Pectin induces apoptosis in human prostate cancer cells: correlation of apoptotic function with pectin structure
Pectin induces apoptosis in human prostate cancer cells: correlation of apoptotic function with pectin structure
Crystal L Jackson 0 1
Tina M Dreaden 0 1
Lisa K Theobald 0 1
Nhien M Tran 0 1
Tiffany L Beal 0 1
Manal Eid 2
Mu Yun Gao 0 1
Robert B Shirley 2
Mark T Stoffel 0 1
M Vijay Kumar 2
Debra Mohnen 0 1
0 Department of Biochemistry and Molecular Biology, The University of Georgia , Athens, GA 30602
1 Complex Carbohydrate Research Center
2 Medical College of Georgia and VA Medical Center , Augusta, GA 30912
1To whom correspondence should be addressed; Tel. þ1 706 542 4458: Fax: þ1 706 542 4412: e-mail: # The Author 2007. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: 805
Received on September 26, 2006; revised on May 9, 2007; accepted on May 11,
Treatment options for androgen-independent prostate
cancer cells are limited. Therefore, it is critical to identify
agents that induce death of both androgen-responsive and
androgen-insensitive cells. Here we demonstrate that a
product of plant cell walls, pectin, is capable of inducing
apoptosis in androgen-responsive (LNCaP) and
androgenindependent (LNCaP C4-2) human prostate cancer cells.
Commercially available fractionated pectin powder (FPP)
induced apoptosis (approximately 40-fold above
nontreated cells) in both cell lines as determined by the
Apoptosense assay and activation of caspase-3 and its
substrate, poly(ADP-ribose) polymerase. Conversely, citrus
pectin (CP) and the pH-modified CP, PectaSol, had little
or no apoptotic activity. Glycosyl residue composition
and linkage analyses revealed no significant differences
among the pectins. Mild base treatment to remove ester
linkages destroyed FPP’s apoptotic activity and yielded
homogalacturonan (HG) oligosaccharides. The treatment
of FPP with pectinmethylesterase to remove
galacturonosyl carboxymethylesters and/or with
endopolygalacturonase to cleave nonmethylesterified HG caused no major
reduction in apoptotic activity, implicating the requirement
for a base-sensitive linkage other than the
carboxymethylester. Heat treatment of CP (HTCP) led to the induction
of significant levels of apoptosis comparable to FPP,
suggesting a means for generating apoptotic pectic
structures. These results indicate that specific structural
elements within pectin are responsible for the apoptotic
activity, and that this structure can be generated, or
enriched for, by heat treatment of CP. These findings
provide the foundation for mechanistic studies of pectin
apoptotic activity and a basis for the development of
pectinbased pharmaceuticals, nutraceuticals, or recommended
diet changes aimed at combating prostate cancer occurrence
Prostate cancer is the most common malignancy and the
second leading cause of death from cancer in American
men. The goal of many cancer therapies, such as antihormone
therapy and chemotherapy, is to induce apoptosis in tumor
cells. Androgen deprivation therapies induce cell death in
androgen-sensitive cells (Colombel and Buttyan 1995,
Bruckheimer et al. 1999, Perlman et al. 1999), whereas
androgen-insensitive cells remain unaffected (Kozlowski et al. 1991;
Santen 1992; Kreis 1995). However, androgen-insensitive cells
are capable of undergoing apoptosis. Thus, the identification of
novel methods to induce apoptosis in prostate cancer cells
irrespective of their androgen response has significant therapeutic
value. In this paper, we demonstrate that pectin, a plant
polysaccharide, induces apoptosis in both androgen-responsive and
androgen-independent prostate cancer cells. This is the first
extensive analysis correlating structural features of pectin
with apoptosis-inducing activity in cancer cells.
The role of dietary components in cancer prevention and
progression is an area of increasing clinical and scientific
interest. Both the American Institute of Cancer Research and the
World Cancer Research Fund estimate that 30 – 40% of
worldwide cancer cases are preventable by dietary means. Pectin is a
natural complex plant polysaccharide present in all higher
plant primary cell walls and, consequently, is a dietary
component of all fruits and vegetables. Pectin accounts for
approximately 30% of the primary walls of all higher plants
except the grass family, where it makes up about 10% of the
primary wall. Pectin has multiple roles in plant growth,
development, and disease resistance (Ridley et al. 2001), and is used
as a gelling and stabilizing agent in the food industry (Thakur
et al. 1997).
Previous research has shown that pectin can suppress
colonic tumor incidence in rats (Heitman et al. 1992) and
inhibit cancer cell metastasis in mice and rats (Platt and Raz
1992; Pienta et al. 1995; Nangia-Makker et al. 2002). Pectin
has been shown to bind to B16-F1 melanoma cells in vitro
(Platt and Raz 1992). Furthermore, when injected
intravenously in mice, relatively large commercial pectin increased
homotypic cell – cell aggregation and metastasis to the lung
while pH-modified, relatively small pectin inhibited lung
metastasis (Platt and Raz 1992), demonstrating a differential
response depending upon the type of pectin used. Oral
administration of a pH-modified citrus pectin (CP) significantly
reduced metastasis of rat prostate adenocarcinoma
MATLyLu to the lung (Pienta et al. 1995). It is noteworthy that
those anti-metastatic effects of pectins occurred in the absence
of cell toxicity (Inohara and Raz 1994). From such data, it
has been hypothesized that pectins can bind to cancer cell
surface galectins (galactose-binding lectins) and interfere
with cell – cell or cell – matrix adhesion, inhibiting metastatic
lesions (Inohara and Raz 1994).
Several studies have indicated that pectins not only inhibit
metastatic lesions, but also induce apoptosis in cancer cells.
Azoxymethane-injected rats fed a citrus pectin or fish oil/
pectin diet had a greater number of apoptotic cells per
colon crypt column compared with rats fed corn oil and/or
cellulose (Chang W-CL et al. 1997). Furthermore, pectin/
fish oil-fed rats had a lower incidence of adenocarcinoma
(51.5%) than animals fed cellulose/corn oil (75.6%) (Chang
W-CL, et al. 1997; Chang WC, et al. 1997). The apoptotic
index in the distal colon of pectin-fed rats was higher than that
in rats fed a standard diet. This was accompanied by reduced
expression of the anti-apoptotic protein Bcl-2 and activation
of caspase-1 and poly(ADP-ribose) polymerase (PARP),
substrates of caspases (Avivi-Green, Madar, et al. 2000;
AviviGreen, Polak-Charcon 2000b, c). Similarly, the administration
of a pectin-rich 15% orange-pulp diet to
dimethylhydrazineinjected Sprague-Dawley rats resulted in a decreased
number of endophytic tumors, an activation of caspase-3,
and an increased activity of T-cell killers in the tumors; all
characteristic anti-tumor effects (Kossoy et al. 2001). In
human colon adenocarcinoma HT29 cells, caspase-3 activity
increased significantly when cells were treated with 10 mg/
mL low-methylated apple pectin (Olano-Martin et al. 2003).
Preclinical studies using a modified a citrus pectin, GCS-100,
showed induction of apoptosis in human multiple myeloma
cell lines that are resistant to conventional and bortezomib
therapies (Chauhan et al. 2005). While GCS-100 did not alter
normal lymphocyte cell viability, in the myeloma lines it
induced DNA fragmentation and the activation of caspase-8,
caspase-3, and PARP indicating that GCS-100 triggered
apoptosis primarily through the intrinsic pathway. Interestingly,
the GCS-100 also inhibited the growth of multiple myeloma
cells directly purified from patients who had relapsed
following multiple therapies with dexamethasone, bortezomib, and
thalidomide (Chauhan et al. 2005), providing evidence that
GCS-100 can induce apoptosis in chemo-resistant myeloma
cells. Taken together, these results suggest that exposure of
malignant cells to pectin induces apoptosis and reduces
Although the usefulness of pectins in cancer therapy is
beginning to be appreciated, the mechanism of induction
of apoptosis by pectins is not known. The elucidation of
the mechanism(s) of action of pectin is complicated by (i)
the structural complexity of this plant-derived cell wall
polysaccharide, (ii) the modifications in pectin structure
resulting from the process of its extraction from plants,
and (iii) the additional modifications of pectin structure
that result from the diverse fragmentation techniques used
to produce specialized pectins [e.g. high-pH (base)
treatment (Platt and Raz 1992; Pienta et al. 1995; Eliaz 2001;
Nangia-Makker et al. 2002)]. Pectin is a family of
complex polysaccharides that contain 4-linked
a-D-galacturonic acid residues (O’Neill et al. 1990). It is generally
accepted that three types of polysaccharides comprise
pectin: a linear homopolymer known as homogalacturonan
(HG), the branched polymer rhamnogalacturonan I (RG-I),
and the substituted galacturonans of which the ubiquitous
member is rhamnogalacturonan II (RG-II) (Albersheim
et al. 1996; Ridley et al. 2001).
