Genotoxic factors associated with the development of receptor-negative breast cancer: potential role of the phenomenon of switching of estrogen effects
Aim: About 30–40% of breast cancers lack steroid receptors (ER and/or PR) at diagnosis that worsen prognosis and limit the usage of hormone therapy. The aim of this paper has been to study the role of DNA-damaging factors as the potential modifiers of the receptor-negative tumors incidence. Material...
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
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Berstein, L.M. Tsyrlina, E.V. Poroshina, T.E. Levina, V.V. Vasilyev, D.A. Kovalenko, I.G. Semiglazov, V.F. 2018-06-13T16:38:05Z 2018-06-13T16:38:05Z 2006 Genotoxic factors associated with the development of receptor-negative breast cancer: potential role of the phenomenon of switching of estrogen effects / L.M. Berstein, E.V. Tsyrlina, T.E. Poroshina, V.V. Levina, D.A. Vasilyev, I.G. Kovalenko, V.F. Semiglazov // Experimental Oncology. — 2006. — Т. 28, № 1. — С. 64-69. — Бібліогр.: 32 назв. — англ. 1812-9269 https://nasplib.isofts.kiev.ua/handle/123456789/134529 Aim: About 30–40% of breast cancers lack steroid receptors (ER and/or PR) at diagnosis that worsen prognosis and limit the usage of hormone therapy. The aim of this paper has been to study the role of DNA-damaging factors as the potential modifiers of the receptor-negative tumors incidence. Materials and Methods: The investigation consisted of two principal parts. In one of them ER and PR content was measured in breast cancer samples from 2284 primary patients (350 of them — current or previous smokers). In separately studied subgroup of 1010 patients 95 suffered with diabetes mellitus type II. Results: As it was shown, smokers and diabetics carry more frequently (p < 0.05) tumors with phenotypes ER+PR- and PR- only in the group of women with conserved menstrual cycle that is in case of relatively higher estrogenic stimulation. In another part of the investigation immunohistochemical study of DNA damage marker — 8-hydroxy-2-deoxyguanosine (8-OH-dG) in 16 R(-) and 18 R(+) breast cancer specimens demonstrated more frequent positive staining in the former group of samples (p = 0.05). Besides, as it was revealed in breast cancer cell line MCF-7 the combination of estradiol with aryl hydrocarbonic receptors agonist beta-naphtoflavone induced pronounced genotoxic damage (by 8-OH-dG content) in association with the loss of ER. Conclusion: Thus, pro-genotoxic status (smoking, diabetes) and direct signs of genotoxic injury, in accordance with regularities of the phenomenon of switching of estrogen effects. can be reckoned among the factors promoting the development of receptor-negative breast cancer. Цель исследования: примерно в 30–40% случаев рака молочной железы (РМЖ) рецепторы стероидных гормонов (ЭР и/или ПР) не выявлены, что оказывает неблагоприятное влияние на прогноз заболевания и ограничивает применение гормонотерапии. Задачей настоящей работы было изучение роли факторов, ассоциированных с повреждением ДНК, как потенциальных модификаторов частоты возникновения рецепторнегативных новообразований. Материалы и методы: исследование состояло из двух основных частей. У 2284 первичных больных РМЖ (350 из них — курящие или курившие ранее) содержание ЭР и ПР в опухолевой ткани определяли рецепторным методом. В отдельно проанализированной подгруппе из 1010 больных у 95 пациенток диагностировали сахарный диабет ІІ типа. Результаты: установлено, что в отличие от пациенток в постменопаузальный период с сохраненной менструальной функцией (то есть в условиях более высокой эстрогенной стимуляции) отмечается достоверное преобладание опухолей фенотипа ЭР + ПР — у курящих по сравнению с некурящими (t = 2,18, p < 0,05), а также у больных сахарным диабетом по сравнению с больными без него (t = 2,01, p = 0,05). Кроме того, в репродуктивный период у больных сахарным диабетом доля ПР(-)-опухолей достоверно превосходила таковую у пациенток без него (t = 2,17, p < 0,05). Во второй части исследования с помощью иммуногис- тохимического метода изучено содержание показателя повреждения ДНК 8-гидрокси-2-дезоксигуанозина (8-OH-dG) в 16 рецепторнегативных и 18 рецепторпозитивных опухолях молочной железы. Выявлено, что в первой группе позитивное окрашивание наблюдали в 81,3 ± 9,8%, а во второй — в 50,0 ± 11,7% случаев, p = 0,05. Помимо этого, результаты экспе- римента на клеточной линии МСF-7 рака молочной железы свидетельствуют, что при комбинации эстрадиола с агонистом арилгидрокарбоновых рецепторов бета-нафтофлавоном значительное усиление генотоксического повреждения (по данным