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

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, whereas dermal fibroblasts predominantly express Fp I.

#### **3.2. H2 O2 production from the complex II in isolated mitochondria**

As shown by the first bar of **Figure 7a–c**, mitochondria isolated from all three cell lines generated H2 O2 when incubated with succinate in the absence of any respiratory chain inhibitors. This result

**Figure 6.** Mitochondrial complex II isoforms expression pattern in HT-29, DLD-1, and dermal fibroblasts. Ethidium bromide-stained agarose gels show the RT-PCR products obtained with Fp-specific primers using the RNA isolated from HT-29, DLD-1, and dermal fibroblasts. Isoform I is comprised of Fp I and isoform II is comprised of Fp II.

shows that mitochondrial complex II in all three cell lines under investigation can rapidly oxidize succinate and feed electrons to their respiratory chains and a portion of those electrons are leaked to O2 to generate ROS.

The second bar in **Figure 7a–c** shows that addition of atpenin A5, an inhibitor of the Q-binding site of complex II, increases the H2 O2 production in mitochondria isolated from HT-29 and DLD-1 cells but not in the mitochondria isolated from dermal fibroblasts. It shows that when electron flow through the Q-binding site is blocked, increase in the proton motive force in the respiratory chain proximal to the complex II Q site leads to leakage of electrons from the respiratory chain to oxygen in mitochondria isolated from cancer cells but not in mitochondria isolated from normal cells. This observation implies that a site proximal to the Q-binding site of complex II is responsible for leakage of electrons in cancer cell respiratory chains, which is different from that in normal cells. As shown by the third bar of the **Figure 7a–c**, addition of NPA, an inhibitor of the dicarboxylate-binding site of the mitochondrial complex II, along with atpenin A5 almost completely abolished the succinate-dependent H2 O2 production in all three cell types. According to this observation, the increment in H2 O2 production from the mitochondria isolated from cancer cells in the presence of atpenin A5 is attributed to a leakage of electrons from a site upstream of the Q site and downstream of the dicarboxylate-binding site. However, one can argue that the difference observed in H<sup>2</sup> O2 production between mitochondria of cancer cells and normal cells in the presence of atpenin A5 is due to the difference in the antioxidant defense systems between the two cell types. Therefore, we observed the H2 O2 production in isolated mitochondria in the presence of antimycin A, an inhibitor of the Qi site of complex III that is reported to induce ROS generation from the respiratory chain complex III [32]. As shown by the fourth bar of **Figure 7a–c**, addition of antimycin A to the reaction mixture could increase the H2 O2 production from mitochondria isolated from cancer cells as well as from the normal cells, indicating that the difference in the antioxidant levels is not the case.

#### **3.3. Superoxide production from the complex II in live cells**

When HT-29 and DLD-1 cells grown in RPMI medium containing glucose were treated with atpenin A5, their confocal images showed a clear red fluorescence in the cytoplasm while the untreated cells were devoid of such red fluorescence. To verify the localization of this fluorescence to mitochondria, the cells were simultaneously loaded with MitoTracker Green, a

**Figure 7.** H2

O2

different from control cells (*P* < 0.05).

production from the mitochondria isolated from HT-29 cells (a), DLD-1 cells (b), and dermal fibroblasts

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(c) oxidizing 0.75 mM succinate. Results are expressed as mean ± SEM of three independent experiments. \*Significantly

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shows that mitochondrial complex II in all three cell lines under investigation can rapidly oxidize succinate and feed electrons to their respiratory chains and a portion of those electrons are leaked

**Figure 6.** Mitochondrial complex II isoforms expression pattern in HT-29, DLD-1, and dermal fibroblasts. Ethidium bromide-stained agarose gels show the RT-PCR products obtained with Fp-specific primers using the RNA isolated from HT-29, DLD-1, and dermal fibroblasts. Isoform I is comprised of Fp I and isoform II is comprised of Fp II.

