The genotoxicity study of resveratrol in primary gastric adenocarcinoma cell cultures.

Abstract

Gastric cancer is the second most common cause of cancer-related death in the world. Some studies indicate that polyphenolic compounds and antioxidants exert a protective action against gastric cancer. Among the polyphenolic compounds tested and proven effective against gastric cancer is resveratrol, a natural polyphenol present in red wines and various human food items. Resveratrol has been shown to suppress proliferation of a wide variety of tumor cells. We tested the genotoxic activity of resveratrol in primary cell cultures from gastric adenocarcinoma, obtained by mucosal biopsy at upper digestive endoscopy. The adenocarcinoma cells were analyzed for the presence of micronuclei at different concentrations of resveratrol at 48 hours and at 72 hours. The results showed that resveratrol induced micronuclei dose-dependently. The frequency of micronuclei increased progressively with the dose of resveratrol used, the high frequency is in the primary culture initiated from gastric adenocarcinoma: signet ring cell type. The high frequency of micronuclei is at 72 hours at the 20 microg/mL resveratrol and is decreased at low concentrations (5 microg/mL, 10 microg/mL resveratrol). This results show the genotoxic activity of resveratrol in adenocarcinoma gastric cell and the anticancer property of this substance.

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Romanian Journal of Morphology and Embryology 2009, 50(3):429–433

O OR RI IG GI IN NA AL L P PA AP PE ER R
The genotoxicity study of resveratrol
in primary gastric adenocarcinoma
cell cultures
P. MITRUŢ1), FL. BURADA2), AURELIA ENESCU1), R. SCOREI3),
DANIELA BADEA4), AMELIA GENUNCHE-DUMITRESCU1),
I. ROGOZ2), MĂDĂLINA MANEA5)
1)Department of Internal Medicine
2)Department of Genetics
Faculty of Medicine, University of Medicine and Pharmacy of Craiova
3)Department of Biochemistry, University of Craiova
4)Department of Physiology, Faculty of Medicine
5)Department of General Nursing, Faculty of Nursing and Midwives
University of Medicine and Pharmacy of Craiova
Abstract
Gastric cancer is the second most common cause of cancer-related death in the world. Some studies indicate that polyphenolic
compounds and antioxidants exert a protective action against gastric cancer. Among the polyphenolic compounds tested and proven
effective against gastric cancer is resveratrol, a natural polyphenol present in red wines and various human food items. Resveratrol has
been shown to suppress proliferation of a wide variety of tumor cells. We tested the genotoxic activity of resveratrol in primary cell cultures
from gastric adenocarcinoma, obtained by mucosal biopsy at upper digestive endoscopy. The adenocarcinoma cells were analyzed for the
presence of micronuclei at different concentrations of resveratrol at 48 hours and at 72 hours. The results showed that resveratrol induced
micronuclei dose-dependently. The frequency of micronuclei increased progressively with the dose of resveratrol used, the high frequency
is in the primary culture initiated from gastric adenocarcinoma: signet ring cell type. The high frequency of micronuclei is at 72 hours at the
20 µg/mL resveratrol and is decreased at low concentrations (5 µg/mL, 10 µg/mL resveratrol). This results show the genotoxic activity of
resveratrol in adenocarcinoma gastric cell and the anticancer property of this substance.
Keywords: gastric cancer, adenocarcinoma, resveratrol, cell culture, micronuclei.
? Introduction
Gastric cancer is the second most common cause of
cancer-related death in the world. Although the
incidence of gastric cancer is on the decline, this disease
remains a major health problem and a common cause of
cancer mortality worldwide, because the disease is
usually detected at an advanced stage, and the currently
available chemotherapeutic agents are not highly
effective. Adenocarcinoma of the stomach constitutes
90–95% of all gastric malignancies and the second
most common gastric malignancies are lymphomas.
At present, there are limited experimental data regarding
specific agents that prevent or retard gastric carcino-
genesis. Some studies indicate that polyphenolic
compounds and antioxidants exert a protective action
against gastric cancer. Among the polyphenolic
compounds tested and proven effective against gastric
cancer is resveratrol. Resveratrol (3,5,4’-trihydroxy-
stilbene) is a natural polyphenol produced by a wide
variety of plants such as grapes (Vitis vinifera), peanuts
(Arachis hypogaea), and mulberries in response to
stress, injury, ultraviolet (UV) irradiation, and fungal
(e.g., Botrytis cinerea) infection [1–3]. The resveratrol
was shown to inhibit the induction, promotion, and
progression of experimentally induced cancer [4].
Moreover, resveratrol inhibits transcription and activity
of cyclooxygenase-2 [5, 6], an enzyme found to be
upregulated in a number of transformed cells and
various forms of cancer. Resveratrol has been shown to
suppress proliferation of a wide variety of tumor cells,
including lymphoid and myeloid cancers, breast, colon,
pancreas, stomach, prostate, ovary, liver, lung and
cervical cancers, melanoma, and muscles [7].
Resveratrol also exhibits antibacterial effects, including
inhibition of growth of different strains of Helicobacter
pylori [8, 9]. Besides inhibiting proliferation, resveratrol
also induces apoptosis either through the caspase-8-
dependent pathway (receptor-mediated; type I) or the
caspase-9-dependent pathway (mitochondrial; type II),
or both [7].
? Material and Methods
We tested the resveratrol in primary adenocarcinoma
cell culture and we analyzed the frequency of micro-
nuclei at different concentrations of the resveratrol.
The adenocarcinoma fragments were obtained by

