Hexamethoxylated Monocarbonyl Analogues of Curcumin Cause G2

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Hexamethoxylated mono-carbonyl analogs of curcumin cause G2/M cell cycle arrest in NCI-H460 cells via Michael acceptor-dependent redox intervention Yan Li, Li-Ping Zhang, Fang Dai, Wen-Jing Yan, Hai-Bo Wang, Zhi-Shan Tu, and Bo Zhou J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b02011 • Publication Date (Web): 08 Aug 2015 Downloaded from http://pubs.acs.org on August 18, 2015

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Hexamethoxylated mono-carbonyl analogs of curcumin cause G2/M cell cycle

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arrest in NCI-H460 cells via Michael acceptor-dependent redox intervention

3 4

Yan Li,†,§ Li-Ping Zhang,‡ Fang Dai,† Wen-Jing Yan,† Hai-Bo Wang,† Zhi-Shan Tu,†

5

Bo Zhou*,†

6 7 8 9



State Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou,

Gansu 730000, China ‡

Gansu provincial Hosipital, Lanzhou, Gansu 730000, China

10

§

11

Institute of Applied Chemistry, Shaoxing University, Shaoxing, Zhejiang, 312000,

12

China

Zhejiang Key Laboratory of Alternative Technologies for Fine Chemicals Process,

13 14 15 16 17 18 19 20 21

* Corresponding authors.

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E-mail: [email protected]

23

Fax: +86-931-8915557 1

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ABSTRACT

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Curcumin, derived from dietary spice turmeric, holds promise for cancer prevention.

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This prompts much interest in investigating the action mechanisms of curcumin and

27

its analogs. Two symmetrical hexamethoxy-diarylpentadienones (1 and 2) as cucumin

28

analogs were reported to possess significantly enhanced cytotoxicity compared with

29

this parent molecule. However, the detailed mechanisms remain unclear. In this study,

30

compounds 1 and 2 were identified as the G2/M cell cycle arrest agents to mediate the

31

cytotoxicity toward NCI-H460 cells via Michael acceptor-dependent redox

32

intervention. Compared with curcumin, they could more easily induce a burst of

33

reactive oxygen species (ROS) and collapse of the redox buffering system. One

34

possible reason is that they could more effectively target intracellular TrxR to convert

35

this antioxidant enzyme into a ROS promoter. Additionally, they caused up-regulation

36

of p53 and p21, and down-regulation of redox sensitive Cdc25C along with cyclin

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B1/Cdk1 in a Michael acceptor- and ROS-dependent fashion. Interestingly, in

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comparison with compound 2, compound 1 displayed a relatively weak ability to

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generate ROS but the increased cell cycle arrest activity and cytotoxicity probably due

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to its Michael acceptor-dependent microtubule-destabilizing effect and greater

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GST-inhibitory activity, as well as its enhanced cellular uptake. This work provides

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useful information for understanding Michael acceptor-dependent and redox-mediated

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cytotoxic mechanisms of curcumin and its active analogs.

44 45

KEYWORDS

2

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Cell cycle; curcumin; reactive oxygen species; redox modulation; thioredoxin

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reductase

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INTRODUCTION

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Intracellular redox homeostasis plays a critical role in many cell signaling pathways,

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and is strictly maintained by the production of reactive oxygen species (ROS) and

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their removal based on a sophisticated antioxidant defense system.1 Now it is well

53

established that cancer cells, compared to normal cells, characterize higher ROS

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levels and aberrant redox homeostasis to maintain their malignant phenotypes such as

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uncontrolled proliferation, invasion, angiogenesis and metastasis.2-5 Thus, cancer cells

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as a kind of already stressed cells, are more vulnerable to further ROS production

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reaching the “toxic threshold” or effective inhibition against antioxidant system.2-5

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This vulnerability illustrates the redox Achilles of cancer cells and can be exploited as

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an attractive anticancer strategy by using ROS-generating agents (prooxidants) to

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disrupt redox homeostasis of cancer cells.2-5 Our previous works also provided useful

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information to support this strategy by highlighting the prooxidative scenario in

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chemical detail for polyphenols as cupric ion-dependent prooxidants.6-8

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According to the “Hard and soft acids and bases” theory, redox intervention in

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cancer cells can be achieved by using α,β-unsaturated carbonyl compounds, often

65

referred to as Michael acceptors (soft electrophiles), to covalently modify cysteine

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residues (soft nucleophiles) in redox-sensitive target proteins. Consequently,

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α,β-unsaturated carbonyl compounds represent a class of important prooxidants by

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virtue of their electrophilicity. Curcumin (Figure 1A) is such a dietary Michael

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acceptor molecule isolated from the rhizome of Curcuma longa Linn., and has

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attracted considerable interest as a potential therapeutic agent especially for human

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cancer treatment, due to its multi-targeted activity and its safety for human use.9-11

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Much evidence indicates that curcumin-mediated ROS accumulation is responsible

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for its apoptosis-inducing activity in various cancer cells.12 How does curcumin

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promote the ROS-generation? Holmgren, Fang and colleagues have pointed out that

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curcumin, as a Michael acceptor molecule, could irreversibly modify thioredoxin

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reductase (TrxR), a seleonoenzyme and pivotal player in maintaining intracellular

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redox homeostasis, rendering this enzyme become a prooxidant and show a strongly

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induced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity to

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produce ROS.13,14

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Despite of the attractive benefits of curcumin as mentioned above, its relatively low

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potency, poor stability and bioavailability severely limit its clinical utility.15

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Consequently, extensive effort has been devoted to synthesis of new curcumin analogs

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to overcome its flaws.15,16 Among these analogs, a suite of diarylpentadienones have

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turned out to be successful in achieving this aim.17-23 We have recently found out that

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compared

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diarylpentadienone

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apoptosis-inducing activity in A549 cells via ROS-mediated mechanisms; this

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compound could effectively and irreversibly modify the TrxR based on its

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electrophilicity, geometry and well cellular uptake, and convert this antioxidant

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enzyme into a ROS promoter, resulting in a burst of the intracellular ROS and falling

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apart of redox buffering system.24 Additionally, we have also noted from published

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data that two symmetrical hexamethoxy-diarylpentadienones, 120,

with

curcumin, displays

a the

double

ortho-trifluoromethyl

significantly

increased

substituted

cytotoxicity

21

and 219,

and

21, 23

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(Figure 1A) are more potent in inducing cancer cell death than curcumin. However,

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the action mechanisms are unclear. As part of our research project in

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prooxidant-mediated cancer chemoprevention,6-8,24 we thus selected the two

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compounds to investigate their cytotoxic mechanisms. To probe the structure–activity

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relationships (SAR) and possibility of Michael acceptor-dependent redox intervention,

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we also synthesized their analogs 3-5 and reduced compounds 1R and 2R that lack

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the Michael acceptor units (Figure 1A). Based on preliminary screening experiments,

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we found that all the test cell lines, including human hepatoma (HepG2 and

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SMMC-7721) and lung carcinoma (A549 and NCI-H460) cells, showed more

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significant response to compounds 1 and 2 than curcumin, with the NCI-H460 being

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the most sensitive cancer cell line for all the test compounds. Additionally, we noted

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with interest that cytotoxicity of compounds 1 and 2 toward NCI-H460 cells was

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mainly mediated by inducing cell cycle arrest. This is different from our previous

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results24 showing that pro-apoptosis ability of another series of diarylpentadienones

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was responsible for their cytotoxicity in A549 cells. More interestingly, in comparison

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with compound 2, compound 1 displayed a relatively weak ability to generate ROS

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but the increased cell cycle arrest activity and cytotoxicity in NCI-H460 cells.

