Full text of DOI:10.1111/j.1349-7006.2002.tb01273.x

Jpn. J. Cancer Res. 93, 417–425, April 2002
Betulinic Acid Inhibits Growth Factor-induced in vitro Angiogenesis via the
Modulation of Mitochondrial Function in Endothelial Cells
Ho Jeong Kwon,1, 4 Joong Sup Shim,1 Jin Hee Kim,1 Hyun Young Cho,2 Young Na Yum,2
Seung Hee Kim2 and Jaehoon Yu3
1Department of Bioscience and Biotechnology, Institute of Bioscience, Sejong University, 98 Kunja-dong,
Kwangjin-gu, Seoul 143-747, Korea, 2National Institute of Toxicological Research, Korea Food and
3
Drug Administration, 5 Nokbeon-dong, Eunpyung-gu, Seoul 122-704, Korea and Life Science Division,
Korea Institute of Science and Technology, P. O. Box 131, Cheongryang, Seoul 136-650, Korea
Betulinic acid (BetA), a pentacyclic triterpene, is a selective apoptosis-inducing agent that works
directly in mitochondria. Recent study has revealed that BetA inhibits in vitro enzymatic activity of
aminopeptidase N (APN, EC 3.4.11.2), which is known to play an important role in angiogenesis,
but the anti-angiogenic activity of BetA has not been reported yet. Data presented here show that
BetA potently inhibited basic fibroblast growth factor (bFGF)-induced invasion and tube forma-
tion of bovine aortic endothelial cells (BAECs) at a concentration which had no effect on the cell
viability. To access whether the anti-angiogenic nature of BetA originates from its inhibitory action
against aminopeptidase N (APN) activity, the effect of BetA on APN was investigated. Surprisingly,
BetA did not inhibit in vivo APN activity in endothelial cells or APN-positive tumor cells. On the
other hand, BetA significantly decreased the mitochondrial reducing potential, and treatment with
mitochondrial permeability transition (MPT) inhibitors attenuated BetA-induced inhibition of
endothelial cell invasion. These results imply that anti-angiogenic activity of BetA occurs through a
modulation of mitochondrial function rather than APN activity in endothelial cells.
Key words: Betulinic acid — Angiogenesis — Aminopeptidase N — Mitochondrial permeability tran-
sition
Betulinic aicd (BetA), a pentacyclic triterpene, has been
isolated from the stembark of Betula ssp. and from many
other plants.1, 2) It was first identified as a selective apopto-
sis-inducing agent in human melanoma cells.3) In an in
vivo animal model system, BetA showed highly effective
tumor growth inhibition in mice injected subcutaneously
with human melanoma MEL-2 cells. Furthermore, it also
induces apoptosis in neuroectodermal tumor and malignant
glioma cells.4, 5) Extensive studies have shown that BetA-
induced apoptosis occurs through the perturbation of mito-
chondrial function, such as loss of membrane potential
(∆ψm), reactive oxygen species (ROS) production, and
permeability transition (PT) pore opening.5) These mito-
chondrial events trigger a sequential apoptotic cascade
including the release of mitochondrial apoptogenic factors,
activation of caspases, and DNA fragmentation.6) All of
these effects are CD95- and p53-independent, suggesting
that BetA may induce apoptosis via a direct effect on
mitochondria.7, 8)
ous studies have shown that APN, as a zinc-dependent
metalloproteinase, plays an important role in metastatic
tumor invasion.12, 13) Recently, Bhagwat et al. reported that
APN was activated by angiogenic stimuli and was essen-
tial for the endothelial cell tube formation.14) Several
inhibitors of APN significantly inhibited retinal neovascu-
larization, in vivo angiogenesis of chorioallantoic mem-
brane, and xenograft tumor growth, suggesting that APN
plays an important role in the progression of tumor vascu-
logenesis and may be involved in a crucial step of the
angiogenic process.15)
These interesting results led us to investigate whether
BetA, a mitochondrial function disruptor as well as an
inhibitor of APN, could inhibit growth factor-induced
angiogenesis. We performed in vitro angiogenesis assays
including cell proliferation, chemoinvasion and tube for-
mation of endothelial cells. Data presented here show that
BetA potently inhibits growth factor-induced angiogenesis
in vitro without showing any cytotoxic effect on endothe-
lial cells. Surprisingly, in vivo enzymatic analysis of APN
revealed that BetA does not inhibit the enzyme activity in
endothelial cells or APN-positive tumor cells. These
results indicate that anti-angiogenic activity of the com-
pound is not derived from the inhibition of APN. Instead,
we found that mitochondrial function in endothelial cells
is major target of BetA.