The second bar in **Figure 7a–c** shows that addition of atpenin A5, an inhibitor of the Q-binding

DLD-1 cells but not in the mitochondria isolated from dermal fibroblasts. It shows that when electron flow through the Q-binding site is blocked, increase in the proton motive force in the respiratory chain proximal to the complex II Q site leads to leakage of electrons from the respiratory chain to oxygen in mitochondria isolated from cancer cells but not in mitochondria isolated from normal cells. This observation implies that a site proximal to the Q-binding site of complex II is responsible for leakage of electrons in cancer cell respiratory chains, which is different from that in normal cells. As shown by the third bar of the **Figure 7a–c**, addition of NPA, an inhibitor of the dicarboxylate-binding site of the mitochondrial complex II, along

mitochondria isolated from cancer cells in the presence of atpenin A5 is attributed to a leakage of electrons from a site upstream of the Q site and downstream of the dicarboxylate-binding

chondria of cancer cells and normal cells in the presence of atpenin A5 is due to the difference in the antioxidant defense systems between the two cell types. Therefore, we observed the

cells as well as from the normal cells, indicating that the difference in the antioxidant levels

When HT-29 and DLD-1 cells grown in RPMI medium containing glucose were treated with atpenin A5, their confocal images showed a clear red fluorescence in the cytoplasm while the untreated cells were devoid of such red fluorescence. To verify the localization of this fluorescence to mitochondria, the cells were simultaneously loaded with MitoTracker Green, a

 production in isolated mitochondria in the presence of antimycin A, an inhibitor of the Qi site of complex III that is reported to induce ROS generation from the respiratory chain complex III [32]. As shown by the fourth bar of **Figure 7a–c**, addition of antimycin A to the

production in mitochondria isolated from HT-29 and

O2

production between mito-

O2

O2

production from mitochondria isolated from cancer

production in

production from the

O2

with atpenin A5 almost completely abolished the succinate-dependent H2

O2

all three cell types. According to this observation, the increment in H2

site. However, one can argue that the difference observed in H<sup>2</sup>

**3.3. Superoxide production from the complex II in live cells**

to O2

96 Mitochondrial Diseases

H2 O2

is not the case.

to generate ROS.

site of complex II, increases the H2

reaction mixture could increase the H2

**Figure 7.** H2 O2 production from the mitochondria isolated from HT-29 cells (a), DLD-1 cells (b), and dermal fibroblasts (c) oxidizing 0.75 mM succinate. Results are expressed as mean ± SEM of three independent experiments. \*Significantly different from control cells (*P* < 0.05).

C 1

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Atpenin+NPA

0 min

C 2

Atpenin+NPA

30 min

Atpenin 0 min

D2

Atpenin 30 min

DLD-1 cells

D1

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MitoTracker Green MitoS OX R ed Merge

A1

A2

Atpenin 30 min

B 1

Atpenin+SOD

0 min

B 2

Atpenin+SOD

30 min

Atpenin 0 min

HT-29 cells

98 Mitochondrial Diseases

G 1

Dermal F ibroblasts

Atpenin 0 min

Atpenin 30 min

G 2

H1

H2

**Figure 8.** Confocal laser scanning microscopic images showing the O2

florescence and other panels have a green florescence.

−

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live cells. Panels A, D, and G show the time lapse images of the cells preincubated with DMSO and treated with atpenin A5. Panels B and E show the cells preincubated with SOD-PEG and treated with atpenin A5. Panels C and F show the cells preincubated with NPA and treated with atpenin A5. Cells treated with antimycin A are shown in panel H. The cells in the first column have a green florescence. The cells in the second column have a red florescence. Out of the cells in the third column, A2, D2 and H2 panels have a dark orange florescence and B2, C2 and E2 panels have a light orange

production from the mitochondrial complex II in

Antimycin A 30 min

Antimycin A 0 min

#### Dermal F ibroblasts

E1

Atpenin+SOD

100 Mitochondrial Diseases

0 min

E 2

Atpenin+SOD

30 min

F 1

0 min

F 2

30 min

Atpenin+NPA

Atpenin+NPA

**Figure 8.** Confocal laser scanning microscopic images showing the O2 − production from the mitochondrial complex II in live cells. Panels A, D, and G show the time lapse images of the cells preincubated with DMSO and treated with atpenin A5. Panels B and E show the cells preincubated with SOD-PEG and treated with atpenin A5. Panels C and F show the cells preincubated with NPA and treated with atpenin A5. Cells treated with antimycin A are shown in panel H. The cells in the first column have a green florescence. The cells in the second column have a red florescence. Out of the cells in the third column, A2, D2 and H2 panels have a dark orange florescence and B2, C2 and E2 panels have a light orange florescence and other panels have a green florescence.

