Evaluation of cancer-preventive activity and structure-activity relationships of 3-demethylubiquinone Q2, isolated from the ascidian Aplidium glabrum, and its synthetic analogs

Article abstract of DOI:10.1007/s11095-005-8813-4

Purpose. 3-Demethylubiquinone Q₂ (1) was isolated from the ascidian Aplidium glabrum. The cancer-preventive properties and the structure-activity relationship for 3-demethylubiquinone Q₂ (1) and 12 of its synthetic analogs (3-14) are reported. Methods. Compounds 3-14, having one or several di- or triprenyl substitutions and quinone moieties with methoxyls in different positions, were synthesized. The cancer-preventive properties of compounds 1 and 3-14 were tested in JB6 Cl41 mouse skin cells, using a variety of assessments, including the methanethiosulfonate (MTS) assay, flow cytometry, and soft agar assay. Statistical nonparametric methods were used to confirm statistical significance. Results. All quinones tested were shown to inhibit JB6 Cl41 cell transformation, to induce apoptosis, AP-1, and NF-κB activity, and to inhibit p53 activity. The most promising effects were indicated for compounds containing two isoprene units in a side chain and a methoxyl group at the para-position to a polyprenyl substitution. Conclusions. Quinones 1 and 3-14 demonstrated cancer-preventive activity in JB6 Cl41 cells, which may be attributed to the induction of p53-independent apoptosis. These activities depended on the length of side chains and on the positions of the methoxyl groups in the quinone part of the molecule.

Full text of DOI:10.1007/s11095-005-8813-4

Pharmaceutical Research, Vol. 23, No. 1, January 2006 (# 2006)
DOI: 10.1007/s11095-005-8813-4
Research Paper
Evaluation of Cancer-Preventive Activity and Structure–Activity Relationships
of 3-Demethylubiquinone Q , Isolated from the Ascidian Aplidium glabrum,
and its Synthetic Analogs
2
1
Sergey N. Fedorov, Oleg S. Radchenko, Larisa K. Shubina, Nadezhda N. Balaneva, Ann M. Bode,
,2
1
1
1
2
1
Valentin A. Stonik, and Zigang Dong
2,3
Received July 13, 2005; accepted September 28, 2005
Purpose. 3-Demethylubiquinone Q (1) was isolated from the ascidian Aplidium glabrum. The cancer-
2
preventive properties and the structureYactivity relationship for 3-demethylubiquinone Q
its synthetic analogs (3Y14) are reported.
2
(1) and 12 of
Methods. Compounds 3Y14, having one or several di- or triprenyl substitutions and quinone moieties
with methoxyls in different positions, were synthesized. The cancer-preventive properties of compounds
1
and 3Y14 were tested in JB6 Cl41 mouse skin cells, using a variety of assessments, including the
methanethiosulfonate (MTS) assay, flow cytometry, and soft agar assay. Statistical nonparametric
methods were used to confirm statistical significance.
Results. All quinones tested were shown to inhibit JB6 Cl41 cell transformation, to induce apoptosis,
AP-1, and NF-kB activity, and to inhibit p53 activity. The most promising effects were indicated for
compounds containing two isoprene units in a side chain and a methoxyl group at the para-position to a
polyprenyl substitution.
Conclusions. Quinones 1 and 3Y14 demonstrated cancer-preventive activity in JB6 Cl41 cells, which may
be attributed to the induction of p53-independent apoptosis. These activities depended on the length of
side chains and on the positions of the methoxyl groups in the quinone part of the molecule.
KEY WORDS: apoptosis; cancer prevention; marine prenylated quinones; nuclear factor; structureY
activity relationship.
INTRODUCTION
quinones have been isolated from brown algae of the order
Fucales (10Y13), sponges (14Y17), alcyonaceans (18), gorgo-
Interest in developing effective cancer therapies led to naceans (19), and ascidians belonging to the genus Aplidium
the discovery of new important technologies and biomole- (20Y25). Brown algae contain diprenyl-, triprenyl-, and
cules, including viral vaccines, microbial-based therapy, tetraprenylquinones and hydroquinones (10Y13). Sponges
interferons, interleukins, and a number of new promising contain prenylated 1,4-benzoquinones and hydroquinones
antitumor agents (1Y4). With respect to antitumor agents, with linear and longer (up to nine prenyl units) terpenoid
some marine secondary metabolites are known to be among side chains (14Y17). Ascidians of the genus Aplidium have
the most promising for chemotherapy of cancer (5Y7). previously yielded about a dozen prenylated quinones and
Polyprenylated 1,4-benzoquinones and hydroquinones are related compounds (20Y25).
commonly found in a variety of organisms and play an im-
The purpose of the present work was to study the
portant role in photosynthesis, electron transport, and as cancer-preventive activity and to establish the corresponding
antioxidants (8,9). The previously described marine polypre- structureYactivity relationships (SARs) of a group of preny-
nylbenzoquinones and hydroquinones contain a terpenoid lated quinones resembling ubiquinones in their structures,
portion ranging from one to nine prenyl units. These but differing in that they have shorter polyprenyl side chains
and modified quinone moieties. Two of these compounds,
1
0 0
-demethylubiquinone Q (1) and its 2 ,3 -cis-isomer, 2,3-
2
Hormel Institute, University of Minnesota, 801 16th Avenue NE,
Austin, Minnesota 55912, USA.
3
0
0
0
0
dimethoxy-5-(3 ,7 -dimethyl-octa-2 (Z),6 -dienyl)-[1,4]benzo-
quinone (2), were isolated from the Far Eastern ascidian
Aplidium glabrum (26), whereas compounds 3Y14 are syn-
thetic analogs of 1. Distinct from previous investigations on
related compounds, we studied the proapoptotic properties
and the cancer-preventive activities of these compounds by
2
Pacific Institute of Bioorganic Chemistry, 159 Prospect 100-let
Vladivostoku, Vladivostok, 690022, Russia.
3
To whom correspondence should be addressed. (e-mail: zgdong@
hi.umn.edu)
ABBREVIATIONS: 3-demethylubiquinone Q2, 2,3-dimethoxy-5-
0
0
0
0
(
3 ,7 -dimethyl-octa-2 (E),6 -dienyl)-[1,4]benzoquinone (1); EGF,
epidermal growth factor; FBS, fetal bovine serum; MEM, minimum using mouse cell lines and methods of flow cytometry or
essential medium; TPA, 12-O-tetradecanoyl-phorbol-13-acetate.
DNA laddering for assessing apoptosis and the well-accepted
0
724-8741/06/0100-0070/0 # 2006 Springer Science + Business Media, Inc.
70

Cancer-Preventive Marine Quinones
71
+
anchorage-independent assay using JB6 P Cl41 cells in soft Syntheses of Quinones 1–14
agar for investigation of the cancer-preventive activity.
