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. 2014 Sep 26;19(1):49.
doi: 10.1186/s40001-014-0049-5.

The roles of mitoferrin-2 in the process of arsenic trioxide-induced cell damage in human gliomas

Chunlei Wang, Xiaofeng Chen, Huichao Zou, Xin Chen, Yaohua Liu, Shiguang Zhao

The roles of mitoferrin-2 in the process of arsenic trioxide-induced cell damage in human gliomas

Chunlei Wang et al. Eur J Med Res. .

Abstract

Background: Among glioma treatment strategies, arsenic trioxide (As2O3) has shown efficacy as a therapeutic agent against human gliomas. However, the exact antitumor mechanism of action of As2O3 is still unclear. Mitochondria are considered to be the major source of intracellular reactive oxygen species (ROS), which are known to be associated with As2O3-induced cell damage. Therefore, we investigated whether mitoferrin-2, a mitochondrial iron uptake transporter, participates in As2O3-induced cell killing in human gliomas.

Methods: Human glioma cell lines were used to explore the mechanism of As2O3's antitumor effects. First, expression of mitoferrin-2 was analyzed in glioma cells that were pretreated with As2O3. Changes in ROS production and apoptosis were assessed. Furthermore, cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).

Results: In the present study we found that As2O3 induced ROS production and apoptosis in glioma cells. In addition, gene expression of mitoferrin-2, a mitochondrial iron uptake transporter, was increased 4 to 5 fold after exposure to As2O3 (5 μM) for 48 hours. Furthermore, apoptosis and cytotoxicity induced by As2O3 in glioma cells were decreased after silencing the mitoferrin-2 gene.

Conclusions: Our findings indicated that mitoferrin-2 participates in mitochondrial ROS-dependent mechanisms underlying As2O3-mediated damage in glioma cells.

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Figures

Figure 1
Figure 1
After exposure of human glioma cell lines U87MG (A) and T98G (B) to As 2 O 3 at different doses for 48 hours, the cytotoxicity of As 2 O 3 was analyzed by MTT. Student’s t-test was used for evaluation statistical significance (*P < 0.05).MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
Figure 2
Figure 2
After exposure of human glioma cell lines U87MG (A) and T98G (B) to As 2 O 3 at different times, the reactive oxygen species (ROS) production of As 2 O 3 was analyzed by flow cytometry. Student’s t-test was used for evaluation statistical significance (*P < 0.05).
Figure 3
Figure 3
Treatment with As 2 O 3 (5 μM, 4 8hours) significantly increased intracellular mitoferrin-2 expression in glioma cells U87MG (A) and T98G lines (B). Data are presented as mean ± SEM for the separate experiments performed in duplicate. Student’s t-test was used to evaluate statistical significance (*P < 0.05).
Figure 4
Figure 4
Silencing mitoferrin-2 by siRNA for 48 hours was shown in U87MG (A) and T98G (B) lines. Expression of mitoferrin-2 was decreased after using siRNA interferencing in both U87MG and T98G. Data are presented as mean ± SEM for the separate experiments performed in duplicate. Student’s t-test was used to evaluate statistical significance (*P < 0.05).
Figure 5
Figure 5
The expressions of mitoferrin-2 in cell lysates were detected by Western blotting. β-actin was used as an internal control. Pretreatment with As2O3 (5 μM, 48 hours) significantly increased intracellular mitoferrin-2 expression in glioma cell lines U87MG and T98G. Silencing mitoferrin-2 by siRNA for 48 hours was shown in both U87MG and T98G lines.
Figure 6
Figure 6
Reactive oxygen species (ROS) production was measured by flow cytometry in human glioma cell lines U87MG (A) and T98G (B). The ROS production in the silenced mitoferrin-2 group following pretreatment with As2O3 was decreased compared with the As2O3 (5 μM) group. Fluorescence signals represent ROS production. Data are presented as mean ± SEM for the separate experiments performed in duplicate. Student’s t-test was used to evaluate statistical significance (*P < 0.05).
Figure 7
Figure 7
Apoptosis in As 2 O 3 -pretreated human glioma cells was measured by flow cytometry. The apoptosis rate was decreased in the silenced mitoferrin-2 group (48 hours) following pretreatment with As2O3 (5 μM, 48 hours) compared with the As2O3 (5 μM, 48 hours) group in both human glioma cell lines U87MG (A) and T98G (B). Student’s t-test was used to evaluate statistical significance (*P < 0.05).
Figure 8
Figure 8
Proliferation in As 2 O 3 -pretreated human glioma cells was measured by MTT. Proliferation rates were not changed in either human glioma cell lines U87MG (A) or T98G (B) after silencing mitoferrin-2 for 48 hours. Proliferation rates were increased in the silenced mitoferrin-2 group following pretreatment with As2O3 for 48 hours compared with the As2O3 (5 μM) group in both human glioma cell lines U87MG (C) and T98G lines (D). Student’s t-test was used to evaluate statistical significance (*P < 0.05). MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
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