Homogalacturonan accounts for 57 – 69% of pectin
(Mohnen 2002) and is a linear polymer of 1,4-linked
a-Dgalactopyranosyluronic acid (GalA) in which some 8 – 74%,
(Voragen et al. 1986) of the carboxyl groups may be methyl
esterified. HG may also be partially O-acetylated at C-3 or
C-2. The length of HG remains unclear, but degrees of
polymerization of 30 – 200 have been reported (reviewed in
Mohnen 1999). Rhamnogalacturonan-I (RG-I) is a family of
pectic polysaccharides that accounts for 7 – 14% of pectin
and consists of a backbone of the repeating disaccharide
[!4)-a-D-GalpA-(1 ! 2)-a-L-Rhap-(1 ! ]. Roughly 20 –
80% of the rhamnoses of RG-I are substituted by L-arabinose,
D-galactose, L-arabinans, galactans, or arabinogalactans
(O’Neill et al. 1990; Mohnen 1999; Ridley et al. 2001). The
side branches include a-1,5- and a-1,3-linked arabinans,
b-1,4-linked or b-1,3 and b-1,6-linked galactans, and
arabinogalactans of diverse linkages (reviewed in Mohnen 1999). The
average MW of sycamore RG-I is estimated to be 105 – 106 Da
(O’Neill et al. 1990). RG-II is a substituted galacturonan that
accounts for 10 – 11% of pectin and whose structure is highly
conserved across plant species. RG-II consists of a HG
backbone with four side branches of complex structure. RG-II is
arguably the most complicated polysaccharide in nature,
consisting of 12 different types of sugars joined in more than 20
different linkages and contains unusual sugars such as
2-Omethyl xylose, 2-O-methyl fucose,
3-deoxy-D-lyxo-2-heptulopyranosylaric acid and apiose (O’Neill et al. 2004).
HG, RG-I, and RG-II are generally purified from
intact-purified cell walls by treatment with the enzyme
endopolygalacturonase, which cleaves a stretch of four or more contiguous
non-methylesterified a-1,4-linked galacturonic acid residues
in HG (Chen and Mort 1996). The ability to cleave the
pectic polysaccharides from the intact wall has been used as
evidence to support the model that the backbones of the
three pectic polysaccharides are covalently linked together in
the wall. Nakamura et al. (2002) have provided structural
evidence to support this linkage. Additional mechanisms for the
association of the pectic polysaccharides include the formation
of borate ester cross-linked RG-II dimers (Kobayashi et al.
1996; O’Neill et al. 1996), HG – Ca2þ interchain salt bridges
(Morris et al. 1982), and possible feruloyl polysaccharide
ester crosslinks (Fry 1982; Ishii 1997). However, a complete
understanding of the larger 3D structure of pectin in the
wall, and how the polymers are associated in the wall, is still
lacking. For example, the role of proposed ester crosslinks
between the carboxyl moiety of galacturonic acid in HG and
other pectic or wall polymers remains unclear (Kim and
Carpita 1992; Brown and Fry 1993; Iiyama et al. 1994; Hou
and Chang 1996; Djelineo 2001). Although the structural
identity of the proposed ester(s) linkage at the carboxyl group of
galacturonic acid in pectin is not known, the data presented
in this paper, for the first time, suggest that this linkage may
be involved in the apoptotic activity of pectin.
Whereas the structure of RG-II is conserved, the structures
of HG and RG-I are more variable in different plants and
cell types because of differences in polymer size, in the
patterns of acetylation, methylation, and other modifications
of GalA in the backbone of HG, and of variations in the
length and type of side branches on the RG-I backbone.
Furthermore, the production of commercial pectin usually
involves an acid extraction of pectin from dried citrus peels or
apple pomace (Thakur et al. 1997), a process which results in
the destruction and loss of RG-II and of some RG-I. In some
cases, commercial CP may be further treated with base or heat
to yield partially fragmented, and structurally modified, pectin.
Thus, an evaluation of the biological effectiveness of pectin
must be accompanied by an understanding of the structure of
the biologically active pectin.
The goals of the present research were two-fold: (i) to
examine the potential use of pectin in cancer therapy and
(ii) to correlate the structural features of pectin with its
apoptosis-inducing activity. The rationale is that pectins have
multiple health promoting effects (Yamada et al. 2003;
Yamada 1996) and induce apoptosis in several types of cancer
cells. The extraordinary structural complexity of pectin
(Ridley et al. 2001) makes it a potential multi-functional
therapeutic agent. We report that fractionated pectin powder
(FPP), a commercially available pectin generated by heat
modification of CP, as well as heat-treated CP created in our
laboratory, induce apoptosis in prostate cancer cells.
Furthermore, by manipulating the structure of pectin, we
demonstrate that a base-sensitive linkage is necessary for the
apoptotic activity of pectin.
Effect of three commercial pectins on apoptosis in human
prostate cancer cell lines LNCaP and LNCaP C4-2
The androgen response of prostate cancer cells is an important
criterion for devising appropriate therapy. Since the cell line
was developed, LNCaP cells have extensively been utilized as
an androgen-responsive cell line in prostate cancer research.
The sister cell line LNCaP C4-2 was derived from LNCaP
cells by passing twice through mice and is androgen dependent.
As discussed earlier, the structure of commercially available
pectin differs depending on the source and method of its
preparation. Therefore, to examine whether pectins prepared using
different extraction protocols have similar biological effects,
prostate cancer cells were treated with several commercially
available pectin preparations: a citrus pectin (CP), Pectasol
(PeS), and fractionated pectin powder (FPP). CP represents
the starting pectin (i.e. “mother pectin”) used to make the
modified pectins. PeS is a pH-modified pectin generated by base
treatment of CP. PeS is similar to the pH-modified pectins
used in many of the previously published studies reporting
anti-cancer effects of pectin (Platt and Raz 1992; Pienta et al.
1995; Chauhan et al. 2005). FPP represents another type of
commercial pectin generated by heat treatment of CP (HTCP)
at 100 – 132 8C for 20 min to 5.5 h. Cells incubated in media
devoid of pectin served as negative controls, whereas cells
treated with thapsigargin, a compound that induces apoptosis
in these prostate cancer cells, were the positive controls.
Figure 1 shows that, among the pectins tested, FPP induced
significant apoptosis in both LNCaP (Figure 1A) and LNCaP
C4-2 (Figure 1B) cells, whereas PeS and CP induced little or
no apoptosis. As expected, thapsigargin, utilized as a positive
control, induced significant apoptosis. In the above experiments,
apoptosis was quantified using an enzyme-linked
immunosorbent assay (ELISA) to measure the generation of an
apoptosisspecific neoepitope of cytokeratin-18, a substrate of activated
caspase. To confirm the induction of apoptosis further, cell
extracts were analyzed by immunoblots, which showed
activation of caspase-3, an inducer of apoptosis, in both LNCaP
and LNCaP C4-2 cells treated with FPP (Figure 1C). The
35 kDa procaspase-3 was cleaved into 19, 17, and 12 kDa
products when treated with FPP, confirming significant apoptotic
response. None of the other pectins induced a similar response,
supporting the ELISA assay data. As expected,
thapsigargintreated cells showed activation of caspase-3.
The analysis of the cell extracts for the presence of PARP, a
substrate of activated caspases, showed the presence of an
85 kDa cleaved product in the positive control
(thapsigargintreated cells) and in the cells treated with FPP (Figure 1D),
but not with PeS or CP. Thus, we confirmed the induction of
apoptosis by FPP using three lines of evidence: (i) activation
of capase-3 and effect of the activated caspase on two
substrates, (ii) PARP, and (iii) cytokeratin-18.
As the above results showed that among the pectins tested,
only FPP induced appreciable apoptosis, experiments were
conducted to identify the effective dose of FPP required for
apoptosis. The treatment of LNCaP cells with increasing
concentrations of FPP showed that 3 mg/mL FPP induced
maximum apoptosis (Figure 2). Lower concentrations of 0.01
and 0.10 mg/mL of FPP did not affect LNCaP cells,
whereas 1.0 mg/mL induced significant apoptosis. As no
significant differences in apoptosis were noted between 1 and
3 mg/mL, subsequent experiments were conducted using
1 mg/mL FPP.
Defining the pectic structure(s) in FPP that induce apoptosis
The above results showed that, among the tested compounds,
only FPP induced apoptosis. Therefore, it was of interest to
analyze the structure of FPP to identify the components(s)
that imparted appreciable apoptosis-inducing activity. Pectin
is a complex polymer consisting of the polysaccharides HG,
RG-I, and RG-II that are held together in the plant wall by
incompletely defined covalent and/or noncovalent
interactions. We hypothesized that the apoptosis-inducing activity
resided in one or more of the pectic polysaccharides HG,
RG-I, and RG-II. To test this hypothesis and to determine
which structural features of pectin were required to induce
apoptosis, LNCaP and LNCaP C4-2 cells were treated for
48 h with 1 mg/mL of pectin fractions enriched for HG,
RG-I, or RG-II. The HG fraction consisted primarily of HG
oligosaccharides (oligogalacturonides; OGAs) of degrees of
polymerization of 7 – 23. Figure 3 shows that none of the
purified pectic polysaccharides induced appreciable apoptosis in
either the LNCaP (Figure 3A) or the LNCaP C4-2 cells
(Figure 3B), suggesting that the apoptosis-inducing activity
did not reside in the individual HG, RG-I, or RG-II
polysaccharides. Thus, we hypothesized that some aspect of pectin
structure lost during the preparation of the purified RG-I,
RG-II, and HG was responsible for the apoptosis-inducing
activity in FPP.
To determine whether major structural differences among
FPP, CP, and PeS accounted for the apoptosis-inducing activity,
the relative sizes of FPP, PeS, and CP were established by
separation over a Superose 12 HR10 size exclusion
chromatography (SEC) column in 50 mM sodium acetate and 5 mM
ethylenediaminetetraacetic acid (EDTA). The eluted pectins
were detected using an uronic acid colorimetric assay.
