Life Sciences 188 (2017) 53–67

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Life Sciences
journal homepage: www.elsevier.com/locate/lifescie

Review article

Autophagy: The spotlight for cellular stress responses T

⁎,1 1
Palaniyandi Ravanan , Ida Florance Srikumar , Priti Talwar
Apoptosis and Cell Survival Research Laboratory, Department of Biosciences, School of Biosciences and Technology, VIT University, Vellore, Tamil Nadu-632014, India

A R T I C L E I N F O A B S T R A C T

Keywords: Autophagy is an essential cellular mechanism which plays “housekeeping” role in normal physiological pro-
Autophagy cesses including removing of long lived, aggregated and misfolded proteins, clearing damaged organelles,
Inflammation growth regulation and aging. Autophagy is also involved in a variety of biological functions like development,
Apoptosis cellular differentiation, defense against pathogens and nutritional starvation. The integration of autophagy into
NF-KB
these biological functions and other stress responses is determined by the transcriptional factors that undertake
HIFα
the regulatory mechanism. This review discusses the machinery of autophagy, the molecular web that connects
Cancer
Neurodegenerative diseases autophagy to various stress responses like inflammation, hypoxia, ER stress, and various other pathologic
Stress responses conditions. Defects in autophagy regulation play a central role in number of diseases, including neurodegen-
erative diseases, cancer, pathogen infection and metabolic diseases. Similarly, inhibiting autophagy would
contribute in the treatment of cancer. However, understanding the biology of autophagy regulation requires
pharmacologically active compounds which modulate the autophagy process. Inducers of autophagy are cur-
rently receiving considerable attention as autophagy upregulation may be a therapeutic benefit for certain
neurodegenerative diseases (via removal of protein aggregates) while the inhibitors are being investigated for
the treatment of cancers. Both induction and inhibition of autophagy have been proven to be beneficial in the
treatment of cancer. This dual role of autophagy in cancers is now getting uncovered by the advancement in the
research findings and development of effective autophagy modulators.

1. Presenting autophagy constitutively at basal rate under normal cellular conditions to maintain
homeostasis for intracellular recycling and metabolic regulation.
In order to maintain a continuous protein turnover, there exists a Autophagy is stimulated during various pathological and physiological
constitutive proteasomal degradation system of proteins within the cell. conditions including starvation. The cells adapt to these conditions by
But the system is known to be restricted to short-lived proteins present converting these signals into several anabolic and catabolic responses.
in the cell. Surprisingly, most of the cellular components are long lived. The cell catabolizes the damaged cellular components for the purpose
Therefore there must be an alternative recycling mechanism for the of generating substrates for sustaining adenosine triphosphate (ATP)
degradation of these macromolecules to promote adaptation and cell production during times of nutrient deprivation [152]. Such type of
survival under adverse conditions. And autophagy was known to be autophagy is called as starvation induced autophagy. The pathway in-
very promising in playing this alternative role. Autophagy occurs volves the formation of unique structures that sequesters the target

Abbreviations: HSC70, Heat Shock 70KDa protein; AMPK, AMP-activated protein kinase; ATG, autophagy-related genes; Beclin-1, coiled-coil, myosin-like BCL2-interacting protein; CQ,
chloroquine; DNA-PK, DNA-dependent protein kinase; FYVE, Fab1, YOTB, Vac1 and EEA1; GFP, green fluorescent protein; HCQ, hydroxychloroquine; LC3, Microtubule-associated
protein light chain 3; mTOR, Mammalian target of rapamycin; mTORC1, Mammalian target of rapamycin complex 1; RICTOR, raptor independent companion of mTOR; mSIN1,
mammalian stress-activated protein kinase interacting protein 1; PROTOR-1, protein observed with rictor-1; RAPTOR, regulatory associated protein of mTOR; MEF, mouse embryonic
fibroblasts; ZFYV1, zinc Finger FYVE-Type Containing 1; WIPI, WD-repeat PtdIns(3)P effector protein; FIP200, FAK family kinase-interacting protein of 200 kDa; PI3K, phosphoinositide
3-kinase; PIK3C3, phosphatidylinositol 3-kinase, catalytic subunit type 3; PI3K, phosphatidylinositol 3-kinase; ULK, Unc-51-like kinase; ULK1, Unc-51-like kinase 1; Vps34, vacuolar
protein sorting 34; UVRAG, UV resistance-associated gene; AMBRA 1, Autophagy And Beclin 1 Regulator 1; LC3, microtubule-associated protein 1A/1B-light chain 3; PE, phosphati-
dylethanolamine; BH3, Bcl2 homology domain; HS1BP3, HCLS1 binding protein 3; GABARAP, gamma-aminobutyric acid receptor-associated protein; SNARES, soluble NSF attachment
protein receptors; NF-κB, nuclear factor κB; PAMP, Pathogen-associated molecular patterns; MPT, mitochondrial permeability transition; PT pores, Permeability Transition pores; IP3R,
Inositol triphosphate Receptor; IRE1α, Inositol Requiring Enzyme 1α; ATF6, Activating Transcription Factor 6; PERK, protein kinase R-like ER Kinase; NF-KB, nuclear factor κ-light-chain-
enhancer of activated B cells; ]ZKSCAN3, zinc finger protein with KRAB and SCAN domains 3; TFEB, transcription factor EB; TNF, Tumor Necrosis Factor; IRF(3,7), interferon regulatory
factor; OPTN, Optineurin; TKB1, TANK binding kinase 1
⁎
Corresponding author.
E-mail address: [email protected] (P. Ravanan).
1
Equal contribution.

http://dx.doi.org/10.1016/j.lfs.2017.08.029
Received 29 June 2017; Received in revised form 5 August 2017; Accepted 28 August 2017
Available online 01 September 2017
0024-3205/ © 2017 Elsevier Inc. All rights reserved.
P. Ravanan et al. Life Sciences 188 (2017) 53–67

