Overexpressing microRNA-203 alleviates myocardial infarction via interacting with long non-coding RNA MIAT and mitochondrial coupling factor 6.

Myocardial infarction (MI) is one of the leading causes of high mortality worldwide. Long non-coding RNA myocardial infarction associated transcript (MIAT) and mitochondrial coupling factor 6 (CF6) aggravate MI. This study aimed to elucidate whether miR-203 interacted with MIAT and CF6 in MI. Results revealed that MIAT and CF6 expressions were upregulated and that miR-203 was downregulated in mouse myocardial tissues after MI, as well as in hypoxic mouse cardiomyocytes. The overexpression of MIAT in mouse cardiomyocytes raised CF6 expression, whereas the knockdown of MIAT had the opposite effect. Mechanistically, the luciferase reporter and RNA pull-down assays corroborated the binding between miR-203 and CF6 3'UTR and between miR-203 and MIAT. The simultaneous overexpression of miR-203 and MIAT restored the reduction of CF6 caused by miR-203 overexpression alone, and the overexpression of miR-203 diminished the percentage of infarct area and the apoptosis of cardiomyocytes in vivo. Our findings corroborate that overexpressing miR-203 alleviates MI via interacting with MIAT and CF6.

A comparative study of mitochondrial respiration in circulating blood cells and skeletal muscle fibers in women.

Skeletal muscle mitochondrial respiration is thought to be altered in obesity, insulin resistance, and type 2 diabetes; however, the invasive nature of tissue biopsies is an important limiting factor for studying mitochondrial function. Recent findings suggest that bioenergetics profiling of circulating cells may inform on mitochondrial function in other tissues in lieu of biopsies. Thus, we sought to determine whether mitochondrial respiration in circulating cells [peripheral blood mononuclear cells (PBMCs) and platelets] reflects that of skeletal muscle fibers derived from the same subjects. PBMCs, platelets, and skeletal muscle (vastus lateralis) samples were obtained from 32 young (25-35 yr) women of varying body mass indexes. With the use of extracellular flux analysis and high-resolution respirometry, mitochondrial respiration was measured in intact blood cells as well as in permeabilized cells and permeabilized muscle fibers. Respiratory parameters were not correlated between permeabilized muscle fibers and intact PBMCs or platelets. In a subset of samples (n = 12-13) with permeabilized blood cells available, raw measures of substrate (pyruvate, malate, glutamate, and succinate)-driven respiration did not correlate between permeabilized muscle (per mg tissue) and permeabilized PBMCs (per 106 cells); however, complex I leak and oxidative phosphorylation coupling efficiency correlated between permeabilized platelets and muscle (Spearman's ρ = 0.64, P = 0.030; Spearman's ρ = 0.72, P = 0.010, respectively). Our data indicate that bioenergetics phenotypes in circulating cells cannot recapitulate muscle mitochondrial function. Select circulating cell bioenergetics phenotypes may possibly inform on overall metabolic health, but this postulate awaits validation in cohorts spanning a larger range of insulin resistance and type 2 diabetes status.

Novel anti-aging gene NM_026333 contributes to proton-induced aging via NCX1-pathway.

Diet-induced metabolic acidosis is associated with the impairment of bone metabolism and an increased risk of a number of chronic noncommunicable diseases, such as type 2 diabetes mellitus and hypertension. Low serum bicarbonate is associated with high mortality in healthy older individuals. Recently, we demonstrated that both coupling factor 6 (CF6)-overexpressing transgenic (TG) and high salt-fed mice which had sustained intracellular acidosis, due to enhanced proton import through ecto-F1Fo complex and/or reduced proton export through Na+-K+ ATPase inhibition, displayed shortened lifespan and early senescence-associated phenotypes such as signs of hair greying and alopecia, weight loss, and/or reduced organ mass. In this study, we searched causative genes of proton-induced aging in CF6-overexpressing TG and high salt-fed mice. We discovered NM_026333 as a novel anti-aging gene which was downregulated in the heart and kidney in both types of mice. NM_026333 protein consists of 269 amino acids with transmembrane region (90-193aa). Induction of NM_026333 or recombinant protein rescued TG cells and CF6-treated human cells from aging hallmarks of impaired autophagy, genomic instability, and epigenetic alteration. NM_026333 protein directly bound plasma membrane Na+-Ca2+ exchanger 1 (NCX1) to suppress its reverse mode, and cancelled proton-induced epigenetic regression of Atg7 that was caused by H3K4 and H4K20 tri-methylation via suppression of demethylase and H4K5 acetylation via suppression of nuclear HDAC3-HDAC4-emerin system. NM_026333 also attenuated proton-induced impaired formation of autolysosome, an increase in nuclear acetylated LC3 II, and acetylation of Atg7. These effects reappeared by NCX1 inhibitor. Furthermore, NCX1 inhibitor extended lifespan compared with vehicle-treatment in TG mice. This study will shed light on novel aging mechanism and provide implications in a target for anti-aging therapy.