Melzig and Bormann reported that BetA inhibited in
vitro enzymatic activity of aminopeptidase N (APN, EC
3.4.11.2).9) APN is identical to CD13 (gp150), a myeloid
cell surface glycoprotein, and is a widely distributed mem-
brane-bound, zinc-dependent metalloproteinase.10, 11) Previ-
4 To whom correspondence should be addressed.
E-mail: kwonhj@sejong.ac.kr
417

Jpn. J. Cancer Res. 93, April 2002
with bFGF (30 ng/ml). Then, BetA was added and incuba-
tion was continued for 6–18 h. The morphological
changes in the cells and tubes formed were observed under
a microscope and photographed at ×100 magnification
using JVC digital camera (Victor, Yokohama).
MATERIALS AND METHODS
Materials BetA was purchased from Aldrich Chem. Co.
(Milwaukee, WI). The basic fibroblast growth factor
(bFGF) was obtained from Upstate Biotechnology (Lake
Placid, NY), bestatin, aminopeptidase N, ala-7-amido-
4-methylcoumarin, and 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) from Sigma (St.
Louis, MO), cell culture media from Life Technology
(Grand Island, NY), the Matrigel from Collaborative Bio-
medical Products (Bedford, MA), and the Transwell plate
from Corning Costar (Cambridge, MA). All chemicals
used in this study were of the highest grade commercially
available.
Cell culture The early passages (5–7 passage) of bovine
aortic endothelial cells (BAECs) and human umbilical
vein endothelial cells (HUVECs) were kindly provided by
Dr. Jo at the NIH of Korea and by Dr. Kwon at Kangwon
Natl. Univ., respectively. BAECs were grown in MEM
supplemented with 10% fetal bovine serum (FBS).
HUVECs were maintained in M199 supplemented with
20% FBS, heparin, and 1.5 ng/ml bFGF. HT1080 and
MDA-MB-231 cells were maintained in Dulbecco’s modi-
fied Eagle’s medium (DMEM), and B16/BL6 and
HCT116 cells in RPMI1640 containing 10% FBS. All
cells were grown at 37°C in a humidified atmosphere of 5%
CO2.
Cell growth assay BAECs were seeded at a density of
2×104 cells/well in a 48-well plate. The cells were incu-
bated in growth media for 24 h. Various concentrations of
BetA were added to the wells and incubation was contin-
ued for 24 or 48 h. The cells in each well were trypsinized
and pelleted by centrifugation. Each pellet was resus-
pended in 10 µl of phosphate-buffered saline (PBS) and
trypan blue dye was added. Cells were observed under a
microscope and counted with a hemocytometer. Cell via-
bility was accessed as unstained cells/total cells×100.
Chemoinvasion assay The invasiveness of the endothe-
lial cells was examined in vitro using a Transwell chamber
system with 8.0-µm-pore polycarbonate filter inserts. The
lower side of the filter was coated with 10 µl of gelatin (1
mg/ml), and the upper side was coated with 10 µl of
Matrigel. Cells (1×105 cells) were placed in the upper part
of the filter and BetA was applied to the lower part for 30
min at room temperature before seeding. The chamber was
then incubated at 37°C for 18 h. The cells were fixed with
methanol and stained with hematoxylin/eosin. The cell
invasion was determined by counting whole cells on the
lower side of the filter using an optical microscope at
×100 magnification.
RNA preparation and reverse transcriptase-poly-
merase chain reaction (RT-PCR) Total cellular RNA
was isolated from cultured cells with an RNeasy mini kit
(Qiagen, Inc., Valencia, CA) and reverse-transcribed by
Molony murine leukemia virus reverse transcriptase (Life
Technologies, Rockville, MD) using Oligo-d(T)15 primer
(Life Technologies). For the determination of APN mRNA
content in each cell line, a standard PCR was performed
using 5′-CCTTCAACCTGGCCAGTGC-3′ and 5′-CGT-
CTTCTCCAGGGCTTGCTCC-3′ (sense and antisense
primers common to murine and human aminopeptidase
N (APN)) as primers. GAPDH mRNA was quantified
using the RT primer pair commercially available from
Stratagene (Heidelberg, Germany) and used to normalize
the cDNA content. The PCR products were resolved by
1% agarose gel electrophoresis and visualized by ethidium
bromide staining.
