Open access peer-reviewed chapter

Autophagy Modulation for Organelle-Targeting Therapy

Written By

Waleska K. Martins and Mauricio S. Baptista

Submitted: 23 November 2015 Reviewed: 27 April 2016 Published: 10 November 2016

DOI: 10.5772/63976

From the Edited Volume

Autophagy in Current Trends in Cellular Physiology and Pathology

Edited by Nikolai V. Gorbunov and Marion Schneider

Chapter metrics overview

1,979 Chapter Downloads

View Full Metrics


Autophagy is a crucial metabolic pathway that sustains cellular homeostasis in health and that can also play either a protective or a destructive role in disease. During the last decade, progress made in understanding of the molecular basis of autophagy has uncovered an exciting opportunity to target it for the treatment of several human illnesses. In fact, there is emerging interest in autophagy-modulating and autophagy-targeted therapy with a variety of pharmacologic agents. However, to develop effective autophagy-targeted therapy, it is essential to identify the pharmacologic key targets in the autophagy pathway. In this chapter, we reviewed the cases of success and pitfalls of activating or inhibiting autophagy attempting therapeutic intervention of diseases, including cancer, neurologic disorders, and infectious diseases. In all these histopathologic states, autophagy is considered as the principal cellular mechanisms of defense and immunochemical homeostasis. In the last section of this chapter, we discuss main directions that may be of particular use in the future investigations, including a promissory avenue for autophagy modulation for organelle-targeting therapy through a promotion of parallel damage in lysosomal and mitochondrial membranes.


  • autophagy-targeted therapy
  • activation/inhibition of autophagy
  • triterpenoids
  • lysosomal and mitochondrial membranes

1. Introduction

The impact of autophagy in human pathogenesis comprises its critical function for the degradation and recycling of long-lived proteins, lipid droplets, protein aggregates, mature ribosomes, glycogen, and even entire organelles such as the endoplasmic reticulum, mitochondria, and Golgi apparatus [1, 2]. For example, when the efficiency of mitophagy (a type of autophagy that specifically targets dysfunctional mitochondria [27]) is reduced, the maintenance of cellular homeostasis decreases, leading to cell aging, genomic instability, and senescence [4, 8, 9]. However, the molecular mechanism by which deficient mitophagy jeopardizes genomic stability are unclear [10].

To develop effective autophagy-based therapy, it has been essential to identify the pharmacologic key targets in the autophagy pathway for the development of new therapeutic agents. As discussed earlier in this book, autophagy can play either a protective or a destructive role in disease states and thus for therapeutic purposes is valuable to identify and develop pharmacologic agents that might activate or inhibit this cellular process [11].

We will evolve this part of the chapter to review the cases of success and pitfalls of activating or inhibiting autophagy attempting therapeutic intervention of diseases, including cancer, neurologic disorders, and infectious diseases. In the following sections, we discuss a few directions that may be of particular use in future investigations. As recently proposed by our group, the promotion of parallel damage in lysosomes and mitochondria represents a promissory avenue for therapeutic autophagy targeting and aiding controlled cell death and senescence [12, 13].


2. Autophagy-modulating drugs

During the last decade, progress made in understanding of the molecular basis of autophagy has uncovered an exciting opportunity to target it for the treatment of human illnesses [14]. In principle, understanding the role of autophagy in diseases has helped identify new avenues of pharmacologic modulation of autophagy as novel therapeutic intervention. Thus, knowing the process of autophagy targeting might facilitate the search of new drugs or concepts for the treatment of several types of diseases whose etiology or progression is associated with autophagy including cancer [8, 1517], degenerative diseases 25 [1823], and lysosomal storage disorders [2426].

2.1. Autophagy inhibitors

The pharmacologic inhibition targeting the early or later autophagy process has been demonstrated to play pivotal roles in cellular outcome and may affect disease processes. Because inhibition of autophagy by pharmacologic agents also may have some off-target effects on cellular functions, the question of whether, for example, cell death can truly occur due to autophagy alone remains to be clarified [11, 27]. Table 1 lists the compounds identified as inhibitors of autophagy.

The inhibitors that target the early stage of autophagy include 3-MA, Wortmannin, and LYS294002, all of which inhibit the class III PI3K (VSP34) and disable the formation of autophagosome. The inhibitors act on the later stage of autophagy, including compounds (see Table 1) that are capable of preventing lysosomal degradation or blocking the fusion of autophagosomes with lysosomes. For example, the neutralization of intralysosomal pH by lysosomotropic agents such as Bafilomycin A1, Chloroquine (CQ), Hydroxychloroquine (HCQ), or NH4Cl prevents the digestive activity of hydrolases, leading to inhibition of degradative activity of autolysosomes [33, 34, 37, 44]. In their unprotonated form, CQ and HCQ can diffuse across cell membranes to become protonated and accumulated only in acidic organelles. Once trapped within lysosomes, they interfere with prosurvival autophagy, resulting in controlled cell death [5760]. This unique property has established CQ as the most widely used drug to inhibit autophagy in vitro and in vivo. Bafilomycin A1 is a selective vacuolar-type H+-ATPase [V-ATPase] inhibitor responsible for acidifying lysosomes and endosomes [33, 34]. Of note Bafilomycin A1 blocks the fusion of autophagosomes with lysosomes, which results from inhibition of ATP2A/SERCA activity independently of its effect on intralysosomal pH [35, 61].

Compounds Autophagy signaling pathway
3-methyladenine  An inhibitor of autophagic/lysosomal protein degradation [28], but not a specific
autophagy inhibitor [29] may also inhibit the activity of Phosphatidylinositol
3-kinase [30], effectively blocking the early stage of autophagy. 3-MA does not
inhibit BECN1-independent autophagy [29].
ARN5187 4-[[[1-(2-fluorophenyl)cyclopentyl]amino]methyl]-2-[(4-methylpiperazin-1
-yl)methyl]phenol, 1 is a lysosomotropic compound with a dual inhibitory activity
against the circadian regulator NR1D2/REV-ERBβ and autophagy [31, 32].
Bafilomycin A1 A V-ATPase inhibitor that causes an increase in lysosomal/vacuolar pH and,
ultimately, blocks fusion of autophagosomes with the vacuole; the latter may result
from inhibition of ATP2A/SERCA [3335].
Betulinic acid A pentacyclic triterpenoid that promotes parallel damage in mitochondrial and
lysosomal compartments and, ultimately, triggers autophagy associated
cell death in human keratinocytes [12].
CA074 N-(l-3-trans-propylcarbamoyloxirane-2-carbonyl)-l-isoleucyl-l-proline is a potent
and specific inhibitor of cathepsin B in vitro [36].
Chloroquine Chloroquine and its analog Hydroxychloroquine are lysosomotropic compounds
that elevate/neutralize the lysosomal/vacuolar pH [37].
Colchicine A microtubule depolarizing agent that may block autophagosome maturation to
autolysosomes and increased LC3II protein levels [38].
-Amine, an active metabolite of clomipramine inhibits late autophagy through a
significant blockage of the degradation of autophagic cargo [39]; may induce an
increase in the steady-state levels of p62/SQSTM1 by inhibiting the autophagic
flux as opposed to an activation of the autophagic pathway [39].
E64d Inhibits papain-like cathepsin cysteine proteases and calpain-activated neutral
proteases [40]; should be used in combination with pepstatin A to inhibit lysosomal
protein degradation [29].
Eflornithine 2,5-diamino-2-(difluoromethyl)pentanoic acid, an irreversible inhibitor of ODC1
(ornithine decarboxylase 1) that blocks spermidine synthesis and ATG5 gene
expression acting as a novel autophagy inhibitor [41].
Leupeptin An inhibitor of cysteine, serine, and threonine proteases that causes significant
inhibition of the intracellular maturation of cathepsin B, L, and H [37, 42, 43]; decreases the
degradation of short- and long-lived proteins [44]; should be used in combination with
pepstatin A and/or E-64d to block lysosomal protein degradation [29].
Lucanthone Interferes with lysosomal function and leads to the accumulation of undegraded
proteins and induces a cathepsin D-mediated apoptosis [45].
LYS294002 2-(4-morpholi-nyl)-8-phenylchromone may prevent autophagic sequestration by
inhibiting phosphatidylinositol 3-kinase activity [30].
Monensin An inhibitor of protein transport, acts as proton exchange for potassium or sodium and
inhibits autophagy by preventing the fusion of the autophagosome with
the lysosome [46].
NH4Cl Lysosomotropic compound that elevate/neutralize the lysosomal/vacuolar pH,
inhibiting the lysosomal pathway of protein degradation [37].
Nocodazole A depolymerizer of nonacetylated microtubules and impairs tubulin acetylation
but does not affect polymerized acetylated microtubules; may impair the conversion
of LC3I to LC3II but does not block the degradation of LC3II-associated
autophagosomes [47].
Pepstatin A An aspartyl protease inhibitor that can be used to partially block lysosomal
degradation [44]; should be used in combination with other inhibitors such as E-64d [29].
PES 2-Phenyl-ethynesulfonamide , a small molecule inhibitor of heat shock protein 70
(HSP70), impairs autophagy through its inhibitory effects on lysosomal functions
showing an accumulation of the precursor procathepsin L and a markedly reduced
abundance of the smaller, mature form of the enzyme [48].
Propofol May exert protective effects on neuronal cells and cardiomyocytes, in part through
the inhibition of early autophagy [4951].
Spautin A specific and potent autophagy inhibitor 1 that promotes degradation of
the Vps34 (a phosphoinositide 3-kinase class III isoform) via inhibiting ubiquitin-
specific processing protease 10 (USP10) and USP13, two ubiquitin-specific peptidases
that target the deubiquitination of Beclin1 [52].
Thapsigargin A sarco/endoplasmic reticulum Ca (2+)-ATPase inhibitor that inhibits autophagic sequestration of cytosolic material through the depletion of intracellular Ca2+ stores [53, 54]; also may lead to the accumulation of mature autophagosomes by blocking autophagosome fusion with the endocytic system by interfering with the recruitment of RAB7 [55].
Vacuolin-1 2-N-[(3-iodophenyl)methylideneamino]-6-morpholin-4-yl-4-N,4-N-diphenyl-
1,3,5-triazine-2,4-diamine, an activator of RAB5A GTPase activity that
potently and reversibly inhibits autophagosome-lysosome fusion; also
may alkalinize lysosomal pH and decrease lysosomal Ca2+ content [56].
Vinblastine A depolymerizer of both nonacetylated and acetylated microtubules that interferes
with both LC3I-LC3II conversion and LC3II-associated autophagosome
fusion with lysosomes [47].
Wortmannin An inhibitor of PI3K and PtdIns3K that blocks autophagy, but not a specific inhibitor
that prevents autophagic sequestration such as 3-methyladenine [30].

Table 1.

Compounds known to inhibit autophagy.

Other inhibitors of autophagy that impair the autolysosome formation include the antidepressant drug Desmethylclomipramine, the anti-schistome agent Lucanthone, Eflornithine, Monensin, PES, Spautin, Thapsigargin, Vacuolin-1, and Vinblastine (see Table 1).

The digestive phase of autophagy may also be blocked by lessening lysosome-mediated proteolysis such as the cysteine protease inhibitor E-64d; the aspartic protease inhibitor Pepstatin A; the active inhibitor of cathepsin B CA074; and the cysteine, serine, and threonine protease inhibitor Leupeptin [37, 44, 62]. Autophagosomes and lysosomes move along the microtubules to fuse, so microtubule-disrupting agents, including taxanes, Nocodazole, Colchicine, and Vinca alkaloids, may inhibit the fusion of autophagosomes with lysosomes [11, 63, 64].

2.2. Autophagy activators

It is now generally believed that modulating the activity of autophagy through targeting specific regulatory molecules in the autophagy machinery may improve clinical outcome for diverse diseases [11, 14]. In this context, mTOR inhibitors has been considered as the most potent activators of autophagy by playing pivotal key negative regulatory role [29]. Table 2 lists the compounds that have been identified as activators of autophagy.

Compounds Autophagy signaling pathway
10-NCP 10-(4′-N-diethylamino)butyl)-2-chlorophenoxazine that may promote potential
and safe upregulation of autophagy in neurons in an AKT-
and mTOR-independent fashion [65].
17-AAG 17-Allylamino-17-Demethoxygeldanamycin that may inhibit the HSP90 CDC37
chaperone complex activating autophagy in certain systems (e.g., neurons),
but impairs starvation-induced autophagy and mitophagy
in others by promoting the turnover of ULK1 [66].
Akti-1/2 Akt inhibitor VIII isozyme-selective Akti-1/2 can promote allosteric inhibition of
AKT1 and AKT2 and activates autophagy in B-cell lymphoma [67].
AUTEN-67 An inhibitor of MTMR14, a myotubularin-related phosphatase that may antagonize
the formation of autophagic membrane [68].
AZD8055 5-[2,4-bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3- d]pyrimidin-7-yl]-2-methoxyphenyl
methanol that may inhibit both mTORC1 and mTORC2 [69].
Everolimus An inhibitor of mTORC1 that induces both autophagy and apoptosis in B-cell lymphoma
primary cultures [67].
KU-0063794 A specific mTOR inhibitor that may bind the catalytic site and activates autophagy [70, 71].
MLN4924 A small molecule inhibitor of NEDD8 activating enzyme (NAE) [72] that triggers
autophagy through the blockage of mTOR signals via DEPTOR as well as the HIF1A-
DDIT4/REDD1-TSC1/2 axis [73] as a result of inactivation
of cullin-RING ligases [74].
Oleanolic acid A pentacyclic triterpenoid that promotes damage in mitochondrial
compartments, and ultimately, activates prosurvival autophagy in human
keratinocytes [12].
NAADP-AM Nicotinic acid adenine dinucleotide phosphate (NAADP) can mobilize
Ca2+ from acidic Ca2+ stores through lysosomal
two-pore channels (TPCs) in primary cultured rat astrocytes and present evidence
that NAADP-evoked Ca2+ signals regulate autophagy [75].
NVP-BEZ235 NVP-BEZ235 is an imidazo[4,5-c]quinoline derivative that can inhibit
the activity of target proteins in the PI3K/AKT/mTOR cascade and
activates autophagy in human gliomas [76, 77].
PMI Is a pharmacological P62-mediated mitophagy inducer (PMI) that activates
mitophagy without recruiting Parkin or collapsing the mitochondrial
membrane potential [78].
PP242 2-(4-amino-1-isopropyl-1H- pyrazolo[3,4-d]pyrimidin-3-yl)-1H-indol-5-ol is a
ATP-competitive inhibitor of mTORC1 and mTORC2 [79, 80]; should be more effective mTORC1
inhibitor than rapamycin [81].
PP30 3-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-N-(4,5-dihydrothiazol-2-yl)
benzamide is a ATP-competitive inhibitor of mTORC1 and mTORC2 [81].
Rapamycin Binds to FKBP1A/FKBP12 and inhibits mTORC1; the complex binds to the FRB domain
of mTOR and limits its interaction with RPTOR, thus inducing autophagy,
but only providing partial mTORC1 inhibition [82].
Resveratrol A natural polyphenol that affects many proteins [83] via both AMPK activation
and JNK-mediated p62/SQSTM1 expression activates autophagy [84, 85].
Ridaforolimus Binds to and inhibits the mammalian target of Rapamycin (mTOR), which may
result in cell cycle arrest and, consequently, the inhibition of tumor cell growth
and proliferation.
RSVAs Synthetic small-molecule analogs of resveratrol that potently activate
AMPK and induce autophagy [86].
Saikosaponin-d A natural small-molecule inhibitor of ATP2A/SERCA that induces autophagy
via direct inhibition of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase
(SERCA), leading to the increase of intracellular calcium ion levels and
activating the Ca2+/calmodulin-dependent kinase kinase-b (CaMKKβ)/AMPK
signaling cascade [87].
Tat-Beclin 1 A cell penetrating peptide that potently induces autophagy [88, 89].
Temsirolimus Is Rapamycin ester analog CCI-779 with better stability and pharmacological
properties compared to rapamycin that activates autophagy in neurons in
Alzheimer's disease [9092].
TMS Trans-3,5,4-trimethoxystilbene upregulates the expression of the transient receptor potential
canonical channel 4 (TRPC4), resulting in mTOR inhibition and autophagy activation [93].
Torin1 1-[4-(4-propanoylpiperazin-1-yl)- 3-(trifluoromethyl)phenyl]-9-quinolin-3-ylbenzo
[h][1, 6]naphthyridin-2-one, a catalytic mTORC1 and mTORC2
inhibitor that induces autophagy [94].
Trehalose mTOR-independent, autophagic enhancer that may be relevant for the treatment of
different neurodegenerative diseases [20, 95, 96].
Tunicamycin A glycosylation inhibitor that induces autophagy due to endoplasmic reticulum
stress [97].
WYE-125132 1-[4-[1-(1,4-dioxaspiro[4.5]decan- 8-yl)-4-(8-oxa-3- azabicyclo[3.2.1]octan-3-yl)pyrazolo
[3,4- d]pyrimidin-6-yl]phenyl]-3-methylurea is an ATP-competitive and
specific inhibitor of mTORC1 and mTORC2 [98].

