Modulation of Tumour-Related Signaling Pathways by

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REVIEW ARTICLE

Modulation of Tumour-Related Signaling Pathways by Natural Pentacyclic Triterpenoids and their Semisynthetic Derivatives Andrey V. Markov, Marina A. Zenkova and Evgeniya B. Logashenko* Institute of Chemical Biology and Fundamental Medicin e, Siberian Branch Russian Academy of Sciences, Novosibirsk, Russian Federation Abstract: Pentacyclic triterp enoids are a large class of natural isoprenoids that are widely

ARTICLE HISTORY Received : September 22.2016 Revised: December 08. 2016 Accepted: December 27,20 16

00/: 10.217410929867324666/70//2/1531 J

biosynthes ized in higher plants. These compounds are potent anticancer agents that exhibit antiproliferative, antiangiogenic , antiinflammatory and proapoptotic activities. Although their effects on multipl e pathways have been reported, unifyi ng mechanisms of action have not yet been es tablish ed. To date, a huge number of semisynthetic derivatives have been synthesized in different laboratories on the basis of triterpenoid scaffo lds, and many have been assayed for their biological activities. The present review focuses on natural triterpenoids of the oleanane-, ursane- and lupane-types and their semi synthetic derivatives. Here, we summarize the diverse cellular and molecular targets of these compounds and the signal pathways involved in the performance of their antitumour actions. Among the most relevant mechanisms involved are cell cycle arrest, apoptosis and autophagy triggered by the effect of triterpenoids on TGF-~ and HER cell surface receptors and the downstream PI3KAkt-mTOR and IKKINF-kB signaling axis, STAn pathway and MAPK cascades.

Keywords: Pentacyclic triterpenoids, semisynthetic derivatives, antitumour, mechanism of action, signaling pathways, molecular targets.

1. INTRODUCTION Natural products play a significant role in antitumour drug development. It has been estimated that approximately 80% of anti tumour agents used in clinics are of natural origin or are stimulated by natural product structures [1]. atural compounds are characterized by a large diversity in chemical structures and a wide spectrum of anti tumour mechanisms, including antiangiogenesis [2], telomerase inhibition [3] and interference with signal transduction [4]. Although cancer chemotherapeutics has witnessed tremendous successes over the past century, great efforts are still needed to design anti tumour agents with novel structures and mechanisms of action. Triterpenoids are one of the most extensively investigated classes of natural compounds, including more

*Add ress cOITespondence to this autho r at the Siberian Branch Russian Academy of Sciences, Institute of Chem ical Biology and Fundamental Medicine, 630090, Novosibirsk, Russian Federation; Tel : +7-3 83-363-5161 , Fax : +7 -3 83 -363-5 153; E-mai l: [email protected] sc.ru 0929-8673 /17 $58.00+.00

than 4000 molecules, which are synthesized by 2,3oxidosqualene cyclization in many plants and exist both as free compounds or their conjugates with sugar residues (saponins) [5]. Triterpenoids are characterized by molecular backbones consisting of six isoprene links, which form four (lanostane and damarane triterpenoids) or five (pentacycl ic triterpenoids) hydrocarbon rings [6] (Fig. 1). Pentacyclic triterpenoids, in turn, are divided into 23 types depending on their molecular backbone structures. The most abundant types of pentacyclic triterpenoids are the oleanane- (oleanolic, glycyrrhetinic, aboswellic, arjunolic, maslinic acids, glycY1Thizin), ursane- (ursolic, asiatic, corosolic, ~-boswellic, ll-keto~-boswellic, acetyl II-keto-~-boswel1ic, pomolic, plectranthoic acids) and lupane- (lupeol, betulin, betulinic acid, dimethyl melaleucate) types (Fig. 2) . They have attracted a lot of attention from researchers for their diverse biological activities, including anti tumour, antiviral, hepatoprotective, antiinflammatOlY and immunomodulatory activities [7]. The investigation of the © 2017 Bentham Science Publishers

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antitumour activity of triterpenoids is still the most common area of study. In a wide range of works it was shown, that these compounds display antiproliferative activity against a huge diversity of tumour cell lines, causing cell cycle arrest and triggering autophagic and apoptotic processes. Besides their anti tumour activity in vitro, triterpenoids inhibit growth and metastasis of tumours on different murine transplantab le, xenograft and carcinogen-induced tumour models in vivo. It is known that these compounds act on diverse intracellular targets and therefore are characterized not only by direct inhibitory effects on tumour cells, but also by effects on the tumour microenvironment (e.g. suppressing tumour-related inflammation, angiogenesis, etc.). But these molecules are characterized by relatively lower efficiency in comparison with current anti tumour drugs. To improve the anticancer activity of natural triterpenoids, many researchers have tried to enhance its potency and selectivity of their action by various derivatizations. To date, hundreds of semisynthetic triterpenoid derivatives have been synthesized and characterized, and many of them are considered as promising pharmaceuticals. One of the most important areas of research involves identifying the molecular mechanism of triter-

penoids' antitumour actIVIty. To date, there are many publications covering this research area; however, it is still not clear which proteins are the primary targets of triterpenoids and which master regulators are switched on in response to these compounds. In this review, we summarize and analyze the data on the mechanism of anti tumour activity of natural pentacyclic triterpenoids of the oleanane-, ursane- and lupane- types, and their semisynthetic derivatives, with emphasis on signali ng pathways, modulated by these compounds, and the molecular targets with which tri terpenoids directly interact.

2. ANTITUMOUR ACTIVITY CYCLIC TRITERPENOIDS

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A large number of studies have demonstrated that triterpenoids display marked antiproliferative activity against various tumour cells in vitro and inhibit tumour growth in vivo [8-50]. Triterpenoids were shown in vitro to cause death of tumour cells of different histogenetic origin, including melanoma [8-10], breast [10-15], pancreatic [16], prostate [10,17,18], thyroid [10], salivary gland [19] , organspecific (hepatocellular [20-24] , non-small cell lung [9,10,25-27], ovarian [9,10,28-30] , cervical

Modulation of Cell Signaling by Penta cyclic Triterpenoids

Current Medicinal Chemistry, 2017, Vol. 24, No.l3

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[10,24,31], oral [19] , colon [9,10,24] , bladder [32], stomach [10 ,20,33,34] carcinomas) and mesenchymal (osteosarcoma [35]), central nervous system [9 ,10,3 6-

38] and haematologic (leukemia [10,20,34,39,40,41], lymphoma [41]) tumours .

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The antitumour activity of triterpenoids was confirmed in animal experiments using carcinogeninduced tumour models such as a mouse skin tumour, promoted by 12-0-tetradecanoylphorbol-13-acetate [42-44] and 7,12-dimethylbenz[a]anthracene [43,45], lung carcinoma, induced by 4-nitroquinoline I-oxide [46] and colon carcinoma, induced by 1,2dimethylhydrazine [47]. Glycyrrhetinic and betulinic acids were shown to inhibit the growth of OberlingGuerin rat myeloma [48] and FSall murin e fibrosarcoma [49], respectively. Oleanolic and ursolic acids were also effective against xenograft models of human hepatocellular carcinoma HepG2 [22] and melanoma MEL-2 [50], respectively. Inhibition of tumour growth by triterpenoids is linked not only with their direct cytotoxic activity against tumour cells, but also with their suppressive effect on angiogenesis and metastasis .

3. ANTIANGIOGENIC ACTIVITY OF PENT ACYCLIC TRITERPENOIDS Angiogenesis is a process that leads to the formation of new blood vessels and plays an important role in a range of physiological (e.g. wound healing or revascularization of ischemic tissues) and pathological (e. g. chronic inflammation or tumour progression) conditions. In the case of tumour, the triggering of angiogenesis leads to an increase in nutrient and oxygen supply to tumour cells, and is associated with intensification of tumour growth and malignancy [51]. This process can be divided into five key steps: (1) acquisition of angiogenic signals such as vascular endothelial growth factor (VEGF) by vascular endothelial cells (VECs); (2) deve lopment of the ability of VECs to damage basal membrane and reconstruct extracellular matrix (a process mediated by matrix metalloproteinases such as MMP-2 and MMP-9); (3) migration, invasion and proliferation of VECs, and (4) formation of a three-dimensional network of capillary-like tubular structures from VECs [52] (aminopeptidase N is an important regulator of this process [53]) . Triterpenoids have been shown to suppress angiogenesis by acting on all of the aforementioned steps. These compounds inhibit the expression VEGF and the metalloproteinases MMP-2 and MMP-9 by tumour cells [21 ,54-59] and effectively decrease the proteolytic activity of MMPs [60,6 1]. Treatment of VECs with triterpenoids was found to significantly inhibit migration and invasion of these cells [52,56]. Interestingly, the effect of triterpenoids on the proliferation of VECs may be compound-dependent. For example, ursolic and

Markov et al.

oleanolic acid were found to decrease the viability of bovine aortic BAE and HUVEC cells [62,63], whereas glycyrrhetinic and betulinic acids were nontoxic to HUVEC cells [64,65]. As a result of listed effects, triterpenoids can inhibit the formation of capillary-like structures from VECs, as demonstrated on a Matrigel model [55,56]) where the suppression of the enzymatic activity of aminopeptidase N by betulinic acid played an important role in inhibiting angiogenesis [66]. The antiangiogenic activity of triterpenoids was confirmed in vivo on a model of chick embryo chorioallantoic membrane [62] and murine models of hepatoma Hepal-6 [21] , renal adenocarcinoma RENCA [56] and melanoma B16FIO [67], where the density of blood vessels was significantly diminished by triterpenoid treatment.