Mitochondrial inhibitory factor protein 1 attenuates coupling factor 6-induced aging signal.

Coupling factor 6 (CF6) forces a counter-clockwise rotation of plasma membrane F1 Fo complex, resulting in proton import and accelerated aging. Inhibitory factor peptide 1 (IF1) suppresses a unidirectional counter-clockwise rotation of F1 Fo complex without affecting ATP synthesis. We tested the hypothesis that IF1 may attenuate CF6-induced aging signaling in CF6-overexpressing transgenic (TG) cells. In IF1-GFP overexpressing wild type (WT) cells, the diffuse peripheral staining of tubular mitochondria was observed with a dense widely distributed network around the nucleus. In TG cells, however, the only peri-nuclear network of fragmented mitochondria was observed at 24 h, but it was developed to a widely distributed mitochondrial network of tubular mitochondria at 72 h. TG cells displayed aging hallmarks of telomere attrition, epigenetic alterations, defective proteostasis, and genomic instability with a decrease in emerin and lamin and loss of heterochromatin. IF1 induction rescued TG cells from telomere attrition, expression of genomic instability with the increase in emerin and lamin, and that of epigenetic alterations with recovery of heterochromatin. In defective proteostasis, IF1 induction restored a potent peri-nuclear staining of autolysosomes compared with the baseline weak staining. The decrease in Atg7 was restored, whereas the increase in P62 was abolished. We conclude that genetic disruption of proton signals by IF1 induction suppressed CF6-induced expression of aging hallmarks such as telomere attrition, epigenetic alterations, defective proteostasis, and genomic instability. Given the widespread biological actions of CF6, the physiological and pathological actions of IF1 may be complex.

Identification of candidate genes and long non-coding RNAs associated with the effect of ATP5J in colorectal cancer.

The incidence and development of colorectal cancer (CRC) is a process with multiple gene interactions. We have previously demonstrated that ATP synthase-coupling factor 6, mitochondrial (ATP5J) is associated with CRC migration and 5-fluorouracil resistance; nevertheless, the exact molecular mechanism remains unclear. The following study uses microarray and bioinformatics methods to identify candidate genes and long non-coding RNAs (lncRNAs) in CRC cells (two pairs) with upregulated and downregulated ATP5J. Briefly, a total of 2,190 differentially expressed mRNAs (DEmRNAs) were sorted. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed for 4 DEmRNAs to validate the results of microarray analysis. Functional annotation and pathway enrichment were analyzed for DEmRNAs using the Database for Annotation, Visualization and Integrated Discovery. Significantly enriched pathways included the regulation of gene expression and cell growth. The protein­protein interaction network was constructed, and AKT serine/threonine kinase 2 (AKT2) was considered as one of the hub genes. For further analysis, 51 DEmRNAs and 30 DElncRNAs were selected that were positively or negatively associated with the expression of ATP5J in the two cell pairs. X-inactive specific transcript (XIST), premature ovarian failure 1B (POF1B) and calmin (CLMN) were identified in the DEmRNA-DElncRNA co-expression network. The expression of AKT2 and XIST in CRC cells was confirmed by RT-qPCR. To sum up, the candidate genes and lncRNAs, as well as potential signaling pathways, which were identified using integrated bioinformatics analysis, could improve the understanding of molecular events involved in the function of ATP5J in CRC.

Marine collagen peptides reduce endothelial cell injury in diabetic rats by inhibiting apoptosis and the expression of coupling factor 6 and microparticles.

