Autophagy Reduces Acute Ethanol-Induced Hepatotoxicity and Steatosis in Mice Wen-Xing Ding 1,2, Min Li 1, Xiaoyun Chen 1, Hong-Min Ni 1,2, Chie-Wen Lin 1, Wentao Gao 1,Binfeng Lu 3, Donna B Stolz 4, Dahn L. Clemens 5, and Xiao-Ming Yin 1,*1Department of Pathology, University of Pittsburgh School of Medicine, PA 152612Department of Pharmacology, Toxicology and Therapeutics, the University of Kansas Medical Center, Kansas City, Kansas 661603Department of Immunology, University of Pittsburgh School of Medicine, PA 152614Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, PA 152615Department of Internal Medicine, University of Nebraska, and VAMC, Omaha, NE 68105Abstract Background & Aims—Alcohol abuse is a major cause of liver injury. The pathologic features of alcoholic liver disease develop over prolonged periods, yet the cellular defense mechanisms
against the detrimental effects of alcohol are not well understood. We investigated whether
macroautophagy, an evolutionarily conserved cellular mechanism that is commonly activated in
response to stress, could protect liver cells from ethanol toxicity.
Methods—Mice were acutely given ethanol by gavage. The effects of ethanol on primary
hepatocytes and hepatic cell lines were studied in vitro.
Results—Ethanol-induced macroautophagy in the livers of mice and cultured cells required
ethanol metabolism, generated reactive oxygen species, and inhibited mTOR signaling.
Suppression of macroautophagy with pharmacological agents or small interfering RNAs
significantly increased hepatocyte apoptosis and liver injury; macroautophagy therefore protected
cells from the toxic effects of ethanol. Macroautophagy induced by ethanol seemed to be selective
for cells with damaged mitochondria and accumulated lipid droplets, but not long-lived proteins,
? 2010 The American Gastroenterological Association. Published by Elsevier Inc. All rights reserved.
*Corresponding author: 412-648-8436 (phone), 412-648-9564 (fax), xmyin@https://www.doczj.com/doc/222142613.html,.
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Contribution of each author
W-XD: study concept and design; data acquisition, analysis and interpretation; manuscript drafting.
ML, XC, H-MN, C-WL, WG, BL, DBS: technical and/or materials support
DLC: material support, manuscript review
X-MY: funding, study concept, design and supervision, data analysis and interpretation, manuscript writing
NIH Public Access
Author Manuscript
Gastroenterology . Author manuscript; available in PMC 2014 August 12.
Published in final edited form as:
Gastroenterology . 2010 November ; 139(5): 1740–1752. doi:10.1053/j.gastro.2010.07.041.NIH-PA Author Manuscript
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which could account for its protective effects. Increasing macroautophagy pharmacologically
reduced hepatotoxicity and steatosis associated with acute ethanol exposure.
Conclusions—Macroautophagy protects against ethanol-induced toxicity in livers of mice.Reagents that modify macroautophagy might be developed as therapeutics for patients with alcoholic liver disease.Keywords GFP-LC3; bodipy staining; autophagy flux; long-lived protein degradation Introduction Alcohol is widely consumed around the world. While a moderate use of alcohol may be beneficial to human health, excessive drinking, whether in a binge or chronic fashion, can lead to considerable health concerns. Binge drinking can cause acute glycogen depletion,hypoglycemia and acidosis in addition to behavioral and psychological changes 1. At the cellular level, binge drinking results in mitochondria damage, generation of free radicals,inhibition of insulin signaling, and steatosis, although cell death is usually minor unless other susceptibility factors exist 2–7. In chronic ethanol abuse, the histological manifestation of the liver injury can progress from steatosis to focal inflammation, focal necrosis, fibrosis and cirrhosis, resulting in alcoholic liver disease 1, 2, 5.Ethanol pathogenesis is contributed by multiple factors 1, 2, 5, 7, 8. Early events such as mitochondrial damage, ROS generation and steatosis seem to be the direct outcome of ethanol metabolism, which are common features of acute and chronic alcohol exposure.
While greater efforts have been spent on the understanding of these mechanisms, little is
known about the cellular protective mechanism against the detrimental effects of ethanol.
We hypothesized that a cellular protective response to ethanol exposure is critical to the
control of ethanol-induced liver pathology.
Among the potential protective mechanisms, we investigated whether macroautophagy
could play a role in limiting ethanol-induced liver injury. Macroautophagy (hereafter
referred to as autophagy) is a bulk intracellular degradation system responsible for the
degradation of macromolecules and subcellular organelles 9. Autophagy involves the formation of double-membraned autophagosomes, which envelop the substrates and fuse
with the lysosomes for degradation. More than thirty Atg genes have been defined in yeast
that participate in autophagy, many of which have mammalian homologues 9. The functions
of Atg8/LC3, Atg7, and Atg6/Beclin 1 are among the best characterized in mammalian cells.
The Atg6/Beclin 1 forms a complex with the Class III phosphoinositide 3-kinase (PI3K)
complex, VPS34 and VPS15, which leads to autophagy specific generation of
phosphatidylinositol-3-phosphate (PtdIns3P). PtdIns3P is required for a series of events that
ultimately contributes to the conjugation of Atg8/LC3 to phosphatidylethanolamine (PE) at
the autophagosomal membranes, which also requires Atg7. PE-conjugated Atg8/LC3, also
known as LC3-II (in contrast to the unlipidated LC3-I), is important in autophagosome
formation 9.
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Autophagy is now known to be widely involved in the pathogenesis of many diseases and is
activated under a variety of stress conditions. Autophagy seems to constitute an effective cellular defense system against multiple pathological insults. Thus, we investigated whether autophagy is activated in response to ethanol exposure and whether it plays a key role in mitigating ethanol-induced pathology. Using an ethanol binge model and primary
hepatocyte culture in which the ethanol-induced early pathological events are well defined,we determined that ethanol could induce a selective autophagy process in primary
hepatocytes dependent on its metabolism, ROS production and mTOR inhibition.Furthermore, autophagy protects hepatocytes against the detrimental effects of ethanol likely by removing damaged mitochondria and accumulated lipid droplets.Materials and Methods Animals experiments Wild type C57BL/6 mice and GFP-LC3 transgenic mice 10 were used in this study. All animals received humane care. All procedures were approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh.Ethanol binge was conducted as previously described 11. This model was designed to achieve blood alcohol levels, behavioral effects, and physiological effects comparable to human binge drinking. After 6 hours of fasting, male mice were given 33% (v/v) ethanol at a total accumulative dosage of 4.5 g/kg body weight by four equally divided gavages in 20-minute intervals. Control mice received the same volume of water. For autophagy inhibition or induction, chloroquine (CQ, 60 mg/kg) or rapamycin (2 mg/kg) were given (i.p.) to the mice 30 minutes before the administration of ethanol. Mice were analyzed 16 hours later. For
inhibition of Atg7 expression in murine livers, siRNA (see supplemental information) was
injected (0.7 nmol/g) through the tail vein using the hydrodynamic technique. Forty-eight
hours later, the mice were subjected to the ethanol treatment.
Cell culture
Murine hepatocytes were isolated and cultured as previously described 12. After initial
culture in William’s medium E with 10% FCS for attachment, cells were cultured in serum-
free William’s medium E overnight before treatment. HepG2 and VL-17A cells 13 were
maintained in DMEM with 10% FCS and other standard supplements. Ethanol and the
following chemicals were applied as indicated in the figure legends: 3-MA (10 mM), CQ
(10 μM), rapamycin (10 μM), 4-MP (4 mM), MnTBAP (1 mM) and NAC (20 mM).
Microscopic analysis
Cells (2 × 105/well in 12-well plate) were infected with adenoviral GFP-LC3 overnight or
loaded with MitoTracker Red (50 nM) for 15 minutes before other treatments. Apoptotic
cell death was determined by nuclear staining with Hoechst 33342 or TUNEL staining.
Cryosection of GFP-LC3 transgenic livers were performed as previously described 10.
