Rottlerin protected dopaminergic cell line from cytotoxicity of 6-hydroxydopamine by inhibiting PKCδ phosphorylation

Abstract

Objective The present study aims to investigate the role of protein kinase C δ subtype (PKCδ) phosphorylation in the process of 6-hydroxydopamine (6-OHDA)-induced dopaminergic cell death, and demonstrate the molecular basis of neurological disorders, such as Parkinson's disease. Methods The pheochromocytoma (PC12) cell line was employed in the present study. Cells were treated with 2 μmol/L PKCδ inhibitor Rottlerin, 10 nmol/L protein kinase C α subtype (PKCα) inhibitor bisindolylmaleimide I, or 5 nmol/L Gö6976 that could specifically inhibit the calcium-dependent PKC isoforms, respectively. PKCδ activator phorbol-12-myristate-13-acetate (PMA, 100 nmol/L) was also used in this study. All these agents were added to the medium before cells were incubated with 6-OHDA. Cells with no treatment served as control. The cytotoxicity of 6-OHDA was determined by methyl thiazolyl tetrazolium (MTT) reduction assay and PKCδ phosphorylation levels in various groups were measured by western blotting. Results Bisindolylmaleimide I and Gö6976 exerted no significant attenuation on the cytotoxicity of 6-OHDA, nor any effects on PKCδ phosphorylation in PC12 cells. However, Rottlerin could inhibit the phosphorylation of PKCδ and attenuate 6-OHDA-induced cell death, and the cell viability was raised to 69.6±2.63% of that in control group (P < 0.05). In contrast, PMA induced a significant increase in PKCδ phosphorylation and also strengthened the cytotoxic effects of 6-OHDA. The cell viability of PMA-treated PC12 cells decreased to 49.8±5.06% of that in control group (P < 0.001). Conclusion Rottlerin can protect PC12 cells from cytotoxicity of 6-OHDA probably by inhibiting PKCδ phosphorylation. The results suggest that PKCδ may be a key regulator of neuron loss in Parkinson's disease.

Enhanced chemosensitization of anoikis-resistant melanoma cells through syndecan-2 upregulation upon anchorage independency

“…We also examined whether the increased SDC2 expression in suspended melanoma cells was also mediated by PKCδ activation. Specific inhibitor on PKCα/β, Gö6976 [ 28 ], and inhibitor on PKCδ, Rottlerin [ 28 ], were used to treat cells and the level of SDC2 expression was characterized by qPCR. As seen in Figure 2 , PKCα/β inhibitor, but not PKCδ inhibitor, reduced SDC2 expression in adherent melanoma cells.…”

Wogonin enhances intracellular adiponectin levels and suppresses adiponectin secretion in 3T3-L1 adipocytes

“…Phosphorylation statues of PKCδ have been suggested to play a critical role in determining its functional properties [34,35,38]. For example, the catalytic activity of membrane-associated allosterically activated PKCδ is increased by its Thr505 phosphorylation [34][35][36][37]. The impact of wogonin on PKCδ phosphorylation is dependent on cell types and their physiological or pathophysiological condition.…”

“…firstly examined the impact of wogonin on PKCδ phosphorylation at Thr505 residue, which reportedly increases the catalytic activity of membrane-associated allosterically activated PKCδ [34][35][36][37]. Mature 3T3-L1 adipocytes were starved serum for 4 h and then treated with 5 μmol/L, 10 μmol/L, 20 μmol/L of wogonin for 24 h, or 10 μmol/L of wogonin for 6 h, 12 h, or 24 h. The cells treated with 10 μmol/L PKCδ specific inhibitor rottlerin for 24 h serviced as positive controls, because it has been shown to inhibit both PKCδ phosphorylation and its action [37].…”

“…Mature 3T3-L1 adipocytes were starved serum for 4 h and then treated with 5 μmol/L, 10 μmol/L, 20 μmol/L of wogonin for 24 h, or 10 μmol/L of wogonin for 6 h, 12 h, or 24 h. The cells treated with 10 μmol/L PKCδ specific inhibitor rottlerin for 24 h serviced as positive controls, because it has been shown to inhibit both PKCδ phosphorylation and its action [37]. We found that wogonin treatment reduced PKCδ-Thr505 phosphorylation in a dose-and time-dependent manner ( Fig.…”

Distinct PTPmu-associated signaling molecules differentially regulate neurite outgrowth on E-, N-, and R-cadherin

“…After this initial publication, it was reported that Rottlerin is ineffective at directly inhibiting PKCδ activity but likely inhibits the enzyme by an indirect mechanism via ATP depletion, reactive oxygen species generation and altering PKCδ tyrosine phosphorylation (Soltoff, 2007). Additional studies have demonstrated, however, that Rottlerin does decrease PKCδ activity (Chaudhuri et al, 2005; Fan et al, 2006; Fan et al, 2009). In addition, treatment of various neuronal cell types with Rottlerin results in data comparable to that of the same neuronal cell types treated with dominant negative PKCδ (Zhang et al, 2007), neurons from PKCδ knockout mice (Zhang et al, 2007; Zhang et al, 2009), PKCδ-small interfering RNA (Zhang et al, 2007) or catalytically inactive PKCδK376R (Kanthasamy et al, 2008) supporting the assertion that Rottlerin inhibits PKCδ activity.…”