Chapter 2 Flavones: Promising Basis for Drug Development of Caspase Activators

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1 Chapter 2 Flavones: Promising Basis for Drug Development of Caspase Activators Joana P Moreira 1, Helena Ramos 2,3, Sofia Salazar 2, Madalena M Pinto 1,4, Lucília Saraiva 2,3,* and Honorina Cidade 1,4 * 1 Departamento de Ciências Químicas, Laboratório de Química rgânica e Farmacêutica, Faculdade de Farmácia, Universidade do Porto, Portugal 2 Departamento de Ciências Biológicas, Laboratório de Microbiologia, Faculdade de Farmácia, Universidade do Porto, Portugal 3 UCIBI/REQUIMTE, Faculdade de Farmácia, Universidade do Porto, Portugal 4 Centro Interdisciplinar de Investigação Marinha e Ambiental (CIIMAR/CIMAR), Universidade do Porto, Portugal. Copyright: 2017 Honorina Cidade, et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source. * Corresponding Author: Lucília Saraiva, UCIBI/ REQUIMTE, Laboratório de Microbiologia, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Portugal, Rua Jorge Viterbo Ferreira nº 228, Porto, Portugal, Tel: ; Fax: ; lucilia. saraiva@ff.up.pt Honorina Cidade,Departamento de Ciências Químicas, Laboratório de Química rgânica e Farmacêutica, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira 228, Porto, Portugal, Tel: ; Fax: ; hcidade@ff.up.pt First Published July 04, 2017 Acknowledgements: This work was partially supported through national funds provided by FCT/MCTES - Foundation for Science and Technology from the Minister of Science, Technology and Higher Education (PIDDAC) and European Regional Development Fund (ERDF) through the CMPETE Programa peracional Factores de Competitividade (PFC) programme, under the Strategic Funding UID/Multi/04423/2013 and by Foundation for Science and Technology (FCT) and CMPETE under the project (3599-PPCDT) PTDC/ DTP-FT/1981/2014 PCI FEDER and PT2020UID/MULTI/04378/2013. Helena Ramos has a PhD grant (SFRH/BD/119144/2016) from FCT. 2 3

2 Abstract Apoptosis is a highly regulated programmed cell death with a central role in cell development, homeostasis and integrity of multicellular organisms. Accordingly, deficient apoptosis, namely due to depleted expression or inactivation of executioner caspases, is commonly associated with severe pathologies, including cancer. Activators of executioner caspases have therefore been considered promising anticancer therapeutic opportunities. Accumulated data have shown a strong correlation between the potent antitumor activity of flavones and their ability to trigger apoptosis through a caspase-dependent manner. Actually, selective activators of executioner caspases have recently emerged from this family of compounds. Collectively, this review highlights the flavone scaffold as promising starting point for the development of new anticancer therapeutic opportunities based on caspase activation. General considerations regarding structure-activity relationship (SAR) studies of flavones as inducers of caspasedependent apoptotic pathway are also pointed out. Keywords Apoptosis; Cancer; Caspases; Flavones; Flavonoids; Chemotherapy Introduction Flavones belong to a large group of naturally occurring compounds with a 2-phenylbenzopyran-3-one (2-phenylchroman) nucleus (Figure 1), which occurs in a wide range of structural forms with different substitution patterns, including hydroxyl, methoxy, and isoprenyl groups, among others [1]. Figure 1: Flavone scaffold. This structural diversity is the basis of the widely recognized variety of biological activities that has been attributed to flavones, namely antioxidant, antitumor, antimicrobial, anti-inflammatory, antiplatelet, and antithrombotic [2]. Furthermore, the ability of flavones to modulate the activity of several enzymes and to modify the behaviour of many cell systems is well-recognized [2]. Among the activities reported for this class of compounds, antitumor is one of the most studied. This activity has been mostly associated with their ability to induce apoptosis by interfering with the activity of a wide range of molecules involved in the apoptotic pathway, such as caspases. Caspases (cysteine-aspartic-acid-proteases) are a cysteine protease/peptidases family that uses the cysteine 4 5

3 residue as catalytic nucleophile to cleave target proteins in sites after aspartic acid residues [3]. The family of caspases consists in fifteen different proteins, being eleven of them expressed in human (caspases-1 to -10 and -14) [4]. In physiological conditions, caspases can be found in cells in their inactive form, as zymogen (procaspase) precursor. These procaspases are single strand proteins, with a variable size N-terminal pro-domain, a central large subunit (p20), with the cysteine residue active site, and a C-terminal small subunit (p10; ~10 KDa) (Figure 2). The catalytic domain includes the large subunit (p20; ~20 KDa) and the small subunit, which has essential residues for caspases activity (2) [4]. Figure 2: Schematic representation of structural features and domain organization of human procaspases from different subfamilies. CARD: Caspases-recruitment domain; DED: death effector domain. These proteolytic enzymes have been mainly involved in induction and execution of apoptosis, and have been grouped into initiator caspases (caspases-2, -8, -9, and -10) and executioner caspases (caspases-3, -6 and -7) (Figure 3) [4]. Additionally, some caspases, called inflammatory caspases (caspases -1, -4, and -5) have been critical mediators of innate immune responses to various internal and external insults [5]. Despite the relevance of caspases for maintaining the integrity and homeostasis of multicellular organisms, its deregulation underlies severe human diseases, including cancer [6-8]. Particularly, the depletion of caspases activity is a common event in apoptosis resistance, which is one of the hallmarks of cancer [9]. Subsequently, the identification of activators of this family of proteins has been considered a promising strategy in anticancer drug discovery [10]. Actually, several reports have highlighted the antitumor potential of some caspase activators, including the procaspase-3 activator compound PAC-1 [10], and compound 1541 as well as their analogues (Figure 4) [11-14]. The present review provides an overview of flavones with caspase-dependent proapoptotic effects reported since The potential exploitation of these compounds in the development of caspase activators is also highlighted. Finally, considerations about SAR of flavone derivatives essential for caspase-dependent apoptotic effect are discussed. 6 7

4 Figure 3: Caspase cascades in intrinsic and extrinsic apoptotic pathways. In the extrinsic pathway, the bounding of tumor necrosis factor (TNF) and other ligands with the transmembrane death receptors leads to the recruitment of intracellular adaptor proteins (Fas-associated death domain, FADD), which in turn recruit the initiator procaspases-8 and -10, forming death-induced signaling complexes (DISC). It is inside this DISC that the initiator procaspases-8 and -10 are activated through induced proximity dimerization, resulting in the activation of procaspases-3, -6 and -7. In the intrinsic pathway, several stimuli like oxidative stress, thermal shock, and DNA damage trigger the release of mitochondria cytochrome c to the cytoplasm, because the mitochondrial outer membrane permeabilization (MMP) is altered. The MMP is highly controlled primarily through interactions between pro- and anti-apoptotic members of the BCL- 2 protein family (anti-apoptotic: Bcl-2 and Bcl-XL; pro-apoptotic: Bax, Bak and Bid). After the release of cytochrome c the apoptosome is formed and the procaspase-9 is activated. The caspase-9 then activates downstream executioner procaspases-3, -6, and -7 to induce apoptosis. Beyond the cytochrome c, a secondary mitochondrial activator of caspases/direct IAP-binding protein (Smac/DIABL) is released into the cytoplasm. Smac/DIABL interacts with the antiapoptotic protein X chromosome-linked inhibitor of apoptosis protein (XIAP). Caspases-3, -7 and -9 are inhibited by XIAP. If Smac/ DIABL is released, XIAP is inhibited and caspases stay active. Bax: Bcl-2 associated X protein; Bcl-2: B cell lymphoma 2; Bcl-XL: B cell lymphoma-extra large; Bak: Bcl-2 antagonist/killer; Bid: BH3-interacting domain death agonist; Smac/DIABL: second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pi; tbid: truncated Bid; XIAP: X chromosome-linked inhibitor of apoptosis protein. Figure 4: Structures of PAC-1 and compound Natural Flavones Inducers of a Caspase-Dependent Apoptotic Pathway Flavones are present in fruits and vegetables included in our daily diet and can exert many beneficial effects on human health including on cancer prevention and therapy [2,15]. These chemopreventive and antitumor activities are mostly mediated by modulation of cell death pathways, including apoptosis. Table 1 summarizes the natural flavones reported as inducers of a caspase-dependent apoptotic pathway since To understand the molecular basis of the antitumor activity of flavonoids, the effect of the flavone itself (1) on proliferation, differentiation, and apoptosis of HT-29 human colon cancer cells was investigated using in vitro [16,17] and in vivo assays [16]. Flavone 1 was found to inhibit cell proliferation through regulation of the mrna levels of cell cycle- and apoptosis-related genes [17]. In addition, flavone (1) increased caspase-3 activity [16, 17]. In fact, it was shown an attenuation of the cytotoxic ef- 8 9