Sections were stained with Bodipy 581/591-C11 (1 μM) or Bodipy 493/503 (0.1 μM) for 15
minutes before analysis. All fluorescence images were digitally acquired with an Olympus
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Fluoview 1000 confocal microscope. Electron microscopy was conducted with standard
procedures using a JEM 1016CX electron microscope.
Biochemical analysis Serum ALT level was determined using a kit from Biotron Diagnostics (Hemet, CA).Hepatic triglyceride was determined as in reference 14. Caspase-3 activity was determined using Ac-DEVD-AFC as the substrate 12. For inhibition of Beclin 1 expression in VL-17A cells, siRNA (see supplemental information) were transfected into VL-17A cells at the concentration of 0.12 μM per 1×106 cells using Oligofectamine. Cells were subjected to other treatment 36 hours later. Densitometry of LC3-II and p62 bands was conducted and the values were normalized to the loading control (β-actin or LAMP1) and then converted to relative units to the untreated control. Autophagy flux was conducted as described in Table S1. Long-lived proteins degradation assay was performed as described previously 9, 15.Statistical analysis Quantitative data are subjected to one way ANOVA with Holm-Sidak analysis, Student’s t test or z test where appropriate using SigmaStat 3.5 (Systat Software, Inc., San Jose, CA),Results Acute ethanol exposure induced autophagy Activation of autophagy by ethanol was first examined in GFP-LC3 transgenic mice 10.Binge ethanol treatment induced a significant elevation of GFP-LC3 puncta in the liver (Fig.1A–B), which represented autophagosomes. Immunoblot analysis confirmed the increase of
the membrane-associated PE-conjugated form of GFP-LC3 (GFP-LC3-II) and the
endogenous LC3 (LC3-II) in ethanol-treated livers (Fig. 1C–D). The lipidated LC3 was
particularly enriched in the LAMP1-positive heavy membrane fraction, where the lysosome
was located, reflecting its destination as the autophagosome fused with the lysosome to form
the degradative autolysosomes. Importantly, electron microscopic (EM) analysis indicated
an apparent accumulation of autophagosomes following ethanol treatment (Fig. 1E–F),
which could be suppressed by siRNA-mediated knockdown of Atg7 (see below).
Pre-treatment of mice with the lysosome-inhibitor chloroquine (CQ) further increased the
levels of GFP-LC3 puncta (Fig. 1A–B), the LC3-II form (Fig. 1D), and the number of
autophagosomes (Fig. 1F), suggesting that at least a portion of autophagosomes resulted
from this elevation was degraded in the lysosome. The appearance of the single GFP moiety
(Fig. 1C), due to the breakdown of GFP-LC3, and the reduction of p62/SQSTM1 (Fig. 1D),
a selective autophagy adaptor molecule that brings ubiquitinated autophagy substrates to
autophagosomes for degradation together 9, 16, in the ethanol-treated livers would also
support an enhanced autophagic degradation by ethanol. Indeed, detailed flux analysis
(Table S1) indicated that there was not only an apparent increase in autophagosome
synthesis, but also an increased autophagic degradation based on changes in GFP-LC3 dots
(Fig. 1B), autophagosome number (Fig. 1F), and LC3-II level in the heavy membranes (Fig.
1D). Such analysis with LC3-II in the total lysates still indicated an increase in synthesis, but
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not in degradation, perhaps due to a lower enrichment of LC3-II in the total lysates than in
the heavy membranes as mentioned above, which reduced the sensitivity of the assay.We next examined the effect of ethanol on cultured primary hepatocytes. Consistent with the in vivo observations, ethanol treatment in vitro also caused a dose-dependent increase in GFP-LC3 puncta (Fig. 2A–B), LC3-II formation (Fig. 2C), and the formation of more and larger autophagosomes (Fig. S1). LC3 punctation was suppressed by 3-MA (Fig. 2A, D), an inhibitor of the Class III PI3K, VPS34. Conversely, these changes could be enhanced by the co-treatment with CQ (Fig. 2A, E). Flux analysis (Table S1) indicated an increased autophagosome synthesis based on both GFP-LC3 puncta and LC3-II levels, although the increased degradation was only evident based on the level of GFP-LC3 puncta, but not based on the LC3-II level in the total lysate, likely due to the same reason discussed above.To further look into the ability of ethanol to promote autophagy degradation, we examined whether it could affect the clearance of long-lived proteins, commonly associated with the general non-selective autophagy 9. Markedly, ethanol did not inhibit long-lived protein degradation, nor did it promote such a degradation in hepatocytes, at either basal level or under starvation (Fig. 2F). This suggested that ethanol-induced elevation in autophagosomal markers and autophagosomes were not due to the suppression of a general autophagic degradation. On the other hand, the lack of enhanced long-lived protein degradation,coupled with a significant reduction of p62/SQSTM1 (Fig. 1D), led us to suspect that ethanol might induce mainly a selective autophagy process that targeted to organelles rather than long-lived proteins (see below).Ethanol induced autophagy via metabolic intermediates, ROS and mTOR inhibition
Most ethanol-mediated effects depend on its metabolism. The first step of ethanol
oxidization is catalyzed mainly by alcohol dehydrogenase (ADH), and to a lesser extent, by
cytochrome P450 2E1 (CYP2E1) and catalase. Ethanol oxidation results in the generation of
ROS, particularly by the later two enzymatic reactions. The production of ROS is considered
a major factor in ethanol-induced pathologies 1, 3, 7. We found that ethanol-induced GFP-
LC3 puncta was significantly diminished in the presence of 4-methyl pyrazole (4-MP), an
inhibitor of both ADH and CYP2E1, or MnTBAP or N-acetylcysteine (NAC), two
antioxidants (Fig. 3A), indicating that ethanol-induced autophagy required ethanol
metabolism and ROS production. To further confirm this requirement, we used HepG2, a
human hepatoma cell line that has a very weak capability of metabolizing ethanol, and
VL-17A, a cell line derived from HepG2, which over-expresses both ADH and CYP2E1 and
efficiently metabolizes ethanol 13. We found that ethanol treatment significantly increased
the number of GFP-LC3 puncta in VL-17A cells, but not in HepG2 cells (Fig. 3B). This
effect could be suppressed by 3-MA (Fig. 3C), but further enhanced in the presence of
lysosomal inhibitors (data not shown). In addition, the levels of endogenous LC3-II and
GFP-LC3-II, as well as the level of free GFP moiety, were all increased in VL-17A cells,
but not in HepG2 cells (Fig. 3D). Thus, ethanol-induced autophagy required ethanol
metabolism and ROS production in hepatocytes.
The mTOR pathway is a major regulatory pathway suppressing the activation of autophagy.
We found that in primary hepatocytes ethanol caused a dose-dependent inhibition of mTOR,
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as manifested by the reduction of both phosphorylated p70 S6 kinase and 4E-BP1, two major downstream targets of mTOR (Fig. 3E). Interestingly, the total levels of these molecules were also reduced, perhaps reflecting an outcome of mTOR inhibition in protein
synthesis. Suppression of mTOR was also observed in the livers of ethanol-treated mice
(data not shown). Notably, the presence of 4-MP or NAC could reverse the inhibition of
mTOR by ethanol (Fig. 3F). Taken together, these findings supported the notion that ethanol
could induce autophagy by inhibiting mTOR activity via ROS.
Inhibition of autophagy enhanced ethanol-induced hepatocyte apoptosis and liver injury
Ethanol is able to induce hepatocyte apoptosis and liver injury, which, however, is
minor 17, 18, 19. Autophagy could be a major protective mechanism limiting ethanol toxicity.
Indeed, while only about 15% of hepatocytes were apoptotic when treated with ethanol
alone, 3-MA co-treatment almost doubled the percentage of cells undergoing apoptosis (Fig.
4A–B). An even stronger enhancement of apoptosis and caspase activation by 3-MA or by a
Beclin 1-specifiic siRNA could be observed in ethanol-treated VL-17A cells (Fig. 4C–F,
S2). On the other hand, ethanol did not seem to noticeably promote necrosis in this model,
although autophagy could have an independent effect (Fig. S2).