определения 8-ОН-dG) отмечается уже к 60 мин эксперимента, когда выявляют и снижение концентрации ЭР. Выводы: прегенотоксический статус (курение, сахарный диабет), равно как и признаки прямого генотоксического повреждения, в соответствии с представлениями о феномене переключения эстрогенного эффекта могут быть причислены к факторам, способствующим развитию рецепторнегативного РМЖ. These studies were partly supported by grant from RFBR (03-04-49282). en Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України Experimental Oncology Original contributions Genotoxic factors associated with the development of receptor-negative breast cancer: potential role of the phenomenon of switching of estrogen effects Генотоксические факторы, ассоциированные с возникновением рецептор-негативного рака молочной железы: потенциальная роль феномена переключения эстрогенного эффекта Article published earlier |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine |
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DSpace DC |
| title |
Genotoxic factors associated with the development of receptor-negative breast cancer: potential role of the phenomenon of switching of estrogen effects |
| spellingShingle |
Genotoxic factors associated with the development of receptor-negative breast cancer: potential role of the phenomenon of switching of estrogen effects Berstein, L.M. Tsyrlina, E.V. Poroshina, T.E. Levina, V.V. Vasilyev, D.A. Kovalenko, I.G. Semiglazov, V.F. Original contributions |
| title_short |
Genotoxic factors associated with the development of receptor-negative breast cancer: potential role of the phenomenon of switching of estrogen effects |
| title_full |
Genotoxic factors associated with the development of receptor-negative breast cancer: potential role of the phenomenon of switching of estrogen effects |
| title_fullStr |
Genotoxic factors associated with the development of receptor-negative breast cancer: potential role of the phenomenon of switching of estrogen effects |
| title_full_unstemmed |
Genotoxic factors associated with the development of receptor-negative breast cancer: potential role of the phenomenon of switching of estrogen effects |
| title_sort |
genotoxic factors associated with the development of receptor-negative breast cancer: potential role of the phenomenon of switching of estrogen effects |
| author |
Berstein, L.M. Tsyrlina, E.V. Poroshina, T.E. Levina, V.V. Vasilyev, D.A. Kovalenko, I.G. Semiglazov, V.F. |
| author_facet |
Berstein, L.M. Tsyrlina, E.V. Poroshina, T.E. Levina, V.V. Vasilyev, D.A. Kovalenko, I.G. Semiglazov, V.F. |
| topic |
Original contributions |
| topic_facet |
Original contributions |
| publishDate |
2006 |
| language |
English |
| container_title |
Experimental Oncology |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| format |
Article |
| title_alt |
Генотоксические факторы, ассоциированные с возникновением рецептор-негативного рака молочной железы: потенциальная роль феномена переключения эстрогенного эффекта |
| description |
Aim: About 30–40% of breast cancers lack steroid receptors (ER and/or PR) at diagnosis that worsen prognosis and limit the usage of hormone therapy. The aim of this paper has been to study the role of DNA-damaging factors as the potential modifiers of the receptor-negative tumors incidence. Materials and Methods: The investigation consisted of two principal parts. In one of them ER and PR content was measured in breast cancer samples from 2284 primary patients (350 of them — current or previous smokers). In separately studied subgroup of 1010 patients 95 suffered with diabetes mellitus type II. Results: As it was shown, smokers and diabetics carry more frequently (p < 0.05) tumors with phenotypes ER+PR- and PR- only in the group of women with conserved menstrual cycle that is in case of relatively higher estrogenic stimulation. In another part of the investigation immunohistochemical study of DNA damage marker — 8-hydroxy-2-deoxyguanosine (8-OH-dG) in 16 R(-) and 18 R(+) breast cancer specimens demonstrated more frequent positive staining in the former group of samples (p = 0.05). Besides, as it was revealed in breast cancer cell line MCF-7 the combination of estradiol with aryl hydrocarbonic receptors agonist beta-naphtoflavone induced pronounced genotoxic damage (by 8-OH-dG content) in association with the loss of ER. Conclusion: Thus, pro-genotoxic status (smoking, diabetes) and direct signs of genotoxic injury, in accordance with regularities of the phenomenon of switching of estrogen effects.
can be reckoned among the factors promoting the development of receptor-negative breast cancer.