proteins (cargo), engulfs and delivers them into the lysosome for de- entire mechanism.
gradation. Based on the mode of delivery of cargo into the lysosome,
three forms of autophagy have been identified: microautophagy, cha- 2.1. Autophagosome formation
perone mediated autophagy and macroautophagy. Microautophagy is
the degradation process where the lysosomal membrane extends to Macroautophagy is initiated by the formation of autophagosome
invaginate the cellular contents. A direct relation of the number of whose role is to deliver the protein aggregates and other target mate-
microautophagic structures with the protein turnover was identified rials such as macromolecules or cell organelles to the lysosome for
using electron microscopy. The second form of autophagy is the cha- degradation. As description in a step by step manner would yield a
perone-mediated autophagy (CMA). This mechanism requires a KFERQ better understanding, the process of autophagosome formation itself is
sequence in proteins for degradation. The proteins having this con- divided into three successive stages- initiation, elongation and nuclea-
sensus sequence are recognized by HSC70, a chaperone protein that tion.
brings them to the lysosomal membrane. The proteins are then trans-
ported into the lysosome by a membrane receptor called LAMP-2A and 2.1.1. Initiation
are degraded in the lysosomal lumen. This process is highly specific as 2.1.1.1. Membrane source. Searching the membrane source for
the degradation is only for the proteins that have the consensus se- phagophore has revealed a strong connection with endoplasmic
quence. CMA controls the proteasomal degradation of MYC/c-Myc reticulum [255]. However, mitochondria [67], ER-Golgi intermediate
protein and regulate their cellular levels [60]. Macroautophagy is the compartment (ERGIC) [58], Golgi apparatus, recycling endosomes
third form of autophagy which involves the formation of special [164] and even plasma membrane [185] have also been proposed to
structures called autophagosomes to entrap the cellular contents and be the membrane source for autophagosomes. COPII vesicles generated
targeted proteins. The autophagosome then fuses with lysosome and from ERGIC serve as membrane template for the lipidation process
delivers its contents into the lumen for degradation. The mechanism is [58]. Sec 24, a COP II coat subunit interacts with Atg9 to regulate the
usually comprised of series of events with respect to the growth and formation of autophagosomes [241].
development of the autophagosome, its fusion with the lysosome and
eventually the degradation [47]. Among the three types, macro- 2.1.1.2. The mTOR complex. mTOR is a serine/threonine kinase that
autophagy is the major or prevalent pathway used by the cells in order forms 2 functionally different complexes: mTORC1 and mTORC2. The
to remove the damaged cellular organelles and other related debris and presence of few subunits differentiates mTORC1 and mTORC2
henceforth, the term “autophagy” indicates macroautophagy. structurally. mTORC2 comprises of subunits like mLST8, RICTOR,
Boya et al. has demonstrated that inhibition of macroautophagy mSIN 1 and PROTOR. And mTORC1 complex constituting subunits
either by chemical inhibitors or RNA interference leads to apoptotic like ULK1, Atg13, Atg101, FIP200 and RAPTOR is more sensitive to
type of cell death [20]. Research findings like systemic autophagy de- nutrient signals [59,78,254]. In most cases autophagy takes place in an
ficiency in mice die within a day after birth [115,118] and oocyte mTORC1 dependent manner through which all the stress signals are
specific autophagy deficiency results in lethality during early embry- integrated into the autophagy pathway [95,261]. Under such starving
ogenesis [231] clearly indicate the principal role of autophagy during conditions, mTORC1 gets inhibited to activate autophagy. And this
development and differentiation in lower eukaryotes as well as in autophagy induction in mammals through mTORC1 inhibition is said to
mammals. Autophagy has been shown to be an essential process for be conserved from yeasts [156].
efficient meiosis progression and proper meiotic chromosome segre-
gation in fission yeast [144]. Autophagy prevents the accumulation of 2.1.1.3. mTORC1. Atg13 and FIP200 interact with ULK1 at its C-
lipids in the liver and thereby lipotoxicity [218]. Upregulating autop- terminal region. Atg101 binds to ULK1 through the N- terminus of
hagy will not only facilitate the clearance of intracellular mutated Atg13 [75]. All these subunits are very crucial for the initiation of the
proteins but can also protect the neurons from apoptotic insults autophagy and the functioning of this complex as whole. The fact that
[89,163]. Hereby it is very clear that the upregulation of this self-eating ULK1 inhibition alone was able to inhibit autophagy, very clearly
mechanism serves as a pro-survival pathway against the factors like cell shapes out the role of ULK1 from ULK2 [26]. Similarly, the WF finger in
damage, hypoxic and starving conditions. Autophagy also plays helpful Atg13 is highly essential for recruiting DCFP1/ZFYV1 and WIPI1 (PI3P
roles in host-pathogen interactions, where autophagic vesicles help to binding proteins) to the isolation membrane [186].
engulf intracellular virus particles and bacteria, targeting these for ly-
sosomal destruction [126,215]. Recent reports have demonstrated that 2.1.1.4. Normal condition. Rag GTPase tethers mTORC1 to the
autophagy is crucial for bone growth [32], epidermal keratinization lysosome which contains v-ATPase essential for the kinase activity of
and hair growth [257], T cells and invariant natural T killer cells de- mTORC1 [265]. A regulator complex tethers Rag GTPase (which are
velopment [170,200], cartilage development [242], osteoblast and er- unbound themselves) to the lysosome [201]. At the lysosome, mTORC1
ythroid differentiation [21,99] and myogenic differentiation [217] and is activated by Rheb (an essential factor for mTORC1 activation)
genomic stability [102]. Besides eliminating the invading pathogens, [56,82,227]. Active mTORC1 phosphorylates ULK1 and Atg13
autophagy is also required for antigen presentation by MHCII [127]. through RAPTOR (regulatory molecule) to inactivate the kinase ULK1
Additionally, there are mounting reports that identified mutations in complex. RAPTOR binds to ULK1 to mediate the interaction of mTORC1
genes involved in autophagy in various diseases including neurode- with ULK1–mAtg13–FIP200 [78]. Thus, under normal condition, active
generative disease, infectious disease and cancer [90]. These essential mTORC1 remains associated with lysosome and activates a
roles played by autophagy and involvement in diseases have gained it a phosphorylation cascade leading to cell survival and proliferation.
lot of research interests to be viewed as a therapeutic target for the
treatment of various diseases. 2.1.1.5. Starving conditions. But under starving conditions, mTORC1
gets inhibited and the lysosome- mTORC1 association gets distorted.
2. The autophagy machinery The starving condition could be the shortage or unavailability of amino
acids, growth factor, oxygen and ATP and each of them inhibit mTOR in
The whole mechanism of autophagy takes place in a well-known different ways. The ULK1 complex gets dissociated from mTORC1 upon
series of steps (Fig. 1) namely, Autophagosome formation, Selection of its inhibition. This dissociation alone cannot activate ULK1 to initiate
cargo, Autophagolysosome formation and Degradation. All these steps autophagy. It requires an additional direct activation by AMPK. This
are very crucial for the autophagy process to take place successfully. activated complex then initiates the autophagosome formation. AMPK
Disruption of both earlier and later stages would equally affect the also induces autophagy by inhibiting mTORC1 via phosphorylation of

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P. Ravanan et al. Life Sciences 188 (2017) 53–67

Fig. 1. Schematic model of the core machinery of autophagy.

Autophagy takes place in a series of steps. The first step is the initiation where the ULK1 complex dissociates from the mTORC1 under starving conditions and becomes activated to initiate
the formation of the isolation membrane also known as phagophore. This is followed by the nucleation step that is driven by the PI3K complex. Here the phagophore almost encapsulates
the cargo to be degraded. The phagophore then elongates and seals encompassing the cargo. The elongation step is carried out by the Atg5-Atg 12-Atg16L and LC3II-PE conjugates. This
results in the formation of autophagosomes. The autophagosomes then fuse to the lysosomes to form autophagolysosomes. The cargo present within the autophagosome is then delivered
into the lysosomes where they get degraded by the lysosomal enzymes.

Rheb and RAPTOR [66]. Active ULK1 activates Atg13 and FIP200 by indicative of the contribution of the defective autophagy to the aging
phosphorylating them [54]. This ULK1 complex activation leads to the process.
initiation of a cup shaped structure called phagophore or isolation
membrane. ULK1 is found to phosphorylate beclin-1 of PI3K complex at 2.1.3. Elongation
Ser14 residue paving way for the progression of autophagy [198]. The entire elongation mechanism is undertaken by 2 ubiquitin like
conjugation system. One is the Atg5-Atg12 conjugating system and the
2.1.2. Nucleation other is the LC3- PE system. The Atg5-Atg12 conjugation system in-
The nucleation step is mainly carried out by VPS34 and Beclin-1 volves other members like Atg7 (E1 like enzyme), Atg10 (E2 like en-
complex. VPS34, the only enzyme to be assigned as class III phospho- zyme) and Atg16L1 and results in the formation of a trimeric or mul-
inositol kinase in mammals, whose solitary substrate is PI, generates a timeric complex Atg5-Atg12-Atg16L1. This complex adds to the proper
vital component called PI3P for the nucleation and growth of the curvature of the phagophore and is essential for the LC3 lipidation
phagophore [59]. VPS34 generates this PI3P by phosphorylating PI [165]. This complex also promotes the second ubiquitin like con-
(Phosphatidyl Inositol) at its D3 position. Beclin-1 extends its role be- jugating system LC3B-PE. The phosphatidic acid content of the autop-
yond this degradative pathway even unto pathways of endosome and hagosome precursor membranes that are Atg16L positive are regulated
cell death. But its role in autophagy is performed by it being a part of by HS1BP3, which thereby is proposed to regulate autophagy [73]. The
the VPS34 complex. AMBRA1 is phosphorylated by ULK1 after which it LC3-PE system comprises Atg4B (cysteine protease), Atg7 (E1 like en-
dissociates from the dynein motor complex and gets translocated to the zyme), Atg 3 (E2 like enzyme). This results in the formation of LC3B-I-
endoplasmic reticulum to bind Beclin-1 in the phagophore formation PE conjugate. This elongates and seals the phagophore to form a double
step [38]. The activity of VPS34- Beclin-1 complex is regulated in a membrane structure called autophagosome. The LC3B-II is then re-
Beclin-1 dependent manner by proteins like Barkor and UVRAG [224]. cruited to the inner and outer surface of the autophagosome in an Atg5-
Another member of the complex p150, that resembles VPS15 protein in Atg12 dependent manner. LC3B-II remains associated to the autopha-
yeast, is a typical VPS34 regulator. Its interaction with PI3K also in- gosomal membrane till the fusion with lysosome takes place. After this,
creases the lipid kinase specific activity [169]. Similarly, the WD re- the LC3B-II at the outer surface of the membrane gets dissociated while
peats in p150 are also essential for a variety of functions within a cell those that are associated with the membrane at the inner surface get
[154,168]. Sharing identity and similarity with the yeast VPS15, p150 degraded along with the cargo [226]. Absence of LC3 and GABARAP
is also expected to play similar roles in mammals and that again needs had no inhibitory effect on the autophagosome formation. But the size,
to be evidenced experimentally. A major depletion of B and T cell efficiency of formation and fusion with lysosome were affected [166].
precursors were observed under Beclin-1 deficient condition indicating
the essentiality of Beclin-1 for the development of immune cells [154]. 2.2. Selection of cargo
In the brain, the level of Beclin-1 drops with the increase in age and
hence low levels of VPS34-Beclin-1 complex formation. This is Besides the fact that autophagy is an indiscriminative degradation