Table 2.

Compounds known to activate autophagy.

There are several other agents that negatively regulate autophagy, such as inositol 1,4,5-trisphosphate (IP3), epidermal growth factor receptor (EGFR), Bcl-2, and Bcl-xl. The mTORC1 inhibitor Rapamycin and its analogs Temsirolimus (CCI-79, Torisel), Everolimus (RAD001, Afinitor), and Ridaforolimus (AP-23573, Deferolimus, MK-8669) are strong inducers of autophagy [14], as are the ATP-competitive inhibitors of mTOR such as Torin 1 [94], PP242 [79, 80], PP30 [81] and AZD8055 [69], and WYE-125132 [98], but their autophagy-inducing efficacy has not been well documented [11]. Other autophagic enhancers that induces autophagy via a mTOR-independent pathway, include AUTEN-67 [68], 10-NCP [65], PMI [78], Resveratrol [84, 85], Trehalose [20, 95, 96], and Tunicamycin [97].


3. Modulation of autophagy as a cancer therapy

The human cancer represents a significant worldwide public health problem considered as the main cause of death [99]. Its worldwide incidence is expected to show more than 21 thousand million new cases in 2030 [100]. To deal with such increased incidence, 47,608 clinical 15 trials have been currently carried out according to Clinical Trials.Gov [101]. Therapeutic targeting of the autophagy pathway as a new anticancer strategy has been under extensive investigation [11, 64, 102, 103]. Several data indicate that prosurvival autophagy confers a tumor growth advantage through the supplementation of required nutrition of growth, and thus it represents a novel therapeutic target [46, 104]. Actually, autophagy may represent a major impediment to successful cancer therapy by radiation, drugs (e.g., Doxorubicin, Temozolomide, and Etoposide), histone deaceltylase inhibitors, Arsenic trioxide, TNF-alpha, IFN-gamma, Imatinib, and Rapamycin and the anti-estrogen hormonal therapy Tamoxifen as reviewed [46, 104, 105]. Dalby and colleagues propose that inhibitors of autophagy may either enhance the efficacy of anti-tumor therapy or promote cell death not only in primary cancer types but also in advanced-stage cancers and metastatic tumors that are considered drug resistant or apoptosis resistant, such as chemotherapy-resistant cancer [105]. However, depending on the context, such as tumor type or stage, the autophagy-enhancing agents believed to induce a type II programmed cell death mechanism through an extensive autophagic degradation of intracellular content may also elicit beneficial effects in the treatment of cancer [105]. Both the approaches, inhibition of either the prosurvival or induction of prodeath mechanism of autophagy, will be discussed further.

3.1. Use of autophagy activators in cancer treatment

Several evidence have suggested that increased autophagy may kill cells. However, the weakness of many studies has been that the demonstration of autophagy after a cytotoxic treatment does not prove that autophagy contributed to cell death, only that it was associated with it [11]. It is equally plausible that increased autophagy in these settings was more a failed effort to maintain cell survival than triggering per se cell death. If autophagy would act more definitively as a prodeath cell role than a prosurvival one, its inhibition would have to increase survival. In fact, most studies have showed that a cell death had been counterbalanced by an autophagic salvage response rather than demonstrate a causative role for autophagy in the promotion of cell death [106, 107]. Nonetheless, induction of autophagy-associated cell death has been suggested as a potential strategy to eradicate human cancers [108]. In fact, several anticancer drugs have been reported to kill tumor cells through autophagy-mediated mechanisms, include Photodynamic Therapy, Cisplatin, 5-Fluoroacil, Etoposide, Imatinib, and Paclitaxel, as reviewed [109].

Rapamycin and its more soluble analog Temsirolimus trigger autophagy, as does KU-63794, whose selective mTOR inhibition has been attributed to its antitumor mechanism regardless apoptosis induction [110112] in which the disruption of the PI3K/Akt signaling pathway might greatly enhance the effectiveness of mTOR inhibitors [110]. Likewise, the inhibitory effect of the mTOR inhibitor Everolimus on acute lymphoblastic leukemia was associated with autophagy activation [113]. The combination of Temsirolimus, an mTOR inhibitor, and HCQ, an autophagy inhibitor, augments cell death in preclinical models [114].

The ATP-competitive inhibitors of mTORC1/mTORC2, WYE-125132 [98], and AZD8055 [69] have demonstrable anticancer activity by growth inhibition, and potentially autophagy both in vitro and in vivo. In case of rapamycin-resistant T37/46 phosphorylation sites on 4E-BP1, AZD8055 may fully inhibit mTOR [69]. AZD8055 is currently in Phase I clinical trials as an antitumor agent (NCT00973076, NCT01316809, NCT00999882 and NCT00731263). The tyrosine kinase inhibitor Dasatinib (BMS-354825) has been reported to enhance the antiglioma effect of Temozolomide through triggering significant decrease in cell proliferation while simultaneously increasing autophagy, and this action can be antagonized by the autophagy inhibitor 3-MA [115].

Other types of drugs possessing an autophagy-inducing effect have also found their potential application in cancer treatment (see Table 3). For instance, autophagy-associated cell death may contribute to the anticancer actions of the histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (Vorinostat) [116118]. Vorinostat may induce autophagy through downregulation of Akt/mTOR signaling and induction of ER stress response, whose biological effects might be antagonized by the autophagy inhibitor 3-MA [117]. Coadministration of Vorinostat and a poly (ADP-ribose) polymerase (PARP) inhibitor Olaparib synergistically inhibits the growth of triple-negative breast cancer cells through increased apoptotic and autophagy-associated cell death [119]. In Tamoxifen-resistant MCF-7 breast cancer cells the HDAC inhibitor MHY218 induces apoptosis or autophagy-related cell death [120]. The estrogen receptor antagonist Raloxifene induces autophagy via the activation of AMPK by sensing decreases in ATP, leading to a nonapoptotic autophagy-associated cell death in breast cancer [121]. However, it has been proposed that autophagy is sterol-dependent and is associated with cell survival rather than cytotoxicity [122]. The natural products Resveratrol [84], triterpenoids Ursolic acid [123125], and Saponin [126] promote cancer cell death associated with activation of autophagy. It is conceivable that some autophagy-inducing agents may also be useful in cancer therapies because of their ability to trigger autophagy-associated cell death [11, 113, 115]. The same attention given to inhibitors of autophagy should be given to autophagy-inducing or autophagy-enhancing agents [114, 127131].

Cancer Type Identifier Study Phase Status
Renal cell
NCT00830895  Everolimus for nonclear cell renal cell carcinoma (RCC) II 1  [132]
NCT01090466  Gemcitabine Hydrochloride, Cisplatin, and Temsirolimus
as first-line therapy to treat patients with locally
advanced and/or metastatic transitional cell cancer
of the urothelium
I/II 1
NCT01313559  Pasireotide (SOM230) with or without Everolimus to
treat patients with hormone-resistant chemotherapy
naive prostate cancer
II 1
NCT00574769  Docetaxel with Everolimus and Bevacizumab in men
with advanced prostate cancer
I/II 1
NCT02339168  Enzalutamide and Metformin Hydrochloride to treat
patients with hormone-resistant prostate cancer
I 2
NCT01748500  Pantoprazole and Docetaxel for men with metastatic
castration-resistant prostate cancer
II 2
NCT01497925  ADIPEG 20 and Docetaxel in solid tumors with emphasis on
prostate cancer and nonsmall cell lung cancer
I 2
Breast cancer NCT00411788  Rapamycin and Trastuzumab for patients with HER-2
receptor positive metastatic breast cancer
II 3
NCT01111825  Temsirolimus and Neratinib for the treatment of patients
with metastatic HER2-amplified or triple negative
breast cancer
I/II 2
NCT00736970  Ridaforolimus in combination with Trastuzuma in patients
with metastatic, HER2-positive breast cancer who
have developed resistance to Trastuzumab.
III 1  [133]
NCT01605396  Ridaforolimus and Exemestane, compared with
Ridaforolimus, Dalotuzumab and Exemestane to
treat breast cancer
II 2
NCT01234857  Ridaforolimus in combination with Dalotuzumab
compared to the standard of care treatment
in estrogen receptor positive
breast cancer patients
II 1
Nonsmall cell
lung cancer
NCT00079235  Temsirolimus to treat patients with stage III-B
(with pleural effusion) or stage IV nonsmall cell
lung cancer
II 1
NCT00923273  Sirolimus and Pemetrexed to treat nonsmall cell lung cancer I/II 1
Small cell
lung cancer
NCT00374140  Everolimus in previously treated small cell lung cancer II 1
NCT01079481  Combination anticancer therapy of Everolimus and
Paclitaxel for relapsed or refractory small cell
lung cancer
I/II 1
NCT01648465  Everolimus to treat newly diagnosed patients with advanced
gastrointestinal neuroendocrine tumors
II 4
NCT01537107  Sirolimus and Vismodegib to treat patients with solid
tumors or pancreatic cancer that is metastatic
or cannot be removed by surgery
I 5
Glioblastoma NCT00329719  Temsirolimus and Sorafenib to treat patients with recurrent
I/II 1
NCT01062399  Everolimus, Temozolomide, and Radiation therapy to treat
patients with newly diagnosed glioblastoma multiforme
I/II 2
NCT01956734  Virus DNX2401 and Temozolomide to treat recurrent
I 2
NCT00522665  Second-line therapy with Irinotecan, Cetuximab,
and Everolimus to treat colorectal cancer
I/II 1
NCT01154335  Everolimus and Linsitinib to treat patients with refractory
metastatic colorectal cancer
I 1  [134]
NCT01188889  Everolimus to treat chronic phase chronic myeloid leukemia
with persistent molecular disease.
I/II 6
NCT00935792  Everolimus and Alemtuzumab to treat patients with recurrent
chronic lymphocytic leukemia or small lymphocytic
I/II 1
NCT00849550  Everolimus in combination with current standard treatment
of XELOX-A (Bevacizumab, Oxaliplatin, Capecitabine)
to treat advanced solid tumors
I 1
NCT01020305  Temsirolimus to reverse androgen insensitivity for
castration-resistant prostate cancer
I/II 1
NCT00657982  Everolimus in a neoadjuvant setting in men with
intermediate or high risk prostate cancer
II 4
NCT01155258  Temsirolimus and Vinorelbine Ditartrate to treat patients
with unresectable or metastatic solid tumors
I 2
NCT01295632  Ridaforolimus with MK-2206 or MK-0752 for participants
with advanced cancer
I 1  [135]
NCT01169532  Ridaforolimus and the HDAC inhibitor Vorinostat to
treat patients with advanced cancer
I 1  [136]
NCT00781846  Ridaforolimus in combination with Bevacizumab for
patients with advanced cancers
I 1  [137]
NCT00739830  Ridaforolimus in advanced endometrial carcinoma II 1  [138]
NCT00693433  Temsirolimus and Dexamethasone to treat patients with
recurrent or refractory multiple myeloma
I 1
NCT00398515  Temsirolimus and Lenalidomide to treat patients with
previously treated multiple myeloma
I 1
NCT00918333  Everolimus and Panobinostat to treat patients with
recurrent multiple myeloma, non-Hodgkin lymphoma, or
Hodgkin lymphoma
I/II 2
NCT00474929  Everolimus and Sorafenib to treat patients with
relapsed or refractory lymphoma or multiple myeloma
I/II 2
NCT01460979  Temsirolimus to treat ovarian cancer of women who
progressed during previous platinum chemotherapy
or within 6 months after therapy or advanced
endometrial carcinoma
II 1
NCT00982631  Temsirolimus and Pegylated Liposomal Doxorubicin to
treat advanced or recurrent breast, endometrial
and ovarian cancer
I 3
NCT01196429  Temsirolimus, Carboplatin, and Paclitaxel as first-line
therapy to treat patients with newly diagnosed
stage III–IV clear cell ovarian cancer
II 1
NCT01010126  Temsirolimus and Bevacizumab to treat advanced endometrial,
ovarian, liver, carcinoid, or islet cell cancer
II 2
NCT01031381  Everolimus and Bevacizumab to treat recurrent ovarian,
peritoneal, and fallopian tube cancer
II 1
NCT01281514  Everolimus and Carboplatin, Pegylated Liposomal Doxorubicin
Hydrochloride to treat patients with relapsed ovarian
epithelial, fallopian tube, or peritoneal cavity cancer
I 4
Melanoma NCT01166126  Temsirolimus and AZD 6244 to treat naive with BRAF
mutant unresectable stage IV
II 1
NCT01014351  Everolimus with Paclitaxel and Carboplatin to treat
metastatic melanoma
II 1
NCT01092728  Dasatinib to treat acral lentiginous, mucosal, or chronic
sun-damaged melanoma
II 1
Sarcoma NCT00112372  Ridaforolimus to treat patients with refractory or advanced
malignancies and sarcomas
I/II 1  [139]
NCT00093080  Ridaforolimus to treat patients with
advanced sarcoma
II 1

Table 3.

