Blueberry flavonoids inhibit matrix
metalloproteinase activity in DU145
human prostate cancer cells
Michael D. Matchett, Shawna L. MacKinnon, Marva I. Sweeney,
Katherine T. Gottschall-Pass, and Robert A.R. Hurta
Abstract: Regulation of the matrix metalloproteinases (MMPs), the major mediators of extracellular matrix (ECM)
degradation, is crucial to regulate ECM proteolysis, which is important in metastasis. This study examined the effects
of 3 flavonoid-enriched fractions (a crude fraction, an anthocyanin-enriched fraction, and a proanthocyanidin-enriched
fraction), which were prepared from lowbush blueberries (Vaccinium angustifolium), on MMP activity in DU145 human
prostate cancer cells in vitro. Using gelatin gel electrophoresis, MMP activity was evaluated from cells after 24-hr ex-
posure to blueberry fractions. All fractions elicited an ability to decrease the activity of MMP-2 and MMP-9. Of the
fractions tested, the proanthocyanidin-enriched fraction was found to be the most effective at inhibiting MMP activity
in these cells. No induction of either necrotic or apoptotic cell death was noted in these cells in response to treatment
with the blueberry fractions. These findings indicate that flavonoids from blueberry possess the ability to effectively de-
crease MMP activity, which may decrease overall ECM degradation. This ability may be important in controlling tumor
Key words: blueberry flavonoids, MMP activity, prostate cancer cells.
Résumé : La régulation de l’activité des métalloprotéases de la matrice (MMP), les médiateurs principaux de la dégra-
dation de la matrice extracellulaire (MEC), est cruciale afin de contrôler la protéolyse qui est importante dans le pro-
cessus métastatique. Cette étude a examiné les effets de trois fractions enrichies en flavonoïdes, soit un extrait brut,
une fraction enrichie en anthocyanine, ainsi qu’une fraction enrichie en proanthocyanidine, préparées à partir de bleuet
nain (ou airelle à feuille étroite) (Vaccinium angustifolium), sur l’activité des MMP des cellules cancéreuses de la pros-
tate humaines DU145, après un traitement de 24 heures avec les extraits de bleuets. Toutes les fractions ont démontré
une capacité à diminuer l’action de la MMP-2 et de la MMP-9. Des fractions testées, la fraction enrichie en proantho-
cyanidine s’est révélée la plus efficace quant à l’inhibition de l’activité MMP dans ces cellules. Aucune induction de
mort cellulaire par apoptose ou par nécrose n’a été notée en réponse au traitement avec les fractions isolées du bleuet.
Ces résultats indiquent que les flavonoïdes du bleuet ont la capacité de diminuer de façon efficace l’activité MMP, ré-
sultant en une diminution de la dégradation de la MEC, ce qui pourrait être important dans le contrôle de la formation
des métastases tumorales.
Mots clés : flavonoïdes du bleuet, activités des métalloprotéases de la matrice (MMP), cellules cancéreuses de la prostate.
[Traduit par la Rédaction]
Matchett et al. 643
The role of extracellular matrix (ECM) integrity and ECM-
degrading enzymes, such as the matrix metalloproteinases
(MMPs or matrixins), in the process of cancer metastasis has
been shown to be substantive, because ECM degradation is
essential for tumor metastasis to occur (Stetler-Stevenson
and Yu 2001; Pupa et al. 2002; Lynch and Matrisian 2002;
Freije et al. 2003). MMP expression and activity are tightly
regulated processes, indicating their importance in regular
cellular dynamics and interactions (Pupa et al. 2002; Visse
and Nagase 2003). Although not the only contributing factor
to tumor metastasis, it has been clearly shown that many
cancer cells demonstrate a pronounced increase in MMP
Biochem. Cell Biol. 83: 637–643 (2005)doi: 10.1139/O05-063© 2005 NRC Canada
Received 13 October 2004. Revision received 5 April 2005. Accepted 13 April 2005. Published on the NRC Research Press Web
site at http://bcb.nrc.ca on 28 September 2005.
M.D. Matchett, M.I. Sweeney, and R.A.R. Hurta.1Department of Biology, University of Prince Edward Island, 550 University
Ave., Charlottetown, PE C1A 4P3, Canada.
K.T. Gottschall-Pass. Department of Family and Nutritional Sciences, University of Prince Edward Island, 550 University Ave.,
Charlottetown, PE C1A 4P3, Canada.