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system, there are evidences on its selectivity of substrate. LC3B-II pre- factors by the immune cells. PPARα suppresses the activation of NF-κB
sent on the inner and outer surface of the autophagosomal membrane is (key player in the inflammatory pathway) by elevating the expression
proposed to interact with the constitutively expressed adaptor molecule levels of IκBα [262]. Growing body of evidences suggest that the ex-
P62/SQSTM1 that contains an ubiquitin binding domain to sequester pression level of IkBα decreases in the absence of PPARα. Similarly,
ubiquitinated proteins and LC3-interacting region (LIR domain) to de- PPAR ligands (Wy14,649 and clofibrate), were shown to inhibit NF-κB
liver sequestered proteins [59,261]. The transcriptional regulation of and increase IkBα levels thereby inhibiting inflammation indicating
p62 is controlled by a redox sensing factor NRF2 [18,84]. ALFY another that the pro-inflammatory genes of NF-κB can be targeted by these
ubiquitin binding protein binds to PI3P, p62 and Atg5 to secure the PPAR ligands [25]. Activation of PPARα inhibits Bile Acid-Induced
proteins present in the autophagosome [6]. NBR1, an ortholog of yeast apoptosis [229]. Jiao et al. demonstrated that PPARα inhibits the in-
Atg19, containing 2 LIR domain and an ubiquitin binding domain re- flammatory responses by promoting autophagy [92]. TNFα regulates
cruits protein aggregates in p62 independent manner [107]. the expression of PPARα. It was demonstrated that, the mRNA levels of
PPARα decreases with increasing concentration of TNFα in NF-κB de-
2.3. Formation of autophagolysosome pendent manner. This down regulation of PPARα is inhibited by the
suppression of NF-κB pathway, making obvious the PPARα- NF-κB's
Autophagosome fuses with early and late endosomes to form am- ability to trans‑repress each other's activity [131].
phisome which then is thought to fuse with the lysosome to form au- In addition, TBK1 (Tank Binding Kinase 1), belonging to the family
tolysosome. Similarly, formation of autolysosomes simply by the fusion of IKK kinases is involved both in neuroinflammation and autophagy. It
of autophagosome and lysosome is possible too [53]. PLEKHM1, a Rab7 is effectively involved in the phosphorylation of autophagy adaptors
effector protein regulates the fusion of autophagosome and lysosome and selection of cargo. It phosphorylates the LC3 binding site of opti-
through HOPS complex and LC3/GABARAP protein [146]. HOPS neurin (OPTN), an autophagy receptor and P62/SQSTM1 [191]. TBK1
complex mediates the fusion by interacting with the autophagosomal is also an important mitophagy (selective degradation of mitochondria)
SNARE, syntaxin 17 (STX17) as confirmed by the co-precipitation of player. The regulatory effect of TBK1 on the P62/SQSTM1 phosphor-
HOPS components, VPS33A and VPS16 along with STX17 [91]. The ylation mediated autophagic engulfment of polyubiquitinated mi-
process is also mediated by SNARES, membrane anchored proteins that tochondria was also experimentally demonstrated [145]. Mutation in
play a vital role in vesicular transport [85]. Microtubules are likely to the TBK1 gene has been found to contribute in Amyotrophic Lateral
facilitate the fusion process as well. Factors like MFN2 [264], pre- Sclerosis (ALS). ALS is a neurodegenerative disorder in which the motor
senilins [157], TECPR1 [29], myosin VI and Tom1 [233] and DRAM1 neurons that control the voluntary action get affected. The disease is
[263] are also required to promote the fusion of autophagosome and characterized by the cytoplasmic accumulation of protein and RNA
lysosome. aggregates in motor neurons [182]. In the neuroinflammatory pathway,
TBK1 gets activated via TLR3 AND TLR4 mediated TRAF3 activation.
3. Integration of autophagy into various stress responses Active TBK1 induces the formation of IRF-3/IRF-7 homodimers which
then translocate to the nucleus to act as transcription factors for IFNα
3.1. NF-κB links autophagy and inflammation and IFNβ and thus a pro-inflammatory and immune response. The TBK1
that is active in autophagy was not found to activate any of its down-
Mutations in the autophagy related genes were observed in in- stream targets of the neuroinflammatory pathway or innate immunity
flammatory disease like Crohn's disease and other inflammation asso- signaling. Activation of p62 and OPTN adaptor proteins requires in-
ciated metabolic diseases [128]. During inflammation, there occurs an teraction with TBK1 at its CCD2 domain which is mutated in most of the
over expression of NF-κB (nuclear factor κ-light-chain-enhancer of ac- ALS patients. Similarly, the impaired maturation and formation of au-
tivated B cells) at the inflamed sites which is induced by the tran- tophagosomes and autolysosomes in TBK1 knockdown cells is in-
scription of pro-inflammatory genes to produce cytokines like TNFα dicative of the role of TBK1 in autophagy [104,161].
and IL-1 [121]. Under normal condition, cells inactivate IKK, a major
regulator of NF-κB to prevent NF-κB from being constitutively activated 3.3. HIFα-NF-κB cross talk in hypoxia and autophagy
[223]. This regulatory function of IKK is responsible for it being tar-
geted for the treatment of various inflammatory diseases. Amino acid depletion, nutrient depletion and hypoxia are few stress
P62, the adaptor protein essential for the sequestration of cargo in signals that serve as stimuli for autophagy induction. In case of hypoxia,
the autophagy pathway, also functions to carry out the CD40 mediated HIF-1α and HIF-2α are the mediators of the hypoxic stress signal. The
activation of NF-κB by the recruitment of TNF-receptor-associated HIF-dependent induction of autophagy by hypoxia was also demon-
factors (TRAFs) to the TRAF binding sites in CD40 [207]. The expres- strated by Bellot et al. [15]. Upon an hypoxia mediated autophagy in-
sion of p62 in oxidative stress triggered autophagy was found to be duction, the HIF-1α knocked out cells showed a decrease in the levels of
dependent on NF-κB [220]. Chang et al. demonstrated that the silencing the well-known autophagy markers, beclin-1 and LC3B-II [37]. HIF
of p62/SQSTM1 resulted in the inhibition of NF-κB degradation [27]. activates autophagy through BNIP3 (Bcl-2/E1B 19 kDa-interacting
NF-κB triggers the production of pro-inflammatory cytokines upon ra- protein 3) to protect the cells from damage (Fig. 2). BNIP3 functions to
pamycin induced mTOR inhibition. This indicates that NF-κB is nega- release the Bcl-2 and Bcl-XL bound beclin-1 by competing with them
tively regulated by mTOR [245]. When Hsp90 (heat shock protein 90) [212,230]. As a response to infection, the transcriptional activation of
is inhibited, its substrates are targeted by autophagy for degradation. HIF-1α requires NF-κB (a key player in the inflammatory pathway)
Similarly, NIK, the HSP90 interacting protein which functions to acti- whose activity is usually high during hypoxia. In rheumatoid arthritis
vate IKKα, is also degraded by autophagy under geldanamycin induced synovial tissue (hypoxic), the inflammatory response is contributed by
disruption of HSP90-NIK interaction [187]. HIF-1α. The NF-κB, induced by HIF-1α under hypoxic conditions reg-
Autophagy is inhibited upon TNFα (tumor necrosis factor α) ulates the pro-inflammatory functions and survival of neutrophil, the
mediated activation of NF-κB [39,77]. The outcomes of many studies innate immune cell [167,238] (Fig. 2). Similarly, NF-κb also binds to
prove that autophagy is required for NF-κB activation and vice versa. the promoter region of HIF-1α gene and aid in the expression of HIF-1α
[19,61]. Unlike in the case of NF-κB, mTOR positively regulates HIF-1α,
3.2. The PPARα-NF-κB axis in autophagy and inflammation wherein it enhances the transcriptional activity of HIF-1α during hy-
poxic conditions [120]. It was also demonstrated that in response to
During anticancer therapy, the inflammatory signals need to be hypoxia, HIF-1α and p65, a subunit of NF-κB bind to the cycloox-
inhibited in order to decrease the release of cytokines and growth ygenase-2 promoter [48]. Hence it is very clear that there exists a