Step A
MATERIALS AND METHODS
Boric trifluoride etherate (0.5 ml) was added to the
stirred mixture containing 1 mmol of the corresponding
phenol (15Y17) and 4 mmol of trans-geraniol (18) or trans-
General Procedures
1
13
H- and C-nuclear magnetic resonance (NMR) spectra farnesol (19) in 10 ml of absolute ether. The mixture was kept
were recorded on a Bruker WM-250 spectrometer at 250 and for 12 h at room temperature, and then 30 ml of water was
2.9 MHz and on a Bruker DPX 300 at 300 and 75 MHz, added to the mixture and products were extracted with ether
6
respectively. High-resolution electron impact mass spec- (3 ꢀ 15 ml). The extract was washed with 10% NaCl and dried
trometry (HREIMS) was obtained on an AMD-604S mass over Na SO . The solvent was removed, and the residue was
2
4
spectrometer. High-performance liquid chromatography separated using column chromatography on silica gel. Preny-
HPLC) separations were conducted on a DuPont 8800 lated phenols were eluted with the solvent system gradient
(
chromatograph equipped with differential refractometer using hexane/acetone, 50:1 Y 20:1. The yield of mixtures of the
an Ultrasphere Si column. The infrared (IR) spectra were purified prenylphenols was about 60%. These mixtures were
measured on a Bruker FT-IR BVector 22^ spectrophotometer. used for step 2 of the syntheses. The prenylated phenols 20,
4
Ultraviolet (UV) spectra were determined in CCl on a Cecil 21, 23, and 28 were isolated as individual compounds by
CE 7200 spectrophotometer. The onset of apoptosis was HPLC on an Ultrasphere Si (Altex, Berkeley, CA, USA), 4.6
analyzed by flow cytometry using the Becton Dickinson mm ꢀ 25 cm column in the system hexane/ethyl acetate, 7:1.
FACSCalibur (BD Biosciences, San Jose, CA, USA). The
MTS reduction assay to determine cell viability was measured Step B
using the Multiskan MS microplate reader (Labsystems,
Helsinki, Finland). Cell colonies in the anchorage-indepen-
dent transformation assay were scored using the LEICA DM mmol) in 3 ml of the mixture CH
IRB inverted research microscope (Leica Mikroskopie und to the cooled (0-C) stirred solution of the corresponding
Systeme GmbH, Wetzlar, Germany) and Image-Pro Plus prenylated phenol in 7 ml of CH CN. After being stirred at
A solution of cerium-ammonium nitrate (CAN; 0.9
3
CN/H O, 1:2, was added
2
3
software, version 3.0 for Windows (Media Cybernetics, Silver 0-C for 1Y2 h, the mixture was poured into 25 ml of 10%
Spring, MD, USA). The luminescence assay for p53, AP-1, NaCl and extracted with ether (3 ꢀ 15 ml).
and NF-kB nuclear factor-dependent transcriptional activity
2 4
The extract was dried over Na SO and evaporated. The
was measured using the Luminoscan Ascent Type 392 micro- corresponding prenylated [1,4]-benzoquinones 1Y10 and
plate reader (Labsystems).
12Y14 were separated by preparative thin-layer chromatog-
raphy on silica gel in the system hexane/acetone, 8:1. Yields
of target products were about 70% at this stage. Total yield
calculated for two stages was about 45%.
Reagents
Minimum essential medium (MEM) and Dulbecco’s
modified Eagle’s medium were from Gibco Invitrogen
Step C (Synthesis of Prenylated Hydroquinone 11)
(
Carlsbad, CA, USA). Fetal bovine serum (FBS) was from
A solution of Na S O (3 mmol) in 3 ml of water was
2
2
4
Gemini Bio-Products (Calabasas, CA, USA). Penicillin/
streptomycin and gentamicin were from Bio-Whittaker
(
added to 1 mmol of the prenylquinone 7 in 7 ml of acetone.
The mixture was stirred for 1 h, diluted with water, and
extracted with ether (3 ꢀ 15 ml). The extract was dried over
Walkersville, MD, USA); L-glutamine was from Mediatech,
Inc. (Herndon, VA, USA). Epidermal growth factor (EGF)
was from Collaborative Research (Bedford, MA, USA).
Luciferase assay substrate and Cell Titer 96 Aqueous One
Solution Reagent (MTS) for the cell proliferation assay were
from Promega (Madison, WI, USA). The Annexin V-FITC
Apoptosis Detection Kit was from Medical & Biological
Laboratories (Watertown, MA, USA). Silica gel L (40/100
mm) for low-pressure column liquid chromatography was
from Chemapol (Prague, Czech Republic). Silica gel plates
for thin-layer chromatography (4.5 ꢀ 6.0 cm, 5Y17 mm) were
from Sorbfil (Sorbpolymer, Krasnodar, Russia).
Na
obtained.
-Demethylubiquinone Q or 2,3-dimethoxy-5-(3 ,7 -di-
2 4
SO and evaporated. As a result, hydroquinone 11 was
0
0
3
2
0
0
methyl-octa-2 (E),6 -dienyl)-[1,4]benzoquinone (1): Yellow
+
oil, HREIMS m/z 304.1655 [M] , calcd. for C18
24 4
H O
1
3
2
3 3
04.1675, IR (CHCl ): 1675, 1657, 1603. H-NMR (CDCl ,
0
50 MHz) d: 6.34 (t, J = 1.7, 1H, H-6); 5.13 (m, 1H, H-2 ); 5.08
0
(m, 1H, H-6 ); 4.02 (s, 3H, OMe); 4.00 (s, 3H, OMe); 3.10 (dd,
0
J = 7.3, 1.7, 2H, H-1 ); 2.09 (m, 2H, H-5 ); 2.08 (m, 2H, H-4 );
.70 (d, J = 1.2, 3H, H-8 ); 1.62 (d, J = 1.2, 3H, H-10 ); 1.60
, 62.9 MHz) d: 16.20 (q, C-
0
0
0
0
1
(
0
13
br. s, 3H, H-9 ). C-NMR (CDCl
3
0
0
0
0
1
0
0 ), 17.79 (q, C-9 ), 25.77 (q, C-8 ), 26.52 (t, C-5 ), 27.17 (t, C-
0
Cell Culture
1 ), 39.72 (t, C-4 ), 61.20 (q, OMe), 61.30 (q, OMe), 117.78 (d,
0
0
0
C-2 ), 123.98 (d, C-6 ), 130.45 (d, C-6), 131.95 (s, C-7 ), 140.17
+
The JB6 P Cl41 mouse epidermal cell line and its stable (s, C-3 ), 144.91 (s, C-2 or C-3), 145.16 (s, C-3 or C-2), 146.92
0
transfectants Cl41YNF-kB, Cl41YAP-1, and Cl41Yp53 (PG- (s, C-5), 184.38 (s, C-4 or C-1), 184.54 (s, C-1 or C-4).
0 0 0 0
2,3-Dimethoxy-5-(3 ,7 -dimethyl-octa-2 (Z),6 -dienyl)-
1
3) were cultured in monolayers at 37-C and 5% CO2 in
MEM containing 5% FBS, 2 mM L-glutamine, 100 U/ml [1,4]benzoquinone (2): Yellow oil, HREIMS m/z 304.1662
+
1
penicillin, and 100 mg/ml streptomycin.
24 4 3
[M] , calcd. for C18H O 304.1675, H-NMR (CDCl , 250

7
2
Fedorov et al.
