Autophagy inducers in cancer

Accepted Manuscript Autophagy Inducers in Cancer Maria Russo, Gian Luigi Russo PII: DOI: Reference:

S0006-2952(18)30064-9 https://doi.org/10.1016/j.bcp.2018.02.007 BCP 13052

To appear in:

Biochemical Pharmacology

Received Date: Accepted Date:

10 December 2017 7 February 2018

Please cite this article as: M. Russo, G.L. Russo, Autophagy Inducers in Cancer, Biochemical Pharmacology (2018), doi: https://doi.org/10.1016/j.bcp.2018.02.007

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Autophagy Inducers in Cancer

Maria Russo and Gian Luigi Russo § Institute of Food Sciences, National Research Council, 83100, Avellino, Italy

§

To whom correspondence should be addressed:

Gian Luigi Russo Institute of Food Sciences National Research Council Via Roma, 64 83100 – Avellino Italy Office: +39 0825299 331 Mobile: +39 3299064414 E-mail: [email protected]

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Abstract Autophagy is a complex, physiological process devoted to degrade and recycle cellular components. Proteins and organelles are first phagocytized by autophagosomes, then digested in lysosomes, and finally recycled to be utilized again during cellular metabolism. Moreover, autophagy holds an important role in the physiopathology of several diseases. In cancer, excellent works demonstrated the dual functions of autophagy in tumor biology: autophagy activation can promote cancer cells survival (protective autophagy), or contribute to cancer cell death (cytotoxic/nonprotective autophagy). A better understanding of the dichotomy roles of autophagy in cancer biology can help to identify or design new drugs able to induce/enhance (or block) autophagic flux. These features will necessary be tissue-dependent and confined to a specific time of treatment. The intent of this review is to focus on the different potentialities of autophagy inducers in cancer prevention versus therapy in order to elicit a desirable clinical response. Few promising synthetic and natural compounds have been identified and the pros and cons of their role in autophagy regulation is reviewed here. In the complex framework of autophagy modulation, “connecting the dots” is not a simple work and the lack of clinical studies further complicates the scenario, but the final goal to obtain clinically relevant autophagy inducers can reveal an unexpected landscape.

Key words: Autophagy; Cancer; Autophagy inducers

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1. Introduction The first use of the term “autophagy” by a French physiologist, M. Anselmier, in a short article describing the effects of fasting in mice published in 1859, took place almost a century before Christian De Duve described it from a mechanistic point of view in a symposium on lysosomes in 1963 (reviewed in [1]). Currently, a search on PubMed database can retrieve more than 30.000 scientific articles containing the term “autophagy”, witnessing the importance of such biological process in life sciences. The scientific interest in autophagy research lastly culminated in 2016 with the award of Nobel Prize for Medicine and Physiology to Professor Y. Oshumi for the discovery, in the early ‘90, of ATG genes (autophagy-related genes), controlling and regulating autophagy in yeasts [2]. From the original Anselmier's farsighted description, to the current “autophagy molecular dissection”, scientists learned that autophagy plays a preeminent role in cellular homeostasis of specific tissues (mainly liver, brain, muscles). Its functions regard cell survival regulation (response to metabolic alterations, recycling damaged macromolecules and organelles) and various programmed forms of cell death (type II cell death), different from apoptosis, which occur in physiological (aging) or pathological conditions, or in response to drug and ionizing radiations [3]. The sometimes-paradoxical effects of autophagy in physiology and pathology are examples of its levels of complexity. However, its dual role in sustaining cell survival or inducing cell death is largely observed in cancer, which, per se, represents an extremely complex disease from the molecular and clinical point of view. Among the different forms of autophagy, the present article focuses on “macroautophagy”, a process which involves the formation of “autophagosomes”, dedicated vesicles that occupy large regions of the cytoplasm, Other variants, such are microautophagy and chaperon-mediated autophagy are not associated with major morphological changes in vesicular compartments [4]. Although very interesting and promising, the study of the modulation of the latter autophagy

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processes in cancer are too preliminary to be analyzed here; however, we predict that in few years the impact of microautophagy and chaperon-mediated autophagy in cancer will significantly grow.

2. Connecting the dots between autophagy and cancer According to International Agency for Research on Cancer (IARC), by 2030 cancer could be the leading cause of death worldwide with 13 million potential cancer-related deaths [5]. This will lead to a 60% increase from 2014 with a further 21.7 million new cancer cases each year and the largest economic cost on a global scale due to life expectancy and productivity loss, together with cancerrelated disabilities1. Cancer is expected to surpass cardiovascular disease as the leading cause of death in the world. Despite this warring picture, there have been significant progress in the understanding of cancer biology, identification of risk factors, new treatments and early diagnosis of some types of cancer. Although still insufficient in terms of worldwide cancer prevention and therapy, progress have been made in reducing cancer mortality [6, 7] and new challenges can arise from pursuing multiple strategies including the revisiting of the anticancer effects of drugs already present in clinics to cure diseases different than cancer. This apparently paradoxical approach finds its rationale considering the existence of complex biological processes, which can support or delay cancer growth and development depending on a significant number of “external” factors including diet, genetic background, predisposition to other chronic and degenerative diseases. One of these cellular processes is autophagy. The metaphoric definition of “double-edged sword” (Fig. 1) is recurrent in many scientific articles and describes the opposite role of autophagy in cancer. These studies are largely based on: 1. genetically engineered mouse models (GEMM); 2. detection of DNA mutations which allowed the classification of different ATG genes, which are directly involved in biochemical regulation of autophagy [8], as both “tumour suppressors” or “oncogenes”. “Macroautophagy”, the process in 1