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Fig. 2. Interdependence of HIFα and NF- κB in the hypoxic in-

flammation and autophagy.
The HIFα and IKK mediate the inflammatory and hypoxic signals
and activate a cascade of signaling downstream them to activate
inflammation and autophagy as stress responses. NF-κB links au-
tophagy and inflammation via HIFα. mTOR inhibition activates
NF-κB in the inflammatory pathway while mTOR activation acti-
vates the transcriptional activity of HIFα.

strong interdependence between HIF-1α and NF-κB in the hypoxic in- undergoes death by turning on apoptosis. Depending on the type of
flammation and autophagy. stress sensed and the ability of the available adaptive mechanisms, the
cell decides to live or die. This topic of the review discusses about the
3.4. The PPARα-AMPK axis in autophagy and lipid metabolism interplay between autophagy, ER stress and apoptosis as an example to
explain how a cell manages a stress (shown in Fig. 3).
Lately, the ligands of PPARs (Peroxisome proliferator-activated re- The ER (Endoplasmic Reticulum) is the major site of protein
ceptors) are being considered as therapeutic targets for the treatment of synthesis. The proteins secreted by the ER are checked by various
cancer because of their low systemic toxicity and high potency in anti- mechanisms for their correct folding and assembly. When this ma-
inflammatory and anti-proliferative properties. PPARα is the nuclear chinery is overpowered, accumulation of misfolded and unfolded pro-
receptor that senses the fatty acids and transcriptionally activates their tein takes place. And now the cell is said to experience ER stress. When
β-oxidation. The activation of this process also involves AMPK (AMP the misfolded proteins accumulate in the ER upon ER stress, the un-
dependent protein kinase), a key player in the autophagy that functions folded proteins are translocated to the cytosol in order to be degraded
to sense the availability of AMP/ATP and activate pro-survival ma- by the proteasome. The ER attempts to remove these proteins by acti-
chinery like autophagy. PPARα regulates such non-autophagic roles of vating UPR system that constitutes series of functions initiated by stress
AMPK [63,256]. In autophagy, AMPK activates autophagy via TSC1/2 transducers like PERK, IRE1 and ATF6 [71,234]. But this adaptive re-
by inhibiting the Akt mediated mTOR activation. Chronic activation of sponse is limited for a short term stress [30,81]. When this exceeds the
the nuclear receptor PPARα with PPAR ligand decreased the expression UPR clearance capacity, the cell utilizes autophagy as a pro-survival
of the autophagy genes like Atg3, Atg4B, Atg7, Atg5 and beclin-1 as mechanism to degrade these accumulated misfolded or unfolded pro-
well as the expression of FoxO1. These research outcomes suggest that teins. The key player contributing to the interplay is Bcl-2 which lo-
persistent activation of PPARα down regulates the autophagic genes in calizes both at mitochondria and endoplasmic reticulum. The ER lo-
a FoxO1 dependent manner [93]. FOXO1 (Forkhead box protein O1) is calized Bcl-2 bound with the autophagy member beclin-1 is proposed to
a transcriptional factor that is involved in the regulation of autophagy aid protection against ER stress by decreasing the steady state levels of
proteins. AMPK was found to be inhibited in Tak1−/− condition. Ca2 + in the ER via IP3R. It is this ER localized Bcl-2 that disrupts the
During non-starving conditions, AMPK remains inactive. This activates beclin-1-VPS34 complex to negatively regulate the beclin-1 dependent
mTORC1 which then functions to synthesize lipids via SREBP-1c– and autophagy [139,176]. Interaction of beclin-1 with the PI3K complex of
PPARγ. The active mTORC1 also inactivates PPARα and hence fatty the autophagy pathway requires dissociation of the beclin-1-Bcl-2
acid β-oxidation. AMPK, LKB1 activation, increased levels of LC3B-II complex. This is carried out by IRE1α, an important ER stress sensor
are usually observed during starvation induced autophagy. But all these localized at the ER membrane. IRE1α activates JNK pathway by in-
responses were attenuated in the Tak1−/− starving condition. This teracting with TRAF2 to phosphorylate Bcl-2 and release beclin-1. A-
clearly shows that TAK1, whose activity is high during autophagy is TF6α, another ER stress sensor generates survival signal via ATF6α-
essential for the activation of starvation induced autophagy [83]. This Rheb-mTOR pathway in dormant carcinoma cells [205]. The Ca2 +
regulatory function of Tak1 towards AMPK connects the role of AMPK activates CamKK/AMPK dependent pathway. Activation of AMPK in-
in autophagy, lipid synthesis and metabolism. duces autophagy via mTOR inhibition [62]. In contrast, the anti-
apoptotic Bcl-2 members, localized at mitochondria bind to the pro-
apoptotic members and inhibit them from inducing apoptosis. They
3.5. Autophagy, ER stress and apoptosis: interplay inhibit the cytochrome c release from the mitochondria via Bax and Bak
[171]. The anti-apoptotic function of Bcl-2 is lost when it gets phos-
When a cell senses the stress signal it responds to it either by phorylated by JNK. Phosphorylated Bcl-2 fails to bind to the pro-
adapting to the stress or by undergoing death. The adaptive response is apoptotic members and also increases the ER release of Ca2 +. This also
rendered when the cell turns on a pro-survival mechanism but when the results in the mitochondrial uptake of Ca2 +, thereby leading to
adaptive mechanisms fail to protect the cells from stress, the cell

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Fig. 3. Schematic model of interplay between ER

stress, autophagy and apoptosis as an example of
how a cell manages a stress.
A cell undergoes ER (Endoplasmic Reticulum)
stress. It then activates the UPR (Unfolded
Protein Response) system to clear the accumu-
lating proteins. If the protein accumulation ex-
ceeds the clearance capacity of the UPR system,
the cell is then to decide between death and
survival. Based on the severity of the impact of
the stress, the cell turns on autophagy, the pro-
survival mechanism or apoptosis, the pro-
grammed cell death mechanism to survive or to
die. The key molecule that bridges between these
two mechanism is Bcl- 2 that resides (B-cell
lymphoma 2) at the ER. Bcl-2 either gets re-
cruited to the PI3K complex of autophagy
pathway to aid in cell survival or gets phos-
phorylated by the JNK pathway resulting in the
activation of pro- apoptotic members leading to
cell death.

