mtDNA gene expression and stability.

<p>Gene expression of <i>7S</i>, 12S (<i>RNR1</i>) and 16S (<i>RNR2</i>) transcripts (A-B) along with SSBP1 and TFAM protein levels (C-D) in MDA-MB-231 (A,C) and H23 (B,D) cell lines after 24 hours exposure to 0.02% DMSO or MitoT (dark gray bars), MitoQ (gray bars) or MitoCA (light gray bars) at 2μM. In A-B, bars represent the average log2 fold change normalized to <i>GAPDH</i> and the DMSO control. Statistical significance is expressed as asterisks at p<0.05 relative to the DMSO control. In C-D, bars signify the mean densitometry normalized to the corresponding DMSO treatment. For each endpoint, two independent experiments were performed (n = 3). Error bars signify +/-1 SEM.</p

Mitochondrial TCA cycle aconitase activity.

<p>Aconitase activity was determined with the colorimetric aconitase enzyme activity assay in MDA-MB-231 (A) and H23 (B) cell lines exposed to either DMSO (0.02%), MitoT, MitoQ or MitoCA at 2μM or Antimycin A (Am-A) at 40μM for 2 (light) or 24 (dark) hours. Bars represent the relative mitochondrial aconitase activity normalized to mitochondrial protein and the DMSO control +/-1 SEM. Statistical significance is represented by asterisks with a p<0.05 relative to the DMSO control. In C-D, immunoblots of mitochondrial extracts were probed with anti-aconitase 2, and quantitated using densitometry and then normalized to VDAC and the DMSO treatment.</p

Therapeutic Targeting of the Mitochondria Initiates Excessive Superoxide Production and Mitochondrial Depolarization Causing Decreased mtDNA Integrity

<div><p>Mitochondrial dysregulation is closely associated with excessive reactive oxygen species (ROS) production. Altered redox homeostasis has been implicated in the onset of several diseases including cancer. Mitochondrial DNA (mtDNA) and proteins are particularly sensitive to ROS as they are in close proximity to the respiratory chain (RC). Mitoquinone (MitoQ), a mitochondria-targeted redox agent, selectively damages breast cancer cells possibly through damage induced via enhanced ROS production. However, the effects of MitoQ and other triphenylphosphonium (TPP⁺) conjugated agents on cancer mitochondrial homeostasis remain unknown. The primary objective of this study was to determine the impact of mitochondria-targeted agent [(MTAs) conjugated to TPP⁺: mitoTEMPOL, mitoquinone and mitochromanol-acetate] on mitochondrial physiology and mtDNA integrity in breast (MDA-MB-231) and lung (H23) cancer cells. The integrity of the mtDNA was assessed by quantifying the degree of mtDNA fragmentation and copy number, as well as by measuring mitochondrial proteins essential to mtDNA stability and maintenance (TFAM, SSBP1, TWINKLE, POLG and POLRMT). Mitochondrial status was evaluated by measuring superoxide production, mitochondrial membrane depolarization, oxygen consumption, extracellular acidification and mRNA or protein levels of the RC complexes along with TCA cycle activity. In this study, we demonstrated that all investigated MTAs impair mitochondrial health and decrease mtDNA integrity in MDA-MB-231 and H23 cells. However, differences in the degree of mitochondrial damage and mtDNA degradation suggest unique properties among each MTA that may be cell line, dose and time dependent. Collectively, our study indicates the potential for TPP⁺ conjugated molecules to impair breast and lung cancer cells by targeting mitochondrial homeostasis.</p></div

Mitochondrial replication machinery.

<p>Gene expression (A,C) and protein levels (B,D) for TWINKLE, POLG and POLRMT in MDA-MB-231 (A,C) and H23 (B,D) cell lines determined by qPCR and immunoblotting. Cells were exposed to DMSO (0.02%), MitoT (dark gray bars), MitoQ (gray bars), and MitoCA (light gray bars) at 2μM for 24 hours. In A-B, bars represent the average log2 fold change normalized to <i>GAPDH</i> and the DMSO control. Statistical significance is expressed as asterisks at p<0.05 relative to the DMSO control. In C-D, bars denote the mean densitometry (+/-1 SEM) of immunoblots of mitochondrial fractions normalized to VDAC and the corresponding DMSO treatment. For each assay, two independent experiments were performed (n = 3).</p

Mitochondrial DNA integrity.

<p>mtDNA damage (A-B) and copy number (C-D) in MDA-MB-231 (A,C) and H23 (B,D) cells after exposure to DMSO [(0.02%) black bars], MitoT (dark gray bars), MitoQ (gray bars) or MitoCA (light gray bars) at 2μM for 24 hours. mtDNA fragmentation (A-B) was evaluated using PCR amplification of a long mitochondrial sequence relative to a short mitochondrial sequence. Band intensities of PCR products were quantitated using densitometry. In A-B, gels are representative images for PCR products. In C & D, mitochondrial copy number was assessed by amplification of short regions of house-keeping genes in both nDNA and mtDNA. Bars depict the mean ratio of long to short band intensities (A-B) or the mean ratio of mtDNA:nDNA (C-D) relative to the DMSO treatment +/-1 SEM. Asterisks show statistical significance at p<0.05 relative to the DMSO control at each time. For each assay, two independent experiments were performed (n = 3).</p

Mitochondrial respiration.

<p>The mRNA (A-B) and protein levels (C-D) levels for mitochondrial respiratory chain subunits were assessed using qPCR and immunoblotting. Mitochondrial bioenergetics were measured using a Seahorse XF^e96 flux analyzer (E-F). MDA-MB-231 (A,C,E) and H23 (B,D,F) cells were exposed to either DMSO (0.02%), MitoT (dark gray bars), MitoQ (gray bars) or MitoCA (light gray bars) at 2μM for 24 hours. In C-D, mitochondrial complexes were probed with an antibody cocktail containing antibodies against all five mitochondrial complexes. Each band represents a different subunit of a mitochondrial complex. Band intensities were quantitated using densitometry. Bars denote the average mRNA log2 fold change normalized to <i>GAPDH</i> (A-B), the mean densitometry normalized to VDAC [(C-D) represented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168283#pone.0168283.g004" target="_blank">Fig 4</a>], the average oxygen consumption rate [OCR (E-F<i>i</i>)] or the average extracellular acidification rate [ECAR (E-F<i>ii</i>)] relative to the DMSO control +/-1 SEM. In A-B and E-F, statistical significance (p<0.05) with respect to the DMSO control is expressed with asterisks above each bar. For each assay, two independent experiments were performed (n = 3).</p