Expression Of mtTFA Research Articles

Anticancer approach of zinc oxide nanocomposite prepared by gamma radiation via induction of apoptosis and PGC‐1α pathways in hepatocellular carcinoma in rats

Zinc oxide nanoparticles (ZnO NPs) are attracting the interest of researchers due to their potential applications in cancer treatment. It was recently shown that ZnO NPs specifically destroy cancer cells. However, the underlying molecular processes that drive ZnO NPs' anticancer activity are unclear. Radical polymerization was used for synthesizing zinc oxide nanoparticles using gamma radiation. Polyvinylpyrrolidone (PVP) was employed as a stabilizer in the production of zinc oxide nanoparticles using zinc nitrate. PVP@ZnO nanostructures were characterized using a number of techniques, including high‐resolution transmission electron microscopy (HRTEM) and UV–visible spectroscopy. Fourier‐transform infrared spectroscopy (FTIR) was used to study the functional groups of the produced nanostructures. Moreover, the surface morphology of prepared zinc oxide nanoparticles (ZnO NPs) and PVP@ZnO nanostructures was shown using SEM. In addition, the crystalline structure of the aforementioned samples was identified and investigated via XRD. The current work examined the anticancer effect of ZnO nanocomposite against rat hepatocellular carcinoma (HCC). The results demonstrated that ZnO nanocomposite exert distinct effects on apoptosis and PGC‐1α pathways in hepatocellular carcinoma in rats. mRNA levels of tumor suppressor gene p53 and antiapoptotic gene BCL‐2 were downregulated, while the apoptotic gene Bax was upregulated and Caspase‐3 activity increased in diethylnitrosamine (DEN) + ZnO nanocomposite‐treated rats in comparison with DEN‐treated rats. ZnO nanocomposite were also found to have an effect on liver function (ALT, AST, ALP, and GGT) and total antioxidant capacity (TAC). Treatment with DEN manifested a significant decrease in the expression of mtTFA and PGC‐1α (mtTFA: 0.54 fold and PGC‐1α: 0.31 fold in respect to the normal control), while ZnO nanocomposite treatment induced a remarkable increase in their expression. In conclusion, the present data demonstrated that ZnO nanocomposite showed anticancer activity via induction of apoptosis and PGC‐1α pathways. In addition, improvements in liver function and antioxidant capacity.

Shengxian decoction protects against chronic heart failure in a rat model via energy regulation mechanisms

BackgroundChronic heart failure (CHF) is actually a disease caused by an imbalanced energy metabolism between myocardial energy demand and supply, ultimately resulting in abnormal myocardial cell structure and function. Energy metabolism imbalance plays an important role in the pathological process of chronic heart failure (CHF). Improving myocardial energy metabolism is a new strategy for the treatment of CHF. Shengxian decoction (SXT), a well-known traditional Chinese medicine (TCM) formula, has good therapeutic effects on the cardiovascular system. However, the effects of SXT on the energy metabolism of CHF is unclear. In this study, we probed the regulating effects of SXT on energy metabolism in CHF rats using various research methods.MethodsHigh-performance liquid chromatography (HPLC) analysis was used to perform quality control of SXT preparations. Then, SD rats were randomly assigned into 6 groups: sham, model, positive control (trimetazidine) and high-, middle-, and low-dose SXT groups. Specific reagent kits were used to detect the expression levels of ALT and AST in rats’ serum. Echocardiography was used to evaluate cardiac function. H&E, Masson and TUNEL staining were performed to examine myocardial structure and myocardial apoptosis. Colorimetry was used to determine myocardial ATP levels in experimental rats. Transmission electron microscopy was used to observe the ultrastructure of myocardial mitochondria. ELISA was used to estimate CK, cTnI, and NT-proBNP levels, and LA、FFA、MDA、SOD levels. Finally, Western blotting was used to examine the protein expression of CPT-1, GLUT4, AMPK, p-AMPK, PGC-1α, NRF1, mtTFA and ATP5D in the myocardium.ResultsHPLC showed that our SXT preparation method was feasible. The results of ALT and AST tests indicate that SXT has no side effect on the liver function of rats. Treatment with SXT improved cardiac function and ventricular remodelling and inhibited cardiomyocyte apoptosis and oxidative stress levels induced by CHF. Moreover, CHF caused decrease ATP synthesis, which was accompanied by a reduction in ATP 5D protein levels, damage to mitochondrial structure, abnormal glucose and lipid metabolism, and changes in the expression of PGC-1α related signal pathway proteins, all of which were significantly alleviated by treatment with SXT.ConclusionSXT reverses CHF-induced cardiac dysfunction and maintains the integrity of myocardial structure by regulating energy metabolism. The beneficial effect of SXT on energy metabolism may be related to regulating the expression of the PGC-1α signalling pathway.