fluorescent probe that is specifically targeted to mitochondria. The resulting confocal images revealed that the green fluorescence of MitoTracker Green colocalize with the red fluorescence of MitoSOX Red indicating that MitoSOX Red is oxidized within mitochondria (**Figure 8**). To determine whether MitoSOX oxidation is a superoxide-dependent process, the cells were preincubated with PEG-SOD, which can scavenge O<sup>2</sup> − generated within cells. In the presence of PEG-SOD, MitoSOX Red fluorescence was negligible (**Figure 8**, panels B and E). This result indicates that the majority of the atpenin A5-induced MitoSOX fluorescence is superoxide dependent. Next, we attempted to determine whether the atpenin A5-induced O<sup>2</sup> − production is a succinate-dependent process by preincubating the cells with NPA before adding atpenin A5. As shown in **Figure 8** panels C and F, the MitoSOX Red fluorescence intensity in the NPAtreated cells was markedly reduced indicating that the atpenin A5-induced MitoSOX Red oxidation is a succinate-dependent event. Collectively, these data indicate that atpenin A5 induces succinate-dependent O2 − production from the mitochondrial complex II within intact DLD-1 and HT-29 cells. In agreement with the results from the isolated mitochondria, addition of atpenin A5 to the MitoSOX Red-loaded dermal fibroblasts did not display any red fluorescence (**Figure 8** panel G) indicating that atpenin A5 does not induce O2 − production in mitochondria of dermal fibroblasts. But dermal fibroblasts showed an increment in red fluorescence with antimycin A (**Figure 8** panel H). This observation shows that the mitochondria of the dermal fibroblasts can produce O<sup>2</sup> − when the Q site of complex III is inhibited by antimycin A.

preventing the electron leak into oxygen. It appears that differences in the three-dimensional structure of the capping domain of the enzyme contribute to the difference in ROS production in cancer cells and normal cells. In fact, previous studies in our laboratory have shown that DLD-1 and HT-29 cells display FRD activity associated with the phosphorylation status of the Fp subunit of complex II and the degree of phosphorylation has been increased under hypoxic and hypoglycemic conditions [11]. If phosphorylation of amino acid residues in the Fp subunit can affect the correct movement of the capping domain during succinate oxida-

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Our observation of a 1.5-fold higher ROS production from isolated mitochondria of DLD-1 cells than that of HT-29 cells may also have resulted due to the difference in the phosphorylation status of the two isoforms because the amino acids, which differ in the two isoforms, are found in the C-terminal domain of the Fp subunit, which plays an important role in opening and closing of the active site of the complex II enzymes [9] Substitution of tyrosine 629 in Fp I with phenylalanine in Fp II may affect the phosphorylation state of the C-terminal domain since tyrosine is a possible phosphorylation site while phenylalanine is not. This difference may affect the closing of the active site in Fp II during enzyme catalysis and allow more electrons to leak to oxygen from FAD. However, it is essential to resolve the crystal structure of the two isoforms of the human mitochondrial complex II with both the open and closed states of the capping domain and to establish the relationship between the phosphorylation status

In summary, complex II Q site inhibitor atpenin A5 differentially affects the ROS production in normal cells and cancer cells. This difference may be utilized for selective destruction of cancer cells using complex II-inhibiting mitocans. We hypothesize that the difference in ROS production observed in this study is attributed to the difference in posttranslational modifications of mitochondrial complex II between normal cells and cancer cells, which may be more pronounced under hypoxic and hypoglycemic tumor microenvironments.