Figure 3C shows that FPP was intermediate in size between the
polydisperse and large CP (Figure 3E), which has an estimated
molecular mass range of 23 – 71 kDa and the relatively
uniformly sized and low-molecular-weight PeS (Figure 3D),
which has a molecular mass range of 10 – 20 kDa. The
polydisperse and intermediate size of FPP (Figure 3C) may indicate
that the apoptosis-inducing activity requires an intermediate
size polymeric structure, although proof of this requires
elucidation of the specific apoptosis-inducing moiety.
The individual pectic polysaccharides HG, RG-I, and RG-II
are purified from wall-derived pectin by a combination of
chemical and enzyme treatments. A common initial
purification step to isolate HG, RG-I, and RG-II is to subject
plant walls to a mild base treatment to remove ester linkages
within pectin. Such a treatment removes, for example,
methyl esters on the GalA residues in HG rendering the
pectin more accessible to the action of enzymes, such as
endopolygalacturonases (EPGs). EPGs cleave HG in regions with
contiguous nonesterified GalA residues (Chen and Mort 1996),
thereby fragmenting the pectin and releasing RG-I and RG-II.
Therefore, to determine whether ester linkages are required for
the apoptosis-inducing activity, FPP was deesterified by mild
base treatment. FPP was brought to pH 12 for 4 h at 2 8C on
ice, neutralized, and repeatedly lyophilized against water, as
described in the Materials and methods section.
Alternatively, the FPP or the deesterified FPP was treated
with EPG to fragment the pectin. LNCaP cells were treated
with the enzyme-treated FPP to determine whether cleavage
of an HG-containing region of FPP affected its
apoptosisinducing activity. Apoptosis assays showed that EPG treatment
Fig. 2. Concentration curve for apoptosis-inducing effect of FPP. LNCaP cells
were treated for 48 h with increasing concentrations (0.01 – 3 mg/mL) FPP,
with 1 mM Thapsigargin (Pos) or with media alone (Neg). Apoptosis was
measured using the M30 Apoptosense assay. Data are the average of duplicate
apoptosis assays of duplicate cell extracts (29 mg protein) + SEM. Comparable
results were obtained in two independent experiments.
of FPP had little or no effect on its apoptosis-inducing activity
(Figure 3F). On the contrary, mild base treatment to remove
ester linkages almost completely abolished the
pectininduced apoptotic response, indicating that one or more
base-sensitive linkages (e.g. ester linkages) of FPP is
necessary for its apoptotic function.
To determine whether the apoptotic effects of FPP were due
to uniquely enriched specific pectic carbohydrates, the
glycosyl residue composition of FPP, PeS, and CP were compared.
Figure 4 shows the average mole percent glycosyl residue
composition of unmodified and base-treated FPP, CP, and
PeS. As expected, GalA was the major component in all
three pectins tested. No consistent correlation was found
between the apoptosis-inducing activity and the glycosyl
residue composition of FPP compared with unmodified CP
and PeS. For example, FPP was arabinose-rich compared
with PeS, but had comparable amounts of Ara to CP.
Likewise, both FPP and PeS had less Gal, slightly less Rha,
and more GalA than CP.
As noted in Figure 3F, mild base treatment to remove ester
linkages in FPP destroyed its apoptosis-inducing activity. To
determine whether this treatment altered the glycosyl residue
composition, FPP, CP, and PeS were brought to pH 12 for
4 h, neutralized, and lyophilized (as described in the
Materials and methods section), and the glycosyl residue
composition was determined. Surprisingly, the deesterification step
led to a large reduction (90%) in the amount of Ara recovered
in FPP (Figure 4), but did not alter Ara significantly in CP and
PeS. At this time, the reason for the loss of Ara in deesterified
FPP and its importance for biological activity are unclear.
Fig. 4. Glycosyl residue composition analyses of unmodified and base-treated
FPP, PeS, and CP. Composition analyses were done by GC – MS of TMS
derivatives of methyl glycosides produced by acid methanolysis (York et al.
1985). Data are the average + SEM mole percentage of each sugar from
duplicate analyses from two separate experiments (N ¼ 4).
To evaluate the fine structural differences among FPP, CP,
and PeS, and in an effort to identify the pectic structure(s) in
FPP responsible for its biological activity further, the
glycosyl residue linkages present in FPP, CP, and PeS were
determined using both a single (Table I) and double (Table II)
methylation procedure. The single methylation method has
the disadvantage of yielding incomplete methylation with
the resulting incomplete linkage results, but has the
advantage of avoiding or reducing fragmentation of the pectin
because of b-elimination of the glycosyluronic acid linkages
in the HG. On the other hand, the double methylation
method leads to more complete methylation, but can lead
to b-elimination of the glycosyluronic acid linkages (York
et al. 1985), and thus, to an apparent increase in the
amount of terminal GalA and a loss in the apparent
4-linked GalA (Table II). Comparison of the linkage data
for the unmodified pectins showed that all three pectins
contain primarily HG (the presence of 4-linked GalA) and
RG-I (2-linked Rha and 2,4-linked Rha). FPP and CP
contained 5-linked arabinan and 4-linked galactan, which are
known to occur as side chains of RG-I, whereas these
linkages were absent or greatly reduced in PeS. As expected,
in the double methylation procedure (Table II), there was
an unrealistically high apparent terminal-GalA content,
likely due to fragmentation of the HG because of
b-elimination. The amount of terminal GalA was significantly less
in the single methylation method. Likewise, as expected,
there was more apparent undermethylation of the pectin in
the single methylation method, identified as apparent
2,3,4,-linked GalA. Undermethylation likely also explains
the greater amounts of 2,3-linked and 3,4-linked GalA
obtained in the single methylation method compared with
the double methylation method. The only linkage data that
correlated with the apoptosis-inducing activity of FPP was
the higher amounts of terminal and 5-linked Ara in FPP.
These linkages were lost in the base-treated FPP. Whether
these glycosyl residues are involved in the apoptosis response
remains to be shown.
Linkage analysis was carried out as explained in Table I, except that following the first methylation the permethylated material was reduced by super-deuteride
and the reduced sample was re-methylated using the NaOH/MeI method (see the Materials and methods section). Data are the average percentage of glycosyl
residues with the specified linkages + SEM from duplicate analyses (N ¼ 2). aU represents undermethylated; apparent 2,3,4,-linked GalA. bT represents
Evidence for a base-sensitive cross-link in FPP required
for apoptotic activity
As described earlier, the mild base treatment of FPP to
remove ester-linked moieties in pectin destroyed its
apoptosisinducing activity, while treatment with EPG had little or no
effect on its apoptosis-inducing activity (Figure 3F). To
determine how the base treatment affected the function of FPP,
intact, EPG-treated, base-treated (deesterified), and deesterified
plus EPG-treated FPP were separated over high percentage
polyacrylamide gels. These gels are particularly useful to
separate HG oligosaccharides (OGAs) (Djelineo 2001). The
polyacrylamide gel electrophoresis (PAGE) gels were stained
with alcian blue and silver stain to detect the pectins. The
alcian blue is a positively-charged dye that binds the negatively
charged GalA in the pectin. PAGE showed that FPP separated as
a smear of dark staining polymeric pectin near the top of the gel
(Figure 5A, lane 1). These lanes also contained discrete bands in
the bottom third of the gel that represent OGAs of degrees of
polymerization (DP) of approximately 6 – 14. EPG-treated FPP
looked similar to unmodified FPP except for the loss of OGAs
at the bottom of the gel (Figure 5A, lane 2) as a result of their
cleavage by EPG into mono-, di-, and tri-galacturonic acid
(Doong et al. 1995), which cannot be visualized in the alcian
stained gels. The similarity of the larger polymeric portion of
untreated and EPG-treated FPP suggests that the bulk of the
polymeric portion of FPP was not accessible to cleavage by
EPG, possibly because of esterification. Unexpectedly, the
treatment of FPP with mild base to deesterify the pectin led to a major
change in the appearance of the FPP as determined by PAGE.
Mild base treatment of FPP resulted in the loss of the large
polymeric alcian-blue stained material and the appearance of
dark-staining bands near the bottom of the gel (compare
lanes 3 and 1 in Figure 5A). To test whether these bands
represented HG oligosaccharides (i.e. OGAs), the deesterified
FPP was subsequently treated with EPG, which eliminated
the dark-staining bands at the bottom of the gel (compare
lanes 4 and 3 of Figure 5A). These results confirm that the
dark-staining bands were indeed OGAs. Since the base
treatment also led to the loss of the apoptosis-inducing activity
(Figure 3F), we conclude that the linkage of the OGAs into
a polymeric structure and/or the specific base-sensitive
linkage itself is required for the apoptosis-inducing activity
Since FPP had significant apoptotic activity, but PeS or CP
did not, intact and mild base treated FPP were compared with
treated and untreated PeS and CP to identify any structural
differences that correlated with FPP’s apoptotic activity.
PAGE analysis of PeS revealed a narrow-range dark-staining
smear near the center of the gel and a series of OGAs, consistent
with its relative low mass of 10 – 20 kDa (Figure 5B, lane 5).
