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ABSTRACT: Less is more: A minimalist "clickable" photo-crosslinker was incorporated with numerous small-molecule kinase inhibitors. The resulting probes were used for both in vitro (cell lysates) and in situ (live cells) proteome profiling, for large-scale identification of their potential cellular kinase targets, showing improved outcomes over previous probes.
Angewandte Chemie International Edition 06/2013; · 13.45 Impact Factor
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ABSTRACT: The lysosome is a key subcellular organelle that receives and degrades macromolecules from endocytic, secretory and autophagic pathways. Lysosomal function is thus critical for an efficient autophagic process. However, the molecular mechanisms mediating lysosomal function upon autophagic induction are largely unknown. Our laboratory recently discovered that upon autophagy activation, the lysosome is activated, and this functional activation is dependent on MTORC1 suppression, suggesting that MTORC1 exerts a suppressive effect on lysosomal function. Therefore, data from our study demonstrate that MTORC1 exerts a dual inhibitory effect on autophagy, blocking autophagy not only at the initiation stage via suppression of the ULK1 complex, but also at the degradation stage via inhibition of lysosomal function. We think that understanding the negative regulatory effect of MTORC1 on lysosomal function expands the functional scope of MTORC1 in autophagy regulation, and offers new clues for developing novel interventional strategies in autophagy- and lysosome-related diseases.
Autophagy 02/2013; 9(5). · 7.45 Impact Factor
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ABSTRACT: Lysosome is a key subcellular organelle in the execution of the autophagic process and at present little is known whether lysosomal function is controlled in the process of autophagy. In this study, we first found that suppression of mammalian target of rapamycin (mTOR) activity by starvation or two mTOR catalytic inhibitors (PP242 and Torin1), but not by an allosteric inhibitor (rapamycin), leads to activation of lysosomal function. Second, we provided evidence that activation of lysosomal function is associated with the suppression of mTOR complex 1 (mTORC1), but not mTORC2, and the mTORC1 localization to lysosomes is not directly correlated to its regulatory role in lysosomal function. Third, we examined the involvement of transcription factor EB (TFEB) and demonstrated that TFEB activation following mTORC1 suppression is necessary but not sufficient for lysosomal activation. Finally, Atg5 or Atg7 deletion or blockage of the autophagosome-lysosome fusion process effectively diminished lysosomal activation, suggesting that lysosomal activation occurring in the course of autophagy is dependent on autophagosome-lysosome fusion. Taken together, this study demonstrates that in the course of autophagy, lysosomal function is upregulated via a dual mechanism involving mTORC1 suppression and autophagosome-lysosome fusion.Cell Research advance online publication 22 January 2013; doi:10.1038/cr.2013.11.
Cell Research 01/2013; · 8.19 Impact Factor
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ABSTRACT: In this study we aim to elucidate the signaling pathway and biological function of autophagy induced by MNNG, a commonly used DNA alkylating agent. We first observed that MNNG is able to induce necrotic cell death and autophagy in Bax-/- Bak-/- double knockout MEFs. We analyzed the critical role of PARP-1 activation and ATP depletion in MNNG-mediated cell death and autophagy via AMPK activation and mTOR suppression. We provide evidence that suppression of AMPK blocks MNNG-induced autophagy and enhances cell death, suggesting the pro-survival function of autophagy in MNNG-treated cells. Taken together, data from this study reveal a novel mechanism in controlling MNNG-mediated autophagy via AMPK activation downstream of PARP-1 activation and ATP depletion.