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Therefore, we report herein the detailed mechanistic study on cytotoxicity of

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compounds 1 and 2 toward NCI-H460 cells, with emphasis placed on elucidating

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Michael acceptor-dependent redox intervention, clarifying the reason why compound

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1 is a stronger G2/M cell cycle arrest agent than compound 2, and emphasizing the

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possibility of multiple targets involved in the processes.

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MATERIALS AND METHODS

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Materials. Roswell Park Memorial Institute (RPMI)-1640, sulforhodamine B

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(SRB), 2',7'-dichlorofluorescin diacetate (DCFH-DA), L-glutathione reduced (GSH),

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L-glutathione oxidized (GSSG), glutathione reductase (GR), 2-vinylpyridine,

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5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), TrxR (from rat liver), Triton X-100, BSA,

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anti-α-tubulin-FITC antibody and 4',6-diamidino-2-phenylindole dihydrochloride

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(DAPI) were purchased from Sigma-Aldrich (St. Louis, MO, USA). NADPH was

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from Roche Diagnostics GmbH (Mannheim, Germany). BCA protein assay kit,

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antifade mounting medium, radioimmunoprecipitation assay buffer and PMSF were

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from Beyotime Institute of Biotechnology (Jiangsu, China). The antibodies against

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p53, p21, cyclin B1, cyclin-dependent kinase 1 (Cdk1), glyceraldehyde 3-phosphate

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dehydrogenase (GAPDH), and TrxR1 were from Cell Signaling Technology (Beverly,

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MA, USA). The antibody against Cdc25C was purchased from Millipore (Billerica,

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MA, USA). TRFS-green was a generous gift from Prof. Jianguo Fang (State Key

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Laboratory of Applied Organic Chemistry, Lanzhou University). DNs-CV, a highly

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fluorogenic probe for glutathione S-transferases (GSTs) was synthesized as previously

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reported by Zhang.25 Curcumin and compound 5 were prepared in our previous

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study.24,26 All other chemicals were of the highest quality available.

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Synthesis. Compounds 1-4 were synthesized from appropriate benzaldehydes and

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acetone under alkaline conditions, and compounds 1R and 2R were synthesized by

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catalytic hydrogenation of 1 and 2 over Pd/C according to our previous works.24,26

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Their structures were characterized by m.p., IR, 1H NMR,

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HRMS (ESI) /MS (EI) analysis.

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C NMR spectra and

(1E,4E)-1,5-bis(2,4,6-trimethoxyphenyl)penta-1,4-dien-3-one (1): a yellow solid;

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yield: 71%. M.p.: 222-224 °C. IR (KBr): 2943, 2834, 1626, 1550, 1458, 1336, 1206,

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1149, 1118, 1028 cm-1. 1H NMR (400 MHz, CD3Cl, 25 °C, TMS): δ 3.86 (s, 6H), 3.91

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(s, 12H), 6.14 (s, 4H), 7.48 (d, J = 16 Hz, 2 H), 8.14 (d, J = 16 Hz, 2 H) ppm;

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NMR (100 MHz, CD3Cl, 25 °C, TMS): δ 55.6 (2C), 56.0 (4C), 90.8 (4C), 107.0 (2C),

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126.7 (2C), 133.7 (2C), 161.7 (4C), 162.9 (2C), 192.7 (1C) ppm; HRMS (ESI): m/z:

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calcd. for C23H26O7 [M+H]+: 415.1751; found: 415.1746, error = 1.2 ppm.

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C

(1E,4E)-1,5-bis(3,4,5-trimethoxyphenyl)penta-1,4-dien-3-one (2): a yellow solid;

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yield: 75%. M.p.: 128-130 °C. IR (KBr): 2940, 2837, 1622, 1582, 1502, 1457, 1414,

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1314, 1276, 1238, 1123 cm-1. 1H NMR (400 MHz, DMSO-d6, 25 °C, TMS): δ 3.71 (s,

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6H), 3.85 (s, 12H), 7.13 (s, 4H), 7.32 (d, J = 16 Hz, 2 H), 7.71 (d, J = 16 Hz, 2 H)

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ppm;

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(4C), 125.2 (2C), 130.3 (2C), 139.6 (2C), 142.9 (2C), 153.2 (4C), 188.3 (1C) ppm;

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HRMS (ESI): m/z: calcd. for C23H26O7 [M+H]+: 415.1751; found: 415.1748, error =

153

0.7 ppm.

13

C NMR (100 MHz, DMSO-d6, 25 °C, TMS): δ 56.1 (4C), 60.2 (2C), 106.1

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(1E,4E)-1,5-bis(2,6-dimethoxyphenyl)penta-1,4-dien-3-one (3): a yellow solid;

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yield: 69%. M.p.: 152-154 °C. IR (KBr): 2939, 2834, 1641, 1570, 1472, 1324, 1258,

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1178, 1107, 1028 cm-1. 1H NMR (400 MHz, DMSO-d6, 25 °C, TMS): δ 3.90 (s, 12H),

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6.73 (d, J = 8 Hz, 4 H), 7.36 (d, J = 8 Hz, 2 H) , 7.47 (d, J = 16 Hz, 2 H) , 8.02 (d, J =

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16 Hz, 2 H) ppm; 13C NMR (100 MHz, DMSO-d6, 25 °C, TMS): δ 56.0 (4C), 104.2

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(4C), 111.5 (2C), 128.2 (2C), 132.1 (2C), 132.9 (2C), 159.8 (4C), 190.3 (1C) ppm;

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HRMS (ESI): m/z: calcd. for C21H22O5 [M+H]+: 355.1540; found: 355.1538, error =

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0.6 ppm.

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(1E,4E)-1,5-bis(2,4-dimethoxyphenyl)penta-1,4-dien-3-one (4): a yellow solid;

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yield: 77%. M.p.: 140-142 °C. IR (KBr): 2947, 2838, 1643, 1613, 1581, 1499, 1462,

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1326, 1268, 1186, 1112, 1031 cm-1. 1H NMR (400 MHz, DMSO-d6, 25 °C, TMS): δ

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3.83 (s, 6H), 3.89 (s, 6H), 6.61 (d, J = 8 Hz, 2 H), 6.63 (s, 2 H), 7.15 (d, J = 16 Hz, 2

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H) , 7.73 (d, J = 8 Hz, 2 H) , 7.86 (d, J = 16 Hz, 2 H) ppm;

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DMSO-d6, 25 °C, TMS): δ 55.5 (2C), 55.8 (2C), 98.4 (2C), 106.3 (2C), 115.9 (2C),

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123.9 (2C), 130.0 (2C), 136.8 (2C), 159.8 (2C), 162.8 (2C), 188.2 (1C) ppm; HRMS

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(ESI): m/z: calcd. for C21H22O5 [M+H]+: 355.1540; found: 355.1546, error = 1.7 ppm.

