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, 14 (1), 45-55

Overexpression of Cyclin B1 Antagonizes Chemotherapeutic-Induced Apoptosis Through PTEN/Akt Pathway in Human Esophageal Squamous Cell Carcinoma Cells

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Overexpression of Cyclin B1 Antagonizes Chemotherapeutic-Induced Apoptosis Through PTEN/Akt Pathway in Human Esophageal Squamous Cell Carcinoma Cells

Yunwei Ou et al. Cancer Biol Ther.

Abstract

The role of cyclin B1 in the clinical therapeutic sensitivity of human esophageal squamous cell carcinoma (ESCC) remains to be defined. In this study, we found that elevated cyclin B1 expression attenuated the apoptosis induced by cisplatin or paclitaxel, while knockdown of cyclin B1 enhanced cisplatin or paclitaxel sensitivity in ESCC cells. Cyclin B1-mediated apoptosis may rely on the Bcl-2-dependent mitochondria-regulated intrinsic death pathway, and the antagonizing effect of cyclin B1 on chemotherapeutic agent-induced apoptosis was through PTEN/Akt pathway. Therefore, cyclin B1 might be a therapeutic target for the development of specific and efficient approaches in the treatment of ESCC.

Figures

<b>Figure 1A–C</b>.
Figure 1A–C.
Overexpression of cyclin B1 contributes to KYSE150 cells resistance to cisplatin or paclitaxel. (A) Western blot analysis of the protein levels of cyclin B1 and actin in KYSE150/pcDNA3.1 and High-CycB1 1–2 cells. (B) Effects of cyclin B1 overexpression on the viability in the KYSE150/pcDNA3.1, High-CycB1–1 and High-CycB1–2 cell lines after treatment with cisplatin, paclitaxel or a control reagent by MTS assay. (C)(a) KYSE150/pcDNA3.1, High-CycB1–1 and High-CycB1–2 cells were not treated or treated with cisplatin and paclitaxel for 24 h, and the cells stained with annexin V and propidium iodide (PI) were analyzed by flow cytometry. The lower left quadrant contains the vital (annexin V-/PI-) population, the upper left quadrant contains the damaged (annexin V-/PI+) population, the upper right quadrant contains the late apoptotic (annexin V+/PI+) cells and the lower right quadrant contains the early apoptotic (annexin V+/PI-) cells. (C)(b) The bar chart shows the percentage of apoptotic cells (early apoptotic + late apoptotic).
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Figure 1D and E. (D)(a) KYSE150/pcDNA3.1, High-CycB1–1 and High-CycB1–2 cells were treated with cisplatin, paclitaxel or a control reagent for 24 h and apoptotic cells were analyzed by DAPI stain (200 × ). (D)(b) The bar chart shows the percentage of apoptotic cells. (E)(a) Overexpression of cyclin B1 inhibits PARP cleavages induced by cisplatin and paclitaxel. KYSE150/pcDNA3.1, High-CycB1–1, High-CycB1–2 cells were treated with cisplatin, paclitaxel or a control reagent for 24 h. PARP were detected by western blot analysis. Actin was used as an equal loading control. (E)(b) The bar chart shows the percentage of PARP-cleaved [PARP-cleaved / (PARP-cleaved + full-length PARP)]. All experiments were performed at least three times with consistent and repeatable results. Each value is expressed as mean ± SD (n = 3). * and ※ p < 0.05 as compared with the control.