0
0 0 0 0 0 0
2-Methoxy-3-(3 ,7 ,11 -trimethyl-dodeca-2 ,6 ,10 -tri-
H, H-6 ); 4.02 (s, 3H, OMe); 4.00 (s, 3H, OMe); 3.11 (br. d, enyl)-[1,4]benzoquinone (10): Yellow oil, HREIMS m/z
MHz) d: 6.37 (t, J = 1.7, 1H, H-6); 5.13 (m, 1H, H-2 ); 5.07 (m,
0
1
0
0
0
+
1
J = 7.1, 1.2, 2H, H-1 ); 2.04 (m, 2H, H-5 ); 2.04 (m, 2H, H-4 ); 342.2212 [M] , calcd. for C H O 342.2195, H-NMR (250
2
2
30
3
0
0
1
.75 (q, J = 1.2, 3H, H-10 ); 1.66 (br. d, J = 1.2, 3H, H-8 ); 1.59 MHz, CDCl ) d: 6.68 (d, J = 10.0, 1H, H-6); 6.57 (d, J = 10.0,
3
0
13
0
0
0
(
(
(
(
1
1
d, J = 1.2, 3H, H-9 ). C-NMR (CDCl , 62.9 MHz) d: 17.79 1H, H-5); 5.07 (m, 3H, H-2 , H-6 , H-10 ); 4.02 (s, 3H, OMe);
3
0
0
0
0
0
0
0
0
q, C-9 ), 22.76 (q, C-10 ), 25.77 (q, C-8 ), 26.52 (t, C-5 ), 27.27 3.16(br. d, J = 7.3, 2H, H-1 ); 2.01 (m, 8H, H-4 , H-5 , H-8 , H-
0
0
0
t, C-1 ), 32.01 (t, C-4 ), 61.20 (q, OMe), 61.30 (q, OMe), 117.78 9 ); 1.73 (br. s, 3H, Me); 1.67 (br. s, 3H, Me); 1.60 (br. s, 3H,
0
0
0
d, C-2 ), 123.98 (d, C-6 ), 130.45 (d, C-6), 131.95 (s, C-7 ), Me); 1.57 (br. s, 3H, Me).
0
0
0
0
0
0
0
40.17 (s, C-3 ), 144.91 (s, C-2 or C-3), 145.16 (s, C-3 or C-2),
46.92 (s, C-5), 184.38 (s, C-4 or C-1), 184.54 (s, C-1 or C-4).
2,3-Dimethoxy-5-(3 ,7 ,11 -trimethyl-dodeca-2 ,6 ,10 -tri-
enyl)-benzene-1,4-diol (11): Yellow oil, HREIMS m/z
0
0
0
0
+
-Methoxy-3-(3 ,7 -dimethyl-octa-2 ,6 -dienyl)-[1,4]ben- 374.2472 [M] , calcd. for C23
1
374.2457, H-NMR (250
2
H
34
O
4
+
zoquinone (3): Yellow oil, HREIMS m/z 274.1558 [M] , MHz, CDCl ) d: 6.49 (s, 1H, H-6); 5.31 (s, 1H, OH); 5.30
3
1
0
0
0
calcd. for C H O 274.1569, H-NMR (250 MHz, CDCl ) d: (m, 1H, H-2 ); 5.17 (s, 1H, OH); 5.12 (m, 2H, H-6 , H-10 );
17
22
3
3
6
.68 (d, J = 10.0, 1H, H-5); 6.59 (d, J = 10.0, 1H, H-6); 5.05 3.91 (s, 3H, OMe); 3.88 (s, 3H, OMe); 3.28 (br. d, J = 7.6, 2H,
0 0 0 0 0 0 0
(
2
m, 2H, H-2 , H-6 ); 4.02 (s, 3H, OMe); 3.15 (br. d, J = 7.3, H-1 ); 2.05 (m, 8H, H-4 , H-5 , H-8 , H-9 ); 1.70 (br. s, 3H,
0 0 0
H, H-1 ); 2.01 (m, 4H, H-4 , H-5 ); 1.73 (br. s, 3H, Me); 1.65 Me); 1.68 (br. s, 3H, Me); 1.60 (br. s, 6H, 2Me).
0
0
0
0
(
br. s, 3H, Me); 1.58 (br. s, 3H, Me).
0
2,3-Dimethoxy-5,6-bis-(3 ,7 -dimethyl-octa-2 ,6 -dienyl)-
-Methoxy-6-(3 ,7 -dimethyl-octa-2 ,6 -dienyl)-[1,4]ben- [1,4]benzoquinone (12): Yellow oil, HREIMS m/z 440.2944
0
0
0
2
zoquinone (4): Yellow oil, HREIMS m/z 274.1576 [M] , [M] , calcd. for C H O , 440.2927, 1H-NMR (250 MHz,
+
+
2
8
40
4
1
274.1569, H-NMR (250 MHz, CDCl
0 00 0 00
calcd. for C17
6
1
H
.45 (q, J = 2.1, 1H, H-5); 5.87 (d, J = 2.4, 1H, H-6); 5.15 (m, 3.99 (s, 6H, 2OMe); 3.19 (br. d, J = 6.8, 2H, H-1 , H-1 ); 2.00
22
O
3
3 3
) d: CDCl ) d: 5.04 (m, 2H, H-2 , H-2 ); 4.94 (m, 2H, H-6 , H-6 );
0
00
0
0
0
0
00
00
H, H-2 ); 5.08 (m, 1H, H-6 ); 3.82 (s, 3H, OMe); 3.14 (br. d, (m, 8H, H-4 , H-5 , H-4 , H-5 ); 1.73 (br. s, 6H, 2Me); 1.66
0
0
0
J = 7.3, 2H, H-1 ); 2.07 (m, 4H, H-4 , H-5 ); 1.70 (br. s, 3H, (br. s, 6H, 2Me); 1.58 (br. s, 6H, 2Me).
0
0
0
0
0
0
Me); 1.63 (br. s, 3H, Me); 1.60 (br. s, 3H, Me).
0
2-Methoxy-5,6-bis-(3 ,7 ,11 -trimethyl-dodeca-2 ,6 ,10 -
-Methoxy-5-(3 ,7 -dimethyl-octa-2 ,6 -dienyl)-[1,4]ben- trienyl)-[1,4]benzoquinone (13): Yellow oil, HREIMS m/z
0
0
0
2
zoquinone (5): Yellow crystals, HREIMS m/z 274.1582 [M] , 546.4048 [M] , calcd. for C H O , 546.4073, H-NMR (250
+
+
1
3
7
54
3
1
274.1569, H-NMR (250 MHz, CDCl
0 0
calcd. for C17
H
.46 (t, J = 1.7, 1H, H-6), 5.92 (s, 1H, H-3), 5.15 (m, 1H, H-2 ), H-10 , H-2 , H-6 , H-10 ); 3.79 (s, 3H, OMe); 3.22 (br. d, J = 6.8,
22
O
3
3 3
) d: MHz, CDCl ) d: 5.87 (s, 1H, H-3); 5.00 (m, 6H, H-2 , H-6 ,
0
0
00
00
00
6
5
0
0
00
0
0
0
0
00
00
.08 (m, 1H, H-6 ), 3.82 (s, 3H, OMe), 3.14 (br. d, J = 7.3, 2H, 4H, H-1 , H-1 ); 2.01 (m, 16H, H-4 , H-5 , H-8 , H-9 , H-4 , H-5 ,
0
0
0
00
00
H-1 ), 2.08 (m, 4H, H-4 , H-5 ), 1.70 (br. s, 3H, Me), 1.62 (br. H-8 , H-9 ); 1.73 (m, 3H, Me); 1.67 (m, 9H, 3Me); 1.60 (m,
s, 3H, Me), 1.60 (br. s, 3H, Me). 12H, 4Me).