4. ANTIMETASTATIC ACTIVITY OF PENTACYCLIC TRITERPENOIDS Metastasis is a multistep process that controls the migration of tumour cells away from primary tumour nodes and the subsequent formation of secondary tumour sites in distant organs. This process is one of the general causes of mortality in tumour patients [68]. The process of metastasis can be divided into the following steps: 1) migration of tumour cells away from the site of primary localization; 2) their invasion through the basal membrane (in this case, proteol ytic enzymes like MMP-2/9 or urokinase-type plasminogen activator (uP A), which are able to destruct basal membrane and extracellular matrix, play an important role); 3) intravasation of tumour cells into the lymphatic system or vasculature; 4) their circulation with lymph or blood; 5) escape of tumour cells from capillaries to distant tissues (extravasation) and, as a result; 6) formation of new sites of tumour growth [69] . Triterpenoids were found to effectively inhibit migration and invasion of a wide range of tumour cells in vitro [55 ,58 ,60,70-73] and decrease the expression of MMP-2, MMP-9 [55 ,7 1-75], uPA [71 ,72] and the adhesion molecule ICAM-l [75], which regulates the binding of tumour cel ls to VECs immediately before intravasation. Oleanolic and betulinic acid were also shown to inhibit the epithelial-mesenchymal transition (EMT) - the process of switching cell phenotypes from epithelial to mesenchymal, leading to an increase in cell mobility and invasion [76] - in hepatocellular carcinoma [77] and melanoma [78] cells. The antimetastatic activity of triterpenoids was confirmed in murine tumour models in vivo. These compounds were shown to effectively inhibit the metastasis

Modulation o/Cell Signaling by Penta cyclic Triterpenoids

of different types of tumours from primary sites to lung tissue. Reduction of metastasis was observed on glycyrrhetinic acid-treated mice bearing MDA-MB-231 human breast cancer xenografts [60] or ursolic acidtreated mice with TRAMP transgenic prostate adenocarcinoma [79]. Glycyrrhizin and ursolic acid were effective in treating a B 16 melanoma metastasis model [80,81] and oleanolic acid and combined injection of betulinic acid with vincristine were shown to effectively inhibit metastasis of melanoma B 16F I 0 cells [27,82].

5. SEMISYNTHETIC DERIVATIVES OF PENTACYCLIC TRITERPENOIDS Although natural pentacyclic triterpenoids are currently being widely investigated and considered as promising pharmaceuticals, there has been an increasing research effort devoted to creating their chemical derivative, with the goal of obtaining more pronounced biological activity and selectivity of action. To date, hundreds of triterpenoid derivatives have been synthesized and characterized. One of the largest successes in the field of chemical transformation of triterpenoids was achieved by Dr. Michael Sporn's group from Dartmouth Medical School (Hanover, USA), who developed semisynthetic derivatives of oleanolic acid CDDO and its methyl (CDDO-Me) and imidazole (CDDO-Im) esters [83] (Fig. 3) - which bear cyano enone functionality in ring A and are characterized by very high anti tumour potential in vitro and in vivo. CDDO and its esters were shown to effectively inhibit proliferation and cause death of a vast numb er of tumour cells (see review [84]), as well as inhibiting their migration, invasion [85] and angiogenesis [86], and significantly suppressing tumour growth in murine models of transplantable, carcinogen-induced and human xenograft tumours [84]. The preclinical success of CDDO and its esters stimulated Dr. Stephen Safe's (Texas, USA) and Dr. Nariman Salakhutdinov 's (Novos ibirsk, Russia) groups to use a glycyrrhetinic acid scaffold to synthesize structural analogues of CDDO-Me - methyl 2-cyano-3,lldioxo-18~-0Iean-l , 12-dien-30-oate (CDODA-Me) [87] and methyl 2-cyano-3, 12-dioxo-18~H-olean9( II), I (2)-dien-30-oate (Soloxolone methyl) [88] (Fig. 3), correspondingly - which were characterized by high antiproliferative activity in vitro. CDODA-Me was shown to effectively inhibit the growth of colon [87,89], bladder [90] , prostate [91] and pancreatic [92] tumours and leukemia [93] cells, whereas Soloxolone methyl caused the death of hepatocellular carcinoma,

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neuroblastoma, cervical, colon and breast cancer cells [88, 94]. The level of activity of these derivatives was comparable with CDDO-Me. For example, the IC so values (concentration of compound that causes 50% cell death) of CDODA-Me and CDDO-Me on SW480 were 0.2 - 0.5 ).1M and:::: 0.2 ).1M, respectively [87] for colon cancer cells, and the IC so values of Soloxolone methyl and CDDO-Me on KB-3-1 cervical carcinoma cells were 0.3 ).1M and l.2 ).1M, respectively [88]. CDODA-Me and Soloxolone methyl were also found to effectively inhibit tumour growth in vivo, in RKO colon cancer xenograft [89] and murine carcinoma Krebs-2 (our unpublished results) models, respectively. To date, many other structural analogues of CDDOMe have been synthesized on the basis of the betulinic [95], u- and ~-boswellic [96], ll-keto-~-boswellic [97], arjunolic [96] and ursolic [98] acids scaffolds. Interestingly, betulinic and ll-keto-~-boswellic acid analogues showed similar levels of bioactivity as compared with CDDO-Me, whereas cyano enone-containing derivatives of residual triterpenoids displayed a lower activity. All of mentioned derivatives have very high cytotoxic potential agai nst a wide range of tumour cells, and therefore have attracted the attention of researchers as promising anticancer agents. However, only CDDOMe and its fluorine-containing analogue RTA408 (omaveloxolone) (Fig. 3) have currently reached the clinical trial stage. CDDO-Me (later called bardoxolone methyl) and RT A408 have successfully completed Phase I trials for the treatment of advanced solid tumours and lymphoid malignancies [99] and nonsmall cell lung carcinoma and melanoma [100], respectively. Currently, RTA408 is being examined in a Phase II study involving patients with melanoma [101]. Other highly active derivatives of triterpenoids, whose mechanism of action are analyzed in this review, include well-known clinically approved drug carbenoxolone and a range of recently synthesized novel semisynthetic compounds [l 02-1 04] (Fig. 3).

6. MECHANISMS OF TRITERPENOIDINDUCED TUMOUR CELL DEATH Many studies have shown that natural and semisynthetic triterpenoids cause tumour cell death mainly through the activation of apoptosis and, to a lesser extent, by autophagy. Only one publication has demonstrated the ability of triterpenoids to induce necrosis [105].

6.1. Apoptosis-Inducing Activity of Triterpenoids Apoptosis is an energy-dependent process of programmed cell death that leads to the removal of defec-

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tive cells (damaged, infected or mutant) from an organism. Triterpenoids have been shown to induce tumour cell death mainly through the induction of apoptosis (see reviews [106-108]).

Apoptosis may be activated by cell-extrinsic or intrinsic signals. The extri nsic, or receptor-dependent, pathway is triggered by the interaction of the death receptors Fas (APO-lICD95), TRAIL (DR4 and DR5) and TNFRl, expressed on the cell surface, with their

Modulation o/Cell Signaling by Pentacyclic Triterpenoids

ligands FasL, Ap02L1TRAIL and TNF-a, respectively. This interaction is followed by receptor activation and the subsequent formation of the death-inducing signaling complex (DISC). This complex recruits procaspases 8 and 10 via the adaptor proteins FADD (in the case of Fas and DR4/5) or TRADD (in the case of TNFR1) , resulting in the activation of caspases 8 and 10, which in tum activate the apoptosis executors caspases 7 and 3 [109]. The intrinsic, or mitochondrial , pathway of apoptosis may be triggered in response to intracellular insults such as DNA damage [110] , oxidative stress [Ill] and an increase in intracellular Ca2+ level [112]. The central event of this pathway is an increase in the permeability of the outer mitochondrial membrane leading to rapid dissipation of the mitochondrial transmembrane potential, mitochondrial swelling, disruption of the mitochondrial outer membrane and, as a result, escape of proapoptotic proteins (cytochrome C, SmacIDIABLO , Omi/HTRA2 , AIF) from the mitochondrial intermembrane space to the cytoplasm. The key participants in this process are members of the Bcl-2 family of proteins - proapoptotic Bax and Bak, wh ich form highpermeability pores in the mitochondrial outer membranes, and antiapoptotic Bcl-2 , Bcl-xL and Mcl-1 , which bind to Bax and Bak and hold them in an inactive complex in the cytoplasm. Cytochrome C, which is translocated to the cytoplasm from mitochondria, causes activation of caspase 9 and subsequently acti vates caspases 3 and 7 [109] , wh ich in tum cleave more than 280 proteins, many of which are key regulators of cell viability [113], ultimately leading to cell death. Many studies have shown that the pentacyclic triterpenoids activate apoptosis in tumour cells mainly through the intrinsic (mitochondrial) pathway, leading to an increase the intracellular ratio of proapoptotic to antiapoptotic Bcl-2 family proteins, leading to increased mitochondrial perturbation and exclusion of proapoptotic proteins from the mitochondrial intermembrane space (see review [106-108]) (Fig. 4). It has been suggested that this processes may also occur independent of the expression of Bax and Bak proteins . A number of papers have described the ability of betulinic [114-116] , ursolic [11 7] and glycyrrhetinic [118] acids and carbenoxolone [102] to stimulate the opening of the mitochondrial pelmeability transition pore (mPTP) (Fig. 4). The classical mPTPs are the protein complexes that traverse both mitochondrial membranes and form a channel which is normally closed [119]. Opening of the mPTP is a regulated process and can be induced by high concentrations of reactive oxygen