S.L. MacKinnon. Institute for Marine Biosciences, National Research Council of Canada, 1411 Oxford St., Halifax, NS B3H 3Z1,
1Corresponding author (e-mail: firstname.lastname@example.org).
activity, and that overexpression of MMPs may be an impor-
tant factor in both tumor invasion and tumor angiogenesis
into foreign tissues (Stetler-Stevenson and Yu 2001; Pupa et
al. 2002). Because of the pivotal role MMPs play in tumori-
genesis and metastasis formation, MMP expression and
regulation may prove to be a strategic target for the develop-
ment of methods to combat and treat cancer (Pupa et al.
2002). Recent studies have shown that lowbush blueberries
(Vaccinium angustifolium) have beneficial effects against
certain chronic diseases (Bomser et al. 1996; Knekt et al.
2002). Blueberries contain relatively high concentrations of
polyphenolic compounds, such as flavonoids and phenolic
acids (Smith et al. 2000; Kalt et al. 2000). Flavonoids have
been shown to have antioxidant effects in many different
models, ranging from free-radical quenching to protection
during hypoxia–ischemia insults (Kandaswami and Middleton
1995; Sweeney et al. 2002; Kahkonen and Heinonen 2003).
Furthermore, flavonoids from Vaccinium species (lingonberry,
bilberry, cranberry, and lowbush blueberry) have anticarcino-
genic properties; they regulate ornithine decarboxylase, the
activity or expression of which is altered in many tumor
types (Bomser et al. 1996). In in vitro studies, green tea
(Camellia sinensis) flavonoids, such as epigallocatechin, have
also been shown to modify MMP activity and to modulate
ornithine decarboxylase activity (Gupta et al. 1999; Garbisa
et al. 2001).
In this study, 3 fractions from lowbush blueberry were
tested to examine their effects on MMP activity in DU145
human prostate cancer cells. These fractions represent the
major groups of flavonoids in these fruits: a crude fraction,
which contains all flavonoids; an anthocyanin-enriched fraction
(AN); and a proanthocyanidin-enriched fraction (PAC). The
anthocyanins and the proanthocyanidins are groups of
flavonoids that are believed to be the major active agents
responsible for the anticarcinogenic properties of many
flavonoid-containing fruits and vegetables (Bomser et al.
1996; Sartor et al. 2002; Sato et al. 2002). Therefore, in this
study, we hypothesized that the gelatinolytic activity of MMPs
in DU145 cells would be inhibited after exposure to the various
flavonoid-containing fractions, which would indicate that
these flavonoid-enriched fractions isolated from lowbush blue-
berry can affect and regulate the activity of MMPs.
Materials and methods
Preparation of blueberry fractions
The pressed juice from organic lowbush blueberries
(V. angustifolium) was loaded onto a preconditioned C18
column, which was subsequently washed with water to
remove sugars. The C18 column was then eluted with
100% methanol, containing 1% formic acid, until no more
color was eluting off the column. The resultant fraction,
referred to as the crude blueberry (CB) extract, was loaded
onto an LH-20 column that had been equilibrated in 50%
methanol in water. Elution with 60% methanol, 35% water,
and 5% formic acid yielded coloured fraction no. 1; further
elution of the column with 70% acetone and 30% water
yielded coloured fraction no. 2.
HPLC analysis of fractions
Coloured fractions no. 1 and no. 2 were chromatographed
on an Agilent 110 Series HPLC (Agilent Technologies,
Kirkland, Que.), which was equipped with a quaternary pump-
ing system, a temperature controlled autoinjector, a column
heater, a diode array detector (DAD), a fluorescence detector,
and an HP ChemStation for data storage and manipulation.
