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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
2
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.
22
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
37
B1/Cdk1 in a Michael acceptor- and ROS-dependent fashion. Interestingly, in
38
comparison with compound 2, compound 1 displayed a relatively weak ability to
39
generate ROS but the increased cell cycle arrest activity and cytotoxicity probably due
40
to its Michael acceptor-dependent microtubule-destabilizing effect and greater
41
GST-inhibitory activity, as well as its enhanced cellular uptake. This work provides
42
useful information for understanding Michael acceptor-dependent and redox-mediated
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cytotoxic mechanisms of curcumin and its active analogs.
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KEYWORDS
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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
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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
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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 =
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0.7 ppm.
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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 =
158
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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13
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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
186
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
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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
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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
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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
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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.,
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Männedorf, Switzerland). Whereas for the GSSG assay, the test solutions contained
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40 µL sample and 200 µL assay buffer were used. Intracellular total GSH and GSSG
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concentrations were estimated using a GSH or GSSG standard curve, respectively,
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and normalized to the determined protein concentrations. Intracellular GSH
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concentration was calculated from the following formula: [GSH] = [Total GSH] − 2 ×
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[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
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(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
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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.
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Intracellular TrxR-inhibitory activity was imaged in living cells using a fluorescent
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probe for mammalian TrxR, TRFS-green, kindly provided by Prof. Fang from
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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
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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
258
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
260
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
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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
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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
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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
281
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
286
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
289
cells/dish were treated with the test compounds (30 µM) for 0.5, 1, 2, 4, 6 or 8 h. After
290
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
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PBS in turns by centrifuging. Ice cold methanol (500 µL) was then added and
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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
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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
29
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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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640
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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
814
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
824
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