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Mitochondrial reactive oxygen species regulate adipocyte differentiation of mesenchymal stem cells in hematopoietic stress induced by arabinosylcytosine.

Wang W, Zhang Y, Lu W, Liu K - PLoS ONE (2015)

Bottom Line: ROS levels were detected using the CM-H2DCFDA probe and Mito-SOX dye.The elevated ROS levels induced by Ara-C were caused by both over-generation of mitochondrial ROS and reduction of antioxidant enzymes (Cu/Zn Superoxide dismutase and catalase).Our findings suggest that a mitochondrial-targeted antioxidant could diminish adipocyte differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology, Peking University People's Hospital, Beijing, China; Institute of Hematology, Peking University, Beijing, China.

ABSTRACT

Objective: The increase in adipocytes induced by chemotherapeutic drugs may play a negative role in hematopoietic recovery. However, the mechanism underlying adipocyte differentiation of mesenchymal stem cells (MSCs) in hematopoietic stress is still unknown. Hence, the involvement of reactive oxygen species (ROS) in adipocyte differentiation under hematopoietic stress was investigated in vitro and in vivo.

Methods: The roles of cellular ROS in adipogenesis were investigated in vivo through an adipocyte hyperplasia marrow model under hematopoietic stress induced by arabinosylcytosine (Ara-C) and in vitro via adipocyte differentiation of human MSCs. ROS levels were detected using the CM-H2DCFDA probe and Mito-SOX dye. Adipogenesis was evaluated by histopathology and oil red O staining, whereas detection of mRNA levels of antioxidant enzymes and adipogenesis markers was performed using quantitative real-time polymerase chain reaction analysis.

Results: ROS were found to play an important role in regulating adipocyte differentiation of MSCs by activating peroxisome proliferator-activated receptor gamma (PPARγ,) while the antioxidant N-acetyl-L-cysteine acts through ROS to inhibit adipocyte differentiation. The elevated ROS levels induced by Ara-C were caused by both over-generation of mitochondrial ROS and reduction of antioxidant enzymes (Cu/Zn Superoxide dismutase and catalase). Our findings suggest that a mitochondrial-targeted antioxidant could diminish adipocyte differentiation.

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The potential mechanism for the elevated ROS levels.(A) Detection of mitochondrial ROS in live cells. Live MSCs were treated with Ara-C (100 mM for 24 hours) or without. Red fluorescence indicates the presence of mitochondrial ROS, while blue fluorescence indicates nuclei stained with Hoechest 33342. (B) Median Fluorescence Intensity (MFI) of mitochondrial ROS in the control and Ara-C groups. (C) MFI of total ROS measured by the CM-H2DCFDA probe in the control and Ara-C groups. (D) Gene expression of NOX2 and NOX4 in the control and Ara-C groups. (E) The mean increase/decrease of mitochondrial ROS and total ROS in Ara-C cells treated with DPI or Mito-Tempo. (F) The expression and activity of major antioxidant enzymes, including Cu/Zn-SOD, Mn-SOD and CAT. (G) The level of the antioxidant molecule GSH in the control and Ara-C groups. (H) The expression of major antioxidant enzymes in vivo. (I) The expression of major antioxidant enzymes on Day 7 and Day 14 of MSC differentiation induced by adipose differentiation medium. *P < 0.05.
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pone.0120629.g003: The potential mechanism for the elevated ROS levels.(A) Detection of mitochondrial ROS in live cells. Live MSCs were treated with Ara-C (100 mM for 24 hours) or without. Red fluorescence indicates the presence of mitochondrial ROS, while blue fluorescence indicates nuclei stained with Hoechest 33342. (B) Median Fluorescence Intensity (MFI) of mitochondrial ROS in the control and Ara-C groups. (C) MFI of total ROS measured by the CM-H2DCFDA probe in the control and Ara-C groups. (D) Gene expression of NOX2 and NOX4 in the control and Ara-C groups. (E) The mean increase/decrease of mitochondrial ROS and total ROS in Ara-C cells treated with DPI or Mito-Tempo. (F) The expression and activity of major antioxidant enzymes, including Cu/Zn-SOD, Mn-SOD and CAT. (G) The level of the antioxidant molecule GSH in the control and Ara-C groups. (H) The expression of major antioxidant enzymes in vivo. (I) The expression of major antioxidant enzymes on Day 7 and Day 14 of MSC differentiation induced by adipose differentiation medium. *P < 0.05.