Mass spectral analysis was carried out on an API-300 triple
quadrupole, equipped with a TurboIonSpray source (MDS
SCIEX, Concord, Ont.) with flow from the HPLC system
Analysis of coloured fraction no. 1 was performed on a
Zorbax SB C18 column (Agilent Technologies), which was
subjected to a gradient elution with H2O (0.2% trifluoroacetic
acid (TFA)) and CH3CN (0.2% TFA). Absorbance was
monitored at 280, 320, 360, and 520 nm. Conditions for
mass spectral analysis in the positive mode included an
IonSpray voltage of 5500 V and a turboprobe temperature of
450 °C. Liquid chromatography/mass spectrometry (LC/MS)-
selective ion monitoring (SIM) was performed for the expected
molecular ions and for respective fragments so that observed
anthocyanins (Prior et al. 2001) and flavonol glycosides (Kader
et al. 1996) could be identified. A chlorogenic-acid standard
(ICN Nutritional Biochemicals, Costa Mesa, Calif.) and an
anthocyanin standard (Extrasynthese, France), containing
cyanidin, delphinidin, malvidin, peonidin, and petunidin-3-
glucosides, aided in the identification of peaks on the LC
Coloured fraction no. 2 was chromatographed on a Luna
Si (2) column (Phenomenex, Torrrance, Calif.). The ternary
solvent system consisted of methanol, dichloromethane, and
acetic acid – water (1:1). Absorbance was monitored at 280,
320, 360, and 520 nm; on the fluorescence detector, the
excitation was set at 276 nm and the emission at 316 nm.
Conditions for mass spectral analysis in the negative mode
included an IonSpray voltage of –5000 V and a turboprobe
temperature of 300 °C. Before the source was introduced,
0.75 mol ammonium hydroxide/L was introduced to the
column effluent through a tee, at a flow rate of 0.04 mL/min.
Assignments were accomplished by LC/MS SIM of the
expected molecular ions or prominent fragments (Lazarus et
al. 1999). Both epicatechin and catechin standards were
obtained using Fluka (Sigma–Aldrich Corp., St. Louis, Mo.).
Human DU145 prostate adenocarcinoma cells (ATCC,
Rockville, Md.) were cultured on 100-mm Falcon plastic
tissue-culture dishes (Becton Dickinson Labware, Franidin
Lakes, N.J.) in alpha minimal essential medium (Invitrogen,
Burlington, Ont.), supplemented with 10% (v/v) fetal bovine
serum (Hyclone/VWR Canlab, Mississauga, Ont.), at 37 °C
in 5% CO2. Defined medium (DM), which is a serum-free
medium, was also used. DM consisted of alpha minimal
essential medium supplemented with transferrin (Sigma,
Oakville, Ont.) and insulin (Sigma). Blueberry-enriched DM
was prepared by dissolving each of the fractions from low-
bush blueberry (CB, coloured fraction no. 1, and coloured
fraction no. 2) in DM, to a final concentration of 0.1, 0.5,
and 1.0 mg/mL, respectively. Subconfluent cells were exposed
© 2005 NRC Canada
638Biochem. Cell Biol. Vol. 83, 2005
to blueberry-enriched DM for 24 h, and the resulting condi-
tioned medium was analyzed for MMP activity.
Gelatin gel electrophoresis
Gelatinolytic activity was analyzed as described elsewhere
(Samuel et al. 1992). Briefly, an aliquot of conditioned medium
was mixed (4:1) with sample buffer, which consisted of 10%
SDS and 0.1% bromophenol blue in 0.3 mol Tris–HCl/L
(pH 6.8), and then incubated at 37 °C for 5 min. Aliquots of
each sample were loaded into wells of a 5% stacking gel and
resolved at a constant current at ambient temperature. The
10% resolving gel contained Type A gelatin (Sigma) to a final
concentration of 1 mg/mL. After electrophoresis, gels were
washed in 0.05 mol Tris–HCl/L (pH 7.4) and 2% Triton X-100
for 1 h at room temperature, followed by a 30-min wash in
0.05 mol Tris–HCl/L (pH 7.4) at room temperature. Gels
were then incubated at 37 °C for 24 h in a substrate buffer
containing 0.05 mol Tris–HCl/L (pH 7.4), 1% Triton X-100,
and 0.005 mol CaCl2/L. Following this incubation period,
gels were stained with 0.1% Coomassie Brilliant Blue R-250
in a solution of acetic acid, methanol, and water (5:10:85 by
volume). Prestained molecular-weight markers (Bio-Rad,
Hercules, Calif.) were also resolved on the same gel. Gelatinase
activity appeared as zones of clearing (due to gelatin degra-
dation) against a blue background. As a loading control,
identically loaded complementary Coomassie Brilliant Blue –
stained polyacrylamide gels without gelatin were used (Oetken
et al. 1992). Staining of these gels produces a number of
protein bands for each lane. The intensity of these stained
bands is used to ensure that an equal amount of protein was
added per lane for each individual zymogram (data not shown).