FEBS letters 11/2012; · 3.54 Impact Factor
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Tiejun Zhang,
Yuwen Li,
Kyeong Ah Park,
Hee Sun Byun,
Minho Won,
Juhee Jeon,
Yoonjung Lee,
Jeong Ho Seok,
Seung-Won Choi,
Sang-Hee Lee,
Jin Man Kim,
Ji Hoon Lee,
Chang Gue Son,
Zee-Won Lee, Han-Ming Shen,
Gang Min Hur
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ABSTRACT: Targeted disruption of STAT3 function has proven to be a useful cancer therapeutic approach by inducing apoptotic cell death. Cucurbitacin is currently under development as a small molecule of STAT3 inhibitor to trigger cell death in many cancers. Here, we systematically studied the molecular mechanisms underlying cucurbitacin-induced cell death, in particular the involvement of autophagy. Treatment with cucurbitacin resulted in non-apoptotic cell death in a caspase-independent manner. Notably, cucurbitacin enhanced excessive conversion of lipidated LC3 (LC3-II) and accumulation of autophagosomes in many cell types. Such autophagy and cell death induced by cucurbitacin were independent of its ability to inhibit STAT3 function, but mainly mediated by enhanced production of mitochondrial-derived reactive oxygen species (ROS), and subsequently activation of extracellular signal-regulated kinase (ERK) and c-jun NH2-terminal kinase (JNK). Interestingly, both the autophagy inhibitor wortmannin and knockdown of Atg5 or Beclin 1 failed to rescue the cells from cucurbitacin-induced cell death, as suppression of autophagy induced the mode of cell death to shift from autophagic cell death to caspase-dependent apoptosis. Thus the present study provides new insights into the molecular mechanisms underlying cucurbitacin-mediated cell death and supports cucurbitacin as a potential anti-cancer drug through modulating the balance between autophagic and apoptotic modes of cell death.
Autophagy 04/2012; 8(4):559-76. · 7.45 Impact Factor
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ABSTRACT: Lipotoxicity refers to the cytotoxic effects of excess fat accumulation in cells and has been implicated as one of the contributing factors to diseases like obesity, diabetes, and non-alcoholic fatty liver. In this study we sought to examine effects of palmitic acid (PA) and oleic acid, two of the common dietary fatty acids on the autophagic process. We found that PA, but not oleic acid, was able to cause an increase in autophagic flux, evidenced by LC3-II accumulation and formation of GFP-LC3 puncta. Notably, PA-induced autophagy was found to be independent of mTOR regulation. Next, in search of the mechanism mediating PA-induced autophagy, we found increased levels of diacylglycerol species and protein kinase C (PKC) activation in PA-treated cells. More importantly, inhibition of classical PKC isoforms (PKC-α) was able to effectively suppress PA-induced autophagy. Finally, we showed that inhibition of autophagy sensitized the cells to PA-induced apoptosis, suggesting the pro-survival function of autophagy induced by PA. Taken together, results from this study reveal a novel mechanism underlying free fatty acid-mediated autophagy. Furthermore, the pro-survival function of autophagy suggests modulation of autophagy as a potential therapeutic strategy in protection of cells against lipotoxicity and lipid-related metabolic diseases.
Journal of Biological Chemistry 03/2012; 287(18):14364-76. · 4.77 Impact Factor
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ABSTRACT: Suppression of autophagy has been increasingly recognized as a novel cancer therapeutic approach. Andrographolide (Andro), a diterpenoid lactone isolated from an herbal plant Andrographis paniculata, is known to possess anti-inflammatory and anticancer activity. In this study, we sought to examine the effect of Andro on autophagy, and to evaluate whether such effect is relevant to the sensitization effect of Andro on apoptosis induced by DNA damage agents in cancer cells. First, we found that Andro is able to significantly enhance autophagic markers in various cancer cell lines, including GFP-LC3 puncta and LC3-II level. Interestingly, Andro treatment also led to marked increase of p62 protein level and addition of chloroquine (CQ) failed to further enhance either LC3-II or p62 level, indicating that Andro is likely to suppress autophagic flux at the maturation and degradation stage. Next, we provided evidence that Andro inhibits autophagosome maturation not by affecting the lysosomal function, but by impairing autophagosome-lysosome fusion. Lastly, we demonstrated that treatment with cisplatin, a DNA damage agent, induces autophagy in cancer cells. Importantly, Andro is capable of sensitizing cisplatin-induced cell killing determined with both short-term apoptosis assays and long-term clonogenic test, via suppression of autophagy, a process independent of p53. In summary, these observations collectively suggest that Andro could be a promising anti-cancer agent in combination therapy via its potent inhibitory effect on autophagy by disrupting autophagosome-lysosome fusion.
