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

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

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

A Review of Cyanophage&ndash;Host Relationships: Highlighting Cyanophages as a Potential Cyanobacteria Control Strategy

Harmful algal blooms (HABs) are naturally occurring phenomena, and cyanobacteria are the most commonly occurring HABs in freshwater systems. Cyanobacteria HABs (cyanoHABs) negatively affect ecosystems and drinking water resources through the production of potent toxins. Furthermore, the frequency, duration, and distribution of cyanoHABs are increasing, and conditions that favor cyanobacteria growth are predicted to increase in the coming years. Current methods for mitigating cyanoHABs are generally short-lived and resource-intensive, and have negative impacts on non-target species. Cyanophages (viruses that specifically target cyanobacteria) have the potential to provide a highly specific control strategy with minimal impacts on non-target species and propagation in the environment. A detailed review (primarily up to 2020) of cyanophage lifecycle, diversity, and factors influencing infectivity is provided in this paper, along with a discussion of cyanophage and host cyanobacteria relationships for seven prominent cyanoHAB-forming genera in North America, including: Synechococcus, Microcystis, Dolichospermum, Aphanizomenon, Cylindrospermopsis, Planktothrix, and Lyngbya. Lastly, factors affecting the potential application of cyanophages as a cyanoHAB control strategy are discussed, including efficacy considerations, optimization, and scalability for large-scale applications

Cyanobacterial Bloom Phenology in Green Bay Using MERIS Satellite Data and Comparisons with Western Lake Erie and Saginaw Bay

Cyanobacteria blooms have been reported to be increasing worldwide. In addition to potentially causing major economic and ecological damage, these blooms can threaten human health. Furthermore, these blooms can be exacerbated by a warming climate. One approach to monitoring and modeling cyanobacterial biomass is to use processed satellite imagery to obtain long-term data sets. In this paper, an existing algorithm for estimating cyanobacterial biomass previously developed for MERIS is validated for Green Bay using cyanobacteria biovolume estimates obtained from field samples. Once the algorithm was validated, the existing MERIS imagery was used to determine the bloom phenology of the cyanobacterial biomass in Green Bay. Modeled datasets of heat flux (as a proxy for stratification), wind speed, water temperature, and gelbstoff absorption along with in situ river discharge data were used to separate bloom seasons in Green Bay from bloom seasons in nearby cyanobacteria bloom hotspots including western Lake Erie and Saginaw Bay. Of the ten-year MERIS dataset used here, the highest five years were considered &ldquo;high bloom&rdquo; years, and the lowest five years from biomass were considered &ldquo;low bloom&rdquo; years and these definitions were used to separate Green Bay. Green Bay had a strong relationship with gelbstoff absorption making it unique among the water bodies, while western Lake Erie responded strongly with river discharge as previously reported. Saginaw Bay, which has low interannual bloom variability, did not exhibit a largely influential single parameter

Cyanobacterial Bloom Phenology in Green Bay Using MERIS Satellite Data and Comparisons with Western Lake Erie and Saginaw Bay

Cyanobacteria blooms have been reported to be increasing worldwide. In addition to potentially causing major economic and ecological damage, these blooms can threaten human health. Furthermore, these blooms can be exacerbated by a warming climate. One approach to monitoring and modeling cyanobacterial biomass is to use processed satellite imagery to obtain long-term data sets. In this paper, an existing algorithm for estimating cyanobacterial biomass previously developed for MERIS is validated for Green Bay using cyanobacteria biovolume estimates obtained from field samples. Once the algorithm was validated, the existing MERIS imagery was used to determine the bloom phenology of the cyanobacterial biomass in Green Bay. Modeled datasets of heat flux (as a proxy for stratification), wind speed, water temperature, and gelbstoff absorption along with in situ river discharge data were used to separate bloom seasons in Green Bay from bloom seasons in nearby cyanobacteria bloom hotspots including western Lake Erie and Saginaw Bay. Of the ten-year MERIS dataset used here, the highest five years were considered “high bloom” years, and the lowest five years from biomass were considered “low bloom” years and these definitions were used to separate Green Bay. Green Bay had a strong relationship with gelbstoff absorption making it unique among the water bodies, while western Lake Erie responded strongly with river discharge as previously reported. Saginaw Bay, which has low interannual bloom variability, did not exhibit a largely influential single parameter

Doxorubicin-induced cardiotoxicity is suppressed by estrous-staged treatment and exogenous 17β-estradiol in female tumor-bearing spontaneously hypertensive rats

Abstract Background Doxorubicin (DOX), an anthracycline therapeutic, is widely used to treat a variety of cancer types and known to induce cardiomyopathy in a time and dose-dependent manner. Postmenopausal and hypertensive females are two high-risk groups for developing adverse effects following DOX treatment. This may suggest that endogenous reproductive hormones can in part suppress DOX-induced cardiotoxicity. Here, we investigated if the endogenous fluctuations in 17β-estradiol (E2) and progesterone (P4) can in part suppress DOX-induced cardiomyopathy in SST-2 tumor-bearing spontaneously hypersensitive rats (SHRs) and evaluate if exogenous administration of E2 and P4 can suppress DOX-induced cardiotoxicity in tumor-bearing ovariectomized SHRs (ovaSHRs). Methods Vaginal cytology was performed on all animals to identify the stage of the estrous cycle. Estrous-staged SHRs received a single injection of saline, DOX, dexrazoxane (DRZ), or DOX combined with DRZ. OvaSHRs were implanted with time-releasing pellets that contained a carrier matrix (control), E2, P4, Tamoxifen (Tam), and combinations of E2 with P4 and Tam. Hormone pellet-implanted ovaSHRs received a single injection of saline or DOX. Cardiac troponin I (cTnI), E2, and P4 serum concentrations were measured before and after treatment in all animals. Cardiac damage and function were further assessed by echocardiography and histopathology. Weight, tumor size, and uterine width were measured for all animals. Results In SHRs, estrous-staged DOX treatment altered acute estrous cycling that ultimately resulted in prolonged diestrus. Twelve days after DOX administration, all SHRs had comparable endogenous circulating E2. Thirteen days after DOX treatment, SHRs treated during proestrus had decreased cardiac output and increased cTnI as compared to animals treated during estrus and diestrus. DOX-induced tumor reduction was not affected by estrous-staged treatments. In ovaSHRs, exogenous administration of E2 suppressed DOX-induced cardiotoxicity, while P4-implanted ovaSHRs were partly resistant. However, ovaSHRs treated with E2 and P4 did not have cardioprotection against DOX-induced damage. Conclusions This study demonstrates that estrous-staged treatments can alter the extent of cardiac damage caused by DOX in female SHRs. The study also supports that exogenous E2 can suppress DOX-induced myocardial damage in ovaSHRs