**2. Materials and methods**

#### **2.1. Culture of cells**

cells but not in complex II mutant cells [27, 28], indicating the importance of a wild-type complex II for its action. More importantly, this complex II inhibitor has caused selective death of

**Figure 4.** Diagrammatic representation of the production of the reactive oxygen species (ROS). When oxygen is reduced

O2

is converted into hydroxyl radicals (OH•) by the Fenton reaction.

by the enzyme superoxide dismutase. In the

cancer cells, but the exact reason for this selectivity is not yet understood [29].

is formed. Superoxide is converted to H2

O2

with a single electron, O2

92 Mitochondrial Diseases

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presence of transition metals such as Fe2+, H2

Human colon cancer cell lines HT-29, which predominantly express Fp I in its complex II, and DLD-1, which predominantly express Fp II in its complex II, were obtained from ETCC and grown in glucose-free Roswell Park Memorial Institute (RPMI) medium supplemented with 1 g/L of glucose with 10% heated fetal bovine serum, 100 units/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B at 37°C and 5% CO2 in a humidified incubator. The medium was renewed in 48 h intervals, and the cells were harvested at 70% confluence. Human dermal fibroblasts, which predominantly express Fp I in its complex II, were obtained from Cell Applications Inc., Japan, and grown in Dulbecco's Modified Eagle Medium Nutrient Mixture F-12 (DMEM-F12).

H2 O2

oxidized by O2

**2.5. Superoxide assay in intact cells**

−

OR) in the dark at 37°C in the CO2

**2.6. Statistical analysis**

O2

**3.1. Complex II isoforms in cell lines**

whereas dermal fibroblasts predominantly express Fp I.

Results of the H2

**3. Results**

**3.2. H2 O2**

H2 O2

 generation assays were also performed in the presence of respiratory chain inhibitors-10mM nitropropionic acid (NPA), which blocks the dicarboxylate-binding site of complex II; 10 μM atpenin A5, which blocks the Q site of complex II; and 10 μM antimycin A, which blocks the Qi site of complex III in order to dissect the specific ROS-producing site of the respiratory chain.

Differential Effect of Atpenin A5 on ROS Production from Wild-Type Mitochondrial Complex II…

MitoSOX Red is a fluorescent probe that is targeted to the mitochondria of intact cells and

in the intact cells as previously described with slight modifications [33, 34]. Briefly, DLD-1 and HT-29 colon epithelial cells and the human dermal fibroblasts were cultured on cover slips and loaded with 5 μM MitoSOX Red and 50 nM MitoTracker Green (Molecular Probes, Eugene,

with prewarmed phosphate-buffered saline (PBS), three cover slip cultures each from DLD-1 and HT-29 cells were treated with the vehicle (0.2% DMSO), PEG-SOD or 10 mM NPA and incubated for 30 min. Thereafter, 10 μM atpenin A5 was added to all the cover slip cultures and imaged at 488 nm/455 nm excitation and emission wavelengths for MitoTracker Green and 633/650 nm for MitoSOX Red immediately (0 min images) and after 30 min (30 min images), using a confocal laser scanning microscope (LSM 510, Carl Zeiss, Germany). The two cover slip cultures of dermal fibroblasts were treated with 0.2% DMSO for 30 min, and subsequently one was treated with 10 μM atpenin A5 and the other one was treated with 1 μM antimycin A. Image acquisition of dermal fibroblasts was done similar to those of DLD-1 and HT-29 cells.

The data were analyzed using Shapiro-Wilks test followed by one way ANOVA. Subsequently, Levene's test and Tuckey's post hoc tests were performed. The level of significance was

cDNA from the two cancer cell lines (HT-29 and DLD-1) and the normal cell line (dermal fibroblasts) were amplified using Fp I- and Fp II-specific primers to analyze the degree of expression of the two Fp types in complex II isoforms. As shown in **Figure 6**, the results revealed that HT-29 and DLD-1 cells predominantly express Fp I and Fp II, respectively, in their complex II,

As shown by the first bar of **Figure 7a–c**, mitochondria isolated from all three cell lines generated

when incubated with succinate in the absence of any respiratory chain inhibitors. This result

P < 0.05. Graph Pad Prism software (version 7, USA) was used for the analysis.

 **production from the complex II in isolated mitochondria**

produced within mitochondria. This probe was used to detect O2

incubator for 15 min. After washing the cells for three times

assay are expressed as means ± SEM of three independent experiments.

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http://dx.doi.org/10.5772/intechopen.71638

production

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