Cell Mitochondrial Mass Research Articles

Kaempferol induces mitochondrial dysfunction and mitophagy by activating the LKB1/AMPK/MFF pathway in breast precancerous lesions.

Kaempferol has been suggested to be an effective anticancer agent in several malignant tumors. However, its function and mechanisms in breast precancerous lesions remain largely elusive. Here, we showed that kaempferol induced excessive mitochondrial fission and mitochondrial damage with activated mitochondrial fission factor (MFF)-mediated dynamin-related protein (DRP) 1 mitochondrial translocation. As a result, the PTEN-induced putative kinase 1 (PINK1)/Parkin signaling pathway was activated, accompanied by excessive mitophagy and reduced mitochondrial mass in cells. We also revealed that kaempferol-induced lethal mitophagy contributed to inhibiting breast precancerous lesion growth in vitro and in vivo. Furthermore, we verified serine/threonine kinase 11 (STK11/LKB1)/AMP-activated protein kinase (AMPK) pathway deficiency in breast precancerous lesions. Moreover, LKB1/AMPK pathway reactivation by kaempferol was required for excessive mitochondrial fission and lethal mitophagy. Taken together, our findings shed new light on the molecular mechanisms related to breast cancer prevention by kaempferol and provide evidence for its potential clinical application.

Mitochondrial Mass of Naïve T Cells Is Associated with Aerobic Fitness and Energy Expenditure of Active and Inactive Adults.

Chronic exercise training is known to induce metabolic changes, but whether these adaptations extend to lymphocytes and how this may affect immune function remains largely unknown. This study was conducted to determine the extent to which mitochondrial characteristics of naïve T cells differ according to fitness status and to further examine the energy production pathways of cells from aerobically trained and inactive participants. Blood was collected from 30 aerobically active (>6 h·wk -1 ) or inactive (<90 min·wk -1 ) men and women. Naïve T cell mitochondrial mass, membrane potential, and biogenesis were assessed with flow cytometry. Participants completed a treadmill maximal oxygen consumption (V̇O 2peak ) test and wore a physical activity monitor for 1 wk. In a subset of participants, naïve CD8 + T cell activation-induced glycolytic and mitochondrial ATP production was measured. Active participants exhibited 16.7% more naïve CD8 + T cell mitochondrial mass ( P = 0.046), 34% greater daily energy expenditure ( P < 0.001), and 39.6% higher relative V̇O 2peak ( P < 0.001), along with 33.9% lower relative body fatness ( P < 0.001). Among all participants, naïve CD8 + T cell mitochondrial mass was correlated with estimated energy expenditure ( r = 0.36, P = 0.048) and V̇O 2peak ( r = 0.47, P = 0.009). There were no significant differences in ATP production, mitochondrial biogenesis, or mitochondrial membrane potential between active and inactive groups. This is the first study to examine the effects of aerobic exercise training status on metabolic parameters within human naïve T cells. Findings suggest that mitochondrial adaptations in certain immune cell types are positively associated with aerobic fitness and energy expenditure. This study provides a foundation for future development of prophylactic and therapeutic interventions targeting specific immune cell subsets to improve the immune response and overall health.

RNA Aptamers Rescue Mitochondrial Dysfunction in a Yeast Model of Huntington’s Disease

Huntington’s disease (HD) is associated with the misfolding and aggregation of mutant huntingtin harboring an elongated polyglutamine stretch at its N terminus. A distinguishing pathological hallmark of HD is mitochondrial dysfunction. Any strategy that can restore the integrity of the mitochondrial environment should have beneficial consequences for the disease. Specific RNA aptamers were selected that were able to inhibit aggregation of elongated polyglutamine stretch containing mutant huntingtin fragment (103Q-htt). They were successful in reducing the calcium overload, which leads to mitochondrial membrane depolarization in case of HD. In one case, the level of Ca2+ was restored to the level of cells not expressing 103Q-htt, suggesting complete recovery. The presence of aptamers was able to increase mitochondrial mass in cells expressing 103Q-htt, along with rescuing loss of mitochondrial genome. The oxidative damage to the proteome was prevented, which led to increased viability of cells, as monitored by flow cytometry. Thus, the presence of aptamers was able to inhibit aggregation of mutant huntingtin fragment and restore mitochondrial dysfunction in the HD cell model, confirming the advantage of the strategy in a disease-relevant parameter.