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430
mucosal biopsy at upper digestive endoscopy. Esopha-
gogastroduodenoscopy has a diagnostic accuracy of
95%. This relatively safe and simple procedure provides
a permanent color photographic record of the lesion.
This procedure is also the primary method to obtain
tissue for diagnosis the suspected lesions. Biopsy of any
ulcerated lesion should require at least six biopsies taken
from around the lesion because of variable malignant
transformation.
The histopathological examination was made in the
Department of Pathology, Emergency County Hospital
of Craiova. The pieces were processed in Pathology Lab
of the same hospital. All specimens were fixed in 15%
buffered neutral pH formaldehyde and paraffin
embedded. Histological sections were stained using
current techniques: Hematoxylin–Eosin, trichromic
van Gieson and Giemsa (for Helicobacter pylori).
We used Laurén histological classification with two
main types of gastric carcinoma: type I (intestinal)
or type II (diffuse). Adenocarcinoma of the stomach
is subclassified according to histological description as
follows: tubular, papillary, mucinous, or signet-ring
cells and undifferentiated lesions.
The gastric adenocarcinoma fragments obtained by
gastric biopsy in upper digestive endoscopy were
quickly transferred to a cell culture laboratory in RPMI
1640 that contain antibiotics. The adenocarcinoma
tumor fragments were minced into very small pieces
with sterile scissors. The tumor cells were carefully
harvest after washing with phosphate buffered saline
(PBS), centrifugation, and were cultivated into a sterile
culture flasks 25 cm2 containing RPMI 1640 medium
(Biochrom AG) supplemented with 10% heat inactivated
fetal bovine serum and antibiotic. The flasks were
maintained in a humidified incubator at 370C in an
atmosphere of 5% CO2. Isolation of cancer cells was
performed by trypsinization when heavy tumor-cell
growth is observed. The cell cultures were examined at
an inverted microscope (Carl Zeiss Citoval) to
approximately 12–24 hours, the success of every culture
is cell proliferation and the appearance of normal
culture medium. The resveratrol (Sigma Aldrich) diluted
in DMSO (dimethyl-sulfoxide) was administrated in
adenocarcinoma cell cultures, in different concentrations
(5 µg/mL, 10 µg/mL, and 20 µg/mL) at each passage
and maintained for 48 hours and 72 hours. In the control
culture was administered DMSO without resveratrol.
After 48 hours, and respectively 72 hours, we
performed the standard cell preparation and staining
with 10% Giemsa stain, and the microscopic slides were
examined with an optical microscope Nikon Eclipse
E200, images were taken with a digital camera (Canon
A650). A total of 1000 tumoral cell were scored per
slide at different resveratrol doses.
? Results
The initiation of primary cell cultures was made
from a case with advanced Borrmann type I gastric
cancer (protruded, protrusive) located on the gastric
fornix (Figure 1), which histopathologically was gastric
adenocarcinoma: signet ring cell type (Figure 2).

Figure 1 – Advanced Borrmann type I gastric cancer
(protruded, protrusive) located on the gastric fornix.

Figure 2 – Gastric adenocarcinoma: signet ring cell
type.
This case was a 69-year-old male, admitted in the
IInd Medical Clinic of the Emergency County Hospital
of Craiova for upper abdominal discomfort. Endoscopic
examination revealed an advanced Borrmann type I
gastric cancer (protrusive). Distal gastrectomy with
sufficient duodenal resection was performed. The patient
was infected with Helicobacter pylori.
Also, another primary cell culture was made from
advanced Borrmann type III gastric cancer (ulcero-
infiltrative) (Figure 3), which histopathologically was
gastric adenocarcinoma papillary type (Figure 4).
This case was a case of a 76-year-old woman.
We scored a total 1000 tumoral cells per concen-
tration of resveratrol to emphasize the presence of
micronuclei from each culture. As negative control,
we used a cell culture treated with solution of DMSO.