5 fect of flavone 1 on cells treated with the caspase-3 inhibitor Ac-DEVD-FMK, but not with the caspase-1 inhibitor Ac-YVAD-FMK, which evidenced a caspase-3-dependent apoptosis [16]. In vivo studies revealed that mg/kg of flavone (1) inhibited tumour formation in xenografts of CL205 cells through induction of apoptosis [16]. Later, it was reported that flavone 1 inhibited the proliferation of HCT116 colon cancer cells, in a concentration-dependent manner, and through promotion of a G2/M cell cycle arrest and induction of an apoptotic pathway involving the activation of caspases-2, -3, -8, -9 and -10, and a decrease of Bcl-2 protein levels [18]. In addition to the unsubstituted flavone (1), several other flavones, with different substitution patterns, including flavones with hydroxyl, methoxy, and prenyl groups, as well as those with a non-aromatic B-ring, have been reported for their promising apoptotic activity. Table 1: Natural Flavones Inducers of A Caspase-Dependent Apoptotic Pathway. H FLAVNES 1: Flavone H 2: chrysin Molecular targets in apoptotic pathway Bcl-2 family: decrease of Bcl-xL, Bcl-2 expression caspases-2, -3, -8, -9, and -10 RS generation Decrease of NF-kB expression Decrease of CX-2 expression Increase of p21 expression Bcl-2 family: increase of Bax, and decrease of Bcl-2 and Mcl-1 expression caspase-3 Decrease of XIAP expression Citotoxic effect (# )/ caspase activation/ ref HT-29 [17] =54.8±1.3 µm Caspase activity*=150 µm HT-29 [16] =n.d. Caspase activity*=200 µm HCT-116 [18] =n.d. Caspase activity*=100 µm HTH7 and KAT18 [19] =n.d. Caspase activity*=25 µm Hl-60 [20] Caspase activity*=40 µm E33 [22] Caspase activity*=80 µm KYSE-510 [23] H H 3: 7,8-DHF Bcl-2 family: increase of Bax and Bid expression caspases-3, -8, and -9 Increase of cyt c release Increase of TRAIL, DR4, Fas and FasL expression Caspase activity*=80 µm U937 [21] Caspase activity*=70 µm Decrease AIP expression MAPK pathway: activation of JNK and ERK 10 11

6 H Table 1 (contd.) H 4: Apigenin H Bcl-2 family: increase of Bax, and decrease of Bid, Bcl-2 and Bcl-x L expression caspases-3, -6, -7, -8 and -9 Δψ m dissipation Increase of cyt c release Increase of TNF-R expression RS generation Increase p53, and p27 expression Increase of AIF expression Increase of Ca 2+ generation Decrease cyclin D1, D3 and CdK4 expression Decrease glutathione levels MAPK pathway: Activation of ERK and p38 MDA-MB-453 [26] =12.12±0.86 µm Caspase activity*=20 µm NUB-7 [27] =35 µm Caspase activity*=15 µm LAN-5 [27] =22 µm Caspase activity*=15 µm THP-1 [28] (24h # )=37.8±4.2 µm (48h # )=31.9±3.2 µm (72h # )=31.9±2.7 µm (96h # )=27.8±3.9 µm Caspase activity*=15 µm 22Rv1 [29] (24h # )=80 µm (48h # )=20 µm Caspase activity*=20 µm MDA-MB-453 [30] (24h # )=59.44 µm (72h # )=35.15 µm Caspase activity*=31.15 µm MH7A [31] =100 µm Caspase activity*=100 µm Table 1 (contd.) H H H 5: baicalein H H H H H H H 6: Baicalin H H 7: norwogonin Bcl-2 family: increase of Bax and Bim, and decrease of Bcl-2 and Bcl-x L expression caspases-3, -8, and -9 Increase of Fas and FasL expression caspases-3, -7-8, and -9 RS generation Δψ m dissipation Increase of cyt c release caspase-3 RS generation HL-60 [35] Caspase activity*=5 µm CH27 [36] (2 days # )=80 µm (3 days # )=50 µm Caspase activity*=50 µm MG-63 [37] Caspase activity*=50 µm U2-S [37] Caspase activity*=50 µm HeLa [38] =96.51 µg/ml Caspase activity*=50 µg/ml HCT116 [43] =40.1 µm Caspase activity*=40 µm DU145 [39] =150 µm Caspase activity*=400 µm Junkat [40] (18h # )=200 µg/ml Caspase activity*=50 µm SW6620 [41] =200 µm Caspase activity*=100 µm CA46 [42] =10 µm Caspase activity*=40 µm HL-60 [20] =21.7±1.5 µm Caspase activity*=10 µm 12 13

7 Table 1 (contd.) H H Table 1 (contd.) H H 8: Luteolin 9: tricetin H H H H H Bcl-2 family: increase of Bax and Bad, decrease of Bcl-2, Bcl-x L and Mcl-1 expression caspases-3, -8, -9 and -10 Δψ m dissipation Increase of cyt c release Increase of Fas and DR5 expression Increase p21 expression Inhibition of AKt MAPK pathway: inhibition of ERK Bcl-2 family: increase of Bax and Bak and decrease of Bcl-2 and Bclx L expression caspase-9 RS generation HL-60 [48] =100 µm Caspase activity*=60 µm PLC/PRF/5 [49] =7.29±2.09 µg/ml Caspase activity*=15 µg/ml Hep3B [49] =15.04±0.17 µg/ml HepG2 [49] =24.97±0.11 µg/ml HA22T/VGH [49] =22.78±0.57 µg/ml SK-Hep-1 [49] =32.59±0.68 µg/ml LCC [47] =12 µm Caspase activity*=40 µm HT-29 [51] =60 µm Caspase activity*=60 µm MG-63 [50] n.d. Caspase activity*=2.5 µg/ml HeLa [52] MCF-7 [53] =23.17µM Caspase activity*=40 µm R 7 R 2 ' R 3 R 5 R 4 ' R 5 ' 10: R 3,R 4,R 5 =H, R 5,R 7 =CH 3 11: R 3,R 5 =H, R 5,R 7,R 4 =CH 3 12: R 3,R 5,R 7,R 5, R 4 =C H 3 13: Tangeretin H 14: 5-Demethyltangeretin Bcl-2 family: increase of Bax, decrease of Bid and Mcl-1 expression caspases-2, -3, -7, and -8 RS generation Increase of DR expression Decrease of cflip expression Bcl-2 family: increase of Bax, Bid and tbid, and decrease of Mcl-1 and Bcl-x L expression caspases-3, -8, and -9 Δψ m dissipation Increase of Fas and FasL expression RS generation Increase of p53 and p21 expression caspase-3 Increase of p53 and p21 expression 10: U937 [58] =24.56±2.80 µm Caspase activity*=n.d. MLT-4 [58] =48.13±5.12 µm Caspase activity* 11: U937 [58] =12.95±0.48 µm Caspase activity* MLT-4 [58] =30.44±0.52 µm Caspase activity* 12: U937 [58] =19.28±0.55 µm Caspase activity* MLT-4 [58] =31.16±2.22 µm Caspase activity* K562 [61] (72h # )=42.47±13.6 mm (96h # )=31.27±6.4 mm Caspase activity*=100 µm AGS [62] Caspase activity*=10 µm H460 [63] (72h # )=1.27 µm Caspase activity*=2 µm H1299 [63] (72h # )=1.02 µm Caspase activity*=2 µm A549 [63] (72h # )=0.99 µm Caspase activity*=2 µm 14 15

8 Table 1 (contd.) 15: Nobiletin H 16: 5-Hydroxy-7-methoxyflavone (HMF) Bcl-2 family: decrease of Bcl-2 and increase of Bax expression caspases-3, -8, and -9 Decrease CX expression MAPK pathway: Inhibition of JNK and Activation of p38 Bcl-2 family: increase of Bax, Bid and decrease of Bcl-2 expression caspase-3 Δψ m dissipation Increase of cyt c release RS generation SNU-16 [64] Caspase activity*=25 µm HL-60 [65] Caspase activity*=40 µm SMMC-7721 [66] =26.51 mg/l Caspase activity*=25 mg/l HCT-116 [67] Caspase activity*=25 µm H Table 1 (contd.) H 18: Acacetin Bcl-2 family: increase of Bax and Bad, decrease of Bid and Bcl-2 expression caspases-3, -7, -8, and -9 Δψ m dissipation Increase of cyt c release Increase AIF expression Increase of Apaf-1 expression Increase of FADD, FAF1, FasL and Fas expression RS generation Increase of p53 expression MAPK pathway: Activation of SAPK/JNK1/2 and c-jun Junkat [74] =25.77 µm Caspase activity*=40 µm MLT-4 [74] =59.07 µm Caspase activity*= 40 µm U932 [74] =45.71 µm Caspase activity*=40 µm AGS [75] Caspase activity*=60 µm MCF-7 [76] H H 17: Wogonin MAPK pathway: Activation of JNK Bcl-2 family: increase of Bax, HL-60 [20] Bad and decrease of Bcl-2, BclxL and Mcl-1 expression =67.5±2.1 µm Caspase activity*=20 µm caspases-3, -4, -8, and -9 U-2S [69] Δψ m dissipation Caspase activity*=75 µm Increase of cyt c release MCF-7 [70] RS generation (48h # )=92.12±3.47 µm Increase p53 expression (72h # )=71.32±2.74 µm H H 19: Hispidulin H Bcl-2 family: increase of Bax and decrease of Bcl-2, Bcl-x L and Mcl-1 expression caspases-3 and -8 Δψ m dissipation Increase of cyt c release Inhibition of P13k/AKT =26.4±0.7 µm Caspase activity*=50 µm SKV3[89] Caspase activity*=40 µm HepG2 [90] Caspase activity*=100 µm Decrease of PI3K/ Akt Caspase activity*=30 µm RS generation Increase ca2+ levels U251 and U87 [71] MAPK pathway: increase of ERK =25 µm Caspase activity*=25 µm HL-60 [72] Caspase activity*=40 µm SK-HEP-1 [73] Caspase activity*=40 µm 16 17