Цель исследования: примерно в 30–40% случаев рака молочной железы (РМЖ) рецепторы стероидных гормонов (ЭР
и/или ПР) не выявлены, что оказывает неблагоприятное влияние на прогноз заболевания и ограничивает применение
гормонотерапии. Задачей настоящей работы было изучение роли факторов, ассоциированных с повреждением ДНК, как
потенциальных модификаторов частоты возникновения рецепторнегативных новообразований. Материалы и методы:
исследование состояло из двух основных частей. У 2284 первичных больных РМЖ (350 из них — курящие или курившие
ранее) содержание ЭР и ПР в опухолевой ткани определяли рецепторным методом. В отдельно проанализированной
подгруппе из 1010 больных у 95 пациенток диагностировали сахарный диабет ІІ типа. Результаты: установлено, что в
отличие от пациенток в постменопаузальный период с сохраненной менструальной функцией (то есть в условиях более
высокой эстрогенной стимуляции) отмечается достоверное преобладание опухолей фенотипа ЭР + ПР — у курящих по
сравнению с некурящими (t = 2,18, p < 0,05), а также у больных сахарным диабетом по сравнению с больными без него
(t = 2,01, p = 0,05). Кроме того, в репродуктивный период у больных сахарным диабетом доля ПР(-)-опухолей достоверно
превосходила таковую у пациенток без него (t = 2,17, p < 0,05). Во второй части исследования с помощью иммуногис-
тохимического метода изучено содержание показателя повреждения ДНК 8-гидрокси-2-дезоксигуанозина (8-OH-dG) в
16 рецепторнегативных и 18 рецепторпозитивных опухолях молочной железы. Выявлено, что в первой группе позитивное
окрашивание наблюдали в 81,3 ± 9,8%, а во второй — в 50,0 ± 11,7% случаев, p = 0,05. Помимо этого, результаты экспе-
римента на клеточной линии МСF-7 рака молочной железы свидетельствуют, что при комбинации эстрадиола с агонистом
арилгидрокарбоновых рецепторов бета-нафтофлавоном значительное усиление генотоксического повреждения (по данным
определения 8-ОН-dG) отмечается уже к 60 мин эксперимента, когда выявляют и снижение концентрации ЭР. Выводы:
прегенотоксический статус (курение, сахарный диабет), равно как и признаки прямого генотоксического повреждения,
в соответствии с представлениями о феномене переключения эстрогенного эффекта могут быть причислены к факторам,
способствующим развитию рецепторнегативного РМЖ.
|
| issn |
1812-9269 |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/134529 |
| citation_txt |
Genotoxic factors associated with the development of receptor-negative breast cancer: potential role of the phenomenon of switching of estrogen effects / L.M. Berstein, E.V. Tsyrlina, T.E. Poroshina, V.V. Levina, D.A. Vasilyev, I.G. Kovalenko, V.F. Semiglazov // Experimental Oncology. — 2006. — Т. 28, № 1. — С. 64-69. — Бібліогр.: 32 назв. — англ. |
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2025-11-24T21:37:40Z |
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| fulltext |
64 Experimental Oncology 28, 64–69, 2006 (March)
Estrogen and progesterone help regulate growth
and differentiation of normal breast tissue, and they are
considered important in the development and progres-
sion of breast cancer [12, 24]. Estrogen receptors (ER)
and progesterone receptors (PR) are nuclear recep-
tors. The estrogen-ER complex binds directly to DNA
and influences the expression of estrogen-responsive
genes, including the gene for PR. In other words, since
PR is an estrogen-dependent protein, its expression
depends on the both: the existence of ER and transfer
of estrogen signal as well [12, 29].
The expression of steroid receptors has important
implications for biology of breast tumors and their
treatment. About 30–40% of breast cancers lack ster-
oid receptors (ER and/or PR) at diagnosis that worsens
prognosis and limits the usage of hormone therapy.
Opinions differ as to whether those cancers which lack
expression of receptors arise from R(-) compartment
within the mammary epithelium or represent evolution
from R(+) to R(-) state. Recently evidence in support
of the idea on distinct etiologic pathways rather than
different stages in the natural history of breast cancer
has been growing [13, 32]. Contemporary concept
says that receptor-positive and receptor-negative
breast cancer (BC) subtypes may have associations
with distinctive risk factors and mechanistic hetero-
geneity by hormone-receptor status related to initial
existence of the two separate types of cancer (R+ and
R-) is rather possible [28].
