INTRODUCTION TO ANTHOCYANINS
Relationship between chemical structure of anthocyanins and its cancer
preventive effect
The chemical structure of anthocyanin is crucial to their biological activities.
However, anthocyain structure-function relationships are not well established because of
difficulties to assess from the studies conducted with so many different anthocyanin from
different sources. Although the relationship could be different depending on experimental
models, several possible relationships can be inferred by comparing the studies about cancer
preventive effect of anthocyanins.
According to many study results, delphinidin presents the strongest inhibitory effect
on cancer cell growth and survival among 6 anthocyanidins. It seems like the hydroxy groups on
ring B of the anthocyanin molecule may have potency on anti-proliferation activity.
Also, Marko et al. compared the abilities of anthocyanidin to inhibit epidermal growth factor
receptor(EGFR) tyrosin kinase which is associated with cancer development. They demonstrated
that anthocyanidins decreased EGFR tyrosine kinase in the order delphinidin = cyaniding >
pelagonidin > peonidin > malvidin. It suggests that potency might be related to the presence
of hydroxy functions in positions 3’ and 5’ of the B ring of the anthocyanin molecule.
In contrast, the presence of methoxy groups in those positions may weaken the abilities.
In a study on human leukemia cells, anthocyanidins possessing hydroxyl group in ring B also
present higher pro-apoptotic activity including altering cell cycle than methoxy groups.
However, another study reported that malvidin, which possesses only methoxy groups in 3’ and
5’ positions of ring B, exerted the greatest anti- proliferation activity among 6 anthocyanidins in
stomach, colon, lung, breast, and central nerve system cancer cells.
Acylation and glycosidic patterns also effect on the biological activities of
anthocyanins. In HT29 colon cancer cells, anthocyanins without acylation were more effective
inhibitors than acylated anthocyanins. The growth inhibitory effects of delphinidin-3-
galactoside and delphinidin-3- glucoside which were purified from bilberry extract were lower
than those of the aglycon delphinidin on HL60 human leukemia cells and HCT116 human
carcinoma cells. Anthocyanin triglycosides had less anti-proliferation ability than
diglycoside in HT29 colon cancer cells, which might imply the importance of glycoside
residue of anthocyanins on their activities.
In terms of radical scavenging activity of anthocyanidins, it might be responsible for
the presences of hydroxyl groups in position 3’ of ring C and also in the 3’, 4’, and 5’ positions
in ring B of the molecules. Generally, the antioxidant activity of anthocyanidins is higher than
that of anthocyanins, and it decreases as the number of sugar moieties increase.
Potential mechanisms of anthocyanins in cancer prevention
Anthocyanins also have demonstrated their strong antioxidant activity, which may be
involved prevention of tumor development caused by excessive oxidative stress. The
phenolic structure of anthocyanins may be related to their antioxidant and anti-carcinogenic
effects. The phenolic structure appear to help scavenging reactive oxygen species (ROS),
increasing the oxygen-radical absorbing capacity of cells, stimulating the expression of
Phase II detoxification enzymes, reducing the formation of oxidative adducts in DNA,
and decreasing lipid peroxidation by modulating signal transduction pathways.
Inflammation may play a role in the promotion of some types of cancer. Abnormal up-
regulation of inflammatory proteins such as nuclear factor-kappa B(NF-κB) and cyclooxygenase-
2(COX-2) is commonly present in many cancers, and inhibitors of those proteins showed
significant cancer preventive effect. Anthocyanins inhibit mRNA or protein expression
levels of NF-κB, COX-2, and various interleukins.
Dysregulated apoptosis also plays major role in inducing cancers. However, the
involved mechanisms are not conclusive and seem to depend on the cell lines and selected
anthocyanin or anthocyanidins. For example, in a recent study, the anthocyanins isolated from
Vitis coignetiae Pulliat induced apoptosis in colon cancer cells by activating p38-MAPK and
suppressing Akt. Other researchers reported that the same anthocyanins induced apoptosis
of human leukemia cells by being associated with modulation of expression of Bcl-2 and IAP
family members and proteolytic activation of caspase-3, -8 and -9. In another study, data
showed that delphinidin induced apoptosis of human colon cancer cells via suppressing of NF-
κB pathway.
Anthocyanins have proved their effect on cell-cycle regulation. By interrupting the
cell cycle at G1 and G2/M, they may induce apoptosis and inhibit cancer cell proliferation.
Anthocyain-rich blackberry extracts significantly reduced the G1 phase and increased proportion
of cells in the sub G1 phase, indicating apoptosis. Delphinidin induced G2/M cell cycle
arrest in human colon cancer cells. In human breast cancer cells, bilberry extract did cause
an increase in the fraction of cells at the G2/M phase of the cell cycle.