Ministry of Education, Culture and Sports in Japan is gratefully acknowledged for their financial

\* and Kiyoshi Kita2

1 Department of Basic Sciences, Faculty of Dental Sciences, University of Peradeniya,

2 School of Tropical Medicine and Global Health, Nagasaki University, Nagasaki, Japan

\*Address all correspondence to: madhaviparanagama@gmail.com

tion, it will be possible for the electrons to leak to oxygen from the FAD site.

of the Fp subunit and the ROS production.

**Acknowledgements**

support.

**Author details**

Madhavi P. Paranagama<sup>1</sup>

Peradeniya, Sri Lanka

#### **4. Discussion**

ROS are produced from the mitochondrial complex II of the cancer cells (DLD-1 and HT-29) but not from the normal cells (dermal fibroblasts) when the entry of electrons to its quinonebinding site is blocked with 10 μM atpenin A5. However, previous studies in our laboratory have revealed that quinone reduction in the mitochondrial complex II can be completely inhibited at the tested concentration of atpenin A5 [8, 10]. Therefore, the difference in ROS production between cancer cells and normal cells may be attributed to a difference in the complex IIs that is upstream of the atpenin A5-binding site. Since the dicarboxylate-binding site inhibitor, NPA, could inhibit the ROS production induced by atpenin A5 in cancer cells, it is concluded that the ROS-producing site must be within the complex II, upstream of the atpenin A5 inhibition site(Q site) and downstream of NPA-binding site (dicarboxylate-binding site). According to previous evidence, the most likely ROS-producing sites of mitochondrial complex II are FAD [35–37] site and the Q site [37–39]. Observations of this study show that the ROS-generating site of complex II in atpenin A5-treated cancer cells is FAD but not Q site.

In order to explain this observation, we analyzed the published data on the crystal structures of flavoproteins, which are available in both the substrate bound and unbound forms [40–42]. They have shown that the capping domain in Fp subunit rotates during the enzyme catalysis. When the active site is not occupied by the substrate, the capping domain is rotated away from the active site creating a solvent channel linking the active site and the surrounding aqueous environment. In the substrate bound state, the capping domain is rotated inward concealing the active site, preventing the access of solvent to the active site [37]. Thus, closed conformation acquired during enzyme catalysis appears to be vital for preventing the electron leak into oxygen. It appears that differences in the three-dimensional structure of the capping domain of the enzyme contribute to the difference in ROS production in cancer cells and normal cells. In fact, previous studies in our laboratory have shown that DLD-1 and HT-29 cells display FRD activity associated with the phosphorylation status of the Fp subunit of complex II and the degree of phosphorylation has been increased under hypoxic and hypoglycemic conditions [11]. If phosphorylation of amino acid residues in the Fp subunit can affect the correct movement of the capping domain during succinate oxidation, it will be possible for the electrons to leak to oxygen from the FAD site.

Our observation of a 1.5-fold higher ROS production from isolated mitochondria of DLD-1 cells than that of HT-29 cells may also have resulted due to the difference in the phosphorylation status of the two isoforms because the amino acids, which differ in the two isoforms, are found in the C-terminal domain of the Fp subunit, which plays an important role in opening and closing of the active site of the complex II enzymes [9] Substitution of tyrosine 629 in Fp I with phenylalanine in Fp II may affect the phosphorylation state of the C-terminal domain since tyrosine is a possible phosphorylation site while phenylalanine is not. This difference may affect the closing of the active site in Fp II during enzyme catalysis and allow more electrons to leak to oxygen from FAD. However, it is essential to resolve the crystal structure of the two isoforms of the human mitochondrial complex II with both the open and closed states of the capping domain and to establish the relationship between the phosphorylation status of the Fp subunit and the ROS production.

In summary, complex II Q site inhibitor atpenin A5 differentially affects the ROS production in normal cells and cancer cells. This difference may be utilized for selective destruction of cancer cells using complex II-inhibiting mitocans. We hypothesize that the difference in ROS production observed in this study is attributed to the difference in posttranslational modifications of mitochondrial complex II between normal cells and cancer cells, which may be more pronounced under hypoxic and hypoglycemic tumor microenvironments.