The mechanisms leading to the development of
receptor-negative BC warrant further studies. Existing
interpretations are not abundant and can be reduced
to the role of several genetic (including BRCA1 and
BRCA2) and epigenetic factors, interrelations with the
presence of EGF and erbB2/HER-2/neu receptors in
tumor tissue and certain features of endocrine (repro-
ductive) system embracing level of estrogenemia and
intratumoral aromatase/estrogen synthetase activity
[3, 10, 15, 16, 25]. Taking into account principal char-
acteristics of the phenomenon of switching of estro-
gen effects (PSEE) described by us earlier [1, 4], the
assumption has been made that peculiar to this phe-
nomenon weakening of hormonal and strengthening of
genotoxic activity of estrogens may be of importance
in predisposing to the disturbances in estrogen signal
transduction and formation of receptor-negative BC,
that needs further consideration.
One of the PSEE inductors is displayed by tobacco
smoke. Chemical substances that its contains have
rather often the direct DNA-damaging capacity and,
in the same time, are able to modify the metabolism of
estrogens into direction of the increased formation of
estrogenic catechol derivatives, which give in their turn
an impulse for the generation of different free-radical
compounds [1, 27, 30]. Pro-genotoxic oxidative stress
is characteristic also for diabetes mellitus type II [7],
the disease, which through variety of mechanisms
(including increase in free estradiol fraction) is related
to the risk of breast cancer [18]. As a consequence,
the first task of this investigation consisted in the
evaluation of the effect of smoking and diabetes on the
Genotoxic factors associated with the development
of receptor-neGative breast cancer: potential role
of the phenomenon of switchinG of estroGen effects
L.M. Berstein1,*, E.V. Tsyrlina1, T.E. Poroshina1, V.V. Levina2, D.A. Vasilyev1, I.G. Kovalenko1, V.F. Semiglazov1
1Prof. N.N.Petrov Research Institute of Oncology, St.Petersburg, Russia
2Institute of Cytology of RAS, St.Petersburg, Russia
Aim: About 30–40% of breast cancers lack steroid receptors (ER and/or PR) at diagnosis that worsen prognosis and limit the us-
age of hormone therapy. The aim of this paper has been to study the role of DNA-damaging factors as the potential modifiers of the
receptor-negative tumors incidence. Materials and Methods: The investigation consisted of two principal parts. In one of them ER
and PR content was measured in breast cancer samples from 2284 primary patients (350 of them — current or previous smokers). In
separately studied subgroup of 1010 patients 95 suffered with diabetes mellitus type II. Results: As it was shown, smokers and diabet-
ics carry more frequently (p < 0.05) tumors with phenotypes ER+PR- and PR- only in the group of women with conserved menstrual
cycle that is in case of relatively higher estrogenic stimulation. In another part of the investigation immunohistochemical study of DNA
damage marker — 8-hydroxy-2-deoxyguanosine (8-OH-dG) in 16 R(-) and 18 R(+) breast cancer specimens demonstrated more
frequent positive staining in the former group of samples (p = 0.05). Besides, as it was revealed in breast cancer cell line MCF-7 the
combination of estradiol with aryl hydrocarbonic receptors agonist beta-naphtoflavone induced pronounced genotoxic damage (by
8-OH-dG content) in association with the loss of ER. Conclusion: Thus, pro-genotoxic status (smoking, diabetes) and direct signs of
genotoxic injury, in accordance with regularities of the phenomenon of switching of estrogen effects.
can be reckoned among the factors promoting the development of receptor-negative breast cancer.
Key Words: breast cancer, steroid receptors, smoking, diabetes, genotoxicity, 8-hydroxy-2-deoxyguanosine (8-OH-dG).
Received: November 2, 2005.
*Correspondence: Fax: 7-812-596-8947;
E-mail: levmb@endocrin.spb.ru
Abbreviations used: AhR — aryl hydrocarbonic receptors; ER —
estrogen receptors; 8-OH-dG — 8-hydroxy-2’-deoxyguanosine;
PR—progesterone receptors; PSEE— phenomenon of switching
of estrogen effects.