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C NMR (100 MHz,

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1,5-bis(2,4,6-trimethoxyphenyl)pentan-3-one (1R): a white solid; yield: 21%.

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M.p.: 73-75 °C. IR (KBr): 3002, 2943, 2839, 1710, 1600, 1500, 1462, 1359, 1237,

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1203, 1158, 1120, 1029 cm-1. 1H NMR (400 MHz, DMSO-d6, 25 °C, TMS): δ 2.49 (t,

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J = 8 Hz, 4H), 2.76 (t, J = 8 Hz, 4H), 3.78 (s, 18H), 6.21 (s, 4 H) ppm; 13C NMR (100

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MHz, DMSO-d6, 25 °C, TMS): δ 18.2 (2C), 43.9 (2C), 55.5 (2C), 56.0 (4C), 91.5

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(4C), 110.0 (2C), 159.6 (4C), 160.8 (2C), 206.1 (1C) ppm; MS (EI): m/z: 419.0

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[M+H]+.

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1,5-bis(3,4,5-trimethoxyphenyl)pentan-3-one (2R): a white solid; yield: 36%.

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M.p.: 79-81 °C. IR (KBr): 2998, 2938, 2839, 1718, 1586, 1509, 1457, 1421, 1329,

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1241, 1179, 1118, 1001 cm-1. 1H NMR (400 MHz, DMSO-d6, 25 °C, TMS): δ 2.72 (t,

180

J = 8 Hz, 4H), 2.85 (t, J = 8 Hz, 4H), 3.82 (s, 6H), 3.84 (s, 12H), 6.39 (s, 4 H) ppm;

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(2C), 105.3 (4C), 136.4 (2C), 136.8 (2C), 153.2 (4C), 209.0 (1C) ppm; MS (EI): m/z:

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419.3 [M+H]+.

C NMR (100 MHz, DMSO-d6, 25 °C, TMS): δ 30.2 (2C), 44.7 (2C), 56.1 (4C), 60.9

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Cell Culture. NCI-H460 (human non-small cell lung cancer) cells, obtained from

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the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of

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Sciences, were cultured in RPMI-1640 medium supplemented with fetal bovine serum

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(10%, v/v), NaHCO3 (2 g/L), glutamine (2 mM), penicillin (100 kU/L) and

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streptomycin (100 kU/L) and maintained in a humidified 5% CO2 atmosphere at

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37 °C.

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Cytotoxicity assay. The SRB assay was used to determine cytotoxicity of the test

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compounds against NCI-H460 cells. The cells (3 × 103/well) treated with graded

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concentrations of the test compounds for 48 h were fixed with trichloroacetic acid and

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stained with SRB (0.4%) for 30 min. When necessary, the cells were pretreated with

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NAC (10 mM) for 1 h before adding the test compounds. Cells were washed with

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acetic acid (1%) and added with Tris base solution (10 mM) for absorbance

196

measurements at 570 nm using a microplate reader (Bio-Rad Model 550). The cell

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viability was expressed as the percentage of the control.

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Cell cycle and apoptosis analysis. The cell cycle distribution and induction of

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apoptosis in NCI-H460 cells treated with the test compounds for the indicated

200

durations were analyzed using a FACSCanto flow cytometer (Becton-Dickinson, San

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Jose, CA, USA), as described in detail in our previous work.27 A total of 10,000 cells

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were acquired per sample and data were analyzed using FACSDiva software in both

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cell cycle and cell apoptosis assays. When necessary, the cells were pretreated with

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NAC (10 mM) for 1 h before adding the test compounds.

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Intracellular ROS assay. NCI-H460 cells (4 × 105/well), incubated with the test

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compounds (5 or 10 µM) for 3 or 9 h, were harvested, stained with DCFH-DA (3 µM)

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for 30 min at 37 °C in dark, then washed with PBS and subjected to immediate

208

analysis by flow cytometry. When necessary, the cells were pretreated with NAC (10

209

mM) for 1 h before adding the test compounds. The ROS levels were calculated

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related to the intensity of FITC fluorescence and expressed as fold increase relative to

211

control.

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Intracellular glutathione assay. A modified GR-DTNB recycling assay method

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was used to measure intracellular glutathione levels.28 Briefly, NCI-H460 cells (4 ×

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105/well) treated with the test compounds were harvested, resuspended in ice-cold

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HCl (10 mM, 500 µL), and lysed by three freeze-thaw cycles. When necessary, the

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cells were pretreated with NAC (10 mM) for 1 h before adding the test compounds.

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The protein concentrations were determined by the BCA protein assay reagent kit.

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Precooled 5-sulfosalicylic acid (6.5%, 120 µL) was added to the 460 µL residual

219

lysates, vortexed and kept at 4 °C for 10 min. After centrifugation (8000 g, 4°C, 15

220

min), the supernatant was divided into two groups: one as the total GSH sample (no

221

treatment), and the other as the GSSG sample (100-µL supernatant added with 5-µL

222

2-vinylpyridine, shaken for 1 h to dislodge the GSH). For the total GSH assay, the test

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solutions contained 5 µL sample, 35 µL PBS and 200 µL assay buffer (1 mM DTNB,

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340 µM NADPH, in PBS) were pre-incubated in a 96-well plate for 5 min and then

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added with 40 µL GR (8.5 IU/mL, in PBS). The absorbance change at 412 nm was

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monitored for 5 min using an Infinite M200 microplate reader (Tecan Group Ltd.,

227

Männedorf, Switzerland). Whereas for the GSSG assay, the test solutions contained

228

40 µL sample and 200 µL assay buffer were used. Intracellular total GSH and GSSG

229

concentrations were estimated using a GSH or GSSG standard curve, respectively,

230

and normalized to the determined protein concentrations. Intracellular GSH

231

concentration was calculated from the following formula: [GSH] = [Total GSH] − 2 ×

232

[GSSG]. Results were expressed as the percentage of the control.

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In vitro and intracellular TrxR-inhibitory activity assays. The NADPH-reduced

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TrxR (120 nM) was incubated with the test compounds (50 µM) in TE buffer (50 mM

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Tris-HCl pH 7.4, 1 mM EDTA, 100 µL) for 1 h and added with a mixture of DTNB

236

(2.25 mM) and NADPH (200 µM) in TE buffer (800 µL). TrxR activity was

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calculated by measuring the slope for the linear increase in absorbance at 412 nm in

238

the initial 90 s using a TU-1901 UV-Vis spectrophotometer (Beijing Purkinje General

239

Instrument Co. Ltd., Beijing, China).29 The inhibitory activity was expressed as the

240

percentage of the control.