ANTHOCYANIN ENRICHED PURPLE-FLESHED SWEET
POTATO FOR POTENTIAL CANCER PREVENTION
abstract
Previously, we selected a purple-fleshed sweet potato clone, P40, from seeds obtained by
crossbreeding. This study is to identify the chemopreventive effect of anthocyanins from purple
sweet potato, P40. We treated SW480 human colon cancer cells with 0 - 40µM of peonidin-3-
glucoside or P40 extract containing corresponding amount of anthocyanins. Both of the
treatments inhibited cell growth in a dose-dependent manner, however, cells treated with P40
extract tends to survive significantly less than those treated with peonidin-3-glucoside. However,
there was no cytotoxicity occurrence during/after treatment. By checking the cell cycle changes,
we found the growth inhibition was not due to cytotoxicity, but due to cytostatic mechanism with
increased number of cells arrested at G1 phase. We also assessed cancer preventive effect of
purple sweet potato diet by using azoxymethane (AOM)-induced aberrant crypt foci (ACF) in
mice. AOM or saline injected mice were fed basal AIN-93M diet or diets containing 10~30% of
P40, 20% O’ Henry or 20% NC Japanese for 6 weeks. After the dietary treatment, ACF
multiplicity was significantly inhibited by 10~30% of P40 diet. Results of imunohistochemistry
in colonic mucosa showed that the expression level of apoptosis marker, caspase-3, was
significantly induced in the mice fed 20% of NC Japanese or 10~20% of P40 diet. Also, PCNA
expression level, which is proliferation marker, was significantly inhibited by 30% of P40 diet
compared to basal diet fed mice. Both in vitro and in vivo results suggest a promising
chemopreventive effect of P40 in cancers.
Introduction
Studies on the biological and nutraceutical properties of sweet potatoes tend towards focusing on
purple sweet potato. Studies have shown the free radical scavenging (1), antidiabetic (2), and
chemopreventive activity of purple sweet potato roots and leaves (3,4). These biological effects
of purple sweet potato may be due to the phenolic pigment "anthocyanin".
45
Anthocyanins are polyphenolic compounds, which are responsible for the intense colors of
many fruits and vegetables such as red grapes, berries, red cabbages and purple sweet potato
(5,6). Anthocyanins not only plays important role in industry as a natural food colorant, but also
provides various health benefits including antioxidant and anti-inflammatory effects (7-9). They
may also reduce the risk of cardiovascular disease (10), diabetes (11), and age-related
neurodegenerative diseases (12).
Anthocyanins or anthocyanin-rich extracts have exhibited inhibitory effect on cancer cell growth
or tumor-inducing cellular events in variety of cancer cells such as lung (13), breast (14),
prostate (15), liver (16), and colon (17) cancers, etc. Also, animal studies have been conducted to
prove their anti-cancer activities by using carcinogen-treated animal models. Those studies have
shown that a anthocyanin-rich diet induced apoptosis and inhibited cell proliferation,
inflammation, and angiogenesis, aberrant crypt foci (ACF) multiplicity, total tumor multiplicity,
tumor burden, and adenocarcinoma multiplicity in tumor tissues of cancer induced animals
(18,19).
Studies have suggested that several anti-cancer mechanisms of anthocyanins may be involved
such as their strong antioxidant, anti-inflammatory properties, and apoptosis induction by
regulating cell cycle in cancers. Phenolic structure of anthocyanins may act as an antioxidant and
inhibit tumor development caused by excessive oxidative stress (20-25). Also, anti-inflammatory
effect of anthocyanins may play an important role in cancer prevention. Abnormal up-regulation
of inflammatory proteins such as nuclear factor-kappa B (NF-κB) and cyclooxygenase-2 (COX-
2) is commonly present in many cancers, and inhibitors of those proteins showed significant
cancer preventive effect (26). Inhibitory effects of anthocyanins on mRNA or protein expression
levels of NF-κB, COX-2, and various inflammatory interleukins have been reported (27,28).
Studies showed that anthocyanin treatments may inhibit cell growth and induce apoptosis in
cancer cells by interrupting cell cycle at G1 and G2/M phase (29,30). However, the involved
mechanisms are still not conclusive and results differ depending on tested anthocyanins from
different sources.