Autophagy 03/2012; 8(3):338-49. · 7.45 Impact Factor
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ABSTRACT: Macroautophagy or autophagy is a self-digesting mechanism that the cellular contents are engulfed by autophagosomes and delivered to lysosomes for degradation. Although it has been well established that autophagy is an important protective mechanism for cells under stress such as starvation via provision of nutrients and removal of protein aggregates and damaged mitochondria, there is a very complex relation between autophagy and cell death. At present, the molecular cross-talk between autophagy and apoptosis has been well discussed, while the relationship between autophagy and programmed necrotic cell death is less understood. In this review we focus on the role of autophagy in necrotic cell death by detailed discussion on two important forms of necrotic cell death: (i) necroptosis and (ii) poly-(ADP-ribose) polymerase (PARP)-mediated cell death. It is believed that one important aspect of the pro-survival function of autophagy is achieved via its ability to block various forms of necrotic cell death.
Experimental Cell Research 02/2012; 318(11):1304-8. · 3.58 Impact Factor
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ABSTRACT: Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) has been intensively studied as a cancer therapeutic agent due to its unique ability to induce apoptosis in malignant cells but not in normal cells. However, as more human cancer cells are reported to be resistant to TRAIL treatment, it is important to develop new therapeutic strategies to overcome this resistance. p53 is an important tumor suppressor that is widely involved in cellular responses to various stresses. In this mini-review, we aim to provide an overview of the intricate relationship between p53 and the TRAIL-mediated apoptosis pathway, and to summarize the current approaches of targeting p53 as a therapeutic strategy to sensitize TRAIL-induced apoptosis in human cancer cells. Although in some cases TRAIL kills cancer cells in a p53-independent manner, it is believed that in cancers with wild-type and functional p53, targeting p53 may be an important strategy for overcoming TRAIL-resistance in cancer therapy.
Cancer letters 01/2012; 314(1):8-23. · 4.86 Impact Factor
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ABSTRACT: It has been well documented that cells deficient in either TSC1 or TSC2 are highly sensitive to various cell death stimuli. In this study, we utilized the TSC2 (-/-) mouse embryonic fibroblasts (MEFs) to study the involvement of autophagy in the enhanced susceptibility of TSC2-null cells to cell death. We first confirmed that both TSC1-null and TSC2-null MEFs are more sensitive to apoptosis in response to amino acid starvation (EBSS) and hypoxia. Second, we found that both the basal and inducible autophagy in TSC2 (-/-) MEFs is impaired, mainly due to constitutive activation of mTORC1. Third, suppression of autophagy by chloroquine and Atg7 knockdown sensitizes TSC2 (+/+) cells, but not TSC2 (-/-) cells, to EBSS-induced cell death. Conversely, the inhibition of mTORC1 by raptor knockdown and rapamycin activates autophagy and subsequently rescues TSC2 (-/-) cells. Finally, in starved cells, nutrient supplementations (insulin-like growth factor-1 (IGF-1) and leucine) enhanced cell death in TSC2 (-/-) cells, but reduced cell death in TSC2 (+/+) cells. Taken together, these data indicate that constitutive activation of mTORC1 in TSC2 (-/-) cells leads to suppression of autophagy and enhanced susceptibility to stress-mediated cell death. Our findings thus provide new insights into the complex relationships among mTOR, autophagy and cell death, and support the possible autophagy-targeted intervention strategies for the treatment of TSC-related pathologies.
Autophagy 10/2011; 7(10):1173-86. · 7.45 Impact Factor
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Katherine C. M. Chew,
Eng-Tat Ang,
Yee Kit Tai,
Fai Tsang,
Shun Qiang Lo,
Elijah Ong,
Wei-Yi Ong, Han-Ming Shen,
Kah-Leong Lim,
Valina L. Dawson,
Ted M. Dawson,
Tuck Wah Soong
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ABSTRACT: Parkinson disease (PD), a prevalent neurodegenerative motor disorder, is characterized by the rather selective loss of dopaminergic
neurons and the presence of α-synuclein-enriched Lewy body inclusions in the substantia nigra of the midbrain. Although the
etiology of PD remains incompletely understood, emerging evidence suggests that dysregulated iron homeostasis may be involved.
Notably, nigral dopaminergic neurons are enriched in iron, the uptake of which is facilitated by the divalent metal ion transporter
DMT1. To clarify the role of iron in PD, we generated SH-SY5Y cells stably expressing DMT1 either singly or in combination
with wild type or mutant α-synuclein. We found that DMT1 overexpression dramatically enhances Fe2+ uptake, which concomitantly promotes cell death. This Fe2+-mediated toxicity is aggravated by the presence of mutant α-synuclein expression, resulting in increased oxidative stress
and DNA damage. Curiously, Fe2+-mediated cell death does not appear to involve apoptosis. Instead, the phenomenon seems to occur as a result of excessive
autophagic activity. Accordingly, pharmacological inhibition of autophagy reverses cell death mediated by Fe2+ overloading. Taken together, our results suggest a role for iron in PD pathogenesis and provide a mechanism underlying Fe2+-mediated cell death.