9 Table 1 (contd.) Table 1 (contd.) H H 20: Skullcapflavone I H H caspases-3 and -9 Bcl-2 family: increase of Bak, Bax and decrease of Bcl-2 expression caspase-3 T-HSC/Cl-6 [91] Caspase activity*=20 µm HepG2 [92] (24h # )= 11.60±1.71 µg/ml Caspase activity*=10 µg/ml H H 24: Eupatilin Bcl-2 family: increase of Bax and decrease of Bcl-2 expression caspases-3, -7 and -9 Δψ m dissipation Increase of cyt c release Increase of p53 and p21 expression Inhibition of AKt HL-60 [95] Caspase activity*= 100 µm MCF-7 [96] =22.9 µg/ml Caspase activity*=22.9 µg/ml H MAPK pathway: Inhibition of ERK H 21: Diosmetin H H Bcl-2 family: increase of tbid, and decrease of Bcl-2 expression caspase-3, -6, -7, -8 and -9 HeLa [93] =26.75 µm Caspase activity*=20 µm H H caspases-3 and -9 Δψ m dissipation Increase of cyt c release Caov-3, Skov-3, Hela, PC3 [97] Caspase activity*=25 µm H H 22: Eupafolin H 23: Jaceosidin H Δψ m dissipation Increase of cyt c release Bcl-2 family: increase of Bax, and decrease of Bcl-2 expression caspase-3 RS generation Increase of p53 and p21 expression MAPK pathway: Inhibition of ERK MCF10A-ras [94] Caspase activity*=100 µm 25: Cirsilineol H H 26: Eupatorin Bcl-2 family: increase of Bax and HL-60 [98] Bid, and decrease of Bcl-2 expression caspases-3, -7, -8 and -9 Caspase activity*=3 µm U937 [98] Increase of cyt c, AIF and Smac/ DIABL release Caspase activity*=3 µm RS generation Molt-3 [98] MAPK pathway: Activation of JNK IC and ERK 50 Caspase activity*=1 µm 18 19

10 Table 1 (contd.) Table 1 (contd.) H H R Bcl-2 family: increase of Bax and decrease of Bcl-2 and Bcl-x L expression caspases-3, -8, and MDAH-2274 [106] =0.69±0.92 µm Caspase activity*=5 µm H R 3 27: R 3,R 4 = CH 3 ; 5HPMF R 4 28: R 3 =H, R 4 =CH 3 ; Gardenin B Bcl-2 family: increase of 27 Bax expression and decrease of Mcl-1 expression H1299 [99] =16.5 µm Caspase family: activation Caspase activity*=10 µm of caspases-3 and -8 HCT116 [100] IC 50 =8.7 µm Caspase activity*=8 µm Decrease of ins and HT-29 [100] CX-2 expression =22.0 µm Increase of p21 expression Caspase activity*=8 µm 28 HCT116 [100] =1.6 µm 31: R= H Protoapigenone H 32: R= H DEDC Δψ m dissipation Increase of cyt c release Increase of RS levels MAPK pathway: Activation of ERK, JNK 1/2 and p38 SKV3 [106] =0.78±0.28 µm Caspase activity*=5 µm HSE6-3 [106] =8.98±0.33 µm Caspase activity*=5 µm HSE u-12 [107] =10.1±0.53 µm Caspase activity*=5 µm MDA-MB-213 [108] (24h # )=4.7µM (48h # )=1.6 µm Caspase activity*=3 µm 32 HepG2 [109] H H 29: Artonin B H H H 30: Gancaonin Q H H Caspase activity*=1.5 µm HT29 [100] =4.04 µm Caspase activity*=1.5 µm Bcl-2 family: increase of CCRF-CEM [104] Bak and Bax, and decrease IC of Bcl-2 expression 50 = 3.45±0.50 µm Caspase activity*= 5µM Caspase family: activation of caspase-3 Δψ m dissipation Increase of cyt c releas Caspase family: activation of caspases-3 and -7 CCRF-CEM[105] =39.8±9.8 µm Caspase activity*=79.6 µm H H 33: DIC H H H 34: DHEC Bcl-2 family: increase of Bax and decrease of Bcl-2 expression caspases-3 and -9 Δψ m dissipation Increase of RS levels Increase of cyt c release Bcl-2 family: increase of Bax and Bad and decrease of Bcl-2 and Bclx L expression caspases-3, -8, and -9 Caspase activity*=2.5 µm HepG2 [110] (24h # )=4.75 µg/ml (48h # )=6.26 µg/ml Caspase activity*=5 µg/ml HT-29 [111] =5.25 µg/ml Caspase activity*=7 µg/ml Δψ m dissipation Increase of cyt c release Increase of RS levels MAPK pathway: activation of JNK 1/2 and p38, and inhibition of ERK # : concentration that causes a growth inhibitory effect of 50% after 24, 48 or 72 hours or 2, 3, or 6 days of treatment. n.d.: not determined. *Lowest concentration that showed caspase activation

11 Hydroxylated Flavones The naturally occurring dihydroxyflavones chrysin (2) and 7,8-dihydroxyflavone (3) have been reported as potent inhibitors of the growth of several human tumour cell lines, with an effect mostly attributed to the induction of a caspase-dependent apoptotic response [19-23]. Particularly, it was shown that chrysin (2) has the ability to induce caspase-dependent apoptosis in anaplastic thyroid cancer (HTH7 and KAT18) [19] and oesophageal carcinoma (E33 and KYSE-519) cell lines [22, 23]. Considering HTH7 and KAT18 cells, the results demonstrated that chrysin-induced growth inhibitory effect was mediated by increased PARP and caspase-3 cleavage [19]. In addition to caspase-3 activation, the apoptotic effect of this compound was mediated through the activation of caspase-8, but not of caspase-9 in E33 and KYSE-519 cells [22, 23]. Chow et al. (2008) compared the apoptotic effect of chrysin (2) to that of 5-hydroxyflavone and 7- hydroxyflavone on leukemia HL-60 cells [20]. Contrary to chrysin (2), neither 5-hydroxyflavone nor 7-hydroxyflavone induced apoptosis in HL-60 cells, suggesting the presence of both hydroxyl groups has a determinant factor for the induction of apoptosis by these small molecules. In 2013, it was found that 7,8-dihydroxyflavone (3) inhibited cell growth of U937 human leukemic cells, in a concentration and time-dependent manner, an effect associated with the activation of a mitochondria- and caspase-dependent apoptotic pathway, as well as the inhibition of extracellular-regulated kinase (ERK) and c-jun N-terminal Kinase (JNK) [21]. Many studies have shown that trihydroxyflavones, such as apigenin (4), baicalein (5), and norwogonin (6), have also potent antiproliferative activity in several human tumour cell lines. Among these flavones, apigenin (4), commonly found in fruits and vegetables, has been con-sidered as a chemopreventive agent [24, 25]. Flavone 4 induced apoptosis through activation of caspase-3 and subsequent cyt c release in HER2/neu-overexpressing breast cancer cells (MDA-MB-453 cells) [26]. Additionally, apigenin (4) inhibited the proliferation and induced apoptosis in NUB-7 and LAN-5 neuroblastoma cells, being this effect mediated by p53 and caspase-3 activation [27]. Actually, in NUB-7 cells, Z-VAD-FMK, a caspase-3 inhibitor, rescued these tumour cells from apigenin-mediated apoptosis [27]. Moreover, in NUB-7 tumour cells, the comparison of the antiproliferative activity of apigenin (4) with that one of chrysin (2), diosmetin (21), baicalein (5), and naringenin (5,7,4 -trihidroxyflavanone), showed that the presence of the C2-C3 double bond and of the 4 -H group on the flavonoid structure were determinant for the increased tumour growth-inhibitory activity of apigenin (4) [27]. In additional work, it was studied the antiproliferative activity of apigenin (4) against several leukemia 22 23

12 (THP-1, U937, HL60, Jurkat, K562), lung (A549), breast (MCF-7), and fibroblast NIH-3t3 cells [28]. Apigenin (4) exhibited selective antiproliferative and apoptotic effect for the leukemia cells. Nevertheless, when the same cell lines were treated with the related flavonoid naringenin, only a slight effect on cell viability was detected [28]. These results reinforced the relevance of the double bond between C2-C3 for the antitumor activity. In that study, it was further demonstrated that apigenin-induced apoptosis, in human monocytic leukemia THP-1 cells, involved the activation of caspase-3 and -9 pathways. The induction of a mitochondrial- and caspase-dependent apoptotic pathway by apigenin (4) was also evidenced in prostate 22Rv1 cancer cells [29]. Similarly, in human MDA-MB-453 breast cancer cells, apigenin caused tumour growth inhibition, after 24 and 72 h treatment, through induction of a mitochondrial apoptotic pathway involving the activation of caspases-3, -6, -7, -8, and -9 [30]. In the same year, the activation of a similar apoptotic pathway was reported for apigenin in MH7A cell lines, but only involving caspase-3 and -7 activation [31]. Further studies, recently reinforced the stimulation by apigenin (4) of an apoptotic pathway associated with caspases activation, namely in lung cancer H460 cells (increase of caspase-3 and decrease of procaspase-9 expression) [32], human A431 epidermoid carcinoma cell line (increase of caspase-3 expression) [33], melanoma (A375) [34], lung carcinoma (A549) [34], and oesophageal (E33 and KYSE-510) cell lines (increase of caspases-3 and -9 expression)[22, 23]. Among trihydroxyflavones, the antitumor potential of baicalein (5) and of the respective 7-D-β-glucuronate (6) is well recognized. In 2004, Li et al. confirmed that baicalein (5) inhibited the growth of human promyelocytic leukemia HL-60 cells through induction of apoptosis with activation of caspase-3 and [35]. Later, similar results were obtained with baicalein (5), in human lung squamous carcinoma CH27 [36] and osteosarcoma [37] cells. It must be highlighted that, in MG-63 and U2-S osteosarcoma cells, it was verified that baicalein (5) reduced cell adhesion, migration and invasion in vitro [37], what emphasizes the antitumor potential of this small molecule. More recently, it was shown that baicalein (5) inhibited the proliferation of human cervical cancer HeLa cells by triggering apoptosis in a caspase-3-dependent way, and through upregulation of caspase-8 [38]. Regarding baicalin (6), several studies have reported the antitumor activity of this compound, which acts as prooxidant and inducer of apoptosis via mitochondrial pathway and through caspase-3 activation, namely in several human prostate cancer cells (DU145, PC-3 and LNCaP) [39], and in Junkat leukemia-derived T cells [40]. In 2012, it was shown that baicalin (6) inhibited the proliferation of human colorectal carcinoma SW6620 cells through induction of apoptosis associated with caspases-3, -8, and -9 activation [41], as well as of CA46 Burkitt lymphoma cells, through apoptosis induction with increased caspases-3 and -9, and [42]. A comparison between 24 25