Furthermore, pretreatment of mice with CQ significantly enhanced ethanol-induced liver
injury as measured by blood ALT level, hepatic caspase-3 activity and TUNEL staining
(Fig. 5A–B, S3). To confirm the specificity of CQ-enhanced liver injury related to
autophagy, we pretreated mice with a specific siRNA against Atg7, which effectively
knocked down the liver Atg7 expression (Fig. 5C). Subsequent ethanol binge exposure
resulted in a reduction in LC3-II level and autophagosome formation (Fig. 5C–D), enhanced
elevation of blood ALT and caspase activation in the liver (Fig. 5E–F). Overall, while
ethanol alone caused about a 3–5-fold increase in blood ALT levels, suppression of
autophagy during ethanol treatment led to an additional 3-fold or more increase in blood
ALT levels (Fig. 5A, E), despite that gross parenchymal alterations an cell death were
minimal (Figs. S4–S5). These findings thus indicated that autophagy played an active role in
limiting ethanol-induced cellular injury.
Ethanol-induced autophagy was selective for the mitochondria and lipid droplets
To understand the mechanisms by which autophagy protected against ethanol-induced
hepatocyte injury, we examined the nature of the autophagy induced by ethanol. Studies
presented above had suggested that ethanol could mainly induce selective autophagy (Fig.
1D, 2F). We observed by EM that a notable fraction of ethanol-induced autophagosomes in
the liver contained mitochondria, which was further increased by pre-treatment with CQ
(Fig. 6A). These mitophagic vesicles were particularly obvious in primary hepatocytes
cultured in the presence of ethanol (Fig. 6B). The engulfed mitochondria seemed to be
damaged (Fig. 6A), and appeared to be much smaller than the nonengulfed mitochondria
(Fig. 6B), suggesting that they could have been fragmented.
In untreated primary hepatocytes infected with adenoviral-GFP-LC3 and loaded with
MitoTracker Red (MTR)20, there were very few GFP-LC3 punctated structures and the
mitochondria exhibited normal spherical morphology (Fig. 6C, panels a–c ). Ethanol NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
treatment greatly increased GFP-LC3 positive punctated structures, and some of them were
co-localized with the MTR signals (Fig. 6C). More importantly, mitochondria engulfed by
the GFP-LC3 rings appeared smaller than the nonengulfed mitochondria, implying that they
might be fragmented, consistent with the EM analysis (Fig. 6B). Quantitative analysis
indicated that the number of GFP-LC3 puncta colocalized with MTR in ethanol-treated
hepatocytes was increased in a dose-dependent manner (Fig. 6D), and could be further
enhanced by the presence of CQ (Fig. 6C, E). Indeed, flux analysis indicated that both the
synthesis and the degradation of the mitochondria-containing autophagosomes were
significantly increased by ethanol treatment (Fig. 6A, 6E, Table S1). Together these
observations indicated that ethanol promoted autophagic degradation of mitochondria, or
mitophagy. Furthermore, this process could be suppressed by 4-MP or NAC (Fig. 6F),
supporting the role of ethanol metabolism and ROS in promoting mitophagy.Another significant feature of ethanol-induced liver pathology is microvesicular steatosis 1, 2, 5, which is considered pathogenic. By using the lipid-binding compound,Bodipy 581/591-C11 (Fig. 7A) or Bodipy 493/503 (Fig. S6), we confirmed the accumulation of lipids in the livers of mice given ethanol binge treatment. In addition, GFP-LC3 puncta or
ring-like structures were found to colocalize with Bodipy-positive lipid droplets (LDs) (Fig.
7A), suggesting the activation of lipophagy, or autophagic removal of lipid droplets. While
LDs that were completely encircled by GFP-LC3 rings (Pattern I) were rare, LDs that were
in contact with one (Pattern II) or more (Pattern III) GFP-LC3 dots were much more
common (Fig. 7B).
To determine whether autophagy was functionally involved in the control of lipid
homeostasis in the liver following ethanol treatment, we pre-treated mice with rapamycin
and found that this dramatically reduced the number of LDs in the ethanol-treated livers
based on Bodipy staining (Fig. 7C–D) and EM analysis (Fig. 7E), and the microvesicular
changes in hepatocytes (Fig. S4). Conversely, blockage of lysosomal function with CQ
significantly increased the number of LDs (Fig. 7C–D) and the histological alterations (Fig.
S4). As the result, the total hepatic triglyceride levels that were elevated due to ethanol
treatment were further increased by CQ pre-treatment, but was greatly reduced by
rapamycin pre-treatment (Fig. 7D-c). We confirmed the specificity of the effects of these
pharmacological modulations in terms of autophagy regulation by knocking down Atg7 in
the liver. These mice exhibited a greatly reduced number of autophagosomes (Fig. 5E), a
significantly increased number of LDs (Fig. 7F) and increased microvesicular changes (Fig.
S5) following ethanol binge treatment. Consistent with this, the levels of hepatic
triglycerides were significantly increased (Fig. 7F). These findings indicated that removal of
lipid droplets by a selective autophagy process played an important role in mitigating
ethanol-induced hepatic steatosis and therefore the injury 2, 5, 8.Discussion
Acute ethanol treatment activates a selective autophagy process
The present study demonstrates that acute ethanol exposure strongly induced
autophagosome synthesis and promoted a selective autophagy process to removed damaged
mitochondria and hepatic lipids droplets. The evidence for increased synthesis is based on
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the increase in the autophagosome number and size, and the positive changes in autophagosomal marker, LC3. The increased level of GFP-LC3 puncta was suppressed by 3-MA, and the formation of LC3-II and the increase in autophagosome number were inhibited by Atg7 siRNA, indicating that these events were induced in response to upstream autophagy signals.Several lines of evidence also supported that ethanol treatment resulted in increased, rather than decreased, autophagic degradation through the lysosome. Ethanol did not inhibit general autophagy at the basal state or under starvation in hepatocytes (Fig 2F) or in VL-17A cells (Fig. S7). Ethanol-promoted formation of LC3-II and LC3 puncta, and the generation of autophagosomes were all further enhanced by CQ co-treatment. Although not without its own limitations, flux analysis (Table S1) confirmed elevated autophagy degradation in the presence of ethanol based on LC3 puncta, LC3-II formation in the heavy membrane fraction and autophagosome number. Notably, when a more specific set of autophagosomes that were associated with mitochondria was analyzed for this purpose, the ethanol-promoted degradation was even more evident.This last observation suggested that ethanol could particularly activate autophagy targeting to specific substrates, i.e., a selective autophagy process. Consistently, ethanol did not promote long-lived protein degradation, commonly seen in non-selective general autophagy (Fig. 2F, S7). In contrast, ethanol did enhance the degradation of p62/SQSTM1 (Fig. 1D), a key adaptor molecule associated with selective autophagy 16. p62/SQSTM1 binds to both LC3 and ubiquitinated autophagic substrates so that they could be taken up by the autophagosome for degradation.Activation or deactivation of selective autophagy may not affect the degradation of targets
of the non-selective autophagy, such as the long-lived proteins 21. Subcellular organelles are
the major targets of selective autophagy 16, which can be activated in response to the changes
in these organelles. Notably, a major target of ethanol toxicity is the mitochondria.
Functional and structural alternations of mitochondria have been observed in hepatocytes
following acute or chronic ethanol treatment 3, 22. Damaged mitochondria could trigger, and
become targets of, autophagy. Indeed we found that ethanol treatment induced significant
autophagic engulfment of damaged and fragmented mitochondria based on both
fluorescence and electron microscopy (Fig. 6), indicating the activation of mitophagy.
Another subcellular organelle that would be subjective to selective autophagy is lipid
droplets, which are significantly elevated in ethanol-treated livers. Autophagy was recently
found to be important in controlling lipid accumulation in hepatocytes subject to abnormal
lipid metabolism conditions 23. We demonstrated the existence of selective autophagic
removal of lipid droplets (lipophagy), primarily based on the double staining of LC3 and
Bodipy (Fig. 7A–B), the quantification of lipid droplets and hepatic triglyceride levels (Fig.
7D–F), and the histological observations of the microvesicular changes in the liver (Figs.
