Induction of autophagy in malignant rhabdoid tumor cells by the histone deacetylase inhibitor FK228 through AIF translocation.

Malignant rhabdoid tumors (MRT) exhibit a very poor prognosis because of their resistance to chemotherapeutic agents and new therapies are needed for the treatment of this cancer. Here, we show that the histone deacetylase (HDAC) inhibitor FK228 (depsipeptide) has an antitumor effect on MRT cells both in vitro and in vivo. FK228 is a unique cyclic peptide and is among the most potent inhibitors of both Class I and Class II HDACs. FK228 inhibited proliferation and induced apoptosis in all MRT cell lines tested. Preincubation with the pancaspase inhibitor zVAD-fmk did not completely rescue FK228-induced cell death, although it did inhibit apoptosis. Transmission electron microscopy (TEM) showed that FK228 could stimulate MRT cells to undergo apoptosis, necrosis or autophagy. FK228 converted unconjugated microtubule-associated protein light chain 3 (LC3-I) to conjugated light chain 3 (LC3-II) and induced localization of LC3 to autophagosomes. Apoptosis inducing factor (AIF), which plays a role in caspase-independent cell death, translocated to the nucleus in response to FK228 treatment. Moreover, small interfering RNA (siRNA) targeting of AIF prevented the morphological changes associated with autophagy and redistribution of LC3 to autophagosomes. Disrupting autophagy with chloroquine treatment enhanced FK228-induced cell death. In vivo, FK228 caused a reduction in tumor size and induced autophagy in tumor tissues. Using immunoelectron microscopy, we confirmed AIF translocation into the nucleus of FK228-induced autophagic cells in vivo. Thus, FK228 is a novel candidate for an antitumor agent for MRT cells.

Involvement of extracellular signal-regulated kinase activation in human osteosarcoma cell resistance to the histone deacetylase inhibitor FK228 [(1S,4S,7Z,10S,16E,21R)-7-ethylidene-4,21-bis(propan-2-yl)-2-oxa-12,13-dithia-5,8,20,23-tetraazabicyclo[8.7.6]tricos-16-ene-3,6,9,19,22-pentone].

The histone deacetylase inhibitor depsipeptide [(1S,4S,7Z,10S, 16E,21R)-7-ethylidene-4,21-bis(propan-2-yl)-2-oxa-12,13-dithia-5,8,20,23-tetraazabicyclo[8.7.6]tricos-16-ene-3,6,9,19, 22-pentone] (FK228) has attracted a great deal of interest because of its antiproliferative and apoptotic properties in various malignancies. Histone deacetylase inhibitors induce the expression of the multidrug resistance transporter P-glycoprotein (P-gp), and FK228 is a known P-gp substrate. Thus, FK228 seems to induce its own mechanism of drug resistance by up-regulating P-gp. The goal of this study was to establish human FK228-resistant osteosarcoma cell lines and to investigate whether there are mechanisms of FK228 resistance in addition to P-gp up-regulation. After 72 h in culture, the 50% inhibitory concentrations (IC(50)) of FK228 were 4.8 and 991 nM in HOS and HOS/FK8 cells, respectively, and 3.6 and 1420 nM in U2OS and U2OS/FK11 cells, respectively. Increased histone H3 acetylation was observed in FK228-resistant cell lines after a 1-h treatment with 10 nM FK228. Unlike in parental cells, significant P-gp overexpression was detected in FK228-resistant cells, and 10 nM FK228 treatment activated the mitogen-activated protein kinase (MAPK) pathway but did not induce Fas ligand (FasL) up-regulation or c-FLIP down-regulation. However, treatment of FK228-resistant cells with a combination of FK228 and mitogen-activated protein kinase kinase (MEK) inhibitors induced apoptosis, up-regulated FasL, and down-regulated c-FLIP. The expression and function of P-gp were unaltered by treatment with MEK inhibitors. These results indicate that the FK228 resistance of osteosarcoma cells is related to P-gp overexpression and MAPK pathway activation by FK228. MEK or P-gp inhibitors may be useful in overcoming this resistance.

Antioxidant, EUK-8, prevents murine dilated cardiomyopathy.