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species (ROS) [120], which are synthesized in cells in response to triterpenoid treatment. The direct interaction oftriterpenoids with components ofthe mPTP may also lead to the opening of the pore. This was shown for betulinic acid [121] and carbenoxolone [102]. Ursolie acid was shown to cause the opening of mPTP [117] by inducing the translocation of cofilin-l protein to the mitochondria. Interestingly, unlike the compounds mentioned above, CDDO and its derivatives lead to the formation of non-classical mPTP pores [122]. These synthetic triterpenoids selectively inhibit the activity of the mitochondrial protease Lon, the main function of which is the degradation of oxidized and misfolded mitochondrial proteins. As a result, large conglomerates of proteins accumulate within the mitochondria and form the constitutively opened channels [123]. In the end, the opening of mPTP pores leads to the leakage of protons, functional disruption of the respiratory chain, mitochondrial swelling and as a result, disruption of the outer mitochondrial membrane and the exclusion of mitochondrial apoptogenic proteins from the cytoplasm (Fig. 4). Triterpenoids are also able to activate the extrinsic apoptotic pathway (Fig. 4). This effect is likely to depend on the cell line used and has been described only in a few studies [87 ,124-1 29]. The extrinsic pathway of apoptosis has been shown to be triggered by glycyrrhetinic and urso lic acids, and the oleanolic acid derivatives CDDO and CDDO-Im. These compounds enhanced the expression of the death receptor Fas and its ligand FasL [28 , 72, 126] and death receptors DR4 and DR5 [128, 129], respectively. Triterpenoids can also cause the activation ofcaspase 8 [29,87,124, 125, 129] and the subsequent fragmentation of the proapoptotic Bid protein by this enzyme [33 , 127]. Triterpeno ids can also inhibit the expression of c-FLIP [128], which is the key inhibitor of DISC formation [130]. 6.2. Autophagy-Inducing Activity of Triterpenoids Autophagy is a regulated and evolutionarily conservative lysosome-dependent pathway involved in the degradation of intracellular components including damaged and obsolete proteins and organelles [131] . During autophagy, areas of cytoplasm are isolated by bilayer membrane resulting in autophagosome formation. Autophagosomes then fuse with lysosomes to generate autophagolysosomes that are capable of enzymatically degrading their contents [131]. Two forms of autophagy exist - basal and induced. Basal autophagy is a housekeeping mechanism that controls intracellular protein quality by removing de-

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Fig. (4). Pentacyclic triterpenoids trigger apoptosis of tumour cells mainly via activat ion of intrinsic (mitochondrial-dependent) and less via activation of extrinsic (receptor-dependent) pathways. Thick arrows indicate the proteins, expression level of which is significantly changed under triterpenoid treatment. Blackfilled boxes contain abbreviations of triterpenoids that cause mPTP opening. For expl anation of abbreviations see the list of abbreviation or Fig. (2) and Fig. (3) .

fective molecules [132 , 133]. Induced autophagy is triggered in cells in response to a number of intrinsic and extrinsic signals (e.g. oxidative stress, hypoxia, infection, lack of nutrients, etc.) and can perform two opposite function. In most cases, induced autophagy fulfills a reparative function , leading to the removal of defective organelles and unfolded and aggregated proteins, restoration of cellular energetic balance and, as a result, cell survival [134]. However, in the case of strong and prolonged stress, cells can autophagocytize up to half of their cytoplasm, which causes a collapse of cell functions and eventually cell death [135 ]. Ursolic, glycyrrhetinic, oleanolic and betulinic acids and CDDO-Me have been shown to induce autophagy in tumour cells [136-145]. Treatment of tumour cells with these compounds caused autophagosome formation (which was identified by transmission electron microscopy [141 ,143] or by immunodetection of LC3II [136-145], a specific protein marker of autophagosome membranes) and led to the formation of

acidic vesicular organelles, corresponding to auto phagolysosomes [138 , 139]. Interestingly, natural triterpenoids (ursolic, glycylThetinic, oleanolic and betulinic acids) mainl y caused reparative autophagy. Pretreatment of tumour cells with autophagy inhibitors (3methyladenin [136,140,145] , wOltman in [136, 143], chloroquine [138, 144], bafilomycin Al [138]) or knockdown of the autophagy-related genes Atg5 [142] and Atg7 [145] was shown to potentiate the proapoptotic activity of these compounds. We suppose that this effect is cell context-dependent. For example, ursolic acid could trigger not only reparative autophagy, which was found in human breast [136] and prostate [140] tumour cells and gliob lastoma [139] cells, but also autophagy-dependent cell death, as detected in TC-l murine cervical tumour cells [143]. Interestingly, chemical derivatives of triterpenoids mainly caused autophagic cell death, as shown for syn thetic analogues of oleanolic (CDDO-Me [137 ,141] , HIMOXOL [146], SZC014 [147] , SZC015 [148] ,) and

Modulation of Cell Signaling by Penta cyclic Tritelpenoids

betulinic (BI0 [149]) acids. BA145 , a chemical derivative of ll-keto-~-boswellic acid, was found to induce cytoprotective autophagy [150] .

7. SIGNALING PATHWAYS RESPONDING TO THE ANTITUMOUR ACTIVITY OF PENT ACYCLIC TRITERPENOIDS The macroscopic events described above correspond to the final stage of triterpenoid action on multiple intracellular signal transduction pathways. Most likely, triterpenoids should be considered as primary chemical signals which are able to either activate cell surface receptors or modulate the activity of intracellular enzymes located at the top of signal trasduction cas cades. The interaction of triterpenoids with their molecular targets leads to the generation of second messengers which further transduce the signal via some specific pathways to effector proteins engaged in apoptosis execution. The cytotoxic effects of triterpenoids on tumour cells have been shown to result mainly from their influence on the cell surface receptors of the transforming growth factor-beta (TGF-~) and the human epidermal growth factor receptors (HER) families , phosphoinositid-3-kinase (PI3K), IKKlNF-kB, JAKSrc-ST AT and mitogen-activated protein kinase (MAPK) signaling pathways. Despite the fact that cyano-enone bearing derivatives of oleanolic and glycyrrhetinic acids (CDDO-Me and its analogs, CDODAMe) are characterized by molecular mechanisms of action different from those of natural triterpenoids, the mode of their action on aforementioned receptors and signaling cascades was found to be si milar. Thus, we decided to integrate information about mechanisms of action of natural and semi-synthetic triterpenoids in order to get a whole picture. All analyzed data about modulation of tumor-related signaling pathways by triterpenoids is tabulated in Table 1. 7.1. Effect of Triterpenoids on Receptor Tyrosine Kinases

7.1.1. Effect of Penta cyclic Triterpenoids on the TGFp/Smad Signaling Pathway Cytokines of TGF-~ superfamily include more than 40 growth factors (TGF-~ 112/3, bone morphogenetic proteins (BMPs), activins, growth differentiation factors (GDFs) and others) and control numerous biological functions such as proliferation, apoptosis, cell differentiation, migration and adhesion. TGF-~ cytokines stimulate the formation of heterotetrameric receptor complexes consisting of TGF-~ receptor I (T~RI) and TGF-~ receptor II (T~RII) di-

ClIrrent Medicinal Chemistry, 2017, Vol. 24, No. 13

1285

mel's on the plasma membrane, which then initiate phosphorylation of the receptor-regulated transcription factors Smads (R-Smads) - TGF-~s and activins lead to phosphorylation of Smad2 and Smad3 (TGF-~ signaling), whereas BMPs and GDFs activate Smadl , Smad5 and Smad8 (BMP signaling). Phosphorylated R-Smads then bind to Smad4 forming an active Smad complex which then translocates to the nucleus where it can regulate gene transcription [lSI] (Fig. 5). Malfunction in the signaling downstream of TGF-~ is implicated in serious human diseases including cancer, fibrosis and wound-healing disorders [151]. Mutations in T~RII cause resistance of cells to TGF-~ and enhancement of their tumourigenic status [152]. Moreover, many types of tumour cells are characterized by decreased expression of T~RII or failure of the TGF-~/Smad signaling pathway [153]. However, this signaling has a dual role in tumour cells : on the one hand, numerous studies have demonstrated that T~RII and TGF-~ have a tumour suppressor activity [153] , but on the other hand, high expression of TGF-~ at the late stage of cancer development promotes proinvasive and prometastatic phenotypes of tumour cells [154]. Thus, the TGF-~/Smad signaling cascade has a complex role in the regulation of both tumour suppression and progression depending on the cell context. For a comprehensi ve analysis of the mechanisms of signaling modulation by triterpenoids, the data on both tumour [155-158] and non-tumour (normal cells [85 ,159-161], hepatic stellate cells [162-164] and rodent CCl4 -induced liver fibrosis [163 ,165 ,166]) models were considered.

TGF-~/Smad

To date, only a few published studies have linked the anti tumour activity of triterpenoids with their effect on TGF-WSmad signaling. Ursolic and arjunolic acids were shown to inhibit this pathway in human glioblastoma U251 cells [.158] and Ehrlich ascites carcinoma [157] cells, respectively, resulting in a significant decrease in tumour cell viability. In contrast, CDDO and CDDO-Im activate TGF-~/Smad signaling in human promonocytic (THP-I and U937) [ISS] and promyelocytic (HL-60) [15 6] leukemia cells, inducing their monocytic differenti ation and promoting the restoration of a normal cell phenotype and sensitivity to apoptosis. In non-tumour models, triterpenoids have been shown to either activate TGF-WSmad signaling, resulting in initiation of MC3T3-El preosteoblast differentiation [161] , inhibition of TGF -~-induced migration of Rat2 fibroblasts [85] , and proliferation of lung epithelial Mv 1Lu cells [160] , or to suppress this signaling cascade, causing a decrease in collagen synthesis in

1286

Current Medicinal Chemistry, 2017, Vo l. 24, No. 13

Markov et al.

