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7-Ketocholesterol (7-KC) is found at an elevated level in patients with cancer and chronic liver disease. The up-regulation of an efflux pump, P-glycoprotein (P-gp) leads to drug resistance. To elucidate the effect of 7-KC on P-gp, P-gp function and expression were investigated in hepatoma cell lines Huh-7 and HepG2 and in primary hepatocyte-derived HuS-E/2 cells. At a subtoxic concentration, 48-h exposure to 7-KC reduced the intracellular accumulation and cytotoxicity of P-gp substrate doxorubicin in hepatoma cells, but not in HuS-E/2 cells. In Huh-7 cells, 7-KC elevated efflux function through the activation of phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway. 7-KC activated the downstream protein synthesis initiation factor 4E-BP1 and induced P-gp expression post-transcriptionally. The stimulation of efflux was reversible and could not be prevented by N-acetyl cysteine. Total cellular ATP content remained the same, whereas the lactate production was increased and fluorescence lifetime of protein-bound NADH was shortened. These changes suggested a metabolic shift to glycolysis, but glycolytic inhibitors did not eliminate 7-KC-mediated P-gp induction. These results demonstrate that 7-KC induces P-gp through PI3K/mTOR signaling and decreased the cell-killing efficacy of doxorubicin in hepatoma cells., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Sirt3, a mitochondrial NAD(+)-dependent deacetylase, is regarded as a potential regulator in cellular metabolism. However, the role of Sirt3 in the regulation of mitochondrial F(o)F(1)ATPase and the linkage to mitochondrial diseases is unclear. In this study, we demonstrated a role of Sirt3 in the regulation of F(o)F(1)ATPase activity in human cells. Knockdown of Sirt3 in 143B cells by shRNA transfection caused increased acetylation levels of the α and OSCP subunits of F(o)F(1)ATPase. We showed that Sirt3 physically interacted with the OSCP and led to its subsequent deacetylation. By incubation of mitochondria with the purified Sirt3 protein, Sirt3 could regulate F(o)F(1)ATPase activity through its deacetylase activity. Moreover, suppression of Sirt3 reduced the F(o)F(1)ATPase activity, consequently decreased the intracellular ATP level, diminished the capacity of mitochondrial respiration, and compromised metabolic adaptability of 143B cells to the use of galactose as the energy source. In human cells harboring ≅85% of mtDNA with 4977bp deletion, we showed that oxidative stress induced a reduction of Sirt3 expression, and an increased acetylation of the OSCP subunit of F(o)F(1)ATPase. Importantly, the expression of Sirt3 was also decreased in the skin fibroblasts from patients with CPEO syndrome. We further demonstrated that oxidative stress induced by 5-10μM of menadione impaired the Sirt3-mediated deacetylation and activation on F(o)F(1)ATPase activity through decreasing the protein level of Sirt3. Our findings suggest that increased intracellular ROS levels might modulate the expression of Sirt3 which deacetylates and activates F(o)F(1)ATPase in human cells with mitochondrial dysfunction caused by a pathogenic mtDNA mutation., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Norcantharidin is the demethylated analog of cantharidin isolated from blister beetles (Mylabris phalerata Pall.). In this study, we evaluated whether norcantharidin exhibits anticancer effects against the human non-small cell lung cancer cell lines A549 (epidermal growth factor receptor (EGFR) mutation-negative) and PC9 (EGFR mutation-positive). Our results revealed that norcantharidin dose-dependently retards cell growth, arrests cell cycle at G2/M phase, reduces cell migration, and even induces apoptosis at the concentration of 100 µM. Moreover, we found that norcantharidin enhances the anticancer effects of gefitinib and cisplatin. Norcantharidin exhibited similar potency of anticancer effects against the two cell lines with different EGFR mutation status and did not affect EGF-induced EGFR phosphorylation, suggesting that the EGFR signaling may not be the target of norcantharidin. In conclusion, our results suggest that norcantharidin exhibits anticancer effects against non-small cell lung cancer cells in vitro and support its potential as a chemotherapeutic agent for treating non-small cell lung cancer.