241

Intracellular TrxR-inhibitory activity was imaged in living cells using a fluorescent

242

probe for mammalian TrxR, TRFS-green, kindly provided by Prof. Fang from

243

Lanzhou University, China.30 NCI-H460 cells seeded on glass coverslips at 2 × 105

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cells /coverslip were treated with the test compounds (30 µM) for 4 h, and incubated

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with TRFS green (5 µM) in fresh medium for another 3 h. When necessary, the cells

246

were pretreated with NAC (10 mM) for 1 h before adding the test compounds. The

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coverslips were rinsed with PBS and inverted onto glass slides with antifade mounting

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medium. The fluorescence images were acquired by a fluorescent microscope Leica

249

DM 4000B (Leica Microsystems CMS GmbH, Wetzlar, Germany) with a × 40

250

objective lens.

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NADPH oxidase activity assay. NADPH-reduced TrxR (120 nM), NADPH (200

252

µM) in TE buffer (200 µL) was incubated with the test compounds (50 µM) for 30

253

min at room temperature. After incubation, the rate of oxidation of NADPH was

254

determined by measuring the absorbance decay at 340 nm using an Infinite M200

255

microplate reader.13 The activity was expressed as fold increase relative to control.

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Western Blot analysis. NCI-H460 cells (2 × 106 cells/dish) treated with the test

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compounds (2.5, 5 µM) for 24 h were lysed with the immunoblotting assay buffer

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containing 1% PMSF at 4 °C. Protein, 40 µg/lane, was used for SDS-PAGE, and

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electroblotted onto PVDF membrane (Bio-Rad Laboratories, Hercules, CA, USA) at

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4 °C. The membranes were blocked with 5% skim milk followed by incubation with

261

appropriate primary antibody at 4 °C overnight, then washed and probed with the

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corresponding horseradish peroxidase-conjugated secondary antibodies for 1 h at

263

room temperature. The signals were finally detected using an enhanced ImageQuant

264

chemiluminescence system (GE Healthcare, Pittsburgh, PA, USA).

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Whole cell analysis of tubulin polymerization. Whole cell analysis of tubulin

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polymerization was conducted according to the established procedures.31 Specifically,

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NCI-H460 cells (4 × 105/well) incubated with the test compounds for 24 h were

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harvested, fixed in 4% paraformaldehyde for 10 min, and then pelleted (500 g, 8 min),

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followed by 10 min of permeabilization in ice-cold 90% methanol at -20 °C. When

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necessary, the cells were pretreated with NAC (10 mM) for 1 h before adding the test

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compounds. The permeablized cells were washed with microtubule stabilizing buffer

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consist of 80 mM Pipes (pH 6.8), 1 mM MgCl2, 5 mM EDTA, and 0.5% Triton X-100

273

and blocked in antibody diluting solution containing PBS (pH 7.4), 0.2% Triton

274

X-100, 2% BSA, and 0.1% NaN3 for 1 h. The cells were then incubated with

275

anti-α-tubulin-FITC antibody (1:50 diluted) in the dark for 3 h, and subjected to

276

analysis by flow cytometry.

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Intracellular GST-inhibitory activity assays. The GST activity was evaluated in

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NCI-H460 cells by imaging experiments using DNs-CV, a previously reported

279

fluorescent probe for GSTs.25 NCI-H460 cells seeded on glass coverslips at 2 × 105

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cells /coverslip were treated with or without the test compounds (30 µM) for 4 h

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followed by their removal and incubation with DNs-CV (2 µM) in serum-free medium

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without phenol red at 37 °C for 30 min. The cells were then fixed with 2%

283

formaldehyde for 15 min and stained with 0.5 µg/mL DAPI for 10 min at room

284

temperature. The coverslips were rinsed with PBS and inverted onto glass slides with

285

antifade mounting medium. The fluorescence images were captured using a

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fluorescent microscope Leica DM 4000B (Leica Microsystems CMS GmbH, Wetzlar,

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Germany) with a × 40 objective lens.

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Cellular uptake assay. NCI-H460 cells seeded in 100-mm dishes at 2 × 106

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cells/dish were treated with the test compounds (30 µM) for 0.5, 1, 2, 4, 6 or 8 h. After

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incubation, the medium was aspirated and the cells were rapidly washed thrice with

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excess of ice cold PBS, collected by trypsin and further washed with medium and

292

PBS in turns by centrifuging. Ice cold methanol (500 µL) was then added and

293

incubated for 10 h at 4 °C to lyse the cells. The cell lysates were centrifuged for 10

294

min at 10000 rpm at 4 °C, and the compound concentrations in the supernatants were

295

then measured using a M850 fluorescence spectrophotometer (Hitachi, Japan;

296

curcumin: Ex = 422 nm, Em = 536 nm; compound 1 and 2: Ex = 366 nm, Em = 512

297

nm).32 The uptake was expressed as nmol per million cells.

298

Statistical Analysis. Data are expressed as mean ± SD. Statistical comparisons

299

among the results were performed using analysis of variance. Significant differences

300

(P < 0.05) between the means of two groups were analyzed by Student's t-test.

301 302

RESULTS

303

Cytotoxicity of compounds 1 and 2 was mediated by G2/M cell cycle arrest. We

304

initially assessed the cytotoxicity of curcumin and its analogs (1-5) towards

305

NCI-H460 cells by the SRB assay. The IC50 values (concentration of the test agent

306

that is required for 50% inhibition of the cell viability) were obtained from a series of

307

dose-response curve (Figure 1B). All the tested mono-carbonyl compounds (1-5) were

308

more cytotoxic than the leading curcumin (IC50 = 38.5 ± 1.1 µM), with the IC50 values

309

being 1.9 ± 0.1, 3.4 ± 0.1, 3.8 ± 0.2, 6.1 ± 0.3 and 9.8 ± 0.3 µM, respectively. Among

310

the test compounds, the symmetrical hexamethoxy-diarylpentadienones (1 and 2)

311

were the most active ones, and displayed 20- and 11-fold more potent than curcumin,

312

respectively. In contrast, their reduced analogs 1R and 2R, where the Michael

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313

acceptor units are completely abolished, showed no obvious effect on the cell viability

314

(IC50 > 100 µM, Figure 1B), Additionally, pretreatment of N-acetylcysteine (NAC),

315

acting as both a ROS scavenger and a sulfhydryl-containing nucleophile to react

316

preferentially with the Michael acceptor units, almost completely abrogated the

317

cytotoxicity induced by compounds 1 and 2 (Figure 1B). The above results indicate an

318

indispensable role of the Michael acceptor units and importance of the

319

ROS-generation in determining the cytotoxicity of compounds 1 and 2. Figure 1 here

320 321

To investigate the possible mechanisms by which curcumin and its active analogs 1

322

and 2 exhibit the cytotoxicity, we further analyzed their effects on cell cycle

323

distribution by flow cytometry. As shown in Figure 1C, 24 h of treatment with

324

compounds 1 and 2 induced a remarkable accumulation of cells in G2/M phase in a

325

dose-dependent fashion. Specifically, compound 1 with increasing concentrations

326

from 1 to 5 µM awaked a successively increased accumulation of cells in G2/M phase,

327

from ~23% to ~86% of the total cell count. However, under the same conditions, 5

328

µM of curcumin exhibited no appreciable effect on the cell cycle distribution. The cell

329

cycle arrest activity follows the sequence of 1 > 2 > curcumin, in line with the results

330

obtained by the cytotoxicity assay.