Iron Loading Exaggerates the Inflammatory Response to the Toll-like Receptor 4 Ligand Lipopolysaccharide by Altering Mitochondrial Homeostasis.

Perioperative and critically ill patients are often exposed to iron (in the form of parenteral-iron administration or blood transfusion) and inflammatory stimuli, but the effects of iron loading on the inflammatory response are unclear. Recent data suggest that mitochondrial reactive oxygen species have an important role in the innate immune response and that increased mitochondrial reactive oxygen species production is a result of dysfunctional mitochondria. We tested the hypothesis that increased intracellular iron potentiates lipopolysaccharide-induced inflammation by increasing mitochondrial reactive oxygen species levels. Murine macrophage cells were incubated with iron and then stimulated with lipopolysaccharide. C57BL/6 wild-type mice were intraperitoneally injected with iron and then with lipopolysaccharide. Markers of inflammation and mitochondrial superoxide production were examined. Mitochondrial homeostasis (the balance between mitochondrial biogenesis and destruction) was assessed, as were mitochondrial mass and the proportion of nonfunctional to total mitochondria. Iron loading of mice and cells potentiated the inflammatory response to lipopolysaccharide. Iron loading increased mitochondrial superoxide production. Treatment with MitoTEMPO, a mitochondria-specific antioxidant, blunted the proinflammatory effects of iron loading. Iron loading increased mitochondrial mass in cells treated with lipopolysaccharide and increased the proportion of nonfunctional mitochondria. Iron loading also altered mitochondrial homeostasis to favor increased production of mitochondria. Acute iron loading potentiates the inflammatory response to lipopolysaccharide, at least in part by disrupting mitochondrial homeostasis and increasing the production of mitochondrial superoxide. Improved understanding of iron homeostasis in the context of acute inflammation may yield innovative therapeutic approaches in perioperative and critically ill patients.

Abstract 3510: N-Myc positively influences mitochondrial biogenesis in neuroblastoma

Abstract Background: Neuroblastoma is the most common extracranial tumor of childhood., accounting for approximately 7% of all cancer in children. The morbidity and mortality are affected by a number of factors - perhaps most significantly by amplification of the oncogene, N-MYC. Advanced stage disease with N-MYC overexpression confers the highest mortality. The N-MYC gene product (N-Myc) regulates the transcription of many genes, and when overexpressed is thought to enhance the cell cycle and dysregulate apoptosis. The function of N-Myc is virtually identical to another oncoprotein of the same family, c-Myc, yet is restricted by developmental stage and cell type. Overexpression of c-Myc has been shown to increase mitochondrial biogenesis by alterations in mitochondrial dynamics. It has been proposed that these changes may contribute to c-Myc induced tumorigenesis. We hypothesize that N-Myc amplification will have similar effects on mitochondrial biogenesis in neuroblastoma. Design/Methods: To perform these studies we utilized a cell line with endogenous levels of N-Myc (SK-N-SH). In these cells we have ectopically overexpressed N-Myc and examined the cells for changes in the mitochondria with various assays, including: Western blots, electron microscopy, flow cytometry, ATP assay and confocal microscopy. Results: We found a X-fold increase in mitochondrial mass in cells that overexpress N-Myc,, as well as a more tubular appearance with less organized cristae. Additionally, N-Myc changed the expression of various mitochondrial fusion and fission proteins. The most dramatic change was seen in the expression of the fusion protein Opa1, which changed by ten-fold. This, combined with a decrease in expression of fission proteins DRP1 and MFF, may account for the more tubular appearance of mitochondria in N-Myc overexpressing cells. Conclusions N-Myc amplification appeared to increase mitochondrial mass. This may occur as a result of increased expression of mitochondrial fusion proteins, as previously observed in c-Myc overexpressing cells. Additionally, mitochondrial function is influenced by N-Myc overexpression. Therefore, N-Myc amplification appears to positively influence mitochondrial biogenesis in neuroblastoma. Further studies are needed to determine if this increase in mitochondrial mass and function is necessary for the development and maintenance of neuroblastoma. Citation Format: Manika Sharma, James A. Graves. N-Myc positively influences mitochondrial biogenesis in neuroblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3510. doi:10.1158/1538-7445.AM2014-3510

Abstract 2169: SapC-DOPS induces lethal mitophagy in glioblastoma.