http://gco.iarc.fr/today/home

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which cellular contents are degraded by lysosomes or vacuoles and recycled, includes several phases regulated by different ATG genes controlling the complete autophagy pathway. The final destiny is the formation of double-membrane vesicles (phagophore, autophagosome) that finally fuse with lysosomes where acidic hydrolases are able to degrade and recycle their cargo [4, 9, 10]. Until now, 20 “core ATG genes” and highly conserved Atg proteins have been identified in yeast and mammals. The different members of the Atg family members can be classified considering their specific “space-temporal” role in autophagy. Initiation depends upon Atg1/Ulk1 kinase and its regulators, Atg13, Atg17, Atg29, Atg31 [11-13]. The Atg6/Beclin-1-Atg14/Atg14L-Vps34-Vps15 [14, 15] and Beclin-1-UVRAG-Bif-1-Vps34-Vps15 [16, 17] complexes are required for phagophore formation/expansion; autophagosome maturation is regulated by the Atg12 conjugation system, Atg5, Atg7, Atg10, Atg12, Atg16 [18, 19]. Finally, fusion and degradation in lysosomes and cargo efflux in the cytoplasm is controlled by the Atg8/LC3 conjugation system comprising Atg3, Atg4, Atg7, Atg8; Atg9, and the Atg2-Atg18 complex ([20-23]; also reviewed in [4, 24-26] and figures and schemes therein). From a biochemical point of view, this picture is even more complex since Atg proteins are at the crossroads of important metabolic pathways: amino-acids sensing regulated by mTOR kinase complex (mammalian Target of Rapamycin), ATP intracellular content controlled by AMPK (AMP-activated kinase) [27, 28] (Fig. 2) and stress signalling mechanism by HIF (hypoxia inducing factor) [29]. All these pathways could turn on/off Atg proteins, in order to obtain a “homeostatic effect”. In other terms, autophagy “basal state” in a cell is strictly dependent upon metabolic/energy or environmental stress. In cancer, it is extremely important to assess whether malignant cells depend on autophagy to overcome metabolic and energy stress during carcinogenesis or, on the opposite, autophagy (and autophagy associated cell death) is an essential process to block carcinogenesis. Recently, the neologism “oncophagy” has coined to describe the role of autophagy in cancer, referring to the close connection between cancer biology/therapy and autophagy [30] (Fig. 2).

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The first description of autophagy as a tumour suppression process arises from the observation that the initial step regulatory gene, Atg6/BECN1, was monoallelically lost in 40% to 75% of human prostate, breast, and ovarian cancers [31]. However, while BECN1 heterozygous mutant mice develop, with long latency, lymphomas, liver and lung tumours, deletion of other essential autophagy genes, such as ATG5 or ATG7 in mice, specifically in liver and pancreatic tissues, produces only benign adenoma in these tissues (Fig. 1). The concept that BECN1 could be a tumour suppressor (based on the studies demonstrating its allelic loss in tumour tissues) could be confused by the presence, in the same locus on human chromosome 17q21, of another tumour suppressor, BRCA1 (breast and ovarian tumour suppressor breast cancer 1). BRCA1 mutations are involved in the early onset of human breast and ovarian cancers. In fact, the current explanation is that the driver mutation in hereditary ovarian and breast cancers are dependent from germline BRCA1 missense mutation and the subsequent somatic loss of wild-type allele, that sometimes include (or not include) BECN1 deletion. BECN1 mutation can be considered in this scenario only a passenger mutation. To confirm this observation, exploring the large-scale genomic analysis of human cancers (Cancer Genome ATLAS, TCGA), no recurrent mutations in BECN1 or other essential autophagy genes are found, with few exceptions [32] [33]. Sequencing over 10.000 human cancers and matching the DNA sequence of normal tissues in TCGA database, large deletions were found on chromosome 17 comprising BRCA1 and BECN1 sequences in breast and ovarian tumours or deletion of only BRCA1, not BECN1, confirming that the driver mutation is BRCA1. The DNA sequencing data indicate that the loss of BECN1 in human cancers is strictly related to the loss of BRCA1 and, finally, that BECN1 could not be a tumour suppressor in most human cancers [33]. Nevertheless, autophagy could be still considered a tumour suppressor process in specific tissues, such as liver and pancreas, because autophagy-deficient mice (systemic mosaic deletion of ATG5 and liver-specific ATG7-/-) develop benign liver and pancreas adenomas [34] (Fig. 1). This could be explained considering autophagy an essential process to suppress the initial stages of liver or pancreas carcinogenesis for its role in controlling organelle integrity and protein quality and to 6

suppress inflammation [29]. To reinforce this hypothesis, another suppressor gene strictly correlated to autophagy is Parkin, an E3 ubiquitin ligase, essential for the clearance of damaged mitochondria (a process called “mitophagy”). This gene is frequently deleted in human cancer on chromosome 6q25-q26, even if another Parkin function is related to cyclin D and E stability, in the context of cell cycle control, so this latter function could also explain its role in tumour suppression [35]. An important autophagy receptor is p62/SQSTM1 (p62, a complex “adapter” protein characterized by a multi-domain structure) which includes a LIR domain (LC3 Interacting Domain) essential to drive cargo and lipidated LC3 to autophagosome [36] (Fig. 2). This function explains why p62 accumulates in cells when autophagy flux is blocked or in autophagy deficient cells. In animal models lacking p62, this genetic alteration inhibited the development of lung cancer, while its gain of function (p62 amplification on chromosome 5q) is linked to renal cancer tumorigenesis [37]. In addition to its role in autophagy, p62 can activate two transcription factors: Nrf2 (through KIR domain binding with its negative regulator Keap-1) and NF-B (through TB domain binding with TRAF6). The final effect is a ROS (reactive oxygen species)-mediated stress response which enhances cell proliferation and induces stress resistance in cancer cells, favouring carcinogenesis [36]. These data reinforce the vision of autophagy as an essential process to block cancer formation at its initial stage (Fig. 1). However, there are studies revealing the “dark side” of autophagy at later stages in cancers, when oncogenes (mainly K-ras and B-raf) are activated and/or tumour suppressors such as PTEN and p53 are inactivated. These studies are based on the use of GEMM and deletions of essential autophagy genes (ATG7 or ATG5) in K-rasG12D [38] [39] or B-raf V600E-driven cancers (primarily in melanoma, pancreas and lung cancers) [39, 40] (Fig. 1). These experimental models have great advantages respect to xenograft mice since they allow the study of cancer progression and the effects of autophagy deficiency only in tumour tissues of animals with an intact immune system. Guo et al. demonstrated that in the absence of an essential autophagy gene (ATG7) in tumour epithelial cells in GEMM expressing K-rasG12D in lung, the mice showed defective mitochondria 7