Synergistic effect of curcumin and chitosan nanoparticles on nano-hydroxyapatite-induced reproductive toxicity in rats.

Although the toxicity/biocompatibility of hydroxyapatite nanoparticles (HAPNPs), a prospective nano-biomaterial, is extensively studied, its interaction on the reproductive system following exposure is less exploited. In the present study, male rats were exposed to HAPNPs (300 mg/kg BW) to determine its possible reproductive toxicity. Also, the protective effects of chitosan(CSNPs, 280 mg/kg BW) and/or curcumin (CurNPs, 15 mg/kg BW) nanoparticles against HAPNPs-induced reproductive toxicity were studied. Animals were orally gavage daily with respective doses for 45 consecutive days. The obtained results indicated that HAPNPs caused a significant decrease in sperm count, sperm motility, testosterone hormone, steroidogenic enzymes (17-ketosteroid reductase and 17β-hydroxysteroid dehydrogenase), and antioxidant enzymes (glutathione peroxidase, glutathione S-transferase, catalase, and superoxide dismutase) in addition to total antioxidant capacity and reduced glutathione. LH and FSH, abnormal sperm, oxidative stress parameters (thiobarbituric acid-reactive substances (TBARS), nitric oxide (NO), and 8-hydroxy-deoxyguanosine (8-OHdG)), p53, TNFα, and interleukin-6 were significantly increased. The DNA damage was also analyzed by assaying 8-OHdG level which is considered as an indicator of genotoxicity and also suppression of the gene expression of mtTFA, induction of UCP2. Similarly, the histopathological evaluation was also changed following exposure to HAPNPs. The antioxidant activity of CSNPs and CurNPs showed mitigating effect against reproductive deterioration induced by HAPNPs throughout improvements in semen characteristics, sex hormones, inflammatory factors, and antioxidant status. The present study concluded that HAPNPs induced reproductive toxicity and it is important to use nano-antioxidants CSNPs and CurNPs as protective agents.

Dose-dependent reproductive toxicity of sodium benzoate in male rats: Inflammation, oxidative stress and apoptosis

The synthetic food preservative sodium benzoate (SB) is widely used in both food and pharmaceutical industries. A growing body of evidence highlights the adverse effects of SB on human health; however, effect of the prolonged intake of SB on the reproductive system is not fully elucidated. The current study investigates the effect of different doses of SB (0−1000 mg/kg BW) on the reproductive system of male rats administered oral SB for 90 consecutive days. Results revealed that increasing doses of SB significantly altered the weight of reproductive organs, decreased sperm count and motility and enhanced the percentage of abnormal sperms. This was concomitant with significant decline in plasma testosterone and FSH levels, increase in plasma LH and decrease in the activities of 17β-HSD and 17-KSR enzymes in the testes. Inflammation and oxidative stress were induced as indicated by the significant increase in TNF-α and IL-6 levels, inhibition of antioxidant enzymes activity and levels of GSH, increase in the levels of NO and TBARS and enhanced protein expression of mtTFA and UCP2 in the testes. Interestingly, p53 expression and caspase-3 activity were upregulated in the testes suggesting induction of apoptosis. Histopathological examination of the testes confirmed apoptosis and revealed degenerative alterations of the testes’ architecture and perturbation of spermatogenesis. Based upon these findings, the no-observed-adverse-effect level of SB on the reproductive system was determined to be less than 1 mg/kg BW/day, highlighting the risks of long-term exposure to low as well as high doses of SB on male reproductive health.

Pro-renin receptor suppresses mitochondrial biogenesis and function via AMPK/SIRT-1/ PGC-1α pathway in diabetic kidney.

Abnormal mitochondrial biogenesis and function has been linked to multiple diseases including diabetes. Recently, we demonstrated the role of renal (Pro)renin receptor (PRR) in the dysregulation of mitochondria. We hypothesized that PRR contributes to the reduction of mitochondrial biogenesis and function in diabetic kidney via PGC-1α/AMPK/SIRT-1 signaling pathway. In vivo and in vitro studies were conducted in C57BL/6 mouse and mouse renal mesangial cells (mRMCs). Control and streptozotocin-induced diabetic mice were injected with scramble or PRR shRNA and followed for a period of eight weeks. PRR mRNA and protein expression increased by 44% and 39% respectively (P<0.05) in kidneys of diabetic mice, and in mRMCs exposed to high glucose by 43 and 61% respectively compared to their respective controls. These results were accompanied by reduced mRNA and protein expressions of PGC-1α (67% and 75%), nuclear respiratory factors (NRF-1, 48% and 53%), mitochondrial transcriptional factor A (mtTFA, 56% and 40%), mitochondrial DNA copy number by 75% (all, P<0.05), and ATP production by 54%, respectively in diabetic kidneys and in mRMCs exposed to high glucose. Compared to non-diabetic control mice, PRR knockdown in diabetic mice and in mRMCs, not only attenuated the PRR mRNA and protein expression but also normalized mRNA and protein expressions of PGC-1α, NRF-1, mtTFA, mitochondrial DNA copy number, and ATP production. Treatment with AMPK inhibitor, Compound C, or SIRT-1 inhibitor, EX-527, alone, or combined with PRR siRNA caused marked reduction of mRNA expression of PGC-1α, NRF-1 and mtTFA, and ATP production in mRMCs exposed to high glucose. In conclusion, our study demonstrated the contribution of the PRR to the reduction of mitochondrial biogenesis and function in diabetic kidney disease via decreasing AMPK/SIRT-1/ PGC-1α signaling pathway.