In this study, the protective effect of melatonin on kainic acid (KA)-induced neurotoxicity involving autophagy and α-synuclein aggregation was investigated in the hippocampus of C57/BL6 mice. Our data showed that intraperitoneal injection of KA (20 mg/kg) increased LC3-II levels (a hallmark protein of autophagy) and reduced mitochondrial DNA content and cytochrome c oxidase levels (a protein marker of mitochondria). Atg7 siRNA transfection prevented KA-induced LC3-II elevations and mitochondria loss. Furthermore, Atg7 siRNA attenuated KA-induced activation of caspases 3/12 (biomarkers of apoptosis) and hippocampal neuronal loss, suggesting a pro-apoptotic role of autophagy in the KA-induced neurotoxicity. Nevertheless, KA-induced α-synuclein aggregation was not affected in the Atg7 siRNA-transfected hippocampus. The neuroprotective effect of melatonin (50 mg/kg) orally administered 1 hr prior to KA injection was studied. Melatonin was found to inhibit KA-induced autophagy-lysosomal activation by reducing KA-induced increases in LC3-II, lysosomal-associated membrane protein 2 (a biomarker of lysosomes) and cathepsin B (a lysosomal cysteine protease). Subsequently, KA-induced mitochondria loss was prevented in the melatonin-treated mice. At the same time, melatonin reduced KA-increased HO-1 levels and α-synuclein aggregation. Our immunoprecipitation study showed that melatonin enhanced ubiquitination of α-synuclein monomers and aggregates. The anti-apoptotic effect of melatonin was demonstrated by attenuating KA-induced DNA fragmentation, activation of caspases 3/12, and neuronal loss. Taken together, our study suggests that KA-induced neurotoxicity may be mediated by autophagy and α-synuclein aggregation. Moreover, melatonin may exert its neuroprotection via inhibiting KA-induced autophagy and a subsequent mitochondrial loss as well as reducing α-synuclein aggregation by enhancing α-synuclein ubiquitination in the CNS., (© 2011 John Wiley & Sons A/S.)

Aging is a degenerative process that is associated with progressive accumulation of deleterious changes with time, reduction of physiological function and increase in the chance of disease and death. Studies in several species reveal a wide spectrum of alterations in mitochondria and mitochondrial DNA (mtDNA) with aging, including (1) increased disorganization of mitochondrial structure, (2) decline in mitochondrial oxidative phosphorylation (OXPHOS) function, (3) accumulation of mtDNA mutation, (4) increased mitochondrial production of reactive oxygen species (ROS) and (5) increased extent of oxidative damage to DNA, proteins, and lipids. In this chapter, we outline the common alterations in mitochondria of the aging tissues and recent advances in understanding the role of mitochondrial H(2)O(2) production and mtDNA mutation in the aging process and lifespan determination. In addition, we discuss the effect of caloric restriction on age-associated mitochondrial changes and its role in longevity. Taking these findings together, we suggest that decline in mitochondrial energy metabolism, enhanced mitochondrial oxidative stress, and accumulation of mtDNA mutations are important contributors to human aging.

Purpose: To examine the effects of ascorbic acid (AA) administrated during cardiopulmonary resuscitation (CPR) on the myocardial injury in a rat model of ventricular fibrillation (VF) and electrical shock (ES)., Methods: VF was induced in male Wistar rats and left untreated for 5 min, followed by 1 min of CPR, and then one ES of 5 J. At the start of CPR, animals received either intravenous administration of AA (100 mg/kg) or Tempol (30 mg/kg), two antioxidants, or 0.9% saline (VF + ES group). After ES, animals were immediately killed. Myocardial lipoxidation was determined by malondialdehyde (MDA) assay. The histology and ultrastructural changes of myocardium were also evaluated. The mitochondrial permeability transition pore (mPTP) opening was measured based on the mitochondrial swelling rate. The complex activities and respiration of mitochondria were assessed, too., Results: Increased myocardial injury and mitochondrial damage in the VF + ES group were noted. AA and Tempol alleviated such damages. Both AA and Tempol improved accelerated mitochondrial swelling; decreased complex activities and respiratory dysfunction occurred in the VF + ES group. The animals receiving AA and Tempol during CPR had better successful resuscitation rates and 72-h survival than the VF + ES group., Conclusions: Intravenous administration of AA and Tempol at the start of CPR may reduce lipid peroxidation and myocardial necrosis, diminish mitochondrial damage, facilitate resuscitation, and improve outcomes after VF + ES.