and protein aggregates leading to cancer cell growth arrest or cell death and more benign disease (oncocytoma) respect to control mice expressing Atg7 [39]. Histological analysis of benign oncocytoma revealed the presence of defective mitochondria and lipid accumulation since the absence of ATG7 impaired mitochondrial respiration and fatty acids oxidation. The explanation was that in K-rasG12D-driven lung cancer autophagy is essential to support metabolism and growth of tumour cells. In fact, Atg7 also suppresses p53 activation contributing to cancer cell growth and progression [38, 39]. A similar observation was revealed in the same context of lung carcinogenesis driven by B-rafV600E. Here, the authors demonstrated that the initial phenotype in the absence of ATG7 in GEMM expressing B-rafV600E consisted in Nrf2 defective antioxidant defence response driving initial steps of carcinogenesis. These mice showed benign lung oncocytoma characterized by defective mitochondria and altered metabolism, which finally compromised tumour growth. In fact, at later time, the anticancer effect due to the absence of ATG7 became the dominant phenotype and the mice experienced lifespan extension and a reduction of tumour mass due to the same histological and metabolic alterations seen in in K-rasG12D mutated lung cancer [41] (Fig. 1). B-raf mutations are also common in melanoma (80-90% cases), a tumour characterized by high levels of basal autophagy. Targeted therapy against this oncogenic alteration usually shows a limited efficacy and resistance is often developed. Using a mice model of B-rafV600E-driven melanoma, in the presence of PTEN tumour suppressor deletion, Xie et al. [40] studied the functional consequences of ATG7 deficiency. The absence of ATG7 prevented melanoma development, indicating an essential role of autophagy in cancer onset. The common phenotype, also seen in lung cancer, was the accumulation of autophagy substrates and growth defects which finally extended mice lifespan since cancer cells showed increased oxidative stress and senescence, which represent well known barrier against carcinogenesis [42]. The data reported above referring to solid tumours are corroborated by parallel results in blood cancers. In fact, recent studies confirm that autophagy contributes to block initial leukemogenesis 8

for its crucial role in hematopoietic stem cells (HSC) maintenance, correct differentiation of myeloid and lymphoid progenitors and elimination of oncogenic proteins such as PML-RAR and BCR-ABL [43, 44]. Autophagy, at later stages, is always functionally linked to drug resistance. Deleting ATG7 gene or blocking the expression of Ulk-1-interacting protein, FIP200, mice showed severe damaged in HSC with defective mitochondria and DNA damage followed by a lethal preleukemic phenotype [45] [46]. This observation was confirmed applying a different approach. Using human acute myeloid leukemia (AML) cells isolated from patients, the loss of ATG5 or ATG7 resulted in an impaired autophagic flux respect to normal HSC with accumulation of damaged mitochondria and ROS increase, confirming the protective role of autophagy against leukemogenesis [47]. Imatinib is a well-known anticancer drug used in Chronic Myeloid Leukemia (CML) against the altered expression/activity of tyrosine kinase resulting from fusion of BCR-ABL in leukemic blasts. It has been demonstrated that Imatinib can induce autophagy in BCR-ABL expressing cells (K562 cell line and blast cells isolated from CML patients) where the oncogenic protein is sequestered in autophagosomes. This observation represents a further example of the tumour suppressive role of autophagy in leukemia [48]. All Trans Retinoic Acid (ATRA) in combination with arsenic trioxide (ATO) represents a successful therapeutic treatment against Acute Promyelocitic Leukemia (APL). In this case, the combined treatment induced terminal differentiation in leukemic blasts exhibiting PML-RAR translocation and the autophagic degradation of this oncogenic protein contributed to explain the pharmacological efficacy of this targeted therapy [49]. These studies, however, show only one side of the story. If Imatinib is necessary to induce autophagosome degradation of BCRABL, following drug withdrawal, it also promotes leukemic cell recovery [50]. This can be explained by admitting that autophagy induced by Imatinib in residual blast cells, narrowed in bone marrow, resulted to be protective, supporting an unwanted drug resistance [51]. These studies suggest the need to identify a close space-temporal window where autophagy must to be suppressed to avoid leukemia resistance [52]. Results from current human clinical trials involving autophagy 9

modulators such as hydroxychloroquine in CML and MM (multiple myeloma) may contribute to bypass these obstacles and improve the anticancer therapy efficacy [52].

3. Autophagy in tumour microenvironment and immune system If genetic studies are essential to understand in deep tumour biology, this approach only reveals some aspects of carcinogenesis, because cancer is not only a “genetic disease”. The real complexity of tumour biology has been excellently explained in pivotal scientific articles describing the “emerging” hallmarks of cancer, including the alterations of immune function and the role of tumour microenvironment. If cancer develops and spreads in the organism, this is due to a few initial genetic drivers mutations followed by other non-genotoxic events, such as tumour immunesurveillance failure [42] [53]. Considering cancer biology in this wider scenery, the opposite role of autophagy in sustaining or inhibiting cancer progression can be better understood. To this regard, an interesting study demonstrated that epithelial cancer cells use oxidative stress to induce autophagy in tissue microenvironment, with the aim to obtain recycled molecules and a net energy transfer from tumour stroma to promote cancer growth. This new paradigm has been called “The Autophagic Tumour Stroma Model of Cancer Cell Metabolism” or “Battery-Operated Tumour Growth” [54]. If this model is correct and confirmed by other studies, a way to avoid cancer resistance should foresee the induction of autophagy in epithelial cancer cells to prevent the use of recycled nutrient and the suppression of autophagy in surrounding stromal cells to obtain an effective starvation of neoplastic cells. The unsolved problem, however, is how to modulate autophagy with a correct and efficient combination of inducers and inhibitors in clinics. To complete the final picture resulting from “oncophagy”, it is worthwhile to remember that anticancer drugs are more efficacious if they induce “immunogenic cell death”, in other words stimulating an antineoplastic immune response. Michaud et al. demonstrated that autophagy is often dysfunctional in cancer cells, is not essential to induce cell death by chemotherapeutic drugs, but is required to attract immune cells (dendritic cells and T-lymphocytes) in tumour microenvironment 10

[55]. The authors showed that ATP extracellular concentration is essential to induce immunogenic anticancer response because suppression of autophagy inhibited ATP efflux from dying tumour cells. On the opposite, increasing extracellular ATP by inhibition of degrading enzymes recruits immune cells and improve the efficacy of antineoplastic drugs in cancer where autophagy is disabled [55]. The final message is that “autophagy is essential for the immunogenic release of ATP from dying cells and to improve the efficacy of antineoplastic chemotherapies when autophagy is disabled” [55] [56].