The present study aimed to elucidate the role of marine collagen peptides (MCPs) in protection of carotid artery vascular endothelial cells (CAVECs) in type 2 diabetes mellitus (T2DM), and the mechanism underlying this process. In an in vivo experiment, diabetic Wistar rats were divided randomly into four groups (n=10/group): Diabetes control, and three diabetes groups administered low, medium and high doses of MCPs (2.25, 4.5 and 9.0 g/kg body weight/day, respectively). Another 10 healthy rats served as the control. In an in vitro experiment, human umbilical­vein endothelial cells (HUVECs) were incubated in normal and high concentrations of glucose with or without MCPs (3.0, 15.0 and 30.0 mg/ml, respectively) for 24, 48 or 72 h. Blood vessel/endothelial construction, inflammatory exudation and associated molecular biomarkers in CAVECs were detected and analyzed. The results of the present study demonstrated that in rats, MCP treatment for 4 weeks significantly lowered blood glucose and attenuated endothelial thinning and inflammatory exudation in carotid­artery vascular endothelial cells. In vitro, the high­glucose intervention significantly increased cell apoptosis in HUVECs, and medium and high doses of MCPs (4.5 and 9.0 g/kg body weight/day, respectively) partially ameliorated this high glucose­mediated apoptosis and decreased levels of apoptosis biomarkers. In conclusion, a moderate oral MCP dose (≥4.5 g/kg body weight/day) may be a novel therapeutic tool to protect against early cardiovascular complications associated with T2DM by inhibiting apoptosis and reducing the expression of coupling factor 6 and microparticles.

Nutritional regulation of coupling factor 6, a novel vasoactive and proatherogenic peptide.

High sodium, high glucose, and obesity are important risk factors for age-related diseases such as cardiovascular disease (CVDs), stroke, and cancer. Coupling factor 6 (CF6) is released from vascular endothelial cells and functions as a circulating peptide that inhibits prostacyclin and nitric oxide generation by intracellular acidosis. High glucose elevates CF6 by activation of protein kinase C and p38 mitogen-activated protein kinase, whereas CF6 causes type 2 diabetes mellitus, resulting in a high glucose vicious cycle. Low glucose increases inhibitory factor peptide 1, an endogenous inhibitor of CF6. High salt intake increases CF6 through nuclear factor κB signaling, whereas CF6 induces salt-sensitive hypertension and salt-induced congestive heart failure. Oral administration of vitamin C cancels salt-induced increase in CF6, and estrogen replacement leads to the delayed onset of CF6-induced salt-sensitive hypertension and the rescue from cardiac systolic dysfunction. Because CF6 contributes to the onset of CVDs, nutritional regulation of CF6 will shed light on the understanding of preventive strategy and mechanisms for CVDs and a target for therapy.

ATP5J and ATP5H Proactive Expression Correlates with Cardiomyocyte Mitochondrial Dysfunction Induced by Fluoride.

To investigate the effect of excessive fluoride on the mitochondrial function of cardiomyocytes, 20 healthy male mice were randomly divided into 2 groups of 10, as follows: control group (animals were provided with distilled water) and fluoride group (animals were provided with 150 mg/L F- drinking water). Ultrastructure and pathological morphological changes of myocardial tissue were observed under the transmission electron and light microscopes, respectively. The content of hydrolysis ATP enzyme was observed by ATP enzyme staining. The expression levels of ATP5J and ATP5H were measured by Western blot and quantitative real-time PCR. The morphology and ultrastructure of cardiomyocytes mitochondrial were seriously damaged by fluoride, including the following: concentration of cardiomyocytes and inflammatory infiltration, vague myofilaments, and mitochondrial ridge. The damage of mitochondrial structure was accompanied by the significant decrease in the content of ATP enzyme for ATP hydrolysis in the fluoride group. ATP5J and ATP5H expressions were significantly increased in the fluoride group. Thus, fluoride induced the mitochondrial dysfunction in cardiomyocytes by damaging the structure of mitochondrial and interfering with the synthesis of ATP. The proactive ATP5J and ATP5H expression levels were a good response to the mitochondrial dysfunction in cardiomyocytes.