2-Methoxy-3,5-bis-(3 ,7 ,11 -trimethyl-dodeca-2 ,6 ,10 -
0 0 0 0
-(3 ,7 -Dimethyl-octa-2 ,6 -dienyl)-[1,4]benzoquinone
0
0
0
0
0
0
2
+
(
C
6): Yellow oil, HREIMS m/z 244.1454 [M] , calcd. for trienyl)-[1,4]benzoquinone (14): Yellow oil, HREIMS m/z
+
1
244.1463, H-NMR (250 MHz, CDCl
1
, 546.4073, H-NMR (250
16
H
20
O
2
3
) d: 6.77 (d, 546.4052 [M] , calcd. for C37
H
54
O
3
0
J = 10.2, 1H, H-6); 6.69 (dd, J = 10.2, 2.3, 1H, H-5); 6.53 (q, J = MHz, CDCl ) d: 6.33 (t, J = 1.2, 1H, H-6); 5.09 (m, 6H, H-2 ,
1.9, 1H, H-3); 5.15 (m, 1H, H-2 ); 5.07 (m, 1H, H-6 ); 3.13 (br. H-6 , H-10 , H-2 , H-6 , H-10 ); 4.00 (s, 3H, OMe); 3.14 (m,
d, J = 7.5, 2H, H-1 ); 2.08 (m, 4H, H-4 , H-5 ); 1.69 (br. s, 3H, 4H, H-1 , H-1 ); 2.04 (m, 16H, H-4 , H-5 , H-8 , H-9 , H-4 ,
3
0 0 0 0 00 00 00
0
0
0
0
00
0
0
0
0
00
0
0
00
00
Me); 1.62 (br. s, 3H, Me); 1.60 (br. s, 3H, Me).
H-5 , H-8 , H-9 ); 1.74 (m, 3H, Me); 1.68 (m, 9H, 3Me); 1.60
,3-Dimethoxy-5-(3 ,7 ,11 -trimethyl-dodeca-2 ,6, 10 -tri- (m, 12H, 4Me).
enyl)-[1,4]benzoquinone (7): Yellow oil, HREIMS m/z 2,3,4-Trimethoxy-6-(3 ,7 -dimethyl-octa-2 ,6 -dienyl)-
0
0
0
0
0
0
2
0
0
0
0
+
1
+
3
72.2316 [M] , calcd. for C23
H
32
O
4
372.2300, H-NMR (250 phenol (20): Pale yellow oil, HREIMS m/z 320.1974 [M] ,
0
j1 1
MHz, CDCl ) d: 6.34 (t, J = 1.8, 1H, H-6); 5.10 (m, 3H, H-2 , calcd. for C H O 320.1987, IR (CCl ): 3541, 2935, 1498,
3
19 28
4
4
0
0
H-6 , H-10 ); 4.02 (s, 3H, OMe); 4.00 (s, 3H, OMe); 3.11 (br. 1464 cm . H-NMR (CDCl
0
3
, 250 MHz) d: 6.44 (s, 1H, H-5),
0
0
0
0
0
0
d, J = 7.3, 2H, H-1 ); 2.05 (m, 8H, H-4 , H-5 , H-8 , H-9 ); 1.68 5.45 (s, 1H, OH), 5.31 (m, 1H, H-2 ), 5.11 (m, 1H, H-6 ), 3.95
br. s, 3H, Me); 1.62 (br. s, 3H, Me); 1.60 (br. s, 6H, 2Me). (s, 3H, OMe), 3.86 (s, 6H, OMe), 3.79 (s, 3H, OMe), 3.31 (br.
(
0 0 0 0 0 0 0 0 0
2
-Methoxy-6-(3 ,7 ,11 -trimethyl-dodeca-2 ,6 ,10 -tri- d, J = 7.1, 2H, H-1 ), 2.07 (m, 4H, H-4 , H-5 ), 1.72 (d, J = 1.2,
enyl)-[1,4]benzoquinone (8): Yellow oil, HREIMS m/z 3H, Me), 1.67 (d, J = 1.2, 3H, Me), 1.60 (d, J = 0.7, 3H, Me).
+
1
42.2182 [M] , calcd. for C H O 342.2195, H-NMR (250
13
0
C-NMR (CDCl , 62.9 MHz) d: 16.12 (q, C-10 ), 17.66 (q, C-
3
3
2
2
30
3
0
0
0
0
0
MHz, CDCl ) d: 6.45 (q, J = 2.0, 1H, H-5); 5.87 (d, J = 2.4, 9 ), 25.66 (q, C-8 ), 26.73 (t, C-5 ), 27.90 (t, C-1 ), 39.75 (t, C-4 ),
3
0
0
0
1
H, H-3); 5.15 (m, 1H, H-2 ); 5.10 (m, 2H, H-6 , H-10 ); 3.81 56.62 (q, OMe), 60.89 (q, OMe), 61.16 (q, OMe), 108.30 (d, C-
0
0
0
0
(
4
1
s, 3H, OMe); 3.14 (br. d, J = 7.3, 2H, H-1 ); 2.06 (m, 8H, H- 5), 121.61 (s, C-6), 121.98 (d, C-2 or C-6 ), 124.20 (d C-6 or
0
0
0
0
0
0
, H-5 , H-8 , H-9 ); 1.67 (br. s, 3H, Me); 1.63 (br. s, 3H, Me); C-2 ), 128.89 (s, C-1), 131.41 (s, C-7 ), 136.59 (s, C-4), 140.04
0
.60 (br. s, 6H, 2Me).
(s, C-3 ), 140.81 (s, C-3 or C-2), 146.14 (s, C-2 or C-3).
0 0 0 0 0 0
0
0
0
0
0
0
2
enyl)-[1,4]benzoquinone (9): Yellow crystals, HREIMS m/z trienyl)-phenol (21): Pale yellow oil, HREIMS m/z 388.2648
-Methoxy-5-(3 ,7 ,11 -trimethyl-dodeca-2 ,6 ,10 -tri-
2,3,4-Trimethoxy-6-(3 ,7 ,11 -trimethyl-dodeca-2 ,6 ,10 -
+
1
+
1
3
42.2172 [M] , calcd. for C22
MHz, CDCl ) d: 6.47 (t, J = 1.7, 1H, H-6); 5.93 (s, 1H, H-3); CDCl
.16 (m, 1H, H-2 ); 5.10 (m, 2H, H-6 , H-10 ); 3.82 (s, 3H, 7.3, 1H, H-2 ); 5.12 (m, 2H, H-6 , H-10 ); 3.95 (s, 3H, OMe);
H
30
O
3
342.2195, H-NMR (250 [M] , calcd. for C24
36 4
H O 388.2635, H-NMR (250 MHz,
3
3
) d: 6.44 (s, 1H, H-5); 5.45 (s, 1H, OH); 5.32 (br. t, J =
0
0
0
0
0
0
5
0
0
0
OMe); 3.14(br. d, J = 7.3, 2H, H-1 ); 2.05 (m, 8H, H-4 , H-5 , 3.87 (s, 3H, OMe); 3.79 (s, 3H, OMe); 3.31(br. d, J = 7.3, 2H,
0
0
0
0
0
0
0
H-8 , H-9 ); 1.68 (br. s, 3H, Me); 1.62 (br. s, 3H, Me); 1.60 (br. H-1 ); 2.06 (m, 8H, H-4 , H-5 , H-8 , H-9 ); 1.72 (br. s, 3H,
s, 6H, 2Me). Me); 1.67 (br. s, 3H, Me); 1.60 (br. s, 6H, 2Me).