BACKGROUND: Mice lacking manganese-superoxide dismutase (Mn-SOD) activity exhibit the typical pathology of dilated cardiomyopathy (DCM). In the present study, presymptomatic and symptomatic mutant mice were treated with the SOD/catalase mimetic, EUK-8. METHODS AND RESULTS: Presymptomatic heart/muscle-specific Mn-SOD-deficient mice (H/M-Sod2(-/-)) were treated with EUK-8 (30 mg x kg(-1) . day(-1)) for 4 weeks, and then cardiac function and the reactive oxygen species (ROS) production in their heart mitochondria were assessed. EUK-8 treatment suppressed the progression of cardiac dysfunction and diminished ROS production and oxidative damage. Furthermore, EUK-8 treatment effectively reversed the cardiac dilatation and dysfunction observed in symptomatic H/M-Sod2(-/-) mice. Interestingly, EUK-8 treatment repaired a molecular defect in connexin43. CONCLUSIONS: EUK-8 treatment can prevent and cure murine DCM, so SOD/catalase mimetic treatment is proposed as a potential therapy for DCM.

Effects of postural change on oxygen saturation and respiration in patients after the Fontan operation: platypnea and orthodeoxia.

The aim of this study was to assess whether platypnea and orthodeoxia occur in Fontan patients. We divided 14 Fontan patients into 2 groups: 8 patients who had pulmonary arteriovenous fistulas and/or intra-atrial shunts (group A) and 6 patients who had neither pulmonary arteriovenous fistulas nor intra-atrial shunts (group B). They were compared with 9 controls (group C). Arterial oxygen saturation, minute ventilation per body weight and ventilatory equivalent for carbon dioxide were measured in the supine and then sitting positions. In group A, 1 patient had platypnea and 3 patients had orthodeoxia (changes in the saturation from the supine position to the sitting position were -4% to -7%) accompanied with slight hyperpnea, and all 4 patients had both pulmonary arteriovenous fistulas and intra-atrial shunts. Contrary, patients in group B had neither platypnea nor orthodeoxia. The saturation was significantly lower and the minute ventilation was significantly higher in the sitting position than in the supine position in group A (p < 0.05). The other groups showed no significant difference in the saturation or the minute ventilation between the 2 positions. All groups showed the ventilatory equivalent was significantly higher in the sitting position than in the supine position (p < 0.05 to 0.01). We demonstrated platypnea and orthodeoxia in Fontan patients with pulmonary arteriovenous fistulas and intra-atrial shunts. We believe platypnea and orthodeoxia should be regard as a complication in Fontan patients with pulmonary arteriovenous fistulas and/or intra-atrial shunts.

Mitophagy is required for mitochondrial biogenesis and myogenic differentiation of C2C12 myoblasts.

Myogenesis is a crucial process governing skeletal muscle development and homeostasis. Differentiation of primitive myoblasts into mature myotubes requires a metabolic switch to support the increased energetic demand of contractile muscle. Skeletal myoblasts specifically shift from a highly glycolytic state to relying predominantly on oxidative phosphorylation (OXPHOS) upon differentiation. We have found that this phenomenon requires dramatic remodeling of the mitochondrial network involving both mitochondrial clearance and biogenesis. During early myogenic differentiation, autophagy is robustly upregulated and this coincides with DNM1L/DRP1 (dynamin 1-like)-mediated fragmentation and subsequent removal of mitochondria via SQSTM1 (sequestosome 1)-mediated mitophagy. Mitochondria are then repopulated via PPARGC1A/PGC-1α (peroxisome proliferator-activated receptor gamma, coactivator 1 alpha)-mediated biogenesis. Mitochondrial fusion protein OPA1 (optic atrophy 1 [autosomal dominant]) is then briskly upregulated, resulting in the reformation of mitochondrial networks. The final product is a myotube replete with new mitochondria. Respirometry reveals that the constituents of these newly established mitochondrial networks are better primed for OXPHOS and are more tightly coupled than those in myoblasts. Additionally, we have found that suppressing autophagy with various inhibitors during differentiation interferes with myogenic differentiation. Together these data highlight the integral role of autophagy and mitophagy in myogenic differentiation.

Ventricular Tachycardia Rotating a Scar of a Total Right Ventricular Exclusion.

