Autophagy論文いくつか

Proc Natl Acad Sci U S A. 2010 Mar 15. [Epub ahead of print]
Mechanisms of acute axonal degeneration in the optic nerve in vivo.

Axonal degeneration is an initial key step in traumatic and neurodegenerative CNS disorders. We established a unique in vivo epifluorescence imaging paradigm to characterize very early events in axonal degeneration in the rat optic nerve. Single retinal ganglion cell axons were visualized by AAV-mediated expression of dsRed and this allowed the quantification of postlesional acute axonal degeneration (AAD). EM analysis revealed severe structural alterations of the cytoskeleton, cytoplasmatic vacuolization, and the appearance of autophagosomes within the first hours after lesion. Inhibition of autophagy resulted in an attenuation of acute axonal degeneration. Furthermore, a rapid increase of intraaxonal calcium levels following crush lesion could be visualized using a calcium-sensitive dye. Application of calcium channel inhibitors prevented crush-induced calcium increase and markedly attenuated axonal degeneration, whereas application of a calcium ionophore aggravated the degenerative phenotype. We finally demonstrate that increased postlesional autophagy is calcium dependent and thus mechanistically link autophagy and intraaxonal calcium levels. Both processes are proposed to be major targets for the manipulation of axonal degeneration in future therapeutic settings.

 ====

Eur J Cancer. 2010 Mar 13. [Epub ahead of print]
Inhibition of autophagy augments 5-fluorouracil chemotherapy in human colon cancer in vitro and in vivo model.

Although 5-fluorouracil (5-FU)-based adjuvant chemotherapy is widely used in the treatment of colorectal cancer, novel therapeutic strategies need to be explored. It has been reported that autophagy is extensively implicated in cancer. However, the function of autophagy is not fully understood. In the present study, apoptosis induced by 5-FU in 3 human colon cancer cell lines (HCT116, DLD-1, and DLD-1/5-FU (a specific 5-FU-resistant sub-line)) was measured using MTT assay, DNA fragmentation assay, Hoechst 33342 staining, and caspase-3 immunoblotting. The autophagy activation induced by 5-FU treatment was revealed by microtubule-associated protein 1 light chain 3 (LC3) immunofluorescence and immunoblotting and p62 immunoblotting. Inhibition of autophagy by 3-methyladenine (3-MA) or small interference RNA targeting Atg7 (Atg7 siRNA) significantly augmented 5-FU-induced apoptosis. This synergistic effect of 5-FU and 3-MA was further confirmed in the DLD-1 xenograft tumour model. Tumour growth was suppressed more significantly with combination treatment than 5-FU treatment alone. In conclusion, autophagy was activated as a protective mechanism against 5-FU-induced apoptosis and its inhibition could be a promising strategy for adjuvant chemotherapy in colon cancer.

 ====

BMC Cancer. 2010 Mar 16;10(1):98. [Epub ahead of print]
Genetic and epigenetic silencing of the beclin 1 gene in sporadic breast tumors.

ABSTRACT: BACKGROUND: : Beclin 1, an important autophagy-related protein in human cells, is involved in cell death and cell survival. Beclin 1 mapped to human chromosome 17q21. It is widely expressed in normal mammary epithelial cells. Although down-regulated expression with mono-allelic deletions of beclin 1 gene was frequently observed in breast tumors, whether there was other regulatory mechanism of beclin 1 was to be investigated. We studied the expression of beclin 1 and explored the possible regulatory mechanisms on its expression in breast tumors. METHOD: S: 20 pairs of tumors and adjacent normal tissues from patients with sporadic breast invasive ductal cancer (IDCs) were collected. The mRNA expression of beclin 1 was detected by real-time quantitative RT-PCR. Loss of heterozygosity (LOH) was determined by real-time quantitative PCR and microsatellite methods. The protein expression of beclin 1, p53, BRCA1 and BRCA2 was assessed by immunohistochemistry. CpG islands in 5' genomic region of beclin 1 gene were identified using MethylPrimer Program. Sodium bisulfite sequencing was used in examining the methylation status of each CpG island. RESULTS: : Decreased beclin 1 mRNA expression was detected in 70% of the breast tumors, and the protein levels were co-related to the mRNA levels. Expression of beclin 1 mRNA was demonstrated to be much higher in the BRCA1 positive tumors than that in the BRCA1 negative ones. Loss of heterozygosity was detected in more than 45% of the breast tumors, and a dense cluster of CpG islands was found from the 5' end to the intron 2 of the beclin 1 gene. Methylation analysis showed that the promoter and the intron 2 of beclin 1 were aberrantly methylated in the tumors with decreased expression. CONCLUSIONS: : These data indicated that LOH and aberrant DNA methylation might be the possible reasons of the decreased expression of beclin 1 in the breast tumors. The findings here shed some new light on the regulatory mechanisms of beclin 1 in breast cancer.

Autophagy & Cancer

Autophagy. 2010 Apr 26;6(3)
Targeting the prodeath and prosurvival functions of autophagy as novel therapeutic strategies in cancer.

Autophagy is an evolutionarily conserved lysosomal pathway for degrading cytoplasmic proteins, macromolecules, and organelles. While autophagy has become one of the most attractive topics in cancer research, the current autophagy literature is often viewed as confusing, because of its association with apparently contradictory roles, such as survival and cell death. Autophagy can serve as a tumor suppressor, as a partial reduction in autophagic capacity or defective autophagy (e.g., heterozygous knockdown BECN1 (+/-) in mice) provides an oncogenic stimulus, causing malignant transformation and spontaneous tumors. In addition, autophagy seems to function as a protective cell survival mechanism against environmental and cellular stress (e.g., nutrient deprivation, hypoxia and therapeutic stress) and causes resistance to antineoplastic therapies. Recent studies have demonstrated that the inhibition of autophagy in cancer cells may be therapeutically beneficial in some circumstances, as it can sensitize cancer cells to different therapies, including DNA-damaging agents, antihormone therapies (e.g., tamoxifen), and radiation therapy. This supports the hypothesis that inhibiting autophagy can negatively influence cancer cell survival and increase cell death when combined with anticancer agents, providing a therapeutic advantage against cancer. On the other hand, the induction of autophagy by the inhibition of anti-autophagic proteins, such as Bcl-2, PKCdelta, and tissue transglutaminase 2 (TG2), may lead to autophagic cell death in some apoptosis-resistant cancers (i.e., breast and pancreatic cancers), indicating that the induction of autophagy alone may also be used as a potential therapy. Overall, the data suggest that, depending on the cellular features, either the induction or the inhibition of autophagy can provide therapeutic benefits to patients and that the design and synthesis of the first-generation modulators of autophagy may provide the tools for proof of concept experiments and the impetus for translational studies that may ultimately lead to new therapeutic strategies in cancer.

＝＝＝

癌細胞によって、survivalに働いたりcell deathに働いたりするので、個々の細胞ごとにeffectを確認しないといけない。

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。

＝＝＝

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）。

＝＝＝

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。。。