331

For further elucidating the cytotoxic mechanisms, we next conducted an apoptosis

332

analysis by flow cytometry with Annexin-V-FITC/propidium iodide (PI) double

333

staining (Figure 1D). High-dose (10 µM) and long-duration (36 h) treatment with

334

compounds 1 and 2 indeed caused obvious cell apoptosis and necrosis. However,

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335

under the condition of low-dose (5 µM) and short-duration (24 h) they induced only a

336

small amount of apoptosis (Figure 1D), but induced significantly the G2/M phase

337

arrest. The above results support the conclusion that the cytotoxicity of compounds 1

338

and 2 towards NCI-H460 cells is predominantly mediated by G2/M cell cycle arrest,

339

even though the arrest further triggers obvious cell apoptosis and necrosis in the case

340

of high-dose or long-duration.

341

In line with those obtained from the cytotoxicity assay, pretreatment of NAC

342

reversed practically the cell cycle arrest and apoptosis induced by compounds 1 and 2.

343

Moreover, 1R and 2R were inactive in inducing the cell cycle arrest and apoptosis

344

(Figures. 1C and 1D). The above results support an indispensable role of the Michael

345

acceptor units and importance of the ROS-generation in inducing the cell cycle arrest

346

and apoptosis.

347

Compounds 1 and 2 caused ROS accumulation and imbalance of cellular

348

redox homeostasis. To further clarify the involvement of ROS in the cell cycle arrest,

349

we also measured the intracellular ROS levels by flow cytometry (Figure 2A).

350

Treatment with either compound 1 or 2 caused dose- and time-dependently a

351

substantial increase in DCFH-DA-reactive ROS, which reached a 6-fold increase in

352

the case of 10 µM compound 2 after 9 h of treatment. In contrast, curcumin was

353

almost inactive in increasing the ROS levels under the same conditions. Moreover, the

354

compounds 1 and 2-enhanced DCF fluorescence intensity was entirely decreased to

355

the control group's levels by pretreating the cells with NAC for 1 h (Figure 2A). This

356

result coupled with the inactivity of 1R and 2R in inducing intracellular ROS

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357

accumulation (Figure 2A) emphasizes a dependence of the ROS-generation on the

358

Michael acceptor units. Noticeably, compound 2 is a stronger ROS generator but a

359

relatively weaker cell cycle arrest and cytotoxic agent than compound 1 (Figures 2A

360

and 1C), implying that in addition to the ROS-generation, there must be other factors

361

contributing to their cell cycle arrest activity and cytotoxicity. We will clarify this

362

point in the following sections. Figure 2 here

363 364

To investigate whether the ROS-generation was associated with collapse of the

365

cellular redox buffering system, we also determined the levels of the reduced and

366

oxidized glutathione (GSH and GSSG). Figures 2B and 2C show a dose- and

367

time-dependent decrease and increase for the GSH and GSSG levels, respectively, in

368

the cells treated with compound 1 or 2. A comparison of Figure 2B with Figure 2C

369

clearly indicates that the alternation is much more pronounced in the GSSG levels

370

than in the GSH levels. According to the measured GSH and GSSG levels, the

371

GSH/GSSG ratios, an important index reflecting the cell redox status, were quantified.

372

As shown in Figure 2D, the test compounds sharply decreased the ratios with the

373

activity order of 2 > 1 > curcumin. Obviously, the activity order accords with their

374

ROS-generating ability, supporting a close connection between the ROS-generation

375

and collapse of intracellular redox buffering system. Likewise, pretreatment of NAC

376

also abolished completely the change of the GSH and GSSH levels induced by

377

compounds 1 and 2, and 1R and 2R were inactive in changing the GSH and GSSH

378

levels (Figures 2B-D).

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379

Compounds 1 and 2 could target TrxR to generate ROS. The thioredoxin (Trx)

380

system, consist of NADPH, TrxR and Trx, is pivotal for maintaining cellular redox

381

balance.33 In this system, TrxR catalyzes the NADPH-dependent reduction of Trx

382

which participates in many redox events.33 It has been reported that curcumin could

383

irreversibly modify TrxR by its Michael acceptor units, and the modified enzyme

384

thereby exhibits the NADPH oxidase-like activity to generate ROS.13 Based on the

385

structure similarity among curcumin and its active analogs, we postulated that

386

compounds 1 and 2 could trigger the ROS accumulation likewise by inhibiting TrxR.

387

To test this possibility, we assessed their TrxR-inhibitory activity in vitro by the

388

DTNB reduction assay.29 After incubation with compounds 1 and 2 for 1 h, the TrxR

389

activity was reduced to ~60% and ~50% of control (Figure 3A), respectively. The lost

390

activity was irrecoverable even after the test compounds were removed (data not

391

shown), indicating an irreversible inhibition. Interestingly, compounds 1 and 2 were

392

weaker in the TrxR-inhibitory activity in vitro than curcumin (Figure 3A), but the

393

enzyme modified by them than by curcumin showed much higher NADPH

394

oxidase-like activity (Figure 3B). Among the test compounds, compound 2 is the most

395

active with a 12.7-fold increased oxidation of NADPH relative to the control,

396

followed by compound 1, whereas curcumin is the worst. The activity is fully in

397

agreement with their ROS-generating ability in NCI-H460 cells. To further elucidate

398

whether they can target intracellular TrxR, we employed a newly reported fluorescent

399

probe (TRFS-green)30 to imaging the TrxR activity in living NCI-H460 cells. On the

400

basis of the bright green fluorescence intensity reflecting the intracellular TrxR

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401

activity (Figure 3C), we can concluded that after only 4 h of treatment, compounds 1

402

and 2 (30 µM) abrogate almost completely the intracellular TrxR activity and are

403

approximately equipotent in the ability, whereas curcumin is obviously inferior to

404

them. The intracellular TrxR-inhibitory activity order is completely different from that

405

obtained from the in vitro DTNB reduction assay. Although the intracellular TrxR

406

activity were significantly inhibited by the above compounds, the cytosolic TrxR1

407

protein levels remained unchanged (Figure 3D). Additionally, consistent with the

408

results obtained from the ROS level assay, complete reversion of the intracellular

409

TrxR-inhibitory activity of compounds 1 and 2 by NAC pretreatment as well as

410

inability of 1R and 2R in the activity (Figure 3C) was also observed, underlining that

411

the Michael acceptor units are necessary for the activity.