S4–S5) under a variety of conditions that modulated autophagy, including pharmacological
and molecular interventions. Direct observations of lipid droplets being completely engulfed
by classical autophagosomes were infrequent with electron microscopy. This may be due to
the fact that the large size of lipid droplets could only allow partial engulfment by the
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autophagosomes 23. Indeed, several patterns of autophagosome/lipid droplet interactions
could be observed as previously described 23. The dynamics of the interaction between lipid
droplets and autophagosome membranes will certainly be an interesting area to explore in
future studies.
Taken together, our studies provide strong evidence that acute ethanol exposure activates a
selective autophagy toward damaged mitochondria and accumulated lipids droplets and
promoted their degradation through the lysosome.
Ethanol metabolism and ROS are required for ethanol-induced autophagy
It is generally accepted that ethanol metabolism is required for ethanol-induced
pathology 1, 2. We found that ethanol-induced autophagy was dependent on ethanol
metabolism as it could be suppressed by 4-MP, an inhibitor of ADH and CYP2E1.
Consistently, ethanol-induced autophagy was only observed in VL-17A cells, which are
reconstituted with ADH and CYP2E1, but not in the parental HepG2 cells that lack these
enzymes and are unable to efficiently metabolize ethanol.
ROS generated by ethanol treatment is a major cause of autophagy induction, as autophagy
could be inhibited by the antioxidants NAC and MnTBAP (Fig. 3). Ethanol-induced ROS
may come from a number of sources, among which ethanol oxidation by CYP2E1 is a major
one 7. In addition, oxidation of acetaldehyde in the mitochondria results in increases of
NADH and its re-oxidation can also contribute to acute ethanol-induced mitochondrial ROS
generation 3. Furthermore, repeated ethanol binge could result in significant alterations in
mitochondrial DNA synthesis, respiration disturbance and ROS generation 22. It will be
informative to further determine the individual importance of these events in autophagy
induction in future studies, since they may constitute additional regulatory checkpoints.
Oxidative stress has been associated with the induction of autophagy in other scenarios, in
which the mitochondria were suspected of being the source of ROS 24, 25. How ROS trigger autophagy is not entirely clear. One possibility is that ROS impair the Akt/mTOR
pathway 25. mTOR is a well defined negative regulator of autophagy 26. Our findings support
that acute ethanol exposure induced autophagy via ROS-dependent inhibition of mTOR
activity. An earlier study did find that acute ethanol administration inhibited Akt activation 6.
Akt is a major upstream activator of mTOR. Thus reduced Akt activity could lead to a
reduced mTOR activity, activating autophagy 26. Our work, however, does not rule out that
ROS can induce autophagy via other mechanisms, which should be explored in the future.
Autophagy could mitigate ethanol-induced hepatotoxicity by removing damaged
mitochondria and accumulated fatty acids
Ethanol-induced cell death in vivo is minor, resulting in a modest increase in blood ALT in
most models of acute or chronic treatment without obvious gross histological changes unless
other susceptibility factors exist (Figs. 5, S4–S5)2, 4, 17–19, 27. Our findings indicated that
autophagy establishes an important threshold for ethanol-induced damage because
suppressing autophagy significantly increased hepatocyte apoptosis. It seems that functional
autophagy in the liver is important to avert the pathological effects of ethanol metabolism
and that without this protective mechanism the detrimental effects of alcohol could be
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significantly amplified. The general molecular mechanisms underlying the pro-survival role
of autophagy are not fully understood, but our studies suggest that the autophagy protection
during acute ethanol treatment could be mediated by the removal of damaged mitochondria
and lipid droplets. Autophagic degradation of mitochondria is well recognized in
hepatocytes during nutrient deprivation and glucagon treatment 28. However, mitophagy may
be more likely involved in constraining detrimental effects associated with the damaged
mitochondria 28. A critical benefit of mitophagy is the elimination of an important source of
ROS, which is associated with the susceptibility of hepatocytes to cellular injury and
death 2, 3, 5, 22, 28. Thus, it is conceivable that autophagic degradation of damaged
mitochondria is a part of the protection mechanism against ethanol toxicity.
Steatosis is a common feature in acute or chronic ethanol treatment 1, 2, 5. Although
reversible upon ethanol withdrawal, hepatocyte steatosis is now considered important for
subsequent pathological changes in ALD, such as cell death, inflammation and fibrosis 5, 8.
Our study now demonstrated that autophagy had a great impact on ethanol-induced steatosis.
We thus propose that lipophagy is an integral part of the autophagy defense system against
ethanol-induced liver injury. A key component of the cellular damage caused by ROS is
lipid peroxidation, which can damage cellular membranes and produce lipid derivatives of
the radicals, such as 4-hydroxynonenal and malondialdehyde, which in turn can mediate
secondary insults to promote more ROS production and inflammation 1, 2, 5, 7, 8. The level of
cellular polyunsaturated fatty acids affects the level of lipid peroxidation and is likely
proportional to the level of lipid droplets (containing triglycerides) accumulated in the cell
through the equilibrium of hydrolysis of triglycerides and esterification of the free fatty
acids with glycerol. Thus, lipophagy may indirectly reduce the level of free fatty acids by
removing lipid droplets.
In summary, this study demonstrates that autophagy protects against ethanol-induced
hepatocyte apoptosis and liver injury. This protection could be mediated via the removal of
damaged mitochondria and accumulated fatty acids, thus concomitantly reducing a major
source of ROS and a major target/amplifier of ROS. These findings imply that a proper
autophagy capability in the liver may be crucial to reduce the detrimental effects of ethanol
consumption and that enhancement of autophagy may be a possible therapeutic strategy to
mitigate the pathology associated with alcoholic liver disease.Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
The authors would like to thank Dr. N. Mizushima (Tokyo Medical and Dental University, Japan) for the GFP-LC3
transgenic mice and Ms M. Sun (University of Pittsburgh) for technical help in EM.
Grant Support
Supported in part by NIH (X.-M. Y.: R01 CA83817, R01 CA111456; D. L. C.: R01 AA11291, W.-X. D.:R21
AA017421) and Dept. VA (D. L. C).
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Abbreviations
3-MA
3-methyladenine 4-MP
4-methyl pyrazole ADH
alcohol dehydrogenase CYP2E1
cytochrome P450 2E1CQ
chloroquine EM
electron microscopy LD
lipid droplets MTR
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Figure 1. Acute ethanol exposure induces autophagy in the liver
(A–C). GFP-LC3 transgenic mice (n=3–5) were treated as indicated and the liver sections
were analyzed by confocal microscopy (A). The number of GFP-LC3 dots (mean+SEM)
were quantified from each animal (B). The total lysates (TL) and the heavy membrane
fraction (HM) of the liver were analyzed by immunoblot assay (C). (D ) Wild type mice were
treated as indicated (n=3) and the liver fractions were analyzed by immunoblot assay.
Densitometry analysis of the LC3-II and p62 was performed for each sample (mean+SEM).
(E ). Wild type liver samples were processed for EM. Arrows denote autophagosomes. N:
nucleus, LD: lipid droplet. (F ). The number of autophagosomes per given area (mean+SEM)
was determined (n=3–4). Scale bar: 20 μm (A), 2 μm (E). *: p<0.05.
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Figure 2. Ethanol induces autophagy in hepatocytes in vitro
(A–C ). Ad-GFP-LC3 infected hepatocytes were treated as indicated (80 mM of ethanol in
A) for 6 hours and examined by confocal microscopy (A) or immunoblot assay (C). Scale
bar: 20 μm. GFP-LC3 dots (mean+SEM) were quantified for each experiment (n=3). (D–E ).
Ad-GFP-LC3 infected hepatocytes were treated with ethanol (80 mM, D, 40 mM, E–F) with
or without 3-MA (D) or CQ (E, F) for 6 hours. GFP-LC3 dots (mean+SEM) were quantified
for each experiment (n=3). Total lysates were subjected to immunoblot assay and
densitometry analysis (mean+SEM) of the LC3-II was performed (n=3). (F ). Hepatocytes
were cultured in William’s medium/10% serum or in EBSS without or with ethanol for 15 hr
and long-lived protein degradation was determined (mean+SEM, n=2–3). *: p<0.001.