13 the antiproliferative effect of baicalein (5) and its 7-D-βglucuronate (baicalin, 6), on three human colorectal cancer cell lines (SW-480, HCT-116, HT-29), non-small cell lung cancer cells (NSCLC), and breast cancer (MCF-7, MDA-MB-231), revealed a lower antiproliferative effect of baicalin when compared to baicalein [43]. In fact, in that study, it was referred that baicalein exhibited a significant tumour growth inhibitory effect on all tested cancer cell lines. Particularly, in HCT116 cells, baicalein-induced growth inhibition was mediated by pro-apoptotic effects with activation of caspases-3 and -9 [43]. Interestingly, in silico modelling it was proposed that baicalein (5) establishes hydrogen interactions with residues of Ser251 and Asp253, at the active site of caspase-3 [43]. Additionally, interactions of baicalein (5) hydroxyl groups with residues Leu227 and Asp228 of caspase-9 were also proposed [43]. Altogether, these results suggest a direct interaction of baicalein (5) with caspases-3 and -9. The antitumor activity of norwogonin (7) was reported by Chow et al. [20]. This flavone exhibited a potent cytotoxic effect on human HL-60 promyelocytic leukemia cells, inducing apoptosis associated with DNA fragmentation, apoptotic bodies, hypodiploid cells, and caspase-3 protein processing [20]. Among the polyhydroxylated flavones, luteolin (8) is one of the most studied mainly due to its potential in cancer prevention and therapy [44]. This flavone can be commonly found in fruits and vegetables [45] and in a variety of plants, including in traditional Chinese medicines [46]. The elucidation of the molecular mechanism associated with luteolin-induced apoptosis in promyelocytic leukemia HL-60, Lewis lung carcinoma (LLC) and oesophageal carcinoma (E33 and KYSE-510) cells revealed the stimulation of a mitochondrial pathway and the activation of caspases-3 and -9 [22,23,47,48]. The antiproliferative activity of luteolin (8) mediated by caspase-3-dependent apoptosis has been reported in several tumour cells, including in five hepatocellular carcinoma (HepG2, SK-Hep-1, PLC/PRF/5, Hep3B, and HA22T/VGH) [49] and MG-63 human osteosarcoma [50] cells. In addition to caspases-3 and -9, the activation of caspase-7 by luteolin (8) has been demonstrated in human HT-29 colon cancer cells [51]. Interestingly, the induction of apoptosis by luteolin (8) through death receptor 5 (DR5) upregulation, along with activation of caspases-3, -8, -9 and -10, was also reported in HeLa cells [52]. The pentahydroxylated flavone tricetin (9) has also been described as inducer of apoptosis in human breast adenocarcinoma MCF-7 cells through caspase-9 activation [53]. In 2004, 22 flavonoids, including isoflavones, flavanones, flavones and flavonols, were tested for their apoptotic activities, in leukemic U937 cells [54]. Several flavones and flavonols, but none of the isoflavones or flavanones tested, induced apoptosis, what highlights the 26 27

14 importance of the flavone and flavonol nucleus for this activity. SAR studies showed that at least two hydroxylations at positions 3, 5, and 7 are essential for apoptosis induction, while hydroxylation at 3 and/or 4 enhances the pro-apoptotic activity. In order to investigate the role of caspase and calpain pathways in flavonoid-induced apoptosis, the genomic DNA fragmentation was analyzed after selective inhibition of caspase- and calpain-dependent signaling pathways. Among flavones, luteolin (8) and chrysin (2) induced apoptosis in a way that required the activation of caspases-3 and -8, but not of caspase-9. n the other hand, an activation of calpains, in addition to caspases, was observed in apoptosis induced by apigenin (4) [54]. The inhibitory growth effect of structurally related flavones and flavonols was also compared in two human oesophageal cells (E33 and KYSE-510) [22, 23]. Among the flavones tested, the order of potency obtained was luteolin (8) > chrysin (2) > apigenin (4). In both type of cells, all flavones induced p53-independent mitochondria-mediated apoptosis through up-regulation of caspases-3 and -9 [22,23]. Methoxylated Flavones Methoxyflavones, particularly polymethoxyflavones, mainly found in citrus plants, have shown a wide range of biological activities, including anti-inflammatory, antiviral, antioxidant, anti-thrombogenic, anti-artherogenic and antitumor activities [55-57]. Regarding antitumor activity, several research works have been conducted in order to clarify their mode of action. In particular, in 2011, the antiproliferative activity of natural methoxyflavone derivatives and their synthetic analogues was evaluated in human leukemia (MLT-4 and U937) and peripheral blood mononuclear cell lines [58]. Flavones revealed to be only cytotoxic in leukemia cells and stimulated apoptosis in TRAIL-resistant leukemia MLT- 4, but not in U937 cells. Tangeretin (13), 5-demethyltangeretin (14) and nobiletin (15) are permethoxylated polymethoxyflavones, commonly found in citrus fruits [59], that have been reported for their growth inhibitory activity in several human cancer cell lines, such as brain, gastric, breast, leukemia, peritoneal, colon and lung cell lines [60]. Tangeretin (13) induced cell death in human K562 erythromyeloblastoid leukemia cell line, which was evidenced by, DNA fragmentation, and activation of the caspase cascade [61]. However, pretreatment with the pan-caspase inhibitor Z-VAD-FMK blocked caspase activation and cell cycle arrest, but did not inhibit apoptosis, suggesting that other cell death mechanisms, like those comprising endoplasmic reticulum (ER) pathways, could be involved [61]. More recently, the apoptotic mechanism of tangeretin (13) was evaluated in human AGS gastric cancer cells [62]. Data showed that tangeretin (13) triggered a p53-dependent mitochondrial apoptotic pathway, involving caspases-3, -8 and -9 activation and the increase of pro-apoptotic protein levels, like Bax, Bid, tbid, and 28 29

15 p53, Fas and FasL. These results therefore suggested that tangeretin (13)-induced apoptosis in AGS cells was mediated by p53-dependent mitochondrial dysfunction and Fas/FasL- extrinsic pathway [62]. The inhibitory effect of tangeretin (13), and of its demethylated analog 5-demethyltangeretin (14), was investigated in three human non small cell lung cancer cells (A549, H460, and H1299) [63]. In cell viability assays, 5-demethyltangeretin (14) exhibited a much higher potency than tangeretin (13). Actually, while extensive G2/M cell cycle arrest and apoptosis was observed for 5-demethyltangeretin (14), a ten-fold higher concentration of tangeretin (13) did not interfere with cell survival [63]. These results suggest the relevance of the 5-hydroxyl group for the antitumor activity of these compounds [63]. Furthermore, cells treated with 5-demethyltangeretin (14) induced activation of a p53-pathway, as well as of caspase-3, and [63]. Interestingly, in 2012, the tumour growth inhibitory activity of nobiletin (15) was studied in p53-mutated SNU-16 human gastric cancer cells [64]. In that study, nobiletin (15) exhibited a higher growth inhibitory effect than other flavonoids, such as baicalein (5), quercetin (3,5,7,3,4 -pentahydroxyflavone) and hesperetin (5,7,3 -trihydroxy-4 -methoxyflavanone). The nobiletin-induced growth inhibitory effect in SNU-16 cells involved apoptosis as evidenced by the increase of the cell population at the sub-g1 phase, appearance of fragmented nuclei, activation of caspases-3 and -9, and [64]. Recently, the molecular mechanisms underlying nobiletin (15) anticancer effects, in acute myeloid leukemia (AML) cells, was investigated [65]. The results showed that nobiletin (15) suppressed cell proliferation in many types of AML cells, being this effect associated with apoptosis induction and caspases-3, -8 and -9 activation in HL-60 cells [65]. In other study, it was shown that the inhibitory effect of nobiletin (15) on hepatocellular carcinoma was associated with apoptosis involving caspase-3 activation, both in in vitro and in vivo antitumor assays [66]. Flavones Carrying Hydroxyl and Methoxy Groups The 5-hydroxy-7-methoxyflavone (16, HMF), a chrysin (2) derivative found in various plant sources, is known to modulate several biological activities. In 2016, Bhardwaj et al. studied the mechanism underlying HMF-induced apoptotic cell death in human colorectal carcinoma HCT116 cells. HMF (16) was shown to be capable of inducing cell death in a dose-dependent manner. Treatment of HCT116 cells with HMF (16) triggered DNA damage and mitochondrial membrane perturbation accompanied by cyt c release, downregulation of Bcl-2, activation of Bid and Bax, and caspase-3-mediated apoptosis [67]. Several reports have revealed in vitro and in vivo tumour therapeutic potential for wogonin (17) [68]. In 2011, the cytotoxic effect of wogonin (17) was investigated in human osteosarcoma cell line (U-2 S). The results pointed to the activation of a mitochondrial apop-totic 30 31

