDE4 and PDE5 as approved drugs. However there is an unmet need to discover new chemical scaffolds as PDE inhibitors. The main drawback of most of PDE inhibitors is their non specificity; owing to their structural resemblance to cAMP or cGMP. Natural product compounds offer high structural diversity hence may provide new PDE inhibitors. We decided to screen our institutional natural product compound library of nearly 900 molecules for PDE5 inhibition and explore the selectivity against PDE1-11 and cytotoxicity of the hit molecule/s. Rottlerin was identified as a PDE5 inhibitor. It was found to inhibit other PDEs with varying specificities. Structure activity relationship data and molecular dynamics studies showed that Tyr612, Asp764, Gln817 and Phe820 in the binding pocket of PDE5 play an important role in the activity of rottlerin. As a pan PDE inhibitor, rottlerin was also found to activate the AMPK pathway and induce neurodifferentiation in IMR-32 cells, with the effect more efficient in samples co-treated with cAMP activator Forskolin. Rottlerin at higher concentrations was shown to induce autophagy, apoptosis and G2/S cell cycle arrest in IMR-32 cells.

Topics: Acetophenones; AMP-Activated Protein Kinases; Autophagy; Benzopyrans; Cell Differentiation; Cell Line, Tumor; G2 Phase Cell Cycle Checkpoints; Humans; Molecular Docking Simulation; Neuroblastoma; Neurons; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Protein Conformation; S Phase Cell Cycle Checkpoints; Signal Transduction

Glutathione (GSH) depletion is widely used to sensitize cells to anticancer treatment inducing the progression of programmed cell death and overcoming chemoresistance. It has been reported that neuroblastoma cells with MYCN amplification are unable to start TRAIL-dependent death and MYCN, in concert with cytotoxic drugs, efficiently induces the mitochondrial pathway of apoptosis through oxidative mechanisms. In this study, we show that GSH loss induced by L-buthionine-S,R-sulfoximine (BSO), an inhibitor of GSH biosynthesis, leads to overproduction of reactive oxygen species (ROS) and triggers apoptosis of MYCN-amplified neuroblastoma cells. BSO susceptibility of SK-N-BE-2C, a representative example of MYCN-amplified cells, has been attributed to stimulation of total SOD activity in the absence of changes in the level and the activity of catalase. Therefore, the unbalanced intracellular redox milieu has been demonstrated to be critical for the progression of neuroblastoma cell death that was efficiently prevented by antioxidants and rottlerin. These results describe a novel pathway of apoptosis dependent on ROS formation and PKC-delta activation and independent of p53, bcl-2, and bax levels; the selective redox modulation of PKC-delta might be suggested as a potential strategy for sensitizing MYCN-amplified cells to therapeutic approaches.

Topics: Acetophenones; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Ascorbic Acid; Benzopyrans; Biphenyl Compounds; Buthionine Sulfoximine; Calpain; Caspases; Catalase; Cell Line, Tumor; DNA Damage; Glutathione; Humans; N-Myc Proto-Oncogene Protein; Neuroblastoma; Nuclear Proteins; Oncogene Proteins; Onium Compounds; Protein Kinase C; Reactive Oxygen Species; Superoxide Dismutase; Superoxide Dismutase-1

To investigate the role of phosphorylation of protein kinase C (PKC) delta in the toxicity of 6-hydroxydopamine (6-OHDA) to the death of dopaminergic neurons.. Human neuroblastoma cells of the line SH-SY5Y were cultured 6-OHDA of the concentrations of 0, 50, 100, 200, and 400 micromol/L was added to observe its toxicity. Rottlerin (PKCdelta inhibitor, 2 micromol/L), bisindolylmaleimide (Bis, general PKC inhibitor, 10 nmol/L), Gö6976 (calcium-dependent PKC inhibitor, 5 nmol/L), and phobol-12-myristate-13-acetate (PMA, PKC activator, 100 nmol/L) were added into the culture fluid of another SH-SY5Y cells respectively, and then (1) culture fluid of equal volume was added for 18 h so as to observe there effects on the survival of the SH-SY5Y cells, or (2) 100 micromol/L 6-OHDA was added to observe the effects of intervention on PKC on the survival of the SH-SY5Y cells by using MTT assay. Cell lysis solution with phosphatase inhibitor was used to lyse the culture cells to extract plasma protein. Western blotting was used to detect the expression of phosphorylated PKCdelta.. MTT assay showed that all different concentrations (50 - 400 micromol/L) of 6-OHDA significantly and dose-dependently caused cell death with an EC50 of 92 micromol/L. Pretreatment with rottlerin and Bis alone did not influence the survival of the cells significantly,. However, the survival rate of the cells pretreated by Gö6976 alone was 92.3% +/- 3.2% that of the control group (P < 0.01), and the survival rate of the cells pretreated by PMA was 49.5% +/- 1.0% that of the control group (P < 0.01) Pretreatment of rottlerin decreased the death rate of the cells treated with 6-OHDA to 30.4% +/- 1.6% and conferred significant protection against 6-OHDA neurotoxicity by 57% +/- 6% compared to that of the cells treated by 6-OHDA alone (P < 0.01). However, Bis and Gö6976 did not affect the 6-OHDA-induced cell damage. Pretreatment of PMA increased the death rate of the cells treated with 6-OHDA to 67.1% +/- 2.2% and significantly aggravated 6-OHDA-induced cell toxicity by 66% +/- 9% (P < 0.01). Western blotting showed that 6-OHDA administration increased the expression of phosphorylated PKCdelta, pretreatment with Rottlerin inhibited such increase, PMA promoted such increase, and Bis and Gö6976 did not influence such increase.. Inhibition of PKCdelta phosphorylation with rottlerin ameliorates the neurotoxicity evoked by 6-OHDA, and activation of PKCdelta phosphorylation by PMA aggravates neurotoxicity, which implicating that this kinase participates in the 6-OHDA-induced neurotoxicity and Parkinsonian neurodegeneration.