Figure 3 – Advanced Borrmann type III gastric
cancer (ulcero-infiltrative).

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431

Figure 4 – Gastric adenocarcinoma: papillary type.
An increased frequency of micronuclei in cell
culture treated with resveratrol in comparison with the
control cell culture indicates that the substance tested
induces chromosomal damage in gastric adenocarcinoma
cell culture.
In gastric adenocarcinoma cell culture signet ring
cell type, the number of micronuclei depends on the
exposure time and the resveratrol dose: in the control
culture the cells number with one micronucleus is 28 at
48 hours (Figure 5), and at 72 hours it is 32; at the
following doses of resveratrol: 5, 10, and 20 µg/mL the
incidence of micronuclei increases progressively
(Table 1).

Figure 5 – Cell with
micronucleus (from
gastric adenocarci-
noma signet ring cell
type).

Table 1 – The frequency of micronuclei in adeno-
carcinoma cell culture signet ring cell type
Cells with one
micronucleus/1000
cells
48 hours 72 hours 48 hours 72 hours
Control culture
28
Cell culture with
5 µg/mL resveratrol
Cell culture with
10 µg/mL resveratrol
Cell culture with
20 µg/mL resveratrol
Cells with two
micronuclei/1000
cells
Cell culture
32 – –
34 44 2 2
40 66 – 2
90 122 7 10
At 48 hours and 72 hours, the frequency of
micronuclei is increased compared with control culture.
The frequency of micronuclei increases progressively
with the dose of resveratrol used, the high frequency is
at 10 and 20 µg/mL resveratrol (Figure 6) at 72 hours,
and the low frequency is at 48 hours at 5 µg/mL
resveratrol.

Figure 6 – The incidence of micronuclei in gastric
adenocarcinoma signet ring cell type at different
resveratrol doses, at 48 hours and 72 hours.
Also, we scored the cell with two micronuclei and
we observed that at 20 µg/mL, resveratrol induces this
kind of cells and at 72 hours the number of cell with two
micronuclei is 10 compared with the control where there
are not cells with two or more micronuclei.
In gastric adenocarcinoma cell culture papillary type
also the number of cells with one micronucleus increases
as follows:
▪ in control cell culture, it is 18 at 48 hours and 26 at
72 hours;
▪ in the culture treated with 5 µg/mL resveratrol, the
number of cells with one micronucleus is 34 at 48 hours
and 32 at 72 hours;
▪ in the culture with 10 µg/mL resveratrol, it is 48 at
48 hours and 66 at 72 hours (Table 2).
Table 2 – The frequency of micronuclei in adeno-
carcinoma cell culture papillary type
Cells with one
micronucleus/1000
cells
48 hours 72 hours 48 hours 72 hours
Control culture
18
Cell culture with
5 µg/mL resveratrol
Cell culture with
10 µg/mL resveratrol
Cell culture with
20 µg/mL resveratrol
Cells with two
micronuclei/1000
cells
Cell culture
26 – –
34 32 1 2
48 66 – 2
81 92 4 7
The high frequency is at 72 hours at 20 µg/mL
resveratrol (Figure 7).

Figure 7 – The incidence of micronuclei in gastric
adenocarcinoma papillary type at different resveratrol
doses, at 48 hours and 72 hours.
The number of micronuclei is slowly increased in
gastric adenocarcinoma cell culture signet ring cell type
compared with adenocarcinoma cell culture papillary
type.
Genotoxic activity of resveratrol is indicated by an
increased incidence of micronucleated adenocarcinoma