In a previous study, BO-1051, an N-mustard linked with a DNA-affinic molecule, was shown to target various types of cancer cell lines. In the present study, we aimed to investigate the cytotoxicity, as well as the underlying mechanism, of BO-1051. We found that BO-1051 simultaneously induced apoptosis and autophagy in hepatocellular carcinoma cell lines. DNA double strand breaks induced by BO-1051 activated the ATM signaling pathway and subsequently resulted in caspase-dependent apoptosis. When autophagy was inhibited in its early or late stages, apoptosis was significantly enhanced. This result indicated autophagy as a cytoprotective effect against BO-1051-induced cell death. We further inhibited ATM activation using an ATM kinase inhibitor or ATM-specific siRNA and found that while apoptosis was blocked, autophagy also diminished in response to BO-1051. We not only determined a signaling pathway induced by BO-1051 but also clarified the linkage between DNA damage-induced apoptosis and autophagy. We also showed that BO-1051-induced autophagy acts as a cytoprotective reaction and downstream target of the ATM-signaling pathway. This research revealed autophagy as a universal cytoprotective response against DNA damage-inducing chemotherapeutic agents, including BO-1051, cisplatin, and doxorubicin, in hepatocellular carcinoma cell lines. Autophagy contributes to the remarkable drug resistance ability of liver cancer., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Mutations and reduced mitochondrial DNA (mtDNA) content are commonly observed in breast cancer, yet their functional significance is not clear. This study aimed to determine whether the mtDNA content in breast cancer plays an important role in modulating the response to anthracycline treatment in vivo and in vitro. The mtDNA content in tumor cells was analyzed using quantitative polymerase chain reaction in 60 Taiwanese breast cancer patients to correlate with their survival. In addition, human breast cancer MDA-MB-231 cells were treated with ethidium bromide to decrease mtDNA copy number. Cell survival was determined by trypan blue exclusion assay and intracellular reactive oxygen species (ROS) were determined by flow cytometry. After an anthracycline-based regimen, the disease-free survival of patients with higher mtDNA content breast cancer was significantly lower than that of patients with lower mtDNA content breast cancer (p = 0.03). Moreover, the MDA-MB-231 cells with low copies of mtDNA had higher sensitivity to doxorubicin treatment and increased ROS production when compared with higher mtDNA parental cells. Our results suggest that the level of mtDNA copy number in breast cancer may be a potential biomarker for prediction of the response to anthracycline-containing regimens in breast cancer patients.

The objective of the study was to o investigate the relationship of the Gly482Ser (G482S) polymorphism in the peroxisome proliferator-activated receptor gamma coactivator-1alpha (PPARGC1A) gene and type 2 diabetes mellitus (T2DM), obesity, and oxidative status in Chinese adults. We enrolled 276 T2DM patients and 1049 nondiabetic subjects aged at least 35 years. The G482S variant was detected using polymerase chain reaction and restriction enzyme digestion. The levels of thiobarbituric acid reactive substance, an indicator of lipid peroxidation, were measured in plasma samples. The homeostasis model assessment-estimated insulin resistance (HOMA-IR) index was determined for nondiabetic subjects. P values were adjusted for age, sex, and body mass index by using a generalized linear model. In this series, there was no association between G482S polymorphism and T2DM and obesity (body mass index >25 kg/m(2)). However, the plasma fasting insulin levels and HOMA-IR indices were significantly higher in nondiabetic subjects harboring the variant (S/S) genotype than in those with the heterozygous (G/S) genotype. With regard to the effect of the different genotypes on body fat distribution, overweight nondiabetic subjects harboring the S/S or G/S genotype had a significantly higher waist-to-hip ratio than those with the wild-type (G/G) genotype. Furthermore, subjects with the S/S genotype had significantly higher serum thiobarbituric acid reactive substance levels than those with the G/G genotype; the diabetic group mainly contributed to this significant association (P < .001). In overweight, nondiabetic Chinese adults, G482S polymorphism in the PPARGC1A gene is associated with hyperinsulinemia, HOMA-IR indices, and abdominal obesity. Furthermore, in hyperglycemia, the S482 allele is related to increased oxidative stress., (Copyright 2010 Elsevier Inc. All rights reserved.)