Assay of APN enzymatic activity The activity of APN
was determined according to Saiki et al.12) Briefly, the
enzyme substrate, ala-7-amido-4-methylcoumarin was dis-
solved (2 mM) in PBS as a stock solution. The substrate
(0.1 mM) was added to PBS with or without inhibitors.
The reaction was started by adding an enzyme solution
(final conc., 0.2 mU) and continued in dark room at 37°C.
After 1 h, the reaction mixture was centrifuged and the
supernatant was collected for measurement of fluorescence
using an FL600 microplate fluorescence reader (Bio-Tek
Instruments, Inc., Winooski, Vermont) at the excitation
wavelength of 360 nm, and the emission wavelength of
460 nm. For in vivo enzyme assay, cells (2×105) were
seeded in a 24-well culture plate and after incubation for
24 h, culture media were replaced with 500 µl of PBS in
each well. Aliquots of substrate (2 mM) were added
directly to each well with or without inhibitors. The plate
was incubated in dark room at 37°C for 1 h. The superna-
tant from each well was collected and enzyme activity was
determined as described above.
MTT reduction assay BAECs were seeded at a density
of 5×103 cells/well in a 96-well plate and incubated for
24 h. Various concentrations of drugs were added to the
wells and incubation was continued for 12 h. After 12 h,
50 µl of MTT (0.4 mg/ml, final conc.) was added and the
plate was incubated for an additional 4 h. After removal of
the culture supernatant, 150 µl of dimethylsulfoxide
(DMSO) was added to dissolve MTT-formazan. The plate
was read at 540 nm using a microplate reader (Bio-Tek
Instruments, Inc., Winooski, VT).
Capillary tube formation assay Matrigel (250 µl, 10
mg/ml) was placed in a 24-well culture plate and poly-
merized for 30 min at 37°C. The BAECs (1×105 cells)
were seeded on the surface of the Matrigel and treated
418

Betulinic Acid Inhibits Angiogenesis
of BetA and stained with trypan blue at each time point.
BetA showed a weak inhibition of BAECs proliferation at
a concentration of 20 µM without showing any cytotoxic-
ity, as in the cell viability assay (Fig. 1B). However, no
inhibition of proliferation was observed at 10 µM BetA up
to 72 h. Therefore, angiogenesis assays were performed in
a concentration range of 1 to 10 µM of BetA within 24 h.
BetA potently inhibits growth factor-induced angiogen-
esis in vitro Angiogenic endothelial cells secrete several
protein-degrading enzymes, including matrix metallopro-
teinases that degrade the basement membrane, favoring
the formation of new blood vessels.16, 17) Thus, endothelial
cell invasion is a crucial step for angiogenesis.18) For in
vitro invasion assay with endothelial cells, BAECs were
starved for 24 h and stimulated by bFGF in the presence
or absence of BetA. The assay was performed using poly-
carbonate-filter Transwells coated with the Matrigel to
prevent the migration of non-invasive cells. BetA potently
inhibited bFGF-induced invasion of BAECs in a dose-
dependent manner (Fig. 2A). An inhibitory effect on
BAECs invasion was seen at a concentration as low as 2
µM BetA (data not shown). We next examined the effect
of BetA on capillary tube formation. In the presence of
bFGF, cultured BAECs on the Matrigel formed an exten-
sive network of thick tubes (Fig. 2C). Treatment of
BAECs with BetA resulted in dose-dependent inhibition of
tube formation induced by bFGF. To rule out the possibil-
ity that the inhibition of tube formation is merely due to a
cytotoxic effect of BetA on BAECs, trypan blue staining
was performed after the complete formation of tubes. The
highest concentration (10 µM) of BetA did not affect the
endothelial cell viability in the formed tubes (Fig. 2C). In
addition, 10 µM BetA did not significantly inhibit the pro-
liferation of BAECs, as shown in Fig. 1B. These results
suggest that BetA selectively and potently inhibits angio-
genesis in vitro, and the anti-angiogenic activity of the
compound may mostly originate from a specific effect on
the angiogenic differentiation of endothelial cells, rather
than anti-proliferative activity.
RESULTS
Effect of BetA on the cell viability and proliferation of
BAECs To determine the optimum dose of BetA in
angiogenesis assays, endothelial cell viability assay was
performed using the trypan blue exclusion method.
BAECs were treated with various doses of BetA for 24 or
48 h and stained with trypan blue. BetA (1–20 µM) for 24
h did not significantly affect the viability of BAECs (Fig.