Hepato-renal toxicity of oral sub-chronic exposure to aluminum oxide and/or zinc oxide nanoparticles in rats.

Aluminum oxide nanoparticles (Al2O3NPs) and zinc oxide nanoparticles (ZnONPs) have been involved in many industries and they are extensively abundant in many aspects of human life. Consequently, concerns have been raised about their potentially harmful effects. However the toxicities of Al2O3NPs and ZnONPs are well documented, the effect of co-exposure to both nanoparticles remains strictly obscure. Therefore, the present study was undertaken to address this issue. Four groups of male Wistar rats (10 rats each) were used; control, Al2O3NPs treated, ZnONPs treated and Co-treated groups. Rats were orally administered their respective treatment daily for 75 days. The effects of each nanoparticle alone or in combination were assessed at different levels including; hepatic and renal function, structure, and redox status, nuclear DNA fragmentation, hepatic expression of mitochondrial transcription factor A (mtTFA) gene and peroxisome proliferator-activated receptor gamma-coactivator 1α (PGC-1α), systemic inflammation, and hematologic parameters. The results confirmed the hepatorenal toxicities of each nanoparticle used at the level of all parameters with suppression of the hepatic expression of mtTFA and PGC-1α. The co-exposure to both nanoparticles results in synergistic effects. From these results, we can conclude that co-exposure to aluminum oxide nanoparticles and zinc oxide nanoparticles results in more pronounced hepatorenal toxicities and systemic inflammation.

PL-020 Establishment of skeletal muscle-specific PGC-1α overexpression model via in vivo local transfection

Objective To establish a skeletal muscle-specific PGC-1α overexpression mouse model via in vivo local transfection.&#x0D; Methods For the PGC-1α in vivo transfection study, the Male FVB/N mice were randomly divided into 2 groups: subject to green fluorescent protein (GFP) transfection (Con-GFP), and subject to PGC-1α in vivo transfection (Con-PGC-1α). Plasmid DNA solution (2.5 mg/ml GFP or 2.7 mg/ml Flag-PGC-1α) were injected into the proximal (6 ml) and distal (6 ml) ends of the muscle belly. Following the injections, electric pulses were applied through 2 stainless steel pin electrodes laid on top of the proximal and distal myotendinous junctions. Then skeletal muscle and myocardium were isolated, and PGC-1α, mtTFA, NF-κB, MnSOD, FNDC5 protein expression were measured with Western blot.&#x0D; Results n skeletal muscle, compared with con-GFP group, the expression of PGC-1α (+125%, p&lt;0.01), mtTFA (+210%, p&lt;0.01), and FNDC5 (+47%, p&lt;0.05) were significantly increased in con-PGC-1α group. However, NF-κB and MnSOD protein level had no change in con-PGC-1α group. In myocardium, compared with con-GFP group, the expression of mtTFA (+130%, p&lt;0.01) and FNDC5 (+55%, p&lt;0.05) were significantly increased in con-PGC-1α group.&#x0D; Conclusions Skeletal muscle-specific PGC-1α overexpression model via in vivo local transfection was established, which was supported by elevated expression of PGC-1α and its downstream FNDC5 and mtTFA. Furthermore, skeletal muscle-specific PGC-1α overexpression induced increase in myocardial mitochondrial biogenesis, while relative mechanism remains to be determined.