Exp Oncol 2006
28, 1, 64–69
Experimental Oncology 28, 64–69, 2006 (March) 65
receptor phenotype of BC patients in the conditions
of variable estrogenic stimulation, i.e. in females of
reproductive and postmenopausal age.
As it was mentioned above, PSEE is characterized
with the decrease in hormonal and the increase in
genotoxic effects of natural estrogens [1, 4]. Similarly
act certain xenoestrogens, which action is realized
through aryl hydrocarbonic receptors, or AhR [23].
Altogether, it stimulated conducting of the another
part of our research which included the comparison
of the content of specific marker of oxidative DNA
damage 8-hydroxy-2´-deoxyguanosine (8-OH-dG)
in human receptor-positive and receptor-negative
BC samples as well as the evaluation of 8-OH-dG, ER
and PR in breast cancer cell line MCF-7 treated with
estradiol, AhR agonist beta-naphtoflavone and their
combination.
materials and methods
The content of ER and PR has been determined
in tumor tissue of totally 2284 primary (without neo-
adjuvant therapy) breast cancer patients, which were
in clinical stages I-IIIa (T0-1N0M0-T1-3N2M0) and in age
varying from 27 to 84 yrs. Among these patients 815
were characterized with preserved menstrual cycle
and remaining 1469 women were in menopause with
the duration not less than 1 year. 350 of these patients
(197 — in reproductive and 153 — in postmenopausal
period) were current or ever smokers consuming daily
from 4–5 to 20–25 cigarettes. In subgroup of 1010
patients (361 with conserved menstrual cycle and 649
postmenopausal), in which the analysis of tumor ste-
roid receptor content was performed, 95 women suf-
fering with diabetes mellitus type II were discovered.
Technically, BC tissue samples collected during opera-
tion were immediately transferred into laboratory and
were placed into liquid nitrogen for further process-
ing. Estrogen and progesterone receptor contents in
tumor tissue were evaluated by the dextrane-charcoal
radioligand assay according to Saez et al. [22] with
small modifications [3] and with the usage of labeled
steroids (2,4,6,7-3H-Oestradiol и 1,2,6,7-3H-Proges-
terone) from “Amersham”, UK. The receptor activity
was expressed as fM/mg protein. Protein content was
determined by the Lowry method. Statistical analysis
of the data was performed by methods allowing for
means and standard errors. The significance of the
differences between the groups was tested using
hi-square approach by computerized programs (Sig-
maPlot, Statistica 6 and MyStat). The differences with
p < 0.05 were considered as significant.
In the second part of the study the content of ER
and PR in human breast tumors and in MCF-7 cell
line was determined with the same method. Immu-
nohistochemical or immunocytochemical evaluation
of 8-OH-dG in the sections of paraffin tumor blocks
or in MCF-7 cells was done according to Bianco et al.
[6] with diluted 1 : 100 antibodies 1F7 from “Trevi-
gen”, USA. Immunostaining was developed by the
peroxidase-antiperoxidase procedure with the usage
of Vecstain ABC kit (Vector Laboratories, USA) and
DAB (3,3´-diaminobenzidine) reaction. As a negative
control, tissue sections or cells were incubated with-
out the primary antibody. In clinical part of this study
material was received from 34 postmenopausal breast
cancer patients with T1-3N0-1M0 stages of the disease.
18 tumors were receptor-positive and 16 — receptor-
negative (with ER and PR values less than 10 fM/mg
protein). Results of 8-OH-dG immunohistochemical
staining were evaluated independently by two inves-
tigators and were presented as + (positive staining), ±
(moderate positive staining) and – (negative staining).
During the processing of the data results in groups +
and ± were combined. In the experiments with cell line
MCF-7, cells were grown in the MEM medium until they
were 70–80% confluent. Then cells were seeded (5 х
104/site) into culture dishes and treated with estradiol
(10-7 М), beta-naphtoflavone (4 х 10-7 М) and their com-
bination in the duration of 1, 24 and 48 h from the start
of incubation. Smears of the cells collected by the end
of the experiment were fixed 10 min in 70% ethanol,
air-dried and kept in hermetic containers at –20 °C until
immunocytochemical reaction performance. Results
of the latter were evaluated on the basis of stepping
procedure by two investigators and presented in the
interval from 0 (no staining) to 1.5 (maximal staining)
with the step equal to 0.25. Statistical analysis of the
data was the same as described above.