Table 1. Effects of triterpenoids on tumour cell signaling Triterpenoid Type Name Lupane

BA

BA

BA

BA

BA

BET

DMM

LUP

LUP

LUP

LUP

(Table 1) contd ....

Tumour cell line (type) KU7 2S3JB-V (bladder) MDA-MB -4S3 BT474 (breast)

RKO SW480 (colon)

IC so ,I1M ~ IO

(72 h)

Biological effects

~7(72h)

t VEGF, tsurv ivin (S - IS 11M, 48h)"

> 10 (48 h) I-S (48 h)

In vitro: apoptosis (I 0 tsurvivin

~S

(48 h)

~ 7(48h)

~lM ,

24 h) ,

In vivo: BT474 xenograft; 20 mg/kg/day (28 day); t tumour growth In vitro: apoptosis, tsurvi vin, t VEGF (S -I S ~IM, 24 h), tlHI' Mb,

U266 (myeloma) MM.IS (myeloma) PC-3 (prostate) DU-14S (prostate) MCF-7 (breast) MDA-MB-231 (breast) PC-3 (prostate)

ND c

In vivo: RKO xenograft; 2S mg/kg/day (every 2 days for 22 days); t tumour growth t Bcl-2, tBcl-xL, tsurv ivin, tcyclinDI (2S 11M, 12-48 h); apoptosis (2S 11M, 24-48 h), jcaspase-3

> 100 (24 h)

Hypox ic PC-3: tHIF-l a , t VEGF, t H UVEC-tube fonnat ion

AS49 NCI-292 (lung) MCF-7 T47D MDA-MB -23 1 (breast)

>S (24 h) >S (24 h)

jp21 , jp27, tcyclin B, Dl , E

» 10 (48 h) » 10 (48 h) > 10(48h) 101 (24 h) 44 (36 h) 46 (48 h)

Absence of apoptosis (10 11M, 24 h); Go arrest (S -1 0 11M, 12-24 h), jROS (T47D : 10 11M, 12 h), tcyclinD 1, jp21, jp27

GBC-SD (gallbladd er)

UPCI:SCC 131 UPCI:SCC084 (OSCC)

LNCaP DUI4S (prostate) HepG2 C3A PLC/PRF5 HUH-7 Hep38 (liver)

26.1 (24 h) 21.4 (24 h)

ND

ND

In vitro: apoptosis (lS-60 11M, 36 h), tcell migrati on and invasio n, tMMP-9 In vivo: GBC-SD xenograft; 30, 60 mg/kg (I time); ltumour growth, lMMP-9, t PCNA In vitro: apoptosis (SO ~IM , 24 h), jcaspase-3; lCOX-2

Ex vivo: primary OSCC tissues from pati ents; l tumour cell s, l Ki6 7 (50 ~lM, 72 h) Cytotoxicity, tCdk2 , lc-myc, tcyc lin DI , lMMP-2 lCyclin DI (SO ~IM , 24-48h), lBcl-xL (50 11M, 48 h), lsurvivin (50 11M, 16-48 h), t VEGF (50 11M, 16-48 h), tMMP -9 (SO 11M, 24-48 h); apoptosis (SO 11M, 1224 h), jBax (50 11M, 12 ,48 h), lBcl-2 (SO 11M, 8-48 h), jcleaved Bid (SO 11M, 8-48 h), jcaspases-3, -9 ; lcell migration and invas ion

Effects on cell signaling

Ref.

tEGFR, tSp-l, 3, 4 (S -I S 11M, 48h)

[184]

tSp-l , 3, 4, t HER-2 , tpHER2 , tp-MAPK, tAkt, tpAkt,t YYI (IOI1M, 48 h), t miR-27a, jZBTBI0 (S -10 11M, 24 h) t Sp-l , 3, 4, t HER-2

[192]

tSp-l , 3, 4 (S-IS 11M, 24-48 h), tEGFR, tNF-kB (P6S) ( IO-I S 11M, 24 h), t miR-27a, jZBTB I0 t Sp-I, 3, 4

[247]

tp-STAT3 (SO-I OO 11M, 4 h), tSTAT3 nuclear translocation and D A binding activity (SO I1M,4 h), t p-c-Src, t p-JAKI (S O 11M, 2-8 h), j SHP-I (S O11M, 2-8 h)

[2S9]

t p-STAT3 , t STAT3 DNA binding activity (S -10 11M, 4 h) , t STAT3 binding to VEGF promoter j p-AMPK, t p-mTOR, tpp70S6K (S 11M, I h)

[276]

t p-P 13K (P4S8) , tp-PDK1, tpAkt, tp-GSK-3~ (10 11M, 1224 h), tp-EGFR (1- 100 11M, 30 min), tp-STAT3 (1 -1 00 11M, 30 min) t p-EG FR, tp-Akt (lS-60 11M, 36 h)

[178]

[233]

[181]

l p-EGFR

l p-EGFR, l p-Akt, lp-IkB, l NF-kB (p50, p65) (50 11M, 30-60 min), lNF-kB (p50, p65) nuclear translocation ND

[185]

t HER-2

[195]

tp-STAT3 (S-50 ~lM , 6h), lSTAT3 DNA binding activity (2S -50 11M, 6 h), tST A T3 nuclear translocation (SO 11M, 6 h), t p-c-Src (50 11M, 4-6 h), l p-JAKI (50 11M, 1-6 h), lpJAK2 (50 11M, 4-6 h), jSHP-1 (SO 11M, 4 h), jSHP-2 (SO ~IM , 1-6 h)

[265]

Modulation a/Cell Signalillg by Pel1lacyclic Triterpenoids Triterpenoid Type Name LUP Ursane

AA

Tumour cell line (type) CD4+ monocytes (non-tumour) RPMJ 8226 (myeloma)

IC so, IlM ND 42.3±4.6 (24 h) 24.9±3.5 I (48 h) ND

AKBWA

U266 MM. IS SCC4 A293 (myeloma)

CA

NCI-N87 (gastric)

26.8±7.9 (7 h)

CA AA UA

IKK~

89.3 (7 h) 95 .0 (7 h) 69 .0 (7 h) 50 (24 h) 90 (24 h) 100 (24 h)

cell free kinase assay

RAW264 .7 (macro phages)

CA

THP-I (macrophages) T98G (g lioma) U373 (g lioma)

CA OA

Human monocyte derived macrophages U373 (g lioma)

Saos2 (osteosarcoma) HSOS-I (osteosarcoma) LM8 (sarcoma)

Current Medicinal Chemistry, 2017, Vol. 24, No. J 3 Biological effects

Effects on cell signaling

1287 Ref.

!M2 macrophages polarization

ND

[273]

G2/M arrest (35-40 f.lM, 24 h)

!FAK, !p-FAK (35-40 f.lM , 24 h)

[212]

!Cyclin D I, !Bcl-xL (50 f.lM, 1224 h), !survivin, !Mcl-1 , ! VEGF (50 ~lM , 2-24 h), !Bcl-2 (50 f.lM, 8-24 h); apoptosis (50 f.lM , 24 h), Icaspase-3

!p-STAn (25-100 f.lM, 4 h or 50 ~lM , 2-12 h), !STA n nuclear translocation and DNA binding activity (50 f.lM, 4 h), ! p-JAK2 (50-100 f.lM, 4 h), !pSrc (25- 100 f.lM , 4h), iSHP-I (10-100 f.lM, 4 h) !HER-2 (25-50 f.lM, 24 h), !pHER2, !p-Akt, !p-ERK 112 (25-50, 12-24 h) Triterpenoids binds to IKK~

[25 8]

Apoptosis (25-50 f.lM, 24 h), Go/G I arrest, IP27 , !cyclin DI , !pro-caspases-3 , -9 ! IKK~ activity

LPS-induced RA W264.7: ! IFN-y

CA: ! p-IKKa/~ , !Akt (20-70 IlM , 90 min), ! F-kB, !IKKa (70 IlM , 90 min) AA, UA: ! p-1KKa/~, !Akt, !NF-kB, ! 1KKa (70-120 IlM, 90 min) Macrophages: !p-STAT3, !pJAK2 , !p-NF-kB (30 IlM, I h) Glioma cells: !p-STAT3, ! pNF-kB (20-30 IlM, 1 h)

[194]

[219]

ND - 100 (24 h) - 100 (24 h) ND

!M2 macrophages polarization

!M2 macrophages polarization

!p-STAT3 (30IlM, 3 h)

CA: -60 (24 h); OA: - 35 (24 h) CA: Saos - 35 (24 h) HSOS-1 - 40 (24 h) LM8 >50 (24 h) - 30 (24 h)

Cytotoxicity

!p-ST A n (20-30 IlM ; 3 h)

Apoptosis (25-50 IlM, 24 h), Icaspase-3 , !pro-caspase-9; jp53, jp21 , ! VEGF (24 h); Compound C (AMPK inhibitor) -7 !UAinduced cytotoxicity, i p-mTOR Go/G 1 arrest, !cyclin B I , D I, D2 , E2 , !Cdk2, 4, 6, I P21 , jp27 (2040 ~lM , 24 h); apoptosis, ! Bcl-2, !Bcl-xL, IBax, jcaspase-3 (20-40 IlM , 24 h); autophagy; Compound C -7 ! PA-induced apoptosis, jpmTOR, IP-S6 Apoptosis (25 IlM, 24 h); stress of endoplasmic reticulum (10-20 ~lM , 24 h); autophagy: jLC3 -11 (15-30 ~lM , 24 h)

j p-AMPK (l0-50 IlM, 24 h), ! p-mTOR, !p-70S6K, !p4EBP1 (l0-50 IlM, 24 h)

[231]

jAMPK activity, jp-AMPK (20 ~lM , 1-24 h), !p-mTOR, !p-S6 (20-40 IlM, 24 h)

[232]

!p-Akt, JERK, jp-ERK (10-20 ~lM , 24 h)

[136]

PA

MCF-7 (breast)

PLA

DUI45 PC3 CWRV I NB26 A375 (prostate)

25.4 (24 h) 32.2 (24 h) 41 (24 h) 53.1 (24 h) 77 (24 h)

UA

MCF-7 (breast)

>30 (24 h)

[261]

[264]

(Table \) contd ....