Journal of Biological Chemistry 09/2011; 286(38):33380-33389. · 4.77 Impact Factor
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Katherine C M Chew,
Eng-Tat Ang,
Yee Kit Tai,
Fai Tsang,
Shun Qiang Lo,
Elijah Ong,
Wei-Yi Ong, Han-Ming Shen,
Kah-Leong Lim,
Valina L Dawson,
Ted M Dawson,
Tuck Wah Soong
[show abstract]
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ABSTRACT: Parkinson disease (PD), a prevalent neurodegenerative motor disorder, is characterized by the rather selective loss of dopaminergic neurons and the presence of α-synuclein-enriched Lewy body inclusions in the substantia nigra of the midbrain. Although the etiology of PD remains incompletely understood, emerging evidence suggests that dysregulated iron homeostasis may be involved. Notably, nigral dopaminergic neurons are enriched in iron, the uptake of which is facilitated by the divalent metal ion transporter DMT1. To clarify the role of iron in PD, we generated SH-SY5Y cells stably expressing DMT1 either singly or in combination with wild type or mutant α-synuclein. We found that DMT1 overexpression dramatically enhances Fe(2+) uptake, which concomitantly promotes cell death. This Fe(2+)-mediated toxicity is aggravated by the presence of mutant α-synuclein expression, resulting in increased oxidative stress and DNA damage. Curiously, Fe(2+)-mediated cell death does not appear to involve apoptosis. Instead, the phenomenon seems to occur as a result of excessive autophagic activity. Accordingly, pharmacological inhibition of autophagy reverses cell death mediated by Fe(2+) overloading. Taken together, our results suggest a role for iron in PD pathogenesis and provide a mechanism underlying Fe(2+)-mediated cell death.
Journal of Biological Chemistry 07/2011; 286(38):33380-9. · 4.77 Impact Factor
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ABSTRACT: The concept of autophagic cell death was first established based on observations of increased autophagic markers in dying cells. The major limitation of such a morphology-based definition of autophagic cell death is that it fails to establish the functional role of autophagy in the cell death process, and thus contributes to the confusion in the literature regarding the role of autophagy in cell death and cell survival. Here we propose to define autophagic cell death as a modality of non-apoptotic or necrotic programmed cell death in which autophagy serves as a cell death mechanism, upon meeting the following set of criteria: (i) cell death occurs without the involvement of apoptosis; (ii) there is an increase of autophagic flux, and not just an increase of the autophagic markers, in the dying cells; and (iii) suppression of autophagy via both pharmacological inhibitors and genetic approaches is able to rescue or prevent cell death. In light of this new definition, we will discuss some of the common problems and difficulties in the study of autophagic cell death and also revisit some well-reported cases of autophagic cell death, aiming to achieve a better understanding of whether autophagy is a real killer, an accomplice or just an innocent bystander in the course of cell death. At present, the physiological relevance of autophagic cell death is mainly observed in lower eukaryotes and invertebrates such as Dictyostelium discoideum and Drosophila melanogaster. We believe that such a clear definition of autophagic cell death will help us study and understand the physiological or pathological relevance of autophagic cell death in mammals.
Autophagy 05/2011; 7(5):457-65. · 7.45 Impact Factor
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ABSTRACT: The protein kinase Akt (also known as protein kinase B) is a critical signaling hub downstream of various cellular stimuli such as growth factors that control cell survival, growth, and proliferation. The activity of Akt is tightly regulated, and the aberrant activation of Akt is associated with diverse human diseases including cancer. Although it is well documented that the mammalian target of rapamycin complex 2 (mTORC2)-dependent phosphorylation of the Akt hydrophobic motif (Ser-473 in Akt1) is essential for full Akt activation, it remains unclear whether this phosphorylation has additional roles in regulating Akt activity. In this study, we found that abolishing Akt Ser-473 phosphorylation stabilizes Akt following agonist stimulation. The Akt Ser-473 phosphorylation promotes a Lys-48-linked polyubiquitination of Akt, resulting in its rapid proteasomal degradation. Moreover, blockade of this proteasomal degradation pathway prolongs agonist-induced Akt activation. These data reveal that mTORC2 plays a central role in regulating the Akt protein life cycle by first stabilizing Akt protein folding through the turn motif phosphorylation and then by promoting Akt protein degradation through the hydrophobic motif phosphorylation. Taken together, this study reveals that the Akt Ser-473 phosphorylation-dependent ubiquitination and degradation is an important negative feedback regulation that specifically terminates Akt activation.