1A). However, the viability of BAECs was slightly
decreased at high concentrations of BetA (15–20 µM) for
48 h. We next examined the effect of BetA on the prolifer-
ation of BAECs. BAECs were treated with various doses
BetA inhibits APN enzymatic activity in vitro but not in
vivo As mentioned above, BetA is an inhibitor of APN, a
potent angiogenesis inducer.14, 15) We examined whether
the anti-angiogenic activity of BetA originates from the
inhibition of enzymatic activity of APN. In vitro APN
activity assay was carried out as previously described by
Saiki et al.12) Partially purified APN (Sigma) was used as
an enzyme source for in vitro analysis. As shown in Fig.
3A, BetA as well as bestatin, a known APN competitive
inhibitor, potently inhibited APN activity with an IC50 of 7
µM. The in vitro inhibition of APN by BetA is consistent
with the report by Melzig and Bormann.9) Next, we inves-
tigated the effect of BetA on the in vivo enzymatic activity
of APN. Endothelial cells were seeded in a 24-well culture
plate and a fluorogenic substrate was added directly to
Fig. 1. Dose response of betulinic acid (BetA) on bovine aortic
endothelial cells (BAECs). (A) Effect of BetA on the viability of
BAECs. Cell viability was determined by trypan blue exclusion
assay as described in “Materials and Methods.”
24 h,
48 h.
(B) Effect of BetA on the proliferation of BAECs. Cells were
treated with BetA (0–20 µM) and cell growth was measured at
various time points.
Control,
BetA (10 µM),
BetA (20
µM).
419

Jpn. J. Cancer Res. 93, April 2002
each well with or without inhibitors. After reaction, the
supernatant from each well was collected to determine in
vivo APN activity. Surprisingly, BetA-treated cells still
showed strong APN activity, at the same level as the drug-
untreated control cells (Fig. 3B), whereas the enzyme
activity from bestatin-treated cells was inhibited. These
results indicate that BetA does not inhibit in vivo APN
activity in endothelial cells.
BetA does not inhibit in vivo APN activity in APN-pos-
itive tumor cells We observed that BetA does not inhibit
in vivo APN activity in endothelial cells. To investigate the
possibility that this lack of effect of BetA is limited to
endothelial cells, we next examined the effect of BetA on
the APN activity in APN-positive tumor cells. RT-PCR
analysis showed that APN is up-regulated in HUVECs in
the presence of bFGF (Fig. 4A). In addition, B16/BL6,
murine melanoma, and HT1080, human fibrosarcoma
cells, constitutively express high levels of APN, whereas
HCT116, colon cancer, and MDA-MB-231, human breast
cancer cells do not express APN (Fig. 4B). So, we exam-
ined the in vivo APN activity of HT1080 cells treated with
various concentrations of BetA. BetA did not inhibit in
vivo APN activity in HT1080 cells, while bestatin potently
inhibited the enzyme activity (Fig. 4C). These data dem-
onstrate that BetA is not an in vivo inhibitor of APN in
tumor or endothelial cells.
BetA strongly decreases mitochondrial reducing poten-
tial in endothelial cells Several investigations have
A
B
Serum free
bFGF
BetA(5 µM)
BetA(10 µM)
bFGF
C
Trypan blue staining
Fig. 2. In vitro anti-angiogenic activity of BetA. (A) Effect of BetA on the basic fibroblast growth factor (bFGF)-induced invasion of
BAECs. Serum-starved cells left untreated in serum-free medium (SF) or treated with BetA were treated with bFGF. (B) Microscopic
observation of invaded BAECs. (C) Effect of BetA on the capillary tube formation of BAECs. Cytotoxic effect of BetA on tube-formed
BAECs was evaluated by trypan blue staining. Actinomycin D, a cytotoxic drug, was used as a control. Figures were selected as repre-
sentative scenes from two independent experiments.
420

Betulinic Acid Inhibits Angiogenesis
shown that mitochondria are likely to be a direct target for
BetA in tumor cells.6–8) This effect of BetA on the mito-
chondrial function may contribute to certain changes in
endothelial cell metabolism. Therefore, we next investi-
gated the effect of BetA on the mitochondrial reducing
potential in BAECs. The assay was performed with MTT,
which is reduced by mitochondrial dehydrogenases, espe-
cially by succinate dehydrogenase.19) BAECs treated with
or without BetA and structurally related compounds
including ursolic acid (UA) and dexamethasone (Dex),
were incubated for 12 h and MTT was added to the cells.