1288

Current Medicinal Chemistry, 2017, Vol. 24, N o. 13

Triterpenoid Type Name UA

Tumour cell line (type)

Markov et al.

IC so ,I1M

Biological effects

Effects on cell signaling

Ref.

U87MG (glioma)

71.0 (24 h) 51.2 (48 h) 45.7 (72 h)

PC3 (prostate) DU145 (prostate)

>40 (24 h) - 30 (24 h)

jp-AMPK (10-40 flM, 24 h), ~p-Akt (10-40 flM, 24 h), ~pmTOR (40 flM, 24 h), ~pp70S6K, ~p-4EBPI (10-40 flM, 24 h), tJNK, jp-JNK (l040 11M, 24 h) ~ p-Akt, ~ p-mTOR (10-40 flM, 24 h), ~ p-p70S6K , ~p-4E -BP I (30-40 ~lM , 24 h)

[139]

UA

UA

U251 (glioblastom a)

- 20 (24 h)

MCF-7 (breast)

- 100 (24 h) - 100 (96 h) - 100 (2 4 h) - 100 (24 h) - 100 (96 h) - 100 (72 h) 26 (24 h) 20(48 h) 18(72 h)

G 1 arrest, ~cyc lin E, Dl , D3 , Kdk4, jp21 , j p27 (10-40 flM, 24 h); autophagy : jLC3-II (10-40 flM, 24 h), formation of acidi c vesicular organelles (40 flM , 24 h), j Ci+ (C)d, jROS Apoptosis (30-40 flM, 24 h); G 1 arrest, ~ cy clin Dl , D3 , ~ Cdk4 , j p21 (30-40 flM, 24 h); au tophagy : j LC3-II (20-40 flM , 24 h) Apoptosis (20 ~lM , 24-72 h), jcaspase-3 activity; ~ miR-2 1 , jPDCD4 (20 flM , 24-72 h) ND

UA GA CBX

MDA-MB-231 (breast)

UA

HT-29 (colon)

F GF - ~I , ~ p-Smad - 2 /3

(20

[140]

[158]

flM, 24-72 h) ~p-EGFR

( 1-100 flM, 30 min), ( 1-100 flM, 30 min)

[178]

(10-40 flM , 24 h), (20-40 flM, 48 h), ~p-JN K (I 0-40 ~lM , 48

[179]

~p-S T AT3

Apoptosis (40 flM, 24-48 h), ~ Bcl-2 , ~ Bcl-xL , j caspase-3 , -9

~p - EGFR

~p-ERK1I2 ~ p-p3 8 ,

h) UA

HCTI1 6 HT29 Cac02 (colon)

9.8 (? h) 8.7 (? h) 6.2 (? h)

In vitro: ~ c-FLlP , ~ Bcl -2 , ~ Bcl xL, ~surviv in , ~cyc lin D I , ~MMP - 9 , ~V EGF , ~ I CAM -I (5 20 JlM, 24 h) In vivo: HCT 116 xenograft ; 250 mg/kg/day (28 days), ~ tum o ur growth, ~ metas tas i s , ~cyc lin D I ,

~ NF-kB

(1 0-20

~lM ,

8 h)

[186]

~ NF - kB , ~ p-STAT3 , ~ p-EGFR

~ c -m y c , ~ B c l-2 , ~ Bcl- xL , ~ s u rv i v in , ~ MMP-9 , ~ VEGF,

UA

K562 (leukemia)

- 20 (24 h)

UA

U937 HL-60 Jurkat (leu kemi a)

ND

BGC-823 (gastric)

- 11 0 (48 h)

~ I CA M-I , j p53, jp2 1 Apoptosis (20-40 flM, 24 h), j caspase-3, -9, j cytoC d (c), j PTEN In vitro: apoptosis (15-20 flM, 612 h), jcaspase-3, -7, -8, -9, ~Mcl-l , jPP2A-C In vivo: U937 xenograft; 50 mg/kg/day (20 days) ; ~ tumour growth , ap optosis, jcaspase-3 ,

~ p - Akt

~ p - Akt,

(20-40 flM, 24 h)

[203]

jp-JNK (10-20 flM , 6-

[214]

12 h), ~p-Akt,

j p-JNK

~ Mcl-l

UA

UA

T24 (bladder)

UA

HepG2 Hep3B (liver)

- 150 flg/ml (24 h) - 50 flg/ml (48 h) 20 (20 h) 58 (20 h)

G 1 arrest, apoptosis (90 fl M, 48 h), j caspase-3, j B ax/Bcl-2, ~ce ll migrati on and in vas ion Apoptosis (50-200 fl g/ml , 24 h), j caspase 3 act ivity, jceram ide level, ~su rv i v in

Apoptosis (15-60 flM , 24 h), j caspase-3; Compound C (AMPK inhibito r) -7 ~ UA - induced apoptosis

~ Akt ,

j miR-1 33a (90 flM, 48

[218]

h) j p-AMPK (200 flg /ml, 1-1 2 h; 50-100 flg/ml ; 12 h); ~p p70S6K, ~p - S6 (50-200 flg/ml ; 24-48 h); jp-JNK, jc-Jun (50200 flg/ml; 24 h) jp-AMPK (30 flM, 8-24 h), ~pAkt (7.5 -30 fl M, 24 h; 30 flM , 8-24 h), jp - GSK3~ (7 .5-30 ~lM, 24 h)

[229]

[230]

(Ta ble 1) contd ....

Modulation o/Cell Signaling by Pel/tacyclic Tritelp enoids Triterpenoid Type Name

Tumour cell line (type)

UA

MCF-7 (breast)

UA

U266 (myeloma)

UA

DUI45 LNCaP (prostate)

UA

UA

UA

HT29 HCTI16 SW480 DLD-I (colon) HT -29 (colon)

IC so, JlM

Current Medicinal Chemistry, 2017, Vol. 24, No. 13 Biological effects

Effects on cell signaling ~l M ,

1289

Ref. [236]

40.4±1.5 (48 h) 26.3±1.55 (72 h) ND

!CyclinD I, !Cdk4 (10-30 flM , 48 h); apoptosis (30 flM, 48 h)

!FoxMI( 10-30

jBax, jBak (50 flM, 6-24 h); !cyclinD I, !Bcl-2, !Bcl-xL, !survivin, ! VEGF, !Mcl- I (50 flM, 12-24 h); apoptosis (50 flM, 12-48 h), jcaspase-3

[254]

>50 (24 h) >50 (24 h)

In vitro : apoptosis (DU 145: 50 flM, 24-72 h; L CaP: 50 flM , 4872 h), ! Bcl-2 , ! Bcl-xL, !XIAP (50 flM , 3-24 h), !survivin , ! VEGF (50 ~lM, 12-24 h)

! STA T3 -DNA binding (25-200 flM, 4h), !p-STAT3 (25-100 flM, 4 b), !STAT3 nuclear translocation (50 flM , 4 h), !pSrc (50 ~lM , 30-240 min), !pJAKI (50 flM, 120-240 min), !p-JAK2 (50 flM , 30-240 min), !p-ERKI /2 (50 flM, 60-240 min), j SHP- I (5- 100 flM, 4 h) ! p-lkB, !p-NF-kB (P65) (5 -50 flM, 4 b), ! NF-kB (P65) (n)f (10-50 ~l M , 4h), !NF-kB DNA binding (10-50 flM , 4h), !plKKa/13 (5 -50 flM, 4 h), !p-Akt (10-50 pM, 4h), !p-PDKI (550 pM, 4h), !p-Src (50 pM , 46 h), !p-JAK2 (50 pM, 1-6 h) ND

! p-ST A T3 (10-50 pM, 4 h)

[262]

ND

ND

AsPC-I MIA PaCa-2 Panc-28 (pancreatic)

ND

UA

TRAMP mice

ND

UA

25 pM (48 h)

UA

HT-29 HCTll6 (colon) HL60 (leukemia)

UA

HeLa (cervical)

32.6±2.4 (5 day) 9.5±1.0 (48 h)