16 pathway, involving reactive oxygen species (RS) production, modification of mitochondrial membrane potential (MMP) and cyt c release, as well as increased caspases-3, -4, -8 and -9 expression [69]. In a later study, the ability of wogonin (17) to induce apoptosis in human MCF-7 breast cancer cells was also investigated [70]. The wogonin (17) pro-apoptotic effect was mediated by activation of the p53-dependent pathway, as well as of caspases-3, -8 and -9 [70]. In addition, it was shown that Z-DEVD-fmk, a selective caspase-3 inhibitor, significantly inhibited wogonin-induced cell apoptosis, what suggested that the pro-apoptotic effect of wogonin (17) was highly dependent on caspases activation in MCF-7 cells [70]. The effect of wogonin (17) was further studied in human U251 and U87 glioma cell lines [71]. In these cells, wogonin (17) induced RS generation, endoplasmic reticulum (ER) stress and cell apoptosis through activation of caspases-3 and -9, and. The antitumor potential of wogonin (17) has also been studied by several research groups in leukemia cells. In particular, the apoptotic activity of wogonin (17), and of its 8-demethylated derivative (7, norwogonin), was evaluated in HL-60 cell lines [20]. DNA fragmentation, apoptotic bodies, and hypodiploid cells, accompanied by the induction of caspase-3 activation, were detected in cells treated with both wogonin (17) and nowogonin (7). Additionally, enzymatic assays for evaluation of caspase-3 activity showed that both flavones activated caspase-3 in HL-60 cells. Despite this, norwogonin (7) exhibited a more potent pro-apoptotic effect than wogonin (17), suggesting that the methylation of the hydroxyl group at C-8 is associated with a depletion of the apoptotic activity [20]. In addition to these flavones, the effect of six structurally related compounds, including 5-hydroxy, 7-hydroxy, 5,7-dihydroxy (2), 5,7-dimethoxy, 7,8-dimethoxy, and 7-methoxy-8-hydroxy flavones, on apoptosis induction in HL-60 cells was also analysed. The obtained results suggested that both hydroxyl groups at C5 and C7 are essential for the apoptotic activity of flavonoids [20]. Using the same human tumor cells, the cytotoxic effect of wogonin (17) and of other structure related flavones (luteolin (8), nobiletin (15), baicalein (5) and apigenin (4)) was evaluated [72]. Wogonin (17), baicalein (5) and apigenin (4) showed pronounced cytotoxic effects on HL-60 cell lines. Despite this, wogonin (17) showed to be the most potent apoptotic inducer, conducting to activation of caspase-3, but not of caspase-1 [72]. In fact, contrary to the caspase-1 inhibitor Ac-YVAD-CH, the caspase-3 inhibitor Ac-DEVD-CH partially reverted the cytotoxic effects of wogonin [72]. Similar effects have been induced by wogonin (17) in SK-HEP-1 hepatocellular carcinoma cells [73]. ther natural trioxygenated flavone with apoptotic inducing effect is acacetin (18), the apigenin -methylated derivative found in several plants. Acacetin (18) inhibited the proliferation of Jurkat cells by inducing mitochondria-mediated apoptosis involving activation of caspases-3, -8 and -9 [74]. Acacetin-induced apoptosis was blocked by inhibitors of caspases-3 and -8, 32 33

17 but not by a caspase-9 inhibitor, suggesting that caspase-9 may not be required for acacetin-induced apoptosis [74]. Similar results were obtained with acacetin (18) in human gastric carcinoma cells [75]. In fact, in these cells, a mitochondrial apoptotic pathway, characterized by mitochondrial transmembrane potential loss, RS generation, and cyt c release, in association with and the activation of caspases-3, -8, and -9, but not of caspase-1, was also induced by acacetin (18). Further evaluate the requirement of caspase-3 activation for acacetin-apoptotic effect, the inhib-itors of caspase-3 (Z-VAD-FMK) and caspase-1 (Ac-YVAD-FMK) were used. The obtained results showed that the inhibitor of caspase-3, but not of caspase-1, significantly inhibited acacetin-induced apoptosis. In addition to the caspase-3-dependent apoptotic effect described above [75], a caspase-7-dependent apoptotic effect was triggered by acacetin (18), in human MCF-7 breast cancer cells [76]. To investigate the relationship between caspase-7 and caspases-8 or -9 activation in acacetin-induced apoptosis, cells were treated with the specific inhibitors of caspase-8 (Z-IETD-FMK) or caspase-9 (Z-LEHD-FMK) [76]. These caspase inhibitors reduced acacetin-induced caspase-7 activity and abolished acacetin-induced growth inhibition, what indicated that caspase-7 activation was mediated by the activation of caspases-8 and -9 [76]. The naturally occurring flavone hispidulin (19) is found in a number of traditional Chinese medicinal herbs [77,78] among other plant species [79]. This flavone (19) has also attained substantial consideration concerning the described biological activities, namely antioxidant, antifungal, anti-inflammatory, anti-thrombosis, anti-epileptic, antimutagenic and antineoplastic properties [80-88]. In 2010, it was found that hispidulin (19) potentiated the TRAIL-induced apoptosis in human ovarian cancer cells, and sensitize TRAIL-resistant cells, being this effect associated with an activation of caspases-3 and -8, and PARP cleavage [89]. This flavone (19) also exhibited cytotoxic effects in hepatoblastoma cancer (HepG2) cells, but not in normal liver cells, through a mitochondria-mediated apoptosis, involving MMP loss and cyt c release, as well as caspase-3 activation [90]. The antiproliferative effect of skullcapflavone I (20) and wogonin (17) was investigated in hepatic stellate cells (HSCs) [91]. nly skullcapflavone I (20) induced apoptosis, being this effect associated with increased activities of caspases-3 and -9. Liu et al. (2016) studied the antitumor effect of diosmetin (21), in hepatocellular carcinoma HepG2 cells. The results demonstrated that diosmetin (21) inhibited cell growth of HepG2 cells, in a concentration and timedependent manner. Diosmetin (21) downregulated Bcl-2 expression and upregulated Bak, Bax, p53 and caspase 3 protein expression [92]

18 Among the natural flavones possessing hydroxyl and methoxy groups concomitantly, highly oxygenated flavones, commonly known as hydroxylated polymethoxyflavones (PMFs), have attracted attention for their broad range of biological activities, particularly antitumor activity. Among these, flavones with five or six hydroxyl or methoxyl groups, such as the structure related flavones and the monodemethylated PMFs 27 and 28, have revealed to be promising pro-apototic agents. The pro-apoptotic activity of eupafolin (22) was investigated in human carcinoma HeLa cells. In these cells, eupafolin (22) showed a cytotoxic effect and induced apoptosis characterized by mitochondrial dysfunction, with loss of MMP and increased cyt c release, as well as activation of caspases-3, -6, -7, -8, and -9 and. Interestingly, the treatment of HeLa cells with the caspase-3 inhibitor z-devd-fmk prevented eupafolinstimulated caspase-8 activation, suggesting a close connection between both caspase pathways [93]. The antiproliferative effect of jaceosidin (23) and eupatilin (24), the 3 --methyl and 3,4 - -dimethyl derivatives of eupafolin (22), was evaluated in ras-transformed human mammary epithelial cells (MCF10Aras). Jaceosidin (22) reduced the cell viability to a greater extent than eupatilin (24). An increased intracellular accumulation of RS was observed in MCF10A-ras cells treated with jaceosidin (23), which was blocked by the antioxidant N-acetylcysteine (NAC), suggesting the involvement of oxidative stress in the antiproliferative activity of this flavone (23). Conversely, eupatilin-induced cell death was not associated with this effect. When MCF10A-ras cells were treated with jaceosidin (23), an stimulation of the p53-pathway with caspase-3 activation and were observed [94]. In human promyelocytic leukemia (HL-60) cells, eupatilin (24) induced a mitochondrial apoptotic pathway with activation of caspases-3, -7 and -9 and [95]. More recently, a similar study with human colorectal tumour cells also showed the activation of an apoptotic pathway by eupatilin (24) with activation of caspases-3 and -7 [96]. The apoptotic effect of cirsilineol (25), the jaceosidin (23) 7--methyl derivative, was studied in Caov-3, Skov- 3, PC3 and Hela cells lines [97]. Particularly, in Caov-3 cells, cirsilineol induced an apoptotic cell death involving mitochondrial dysfunction, with loss of MMP and cyt c release, as well as the activation of caspases-3 and -9 and [97]. Eupatorin (26), the jaceosidin (23) 6--methyl derivative isolated from several medicinal plants, revealed promising antiproliferative activity in multiple human tumour cell lines. In 2014, it was shown that eupatorin (26) markedly inhibited cell proliferation in three human 36 37

19 leukemia cells lines (HL-60, U937, Molt-3) [98]. Treatment of Molt-3, HL-60 and U937 cells with eupatorin (26) induced apoptosis through a mitochondrial pathway, involving cyt c release and increased RS levels, with activation of caspases-3, -7, -8 and -9 [98]. Among PMFs, 5-hydroxyPMFs (5H-PMFs) have attracted attention due to their stronger anticancer activity in comparison with their permethoxylated counterparts [99-101]. The growth inhibitory effect of nobiletin (15), a permethoxylated PMF, and of its monodemethylated analogue 5-hydroxy-6,7,8,3,4 -pentamethoxyflavone (5HPMF, 27) on H1299, H441, and H460 lung cancer cells, was studied [99]. 5HPMF (27) revealed a more potent growth inhibitory activity than nobiletin (15), what suggests that the hydroxyl group at C-5 is essential for the antiproliferative activity. Further studies demonstrated that the monodemethylated flavone 5HPMF (27) downregulated oncogenic proteins (e.g., Mcl-1 and K-ras) and induced apoptosis through activation of caspase-3 and in H1299 cells [99]. In 2010, a similar study with 5-hydroxy-6,7,8,3,4 - pentamethoxyflavone (5HPMF, 27) and other structure related 5-hydroxy PMFs, including gardenin B (28), was carried out in human colon cancer HCT116 and HT29 cells [100]. In that work, all 5-hydroxy PMFs demonstrated more potent growth inhibitory effect on colon cancer cells compared to their permethoxylated analogues. In addition, contrary to permethoxylated PMFs, which did not induce apoptosis neither in HCT116 nor in HT29 cells, 5-hydroxy PMFs significantly induced apoptosis, further suggesting the importance of a 5-hydroxyl group for the pro-apoptotic activity of these compounds. It was also demonstrated that the growth inhibitory effects of these 5-hydroxy PMFs were associated with their ability to modulate key signaling proteins related to cell proliferation and apoptosis, namely caspases-3 and -8 [100]. More recently, the cytotoxic effect of gardenin B (28), in HL-60 and U-937 cells, was demonstrated to be associated with a significant induction of caspases-2, -3, -8 and -9 activities, suggesting that both extrinsic and intrinsic apoptotic pathways were involved in its pro-apoptotic effect [102]. Prenylated Favones During the last years, prenylated flavones have attracted attention of the scientific community mainly because of their significant antitumor activity [103]. In fact, among these compounds some have revealed to be promising pro-apoptotic agents, namely artonin B (29), and gancaonin Q (30). The growth inhibitory effect of artonin B (29) was studied in human acute lymphoblastic leukemia CCRF-CEM cells and in normal epithelia HaCa cells. Artonin B (29) showed antiproliferative activity in 38 39