412

Figure 3 here

413

Molecular mechanisms for compounds 1 and 2-mediated G2/M cell cycle

414

arrest. The progression through the cell cycle phase is orchestrated by promotion of

415

cyclins, cyclin-dependent kinases (Cdks), and Cdc25 phosphatases as well as by

416

inhibition of Cdk inhibitors such as p21.34,

417

compounds 1 and 2-mediated G2/M cell cycle arrest in NCI-H460 cells, we examined

418

their effects on expression of proteins that are critical for G2/M transition, including

419

Cdk1, cyclin B1, Cdc25C, p21 and p53, by Western blotting (Figure 4). When the

420

cells were treated with either compound 1 or 2 for 24 h, a marked dose-dependent

421

decrease of Cdk1, cyclin B1 and Cdc25C expression was observed. Conversely, the

422

p53 and p21 expression increased in a dose-dependent manner. At the same

35

To clarify the mechanisms for

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423

concentration, both the inhibition and stimulation effect on protein express was much

424

more pronounced by compound 1 than compound 2. This outcome is in accordance

425

with their G2/M cell cycle arrest ability. In addition, pretreatment of NAC completely

426

reversed the above changes in protein expression, highlighting a central role of the

427

Michael acceptor units and the ROS-generation in regulating expression of the redox

428

active cell-cycle-regulatory proteins.

429

Figure 4 here

430

Inhibition of tubulin polymerization by compound 1. As mentioned above,

431

compound 2 is a stronger ROS generator but a relatively weaker G2/M cell cycle

432

arrest agent than compound 1. Therefore, we believe that as small molecular

433

compounds, they can target not only TrxR to generate ROS but also other proteins

434

related to the cell cycle arrest by virtue of their Michael acceptor units. Microtubules,

435

as key components of the cytoskeleton, are composed of tubulin and are responsible

436

for mitosis and cell division, and interfering with microtubule dynamics could induce

437

cell cycle arrest during the M phase.36, 37 Some electrophiles such as 6-shagol38 and

438

dially trisulfide39 can target sulfhydryl groups of cysteine residues (Cys-12β and

439

Cys-354β) in tubulin, thereby perturbing tubulin polymerization, causing mitotic

440

arrest and triggering cell death. To check whether compounds 1 and 2 impact on the

441

tubulin polymerization, a flow cytometry method was employed to achieve a rapid

442

and quantitative analysis of the whole cell tubulin polymerization levels. Paclitaxel

443

and colchicine were used as the reference compounds with known functions to

444

stabilize and destabilize microtubules, respectively. As shown in Figure 5, compound

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445

1, acting like the microtubule destabilizer colchicine, obviously reduced the

446

anti-tubulin fluorescence signal in a dose-dependent manner, indicating an inhibition

447

of microtubule polymerization. In contrast, compound 2 and 1R failed to exhibit the

448

effect. Furthermore, adding NAC almost blocked the inhibition of microtubule

449

polymerization by compound 1. These results reflect that the inhibitory activity of

450

compound 1 against microtubule polymerization relies on not only the

451

Michael-acceptor units but also the installation mode of methoxy groups on the

452

aromatic rings, and is independent of the ROS-generation.

453

Figure 5 here

454

Higher GST-inhibitory activity by compound 1 than compound 2. GSTs are a

455

family of phase II detoxification enzymes which catalyze the conjugation of GSH to a

456

broad spectrum of endogeneous and exogeneous electrophilic compounds, resulting in

457

their removal from the cells.40,41 GSTs have emerged as a promising therapeutic target

458

since they are overexpressed in a wide variety of tumors and contribute to resistance

459

to chemotherapeutics.40,41 It has been reported that camptothecin-induced S or G2/M

460

arrest of HeLa cells is intensified by silencing Glutathione S-transferase P1 (GSTP1)

461

gene.42 Additionally, curcumin has also been identified as an inhibitor of human

462

GSTs,43 and its pro-apoptosis activity against K562 cells is mediated by inhibiting the

463

GSTP1 expression at the level of transcription.44 Therefore, to further elucidating the

464

reason for the increased G2/M cell cycle arrest activity by compound 1 compared with

465

compound 2, we compared their GST-inhibitory activity together with that of

466

curcumin in NCI-H460 cells by using GST (DNs-CV)25, a red fluorescent probe

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467

applied to the imaging of GST activity in living cells. As illustrated in Figure 6, after

468

only 4 h of treatment, compound 2 decreased obviously the fluorescence signal

469

localized to an area adjacent to the nucleus which reflects the intracellular GST

470

activity, but to a lesser extent than compound 1 did. The order of intracellular

471

GST-inhibitory activity further corresponds to the increased activity of compound 1 in

472

the cell cycle arrest. Besides, the Michael acceptor-dependent effect of compounds 1

473

and 2 in relation to the GST-inhibitory activity was observed as evidenced by both

474

complete blockage of their activity by NAC pretreatment and failure of 1R and 2R in

475

exhibiting the activity. Figure 6 here

476 477

Increased cellular uptake of compound 1 compared with compound 2 and

478

curcumin. Considering that cellular uptake is also a probable factor in determining

479

the

480

microtubule-destabilizing effect and GST-inhibitory activity, we finally assayed

481

intracellular concentrations of curcumin and its active analogs at different time points

482

by fluorescence analysis of methanol extracted cell lysates. As shown in Figure 7,

483

uptake of curcumin reached a maximum value after 2 h of incubation followed by its

484

rapid decay during 8 h. Compound 2 was analogous to curcumin in the time-course of

485

uptake and decay, but appeared a relatively rapid and high peak uptake. Notably,

486

compound 1 exhibited the most efficient uptake, which was almost 6 times greater

487

than that of compound 2, and its decay occurred only after 4 h of incubation.

cell

cycle

arrest

activity

apart

from

the

ROS-generating

ability,

488

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Figure 7 here

489 490 491

DISCUSSION

492

The past two decades have witnessed much interest in investigating cancer

493

chemoprevention and chemotherapy mechanisms of curcumin due to its

494

multi-targeted activity, selectivity and safety for human use as a dietary molecule.9-11

495

Like most polyphenols, although curcumin is a well-known natural antioxidant, it is

496

also a prooxidant able to promote the ROS-generation under special conditions such

497

as high concentrations,12, 45-47 and in the presence of cupric ions.48,49 Another efficient

498

mean of curcumin as a prooxidant is to covalently modify cysteine (selenocysteine)

499

residues in redox-sensitive target proteins such as TrxR13,14 by virtue its Michael

500

acceptor units. More importantly, the prooxidant property of curcumin is responsible

501

for its apoptosis-inducing activity in various cancer cells and thus therapeutic effect.12

502

In this study, we selected symmetrical hexamethoxy-diarylpentadienones, 1 and 2 (as

503

the active and metabolically stable curcumin analogs) to investigate the cytotoxic