Mild base treatment of PeS had no effect on PeS
(Figure 5B, lane 6). This was expected since PeS is generated
from CP by a base treatment, and thus, the further base
treatment did not modify the PeS. A similar analysis of CP revealed
that the bulk of untreated CP barely entered the PAGE gel, as
indicated by the dark staining material at the top of the gel,
although a relatively small amount of OGAs were also
detected (Figure 5B, lane 7). Mild base treatment of CP
led to the appearance of a broad smear of stained material in
the upper half of the gel and to a slight increase in the
amount of OGAs (compare lanes 7 and 8, Figure 5B),
which suggests that (i) the generated material was more
negatively charged because of an increased number of HG
carboxyl groups following the removal of methyl esters
and/or that (ii) base treatment led to a reduction in the size
of the polymer.
Mild base treatment is non-specific and may remove several
types of esters including carboxymethyl, acetyl, and other
esters. Therefore, to achieve specific cleavage of HG
carboxymethyl esters, FPP was treated with pectinmethylesterase
(PME). LNCaP cells were treated with intact FPP, with FPP
treated with PME, or with FPP treated with both PME and
EPG, and the apoptotic activity was assayed. The removal of
methyl esters present in FPP resulted in only a very small
reduction (4%) in the apoptotic activity of FPP (Figure 5C).
Apoptotic activity was reduced 20% when FPP was treated
with both PME and EPG, although the loss of apoptotic
activity was considerably less than the effect of mild base
treatment (compare Figures 5C and 3F). These results
suggest that base-sensitive linkages other than the HG
carboxymethylesters are important for the apoptosis-inducing activity
The effects of the above manipulations on FPP were further
analyzed by PAGE, which showed that PME treatment shifted
the bulk of the dark staining material from the top of the gel
(Figure 5D, lane 3) to the bottom half of the gel (lane 5).
Mild base treatment led to the generation of fast moving
FPP fragments (lane 4) suggesting that this treatment not
only cleaved methyl esters, but also cleaved additional
linkages ( possibly esters) leading to extensive fragmentation of
the FPP and loss of apoptotic activity. Interestingly, although
PME treatment of the FPP generated fragments that moved
faster into the gel, a large proportion of that material migrated
more slowly than the base-treated material (compare lanes 4
and 5 in Figure 5D). Since the PME-treated FPP retained
apoptotic activity (Figure 5C), it is likely that the moderately sized
material near the center of the gel contains fragmented FPP
that is responsible for apoptotic activity.
To determine the effect of EPG on the structure of the
PMEtreated FPP, intact, PME-treated, and base-treated FPP were
treated with EPG and separated by PAGE (Figure 5D, lanes
6 – 8). As expected, the OGAs present in intact FPP
(Fig. 5D, lane 3) were lost upon treatment with EPG
(Figure 5D, lane 6). Likewise, the treatment of deesterified
FPP with EPG resulted in the loss of OGAs (compare lanes
4 and 7, Figure 5D) and the movement of the polymeric
fragments further into the gel. Finally, the removal of the HG
methyl esters by PME treatment produced a broad band of
slow migrating material (Figure 5D, lane 5). The treatment
of this material with EPG led to the loss of OGAs, and the
appearance of a broad band of stained material suggesting
that a significant amount of the pectin remained cross-linked
as a diverse size range of oligosaccharides and polysaccharides
(Figure 5D, lane 8). An important observation was that less
pectic material was cleaved by EPG following PME treatment
of FPP (lane 8, Figure 5D) compared with base treatment
(Figure 5D, lane 7), suggesting that base treatment causes
the loss of linkages in addition to methyl esters. The presence
of an ester-like cross-linking in the HG backbone is further
supported by the observation that EPG treatment led to the
loss of only OGA bands (Figure 5A, lane 2), indicating that
EPG does not possess the ability to degrade the larger pectin
components in FPP, while EPG treatment of base-treated
FPP leads to the loss of the vast majority of the FPP
(Figure 5A, lane 4).
Generation of apoptotic activity by heat treatment
of citrus pectin
As previously described, the most active apoptotic commercial
pectin, FPP, is generated by heat treatment of citrus pectin at
100 – 132 8C for 20 min to 5.5 h. In an attempt to recreate the
apoptotic response of FPP, we subjected CP (Sigma P-9135,
8.1% methylester, 79.5% galacturonic acid) to heat treatment
by autoclaving at 123.2 8C and 17.2 – 21.7 psi for 30 and
60 min (HTCP 30 and HTCP 60, respectively). Figure 6A,
like Figure 1, shows that, as expected, unmodified CP induces
very little apoptosis. Heat modification of the CP significantly
increased its apoptotic response, making HTCP as apoptotically
active as FPP. The application of a longer heat cycle (compare
HTCP 60 with HTCP 30) only slightly increased the apoptotic
response of the treated CP.
Fig. 6. Induction of apoptosis by HTCP. (A) LNCaP cells were treated for
48 h with 3.0 mg/mL CP, with CP that was heat treated for 30 (HTCP30) or 60
(HTCP60) min at 123.2 8C and 17.2– 21.7 psi or with 0.01 mM thapsigargin
( pos). Neg indicates negative control (cells treated with media only).
Apoptosis was measured by the M30 Apoptosense assay using equal amounts
of protein (15 mg). Data are the average of duplicate apoptosis assays of
duplicate cell extracts + SEM. Comparable results were obtained in two
independent experiments. (B) FPP, CP, and HTCP separated by PAGE (see the
Materials and methods section). Lane 1, 0.1 mg OGA of DP 14 (see arrow) and
0.1 mg mixture of OGAs of DP approximately 7 – 23; lanes 2 – 4, 6 mg of CP,
HTCP and FPP, respectively.
The effect of the heat modification on the structure of CP
was analyzed by PAGE. As in previous PAGE analyses,
because of its large polymeric structure, the bulk of the
unmodified CP barely entered the gel (Figure 6B, lane 2).
Heat treatment of citrus pectin facilitated its movement
further into the gel as seen by its separation into a smear of
dark staining polymeric pectin near the top of the gel and
OGAs lower in the gel (Figure 6B, lane 3). The HTCP
appears very similar to FPP (compare Figure 6B, lanes 3 and
4). The ability of both FPP and HTCP to induce significant
levels of apoptosis supports our hypothesis that the apoptotic
response obtained with FPP is due to a particular pectin
structure. Further structure – function studies will be necessary to
understand the structure of the apoptotic pectin.
Effect of apotosis-inducing pectins on human umbilical
vein endothelial cells
Vascular endothelial cells are arranged in a monolayer at the
luminal face of all blood vessels and are, therefore, in direct
contact with the circulating blood and any therapeutic agents
it may contain. It is, therefore, important to determine
whether potential pectin anticancer therapeutics have lethal
effects on normal cells. To determine whether the
apoptosisinducing pectins, FPP and HTCP, reduce the viability of
normal cells, human umbilical vein endothelial cells
(HUVECs) were treated for 48 h with FPP and HTCP, and
the cells were assayed for apoptosis using a Caspase-3
colorimetric assay. HUVECs, unlike prostate cancer cells, do not
express the cytokeratin-18 neoepitope and thus, the
M30Apoptosense ELISA could not be used with these cells.
Cells treated with etoposide, a topoisomerase II inhibitor
known to induce apoptosis in HUVECs, were used as a
positive control. Figure 7 shows that, as expected, the positive
control etoposide induced significant apoptosis. On the
contrary, the treatment of HUVECs with FPP or HTCP did not
induce apoptosis. These results demonstrate that FPP and
Fig. 7. Effect of pectins on normal HUVECs. Apoptosis induced in HUVECs
was measured by a Caspase-3 colorimetric assay. HUVECs were treated for
48 h with 3.0 mg/mL FPP, 3.0 mg/mL heat-treated citrus pectin (HTCP30),
0.1 mM etoposide ( pos) or with media alone (Neg). Caspase-3 activity is
expressed as microgram p-nitroaniline released per hour per microgram
protein. Equal amounts of cell extracts (5 mg protein) were used. Data are the
average of duplicate caspase assays of duplicate cell extracts + SEM.
HTCP induce apoptosis in prostate cancer cells, but not in
Cancer therapy is aimed at either the primary tumor or
metastatic cells. Because of the differences in the response of
primary and metastatic cancers, most therapies do not
address both cancer types. Pectin, a natural plant
polysaccharide present in all higher plant cell walls, and thus in all fruits
and vegetables and in most plant derived foods, is a compound
that appears to be able to inhibit cancer metastasis and primary
tumor growth in multiple types of cancer in animals. Although
pectins were initially recognized as compounds capable of
inhibiting metastatic lesions (Heitman et al. 1992; Platt and
Raz 1992; Pienta et al. 1995; Nangia-Makker et al. 2002),
more recently, pectins have been shown to reduce primary
tumor growth (Nangia-Makker et al. 2002). It has been
suggested that the inhibitory effects of pectin on metastatic
lesions in the lung are mediated through their binding to
galectin-3 (a galactoside-binding lectin). Galectins are specific
carbohydrate-binding proteins present on the surface of
cancer cells. Galectins facilitate cell – cell interactions by
binding to galactose-containing molecules on neighboring
cancer cells. In human colon, stomach and thyroid cancers,
the amount of galectin increased with the progression of
cancer. Blocking galectin-3 expression in highly malignant
human breast, papillary, and tongue carcinoma cells led to
reversion of the transformed phenotype and suppression of
tumor growth in nude mice (Honjo et al. 2000, 2001). It has
been proposed that the pH-modified CP blocks binding of
galectins, and thus, inhibits tumor cell – cell interactions. The
potential impact of blocking galectin action includes inhibition
of the aggregation of cancer cells to each other and to normal
cells, thus inhibiting metastatic lesions. However, LNCaP cells
do not express galectin-3 (Ellerhorst et al. 1999, 2002; Califice
et al. 2004; our unpublished observations), suggesting that the
apoptotic effects of pectins reported here are due to
mechanisms not mediated through galectin-3.