Clinical trials of the effects of autophagy activators on human cancers.

1 Completed or terminated; 2 Active, not recruiting; 3 Unknown; 4 Recruiting; 5 Suspended; 6 Withdrawn.

3.2. Use of autophagy inhibitors in cancer treatment

Autophagy confers stress tolerance that enables tumor cells to maintain metabolic homeostasis and the adaptation to hypoxic, nutrient-limiting, and metabolically stressed environments as well as resistance to therapy-induced stress, such as chemotherapy or radiotherapy [11, 64, 105]. Since autophagy activation confers an advantage to tumor growth, it would be one of the hallmarks of tumor progression [140]. For example, K-RasV12 transforming malignant cells are capable of evading metabolic stress and cell death through activation of autophagy cascades. In an attempt to overcome this advantage of tumor behavior, the treatment with autophagy inhibitors Bafilomycin A1 or 3-MA successfully decreases the growth of human breast epithelial cells in vitro [141]. Also, targeting autophagy inhibition using CQ suppressed growth and tumorigenicity of K-Ras mutation tumor cells leading to prolonged survival in pancreatic cancer xenografts and genetic mouse models [142]. These preclinical results suggest that autophagy might be exploited as a new therapeutic target in the setting of tumors driven by oncogenic RAS, which may improve clinical outcome of the patients with RAS-driven tumors, such as pancreatic cancer and malignant melanoma; however, recently reported KRAS-driven tumor lines may not require autophagy for growth [143]. By profiling 47 cell lines with pharmacological and genetic loss-of-function tools, Eng and colleagues suggested that KRAS mutation status would not predict the sensitivity of cancer cells to autophagy inhibition with CQ [143]. Accordingly, oncogenic B-RAF signaling in melanoma impairs the therapeutic advantage of autophagy inhibition [144].

Despite this controversial relation regarding the activation of MAPK pathway and the prediction of the efficacy of autophagy inhibition, the pharmacologic inhibition targeting the early or late autophagy process may increase controlled cell death of several other human tumors during chemotherapy aiding improved clinical outcomes [59, 102, 109]. The therapeutic modulation of autophagy for cancer treatment has been supported by preclinical models in which inhibition of autophagy restored chemosensitivity and enhanced tumor cell death [64]. For example, CQ and its analog HCQ given in combination with chemotherapy suppressed tumor growth and triggered cell death to a greater extent than did chemotherapy alone, both in vitro and in vivo as reviewed [64].

Moreover, suppression of autophagy via use of chemical inhibitors of autophagy such as 3-MA can sensitize tumor cells to the effects of chemotherapeutic drugs [11, 14], 5-Fluorouracil [128, 145], TNF-a [146], proteasome inhibitors [147], and Src family kinase (SFK) inhibitor Saracatinib [148].

The sensitizing effects of inhibiting autophagy on the antitumor efficacy of chemotherapeutic agents have been recapitulated in preclinical models of Myc-induced lymphoma [67, 149], colon cancer [45, 59, 127, 128, 131, 145, 150153], ovarian cancer [154], breast cancer [31, 32, 155, 156] hepatocellular cancer [157], prostate cancer [148, 156], bladder cancer [156], melanoma [114], and glioma [152, 158]. Preclinical evidence reveals the efficacy of CQ to inhibit the genesis and self-renewal of cancer stem cells (CSC) and underlines the impact of this “old drug” as repurposing strategy to open a new CSC-targeted chemoprevention era [153].

Several clinical trials that have been conducted or are in progress have shown favorable effects of CQ as a novel antitumor drug as reviewed [159, 160]. Autophagy inhibition may contribute to the anticancer actions of the histone deacetylase (HDAC) inhibitor Vorinostat [127, 130, 136]. Table 4 compiles recent clinical trials therapeutic targeting autophagy inhibition.

Cancer Type Identifier  Study Phase Status
Renal cell
NCT01144169  Hydroxychloroquine before surgery in
patients with primary renal cell carcinoma
I 1
NCT01480154  Akt Inhibitor MK2206 and Hydroxychloroquine to
treat advanced solid tumors, melanoma,
prostate, or kidney cancer
I 2
NCT01510119 Everolimus and Hydroxychloroquine to
treat renal cell carcinoma
I/II 4
NCT00726596 Hydroxychloroquine to treat patients with
rising PSA levels after local therapy for
prostate cancer
II 2
NCT00786682 Docetaxel and Hydroxychloroquine to treat
metastatic prostate cancer
II 1
NCT01480154 Akt Inhibitor MK2206 and Hydroxychloroquine to treat
advanced solid tumors, melanoma, prostate,
or kidney cancer
I 2
NCT01828476 Navitoclax and Abiraterone with or without
Hydroxychloroquine to treat progressive
metastatic castrate refractory prostate cancer
II 1
Breast cancer NCT01292408 Hydrochloroquine to treat breast cancer patients II 3
NCT00765765 Hydroxychloroquine and Ixabepilone to treat
metastatic breast cancer
I/II 1
NCT01023477 Chloroquine to treat ductal carcinoma in situ I/II 4
NCT02333890 Chloroquine to treat breast cancer II 4
cell lung
NCT00977470 Erlotinib with or without Hydroxychloroquine
in chemo-naive advanced NSCLC and (EGFR) mutations
II 2
NCT00809237 Hydroxychloroquine and Gefitinib to treat lung cancer I/II 3
NCT00933803 Hydroxychloroquine, Carboplatin, Paclitaxel, and
Bevacizumab to treat recurrent advanced
non-small cell lung cancer
I/II 1
NCT01649947 Hydroxychloroquine, Carboplatin, Paclitaxel, and
Bevacizumab to treat advanced/recurrent nonsmall
cell lung cancer
II 4
NCT00728845 Hydroxychloroquine, Carboplatin, Paclitaxel, and
Bevacizumab to treat recurrent advanced non–small
cell lung cancer
I/II 1
Small cell
lung cancer
NCT00969306 Chloroquine to treat stage IV small cell lung cancer I 4
NCT01273805 Hydroxychloroquine to treat patients with metastatic
pancreatic cancer
II 2
NCT01128296 Study of presurgery Gemcitabine and Hydroxychloroquine
to treat stage IIB or III adenocarcinoma
of the pancreas
I/II 2
NCT01506973 Hydroxychloroquine in combination with Gemcitabine/
Abraxane to inhibit autophagy in pancreatic
I/II 4
NCT01978184 Gemcitabine and Abraxane with or without
II 4
Glioblastoma NCT00486603 Hydroxychloroquine, radiation therapy, and
Temozolomide to treat patients with newly
diagnosed glioblastoma multiforme
I/II 1  [161]
NCT00224978 Chloroquine to treat glioblastoma multiforme III 1  [162]
NCT02432417 Chloroquine and chemoradiation to treat glioblastoma II 5
NCT02378532 Chloroquine and Chemoradiation to treat glioblastoma I 5
NCT01206530 FOLFOX, Bevacizumab and Hydroxychloroquine to
treat colorectal cancer
I/II 4
NCT01006369 Hydroxychloroquine, Capecitabine, Oxaliplatin,
and Bevacizumab to treat metastatic
colorectal cancer
II 6
NCT02316340 Vorinostat and Hydroxychloroquine versus Regorafenib
to treat colorectal cancer
II 4
NCT01227135 Imatinib Mesylate with or without Hydroxychloroquine
to treat patients with chronic myeloid leukemia
II 3
NCT00771056 Hydroxychloroquine in untreated B-CLL Patients II 6
solid tumor
NCT00813423 Sunitinib Malate and Hydroxychloroquine to
treat patients with advanced solid tumors
that have not responded to chemotherapy
I 2
NCT00714181 Hydroxychloroquine and Temozolomide to treat
patients with metastatic or unresectable
solid tumors
I 1
NCT00909831 Hydroxychloroquine and Temsirolimus to treat
patients with metastatic solid tumors
that have not responded to treatment
I 2
NCT01023737 Hydroxychloroquine and with histone deacetylase
(HDAC) inhibitor Vorinostat in patients with
advanced solid tumors
I 4
NCT01266057 Sirolimus or Vorinostat and Hydroxychloroquine
in advanced solid tumors
I 4
NCT00568880 Hydroxychloroquine and Bortezomib to treat
patients with relapsed or refractory
multiple myeloma
I/II 3
NCT01689987 Hydroxychloroquine, Cyclophosphamide, Dexamethasone,
and Sirolimus to treat patients with relapsed
or refractory multiple myeloma
I 2
NCT01438177 Chloroquine and VELCADE and Cyclophosphamide to
treat relapsed and refractory multiple myeloma
II 1
Melanoma NCT00962845 Hydroxychloroquine to treat patients with stage
III or stage IV melanoma that can be removed
by surgery
I 2
NCT01480154 Akt Inhibitor MK2206 and Hydroxychloroquine to
treat patients with advanced solid tumors, melanoma,
prostate, or kidney cancer
I 2
NCT02257424 Dabrafenib, Trametinib and Hydroxychloroquine in
patients with advanced BRAF mutant melanoma
I/II 4

Table 4.

Clinical trials of the effects of autophagy inhibitors on human cancers.

1 Completed or terminated; 2 Active, not recruiting; 3 Unknown; 4 Recruiting; 5 Not yet recruiting; 6 suspended.


4. Therapeutic effects of autophagy modulators on cardiovascular diseases

Autophagy plays a dichotomous role on many cardiac pathologic states in which it may exert both protective and detrimental effects through context-dependent mechanisms. As a protective mechanism, autophagy closely protects the heart from myocardial ischemia-reperfusion (I/R) and attenuates cardiac remodeling after myocardial infarction [49, 163]. In fact, as demonstrated in ischemia-reperfusion-induced heart injury, Parkin-mediated mitophagy showed a protective role against the cell death of cardiomyocytes [164]. Moreover, recent evidence indicates that basal levels of autophagy are required for the maintenance of normal cardiovascular function and morphology [165]. However, by contrast, excessive levels of autophagy—or perhaps distinct forms of autophagic flux—contribute to several types of cardiomyopathy by functioning as a controlled cell death pathway [165, 166]. In line with these findings, the selection of activators or inhibitors of autophagy for prevention or treatment of cardiovascular diseases will be complicated. Nevertheless, in practice successful therapeutic approaches that regulate autophagy have been reported recently, suggesting that the autophagic machinery may be properly manipulated to treat heart failure or to prevent rupture of atherosclerotic plaques and sudden death [165, 166]. Whereas there have been no clinical data reporting the efficacy of pharmacologic modulation of autophagy in cardiac diseases, as reviewed in 2011, nine patents have disclosed the pharmacologic modulation of autophagy as a new therapeutic strategy against cardiovascular diseases [166]. In this section, we will review the fundamental use of autophagy modulators on heart diseases, whose biological effects have been identified through both in vitro and in vivo models.


5. Use of autophagy inhibitors in treatment of heart disease

Pharmacologic suppression of autophagy pathway comprises potential new targets for treating cardiac disorders [11]. In vitro and in vivo studies demonstrated that the inhibitor of histone deacetylases Trichostatin A may attenuate both load- and agonist-induced hypertrophic growth and abolish the associated activation of autophagy, reducing pathologic cardiac remodeling during severe pressure overload [167]. Through negative modulation of early stage of autophagy process by inhibiting the expression of Beclin-1 induced by myocardial I/R injury parallel to phosphorylation of mTOR, Propofol reduces autophagy-associated cell death induced by the myocardial I/R injury [49]. Although these strategies for suppressing the excessive activation of autophagy for treating cardiac disorders are in theory promising, a comprehensive view of myocardial autophagy will be obligatory to avoid disrupting homeostatic mechanisms [166]. Thus, although the major challenges remain, patients with heart disease are likely to benefit from these efforts.

In other situations, such as in response to stress, activation rather than suppressing autophagy might be beneficial, since it increases the clearance of misfolded and other harmful proteins. In fact, recent reports had established a requirement for autophagy for cardioprotection in rodent models mediated by a variety of agents including the adenosine A1 receptor agonist Chlorocyclopentyladenosine, Sulfaphenazole, and ischemic preconditioning [168]. On the fate of ischemic-reperfused cardiomyocytes, autophagy plays a protective role [169, 170].

The development of a pharmacological agent to salvage myocardium after an ischemic insult has been explored. For example, attempting to enhance the heart’s tolerance to ischemia-reperfusion through inducing autophagy, the antimicrobial agent Sulfaphenazole might be used [163]. Likewise, chloramphenicol succinate has been shown to activate autophagy and reduce myocardial damage during I/R [169, 170]. In the case of regression of established increase in myocyte cell size (i.e., cardiac hypertrophy) induced by ascending aortic constriction (i.e., pressure overload), the administration of Rapamycin, a mTOR inhibitor, may improve cardiac function [171, 172]. In line with this finding, animal studies suggest that mTOR inhibition attenuates cardiac allograft remodeling secondary to downregulation of mTOR downstream targets and increased autophagy. To increase the paucity of data regarding effect of Sirolimus, a mTOR inhibitor, on human heart remodeling, a current clinical trial Phase 1 has been conducted (NCT01889992).

Macrophages play a central role in atherosclerotic plaque destabilization, leading to acute coronary syndromes and sudden death, and therefore their clearance from atherosclerotic plaques through autophagy has been suggested as an attractive therapeutic strategy for atherosclerosis [173]. In line with these findings, the stent-based delivery of Everolimus was shown to selectively clear macrophages from atherosclerotic plaques in rabbits by activating autophagy without altering smooth muscle cells [174]. As suggested recently, mTOR inhibition represents a promising strategy for stabilization of atherosclerotic plaques [175], as this also prevents adverse left ventricular remodeling and limits infarct size following myocardial infarction [176]. Though the benefits afforded by autophagic activation depend on cardiac pathologic states, vigilance for extra-cardiac effects may be critical [166].


6. Use of autophagy modulators for neurologic disorders

In contrast to other cell types, neurons for being nondividing cells are particularly sensitive to changes in autophagic degradation [177]. As most neurons must survive for the lifetime of the organism, maintenance of organelle function and clearance of aberrant, misfolded, and aggregate proteins are critical processes regulated by autophagy [19]. In fact, many aggregate-prone forms of such proteins, including tau [178], α-synuclein [179, 180], mutant huntingtin [181], and mutant ataxin 3 [178] have a higher dependency on autophagy for their clearance. While autophagy clears these aggregate-prone proteins, upregulation of autophagy may also contribute to amyloid-β pathology [182], as autophagic vacuoles may represent one site of amyloid-β generation. The intracellular accumulation of these aggregate proteins are features of many late-onset neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD), tauopathies, and polyglutamine expansion diseases—such as Huntington’s disease (HD) and various spinocerebellar ataxias (SCAs) [183]. Currently, there are no effective therapeutic strategies capable of attenuating or preventing the neurodegeneration resulting from these diseases in humans.