504

mechanisms towards NCI-H460 cells from a chemical and biological point of view,

505

and

506

acceptor-dependent redox intervention and designing curcumin-inspired anticancer

507

agents by a prooxidant strategy.

tried

to

provide

useful

information

for

understanding

the

Michael

508

The IC50 values of curcumin, compounds 1-5, 1R and 2R against NCI-H460 cells

509

enable us to deduce the activity order of 1 > 2 ~ 3 > 4 > 5 > curcumin > 1R, 2R and

510

allowed us to identify the following SAR: the Michael acceptor units is necessary for

511

the cytotoxicity, whereas the position and number of the methoxy groups on the 24

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512

aromatic rings further contributes to the activity. Abrogating the cytotoxicity of

513

compounds 1 and 2 by NAC (Figure 1B), acting as both an antioxidant and a

514

nucleophile, further supports the Michael acceptor-dependent cytotoxicity and

515

contribution of ROS to the activity. Subsequently, in NCI-H460 cells, compounds 1

516

and 2 were identified as the G2/M cell cycle arrest agents to mediate the cytotoxicity

517

via Michael acceptor- and ROS-dependent mechanisms (Figure 1C and Figure 2A).

518

Similarly, compound 2 has been previously found to induce G2/M cell cycle arrest in

519

HCT116 cells.19 Additionally, the dependency of the ROS-generation on the Michael

520

acceptor units is supported by the fact that NAC blocks completely the stimulatory

521

effect of compounds 1 and 2 on the ROS-generation, as well as 1R and 2R are

522

inactive in inducing ROS accumulation (Figure 2A).

523

Concerning the ROS-generating mechanism, our data (Figure 3) show that

524

compounds 1 and 2, similar to curcumin,13,14 could irreversibly inhibit TrxR

525

depending on their Michael acceptor units, and the resulting covalently modified TrxR

526

serves as an important source of ROS. The in vitro assay shows the weaker

527

TrxR-inhibitory potency of compounds 1 and 2 than that of curcumin (Curcumin >

528

1 > 2, Figure 3A), but the higher NADPH oxidase-like activity of the TrxR modified

529

by them than by curcumin (2 > 1 > curcumin, Figure 3B). In contrast, the in vivo

530

experiments suggest that compounds 1 and 2 are much more effective inhibitors of

531

TrxR in NCI-H460 cells than curcumin (1 ~ 2 > curcumin, Figure 3C). The

532

inconsistent results between the in vitro and in vivo TrxR-inhibitory activity is due, at

533

least in part, to the increased cell uptake of compounds 1 and 2 compared with that of

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534

curcumin (Figure 7D). Notably, the SAR in the NADPH oxidase-like activity of the

535

TrxR modified by curcumin and compounds 1 and 2, as well as their in vivo

536

TrxR-inhibitory activity, correlate well with their ROS-generating activity order (2 >

537

1 > curcumin). The correlation also raises the possibility that TrxR is one of the

538

targets by which compounds 1 and 2 promote the ROS-generation. Additionally, the

539

cytosolic TrxR1 protein levels remained unchanged after exposing cells to curcumin

540

and its active analogs (Figure 3D), suggesting that the decrease of TrxR activity is

541

mediated by directly inhibiting enzyme activity, instead of down-regulating protein

542

expression. The above results also emphasize a broad substrate specificity of TrxR

543

based on the following reasons: its penultimate selenocysteine residue locates at the

544

flexible C-terminal tails and is thus easily accessible for inhibitors; its low pKa value

545

of 5.2 renders it easy to ionize at physiological pH, resulting in formation of a highly

546

active and nucleophilic selenolate.33, 50 Therefore, despite of the different installation

547

mode of methoxy groups on the aromatic rings, both compounds 1 and 2 could

548

effectively target intracellular TrxR.

549

The ROS-generation and collapse of intracellular redox buffering system (decrease

550

in the GSH/GSSG ratios) induced by compounds 1 and 2 occurred almost

551

simultaneously according to the time points of 6 and 9 h (Figure 2). This invites

552

inevitably the questions: do ROS induce reduction of GSH, or does low GSH cause an

553

increase in the ROS-generation? Currently, our cumulated data are not yet sufficient

554

to clarify this point. However, the preferential reactivity of compounds 1 and 2 with

555

TrxR at micromolar concentrations,51 despite the presence of millimolar

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556

concentrations of GSH,52 might result in the priority of the ROS-generation. The

557

preferential reactivity is based on the fact that, as discussed above, selenocysteine is

558

more easily ionized than GSH at physiological pH and nucleophilicity of the resulting

559

selenolate is far stronger than that of thiolate. Furthermore, it could be also supported

560

by our previous results showing that a double ortho-trifluoromethyl substituted

561

diarylpentadienone, structurally similar to compounds 1 and 2, induced a dramatic

562

increase in the GSSG levels while it barely changed the GSH levels in A549 cells.24

563

Besides, almost simultaneous occurrence of the ROS-generation and collapse of the

564

redox buffering system just reflect that the redox intervention style would be a vicious

565

cycle, especially when the intervention occurs in such a double-effect pathway

566

including both irreversibly inhibiting TrxR and converting it into a prooxidant.

567

Cell cycle progression is precisely regulated by cyclins, Cdks and Cdk inhibitors,

568

and is also subject to a redox control fashion because ROS can influence the presence

569

and activity of these proteins.34, 35 The G2/M phase transition and completion of the M

570

phase require binding of cyclin B to Cdk1 and subsequent Cdk1 activation.34,35

571

Western blotting results reveal that both compounds 1 and 2 decrease

572

dose-dependently the expression levels of cyclin B1 and Cdk1 (Figure 4).

573

Additionally, p53 and p21 levels are up-regulated in the G2/M cell cycle arrest

574

(Figure 4). It is generally agreed that p53, a multifunctional tumor suppressor, plays

575

an important role in cell cycle arrest through its downstream mediator p21 especially

576

under a stressed situation.53 When the cellular redox state is shifted toward a

577

more-oxidative condition, the activity of the cyclin B1/Cdk1 complex can be inhibited

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578

through up-regulation of a p53-inducible cell cycle inhibitor p21,54,55 thus causing the

579

G2/M cell cycle arrest. Cdc25C phosphatase is another redox sensitive

580

cell-cycle-regulatory protein which activates the cyclin B1/Cdk1 complex activity by

581

dephosphorylating pThr14 and pTyr15 on Cdk1.56 ROS can target the highly reactive

582

cysteine 330 and 377 at the active site in Cdc25C, leading to the enzyme inactivation,

583

and the ROS-inactivated Cdc25C is expected to prevent cell-cycle progression at

584

G2/M phase via inhibiting the cyclin B1/Cdk1.56 A decrease of Cdc25C expression

585

was also observed in the case of compounds 1 and 2 (Figure 4). In addition, all the

586

alterations of the regulatory proteins can be rescued by NAC (Figure 4). This further

587

supports that compounds 1 and 2, by virtue of their Michael acceptor units, can

588

collapse the cell-cycle-regulatory system in NCI-H460 cells by both inducing a burst

589

of ROS and directly modifying cysteine residues of the above redox-sensitive target

590

proteins. The contribution of the latter can be deduced form the fact that compound 1,

591

compared with compound 2, is more effective to induce the alteration in the cyclin B1,

592

Cdk1, p53, p21 and Cdc25C expression, the G2/M cell cycle arrest activity and thus

593

the cytotoxicity, but is relatively less effective to promoting the ROS-generation.