4. Autophagy inducers Macroautophagy is often seen as a cellular process capable of increasing the fitness of cells and overcome resistance to several forms of stress [9, 10]. As discussed above, it has been proposed by several authors that an effective strategy for enhancing sensitivity of cancer cells to radiotherapy and/or chemotherapy can be the recurrence to autophagic inhibitors, especially in those cancers driven by K-ras mutations [38, 39] and B-raf [40, 41], as reported above (reviewed in [33, 57, 58]) (Fig. 1). However, as pointed out by Kroemer’s group, “sustained long-term effects of successful treatments can only be explained by anticancer immune responses” [56]. The arguments sustained by these authors deal with the need to reconstitute the efficacy of immunosurveillance in malignant cells and organisms affected by cancer. The transition from a healthy cell to its pre-malignant form is normally blocked by a functional immune response since cancer cells are antigenically distinct from their “normal” counterpart and, for this reason, eliminated from the tissue/organism. When immunosurveillance fails, cancer cells escape and initiate the multistep process leading to metastatic tumours. Consequently, chemo- and radiotherapies can be effective if they restore immunosurveillance. Accumulating evidence is going in this direction. As an example, in multiple myeloma, several therapeutic strategies are currently explored to reverse natural killer dysfunctions, which impede killing of transformed plasma cells, such as Pembrolizumab (anti PD-1 receptor) in combination with Lenalidomide, or Daratumumab (CD38 ligand) associated with Bortezomib and 11

Dexamethasone [59]. In general, several forms of radiation and photodynamic therapies, as well as chemotherapeutic drugs, such as oxaliplatin, cyclophosphamide, doxorubicin, trigger immunogenic cell death (ICD) in cancer cells, a specific form of cell death which culminates in the activation of dendritic cells and consequent activation of specific T cell response [60, 61]. In this scenario, the important novelty is represented by the positive role of autophagy. In fact, the activation of this process, not its inhibition, may contribute to restore immunosurveillance in cancer cells. In an excellent review recently published by Pietrocola et al. [62], the authors deeply analyzed the immunological consequences of the involvement of autophagy in cancer therapy. They agree that the inhibition of autophagy may increase cancer cell death following chemotherapy or radiation therapy. A good example is the demonstration in canine patients, applying comparative in vivo oncology-arrayed microinjection technology, doxorubicin induced ICD recruiting immune cell populations (largely macrophages) directly into regions of cell death and accumulating CD3positive T cells surrounding the perimeter of the tumor cell death zone [63]. PS-1001, a dimeric chloroquine autophagy inhibitor [64], was able to bypass the subset of doxorubicin-resistant tumors (about 50% of the total cases), PS-1001 by increasing macrophage recruitment and switching macrophage polarization toward the antitumor M1 macrophage state [63]. However, the point raised by Pietrocola et al. is that inhibiting autophagy may also favor relapse preventing the activation of immune responses against tumor. On the opposite, autophagy inducers may improve the efficacy of the immune system in eradicating cancer cells from the organism [62]. To this regard, a key example is represented by the role of nutrient starvation. In the absence of activating mutations that cause constitutive activation of the phosphatidylinositol-3-kinase (PI3K) pathway, which suppresses autophagy via IGF1R (insulin-like growth factor 1 receptor), tumor progression is prevented in both cellular and mice model where nutrient deprivation is experimentally applied [65]. Similarly, the synergism between chemotherapy and fasting can be reverted or eliminated in not autophagy-competent tumors, like those in which ATG5 had been depleted by transfection with a construct encoding a specific short hairpin RNA (shRNA) [66]. On 12

the opposite, in xenograft mice, starved for 48 h before chemotherapeutic treatment significantly reduced side effects and high-dose toxicity compared to mice fed with standard diets [67]. The safety of 24-72 h short term starvation before chemotherapy administration has been also reproduced in cancer patients, where 8-30% reduction in IGF-1 levels was measured in the fasting cohorts [68]. Since water and short term starvation is a demanding treatment in both mice and patients, the effects of a fasting-mimicking diet, low in proteins, carbohydrates and calories has been tested alone or in combination with chemotherapy, resulting in T-cell-dependent elimination of cancer cells throughout the stimulation of the hematopoietic system and the increased CD8+dependent tumor-cytotoxicity [69]. Several approved and/or experimental drugs, together with natural compounds, have been reported to induce autophagy in different cancer types [70-72] (Table 1). In the next paragraphs, we will review more in detail a group of these agents selected in relation to their promising future outcomes. However, it must be underlined that in the large majority of cases, data are referring to pre-clinical studies and the molecules investigated, especially those present in foods and herbs, are known for their pleiotropic modes of actions, making difficult to identify a specific molecular mechanism. In the examples of drugs already approved by international drug agencies, many of them are on the market as remedies against diseases different from cancer (refer to Table 4 in [70] and Table 1 in [71] and references therein). This is the case of few FDA-approved drugs employed in the treatment of different clinical conditions such as neurodegenerative disorders, aging, metabolic, infectious diseases and also able to induce autophagy. Two of them, metformin and rapamycin, will be further reviewed here for their potential role in cancer treatment (Table 1, Fig. 2).

4.1 BH-3 mimetics As reported above, BECN1/ATG6 is a phylogenetically conserved gene, essential for the initiation of autophagy. Its product, the protein Beclin-1, interacts with the class III PI3K, Vps34 [73]. It has 13