Background: Peroxisome proliferator-activated receptor gamma (PPARgamma) activation has been proposed as a potential therapeutic strategy for various types of human cancer. The aim of this study was to activate PPARgamma and overexpress PGC-1alpha in HepG2 cells in order to analyze their effects on cell motility and E-cadherin expression., Materials and Methods: Adenovirus-mediated gene transfer was performed to overexpress PGC-1alpha in HepG2 human hepatoma cells. Small interference RNA (siRNA) was used to silence the expression of E-cadherin and PPARgamma. Cell motility was assessed by transwell cell migration analysis. Measurements of mRNA and protein expression were done by quantitative RT-PCR and Western blotting., Results: Treatment with synthetic PPARgamma agonists, thiazolidinediones (rosiglitazone; troglitazone), and adenovirus-delivered overexpression of PPARgamma transcriptional coactivator-1alpha (PGC-1alpha) up-regulated E-cadherin expression and reduced motility of HepG2 cells. Using PPARgamma antagonist GW9662, we demonstrated that both PPARgamma-dependent and -independent pathways were involved in PGC-1alpha-induced up-regulation of E-cadherin. In addition, siRNA-mediated knockdown of E-cadherin expression restored the motility of PGC-1alpha-overexpressing HepG2 cells, indicating that up-regulated E-cadherin is responsible for the lower migratory ability of these cells. Intriguingly, siRNA-mediated silencing of PPARgamma abolished E-cadherin protein expression but also reduced the motility of HepG2 cells., Conclusion: PPARgamma/PGC-1alpha pathway plays a crucial role in modulating E-cadherin expression and motility of HepG2 cells and may be a potential target for the prevention of HCC metastasis.

The NAD(P)H:quinone oxidoreductase 1 (NQO1) enzyme is implicated in protection against oxidative stress and carcinogenesis. NQO1 C609T genetic polymorphism was reported to be associated with an increased risk for cancers, including breast cancer. However, there is still lack of evidence whether higher oxidative stress occurs in breast tissues of patients with NQO1 609 C/T and/or T/T genotypes. Mitochondrial DNA (MtDNA) 4,977-bp deletion, the most common mutation of mtDNA, was frequently detected in post-mitotic tissues of aged subjects and associated with oxidative damage. In this study, we detected the mtDNA 4,977-bp deletion in 60 breast cancers and corresponding non-cancerous breast tissues. The incidence of the common 4,977-bp deletion in non-cancerous breast tissues (48.3%) was higher than that in breast cancer (5.0%). Moreover, 63.4% of the breast cancer patients with NQO1 C/T or T/T genotypes had the deletion in their non-cancerous breast tissues. The mtDNA deletion was more frequently detected in breast tissue of NQO1 C/T carriers (65.2%) and T/T carriers (61.1%) as compared with the NQO1 C/C carriers (15.8%, P=0.003). Similar results were observed in the <50 years old (y/o) group (P=0.06) and the > or =50 y/o group (P=0.005). Our findings suggest that mtDNA 4,977-bp deletion associated with NQO1 deficiency is involved in carcinogenesis and progression of breast cancer.

Aging is a biological process that is characterized by the gradual loss of physiological function and increases in the susceptibility to disease of an individual. During the aging process, a wide spectrum of alterations in mitochondria and mitochondrial DNA (mtDNA) has been observed in somatic tissues of humans and animals. This is associated with the decline in mitochondrial respiratory function; excess production of the reactive oxygen species (ROS); increase in the oxidative damage to mtDNA, lipids and proteins in mitochondria; accumulation of point mutations and large-scale deletions of mtDNA; and altered expression of genes involved in intermediary metabolism. It has been demonstrated that the ROS may cause oxidative damage and mutations of mtDNA and alterations of the expression of several clusters of genes in aging tissues and senescent cells. We found that intracellular levels of hydrogen peroxide (H2O2) and oxidative damage to DNA in the tissue cells and skin fibroblasts of old donors were higher than those of young donors. In H2O2-induced senescent skin fibroblasts, we observed an increase in the protein expression and activity levels of manganese-dependent superoxide dismutase and a concurrent decrease in the activity of cytochrome c oxidase and the rate of oxygen consumption. Moreover, the mRNA and protein expression levels of pyruvate dehydrogenase (PDH) were decreased but those of PDH kinase and lactate dehydrogenase were increased in senescent skin fibroblasts. The changes in the expression of these enzymes suggest a metabolic shift from mitochondrial respiration to glycolysis as a major supply of ATP in aging human cells. On the other hand, recent studies on mitochondrial mutant mice, which carry a proofreading deficient subunit of DNA polymerase gamma, revealed that mtDNA mutations accumulated in somatic tissues in the mice that displayed prominent features of aging. Taken together, we suggest that the respiratory function decline and increase in the production of the ROS in mitochondria, accumulation of mtDNA mutation and oxidative damage, and altered expression of a few clusters of genes that culminated in the metabolic shift from mitochondrial respiration to glycolysis for major supply of ATP were key contributory factors in the aging process in the human and animals.