Necrotic and apoptotic cell-death assays
Cell cultures were analyzed for the presence of markers
for either necrotic or apoptotic cell death. Lactate dehydro-
genase (LDH) activity and caspase-3 activity are routinely
used indicators of necrotic and apoptotic cell death (Gay et
al. 1968; Cohen 1997). Commercially available assay kits
were used to detect the activity of LDH (Diagnostic Chemicals
Ltd., Charlottetown, P.E.I.) and caspase-3 (Sigma).
Analysis of fractions
One gram of CB produced 0.75 g of coloured fraction no. 1
and 0.085 g of coloured fraction no. 2. Through HPLC
analysis, coloured fraction no. 1 was found to contain mostly
anthocyanins and flavonol glycosides, with less than 2%
chlorogenic acid. Using LC/MS SIM, 21 anthocyanins were
identified in the profile when monitored at 520 nm (Fig. 1).
Using LC/DAD and a standard that contained cyanidin,
delphinidin, malvidin, peonidin, and petunidin-3-glucosides,
© 2005 NRC Canada
Matchett et al.639
Fig. 1. Liquid chromatography/mass spectrometry (LC/MS)-selective ion monitoring (SIM) analysis of colored fraction no. 1 showed
the presence of the following 21 anthocyanins and 3 flavonol glycosides: (1) delphinidin-3-galactoside, (2) delphinidin-3-glucoside,
(3) cyanidin-3-galactoside, (4) delphinidin-3-arabinoside, (5) cyanidin-3-glucoside, (6) petunidin-3-galactoside, (7) cyanidin-3-arabinoside,
(8) petunidin-3-glucoside, (9) peonidin-3-galactoside, (10) petunidin-3-arabinoside, (11) quercetin-3-glucoside, (12) malvidin-3-galactoside,
(13) peonidin-3-glucoside, (14) quercetin-3-galactoside, (15) peonidin-3-arabinoside, (16) malvidin-3-glucoside, (17) malvidin-3-arabinoside,
(18) quercetin-3-rhamnoside, (19) delphinidin-6-acetyl-3-galactoside, (20) cyanidin-6-acetyl-3-glucoside, (21) petunidin-6-acetyl-3-glucoside,
(22) malvidin-6-acetyl-3-galactoside, (23) peonidin-6-acetyl-3-glucoside, (24) malvidin-6-acetyl-3-glucoside.
the retention times of these 5 anthocyanins were confirmed.
Malvidin-3-glucoside, petunidin-3-glucoside, and delphinidin-
3-glucoside made up 37% of the anthocyanins. Glycosides
of cyanidin and, in particular, peonidin were minor contributors
to the total anthocyanin content of coloured fraction no. 1.
Three flavonol glycosides, quercetin-3-glucoside, quercetin-
3-galactoside, and quercetin-3-rhamnoside, were identified
using LC/DAD monitoring at 360 nm and LC/MS SIM. The
retention time of quercetin-3-glucoside was confirmed with
a standard. Kaempferol glycosides were not detected with
LC/DAD or LC/MS techniques. Chlorogenic acid was iden-
tified at a level of 13.5 µg/mg of coloured fraction no. 1 by
monitoring the HPLC/DAD chromatogram at 320 nm and
with the use of a standard. Thus, coloured fraction no. 1 is
Using LC/MS SIM, coloured fraction no. 2 was found to
contain a range of proanthocyanidins, from monomers to
decamers. Levels of the monomers epicatechin and catechin
were determined to be 0.125 and 0.187 µg/mg, respectively,
using the respective standards and LC/fluorescence detection
analysis. When the chromatographic profile was monitored
at 320 nm and 360 nm, no peaks were observed that corre-
sponded to chlorogenic acid or flavonol glycosides, respec-
tively. Thus, coloured fraction no. 2 is a PAC.
Effects of lowbush-blueberry fractions on MMP activity
Gelatinolytic activity of conditioned medium from DU145
cells after 24 h of treatment with CB fraction was determined.