Yiqi Huoxue prescription can prevent and treat post-MI myocardial remodeling through promoting the expression of AMPK signal pathway

ObjectiveTo investigate how Yiqi Huoxue (YQHX) prescription regulates mitochondrial biosynthesis and ATP synthesis via AMP-activated protein kinase (AMPK) and to reveal its molecular mechanism in preventing and treating post-MI myocardial remodeling. MethodsThe MI animal model of myocardial infarction were established by ligating Sprague Dawley (SD) rats' left anterior descending coronary arteries; the animals were randomly divided into MI group, YQHX prescription group and perindopril group, and a sham operation group was set at the same time. Related drug intervention was administered on the 2nd day after surgery, the YQHX group was given Astragalus, angelica, ginseng, Ligusticum wallichii and pseudoginseng, provided by the Dongzhimen Hospital of Beijing University of Chinese Medicine, once per day, at a dose of 21 g/kg body weight/day (the clinical equivalent dose based on a previous study), and the changes in relevant indicators were observed at 1 week and 4 weeks. Echocardiography (ECG) was used to observe the changes in rat cardiac structure and functions; the morphology-based technique was used to observe the changes in myocardial cells and mitochondria; the expression of AMPK signal pathway-related proteins and mRNA was detected using western blotting and real-time fluorescence quantification respectively, while fluorescence enzyme-labeled method was used for detecting ATP synthesis. ResultsCardiac structure and functions: Compared with the MI group at 1 week, the YQHX prescription group and the perindopril group exhibited increased LVEF and LVFS (Pall < .05); their LVEDs and LVEDd were reduced but suggested no statistically significant differences (F = 2.258, F = 0.3464, Pall > .05). At 4 weeks, both LVEF and LVFS were elevated in the YQHX prescription group (P = .008, .009) and the perindopril group (P = .279, .333), where differences in the later group indicated no statistical significance; the YQHX prescription group and the perindopril group were also featured by reduced LVEDs and LVEDd (Pall < .05). Morphology: Compared with the MI group at two time nodes, the YQHX prescription group and the perindopril group exhibited significant improvement in the pathological changes in myocardial cells and mitochondrial structure. Expression of AMPK signal pathway-related proteins and mRNA: The expression of both pLKB1 and pAMPK proteins followed a rising trend in the YQHX prescription group and the perindopril group at 1 week. The expression of LKB1mRNA and AMPKmRNA was elevated (Pall < .05). The increased expression of PGC-1α, NRF1 and mtTFA proteins demonstrated statistically significant differences (Pall < .05). The expression of mtDNA protein followed an increasing trend. At 4 weeks, the expression of both pLKB1 and pAMPK proteins was elevated (Pall < .05). Heightened expression was also reported in LKB1mRNA and AMPKmRNA (Pall < .05). The increased expression of PGC-1α, NRF1, mtTFA and mtDNA proteins demonstrated statistically significant differences (Pall < .05). ATP synthesis: ATP synthesis was increased in the YQHX prescription group and the perindopril group at both 1 week and 4 weeks (Pall < .001). ConclusionThe possible mechanism of YQHX prescription for preventing and treating post-MI myocardial remodeling may function through strengthening the activation AMPK signal pathway by LKB1, thus further increasing the expression of downstream transcription factor proteins and initiating mitochondrial replication and transcription; as a result, the YQHX prescription can improve the post-MI damage in mitochondrial morphological structure in the tissue at heart marginal zone as well as enhance mitochondrial biosynthesis and ATP synthesis.

T-2 toxin inhibits murine ES cells cardiac differentiation and mitochondrial biogenesis by ROS and p-38 MAPK-mediated pathway.

To investigate the effect of T-2 toxin on murine embryonic stem cells (ESCs) cardiac differentiation and mitochondrial biogenesis in vitro. Cardiac differentiation of the mouse ESCs was initiated by embryoid bodies (EBs) formation in hanging drops. EBs were exposed to 0.5ng/ml T-2 toxin for 24, 72 and 120h. Cultures were observed daily for the appearance of contracting clusters, and cardiac-specific protein (α-actiniin) were measured by Western blot and immunocytochemistry. Mitochondrial ultrastructure was observed by confocal laser scanning microscopy and transmission EM photography. Reactive oxygen species (ROS) was monitored by H2-dichlorofluorescein-diacetate (H2DCF-DA). The phosphorylation of the p38 (p-p38) and p38 mitogen-activated protein kinase (MAPK) and the expression of mitochondrial biogenesis proteins, including peroxisome proliferator activated receptor coactivator-1 alpha (PGC-1α), nuclear respiratory factor 1 (NRF-1), mitochondrial transcription factor A (mtTFA), and mitochondrial respiratory chain complex IV (COXIV) were analyzed using Western blot. In some experiments, mESCs were pre-treated with the antioxidant Trolox (200μM) for 30min, then exposed to Trolox (200μM) and T-2 toxin (0.5ng/ml) for 72h. Contracting clusters were observed under the microscope light and cardiac-specific protein (α-actinin) expressed positively indicated mESCs directly differentiated in cardiomyocytes. However, the cardiac differentiation was inhibited by T-2 toxin treatment 72 and 120h. ROS accumulated in murine ES cells in a time-dependent manner. The expression of p-p38 significantly increased in 24h group and decrease in 72 and 120h groups. The decrease of mitochondrial number and the mitochondrial biogenesis-related proteins expression, including PGC-1α, NRF-1, mtTFA, and COXIV decreased in a time-dependent manner with T-2 toxin treatment. However, the inhibition of mitochondrial biogenesis by T-2 toxin in differentiated mESCs was recovered significantly in the presence of the antioxidant Trolox. Taken together, T-2 toxin decreased the expression of PGC-1α, NRF-1, and mtTFA, inhibited mitochondrial biogenesis, and then inhibited the cardiac differentiation of murine ES cells, and the effect was partly responsible for the p38 MAPK mediated by ROS.

