Yoshimi Hiraumi

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.

The Bcr-Abl kinase inhibitor INNO-406 induces autophagy and different modes of cell death execution in Bcr-Abl-positive leukemias

Bcr-Abl tyrosine kinase (TK) inhibitors are promising therapeutic agents for Bcr-Abl-positive (Bcr-Abl+) leukemias. Although they are known to promote caspase-mediated apoptosis, it remains unclear whether caspase-independent cell death-inducing mechanisms are also triggered. Here we demonstrated that INNO-406, a second-generation Bcr-Abl TK inhibitor, induces programmed cell death (PCD) in chronic myelogenous leukemia (CML) cell lines through both caspase-mediated and caspase-independent pathways. The latter pathways include caspase-independent apoptosis (CIA) and necrosis-like cell death (CIND), and the cell lines varied regarding which mechanism was elicited upon INNO-406 treatment. We also observed that the propensity toward CIA or CIND in cells was strongly associated with cellular dependency on apoptosome-mediated caspase activity. Cells that undergo CIND have a high apoptosome activity potential whereas cells that undergo CIA tend to have a lower potential. Moreover, we found that INNO-406 promotes autophagy. When autophagy was inhibited with chloroquine or gene knockdown of beclin1 by shRNA, INNO-406-induced cell death was enhanced, which indicates that the autophagic response of the tumor cells is protective. These findings suggest new insights into the biology and therapy of Bcr-Abl+ leukemias.

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.

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 β1, 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 β1 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.

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 (IC50) 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.

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.

Myogenic Progenitor Cell Differentiation Is Dependent on Modulation of Mitochondrial Biogenesis through Autophagy

Over the last decade, stem/progenitor cell therapy has emerged as an innovative approach to promote cardiac repair and regeneration. However, the therapeutic prospects of are currently limited by inadequate means to regulate cell proliferation, homing, engraftment, and differentiation. Autophagy, a lysosome-mediated degradation pathway for recycling organelles and protein aggregates, is recognized as important for facilitating cell differentiation. Studies have shown that induced pluripotent stem cells (iPCs), which exhibit a predominantly glycolytic metabolism, shift toward oxidative mitochondrial metabolism as a prerequisite for the formation of sarcomeres and differentiation into cardiomyocytes. C2C12 myoblasts are a mouse-derived myogenic progenitor cell line which can be induced to differentiate into myotubes. We hypothesize that autophagy is essential in coordinating transcription factor activity and metabolic reprogramming of mitochondria to support myocyte differentiation.