In the present study, we bred a new variety of purple-fleshed sweet potato clone, P40, from
seeds obtained by crossbreeding from the International Potato Center in Lima, Peru. We
hypothesized that P40 may have high anthocyanin content and have health beneficial activities
compared to other sweet potato cultivars. To prove this, we included two cultivars as controls,
which are white- (O’ Henry) and yellow-fleshed (NC Japanese) sweet potato. Chemopreventive
effect of anthocyanin from purple sweet potato was tested on SW480 human colon cancer cells
and azoxymethan-induced aberrant crypt foci in mice. Finally, we investigated the potential
mechanisms involved in this inhibition.
Discussion
Colon cancer is the one of the most common cancers and the third leading cause of cancer death
for both men and women in the United States. The need for new chemopreventive agents
without adverse effects have led researchers’ interest to identifying phytochemicals for their
potential use. Studies have constantly shown that preventive effects of phytochemical
anthocyanins and their aglycons on colon cancer. As part of those
efforts, we evaluated SW480 colon cancer cell growth and cell cycle changes by treating
anthocyanin-rich purple sweet potato, P40, extract or its major anthocyanin, peonidin 3-
glucoside. After 48h incubation, both of the treatments significantly inhibited cancer cell growth
without cytotoxitity.
Interestingly, despite the fact we treated the cells with the same
level of anthocyanins in both treatments, the inhibitory effect was much higher in the cells
treated with P40 extract than those with peonidin 3-glucoside itself. P40 extract may contain
various chemopreventive phytochemicals other than anthocyanins such as phenolics. We also
learned that 48h treatment with peonidin 3-glucoside significantly arrested cancer cells increased
at G1 phase.
These findings suggest that P40 may inhibit cancer cell growth by
inducing apoptosis, not by cytotoxicity.
Azoxymethane (AOM) is a classic carcinogen that has been used to initiate and promote tumor
development in animal models that has characteristics similar to human colonic tumors (37).
Aberrant crypt foci (ACF) system has been used extensively to identify modulators of colon
carcinogenesis as the earliest identifiable putative premalignant precursors of animal or human
colon cancers (32, 38-40). In agreement with others, we used AOM-induced mice model to test
the chemopreventive effect of purple sweet potato diet. We fed the mice with formulated diet
high in sweet potato (10~30% P40, 20% O’ Henry, or 20% NC Japanese based on AIN93M diet
, and then evaluated the changes in ACF formation. 10-30% P40 diets effectively
inhibited ACF growth indicating preventive effect of anthocyanin diet on tumor development in
colon during the initiation phase by blocking ACF development. The result also showed 20% NC
Japanese diet significantly inhibited large ACF formation in mice colons (p < 0.01).
It could be because of small amount of anthocyanins which came from its purple cortex. It is also
possible that the inhibition effect might be associated with beta-carotenoids, which usually exist
in yellow-or orange-fleshed sweet potatoes (41). In this study, we only evaluated inhibitory
effects of P40 on ACF formation. However, we are planning to do an extended study for
observing the effect of anthocyanin on tumor endpoint.
Subsequently, we analyzed the mice colon tissues for protein expressions by
immunohistochemistry. Studies have used various immunohistochemical markers as prognostic
indices of tumors, for the most part carcinoma. Among those markers, we used caspase-3 and
PCNA. As critical mediators of mitochondrial events of apoptosis, caspase 3 staining is an
important method for indicating apoptosis level induced by a wide variety of apoptotic signals
(42). PCNA is associated with most of the carcinogenesis, which develops proliferative
abnormalities, thus it is often used as a cell proliferation marker (43). Our results showed that
20% NC Japanese diet and 20% P40 diet significantly induced caspase 3 expression in mice
colon compared to control diet.
However, there was no induction of caspase 3
expression in 30% P40 diet group. We assume it might be experimental error since
immunohistochemistry only gives semi-quantitative data. We will confirm the data with further
studies. In the colon of mouse fed 30% P40 diet, immunohistochemical staining level of PCNA
decreased statistically significantly compared to control diet group (Figure 3.5.C). These findings
suggest that 20% P40 diet may effectively upregulate Caspase 3 expression in mice colon
tissues, thus, promote cell apoptosis, and 30% P40 diet showed inhibitory effect on cell
proliferation in mice colon crypts.
In conclusion, we demonstrated the anticancer activity of anthocyanin-rich purple sweet potato,
P40 in vitro and in vivo. In vitro, we showed cell growth inhibition effect of P40 due to its high
level of anthocyanins content and suggested potential mechanism involved; high antioxidant
capacity of P40 and ability of arresting cancer cells at G1phase. In vivo, P40 diet significantly
inhibited ACF multiplicity by inducing apoptosis and inhibiting cell proliferation in AOM-
induced mice crypts. These findings suggest that new purple-fleshed sweet potato, P40 may be
an excellent therapeutic agent for preventing colon cancer in human.
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by Galileo Comba