Lung-specific RNA interference of coupling factor 6, a novel peptide, attenuates pulmonary arterial hypertension in rats.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a progressive and life-threatening disease associated with high morbidity and mortality rates. However, the exact regulatory mechanism of PAH is unknown. Although coupling factor 6 (CF6) is known to function as a repressor, its role in PAH has not been explored. Here, we investigated the involvement of endogenous CF6 in the development of PAH. METHODS: PAH was induced with monocrotaline (MCT), as demonstrated by significant increases in pulmonary artery pressure and vessel wall thickness. The adeno-associated virus (AAV) carrying CF6 short hairpin RNA (shRNA) or control vector (2×10(10) gp) was intratracheally transfected into the lungs of rats 2 weeks before or after MCT injection. RESULTS: A 2-6-fold increase in CF6 was observed in the lungs and circulation of the MCT-injected rats as confirmed by qRT-PCR and ELISA. Immunohistochemistry analysis revealed a small quantity of CF6 localized to endothelial cells (ECs) under physiological conditions spread to surrounding tissues in a paracrine manner in PAH lungs. Notably, CF6 shRNA effectively inhibited CF6 expression, abolished lung macrophage infiltration, reversed endothelial dysfunction and vascular remodeling, and ameliorated the severity of pulmonary hypertension and right ventricular dysfunction at 4 weeks both as a pretreatment and rescue intervention. In addition, the circulating and lung levels of 6-keto-PGF1a, a stable metabolite of prostacyclin, were reversed by CF6 inhibition, suggesting that the effect of CF6 inhibition may partly be mediated through prostacyclin. CONCLUSIONS: CF6 contributes to the pathogenesis of PAH, probably in association with downregulation of prostacyclin. The blockage of CF6 might be applied as a novel therapeutic approach for PAH and PA remodeling.

Conjugated linoleic acid and nitrite attenuate mitochondrial dysfunction during myocardial ischemia.

Cardiovascular health is influenced by dietary composition and the western diet is composed of varying types/amounts of fat. Conjugated linoleic acid (cLA) is an abundant dietary unsaturated fatty acid associated with health benefits but its biological signaling is not well understood. Nitrite is enriched in vegetables within the diet and can impact signaling of unsaturated fatty acids; however, its role on cLA signaling is not well understood. Elucidating how nitrite may impact the biological signaling of cLA is important due to the dietary consumption of both cLA and nitrite in the western diet. Since co-administration of cLA and nitrite results in cardioprotection during myocardial infarction (MI), it was hypothesized that cLA and nitrite may affect cardiac mitochondrial respiratory function and complex activity in MI. C57BL/6J mice were treated with cLA and nitrite for either 10 or 13days, where MI was induced on day 3. Following treatment, respiration and complex activity were measured. Among the major findings of this study, cLA treatment (10days) decreases state 3 respiration in vivo. Following MI, nitrite alone and in combination with cLA attenuates increased state 3 respiration and decreases hydrogen peroxide levels. Further, nitrite and cLA co-treatment attenuates increased complex III activity after MI. These results suggest that cLA, nitrite and the combination significantly alter cardiac mitochondrial respiratory and electron transport chain activity in vivo and following MI. Overall, the daily consumption of cLA and nitrite in the diet can have diverse cardiovascular implications, some of which occur at the mitochondrial level.

ATP5A1 and ATP5B are highly expressed in glioblastoma tumor cells and endothelial cells of microvascular proliferation.

Glioblastoma (GBM) is the most common primary malignant brain tumor. Microvascular proliferation is one of the characteristic pathologic features of GBM. Mitochondrial dysfunction plays an important role in the pathogenesis of GBM. In this study, microvascular proliferation from GBM and normal brain blood vessels were laser microdissected and total RNA was isolated from these microvasculatures. The difference of mRNA expression profiles among GBM microvasculature, normal brain blood vessels and GBM tumor cells was evaluated by mitochondria and metabolism PCR gene arrays. It was found that the mRNA levels of ATP5A1 and ATP5B in GBM tumor cells as well as microvascular proliferation were significantly higher compared with normal brain blood vessels. Immunohistochemical stains with anti-ATP5A1 antibody or anti-ATP5B antibody were performed on tissue microarray, which demonstrated strongly positive expression of ATP5A1 and ATP5B in GBM tumor cells and GBM microvascular proliferation while normal blood vessels were negative. By analyzing The Cancer Genome Atlas data sets for GBM and other cancers, genomic DNA alterations (mutation, amplification or deletion) were less likely the reason for the high expression of ATP5A1 and ATP5B in GBM. Our miRNA microarray data showed that miRNAs that target ATP5A1 or ATP5B were down-regulated, which might be the most likely reason for the high expression of ATP5A1 and ATP5B in GBM tumor cells and microvascular proliferation. These findings help us better understand the pathogenesis of GBM, and agents against ATP5A1 and/or ATP5B might effectively kill both tumor cells and microvascular proliferation in GBM. MiRNAs, such as Let-7f, miR-16, miR-23, miR-100 and miR-101, that target ATP5A1 or ATP5B, might be potential therapeutic agents for GBM.