Cancer-Preventive Marine Quinones
73
0
0
0
0
+
,4-Dimethoxy-6-(3 ,7 -dimethyl-octa-2 ,6 -dienyl)-phe- activated with EGF) or 2 weeks (JB6 P Cl41 cells, activated
3
nol (23): Pale yellow oil, HREIMS m/z 290.1876 [M] , calcd. with TPA). Cell colonies were then scored using the LEICA
+
1
for C H O 290.1882, H-NMR (250 MHz, CDCl ) d: 6.73 DM IRB inverted research microscope (Leica Mikroskopie und
18
26
3
3
0
(
s, 1H, H-2); 6.49 (s, 1H, H-5); 5.27 (br t, J = 7.3, 1H, H-2 ); Systeme GmbH) and Image-Pro Plus software, version 3.0 for
0
5
.15 (s, 1H, OH); 5.10 (m, 1H, H-6 ); 3.87 (s, 3H, OMe); 3.79 Windows (Media Cybernetics). For each compound, two
0
(
4
s, 3H, OMe); 3.24 (br. d, J = 7.3, 2H, H-1 ); 2.05 (m, 4H, H- independent experiments in triplicate for each concentration
0
0
, H-5 ); 1.68 (br. s, 6H, 2Me); 1.60 (br. s, 3H, Me).
0
were performed. (see Table S2, available online at www.
,4-Dimethoxy-6-(3 ,7 -dimethyl-octa-2 ,6 -dienyl)-phenol springerlink.com; search for DOI: 10.1007/s11095-005-8813-4;
0
0
0
2
28): Pale yellow oil, HREIMS m/z 290.1898 [M] , calcd. for Electronic Supplementary Materials can be found at the end
+
(
1
18 26 3 3
C H O 290.1882, H-NMR (250 MHz, CDCl ) d: 6.36 (d, of the article).
J = 2.7, 1H, H-3 or H-5); 6.31 (d, J = 2.7, 1H, H-3 or H-5); 5.33
0
(
br. t, J = 7.1, 1H, H-2 ); 5.26 (s, 1H, OH); 5.11 (br. t, J = 6.6,
0
1
J = 7.3, 2H, H-1 ); 2.08 (m, 4H, H-4 , H-5 ); 1.72 (br. s, 3H,
Me); 1.67 (br. s, 3H, Me); 1.60 (br. s, 3H, Me).
H, H-6 ); 3.86 (s, 3H, OMe); 3.75 (s, 3H, OMe); 3.35 (br. d, Apoptosis Assay Using Flow Cytometry
0
0
0
+
5
JB6 P Cl41 cells (3 ꢀ 10 cells/dish) were grown in 6-cm
dishes for 24 h in 5% FBS-MEM. Then cells were treated
with different concentrations of the compounds dissolved in
0.1% FBS medium for 3 h. Then the medium was removed,
and attached cells were harvested with 0.025% trypsin in
Cell Viability Assay
+
JB6 P Cl41 cells were cultured overnight in 96-well 0.1% ethylenediaminetetraacetic acid (EDTA) in phosphate-
plates (6000 cells/well) using 5% FBS-MEM. Then the buffered saline (PBS). Trypsinization was stopped by adding
medium was replaced with 0.1% FBS-MEM containing the 2 ml of 5% FBS in PBS. Cells were then washed by
quinones at different concentrations in a volume of 0.1 ml, centrifugation at 1000 rpm (170 rcf) for 5 min and
and the cells were incubated with the quinone solutions for processed for detection of apoptosis using Annexin V-FITC
2
2 h. Then 20 ml of the MTS reagent was added into each and propidium iodide staining according to the manu-
5
well. The MTS reduction was measured 2 h later spectro- facturer’s protocol. In brief, 1Y5 ꢀ 10 cells were collected
photometrically at 492 and at 690 nm as a background using after centrifugation and resuspended in 500 ml of 1ꢀ binding
the Multiskan MS microplate reader (Labsystems). For each buffer (Annexin V-FITC Apoptosis Detection Kit, Medical
compound, two independent experiments with five samples & Biological Laboratories). Then, 5 ml of Annexin V-FITC
for each concentration were performed. (see Table S1, and 5 ml of propidium iodide were added, and the cells were
available online at www.springerlink.com; search for DOI: incubated at room temperature for 5 min in the dark and
1
rials can be found at the end of the article).
0.1007/s11095-005-8813-4; Electronic Supplementary Mate- were analyzed by flow cytometry. For each compound, two
independent experiments in duplicate for each concentration
were performed. (see Table S3, available online at www.
springerlink.com; search for DOI: 10.1007/s11095-005-8813-4;
Electronic Supplementary Materials can be found at the end
of the article).
Anchorage-Independent Assay
The cancer-preventive effects of the quinones were evalu-
+
ated in 6-well plates using JB6 P Cl41 cells, activated with EGF
(
10 ng/ml) or 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 20 Apoptosis Assay Using DNA Ladder Method
3
+
ng/ml). JB6 P Cl41 cells (8 ꢀ 10 /ml) were treated with the
indicated concentrations of the quinones in 1 ml of 0.33% basal
JB6 Cl 41 cells were grown in 10-cm dishes and treated
medium Eagle (BME) agar containing 10% FBS over 3.5 ml of with 3-demethylubiquinone Q
.5% BME agar containing 10% FBS and indicated confluent. The cells were incubated with quinone 1 for 24 h.
concentrations of the quinones. The cultures were maintained Then, both detached and attached cells were harvested
2
(1) when cells were 80%
0
+
in a 37-C, 5% CO
2
incubator for 1 week (JB6 P Cl41 cells, by scraping followed by centrifugation. The obtained cells
Scheme 1. The general scheme of synthesis of quinones 1Y14.

7
4
Fedorov et al.
were disrupted with lysis buffer (5 mM TrisYHCl, pH 8.0, MEM were added into each well of a 96-well plate. Plates
2
4
0 mM EDTA, and 0.5% Triton X-100) and left on ice for were incubated for 24 h and then treated with various con-
5 min. After centrifugation at 14,000 rpm (45 min, 4-C), the centrations of quinones in 100 ml of 0.1% FBS-MEM. After
supernatant fraction containing fragmented DNA was incubation with quinones for 24 h, the cells were extracted
extracted twice with phenol/chloroform/isopropyl alcohol for 1 h at room temperature with 100 ml/well of lysis buffer
(
25:24:1, v/v/v) and once with chloroform. Then the frag- [0.1 M potassium phosphate buffer at pH 7.8, 1% Triton
mented DNA was precipitated overnight at j20-C after X-100, 1 mM dithiothreitol (DTT), 2 mM EDTA]. Then 30 ml of
addition of two volumes of 100% ethanol and 1/10 volume of lysate from each well was transferred in a plate used for
5
1
M NaCl. The DNA pellet was saved by centrifugation at luminescent analysis, and luciferase activity was measured
4,000 rpm for 45 min, washed once with 70% ethanol, dried, using 100 ml/well of the luciferase assay buffer [1 mM D-
and resuspended in TrisYEDTA buffer (10 mM TrisYHCl, luciferase, pH 6.1Y6.5; 40 mM Tricin, 2.14 mM magnesium
mM EDTA, pH 8.0). After addition of 100 mg/ml RNAse A carbonate (MgCO ) IMg(OH) I5H O, 5.34 mM MgSO I7H O,
1
3
4
2
2
4
2
(
Sigma), the mixture was incubated at 37-C for 2 h. The DNA O, 66.6 mM DTT, 1.06 mM adenosine triphosphate, 0.54 mM
fragments were separated by 1.8% agarose gel electrophore- coenzyme A, 0.2 mM EDTA, pH 7.8] and the Luminoscan
sis. DNA laddering in the gel was stained with ethidium Ascent Type 392 microplate reader (Labsystems). For each
bromide and photographed under UV light. Two indepen- compound, two independent experiments with five samples for
dent experiments were performed.
each concentration were performed. (see Table S4, available
online at www.springerlink.com; search for DOI: 10.1007/
s11095-005-8813-4; Electronic Supplementary Materials can be
found at the end of the article).