We describe a 15-year-old postoperative girl who underwent surgical 3-dimensional mapping and ablation of hemodynamically unstable ventricular tachycardia (VT) with an on-pump beating heart surgical technique. She had previously received a tricuspid valve closure, entire right ventricular free wall resection, and finally Fontan operation with an extracardiac conduit to treat a severe Ebstein's anomaly. Activation mapping revealed a VT rotating around a large right ventricular free wall incisional scar with a narrow conduction channel between the scar and a tricuspid annulus (TA). A linear radiofrequency ablation connecting the scar and the TA terminated the VT.

Mesenchymal mode of migration participates in pulmonary metastasis of mouse osteosarcoma LM8.

The outcomes of osteosarcoma patients still remain poor because of intractable pulmonary metastasis. We previously established a highly metastatic osteosarcoma cell line, LM8 from Dunn mouse osteosarcoma by in vivo selection. We herein aimed to clarify the characteristic biological features related with high metastatic potential and new target molecules to suppress pulmonary metastasis of osteosarcoma, using this syngeneic spontaneous metastatic model. LM8 cells acquired fibroblastic morphology with striking filopodia on the cell surface. Immunostaining showed faint stress fiber formation and peripherally localized integrin ß1, and biochemical analyses showed the activated Cdc42 and autophosphorylation of focal adhesion kinase (FAK) in LM8 cells when compared to Dunn cells. LM8 cells had activated motility in single cell migration mode. LM8 migration was increased by a Rho-associated kinase (ROCK) inhibitor, Y-27632, while decreased by Cdc42 silencing using RNA interference system. We found that a clinically approved camptothecin analog, irinotecan suppressed the migration, Cdc42 activity, and autophosphorylation of FAK, and attenuated integrin ß1 distribution selectively in LM8 cells. Daily oral administration of irinotecan significantly reduced the rate and size of pulmonary metastasis in syngeneic C3H mice. The fibroblastic morphology and activated cell migration with the dependency on Cdc42 but not Rho-ROCK signaling pathway argued that LM8 moved in mesenchymal mode of cell migration. This activated mesenchymal migration was a key component of the pulmonary metastasis of LM8 cells. The inhibition of mesenchymal migration by irinotecan, in addition to its cytotoxic effects, might be effective in preventing pulmonary metastasis of osteosarcoma.

Prognostic significance of the BAALC isoform pattern and CEBPA mutations in pediatric acute myeloid leukemia with normal karyotype: a study by the Japanese Childhood AML Cooperative Study Group.

High BAALC (brain and acute leukemia, cytoplasmic) gene expression may indicate an adverse prognosis for adults who have acute myeloid leukemia (AML) and a normal karyotype, but its prognostic significance for pediatric AML cases is unclear. Whether different BAALC isoform patterns are of prognostic significance is also unclear. Newly diagnosed AML patients with normal karyotype who were treated by the Japanese Childhood AML Cooperative Treatment Protocol AML 99 were analyzed in terms of their BAALC expression levels (n = 29), BAALC isoforms (n = 29), and CEBPA mutations (n = 49). Eleven and 18 patients exhibited high and low BAALC expression, respectively, but these groups did not differ significantly in terms of overall survival (54.6 vs. 61.1%, P = 0.55) or event-free survival (61.4 vs. 50.0%, P = 0.82). Three of these 29 patients (10.3%) expressed the exon 1-5-6-8 BAALC isoform along with the expected 1-6-8 isoform and had adverse clinical outcomes. Novel CEBPA mutations were also identified in four of 49 patients (8.2%). All four patients have maintained complete remission for at least 5 years. Thus, 1-5-6-8 isoform expression may be associated with an adverse prognosis in pediatric AML with normal karyotype. CEBPA mutations may indicate a favorable prognosis.

Granulocyte colony-stimulating factor protects cardiac mitochondria in the early phase of cardiac injury.