The major goals of the present research were two-fold: (i) to
identify pectins that are capable of inducing death of prostate
cancer cells and (ii) to determine the structure of the pectin
that is responsible for such biological effects. Initial
experiments demonstrated that among the pectins tested, only FPP
induced apoptosis. The pH-modified CP, PeS, is similar to
the modified CP that has been shown to inhibit metastatic
lesions and to induce apoptosis in multiple myeloma cells
(Chauhan et al. 2005). Significantly, PeS did not induce
appreciable apoptosis in prostate cancer cells, agreeing with
published data showing no cytotoxic effects of pH-modified
pectin on prostate cancer cells and xenografts (Pienta et al.
1995). Thus, the main thrust of the present research was to
characterize the structure – function relationships of the
apoptotic pectin FPP. Most of the published reports on the anticancer
effects of pectin utilized pectins that were modified by
alterations in pH in an effort to fragment pectin structure to facilitate
biological effects in the systems under study. This procedure,
in addition to fragmenting the pectin, may affect the structure
of the pectin and thus its function. FPP was not produced by
pH treatment, but rather was produced by heating CP. We
therefore utilized several methods to correlate the biological
function of FPP with its structure and compared FPP structure
with that of PeS and CP, which were not apoptotic.
As a first step, the size and glycosyl residue composition and
linkage of the different pectins were compared. Our results did
not show any significant differences between the glycosyl
residue composition or linkages of FPP compared with PeS
and CP, indicating that differences in apoptotic activity
among the three pectin preparations were not due to
differences in the major types of pectin present. Also, there was
no correlation in the size of the pectins and their apoptotic
activity. As pectins consist of HG and RG, we hypothesized
that one or more of these polysaccharides in FPP was
responsible for its apoptotic effects. However, experiments testing the
apoptotic effects of HG, RG-I, and RG-II, indicated that these
individual structural components were not by themselves
responsible for the apoptosis-inducing activity of FPP. We
therefore conclude that the apoptosis-inducing activity of
FPP is related to some structural constituent not detected by
the above analyses.
To further understand the structure – function relationship of
apoptotic pectin, FPP was specifically fragmented using
endopolygalacturonase (EPG), which cleaves HG at contiguous
nonesterified GalA residues. The cleavage of FPP with EPG
did not have a major effect on its apoptotic activity. Thus,
two methods were used to remove ester linkages from FPP
prior to EPG treatment and thereby, make FPP more
susceptible to EPG cleavage: chemical deesterification by mild
base treatment and specific enzymatic hydrolysis of methyl
esters by treatment with pectinmethylesterase (PME).
Chemical deesterification of FPP resulted in significant loss
of apoptosis suggesting that a base-sensitive structure, such
as an ester linkage, is necessary for the apoptotic activity of
FPP. Since specific cleavage of methyl esters by PME did
not destroy FPP’s apoptosis inducing activity, linkages other
than methyl esters are required for apoptotic activity.
Furthermore, PAGE analysis of intact and treated FPP
showed that a polymeric/oligomeric FPP structure containing
a base-sensitive linkage, and/or the specific base-sensitive
linkage itself, is required for the apoptotic activity of FPP.
Taken together the results suggest that an ester-based (or
related) cross-link in pectin is important for the
apoptosisinducing activity of FPP.
The incubation of FPP with a nonspecific protease, with
endo-a1,5-arabinase or with RNAse prior to cell treatment
(data not shown), did not significantly affect FPP’s
apoptosis-inducing ability, suggesting that the apoptotic response is
not due to the presence of proteins, a1,5-arabinan, or RNA
within the pectin preparation. Significantly, we have been
successful in generating the apoptosis-inducing capability in CP
by heat treatment, a critical step in the production of FPP
from the mother pectin. This supports our conclusion that a
specific pectin structure and/or pectin-containing linkage is
responsible for inducing apoptosis. The question of whether
the heat treatment of CP causes a covalent or noncovalent
modification of CP structure that leads to the apoptotic activity
remains to be determined.
In conclusion, we provide the first evidence that specific
structural characteristics of pectin are responsible for inducing
apoptosis in cancer cells. Our results demonstrate that different
extraction protocols may alter the structure of pectin and can
lead to differences in pectin’s apoptosis-inducing activity.
Further experiments to identify the specific apoptotic structure
in pectin will enable us to generate the smallest fragment that
is capable of inducing apoptosis. A detailed understanding of
structure – function relationships of such a fragment may lead
to effective anti-cancer therapy. This is of particular
therapeutic significance, as we have demonstrated that manipulating
the structure of pectin results in a compound that is capable of
inducing apoptosis in both androgen-responsive and
androgenindependent prostate cancer cells.
Materials and methods
Two prostate cancer cell lines, LNCaP and LNCaP C4-2, were
utilized in these experiments. LNCaP obtained from American
Type Culture Collection (Rockville, MD) are
androgenresponsive prostate cancer cells, whereas LNCaP C4-2 cells
were derived from LNCaP cells as androgen-refractory pros- oD
tate cancer cells ( purchased from Grocer Inc., Oklahoma nw
City, OK). HUVECs were from American Type Culture lado
Collection. EBM-2 media and EGM-2 supplements were ed
from Cambrex Bio Science (Walkersville, MD). RPMI-1640 fro
media supplemented with 25 mM HEPES and L-glutamine hm
was purchased from Hyclone (Logan, UT). Fetal bovine :ttp
serum (FBS), penicillin/streptomycin, citrus pectin (P-9135), /l/g
sodium hydroxide, alcian blue, and caspase-3 colorimetric cyo
assay kit were from Sigma-Aldrich (St Louis, MO). .ob
FBuion-gRizaodnPerowteaisn Aobsstaaiyneddye frreoamgenItncvoitnrocegnetnrat(eCwaralssbpaudr,chCasAe)d. jfrxood
from Bio-Rad (Hercules, CA). M-30 Apoptosense ELISA was run
from Peviva AB (Sweden). Sodium carbonate and sodium .lsa
acetate were purchased from J.T. Baker (Phillipsburg, NJ) rgo
and acetic acid from EM Science (Gibbstown, NJ). PeS was /by
purchased from EcoNugenics (Santa Rosa, CA), FPP from ug
Thorne Research (Dover, ID), and CP (P-9135) from Sigma- tse
Aldrich. Purified PME (from Aspergillus niger 2.2 mg/mL, on
1.0 U/mg, 1 U ¼ 1 mmol/min) and EPG (from A. niger, ovN
0.5 mg/mL, 1.2 U/mL, 1 U ¼ 1 mmol/min) were obtained em
from Carl Bergmann (Complex Carbohydrate Research reb
Center, University of Georgia, Athens, GA). All other chemi- ,
cals, unless otherwise stated, were from Fisher Scientific. 021
Purifed HG, RG-I, and RG-II were a gift of Stefan Eberhard
(Complex Carbohydrate Research Center, University of
Georgia, Athens, GA). The HG was a mixture of
oligogalacturonides of degrees of polymerization of approximately 7 –
23 that were produced by partial endopolygalacturonase
treatment of commercial polygalacturonic acid as described by
Spiro et al. (1993). RG-I was isolated from sycamore (Acer
pseudoplatanus) suspension culture cells as described in
Marfa` et al. (1991). RG-II was isolated from red wine as
described by Pellerin et al. (1996).
Endopolygalacturonase treatment of pectins
Ammonium formate, pH 4.5, was added to 500 mL of 20 mg/
mL deesterified FPP (Des FPP) and FPP to give a final
ammonium formate concentration of 10 mM and 2 mL
of 1.2 U/mL, 0.5 mg/mL A. niger endopolygalacturonase
(EPG) was added. As a negative control, 500 mL of 20 mg/mL
FPP in 10 mM ammonium formate was also prepared. The
three FPP samples were incubated overnight at room
temperature (RT), frozen at 280 8C, and lyophilized to dryness. The
dry samples were analyzed by high-percentage acrylamide
PAGE and tested for apoptotic activity in an M-30
Pectinmethylesterase (PME) treament of pectins
One microliter of 1 U/mg, 2.2 mg/mL A. niger PME was
added to 1 mL of 20 mg/mL FPP in 10 mM ammonium
formate. One milliliter of 20 mg/mL FPP in 10 mM
ammonium formate served as a negative control. PME activity
was confirmed by the detection of methanol released using the
method of Klavons and Bennett (1986). A combined PME þ
EPG treatment involved mixing 2 mL of 1.2 U/mL, 0.5 mg/
mL A. niger EPG and 1 mL of 1 U/mg, 2.2 mg/mL A. niger
PME in 10 mM ammonium formate. The FPP samples were
incubated overnight at RT, frozen at 280 8C, and lyophilized
to dryness. The dry samples were analyzed by high-percentage
acrylamide PAGE and tested for apoptotic activity in an M-30
Fifty milligrams pectin was dissolved in 50 mL of de-ionized
water and placed on ice. The starting pH of the solution was
measured and the pH was adjusted to 12 by adding 1.0 M
cold NaOH. A pH of 12 was maintained for 4 h by the addition
of 0.1 M NaOH, and the solution was kept on ice to retain a
temperature of 2 8C. After 4 h, the pH was adjusted to 5.2
by the addition of glacial acetic acid. The deesterified pectin
solution was frozen at 280 8C, and lyophilized. The dry
material was dissolved again in water, frozen, and
Fractionation of citrus pectin by heat treatment
A 0.1% aqueous pectin dispersion was prepared by dissolving
200 mg of unmodified CP in 200 mL of de-ionized, filtered
water. The solution was autoclaved at 123.2 8C at 17.2 –
21.7 psi for 30 min. At the end of the heat treatment, the
solution was allowed to cool to RT and stored overnight at 4 8C,
allowing formation of a gel-like precipitate. The next day, the
aqueous phase was collected, frozen at 280 8C and
lyophilized. An alternative heat treatment was also carried out by
autoclaving the pectin dispersion at 123.2 8C at 17.2 –
21.7 psi for 60 min.