DU145 cells were exposed to 0.1, 0.5, and 1.0 mg/mL CB
fraction. As shown in Fig. 2, in control cells not exposed to
the CB fraction, discrete bands of gelatinolytic activity were
noted at about 92 kDa and ~62–72 kDa. These gelatinolytic
activities correspond to MMP-9, activated forms of MMP-2
and pro-MMP-2. Activity of MMP-9 was unaffected by
0.1 mg/mL CB fraction, but was progressively inhibited by
0.5 and 1.0 mg/mL (Fig. 2). The proteolytic activity of both
the proenzyme forms of MMP-2 and activated isoforms of
MMP-2 were apparently unaffected by 0.1 mg/mL CB fraction,
but were progressively inhibited by 0.5 and 1.0 mg/mL. In
this regard, 1.0 mg/mL CB fraction results in a pronounced
decrease in gelatinolytic activity of the activated isoforms of
MMP-2, and a complete inhibition of the activity of pro-
MMP-2 (Fig. 2).
Gelatinolytic activity of conditioned medium from DU145
cells after 24 h of treatment with 0.1, 0.5, and 1.0 mg/mL
AN fraction is shown in Fig. 3. Figure 3 also shows that
MMP activity is differentially modulated after 24 h of
exposure to the AN fraction from lowbush blueberry. MMP-9
activity was unaffected after exposure to either 0.1 or
0.5 mg/mL AN fraction, but was inhibited in the presence
of 1.0 mg/mL. MMP-2 activity is unaffected by 0.1 mg/mL
AN fraction, but is affected by 0.5 and 1.0 mg/mL. In this
regard, pro-MMP-2 activity is virtually abolished in the pres-
ence of 1.0 mg/mL AN fraction.
Gelatinolytic activity of conditioned medium from DU145
cells after 24 h of exposure to 0.1, 0.5, and 1.0 mg/mL PAC
fraction from lowbush blueberry is shown in Fig. 4. Figure 4
also shows that all MMP activity is dramatically reduced in
the presence of the PAC fraction. Activity of MMP-9 is
markedly reduced in the presence of 0.1 mg/mL of PAC, and
is completely abolished in response to 0.5 and 1.0 mg/mL.
MMP-2 activity is also virtually eliminated after exposure to
0.1 mg/mL PAC, and is completely abolished in response to
0.5 and 1.0 mg/mL. To further characterize the effect of the
PAC fraction on MMP activity, the effects of the PAC fraction
at lower concentrations was evaluated. Figure 5 shows the
effect of 24-h exposure to 0.01 mg/mL and to 0.05 mg/mL
© 2005 NRC Canada
640Biochem. Cell Biol. Vol. 83, 2005
Fig. 2. Effects of crude fraction from lowbush blueberry (CB) on
MMP activity in DU145 cells. Gelatin gel electrophoresis was
performed on aliquots of conditioned medium from DU145 cells
after 24-h exposure to CB fraction. Gelatinolytic activity in cells
cultured in the absence of CB fraction is shown in lane 1, and
gelatinolytic activity in cells after 24-h exposure to 0.1 mg/mL
CB fraction is shown in lane 2, to 0.5 mg/mL is shown in lane 3,
and to 1.0 mg/mL is shown in lane 4.
Fig. 3. Effects of anthocyanin-enriched fraction from lowbush
blueberry (AN) on MMP activity in DU145 cells. Gelatin gel
electrophoresis was performed on aliquots of conditioned me-
dium from DU145 cells following 24 h exposure to AN frac-
tion. Control cells (lane 1), and cells exposed to 0.1 mg/mL
AN (lane 2), 0.5 mg/mL AN (lane 3), and 1.0 mg/mL AN frac-
tion (lane 4).
Fig. 4. Effects of proanthocyanidin-enriched fraction from
lowbush blueberry (PAC) on MMP activity in DU145 cells. Gel-
atin gel electrophoresis was performed on aliquots of conditioned
medium from DU145 cells after 24-h exposure to PAC fraction.
Control cells (lane 1), and cells exposed to 0.1 mg/mL PAC
(lane 2), 0.5 mg/mL PAC (lane 3) and 1.0 mg/mL PAC fraction
PAC fraction on MMP activity in DU145 cells. At these
concentrations, the PAC fraction was unable to inhibit MMP
activity in these cells, suggesting that a threshold value of
the PAC fraction is required for the inhibitory effect to be
expressed. For comparison, the remaining lanes in Fig. 5 are
replicate lanes of 0.1, 0.5, and 1.0 mg/mL PAC fraction,
respectively, shown in Fig. 4. Results presented in Figs. 2–5
are representative of observations noted from, at a minimum,
3 separate experiments.