Although granulocyte colony-stimulating factor (G-CSF) reportedly plays a cardioprotective role in several models of cardiac injury, clinical use of this drug in cardiac patients has been controversial. Here, we tested, in vivo and in vitro, the effect of G-CSF on cardiac mitochondria, which play a key role in determining cardiac cellular fate and function. Mild stimulation of C57/BL6 mice with doxorubicin (Dox) did not induce cardiac apoptosis or fibrosis but did induce damage to mitochondrial organization of the myocardium as observed through an electron microscope. Cardiac catheterization and echocardiography revealed that Dox did not alter cardiac systolic function or left ventricular size but did reduce diastolic function, an early sign of cardiac damage. Treatment with G-CSF attenuated significantly the damage to mitochondrial organization and rescued diastolic function. In an in vitro model for rat neonatal cardiomyocytes, a subapoptotic dose of Dox induced severe mitochondrial damage, including marked swelling of the cardiac mitochondria and/or decreased mitochondrial membrane potential. These mitochondrial changes were completely blocked by pretreatment with G-CSF. In addition, G-CSF dramatically improved ATP generation, which rescued Dox-impaired mitochondrial electron transport and oxygen consumption mainly through complex IV. These findings clearly indicate that G-CSF protects cardiac mitochondria, which are key organelles in the determination of cardiac cellular fate, in the early phase of cardiac injury.

Generation of cardiac and endothelial cells from neonatal mouse testis-derived multipotent germline stem cells.

Multipotent germline stem (mGS) cells have been established from neonatal mouse testes. Here, we compared mGS, embryonic stem (ES), and embryonic germ (EG) cells with regard to their ability to differentiate into mesodermal cells, namely, cardiomyocytes and endothelial cells. The in situ morphological appearances of undifferentiated mGS, ES, and EG cells were similar, and 4 days after being induced to differentiate, approximately 30%-40% of each cell type differentiated into Flk1(+) cells. The sorted Flk1(+) cells differentiated efficiently into cardiomyocytes and endothelial cells. By day 10 after differentiation induction, the three cell types generated equal number of endothelial colonies. However, by day 13 after differentiation induction, the Flk1(+) mGS cells generated more contractile colonies than did the Flk1(+) ES cells, whereas the Flk1(+) EG cells generated equivalent numbers as the Flk1(+) mGS cells. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis of differentiation markers such as Rex1, FGF-5, GATA-4, Brachyury, and Flk1 revealed that mGS cells expressed these markers more slowly during days 0-4 after differentiation induction than did ES cells, but that this mGS cell pattern was similar to that of the EG cells. RT-PCR analysis also revealed that the three differentiation cell types expressed various cardiac markers. Moreover, immunohistochemical analysis revealed that the contractile colonies derived from Flk1(+) mGS cells express mature cardiac cell-specific markers. In conclusion, mGS cells are phenotypically similar to ES and EG cells and have a similar potential to differentiate into cardiomyocytes and endothelial cells. Disclosure of potential conflicts of interest is found at the end of this article.

Doppler echocardiographic differentiation of functional from anatomical pulmonary atresia: analysis using quantitative parameters.

Functional pulmonary atresia must be distinguished from anatomical atresia, which has an intact ventricular septum, to avoid inappropriate treatment, but there is a paucity of data regarding the echocardiographic features that differentiate these conditions. Echocardiographic findings in 5 neonates with functional atresia were compared to those in 5 with anatomical atresia. The left and right ventricular end-diastolic dimensions (LVDd, RVDd), percent of normal predicted LVDd, RVDd/LVDd, tricuspid valve ring diameter (TVD), percent of normal predicted TVD, grade of tricuspid regurgitation (TR), peak TR velocity, pulmonary valve ring diameter (PVD), percent of normal predicted PVD, the minimum diameter of the ductus and the peak velocity through it (PDA velocity) were measured. In addition, systolic pulmonary (PAp) and right ventricular pressure (RVp) from either PDA velocity or TR velocity, and calculated PAp/RVp were also estimated. There were significant differences in RVDd/LVDd, %TVD, and peak TR velocity between the 2 groups. All functional patients showed RVDd/LVDd >0.6, %TVD >100%, estimated RVp <50mmHg, PAp/RVp >0.85, and peak TR velocity <4m/s, whereas the findings in anatomical atresia patients were completely the opposite. In conclusion, a large RVDd/LVDd, TVD, PAp/RVp, low RVp and small TR velocity all suggest functional rather than anatomical pulmonary atresia, although there may be some exceptions such as severe Ebstein anomaly.