apoptosis. Another key interaction between ER stress and apoptosis led to the localization of ZKSCAN3 in the cytoplasm (Fig. 4). Similarly,
occurs via CHOP. Under ER stress, the expression level of Bcl-2 is de- overexpression of ZKSCAN3 repressed the rapamycin induced autop-
creased by CHOP to sensitize the cell to undergo apoptosis [130]. hagy while silencing ZKSCAN3 increased the level of the autophagy
Thus a cell that undergoes ER stress operates between autophagy marker, LC3-II [28]. In addition, the TFEB target genes are regulated by
and apoptosis to adapt and survive or to die. This is not specific for ER the Krüppel-associated box (KRAB) and SRE-ZBP/CTfin-51/AQ-1/
stress but is common to all the stress faced by a cell. But the decision to Number 18 cDNA-homology (SCAN) domains of ZKSCAN3 [31]. Com-
choose between the survival and death depends on the effect of stress plementarily, TFEB (transcription factor EB), a member of the basic
on the cell and the effect at which the adaptive responses work. helix–loop–helix Leu zipper family is known to positively regulate au-
tophagy by upregulating the expression of ATG9B, LC3, and SQSTM1.
Unlike ZKSCAN3, during non-stressed conditions, TFEB resides inactive
4. The ZKSCAN3-TFEB rival in autophagy regulation in the cytoplasm while it gets translocated to the nucleus during au-
tophagy [51,209]. This nuclear translocation is regulated by in-
ZKSCAN3 (Zinc finger protein with KRAB and SCAN domains 3) is a tracellular Ca2+ levels [147]. Lysosome is the major cell organelle in-
DNA-binding protein belonging to the zinc-finger family. It is involved volved in the autophagy process. The role of ZKSCAN3 and TFEB
in the transcriptional regulation of autophagy in that it represses the extends also to the lysosomal biogenesis. Stable repression of ZKSCAN3
autophagy transcriptionally. Mounting evidences suggest that proteins resulted in an increase in the level of the well-known lysosomal marker,
functioning in autophagy (encoded by more than 60 genes) are tran- LAMP1 while its overexpression decreases the lysososmal number [28].
scriptionally modulated by ZKSCAN3. Under normal conditions, CLEAR element is the region on the promoter of lysosomal genes
ZKSCAN3 localizes in the nucleus. In contrast, autophagy induction has

Fig. 4. Schematic representation of autophagy regulation by

TFEB and ZKSCAN3.
The figure explains the opposing roles of TFEB and ZKSCAN3 in
regulating autophagy and change in their localization and ex-
pression levels under basal and starving conditions.

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P. Ravanan et al. Life Sciences 188 (2017) 53–67

enriched with TCACGTGA motif and is required for the lysosomal Leukemia (AML) cells that are chemo resistant as conferred by their
function. Suppression of TFEB results in the down regulation of the hypoxic bone marrow microenvironment. It was experimentally de-
CLEAR element indicating the requirement of an active TFEB for the monstrated that the inhibition of autophagy via ATG7 knock down in
lysosomal functioning [202,203] (Fig. 4). TFEB promotes autophagy by AML cells increased their sensitivity to chemotherapy [179]. A similar
inhibiting Akt and mTOR via PTEN [140,181]. Interestingly, TFEB is response was observed in Acute promyelocytic leukemia (APL) cells
also regulated by the mTORC1 residing at the lysosomal surface [147]. upon inhibiting autophagy via siRNA mediated knock down of a cal-
All these research findings and facts strongly indicate that ZKSCAN3 cium-and zinc-binding protein, S100A8 [252]. In addition, inhibition of
and TFEB counteract and transcriptionally regulate autophagy. autophagy was found to sensitize Chronic Myeloid Leukemia (CML)
cells to treatment with tyrosine kinase inhibitors (TKIs) [14,158].
5. Functioning of autophagy in diseases Apilimod, selective inhibitor of phosphatidylinositol-3-phosphate 5 ki-
nase (PIKfyve) inhibits autophagic flux and induces cytotoxicity in B-
5.1. Autophagy and cancer cell non-Hodgkin lymphoma (NHL) [9,57]. De Leo et al. demonstrated
the treatment of EBV-positive Burkitt's lymphoma via autophagy in-
Most of the autophagy regulators are oncogenes and tumor sup- hibition [34]. These research findings collectively indicate that in-
pressors. And this is the reason behind the yet unknown role of au- hibition of autophagy helps the effective treatment of blood cancers.
tophagy in cancer [127]. A cell needs to undergo a change in the me- Alternatively, cancer cell whose apoptotic machinery is mutated,
tabolic machinery to become cancerous. Autophagy is found to be down tend to resist anticancer therapies. For such apoptosis deficient cells
regulated in carcinogenesis. Cancer cells express a set of anti-autop- autophagy serves as an alternative form of death. But there are tumors
hagic genes like Bcl-2, AKT and PI3KC1 which makes it clear that au- that lack the autophagic machinery too and this is the reason for their
tophagy serves as a hindrance for a normal cell in getting transformed high resistivity against several anticancer therapies [258]. However,
into a cancerous one. Now the fact that lysosomes actively traffic in the ability of autophagy to modulate a tumor's response to anticancer
tumorigenic condition, not only puts forth the dual role of autophagy therapies is determined by the cellular context [180].
but also insists the need for the clearance of doubts on whether au-
tophagy leads to cell survival or cell death in cancer [117,153]. The 5.2. Autophagy impairment in neurodegenerative diseases
autophagically inhibitory activity of certain factors like AKT and Ras
are found to be tumor promotive, that is oncogenic and the tumor Stimulation of autophagy has been shown to reduce the severity of
suppressors like PTEN, DAPK, and p53 are autophagy inductive. neurodegenerative diseases. Mounting evidences suggest that, autop-
A cell requires activation of PI3K/AKT/MTOR as a first step to be- hagy dysregulation play a crucial role in certain diseases like neuro-
come cancerous. This activation then leads to cell proliferation and degenerative disorders, cancer, metabolic diseases and infectious dis-
synthesis of proteins which would obviously create energy demand for eases. Autophagy is impaired in the case of neurodegenerative diseases
the inner cells of the tumor and hence they tend to activate autophagy, wherein toxic accumulation of aggregate prone proteins is observed.
the alternative source of energy. Also the induction and upregulation of Upregulation of autophagy will facilitate clearance of these in-
autophagy in cells that face metabolic stress has also been experimen- tracellular mutated proteins [17,149,188,189,244]. As neurons are
tally proven by studies utilizing three dimensional morphogenesis as- non-dividing cells, there exists the inability for these cells to reduce the
says in mammary epithelial cells. Metabolic stress combined with in- toxicity of the accumulating protein aggregates through cell division.
activated autophagy in cells that are deficient in apoptosis resulted in Therefore, at certain point of time, these accumulating proteins tend to
cell death necrotically via the activation of pro-inflammatory factors exceed the recognizing and clearing ability of the cellular degradation
that recruit macrophages. Alternatively induction of autophagy under system which is why accumulation of protein aggregates is considered
same conditions of apoptosis deficiency and metabolic stress suppressed as the hallmark of most neurodegenerative diseases [127]. Excessive or
necrosis [36]. Similarly, autophagy inhibition leads to apoptosis [20]. insufficient autophagic activity can influence neuronal survival and
Tumor cells in melanoma patients exhibited low levels of Atg5, a key cause neurodegeneration [105]. Autophagy has been implicated in
player of autophagy machinery. And depleting the Atg5 levels also re- eliminating protein aggregates which are not removed via proteosomal
sulted in growth of tumor [134]. Also, down regulation of autophagy in degradation and therefore is performing an important “housekeeping”
MCF7 cell line resulted in the growth of breast cancer cells [162]. function, representing its role in the prevention of neurodegenerative
In case of cancer and the treatment strategy, both autophagy in- diseases. Most neurodegenerative disease associated proteins that form
hibitors and inducers are currently being investigated. The role of au- intracytoplasmic aggregates are autophagy substrates [17], indicating
tophagy in cell survival has stimulated interests of researchers to de- the importance of autophagy in neurodegenerative diseases.
termine whether autophagy may promote therapeutic resistance to Conditional knockout of genes involved in autophagy such as atg5
cytotoxic therapy. Yang et al. and others [10,88,138,208,253] have and atg7 in the mouse brain resulted in neurodegenerative phenotype
shown that autophagy inhibition (via 3-methyl adenine treatment/ge- and accumulation of protein aggregates [68,114]. Furthermore, the
netic knockouts of autophagy genes/chloroquine) will sensitize the clearance of protein aggregates is delayed when autophagy is impaired
tumor cells to cell death induced by cytotoxic agents. Also, tumors and most importantly, their turnover is enhanced when autophagy is
formed by autophagy deficient cells have been shown to display upregulated [213]. Therefore, autophagy upregulation is desired in
genomic instability and DNA damage, which is in part mediated by ROS case of neurodegenerative diseases. Induction of autophagy using che-
[142,143]. Defects in autophagy can occur at different stages of the mical inducers could be a therapeutic opportunity for number of neu-
pathway in different diseases, and this may influence treatment stra- rodegenerative diseases.
tegies as well [141,197]. Defects in autophagosomes formation may be
amenable to drugs that enhance autophagosomes biogenesis. For ex- 5.3. Protective role of autophagy in other diseases
ample, laforin mutations (causing Lafora epilepsy) impair autophago-
somes formation by enhancing mTOR activity. Rapamycin, an autop- Down regulation of autophagy increases the susceptibility to several
hagy inducer (mTOR inhibitor) may therefore be beneficial in this disorders like neurodegenerative diseases, cancer, aging, metabolic
context [8]. Autophagy is also found to be upregulated during metas- disorders and other infectious diseases. In the view of the role of au-
tasis [52,123,155]. Advanced tumor cells exhibit upregulated autop- tophagy in the maintenance of metabolic homeostasis, disease initiation
hagy that serves as a pro-survival pathway helping them to escape the and progression is found to be contributed by autophagy deficiency.
anticancer therapies. Therefore, autophagy inhibition is desirable in Loss of autophagy in mice, achieved by deleting Atg7 of beta cells re-
such cases. This phenomenon is similar in the case of Acute Myeloid sulted in the impairment of glucose tolerance and serum level of insulin