Abstract The goal of this study is to evaluate SapC-DOPS, a novel cancer nanotherapeutic, for glioblastoma multiforme (GBM). SapC-DOPS delivered intravenously (i.v) was found to specifically target intracranial tumors in mice bearing spontaneous brain tumor, as well in nude mice intracranially implanted with human GBM cells. Treatment of tumor bearing mice with SapC-DOPS (i.v.) significantly increased survival: 25% and 75% long-term survivors in U87ΔEGFR-Luc and X12v2 implanted mice, respectively (P&amp;lt;.0001). Western blot analysis of primary GBM neurospheres treated with SapC-DOPS did not show activation of apoptosis as measured by cleaved caspase 9 and cleaved PARP, and lacked activation of DNA damage markers phosphor-ATM and γ-H2AX. Consistent with this, treatment with a pan-caspase inhibitor Z-VAD-FMK did not rescue SapC-DOPS-induced killing (P&amp;gt;0.05). In contrast, SapC-DOPS treatment increased levels of an autophagic marker LC3-II via western blot. Autophagosome formation was also confirmed through transmission electron microscopy. Utilizing a stable GBM cell line expressing a GFP-LC3 fusion protein, we observed punctuated GFP expression following treatment, indicative of autophagosome formation. Quantification of GFP punctated cells showed a significant increase in SapC-DOPS treated cells compared to control (P&amp;lt;.001). Analysis of red/green fluorescence following acridine orange staining showed an induction of acidic vesicular organelles indicative of autophagolysosomes. In addition, inhibition of autophagosome formation using 3-methyladeneine or inhibition of auotphagic vacuole maturation with bafilomycin A1 resulted in a significant rescue of SapC-DOPS-induced killing (P&amp;lt;.001). Knockdown of ATG5 using siRNA also resulted in a rescue of SapC-DOPS-induced cell death and autophagy induction (P&amp;lt;.001). Interestingly, we did not observe a decrease in the activation of mTOR as determined by the phosphorylation of 4EBP1 and p70S6K which are typically induced during autophagy. This led us to test the use of rapamycin, a known inhibitor of mTOR and inducer of autophagy, in combination with SapC-DOPS. By using the Chou Talalay analysis, we observed strong synergy for multiple drug combinations in primary GBM neurospheres (combination index &amp;lt; .4). To investigate whether SapC-DOPS induced autophagy could be preferentially targeting mitochondria (mitophagy), we utilized MitoTracker Green (mitochondria) and LysoTracker Red (autophagolysosomes). By using confocal microscopy analysis, we were able to observe a decrease in mitochondrial mass in cells treated with SapC-DOPS as well as co-localization of mitochondria with autophagolysosomes. In addition to this, we observed a significant decrease in ATP levels following SapC-DOPS treatment (P&amp;lt;.01). These findings suggest therapeutic implications for treating GBM by using SapC-DOPS alone and in combination with an AKT/mTOR inhibitor. Citation Format: Jeffrey Wojton, Naduparambil K. Jacob, Nicholas Denton, Nina Dmitrieva, Hiroshi Nakashima, Chang-Hyuk Kwon, Lionel Chow, Ennio A. Chiocca, Arnab Chakravarti, Balveen Kaur, Xiaoyang Qi. SapC-DOPS induces lethal mitophagy in glioblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2169. doi:10.1158/1538-7445.AM2013-2169

Increased Mitochondrial Mass in Cells with Functionally Compromised Mitochondria after Exposure to both Direct γ Radiation and Bystander Factors

The bystander effect describes radiation-like damage in unirradiated cells either in the vicinity of irradiated cells or exposed to medium from irradiated cells. This study aimed to further characterize the poorly understood mitochondrial response to both direct irradiation and bystander factor(s) in human keratinocytes (HPV-G) and Chinese hamster ovarian cells (CHO-K1). Oxygen consumption rates were determined during periods of state 4, state 3 and uncoupled respiration. Mitochondrial mass was determined using MitoTracker FM. CHO-K1 cells showed significantly reduced oxygen consumption rates 4 h after exposure to 5 Gy direct radiation and irradiated cell conditioned medium (ICCM) and an apparent recovery 12-24 h later. The apparent recovery was likely due to the substantial increase in mitochondrial mass observed in these cells as soon as 4 h after exposure. HPV-G cells, on the other hand, showed a sustained increase in oxygen consumption rates after ICCM exposure and a transient increase 4 h after exposure to 5 Gy direct radiation. A significant increase in mitochondrial mass per HPV-G cell was observed after exposure to both direct radiation and ICCM. These findings are indicative of a stress response to mitochondrial dysfunction that increases the number of mitochondria per cell.