Cell culture, pectin treatments, and quantification
LNCaP and LNCaP C4-2 cells were grown in RPMI-1640
medium supplemented with 25 mM HEPES, 2.0 mM
L-glutamine, 10% heat-inactivated FBS, 50 U/mL penicillin,
0.05 mg/mL streptomycin, and 0.25 mg/mL fungizone in the
presence of 5% CO2 at 37 8C. Cells were maintained in
logarithmic growth phase by routine passage every 10 – 12 days
(LNCaP) or 6 – 7 days (LNCaP C4-2). Cells were plated at a
density of 1.6 105 cells per well in 6-well culture plates
and allowed to adhere for 24 h. The medium was removed
and replaced with media containing filter-sterilized pectin
(0.20 mm nylon filters; Fisher Scientific). Treated cells were
incubated in media containing the following compounds as
indicated: FPP, PeS, CP, Des FPP, PME-treated fractionated
pectin powder (FPPþPME), EPG-treated fractionated pectin
powder (FPPþEPG), PME-treated Des FPP, EPG-treated
Des FPP, combined enzyme treatments, RG-I, RG-II, purified
HG, and positive control Thapsigargin (Sigma). The negative
controls were untreated cells cultured in media alone. Cells
were harvested after 48 h and lysed in ice-cold Lysis Buffer
(10 mM Tris – HCL, pH 7.4, 10 mM MgCl2, 150 mM NaCl,
0.5% Nonidet P-40), incubated on ice for 5 min, and soluble
protein was collected by centrifugation at 4 8C. The protein
concentration was determined in triplicate using a Bradford/
Bio-Rad Protein Assay (Bio-Rad Protein Assay Dye Reagent
Concentrate). Apoptotic activity was assayed using the M-30
Apoptosense ELISA (see the Apoptosense assay section).
HUVECs were grown in EBM-2 Complete Media in the
presence of 5% CO2 at 37 8C. The media was renewed every other
day, and cells were subcultured when 70 – 80% confluent,
approximately every 5 days. Cells were plated at a density of
1.6 105 cells per well in 6-well culture plates and allowed
to adhere for 24 h. After 24 h, the cells were treated with
medium containing filter-sterilized pectin (0.20 mm nylon
filters; Fisher Scientific) and harvested after another 48 h.
The pectin treatments applied were as follows: FPP, CP, PeS,
HTCP, positive control (the DNA synthesis inhibitor
Etoposide; Sigma) and the negative control (untreated cells
cultured in media alone). Apoptotic activity in extracts from
HUVECs was assayed using the Caspase 3 colorimetric
Assay (see the Caspase 3 Colorimetric assay section).
Cells grown in culture flasks and treated as described earlier
were harvested and total protein extracted as described
earlier. Protein was assayed for the presence of the
apoptosis-specific cytokeratin-18 neoepitope (generated by cleavage
of cytokeratin-18 by caspases activated in response to
treatment) using the M30-Apoptosense ELISA (Peviva AB,
Sweden). In brief, protein extract was added to 96-well
plates coated with mouse monoclonal anti-cytokeratin-18
M30 antibody. Horseradish peroxidase tracer solution was
added to the wells in a dark room illuminated with a green
safety light, and the plate was incubated with agitation for
4 h at RT. Color was developed by adding tetramethyl
benzidine solution and incubating in darkness for 20 min. Optical
density (OD) was determined at 450 nm using Spectra MAX
340 (Molecular Devices, Menlo Park, CA) or Finstruments
Model 347 (Vienna, VA) microplate readers. The amount of
cytokeratin-18 neoepitope was determined based on standard
curves generated using standards provided by the
Caspase 3 colorimetric assay
HUVEC cells were grown in culture flasks and treated as
described above (see Cell culture, pectin treatments and
quantification of apoptosis section). Cells were harvested
after 48 h by trypsinization; 1 mL 0.25% trypsin – EDTA was
applied to each plate for 3 min at 37 8C to loosen cells
followed by neutralization with media. Cells were collected by
centrifugation at 4 8C and PBS ( phosphate-buffered
saline)washed cell pellets were resuspended in Lysis Buffer. The
soluble protein was collected by centrifugation at 4 8C. The
assay was carried out in a 96-well plate with each well
containing, 5 mL cell lysate, 85 mL of 1X assay buffer (20 mM
HEPES, pH 7.4, 2 mM EDTA, 0.1% CHAPS, 5 mM
dithiothreitol), and 10 mL of the caspase-3 colorimetric substrate,
2 mM Ac-DEVD-p-nitroanilide. Caspase-3 positive controls
and caspase 3 inhibitor controls were prepared according to
the manufacturer’s instructions (Sigma-Aldrich). The 96-well
plate was incubated at 37 8C for 90 min to allow cleavage of
the chromophore p-nitroaniline from Ac-DEVD-pNA by
caspase-3 present in the sample and the OD at 405 nm was
detected using a Bio-Rad 680 microplane reader. Caspase-3
activity was proportional to the amount of yellow color
produced upon cleavage. Protein concentration was determined
using a Bradford/Bio-Rad Protein Assay (Bio-Rad Protein
Assay Dye Reagent Concentrate). Caspase-3 activity was
expressed as micromoles of p-nitroaniline released per hour
per microgram protein.
Preparation of cell lysates and western blotting
Cells were harvested by trypsinization and the washed cell
pellets were resuspended in lysis buffer (1X PBS, 1% Triton
X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl
sulfate (SDS), 1 mM EDTA, 0.5 mg/mL leupeptin, 1 mg/mL
pepstatin, 1 mg/mL phenylmethyl sulfonyl fluoride, and
1 mg/mL aprotinin) and incubated on ice for 30 min. The
lysed cells were centrifuged at 10 000 g for 10 min at 4 8C
and the supernatant was collected. Protein concentration was
determined as described above.
Proteins (50 mg, unless stated otherwise) were separated on
NuPAGE 10% Bis-Tris gels (Novex pre-cast mini gels,
Invitrogen, Carlsbad, CA) at 100 V for 1 h in the presence of
1X 2-(N-morpholino) ethanesulfonic acid (MES)-SDS
running buffer (Invitrogen, Carlsbad, CA). Separated proteins
were transferred to (Polyvinylidenedifluoride; PVDF)
membranes (Bio-Rad Laboratories, Hercules, CA) at 42 V for
2.5 h using a Novex XCell II blotting apparatus in MES
transfer buffer in the presence of NuPAGE antioxidant. Transfer of
the proteins to the PVDF membrane was confirmed by staining
with Ponceau S (Sigma). The blots were blocked in 5% nonfat
dry milk in tris-buffered saline (TBS), washed twice for
10 min each with TBS containing 0.01% Tween-20 and
incubated for 2 h at RT with primary antibody diluted in TBS
containing 0.5% milk. The following antibodies were used in the
immunoblots: rabbit polyclonal anti-caspase-3 antibody (BD
Pharmingen, San Diego, CA), rabbit polyclonal anti-PARP
antibody (Cell Signaling Technology, Beverly, MA), and
anti-actin antibody (Sigma). Immunoreactive bands were
visualized using the ECL detection system (Amersham, Pharmacia
Biotech, Arlington Heights, IL) and signals were developed
after exposure to X-ray film (X-Omat films, Eastman Kodak
Company, Rochester, NY).
Size exclusion chromatography
Five milligrams of FPP, PeS, and CP were separated at 0.5 mL/
min in 50 mM sodium acetate and 5 mM EDTA over a
Superose 12 HR10 (10 – 300 mm) SEC column using a Dionex
DX500 system. The eluted pectin was detected using an
uronic acid colorimetric assay (Blumenkrantz and
Glycosyl residue composition analysis
Pectin samples were analyzed for glycosyl residue composition
at the Complex Carbohydrate Service Center, the University of
Georgia, Athens by combined gas chromatography – mass
spectrometry (GC – MS) of the per-O-trimethylsilyl (TMS)
derivatives of the monosaccharide methyl glycosides produced
from the sample by acid methanolysis (York et al. 1985).
Methyl glycosides were prepared by methanolysis in 1 M HCl
in methanol at 80 8C for 18 – 22 h, followed by re-N-acetylation
with pyridine and acetic anhydride in methanol for the detection
of amino sugars. The samples were per-O-trimethylsilylated by
treatment with Tri-Sil (Pierce) at 80 8C for 0.5 h. The GC – MS
analysis of the TMS methyl glycosides was performed on an
HP 5890 GC interfaced to a 5070 MSD using a Supelco DB1
fused silica capillary column.