Autophagy has been considered as a potentially novel approach for treating neurodegenerative disorders [160, 183], although its role in neurodegenerative disorders remains unclear [11]. Even though autophagy may be initially induced as a neuroprotective response, due to excessive, imbalanced induction or defects in completing degradation, it may also contribute to neuronal atrophy, neurite degeneration, and cell death [19, 177]. It is noteworthy that the failed attempt of autophagy at neuron survival has been closely associated with age-related autophagy insufficiency and lysosomal aging [19]. In fact, several evidences recently suggest a possible role for autophagic dysfunction in the pathogenesis of neurodegenerative diseases [184]. Conversely, autophagy also has the ability to decrease the accumulation of toxic, aggregate-prone proteins that cause neurodegeneration [11, 183]. As summarized in the following paragraph, multiple studies provide proof of principle for the activation of autophagy as a therapy for neurodegenerative disease. To date, there are still very few reported clinical results demonstrating that modulation of autophagy indeed represents an effective therapeutic intervention for these devastating diseases [11].

HD disease is caused by a polyglutamine expansion mutation in the huntingtin protein (polyQ-expanded Htt) that confers a toxic gain-of-function and causes the protein to become aggregate-prone proteins, which are cleared by autophagy. It is noteworthy that upregulating this process by Rapamycin attenuates their toxicity in various HD models [181, 185]. The autophagy inducer Rapamycin or its analog CCI-779 has been reported to promote autophagic clearance of polyQ-expanded Htt protein [178, 181, 186]. Likewise, Rapamycin increases the clearance of α-synuclein and lessens the formation of aggregates (Lewy bodies) in neurons [179]. Rapamycin in combination with lithium showed a greater protection against neurodegeneration in an HD fly model [185]. Interestingly, the disaccharide bilayer membrane-protector Trehalose [187] accelerates the autophagic clearance of mutant Huntingtin and α-synuclein through an mTOR-independent pathway [95]. However, in a combination of mTOR inhibitor, Rapamycin Trehalose’s effect on autophagic activity increases, resulting in an additive effect on the clearance of the above proteins [95]. Together these studies demonstrate that autophagy upregulation and promotion of aggregation-prone protein degradation ameliorate neurodegenerative pathology, but conversely autophagy inhibition enhances the toxicity of these proteins [184].


7. Autophagy modulators for treatment of other diseases

Similar to the details outlined above for neurodegenerative disorders, autophagy upregulation may enhance the clearance of a range of infectious agents, including multidrug-resistant (MDR) strains of Mycobacterium tuberculosis. In some cases, mouse models and preclinical data have strengthened the protective role of autophagy against microbial infections, as summarized in Table 5.

AR-12 induces autophagic clearance of Francisella tularensis from the human leukemic cell line THP-1 macrophages [188] and Salmonella enterica serovar Typhimurium in murine macrophages, both in vitro and in vivo [189]. Additionally, experimental findings underscore the importance of host autophagy in orchestrating successful antimicrobial responses to Mycobacterium tuberculosis during chemotherapy with Isoniazid and Pyrazinamide [190]. Likewise, the most active form of vitamin D 1,25D3 may inhibit replication of human immunodeficiency virus (HIV) in human macrophages through autophagy activation [191].

Based on these preclinical findings, researchers have raised the possibility that some antimyco-bacterial chemotherapies already used in clinical for the treatment of infectious diseases anti-infection effects, at least partially, via inducing autophagy. However, it is still unclear whether those findings can be translated into the clinical treatment of certain infections [11]. Stimulation of autophagy with Rapamycin reduces intracellular survival of mycobacteria in macrophages [192].

Drugs Effects
1,25D3 1α,25-dihydroxycholecalciferol inhibits HIV replication and mycobacterial growth [191].
AR-12 2-amino-N-4-5-(2 phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl phenyl-acetamide inhibits activity of phosphoinositide-dependent kinase-1 and promotes autophagic clearance of bacteria in human and murine macrophages [188, 189].
Carbamazepine Induces antimicrobial autophagy through a mTOR-independent pathway controlled by cellular depletion of myo-inositol [193].
Fluoxetine A selective serotonin reuptake inhibitor, enhances secretion of proinflammatory cytokine TNF-α and induces autophagy in Mycobacterium tuberculosis–infected macrophages [194].
Gefitinib An inhibitor of the Epidermal Growth Factor Receptor (EGFR), activates autophagy, and restricts growth of Mycobacterium tuberculosis in the lungs of infected mice [194].
Isoniazid or pyrazinamide Reduces Mycobacterium tuberculosis (Mtb)–induced proinflammatory responses by promoting autophagy activation and phagosomal maturation in Mtb-infected host cells [190].
Nitozoxanide Nitazoxanide and its active metabolite Tizoxanide strongly stimulate autophagy and inhibit mTORC1 signaling and intracellular proliferation of Mycobacterium tuberculosis [195].
Nortriptyline Induces the formation of autophagosomes that progressively acidify over time and become competent for Mycobacterium tuberculosis degradation in infected macrophages [196].
Prochlorperazine edisylate Modulates autophagy that correlates with delivery of Mycobacterium tuberculosis to lysosomes leading to mycobacterial degradation [196].
Rapamycin Induces autophagy and suppresses intracellular survival of M. tuberculosis [192].
Statins Enhances autophagy and phagosome maturation leading to reduction the Mycobacterium tuberculosis burden in human macrophages and in mice [197].
Valproic acid Stimulates autophagic killing of intracellular Mycobacterium tuberculosis within primary human macrophages [193].

Table 5.

Preclinical studies of the effects of autophagy activators on infectious diseases.

Remarkably, several FDA-approved drugs counter M. tuberculosis infection, possibly through autophagy, which disrupts the host-pathogen equilibrium in favor of the host (see Table 5). The antidepressants Fluoxetine [194] and Nortriptyline [196], the anticonvulsants Carbamazepine and Valproic acid, and the antipsychotic Prochlorperazine edisylate reveal relevant antimycobacterial properties by targeting autophagy in the host (i.e., infected macrophages). Notably, in mice infected with a highly virulent MDR-strain of Mycobacterium tuberculosis, Carbamazepine reduces bacterial burden, improve lung pathology, and stimulate adaptive immunity [193]. Furthermore, the tyrosine kinase inhibitor (Gefitinib) also activates autophagy and suppress Mycobacterium tuberculosis in macrophages and, to some extent, in infected mice [194]. Other autophagy-inducing candidate drugs attempting to Antituberculosis Host-Directed Therapy (HDT) include antiprotozoal drug Nitozoxanide [195] and cholesterol-lowering drugs, i.e., Statin [197]. Together these findings support that autophagy enhancement by repurposed drugs provides an easily implementable potential therapy for the treatment of multidrug-resistant mycobacterial infection.


8. Promising future therapeutic strategies

8.1. The case of the pentacyclic triterpenoids, the working hypothesis

After decades of scientific discoveries and discussions [10, 198] the general agreement is that autophagy associated cell-death is commonly linked to failure in either the fusion of autophagosomes with lysosomes or in the digestion activity of autolysosomes [27, 198]. Whereas the understanding of this process at the molecular level needs a deeper knowledge of the competition between its activation and inhibition pathways, autophagy has been explored as a potential therapeutically target for treating several diseases [11, 14]. Consequently, the impact of activating mitophagy on the condition of autophagy impairment is a noteworthy subject to explore. A recent work has proposed that by modulating parallel damage in membranes of mitochondria and lysosome, autophagy turns into a destructive process [12]. Comparative analysis of the biological effects of two chemical isomers, i.e., pentacyclic triterpenoids Betulinic and Oleanolic acids (BA and OA, respectively), Martins and colleagues showed that the main differences between the activity of BA and OA is due to their efficiency in interacting and damaging membranes [12] (see Figure 1). These triterpenoids are new promising drugs with various pharmacological actions (anti-inflammatory, antiviral, antifungal, antimalarial, among others), being easily extracted from plants [199]. So far, about 2167 patents for AB and 1018 for OA have been deposited.

Figure 1.

Modulation of membrane damage provided insights into biological effects of triterpenoids in vitro (Figure kindly supplied by Martins WK).

By comparing these triterpenoids, it was realized that the fate of autophagy may depend on the extent of lysosomal and mitochondrial membrane damage. In case of OA, there is marked cytoplasmic vacuolization and mitochondria shrinkage with remarkable cellular recovery that was intrinsically associated with autophagy activation. However, cell recovery failed upon concomitant lysosome inhibition with CQ or Bafilomycin A1 [12]. Of note, the lysosomal damage BA-mediated is per se capable of compromising autophagy, without any incremental damage when lysosomal function was deeply altered by lysosomal inhibitors, such as CQ and Bafilomycin A1.

BA and OA differ significantly on their ability to penetrate membranes, which appears to be mainly related to the twisted backbone structure of OA, in contrast to the fully planar structure of BA. Interestingly, this stronger efficiency in interacting and damaging membrane mimics ascribed to BA correlates with a higher ability of disturbing mitochondrial and lysosomal membranes of human keratinocytes [12]. The ability of BA to disturb the mitochondrial membrane is in agreement with other published results [106, 200, 201]. For example, by inhibiting the activity of steroyl-CoA-desaturase (SCD-1) BA may also directly and rapidly impact on the saturation level of cardiolipin (CL), a specific mitochondrial phospholipid lipid that has important structural and metabolic functions, and at the same time regulates mitochondria-dependent cell death [202]. Interestingly, thermodynamic analyses of Langmuir monolayers and AFM study of Langmuir-Blodgett monolayers provide insights into the ability of BA interacting with CL-enriched membranes. BA may orient nearly perpendicularly with hydroxyl group toward water, which causes phase separation and changes the permeability of CL film [203]. BA was also shown to disrupt membranes of human red blood cells (RBC) in vitro, with release of calcein from the RBC ghosts in a way similar to Digitonin in membrane permeabilization experiments [204].

Of note, the damage in lysosomal function caused by BA may not be explained by traditional justifications (lack of lysosome acidification or neutralization of its internal pH). Otherwise, BA disturbs lysosome’s membrane integrity that dramatically jeopardizes the lysosomal function, leading to a lysosomal-mitochondrial axis of cellular stress that causes autophagy-associated cell death [12]. Remarkably, in the survival of BA-challenged cells occurs sustained formation of reactive oxygen species (ROS) inside nonfunctional lysosomes, which in the long-term response leads to lipofuscinogenesis, genomic instability, and cell senescence [13]. Thus, promotion of concomitant damage in mitochondrial and lysosomal membranes seems to be an efficient strategy for inducing autophagy-associated cell death and cell aging.

The AB’s ability to promote parallel damage in lysosomes and mitochondria could be the explication for the positive synergistic action of BA in different antitumor protocols including radiation [205, 206], chemotherapy drugs, such as Cisplatin [207] and Vincristine [208]. Therefore, the possible increase of cell death potentially relates to the AB ability of suppressing prosurvival autophagy. The knowledge of this premise at molecular level may contribute to the development of new autophagy modulators.

Since 1995, BA has been considered as a highly promising anticancer drug showing remarkable antitumor effects against several human tumors [106, 208222]. In addition, in the last decade, many studies have shown further effects that justify the expectation that triterpenes and synthetic analogs are useful to treat cancer by several modes of action [223]. For example, BA acid derivatives are under evaluation as chemotherapeutic agent against several types of human tumors in vitro and in vivo [152, 199, 223235]. The synthetic analog of OA [2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO)] is currently under clinical Phase I study (NCT00322140) for treating solid tumors and lymphoma [101]. Introduced in 2009, the new semisynthetic candidate drug designated NVX-207 (3-acetyl-betulinic 2-amino-3-hydroxy-2-hydroxymethyl-hi-ethyl propanoate) enhanced apoptosis-inducing activity and dramatically enhanced solubility over BA [230]. However, limited solubility and often ultimately modest efficacy have hampered the development of this class of compounds [230]. Thus, scientific efforts focused on the elucidation of molecular mechanisms triggered by these triterpenoids, attending the interests of the scientific community as well as of the pharmaceutical industry.


9. Perspectives for drug development

During the last decade, important progress was made in understanding the molecular basis of autophagy uncovering its potential in anticancer therapies [105, 236, 237]. Because the abrogation of autophagy via knockdown of autophagy-related molecules increases the sensibility of therapy-resistant cancer cells to conventional cancer therapies, there has been great interest in developing clinically relevant autophagy inhibitors [238]. As reviewed by Yang and colleagues, multiple studies have shown that genetic knockdown of autophagy-related genes (Atgs) or pharmacological inhibition of autophagy can effectively enhance tumor cell death induced by diverse anticancer drugs in preclinical models [64].

Figure 2.

Modulation of membrane damage provided insights into biological effects after PDT (Figure kindly supplied by Martins WK).

Photodynamic therapy (PDT) is a procedure that has applications in the selective eradication of cancer where sites of tumor lesions are clearly delineated. It is a two- step process whereby cells are first incubated with photosensitizers and then photoirradiated. This results in the formation of singlet molecular oxygen and other reactive oxygen species (ROS) that can cause photodamage at sites where the photosensitizing agent has localized [239]. Photosensitizers found to be clinically useful, showing affinity for the endoplasmic reticulum, mitochondria, lysosomes, or combinations of these sites [240]. The induction of cell death triggered by apoptosis and/or autophagy in photosensitized cells is a common outcome of PDT [239242]. Therefore, the photosensitizers are drugs that are used to treat a series of different diseases. Our group has addressed the concept of parallel photodamage in mitochondria and lysosome with the consequent induction of cell death and senescence after PDT (Figure 2). In near future, we will exploit the possible use of this concept in the development of new photosensitizers targeting autophagy as cell death mechanism.