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P. Ravanan et al. Life Sciences 188 (2017) 53–67

[97]. Autophagy regenerates the lipids and amino acids, the so called biosynthesis and autophagy. Inhibition of mTOR mimics cellular star-
metabolic precursors during the degradation process. The re- vation by blocking signals required for cell growth and proliferation.
noprotective role played by autophagy gained a lot of interest too. The mTOR is also a downstream target of PI3K, an important mediator of
renal tubular epithelial cells take up an excessive amount of energy as TLR signaling [96,160]. Everolimus, a derivative of rapamycin is also a
they function in active transport and hence are more prone to hypoxic known inducer of autophagy which acts by inhibiting mTOR signaling.
conditions. Acute Kidney Injury (AKI) is a condition where these cells Torin1 is one another inducer of autophagy, a selective ATP-competi-
undergo acute cell death, accompanied by inflammation. During this tive small molecule mTOR inhibitor. Torin1 has been shown to induce
process, organelles like mitochondria, lysosomes and others are da- autophagy much more potently than rapamycin. KU0063794 induces
maged and this is where the autophagy comes into play to clear them autophagy by inhibiting both the mTOR complexes mTORC1 and
[43]. The protective effect of autophagy against glucose intolerance has mTORC2. Deforolimus (AP23573), an analogue of rapamycin inhibits
also been studied using mouse model [70]. Dysregulation in the way of the activity of mTOR and induces autophagy [192]. Autophagy can also
autophagosomes to lysosomes was distinctively observed in patholo- be induced in mTOR independent manner. Metformin belongs to the
gical conditions like bacterial infection, neurodegenerative diseases and biguanide class of antidiabetic drug. Metformin activates AMPK, an
cancer. Misfolded proteins tend to aggregate under impaired conditions enzyme that regulates autophagy. Activation of AMPK leads to the in-
of autophagy. Many reasons contribute to the impairment of autop- hibition of mTOR signaling without involving AKT. Metformin can be
hagy. Failures of recognition of cargo by autophagosomes and ag- used to induce autophagy in AMPK dependent manner. Epidemiologic
gregates induced damage to the autophagy pathway make the move- studies have reported that metformin treatment is associated with a
ment of autophagosomes within the cell impossible. And this remains decreased incidence of various cancers [10,175,246]. AICAR inhibits
the most possible reason behind the failure of autophagy in certain the activity of mTOR by phosphorylating raptor [124]. Trehalose is a
disease conditions. Improper fusion of autophagosomes to the lyso- disaccharide found in plants and is able to induce autophagy in mTOR
somes which occurs due to the change in pH resulting from various independent manner [16,204]. Perifosine, an alkylphospholipid, in-
diseases is considered to be another cause of such flawed fusion. This duces cell cycle arrest and apoptosis through the inhibition of Akt [72].
also affects the activity of the hydrolytic enzymes within the lysosomes mTOR is a target of Akt whose activation suppresses autophagy.
[33]. Downregulation of autophagy resulted in CYP2E1 mediated Therefore, by suppressing Akt, perifosine induces autophagy either
toxicity by increasing oxidative stress and p38 activation [260]. The alone or in combination with rapamycin. A plant derivative of poly-
loss of autophagy also leads to the accumulation of genomic damage phenol compound 3, 4′, 5-trihydroxy-trans‑stilbene has also been
where autophagy usually decreases the DNA repair occurring by shown to induce autophagy by modulating mTOR and AMPK signaling.
homologous recombination. The DNA repair occurs via non-homo- Estrogen receptor antagonist tamoxifen can also promote autophagy by
logous end joining resulting in genomic damages [133]. Similarly, au- stimulating the expression of Atg genes and activating mTOR signaling.
tophagy deficiency induced by tissue specific deletion of Atg7 caused Histone deacetylase inhibitors, such as SAHA, trichostatin A and val-
protection against insulin resistance and obesity [106]. Downregulation proic acid can induce the expression of Atg and LC3 proteins and induce
of autophagy by Bcl-2 in motor neurons extended the life of mice with a autophagy. N(10) substituted phenoxazine, a small molecule scaffold
lethal disease called Amyotropic Lateral Scelerosis (ALS) [76]. It is a has been effective in the treatment of Huntington disease (HD) by in-
well-known fact that the accumulation of aggregates of protein is the ducing autophagy in neuronal cells. In a HD model, this compound was
key reason behind most of the neurodegenerative diseases. Autophagy neuroprotective and decreased the accumulated protein aggregates
is also found be dysregulated in such cases: misfolded transthyretin [232]. The PI3KC1 complex activates Akt under nutrient rich condition
(TTR) aggregates and TTR- related amyloidoses resulted in the partial thereby activating mTORC1 subsequently. Therefore autophagy can be
impairment of autophagy [228]. Therapeutic inventions increasing in induced by inhibiting PI3KC1. There are many PI3KC1 inhibitors
the view of caspase inhibition, oxidative stress and autophagy are identified and each of them are at different phases of clinical trials
proven to be more beneficial for treating these diseases [43]. [194]. The preclinical inhibitor IC87114 inhibits the PI3Kδ isoform and
Although there are evidences linking autophagy with diseases, the distinguishes class I isoforms. It is also found to inhibit AML pro-
role of autophagy in diseases still remains an open research problem liferation [80,199]. A study in malignant glioma cells confirms the
because of the limited availability of autophagy specific chemical ability of another compound called PX-886 to induce autophagy by
modulators [216]. In recent years, researchers have begun to screen inhibiting PI3K [116]. 6 Bio, a novel autophagy inducer has recently
compound libraries in search of autophagy modulators. As a result, been identified to induce autophagic degradation of alpha synuclein
there are some chemical modulators of autophagy identified, to mainly [222].
aid in research. However, addressing the therapeutic potential of In addition, curcumin analog C1 was identified to directly activate
modulating autophagy signaling in human diseases requires active TFEB and regulate the degradation of amyloid precursor proteins, tau
chemicals with pharmacologically desirable properties. and α- synuclein in Alzheimer's and Parkinson's disease. This discovery
is attracting many researchers for the exploration of the mechanism of
6. Autophagy modulators at a glance autophagy induction because the induction is mTOR independent
[221]. VJR-TZ-18, a novel PI3K δ inhibitor exerts antitumor activity
Recently, the development of autophagy modulators has become against in vivo and in vitro anticancer models via induction of autophagy
interests of many researchers to contribute to the better understanding [119]. DNLA (Dendrobium nobile Lindl alkaloid), was found to induce
of autophagy mechanism. Such research findings gave rise to effective autophagy flux in hippocampus neurons in vitro [129].
modulators that are being successful in various stages of clinical trials. Autophagy impairment is connected to various health issues and
disease conditions. This gave rise to the thought that autophagy in-
6.1. Chemical inducers of autophagy ducers can be used to induce autophagy in such cases. And the inducers
being developed are expected to have maximum clinical benefits with
Many anticancer agents such as tamoxifen, rapamycin, etoposide, minimum toxic effects which remain difficult for most of the currently
arsenic trioxide, histone deacetylase inhibitors, ionizing radiation and existing inducers. However, studies on these novel inducers besides
vitamin D analogues have been shown to induce autophagic type of cell benefiting in the treatment of various diseases help us understand the
death [214]. Among the autophagy inducers, rapamycin was the first different roles of autophagy. Table 1 lists the chemical inducers of
drug tfo be found to induce autophagy. Rapamycin is an inhibitor of autophagy and their therapeutic status (Table 1).
Serine/Threonine protein kinase, mTOR signaling which is a major
regulator of cell growth, survival, protein synthesis, ribosome