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Figure 3. Ethanol-induced autophagy requires ethanol metabolism, ROS and mTOR inhibition
(A ). Ad-GFP-LC3-infected hepatocytes were treated with ethanol (80 mM) with or without
4-MP, MnTBAP or NAC for 6 hours. GFP-LC3 dots (mean+SEM) were quantified from
each experiment (n=3). (B–D ). Ad-GFP-LC3 infected HepG2 (B, D) and VL-17A (B, C, D)
cells were treated with ethanol (40 mM unless indicated) and other agents for 24 hours.
GFP-LC3 dots per cell (mean+SEM) were quantified from each experiment (B, C, n=3) and
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immunoblot analysis conducted (D). (E–F). Primary hepatocytes were treated as indicated
for 6 hours and total lysate were subjected to immunoblot analysis. Scale bar: 10 μm (A–B).
*: p<0.05.
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帕金森病中的自噬途径与关键药物靶点 欧阳亮, 张岚, 刘博* (四川大学华西医院, 生物治疗国家重点实验室, 生物治疗协同创新中心, 四川成都 610041) 摘要: 帕金森病 (PD) 是一种常见的神经退行性疾病。在过去几十年中, 对PD的发病机制的探索已有了较大的进步, 环境因素和遗传因素都会导致PD的发生, 然而它的具体发病机制仍然未知。最近的研究表明自噬过程或许与PD密切相关, 在许多PD患者和动物模型中都观察到了异常的自噬水平。此外, 一些PD相关蛋白, 如α-synuclein、Parkin和PINK1等都被发现参与自噬的调控, 被认为与PD的发病机制相关。本文综述了几种重要PD相关蛋白在自噬途径中的作用, 同时概述了通过调节自噬过程来治疗PD的潜在策略。 关键词: 自噬; 帕金森病; α-突触核蛋白; 线粒体自噬; 帕金森病治疗 中图分类号: R966 文献标识码:A 文章编号: 0513-4870 (2016) 01-0009-09 Autophagy pathways and key drug targets in Parkinson’s disease OUYANG Liang, ZHANG Lan, LIU Bo* (State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, and Collaborative Innovation Center of Biotherapy, Chengdu 610041, China) Abstract: Parkinson’s disease (PD) is a common neurodegenerative disorder associated with aging. Great progresses have been made toward understanding the pathogenesis over the past decades. It seems that both genetic factors and environmental factors contribute to PD, while the precise pathogenesis still remains unknown. Recently, increasing evidence has suggested that autophagy dysregulation is closely related to PD. Dysregula-tion of the autophagic pathways has been observed in the brains of PD patients or in animal models of PD, and a number of PD-associated proteins, such as α-synuclein, Parkin and PINK1, were found to involve in autophagy, suggesting a link between autophagy and pathogenesis of PD. In this review, we summarized the role of PD-associated proteins in autophagy pathways. In addition, we described the efficacy of autophagy-modulating compounds in PD models and discussed promising strategies for PD therapy. Key words: autophagy; Parkinson’s disease; α-synuclein; mitophagy; Parkinson’s disease therapy 1 帕金森病概述 帕金森病 (Parkinson’s disease, PD) 是居于阿兹海默病后的第2位最常见的神经退行性疾病, 其主要病理学特征是在黑质中多巴胺能神经元细胞死亡, 黑质纹状体通路退化[1]。此外, 受损的多巴胺能神经元胞浆内存在着路易小体 (Lewy body, LB), 收稿日期: 2015-08-11; 修回日期: 2015-10-14. 基金项目: 国家自然科学基金资助项目 (81473091, 81260628). *通讯作者 Tel / Fax: 86-28-85503817, E-mail: liubo2400@https://www.doczj.com/doc/222142613.html, DOI: 10.16438/j.0513-4870.2015-0706 其内主要包含异常的或者聚集体形式的α-突触核蛋白(α-synuclein)[2]。脑内产生多巴胺的细胞逐渐丧失了影响神经系统的功能, 使患者控制肌肉的能力受限。在临床上, PD具有一些核心运动症状, 统称为震颤性麻痹, 包括静止性震颤、运动迟缓、肌强直、姿势不稳和步态障碍。此外, 临床描述的PD还包括几种非运动性症状, 如执行功能障碍、自主神经系统功能障碍、睡眠障碍、行为和精神方面的改变以及嗅觉障碍等[2?4]。据2013年美国国立PD基金会统计, 全世界有400万～600万名PD患者, 在工业化国家中流