Journal of Biological Chemistry 02/2011; 286(16):14190-8. · 4.77 Impact Factor
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ABSTRACT: Luteolin, a plant flavonoid is known to possess multiple biological activities such as anti-inflammation, anti-allergy, anti-oxidant as well as anti-cancer. At present, the anti-proliferative potential of luteolin has not been fully understood. In this study, we focused on the effect of luteolin on cell cycle regulation in human nasopharyngeal carcinoma (NPC) cells. First, we found that luteolin inhibited cell cycle progression at G1 phase and prevented entry into S phase in a dose- and time-dependent manner. Next, it was found that luteolin treatment led to down-regulation of cyclin D1 via enhanced protein phosphorylation and proteasomal degradation, leading to reduced CDK4/6 activity and suppression of retinoblastoma protein (Rb) phosphorylation, and subsequently inhibition of the transcription factor E2F-1. In search of the molecular mechanisms underlying luteolin-mediated cyclin D1 down-regulation, it was found that luteolin was capable of suppressing Akt phosphorylation and activation, resulting in de-phosphorylation and activation of glycogen synthase kinase-3β (GSK-3β). Activated GSK-3β then targeted cyclin D1, causing phosphorylation of cyclin D1 at Thr(286) and subsequent proteasomal degradation. The above findings were reinforced by the fact that luteolin was able to abrogate the effect of insulin on the Akt/GSK-3β/Cyclin D1 pathway, resulting in suppression of insulin-induced cell proliferation. Since Akt is often over-activated in many human cancers including NPC, it is thus believed that data from this study support the potential application of luteolin as a chemotherapeutic or chemopreventive agent in human cancer.
Cancer letters 12/2010; 298(2):167-75. · 4.86 Impact Factor
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ABSTRACT: Chrysin exists widely in plants, honey and propolis. The anti-cancer property of chrysin has been demonstrated though the molecular mechanism is not clear. In this study, we found that pre-treatment with chrysin could promote the cell death induced by TRAIL according to the morphological changes and appearance of sub-G1 peak in four human cancer cell lines. In HCT-116 cells, the results of flow cytometry analysis showed that the percentage of sub-G1 reached (38.89 ± 3.78) % when pre-treatment of chrysin was used at 40 μM, but that was only (2.53 ± 0.10) % in the untreated group and (13.22 ± 0.20) % in TRAIL alone group. The differences between the combination and the untreated or TRAIL alone group were all significant (P<0.05) and dose-dependent effect was obvious. Similar results were obtained in CNE1 cells. In the search of molecular mechanisms, we found that pre-treatment with chrysin could increase TRAIL-induced degradation of caspase 3, caspase 8, PARP proteins. Z-VAD-fmk, which is a pan-caspase inhibitor, could inhibit the apoptosis enhanced by the combination of chrysin and TRAIL. All data indicate that chrysin can enhance the apoptosis induced by TRAIL, and the apoptosis is caspase-dependent and related to the activation of caspase 8.
Toxicology in Vitro 12/2010; 25(3):630-5. · 2.78 Impact Factor
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ABSTRACT: We would like to add a note of concern to our manuscript published last year in Autophagy (Huang Q, Shen H-M. To die or to live: The dual role of poly(ADP-ribose) polymerase-1 in autophagy and necrosis under oxidative stress and DNA damage. Autophagy 2009; 5:273-6). Our concern is due to the retraction of the related article, to which our article was an addendum:: : Huang Q, Wu YT, Tan HL, Ong CN, Shen HM. A novel function of poly(ADP-ribose) polymerase-1 in modulation of autophagy and necrosis under oxidative stress. Cell Death Differ 2009; 16:264-77: : The decision for the retraction is based on the discovery of some technical errors in the data, although we are still confident about the overall validity of the conclusion of the study. : Qing Huang and Han-Ming Shen.