In vivo: DU 145 xenograft; 200 mg/kg (twice a week for 6 weeks); ! tumour growth, ! VEGF, !caspase-3 In vitro: jcaspase-3, !tumour sphere-fonl1ing capacity in vivo: HCT 116 xenograft; 10 mg/kg/day (13 day); tumour growth in vitro: !angiogenesis, ! VEGF A,lbFGF in vivo: (a) HT-29 xenograft; 12.5 mg/kg/day (6 day a week for 16 day); ! tumour growth; (b) chick chorioallantoic membrane, !angiogenesis, ! VEGF-A, !bFGF in vitro : Apoptosis (20 flM, 24 h), !Bcl-2, !XIAP, !cIAP-I , !cFLIP, !cMyc, !cyclin 01 , !MMP-9, ! ICAM-l !VEGF Tn vivo : Panc-28 xenograft; 250 mg/kg/day (4 weeks); !tumour growth, !metastasis, ! Bcl-2, ! ICAM-I , !MMP-9, !cyclin DI , !COX-2, ! VEGF In vivo : TRAMP mice; 1% w/w diet (6 or 12 week); prostatic intraepithelial neoplasia, tumour growth,! T F-a, !IL-6, !cyclin D I, lCOX-2 , jcaspase-3 Apoptosis (20-30 pM, 24-48 h), jcaspase-3 activtiy, jCOX-2

Induction of HL60 monocytic differentiation (30 flM, 5 days) Apoptosis (40-60flM, 48 h) , jcaspases-9, -3 activities, jcytoC (c), jBax, jBak, !Bcl-2 , !Bcl-xL, jDUSP 1, 2, 4, 5, 6, 7, !DUSP 9, 10

48 h)

[243]

ND

!p-STAT3, !p-Akt. !p-p70S6K

[263]

! p-STA T3, !p-Akt, ! p-p70S6K

!p-NF-kB DNA binding activity (5-20 pM, 8 b)

[246]

! p-STAT3 , !NF-kB nuclear translocation

! p-Akt, ! lKKa/l3 , !STAT3 , !NK-kB (P65)

[272]

! p-ERK, j p-p38 (20-30 flM , 14 h)

[280]

j p-ERK (30 pM, 15 min - 3 h)

[28 1]

! p-p3 8, !p-ERKI I2 (40-60 11M, 48 h)

[282]

(Ta ble 1) contd ....

1290

Markov et al.

Current Medicinal Chemistry, 2017, Vol. 24, No. 13

Triterpenoid Type Name

Oleanane

Tumour cell line (type)

IC so,IlM

UA

HT-29 (colon) DUl45 (prostate)

AA-der

A549 (lung) PC9/G (lung)

KBWAderl

PC-3 (prostate) HUVEC (nontumour)

KBWAder2

HL-60 Molt-4 THPI (leukemia)

0.67 (48 h) 1 (48 h) 1.5 (48 h)

ARJA

Ehrlich ascites carCll10ma

-50 (48 h)

GA

MCF -7 (breast)

GA

LNCaP (prostate)

GA

LNCaP DUI45 (prostate)

GA

BT549 Hs5 78T MDA-MBMDA-MB-436 BT20 MCF-7 T47 D (breast)

ND

26.0±2.5 (48 h) 25.6±0.5 1(48h) 40 (24 h) 8 (24 h)

- 100 (24 h) 32.6 (48 h) >25 (24 h)

Biological effects

Effects on cell signaling

Ref.

jATP (25 IlM, 1-10 min), jCOX2

jP2Y 2 (25 IlM, 10-20 min), jpp38 (25 IlM, 1-4 h); chelerythrine (inhibitor ofPKC) ~ tUA-induced p-p38 ; jp-Src (25 ~IM , 10-30 min) tp-EGFR, tp-ERK1I2 (10 IlM, 48 h)

[289]

tp-Akt, t p-mTOR, tRaptor, t Rictor, t p-p70S6K (20-40 11M, 24h)

[150]

tp-Akt(2IlM , 6-1 8 h), j IkB (2 11M, 3-18 h), tNF-kB (p65, p50) nuclear translocation (2 IlM, 6-18 h)

[97]

F~RI , FGF-~I

[157]

tCyclin B, E, tCkdl, 2 (I 0 48 h)

~IM ,

Apoptosis (30-40 ~IM , 24h), tllljlM, jcaspase-3 , PARP-I cleavage, lcellmigration; autophagy: jLC3-II Apoptosis (2 IlM, 3-18 h); jcaspase-9 activity, jcaspase-3, tllljlM, jcytoC (c), jSmac/DlABLO (c), jAIF (c), jBax/Bcl-2 , tBcI-xL, tBid, jDR5 In vitro: toxidative stress, t TNFa,l IL-IP, IL-IO ; apoptosis icaspase-3 (10-100 IlM, 48 h) In vivo : 100 and 250 mg/kg, l tumour growth, loxidative stress, jcaspase-3 Apoptosis ( 100 IlM, 24-72 h): lllljlM, j cytoC (c), jcaspase-9, jBaxlBcl-2, jBim Absence of apoptosis (20 IlM, 24h), lMMP-9 activity, tcell invasion , 1VEGF

- 200 (48 h) - 500 (48 h)

Apoptosis (100 IlM, 48 h) ; lcell invasion; ltube forma tion fro m HUVEC; lVEGF, lMMP-9 ( 100 IlM, 24 h); l HMGB I, lTL-6, l lL8, jNAG-I (100 IlM, 48 h) 77.6 (24 h) In vitro: lcell invasion ( 12.5 -50 77.3 (24 h) IlM, 24h); 1cell migration (12.5 50 IlM, 72 h); lMMP-2/9 activi120.9(24 h) ties, 1 MMP-2/9 (12.5-50 IlM, 24 97.5 (24 h) h) >100 (24 In vivo: MDA-MB-23I xenograft; h) 40 mgikg (every 2 daysl2 >100 (24 months): lxenograft growth, h) t metastasis > 100 (24 h) > 100 (24 TNF-a-induced HUVEC: h) l ICAM-I , FHP-I cell adhesion to HUVEC (50-100 IlM, 3 h)

GA

HUVEC (non-hlmour)

GA

HepG2 Hep3B (liver)

>60 (24 h)

GA

NCI-H460 A549 (lung)

62 Ilg/ml (7 h) 78 Ilg/ml (7 h)

j Bax, jcaspase-3 (20-60 IlM, 24 h); autophagy: j LC3-11, formation of acidic vesicu lar organelles (4060 IlM, 24 h) Apoptosis (12.5-50 11M, 7 h), jcaspases-3 , -9 , l Bcl-xL, l Bcl-2 , lcyclin DI , E

(50-1 00 IlM,

[183]

48 h) F~RI , FGF - ~I

tp-Akt, t p-GSK-3, jFOX03a (100 IlM, 18h)

[I I]

lNF-kB luciferace activity (20 ~IM, 24 h); lNF-kB DNA binding, lNF-kB (P65), lp-lkB , lpPT3K, lp-Akt (20 IlM, I h) tNF-kB (P65) (100 IlM, 24 h)

[54]

[55]

MDA-MB -231: l p-p38, tNFkB activity (25-50 11M, 24 h)

ND

lp-JNK, lp-c-Jun (50 IlM, 3 h); jIkB, lNF-kB (P65) nuclear translocation (15-50 IlM, 3 h) ; tNF-kB DNA binding activity (50 ~IM , 3 h) jp-ERK (20-60 IlM, 24 h)

[64]

lp-PKC alP II, jp-PKC 1) (12.5-50 IlM, 7 h), lp-ERK, jp-JNK (25-50 IlM, 7 h)

[278]

[138]

(Table 1) contd ....

Modulation o/Cell Signaling by Pen/acyclic Triterpenoids Triterpenoid Type Name GA

Tumour cell line (type) MMQ GH3 (pituitary adenoma)

Biological effects

Effects on cell signaling

Ref.

111.5 (24 h) 69.6 (24 h)

In vitro: apoptosis (GH3: 60 IlM , 24 h; MMQ: 100 IlM , 24 h), GOIG 1 arrest, ~L'1Ij1M' jcaspase-3 , jROS In vivo: GH3 xenograft; 20 mg/kg (evelY 2 days for 12 days); ~tumour growth , ~Bc l-2, jBax, jcaspase-3 In vitro: apoptosis ( I mM, 72 h),

jp-JNK, jp-p38 (? IlM, 0.5-6 h), jp-CaMKII (? IlM, 0.5 -3 h)

[285]

~

GLZ

TF-I (leukemia)

16 (24 h)

GLZ

Bone man'owderived macrophages (nontumour)

>400 Ilg/l111 (? h)

GLZ-der

HepG2 (liver) HeLa (cervical) A549 (lung)

6.7 (? h) 7.4 (? h) 15.8 (? h)

OA

HepG2 (li ver)

~40

OA

OA

OA

OA

1000 (72 h) > 1000 (72 h)

U87MG U251MG Primary glioma ce ll s (glioma) A375 (skin melanoma)

~80

MG63 Saos-2 (osteosarcoma) MKN28 (gastric)

~7 5

HL-60 SUP-BIS THP-I KS62 (leukemi a)

(24 h)

(48 h)

40.7±0.2 (24 h)

~7 5

(72 h) (72 h)

~90(12h)

~32

OA

1291

ICso, /-lM

A549 NCI -H23 (l ung)

GLZ

Current Medicinal Chemistry, 2017, Vol. 24, No. 13

NO

(24 h)

NO

NO

[215]

~COX-2

In vivo : A549 xenogra ft ; 50 mglkg (every 2 days for 8 weeks); !tumour growth In vitro: !cell migration and invasion , apop tosis (5 -20 IlM, 24 h), jcaspase-3 , lcyclinO I , ~s u rvivin In vivo: TF-I solid tumour; 100 mg/kg/day (25 days); ~tumour growth, jcaspase-3 ~M2 macrop hages polarization ( 100 Ilg/l111 , 6h, 48 h)