Complex-I Alteration and Enhanced Mitochondrial Fusion Are Associated With Prostate Cancer Progression.

Mitochondria (mt) encoded respiratory complex-I (RCI) mutations and their pathogenicity remain largely unknown in prostate cancer (PCa). Little is known about the role of mtDNA loss on mt integrity in PCa. We determined mtDNA mutation in human and mice PCa and assessed the impact of mtDNA depletion on mt integrity. We also examined whether the circulating exosomes from PCa patients are transported to mt and carry mtDNA or mt proteins. We have employed next generation sequencing of the whole mt genome in human and Hi-myc PCa. The impact of mtDNA depletion on mt integrity, presence of mtDNA, and protein in sera exosomes was determined. A co-culture of human PCa cells and the circulating exosomes followed by confocal imaging determined co-localization of exosomes and mt. We observed frequent RCI mutations in human and Hi-myc PCa which disrupted corresponding complex protein expression. Depletion of mtDNA in PCa cells influenced mt integrity, increased expression of MFN1, MFN2, PINK1, and decreased expression of MT-TFA. Increased mt fusion and expression of PINK1 and DNM1L were also evident in the Hi-myc tumors. RCI-mtDNA, MFN2, and IMMT proteins were detected in the circulating exosomes of men with benign prostate hyperplasia (BPH) and progressive PCa. Circulating exosomes and mt co-localized in PCa cells. Our study identified new pathogenic RCI mutations in PCa and defined the impact of mtDNA loss on mt integrity. Presence of mtDNA and mt proteins in the circulating exosomes implicated their usefulness for biomarker development.

Recombinant human augmenter of liver regeneration protects hepatocyte mitochondrial DNA in rats with obstructive jaundice

BackgroundHepatocyte mitochondrial DNA (mtDNA) damage is an important cause of mitochondrial and hepatic function impairment in obstructive jaundice (OJ). This study investigated the protective effect of recombinant human augmenter of liver regeneration (rhALR) on hepatocyte mtDNA in rats with OJ. Materials and methodsWistar rats were randomly divided into three groups as follows: sham-operation, biliary obstruction and recanalization with rhALR treatment (BDO-RBF-rhALR), and BDO-RBF-Vehicle (n = 48 per group). After biliary obstruction, rats were intraperitoneally injected with 40 μg/kg rhALR in BDO-RBF-rhALR group and same volume of normal saline in other two groups once every 12 h, until sacrifice. Mitochondrial transcription factor A (mtTFA) and nuclear respiratory factor-1 (NRF-1) expression in hepatocytes were detected by real-time reverse transcription-polymerase chain reaction and Western blot. Hepatocyte mtDNA damage was evaluated by real-time-polymerase chain reaction. Mitochondrial and hepatic functions were also assessed. ResultsAfter biliary obstruction, hepatic function was clearly impaired, as shown by the increases in serum alanine aminotransferase, aspartate aminotransferase, and total bilirubin levels, and the decrease in albumin level. Mitochondrial respiratory control ratio, phosphorus oxygen ratio, and ATP levels (all indicators of mitochondrial function) were decreased. The relative amount of total mtDNA, mtTFA, and NRF-1 expression in rat liver tissues were decreased, whereas the relative amount of deleted mtDNA was increased. However, the damage was significantly improved in the BDO-RBF-rhALR group. After recanalization, these changes were gradually restored, but the recovery was faster in the BDO-RBF-rhALR group than in BDO-RBF-Vehicle group. ConclusionsrhALR may protect and improve mitochondrial and hepatic functions in rats with OJ by promoting the expression of mtTFA and NRF-1 and by protecting and repairing damaged mtDNA.

The combination of strong immunohistochemical mtTFA expression and a high survivin index predicts a shorter disease-specific survival in pancreatic ductal adenocarcinoma.