been reported that the affinity of the wild-type Beclin-1 (the BH3 peptide) for Bcl-XL was significantly high, with a Kd 203±6 nM, similar to Bax-BH3 or Bak-BH3 [74]. ABT-737 is a founder of a class of pro-apoptotic, pharmacological agents, so-called BH-3 mimetics, able to bind with high affinity anti-apoptotic Bcl-2, Bcl-XL and Bcl-w, but does not antagonize other antiapoptotic members, such as Mcl-1 or Bfl-1/A1 which determine resistance to ABT-737 in several cancers including chronic lymphocytic leukemia [75-77]. ABT-737 inhibits competitively the binding of Beclin-1 BH3 peptide to Bcl-XL with an IC50 in the micromolar range. This effect was specific since no inhibition was measured on the interaction between Mcl-1 and Beclin-1 [74]. Consequently, Beclin-1-dependent autophagy was increased as determined by the appearance of autophagic vacuoles, which were suppressed after treatment with Beclin-1-specific siRNAs. In addition, ABT-737 mimicked nutrient withdrawal in affecting the interaction between Beclin-1 and Bcl-2 located in the ER. However, the functional relevance of this interaction in term of anticancer effects was not described. More recently, (-)-gossypol, a pan-BH3 mimetic with a range of inhibition wider than ABT-737 and its derivatives (ABT-263 and ABT-199), showed in vitro and in vivo anticancer effects in glioblastoma cells throughout a mechanism involving autophagy-like cell death [78, 79]. In fact, (-)-gossypol triggered the formation of autophagosomes and lysosomes and cytochrome c release, but cell death was caspase-independent and occurred via lysosomal damage. In addition, knocking-down BECN1 and ATG5 using lentivirus infection, strongly reduced (-)gossypol mediated cell death. It is also worthwhile to note that (-)-gossypol in combination with Temozolomide (an alkylating agent) potentiated apoptosis in apoptosis-resistant malignant glioma cells [78]. Obatoclax mesylate (GX15-070) is a small molecule antagonist of Bcl-2 family members (Bcl-2, Bcl-XL, Bcl-w and Mcl-1) which showed in clinical trials anticancer activity against several hematologic cancers [80, 81]. The role of Obatoclax in cancer is controversial. Earlier works indicated that the primary mechanism triggered by this drug was the induction of autophagy. Loss of Beclin-1 expression suppressed the ability of Obatoclax, as a single agent or in combination with 14

HDACi (Histone Deacetylase Inhibitors) plus Sorafenib, to cause cell death, inducing a so-called “toxic mitochondrial form of autophagy” [82]. In leukemic cell lines, Obatoclax disrupted the complex between Beclin-1 and Mcl-1, inducing an autophagy-dependent cell death pathway. In fact, autophagy was required for glucocorticoids sensitization by Obatoclax, as evidenced by autophagosome formation after exposure to low-dose of Obatoclax in combination with dexamethasone and the concomitant generation of endogenous LC3-II [83]. Obatoclax also stimulates autophagy to necroptosis triggering massive accumulation of autophagosomes. The blockade of autophagosome formation, by silencing of ATG5 or ATG7, abolished obatoclaxmediated cell death in rhabdomyosarcoma cells. The mechanism of action suggested that Obatoclax stimulates the interaction of necrosome components, such as FADD, RIP1 and RIP3 with Atg5, a constituent of autophagosomal membranes [84]. More recently, it has been reported that Obatoclax induced autophagy-dependent necroptosis via Mcl-1 inhibition. In fact, suppressing the expression of ATG5 by using siRNA significantly reduced the levels of LC3-II in Obatoclax-treated human oral cancer cells. In addition, Obatoclax favored the interaction between key components of the necrosome, such as RIP1K and RIP3K, with p62. On the opposite, the inhibition of RIP1 kinase by necrostatin-1 significantly protected cells from Obatoclax-induced cell death [85]. The multifaceted anticancer activity of Obatoclax also includes modulation of autophagic flux. In cisplatin-sensitive and -resistant esophageal cancer cells, this agent blocked autophagic flux as demonstrated by increased levels of p62 accompanied by elevated numbers of LC3-positive puncta. The addition of the lysosome inhibitor chloroquine did not increase LC3- II protein expression following Obatoclax treatment, indicating a block in the lysosomal degradation or autophagosomelysosome fusion and/or rather than increased autophagosome formation [86]. In a different model represented by colorectal cancer cells, Obatoclax inhibited autophagic flux and induced cell death. In this work, as above for esophageal cancer cells, Obatoclax caused accumulation of p62 and LC3II, but its effect was independent of the pro-autophagy proteins Beclin-1, Atg7 and Atg12. In fact,

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Obatoclax-induced increase of LC3-II was not affected after ATG7, ATG12 and BECN1 knockdown using siRNA [87].

4.2 Metformin Metformin was firstly used in the popular medicine as a synthetic derivative of guanidine against symptoms of diabetes and soon became a first-line drug against type 2 diabetes (T2D) with a mechanism of action involving the pharmacological activation of AMPK. Later, analyses of retrospective data from patients affected by T2D indicated that metformin was associated with a 30% reduction in cancer incidence [27, 88, 89]. As extensively recently reviewed [71], the anticancer effects of metformin was associated with autophagy induction in malignant cell lines and mice models. As reported above for the BH-3 mimetics, the classical experimental approach to demonstrate the activation of autophagy by metformin has been the silencing of ATG genes, which attenuated or reverted the cytotoxic effects of the drug. Similar results were obtained using pharmacological inhibitors of the autophagic flux, which partially or totally counteracted the metformin-mediated antiproliferative effects. The exact molecular mechanisms by which metformin exerts its anticancer capacity has not been fully elucidated, being involved AMPK-dependent and AMPK-independent pathways. In the first case, activation of AMPK leads to mTOR inhibition, a negative regulator of autophagy, ending with apoptosis or cell cycle arrest [90]. AMPK activates the pro-autophagy Vps34 complex by phosphorylating Beclin-1 in Ser-91 and Ser-94 and inducing autophagy [91]. Similarly, when the energy level is limited, AMPK activation promotes autophagy by direct phosphorylation of Ulk1, a homologue of yeast ATG1, on Ser 317 and Ser-777 and parallel inhibition of mTORC1 complex through the phosphorylation of TSC2 and Raptor [92] (Fig. 2). Alternatively, metformin can enhance TRAIL (Tumor necrosis factor (TNF)-related apoptosis-inducing ligand) induced cell death in TRAIL-resistant lung cancer cells by activating the autophagy flux, as shown by a dosedependent accumulation of LC3-II and decrease in the p62 protein levels. Blocking the autophagic 16

flux by silencing ATG5 gene abolished the enhancing effect of metformin on TRAIL-mediated apoptosis. This mechanism did not appear to be strictly dependent upon AMPK activation [93].