Topics: Acetophenones; Benzopyrans; Blotting, Western; Carbazoles; Cell Line, Tumor; Cell Survival; Dopamine; Enzyme Activators; Humans; Indoles; Maleimides; Neuroblastoma; Oxidopamine; Phosphorylation; Protein Kinase C-delta; Signal Transduction; Tetradecanoylphorbol Acetate

Protein kinases C (PKCs) are a family of isoenzymes sensitive to oxidative modifications and involved in the transduction signal pathways that regulate cell growth. As such, they can act as cellular sensors able to intercept intracellular redox changes and promote the primary adaptive cell response. In this study, we have demonstrated that PKC isoforms are specifically influenced by the amount of intracellular glutathione (GSH). The greatest GSH depletion is associated with a maximal reactive oxygen species (ROS) production and accompanied by an increase in the activity of the delta isoform and a concomitant inactivation of alpha. ROS generation induced early morphological changes in GSH-depleted neuroblastoma cells characterized, at the intracellular level, by the modulation of PKC-delta activity that was involved in the pathway leading to apoptosis. When cells were pretreated with rottlerin, their survival was improved by the ability of this compound to inhibit the activity of PKC-delta and to counteract ROS production. These results define a novel role of PKC-delta in the cell signaling pathway triggered by GSH loss normally associated with many neurodegenerative diseases and clinically employed in the treatment of neuroblastoma.

Topics: Acetophenones; Antioxidants; Apoptosis; Ascorbic Acid; Benzopyrans; Buthionine Sulfoximine; Calcium-Calmodulin-Dependent Protein Kinases; Enzyme Inhibitors; Glutathione; Humans; Malondialdehyde; Neuroblastoma; Oxidation-Reduction; Phosphorylation; Protein Kinase C; Protein Kinase C-delta; Protein Transport; Reactive Oxygen Species; Signal Transduction; Tumor Cells, Cultured

Post-translational modification of neural cell adhesion molecule (NCAM) with alpha2,8-linked polysialic acid, which regulates homophilic adhesion and/or signal transduction events, is crucial to synaptic plasticity in the developing and adult brain. Evidence from in vitro models has implicated polysialylation in the regulation of cell growth, migration, and differentiation. Here, using two in vitro models, we demonstrate that polysialylation is downregulated by cell-cell contact and correlated with a state of neuronal differentiation. Furthermore, we report a role for protein kinase C delta (PKCdelta) in the regulation of NCAM polysialylation. Pharmacological studies using the PKC activator, phorbol myristate acetate, and inhibitors, calphostin-C, and staurosporine, demonstrated PKC activity to be inversely related to NCAM polysialylation in the mouse neuro-2A cell line. Isoform-specific immunoblot studies indicated this effect to be mediated by the calcium-independent PKCdelta isozyme, as its expression was inversely related to NCAM polysialylation state in both neuro-2A and rat PC-12 cell lines. Isoform specificity was further confirmed using the PKCdelta-selective inhibitor rottlerin, which produced a marked increase in PSA expression (36.9+/-5.25 a.u. vs. 24.7+/-0.80 arbitrary units control) coupled with a neuritogenic response. Likewise, decreased expression of PKCdelta was seen in nerve growth factor (NGF)-differentiated PC-12 cells. These findings suggest that the neuronal differentiation process may involve inhibition of PKCdelta, resulting in enhanced morphological plasticity, as evidenced by activation of NCAM polysialylation.

Topics: Acetophenones; Animals; Benzopyrans; Binding Sites; Binding, Competitive; Cell Communication; Cell Differentiation; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Isoenzymes; Mice; Neural Cell Adhesion Molecule L1; Neural Cell Adhesion Molecules; Neuroblastoma; Neuronal Plasticity; Neurons; PC12 Cells; Polysaccharides; Protein Kinase C; Protein Kinase C-delta; Rats; Sialic Acids; Sialyltransferases; Tumor Cells, Cultured