In vitro: cytotoxicity /17 vivo: (a) S 180 mice sarcoma, HepG2 xenograft; 60 l11glkg/day ( 10 day); ! tumour growth ; (b) Ehrlich ascites carcinoma; 60 mglkg/day (10 day); jsurvival /17 vitro: apoptosis (40 IlM, 16-24 h), ~L'1Ij1 M, jBaxlBcl-2, jcytoC, j caspases-9 and -3 activities, JATP (40 IlM, 8-16h), j ROS (40IlM, 16h), jCOX-2 (40 IlM , 816h), G 2/M arrest, jp53 (40 IlM , 8- 16h) 117 vivo: HepG2 xenograft; 75 or ISO mglkg/day (21 day): ~xen ograft growth, apoptosis, j BaxlBcl-2, jcytoC, jcaspases-9 and -3 activities, jCOX-2, G 2iM arrest, jp53 Apoptosis (50-200 IlM, 48 h) ; ~cell migration and invasion, ~epith elia l-m esench y mal transition (20-50 flM, 48 h) Apoptosis (20-40. 7 IlM , 24 h), GoIG I arrest, L'1Ij1M. j p53. jBaxlBcl-2, jcytoC (c), jcaspase3 Apoptosis (2S -1 00 IlM, 24 h), jcaspase 3; GOIG 1 arrest, ~cyc lin OI ( 12.5 -1 00 Il M, 24 h) Apoptosis (5-20 IlM , 24 h), !L'1Ij1M, jcaspase-3, -9 , i Apaf-I Apoptosis (80 IlM , 24 h), G 1 arrest

~ PT EN

! p-Akt ( 10-20 IlM , 24 h), ~pmTOR (20 IlM, 24 h), !pST A T3 (5-20 IlM, 24 h) ! p-Akt, !p-mTOR, ~p-STA T3

[266]

Inhibitors NF -kB (Bay 110782) and JNK (SP600125) decreased GLZ-induced productiol1 of M I l11acrophagerelated cytokines ~EGF R activity (cell free kinase assay)

[272]

~ p-Akt (40 IlM, 8-24h), !pmTOR (40 IlM, 16-24h), jpERK (40 IlM, 16-24 b)

[188]

[22]

!p-Akt, !p-mTOR, jp-ERK

~ p- E RK

(50 IlM, 48 h)

[70]

!EG FR, !p-EGFR (40.7 IlM , 3-6 h), ~p-Ak t , ~E RKl /2, ! pSTAT5(20-40. 7 IlM, 24 h)

[180]

~p -Akt , ~p -p 70S6K I ,

[20 1]

! p-S 6

(2S-100 IlM , 24 h) jp-JNK, !p-Akt ( 10-20 IlM , I h) ~ PI3K (PI lOa), ~ pAkt, ! mTOR (80 IlM , 24 h)

[202] [213]

(Ta ble 1) cootd ....

1292

Current Medicinal Chemistry, 2017, Vo l. 24, No. 13

Triterpenoid Type Name OA

Tumour cell line (type) PC-3 (prostate) MCF-7 (breast)

OA I

OA

MCF7IHER2 MDA-MB43SIHER2 (breast)

CDOOMe

LNCaP PC-3 OU14S (prostate)

CDDOMe COOO1m CDDOMe

CODOMe

CODOMe CDDOMe

CDDOMe

(Table I) contd ....

ND

THP-I (macroNO phages) AS49 (lung) BXPC-3 (pancreatic) PANC-I (pancreatic) U20S (osteosarcoma)

CDDO

CDDOMe

IC so , I1M

3.S±0.2 (72 h) 3.9±0.2 (72 h)

I]IM, ! BcI -2, ! Bcl-xL, XIAP

t p-p38, tp-lNK (SO -100 ~lg/ ml , 3h), tp-ERK I12 (25-100 ~lg/ml , 3h), tp-ASK I (100 flg /ml , 3-12 h), ND

[279]

! p-HER2, ! HER-2 activity (110 11M, I h) ! p-HER2

[193 ]

! p-Akt, ! p-mTOR, !NF-kB (P6S) nuclear translocation ( 1.25-10 flM , 20 h)

[124]

ND

[128]

!p-HER-2 (0.1-1 flM , 24 h), direct interaction with HER-2 !HER-2, !p-HER2

[196]

!p-STAT3, !p-Akt, !p-Src (500 nM, 2 h)

[204]

I]IM, !cytoC (m), l ROS Apoptosis (2.S-1 0 flM, 20 h), procaspases-3, -8, -9, !t>I]IM, ! BcI-2 , ! Bcl-xL, !cIAP I, !survivin tROS, apoptosis (2.S -5 ~IM , 24 h), ! procaspases-3 , -8 , !t>I]IM

t p-AMPK, !p-mTOR

ND

! p-Akt, ! p-FOX03a, !pmTOR, ! p-p70S6KI , !p-eIF4E, ! p-4E-BP I, !NF-kB (1.2S10 flM, 24 h) ND

[205]

! p-Akt, !p-mTOR, !NF-kB (P6S) ( 1.2S-5 ~IM , 20h)

[206]

!p-Akt, !p-mTOR, !NF-kB (S10 flM , 20 h)

[207]

!p-Akt, !p-mTOR, !NF-kB (1.2S-5 ~IM, 24 h)

[208]

Modulation o/Cell Signaling by Pentacyclic Triterpenoids

Triterpenoid Tumour cell lin e (type) Type Name CDDO-Me Mia-PaCa-2 Panc- l (pancreatic) CDDO-Me Mia-PaCa-2 Panc-I I (pancreatic) CDDO-Me LNCaP PC-3 (prostate)

CDDO-Me Ec109 KYSE 70 (ESCC)

IC so, I1M - 1.25 (72 h)

- 1.25 (72 h) - I (72 h) - 1.25 (72 h) ND

Current Medicinal Ch emistry, 2017, Vol. 24, N o. 13

Biological effects

Effects on cell signaling tp-Akt, tp-mTOR, tNF-kB (P65), jp-ERK I/2 (1.25 -5 flM, 20 h)

[209]

Apoptosis (0 .63-5 flM, 48 h), ttelomerase activity, tc-Myc

tSpl , tNF-kB, tp-STAn , tSTAT3, tp-Akt, tAkt (0.63 -5 flM, 48 h) tp-Akt, tp-mTOR, tNF-kB (1.25 -10 flM , 20 h)

[210]

[228]

tp-GSK-3a, apoptosis (0.6-5 flM , 20h), tp-caspase-9, tp-Bad, t pFOX03a (0.6 -5 ~lM , 20 h), t PTEN (0 .6-5 flM, 20 h), tPP2A (1.25-5 flM, 20 h), t PHLPPI (0.3-5flM, 20h) 0.78 (24 h) Gi M arrest, jp2 1, j p53 ; apoptosis 1.21 (24 h) (Ec109: I flM, 24 h; KYSE70: 0.25 -1 flM, 24 h), t Bcl-xL, j BaxlBcl-2 , j PUMA, jcytoC (c), j caspase-3 ; autophagy; tcell invasion, tepithelia l-mesenchymal transition

CDDO-Me U937 (leukemia)

ND

t Bcl-xL, t Bcl-2 (1 fl M, 24 h), tpIkB

CDDO-Me A549 (lung) CDDO-EA

ND

in vitro: Apoptosis (1 flM, 24 h) In vivo: Vinyl carbamate-induced

ND

ND

CDDO-Me SKOV -3TR (ovarian) ND OVCAR-8TR (ovarian) MDA-MB-468 I (breast) CDDO-Me PDA 4964 (pancre- ND CDDO-EA atic) PanAsc 2159 (developed fi-om KPC mice)

CDDO-Me Smad4 Tko mice

ND

Ref.

jROS, apoptosis (1.25 -5 flM, 20 h), tpro-caspases-3,-8,-9, tll'l'M, tcytoC (m)

Ec 109: t p-PI3K, jp-p38 (1 flM, 24 h), j p-AMPK, tp-Akt, j PTEN (0.25-1 flM, 24 h), tpmTOR (0.5 -1 flM , 24 h) KYS E70 : t p-PI3K, t p-AMPK, j p-p38 (1 flM , 24 h), tp-mTOR (0.25-1 flM , 24 h) , jPTEN, tpAkt (0.5-1 flM, 24 h) tNF-kB (P65) nuclear trans location (0.25-1 flM, 6h), direct inh ibition of IKK~ kinase activity (0.25 -0.5 flM) tp-STAT3 (0.3-1 flM, 24 h)

CDDO-Me HeLa (cervical) MDA-MB-468 (breast)

1293

lung tumour; 60 (CDDO-Me) or 400 (CDDO-EA) mg/kg diet (15 weeks); t tumour fonnation tcyclinD 1, tsurvivin (1 flM ,2 -6h)

tIL-6 (0.05 -3 flM, 96 h) , apoptosis (1 flM, 8 h), tBcl-xL, tsurvivin

[21 1]

[242]

[255]

IL-6-induced HeLa: t p-JAKI [256] (HeLa: 1 flM, 6h), t p-STA n (12 flM , 6 h), tSTAn (n) ( 1-2 flM, 6 h). t ST AT3 dimerization (I fl M, 2h) MDA-MB-468: t p-JAKI (0 .5-2 flM , 6h), t p-STAT3 (0.5-2 flM , 6h or I flM , 2-6 h), t ST AT3 (n) (1-2 flM , 6h), tSTAn dimerization ( I flM , 2h) tST AT3 nuclear trans location (1 [257] flM, 4 h), tp-STAn (I flM, 824 h), tp-JAK2 , tp-Src (I flM, 24 h)

In vitro: apoptosis (CDDO-Me : 1

PDA 4964: Biotiny lated CDDO- [244] flM, 24 h; CDDO-EA: 3 flM, 24 h) Me directly interacts with STAn, IKK, RXRa, CREB, EGFR, HER-2 and a-tubulin PancAsc 2159: Biotinylated CDDO-Me directly interacts with ST AT3 and IKK; t pST AT3 (I flM , 24 h), jIkB (1-2 ~lM , 24 h) In vivo: (a) colitis-associated colon tp-STATl , t p-STAn [269] tumour; 200 ng/mouse (3 times/week for 1 moth); jsurvival, tIL-6, t IFNy, t iNOS; (b) AOMlDSS-induced colon carcinoma; 25 0 ng/mouse (3 times/week fo r 4 week), t intlammation, tIL-6, tIFN-y (Table 1) contd ....