  1. 1. Glick D, Barth S, Macleod KF. Autophagy: cellular and molecular mechanisms. J Pathol. 2010 May;221(1):3–12.
  2. 2. Reggiori F, Komatsu M, Finley K, Simonsen A. Autophagy: More Than a Nonselective Pathway. Int J Cell Biol. 2012 Jan;2012 (Figure 1):1–18.
  3. 3. Ding W-X, Yin X-M. Mitophagy: mechanisms, pathophysiological roles, and analysis. Biol Chem. 2012 Jan 1;393(7):547–64.
  4. 4. May AI, Devenish RJ, Prescott M. The many faces of mitochondrial autophagy: making sense of contrasting observations in recent research. Int J Cell Biol. 2012;2012:431684.
  5. 5. Lemasters JJ. Selective mitochondrial autophagy, or mitophagy, as a targeted defense against oxidative stress, mitochondrial dysfunction, and aging. Rejuvenation Res. 2005 Mar;8(1):3–5.
  6. 6. Reggiori F, Komatsu M, Finley K, Simonsen A. Selective types of autophagy. Int J Cell Biol. 2012 Jan;2012:156272.
  7. 7. Youle RJ, Narendra DP. Mechanisms of mitophagy. Nat Rev Mol Cell Biol. Nature Publishing Group; 2011 Jan;12(1):9–14.
  8. 8. Vessoni AT, Filippi-Chiela EC, Menck CF, Lenz G. Autophagy and genomic integrity. Cell Death Differ. 2013 Nov;20(11):1444–54.
  9. 9. Lu H, Li G, Liu L, Feng L, Wang X, Jin H. Regulation and function of mitophagy in development and cancer. Autophagy. 2013 Nov 1;9(11):1720–36.
  10. 10. Levine B, Kroemer G. Autophagy in the pathogenesis of disease. Cell. Elsevier; 2008 Jan 11;132(1):27–42.
  11. 11. Cheng Y, Ren X, Hait WN, Yang J-M. Therapeutic targeting of autophagy in disease: biology and pharmacology. Pharmacol Rev. 2013;65(4):1162–97.
  12. 12. Martins WK, Costa ÉT, Cruz MC, Stolf BS, Miotto R, Cordeiro RM, et al. Parallel damage in mitochondrial and lysosomal compartments promotes efficient cell death with autophagy: The case of the pentacyclic triterpenoids. Sci Rep. 2015 Jul 27;5:12425.
  13. 13. Martins WK, Gomide AB, Junqueira H, Stolf BS, Itri R, Baptista MS. Membrane damage by Betulinic Acid provides insights into cellular aging. Biochimica et Biophysica Acta (BBA) - General Subjects, in press, 10.1016/j.bbagen.2016.10.018
  14. 14. Rubinsztein DC, Levine PC and B. Autophagy modulation as a potential therapeutic target for diverse diseases. Nat Rev Drug Discov. 2012;29(6):709–30.
  15. 15. Pellegrini P, Strambi A, Zipoli C, Hägg-Olofsson M, Buoncervello M, Linder S, et al. Acidic extracellular pH neutralizes the autophagy-inhibiting activity of chloroquine: Implications for cancer therapies. Autophagy. 2014;10(4):1–10.
  16. 16. Mathew R, Karantza-Wadsworth V, White E. Role of autophagy in cancer. Nat Rev Cancer. 2007;7(12):961–7.
  17. 17. Choi KS. Autophagy and cancer. Exp Mol Med. 2012;44(2):109.
  18. 18. Johri A, Beal MF. Mitochondrial dysfunction in neurodegenerative diseases. J Pharmacol Exp Ther. 2012 Sep;342(3):619–30.
  19. 19. Cherra SJ, Dagda RK, Chu CT. Review: Autophagy and neurodegeneration: Survival at a cost? Vol. 36, Neuropath Appl Neuro. 2010. p. 125–32.
  20. 20. Casarejos MJ, Solano RM, Gómez a., Perucho J, De Yébenes JG, Mena M a. The accumulation of neurotoxic proteins, induced by proteasome inhibition, is reverted by trehalose, an enhancer of autophagy, in human neuroblastoma cells. Neurochem Int. 2011;58(4):512–20.
  21. 21. Nixon R a. The role of autophagy in neurodegenerative disease. Nat Med. Nature Publishing Group; 2013;19(8):983–97.
  22. 22. Batlevi Y, La Spada AR. Mitochondrial autophagy in neural function, neurodegenerative disease, neuron cell death, and aging. Vol. 43, Neurobiol Dis. 2011. p. 46–51.
  23. 23. Sarkar S, Ravikumar B, Rubinsztein DC. Chapter 5 Autophagic clearance of aggregate-prone proteins associated with neurodegeneration. 1st ed. Vol. 453, Methods in Enzymology. Elsevier Inc.; 2009. 83–110 p.
  24. 24. Kiselyov K, Jennigs JJ, Rbaibi Y, Chu CT. Autophagy, mitochondria and cell death in lysosomal storage diseases. Autophagy. 2007;3(3):259–62.
  25. 25. Appelqvist H, Wäster P, Kågedal K, Öllinger K. The lysosome: from waste bag to potential therapeutic target. J Mol Cell Biol. 2013 Aug;5(4):214–26.
  26. 26. Ordonez MP. Defective mitophagy in human Niemann-Pick type C1 neurons is due to abnormal autophagy activation. Autophagy. 2012;8(7):1157–8.
  27. 27. Kroemer G, Levine B. Autophagic cell death: the story of a misnomer. Nat Rev Mol Cell Biol. 2008 Dec;9(12):1004–10.
  28. 28. Seglen PO, Gordon PB. 3-Methyladenine: specific inhibitor of autophagic/lysosomal protein degradation in isolated rat hepatocytes. Proc Natl Acad Sci U S A. 1982;79(6):1889–92.
  29. 29. Klionsky DJ, Abdelmohsen K, Abe A, Abedin MJ, Abeliovich H, Acevedo Arozena A, et al. Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy. 2016;12(1):1–222.
  30. 30. Blommaart EF, Krause U, Schellens JP, Vreeling-Sindelárová H, Meijer a J. The phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 inhibit autophagy in isolated rat hepatocytes. Eur J Biochem. 1997;243:240–6.
  31. 31. De Mei C, Ercolani L, Parodi C, Veronesi M, Lo Vecchio C, Bottegoni G, et al. Dual inhibition of REV-ERBβ and autophagy as a novel pharmacological approach to induce cytotoxicity in cancer cells. Oncogene. 2015 May 14;34(20):2597–608.
  32. 32. Torrente E, Parodi C, Ercolani L, De Mei C, Ferrari A, Scarpelli R, et al. Synthesis and in vitro anticancer activity of the first class of dual inhibitors of REV-ERBβ and autophagy. J Med Chem. 2015 Aug 13;58(15):5900–15.
  33. 33. Yoshimori T, Yamamoto A, Moriyama Y, Futai M, Tashiro Y. Bafilomycin A1, a specific inhibitor of vacuolar-type H + −ATPase, inhibits acidification and protein degradation in lysosomes of cultured cells. J Biol Chem. 1991;266(26):17707–12.
  34. 34. Yamamoto a, Tagawa Y, Yoshimori T, Moriyama Y, Masaki R, Tashiro Y. Bafilomycin A1 prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes in rat hepatoma cell line, H-4-II-E cells. Cell Struct Funct. 1998;23(1):33–42.
  35. 35. Klionsky DJ, Elazar Z, Seglen PO, Rubinsztein DC. Does bafilomycin A1 block the fusion of autophagosomes with lysosomes? Vol. 4, Autophagy. 2008. p. 849–50.
  36. 36. Murata M, Miyashita S, Yokoo C, Tamai M, Hanada K, Hatayama K, et al. Novel epoxysuccinyl peptides. Selective inhibitors of cathepsin B, in vitro. FEBS Lett. 1991 Mar 25;280(2):307–10.
  37. 37. Seglen PO, Grinde B, Solheim a E. Inhibition of the lysosomal pathway of protein degradation in isolated rat hepatocytes by ammonia, methylamine, chloroquine and leupeptin. Eur J Biochem. 1979;95(2):215–25.
  38. 38. Ju JS, Varadhachary AS, Miller SE, Weihl CC. Quantitation of “autophagic flux” in mature skeletal muscle. Autophagy. 2010;6(7):929–35.
  39. 39. Rossi M, Munarriz ER, Bartesaghi S, Milanese M, Dinsdale D, Guerra-Martin MA, et al. Desmethylclomipramine induces the accumulation of autophagy markers by blocking autophagic flux. J Cell Sci. 2009;122(18):3330–9.
  40. 40. Tamai M, Matsumoto K, Omura S, Koyama I, Ozawa Y, Hanada K. In vitro and in vivo inhibition of cysteine proteinases by EST, a new analog of E-64. J Pharmacobiodyn. 1986 Aug;9(8):672–7.
  41. 41. Vanrell MC, Cueto J a, Barclay JJ, Carrillo C, Colombo MI, Gottlieb R a, et al. Polyamine depletion inhibits the autophagic response modulating Trypanosoma cruzi infectivity. Autophagy. 2013;9(7):1080–93.
  42. 42. Nishimura Y, Kato K, Furuno K, Himeno M. Inhibitory effect of leupeptin on the intracellular maturation of lysosomal cathepsin L in primary cultures of rat hepatocytes. Biol Pharm Bull. 1995 Jul;18(7):945–50.
  43. 43. Kirschke H, Langner J, Wiederanders B, Ansorge S, Bohley P, Broghammer U. Intracellular protein breakdown. VII. Cathepsin L and H; two new proteinases from rat liver lysosomes. Acta Biol Med Ger. 1976;35(3–4):285–99.
  44. 44. Ahlberg J, Berkenstam A, Henell F, Glaumann H. Degradation of short and long lived proteins in isolated rat liver lysosomes. Effects of pH, temperature, and proteolytic inhibitors. J Biol Chem. 1985;260(9):5847–54.
  45. 45. Carew JS, Espitia CM, Esquivel J a., Mahalingam D, Kelly KR, Reddy G, et al. Lucanthone is a novel inhibitor of autophagy that induces cathepsin d-mediated apoptosis. J Biol Chem. 2011;286(8):6602–13.
  46. 46. Kondo Y, Kanzawa T, Sawaya R, Kondo S. The role of autophagy in cancer development and response to therapy. Nat Rev Cancer. 2005;5(9):726–34.
  47. 47. Xie R, Nguyen S, McKeehan WL, Liu L. Acetylated microtubules are required for fusion of autophagosomes with lysosomes. BMC Cell Biol. 2010;11(1):89.
  48. 48. Leu JI-J, Pimkina J, Frank A, Murphy ME, George DL. A small molecule inhibitor of inducible heat shock protein 70. Mol Cell. Elsevier Ltd; 2009;36(1):15–27.
  49. 49. Noh HS, Shin IW, Ha JH, Hah Y-S, Baek SM, Kim DR. Propofol protects the autophagic cell death induced by the ischemia/reperfusion injury in rats. Mol Cells. 2010 Nov;30(5):455–60.
  50. 50. Cui D, Wang L, Qi A, Zhou Q, Zhang X, Jiang W. Propofol prevents autophagic cell death following oxygen and glucose deprivation in PC12 cells and cerebral ischemia-reperfusion injury in rats. PLoS One. 2012;7(4).
  51. 51. Cui DR, Wang L, Jiang W, Qi AH, Zhou QH, Zhang XL. Propofol prevents cerebral ischemia-triggered autophagy activation and cell death in the rat hippocampus through the NF-kB/p53 signaling pathway. Neuroscience. 2013 Aug 29;246:117–32.
  52. 52. Liu J, Xia H, Kim M, Xu L, Li Y, Zhang L, et al. Beclin1 controls the levels of p53 by regulating the deubiquitination activity of USP10 and USP13. Cell. Elsevier Inc.; 2011;147(1):223–34.
  53. 53. Gordon PB, Holen I, Fosse M, Røtnes JS, Seglen PO. Dependence of hepatocytic autophagy on intracellularly sequestered calcium. J Biol Chem. 1993;268(35):26107–12.
  54. 54. Engedal N, Torgersen ML, Guldvik IJ, Barfeld SJ, Bakula D, Saetre F, et al. Modulation of intracellular calcium homeostasis blocks autophagosome formation. Autophagy. 2013;9(10):1475–90.
  55. 55. Ganley IG, Wong P-M, Gammoh N, Jiang X. Distinct autophagosomal-lysosomal fusion mechanism revealed by thapsigargin-induced autophagy arrest. Mol Cell. 2011 Jun;42(6):731–43.
  56. 56. Lu Y, Dong S, Hao B, Li C, Zhu K, Guo W, et al. Vacuolin-1 potently and reversibly inhibits autophagosome-lysosome fusion by activating RAB5A. Autophagy. 2014;10(11):1895–905.
  57. 57. Yoon YH, Cho KS, Hwang JJ, Lee SJ, Choi J a., Koh JY. Induction of lysosomal dilatation, arrested autophagy, and cell death by chloroquine in cultured ARPE-19 cells. Investig Ophthalmol Vis Sci. 2010;51(11):6030–7.
  58. 58. Maycotte P, Aryal S, Cummings CT, Thorburn J, Morgan MJ, Thorburn A. Chloroquine sensitizes breast cancer cells to chemotherapy independent of autophagy. Autophagy. 2012;8(2):200–12.
  59. 59. Schonewolf C a, Mehta M, Schiff D, Wu H, Haffty BG, Karantza V, et al. Autophagy inhibition by chloroquine sensitizes HT-29 colorectal cancer cells to concurrent chemoradiation. World J Gastrointest Oncol. 2014;6(3):74–82.
  60. 60. Geng Y, Kohli L, Klocke BJ, Roth K a. Chloroquine-induced autophagic vacuole accumulation and cell death in glioma cells is p53 independent. Neuro Oncol. 2010 May;12(5):473–81.
  61. 61. Mauvezin C, Nagy P, Juhász G, Neufeld TP. Autophagosome—lysosome fusion is independent of V-ATPase-mediated acidification. Nat Commun. 2015;6(May):7007.
  62. 62. Montaser M, Lalmanach G, Mach L. CA-074, but not its methyl ester CA-074Me, is a selective inhibitor of cathepsin B within living cells. Biol Chem. 2002;383(7–8):1305–8.
  63. 63. Amaravadi RK, Lippincott-Schwartz J, Yin XM, Weiss W a., Takebe N, Timmer W, et al. Principles and current strategies for targeting autophagy for cancer treatment. Clin Cancer Res. 2011;17(4):654–66.
  64. 64. Yang ZJ, Chee CE, Huang S, Sinicrope F a. The role of autophagy in cancer: therapeutic implications. Mol Cancer Ther. 2011;10(9):1533–41.
  65. 65. Tsvetkov AS, Miller J, Arrasate M, Wong JS, Pleiss M a, Finkbeiner S. A small-molecule scaffold induces autophagy in primary neurons and protects against toxicity in a Huntington disease model. Proc Natl Acad Sci U S A. 2010;107(39):16982–7.
  66. 66. Joo JH, Dorsey FC, Joshi A, Hennessy-Walters KM, Rose KL, McCastlain K, et al. Hsp90-Cdc37 chaperone complex regulates Ulk1- and Atg13-mediated mitophagy. Mol Cell. 2011 Aug;43(4):572–85.
  67. 67. Colomer D, Rosich L. Counteracting autophagy overcomes resistance to everolimus in mantle cell lymphoma. Clin Cancer Res. 2012;18(19):5278–89.
