Autophagy関連論文いくつか

AMPK-mediated autophagy inhibits apoptosis in cisplatin-treated tumour cells
Journal of Cellular and Molecular Medicine, 13:3644 - 3654, 2010

The role of autophagy in cisplatin anticancer action was investigated using human U251 glioma, rat C6 glioma and mouse L929 fibrosarcoma cell lines. A dose- and time-dependent induction of autophagy was observed in tumour cells following cisplatin treatment, as demonstrated by up-regulation of autophagy-inducing protein beclin-1 and subsequent appearance of acridine orange-stained acidic autophagic vesicles. The presence of autophagosomes in cisplatin-treated cells was also confirmed by electron microscopy. Inhibition of autophagy with lysosomal inhibitors bafilomycin A1 and chloroquine, or a PI3 kinase inhibitor wortmannin, markedly augmented cisplatin-triggered oxidative stress and caspase activation, leading to an increase in DNA fragmentation and apoptotic cell death. The mechanisms underlying the protective effect of autophagy apparently involved the interference with cisplatin-induced modulation of Bcl-2 family proteins, as inhibition of autophagy potentiated cisplatin-mediated up-regulation of proapoptotic Bax and down-regulation of anti-apoptotic Bcl-2. Autophagy induction in cisplatin-treated cells was preceded by activation of adenosine monophosphate-activated protein kinase (AMPK) and concomitant down-regulation of mammalian target of rapamycin (mTOR)-mediated phosphorylation of p70S6 kinase. The ability of cisplatin to trigger autophagy was reduced by small interfering RNA (siRNA)-mediated AMPK silencing, while transfection with mTOR siRNA was sufficient to trigger autophagy in tumour cells. Finally, siRNA-mediated AMPK down-regulation and AMPK inhibitor compound C increased cisplatin-induced tumour cell death, while mTOR siRNA and AMPK activator metformin protected tumour cells from cisplatin. Taken together, these data suggest that cisplatin-triggered activation of AMPK and subsequent suppression of mTOR activity can induce an autophagic response that protects tumour cells from cisplatin-mediated apoptotic death.

AMPKとautophagyのリンク。備忘的に。

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Autophagy influences glomerular disease susceptibility and maintains podocyte homeostasis in aging mice

JCI 2010 online in March

Injury and loss of podocytes are leading factors of glomerular disease and renal failure. The postmitotic podocyte is the primary glomerular target for toxic, immune, metabolic, and oxidant stress, but little is known about how this cell type copes with stress. Recently, autophagy has been identified as a major pathway that delivers damaged proteins and organelles to lysosomes in order to maintain cellular homeostasis. Here we report that podocytes exhibit an unusually high level of constitutive autophagy. Podocyte-specific deletion of autophagy-related 5 (Atg5) led to a glomerulopathy in aging mice that was accompanied by an accumulation of oxidized and ubiquitinated proteins, ER stress, and proteinuria. These changes resulted ultimately in podocyte loss and late-onset glomerulosclerosis. Analysis of pathophysiological conditions indicated that autophagy was substantially increased in glomeruli from mice with induced proteinuria and in glomeruli from patients with acquired proteinuric diseases. Further, mice lacking Atg5 in podocytes exhibited strongly increased susceptibility to models of glomerular disease. These findings highlight the importance of induced autophagy as a key homeostatic mechanism to maintain podocyte integrity. We postulate that constitutive and induced autophagy is a major protective mechanism against podocyte aging and glomerular injury, representing a putative target to ameliorate human glomerular disease and aging-related loss of renal function.

●podocyteでbasal autophagyが高い（Fig1)→podocyte cell lineを作製！。

●ubiquitin-proteasome systemとfunctional cross-talk（UPSによるcompensation）があることを示す（Fig3)。が、age-dependentにlate-onset  glomeruosclerosisを生じる（Fig4)。

●ER stress (Calnexin)、oxidized and ubiquitinated protein aggregate（Ub, p62など）の蓄積を伴う（Fig5)。逆に、proteinuric diseaseでautophagyがupregulateしていることを示す（Fig6、BSAのoverloadで人工的にproteinuriaにしたサンプルと、ヒト疾患のbiopsy標本)。

●ATG5ノックアウトを用いて、stress adaptationにautophagyが必須であることを示す（Fig7、puromycin aminonucleoside (PAN)、adriamycinで負荷）。

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Induction of autophagy-dependent necroptosis is required for childhood acute lymphoblastic leukemia cells to overcome glucocorticoid resistance

JCI 2010 online in March

In vivo resistance to first-line chemotherapy, including to glucocorticoids, is a strong predictor of poor outcome in children with acute lymphoblastic leukemia (ALL). Modulation of cell death regulators represents an attractive strategy for subverting such drug resistance. Here we report complete resensitization of multidrug-resistant childhood ALL cells to glucocorticoids and other cytotoxic agents with subcytotoxic concentrations of obatoclax, a putative antagonist of BCL-2 family members. The reversal of glucocorticoid resistance occurred through rapid activation of autophagy-dependent necroptosis, which bypassed the block in mitochondrial apoptosis. This effect was associated with dissociation of the autophagy inducer beclin-1 from the antiapoptotic BCL-2 family member myeloid cell leukemia sequence 1 (MCL-1) and with a marked decrease in mammalian target of rapamycin (mTOR) activity. Consistent with a protective role for mTOR in glucocorticoid resistance in childhood ALL, combination of rapamycin with the glucocorticoid dexamethasone triggered autophagy-dependent cell death, with characteristic features of necroptosis. Execution of cell death, but not induction of autophagy, was strictly dependent on expression of receptor-interacting protein (RIP-1) kinase and cylindromatosis (turban tumor syndrome) (CYLD), two key regulators of necroptosis. Accordingly, both inhibition of RIP-1 and interference with CYLD restored glucocorticoid resistance completely. Together with evidence for a chemosensitizing activity of obatoclax in vivo, our data provide a compelling rationale for clinical translation of this pharmacological approach into treatments for patients with refractory ALL.

薬剤耐性（特にglucocorticoid耐性）ALLにBCL2-familyのputative antagonistであるobatoclaxを用いると、autophagy dependent necroptosisを起こして耐性が解除される、という内容。

●DEXとobatoclaxの組み合わせで、ALL細胞のviabilityが低下、これはBCL-2及びBCL-XLの特異的阻害剤であるABT-737では同様の効果が得られないことから、BCL-2ファミリーのMCL-1によるものと（Fig1、refでMCL-1がglucocorticoid耐性の主要因子であることが示されている)。

●この耐性解除はapoptosis independent、autophagy dependent（Fig2-4）。

●DEX＋obatoclaxでmTOR活性が低下（Fig5、機序は？）。

●ObatoclaxがBeclin-1とMCL-1との結合を抑制することを証明（Fig6)。ここで見られる細胞死がnecroticであることを示す（Fig7A,B、plasma membraneのintegrityが消失することを電顕で示す）。さらに、necrotic deathに関係するRIP-1 kinaseと、その制御因子であるdeUb enzymeのCYLDがこの経路のpositive regulatorであることを示す（Fig7C-G）。

●ヒト検体でこの現象の臨床的重要性を証明（Fig8）。

●Beclin1はhaploinsufficientなoncosupressor→autophagyの癌抑制的な機能を示唆。上流のあの分子と同じだ。。。