20 CCRF-CEM cells, but not in normal epithelia HaCa cells, through induction of a mitochondrial apoptotic pathway, characterized by loss of MMP and cyt c release, with caspase-3 activation [104]. The ability of some naturally occurring flavonoids from the Dorstenia sp, including gancaonin Q (30), to inhibit the proliferation of tumour cells was evaluated using a panel of fourteen cancer cell lines and AML12 normal hepatocytes. All flavonoids showed growth inhibitory activity against most of the human tumor cell lines, but none of the compounds tested exhibited a significant inhibition in AML12 normal hepatocytes cells. Moreover, when CCRF CEM cells were treated with gancaonin Q (30), a significant apoptosis induction was observed, which was associated with the activation of caspases-3 and 7 [105]. Flavones with Non Aromatic B-Ring The antitumor activity of protoapigenone (31) was assessed in ovarian cancer cells [106]. The obtained results showed that protoapigenone (31) had a significant cytotoxicity in human MDAH-2774 and SKV3 ovarian cancer cells, but not in the immortalized non-cancer ovarian epithelial cells. Additionally, in human SKV3 [106] and prostate cancer [107] cells, pro-toapigenone (31) induced apoptosis through activation of caspase-3 and subsequent. In 2011, the mechanism by which protoapigenone (31) caused cell death in human MDA-MB-231 breast cancer cells was investigated. The results demonstrated an activation of a mitochondrial apoptotic pathway, involving loss of MMP and increased levels of RS, as well as caspases-3 and -9 activation and [108]. In fact, when MDA-MB-231 cells were pretreated with the general caspase inhibitor Z-VAD-fmk, protoapigenone-induced was prevented. However, apoptosis caused by this flavone (31) was only partially reversed, suggesting that activation of caspases is not the only mechanism associated with protoapigenone (31) pro-apototic activity [108]. In addition to protoapigenone (31), other structure related flavones ((DEDC (32), DIC (33) and DHEC (34)) have proved to be potent pro-apoptotic agents in several human tumor cells. In human HepG2 hepatoma cells, DEDC (32) induced cell death, an effect involving mitochondrial dysfunction, RS generation, loss of mitochondrial membrane potential, and cyt c release, being these effects associated with caspases-3, -8, and -9 activation [109]. DIC (33) showed antiproliferative effect on human HepG2 hepatoma cells in a dose- and time-dependent manner. Moreover, this flavone induced apoptosis via a RS-mediated mitochondrial pathway, with cyt c release and activation of caspases-3 and -9 [110]

21 Wei et al. (2011) isolated a novel non-aromatic B-ring flavonoid (DHEC, 34) and evaluated its antitumor activity in human colon HT-29 cancer cells [111]. DHEC-treated cells showed an accumulation of intracellular RS, loss of mitochondrial membrane potential, cyt c release, PARP cleavage and activation of caspases-3, -8, and -9. These results suggested that the antitumor activity of this flavone is mediated by induction of a mitochondrial and caspasedependent apoptosis [111]. Synthetic Flavones Inducers of a Caspase-Dependent Apoptotic Pathway Considering the promising biological activities described for natural flavones, several synthetic analogues possessing different substitution patterns have been obtained and studied for their antitumor effects. Table 2 summarizes the synthetic flavones reported as inducers of caspase-dependent apoptotic pathways since The antiproliferative activity of synthetic analogues 35 and 36 of natural polymethoxyfla-vones was evaluated in human leukemia (MLT-4 and U937) and peripheral blood mononuclear cells [58]. All flavones showed to be cytotoxic in MLT-4, U937 and peripheral blood mononuclear cells and stimulated apoptosis in TRAILresistant leukemia MLT-4, but not in U937 cells. Furthermore, 2 -methoxyflavone (36) and 5-methoxyflavone (35) enhanced TRAIL-induced apoptosis through the upregulation of DRs and activation of caspases-3 and - 8 [58]. The apoptotic effect observed for 5-methoxyflavone (35), in MLT-4 cell lines, was in accordance with that observed in human HCT116 colon cancer cells. In fact, 5-methoxyflavone (35) strongly inhibited the growth and clonogenicity of HCT116 tumor cells, activated the DNA damage responses activity, and increased the cleavage of caspases-2 and -7, leading to apoptosis [112]. Two baicalein analogues with a benzyl and amine side chains at C-6 and C-7 (compounds 37 and 38, respectively), significantly inhibited the growth of various cancer cells and induced apoptosis in HepG-2 cells, in a concentration-dependent manner [113,114]. In addition, both flavones induced mitochondrial-mediated apoptosis associated with activation of caspases-3 and -9 and RS generation [113,114]. DHF-18 (39), a baicalein derivative (5) with a piperazine substitution at C-7, has shown potent in vivo and in vitro antitumor activities [115]. It was demonstrated that DHF-18 (39) inhibited tumour growth in mice inoculated with Heps hepatoma cells without evident toxicity. Moreover, DHF-18 (39) was able to induce apoptosis in HepG2 cells, being this effect associated with an increase of intracellular RS, loss of mitochondrial membrane potential, and cyt c release from mitochondria, followed by activation of caspases-3 and -9. The increase of TNF-R1, 42 43

22 activation of caspase-8 and the subsequent cleavage of Bid suggested that the DHF-18 (39) may also stimulated an extrinsic apoptotic pathway [115]. In addition to DHF-18 (39), the pro-apoptotic effect of other flavones with amino groups at C-7 has been described. LYG-202 (40), a DHF-18 (39) analogue possessing an additional methoxy substituent at C-8, was reported for its antiproliferative activity against HepG2, MCF-7, HeLa, BGC-823, MDA-MB-435, and HCT-116 tumour cells [116]. The activation of both extrinsic and intrinsic pathways of the apoptotic cascade, including activation of caspases-3, -8, and -9 was demonstrated [116]. Moreover, LYG-202 (40) induced apoptosis in human colorectal carcinoma HCT116 cells with and activation of caspases-3, -8 and -9, suggesting that both intrinsic and extrinsic apoptotic pathways may be involved in LYG-202-induced apoptosis in HCT116 cells [117]. Nevertheless, several evidences support that the p53 pathway may also play a crucial role in LYG-202-induced apoptosis [117]. The pro-apoptotic effect of GL-V9 (41), a wogonin (17) derivative with a pyrrolidin substituent at C-7, was studied in human hepatocellular carcinoma HepG2 cells [118]. The results showed that GL-V9 (41) has a more potent antiproliferative and pro-apoptotic effect than wogonin. In addition, GL-V9 (41) induced cleavage of PARP, and activation of caspases-3 and -9, while caspase-8 remained unchanged [118], what suggests that GL-V9-induced apoptosis in HepG2 cells was achieved through a mitochondrial signalling pathway. The V8 compound (42) is another derivative of wogonin (17) with amino groups, which has demonstrated to be a more potent antitumor agent than its precursor. In fact, in hepatocellular carcinoma, V8 exhibited a higher apoptosis inducing effect than wogonin [119]. In these tumor cells, V8-induced apoptosis was mediated by a mitochondrial pathway, with increase RS generation, loss of mitochondrial membrane potential, cyt c release, and activation of caspases-3 and -9. verall, the results obtained for both wogonin derivatives GL-29 (41) and V8 (42) suggest that the inclusion of amino groups on the 7-hydroxyl group can be an important strategy to improve the antitumor activity. Pan et al. synthesized a new 8-aminated flavone (LW- 214, 43) by molecular modification of chrysin (2), and studied its in vitro and in vivo antitumor effect [120]. In human MCF-7 breast cancer cells, LW-214 (43) triggered a mitochondrial apoptotic pathway, characterized by loss of MMP and cyt c release, associated with RS generation by down-regulated Trx-1 function, caspase-9 activation, and [120] 44 45

23 The flavone amino derivate flavopiridol (44), in addition to its well-known effect as CDK inhibitor, have also revealed pro-apoptotic effect on human tumour cell lines. Actually, it was reported that flavopiridol (44) induced apoptosis in B cell chronic lymphocytic leukemia (CLL) cells ex vivo by downregulation of both the inducible nitric oxide (N) synthase and the CDK inhibitor p27kip 1 [121]. Later, the same research group demonstrated that ins downregulation was caspase-dependent [122]. The induction of apoptosis by flavopiridol (44) was further described in prostate cancer stem cells (CSCs), being this effect associated with activation of p53 and caspases-3, -8 [123]. The antitumor activity of 3,3 -diamino-4 -methoxyflavone (DD1, 45) was evaluated in acute myeloid leukemia (AML) cells [124]. When these cells were treated with DD1 (45) an inhibition of proliferation, through apoptosis induction, was detected. nce again, the apoptotic effect induced by this flavone (45) was associated with recruiting of mitochondria activation of caspases-3, -8 and -9 and. It was also detected the decrease of Thr389-phosphorylation and of the protein levels of the caspase-3 substrate P70 ribosomal S6 kinase (P70S6K), being both effects prevented by caspase inhibitors [124]. In 2015, a series of oxime/amide-containing flavone derivatives were synthesized and tested for their antitumor activity. WTC-01 (46) revealed potent antiproliferative effects in human nasopharyngeal carcinoma cells (NPC-TW01 and HNE-1), being this effect associated with an increase of caspase-3 and Bax, and a decrease of caspase-9 and Bcl-2 expression levels, as well as a loss of MMP [125]. The promising antitumor activity described for chrysin (2) has encouraged the synthesis of several derivatives in order to enhance activity. In addition to the 8-aminated derivative LW-214 (43), described above, derivatives with electron withdrawing effect on A ring have been synthesized and evaluated for their antitumor activity. Particularly, it was shown that 8-bromo-7-methoxychrysin (47, BrMC) inhibited the proliferation of human hepatocellular carcinoma cells. BrMC (47) was a more potent inhibitor than chrysin (2) against HepG2 and Bel-7402 cells. These evidences the importance of methyl groups at C-7 and of halogen atoms at C-8. The treatment of HepG2 cells with BrMC (47) increased the levels of caspase-3, being this effect prevented by the caspase-3-specific inhibitor Z- DEVD-fmk, suggesting that BrMC induced apoptosis in a caspase-dependent manner [126]. ther flavones possessing electron withdrawing groups with promising apoptotic effects include flavones 48, 49, 50 and 51. The 2 -nitroflavone (48) showed to induce apoptosis in HeLa human cervix adenocarcinoma 46 47