Glycosyl residue linkage analysis
Pectin samples were analyzed for glycosyl residue linkage at
the Complex Carbohydrate Service Center at the University
of Georgia, Athens basically as described by York et al.
(1985). Glycosyl residue linkage analyses were conducted
using both single and double methylation procedures. For the
“single methylation” linkage analysis, the samples were
permethylated, depolymerized, reduced, acetylated, and the
resultant partially methylated alditol acetate residues were
analyzed by GC – MS. Specifically, the samples were
permethylated by the Hakomori method (Hakomori 1964),
by treatment with dimethylsulfinyl anion and methyl iodide
(MeI) in dimethyl sulfroxide, the permethylated material
reduced by super-deuteride, hydrolyzed in 2 M trifluoroacetic
acid (TFA) for 2 h at 121 8C, reduced with NaBD4, and
acetylated using acetic anhydride – TFA. The resulting partially
methylated alditol acetates were separated on a 30 m
Supelco 2330 bonded phase fused silica capillary column
and analyzed on a Hewlett Packard 5890 GC interfaced to a
5970 mass detector in selective electron impact ionization
mode. For the “double methylation” linkage analysis, the
methods were as described above for the “single methylation”
method except that following the first methylation the
permethylated material was reduced by super-deuteride and the
reduced sample was re-methylated using the NaOH – MeI
method of Ciucanu and Kerek (1984). The remethylated
samples were hydrolyzed using 2 M TFA and processed as
described above for the “single methylation” method.
PAGE of pectins
Pectin samples were separated by PAGE and analyzed by
alcian blue staining using a modified procedure of Corzo
et al. (1991) and Reuhs et al. (1993, 1998) as described by
Djelineo (2001). Pectin samples were mixed in a 5:1 ratio
with 6X sample buffer (0.63 M Tris-Cl, pH 6.8, 0.05%
phenol red, and 50% glycerol), loaded onto a resolving gel
[0.38 M Tris pH 8.8, 30% (wt/vol) acrylamide (37.5:1
acrylamide: bis-acrylamide, wt/wt)] overlaid with a stacking gel
(5% acrylamide, 0.13 M Tris, pH 6.8) and separated at
17.5 mA for 60 min or until the phenol dye was within 1 cm
of the end of the gel. The gel was stained 20 min with 0.2%
alcian blue in 40% ethanol, washed thrice for 20 s and then
20 min in water. The gel was incubated with shaking in
0.2% silver nitrate containing 0.075% formaldehyde, rinsed
thrice for 20 s with water, and then incubated in 4% sodium
carbonate containing 0.05% formaldehyde until bands
appeared. The carbonate solution was immediately removed
and the gel was stored overnight in 5% acetic acid and then
stored in water or dried.
We thank Stefan Eberhard (CCRC, University of Georgia) for
the gift of purifed HG, RG-I, and RG-II, Carl Bergmann for
the gift of purified A. niger endopolygalacturonase and
pectinmethylesterase, Lance Wells for the HUVECs,
colleagues at the CCRC for helpful discussions, and Alan
Darvill for critical reading of the manuscript. This work was
supported in part by the Georgia Cancer Coalition-Georgia
Department of Human Resources and the University of
Georgia-Medical College of Georgia Joint Intramural Grants
Program (M.V. and D.M.). Carbohydrate analyses performed
by the CCRC Analytical Services supported by the DOE
center grant DE-FG05-93-ER20097.
Conflict of interest
CP, citrus pectin; Des FPP, deesterified fractionated pectin
powder; ELISA, enzyme-linked immunosorbent assay; EPG,
endopolygalacturonase; EDTA, ethylenediaminetetraacetic acid;
FBS, fetal bovine serum; FPP, fractionated pectin powder;
GalA, a-D-galactopyranosyluronic acid; GC-MS, gas
chromatography-mass spectrometry; HG, homogalacturonan; HTCP, heat
treated citrus pectin; HUVEC, human umbilical vein endothelial
cell; LNCaP, lymph node-derived human prostate cancer; MES,
2-(N-morpholino)ethanesulfonic acid; OGAs,
oligogalacturonides; PAGE, polyacrylamide gel eletrophoresis; PARP, poly
(ADP-ribose) polymerase; PeS, pectasol; PME,
pectinmethylesterase; PVDF, polyvinylidenedifluoride; RG-I,
rhamnogalacturonan I; RG-II, rhamnogalacturonan II; SDS, sodium dodecyl
sulfate; SEC, size exclusion chromatography; TBS,
trisbuffered saline; TFA, trifluoroacetic acid; TMS, trimethylsilyl.
Albersheim P , Darvill AG , O'Neill MA , Schols HA , Voragen AGJ . 1996 . An hypothesis: the same six polysaccharides are components of the primary cell walls of all higher plants . In: Visser J , Voragen AGJ, editors. Pectins and Pectinases , 14 . Amsterdam (NL): Elsevier Sciences B.V. ; pp. 47 - 53 .
Avivi-Green C , Madar Z , Schwartz B. 2000 . Pectin-enriched diet affects distribution and expression of apoptosis-cascade proteins in colonic crypts of dimethylhydrazine-treated rats . Int J Mol Med . 6 : 689 - 698 .
Avivi-Green C , Polak-Charcon S , Madar Z , Schwartz B. 2000a. Apoptosis cascade proteins are regulated in vivo by high intracolonic butyrate concentration: correlation with colon cancer inhibition . Oncol Res . 12 : 83 - 95 .
Avivi-Green C , Polak-Charcon S , Madar Z , Schwartz B. 2000b. Dietary regulation and localization of apoptosis cascade proteins in the colonic crypt . J Cell Biochem . 77 : 18 - 29 .
Blumenkrantz N , Asboe-Hansen G. 1973 . New method for quantitative determination of uronic acids . Anal Biochem . 54 : 484 - 489 .
Brown JA , Fry SC . 1993 . Novel O-D-galacturonoyl esters in the pectic polysaccharides of suspension-cultured plant cells . Plant Physiol . 103 : 993 - 999 .
Bruckheimer E , Gjertsen B , McDonnell T. 1999 . Implications of cell death regulation in the pathogenesis and treatment of prostate cancer . J Semin Oncol . 26 ( 4 ): 382 - 398 .
Califice S , Castronovo V , Bracke M , van den Bruˆle F. 2004 . Dual activities of galectin-3 in human prostate cancer: tumor suppression of nuclear galectin-3 vs. tumor production of cytoplasmic galectin-3 . Oncogene. 23 : 7527 - 7536 .
Chang WC , Chapkin RS , Lupton JR . 1997 . Predictive value of proliferation, differentiation and apoptosis as intermediate markers for colon tumorigenesis . Carcinogenesis . 18 : 721 - 730 .
Chang W-CL , Chapkin RS , Lupton JR . 1997 . Fish oil blocks azoxymethaneinduced rat colon tumorigenesis by increasing cell differentiation and apoptosis rather than decreasing cell proliferation . J Nutr . 128 : 491 - 497 .
Chauhan D , Li G , Podar K , Hideshima T , Neri P , He D , Mitsiades N , Richardson P , Chang Y , Schindler J et al. 2005 . A novel carbohydratebased therapeutic GCS-100 overcomes bortezomib resistance and enhances dexamethasone-induced apoptosis in multiple myeloma cells . Cancer Res . 65 : 8350 - 8358 .
Chen EMW , Mort AJ . 1996 . Nature of sites hydrolyzable by endopolygalacturonase in partially-esterified homogalacturonans . Carbohydr Polym . 29 : 129 - 136 .
Ciucanu I , Kerek F. 1984 . A simple and rapid method for the permethylation of carbohydrates . Carbohydr Res . 131 : 209 - 217 .
Colombel M , Buttyan R . 1995 . Hormonal control of apoptosis: the rat prostate gland as a mode system . Methods Cell Biol . 46 : 369 - 385 .
Corzo J , Pe´rez-Galdona R , Leo´n-Barrios M , Gutie´rrez-Navarro AM . 1991 . Alcian Blue fixation allows silver staining of the isolated polysaccharide component of bacterial lipopolysaccharides in polyacrylamide gels . Electrophoresis . 12 : 439 - 441 .
Djelineo I. 2001 . Structural studies of pectin (Ph.D. Thesis ). Athens; The University of Georgia.
Doong RL , Liljebjelke K , Fralish G , Kumar A , Mohnen D. 1995 . Cell free synthesis of pectin: identification and partial characterization of polygalacturonate 4-a-galacturonosyltransferase and its products from membrane preparations of tobacco (Nicotiana tabacum L. cv samsun) cell suspension cultures . Plant Physiol . 109 : 141 - 152 .
Eliaz I. 2001 . The potential role of modified citrus pectin in the prevention of cancer metastasis . Clin. Pract. Altern. Med . 2 : 1 - 7 .
Ellerhorst JA , Nguyen T , Cooper DN , Estrov Y , Lotan D , Lotan R . 1999 . Induction of differentiation and apoptosis in prostate cancer cell line LNCaP by sodium butyrate and galectin-1 . Int J Oncol. 14 : 225 - 232 .
Ellerhorst JA , Stephens LC , Nguyen T , Xu X-C. 2002 . Effects of galectin-3 expression on growth and tumorigenicity of the prostate cancer cell line LNCaP . Prostate 50 : 64 - 70 .
