Cytotoxic Compounds from Crossosoma bigeloVii
Journal of Natural Products, 2009, Vol. 72, No. 5 811
(CH, C-3′), 75.7 (CH, C-5′), 73.1 (CH, C-2′), 72.1 (CH, C-4′′), 70.7
(CH, C-5′′), 70.3 (CH, C-2′′), 70.0 (CH, C-4′), 68.4 (CH, C-3′′), 66.6
(CH2, C-6′), 60.1 (CH3, C-10), 20.0 (CH3, C-9), 17.9 (CH3, C-6′′);
FABMS m/z 569 [M + K]+ (7), 553 [M + Na]+ (15), 531 [M + H]+
(50), 385 (11), 339 (13), 223 (100); HRFABMS m/z 531.1713 [MH]+
(calcd for C23H31O14, 531.1714).
fection of the specific reporter plasmid (pGL2-TK-HRE and pGL3-control,
respectively) with an expression vector carrying the neomycin resistance
gene for selection in mammalian cells (ratio 100:1). Twenty-four hours
after transfection, reagents were removed, and cells were allowed to recover
for 24 h before the addition of selection medium containing the antibiotic
G418 at 500 µg/mL (Invitrogen-Life Technologies, Inc.). Stably transfected
cells were seeded at a concentration of 104 cells/well in 96-well optiplates
(Packard Instrument, Inc., Meriden, CT) the day before treatment and
routinely treated for 16-24 h. Luciferase reporter assays were performed
in 96-well optiplates using Bright Lite Plus luciferase assay reagents (Perkin
Elmer, Waltham, MA).
5-Hydroxy-2-methylchromone-7-O-rutinoside (3): amorphous,
light yellow solid; [R]20 -54.5 (c 0.149, MeOH); UV (MeOH) λmax
D
(log ꢀ) 249 (3.97), 255 (3.97), 284 (3.51), 313 (2.92) nm; + NaOAc
248, 256, 283, 313 nm; + AlCl3 257, 302, 360 nm; + AlCl3/HCl 256,
1
302, 365 nm; IR (KBr) νmax 3404 (br), 1663, 1625, 1070 cm-1; H
NMR (DMSO-d6, 500 MHz) δ 12.78 (1H, s, 5-OH), 6.63 (1H, d, J )
2.0 Hz, H-6), 6.37 (1H, d, J ) 2.0 Hz, H-3), 6.24 (1H, s, H-3), 5.00
(1H, d, J ) 7.0 Hz, H-1′), 4.50 (1H, s, H-1′′), 3.85 (1H, d, J ) 10.0
Hz, H-6′a), 3.64 (1H, H-2′′), 3.58 (1H, dd, J ) 9.0, 9.0 Hz, H-5′),
3.46 (1H, dd, J ) 7.5, 2.5, H-3′′), 3.40 (1H, dd, J ) 9.5, 6.5 Hz, H-5′′),
3.33 (1H, H-6′b), 3.28 (1H, H-3′), 3.27 (1H, H-2′), 3.14 (1H, dd, J )
9.5, 7.5 Hz, H-4′′), 3.09 (1H, dd, J ) 9.0, 9.0 Hz, H-4′), 2.41 (3H, s,
H-9), 1.09 (3H, d, J ) 6.5 Hz, H-6′′); 13C NMR (DMSO, 125 MHz)
δ 182.0 (C, C-4), 169.0 (C, C-2), 162.5 (C, C-7), 161.0 (C, C-5), 157.5
(C, C-8a), 108.2 (C, C-3), 105.2 (CH, C-4a), 100.7 (C, C-1′′), 99.8
(CH, C-1′), 99.6 (CH, C-6), 94.4 (CH, C-8), 76.4 (CH, C-3′), 75.7
(CH, C-5′), 73.0 (CH, C-2′), 72.1 (CH, C-4′′), 70.7 (CH, C-5′′),
70.3 (CH, C-2′′), 69.9 (CH, C-4′), 68.4 (CH, C-3′′), 66.5 (CH2, C-6′),
20.2 (CH3, C-9), 17.8 (CH3, C-6′′); FABMS m/z 523 [M + Na]+
(28), 501 [M + H]+ (13), 355 (26), 193 (100); HRFABMS m/z
501.1613 [MH]+ (calcd for C22H29O13, 501.1608).
Total Extract Preparation and Immunoblotting (Western Blot).
Cells were washed with phosphate-buffered saline and lysed with RIPA
lysis buffer [1 mM NaF, 1 mM sodium vanadate] (Pierce Chemicals,
Rockford, IL) containing a cocktail of protease inhibitors (Roche, Man-
nheim, Germany). Using mechanical scraping, cell lysates then were
collected and rotated at 4 °C for 15 min, and cellular debris was pelleted
by centrifugation at 15000g for 15 min at 4 °C. Protein concentration was
measured by Bradford assay, using bovine serum albumin as a standard.
Protein (100 µg) was separated on a 4-20% Tris-Glycine gel (Invitrogen,
Carlsbad, CA), electroblotted on a polyvinylidene difluoride membrane
(Invitrogen), and subjected to immunoblot analysis. Monoclonal anti-HIF-
1R antibody was purchased from BD-Transduction Laboratories (Lexing-
ton, KY) and used at a 1:300 dilution. Horseradish peroxidase-conjugated
antimouse IgG (1:10 000 dilution) and enhanced chemiluminescence
reagents were obtained from Amersham Pharmacia Biotech (Piscataway,
NJ).