24 cells [127]. verall results indicated that both death receptor and mitochondria-dependent pathways, as well as the activation of caspases-3, -8 and -9, are involved in the apoptotic cell death promoted by this synthetic flavone. Particularly, it was shown that the apoptotic effect of flavone 48 was partially reverted by treatment with inhibitors of caspases-8 or -9 [127], indicating the activation of a caspase-dependent apoptotic cell death. [127]. The in vitro and in vivo effect of flavone 48 has also been studied in murine mammary adenocarcinoma. The 2 -nitroflavone (48) induced apoptosis through a mitochondrial pathway, involving cyt c release to cytosol, and the activation of caspases-3, -8, and -9 [128]. In vivo assays confirmed this pro-apoptotic effect in xenograft mouse models treated with 10 mg/kg of compound 48 [128]. More recently, the antiproliferative activity of 2 -nitroflavone (48) was evaluated in different haematological cancer cells. nce again, it was found that flavone 48 induced an apoptotic response in HL-60 cells through activation of caspases-3, -8 and -9, up-regulation of both the tumor necrosis factorrelated apoptosis-inducing ligand (TRAIL) and its DR5, and cyt c release, indicating the involvement of both death receptor- and mitochondria-dependent apoptotic pathways [129]. A series of new flavone derivatives with potent antiproliferative activity against HepG-2 were synthesized by Liu et al (2010) [130]. Among the synthetic compounds, flavone 49, with a chloride at C-6, was the most active. The induction of death-receptor and mitochondria-dependent apoptosis, through activation of a caspase cascade, was evidenced by the increase of caspases-3, -8 and -9 activity [130]. A set of flavones with A ring methyl esters possessing electron withdrawing and electron donating groups on the B-ring (flavones 50 and 51) were synthesized and their antiproliferative activity against HL-60 human leukemia cell line was evaluated [131]. The results demonstrated that the presence of either a methyl group or a chlorine atom at position 2 was associated with a higher activity. These two flavones (50 and 51) induced activation of caspases-3, -6, -7, -8 and -9, and, being these effects significantly reduced by caspase inhibitors (broadspectrum caspase inhibitor Z-VAD-fmk, caspases-3 and -7 inhibitor Z-DEVD-fmk, caspase-8 inhibitor Z-IETDfmk, and caspase-9 inhibitor Z-LEHD-fmk) [131]. These results indicated the induction of an apoptotic cell death highly dependent on a caspase pathway. The antiproliferative effect of a series of 42 flavonoids (flavones, flavonols, flavanones and chalcones), including chrysin (2) and apigenin (4) derivatives, was evaluated in human HT-29 colonic cells in order to compare the effect of hydroxylation, methoxylation and/or C-alkylation at 48 49

25 various positions in the A- and B- rings. Additionally, the activity of caspases-3 and -7 was assessed in HT-29-treated cells. Flavones and flavonols showed more pronounced activity than other classes of flavonoids. Additionally, C- isoprenylation was the most effective, with substitution at 8-position and longer chains, such as geranyl leading to improved outcomes. Compounds behaved as inducers of a caspasedependent apoptotic pathway, being 8-geranylchrysin (57) and 8-linalylchrysin (58) the most potent inducers of caspases-3 and -7 activity, with a higher potency than chrysin (2) [132]. In 2012, the effect of 7-hydroxyflavone phosphate ester (FP, 61) on HeLa cells was studied. This flavone (62), as well as 7-hydroxyflavone, inhibited tumor proliferation and induced apoptosis in HeLa cells through caspase-3 activation and PARP-1 cleavage [133]. The growth inhibitory effect of WYC02-9 (62), a nonaromatic B ring flavone obtained by molecular modification of protoapigenone (WYC02, 30), was evaluated in human DU145 prostate cancer cells [134]. WYC02-9 (62) inhibited cell growth of DU145 cells more significantly than protoapigenone (30). Additionally, treatment of DU145 cells with WYC02-9 (63) induced an increase in RS generation, being this effect accompanied by an induction of DNA damage, and mitochondria-dependent cell apoptosis [134]. Flavones as Direct Modulators of Caspases In order to streamline the identification of modulators of caspase proteins, yeast-based screening assays, welladaptable to the high-throughput screening, were developed for an independent analysis of the executioner caspases-3 and -7, in a simplified cellular system [135,136]. These assays were based on the heterologous expression in yeast of human procaspases-3 or -7 that, after processing namely by small-molecule activators, lead to an active caspase form with a pronounced cytotoxic activity in yeast [136]. A correlation between the yeast growth inhibitory effect of human caspase and the degree of activation was established in these studies. Besides this system, an additional approach, based on the heterologous expression of active forms of caspase-3 (reverse caspase-3; [135]) and caspase-7 (caspase-7 53 [136] were also developed for the search of activators [136] and inhibitors [135] of caspases-3 and -7 (Figure 5)

26 Figure 5: Yeast caspase screening assays. The expression of human caspases-3 or -7 in yeast is the basis of two cell-based assays for the screening of their modulators. (A) The expression of active forms of caspase-3 (reverse caspase-3) and caspase-7 (caspase-7) is associated with a yeast growth inhibitory effect, increased by activators and reverted by inhibitors. (B) The processing of human procaspases-3 and -7 by activators leads to active caspases with subsequent yeast growth inhibition. PD: prodomain; LS: large subunit; SS: Small subunit. The high number of reports concerning the stimulation of a caspase-dependent apoptotic pathway by flavones has raised great interest for this family of compounds as potential activators of caspases (Tables 1 and 2). In fact, this motivated the use of the yeast-based caspase screening assays in the analysis of a chemical family of flavones. This approach led to the recent identification of selective small-molecule activators of caspase-7 with a flavone scaffold, namely 5,6-dihydroxy-7-prenyloxyflavone (63) (Table 2) [136]. Actually, unlike the known caspases-3 and -7 activator, PAC-1 [137], a selective activation of caspase-7 by flavone 63 was revealed by the yeast assay. In conformity with the results from yeast, an in vitro assay using a recombinant procaspase showed that this compound directly processed the procaspase-7 to the active caspase-7 [136]. Additionally, flavone 63 showed potent growth inhibitory effect in several human tumour cells [136, 138], and a higher potency than PAC-1 in breast adenocarcinoma MCF-7 cells (without caspase-3) [136]. Further supporting the results from yeast and in vitro assays, it was shown that flavone 63 increased caspase-7 activity and processed procaspase-7 to the active caspase-7 in human tumour cells [136]. Interestingly, this compound increased the sensitivity of tumour cells, such as MCF-7, to the effect of chemotherapeutic drugs, such as etoposide [136]. Pereira et al. (2014) claimed that the prenyl group at position 7 of flavone 63 could be associated with its selectivity to caspase-7, since no effect on caspase-7 was observed with the parent compound baicalein (5). Additionally, the ineffectiveness of baicalein (5) on caspase-7 suggests that the presence of the prenyl group at position 7 of flavone 63 is an interesting structural moiety associated with the activation of caspase-7. These data may be crucial for the design of new caspase-7 activators based on the prenylation of the flavone scaffold. Since few templates for caspase activators have been described, flavone 63 may open the way to the structure-based design of new classes of caspase activators with improved antitumor properties

27 Table 2: Synthetic Flavones Inducers of A Caspase-Dependent Apoptotic Pathway. FLAVNES Molecular targets in apoptotic pathway Citotoxic effect (# )/ caspase activation/ ref 35 Table 2 (contd.) U937 [58] =31.61±0.68 µm Caspase activity*=n.d. MLT-4 [58] Bcl-2 family: increase of Bax and decrease of Bcl-2 expression caspases-3 and -9 BEL-7402 [114] (24h#)=18.59±1.01 µm R 7 R 2 ' R 3 R 5 R 4 ' R 5 ' 35: R 3,R 7,R 2 R 4, R 5 =H, R 5 =CH 3 ; 5-MF 36: R 3,R 5,R 7,R 4,R 5 =H, R 2 =CH 3 ; 2 -MF H 37: HQS-3 Bcl-2 family: increase of Bax, decrease of Bid and Mcl-1 expression caspases-2, -3, -7, and -8 RS generation Increase of TRAIL and DR expression Decrease of cflip expression Bcl-2 family: increase of Bax expression and decrease of Bcl- 2 expression caspases-3 and -9 Increase of RS levels =30.11±0.16 µm Caspase activity* HCT116 [112] Caspase activity*=40 µm 36 U937 [58] =46.70±1.60 µm Caspase activity* MLT-4 [58] =26.29±2.22 µm Caspase activity* HepG2 [113] =22.98±2.92 µm Caspase activity*=10 µm N N N H 38 H 39: DHF-18 Δψ m dissipation Increase of cyt c release Increase of AIF release RS generation MAPK pathway: Activation JNK and inhibiton of ERK expression Bcl-2 family: increase of Bax and Bak expression and decrease of Bid and Bcl-2 expression caspases-3, -8, and -9 Δψ m dissipation Increase of RS levels Increase of cyt c release Increase of TNF-R1 expression (48h#)=13.30±1.21 µm Caspase activity*=10 µm HepG2 (24h#)=45.74±4.61 µm (48h#)=24.87±4.57 µm Caspase activity*=10 µm HepG2 [115] =62.4±1.5 µm Caspase activity*=10 mg/kg 54 55

