**1.3. ROS production from mitochondrial complex II**

by recent metabolic profiling studies also on gastric and colon carcinomas [14]. In addition to its role in energy metabolism, mitochondrial complex II has been identified to have a novel function as a tumor suppressor in certain tissues. Mutations in its subunits are reported to cause cancers such as pheochromocytoma (tumors of the chromaffin cells in the adrenal medulla) and paraganglioma (extra adrenal tumors of sympathetic or parasympathetic origin). Moreover, breast, thyroid, and renal carcinomas are also known to be associated with complex II mutations. More recently, mutations in the complex II assembly factors, succinate dehydrogenase assembly factor 1 (SDHAF1) and succinate dehydrogenase assembly factor 2 (SDHAF2), are also reported to be linked with infantile leukoencephalopathy and paragan-

**Figure 3.** A schematic representation of the function of the mitochondrial complex II in the anaerobic respiratory chain. Electrons originating from NADH oxidation in complex I reduce the rhodoquinone (RQ) to rhodoquinol (RQH2) at its Q site. Rhodoquinol is subsequently oxidized to rhodoquinone at the Q site of the complex II. The resulting electrons are transferred via the Fe-S clusters and FAD to reduce fumarate to succinate. Fumarate is the terminal electron acceptor of

glioma (for a recent review, see [2]).

the anaerobic respiratory chain.

90 Mitochondrial Diseases

Reactive oxygen species (ROS) are inevitable byproducts of the aerobic respiratory chain. Major ROS produced by the respiratory chain is superoxide (O2 − ). It is subsequently dismutated into hydrogen peroxide (H2 O2 ) by superoxide dismutase (**Figure 4**). Both O2 − and H2 O2 are known to contribute to physiological and pathological redox signaling [15, 16].

However, conventionally, mitochondrial complex II is not identified as a significant source of ROS. But, two potential ROS-producing sites can be identified in mitochondrial complex II when its sequence of electron transport is considered together with the spatial arrangement of its redox centers. When one molecule of succinate is oxidized by complex II, FAD is reduced with two electrons, but subsequent transfer of electrons through Fe-S clusters occurs one at a time. Hence, when one electron is delivered to the proximal Fe-S cluster from FAD, a flavin radical is formed until the second electron is delivered. Similarly, after sequential transfer of single electrons through Fe-S clusters, quinone is reduced with two electrons. Hence, when one electron arrives at the Q site, until another electron arrives from Fe-S clusters, a quinone radical is formed. These two radicals have the potential to generate ROS, if there is accessibility to oxygen (**Figure 5**).

During the past two decades, complex II has emerged as an important source of ROS. On the one hand, ROS produced from mutated complex II has been proposed to be the underlying cause of complex II–associated human diseases. Thus, numerous studies have been conducted to understand ROS production from human complex II. Most of these studies have been conducted on model organisms such as *Escherichia coli, Saccharomyces cerevisiae, and Caenorhabditis elegans* [17–20]. A hallmark in these studies is the finding that the *mev-1* mutation in the mitochondrial complex II of *C. elegans* results in enhanced O2 − production and oxygen hypersensitivity [19, 20]. Later on, this finding was further reinforced by a study using a transgenic mouse cell line with the equivalent mutation in the *CybL* gene as the *mev-1* mutation demonstrating O2 − overproduction from mitochondria that leads to tumorigenesis [21]. More recently, mutations in the Ip subunit in the *C. elegans* have also been found to display enhanced ROS production [22]. Site-directed mutagenesis of CybS subunit in *E. coli* [23] and the Ip and CybS subunits in *S. cerevisiae* [18, 24] has also shown enhanced ROS production providing further support for the hypothesis that ROS underlies the complex II mutations-associated carcinogenesis. Indeed, some studies have been conducted on human cell lines using pharmacological and genetic interventions to mimic complex II mutations, but the results are controversial. For example, ROS production from human mitochondrial complex II has been shown by Guzy et al. [25] in intact cells of the hepatoma cell line Hep3B by inhibiting the complex II activity by the Q site inhibitor theonyl trifluoroacetone (TTFA) and by RNA interference of Ip subunit. In contrast, RNA interference of the same subunit of complex II in the same cell line by Cervera et al. have failed to detect ROS production [26].

On the other hand, ROS produced from mitochondrial complex II has received considerable attention as a mediator of "mitocans" (a novel class of mitochondrial targeted anticancer drugs) induced cancer cell death [5]. Interestingly, complex II Q site inhibiting mitocan, ɑ-tocopheryl succinate (ɑ-TOS) has been found to induce ROS only in complex II competent

In this chapter, we show that the potent and specific complex II Q site inhibitor atpenin A5 [30] can specifically induce ROS production in two human cancer cell lines HT-29, which predominantly express Fp I, and DLD-1, which predominantly express Fp II in the mitochondrial complex II but not in the noncancerous tissue, human dermal fibroblasts that predominantly express Fp I. This difference in ROS production may be attributed to the difference in the posttranslational modifications of the wild-type mitochondrial complex II's in normal cells and cancer cells. This finding highlights new avenues in developing complex II-targeted mitocans

**Figure 5.** Ribbon model of mitochondrial complex II of *Sus scrofa* (pdb1ZOY) showing the potential ROS production sites. Fp, Ip, Cy*b*L, and Cy*b*S subunits are shown in green, blue, red, and orange, respectively. The redox centers—FAD,

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

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

93

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

The medium was renewed in 48 h intervals, and the cells were harvested at 70% confluence.

in a humidified incubator.

streptomycin, and 0.25 μg/mL amphotericin B at 37°C and 5% CO2

with considerable promise.

**2.1. Culture of cells**

**2. Materials and methods**

iron sulfur clusters, and heme *b—*are labeled in the figure.

**Figure 4.** Diagrammatic representation of the production of the reactive oxygen species (ROS). When oxygen is reduced with a single electron, O2 − is formed. Superoxide is converted to H2 O2 by the enzyme superoxide dismutase. In the presence of transition metals such as Fe2+, H2 O2 is converted into hydroxyl radicals (OH•) by the Fenton reaction.

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

Differential Effect of Atpenin A5 on ROS Production from Wild-Type Mitochondrial Complex II… http://dx.doi.org/10.5772/intechopen.71638 93

**Figure 5.** Ribbon model of mitochondrial complex II of *Sus scrofa* (pdb1ZOY) showing the potential ROS production sites. Fp, Ip, Cy*b*L, and Cy*b*S subunits are shown in green, blue, red, and orange, respectively. The redox centers—FAD, iron sulfur clusters, and heme *b—*are labeled in the figure.

In this chapter, we show that the potent and specific complex II Q site inhibitor atpenin A5 [30] can specifically induce ROS production in two human cancer cell lines HT-29, which predominantly express Fp I, and DLD-1, which predominantly express Fp II in the mitochondrial complex II but not in the noncancerous tissue, human dermal fibroblasts that predominantly express Fp I. This difference in ROS production may be attributed to the difference in the posttranslational modifications of the wild-type mitochondrial complex II's in normal cells and cancer cells. This finding highlights new avenues in developing complex II-targeted mitocans with considerable promise.