results and discussion
Data on distribution of steroid receptor pheno-
types in 2284 breast tumors and in the subgroup of
1010 cancers are presented in Table 1. Statistically
significant difference between groups of interest was
revealed only in reproductive period and only in regard
of ER + PR- tumors which were overrepresented in
smokers vs. non-smokers (t = 2.18, p < 0.05; χ2 = 5.01,
p = 0.025) as well as in patients suffering with diabetes
mellitus type II vs. patients without diabetes, t = 2.01,
p = 0.05; χ2 = 6.38, p = 0.012 (see Table 1). These
results deserve special assessment at least in the two
directions. First of all, smoking and diabetes as a rule
worsen prognosis quo ad vitam in patients with breast
cancer [17, 31]. Since the survival rate is usually worse
in younger patients in comparison with postmeno-
pausal females [11], it may be assumed that tumor
receptor phenotypes discovered in premenopausal
smokers and diabetics additionally predispose to the
more aggressive course of the disease. Secondly
and more importantly in the context of this paper, an
inclination to the predominant formation of the tumors
with phenotype ER+PR- (that evidently reflects impos-
sibility or failure of estrogenic signal transduction and
insufficient induction of estrogen-dependent proteins
including PR [12, 13]) in smoking and suffering with
diabetes females was observed in reproductive pe-
riod and not in menopause, suggesting that smoking
and diabetes realize mentioned effect predominantly
in the case of excessive or non-deficient estrogenic
stimulation. Thus, it may be concluded that combined
66 Experimental Oncology 28, 64–69, 2006 (March)
action of estrogens and genotoxic shifts peculiar to
the diabetes and tobacco smoke influence may cre-
ate conditions favoring the development of receptor-
negative breast cancer.
As it was mentioned above, in another part of the
study the staining of breast tumors and MCF-7 cell line
was performed with antibodies to 8-OH-dG aiming to
demonstrate, whether DNA damage signs are associ-
ated with receptor phenotype. In studied material the
content of ER and PR in R+ breast cancers (n = 18)
varied correspondingly from 20 to 250 fM/mg protein
and from 19 to 470 fM/mg protein while in R(-) tumors
(n = 16) it varied from 0 to 4 fM/mg protein (ER) and
from 0 to 8 fM/mg protein (PR). Specific positive im-
munohistochemical 8-OH-dG staining (Fig. 1) was
revealed in 9 of 18 cases (50.0 ± 11.7%) of R+ cancers
and in 13 of 16 (81.3 ± 9.8%) of R(-) tumors; differ-
ence is statistically significant (t = 2.05, p = 0.05).
Thus, although 8-OH-dG can be found also in recep-
tor-positive BC, the incidence of its discovery in R(-)
tumors is higher presenting the evidence of the greater
significance of genotoxic factors just for the formation
of receptor-deficient tumors.
Immunocytochemical detection of 8-OH-dG in
MCF 7 cells (Fig. 2) demonstrated that whereas in the
cells treated only with estradiol or beta-naphtoflavone
content of 8-OH-dG increased steadily and signifi-
cantly by 48 h (Table 2), the genotoxic damage induced
by the joint action of these two agents have reached
its peak already by 1 h, when simultaneous decrease
in ER content and low level of PR were discovered
(data not shown). So, it can not be excluded, that the
accumulation of DNA damage can be accelerated in
the situation when hormonal effect of estrogens is
modified by additional pro-genotoxic factor (tobacco
smoke, diabetes-associated pro-oxidant metabolic
shifts, activation of AhR pathway etc) and this, in com-
bination, leads to transition of the cells from R+ to the
R(-) state. Although preceding data on the 8-OH-dG
content in receptor-positive and receptor-negative BC
are the matter of some controversy [6, 20], the firm
evidence on the contribution of oxidative stress to an
increasing incidence of ER+PR- tumors with aging are
presented recently [21].
fig. 1. Immunohistochemical detection of 8-hydroxy-2-
deoxyguanosine (8-OH-dG) in human breast cancer sections:
a — receptor-negative tumor, positive 8-OH-dG staining;
b — receptor-positive tumor, negative 8-OH-dG staining
Table 2. The dynamic of the accumulation of 8-hydroxy-2-deoxyguano-
sine (8-OH-dG) in the MCF-7 cells under the influence of estradiol (E2),
beta-naphtoflavone (B) and their combination
Group/Time 1 h 24 h 48 h
Е2 0.625 ± 0.075 1.00 ± 0.05 1.25 ± 0.05*
В 0.25 ± 0.075 0.50 ± 0.10 1.25 ± 0.05*
Е2 + В 1.125 ± 0.025** 1.075 ± 0.025 1.00 ± 0.012
Note: Data of immunocytochemical analysis are presented in conditional
units (see section “Material and methods”).