After 4 h, cells were observed under a microscope and
MTT reduction was measured at 540 nm using a micro-
plate reader. Cell viability assay using trypan blue staining
was performed in parallel. BetA potently decreased MTT
reduction in endothelial cells, while UA and Dex did not
(Fig. 5, A and C). Microscopic observation of cells after
MTT administration showed the mitochondrial localization
in cells as black dots (Fig. 5B). Interestingly, cell prolifer-
ation was not affected at the same concentration of BetA
Fig. 3. Effect of BetA on the enzymatic activity of aminopeptidase N (APN). (A) In vitro analysis of APN activity. Commercially
available leucine aminopeptidase was used as an enzyme source. Bestatin, a competitive inhibitor of APN, was used as a positive con-
trol. (B) In vivo analysis of APN activity. BAECs grown in culture plates were used as an enzyme source and APN activity was deter-
mined as described in “Materials and Methods.”
BetA,
Bestatin.
A
C
bFGF
24(h)
12
APN
GAPDH
B
APN
GAPDH
Fig. 4. Effect of BetA on in vivo APN activity in APN-positive tumor cells. (A) RT-PCR analysis of the expression of APN in endo-
thelial cells with or without bFGF stimulation. (B) Analysis of APN expression in various tumor cells using RT-PCR. (C) Effect of BetA
on in vivo enzymatic activity of APN in HT1080 cells.
BetA,
Bestatin.
421

Jpn. J. Cancer Res. 93, April 2002
that showed a potent decrease in MTT reduction in endo-
thelial cells. In addition, BetA-induced inhibition of MTT
reducibility by endothelial cells was fully reversible (data
not shown). These results suggest that BetA may affect the
mitochondrial redox potential of endothelial cells in
reversible manner without inhibiting cell proliferation.
Mitochondrial permeability transition (MPT) inhibi-
tors attenuate BetA-induced inhibition of endothelial
cell invasion It was previously observed that isolated
mitochondria treated with BetA could induce an apoptotic
cascade, including caspase activation and nuclear frag-
mentation.6) These apoptotic events were preceded by the
induction of mitochondrial permeability transition (MPT)
and sequential loss of mitochondrial membrane potential
(∆ψm). Several MPT inhibitors significantly inhibited
BetA-induced mitochondria-derived apoptosis, suggesting
that the compound is a potent inducer of MPT.20) These
effects of BetA on mitochondria may not be limited in
tumor cells. Thus, we next examined the effect of MPT
inhibitors including bongkrekic acid (BA), an inhibitor of
adenosine nucleotide translocater,21) cyclosporine A (CsA),
a transient inhibitor of MPT,22) and z-VAD-fmk, a broad
range caspase inhibitor, on the BetA-induced inhibition of
endothelial cell invasion. BAECs pretreated with MPT
inhibitors were seeded on the upper chamber of Trans-
wells in the presence or absence of BetA, and invasion
assay was performed as described in “Materials and Meth-
ods.” bFGF stimulated the invasion of BAECs and this
invasiveness was potently inhibited by BetA (Fig. 6). Sur-
prisingly, BA as well as CsA attenuated BetA-induced
A
B
BetA(10 µM)
Control
Cell
Control
morphology
MTT
reduction
BetA
(5 µM)
C
D
Dex
(15 µM)
UA
(10 µM)
Fig. 5. Effect of BetA on the mitochondrial reducing potential in endothelial cells. (A) Effect of BetA on the morphology of BAECs
and their capacity for MTT reduction. Cells treated with BetA for 12 h were exposed to MTT solution and observed under a microscope
at ×100 magnification. In parallel, a group of cells was treated with BetA for 12 h and observed without addition of MTT solution. (B)
Microscopic observation of MTT reduction in BAECs (×400 magnification). Black dots indicated by white arrows represent mitochon-
dria localization in BAECs. BetA (5 µM) significantly reduced MTT reduction. (C) Effect of dexamethasone (Dex) or ursolic acid (UA)
on the MTT reduction in BAECs. (D) Quantitative analysis of MTT reduction in BetA-treated BAECs using a 540 nm filter-equipped
microplate reader. Cell viability was determined in parallel by trypan blue exclusion assay.
Cell viability,
MTT reducibility.