Objective: The aim of this study was to examine the potential influence of insulin resistance (IR), hyperglycemia and oxidative stress on leucocytes mitochondrial DNA (mtDNA) content., Research Design and Method: One hundred twenty-five T2DM, 101 IFG and 70 normal subjects were enrolled in this study. The quantity of relative mtDNA content was measured by a real-time PCR and corrected by simultaneous measurement of the nuclear DNA. Parameters of lipid peroxidation, thiobarbituric acid reactive substance (TBARS), and total free thiols as antioxidative status were measured from serum samples. IR was assessed by homeostasis model assessment in the non-diabetic groups. Relationships among different variables were analyzed by general linear model correlation., Results: In all subjects, after correcting for age, sex and BMI, there were progressive increases of leucocyte mtDNA copy number, TBARS, and total reduced thiols with progressive dysregulation of glucose metabolism (normal vs. IFG vs. T2DM). Furthermore, correlation between mtDNA content and glucose dysregulation persisted after sequential correction for age, sex, BMI and TBARS. The independent predictor of mtDNA content by regression analysis was hyperglycemia. In non-diabetic group, influence of family history of diabetes on mtDNA content turned to non-significant after correcting for fasting plasma glucose (FPG). Correlation study revealed that mtDNA content was correlated with FPG (P<0.001), but not IR., Conclusion: Our results indicate that hyperglycemia, not IR, is associated with an increase of leucocyte mtDNA copy number in cases of glucose dysregulation.

Objective: Since mitochondrial DNA (mtDNA) mutations have been established to associate with the aging process and some degenerative diseases, we investigated the correlation between idiopathic osteoarthritis (OA) and the 4977-bp mtDNA deletion., Design: Cartilage were collected from six sites in knee joints removed from 18 aged patients with idiopathic OA, 10 aged non-OA cadavers, 3 young cadavers (YC), and lateral femoral condyle of 9 young patients. Histopathologic changes were examined and the common 4977-bp mtDNA deletions were analyzed in young and elderly cartilages obtained from different sites in the knee joint. The association of the 4977-bp deletion of mtDNA with idiopathic OA and aging was evaluated., Results: The 4977-bp mtDNA deletion was detected in 17 of the 18 OA patients, 9 of the 10 aged non-OA cadavers, and 1 of the 3 YC. None of the nine specimens collected from the lateral femoral condyle of young patients had a detectable deletion of mtDNA. The 4977-bp mtDNA deletion was not significantly correlated with the severity of OA graded by the Mankin score. The frequencies of occurrence of the 4977-bp mtDNA deletion were significantly different between the OA group and the aged non-OA control group (P=0.004) and between the aged non-OA group and the young control group (P=0.002)., Conclusions: The results suggest that accumulation of the 4977-bp deletion of mtDNA in knee cartilage increases with age and may play a role in the development of idiopathic OA in the knee joint.

Mutations of mitochondrial DNA (mtDNA) have been proposed to be involved in carcinogenesis. In this study, we applied the polymerase chain reaction techniques to investigate the frequency of occurrence and proportion of mtDNA with length mutations (deletions and tandem duplications) in light-associated skin tumors (actinic keratosis, AK; basal cell carcinoma, BCC; and squamous cell carcinoma, SCC) in aged individuals. We demonstrated the existence of multiple mtDNA deletions and tandem duplications in tissues of AK, BCC, SCC and normal skin. We showed that the frequencies of occurrence of the 4,977 bp and 7,436 bp deletions and tandem duplications (200 bp and 260 bp) of mtDNA in light-associated skin tumors were not significantly different from those of sun-exposed normal skin (p>0.05), but higher than those of non-exposed normal skin (p<0.05). In addition, we found that the proportion of the 4,977 bp-deleted mtDNA in the skin of the same individual was also affected by skin pathologies. The proportion of 4,977 bp-deleted mtDNA in relatively rapid growing tumor cells in SCC was lower than that of normal skin cells. We suggest that the existence of these length mutations of mtDNA in normal, precancerous or cancerous human skin may be attributed to the stochastic effect of photo-damage. However, it is unclear whether the mutations of mtDNA have a direct bearing on carcinogenesis in skin. Future investigation is warranted to elucidate the causal relationship between mtDNA mutations and skin cancers and to address the pathophysiological role of mtDNA mutations in skin cancer development.

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., (Copyright 2002 National Science Council, ROC and S. Karger AG, Basel)