  68. 68. Papp D, Kovács T, Billes V, Varga M, Tarnóci A, Hackler L, et al. AUTEN-67, an autophagy-enhancing drug candidate with potent antiaging and neuroprotective effects. Autophagy. 2015 Aug 27;
  69. 69. Chresta CM, Davies BR, Hickson I, Harding T, Cosulich S, Critchlow SE, et al. AZD8055 is a potent, selective, and orally bioavailable ATP-competitive mammalian target of rapamycin kinase inhibitor with in vitro and in vivo antitumor activity. Cancer Res. 2010;70(1):288–98.
  70. 70. García-Martínez JM, Moran J, Clarke RG, Gray A, Cosulich SC, Chresta CM, et al. Ku-0063794 is a specific inhibitor of the mammalian target of rapamycin (mTOR). Biochem J. 2009;421(1):29–42.
  71. 71. Nyfeler B, Bergman P, Triantafellow E, Wilson CJ, Zhu Y, Radetich B, et al. Relieving autophagy and 4EBP1 from rapamycin resistance. Mol Cell Biol. 2011;31(14):2867–76.
  72. 72. Soucy TA, Smith PG, Milhollen MA, Berger AJ, Gavin JM, Adhikari S, et al. An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer. Nature. 2009 Apr 9;458(7239):732–6.
  73. 73. Yang D, Zhao Y, Liu J, Sun Y, Jia L. Protective autophagy induced by RBX1/ROC1 knockdown or CRL inactivation via modulating the DEPTOR-MTOR axis. Autophagy. 2012 Dec 27;8(12):1856–8.
  74. 74. Zhao Y, Xiong X, Jia L, Sun Y. Targeting Cullin-RING ligases by MLN4924 induces autophagy via modulating the HIF1-REDD1-TSC1-mTORC1-DEPTOR axis. Cell Death Dis. Nature Publishing Group; 2012;3(9):e386.
  75. 75. Pereira GJS, Hirata H, Fimia GM, do Carmo LG, Bincoletto C, Han SW, et al. Nicotinic acid adenine dinucleotide phosphate (NAADP) regulates autophagy in cultured astrocytes. J Biol Chem. 2011;286(32):27875–81.
  76. 76. Liu T-J, Koul D, LaFortune T, Tiao N, Shen RJ, Maira S-M, et al. NVP-BEZ235, a novel dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor, elicits multifaceted antitumor activities in human gliomas. Mol Cancer Ther. 2009;8(8):2204–10.
  77. 77. Serra V, Markman B, Scaltriti M, Eichhorn PJ a, Valero V, Guzman M, et al. NVP-BEZ235, a dual PI3K/mTOR inhibitor, prevents PI3K signaling and inhibits the growth of cancer cells with activating PI3K mutations. Cancer Res. 2008;68(16):8022–30.
  78. 78. East DA, Fagiani F, Crosby J, Georgakopoulos ND, Bertrand H, Schaap M, et al. PMI: A ΔΨm independent pharmacological regulator of mitophagy. Chem Biol. 2014;21(11):1585–96.
  79. 79. Apsel B, Blair JA, Gonzalez B, Nazif TM, Feldman ME, Aizenstein B, et al. Targeted polypharmacology: discovery of dual inhibitors of tyrosine and phosphoinositide kinases. Nat Chem Biol. 2008 Nov 12;4(11):691–9.
  80. 80. Hsieh AC, Costa M, Zollo O, Davis C, Feldman ME, Testa JR, et al. Genetic Dissection of the Oncogenic mTOR Pathway Reveals Druggable Addiction to Translational Control via 4EBP-eIF4E. Cancer Cell. 2010 Mar;17(3):249–61.
  81. 81. Feldman ME, Apsel B, Uotila A, Loewith R, Knight ZA, Ruggero D, et al. Active-site inhibitors of mTOR target rapamycin-resistant outputs of mTORC1 and mTORC2. PLoS Biol. 2009;7(2):e38.
  82. 82. Brown EJ, Albers MW, Shin TB, Ichikawa K, Keith CT, Lane WS, et al. A mammalian protein targeted by G1-arresting rapamycin-receptor complex. Nature. 1994 Jun 30;369(6483):756–8.
  83. 83. Pirola L, Fröjdö S. Resveratrol: one molecule, many targets. IUBMB Life. 2008 May;60(5):323–32.
  84. 84. Puissant A, Auberger P. AMPK- and p62/SQSTM1-dependent autophagy mediate resveratrol-induced cell death in chronic myelogenous leukemia. Autophagy. 2010 Jul;6(5):655–7.
  85. 85. Vingtdeux V, Giliberto L, Zhao H, Chandakkar P, Wu Q, Simon JE, et al. AMP-activated protein kinase signaling activation by resveratrol modulates amyloid- peptide metabolism. J Biol Chem. 2010;285(12):9100–13.
  86. 86. Vingtdeux V, Chandakkar P, Zhao H, D’Abramo C, Davies P, Marambaud P. Novel synthetic small-molecule activators of AMPK as enhancers of autophagy and amyloid—peptide degradation. FASEB J. 2011;25(1):219–31.
  87. 87. Wong VK, Li T, Law BY, Ma ED, Yip NC, Michelangeli F, et al. Saikosaponin-d, a novel SERCA inhibitor, induces autophagic cell death in apoptosis-defective cells. Cell Death Dis. 2013 Jul;4(7):e720.
  88. 88. Shoji-Kawata S, Sumpter R, Leveno M, Campbell GR, Zou Z, Kinch L, et al. Identification of a candidate therapeutic autophagy-inducing peptide. Nature. 2013;494(7436):201–6.
  89. 89. Liu Y, Shoji-Kawata S, Sumpter RM, Wei Y, Ginet V, Zhang L, et al. Autosis is a Na+,K + −ATPase-regulated form of cell death triggered by autophagy-inducing peptides, starvation, and hypoxia-ischemia. Proc Natl Acad Sci U S A. 2013 Dec 17;110(51):20364–71.
  90. 90. Frederick C, Ando K, Leroy K, Héraud C, Suain V, Buée L, et al. Rapamycin ester analog CCI-779/Temsirolimus alleviates tau pathology and improves motor deficit in mutant tau transgenic mice. J Alzheimers Dis. 2015;44(4):1145–56.
  91. 91. Jiang T, Yu J-T, Zhu X-C, Zhang Q-Q, Cao L, Wang H-F, et al. Temsirolimus attenuates tauopathy in vitro and in vivo by targeting tau hyperphosphorylation and autophagic clearance. Neuropharmacology. 2014 Oct;85:121–30.
  92. 92. Jiang T, Yu J-T, Zhu X-C, Tan M-S, Wang H-F, Cao L, et al. Temsirolimus promotes autophagic clearance of amyloid-β and provides protective effects in cellular and animal models of Alzheimer’s disease. Pharmacol Res. 2014 Mar;81:54–63.
  93. 93. Zhang L, Dai F, Cui L, Jing H, Fan P, Tan X, et al. Novel role for TRPC4 in regulation of macroautophagy by a small molecule in vascular endothelial cells. Biochim Biophys Acta. 2015 Feb;1853(2):377–87.
  94. 94. Thoreen CC, Kang SA, Chang JW, Liu Q, Zhang J, Gao Y, et al. An ATP-competitive mammalian target of rapamycin inhibitor reveals rapamycin-resistant functions of mTORC1. J Biol Chem. 2009;284(12):8023–32.
  95. 95. Sarkar S, Davies JE, Huang Z, Tunnacliffe A, Rubinsztein DC. Trehalose, a novel mTOR-independent autophagy enhancer, accelerates the clearance of mutant huntingtin and ?-synuclein. J Biol Chem. 2007;282(8):5641–52.
  96. 96. Zhang X, Chen S, Song L, Tang Y, Shen Y, Jia L, et al. MTOR-independent, autophagic enhancer trehalose prolongs motor neuron survival and ameliorates the autophagic flux defect in a mouse model of amyotrophic lateral sclerosis. Autophagy. 2014;10(4):588–602.
  97. 97. Carpenter JE, Jackson W, Benetti L, Grose C. Autophagosome formation during varicella-zoster virus infection following endoplasmic reticulum stress and the unfolded protein response. J Virol. 2011;85(18):9414–24.
  98. 98. Yu K, Shi C, Toral-Barza L, Lucas J, Shor B, Kim JE, et al. Beyond rapalog therapy: preclinical pharmacology and antitumor activity of WYE-125132, an ATP-competitive and specific inhibitor of mTORC1 and mTORC2. Cancer Res. 2010;70(2):621–31.
  99. 99. Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, et al. Cancer incidence and mortality worldwide: Sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer. 2015 Mar 1;136(5):E359–86.
  100. 100. Boyle P LB. World Cancer Report 2008. IARC Press. :510p.
  101. 101. National Institutes of Health. Clinical Trial. Gov: A service of the U.S. National Institutes of Health. 2016.
  102. 102. Amaravadi RK, Lippincott-Schwartz J, Yin XM, Weiss W a, Takebe N, Timmer W, et al. Principles and current strategies for targeting autophagy for cancer treatment. Vol. 17, Clinical Cancer Research. 2011. p. 654–66.
  103. 103. Vakifahmetoglu-norberg H, Xia H, Yuan J. Pharmacologic agents targeting autophagy. J Clin Invest. 2015;125(1):5–13.
  104. 104. Liu J, Lin M, Yu J, Liu B, Bao J. Targeting apoptotic and autophagic pathways for cancer therapeutics. Cancer Lett. 2011 Jan 28;300(2):105–14.
  105. 105. Dalby KN, Tekedereli I, Lopez-Berestein G, Ozpolat B. Targeting the prodeath and prosurvival functions of autophagy as novel therapeutic strategies in cancer. Vol. 6, Autophagy. 2010. p. 322–9.
  106. 106. Potze L, Mullauer FB, Colak S, Kessler JH, Medema JP. Betulinic acid-induced mitochondria-dependent cell death is counterbalanced by an autophagic salvage response. Cell Death Dis. Nature Publishing Group; 2014 Jan;5(4):e1169.
  107. 107. Debnath J, Baehrecke EH, Kroemer G. Does autophagy contribute to cell death? Autophagy. 2005 Jul;1(2):66–74.
  108. 108. White E, DiPaola RS. The double-edged sword of autophagy modulation in cancer. Clin Cancer Res. 2009;15(17):5308–16.
  109. 109. Levy JMM, Thorburn A. Targeting autophagy during cancer therapy to improve clinical outcomes. Pharmacol Ther. Elsevier B.V.; 2011;131(1):130–41.
  110. 110. Takeuchi H, Kondo Y, Fujiwara K, Kanzawa T, Aoki H, Mills GB, et al. Synergistic augmentation of rapamycin-induced autophagy in malignant glioma cells by phosphatidylinositol 3-kinase/protein kinase B inhibitors. Cancer Res. 2005 May 15;65(8):3336–46.
  111. 111. Yazbeck VY, Buglio D, Georgakis G V, Li Y, Iwado E, Romaguera JE, et al. Temsirolimus downregulates p21 without altering cyclin D1 expression and induces autophagy and synergizes with vorinostat in mantle cell lymphoma. Exp Hematol. 2008 Apr;36(4):443–50.
  112. 112. Zhang H, Berel D, Wang Y, Li P, Bhowmick NA, Figlin RA, et al. A comparison of Ku0063794, a dual mTORC1 and mTORC2 inhibitor, and temsirolimus in preclinical renal cell carcinoma models. PLoS One. 2013;8(1):e54918.
  113. 113. Crazzolara R, Cisterne A, Thien M, Hewson J, Baraz R, Bradstock KF, et al. Potentiating effects of RAD001 (Everolimus) on vincristine therapy in childhood acute lymphoblastic leukemia. Blood. 2009;113(14):3297–306.
  114. 114. Rangwala R, Chang YC, Hu J, Algazy KM, Evans TL, Fecher LA, et al. Combined MTOR and autophagy inhibition: phase I trial of hydroxychloroquine and temsirolimus in patients with advanced solid tumors and melanoma. Autophagy. 2014;10(8):1391–402.
  115. 115. Milano V, Piao Y, LaFortune T, de Groot J. Dasatinib-induced autophagy is enhanced in combination with temozolomide in glioma. Mol Cancer Ther. 2009;8(2):394–406.
  116. 116. Shao Y, Gao Z, Marks PA, Jiang X. Apoptotic and autophagic cell death induced by histone deacetylase inhibitors. Proc Natl Acad Sci U S A. 2004;101(52):18030–5.
  117. 117. Liu Y-L, Yang P-M, Shun C-T, Wu M-S, Weng J-R, Chen C-C. Autophagy potentiates the anti-cancer effects of the histone deacetylase inhibitors in hepatocellular carcinoma. Autophagy. 2010 Nov;6(8):1057–65.
  118. 118. Yamamoto S, Tanaka K, Sakimura R, Okada T, Nakamura T, Li Y, et al. Suberoylanilide hydroxamic acid (SAHA) induces apoptosis or autophagy-associated cell death in chondrosarcoma cell lines. Anticancer Res. 2008;28 (3 A):1585–91.
  119. 119. Min A, Im S-A, Kim DK, Song S-H, Kim H-J, Lee K-H, et al. Histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), enhances anti-tumor effects of the poly (ADP-ribose) polymerase (PARP) inhibitor olaparib in triple-negative breast cancer cells. Breast Cancer Res. ???; 2015;17(1):33.
  120. 120. Park JH, Ahn MY, Kim TH, Yoon S, Kang KW, Lee J, et al. A new synthetic HDAC inhibitor, MHY218, induces apoptosis or autophagy-related cell death in tamoxifen-resistant MCF-7 breast cancer cells. Invest New Drugs. 2012 Oct;30(5):1887–98.
  121. 121. Kim DE, Kim Y, Cho D-H, Jeong S-Y, Kim S-B, Suh N, et al. Raloxifene induces autophagy-dependent cell death in breast cancer cells via the activation of AMP-activated protein kinase. Mol Cells. 2015;38(2):138–44.
  122. 122. de Medina P, Silvente-Poirot S, Poirot M. Tamoxifen and AEBS ligands induced apoptosis and autophagy in breast cancer cells through the stimulation of sterol accumulation. Autophagy. 2009 Oct;5(7):1066–7.
  123. 123. Leng S, Hao Y, Du D, Xie S, Hong L, Gu H, et al. Ursolic acid promotes cancer cell death by inducing Atg5-dependent autophagy. Int J Cancer. 2013;133(12):2781–90.
  124. 124. Xavier CPR, Lima CF, Pedro DFN, Wilson JM, Kristiansen K, Pereira-Wilson C. Ursolic acid induces cell death and modulates autophagy through JNK pathway in apoptosis-resistant colorectal cancer cells. J Nutr Biochem. Elsevier Inc.; 2013;24(4):706–12.
  125. 125. Pinto Ribeiro Xavier C, Lima C, Rohde M, Pereira-Wilson C. 253 Autophagy triggered by ursolic acid synergistically enhances 5-fluorouracil induced cell death in HCT15 (MSI p53 mutant) colorectal cancer cells. Eur J Cancer Suppl. Elsevier Ltd; 2010 Jun;8(5):66–7.
  126. 126. Ellington AA, Berhow M, Singletary KW. Induction of macroautophagy in human colon cancer cells by soybean B-group triterpenoid saponins. Carcinogenesis. 2005 Jan;26(1):159–67.