Mentions: First, ROS generation induced by Ara-C was investigated. Intracellular ROS are often generated via the catalytic action of NOX and/or the mitochondrial respiratory chain, which is cell type-dependent. Using MitoSOX, a probe that selectively detects mitochondrial superoxide, it was found that mitochondrial ROS were significantly increased in MSCs treated with Ara-C compared with the control group (Fig. 3A-B). Meanwhile, the total ROS were also significantly increased in the Ara-C group. As shown in Fig. 3C, Ara-C treated cells exhibited an approximate 60% increase in total ROS compared to the control, while mito-SOX exhibited a 48% increase in total ROS compared to the control. To examine the involvement of NOX in the rise of ROS levels induced by Ara-C, real-time PCR was performed to evaluate the NOX isoforms NOX2 and NOX4, which are the main catalytic subunits of the NOX family, in MSCs treated with (100 μM for 24 h) or without Ara-C. As shown in Fig. 3D, expression of both NOX2 and NOX4 was significantly lower in the Ara-C treated- group than in the control. Mitochondrial ROS is another source of intracellular ROS. To further dissect the respective role of NOX and mitochondria as the ROS generator, Ara-C-treated cells were also exposedto DPI and Mito-Tempo, respectively. DPI at the dose used in our experiment (500 nM) was found to block NOX enzymes without affecting mitochondrial ROS production [19], while Mito-Tempo is a well-known mitochondria-targeted antioxidant [13]. As shown in Fig. 3E, only Mito-Tempo led to a significant reduction in mitochondrial ROS and total ROS production. These data indicate that mitochondria, not NOX, are responsible for the over-generation of ROS induced by Ara-C.


Mitochondrial reactive oxygen species regulate adipocyte differentiation of mesenchymal stem cells in hematopoietic stress induced by arabinosylcytosine.

Wang W, Zhang Y, Lu W, Liu K - PLoS ONE (2015)

The potential mechanism for the elevated ROS levels.(A) Detection of mitochondrial ROS in live cells. Live MSCs were treated with Ara-C (100 mM for 24 hours) or without. Red fluorescence indicates the presence of mitochondrial ROS, while blue fluorescence indicates nuclei stained with Hoechest 33342. (B) Median Fluorescence Intensity (MFI) of mitochondrial ROS in the control and Ara-C groups. (C) MFI of total ROS measured by the CM-H2DCFDA probe in the control and Ara-C groups. (D) Gene expression of NOX2 and NOX4 in the control and Ara-C groups. (E) The mean increase/decrease of mitochondrial ROS and total ROS in Ara-C cells treated with DPI or Mito-Tempo. (F) The expression and activity of major antioxidant enzymes, including Cu/Zn-SOD, Mn-SOD and CAT. (G) The level of the antioxidant molecule GSH in the control and Ara-C groups. (H) The expression of major antioxidant enzymes in vivo. (I) The expression of major antioxidant enzymes on Day 7 and Day 14 of MSC differentiation induced by adipose differentiation medium. *P < 0.05.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4359087&req=5