Increase in Mitochondrial Mass in Human Fibroblasts under Oxidative Stress and during Replicative Cell Senescence

Abnormal proliferation of mitochondria generally occurs in muscle of aged individuals and patients with mitochondrial myopathy. An increase in the mitochondrial DNA (mtDNA) copy number has also been observed in aging human tissues. However, the molecular mechanism underlying the increase in mitochondrial mass and mtDNA is still unclear. In a previous study, we demonstrated that sublethal levels of oxidative stress caused an increase in mitochondrial mass in human lung cells. In this communication, we report our recent findings that the mitochondrial mass in human lung fibroblasts (MRC-5) in a later proliferation stage is significantly increased compared to that in the early stages of proliferation. The extent of the increase in mitochondrial mass in the senescent cells was similar to that in cells in the early stages of proliferation that had been treated with low concentrations (≤180 µM) of hydrogen peroxide (H₂O₂). Moreover, we found that the rate of reactive oxygen species (ROS) production was higher in cells in the later proliferation stage compared to cells in the early proliferation stages. A similar phenomenon was also observed in cells in the early proliferation stages under low levels of oxidative stress. On the other hand, the mRNA levels of many nuclear DNA-encoded proteins involved in mitochondrial biogenesis, particularly nuclear respiratory factor-1, were found to increase in cells in later proliferation stages and in cells in early proliferation stages that had been treated with 180 µM H₂O₂. Interestingly, the increase in mitochondrial mass in the cells under oxidative stress could be repressed by treatment with cycloheximide or m-chlorocarbonyl cyanide phenylhydrazone but not by chloramphenicol. Furthermore, the mitochondrial mass of mtDNA-less ρ^o cells was also significantly increased by exposure to low concentrations (e.g. 180 µM) of H₂O₂. These results suggest that the increase in mitochondrial mass in replicative senescent cells may result from an increase in ROS production, and that it is dependent on both de novo synthesis of nuclear DNA-encoded proteins and their import into mitochondria, dictated by the membrane potential of mitochondria.

Increase in mitochondrial mass in human fibroblasts under oxidative stress and during replicative cell senescence.

Abnormal proliferation of mitochondria generally occurs in muscle of aged individuals and patients with mitochondrial myopathy. An increase in the mitochondrial DNA (mtDNA) copy number has also been observed in aging human tissues. However, the molecular mechanism underlying the increase in mitochondrial mass and mtDNA is still unclear. In a previous study, we demonstrated that sublethal levels of oxidative stress caused an increase in mitochondrial mass in human lung cells. In this communication, we report our recent findings that the mitochondrial mass in human lung fibroblasts (MRC-5) in a later proliferation stage is significantly increased compared to that in the early stages of proliferation. The extent of the increase in mitochondrial mass in the senescent cells was similar to that in cells in the early stages of proliferation that had been treated with low concentrations (< or = 180 microM) of hydrogen peroxide (H(2)O(2)). Moreover, we found that the rate of reactive oxygen species (ROS) production was higher in cells in the later proliferation stage compared to cells in the early proliferation stages. A similar phenomenon was also observed in cells in the early proliferation stages under low levels of oxidative stress. On the other hand, the mRNA levels of many nuclear DNA-encoded proteins involved in mitochondrial biogenesis, particularly nuclear respiratory factor-1, were found to increase in cells in later proliferation stages and in cells in early proliferation stages that had been treated with 180 microM H(2)O(2). Interestingly, the increase in mitochondrial mass in the cells under oxidative stress could be repressed by treatment with cycloheximide or m-chlorocarbonyl cyanide phenylhydrazone but not by chloramphenicol. Furthermore, the mitochondrial mass of mtDNA-less rho(o) cells was also significantly increased by exposure to low concentrations (e.g. 180 microM) of H(2)O(2). These results suggest that the increase in mitochondrial mass in replicative senescent cells may result from an increase in ROS production, and that it is dependent on both de novo synthesis of nuclear DNA-encoded proteins and their import into mitochondria, dictated by the membrane potential of mitochondria.