594

To clarify the reason why compound 1, compared with compound 2, displays a

595

relatively weak ability to generate ROS but the increased cell cycle arrest activity and

596

cytotoxicity,

597

microtubule-interfering agents cause cell cycle arrest during the M phase by either

598

stabilizing or destabilizing tubulin.36,37 Notably, compound 1 was an effective

599

microtubule destabilizer, whereas compound 2 and 1R were ineffective (Figure 5). We

we

turn

to

another

target,

tubulin/microtubule.

Most

28

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600

can conclude from the SAR that both the Michael-acceptor units and the installation

601

mode of methoxy groups on the aromatic rings are indispensable for the inhibitory

602

activity of compound 1 against microtubule polymerization. More importantly,

603

compound 2 failed to display the effect despite of its strong ROS-generating ability,

604

clearly indicating that the microtubule-destabilizing effect of compound 1 is

605

independent of the ROS-generation, and contributes to its increased G2/M cell cycle

606

arrest activity. Notably, the abrogative effect of NAC on the microtubule-destabilizing

607

activity of compound 1 should be only due to its function as a nucleophile (not as an

608

antioxidant) to react preferentially with the Michael acceptor acceptors of compound

609

1, thereby inhibiting binding of compound 1 to active cysteine residues in tubulin.

610

Considering that camptothecin-induced S or G2/M arrest of HeLa cells is

611

intensified by silencing GSTP1 gene,42 and curcumin-induced apoptosis of K562 cells

612

is mediated by inhibiting the GSTP1 expression,44 we also investigated the

613

GST-inhibitory activity cucumin and its active analogs 1 and 2. Our results show that

614

the activity is the Michael acceptor-dependent, and compound 1 is the most active,

615

followed by compound 2; curcumin is the worst (Figure 6). This provides another

616

possible explanation for the increased G2/M cell cycle arrest activity of compound 1

617

despite of its relatively weaker ROS-generating ability than that of compound 2.

618

To sum up, in this work, the active curcumin analogs 1 and 2 were identified as the

619

G2/M cell cycle arrest agents to mediate the cytotoxicity toward NCI-H460 cells via

620

Michael acceptor-dependent redox intervention. As summarized in Figure 8, they

621

could act as dual-effective TrxR inhibitors not only inactivating TrxR by their Michael

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622

acceptor units, but also converting this antioxidant enzyme into an ROS promoter

623

with a NADPH oxidase-like activity, leading to a burst in ROS associated with falling

624

apart of the redox buffering system. Depending on the Michael acceptor unit and the

625

ROS-generation, they cause a final G2/M cell cycle arrest in NCI-H460 cells by

626

decreasing the expression levels of cyclin B1 and Cdk1 via up-regulation of p53 and

627

p21, and down-regulation of Cdc25C. Additionally, the increased cell cycle arrest

628

activity of compound 1 compared with compound 2 is derived, at least in part, from

629

its Michael acceptor-dependent microtubule-destabilizing effect and greater

630

GST-inhibitory activity, as well as its enhanced cellular uptake. Figure 8 here

631 632 633

ACKNOWLEDGMENT

634

We are grateful to Prof. Jianguo Fang (Lanzhou University) for the generous gift of

635

TRFS-green. This work was supported by the National Natural Science Foundation of

636

China (Grant No. 21172101), the 111 Project, and the Fundamental Research Funds

637

for the Central Universities (lzujbky-2015-51).

638

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639

REFERENCES

640

(1) Bindoli, A.; Rigobello, M. P. Principles in redox signaling: from chemistry to

641

functional significance. Antioxid. Redox Signal. 2013, 18, 1557-1593.

642

(2) Trachootham, D.; Alexandre, J.; Huang, P. Targeting cancer cells by

643

ROS-mediated mechanisms: a radical therapeutic approach? Nat. Rev. Drug Discov.

644

2009, 8, 579-591.

645

(3) Policastro, L. L.; Ibañez, I. L.; Notcovich, C.; Duran, H. A.; Podhajcer, O. L. The

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tumor microenvironment:

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Figure captions:

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Figure 1. Cytotoxicity of compounds 1 and 2 toward NCI-H460 cells is mediated by

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the G2/M cell cycle arrest. (A) Chemical Structures of curcumin (Cur) and its analogs.

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(B) Cytotoxicity of Cur and its active analogs. Cells (3 × 103/well) were treated with

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the test compounds with serial concentrations for 48 h in the absence or presence of 1

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h pretreatment with NAC, and cell viability was measured by the SRB assay as

815

described in the section of materials and methods. Data represent mean ± SD of three

816

experiments. (C) Michael acceptor- and ROS-dependent G2/M arrest induced by

817

compounds 1 and 2 in NCI-H460 cells. Cells (4 × 105/well) were treated with the test

818

compounds with indicated concentrations for 24 h in the absence or presence of 1 h

819

pretreatment with NAC, and subjected to cell cycle analyses. Representative

820

histograms from three repeat experiments are shown to depict cell cycle distribution.

821

(D) Apoptosis induced by compounds 1 and 2. Cells (3 × 105/well) were treated with

822

the test compounds with indicated concentrations for 24 or 36 h in the absence or

823

presence of 1 h pretreatment with NAC, and subjected to Annexin V-FITC/PI double

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staining assay. Cells show four different cell populations marked as follows: necrotic

825

cells (upper left, Q1), late apoptotic cells (upper right, Q2), live cells (lower left, Q3),

826

and early apoptotic cells (lower right, Q4). Data are representative from three

827

independent experiments.

828 829

Figure 2. Compounds 1 and 2 cause ROS accumulation associated with imbalance of

830

cellular redox homeostasis (A) ROS accumulation induced by curcumin (Cur) and its

831

active analogs in NCI-H460 cells. Cells (4 × 105/well) were treated with the test

832

compounds with indicated concentrations for 6 or 9 h in the absence or presence of 1

833

h pretreatment with NAC, and subjected to DCFH-DA staining assay. (B-D)

834

Alterations of intracellular GSH levels (B), GSSG levels (C), and GSH/GSSG ratios

835

(D) induced by Cur and its active analogs. Cells (4 × 105/well) were treated with the

836

test compounds with indicated concentrations for 6 or 9 h in the absence or presence

837

of 1 h pretreatment with NAC, and subjected to intracellular glutathione assay as

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838

described in the section of materials and methods. Data are expressed as mean ± SD;

839

n = 3, * P