The Effect of Quinones 1 and 3–14 on p53-, AP-1-,
and NF-kB-Dependent Transcriptional Activities
The ability of quinones 1 and 3Y14 to influence AP-1-, Statistics
NF-kB-, and p53-dependent transcriptional activities in the
mouse JB6 Cl41 cell line was evaluated using the luciferase
method. Viable JB6-LucPG-13, JB6-LucAP-1, or JB6- Windows (StatSoft, Inc., Tulsa, OK, USA, 2001) was used
The statistical computer program Statistica 6.0 for
3
LucNF-kB cells (6 ꢀ 10 ) suspended in 100 ml 5% FBS- for analysis of the obtained data. Nonparametric MannY
Scheme 2. Syntheses of quinones 1, 2, 7, 11, 12 (A); 3, 5, 9, 10, 14 (B); and 4, 8, 13 (C).

Cancer-Preventive Marine Quinones
75
Whitney U test was used to compare two independent groups an acidic catalyst as was described earlier (27). Under these
of data. Nonparametric Spearman rank-order correlations conditions, total yields of the alkylation products were
method was used to compute single nonparametric correla- about 60%. The second stage, the oxidative demethylation of
tions. Method of regressions was used to compute IC50 or the fractions obtained with CAN, yielded mixtures of the
inhibition of the number of the colonies C50 (INCC50) in corresponding quinones, which were further separated to
corresponding experiments.
obtain individual compounds. The third stage, reduction of
the obtained quinones with Na , was used only in one case
2 2 4
S O
and yielded the corresponding prenylated hydroquinone (11).
In particular, prenylquinones 1, 2, 7, 11, and 12 were
synthesized from 2,3,4-trimethoxyphenol (15, Scheme 2A),
prenylquinones 3, 5, 9, 10, and 14 from 3,4-dimethoxyphenol
RESULTS
Syntheses of Polyprenylquinones 1 Through 14
(
16, Scheme 2B), and prenylquinones 4, 8, and 13 from 2,4-
Syntheses of polyprenylquinones 1Y14 were carried out dimethoxyphenol (17, Scheme 2C). The previously known
0
0
0
0
in accordance with Scheme 1. The first stage yielded 2-(3 ,7 -dimethyl-octa-2 ,6 -dienyl)-1,4-benzoquinone (6) (not
prenylated phenols through the alkylation of corresponding shown on scheme) was synthesized as earlier described (27).
0 0
available methoxyphenols by trans-geraniol (18) or trans-
farnesol (19) in the presence of boric trifluoride etherate as less than 6% of the corresponding 2 -3 -cis-isomers as
Target 2 -3 -trans-prenylquinones 1 and 3Y14 contained
0
0
Scheme 2. Continued.

7
6
Fedorov et al.
intermediate prenylphenols 20, 21, 23, and 28 were estab-
lished using NMR spectroscopy in comparison with spectra
of 1 and 2 (26).
3
3
-Demethylubiquinone Q and Synthetic Polyprenylquinones
2
+
–14 Inhibit Malignant JB6 P Cl41 Cell Transformation
Both natural and synthetic compounds 1 and 3Y14 were
assayed for cancer-preventive activity using the anchorage-
+
independent JB6 P Cl41 cell transformation assay in a soft
agar. Based on our previous experience (30Y39), inhibition of
cell transformation is a good indication that a compound will
have an effective cancer-preventive activity. Toxicity of each
compound for JB6 Cl41 cells was determined by the MTS
cell viability assay. For one of the quinones, natural 3-
demethylubiquinone Q A (1), the corresponding data are
2
shown in Figs. 1 and 2. Using the obtained data and statistical
computer program Statistica 6.0, the corresponding regres-
Fig. 1. The inhibition of epidermal growth factor (EGF)-induced
+
2
JB6 P Cl41 cell transformation by demethylubiquinone Q (1) in
+
3
soft agar (anchorage-independent assay). JB6 P Cl41 cells (8 ꢀ 10 /
ml in 6-well plates) were activated with EGF (10 ng/ml), treated with
the indicated concentrations of quinone (1), maintained for 1 week,
and cell colonies were then scored as described in BMaterials and
Methods^. Data represent the percentage of EGF-activated, quinone
(1)-treated cell colonies compared to percentage of EGF-activated,
untreated cells. Each bar represents the mean T SD from six samples sions were built, and the IC50 for decreased cell viability and
of two independent experiments. * indicates a significant inhibition the INCC50 for inhibition of cell transformation were
by quinone (1) (p < 0.05) compared to EGF-activated untreated determined for each quinone studied. These data are
control.
summarized in Table I. The obtained results indicated that
all quinones studied inhibited cell transformation induced by
impurities. The formation of these isomers may be explained EGF or TPA in dose-dependent manner in JB6 Cl41 cells.
by the isomerization of geraniol and farnesol or corre- For some of the quinones, the dose that inhibited malignant
sponding products of alkylation at the action of boric transformation by 50% was below that which was toxic
trifluoride etherate at the stage of prenylation of phenols. (Table I). For example, for 3-demethylubiquinone Q , the
2
As a rule, impurities of cis-isomers were not separated from corresponding doses were 7.3 and 11.4 mM. To understand
the major products (trans-isomers) except for the mixture of the possible inhibitory signaling pathway activated by the
1
and 2, which was separated by HPLC. To the best of our quinones studied, we then investigated whether the JB6 cells
knowledge, among all the above-mentioned polyprenylqui- were undergoing apoptosis induced by quinones.
nones, only compounds 1, 6, 7, and 8 were described
previously (22,27Y29). Intermediate prenylated phenols 20,
2
1, 23, and 28 were obtained as individual substances. The The Quinones Induce Apoptosis in JB6 Cl41 Cells
structures of the target prenylquinones 3Y14 as well as the
The ability of the quinone compounds to induce
apoptosis was determined by flow cytometry. The results
indicated that quinones 1 and 3Y14 induced apoptosis in JB6
Table I. IC50 and INCC50 of the Quinones 1 and 3Y14 for JB6 Cl41
+
P
Cells
Compounds
IC50 (mM)
INCC50 (mM)
Structural group 1
Quinone 1
Quinone 3
11.4
45.1
8.3
7.3
15.1
6.6
Quinone 4
Quinone 5
Quinone 6
11.8
23.4
3.1
14.7
Structural group 2
Quinone 7
Quinone 8
+
(1) on JB6 P Cl41 cell
Fig. 2. The effect of demethylubiquinone Q
viability. The cells were cultured in 96-well plates, as described
2
5.1
4.7
19.4
16.7
7.4
in BMaterials and Methods^. Then, the medium was replaced with
Quinone 9
8.6
0
.1% FBS-MEM containing the indicated concentrations of quinone
Quinone 10
Quinone 11
Structural group 3
Quinone 12
Quinone 13
Quinone 14
25.5
4.6
24.6
51.7
(1). The cells were incubated with the quinone (1) for 22 h. The
methanethiosulfonate reagent was then added, and its reduction was
measured spectrophotometrically 2 h later. Data represent the
percentage of quinone (1)-treated viable cells compared to
percentage of untreated control cells. Each data point represents
the mean T SD from ten samples of two independent experiments.
a
>140
29.2
54.7
95.3
a
>15
99.6
*
(
indicates a significant decrease in viability induced by quinone (1)
p < 0.05) compared to untreated control cells.
INCC50: inhibition of the number of the colonies C50.
a
In all calculations the designated numbers were used.