422

Betulinic Acid Inhibits Angiogenesis
A
B
bFGF
Fig. 6. Effect of mitochondrial permeability transition (MPT) inhibitors on BetA-induced inhibition of endothelial cell invasion. (A)
BAECs pretreated with various MPT inhibitors including cyclosporine A (CsA) and bongkrekic acid (BA), and caspase inhibitor, z-
VAD-fmk for 2 h were exposed to BetA and bFGF, and invasion assay was carried out. Invaded cells were observed under a micro-
scope at ×100 magnification. (B) Invaded cells were counted under a microscope and analyzed quantitatively.
inhibition of BAECs invasion. z-VAD-fmk also partially
prevented the effect of BetA. These results suggest that the
anti-angiogenic activity of BetA arises, at least in part,
through modulation of the mitochondrial function in
endothelial cells.
matic activity of APN. This result is consistent with the
previous observation by Melzig and Bormann.9) However,
BetA does not inhibit in vivo enzymatic activity of APN in
endothelial cells or APN-positive tumor cells. These
results demonstrate that BetA is not an in vivo inhibitor of
APN. Although, the reason for the different activity spec-
trum of BetA in vivo and in vitro is not clear, APN may
not be related to the anti-angiogenic activity of the com-
pound.
Several investigations have demonstrated that BetA
might directly target mitochondrial function in various
tumor cells.6–8) Thus, it is probable that BetA directly
affects the mitochondrial function in endothelial cells. The
present data show that BetA strongly decreases the mito-
chondrial reducing potential in BAECs. In addition, sev-
eral MPT inhibitors can attenuate the inhibition of
angiogenesis induced by the compound. These data
strongly support the idea that BetA may inhibit angiogene-
sis via the modulation of mitochondrial function in endo-
thelial cells.
DISCUSSION
Angiogenesis is a key process for the outgrowth of can-
cer cells and their spread into other tissues. Therefore, the
specific inhibition of angiogenesis may be a powerful
means to suppress angiogenesis-related diseases including
cancer. Extensive studies have been carried out to identify
the cellular target proteins for angiogenesis, and several
of these target proteins have been identified, i.e., matrix
metalloproteinases,23) vascular endothelial growth factor
receptors,24, 25) methionine aminopeptidase-2,26, 27) and his-
tone deacetylases.28, 29) APN has been highlighted recently
by many investigators as having possible involvement in
angiogenesis.14, 15) In this respect, the specific inhibition of
APN activity may be a novel approach to angiogenesis
therapy.
The MPT pore is known to possess several redox-sensi-
tive sites.30) An enhanced generation of reactive oxygen
species could induce changes in cellular redox potential.31)
These changes, including depletion of nonoxidized glu-
tathione or of NADPH, facilitate MPT. In this respect, the
The present data show that BetA selectively and
potently inhibits growth factor-induced angiogenesis in
vitro. Furthermore, BetA strongly inhibits in vitro enzy-
423

Jpn. J. Cancer Res. 93, April 2002
BetA-induced decrease in mitochondrial reducing potential
may facilitate MPT in endothelial cells. The induction of
MPT in cells may cause the release of apoptogenic factors
that can directly trigger cell death.32) However, these apo-
ptogenic factors may not contribute the anti-angiogenic
activity of the compound, since the BetA-induced decrease
in the mitochondrial reducing potential is fully reversible
and results in no significant inhibition of the cell viability.
On the other hand, Lee et al. reported very recently that
pyruvate, the end metabolite of glycolysis could induce
angiogenesis both in vivo and in vitro.33) This study shows
that an increase in mitochondrial oxidative phosphoryla-
tion can enhance angiogenic differentiation of endothelial
cells. Thus, it is possible that the effect of BetA on MPT
may cause the reversible inhibition of the mitochondrial
respiration. To investigate this possibility, we examined
the effect of BetA on the enzymatic activity of succinate
dehydrogenase (SDH), a component of mitochondrial res-
piratory chain complex II. BetA did not significantly
inhibit SDH activity in vivo or in vitro (data not shown),
suggesting that SDH is not associated with the inhibition
of mitochondrial reducing potential and the inhibition of
angiogenesis by BetA. Further investigations of the effect
of BetA on other mitochondrial respiratory complexes and
dehydrogenases are needed to account for the anti-angio-
genic activity of the compound.
In conclusion, betulinic acid potently inhibits growth
factor-induced angiogenesis, at least in part through the
modulation of mitochondrial function in endothelial cells.
ACKNOWLEDGMENTS
This work was supported by grant number FG-3-3-01 for the
21C Frontier Functional Human Genome Project from the Minis-
try of Science & Technology of Korea.
(Received November 22, 2001/Revised January 15, 2002/
Accepted January 26, 2002)
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