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. 2008 Jul;10(7):736-44.
doi: 10.1593/neo.08304.

Inhibition of STAT3 Expression and Signaling in Resveratrol-Differentiated Medulloblastoma Cells

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Free PMC article

Inhibition of STAT3 Expression and Signaling in Resveratrol-Differentiated Medulloblastoma Cells

Li-Jun Yu et al. Neoplasia. .
Free PMC article

Abstract

In this study, the potential influence of resveratrol (3,5,4'-trihydroxy-trans-stilbene) in signal transducer and activator of transcription 3 (STAT3) signaling of medulloblastoma cells was evaluated by checking the status of STAT3 signaling and its downstream gene expression in two medulloblastoma cell lines (UW228-2 and UW228-3) with and without resveratrol treatment. The results revealed that resveratrol induced neuronal differentiation of medulloblastoma cells. Signal transducer and activator of transcription 3 expression and phosphorylation were detected in normally cultured UW228-2 and UW228-3 cells that were apparently attenuated after resveratrol treatment. The expression of STAT3 downstream genes, survivin, cyclin D1, Cox-2, and c-Myc, was suppressed but Bcl-2 was enhanced by resveratrol. Meanwhile, the production and secretion of leukemia inhibitory factor, a STAT3 activator, became active in resveratrol-treated cells. To further ascertain the significance of STAT3 signaling for medulloblastoma cells, AG490, a selective inhibitor of STAT3 phosphorylation, was used to treat UW228-3 cells. Phosphorylation of STAT3 was inhibited by AG490 accompanied with growth suppression, differentiation-like changes, and down-regulation of survivin, cyclin D1, Cox-2, and c-Myc. Our data thus suggest the importance of STAT3 signaling in maintenance and survival of medulloblastoma cells. This signaling may be the major target of resveratrol. Enhanced leukemia inhibitory factor and Bcl-2 expressions in resveratrol-treated cells might reflect a compensatory response to the loss of STAT3 function.

Figures

Figure 1
Figure 1
Evaluation of chemosensitivities ofUW228-2 andUW228-3 cells to resveratrol treatment by flow cytometry analysis (A) and trypan blue discrimination of stained (unviable) and unstained (viable) cells (B). 3R indicates resveratrol-treated UW228-3 cells; 2R, resveratrol-treated UW228-2 cells; AD, cells died of apoptosis; G1, cells at G1 phase.
Figure 2
Figure 2
Evaluation of STAT3 phosphorylation and expression of survivin, cyclin D1, and Bcl-2 as well as Cox-2, c-Myc, and synaptophysin in medulloblastoma cells by ICC and IF staining (A), RT-PCR (B), and Western blot analysis (C). NC indicates normally cultured/nontreated cells; Res, 100-µM resveratrol-treated cells. β-Actin was used as a Ruantitative control in RT-PCR and Western blot analysis. The negative data of GFAP examination were not shown.
Figure 3
Figure 3
Analyses of LIF expression and intracellular distribution in UW228-3 cells without (NC) and with (Res) resveratrol treatment by ICC and IF staining (A), immunoelectron microscopic examination (B), and RT-PCR and Western blot analysis (C). LIF secretion activity was checked by Western blot analysis using concentrated condition media of UW228-3 cells cultured for 48 hours without and with resveratrol treatment. The medium containing 10% FBS was used as background control (D). In (A), the arrows indicate the cells shown in the inserts with high magnification. In (B), the magnification scales of electron microscopic illustrations are indicated as x10K and x20K (10,000 and 20,000 times); the black arrows indicate the vesicular structures and the red ones indicate the vesicles shown in the inserts.
Figure 4
Figure 4
Morphologic changes, synaptophysin expression and the state of STAT3 phosphorylation of UW228-3 cells without (NC) and with AG490 treatment. The expression patterns of STAT3 downstream genes in those cells are summarized in Table 2.

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