* The difference with 1 h value is significant (p < 0.02).
** The difference with the data in E2 and B groups is significant (p < 0.05).
Table 1. The phenotypes of tumor steroid receptors in smoking and suffering from diabetes mellitus breast cancer patients
Patients
Steroid receptor phenotypes TotallyER+PR+ ER+PR- ER-PR+ ER-PR-
n % n % n % n % n %
Smokers
All 144 41.1 ± 2.6 92 26.3 ± 3.9 34 9.7 ± 1.6 80 22.9 ± 2.2 350 100
RP 70 35.5 ± 3.4 53 27.0 ± 3.1 23 11.7 ± 2.3 51 26.0 ± 3.1 197 100
MP 74 48.4 ± 4.0 39 25.5 ± 3.5 11 7.2 ± 2.1 29 19.0 ± 3.2 153 100
Non-smokers
All 799 41.3 ± 1.1 441 22.8 ± 0.9 210 10.8 ± 0.7 484 25.0 ± 1.0 1934 100
RP 266 43.0 ± 2.0 120 19.4 ± 1.6 76 12.3 ± 1.3 156 25.2 ± 1.7 618 100
MP 533 40.5 ± 1.3 321 24.4 ± 1.2 134 10.2 ± 0.8 328 25.0 ± 1.2 1316 100
With diabetes mellitus type II
All 39 41.0 ± 5.0 26 27.4 ± 4.6 9 9.5 ± 3.0 21 22.1 ± 4.2 95 100
RP 3 23.0 ± 17.3 6 46.0 ± 13.8 1 8.0 ± 7.5 3 23.0 ± 11.7 13 100
MP 36 44.0 ± 5.5 20 24.0 ± 4.7 8 10.0 ± 3.3 18 22.0 ± 4.6 82 100
Without diabetes mellitus
All 398 43.5 ± 1.6 185 20.0 ± 1.3 100 11.0 ± 1.0 232 25.5 ± 1.4 915 100
RP 159 46.0 ± 2.7 63 18.0 ± 2.0 46 13.0 ± 1.8 80 23.0 ± 2.2 348 100
MP 239 42.0 ± 2.1 122 21.5 ± 1.7 54 9.5 ± 1.2 152 27.0 ± 1.9 567 100
Notes: RP — reproductive period; MP — postmenopausal period. For statistical significance values — see text.
Experimental Oncology 28, 64–69, 2006 (March) 67
fig. 2. Immunocytochemical detection of 8-hydroxy-2-deoxy-
guanosine (8-OH-dG) in MCF-7 cell line: a — positive staining;
b — negative staining
Of note, in normal breast tissue ER’s and markers of
proliferation (e.g. Ki-67) are detected in separate cell
populations, indicating that ER+ cells (which mostly
originate from the luminal type of epithelium) normally
are not dividing, or that the receptor is down-regulated
as cells enter division. In contrast to the normal hu-
man breast, the separation between steroid receptor
expression and proliferation is disrupted starting at
an early stage of breast carcinogenesis, and co-ex-
pression of these two markers (ER and Ki-67) occurs
frequently in tumor cells. Therefore, suggestion is
made that ER+ cells are quiescent stem cells acting
as “steroid hormone sensors” which might secrete
paracrine factors to influence the proliferative activity
of adjacent ER- or progesterone receptor (PR)-nega-
tive cells originating predominantly from the basal/
myoepithelial lineage. According to this assumption,
after R+ cells are transformed, they acquire the abil-
ity to proliferate in breast lesions [8, 25]. On the other
hand, transformed mammary stem or progenitor cells
may undergo aberrant differentiation processes that
result in generation of the phenotypic heterogeneity
[9], which might serve as a basis for the variability
observed in the steroid hormone receptor status of
breast cancer. This corresponds with a notion that
receptor-negativity is not only a result of cancer pro-
gression from a receptor-positive state and that, in
contrast, a distinction in the content of receptors may
reflect different pathogenesis of the disease [28, 32].