4.3 Rapamycin and rapalogs The PI3K/mTOR pathway rapidly became a promising chemotherapeutic target, being almost universally activated in cancers [94-96]. Accordingly, the mTOR naturally-occurring inhibitor rapamycin and its analogs, the so-called rapalogs, have been rapidly identified as potential anticancer drugs. In fact, the rapamycin prodrug Temsirolimus became the first FDA-approved rapalogs for cancer treatment [97]. Recent evidence suggest that, event at relevant concentrations, toxicity of rapamycin and its analogs is low, while the main effect seems the capacity to slowdown cancer cell growth. If this may limit their chemotherapeutic efficacy, rapalogs are seen as promising cancer-preventive drugs and anti-aging compounds [96]. A growing number of articles indicate that mTOR inhibition by rapalogs is associated with stimulation of autophagy [26]. RAD-001, also known as Everolimus, a derivative of rapamycin, sensitized endometrial cancer Ishikawa and HEC1A cells to paclitaxel-induced apoptosis via the activation of autophagy detected by downregulation of AKT/mTOR phosphorylation, accumulation of GFP-LC3 dots, increased expression level of LC3-II protein. Conversely, this process was abolished by shRNA knockdown of ATG5 [98]. In pancreatic carcinoma PC-2 cells, rapamycin activated Beclin-1 gene expression in a dose-dependent manner, increased formation of autophagic vacuoles, resulting in inhibition of proliferation and induction of apoptosis [99]. Also in a preclinical model represented by the combination of oncolytic adenovirus and rapamycin, the co-treatment synergistically reduced lung cancer cell proliferation by autophagy modulation revealed by increased LC3-II/ LC3-I ratio [100]. A similar synergistic mechanism was described to explain the capacity of rapamycin to induce autophagy in malignant glioma cells in combination with PI3K inhibitors, although in this case, the autophagic markers were limited to labeling of autophagic vacuoles with monodansylcadaverine [101]. Despite these experimental indications, as underlined by different authors, the anticancer effects by rapalogs 17

should be taken with caution [71, 96], since not only the large majority of these studies have been performed on cancer cell line, but many of them go in opposite direction, suggesting an inhibitory effects of rapalogs on autophagy [102-104]. In addition, until now, only a limited clinical efficacy of rapalogs has been shown in various cancers [105, 106]. In an ongoing phase I trial clinical trials Everolimus was tested in combination with the autophagic flux inhibitor hydroxychloroquine in women affected by lymphangioleiomyomatosis (NCT01687179; clinicaltrails.gov) and Rapamune (the commercial name for rapamycin) has been given in combination with hydroxychloroquine to patients with advanced cancer (NCT01266057; clinicaltrails.gov).

4.4 Natural compounds A plethora of naturally occurring compounds have been identified as bona fide autophagy inducers. A recent review has been published on this topic [72]. Few of them showed contradictory results, others are more promising but with still vague molecular mechanism of action. In general, data available are limited to studies on cell lines. It has been proposed that resveratrol improve the efficacy of cancer chemotherapeutics, reducing their side effects [107-109]. In this context, a recent article demonstrated that resveratrol was more effective than amino acid starvation in inducing autophagy in ovarian cancer cells [110]. Both starvation and resveratrol freed Bcl-2/Beclin-1 complex, but the association between free Beclin-1 with Vps34, which leads to the production of PI3P, essential for the recruitment of autophagosomal membranes, was clearly detected only in starved cells, while resveratrol apparently induced a noncanonical Beclin-1-independent autophagy, inhibiting PI3K/Akt/mTORC1 pathway and activating AMPK, as suggested by others [111] (Fig. 2). The previous observation that resveratrol metabolites such as resveratrol sulfates can be deconjugated by sulfatases to generate a reservoir of free resveratrol in vitro and in vivo enforces the potential anticancer role of this agent. In fact, in colorectal cells, the antiproliferative effects of resveratrol-sulfates was attributed to increased presence of autophagomes, as demonstrated by their capacity to increase the conversion from LC3-I 18

to LC3-II, which is subsequently recruited to the phagophore membrane to initiate autophagy [112]. In one of the few in vivo study reported in the literature with plant derived bioactive molecules, it has been demonstrated that resveratrol (at low doses) was able to induce AMPK dependent autophagy, increasing p21 expression as a marker of senescence and suppressing adenoma development in a mice model of colorectal cancer (APC min mice) and in human colorectal tissues [113]. Of course, also for resveratrol, contradicting reports are present in the literature where inhibition of autophagy favored by resveratrol induces apoptosis in cancer cells [114, 115]. Curcumin (diferuloylmethane) the most studied bioactive compound from Curcuma species showed therapeutic efficacy in cancer therapy and sensitizing effects to chemotherapy and radiotherapy [116]. In gastric SGC-7901and BGC-823 cancer cell lines, curcumin inhibited dose-dependently proliferation and induced autophagy as resulted by the increased expression of autophagy-related proteins Beclin-1, Atg3 and Atg5; in addition, LC3-I expression was reduced whereas LC3-II promoted. The potentiation of autophagy induced by curcumin was dual and involved the downregulation of the PI3K/Akt/mTOR pathway, already seen above, and the up-regulated expressions of p53 and p21 [117]. A comparable effect was observed by other authors on the same type of cancer cells, but they questioned if curcumin‑ induced autophagy had a protective role or promoted cell death, since the autophagy inhibitor 3-methyladenine (3-MA) significantly promoted apoptosis following curcumin treatment [118]. In castration-resistant prostate cancer cells, curcumin synergized with 3-MA, confirming the induction of a protective form of autophagy [119]. This picture was reverted in A549 human lung adenocarcinoma cell line where curcumin inhibited growth and induced autophagy, but this effect was partly blocked by 3-MA. In this work, curcumin triggered the phosphorylation of AMPK and, blocking pharmacologically or genetically AMPK pathway by compound C or siRNA-mediated AMPKα1, respectively, the antiproliferative effect of curcumin was abolished [120]. Quercetin (3,5,7,3',4'-Pentahydroxyflavone) is a broadly studied natural compound, belonging to the polyphenol class of flavonoids. Its pleiotropic effects against cancer has been largely reviewed 19