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Figure 2A–C. Suppression of cyclin B1 contributes to sensitization of EC9706 cells to cisplatin or paclitaxel. (A) Western blot analysis of the protein levels of cyclin B1 and actin in EC9706 control-siRNA and CycB1 siRNA 1–2 cells; (B) Effects of cyclin B1 suppression on the viability in the EC9706 control-siRNA and CycB1 siRNA 1–2 cells lines after treatment with cisplatin, paclitaxel or a control reagent by MTS assay. (C)(a) EC9706 control-siRNA and CycB1 siRNA 1–2 cells were not treated or treated with cisplatin and paclitaxel for 24 h and the cells stained with annexin V and propidium iodide (PI) were analyzed by flow cytometry. The lower left quadrant contains the vital (annexin V-/PI-) population, the upper left quadrant contains the damaged (annexin V-/PI+) population, the upper right quadrant contains the late apoptotic (annexin V+/PI+) cells and the lower right quadrant contains the early apoptotic (annexin V+/PI-) cells. (C)(b) The bar chart shows the percentage of apoptotic cells (early apoptotic + late apoptotic).
<b>Figure 2D and E</b>.
Figure 2D and E.
(D)(a) EC9706 control-siRNA and CycB1 siRNA 1–2 cells were treated with cisplatin, paclitaxel or a control reagent for 24 h, and apoptotic cells were analyzed by DAPI stain (200 × ). (D)(b) The bar chart shows the percentage of apoptotic cells. (Ea) Suppression of cyclin B1 increases PARP cleavages induced by cisplatin or paclitaxel. EC9706 control-siRNA and CycB1 siRNA 1–2 cells were treated with cisplatin, paclitaxel or a control reagent for 24 h. PARP were detected by western blot analysis. Actin was used as an equal loading control. (E)(b) The bar chart shows the percentage of PARP-cleaved [PARP-cleaved / (PARP-cleaved + full-length PARP)]. All experiments were performed at least three times with consistent and repeatable results. Each value is expressed as mean ± SD (n = 3). *and ※ p < 0.05 as compared with the control.
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Figure 3. Western blot analysis of the protein levels of cleaved-caspase-9, cleaved-caspase-3, cleaved-caspase-8, Bcl-2 and actin in KYSE150/pcDNA3.1, High-CycB1 1–2, EC9706 control-siRNA and CycB1 siRNA 1–2 cells after treatment with cisplatin, paclitaxel or a control reagent for 24 h. All experiments were performed at least three times with consistent and repeatable results.
<b>Figure 4 and B</b>.
Figure 4 and B.
PTEN-PI3K/Akt pathway involves in cyclin B1-mediated chemotherapeutic-induced apoptosis. (A) Western blot analysis of the protein levels of PTEN, p53, Akt, p-Akt and actin in KYSE150, pcDNA3.1, High-CycB1 1–2, EC9706, control-siRNA and CycB1 siRNA 1–2 cells after treatment with cisplatin, paclitaxel or a control reagent for 24 h. (B) Inhibitor of PI3K/Akt (LY 294002) reduces the phosphorylation of Akt (p-Akt). KYSE150, pcDNA3.1, High-CycB1 1–2, EC9706, control-siRNA and CycB1 siRNA 1–2 cells were incubated with LY 294002 (50 μmol/L) for 2 h. Cell lysates were subjected to western blotting with indicated antibodies. Protein expression levels were normalized with actin.
<b>Figure 4 and D</b>.
Figure 4 and D.
(C) Effects of LY 294002 on cyclin B1-mediated chemotherapeutic-induced apoptosis in KYSE150, pcDNA3.1, High-CycB1 1–2, EC9706, control-siRNA and CycB1 siRNA 1–2 cells by MTS assay. Cells were preincubated with LY 294002 (50 μmol/L) for 2 h, then cisplatin or paclitaxel was added and incubated for 24 h. Twenty μl of MTS reagent was added to each well, followed by a 1–4 h incubation at 37°C and 5% CO2. The OD was read at 490 nm with a microplate reader. (D) Effects of LY 294002 on cyclin B1-mediated chemotherapeutic-induced apoptosis in KYSE150, pcDNA3.1, High-CycB1 1–2, EC9706, control-siRNA and CycB1 siRNA 1–2 cells by DAPI stain. Cells were preincubated with LY 294002 (50 μmol/L) for 2 h, then cisplatin or paclitaxel was added and incubated for 24 h and apoptosis was detected by DAPI stain. All experiments were performed at least three times with consistent and repeatable results. Each value is expressed as mean ± SD (n = 3). *and ※ p < 0.05 as compared with the control.

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