Necrotic and apoptotic cell death in DU145 cells in
response to blueberry fractions
To determine whether the decrease in MMP activity in
response to treatment with the blueberry fractions was due
to a toxic effect on the cells, the contribution of generalized
cell death, resulting from cellular necrosis, and programmed
cell death, resulting from cellular apoptosis, was evaluated.
LDH and caspase-3 activities were used as indicators of
necrotic cell death and apoptotic cell death, respectively.
Biochemical assays were performed to quantify the presence
and the activity of LDH in DU145 cells exposed to the highest
concentration of CB, AN, and PAC fractions from lowbush
blueberry, namely 1.0 mg/mL. Figure 6 shows LDH activity
in DU145 cells in the absence and presence of these fractions.
Treatment of these cells with CB, AN, and PAC fractions
(1.0 mg/mL) for 24 h did not result in any more LDH activity
than in control cells, indicating that increased necrotic cell
death was apparently not a contributing factor to the reduction
in MMP activity noted in these cells in response to treatment
with these blueberry fractions. Figure 7 shows caspase-3
activity in DU145 cells in the absence and presence of blue-
berry fractions. Treatment of DU145 cells with CB, AN, and
PAC fractions (1.0 mg/mL) did not result in any more
caspase-3 activity than in control cells, indicating that apoptotic
© 2005 NRC Canada
Matchett et al.641
Fig. 5. Effects of lower concentrations of PAC fraction from lowbush blueberry on MMP activity in DU145 cells. Gelatin gel electro-
phoresis was performed on aliquots of conditioned medium from DU145 cells after 24-h exposure to various concentrations of PAC
fraction. Gelatinolytic activity in control cells (lane 1), and gelatinolytic activity in cells exposed to 0.01 mg/mL (lane 2), 0.05 mg/mL
(lane 3), 0.1 mg/mL (lane 4), 0.5 mg/mL (lane 5), and 1.0 mg/mL (lane 6) PAC fraction, respectively.
Fig. 6. Lactate dehydrogenase (LDH) activity as a measure of necrotic cell death. LDH activity was measured in DU145 cells after 24-h
exposure to defined medium (control cells, lane 1), defined medium supplemented with CB fraction (1.0 mg/mL, lane 2), defined me-
dium supplemented with AN fraction (1.0 mg/mL, lane 3), and defined medium supplemented with PAC fraction (1.0 mg/mL, lane 4).
Results indicated are from duplicate experiments with 5 culture plates per condition tested.
cell death was also not a contributing factor to the reduction
in MMP activity noted in these cells in response to treatment
with these blueberry fractions. These observations suggest
that the decreased expression of MMPs observed in DU145
cells in response to treatment with blueberry fractions were
target-directed and not the result of cellular death.
This study demonstrated that MMP activity from human
prostate cancer cells, specifically DU145 cells, decreases
after exposure to flavonoid-enriched fractions from lowbush
blueberry. These results are in keeping with other studies in-
vestigating the effects of flavonoids in cancer models
(Garbisa et al. 2001; Sartor et al. 2002; Sato et al. 2002).
Although the gelatinolytic activity of the MMPs in DU145
cells decreases in response to treatment with blueberry fractions,
the effects of each flavonoid-enriched fraction on MMP activity
is varied. This is not unexpected; although the anthocyanins
and proanthocyanidins are both flavonoids, they comprise
distinct flavonoid subclasses and, as such, have different
chemical structures. The CB fraction contains approximately
75% anthocyanins and 8.5% proanthocyanidins, which may
have had synergistic or additive effects. To the best of our
knowledge, this study is the first to demonstrate a link
between bioactive-containing fractions isolated from lowbush
blueberry and the inhibition of MMP expression. The PAC
fraction contains a range of proanthocyanidins, from monomers
to decamers (Fig. 1). The AN fraction contains a number of
potentially bioactive compounds, including 4 main glucosides
(malvidin, petunidin, delphinidin and quercetin), quercetin-
3-galactoside, and quercetin-3-rhamnoside (Fig. 1). Antho-
cyanins and proanthocyanidins from other sources have been
shown to affect MMP activity. Delphinidin has been shown
to decrease the activity of MMP-2 (in human neuroblastoma
cells) and MMP-9 (in HT-1080 human fibrosarcoma cells)
(Sartor et al. 2002). Green tea catechins, which are very
similar to proanthocyanidins, have been shown to downregulate
MMP activity in cancer cells (Gupta et al. 1999; Garbisa et
al. 2001). Epigallocatechin-3-gallate has been reported to
inhibit MMP-2 and MMP-9 activity (Sartor et al. 2002).