pone.0120629.g003: The potential mechanism for the elevated ROS levels.(A) Detection of mitochondrial ROS in live cells. Live MSCs were treated with Ara-C (100 mM for 24 hours) or without. Red fluorescence indicates the presence of mitochondrial ROS, while blue fluorescence indicates nuclei stained with Hoechest 33342. (B) Median Fluorescence Intensity (MFI) of mitochondrial ROS in the control and Ara-C groups. (C) MFI of total ROS measured by the CM-H2DCFDA probe in the control and Ara-C groups. (D) Gene expression of NOX2 and NOX4 in the control and Ara-C groups. (E) The mean increase/decrease of mitochondrial ROS and total ROS in Ara-C cells treated with DPI or Mito-Tempo. (F) The expression and activity of major antioxidant enzymes, including Cu/Zn-SOD, Mn-SOD and CAT. (G) The level of the antioxidant molecule GSH in the control and Ara-C groups. (H) The expression of major antioxidant enzymes in vivo. (I) The expression of major antioxidant enzymes on Day 7 and Day 14 of MSC differentiation induced by adipose differentiation medium. *P < 0.05.
Mentions: First, ROS generation induced by Ara-C was investigated. Intracellular ROS are often generated via the catalytic action of NOX and/or the mitochondrial respiratory chain, which is cell type-dependent. Using MitoSOX, a probe that selectively detects mitochondrial superoxide, it was found that mitochondrial ROS were significantly increased in MSCs treated with Ara-C compared with the control group (Fig. 3A-B). Meanwhile, the total ROS were also significantly increased in the Ara-C group. As shown in Fig. 3C, Ara-C treated cells exhibited an approximate 60% increase in total ROS compared to the control, while mito-SOX exhibited a 48% increase in total ROS compared to the control. To examine the involvement of NOX in the rise of ROS levels induced by Ara-C, real-time PCR was performed to evaluate the NOX isoforms NOX2 and NOX4, which are the main catalytic subunits of the NOX family, in MSCs treated with (100 μM for 24 h) or without Ara-C. As shown in Fig. 3D, expression of both NOX2 and NOX4 was significantly lower in the Ara-C treated- group than in the control. Mitochondrial ROS is another source of intracellular ROS. To further dissect the respective role of NOX and mitochondria as the ROS generator, Ara-C-treated cells were also exposedto DPI and Mito-Tempo, respectively. DPI at the dose used in our experiment (500 nM) was found to block NOX enzymes without affecting mitochondrial ROS production [19], while Mito-Tempo is a well-known mitochondria-targeted antioxidant [13]. As shown in Fig. 3E, only Mito-Tempo led to a significant reduction in mitochondrial ROS and total ROS production. These data indicate that mitochondria, not NOX, are responsible for the over-generation of ROS induced by Ara-C.

Bottom Line: ROS levels were detected using the CM-H2DCFDA probe and Mito-SOX dye.The elevated ROS levels induced by Ara-C were caused by both over-generation of mitochondrial ROS and reduction of antioxidant enzymes (Cu/Zn Superoxide dismutase and catalase).Our findings suggest that a mitochondrial-targeted antioxidant could diminish adipocyte differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology, Peking University People's Hospital, Beijing, China; Institute of Hematology, Peking University, Beijing, China.

ABSTRACT

Objective: The increase in adipocytes induced by chemotherapeutic drugs may play a negative role in hematopoietic recovery. However, the mechanism underlying adipocyte differentiation of mesenchymal stem cells (MSCs) in hematopoietic stress is still unknown. Hence, the involvement of reactive oxygen species (ROS) in adipocyte differentiation under hematopoietic stress was investigated in vitro and in vivo.

Methods: The roles of cellular ROS in adipogenesis were investigated in vivo through an adipocyte hyperplasia marrow model under hematopoietic stress induced by arabinosylcytosine (Ara-C) and in vitro via adipocyte differentiation of human MSCs. ROS levels were detected using the CM-H2DCFDA probe and Mito-SOX dye. Adipogenesis was evaluated by histopathology and oil red O staining, whereas detection of mRNA levels of antioxidant enzymes and adipogenesis markers was performed using quantitative real-time polymerase chain reaction analysis.

Results: ROS were found to play an important role in regulating adipocyte differentiation of MSCs by activating peroxisome proliferator-activated receptor gamma (PPARγ,) while the antioxidant N-acetyl-L-cysteine acts through ROS to inhibit adipocyte differentiation. The elevated ROS levels induced by Ara-C were caused by both over-generation of mitochondrial ROS and reduction of antioxidant enzymes (Cu/Zn Superoxide dismutase and catalase). Our findings suggest that a mitochondrial-targeted antioxidant could diminish adipocyte differentiation.

Show MeSH
Related in: MedlinePlus