[121-123]. The recent observation that quercetin can restore sensitivity to B-cells resistant to chemotherapy in chronic lymphocytic leukemia by directly inhibiting PI 3K/Akt pathway [76], indirectly suggest a role of the molecule also in modulating autophagy in cancer cells. An early work, clearly reported that quercetin activated autophagy in gastric cancer cells by inducing the formation of acidic vesicular organelles, autophagic vacuoles, increasing the LC3-II/ LC3-I ratio and recruiting LC3-II to the autophagosomes. In vivo studies confirmed this effect since the expression of LC3 and accumulation of LC3-II were significantly increased in gastric tumor xenograft after quercetin treatment. However, the induced autophagy was “protective” since autophagic inhibition by chloroquine or selective silencing of ATG5 or BECN1 genes increased apoptotic cell death in AGS and MKN28 gastric cancer cells [124]. A more recent paper, convincingly confirmed that quercetin can trigger protective autophagy although in a different cell type and with a different mechanism. In ovarian cancer cells, CaOV3 and in the primary P#1 cells, quercetin inhibited cell growth with an IC50 in the micromolar range via an ER (endoplasmic reticulum) stress-mediated mechanism. ER stress mediates a relatively new mode of cell death with potential implication for cancer therapy [125]. Quercetin triggered ER-stress and induced apoptosis suppressing constitutive STAT3 phosphorylation and down-regulating Bcl-2 whose expression is controlled by STAT3 [126]. In addition, quercetin induced autophagy in both CaOV3 and P#1 cells. However, when the autophagy inhibitor 3-MA was used in combination with quercetin to treat ovarian cancer cells or in xenograft mice injected with CAOV3, the expression of LC3, Atg5 and Beclin-1 was strongly reduced and the cytotoxicity of quercetin significantly amplified [126]. An interesting observation, which highlights the complexity of the autophagy regulation in cancer cells, has been reported for gliomas. Quercetin inhibited proliferation and induced autophagy in glioma cells. When 3-MA, which blocks the early stage of autophagy, was tested in combination with quercetin, the cytotoxic effects were attenuated and, consequently, autophagy activation was necessary to ensure the anticancer effect of quercetin, at least in the early stage. However, cotreatment of quercetin and chloroquine, which inhibits the late autophagy stage, increased the anti20

glioma efficacy of quercetin mono-treatment, confirming its capacity to induce a protective autophagy. These data were strengthened by parallel experiments in glioma xenografts obtained implanting C6 glioblastoma cells intracranially and treating mice with quercetin, chloroquine or both. In the combination group, autophagy was strongly inhibited, as detected by LC3-II accumulation and apoptosis increased as demonstrated by activation of caspase-3 [127]. Finally, to complete the picture on the multifaceted outcomes of quercetin in modulating autophagy, evidence have been published on the capacity of quercetin to activate a non-protective form of autophagy. Similarly to metformin (see above), quercetin can enhance TRAIL-induced cell death in human lung cancer cells and this effect was mediated by induction of autophagy flux and apoptosis. In fact, quercetin treatment increased distribution of LC3 punctate fluorescent distribution in the autophagosomes, decreased p62 and increased LC3-II expressions. In the presence of chloroquine, the autophagic flux was blocked and the capacity of quercetin to enhance TRAIL-induced apoptosis abolished [128].

5. Conclusions We learned from the discussion reported above that the main obstacle in developing autophagy inducers in clinics is represented by the dual and opposite functions of autophagy in cancer cells. In specific circumstances, depending on the genetic background, stage of the tumor, etc. autophagy can be “protective”, reducing the effects of different cellular stresses and promoting an unwanted survival of malignant cells, or, on the opposite, can potentiate different forms of cell death. For almost all the examples discussed above and referring to synthetic or natural compounds, we reported cases where the same molecule activated the autophagic flux, but the result in terms of cancer cell survival resulted controversial. Perhaps, one of the key question regards the appropriateness of the pre-clinical models employed. Especially when we are dealing with pleiotropic compounds, both drugs or natural agents, their low specificity can generate artifacts if the treatment is performed exclusively on cell lines. This criticism is not novel and is shared by the 21

large class of phytochemicals. As an example, we recently reviewed the controversial effects of polyphenols as antioxidants in cancer therapy and prevention ending up with the conclusion that the inappropriate use of cellular and animal model generated a significant part of the confusion and contradictory results present in the literature [129]. Of course, we can suggest a more rational selection of adequate cell lines, where the main autophagic pathways have been identified and characterized, before starting the treatment with potential autophagy inducers in mono-treatment or, as often reported, in combination with other drugs. However, for autophagy, this may not be enough. As briefly reported at the beginning of this article, the field “exploded” in the last few years. The complexity of this process multiplied the number of specific assays and, consequently, the reagents and methods available to better assess the autophagy pathways under investigation. Fortunately, periodically, guidelines are published to help the researchers to better examine macroautophagy and related processes, as well as reviewers to critically evaluate autophagy related manuscripts [4, 130, 131]. As an example, until few years ago, a simple immunoblot showing the change in the expression of LC3 or the ratio LC3-II/LC3-I was considered sufficient to define the presence of an ongoing process of autophagy. Nowadays, it is mandatory to identify the autophagic pathway investigating by gene knockout or RNA interference, in addition to assess the expression of multiple autophagy-related proteins. Moreover, the functional pleiotropy of Atg proteins, which are involved in other cellular pathways, implies that not all of them can be employed as unique and specific marker for a defined autophagic process [130]. As a corollary, the conclusions deriving from articles published before the last 5-7 years and dealing with autophagy regulation should be carefully taken into consideration if the currently accepted methodological criteria have not been applied. If the lack of specificity and the high pleiotropy may represent a problem in view of the potential chemotherapeutic features of autophagy inducing agents, it may switch into an advantage in the case of chemopreventive strategies. In fact, as non-pharmacologic interventions such as limited caloric intake and moderate exercise can induce autophagy and ameliorate health, several approved 22

drugs and natural agents can mimic this effect. This role is emerging for degenerative and infectious diseases and can be also extended to primary and secondary cancer chemoprevention where low dosage and prolonged drug administration is required. In this case, the tolerability of the nonautophagic adverse effects of autophagy-inducing agents can be mild and acceptable with the result that, at the low doses applied, only the desirable autophagy outcomes prevail. To this regard, the observation that the toxicity of rapalogs can be very low and their main effects result in the capacity to slow-down cancer cell growth has been recently confirmed by a recent work from our laboratory where a carotenoid extract triggered nonprotective autophagy by inducing a delay in cell growth [132]. However, this possible interpretation cannot lower the attention on searching for new autophagy inducers characterized by high specificity and limited side effects. A great attention in designing new autophagy inducers in cancer treatment must be given to the interplay between autophagy and other processes regulating cell death and cell survival, such as apoptosis. As an example, new BH3 mimetics may interfere with the binding between Beclin-1 and Bcl-2 members with anti-apoptotic functions, but also influence the binding between the pro-apoptotic and the antiapoptotic Bcl-2 family members resulting in reduced specificity towards autophagy regulation and potentially increased side effects. It would be desirable that, based on the structural determinants involved in the Beclin-1 binding to BH3 domain, only BH3 mimetics highly specific for autophagy induction will be designed. Other autophagic molecular targets may result more promising being, theoretically, less influenced by not specific interactions. To this regard, post-translation modifications (phosphorylations/dephosphorylation, acetylations/deacetylation, ubiquitynations/deubiquitinations) on Ulk1 represents promising targets for new autophagy inducers [133]. As an example, the activation of TIP60, which acetylates and activates Ulk1 [134] and/or the Ulk1 ubiquitylation and activation by AMBRA1/E3-ligase TRAF6 complex [135] may have the advantage to highly specifically enhance autophagy, without influencing other regulatory pathways.

