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. 2012 Oct;4(4):723-726.
doi: 10.3892/ol.2012.805. Epub 2012 Jul 16.

Depletion of Bmi-1 enhances 5-fluorouracil-induced apoptosis and autophagy in hepatocellular carcinoma cells

Jing Wu  1 , Dong Hu, Rongbo Zhang
Affiliations

Depletion of Bmi-1 enhances 5-fluorouracil-induced apoptosis and autophagy in hepatocellular carcinoma cells

Jing Wu et al. Oncol Lett. 2012 Oct.

Abstract

5-fluorouracil (5-FU) is one of the standard chemoradiotherapy regimens for hepatocellular carcinoma (HCC) treatment. B-cell-specific Moloney murine leukemia virus insertion site 1 (Bmi-1) has been demonstrated to regulate proliferation. Additionally, Bmi-1 overexpression has been identified in HCC cell lines and correlates with the advanced invasive stage of tumor progression and poor prognosis. In this study, we examined the effects of 5-FU treatment on cell growth in HCC cells with or without Bmi-1 depletion. The IC(50) values of 5-FU were significantly decreased to a greater extent in cells with Bmi-1 knockdown. Depletion of Bmi-1 increased sensitivity of the cells to 5-FU and increased apoptosis. Knockdown of endogenous Bmi-1 led to a substantial reduction in the levels of phospho-AKT and Bcl-2 with a concomitant increase in the levels of Bax. Additionally, 5-FU induced the conversion/turnover of microtubule-associated protein 1 light chain 3 (LC3). Knockdown of endogenous Bmi-1 led to an increase in the levels of Beclin-1 and the accumulation of LC3-II. Together, these findings reveal that Bmi-1 depletion enhanced the chemosensitivity of HCC cells by inducing apoptosis and autophagy, which is associated with the PI3K/AKT and Bcl-2/Beclin-1 pathways.

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Figures

Figure 1.
Figure 1.
Effect of Bmi-1 downregulation on the chemosensitivity of SK-HEP-1 and SMMC-7721 cells to 5-FU treatment. (A and B) Effect of siBmi1 in SK-HEP-1 and SMMC-7721 cells. Protein samples were isolated from SK-HEP-1-siBmi1 (SK-siBmi1), SK-HEP-1-siNS (SK-siNS), SMMC-7721-siBmi1 (7721-siBmi1) and SMMC-7721-siNS (7721-siNS) cells. The protein expression of Bmi-1 was detected using western blot analysis. Triplicate experiments revealed consistent results. (C and D) Cell viability was determined using an MTT assay. SK-siBmi1, SK-siNS, 7721-siBmi1 and 7721-siNS cells were treated with increasing concentrations of 5-FU for 72 h. All assays were conducted in triplicate. Bmi-1, B-cell-specific Moloney murine leukemia virus insertion site 1; siBmi1, Bmi-1 targeted siRNA oligonucleotide; siNS, negative control siRNA oligonucleotides; OD, optical density; 5-FU, 5-fluorouracil.
Figure 2.
Figure 2.
Effect of Bmi-1 knockdown on 5-FU-induced apoptosis. Flow cytometry was performed to measure apoptotic rates. Percentages of apoptotic cells were identified in (A) untreated SK-siNS, (B) SK-siNS cells treated with 10 mg/l 5-FU for 72 h, (C) untreated SK-siBmi1 and (D) SK-siBmi1 cells treated with 10 mg/l 5-FU for 72 h. 5-FU, 5-fluorouracil; FITC, fluorescein isothiocyanate; siNS, negative control siRNA oligonucleotides; siBmi1, Bmi-1 targeted siRNA oligonucleotide; Bmi-1, B-cell-specific Moloney murine leukemia virus insertion site 1.
Figure 3.
Figure 3.
Effects of depletion of Bmi-1 expression on p-AKT, Bcl-2, Bax, Beclin-1 and LC3 levels. SK-siNS and SK-siBmi1 cells were incubated for 72 h with or without 5-FU treatment. The protein expression of p-AKT, Bcl-2, Bax, Beclin-1 and LC3 was detected by western blot analysis. All assays were conducted in triplicate. siNS, negative control siRNA oligonucleotides; 5-FU, 5-fluorouracil; siBmi1, Bmi-1 targeted siRNA oligonucleotide; Bmi-1, B-cell-specific Moloney murine leukemia virus insertion site 1; LC3, light chain 3.
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