中国组织化学与细胞化学杂志 CHINESE JOURNAL OF HISTOCHEMISTRY AND CYTOCHEMISTRY 第29卷第2期2020年4月 V ol.29.No.2April.2020 〔收稿日期〕2020-01-06 〔修回日期〕2020-04-09 〔基金项目〕国家重点研究计划“重大慢性非传染性 疾病防控研究”重点专项2018年度定向项目子课题（2018YFC1311300）；湖北省第二届医学领军人才工程第二层次基金（[2019]47） 〔作者简介〕高利昆，女（1983年），汉族，在读博士 *通讯作者(To whom correspondence should be addressed)：dr_hongli@https://www.doczj.com/doc/222142613.html, 细胞自噬与肿瘤治疗的研究进展 高利昆1，袁静萍2，洪莉1* （武汉大学人民医院1妇产科，2病理科，武汉430060） 〔摘要〕自噬是一种高度保守的、存在于真核细胞内的自我降解机制，自噬水平异常会破坏细胞内稳态。自噬在肿瘤中既能抑制早期肿瘤发生又可促进肿瘤发展，尤其是其对多种肿瘤治疗有细胞毒性、细胞保护性的双重影响，如何调控自噬用于肿瘤治疗及化疗增敏已成为肿瘤防治领域的研究热点。在本综述中对自噬在肿瘤致病及进展过程中的具体作用展开了总结，重点讨论了自噬对肿瘤治疗的影响及当前靶向自噬改善肿瘤治疗的各种新策略。 〔关键词〕自噬；肿瘤治疗；化疗耐药 〔中图分类号〕R736.1 〔文献标识码〕A DOI ：10.16705/ j. cnki. 1004-1850. 2020. 02. 015 Recent progress in autophagy and tumor therapy Gao Likun 1, Yuan Jingping 2, Hong Li 1* (1Department of Obstetrics and Gynecology, 2Department of Pathology, Renmin Hospital of Wuhan University, Wuhan 430060, China) 〔Abstract 〕 Autophagy is a highly conservative self-degradation mechanism that exists in eukaryotic cells. Abnormal levels of autophagy will destroy intracellular homeostasis. Autophagy can not only suppress early tumorigenesis but also promote tumor devel-opment, especially it has dual effects of cytotoxicity and cytoprotection on various tumor treatments. How to regulate autophagy for tumor treatment and chemotherapy sensitization has become research hotspots in the field of cancer prevention and treatment. In this review, the specific role of autophagy in the pathogenesis and progression of tumors is summarized, focusing on the impact of autoph -agy on tumor treatment and the current various new strategies targeted on autophagy to improve tumor treatment. 〔Keywords 〕Autophagy; tumor therapy; chemoresistance 自噬(autophagy)是真核细胞内发生的一种高度保守的自我降解机制[1]。生理状态下，基础活性下的自噬会降解并清除受损或死亡细胞器、错配蛋白质等物质，维持细胞内稳态[2]，自噬异常则会打破细胞原有平衡而导致肿瘤发生。自噬可分为3种类型：巨自噬（macroautophagy ）、微自噬（microau-tophagy ）及分子伴侣介导的自噬(chaperone-mediated autophagy ，CMA)。巨自噬涉及到特有的双层膜囊泡即自噬体的形成，可捕获自噬相关货物并进一步将货物传送至溶酶体形成自噬溶酶体[1]。微自噬不涉及胞浆内双层膜囊泡的形成，而是由溶酶体内陷或 突出直接吞噬细胞内物质[3]。分子伴侣介导的自噬的特征则是通过热休克蛋白家族与位于溶酶体膜上的溶酶体相关膜蛋白2（LAMP-2A ）共同作用于胞浆内物质后将其转运至溶酶体内发生降解[4]。本文中所讨论的自噬即为巨自噬，目前了解最多。1 自噬发生过程与调控 自噬的发生是一个动态发展的连续过程，主要包括双层膜结构的起始阶段、延伸阶段、成熟降解阶段从而完成自噬体形成融合到溶酶体，进一步形成自噬溶酶体，降解其内包裹的物质并释放得以重新利用[1]。迄今为止发现，自噬全过程由超过30个自噬相关基因（autophagy related gene, ATG ）及相应蛋白严格调控[5]。依据功能和生理上的相互作用机制， ATG 蛋白参与形成五个分子复合体[5]：①ULK1复合体：由ULK1/ULK2、ATG13/101、FIP200组成，该复合体的形成及活化启动自噬的发生。②ATG9复合体：唯一的整体跨膜核心蛋白，促进磷酸的转运。③PI3KC3复合体：包括将PI 转化为PI-3磷酸盐（PI3P ）的催化亚单位真空蛋白34（VPS34）、beclin1和囊泡运输因子p115与ATG14联结一起组
细胞自噬和肿瘤 摘要：近年来，细胞自噬与肿瘤的关系是研究热点。研究表明，自噬是肿瘤的双刃剑；自噬调节剂与细胞毒性药物联用在临床应用上具有很大潜力。对于细胞自噬和癌症发生之间关系还需要更加深入的研究，这将会有助于人类更好地认识并最终攻克癌症。本文将针对细胞自噬在肿瘤发生过程的作用、主要的信号调节通路以及其在治疗中的作用进行简单介绍。 关键词：细胞自噬、肿瘤、信号调节通路、治疗 一、细胞自噬和肿瘤的关系 细胞自噬(autophagy)是指真核生物中的一些受损或衰老的蛋白质以及细胞器被双层膜结构的自噬小泡包裹后，送入溶酶体中进行降解并得以循环利用的过程。细胞自噬过程大致分为4个阶段:首先，细胞浆中出现游离前自噬泡，诱导起始与成核；前自噬泡包裹受损或衰老的蛋白质以及细胞器，逐渐发展成为由双层膜结构形成的自噬泡；自噬泡与溶酶体膜融合后，内膜及其包裹的物质进入溶酶体，被其中的酶水解；最后，溶酶体的物质分解为细胞生存所必需的组成成分，底物降解再循环。 肿瘤发生的机制很复杂，主要表现为细胞过度增殖，具有高代谢的特点，对营养和能量的需求很高；另外肿瘤细胞在恶劣的细胞外环境下（如代谢压力、缺氧、缺乏血供、养分及生长因子不足、抗肿瘤治疗等）具有很强的凋亡抵抗能力，逆境生存的能力极强。这些都有赖于肿瘤细胞的自噬活性。 正常生理情况下，细胞自噬能够及时清除细胞中产生的受损或衰老的蛋白质以及细胞器等，有利于细胞保持自稳状态。细胞自噬可以抑制细胞发生癌变，首先，细胞自噬可以清除坏死的细胞器，调整内源性的压力，从而稳定基因组，减少细胞向癌细胞的转变；其次，细胞自噬既可以加强细胞检验点的检查作用，又起到了稳定基因组的作用，从而减少了细胞的癌变。然而肿瘤一旦形成，细胞自噬又会为癌细胞提供更丰富的营养，促进肿瘤生长。不过，目前大多数研究者还是认为，细胞自噬是一种对抗细胞癌变的机制。(见图1)。[1] 图1、细胞自噬在肿瘤发生和发展中的角色 二、检测细胞自噬与细胞癌变的一般方法
自噬与肿瘤的关系及抗肿瘤药物 张庆余任皓刘洁朱润芝 广东医学院附属医院肝胆外科研究室 湛江市肝胆相关疾病重点实验室 摘要：自噬是将细胞内受损、变性或衰老的蛋白质以及细胞器运输到溶酶体进行消化降解的过程.正常生理情况下，细胞自噬利于细胞保持自稳状态;在发生应激时，细胞自噬防止有毒或致癌的损伤蛋白质和细胞器的累积，抑制细胞癌变;然而肿瘤一旦形成，细胞自噬为癌细胞提供更丰富的营养，促进肿瘤生长.因此，在肿瘤发生发展的过程中，细胞自噬的作用具有两面性.对于细胞自噬和肿瘤发生之间的关系有待深入的研究，这将会有助于人类更好地认识并最终攻克肿瘤。 