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The novel identified compounds must show their effectiveness at physiological and pharmacological concentrations and respond to the methodological priorities discussed above, such as: a) the putative new inducers should be inactivated when autophagy genes are shut-off; b) their specific and direct target(s) must be identified and confirmed showing that their over-expression or knockout enhance or abolish, respectively, the pro-autophagy effects. Partial progress have been obtained by large screenings based on the detection of GFP-LC3 puncta and/or LC3 expression as markers of autophagosomes presence [136-138]. Strategies can also take advantage of bioinformatics approach aimed to characterize protein interactomics and phosphoproteomics in autophagy pathways, or, alternatively, as discussed above, scientists can develop autophagy inducers able to trigger key components in the regulatory autophagy pathway, which are unique and absolutely required to activate downstream events. However, without a robust validation of the biological activity of these candidates in adequate pre-clinical models followed by clinical trials, their validity remains elusive. To this regard, it is worthwhile to note that searching for ongoing clinical studies in cancer prevention or therapy based on the treatment with the potential autophagy inducers reviewed in the present work, we did not retrieve any significant results. Due to the complexity and context dependency of autophagy in cancer progression, the search for efficient autophagy inducers is still in its infancy. However, the enormous scientific and applicative interests in the field allow us to predict that in the near future important progress will be obtained. In fact, this class of synthetic and natural agents may represent the key to understand when and how autophagy sustains cancer survival and progression and when and how autophagy can kill cancer cells stimulating immunosurveillance or other cytotoxic mechanisms.

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Conflict of interest The authors declare no conflict of interest

Acknowledgments This work was been partially supported by the following grants: C.I.S.I.A. project (Innovazione e Sviluppo del Mezzogiorno—Conoscenze Integrate per Sostenibilità ed Innovazione del Made in Italy Agroalimentare—Legge 191/2009) from the Italian Ministry of Economy and Finance and the National Research Council; BenTeN project (Wellness from biotechnologies: New Processes and Products for Nutraceutical, Cosmeceutical and Human Nutrition), within the Biotechnology Network of Campania Region (Italy); CAMPUS-QUARC project, FESR Campania Region programme 2007/2013, objectives 2.1, 2.2.

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Figure Legends: Figure 1. Double-edged sword of autophagy in cancer. Schematic illustration of the concept that autophagy can sustain or inhibit cancer cell growth, as described in the present review and discussed by others [26, 57, 139, 140]. The cartoon summarizes how the presence or absence of autophagy genes can promote or slower, respectively, cancer progression in tumors depending upon K-ras- and B-raf mutations (see text for details).

Figure 2. Autophagy inducers in cancer. Schematic illustration of the central pathways nodes (indicated with numbers 1-5 in the blue circles) influenced by the autophagy inducers described into the

text. mTOR (1) negatively regulates autophagy by phosphorylating Atg13, which reduces its interaction with Ulk1 and inhibits autophagosome formation. In condition of increased intracellular energy, mTOR also inhibits AMPK (2), a metabolic sensor that can initiate autophagy. AMPK can also directly phosphorylate and activate Ulk1. Autophagy initiation from the phagophore involves Vps34, a class III PI3K, in association with Beclin-1, which can interact with several partners, including Bcl-2 anti-apoptotic members, which modulate its binding to Vps34 (3). The complex Beclin-1/Vps34 drives the vehicle nucleation and elongation, which requires LC3-II, the lipidated form of LC3 (4). The incorporation of LC3 into the membrane is a process strongly dependent upon the activation of key Atg members, such as Atg7, Atg3 and Atg5. LC3-II also binds to the adaptor protein p62/sequestosome1, which facilitates the degradation of ubiquitinated aggregates. The process terminates with the formation of mature autophagosomes and, ultimately, in cancer cell death. Autophagic flux can be inhibited by drugs that target late stages, such as cloroquine and bafilomycin A1 (5) (see text for further details).

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34

Table 1 Selected compounds able to induce cytotoxic autophagy in different cancer cell lines. Compounds

Type of cellular model

Mode of autophagy induction

Reference

ABT-737

Cervix adenocarcinoma cell line

Inhibits the binding of Beclin-1 to Bcl-XL

[74]

GX15-070 (Obatoclax mesylate)

Rhabdomyosarcoma cell line Colorectal and esophageal cell lines

Autophagy to necroptosis with accumulation of autophagosomes Block of autophagic flux, increased levels of p62 and numbers of LC3-positive puncta

[84]

Glioblastoma cell line

Formation of autophagosomes, lysosomes and cytochrome c release

[78, 79]

Endometrial cancer cells

[90]

Lung adenocarcinoma cell line

Activation of AMPK, phosphorylation of Beclin-1 and phosphorylation of Ulk1 Activation of the autophagy flux and enhancing TRAILrelated apoptosis

Endometrial, pancreatic, lung cell lines

mTOR inhibition associated with stimulation of autophagy

[26, 99, 100]

Resveratrol

Brest cancer cell line Colorectal cell line

mTOR inhibition associated with stimulation of autophagy; increased autophagomes

[111, 112]

Curcumin

Gastric cell lines,

Increased expression of autophagy-related proteins

[117]

BH-3 mimetics

(-)-gossypol

[86, 87]

Approved drugs Metformin

Rapamycin and rapalogs

[93]

Natural compounds

Lung adenocarcinoma cell line Quercetin

Glioblastoma cell line Lung adenocarcinoma cell line

Beclin1, Atg3 and Atg5 Activation of AMPK Increased expression of autophagy-related proteins Beclin1, Atg3 and Atg5 Activation of the autophagy flux (decreased p62 and increased LC3-II expressions) and enhancing TRAILrelated apoptosis

[120] [127] [128]