Celastrol induces cell cycle arrest by MicroRNA-21-mTOR-mediated inhibition p27 protein degradation in gastric cancer - PubMed (original) (raw)

Celastrol induces cell cycle arrest by MicroRNA-21-mTOR-mediated inhibition p27 protein degradation in gastric cancer

Min Sha et al. Cancer Cell Int. 2015.

Abstract

Objective: Celastrol has anti-cancer effects by increase of apoptosis of gastric cancer cells. However, its role in gastric cancer cell cycle is still unclear. The aim of this study was to investigate the effect and mechanism of celastrol on gastric cancer cell cycle.

Methods: The effects of celastrol on cell cycle in BGC-823 and MGC-803 cells were assayed via flow cytometry analysis. The expression of p27 and mTOR was detected by real-time PCR and western blot. The activity of mTOR and mTORC2 was measured by mTOR and mTORC2 kinase assays. miR-21 mimic was used to up-regulate miR-21 expression and mTOR expression plasmid was used to increase mTOR level in gastric cancer cells.

Results: Celastrol caused G2/M cell-cycle arrest that was accompanied by the down-regulation of miR-21 expression. In particular, miR-21 overexpression reversed cell cycle arrest effects of celastrol. Further study showed that celastrol increased levels of the p27 protein by inhibiting its degradation. miR-21 and mTOR signaling pathway was involved in the increase of p27 protein expression in BGC-823 and MGC-803 cells treated with celastrol. Significantly, miR-21 overexpression restored the decrease of mTOR activity in cells exposed celastrol.

Conclusions: The effect of celastrol on cell cycle arrest of gastric cancer cells was due to an increase of p27 protein level via inhibiting miR-21-mTOR signaling pathway.

Keywords: Celastrol; Cell cycle arrest; mTOR; miR-21; p27.

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Figures

Fig. 1

Fig. 1

Celastrol induced G2/M cycle arrest of gastric cancer cells. Celastrol (2 μM) induced the accumulation of cells in the G2/M phase and decreased the cell population in the G0/G1 phases in BGC-823 (a) and MGC-803 cells (b). **P < 0.01 vs. control group

Fig. 2

Fig. 2

Up-regulation of miR-21 can reverse the effect of celastrol on cell cycle arrest. Flow cytometry analysis revealed that miR-146a mimic reversed the increase of G2/M-phase and the decrease of the G0/G1-phase induced by celastrol in BGC-823 (a) and MGC-803 cells (b). *P < 0.01 vs. Pre-NC

Fig. 3

Fig. 3

Celastrol increased p27 protein level in gastric cancer cells through miR-21. Western blot showed that celastrol significantly increased p27 protein level in BGC-823and MGC-803 cells (a). The real-time PCR revealed that celastrol had no effect on p27 mRNA level in BGC-823and MGC-803 cells (b). BGC-823and MGC-803 cells were treated without (control) or with celastrol for 24 h, at which time cycloheximide (50 μg/ml) was added for the indicated periods of time. All the treated cells were then harvested and lysed for western blot analyses to determine the protein levels of p27 and β-actin (as a loading control). Representative immunoblots and a graph showing protein levels of p27 relative to β-actin are shown (c). Western blot analyses revealed that miR-21 mimic reversed the increase of p27 protein level in BGC-823 and MGC-803 cells treated with celastrol (d). **P < 0.01, indicate significant differences from the respective control groups

Fig. 4

Fig. 4

Celastrol inhibited mTOR activity through miR-21. Western blot showed that celastrol significantly decreased p-mTOR level in BGC-823and MGC-803 cells (a). mTOR and mTORC2 kinase assays revealed that miR-21 mimic reversed the decrease of mTOR (b) and mTORC2 (c) activity. **P < 0.01, indicate significant differences from the respective control groups

Fig. 5

Fig. 5

Down-regulation of miR-21 expression can inhibit mTOR activity. mTOR and mTORC2 kinase assays revealed that miR-21 inhibitor significantly reduced mTOR (a) and mTORC2 (b) activity. **P < 0.01, indicate significant differences from the respective control groups

Fig. 6

Fig. 6

Celastrol increased p27 expression through mTOR. Western blot analyses revealed that mTORSL1+IT reversed the increase of p27 protein level in BGC-823 and MGC-803 cells treated with celastrol (a). The real-time PCR revealed that mTORSL1+IT had no effect of p27 mRNA level in BGC-823 and MGC-803 cells treated with celastrol (b)

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