  127. 127. Carew JS, Medina EC, Esquivel JA, Mahalingam D, Swords R, Kelly K, et al. Autophagy inhibition enhances vorinostat-induced apoptosis via ubiquitinated protein accumulation. J Cell Mol Med. 2010;14(10):2448–59.
  128. 128. Li J, Hou N, Faried A, Tsutsumi S, Kuwano H. Inhibition of autophagy augments 5-fluorouracil chemotherapy in human colon cancer in vitro and in vivo model. Eur J Cancer. 2010 Jul;46(10):1900–9.
  129. 129. Verschooten L, Barrette K, van Kelst S, Rubio Romero N, Proby C, de Vos R, et al. Autophagy inhibitor chloroquine enhanced the cell death inducing effect of the flavonoid luteolin in metastatic squamous cell carcinoma cells. PLoS One. 2012;7(10):1–11.
  130. 130. Poklepovic A, Gewirtz DA. Outcome of early clinical trials of the combination of hydroxychloroquine with chemotherapy in cancer. Autophagy. 2014;10(8):1478–80.
  131. 131. Liang X, Tang J, Liang Y, Jin R, Cai X. Suppression of autophagy by chloroquine sensitizes 5-fluorouracil-mediated cell death in gallbladder carcinoma cells. Cell Biosci. Cell & Bioscience; 2014;4(1):10.
  132. 132. Koh Y, Lim HY, Ahn JH, Lee J-L, Rha SY, Kim YJ, et al. Phase II trial of everolimus for the treatment of nonclear-cell renal cell carcinoma. Ann Oncol. 2013 Apr;24(4):1026–31.
  133. 133. Seiler M, Ray-Coquard I, Melichar B, Yardley DA, Wang RX, Dodion PF, et al. Oral ridaforolimus plus trastuzumab for patients with HER2+ trastuzumab-refractory metastatic breast cancer. Clin Breast Cancer. 2015 Feb;15(1):60–5.
  134. 134. Bendell JC, Jones SF, Hart L, Spigel DR, Lane CM, Earwood C, et al. A phase Ib study of linsitinib (OSI-906), a dual inhibitor of IGF-1R and IR tyrosine kinase, in combination with everolimus as treatment for patients with refractory metastatic colorectal cancer. Invest New Drugs. 2015 Feb;33(1):187–93.
  135. 135. Piha-Paul SA, Munster PN, Hollebecque A, Argilés G, Dajani O, Cheng JD, et al. Results of a phase 1 trial combining ridaforolimus and MK-0752 in patients with advanced solid tumours. Eur J Cancer. 2015 Sep;51(14):1865–73.
  136. 136. Zibelman M, Wong Y-N, Devarajan K, Malizzia L, Corrigan A, Olszanski AJ, et al. Phase I study of the mTOR inhibitor ridaforolimus and the HDAC inhibitor vorinostat in advanced renal cell carcinoma and other solid tumors. Invest New Drugs. 2015 Oct;33(5):1040–7.
  137. 137. Nemunaitis J, Hochster HS, Lustgarten S, Rhodes R, Ebbinghaus S, Turner CD, et al. A phase I trial of oral ridaforolimus (AP23573; MK-8669) in combination with bevacizumab for patients with advanced cancers. Clin Oncol (R Coll Radiol). 2013 Jun;25(6):336–42.
  138. 138. Oza AM, Pignata S, Poveda A, McCormack M, Clamp A, Schwartz B, et al. Randomized phase II trial of ridaforolimus in advanced endometrial carcinoma. J Clin Oncol. 2015 Nov 1;33(31):3576–82.
  139. 139. Mita MM, Poplin E, Britten CD, Tap WD, Rubin EH, Scott BB, et al. Phase I/IIa trial of the mammalian target of rapamycin inhibitor ridaforolimus (AP23573; MK-8669) administered orally in patients with refractory or advanced malignancies and sarcoma. Ann Oncol. 2013;24(4):1104–11.
  140. 140. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. Elsevier Inc.; 2011 Mar 4;144(5):646–74.
  141. 141. Kim MJ, Woo SJ, Yoon CH, Lee JS, An S, Choi YH, et al. Involvement of autophagy in oncogenic K-Ras-induced malignant cell transformation. J Biol Chem. 2011;286(15):12924–32.
  142. 142. Yang S, Wang X, Contino G, Liesa M, Sahin E, Ying H, et al. Pancreatic cancers require autophagy for tumor growth. Genes Dev. 2011;25(7):717–29.
  143. 143. Eng CH, Wang Z, Tkach D, Toral-Barza L, Ugwonali S, Liu S, et al. Macroautophagy is dispensable for growth of KRAS mutant tumors and chloroquine efficacy. Proc Natl Acad Sci U S A. 2016 Jan 5;113(1):182–7.
  144. 144. Armstrong JL, Corazzari M, Martin S, Pagliarini V, Falasca L, Hill DS, et al. Oncogenic B-RAF signaling in melanoma impairs the therapeutic advantage of autophagy inhibition. Clin Cancer Res. 2011 Apr 15;17(8):2216–26.
  145. 145. Li J, Hou N, Faried A, Tsutsumi S, Takeuchi T, Kuwano H. Inhibition of autophagy by 3-MA enhances the effect of 5-FU-induced apoptosis in colon cancer cells. Ann Surg Oncol. 2009;16(3):761–71.
  146. 146. Giampietri C, Petrungaro S, Padula F, D’Alessio A, Marini ES, Facchiano A, et al. Autophagy modulators sensitize prostate epithelial cancer cell lines to TNF-alpha-dependent apoptosis. Apoptosis. 2012 Nov;17(11):1210–22.
  147. 147. Zhu K, Dunner K, McConkey DJ. Proteasome inhibitors activate autophagy as a cytoprotective response in human prostate cancer cells. Oncogene. 2010 Jan 21;29(3):451–62.
  148. 148. Wu Z, Chang P-C, Yang JC, Chu C-Y, Wang L-Y, Chen N-T, et al. Autophagy blockade sensitizes prostate cancer cells towards Src family kinase inhibitors. Genes Cancer. 2010;1(1):40–9.
  149. 149. Amaravadi RK, Yu D, Lum JJ, Bui T, Christophorou MA, Evan GI, et al. Autophagy inhibition enhances therapy-induced apoptosis in a Myc-induced model of lymphoma. J Clin Invest. 2007;117(2):326–36.
  150. 150. Ding W-X, Ni H-M, Gao W, Chen X, Kang JH, Stolz DB, et al. Oncogenic transformation confers a selective susceptibility to the combined suppression of the proteasome and autophagy. Mol Cancer Ther. 2009;8(7):2036–45.
  151. 151. Dikic I, Johansen T, Kirkin V. Selective autophagy in cancer development and therapy. Cancer Res. 2010;70(9):3431–4.
  152. 152. Fischer S, Ronellenfitsch MW, Thiepold AL, Harter PN, Reichert S, Kögel D, et al. Hypoxia enhances the antiglioma cytotoxicity of b10, a glycosylated derivative of betulinic acid. PLoS One. 2014;9 (4).
  153. 153. Vazquez-Martin A, López-Bonetc E, Cufí S, Oliveras-Ferraros C, Del Barco S, Martin-Castillo B, et al. Repositioning chloroquine and metformin to eliminate cancer stem cell traits in pre-malignant lesions. Drug Resist Updat. 2011;14(4–5):212–23.
  154. 154. Le X-F, Mao W, Lu Z, Carter BZ, Bast RC. Dasatinib induces autophagic cell death in human ovarian cancer. Cancer. 2010 Nov 1;116(21):4980–90.
  155. 155. Wilson EN, Bristol ML, Di X, Maltese WA, Koterba K, Beckman MJ, et al. A switch between cytoprotective and cytotoxic autophagy in the radiosensitization of breast tumor cells by chloroquine and vitamin D. Horm Cancer. 2011 Oct 2;2(5):272–85.
  156. 156. Rossi M, Rotblat B, Ansell K, Amelio I, Caraglia M, Misso G, et al. High throughput screening for inhibitors of the HECT ubiquitin E3 ligase ITCH identifies antidepressant drugs as regulators of autophagy. Cell Death Dis. Nature Publishing Group; 2014;5(5):e1203.
  157. 157. Hu T, Li P, Luo Z, Chen X, Zhang J, Wang C, et al. Chloroquine inhibits hepatocellular carcinoma cell growth in vitro and in vivo. Oncol Rep. 2015 Nov 2;43–9.
  158. 158. Fan Q-W, Cheng C, Hackett C, Feldman M, Houseman BT, Nicolaides T, et al. Akt and Autophagy Cooperate to Promote Survival of Drug-Resistant Glioma. Sci Signal. 2010 Nov 9;3(147):ra81–ra81.
  159. 159. Solomon VR, Lee H. Chloroquine and its analogs: A new promise of an old drug for effective and safe cancer therapies. Eur J Pharmacol. Elsevier B.V.; 2009;625(1–3):220–33.
  160. 160. Sui X, Chen R, Wang Z, Huang Z, Kong N, Zhang M, et al. Autophagy and chemotherapy resistance: a promising therapeutic target for cancer treatment. Cell Death Dis. 2013;4:e838.
  161. 161. Rosenfeld MR, Ye X, Supko JG, Desideri S, Grossman SA, Brem S, et al. A phase I/II trial of hydroxychloroquine in conjunction with radiation therapy and concurrent and adjuvant temozolomide in patients with newly diagnosed glioblastoma multiforme. Autophagy. 2014;10(8):1359–68.
  162. 162. Sotelo J, Briceño E, López-González MA. Adding chloroquine to conventional treatment for glioblastoma multiforme: a randomized, double-blind, placebo-controlled trial. Ann Intern Med. 2006 Mar 7;144(5):337–43.
  163. 163. Huang C, Liu W, Perry CN, Yitzhaki S, Lee Y, Yuan H, et al. Autophagy and protein kinase C are required for cardioprotection by sulfaphenazole. Am J Physiol Heart Circ Physiol. 2010;298(2):H570–9.
  164. 164. Huang C, Andres AM, Ratliff EP, Hernandez G, Lee P, Gottlieb RA. Preconditioning involves selective mitophagy mediated by parkin and p62/SQSTM1. PLoS One. 2011;6(6).
  165. 165. Martinet W, Knaapen MWM, Kockx MM, De Meyer GRY. Autophagy in cardiovascular disease. Trends Mol Med. 2007 Nov;13(11):482–91.
  166. 166. Nemchenko A, Chiong M, Turer A, Lavandero S, Hill JA. Autophagy as a therapeutic target in cardiovascular disease. J Mol Cell Cardiol. 2011 Oct;51(4):584–93.
  167. 167. Cao DJ, Wang Z V, Battiprolu PK, Jiang N, Morales CR, Kong Y, et al. Histone deacetylase (HDAC) inhibitors attenuate cardiac hypertrophy by suppressing autophagy. Proc Natl Acad Sci U S A. 2011;108(10):4123–8.
  168. 168. Gottlieb RA, Mentzer RM. Cardioprotection through autophagy: Ready for clinical trial? Autophagy. 2011;7(4):434–5.
  169. 169. Sala-Mercado JA, Wider J, Reddy Undyala VV, Jahania S, Yoo W, Mentzer RM, et al. Profound cardioprotection with chloramphenicol succinate in the swine model of myocardial ischemia-reperfusion injury. Circulation. 2010;122(11 SUPPL. 1):179–85.
  170. 170. Przyklenk K, Undyala VVR, Wider J, Sala-Mercado JA, Gottlieb RA, Mentzer RM. Acute induction of autophagy as a novel strategy for cardioprotection: Getting to the heart of the matter. Autophagy. 2011;7(4):432–3.
  171. 171. Shioi T, McMullen JR, Tarnavski O, Converso K, Sherwood MC, Manning WJ, et al. Rapamycin attenuates load-induced cardiac hypertrophy in mice. Circulation. 2003;107(12):1664–70.
  172. 172. McMullen JR, Sherwood MC, Tarnavski O, Zhang L, Dorfman AL, Shioi T, et al. Inhibition of mTOR signaling with rapamycin regresses established cardiac hypertrophy induced by pressure overload. Circulation. 2004;109(24):3050–5.
  173. 173. Martinet W, Verheye S, De Meyer GRY. Selective depletion of macrophages in atherosclerotic plaques via macrophage-specific initiation of cell death. Trends Cardiovasc Med. 2007 Feb;17(2):69–75.
  174. 174. Verheye S, Martinet W, Kockx MM, Knaapen MWM, Salu K, Timmermans JP, et al. Selective clearance of macrophages in atherosclerotic plaques by autophagy. J Am Coll Cardiol. 2007;49(6):706–15.
  175. 175. Martinet W, De Loof H, De Meyer GRY. mTOR inhibition: a promising strategy for stabilization of atherosclerotic plaques. Atherosclerosis. 2014 Apr;233(2):601–7.
  176. 176. Buss SJ, Riffel JH, Katus HA, Hardt SE. Augmentation of autophagy by mTOR-inhibition in myocardial infarction: When size matters. Autophagy. 2010 Feb;6(2):304–6.
  177. 177. Cherra SJ, Chu CT. Autophagy in neuroprotection and neurodegeneration: A question of balance. Future Neurol. 2008 May;3(3):309–23.
  178. 178. Berger Z, Ravikumar B, Menzies FM, Oroz LG, Underwood BR, Pangalos MN, et al. Rapamycin alleviates toxicity of different aggregate-prone proteins. Hum Mol Genet. 2006;15(3):433–42.
  179. 179. Webb JL, Ravikumar B, Atkins J, Skepper JN, Rubinsztein DC. -Synuclein is degraded by both autophagy and the proteasome. J Biol Chem. 2003 Jun 27;278(27):25009–13.
  180. 180. Spencer B, Potkar R, Trejo M, Rockenstein E, Patrick C, Gindi R, et al. Beclin 1 gene transfer activates autophagy and ameliorates the neurodegenerative pathology in alpha-synuclein models of Parkinson’s and Lewy body diseases. J Neurosci. 2009;29(43):13578–88.
  181. 181. Ravikumar B, Duden R, Rubinsztein DC. Aggregate-prone proteins with polyglutamine and polyalanine expansions are degraded by autophagy. Hum Mol Genet. 2002;11(9):1107–17.
  182. 182. Haung Yu W, Cuervo AM, Kumar A, Peterhoff CM, Schmidt SD, Lee JH, et al. Macroautophagy—A novel ??-amyloid peptide-generating pathway activated in Alzheimer’s disease. J Cell Biol. 2005;171(1):87–98.
  183. 183. Rubinsztein DC, Bento CF, Deretic V. Therapeutic targeting of autophagy in neurodegenerative and infectious diseases. J Exp Med. 2015 Jun 29;212(7):979–90.
  184. 184. Frake RA, Ricketts T, Menzies FM, Rubinsztein DC. Autophagy and neurodegeneration. J Clin Invest. 2015 Jan;125(1):65–74.
  185. 185. Sarkar S, Krishna G, Imarisio S, Saiki S, O’Kane CJ, Rubinsztein DC. A rational mechanism for combination treatment of Huntington’s disease using lithium and rapamycin. Hum Mol Genet. 2008 Jan 15;17(2):170–8.