28 Table 2 (contd.) Table 2 (contd.) HepG2 [116] N Bcl-2 family: increase of Bax and decrease of Bcl-2 expression HepG2 [118] (24h # )=54.3±1.1 µm N N H 40: LYG-202 Bcl-2 family: increase of Bax and decrease of Bcl-2 and Bid expression caspases-3, -8, and -9 Increase of TNF-α activity Increase p53 and p21 expression =4.74±0.80 µm Caspase activity*=10 µm MCF-7 [116] =27.70±1.09 µm Caspase activity*=5 µm HeLa [116] =8.05±0.21 µm Caspase activity*=5 µm BGC-823 [116] =6.42±1.42 µm Caspase activity*= 5µM MDA-MB-435 [116] =6.74±0.71 µm Caspase activity*=5 µm HCT116 [116] R N(H2C)4 R H 41: GL-V9 H 42: R=(CH 2 ) 2 H; V8 (CH2)3N(CH2CH 3)2 caspases-3 and -9 Bcl-2 family: increase of Bax and BH-3 expression, and decrease of Bcl-2 expression caspases-3 and -9 Δψ m dissipation Increase of cyt c release Increase of RS levels Increase of Ca 2+ levels Bcl-2 family: increase of Bax expression, and decrease of Bcl-2 expression caspase-9 Δψ m dissipation (48h # )=35.2±3.2 µm Caspase activity*=10 µm HepG2 [119] =23 µm Caspase activity*=20 µm MCF-7 [120] =5.21±1.99 µm =10.90±0.95 µm H Increase of cyt c release Caspase activity*=5 µm Caspase activity*=5 µm HCT-116 [117] 43: LW-214 Increase of AIF c release Increase of RS levels (12h # )=26.29±0.95 µm (24h # )=17.82±0.63 µm MAPK pathway: Activation of ASK and JNK (48h # )=3.78±0.23 µm Caspase activity*=16 µm 56 57

29 Table 2 (contd.) H H N H Cl H 44: Flavopiridol NH 2 NH 2 45: 3,3 -diamino-4 -methoxyflavone; DD1 N H 46: WTC-01 Br H 47: 8-bromo-7-methoxychrysin ( BrMC) Caspase family: increase of caspases-3 and -8 activities Activation of p53 expression Inhibition of ins expression Bcl-2 family: increase of Bax and Bad expression caspase-3, -8, and -9 Δψ m dissipation Bcl-2 family: increase of Bax, and decrease of Bcl-2 expression caspases-3 and -9 Δψ m dissipation caspase-3 Increase of RS levels MAPK pathway: activation of JNK Bcl-2 family: increase of Bax and decrease of Bcl-2 and Bclx L expression CSC [123] =500 nm Caspase activity*=500 nm AML [124] (48h # )=10 µm Caspase activity*=10 µm NPC-TW01 and HNE-1 [125] Caspase activity*=0.45 µm HepG2 [126] =n.d. Caspase activity*=10 µm Bel-7402 [126] =n.d. Caspase activity*=10 µm HeLa [127] =3±1 µm Cl Table 2 (contd.) 49: 6-chloro-2-(3,5-dimethoxyphenyl)- 4H-chromen-4-one CCH 3 50: R=Cl, methyl 2-(2-chlorophenyl)- 6,7-dimethyl-4-oxo-4H-chromene-8- carboxylate 51: R=CH 3, 1-methyl 6,7-dimethyl- 4-oxo-2-(-tolyl)-4H-chromene-8- carboxylate R Bcl-2 family: increase of Bax and decrease of Bcl-2 expression caspases-3, -8, and -9 Bcl-2 family: decrease of Bcl-2 and Bid expression caspases-3, -6, -7, -8, and -9 Increase of cyt c release MAPK pathway: Activation of ERK HepG2 [130] =1.1±0.3 µm Caspase activity*=50 µm 50 HL-60/MX1 [131] =18.9±3.7 µm Caspase activity*=30 µm HL-60 [131] =10.1±1.1 µm Caspase activity*=30 µm 51 HL-60/MX1 [131] =70.7±0.7 µm Caspase activity*=30 µm HL-60 [131] =9.3±1.2 µm Caspase activity*=30 µm 2 N 48: 2 -Nitroflavone caspases-3, -8, and -9 Increase of cyt c release Increase of Fas, FasL, TRAIL and DR5 expression Caspase activity*=20 µm LeM3 [128] =4±1 µm Caspase activity*=20 µm HL-60 [129] MAPK pathway: Activation of p38 and JNK and inhibiton of ERK expression =1±0.5 µm Caspase activity*=20 µm 58 59

30 Apoptosis Table 2 (contd.) Apoptosis 57 HT-29 [132] (6h # )=18.0±7.4 µm Table 2 (contd.) (24h # )=7.6±4.3 µm Caspase activity*=50 µm HT-29 [132] HT-29 [132] (6h # )=17.0±5.1 µm (6h # )=28.4±10.0 µm (24h # )=19.4±3.2 µm (24h # )=28.1±5.4 µm Caspase activity*=50 µm Caspase activity*=50 µm HT-29 [132] HT-29 [132] R 3 R 4 R 5 (6h # )=90.4±4.2 µm (6h # )=43.3±9.5 µm (24h # )=80.3±3.4 µm (24h # )=24.5±4.5 µm R 2 R 1 Caspase activity*=50 µm Caspase activity*=50 µm 52: R 1 =-3,3 DMA, R 3 =H, R 2,R 4, R 5 =H 54 HT-29 [132] 60 HT-29 [132] 53: R 1 =H, R 3 =-Ger, R 2,R 4, R5=H 54: R 1, R 3 =H, R 4 =-3,3 DMA, R 2, R 5 =H 55: R 1, R 3 =H, R 4 =1,1-DMA, R 2, R 5 =H 56: R 1, R 3 =H, R 2 =Ger, R 4, R 5 =H 57: R 1, R 3 =H, R 4 =Ger, R 2, R 5 =H 58: R 1, R 3 =H, R 4 =Lin, R 2, R 5 =H 59: R 1, R 3 =H, R 4 =Ner, R 2, R 5 =H 60: R 1, R 3, R 4 =H, R 2 = 3,3-DMA, R 5 =H caspases-3 and -7 (6h # )=86.3±8.3 µm (24h # )=75.5±7.8 µm Caspase activity*=50 µm 55 HT-29 [132] (6h # )=76.7±5.5 µm (24h # )=57.7±5.0 µm Caspase activity*=50 µm 56 P 61: Diethyl flavon-7-yl phosphate (FP) caspase-3 (6h # )=67.4±6.1 µm (24h # )=71.7±5.2 µm Caspase activity*=50 µm HeLa [133] (24h # )=48.8 µm (72h # )= 18.5 µm Caspase activity*=20 µm HT-29 [132] (6h # )=58.2±12.7 µm (24h # )=53.9±6.5 µm H caspases-3, and -9 Δψ m dissipation Increase of RS levels DU145 [134] =2.0±0.2µM Caspase activity*=5 µm 62: WYC2-9 (H3C)2HCH 2 C MCF-7 [136] H Caspase family: Selective activation of caspase-7 =8.3±0.4µM H Caspase activity*=6µm 63 # : concentration that causes a growth inhibitory effect of 50% after 24, 48 or 72 hours or 2, 3, or 6 days of treatment. n.d.: not determined. *Lowest concentration that showed caspase activation. DMA: dimethylallyl; Ger: geranyl; Lin: linalyl Ner: nerolidyl

31 SAR Studies Despite the great number of works describing the pro-apototic activity of flavones, most of these studies report a limited number of flavones, what has greatly limited SAR studies. Nevertheless, the comparison of the caspase-dependent apoptotic effect of flavones with other structure related flavonoids such as flavonols, isoflavones and flavanones demonstrated the importance of the flavone nucleus for this activity [132]. Although the nonsubstituted flavone (1) has been described as an inducer of caspase-dependent apoptotic pathway, its effect was quite low in comparison with flavone derivatives. Particularly, the presence of hydroxyl groups at positions 5, 7, 3 and 4, and methoxy groups at positions 5, 6, 7, 8, 3 and 4 seems to improve caspase activation. SAR studies suggest that the introduction of prenyl side chains in the flavone scaffold, such as 3,3-dimethylallyl (3,3-DMA), 1,1-dimethylallyl (1,1-DMA), geranyl (Ger) and linalyl (lin) at C-7 and C-8 is also associated with an enhancement of caspase activation [132]. The preparation of synthetic analogues of natural flavones have allowed the identification of several promising caspase activators. In fact, the introduction of electron withdrawing groups, such as atoms of chlorine and bromine at C-6, C-8 and C-2, revealed an interesting strategy to obtain potent apoptosis inducers [126,130,131]. Likewise, the introduction of amine side chains at C-7 proved to be an important strategy to improve the proapoptotic effect [ ]. Figure 6 summarizes the main conclusions about SAR studies of inducers of a caspasedependent apoptotic pathway with flavone scaffold. Figure 6: SAR schematic considerations concerning flavones as inducers of a caspase-dependent apoptotic pathway. Conclusion Restoration of apoptosis is one of the most appealing strategies in anticancer therapy due to the current impairment of this cell death pathway in cancer cells. Flavones are among the small molecules most frequently reported as apoptotic inducers. Despite the diversity of apoptotic molecular targets reported for this family of compounds the activation of the caspase pathway has been a common event for the wide range of flavones already studied in tumour cells. In fact, an impressive number of studies are presented in this review supporting a targeted mod-ulation of caspase proteins by flavones. The recent identification of flavone 63 as potential direct caspase activator further supports that flavones are promising starting points for the structure-based design 62 63