Autophagy 11/2010; 6(8):1232. · 7.45 Impact Factor
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ABSTRACT: Chrysin (5,7-dihydroxyflavone) is a natural flavonoid commonly found in many plants. The anti-cancer property of chrysin has been demonstrated although the molecular mechanisms remain to be further elucidated. In the present study, we found that, pretreatment with chrysin greatly sensitized various human cancer cells to tumor necrosis factor-alpha (TNFalpha)-induced apoptosis. In the search of the molecular mechanisms responsible for the sensitization effect of chrysin, we discovered that such sensitization is closely associated with the inhibitory effect of chrysin on TNFalpha-mediated nuclear transcription factor-kappaB (NF-kappaB) activation. Pretreatment with chrysin inhibited TNFalpha-induced degradation of Inhibitor of kappaB (IkappaB) protein and subsequent nuclear translocation of p65. As a result, chrysin suppressed the expression of NF-kappaB-targeted anti-apoptotic gene, c-FLIP-L. The role of c-FLIP-L was further confirmed by its ectopic expression, which significantly protected cell death induced by combined treatment with chrysin and TNFalpha. Data from this study thus reveal a novel function of chrysin and enhance the value of chrysin as an anti-cancer agent.
Cancer letters 07/2010; 293(1):109-16. · 4.86 Impact Factor
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ABSTRACT: A group of phosphoinositide 3-kinase (PI3K) inhibitors, such as 3-methyladenine (3-MA) and wortmannin, have been widely used as autophagy inhibitors based on their inhibitory effect on class III PI3K activity, which is known to be essential for induction of autophagy. In this study, we systematically examined and compared the effects of these two inhibitors on autophagy under both nutrient-rich and deprivation conditions. To our surprise, 3-MA is found to promote autophagy flux when treated under nutrient-rich conditions with a prolonged period of treatment, whereas it is still capable of suppressing starvation-induced autophagy. We first observed that there are marked increases of the autophagic markers in cells treated with 3-MA in full medium for a prolonged period of time (up to 9 h). Second, we provide convincing evidence that the increase of autophagic markers is the result of enhanced autophagic flux, not due to suppression of maturation of autophagosomes or lysosomal function. More importantly, we found that the autophagy promotion activity of 3-MA is due to its differential temporal effects on class I and class III PI3K; 3-MA blocks class I PI3K persistently, whereas its suppressive effect on class III PI3K is transient. Because 3-MA has been widely used as an autophagy inhibitor in the literature, understanding the dual role of 3-MA in autophagy thus suggests that caution should be exercised in the application of 3-MA in autophagy study.
Journal of Biological Chemistry 04/2010; 285(14):10850-61. · 4.77 Impact Factor
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[hide abstract]
ABSTRACT: A group of phosphoinositide 3-kinase (PI3K) inhibitors, such as 3-methyladenine (3-MA) and wortmannin, have been widely used
as autophagy inhibitors based on their inhibitory effect on class III PI3K activity, which is known to be essential for induction
of autophagy. In this study, we systematically examined and compared the effects of these two inhibitors on autophagy under
both nutrient-rich and deprivation conditions. To our surprise, 3-MA is found to promote autophagy flux when treated under
nutrient-rich conditions with a prolonged period of treatment, whereas it is still capable of suppressing starvation-induced
autophagy. We first observed that there are marked increases of the autophagic markers in cells treated with 3-MA in full
medium for a prolonged period of time (up to 9 h). Second, we provide convincing evidence that the increase of autophagic
markers is the result of enhanced autophagic flux, not due to suppression of maturation of autophagosomes or lysosomal function.
More importantly, we found that the autophagy promotion activity of 3-MA is due to its differential temporal effects on class
I and class III PI3K; 3-MA blocks class I PI3K persistently, whereas its suppressive effect on class III PI3K is transient.
Because 3-MA has been widely used as an autophagy inhibitor in the literature, understanding the dual role of 3-MA in autophagy
thus suggests that caution should be exercised in the application of 3-MA in autophagy study.
Journal of Biological Chemistry 04/2010; 285(14):10850-10861. · 4.77 Impact Factor