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Table 1
Chemical inducers of autophagy.

Mechanism Inducers Therapeutic status

mTOR inhibition Rapamycin(sirolimus) Renal, advanced endometrial cancer and other types of cancers in clinical trial of phase I/II [7]
Everolimus(RAD- 001) NCT01509560-condition after allogenic peripheral stem cell transplantation (SCT), phase II
[240]
Temsirolimus(CCI-779) NCT00823459-Phase II, Low-grade Glioma [137]
Ridaforolimus(AP23573) NCT00122343-phase II, endometrial cancer [250]
PI3KC1 inhibition IC87114 and analogs Preclinical and phase I, acute myeloid leukemia (AML) [80]
PX-866 Phase II, prostate cancer [2]
Dual PI3K/mTOR inhibition NVP-BEZ235 NCT01343498-phase I, malignant solid tumors [3]
GSK2126458 NCT01725139-phase I, idiopathic Pulmonary Fibrosis [109]
XL765 NCT01082068-phase II, breast cancer [259]
AZD8055 Phase I solid tumors [1]
AMPK activation Metformin NCT02972723-phase IV, chronic hepatitis C infection [103]
AICAR NCT01246778–cardiotoxicity, ischemia heart failure [148]
AKT inhibition Perifosine NCT01049841-phase I, pediatric solid tumors [190]
Triciribine NCT00642031-phase I, hematologic malignancies leukemia [5]
Estrogen receptor Antagonist Arsenic trioxide Phase 1 study for leukemia [193]
Tamoxifen melanoma and Metastatic bladder cancer [113]
Beclin 1 mediated Induction Vitamin D and its analogues Phase 1 trial for advanced solid tumors [87]
Pan-Histone deacetylase Inhibition Suberoylanilide hydroxamic acid (SAHA) Phase 1 trial for colorectal cancer[206]
Valproic acid Phase 2 clinical trial for castration resistant Prostate cancer [211]
GSK3βP inhibition Lithium carbonate Phase 2 trial for amyotrophic lateral sclerosis [49]
other inducers Synthetic triterpenoids Preclinical trials [243]
Trehalose Treatment of vascular aging [204]
Resveratrol Phase II, Type 2 diabetes [4]
Antioxidant TRAIL Phase I/II [125]

6.2. Chemical inhibitors of autophagy degraded structurally. Under glucose-starving conditions, spautin-1
ubiquitinates beclin-1 and degrades it proteasomally. Degradation of
Autophagy can be triggered by various reasons ranging from nu- beclin-1 disrupts the VPS34 complex and results in autophagy inhibi-
trient starvation to microbial invasion. Primarily, the induction of au- tion. Spautin-1 has also been pre-clinically proven to be therapeutically
tophagy involves membrane nucleation, controlled by ULK complex potent in chronic myeloid leukemia [210]. SAR405 was experimentally
and beclin-1. Positive regulators of ULK complex and beclin-1 have demonstrated to inhibit autophagy induced by mTOR inhibition and
been targeted for inhibition of autophagy and have been demonstrated starvation [172,195]. It selectively inhibits the activity of VPS34
to block autophagy. Inhibitors of MAP kinases, JNK1, ERK, p38 have [86,94]. VPS34-IN1, member of bisaminopyrimidine family, was
been shown to inhibit autophagy. The induction of Atg proteins and known to exhibit high selectivity towards VPS34 [12,41]. But its ac-
LC3 protein is required for vesicle formation and expansion. SP600125 tivity on autophagy regulation is not known. Similarly another member
is a potent and selective MAP Kinase (JNK) inhibitor. In general, in- of bisaminopyrimidine family, PIK-III was known to inhibit the de-
hibition of JNK activity leads to the downregulation of beclin-1 and gradation of autophagy substrate receptor under induced conditions of
thereby reduction in autophagy process. Autophagy inhibitors can be autophagy [41]
categorized based on their specificity towards a particular target. Another class of inhibitors is the ULK1 inhibitors. Compound 6
Currently the inhibitors based on their target were grouped as PI3K [177], MRT68921 [122], and SB1-0206965 [45] are known to inhibit
inhibitors, autophagolysosome formation inhibitors, inhibitors of lyso- autophagy via ULK1 inhibition but their specificity affects their in vitro
somal degradative enzymes. Some of them are known to work effec- applicability [173]. All classes of inhibitors discussed so far with re-
tively in combination. spect to their target can also be categorized as “early inhibitors”. As the
3 Methyl Adenine, wortmannin, LY294002 are the well-known class name suggests, they target the early stages of the autophagy pathway
of PI3K inhibitors. 3 Methyl Adenine, known to be the most widely used like phagophore formation, nucleation and elongation. Those that
inhibitor, plays a dual role of inhibiting both PI3K and VPS34. It also target the later stages of autophagy can be categorized as “late in-
has the ability of inhibiting autophagy both under starving and nutrient hibitors”. These inhibitors work by specifically targeting lysosomal
rich condition. But it has been found to be disadvantageous in its po- enzymes and fusion of autophagosome and lysosome. Also autophagy is
tency. Its low potency to inhibit autophagy requires a high concentra- said to have successfully taken place only when the targeted and se-
tion of it which again poses a threat of inhibiting non-targeted kinases questered autophagosomal contents that are delivered into the lyso-
[24,184,249]. Wortmannin, another inhibitor of the same class, inhibits somes are degraded completely. Vacuolar (H+)-ATPase is the enzyme
all 3 classes of PI3K and suppresses autophagy [178]. Similar to 3MA, found in lysosome as well as endosome. Lysosome maintains an internal
wortmannin also inhibits PI3KCI regardless of the nutrient availability pH of about 5 while the pH in the cytoplasm is maintained around a
[247]. The effective inhibition caused by wortmannin is attributed to its neutral range of 7.2. Lysosomal hydrolases require an acidic pH to be
irreversible and covalent binding to the target [183,237]. LY294002 is active. To maintain such an acidic internal environment, an active
the first PI3K inhibitor to be synthetically made [178]. It also has dis- concentration of protons in the lysosome is necessary. This is carried
advantage similar to 3 Methyl Adenine [111]. Other novel PI3K in- out by a proton pump present in the lysosomal membrane. The energy
hibitors like PT210 [112,151] and GSK-2126458 [109], though known requirement of the proton pump to carry out this function is met out by
to effectively inhibit PI3K and its isoform, a lack of clinical report on ATP hydrolysis which is catalyzed by V-ATPase (Vacuolar (H+)-A-
their effect on autophagy hampers them from becoming therapeutically TPase) [74]. Bafilomycin A1 inhibits the proton transport in lysosome
advantageous [173]. Spautin 1, a derivative of quinazoline degrades and inhibits hydrolases [108,248,251]. This poor maintenance of in-
VPS34 complex by inhibiting the ubiquitin specific peptidases, USP10 ternal acidic pH alters the activity of the hydrolases ultimately leading
and USP13 [135]. These enzymes are responsible for the de- ubiquiti- to the autophagy inhibition. Lysosomal proteases like aspartic protei-
nation of beclin-1. Hence VPS34 is not inhibited functionally but nases (D and E) and cysteine proteases (B, H and L) are inhibited by