Real-Time PCR. Total RNA from U251 cells was obtained using RNA
Mini Kit (Qiagen, Inc.). A total of 500 ng of RNA was used to perform
RT-PCR using RT-PCR kit (PE Biosystems, Foster City, CA). The
conditions used for RT-PCR were as follows: 10 min at 25 °C, 30 min at
48 °C, and 5 min at 95 °C.
Cytotoxicity Testing. The human tumor cytotoxicity assays were
performed at the Purdue University Cell Culture Laboratory, Purdue
Cancer Center, using the standard MTT protocol.25 The following
human tumor cell lines were employed: A-549 lung carcinoma; MCF-
7, breast adenocarcinoma; and HT-29, colon adenocarcinoma. Adria-
mycin was used as a reference compound in all the assays. Cells were
maintained in MEM supplemented with 10% fetal calf serum and 0.1 g
L-1 penicillin G + 0.1 g L-1 streptomycin sulfate. The cells were
transferred into 96-well plates one day prior to the addition of the
assayed compounds, and incubated overnight at 37 °C. Compounds
were dissolved in DMSO, diluted with medium at different concentra-
tions, and incubated with the cells for six days. Cell survival was
determined colorimetrically via MTT, a tetrazolium salt, at 547 nm.
Cell Lines and Reagents. U251 human glioma cells are routinely
maintained in RPMI 1640 (Whittaker Bioproducts, Walkersville, MD)
supplemented with 5% heat-inactivated fetal bovine serum (Whittaker),
penicillin (50 IU mL-1), streptomycin (50 g mL-1), and 2 mM glutamine
(all purchased from Invitrogen-Life Technologies, Inc., Carlsbad, CA).
U251-HRE and U251-pGL3 control stable cells were maintained in
RPMI 1640 supplemented with 5% heat-inactivated fetal bovine serum,
penicillin (50 IU mL-1), streptomycin (50 g mL-1), 2 mM glutamine,
and G418 at 100 µg mL-1. Cells were maintained at 37 °C in a
humidified incubator containing 21% O2, 5% CO2 in air (referred to as
normoxic conditions). Hypoxia treatment was performed by placing
cells in a modular incubator chamber (Billupus-Rothemberg Inc., Del
Mar, CA) and then flushing with a mixture of 1% O2, 5% CO2, and
94% N2 for 20 min. The chamber was then placed at 37 °C. Hypoxic
conditions were also achieved in an Invivo2 400 hypoxic workstation
(Ruskinn Technologies, Cincinnati, OH) set to deliver 1% O2 in 5%
CO2 at 37 °C.
To measure human VEGF and CA9 expression, real-time PCR was
performed using an ABI-Prism 7700 sequence detector (Applied Biosys-
tems, Foster City, CA). Typically 5 ng of reverse-transcribed cDNA per
sample was used to perform real-time PCR in triplicate samples. Real-
time PCR cycles started with 2 min at 50 °C, 10 min at 95 °C, and then
40 cycles of 15 s at 95 °C and 1 min at 60 °C. Primers and specific probes
were obtained from Applied Biosystems and are available upon request.
[The following primers and probes were used: human VEGF forward, 5′-
TACCTCCACCATGCCAAGTG-3′; human VEGF reverse, 5′-ATGAT-
TCTGCCCTCCTCCTTC-3′; probe, 5′-FAM-TCCCAGGCTGCAC-
CCATGGC-TAMRA-3′]. Detection of VEGF and 18S rRNA was
performed using TaqMan Universal PCR Master Mix (Applied Biosys-
tems), and CA9 detection was performed using Sybr Green PCR Master
Mix (Applied Biosystems).
Acknowledgment. This project has been funded in whole or in part
with federal funds from the National Cancer Institute, National Institutes
of Health, under contracts R01-CA-49632 (Purdue University) and NO1-
CO-12400 (NCI). The content of this publication does not necessarily
reflect the views or policies of the Department of Health and Human
Services, nor does mention of trade names, commercial products, or
organizations imply endorsement by the U.S. Government. This research
was supported [in part] by the Developmental Therapeutics Program in
the Division of Cancer Treatment and Diagnosis of the National Cancer
Institute. We gratefully acknowledge R. Spjut for the plant collection, J.
Klose for NMR experiments, M. Selby for performing the HIF-1 and pGL3
biological screens, and J. Britt for robotics support.
Plasmids. The pGL2-TK-HRE plasmid was generated by subcloning
three copies of the HRE (5′-GTGACTACGTGCTGCCTAG-3′) from the
inducible nitric oxide synthase promoter into the pGL2-TK promoter vector.
Plasmids were sequenced at the Molecular Technology Laboratory, SAIC-
Frederick, Inc. The pGL3-control (Promega) contains the firefly luciferase
coding sequence under control of the SV40 promoter and enhancer
sequences.
Supporting Information Available: 1D- and 2D-NMR spectra of
1-3 and the HPLC biogram of the organic extract of C. bigeloVii are
Transient Transfection. U251 cells were transfected with pGL2-TK-
HRE plasmid using Effectene Transfection Reagents (Qiagen, Inc.,
Valencia, CA) according to the manufacturer’s instructions. After 16 h of
transfection, the medium was changed and treated with convallatoxin from
1 µΜ to 0.005 µM for 16 h, and luciferase expression was measured using
Steady Glo luciferase assay reagents.
Stable Transfection and Engineered Cell Lines. DNA plasmids
were prepared using a commercially available kit (Endofree Maxi-Prep;
Qiagen,Inc.). Transfections were performed using Effectene Transfection
Reagents (Qiagen, Inc.) according to the manufacturer’s instructions. Stably
transfected cells (U251-HRE, U251-pGL3) were generated by cotrans-
References and Notes
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