As a result, R+ and R(-) breast tumors differ in clinical
aggressiveness and proliferative activity, which fre-
quently is higher just in R(-) carcinomas [11, 28]; the
latter observation is in the agreement with the basic
patterns of the phenomenon of switching of estrogen
effects, or PSEE [1, 4].
What are the practical consequences which can be
deduced from the said above? Since along with the
endocrine and genetic mechanisms genotoxic dam-
age is related to the development of receptor-negative
breast cancer, existing and future prevention measures
should include in a degree greater than before not
only anti-hormonal agents but antigenotoxicants also.
The list of the latter recently somewhat widened and
includes f.e. N-acetylcysteine, melatonin, resveratrol
etc [5, 14]. Additionally, some hypolipidemic and anti-
diabetic drugs can be used with the same aim and as
a supplement to neoadjuvant and adjuvant treatment
especially because some of them (like statins) were
rather efficient in the experiments with receptor-nega-
tive cell lines [2, 19]. Finally, the possibility to improve
situations due to the inductors of steroid receptors
(interferons etc) should not be ignored also, since their
usage had been rather effective in tamoxifen-resistant
receptor-negative breast cancer [26]. It is suggested
that further studies might be based on proteomic
approach and on the taking into account the ratio in
hormonal/genotoxic properties of mammary adipose
tissue surrounding correspondingly receptor-positive
and receptor-negative breast cancers.
acknowledGement
These studies were partly supported by grant from
RFBR (03-04-49282).
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Experimental Oncology 28, 64–69, 2006 (March) 69
Генотоксические факторы, ассоциированные
с возникновением рецептор-неГативноГо рака
молочной железы: потенциальная роль феномена
переключения эстроГенноГо эффекта
Цель исследования: примерно в 30–40% случаев рака молочной железы (РМЖ) рецепторы стероидных гормонов (ЭР
и/или ПР) не выявлены, что оказывает неблагоприятное влияние на прогноз заболевания и ограничивает применение
гормонотерапии. Задачей настоящей работы было изучение роли факторов, ассоциированных с повреждением ДНК, как
потенциальных модификаторов частоты возникновения рецепторнегативных новообразований. Материалы и методы:
исследование состояло из двух основных частей. У 2284 первичных больных РМЖ (350 из них — курящие или курившие
ранее) содержание ЭР и ПР в опухолевой ткани определяли рецепторным методом. В отдельно проанализированной
подгруппе из 1010 больных у 95 пациенток диагностировали сахарный диабет ІІ типа. Результаты: установлено, что в
отличие от пациенток в постменопаузальный период с сохраненной менструальной функцией (то есть в условиях более
высокой эстрогенной стимуляции) отмечается достоверное преобладание опухолей фенотипа ЭР + ПР — у курящих по
сравнению с некурящими (t = 2,18, p < 0,05), а также у больных сахарным диабетом по сравнению с больными без него
(t = 2,01, p = 0,05). Кроме того, в репродуктивный период у больных сахарным диабетом доля ПР(-)-опухолей достоверно
превосходила таковую у пациенток без него (t = 2,17, p < 0,05). Во второй части исследования с помощью иммуногис-
тохимического метода изучено содержание показателя повреждения ДНК 8-гидрокси-2-дезоксигуанозина (8-OH-dG) в
16 рецепторнегативных и 18 рецепторпозитивных опухолях молочной железы. Выявлено, что в первой группе позитивное
окрашивание наблюдали в 81,3 ± 9,8%, а во второй — в 50,0 ± 11,7% случаев, p = 0,05. Помимо этого, результаты экспе-
римента на клеточной линии МСF-7 рака молочной железы свидетельствуют, что при комбинации эстрадиола с агонистом
арилгидрокарбоновых рецепторов бета-нафтофлавоном значительное усиление генотоксического повреждения (по данным
определения 8-ОН-dG) отмечается уже к 60 мин эксперимента, когда выявляют и снижение концентрации ЭР. Выводы:
прегенотоксический статус (курение, сахарный диабет), равно как и признаки прямого генотоксического повреждения,
в соответствии с представлениями о феномене переключения эстрогенного эффекта могут быть причислены к факторам,
способствующим развитию рецепторнегативного РМЖ.
Ключевые слова: рак молочной железы, рецепторы эстрогенов, рецепторы прогестерона, рецепторнегативные опухоли,
курение, диабет, генотоксичность, 8-гидрокси-2-дезоксигуанозин (8-OH-dG).
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