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P. Ravanan et al. Life Sciences 188 (2017) 53–67

pepstatin A and E64d respectively [98]. They inhibit autophagy and are their potency and concentration. The development of new, effective
applicable for the monitoring of autophagic flux [225]. They can also autophagy inhibitors targeting new autophagy players is another
be used in combination [173]. Desmethylclomipramine and lucanthone striking area of research in autophagy.
affect the fusion and membrane permeabilization of lysosome respec-
tively [23,196]. Autophagy is also inhibited by targeting the lysosomal
acidification. Chloroquine and hydroxy chloroquine (HCQ) are the 7. Conclusion
drugs that inhibit autophagy by altering the pH in the lysosomal lumen
[74,173,219]. This results in the weak or no activity of the lysosomal Autophagy serves as a pro-survival mechanism for a cell in many
enzymes and autophagy. HCQ is less potent in low concentration and a cases. The process of autophagy is regulated by many regulators in vivo.
high micromolar concentration is required for the inhibition of autop- And the integration of autophagy in different stress responses com-
hagy in vitro. Another compound called Lys05 was identified to be very pletely relies on the transcription factors that regulate the autophagy
effective compared to the CQs identified so far [11]. Apilimod, a clin- players functionally. The transcriptional factors like NF-κB are involved
ical stage drug was recently identified as an anti- proliferative com- in multiple pathways and serve as better targets to target multiple
pound to treat B-cell non-Hodgkin lymphoma by targeting PIKfyve lipid signaling molecules via them. However, targeting the molecule in a
kinase. The compound has been found to disrupt the lysosomal home- functionally specific manner is a problem of another level. Autophagy is
ostasis and inhibit the autophagy flux [57]. Autophinib, another potent impaired in most of the diseases and autophagy needs to be induced in
autophagy inhibitor was recently discovered to inhibit both starvation such cases. Similarly, there is a need for the hunt of inhibitors that
and rapamycin induced autophagy by targeting VPS34 [236]. 6b and would effectively inhibit autophagy. Developing modulators, especially
IMB-6G are tricyclic sophoridinic analogs that were found to inhibit inhibitors that targets the specific function of an autophagy player
autophagy flux. without affecting its non-autophagic functions would aid in the devel-
They were identified to have potent anti-proliferative property opment of novel therapeutic strategies. Though the experimental evi-
against wide range of cancer cells [136]. Elaiophylin, a natural com- dencing of the impairment of autophagy to be the reason for the pa-
pound inhibits autophagy at the later stage and also possesses anti- thogenesis of many diseases is significantly increasing, there is always a
tumor activity against ovarian cancer cells [55].Various chemical in- need for effective modulators to study the mechanism underlying the
hibitors of autophagy and their targets are listed in Table 2. impairment of autophagy machinery as well as the effect of its upre-
Though the research interests in understanding autophagy are gulation on cellular functioning.
growing day by day, the availability of tools to study autophagy is very
poor. Discovery of 3-Methyl Adenine has brought a remarkable progress
Conflict of interest
of research in the field of autophagy. Also in recent times, many au-
tophagy inhibitors were developed. But there are open questions on
The authors declare no conflict of interest.

Table 2
Chemical inhibitors of autophagy.

S. No Target Inhibitors Therapeutic role/status

1. ULK SB1-0206965 Strongly suppresses AZD8055 induced autophagy in A549 lung cancer cells [45]
MRT67307 Improves the TLR2- stimulated increase in α- synuclein by inhibiting the IκB kinases [44]
MRT68921 The specificity of this compound towards ULK1 to inhibit autophagy was evidenced in MEFs
[46]
2. PI3K/Vps34 3-Methyl Adenine Increased the potential of drugs like pemetrexed and simvastatin to induce apoptosis in MSTO-
211H and A549 cells [79]
Wortmannin Increased the anticancer activity of silver nanoparticles in B16 mouse melanoma cell model
[132]
LY294002 Inhibited virus induced autophagy in Transmissible Gastroenteritis Virus(TGEV) infected ST
cells [64]
PT210 Shows higher IC50 for p110γ (4 μM) than for Vps34 (450 nM) and reversed kinase specificity
[150]
GSK-2126458 Idiopathic pulmonary fibrosis, phase I [110]
Spautin-1 Improves the apoptosis inducing potential of Imatinib in chronic myeloid leukemia [210]
SAR405 Exhibits anti- proliferative effect on ACHN and 786-O renal carcinoma cell lines in combination
with everolimus [172]
PIK-III The link between NCOA4 and autophagy was unveiled via PIK-III mediated autophagy
inhibition [42]
VPS34- IN1 Reveals the regulatory function of VPS34 over SGK3 activity in U20S cell line [13]
Compound 31 The inhibitory effect was tested in jurkat and Ramos cells [174]
3. Sequestration Verteporfin Polyploidal choroidal vasculopathy, phase IV [40]
4. Formation of autolysosome and lysosomal Bafilomycin A1 Autophagy inhibition using bafilomycin increased the anti- cancer efficacy of EGFR inhibitors
degradation [100]
Chloroquinone (CQ) Antineoplastic agent in gastrointestinal malignancies [101]
HydroxyChloroquinone Phase I trial in combination with bortezomib in patients with relapsed/refractory myeloma
[235]
Ammonium chloride Inhibited autophagy and increased the cytotoxicity of epirubucin MDA- MB-231 and SK- BR- 3
breast cancer cell lines [65]
Compound 30 Exhibits antitumor activity in LNCaP cells by suppressing glucose uptake [239]
Pepstatin A Reduced renal fibrosis in the mouse models of chronic kidney disease(CKD) [50]
E-64d Neuroprotective role in seizure induced brain damage [159]
Leupeptin Useful to study the dynamics of Macroautophagy in mice [69]
Lucanthone Preclinical studies report lucanthone to be superior in efficacy and tolerability than CQ [22]
ARN5187 ARN5187 mediated autophagy inhibition exhibited a low cytotoxicity in many non- cancer
cells [35]
Apilimod Treatment of B-cell non-Hodgkin lymphoma [57]

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