关键词：肿瘤，自噬，抗肿瘤药物 Abstract:Autophagy is the major intracellular degradation system by which cytoplasmic materials (denatured protein, damaged organelles) are delivered to and degraded in the lysosome to maintain homeostasis. Once carcinogenesis rising up, autophagy would be also employed by cancer cells. Autophagy plays an important role in cancer cells– both in protecting against cancer as well as potentially contributing to the growth of cancer. However, autophagy can also contribute to cancer by promoting survival of tumor cells that have been starved. The relationship between autophagy and tumorigenesis need to be further researched, which will help humanity better understand and ultimately overcome the cancers. Keywords: Tumor, Autophagy, Anti-tumor drug 1 细胞自噬的概念 1.1 自噬的基本概念 传统的细胞死亡方式分类包括细胞坏死和细胞凋亡2种，后者又称为程序性细胞死亡，而新近的研究发现，除坏死和凋亡之外还存在其他细胞死亡方式，例如自噬[1] 。 细胞自噬是真核生物中进化保守的对细胞内物质进行周转的重要过程。该过程中一些损坏的蛋白或细胞器被双层膜结构的自噬小泡包裹后，送入溶酶体(动物）或液泡（酵母和植物）中进行降解并得以循环利用。细胞自噬最早由Ashford和Ponen于1962年用电子显微镜在人的肝细胞中观察到，是细胞中初级溶酶体处理内源性底物的重要过程，同时参与维持蛋白代谢平衡及细胞内环境的稳定，其在清除废物、结构重建以及细胞生长发育中起重要作用[2]。自噬存在于真核细胞的病理生理过程中，近年随着对自噬研究的不断深入，其在肿瘤中的作用日渐引起广泛关注。目前研究表明自噬功能异常在肿瘤发生、发展中均扮演重要角色[3] 。在生物进化中，细胞自噬是一种保守的过程，从酵母到植物细胞再到哺乳动物，都存在这样的过程，并且其中的很多调节因子在多个生物种中都能找到其同源体[4]。 1.2 与自噬相关的重要细胞器——溶酶体 损坏的蛋白或细胞器在体内降解主要有泛素-蛋自酶体途径（UPS）和溶酶体途径（细胞自噬）。UPS和自噬在降解过程、机制、亚细胞定位、降解底物、系统活性等方面均有区别，细胞自噬是UPS受损时的代偿途径，细胞自噬可以在UPS抑制时激活；细胞自噬的长期抑制可导致UPS功能受损，但UPS不能代偿细胞自噬被抑制的功能[5] 。本文主要讨论溶酶体途径。 自噬是细胞对持续性内外刺激的非损伤性应答反应，以维持细胞结构、代谢和功能的平
Vol.5 No.1Feb. 2019生物化工Biological Chemical Engineering 第 5 卷 第 1 期 2019 年 2 月肿瘤的自噬机制及自噬相关治疗 司新红１，高睿１，商雨婷１，李若谨１，王澈２＊ （１．辽宁师范大学化学化工学院，辽宁大连 １１６０２９；２．辽宁省生物技术与分子药物研发重点实验室，辽宁大连 １１６０８１） 摘?要： 自噬是一种细胞自我降解的过程。当细胞处于应激状态下受损时，细胞启动自噬来清除受损的蛋白质和细胞器，细胞依赖自噬促进细胞内成分的再利用，维持代谢稳态与细胞生长。越来越多的研究表明，自噬在肿瘤细胞中也具有非常重要的作用，一方面适度的自噬可以使受损的肿瘤细胞存活，另一方面过度的自噬则会加速肿瘤细胞的死亡。阐明肿瘤细胞中的自噬机制有助于发现新的抗肿瘤分子靶点，进而开发出有效的基于自噬机制的抗肿瘤药物。本文通过阐述自噬在肿瘤细胞中的发生机制来探讨自噬对肿瘤细胞生存的影响，并进一步探讨自噬在肿瘤治疗中的意义。 关键词： 自噬；肿瘤细胞；肿瘤治疗中图分类号：Ｒ７３０．５ 文献标志码：Ａ Ｔｕｍｏｒ Ａｕｔｏｐｈａｇｙ Ｍｅｃｈａｎｉｓｍ ａｎｄ Ａｕｔｏｐｈａｇｙ Ｒｅｌａｔｅｄ Ｔｒｅａｔｍｅｎｔ Ｓｉ Ｘｉｎ－ｈｏｎｇ１，Ｇａｏ Ｒｕｉ１，ＳｈａｎｇＹｕ－ｔｉｎｇ１，Ｌｉ Ｒｕｏ－ｊｉｎ１，Ｗａｎｇ Ｃｈｅ２＊ （１．Ｓｃｈｏｏｌ ｏｆ Ｃｈｅｍｉｓｔｒｙ ａｎｄ Ｃｈｅｍｉｃａｌ Ｅｎｇｉｎｅｅｒｉｎｇ， Ｌｉａｏｎｉｎｇ Ｎｏｒｍａｌ Ｕｎｉｖｅｒｓｉｔｙ，Ｌｉａｏｎｉｎｇ Ｄａｌｉａｎ １１６０２９； 2.Liaonin ｇ Ｋｅｙ Ｌａｂｏｒａｔｏｒｙ ｏｆ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ ａｎｄ Ｍｏｌｅｃｕｌａｒ Ｄｒｕｇ Ｄｅｖｅｌｏｐｍｅｎｔ， Ｌｉａｏｎｉｎｇ Ｄａｌｉａｎ １１６０８１） Abstract: Ａｕｔｏｐｈａｇｙ ｉｓ ａ ｐｒｏｃｅｓｓ ｏｆ ｃｅｌｌ ｓｅｌｆ－ｄｅｇｒａｄａｔｉｏｎ． Ｗｈｅｎ ｃｅｌｌｓ ａｒｅ ｄａｍａｇｅｄ， ａｕｔｏｐｈａｇｙ ｉｓ ｉｎｉｔｉａｔｅｄ ｔｏ ｒｅｍｏｖｅ ｄａｍａｇｅｄ ｐｒｏｔｅｉｎｓ ａｎｄ ｏｒｇａｎｅｌｌｅｓ． Ｃｅｌｌｓ ｒｅｌｙ ｏｎ ａｕｔｏｐｈａｇｙ ｔｏ ｐｒｏｍｏｔｅ ｔｈｅ ｒｅｕｓｅ ｏｆ ｉｎｔｒａｃｅｌｌｕｌａｒ ｃｏｍｐｏｎｅｎｔｓ， ｍａｉｎｔａｉｎｉｎｇ ｍｅｔａｂｏｌｉｃ ｈｏｍｅｏｓｔａｓｉｓ ａｎｄ ｓｕｒｖｉｖａｌ ｏｆ ｃｅｌｌｓ； Ｉｎｃｒｅａｓｉｎｇ ｌｉｎｅｓ ｏｆ ｅｖｉｄｅｎｃｅ ｈａｖｅ ｓｈｏｗｎ ｔｈａｔ ａｕｔｏｐｈａｇｙ ａｌｓｏ ｐｌａｙｓ ｉｍｐｏｒｔａｎｔ ｒｏｌｅｓ ｉｎ ｃａｎｃｅｒ ｃｅｌｌｓ． Ｏｎ ｔｈｅ ｏｎｅ ｈａｎｄ， ｍｏｄｅｒａｔｅ ａｕｔｏｐｈａｇｙ ｃａｎ ｒｅｓｃｕｅ ｔｈｅ ｄａｍａｇｅｄ ｃａｎｃｅｒ ｃｅｌｌｓ ｔｏ ｓｕｒｖｉｖｅ．Ｏｎ ｔｈｅ ｏｔｈｅｒ ｈａｎｄ， ｅｘｃｅｓｓｉｖｅ ａｕｔｏｐｈａｇｙ ａｃｃｅｌｅｒａｔｅｓ ｃａｎｃｅｒ ｃｅｌｌｓ ｄｅａｔｈ． Ｅｌｕｃｉｄａｔｉｎｇ ｔｈｅ ｍｏｌｅｃｕｌａｒ ｍｅｃｈａｎｉｓｍｓ ｏｆ ａｕｔｏｐｈａｇｙ ｉｎ ｃａｎｃｅｒ ｃｅｌｌｓ ｈｅｌｐｓ ｕｓ ｄｉｓｃｏｖｅｒ ｎｅｗ ｍｏｌｅｃｕｌａｒ ｔａｒｇｅｔｓ ａｎｄ ｄｅｖｅｌｏｐ ｅｆｆｅｃｔｉｖｅ ａｕｔｏｐｈａｇｙ－ｂａｓｅｄ ａｎｔｉ－ｃａｎｃｅｒ ｄｒｕｇｓ． Ｉｎ ｔｈｉｓ ｒｅｖｉｅｗ， ｗｅ ｓｕｍｍａｒｉｚｅｄ ｔｈｅ ｍｏｌｅｃｕｌａｒ ｍｅｃｈａｎｉｓｍｓ ｏｆ ａｕｔｏｐｈａｇｙ ｉｎ ｃａｎｃｅｒ ｃｅｌｌｓ， ａｎｄ ｆｕｒｔｈｅｒ ｄｉｓｃｕｓｓｅｄ ｔｈｅ ｓｉｇｎｉｆｉｃａｎｃｅ ｏｆ ａｕｔｏｐｈａｇｙ ｉｎ ｃａｎｃｅｒ ｔｈｅｒａｐｙ． Keywords: Autophagy; Cancer cells; Cancer therapy １ 细胞自噬的分子机制 细胞自噬是进化过程中高度保守的自我调节机 制，可分为巨自噬（或大自噬，Macroautophagy）、微 自噬（或小自噬，Microautophagy）和分子伴侣介导 的自噬（Chaperone-mediated autophagy）。巨自噬通 过形成具有双层膜结构的自噬体（autophagosome）， 包裹细胞质中的物质并与溶酶体结合形成自噬溶酶 体，继而在自噬溶酶体中将物质降解，最终将降解后的物质用于细胞生存和功能维持；微自噬是通过溶酶体或液泡表面直接吞噬特定的细胞器将其降解；分子伴侣介导的自噬则是通过分子伴侣将胞浆蛋白导入溶酶体内腔进行降解的过程[1-2]。巨自噬与微自噬通常是非选择性的过程，而分子伴侣自噬为选择性降解过程。在分子伴侣自噬过程中，热休克蛋白（HSC70）特异性识别具有KFERQ 基序的底物，只有被识别之后的这一类底物才可以被运送至溶酶体进行降解[3]。 司新红（1993—），女，河南郑州人，硕士，研究方向：有机化学。 王澈（1977—），女，吉林长春人，博士，教授，研究方向：生物技术与分子药物研发。E-mail: wangche@https://www.doczj.com/doc/222142613.html,。文章编号：2096-0387（2019）01-0153-03