Cancer-Preventive Marine Quinones
77
Group 1 consists of quinones 1 and 3Y6, which have two
isoprene units (10 carbon atoms) in their side chains. Group 2
(
quinones 7Y11) contains three isoprene units (15 carbon
atoms) in their terpenoid parts, and group 3 (quinones 12Y14)
have four to six isoprene units (20Y30 carbon atoms) in their
side chains. Significant differences and correlations between
the data regarding the biological activities obtained for
different structural groups of the quinones were determined
using the nonparametric Spearman correlation method and
the MannYWhitney U test and the data in Table I.
Our results indicated that the biological activity of these
quinones depends on the length of the terpenoid side chains
in the molecule. Statistical analysis of data from Table I
indicates that quinones in group 1 averaged an IC50 of 20.0 T
2
Fig. 3. The induction of apoptosis by demethylubiquinone Q (1) in
+
5
JB6 P Cl41 cells measured by flow cytometry. The cells (3 ꢀ 10 /dish)
were grown in 6-cm dishes and treated with the indicated con-
centrations of quinone (1) as described in BMaterials and Methods^.
Cells were harvested and processed for detection of apoptosis using
Annexin V-FITC and propidium iodide staining according to the
manufacturer’s protocol. Each bar represents the mean T SD from four
samples of two independent experiments. * indicates a significant
1
5.2 mM for toxicity in JB6 Cl41 cells. Quinones in group 2
had an average IC50 of 9.7 T 9.0 mM. Finally, the quinones in
group 3 showed an average IC50 of 84.9 T 63.8 mM. These
results suggest that quinones 7Y11 (group 2), with three
isoprene units in their terpenoid portion, are on the whole
increase in apoptosis by quinone (1) (p < 0.05) com-pared to untreated more toxic against JB6 cells than the quinones in group 1
control cells.
(quinones 1 and 3Y6), which have only two isoprene units in
their side chains. The opposite conclusion can be drawn when
comparing the IC50 of quinones in groups 1 and 3 or for those
in groups 2 and 3. The quinones of groups 1 or 2 are
significantly more toxic for JB6 Cl41 cells than those of group
Cl41 cells in a dose-dependent manner (Fig. 3). For the
natural 3-demethylubiquinone Q A (1), apoptosis was also
2
demonstrated by DNA laddering in JB6 Cl41 cells (Fig. 4).
3
. Therefore, we conclude that when the length of the
terpenoid portion increases as from group 1 to group 2, the
toxicity of the quinones of this series also increases; but it
decreases dramatically by further increasing the length of the
terpenoid portion as from group 2 to group 3.
In the statistical analysis of the effect of these quinones
on EGF-induced cell transformation in JB6 Cl41 cells, we
found that the quinones in group 1 displayed an average
Polyprenylquinones 1 and 3–14 Inhibit p53 and Induce
AP-1 or NF-kB Transcriptional Activity
Several key transcription factors, including the p53
tumor suppressor protein, AP-1, or NF-kB, are often
implicated in the induction or inhibition of apoptosis by
various stimuli, including chemopreventive compounds or
drugs. Therefore, we then studied the effect of quinones 1
and 3Y14 on these three transcription factors. JB6 Cl41 cell
lines stably expressing a luciferase reporter gene controlled
by an AP-1, NF-kB, or p53 DNA binding sequence were
used. To study the effect of the substances on the nuclear
factors-dependent transcriptional activity, we used broad
range of the concentrations. In Table II, we showed only
maximal significances of the corresponding induction or
inhibition. These significances were achieved at the concen-
trations of the substances that correlated well with their IC50
for the corresponding cells. Quinones 1 and 3Y14 showed a
significant (up to an 8-fold) induction of AP-1- or NF-kB-
dependent transcriptional activation and a substantial (up to
4
(
-fold) inhibition of p53-dependent transcriptional activity
see Table II).
Structure–Activity Relationships for Polyprenylquinones
and 3–14
1
2
Fig. 4. The induction of apoptosis by demethylubiquinone Q (1) in
JB6 Cl41 cells determined by the method of DNA laddering. JB6
Cl41 cells were grown in 10-cm dishes, treated with the indicated
concentrations of quinone (1) for 24 h, and harvested as described in
BMaterials and Methods.^ The isolated DNA fragments were
separated by 1.8% agarose gel electrophoresis. DNA laddering in
One of the major goals of the present investigation was
to establish the SARs for the quinones studied. The SARs of
quinones 1 and 3Y14 were studied using statistical analysis
(
Statistica 6.0). The quinone compounds 1 and 3Y14 were
divided into three groups according to the number of iso- the gel was stained with ethidium bromide and photographed under
prene units included in their terpenoid parts (see Table I). ultraviolet light. A representative experiment is shown.

7
8
Fedorov et al.
Table II. The Effect of the Quinones 1 and 3Y14 on AP-1-, NF-kB-, and p53-Dependent Transcriptional Activity in JB6 Cl41 Cells
AP-1-dependent transcriptional
activity (% of untreated
control)
NF-kB-dependent transcriptional
activity (% of untreated
control)
p53-dependent transcriptional
activity (% of untreated
control)
Compounds
Structural group 1
Quinone 1
Quinone 3
190.3
159.8
293.3
721.7
880.7
216.9
194.8
197.9
201.2
421.7
71.7
55.8
36.3
31.8
47.0
Quinone 4
Quinone 5
Quinone 6
Structural group 2
Quinone 7
Quinone 8
125.0
97.2
99.2
138.8
223.0
179.3
403.5
24.6
57.9
26.9
31.5
22.8
Quinone 9
252.2
107.7
84.0
Quinone 10
Quinone 11
Structural group 3
Quinone 12
Quinone 13
Quinone 14
a
a
139.5
a
225.7
a
198.5
549.7
36.5
a
Significant differences were not determined.
INCC50 of 9.4 T 5.3 mM, and the quinones in group 2 INCC50 had the following values: for quinones 3 and 10,
averaged an INCC50 of 24.0 T 16.7 mM. Group 3 again 15.1 and 24.6 mM, respectively; for quinones 4 and 8, 6.6
possessed minimal activity among all three groups of com- and 16.7 mM, respectively; and for quinones 5 and 9, 3.1
pounds with an average INCC50 of 59.7 T 33.3 mM (Table I). and 7.4 mM, respectively.
Therefore, based on this analysis and the results of the
Induction of AP-1 transcriptional activity by the ortho
MannYWhitney U test, group 1 quinones had the most potent compounds 3 and 10 averaged 133.8% of control, and for the
effect on inhibition of cell transformation (p = 0.0283 vs. induction by the meta derivatives 4 and 8, the average was
group 2; p = 0.0253 vs. group 3; p = 0.0084 vs. groups 2 and 3), 187.7% of control. The para derivatives 5 and 9 had the
and group 3 was the least effective. A significant correlation highest induction of AP-1 activation at 486.9% of control.
was observed between the length of the terpenoid portion The ortho-disubstituted quinones 3 and 10 are the least active
and INCC50 (p = 0.0002, R = 0.8556). These results indicate compounds not only in the induction of AP-1 transcriptional
that when the length of the terpenoid portion increases, the activity but also in the inhibition of cell transformation
INCC50 values for cell transformation also increase.