Mitochondrial transcription factor A (mtTFA) plays a crucial role in both the transcription and maintenance of mitochondrial DNA. A high expression of mtTFA has been demonstrated in several solid tumors, and is closely associated with cancer cell survival/apoptosis and growth. However, its expression pattern in pancreatic ductal adenocarcinoma (PAC) remains to be elucidated. Additionally, our groups have recently revealed that a subset of apoptosis-related genes is strongly regulated by mtTFA, and that two putative mtTFA binding sites are present in the promoter region of the survivin gene, which is a member of the inhibitor-of-apoptosis protein family. We therefore investigated the correlation of the immunohistochemical mtTFA expression and the survivin index with various clinicopathological variables and the prognosis, using 70 paraffin-embedded tumor samples from patients with surgically-resected PAC. The mtTFA expression or survivin index was considered to be strong or high when ≥30% or 10% of the PAC cells showed positive staining, respectively. Strong mtTFA expression and/or a high survivin index was revealed to have a significant relationship to a pathologically high tumor grading and advanced tumor stage. Moreover, mtTFA showed significantly high co-expression with survivin. Univariate and multivariate analyses demonstrated that both the strong mtTFA expression and high survivin index groups had significantly shorter survival rates, especially within the first two years postoperatively. The combination of strong mtTFA expression and a high survivin index may predict a poor prognosis in patients with PAC, and these new biomarkers might offer useful information for the early clinical management.

Mitochondrial Transcription Factor A Worsens the Clinical Course of Patients With Pancreatic Cancer Through Inhibition of Apoptosis of Cancer Cells

Mitochondrial transcription factor A (mtTFA) is mandatory for both the transcription and maintenance of mitochondrial DNA. This study aimed to investigate the significance of mtTFA expression in pancreatic ductal adenocarcinoma (PDAC). Surgical specimens from 93 patients with PDAC who all underwent pancreatectomy were immunohistochemically stained using a polyclonal anti-mtTFA antibody. The relationship between the expression of mtTFA, clinicopathologic factors, and prognosis of these patients were evaluated. Positive mtTFA expression was significantly associated with lymphovascular invasion and metastatic recurrence in the liver and correlated with an advanced surgical stage. A univariate analysis showed that the patients with positive mtTFA expression had a significantly shorter survival time than those patients with negative mtTFA expression, and a multivariate analysis revealed that mtTFA expression was one of the independent prognostic factors in patients with PDAC. Positive mtTFA expression was significantly correlated with a low apoptotic index but not significantly correlated with the mind bomb homolog-1 (MIB-1) index. The expression mtTFA worsens the clinical course of patients with PDAC through the inhibition of apoptosis of PDAC cells and is an independent marker for the poor prognosis of the patients with PDAC after pancreatectomy. Mitochondrial transcription factor A may be a novel target for the treatment of PDAC.

Expression and Methylation of Mitochondrial Transcription Factor A in Chronic Obstructive Pulmonary Disease Patients with Lung Cancer

BackgroundApoptosis plays a central role in the pathogenesis of chronic obstructive pulmonary disease (COPD), and this process can be regulated by mitochondrial transcription factor A (mtTFA). Epigenetics is involved in the regulation and modification of the genes involved in lung cancer and COPD. In this study, we determined the expression of mtTFA and its methylation levels in the COPD patients with lung cancer.MethodsTwenty-one squamous cell lung cancer patients, 11 with COPD and 10 without COPD, undergoing pneumonectomy were enrolled. The apoptotic index (AI) of pulmonary vascular endothelial cells was analyzed by transferase-mediated deoxyuridine triphosphate-biotin nick end labeling assay. The expression of mtTFA mRNA and protein was measured using PCR, immunohistochemistry and Western-blot. Methylation of the mtTFA promoter was detected using bisulfite sequencing PCR.ResultsCompared to the non-COPD group, the AI was higher, and expression of mtTFA mRNA and protein was lower in the COPD group (P<0.001). Expression of the mtTFA protein was positively correlated with FEV1/Pre (r = 0.892, P<0.001), and negatively correlated with AI (r = −0.749, P<0.001) and smoke index (r = −0.763, P<0.001). Percentage of mtTFA promoter methylation in the COPD patients was significantly higher compared to the non-COPD patients (P<0.05).ConclusionThese results suggest that the expression of mtTFA mRNA and protein is down-regulated in the lung tissue from the COPD patients with squamous cell lung cancer, and the level of mtTFA protein is related to apoptosis of pulmonary vascular endothelial cells. Aberrant mtTFA methylation may also play an important role in the pathogenesis of COPD.