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432
cells for the treatment group compared with the
concurrent control group. In our experiments, the
resveratrol induces micronuclei in adenocarcinoma
gastric cell culture dose-dependently, and the high
incidence of micronuclei is at 20 µg/mL resveratrol.
Micronuclei are formed because of chromosomal
breakage or spindle damage. Fragments of whole
chromosomes may not be included in the nuclei of
the daughter cells following cell division and form
single, or multiple micronuclei in the cytoplasm of
these cells. These results show the genotoxic activity
of resveratrol in adenocarcinoma gastric cell and show
the anticancer property of this substance.
? Discussion
Resveratrol induced micronuclei in other cell
cultures. Schmitt E et al. investigated the genotoxic
potential of resveratrol. In L5178Y mouse lymphoma
cells, they found a reduction in cell proliferation
and in cell viability, as well as an induction of
micronuclei [10].
In the Chinese hamster lung, the resveratrol induced
micronuclei, polynuclei and karyorrhectic cells after
a 48 hours treatment and sister chromatid exchanges
in a dose dependent manner at concentrations up to
10 µg/mL. A slight increase in micronuclei occurred
with the 24 hours treatment and a dose-dependent
increase in micronuclei, polynuclei, and karyorrhectic
cells occurred with the 48 hours treatment up to the
10 µg/mL dose. The resveratrol caused S-phase arrest
and induced apoptosis after a 48 hours treatment [11].
Several compounds show structural similarities to
trans-resveratrol. In mouse L5178Y lymphoma-cells,
resorcinol induced trifluorothymidine resistance in the
absence of S9. With and without S9, resorcinol induced
sister chromatid exchanges in Chinese hamster ovary
(CHO) cells, and only with S9 did it induce chromo-
somal aberrations. Positive results were obtained in the
micronulei test [12].
Resveratrol has been shown to suppress proliferation
of gastric cancer cells [13–15]. Atten MJ et al. reported
that resveratrol inhibited proliferation of nitrosamine-
stimulated human gastric adenocarcinoma KATO-III
and RF-1 cells [13]. It arrested KATO-III cells in the
G0/G1 phase of the cell cycle and eventually induced
apoptotic cell death by utilizing a protein-kinase C
(PKC)-mediated mechanism to deactivate these gastric
adenocarcinoma cells. Holian O et al. demonstrated
that, in gastric adenocarcinoma cell line SNU-1, which
was stimulated by hydrogen peroxide (H2O2), resveratrol
suppressed DNA-synthesis and generation of endogenous
O2, but stimulated NOS activity, which may have been
responsible for inhibition of SNU-1 proliferation [14].
Resveratrol also inhibited the growth of esophageal
cancer cell line EC–9706 [15].
Resveratrol participates in the prevention of carcino-
genesis by inhibition of P450, an enzyme of phase I
[16, 17], and through induction of phase II xenobiotic
metabolizing enzymes [18]. Resveratrol can induce
the activation of p53 and the subsequent apoptosis
occurring through p53-dependent pathway [19], but it
can also induce apoptosis independently of p53 [20].
Resveratrol can modulate signal transduction pathways
by the inhibitory effect on the activation of transcription
factors such as NF-κBand AP1 [18].
Resveratrol, as a selective inhibitor of cyclo-
oxygenase-2 (COX-2), is also a strong inhibitor of
the dioxygenase activity of lipoxygenase (LOX) [21].
Atten MJ et al. demonstrated that resveratrol treatment
significantly inhibited PKC activity of KATO-III human
gastric adenocarcinoma cells and of human recombinant
PKC-a [13]. Holian O et al. found that resveratrol
stimulated NOS (nitric oxide synthase) activity in
human gastric adenocarcinoma SNU-1 cells [14].
They suggested that the antioxidant action of resveratrol
toward gastric adenocarcinoma cells might reside in its
ability to stimulate NOS to produce low levels of
NO (nitric oxide), which, in turn, exerts antioxidant
action. Thus, whether resveratrol induces or inhibits
NO production depends on the cell system, inducer,
and other conditions.
? Conclusions
Polyphenolic compounds and antioxidants exert a
protective action against gastric cancer.
Resveratrol induces micronuclei in adenocarcinoma
cell culture, dose-dependently. An increase number of
micronuclei indicates genotoxic activity of resveratrol,
the high incidence is at 20 µg/mL resveratrol.
Resveratrol is possible to be used as a cancer chemo-
preventive or even cancer therapeutic agent. Therefore,
agents like resveratrol inhibit proliferation of transformed
gastric epithelial cells while remaining relatively
nontoxic to the host may constitute a new and effective
defense against gastric carcinogenesis.
Acknowledgements
The present paper is integral part of the research
grants “Experimental studies on cell cultures and
animals regarding the effect of resveratrol extracted
from vine on gastric neoplasic injuries”, CNCSIS
Program 2007–2008, Contract no. 954, and “Endoscope,
histological and immunohistochemical monitorization
of gastric premalignant lesions and characterization of
precocious stages of oncogene invasion in gastric
cancer”, CEEX Program (2006–2008), financed by
MEdC–ANCS, Contract no. 190.
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Corresponding author
Paul Mitruţ, Assistant Professor, MD, PhD, Department of Internal Medicine, Faculty of Medicine, University of
Medicine and Pharmacy of Craiova, 2–4 Petru Rareş Street, 200349 Craiova, Romania; Phone +40722–724 430,
e-mail: centrul_renasterea@rdscv.ro






Received: March 29th, 2009
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Accepted: June 25th, 2009