Mitochondrial Inhibitory Factor Protein 1 Functions as an Endogenous Inhibitor for Coupling Factor 6.

Coupling factor 6 (CF6) forces a counter-clockwise rotation of plasma membrane F1 Fo complex unlike a proton-mediated clockwise rotation in the mitochondria, resulting in ATP hydrolysis, proton import, and apoptosis. Inhibitory peptide 1 (IF1) inhibits a unidirectional counter-clockwise rotation of F1 Fo complex without affecting ATP synthesis by a clockwise rotation. We tested the hypothesis that IF1 may antagonize the biological action of CF6 in human embryonic kidney 293 cells. We generated mature and immature IF1 expression vectors and those labeled with GFP at the C-terminus. In the immature IF1-GFP overexpressing cells, the mitochondrial network of IF1-GFP was newly found at the plasma membrane after peripheral translocation, whereas in mature IF1-GFP transfected cells, a less punctuate rather homogenous pattern was found in the cytoplasm. IF1 protein was detected in the exosome fraction of culture media, and it was enhanced by mature or immature IF1 transfection. Extracellular ATP hydrolysis was enhanced by CF6, whereas immature or mature IF1 transfection suppressed ATP hydrolysis in response to CF6. Intracellular pH was decreased by CF6 but was unchanged after immature IF1 transfection. CF6-induced increase in apoptotic cells was blocked by immature or mature IF1, being accompanied by protein kinase B (PKB) phosphorylation. IF1 antagonizes the pro-apoptotic action of CF6 by relief of intracellular acidification and resultant phosphorylation of PKB. Given the widespread biological actions of CF6, the physiological and pathological functions of IF1 may be expected to be complex. J. Cell. Biochem. 117: 1680-1687, 2016. © 2015 Wiley Periodicals, Inc.

Mitochondrial ATP synthase activity is impaired by suppressed O-GlcNAcylation in Alzheimer's disease.

Glycosylation with O-linked ß-N-acetylglucosamine (O-GlcNAc) is one of the protein glycosylations affecting various intracellular events. However, the role of O-GlcNAcylation in neurodegenerative diseases such as Alzheimer's disease (AD) is poorly understood. Mitochondrial adenosine 5'-triphosphate (ATP) synthase is a multiprotein complex that synthesizes ATP from ADP and Pi. Here, we found that ATP synthase subunit α (ATP5A) was O-GlcNAcylated at Thr432 and ATP5A O-GlcNAcylation was decreased in the brains of AD patients and transgenic mouse model, as well as Aß-treated cells. Indeed, Aß bound to ATP synthase directly and reduced the O-GlcNAcylation of ATP5A by inhibition of direct interaction between ATP5A and mitochondrial O-GlcNAc transferase, resulting in decreased ATP production and ATPase activity. Furthermore, treatment of O-GlcNAcase inhibitor rescued the Aß-induced impairment in ATP production and ATPase activity. These results indicate that Aß-mediated reduction of ATP synthase activity in AD pathology results from direct binding between Aß and ATP synthase and inhibition of O-GlcNAcylation of Thr432 residue on ATP5A.

Mitochondrial coupling factor 6 upregulation in hypertension-induced cardiac hypertrophy.

BACKGROUND: Mitochondrial coupling factor 6 (CF6) is a constriction factor in cardiac hypertrophy, whose mechanisms are not fully understood. MATERIALS AND METHODS: Here, we established cardiac hypertrophy models for feeding spontaneously hypertensive rats (SHRs) aged 10, 20, and 30 weeks. Hemodynamic monitoring was performed during the feeding program to ensure the success of the model. RESULTS: Cardiac hypertrophy, but not fibrosis, occurred in the 10-, 20-, and 30-week-old SHRs. No significant changes in CF6 gene expression were detected by RT-PCR in any of the SHR groups as compared with the control groups (p > 0.05). ELISA assessment showed that the CF6 protein level in the 20- and 30-week-old SHRs with cardiac hypertrophy was significantly increased (vs. control, p < 0.05). CONCLUSION: CF6 protein was upregulated in cardiac hypertrophy induced by hypertension; further mechanisms involved in this process should be investigated.