MicroRNA-494 regulates mitochondrial biogenesis in skeletal muscle through mitochondrial transcription factor A and Forkhead box j3

MicroRNAs (miRNAs) are important posttranscriptional regulators of various biological pathways. In this study, we focused on the role of miRNAs during mitochondrial biogenesis in skeletal muscle. The expression of miR-494 was markedly decreased in murine myoblast C₂C₁₂ cells during myogenic differentiation, accompanied by an increase in mtDNA. Furthermore, the expression of predicted target genes for miR-494, including mitochondrial transcription factor A (mtTFA) and Forkhead box j3 (Foxj3), was posttranscriptionally increased during myogenic differentiation. Knockdown of miR-494 resulted in increased mitochondrial content and upregulation of mtTFA and Foxj3 at the protein level. A 3'-untranslated region reporter assay revealed that miR-494 knockdown directly upregulated the luciferase activity of mtTFA and Foxj3. All of these observations were reversed by overexpression of miR-494. Furthermore, the miR-494 content significantly decreased after endurance exercise in C57BL/6J mice, accompanied by an increase in expression of mtTFA and Foxj3 proteins. These results suggest that miR-494 regulates mitochondrial biogenesis by downregulating mtTFA and Foxj3 during myocyte differentiation and skeletal muscle adaptation to physical exercise.

Abstract 1118: Protease-integrin regulates mitochondrial biogenesis in glioma xenograft cells

Abstract Various studies have described mitochondrial dysfunction and its association with several diseases, including cancer. Regulation of mitochondrial biogenesis via mitochondrial-nuclear genome encoded protein signaling has been identified in recent literature. In the present study, we explored whether matrix metalloproteinase-9 (MMP-9) shRNA mediates mitochondrial biogenesis and alters glioma proliferation. We found that MMP-9 shRNA initiated mitochondrial biogenesis in glioma cells by increasing the expression of mitochondrial transcription factor (mtTFA). MMP-9 silencing elevated nuclear respiratory factor-1 (NRF1) and -2 (NRF2), which are involved in transcription regulation of mtTFA. Mitochondrial localization of mtTFA was more evident in MMP-9-treated glioma cells as compared to untreated cells. In addition, MMP-9 suppression significantly increased the binding of mtTFA to light strand and heavy strand promoter of mitochondrial genome as determined by mitochondrial chromatin immunoprecipitation assay. MMP-9 transcriptional suppression decreased the expression of phospho Akt and PI3K while increasing PTEN expression in glioma xenograft cells. We also show that dominant negative Akt increased mtTFA, NRF1 and NRF2, and decreased PI3K, integrin αv and integrin β3. Further, MMP-9 and Akt suppression increased the nuclear and mitochondrial expression of mtTFA. As indicated by increased copy number of displacement (D) loop, mitochondrial replication was observed in both MMP-9- and Akt-downregulated glioma cells. As confirmed by immunoprecipitation analysis, interaction of αvβ3 integrin with MMP-9 decreased. Studies with functional blocking of αvβ3 integrin indicated elevation of mtTFA, a decrease in phospho Akt, and overexpression of D-loop; these data indicate MMP-9 mediates mitochondrial biogenesis via αvβ3 integrin-Akt/PTEN, which led to increased mtTFA expression. Earlier studies from our lab also showed apoptosis induction in these glioma cells by MMP-9 shRNA. Moreover, 4910 and 5310 glioma xenograft tissue sections from mice treated with MMP-9 shRNA showed reduced expression of phospho Akt, and elevated PTEN and mtTFA levels. Decreased co-localization of αvβ3 integrin and MMP-9 was associated with MMP-9-suppressed tumor sections. Further, RT-PCR and western blot analyses showed increased expression of proteins involved in mitochondrial biogenesis encoded both by mitochondrial and nuclear genome in MMP-9 shRNA-treated glioma cells. Elevated levels of reactive oxygen species and the results of the FACS analysis show increased mitochondrial organelle stain in MMP-9-silenced glioma cells. Taken together, these data provide new insight into MMP-9-mediated glioma proliferation inhibition by mitochondrial biogenesis induced cell death. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1118. doi:1538-7445.AM2012-1118

Clinical significance of mitochondrial transcription factor A expression in patients with colorectal cancer

Mitochondrial transcription factorA (mtTFA) is necessary for both the transcription and maintenance of mitochondrial DNA (mtDNA). The present study investigated the relationship between clinicopathological factors, prognosis and the immunohistochemical expression of mtTFA in the tumors of patients diagnosed with primary colorectal cancer (CRC). Surgical specimens from 105 colorectal patients were immunohistochemically stained using a polyclonal anti-mtTFA antibody. The relationships among the mtTFA expression, clinicopathological factors and prognosis were evaluated. A total of 47 (44.8%) of the 105 patients with CRC were determined to have positive mtTFA expression. The positive expression of mtTFA significantly correlated with lymph node metastasis, distant metastasis and advanced TNM staging. On the other hand, negative mtTFA expression showed a tendency to correlate with high Ki-67 index. The survival of patients with positive mtTFA expression was significantly worse than that of patients with negative mtTFA expression. The positive mtTFA expression appears to be a useful marker for tumor progression and poor prognosis in patients with CRC.

