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Original Article

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Blood Res 2018; 53(1):

Published online March 31, 2018

https://doi.org/10.5045/br.2018.53.1.53

© The Korean Society of Hematology

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

Taghi Khanzadeh1,2,3, Majid Farshdousti Hagh2*, Mehdi Talebi3,4, Bahman Yousefi1,5, Ako Azimi6, Abbas Ali Hossein pour feizi3,4, and Behzad Baradaran2,4

Author Info. +

1Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran.

2Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.

3Hematology and Oncology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.

4Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.

5Department of Biochemistry, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran.

6Department of Basic Sciences, Maragheh University of Medical Sciences, Maragheh, Iran.

Correspondence to : Majid Farshdousti Hagh, Ph.D. Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. m.farshdousti@gmail.com

Received: February 12, 2017; Revised: December 4, 2017; Accepted: December 12, 2017

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

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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.

Keywords Precursor cell lymphoblastic leukemia-lymphoma, Resveratrol, Prednisolone, BAX, BCL2, Apoptosis

Article

Original Article

Blood Res 2018; 53(1): 53-60

Published online March 31, 2018 https://doi.org/10.5045/br.2018.53.1.53

Copyright © The Korean Society of Hematology.

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

Taghi Khanzadeh1,2,3, Majid Farshdousti Hagh2*, Mehdi Talebi3,4, Bahman Yousefi1,5, Ako Azimi6, Abbas Ali Hossein pour feizi3,4, and Behzad Baradaran2,4

1Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran.

2Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.

3Hematology and Oncology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.

4Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.

5Department of Biochemistry, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran.

6Department of Basic Sciences, Maragheh University of Medical Sciences, Maragheh, Iran.

Correspondence to:Majid Farshdousti Hagh, Ph.D. Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. m.farshdousti@gmail.com

Received: February 12, 2017; Revised: December 4, 2017; Accepted: December 12, 2017

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.

Keywords: Precursor cell lymphoblastic leukemia-lymphoma, Resveratrol, Prednisolone, BAX, BCL2, Apoptosis

Fig 1.

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Figure 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).

Blood Research 2018; 53: 53-60https://doi.org/10.5045/br.2018.53.1.53

Fig 2.

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Figure 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).

Blood Research 2018; 53: 53-60https://doi.org/10.5045/br.2018.53.1.53

Fig 3.

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Figure 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).

Blood Research 2018; 53: 53-60https://doi.org/10.5045/br.2018.53.1.53

Fig 4.

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Figure 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.

Blood Research 2018; 53: 53-60https://doi.org/10.5045/br.2018.53.1.53

Fig 5.

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Figure 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.

Blood Research 2018; 53: 53-60https://doi.org/10.5045/br.2018.53.1.53
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