Adjustments of muscle capillarity but not mitochondrial protein with skiing in the elderly.

Downhill skiing in the elderly increases maximal oxygen uptake (VO2max) and carbohydrate handling, and produces muscle hypertrophy. We hypothesized that adjustments of the cellular components of aerobic glucose combustion in knee extensor muscle, and cardiovascular adjustments, would increase in proportion to VO2max. Nineteen healthy elderly subjects (age 67.5 ± 2.9 years) who completed 28.5 days of guided downhill skiing over 3 months were assessed for anthropometric variables, cardiovascular parameters (heart rate, hematocrit), VO2max, and compared with controls (n = 20). Biopsies of vastus lateralis muscle were analyzed for capillary density and expression of respiratory chain markers (NDUFA9, SDHA, UQCRC1, ATP5A1) and the glucose transporter GLUT4. Statistical significance was assessed with a repeated analysis of variance and Fisher's post-hoc test at a P value of 5%. VO2max increased selectively with ski training (+7 ± 2%). Capillary density (+11 ± 5%) and capillary-to-fiber ratio (12 ± 5%), but not the concentration of metabolic proteins, in vastus lateralis were increased after skiing. Cardiovascular parameters did not change. Fold changes in VO2max and capillary-to-fiber ratio were correlated and were under genetic control by polymorphisms of the regulator of vascular tone, angiotensin converting enzyme. The observations indicate that increased VO2max after recreational downhill ski training is associated with improved capillarity in a mainly recruited muscle group.

Compromised mitochondrial remodeling in compensatory hypertrophied myocardium of spontaneously hypertensive rat.

BACKGROUND: Hypertension leads to cardiac hypertrophy as an adaptive response to increased workload. While initial development of hypertrophy is compensatory when contractile function is maintained, persistent stress on heart leads to deteriorated cardiac function and onset of heart failure. Mitochondrial dysfunction develops in the failing heart; however, whether it presents in compensatory cardiac hypertrophy is controversial. METHODS: Spontaneously hypertensive rats (SHRs) and age-matched normotensive Wistar Kyoto rats were used in the study. Mitochondrial function and remodeling-related mechanisms in the left ventricles were measured by enzyme activity tests, Western blots, and reverse transcriptase polymerase chain reaction. RESULTS: Compensatory cardiac hypertrophy in SHR was indicated by higher heart/weigh ratio, left ventricular systolic pressure and ±dp/dt(max) (P<.001, P<.05, and P<.01, respectively). Enzyme activities of mitochondrial complex I and II were significantly reduced (P<.05 and P<.01) in SHR in concert with decreased expression of complex subunits (P<.01 for NDUFS3, P=.068 for SDHB, and P<.05 for ATP5A1). Mitochondrial fission protein Drp1 was decreased (P<.05), while fusion protein OPA1 was increased (P<.01). Parkin and SirT1/AMPK-PGC-1α signaling, responsible for mitochondrial elimination and biogenesis respectively, were decreased in SHR (P<.01 for Parkin, P<.001 for SirT1 and p-AMPK). CONCLUSION: Our results implicated that mitochondrial function and remodeling, indicated by mitochondrial enzyme activities and remodeling-related molecules, were compromised in compensatory hypertrophied myocardium of the SHR hypertensive model. SUMMARY: Mitochondrial function in compensatory hypertrophied myocardium is controversial. Our present study found mitochondrial dysfunction in the left ventricle of spontaneously hypertensive rats, which was possibly a result of compromised mitochondrial remodeling including mitochondrial dynamics, elimination, and biogenesis.

5-Aminolevulinic acid protects against cisplatin-induced nephrotoxicity without compromising the anticancer efficiency of cisplatin in rats in vitro and in vivo.