Myricetin, which is a proanthocyanidin, also effectively inhibits
the activity of MMPs (Sartor et al. 2002). In addition, the
proanthocyanidin nobiletin has been shown to decrease the
activity of MMP-1 and MMP-9 in vitro (Sato et al. 2002).
The results presented in this study are consistent with these
observations. Studies are currently underway to define the
exact nature of the bioactive compounds found in these frac-
tions isolated from lowbush blueberries.
It is important to note that these blueberry fractions did
not induce necrotic cell death in these cells. These findings
indicate a specificity between the action of the bioactive
components contained in these blueberry fractions and the
inhibition of MMP (MMP-2 and MMP-9) activity in DU145
cells. InStudies looking at the mechanism(s) involved in this
inhibitory process are ongoing.
MMP-2 and MMP-9 are the major mediators of basement
membrane degradation and, as such, are possible targets for
the development of novel anticancer treatments. Our findings
demonstrate that flavonoid-enriched fractions from lowbush
blueberries can downregulate the activities of specific MMPs
in a target-directed manner, suggesting that further under-
standing of the complex properties of flavonoids, in particular
those from lowbush blueberry, may allow for the further
© 2005 NRC Canada
642 Biochem. Cell Biol. Vol. 83, 2005
Fig. 7. Caspase-3 activity as a measure of apoptotic cell death. Caspase-3 activity was measured in DU145 cells after 24-h exposure to
defined medium (control cells, lane 1), defined medium supplemented with CB fraction (1.0 mg/mL, lane 2), defined medium supple-
mented with AN fraction (1.0 mg/mL, lane 3), and defined medium supplemented with PAC fraction (1.0 mg/mL, lane 4). Results in-
dicated are from duplicate experiments with 5 culture plates per condition tested.
development and refinement of the role of flavonoids in the Download full-text
prevention of carcinogenesis and metastasis.
In conclusion, our results are the first to describe a potential
regulatory effect of flavonoids isolated from lowbush blue-
berry on human prostate cancer cells. This regulatory effect
was directed at the activity of MMP-2 and MMP-9. These
activities may be one of several targets of the potential
antiproliferative effects associated with flavonoid-containing
fractions isolated from lowbush blueberries.
This work was supported by research grants from the P.E.I.
Cancer Research Council to M.S., K.G.P., and R.A.R.H. M.D.M.
is the recipient of a graduate student scholarship from the
Canadian Cancer Society (P.E.I. Division) and a Canada
Graduate Scholarship (CGS-M) from N.S.E.R.C. The blue-
berry extracts used in this study were kindly provided by
Dr. W. Kalt, A.A.F.C., Kentville, N.S.
Bomser, J., Madhavi, D.L., Singletary, K., and Smith, M.A.L. 1996.
In vitro anticancer activity of fruit extracts from Vaccinium
species. Planta Med. 62: 212–216.
Cohen, G.M. 1997. Caspases: the executioners of apoptosis (a
review). J. Biochem. 326: 1–16.
Freije, J.M., Balbin, M., Pendas, A.M., Sanchez, L.M., Puente, X.S.,
and Lopez-Otin, C. 2003. Matrix metalloproteinases and tumor
progression. Adv. Exp. Med. Biol. 532: 91–107.
Garbisa, S., Sartor, L., Biggin, S., Salvato, B., Benelli, R., and Albini,
A. 2001. Tumor gelatinases and invasion inhibited by the green tea
flavanol epigallocatechin-3-gallate. Cancer, 91: 822–832.
Gay, R.J., McComb, R.B., and Bowers, G.N. 1968. Optimum
conditions for human lactate dehydrogenase isoenzymes as
they affect total lactate dehydrogenase activity. Clin. Chem.
Gupta, S., Ahmad, N., Mohan, R.R., Husain, M.M., and Mukhtar, H.
1999. Prostate cancer chemoprevention by green tea: in vitro and
in vivo inhibition of testosterone-mediated induction of ornithine
decarboxylase. Cancer Res. 59: 2115–2120.