---------------------------------------------------------------最新资料推荐------------------------------------------------------ 自噬与肿瘤的关系及抗肿瘤药物.doc 自噬与肿瘤的关系及抗肿瘤药物摘要： 自噬是将细胞内受损、变性或衰老的蛋白质以及细胞器运输到溶酶体进行消化降解的过程.正常生理情况下，细胞自噬利于细胞保持自稳状态；在发生应激时，细胞自噬防止有毒或致癌的损伤蛋白质和细胞器的累积，抑制细胞癌变；然而肿瘤一旦形成，细胞自噬为癌细胞提供更丰富的营养，促进肿瘤生长.因此，在肿瘤发生发展的过程中，细胞自噬的作用具有两面性.对于细胞自噬和肿瘤发生之间的关系有待深入的研究，这将会有助于人类更好地认识并最终攻克肿瘤。 关键词： 肿瘤，自噬，抗肿瘤药物 Abstract： Autophagy is the major intracellular degradation system by which cytoplasmic materials （denatured protein， damaged organelles） are delivered to and degraded in the lysosome to maintain homeostasis. Once carcinogenesis rising up，autophagy would be also employed by cancer cells. Autophagy plays an important role in cancer cells? both in protecting against cancer as well as potentially contributing to the growth of cancer. However ， autophagy can also contribute to cancer by promoting survival of tumor cells that have been starved. The relationship between autophagy and tumorigenesis 1 / 16
收稿日期:2006205226 基金项目:国家自然科学基金资助项目(30400531)作者简介:温莹浩(19772),女,江西萍乡人,第二军医大学东方肝胆外科医院硕士生,从事肿瘤基因治疗研究. 自噬与肿瘤 温莹浩,殷正丰 (第二军医大学东方肝胆外科医院分子肿瘤实验室,上海200438) 关键词:肿瘤;自噬;癌基因;基因,抑制,肿瘤 中图分类号:R 730 文献标识码:A 文章编号:100121692(2006)0520482205 细胞稳态依赖于大分子物质生物合成和分解代谢间的平衡。自噬(au tophagy )即是细胞用来达到这种动态平衡的重要机制之一,它是普遍存在于大部分真核细胞中的一种现象,从酵母到人类存在着共同的自噬分子调控机制。 真核细胞的降解主要有两条途径,即蛋白酶降解和自噬,但只有自噬具有降解整个细胞器的效力。自噬是大分子物质和细胞器在溶酶体腔发生的降解途径。自噬的作用主要是清除降解细胞内受损伤的细胞结构、衰老的细胞器,以及不再需要的生物大分子等,同时也为细胞内细胞器的构建提供原料,即细胞结构的再循环。因此,自噬是细胞成分更新、发育、分化及组织重塑的重要调控机制。自噬也与包括肿瘤在内的多种人类疾病有关。有趣的是,自噬能够保护细胞,也能造成细胞损伤。1 自噬的作用及意义 “au tophagy ”源于希腊词语,其语源是“phagy ”=吃与“au to ”=自我,因此自噬是一动态过程。自噬始于包裹膜包裹胞内大分子物质和细胞器后形成具有双层或多层膜的液泡即自噬体(au top hago som e )或自噬液泡(au top hagic vacuo le )。包裹膜可能源于粗面内质网的无核糖区、反面高尔基体网络或其它有膜囊泡体。自噬体膜相关蛋白的缺乏是从酵母到哺乳细胞的共同特征,故难以确定自噬体膜的起源。对大鼠纯化自噬体的分析显示,自噬体膜富集三种也存在于胞液中的蛋白:精氨琥珀酸合酶、32磷酸甘油醛脱氢酶N 2端截断变异体和短链22烯酰CoA 水合酶变异体,但其在自噬体形成中的作用尚未明确[1]。自噬体形成后,通过与溶酶体融合形成自噬性溶酶体(au 2top hago lyso som e ),完成对包裹物质的降解和再循环 利用。 自噬主要有三种作用[2]:(1)自噬是对营养缺乏的一种适应性反应,如氨基酸缺乏在肝脏和培养细胞中可以诱导自噬。自噬对大分子物质的降解能够产生调节新陈代谢和生物合成的氨基酸及其它因子,为细胞提供能量,维持细胞的生命活动。氨基酸是自噬的重要调节因子,缺失氨基酸在肝脏中迅速诱导自噬,最初可观察到自噬体,经历7～8分钟后降解液泡出现。从自噬体的形成到转化成残余体这一完整自噬过程约需20分钟,营养缺乏的肝细胞中自噬途径对胞质蛋白这种非选择性的降解率为3%～4% 小时。(2)自噬能够调节过氧化物酶体、线粒体、内质网的更新,故自噬被认为是一种参与细胞质稳态的看家机制,在许多病理生理和应激刺激状况 下可观察到对这些细胞器选择性的分隔。 (3)自噬参与特定的组织特异性功能。自噬与肺泡表面活性剂 生物合成有关。黑质多巴胺能神经元神经黑色素的生物合成应归于胞质多巴胺2苯醌被分隔入自噬液泡。在红细胞成熟过程中,细胞核排出后需要自噬清除核糖体、线粒体等细胞器。2 肿瘤细胞中自噬的作用 自噬是肿瘤细胞对外源性应激具有不同结果的反应,多项证据表明自噬在肿瘤发生中同时具有促进和抑制作用。 首先,自噬是保护肿瘤细胞避免受到低营养、电离辐射和治疗损伤所诱导的应激的一种保护机制。在肿瘤进展过程中,由于血供减少,肿瘤细胞特别是肿瘤内部细胞在乏氧和营养受限的状况下生存[3]。有趣的是,乏氧和营养缺失均能刺激自噬启动,通过自噬对胞内物质的降解和循环利用而改变新陈代谢。肿瘤细胞对应激作出适应性反应得以生存,同时,自噬对线粒体的分隔可防止促凋亡因子如细胞 色素和凋亡诱导因子(A IF )的扩散。自噬的这种作用能够帮助细胞逃逸凋亡。乳腺、结肠和前列腺癌细
细胞自噬与肿瘤治疗 施晓莹 摘要：细胞自噬是真核生物的一种进化保守的新陈代谢的过程，双层膜结构的自噬小泡包裹损坏的蛋白或细胞器，将其送入动物体的溶酶体或植物体和酵母的液泡中进行降解，使之得以循环利用，来维持细胞内的稳态。现在有研究表明，细胞自噬对肿瘤发生的不同阶段具有促进和抑制的双重作用，可通过对自噬的深入研究研制出治疗肿瘤的药物。 关键字：自噬肿瘤治疗 前言：细胞自噬是高度保守的自我消化和细胞存活维持稳态的过程。本文将对细胞自噬的过程、种类及功能进行简单介绍，并概述目前细胞自噬对治疗肿瘤的研究进展。随着环境的不断恶化和人类对自身健康的忽视，患癌率在不断上升，研制出有效治疗肿瘤的药物已迫在眉睫，探究细胞自噬如何抑制肿瘤的发生是目前科学界最感兴趣的项目之一，对人类今后的生命发展有着重要意义。 细胞自噬（autophagy）是真核生物的一种进化保守的新陈代谢的过程，双层膜结构的自噬小泡包裹损坏的蛋白或细胞器，将其送入动物体的溶酶体或植物体和酵母的液泡中进行降解，使降解产物氨基酸、核苷酸、游离脂肪酸等可得以循环利用，来维持细胞内环境的动态平衡。通常，寿命较短的蛋白质如调控蛋白等通过泛素-蛋白酶体系统进行降解；而寿命较长的蛋白质及细胞结构则通过细胞自噬途径，由溶酶体进行降解。 1.细胞自噬的过程 1.1膜泡的形成 当外界有辐射、低氧、饥饿、高温等因素或细胞内部发生应激反应如遇细胞器损坏或突变蛋白等均会引诱细胞发生自噬，此时来自内质网或细胞质中的双层膜形成膜泡，即前自噬体。 1.2自噬体的形成 膜泡募集自噬蛋白进入到吞噬泡成核中心，泛素化耦联系统对其进行辅助, 募集 Atg3、Atg7、Atg10、Atg8/LC3Atg4到前自噬体结构，使囊泡膜逐渐扩展，包裹细胞内待降解的细胞器或其他细胞内含物，然后闭合形成自噬体。 1.3自噬溶酶体的融合 自噬体酸化与细胞骨架微管系统相互作用，并与溶酶体融合形成自噬溶酶体。 1.4自噬体的降解 溶酶体内的一些酶参与水解自噬体，自噬体膜的受体蛋白能够降解错误折叠的蛋白质多聚体，还能够降解功能失常的整个线粒体、过氧化物体、高尔基体等细胞器，甚至可以清除细胞内的病原体，并且还能提供营养物质和 ATP给细胞再利用[1]。（见图一）