INHIBITION OF CANCER CELL PROLIFERATION IN
VITRO BY DIFFERENT BERRIES AND CORRELATION
WITH THEIR ANTIOXIDANT ACTIVITIES
Shela Gorinstein#1, Yong Seo Park2, Sylwia Flis3, Zenon Jastrzebski3, Patricia
Arancibia-Avila4, Milan Suhaj5, Elena Katrich1, Moshe Weisz1, Zeev Tashma1
1The
Hebrew University of Jerusalem, Faculty of Medicine, School of Pharmacy, The Institute for Drug Research, Jerusalem, Israel
2Department of Horticultural Science, Mokpo National University, Muan, South Korea
3Department
of Pharmacology, National Medicines Institute, Warsaw, Poland
of Basic Sciences, Universidad del Bio-Bio, Chillan, Chile
5Food Research Institute, 824 75 Bratislava, Slovakia
4Department
#This
research was done in memory of my dear brother Prof. Simon Trakhtenberg,
who died in November 2011, who encouraged and supported me during all his life
Our publications- http://www.bashanfoundation.org/shela/shelapub.html
1. Background
The aim of this research was to find out the proliferation inhibition properties in vitro of Chilean and
Polish berries [Myrteola nummularia, ‘Murtilla-like’] vs. well known ‘Murtilla’, blueberries, and
raspberries and the possible correlations with bioactive compounds and the antioxidant activities
(AA). Dimethylsulfoxide (DMSO) extracts of berries on human colorectal cancer (CRC) cell lines
HT-29 and SW48 proliferation were investigated.
Figure 3. Induction of cell
death by berries extracts in
the human CRC cells. Death
cells (%) was determined by
FACS analysis after 72 h of
treatment. Staining - annexin
V-FITC/PI. The induction of
cell death was significant (*)
in comparison with control
(P<0.05). Cells stained with
Calcein-AM and PI (10×).
Green fluorescence indicates
living cells, red fluorescence dead cells. Blueberry Poland
(BBP), blueberry Chile
(BBCh), 'Murtilla' (MT) and
'Murtilla-like' (MTL).
2. Materials and methods
Bioactive compounds and the antioxidant activities in berries were determined by radical
scavenging assays. The interaction between bovine serum albumin (BSA) and quercetin,
and BSA and berry extracts was measured by 3-dimensional fluorescence (3D-FL) and
FTIR (1-4). MTT assay, flow cytometric analyses of cell cycle and apoptosis were used.
3. Results
A
B
Figure 1. A , Box/Whisker and Dot Plots of DPPH-free radical activity of DMSO extracts; B, Principal component
analysis (PCA), based on DPPH measurements of DMSO extracts of MNR- ‘Murtilla’ non-ripe, MLN- ‘Murtilla-like’
non-ripe, BP- blueberry Poland, BC- blueberry Chile, and R- raspberry; Extract concentrations (1- 25 mgml-1) and
DPPH reaction times (1 – 90 min) were used.
DPPH (1, 1-diphenyl-2-picrylhydrazyl radical) kinetic measurements (Fig. 1A) showed
that the quenching ability of the DMSO extracts were comparable between them, with
the exception of higher values of ‘Murtilla’ non-ripe berries. The plot of principal
components (Fig. 1B) illustrates the large variability of the investigated berries. PCA
segregated from the rest berries clearly only one group of ‘Murtilla’ non-ripe with the
highest antiradical activity.
The berry extracts decreased the proliferation of both CRC cells (Figs. 2, 3),
and the effect was concentration dependent. The inhibition effect of growth
of human CRC cells for the highest concentration of the extracts varied 2-3fold among the berries. The lowest IC50 values 751 and 858 µg/ml were for MT
extract on HT-29 and SW48. HT-29 cells treated with MT showed a decrease
in G1 phase cells from 77% to 56%. Death cells (%) treated with MT were 80.1
and 72.5 for SW48 and HT-29 cells, respectively.
Fluorescence emission system of BSA was studied with increased
concentrations of flavonoids and berry extracts and with increased duration
of reaction time (Fig. 4). After interaction with quercetin and berries extracts
FTIR spectrum of BSA showed a shift in the maximum of Amides I and II in
the region of 1650 and 1550 cm-1, respectively.
4. Discussion
There were differences in the contents of the analyzed antioxidants in the
extracts: the highest amount of polyphenols, flavonoids and antioxidant
activities were in 'Murtilla' berry extract (1-4). DPPH kinetic measurements were
used to compare, distinguish and discriminate the antiradical activity among
berry extracts by multivariate analysis. It is known that the interaction between
drugs and serum albumin plays an important role in the distribution and
metabolism of drugs, therefore the interaction between two kinds of flavonoids
(catechin and quercetin) and BSA and berries extracts was investigated by 3D-
A
B
FL and FTIR spectroscopy (4). The results of this study indicated that
flavonoids have strong ability to quench the intrinsic fluorescence of BSA by
forming complexes. The interaction between polyphenol extracts of 'Murtillalike' and BSA showed that the new kind of berries has a strong ability as other
studied samples to decrease the intrinsic fluorescence of BSA.
5. Conclusions
Figure 2. A, Survival of human colorectal cancer (CRC) cells of the SW48 and HT-29 lines. B,
Changes in the cell cycle progression of human CRC cells after 72 h of treatment with berries
extracts: blueberry Poland (BBP), blueberry Chile (BBCh), 'Murtilla' (MT) and 'Murtilla-like' (MTL).
A
B
C
The inhibition of cancer cell proliferation correlated with the levels
of polyphenols, flavonoids and their antioxidant activities.
These findings suggest that the intake of a new kind of berry,
as a source of natural antioxidants, may reduce colon cancer
risk.
6. References
Figure 4. Changes in the fluorescence intensity as a result of binding affinity (A): 2 x 10-5 M/L BSA
(first line from the top) and 8 g ‘Murtilla’ (second line from the top) with addition of 0.88 x 10-5 M/L
quercetin after 20 min (last line from the top). The reaction times: 0, 2, 5, 10, 15, and 20 min; (B): The
same conditions as in (A), but quercetin and ‘Murtilla’ extracts were added immediately to BSA at 0
time (second line from the top); (C) contour map (from 3 D-FL spectra) illustrates the quenching
results after reaction time of 20 min as in (A).
1. Gorinstein, S., Leontowicz, H., Leontowicz, M., Jesion, I., Namiesnik, J., Drzewiecki, J., Park, Y.S,
Ham, K.S., Giordani, E., & Trakhtenberg, S. (2011). Nutrition, 27, 838-846.
2. Park, Y.S., Leontowicz, H., Leontowicz, M., Namiesnik, J., Suhaj, M., Cvikrova, M., Martincova, O.,
Weisz, M., & Gorinstein, S. (2011), Journal of Food Composition and Analysis, 24, 963-970.
3. Gorinstein, S., Poovarodom, S., Leontowicz, H., Leontowicz, M., Namiesnik, J., Vearasilp, S.,
Haruenkit, R., Ruamsuke, P., Katrich, E., & Tashma, Z. (2011). Food Research International, 44,
2222-2232.
4. Leontowicz, H., Leontowicz, M., Jesion, I., Bielecki, W., Poovarodom, S., Vearasilp, S., GonzálezAguilar, G., Robles-Sánchez, M., Trakhtenberg, S., & Gorinstein, S., 2011. European Journal of
Integrative Medicine, 3, 169–181.