Blood Res.  2018 Mar;53(1):53-60. 10.5045/br.2018.53.1.53.

Investigation of BAX and BCL2 expression and apoptosis in a resveratrol- and prednisolone-treated human T-ALL cell line, CCRF-CEM

• Khanzadeh
• Hagh MF
• Talebi M
• Yousefi B
• Azimi A
• Hossein pour feizi AA
• Baradaran

• KMID: 2414365
• DOI: http://doi.org/10.5045/br.2018.53.1.53

Abstract

BACKGROUND
The numerous side effects and chemo-resistance of conventional chemical drugs in the treatment of malignancies have led to consideration of the anti-cancer properties of natural products. In the present study, we aimed to explore the apoptotic effect of two natural components, resveratrol and prednisolone, on the T acute lymphoblastic leukemia (ALL) cell line, CCRF-CEM. Our findings suggested the incorporation of these natural agents into drug regimens to treat patients with ALL.
METHODS
In this study, we investigated the effect of different doses of resveratrol (15, 50 and 100 µM) and prednisolone (700 µM) on BAX (apoptosis promoter) and BCL2 (apoptosis inhibitor) expressions following 24 and 48 hours of treatment on CCRF-CEM cells, using real-time PCR, and on apoptosis induction using flow cytometry.
RESULTS
The results showed a time- and dose-dependent increase in BAX expression and a decrease in BCL2 expression. Apoptosis was induced in CCRF-CEM cells treated with resveratrol and prednisolone for 24 and 48 hours. Combined resveratrol and prednisolone treatment showed synergistic effects on the overexpression of BAX and the downregulation of BCL2. The drug combination had a greater influence on apoptosis induction compared with either drug administered alone after 48 hours of treatment.
CONCLUSION
The results of this study suggested that resveratrol exhibited a remarkable efficacy to improve the influence of glucocorticoids drugs, especially prednisolone, to induce apoptosis in the CCRF-CEM cell line.

Keyword

Precursor cell lymphoblastic leukemia-lymphoma; Resveratrol; Prednisolone; BAX; BCL2; Apoptosis

MeSH Terms

Apoptosis*
Biological Products
Cell Line*
Down-Regulation
Flow Cytometry
Glucocorticoids
Humans*
Precursor Cell Lymphoblastic Leukemia-Lymphoma
Precursor T-Cell Lymphoblastic Leukemia-Lymphoma*
Prednisolone
Real-Time Polymerase Chain Reaction
Biological Products
Glucocorticoids
Prednisolone

Figure

• 

  Fig. 1 Evaluation of the effects of resveratrol (RES) and prednisolone (P) on the BAX expression level using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). CCRF-CEM cells were cultured and treated with resveratrol (15, 50, and 100 µM) and prednisolone (700 µM). BAX was examined in CCRF-CEM cells after 12, 24, and 48 hours of resveratrol and prednisolone treatment. CCRF-CEM cells without treatment were used as a control to evaluate the relative expression of BAX. The data are presented as mean±SD. The relative gene expression of the control was set as 1. (A) The BAX expression level in CCRF-CEM cells after 12 hours of treatment. (B) The BAX expression level in CCRF-CEM cells after 24 hours of treatment. (C) The BAX expression level in CCRF-CEM cells after 48 hours of treatment (a)P<0.05, b)P<0.01).

• 

  Fig. 2 Evaluation of the effects of resveratrol (RES) and prednisolone (P) on the BCL2 expression level using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). CCRF-CEM cells were cultured and treated with resveratrol (15, 50, and 100 µM) and prednisolone (700 µM). BCL2 was examined in CCRF-CEM cells after 12, 24, and 48 hours of resveratrol and prednisolone treatment. CCRF-CEM cells without treatment were used as a control to evaluate the relative expression of BCL2. The data are presented as mean±SD. The relative gene expression of the control was set as 1. (A) The BCL2 expression level in CCRF-CEM cells after 12 hours of treatment. (B) The BCL2 expression level in CCRF-CEM cells after 24 hours of treatment. (C) The BCL2 expression level in CCRF-CEM cells after 48 hours of treatment (a)P<0.05, b)P<0.01).

• 

  Fig. 3 Resveratrol (RES) and prednisolone (P) induced apoptosis in a dose- and time-dependent manner. CCRF-CEM cells were treated with resveratrol (15, 50, and 100 µM) and prednisolone (700 µM) and induced apoptosis in a dose-dependent manner after 24 hours (A) and 48 hours (B) (a)P<0.05, b)P<0.01).

• 

  Fig. 4 Representative data of double staining for annexin V-propidium iodide (PI) uptake from test groups and control by fluorescence activated cell sorting (FACS), after 24 hours of incubation. The percentage of PI-negative and annexin V-positive cells that were in early apoptosis, and PI positive and annexin V-positive cells that were dead or in end-stage apoptosis, are represented in each quadrant. (A) control, (B) 15 µM resveratrol (R), (C) 50 µM R, (D) 100 µM R, (E) 700 µM prednisolone (P), (F) 50 µM R+700 µM P. Ethanol, vehicle control.Abbreviation: FITC, fluorescein isothiocyanate.

• 

  Fig. 5 Representative data of double staining for annexin V-propidium iodide (PI) uptake from test groups and control by fluorescence activated cell sorting (FACS), after 48 hours of incubation. The percentage of PI-negative and annexin V-positive cells that were in early apoptosis, and PI positive and annexin V-positive cells that were dead or in end-stage apoptosis, are represented in each quadrant. (A) control, (B) 15 µM resveratrol (R), (C) 50 µM R, (D) 100 µM R, (E) 700 µM prednisolone (P), (F) 50 µM R+700 µM P. Ethanol, vehicle control.Abbreviation: FL1-H, height in the FL1 channel.

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