1294

Current Medicinal Chemistry, 2017, Vol. 24, No. 13

Triterpenoid Type Name

Tumour cell line (type)

Markov et al. Biological effects

IC so ,I1M

iCHOP, i DR5 (0. 1-5 ~M , 12h or I ic-Jun, ip-c-J un , SP600125 ~M, 2-24 h), apoptosis (0.5- 1~M , 8 (lNK inhibitor) -7 lCHOP; h), stress of endop lasmic reticulum siRNA against CHOP -7 tCDDO -M e induced apoptosis Monocytic differentiation jT~R ll (100 nM, 48 h)

CDDO-Me H1792 HI57 H460 I (lung) THP-I CDDO CDDO-Im U937 (leukemia) CDDO-Im HL60 (leukemia)

ND

ND

Monocytic differentiation (2 0 nM, 48 h)

CDDO-Im

ND

In vivo: MMTV-ErbB2/neu transgen ic mice; 1.6 ~g/kg (3 times a week, 56 week); !mammalY tumouri genes is; ! tumour growth, !cMyc, IP2 1, lcyclinD I , lBcl-2 Biotinylated analog of CD DO-1m physically interacts with mTOR, RAPTOR, PIK3PI Apoptosis (500 nM, 4h)

MMTV -ErbB2/l1eu ce ll s

CDDO-I m

HEK-2 93 (kidney, transformed) PC-3 (prostate) CDDO-Im RPMl 8226 (myeloma) J 3 (mye loma) A549 (lung) MDA-MB-23I OA-der MCF-7 BT-474 T-47D I (breast) OLO-2 SMMC-772I Bel-7402 HepG2 (liver)

CDODAMe

CDODAMe

LNCaP PC3 DUI45 (prostate) Pancl Panc2 8 (pancreatic)

ND

1.2 (48 h)

,ND

Effects on cell signaling

5.5 (24 h) Apoptosis (5 -10 ~M , 24 h), PARP8. 1 (24 h) I and Bid cleavage, jcaspase-3 , -8, 11.5 (24 h) -9 16A (24 h)

Ref. [290]

[155]

ip-ERK I /2 (20 nM, 0.5-48 h), [156] i p-Smad3, i p-Smad 1/5 (20 nM , 24-72 h), iS mad4 (20 nM , 0.572 h), jTGF -~2 , jT~Rll , i BMP6 (20 11M , 24 h) !p-HER2, !p-EGFR, !EGFR, [187] !p-HE R3, !p-ERKI /2, !p-Akt, !p-JAK2, !p-Src, !p-STA T3

j lkB (CD DO-Im: 0.25-0.5 ~M), [221] ! p-Akt, tp-S6, !p-4EBP I, ! pmTOR (0. 1-1 ~, Ih) lp-STAT5 (250 nM , 1-3 h), ! p- [253] STAT3 , !p-TYK2 (250 nM , 0.5I h), iSOCS-I ( I ~M, 4-24 h), jSHP-I (300 nM, 0.5-2 h) [103] !EGFR, !HER-2 (5 -1 5 ~tM, 24 h)

5.1 ±0.8 (24 h) 2.I±OA (24 h) 7A±i.3 (24 h) - 2 (24 h) - OA (96 h) - 0.7 (96 h)

Apoptosis (0 .5-2 ~M , 24 h), G 2/M arrest, !~II' M' j BaxlBcl-2 , icytoC (c), jcaspases-3 , -9, !AIF (m), jROS

ip-p38, jp-lNK (I

Apoptosis (2.5-5 ~M , 24 h); jp27 , jp21, !cyclin DI, jNAG-I (1-5 ~M, 24 h)

jp-Akt (2.5 ~M, 4-18 h) ; jpERK, ip-lNK (2 .5 ~M , 2-24 h)

1.21 (? h) 1.79 (? h)

Apoptosis (5 -7 .5 11M, 24 h); G o/G 2 arrest (0.5-2 .5 ~M, 48 h); p21 , p2 , cyclin DI (0.5-7 .5 ~M , 24 h); iNAG- I (2. 5-7 .5 ~M, 24 h)

jp-Akt, jp-p3 8, i -ERK2 (5 Ih)

~M ,

? h)

[287]

~M,

[91]

[92]

InformatIon , conta llled mto brackets, shows mcubatlon condIllons (concentration of compound, mcuballon penod) under whIch mentIoned bIologIcal effect was observed; (b) ~"'M is defined as mitochondrial membrane potential: 1 tmiR.133a

/ (f -

'-mTORC2

.,MI .'1/""'9 KBWA·der1

~

11!91

~ -u~~~u~a~i:n-~ ~~;e;n: :r-~;s- - ~~- .- :;r;c~ ~i~d~n~ - - - - - - --~

:

~

I

-

,T -

do,,:,,~r~gulation of proteins or miRs;®r.- - inhibition of phosphorylatio~ inhIbItIon of cell processes

- _ - . _ proposed effect

I

------------------------------------------ -- ~

Fig. (7). Schematic diagram of pentacycl ic triterpenoids effects on the PI3K1Akt signaling axis in tumour cells. Triterpenoids inhibit PI3K / Akt signaling at multiple leve ls, causing significant decrease of Akt phosphorylation that subsequently inhibits phosphorylation of Akt downstream targets mTORCl and Fox03a and, as a result, causes suppression of tumour cells proliferation, survival, migration and invasion. Triterpenoid names are marked into black-filled boxes.

no ids, only betulinic acid was characterized by relatively low energy of interaction with PI3K (92.2 kl/mol) [220]. PI3K regulatory subunit 1 (PIK3Rl) was found to be a target of CDDO-Im; however, the interaction of this triterpenoid with PIK3Rl did not affect the kinase activity of PBK [221 ]. (v) inhibition of vital processes into the cells due to induction of apoptosis. In the majority of analyzed works deals with triterpenoids inhibitory effects on PI3K1 Akt signaling axis, used protocols of cell treatment caused not only modulation of mentioned signaling, but also activation of apoptotic process [97,124,140, 150, 181, 192, 194,201,203,205 2ll, 213, 214, 218]. Thus, it is unclear, which of aforementioned processes is switched on first under triterpenoids treatment. The major effectors of the P13K1Akt signal transduction pathway are protein signaling complex mTORCl and transcriptional factors NF-kB and FOX03a. These proteins were also found to be sensitive to triterpenoid action (see below).

7.2.1. Effect of Pentacyclic Triterpenoids on tlte mTORC1 Signaling Axis mTOR is a serine/threonine kinase involved in the regulation of cellular metabolism and cell growth. Dys-

regulation of mTOR signaling is associated with a wide range of diseases including cancer, immunological and neurological disorders , and cardiovascular and metabolic diseases [222]. mTOR interacts with several proteins and forms two distinct signaling complexes mTORCl and mTORC2. Proteins mLST8, DEPTOR and TtiliTel exist in both mTORCI and mTORC2, whereas RAPTOR and PRAS40 are members of mTORC1, and RICTOR, mSinl and PROCTOR1I2 are specific to mTORC2 [223]. mTORCl and mTORC2 participate in PI3K1 Akt signaling. As mentioned above, mTORC2 phosphorylates Akt at Ser473 (see section 6.2), whereas mTORCl is a downstream effector of Akt. Akt activates mTORCI (i) by direct phosphorylation of TscllTsc2 protein complex, leading to its inactivation, followed by converting Rheb into its active GTP-bound state, which directly interact with mTORCl and strongly stimulate its kinase activity [224], or (ii) by phosphorylation of PRAS40, a negative regulator of mTORCl kinase activity, leading to dissociation of PRAS40 from mTORCI [222-224] (Fig. 8) . One of the main physiological functions of mTORCl is to stimulate protein synthesis in cells, leading to enhanced proliferation. mTORCl fulfills this effect via direct phosphorylation of the translational repressor 4E-BPI and ribosomal kinase p70S6K (Fig.

1300

Markov et al.

Current Medicinal Chemistry, 2017, Vol. 24, No. 13

CaMKK

~

~

~ e-

~ ~( \~ '-------1 Ts c 1 ~ AMPK

••

~ • •

~

Tsc2

I

G,?P

Ceramide

*=l#'_

bmI.!M",

~ IIlliII

Zft'"

GTP

9~9

;:ter'.

Wl'MW'~ P

P

Dissociation from mTORC1 Autophagy

.

...

. • Binding to 5'cap of RNA • The assembly of pre-initiation translation complex Proteasome degradation

~

/

t -