BACKGROUND/AIMS: Nephrotoxicity is a frequent and major limitation in cisplatin (CDDP)-based chemotherapy. 5-Aminolevulinic acid (ALA) is widely distributed in animal cells, and it is a precursor of tetrapyrole compounds such as heme that is fundamentally important in aerobic energy metabolism. The aim of this study is to evaluate the protective role of ALA in CDDP-induced acute kidney injury (AKI). METHOD: We used CDDP-induced AKI rat model and cultured renal tubular cells (NRK-52E). We divided four groups of rats: control, CDDP only, CDDP + ALA(post);(ALA 10 mg/kg + Fe in drinking water) after CDDP, CDDP + ALA(pre & post). RESULT: CDDP increased Cr up to 6.5 mg/dl, BUN up to 230 mg/dl, and ALA significantly reduced these changes. ALA ameliorates CDDP-induced morphological renal damages, and reduced tubular apoptosis evaluated by TUNEL staining and cleaved caspase 3. Protein and mRNA levels of ATP5α, complex(COX) IV, UCP2, PGC-1α in renal tissue were significantly decreased by CDDP, and ALA ameliorates reduction of these enzymes. In contrast, Heme Oxigenase (HO)-1 level is induced by CDDP treatment, and ALA treatment further up-regulates HO-1 levels. In NRK-52E cells, the CDDP-induced reduction of protein and mRNA levels of mitochondrial enzymes was significantly recovered by ALA + Fe. CDDP-induced apoptosis were ameliorated by ALA + Fe treatment. Furthermore, we evaluated the size of transplantated bladder carcinoma to the rat skin, and ALA did not change the anti cancer effects of CDDP. CONCLUSION: These data suggested that the protective role of ALA in cisplatin-induced AKI is via protection of mitochondrial viability and prevents tubular apoptosis. Also there are no significant effects of ALA on anticancer efficiency of CDDP in rats. Thus, ALA has the potential to prevent CDDP nephrotoxicity without compromising its anticancer efficacy.

Over-expression of the ATP5J gene correlates with cell migration and 5-fluorouracil sensitivity in colorectal cancer.

Recently we found that ATP5J was over-expressed in tissue samples from patients with colorectal cancer. However, the clinical significance and function of the over-expression of ATP5J in these patients remains unclear. We investigated these issues in the current study. Our results indicated that expression of ATP5J was significantly higher in colorectal cancer tissue than in adjacent tissue, and it was also significantly higher in metastatic lymph nodes than in primary cancer tissue (P<0.05). A correlation between ATP5J expression and tumor differentiation was detected, but no correlation with gender, age, T stage, lymph node metastasis, or survival status was observed. Down-regulation of ATP5J expression attenuated the ability of cell migration and increased the sensitivity to 5-fluorouracil (5-Fu) in cells of the DLD1 cell line. Inversely, up-regulation of ATP5J expression enhanced cell migration and decreased 5-Fu sensitivity, suggesting that the function of ATP5J in colorectal cancer might involve cell migration and 5-Fu sensitivity.

Importance of glycolysis and oxidative phosphorylation in advanced melanoma.

Serum lactate dehydrogenase (LDH) is a prognostic factor for patients with stage IV melanoma. To gain insights into the biology underlying this prognostic factor, we analyzed total serum LDH, serum LDH isoenzymes, and serum lactate in up to 49 patients with metastatic melanoma. Our data demonstrate that high serum LDH is associated with a significant increase in LDH isoenzymes 3 and 4, and a decrease in LDH isoenzymes 1 and 2. Since LDH isoenzymes play a role in both glycolysis and oxidative phosphorylation (OXPHOS), we subsequently determined using tissue microarray (TMA) analysis that the levels of proteins associated with mitochondrial function, lactate metabolism, and regulators of glycolysis were all elevated in advanced melanomas compared with nevic melanocytes. To investigate whether in advanced melanoma, the glycolysis and OXPHOS pathways might be linked, we determined expression of the monocarboxylate transporters (MCT) 1 and 4. Analysis of a nevus-to-melanoma progression TMA revealed that MCT4, and to a lesser extend MCT1, were elevated with progression to advanced melanoma. Further analysis of human melanoma specimens using the Seahorse XF24 extracellular flux analyzer indicated that metastatic melanoma tumors derived a large fraction of energy from OXPHOS. Taken together, these findings suggest that in stage IV melanomas with normal serum LDH, glycolysis and OXPHOS may provide metabolic symbiosis within the same tumor, whereas in stage IV melanomas with high serum LDH glycolysis is the principle source of energy.