J Korean Med Sci.  2014 Nov;29(11):1464-1472. 10.3346/jkms.2014.29.11.1464.

Knockdown of Bcl-xL Enhances Growth-Inhibiting and Apoptosis-Inducing Effects of Resveratrol and Clofarabine in Malignant Mesothelioma H-2452 Cells

• Lee YJ
• Hwang IS
• Lee YJ
• Lee CH
• Kim SH
• Nam HS
• Choi YJ
• Lee SH

• KMID: 2069926
• DOI: http://doi.org/10.3346/jkms.2014.29.11.1464

Abstract

Mcl-1 and Bcl-xL, key anti-apoptotic proteins of the Bcl-2 family, have attracted attention as important molecules in the cell survival and drug resistance. In this study, we investigated whether inhibition of Bcl-xL influences cell growth and apoptosis against simultaneous treatment of resveratrol and clofarabine in the human malignant mesothelioma H-2452 cells. Resveratrol and clofarabine decreased Mcl-1 protein levels but had little effect on Bcl-xL levels. In the presence of two compounds, any detectable change in the Mcl-1 mRNA levels was not observed in RT-PCR analysis, whereas pretreatment with the proteasome inhibitor MG132 led to its accumulation to levels far above basal levels. The knockdown of Bcl-xL inhibited cell proliferation with cell accumulation at G2/M phase and the appearance of sub-G0/G1 peak in DNA flow cytometric assay. The suppression of cell growth was accompanied by an increase in the caspase-3/7 activity with the resultant cleavages of procaspase-3 and its substrate poly (ADP-ribose) polymerase, and increased percentage of apoptotic propensities in annexin V binding assay. Collectively, our data represent that the efficacy of resveratrol and clofarabine for apoptosis induction was substantially enhanced by Bcl-xL-lowering strategy in which the simultaneous targeting of Mcl-1 and Bcl-xL could be a more effective strategy for treating malignant mesothelioma.

Keyword

Mesothelioma, Malignant; Mcl-1; Bcl-xL; Resveratol; Clofarabine; Apoptosis

MeSH Terms

Adenine Nucleotides/*pharmacology
Antimetabolites, Antineoplastic/*pharmacology
Apoptosis/*drug effects
Arabinonucleosides/*pharmacology
Caspase 3/metabolism
Caspase 7/metabolism
Cell Line, Tumor
Cell Proliferation/drug effects
G2 Phase Cell Cycle Checkpoints/drug effects
Gene Knockdown Techniques
Humans
Leupeptins/pharmacology
Lung Neoplasms/metabolism/pathology
M Phase Cell Cycle Checkpoints/drug effects
Mesothelioma/metabolism/pathology
Myeloid Cell Leukemia Sequence 1 Protein/antagonists & inhibitors/genetics/metabolism
RNA Interference
RNA, Messenger/metabolism
RNA, Small Interfering/metabolism
Stilbenes/*pharmacology
bcl-X Protein/antagonists & inhibitors/*genetics/*metabolism
Adenine Nucleotides
Antimetabolites, Antineoplastic
Arabinonucleosides
Leupeptins
Myeloid Cell Leukemia Sequence 1 Protein
RNA, Messenger
RNA, Small Interfering
Stilbenes
bcl-X Protein
Caspase 3
Caspase 7

Figure

• 

  Fig. 1 Effects of resveratrol and clofarabine, alone or in combination, on cell proliferation. (A) Cells were treated with various concentrations of clofarabine (0, 20, 40, 80, 160, and 320 nM) or resveratrol (0, 10, 15, 20, 25, and 30 µM) for 72 hr. (B) Cells were co-treated resveratrol (15 µM) and clofarabine (0, 20, 40, 80, 160, and 320 nM, respectively) for 72 hr. (C) Cells were co-treated with resveratrol (15 µM) and clofarabine (40 nM), alone or in combination, for 1, 2, and 3 days. The percentage of viable cells was determined by using the MTT assay. Error bars represent the mean ± SEM for three independent experiments. *P < 0.05 compared to respective controls. Clo, clofarabine; Res, resveratrol.

• 

  Fig. 2 Effects of resveratrol and clofarabine on Mcl-1 and Bcl-xL protein levels. (A) Cells were co-treated with resveratrol (15 µM) and clofarabine (40 nM), alone or in combination, for 24 hr. (B) Cells were co-treated with resveratrol (15 µM) and clofarabine (40 nM) for 1, 2, and 3 days. (C) Cells were co-treated with resveratrol (5, 10, 15, and 20 µM) and clofarabine (40 nM) for 24 hr. Whole cell lyastes were analyzed by Western blot analysis using antibodies against Mcl-1, Bcl-xL, and β-actin. *P < 0.05 compared to respective controls. Clo, clofarabine; Res, resveratrol.

• 

  Fig. 3 Regulation of Mcl-1 protein level by resveratrol and clofarabine. (A) Cells were seeded in 6-well culture plate and were co-treated with resveratrol (15 µM) and clofarabine (40 nM) for the indicated times, after which the total RNA was isolated and subjected to RT-PCR analysis. GAPDH primers were used to amplify as an internal standard. (B) Cells were treated with MG132 (10 µM) for 2 hr prior to incubation with resveratrol (15 µM) and clofarabine (40 nM) for additional 24 hr. (C) Cells were pretreated with 0.1 µM cycloheximide (CHX) alone or CHX plus 10 µM MG132, for varying intervals as indicated. Whole cell lyastes were obtained and subjected to Western blot analysis using antibodies against Mcl-1, Bcl-xL, and β-actin. Normalized intensity of Mcl-1 or Bcl-xL versus β-actin was presented as the mean value from two independent experiments. Clo/Res, clofarabine plus resveratrol.

• 

  Fig. 4 Effects of Bcl-xL knockdown on proliferation of H-2452 cells. Cells were transfected with 10 nM Bcl-xL siRNA (siBcl-xL) alone or in combination with 10 nM Mcl-1 siRNA (siMcl-1) for 24 hr, and then incubated with resveratrol (15 µM) and clofarabine (40 nM) for the indicated times, after which they were processed for MTT assay (A), for the determination of sub-G0/G1 peak (B) and cell distribution at G1, S, and G2/M phases (C) using a flow cytometry after staining with propidium iodide (20 µg/mL). Error bars represent the mean ± SEM for three independent experiments. *P < 0.05 compared to respective si-C controls; #P < 0.05 compared to respective Clo/Res (-) groups. Clo/Res, clofarabine plus resveratrol.

• 

  Fig. 5 Effects of Bcl-xL knockdown on apoptosis mediated by resveratrol and clofarabine. Cells were transfected with 10 nM Bcl-xL siRNA (siBcl-xL) alone or in combination with 10 nM Mcl-1 siRNA (siMcl-1) for 24 hr, and then incubated with resveratrol (15 µM) and clofarabine (40 nM) for the indicated times, after which they were processed for caspase-3/7 activity as measured by ApoTox-Glo™ Triplex Assay (A), for Western blot analysis using antibodies against Mcl-1, Bcl-xL, PARP, procaspase-3, cleaved caspase-3, and β-actin (B), and for Annexin V binding assay using a Muse cell analyzer. Error bars represent the mean ± SEM for three independent experiments. *P < 0.05 compared to respective si-C controls; #P < 0.05 compared to respective Clo/Res (-) groups. Clo/Res, clofarabine plus resveratrol.

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