Fry SC . 1982 . Phenolic components of the primary cell wall . Feruloylated disaccharides of D-galactose and L-arabinose from spinach polysaccharide. Biochem J . 203 : 493 - 504 .
Hakomori SA . 1964 . A rapid permethylation of glycolipid and polysaccharide catalyzed by methylsulfinyl carbanion in dimethyl sulfoxide . J Biochem . 55 : 205 - 208 .
Heitman DW , Hardman WE , Cameron IL . 1992 . Dietary supplementation with pectin and guar gum on 1,2- dimethylhydrazine-induced colon carcinogenesis in rats . Carcinogenesis . 13 : 815 - 818 .
Honjo Y , Inohara H , Akahani S , Yoshii T , Takenaka Y , Yoshida J-I , Hattori K , Tomiyama Y , Raz A , Kubo T. 2000 . Expression of cytoplasmic galectin-3 as a prognostic marker in tongue carcinoma . J Clin Cancer Res . 6 : 4635 - 4640 .
Honjo Y , Nangia-Makker P , Inohara H , Raz A. 2001 . Down-regulation of galectin-3 suppresses tumorigenicity of human breast crcinoma cells . J Clin Cancer Res . 7 : 661 - 668 .
Hou W-C , Chang W-H. 1996 . Pectinesterase-catalyzed firming effects during precooking of vegetables . J Food Biochem . 20 : 397 - 416 .
Iiyama K , Lam TBT , Stone BA . 1994 . Covalent cross-links in the cell wall . Plant Physiol . 104 : 315 - 320 .
Inohara H , Raz A. 1994 . Effects of natural complex carbohydrates (citrus pectin) on murine melanoma cell properties related to galectin-3 functions . Glycoconjugate J . 11 : 527 - 532 .
Ishii T. 1997 . Structure and functions of feruloylated polysaccharides . Plant Sci . 127 : 111 - 127 .
Kim J-B , Carpita NC . 1992 . Changes in esterification of the uronic acid groups of cell wall polysaccharides during elongation of maize coleoptiles . Plant Physiol . 98 : 646 - 653 .
Klavons JA , Bennett RD . 1986 . Determination of methanol using alcohol oxidase and its application to methyl ester content of pectins . J Agric Food Chem . 34 : 597 - 599 .
Kobayashi M , Matoh T , Azuma J-I. 1996 . Two chains of rhamnogalacturonan II are cross-linked by borate-diol ester bonds in higher plant cell walls . Plant Physiol . 110 : 1017 - 1020 .
Kossoy G , Ben-Hur H , Zusman I , Madar Z. 2001 . Effects of a 15% orangepulp diet on tumorigenesis and immune response in rats with colon tumors . Oncol Rep . 8 : 1387 - 1391 .
Kozlowski J , Ellis W , Grayhack J. 1991 . Advanced prostatic carcinoma . Early versus late endocrine therapy . J Urol Clin North Am . 18 ( 1 ): 15 - 24 .
Kreis W. 1995 . Current chemotherapy and future directions in research for the treatment of advanced hormone-refractory prostate cancer . Cancer Invest . 13 : 296 - 312 .
Marfa ` V, Gollin DJ , Eberhard S , Mohnen D , Darvill A , Albersheim P. 1991 . Oligogalacturonides are able to induce flowers to form on tobacco explants . Plant J . 1 : 217 - 225 .
Mohnen D. 1999 . Biosynthesis of pectins and galactomannans . In: Pinto BM editor. Comprehensive Natural Products Chemistry. Carbohydrates and Their Derivatives Including Tannins , Cellulose, and Related Lignins , 3 . Oxford: Elsevier; pp. 497 - 527 .
Mohnen D. 2002 . Biosynthesis of pectins . In: Seymour GB, Knox JP , editors. Pectins and Their Manipulation . Oxford, (UK) : Blackwell Publishing and CRC Press; pp. 52 - 98 .
Morris ER , Powell DA , Gidley MJ , Rees DA . 1982 . Conformations and interactions of pectins I. Polymorphism between gel and solid states of calcium polygalacturonate . J Mol Biol . 155 : 507 - 516 .
Nakamura A , Furuta H , Maeda H , Takao T , Nagamatsu Y. 2002 . Structural studies by stepwise enzymatic degradation of the main backbone of soybean soluble polysaccharides consisting of galacturonan and rhamnogalacturonan . Biosci Biotechnol Biochem . 66 : 1301 - 1313 .
Nangia-Makker P , Hogan V , Honjo Y , Baccarini S , Tait L , Bresalier R , Raz A. 2002 . Inhibition of human cancer cell growth and metastasis in nude mice by oral intake of modified citrus pectin . J Natl Cancer Inst . 94 : 1854 - 1862 .
O'Neill M , Albersheim P , Darvill A. 1990 . The pectic polysaccharides of primary cell walls . In: Dey PM editor. Methods in Plant Biochemistry . vol 2. London, (UK): Academic Press pp. 415 - 441 .
O'Neill MA , Ishii T , Albersheim P , Darvill AG . 2004 . Rhamnogalacturonan II: structure and function of a borate cross-linked cell wall pectic polysaccharide . Annu Rev Plant Biol . 55 : 109 - 139 .
O'Neill MA , Warrenfeltz D , Kates K , Pellerin P , Doco T , Darvill AG , Albersheim P. 1996 . Rhamnogalacturonan-II, a pectic polysaccharide in the walls of growing plant cell, forms a dimer that is covalently crosslinked by a borate ester-in vitro conditions for the formation and hydrolysis of the dimer . J Biol Chem . 271 : 22923 - 22930 .
Olano-Martin E , Rimbach GH , Gibson GR , Rastall RA . 2003 . Pectin and pectic-oligosaccharides induce apoptosis in in vitro human colonic adenocarcinoma cells . Anticancer Res . 23 : 341 - 346 .
Pellerin P , Doco T , Vidal S , Williams P , O'Neill MA , Brillouet J-M. 1996 . Structural characterization of red wine rhamnogalacturonan II . Carbohydr Res . 290 : 183 - 197 .
Perlman H , Zhang X , Chen M-W , Walsh K , Buttyan R . 1999 . An elevated bax/bcl-2 ratio corresponds with the onset of prostate epithelial cell apoptosis . Cell Death Differ . 6 : 48 - 54 .
Pienta KJ , Naik H , Akhtar A , Yamazaki K , Replogle TS , Lehr J , Donat TL , Tait L , Hogan V , Raz A. 1995 . Inhibition of spontaneous metastasis in a rat prostate cancer model by oral administration of modified citrus pectin . J Natl Cancer Inst . 87 : 348 - 353 .
Platt D , Raz A. 1992 . Modulation of the lung colonization of B16-F1 melanoma cells by citrus pectin . J Natl Cancer Inst . 84 : 438 - 442 .
Reuhs BL , Carlson RW , Kim JS . 1993 . Rhizobium fredii and Rhizobium meliloti produce 3-deoxy-D-manno-2-octulosonic acid-containing polysaccharides that are structurally analogous to group II K antigens (Capsular polysaccharides) found in Escherichia coli . J Bacteriol . 175 : 3570 - 3580 .
Reuhs BL , Geller DP , Kim JS , Fox JE , Kolli VSK , Pueppke SG . 1998 . Sinorhizobium fredii and Sinorhizobium meliloti produce structurally conserved lipopolysaccharides and strain-specific K antigens . Appl Environ Microbiol . 64 : 4930 - 4938 .
Ridley BL , O'Neill MA , Mohnen D. 2001 . Pectins: structure, biosynthesis, and oligogalacturonide-related signaling . Phytochemistry . 57 : 929 - 967 .
Santen R . 1992 . Clinical review 37: endocrine treatment of prostate cancer . J Clin Endocrinol Metab . 75 ( 3 ): 685 - 689 .
Spiro MD , Kates KA , Koller AL , O'Neill MA , Albersheim P , Darvill AG . 1993 . Purification and characterization of biologically active 1,4-linked a-D-oligogalacturonides after partial digestion of polygalacturonic acid with endopolygalacturonase . Carbohydr Res . 247 : 9 - 20 .
Thakur BR , Singh RK , Handa AK . 1997 . Chemistry and uses of pectin-a review . Crit Rev Food Sci Nutr . 37 : 47 - 73 .
Voragen AGJ , Schols HA , Pilnik W. 1986 . Determination of the degree of methylation and acetylation of pectins by h .p. l.c. Food Hydrocolloids . 1 : 65 - 70 .
Yamada H. 1996 . Contributions of pectins on health care . In: Visser J , Voragen AGJ, editors. Pectins and Pectinases. Proceedings of an International Symposium, Wageningen, The Netherlands , 1995 Dec 3 - 7 , vol. 14 ; Amsterdam (NL): Elsevier; pp. 173 - 190 .
Yamada H , Kiyohara H , Matsumoto T. 2003 . Recent studies on possible functions of bioactive pectins and pectic polysaccharides from medicinal herbs on health care . In: Voragen F, Schols H , Visser R , editors. Advances in Pectin and Pectinase Research. Dordrecht, (NL) : Kluwer Academic Publishers; pp. 481 - 490 .
York WS , Darvill AG , McNeil M , Stevenson TT , Albersheim P. 1985 . Isolation and characterization of plant cell walls and cell wall components . Methods Enzymol . 118 : 3 - 40 .