  186. 186. Ravikumar B, Vacher C, Berger Z, Davies JE, Luo S, Oroz LG, et al. Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease. Nat Genet. 2004 Jun;36(6):585–95.
  187. 187. Rodrigues D, Viotto AC, Checchia RG, Gomide AB, Itri R, Severino D, et al. Mechanism of Aloe Vera extract protection against UVA: shelter of lysosomal membrane avoids photodamage. Photochem Photobiol Sci. 2016;
  188. 188. Chiu H-C, Soni S, Kulp SK, Curry H, Wang D, Gunn JS, et al. Eradication of intracellular Francisella tularensis in THP-1 human macrophages with a novel autophagy inducing agent. J Biomed Sci. 2009;16:110.
  189. 189. Chiu HC, Kulp SK, Soni S, Wang D, Gunn JS, Schlesinger LS, et al. Eradication of intracellular Salmonella enterica serovar typhimurium with a small-molecule, host cell-directed agent. Antimicrob Agents Chemother. 2009;53(12):5236–44.
  190. 190. Kim JJ, Lee HM, Shin DM, Kim W, Yuk JM, Jin HS, et al. Host cell autophagy activated by antibiotics is required for their effective antimycobacterial drug action. ?? 2012 Elsevier Inc. Cell Host Microbe. Elsevier Inc.; 2012;11(5):457–68.
  191. 191. Campbell GR, Spector SA. Vitamin D inhibits human immunodeficiency virus type 1 and Mycobacterium tuberculosis infection in macrophages through the induction of autophagy. PLoS Pathog. 2012;8(5).
  192. 192. Gutierrez MG, Master SS, Singh SB, Taylor GA, Colombo MI, Deretic V. Autophagy is a defense mechanism inhibiting BCG and Mycobacterium tuberculosis survival in infected macrophages. Cell. 2004;119(6):753–66.
  193. 193. Schiebler M, Brown K, Hegyi K, Newton SM, Renna M, Hepburn L, et al. Functional drug screening reveals anticonvulsants as enhancers of mTOR-independent autophagic killing of Mycobacterium tuberculosis through inositol depletion. EMBO Mol Med. 2015;7(2):127–39.
  194. 194. Stanley SA, Barczak AK, Silvis MR, Luo SS, Sogi K, Vokes M, et al. Identification of host-targeted small molecules that restrict intracellular mycobacterium tuberculosis growth. PLoS Pathog. 2014;10 (2).
  195. 195. Lam KKY, Zheng X, Forestieri R, Balgi AD, Nodwell M, Vollett S, et al. Nitazoxanide stimulates autophagy and inhibits mTORC1 signaling and intracellular proliferation of Mycobacterium tuberculosis. PLoS Pathog. 2012;8(5).
  196. 196. Sundaramurthy V, Barsacchi R, Samusik N, Marsico G, Gilleron J, Kalaidzidis I, et al. Integration of chemical and RNAi multiparametric profiles identifies triggers of intracellular mycobacterial killing. Cell Host Microbe. Elsevier Inc.; 2013;13(2):129–42.
  197. 197. Parihar SP, Guler R, Khutlang R, Lang DM, Hurdayal R, Mhlanga MM, et al. Statin therapy reduces the mycobacterium tuberculosis burden in human macrophages and in mice by enhancing autophagy and phagosome maturation. J Infect Dis. 2014 Mar 1;209(5):754–63.
  198. 198. Tsujimoto Y, Shimizu S. Another way to die: autophagic programmed cell death. Cell Death Differ. 2005 Nov;12 Suppl 2:1528–34.
  199. 199. Dzubak P, Hajduch M, Vydra D, Hustova A, Kvasnica M, Biedermann D, et al. Pharmacological activities of natural triterpenoids and their therapeutic implications. Nat Prod Rep. 2006 Jul;23(3):394–411.
  200. 200. Fulda S. Activation of mitochondria and release of mitochondrial apoptogenic factors by betulinic acid. J Biol Chem. 1998 Dec 18;273(51):33942–8.
  201. 201. Li Y, He K, Huang Y, Zheng D, Gao C, Cui L, et al. Betulin induces mitochondrial cytochrome c release associated apoptosis in human cancer cells. Mol Carcinog. 2010 Jul;49(7):630–40.
  202. 202. Potze L, Di Franco S, Grandela C, Pras-Raves ML, Picavet DI, van Veen H a, et al. Betulinic acid induces a novel cell death pathway that depends on cardiolipin modification. Oncogene. Nature Publishing Group; 2015 Apr 20; (October 2014):1–11.
  203. 203. Chen Y, Sun R, Wang B. Monolayer behavior of binary systems of betulinic acid and cardiolipin: thermodynamic analyses of Langmuir monolayers and AFM study of Langmuir-Blodgett monolayers. J Colloid Interface Sci. Elsevier Inc.; 2011 Jan 1;353(1):294–300.
  204. 204. Gao M, Lau PM, Kong SK. Mitochondrial toxin betulinic acid induces in vitro eryptosis in human red blood cells through membrane permeabilization. Arch Toxicol. 2014;88(3):755–68.
  205. 205. Eder-Czembirek C, Erovic BM, Czembirek C, Brunner M, Selzer E, Pötter R, et al. Betulinic acid a radiosensitizer in head and neck squamous cell carcinoma cell lines. Strahlenther Onkol. 2010 Mar;186(3):143–8.
  206. 206. Selzer E, Pimentel E, Wacheck V, Schlegel W, Pehamberger H, Jansen B, et al. Effects of betulinic acid alone and in combination with irradiation in human melanoma cells. J Invest Dermatol. 2000 May;114(5):935–40.
  207. 207. Jeremias I, Steiner HH, Benner A, Debatin K-M, Herold-Mende C. Cell death induction by betulinic acid, ceramide and TRAIL in primary glioblastoma multiforme cells. Acta Neurochir (Wien). 2004 Jul;146(7):721–9.
  208. 208. Sawada N, Kataoka K, Kondo K, Arimochi H, Fujino H, Takahashi Y, et al. Betulinic acid augments the inhibitory effects of vincristine on growth and lung metastasis of B16F10 melanoma cells in mice. Br J Cancer. 2004 May 19;90(8):1672–8.
  209. 209. Schmidt ML, Kuzmanoff KL, Ling-Indeck L, Pezzuto JM. Betulinic acid induces apoptosis in human neuroblastoma cell lines. Eur J Cancer. 1997. pp. 2007–10.
  210. 210. Fulda S, Susin SA, Kroemer G, Cells N. Molecular ordering of apoptosis induced by anticancer drugs in neuroblastoma cells molecular ordering of apoptosis induced by anticancer drugs in. 1998;4453–60.
  211. 211. Kasperczyk H, La Ferla-Brühl K, Westhoff MA, Behrend L, Zwacka RM, Debatin K-M, et al. Betulinic acid as new activator of NF-kappaB: molecular mechanisms and implications for cancer therapy. Oncogene. 2005 Oct 20;24(46):6945–56.
  212. 212. Pisha E, Chai H, Lee IS, Chagwedera TE, Farnsworth NR, Cordell GA, et al. Discovery of betulinic acid as a selective inhibitor of human melanoma that functions by induction of apoptosis. Nat Med. 1995 Oct;1(10):1046–51.
  213. 213. Ehrhardt H, Fulda S, Führer M, Debatin KM, Jeremias I. Betulinic acid-induced apoptosis in leukemia cells. Leukemia. 2004 Aug;18(8):1406–12.
  214. 214. Kessler JH, Mullauer FB, de Roo GM, Medema JP. Broad in vitro efficacy of plant-derived betulinic acid against cell lines derived from the most prevalent human cancer types. Cancer Lett. 2007 Jun 18;251(1):132–45.
  215. 215. Mullauer FB, Kessler JH, Medema JP. Betulinic acid induces cytochrome c release and apoptosis in a Bax/Bak-independent, permeability transition pore dependent fashion. Apoptosis. 2009 Feb;14(2):191–202.
  216. 216. Rzeski W, Stepulak A, Szymański M, Sifringer M, Kaczor J, Wejksza K, et al. Betulinic acid decreases expression of bcl-2 and cyclin D1, inhibits proliferation, migration and induces apoptosis in cancer cells. Naunyn Schmiedebergs Arch Pharmacol. 2006 Oct;374(1):11–20.
  217. 217. Yang L, Chen Y, Ma Q, Fang J, He J, Cheng Y, et al. Effect of betulinic acid on the regulation of Hiwi and cyclin B1 in human gastric adenocarcinoma AGS cells. Acta Pharmacol Sin. 2010;31(1):66–72.
  218. 218. Raghuvar Gopal D V., Narkar Aa., Badrinath Y, Mishra KP, Joshi DS. Betulinic acid induces apoptosis in human chronic myelogenous leukemia (CML) cell line K-562 without altering the levels of Bcr-Abl. Toxicol Lett. 2005 Mar 15;155(3):343–51.
  219. 219. Raghuvar Gopal D V., Narkar a. a., Badrinath Y, Mishra KP, Joshi DS. Protection of Ewing’s sarcoma family tumor (ESFT) cell line SK-N-MC from betulinic acid induced apoptosis by alpha-DL-tocopherol. Toxicol Lett. 2004 Dec 2;153(2):201–12.
  220. 220. Wick W, Grimmel C, Wagenknecht B, Dichgans J, Weller M. Betulinic acid-induced apoptosis in glioma cells: A sequential requirement for new protein synthesis, formation of reactive oxygen species, and caspase processing. J Pharmacol Exp Ther. 1999 Jun;289(3):1306–12.
  221. 221. Chadalapaka G, Jutooru I, Burghardt R, Safe S. Drugs that target specificity proteins downregulate epidermal growth factor receptor in bladder cancer cells. Mol Cancer Res. 2010;8(5):739–50.
  222. 222. Hsu T, Wang M, Chen S, Huang S, Yeh Y, Su W, et al. Betulinic acid decreases specificity protein 1 (Sp1) level via increasing the sumoylation of Sp1 to inhibit lung cancer growth. Mol Pharmacol. 2012;82(6):1115–28.
  223. 223. Safe SH, Prather PL, Brents LK, Chadalapaka G, Jutooru I. Unifying mechanisms of action of the anticancer activities of triterpenoids and synthetic analogs. Anticancer Agents Med Chem. 2012;12(10):1211–20.
  224. 224. Fulda S. Betulinic Acid for cancer treatment and prevention. Int J Mol Sci. 2008 Jun;9(6):1096–107.
  225. 225. Nakagawa-Goto K, Yamada K, Taniguchi M, Tokuda H, Lee K-HH. Cancer preventive agents 9. Betulinic acid derivatives as potent cancer chemopreventive agents. Bioorganic Med Chem Lett. Elsevier Ltd; 2009 Jul 1;19 (13):3378–81.
  226. 226. Rajendran P, Jaggi M, Singh MK, Mukherjee R, Burman AC. Pharmacological evaluation of C-3 modified Betulinic acid derivatives with potent anticancer activity. Invest New Drugs. 2008;26(1):25–34.
  227. 227. Yang S, Liang N, Li H, Xue W, Hu D, Jin L, et al. Design, synthesis and biological evaluation of novel betulinic acid derivatives. Chem Cent J. 2012;6(1):141.
  228. 228. Li F, Goila-Gaur R, Salzwedel K, Kilgore NR, Reddick M, Matallana C, et al. PA-457: a potent HIV inhibitor that disrupts core condensation by targeting a late step in Gag processing. Proc Natl Acad Sci U S A. 2003;100(23):13555–60.
  229. 229. Yu D, Morris-Natschke SL, Lee KH. New developments in natural products-based anti-AIDS research. Vol. 27, Med Res Rev. 2007. p. 108–32.
  230. 230. Willmann M, Wacheck V, Buckley J, Nagy K, Thalhammer J, Paschke R, et al. Characterization of NVX-207, a novel betulinic acid-derived anti-cancer compound. Eur J Clin Invest. 2009;39(5):384–94.
  231. 231. Bayer M, Proksch P, Felsner I, Brenden H, Kohne Z, Walli R, et al. Photoprotection against UVAR: Effective triterpenoids require a lipid raft stabilizing chemical structure. Exp Dermatol. 2011;20(11):955–8.
  232. 232. Gonzalez P, Mader I, Tchoghandjian A, Enzenmüller S, Cristofanon S, Basit F, et al. Impairment of lysosomal integrity by B10, a glycosylated derivative of betulinic acid, leads to lysosomal cell death and converts autophagy into a detrimental process. Cell Death Differ. 2012 Aug;19(8):1337–46.
  233. 233. Enzenmüller S, Gonzalez P, Karpel-Massler G, Debatin KM, Fulda S. GDC-0941 enhances the lysosomal compartment via TFEB and primes glioblastoma cells to lysosomal membrane permeabilization and cell death. Cancer Lett. 2013;329(1):27–36.
  234. 234. Csuk R. Betulinic acid and its derivatives: a patent review (2008–2013). Expert Opin Ther Pat. 2014 Aug;24(8):913–23.
  235. 235. Liby K, Honda T, Williams CR, Risingsong R, Royce DB, Suh N, et al. Novel semisynthetic analogues of betulinic acid with diverse cytoprotective, antiproliferative, and proapoptotic activities. Mol Cancer Ther. 2007 Jul;6(7):2113–9.
  236. 236. Morselli E, Galluzzi L, Kepp O, Vicencio J-MM, Criollo A, Maiuri MC, et al. Anti- and pro-tumor functions of autophagy. Biochim Biophys Acta. 2009 Sep;1793(9):1524–32.
  237. 237. Liu EY, Ryan KM. Autophagy and cancer—issues we need to digest. J Cell Sci. 2012 May 15;125(Pt 10):2349–58.
  238. 238. Cufí S, Vazquez-Martin A, Oliveras-Ferraros C, Corominas-Faja B, Cuyàs E, López-Bonet E, et al. The anti-malarial chloroquine overcomes primary resistance and restores sensitivity to trastuzumab in HER2-positive breast cancer. Sci Rep. 2013;3:2469.
  239. 239. Bacellar I, Tsubone T, Pavani C, Baptista M. Photodynamic efficiency: from molecular photochemistry to cell death. Int J Mol Sci. 2015;16(9):20523–59.
  240. 240. Reiners JJ, Agostinis P, Berg K, Oleinick NL, Kessel D. Assessing autophagy in the context of photodynamic therapy. Autophagy. 2010 Jan;6(1):7–18.
  241. 241. Oleinick NL, Morris RL, Belichenko I. The role of apoptosis in response to photodynamic therapy: what, where, why, and how. Photochem Photobiol Sci. 2002 Jan;1(1):1–21.
  242. 242. Inguscio V, Panzarini E, Dini L. Autophagy Contributes to the Death/Survival Balance in Cancer PhotoDynamic Therapy. Vol. 1, Cells. 2012. p. 464–91.

Written By

Waleska K. Martins and Mauricio S. Baptista

Submitted: 23 November 2015 Reviewed: 27 April 2016 Published: 10 November 2016