Kader, F., Rovel, B., Girardin, M., and Metche, M. 1996. Fractionation
and identification of phenolic compounds of highbush blueberries
(Vaccinium corymbosum L.). Food Chem. 55: 35–40.
Kahkonen, M.P., and Heinonen, M. 2003. Antioxidant activity of
anthocyanins and their aglycons. J. Agric. Food Chem. 51:
Kalt, W., McDonald, J.E., and Donner, H. 2000. Anthocyanins,
phenolics, and antioxidant capacity of processed lowbush blue-
berry products. J. Food Sci. 65: 390–393.
Kandaswami, C., and Middleton, E, Jr. 1995. Flavonoids as antiox-
idants. In Natural Antioxidants – Chemistry, Health Effects and
Applications. Edited by F. Shahidi. AOCS Press, Champaign,
Ill. pp. 174–203.
Knekt, P., Kumpulainen, J., Jarvinen, R., Rissanen, H., Heliovaara, M.,
Reunanen, A. et al. 2002. Flavonoid intake and risk of chronic
diseases. Am. J. Clin. Nutr. 76: 560–568.
Lazarus, S.A., Adamson, G.E., Hammerstone, J.F., and Schmitz, H.H.
1999. High-performance liquid chromatography/mass spectrometry
analysis of proanthocyanidins in foods and beverages. J. Agric.
Food Chem. 47: 3693–3701.
Lynch, C.C., and Matrisian, L.M. 2002. Matrix metalloproteinases
in tumor-host cell communication. Differentiation, 70: 9–10,
Oetken, C., Pessa-Morikawa, T., Autero, M., Andersson, L.C., and
Muestelin, T. 1992. Reduced tyrosine phosphorylation in polyamine-
starved cells. Exp. Cell Res. 202: 370–375.
Prior, R.L., Lazarus, S.A., Cao, G., Muccitelli, H., and Hammerstone,
J.F. 2001. Identification of procyanidins and anthocyanins in blue-
berries and cranberries (Vaccinium spp.) using high-performance
liquid chromatography/mass spectrometry. J. Agric. Food Chem.
Pupa, S.M., Menard, S., Forti, S., and Tagliabue, E. 2002. New
insights into the role of extracellular matrix during tumor onset
and progression. J. Cell. Physiol. 192: 259–267.
Samuel, S.K., Hurta, R.A.R., Kondaiah, P., Khalil, N., Turley, E.A.,
Wright, J.A., and Greenberg, A.H. 1992. Autocrine induction of
tumor protease production and invasion by a metallothionein-
regulated TGF-β1. EMBO J. 11(4): 1599–1605.
Sartor, L., Pezzato, E., Dell’Aica, I., Caniato, R., Biggin, S., and
Garbisa, S. 2002. Inhibition of matrix-proteases by polyphenols:
chemical insights for anti-inflammatory and anti-invasion drug
design. Biochem. Pharmacol. 64: 229–237.
Sato, T., Koike, L., Miyata, Y., Hirata, M., Mimaki, Y., Sashida, Y.,
Yano, M., and Ito, A. 2002. Inhibition of activator protein-1
binding activity and phosphatidylinositol 3-kinase pathway by
nobiletin, a polymethoxy flavonoid, results in augmentation of
tissue inhibitor of metalloproteinases-1 production and suppression
of production of matrix metalloproteinases-1 and -9 in human
fibrosarcoma HT-1080 cells. Cancer Res. 62: 1025–1029.
Smith, M.A.L., Marley, K.A., Seigler, D., Singletary, K.W., and
Meline, B. 2000. Bioactive properties of wild blueberry fruits. J.
Food Sci. 65: 352–356.
Stetler-Stevenson, W.G., and Yu, A.E. 2001. Proteases in invasion:
matrix metalloproteinases. Semin. Cancer Biol. 11: 143–152.
Sweeney, M.I., Kalt, W., MacKinnon, S.L., Ashby, J., and Gottschall-
Pass, K.T. 2002. Feeding rats diets enriched in lowbush blueberries
for six weeks decreases ischemia-induced brain damage. Nutr.
Neurosci. 5(6): 427–431.
Visse, R., and Nagase, H. 2003. Matrix metalloproteinases and
tissue inhibitors of metalloproteinases: structure, function, and
biochemistry. Circ. Res. 92(8): 827–839.
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