We then compared toxicity with cell transformation in disubstituted derivatives, quinone 5 having two isoprene units
compared with the meta- and para-analogs. Among the para-
+
JB6 P Cl41 cells and found that the quinones in group 1 in the side chain showed better activities compared with
displayed an average INCC50 (inhibition of cell transforma- quinone 9 having three isoprene units in the side chain.
tion) 1.4Y4 times less than the IC50 (toxicity) for the Indeed, the para-disubstituted quinones 5 and 9 showed an
corresponding cells (Table I). On the other hand, the INCC50 of 3.1 and 7.4 mM, respectively, against EGF-induced
+
majority of quinones in group 2 showed an INCC50 (inhibi- JB6 P Cl41 cell transformation. Quinone 5 also demonstrated
tion of cell transformation) at doses 4Y10 times higher than a higher induction of AP-1 transcriptional activity (721.7%)
the IC50 values (toxicity; Table I). Based on these data, we compared with quinone 9 (252.2%).
can conclude that quinones of group 1 are distinctly more
toxic to transformed JB6 cells than to normal JB6 cells. In
contrast, the quinones in group 2 are more toxic to normal DISCUSSION
JB6 cells than to those that have been transformed.
Therefore, the quinones in group 1 have more potential with
respect to cancer-preventive activity than the quinones from nones and hydroquinones are very common in nature, to the
group 2. best of our knowledge, only one related marine metabolite,
In spite of the fact that polyprenylated 1,4-benzoqui-
We further showed that the activity of these quinones 2-(3-methylbuten-2-yl)-[1,4] hydroquinone (20), was earlier
depends on the position of the methoxy group relative to the studied for its cancer-protective properties. To establish the
terpenoid part. We selected several pairs of structurally cancer-preventive activity of this metabolite, the modified
similar quinones, which have the methoxy groups in the Ames assay for mutagenicity of benzo(a)-pyrene, aflatoxin
same position. These pairs are as follows: (1) ortho-analogs, B1, or UV against Salmonella typhimurium was used (40).
quinones 3 and 10; (2) meta-analogs, quinones 4 and 8; and However, biological tests examining transformation of ani-
(
3) para-analogs, quinones 5 and 9. Based on the data from mal cells after treatment with tumor promoters were not used
Tables I and II, we conclude that the cancer-preventive in this study. For the first time in the present work, the
activity and the effect of quinones on AP-1 transcriptional cancer-preventive properties for a large group of the newly
activity increased in the line of ortho Y meta Y para. The synthesized and earlier known polyprenylated benzoqui-

Cancer-Preventive Marine Quinones
79
+
nones were studied using mouse epithelial JB6 P Cl41 cells these drugs (55,56). NF-kB, a member of a family of highly
and mouse embryonic fibroblasts (MEFs). regulated dimeric transcription factors, is involved in the
In our study the quinones having two isoprene units in activation of a large number of genes that respond to
the side chain showed specific effects against the malignantly infections, inflammation, and other stressful situations. NF-
transformed JB6 Cl41 cells compared with normal cells. The kB is reported to be involved in both induction and in-
active doses differed up to 4-fold.
hibition of apoptosis (52,53,57Y59). Our study therefore
We established structureYactivity relationships for the suggests that apoptosis induced by quinones 1 and 3Y14
quinones studied with respect to cytotoxic or cancer-preventive occurs independently of p53 activation, but instead may be
properties. Our present study indicated that cytotoxicity of related to the induction of AP-1 and NF-kB transcriptional
quinones increased with the number of carbon atoms from activity.
quinones having two prenyl units in their side chain to their
analogs having three prenyl units and then decreased for
compounds with four to six isoprene units. The observed SARs
do not fully correspond to that earlier established for related
nonmethoxylated quinones. A series of nonmethoxylated pre-
CONCLUSION
Our results show that methoxylated polyprenylquinones
and their synthetic analogs represent a new prospective
nylated quinones with side chains containing from one to eight
group of marine secondary metabolites as cancer-preventive
prenyl units was synthesized by an Italian research group and
compounds. They show also cytotoxic properties and induce
studied along with related marine natural compounds (27).
+
apoptosis of JB6 P Cl41 cells and MEFs. The most active of
Resembling our study, the toxicity of these compounds in brine
these compounds are potent inducers of AP-1 and NF-kB
activation and, at the same time, inhibitors of p53 tran-
shrimp and fish lethality assays was shown to first increase and
then again to decrease with the length of the terpenoid part.
scriptional activities.
In our study, therefore, the cancer-preventive effects of
But distinct from our study, quinones containing two isoprene
units in the terpenoid portion were reported to be the most
quinones 1 and 3Y14 (Table I) may be explained by the
induction of p53-independent apoptosis.
We also found that quinones having a side chain of 10-
toxic among all studied compounds (27). No one has previously
established the SARs for any series of prenylquinone com-
pounds with respect to cancer-preventive activity. Our study
carbon atom length showed specificity in the inhibitory effect
showed that cancer-preventive activity decreased when the
+
for transformed JB6 P Cl41 cells in contrast to quinones
polyprenyl side chain became longer. The most active cancer-
preventive polyprenylquinones, among those studied herein,
have a side chain containing two isoprene units.
with 15 or 20Y30 carbon atoms in the side chain. We conclude
therefore that a further search for cancer-preventive agents
may be promising among diprenylated analogs of the com-
No previous evidence exists in the literature that shows
pounds studied. Taking into consideration that these com-
that any polyprenyl quinone induces apoptosis in any cell
pounds were active against transformation of the epithelial
line. Using flow cytometry and the DNA laddering method,
JB6 cells, we suggest that some quinones studied may be used
as themselves or as models for the development of promising
we showed that quinones 1 and 3Y14 induced apoptosis in
JB6 Cl41 cells and MEFs. The tumor suppressor protein p53,
skin cancer-preventive agents.
Taking into account the data from Table I, and SAR
which is a part of the cell’s emergency team and functions to
negatively regulate cell growth following DNA damage, is
based on the position of the methoxy-groups, we also con-
often involved in apoptosis induced by various stimuli
clude that quinone 5, which has a diprenylated side chain in
including chemopreventive agents and drugs (41Y45). How-
the para-position relative to the methoxy-group, is the most
ever, in our study, quinones 1 and 3Y14 did not activate p53,
potent among all the quinones studied with respect to cancer-
preventive effect.
but instead, most of the quinones studied demonstrated
significant inhibition of p53-dependent transcriptional activ-
ity. In addition, these compounds induced a substantial
activation of AP-1- or NF-kB-dependent transcriptional
activities (Table II). The AP-1 transcription factor regulates
ACKNOWLEDGMENTS
a variety of cellular processes, including proliferation,
This work was supported in part by The Hormel
differentiation, and apoptosis, and has been considered
Foundation and National Institutes of Health grants
primarily to be an oncogene (33,36Y38,46,47). Recently, some
CA81064, CA77646, and CA88961. The Russian co-authors
of the AP-1 proteins, such as Jun-B and c-Fos, were shown to
are grateful for financial support by the RFFI Grant no. 05-
have tumor-suppressor activity both in vitro and in vivo
0
7
4-48246, Russian Federation President Grant no.
25.2003.4, Program of Presidium of RAS BMolecular and
(
48,49). Activation of another AP-1 protein, c-Jun, is re-
quired for the induction of Fas L-mediated apoptosis in
PC12 and human leukemia HL-60 cells (50,51). Activation
of both AP-1 and NF-kB nuclear factors is necessary for
DNA damaging agents- and ceramide-induced apoptosis in
T lymphocytes and Jurkat T cells (52,53). The balance
between AP-1 family members, c-Jun, and ATF-2 governs
the choice between differentiation and apoptosis in PC12
cells (54). Anticancer drugs, such as vinblastine, which inhibit
microtubules, activate AP-1 in human KB-3 carcinoma cells.
This activation is required for efficient apoptosis induced by
Cell Biology^ Grant no. 05-I-05-005, and FEB Grant no. 05-
III-A-05-129.
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