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Triptolide inhibits the proliferation of cells from lymphocytic leukemic cell lines in association with downregulation of NF-κB activity and miR-16-1*.

ABSTRACT: AIM: To examine the effects of triptolide (TPL) on T-cell leukemia cells and identify their underlying mechanisms. METHODS: The cytotoxicity of TPL was assessed by MTT assay. Cell apoptosis was determined using annexin V and DAPI staining and analyzed by flow cytometry or fluorescence microscopy. The activation of caspase pathways and the expression of nuclear factor κB (NF-κB) p65 were examined by Western blotting. Differences in microRNA (miRNA) expression in Molt-4 and Jurkat cells before and after TPL treatment were identified using microarrays and real-time RT-PCR, respectively. RESULTS: TPL 20-160 nmol/L treatment potently inhibited cell growth and induced apoptosis in T-cell lymphocytic leukemia cell lines. Molt-4 and Jurkat cells, however, were more sensitive to TPL than L428 and Raji cells. After 24 h of treatment, bortezomib abrogated the growth of Molt-4 and Jurkat cells with an IC(50) of 15.25 and 24.68 nmol/L, respectively. Using Molt-4 cells, we demonstrated that treatment 20-80 nmol/L inhibited the translocation of NF-κB p65 from the cytoplasm to the nucleus and that phosphorylated NF-κB p65 in nuclear extracts was down-regulated in a dose-dependent manner. Similar results were also seen in Jurkat cells but not in L428 cells, as these cells are resistant to TPL and bortezomib (a NF-κB inhibitor). Twenty-three miRNAs were differentially expressed after TPL treatment. Functional analysis revealed that TPL treatment could inhibit expression of miR-16-1* and that transfection of miR-16-1* led to significantly decreased apoptosis induced by TPL. CONCLUSION: Our in vitro studies suggest that TPL might be an effective therapeutic agent for treatment of T-cell lymphocytic leukemia and that its cytotoxic effects could be associated with inhibition of NF-κB and down-regulation of miR-16-1*.

SUBMITTER: Meng HT

PROVIDER: S-EPMC4001980 | biostudies-other | 2011 Apr

REPOSITORIES: biostudies-other

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Project description:BackgroundControlling thrombin-driven microglial activation may serve as a therapeutic target for intracerebral hemorrhage (ICH). Here, we investigated microRNA (miRNA)-based regulation of thrombin-driven microglial activation using an in vitro thrombin toxicity model applied to primary human microglia.MethodsA miRNA array identified 22 differential miRNA candidates. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) identified miR-181c as the most significantly downregulated miRNA. TargetScan analysis identified mixed lineage leukemia-1 (MLL1) as a putative gene target for miR-181c. qRT-PCR was applied to assess tumor necrosis factor-alpha (TNF-α), miR-181c, and MLL1 levels following thrombin or proteinase-activated receptor-4-specific activating peptide (PAR4AP) exposure. Anti-TNF-α antibodies and tumor necrosis factor receptor (TNFR) silencing were employed to test TNF-α/TNFR dependence. A dual-luciferase reporter system and miR-181c mimic transfection assessed whether mir-181c directly binds to and negatively regulates MLL1. Nuclear factor kappa-B (NF-κB)-dependent luciferase reporter assays and NF-κB target gene expression were assessed in wild-type (MLL1+) and MLL1-silenced cells.ResultsThrombin or PAR4AP-induced miR-181c downregulation (p < 0.05) and MLL1 upregulation (p < 0.05) that were dependent upon TNF-α/TNFR. miR-181c decreased wild-type MLL1 3'-UTR luciferase reporter activity (p < 0.05), and a miR-181c mimic suppressed MLL1 expression (p < 0.05). Thrombin treatment increased, while miR-181c reduced, NF-κB activity and NF-κB target gene expression in both wild-type (MLL1+) and MLL1-silenced cells (p < 0.05).ConclusionsThrombin-induced, TNF-α/TNFR-dependent miR-181c downregulation promotes MLL1 expression, increases NF-κB activity, and upregulates NF-κB target gene expression. As miR-181c opposes thrombin's stimulation of pro-inflammatory NF-κB activity, miR-181c mimic therapy may show promise in controlling thrombin-driven microglial activation following ICH.

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Triptolide inhibits the proliferation of cells from lymphocytic leukemic cell lines in association with downregulation of NF-κB activity and miR-16-1*.

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