Differential Expression of PGC-1α and Metabolic Sensors Suggest Age-Dependent Induction of Mitochondrial Biogenesis in Friedreich Ataxia Fibroblasts

BackgroundFriedreich's ataxia (FRDA) is a mitochondrial rare disease, which molecular origin is associated with defect in the expression of frataxin. The pathological consequences are degeneration of nervous system structures and cardiomyopathy with necrosis and fibrosis, among others.Principal FindingsUsing FRDA fibroblasts we have characterized the oxidative stress status and mitochondrial biogenesis. We observed deficiency of MnSOD, increased ROS levels and low levels of ATP. Expression of PGC-1α and mtTFA was increased and the active form of the upstream signals p38 MAPK and AMPK in fibroblasts from two patients. Interestingly, the expression of energetic factors correlated with the natural history of disease of the patients, the age when skin biopsy was performed and the size of the GAA expanded alleles. Furthermore, idebenone inhibit mitochondriogenic responses in FRDA cells.ConclusionsThe induction of mitochondrial biogenesis in FRDA may be a consequence of the mitochondrial impairment associated with disease evolution. The increase of ROS and the involvement of the oxidative phosphorylation may be an early event in the cell pathophysiology of frataxin deficiency, whereas increase of mitochondriogenic response might be a later phenomenon associated to the individual age and natural history of the disease, being more evident as the patient age increases and disease evolves. This is a possible explanation of heart disease in FRDA.

Clinical usefulness of mitochondrial transcription factor A expression as a predictive marker in colorectal cancer patients treated with FOLFOX

We previously reported that mitochondrial transcription factor A (mtTFA) preferentially recognizes cisplatin-damaged DNA via physical interaction with p53 and is upregulated by treatment with cisplatin and fluorouracil (5-FU). The aim of the present study was to evaluate whether expression of mtTFA predicts the clinical outcome in patients with metastatic colorectal cancer treated with modified 5-fluorouracil, leucovorin and oxaliplatin 6 (mFOLFOX6). Fifty-nine patients with metastatic lesions from colorectal cancer treated with mFOLFOX6 were included in this study. The subjects consisted of 25 women and 34 men with a median age of 62 years. The patients were treated with oxaliplatin (85 mg/m(2) ) plus leucovorin (200 mg/m(2) ) as a 2-h infusion on day 1, followed by 5-FU (400 mg/m(2) ) bolus and 46-h continuous infusion of 2400 mg/m(2) . The expressions of mtTFA and p53 of resected primary tumors were examined by immunohistochemical analysis. Of the 59 patients, 33 (complete response 1, partial response 32) achieved a confirmed response to therapy. The positive cytoplasmic staining rate for mtTFA was 44.1% and that for p53 was 59.3%, respectively. Strong expression of mtTFA was detected in eight of 33 complete response/partial response (24.2%) and in 18 of 26 SD/PD (69.2%), indicating that mtTFA expression was significantly correlated with response to chemotherapy (P<0.01). Median overall survival was significantly longer in patients without mtTFA expression (P=0.0493). Multivariate analysis revealed that mtTFA expression significantly affected overall survival (hazard ratio 2.10, P=0.036). Immunohistochemical study of mtTFA may be useful for predicting the clinical outcome of metastatic colorectal cancer patients treated with FOLFOX.

Chronic Exposure to Arsenic Causes Increased Cell Survival, DNA Damage, and Increased Expression of Mitochondrial Transcription Factor A (mtTFA) in Human Prostate Epithelial Cells

Arsenic is a known carcinogen, and its exposure is associated with cancers in multiple target organs including the prostate. Whether arsenic causes cancer by increased cell proliferation or cell survival is not clear. Additionally, mitochondria have been shown to play important roles in arsenic-induced DNA damage and carcinogenesis. However, the mechanism of mitochondrial involvement in arsenic-induced cancer is not clear. Therefore, the objectives of this study were to investigate the effect of arsenic on cell proliferation/survival and genotoxicity, and to determine the effect of arsenic on the expression of mitochondrial transcription factor A (mtTFA) in human prostate epithelial cells, RWPE-1. Results of this study revealed that chronic exposure to arsenic causes increased cell survival. Arsenic also induced nuclear DNA damage and mutations in mitochondrial DNA. Expressions of DNA repair genes ERCC6, XPC, OGG1, and reactive oxygen species (ROS) scavenger MnSOD was also altered in arsenic-exposed cells. Arsenic concentration-dependent increased expression of mtTFA and its regulator NRF-1 was observed in arsenic-exposed cells, suggesting that arsenic regulates mitochondrial activity through an NRF-1-dependent pathway. In summary, this study suggests that chronic exposure to arsenic causes DNA damage and increased cell survival that may ultimately result in neoplastic transformation of human prostate epithelial cells. Additionally, this study also provides evidence that arsenic controls mitochondrial function by regulating mtTFA expression.