RU2708668C1 - Method for drug prevention of breast cancer - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to oncology, and can be used for drug prevention of breast cancer (BC). Method of prevention of BC consists in the fact that of women aged 45–65 years, diagnosed with nodular or diffuse form of mastitis or increased mammographic density, females with a female line of malignant reproductive organs are selected; the selected women are examined for the methylation level of one of the BRCA1, WIF1 and RASSF1A genes, and women are selected in the risk group, the methylation level of one of the genes being not less than 50 %, and women at risk take the preparation based on indole-3-carbinol in dosage of 400 mg to 600 mg of indole-3-carbinol per day for 6 to 8 months annually.

EFFECT: using the invention enables significantly reducing the development of malignant phenomena in the risk groups by BC by creating a new algorithm of preventive measures based on the formation of risk groups for developing BC on the basis of molecular markers-precursors of early carcinogenesis and drug-induced correction of these parameters with subsequent control of BC incidence.

1 cl, 1 tbl, 6 dwg, 2 ex

Description

The invention relates to medicine and can be used for drug prevention of breast cancer.

Breast cancer (breast cancer) is one of the most pressing problems of modern oncology. In most economically developed countries, breast cancer occupies the first place in the structure of the oncological morbidity and mortality of the female population and is one of the main causes of death among middle-aged women (40-55 years old).

Risk factors that increase the likelihood of breast cancer are:

- overweight;

- hormonal and metabolic disorders, in particular, a reduced (<2) ratio of estrogen metabolites: 2-hydroxyestrone and 16α-hydroxyestrone (2-OHE1 / 16α-OHE1);

- violation of menstrual function;

- concomitant gynecological diseases;

- restriction of natural reproductive functions (reduction in the number of births, refusal of breastfeeding);

- stressful situations;

- injuries and operations on the mammary gland;

- a genetic factor, namely the presence of breast cancer in blood relatives along the female line (in mother, grandmother, sister, aunt, etc.).

- It should also be noted that the most important risk factor for the development of breast cancer is age after 40 years (Fig. 1).

Today, in all economically developed countries, health authorities and public organizations interested in cancer prevention are conducting a large-scale expansion and active promotion of a preventive x-ray examination - mammographic screening - among the female population. In this case, mammography is performed to identify early (preclinical) and, as a rule, non-palpable forms of breast cancer.

According to the results of a large number of extensive randomized trials, regular (with an interval of 1-2 years) mammogram screening in women aged 50 to 69 years, conducted with or without physical examination, reduced mortality from breast cancer by about 30%.

The sensitivity of a mammogram depends on the quality of the equipment, the competence of the personnel serving it, as well as the density of the examined breast tissue, which, among other things, depends on age and hormonal status. It is known that the dense breast tissue characteristic of healthy young women is often an obstacle to the successful preclinical diagnosis of breast foci [Haars G, van Noord PA, van Gils CH, Grobbee DE, Peeters PH. Measurements of breast density: no ratio for a ratio. Cancer Epidemiol Biomarkers Prev. (2005); 14 (11 Pt 1): 2634-2640]. There is evidence that increased breast density in young women can be a key risk factor, since it reflects the number of undifferentiated cells most susceptible to malignant transformation before the protective effect of their differentiation during pregnancy and age-related involution appears [Boyd N, Martin L, Chavez S et al (2009) Breast-tissue composition and other risk factors for breast cancer in young women: a cross-sectional study. Lancet Oncol., 10 (6), 569-580].

Closest to the proposed is a method of drug prevention of breast cancer, which consists in the selection of women with a reduced level of expression of the BRCA1 gene / protein, leading to an increased risk of breast cancer, aged 25 to 63 years, which for 4-6 weeks received 3,3'-diindolylmethane (DIM) at a dose of 300 mg / day, evaluated the expression of BRCA1 mRNA at baseline and after 4-6 weeks, while the use of DIM for 4-6 weeks increased the expression of BRCA1 mRNA by an average of 34 % [A.A. Estimator, V.P. Smetnik, V.I. Kiselev. Experience with the use of indole-3 carbinol in the treatment of breast diseases and the prevention of breast cancer. The journal "Obstetrics and Gynecology" No. 2, 2017, p. 106-112].

The demonstration of the possibility of activating the BRCA gene using DIM is an extremely important scientific observation, which shows that indole compounds are able to activate the expression of the tumor suppressor gene. However, it should be noted that from a practical point of view, this result does not prove the preventive effect of this approach, since the fate of these women is not traced, it is not known whether they developed breast cancer. In addition, hereditary breast cancer, dependent on a mutation in the BRCA gene, accounts for only 3-5% of total breast cancer. Therefore, even if a preventive effect is shown, it concerns a small proportion of all patients and does not solve the problem of sporadic cancer, which accounts for about 95% of all cases.

The technical problem solved by the present invention was the creation of a new algorithm of preventive measures based on the formation of risk groups for the development of breast cancer based on molecular markers-precursors of early carcinogenesis and drug correction of these parameters with subsequent control of the incidence of breast cancer.

The technical problem is solved by the method of preventing breast cancer, which consists of selecting women with a malignant or diffuse form of mastopathy or increased mammographic density from women aged 45-65 who have malignant formations of the reproductive organs on the female side and conduct a study on selected women the methylation level of one of the BRCA1, WIF1, and RASSF1A genes is selected at risk for women whose methylation level of one of the genes is at least 50% and women at risk take the drug and based on the indole-3-carbinol in a dosage of 400 mg to 600 mg of indole-3-carbinol per day for a period of from 6 to 8 months of each year. Recommended for life.

It is well known that genetic mutations play a huge role in the development of cancer. Many of these mutations and the degree of risk of developing neoplasias associated with them are described. However, despite the critical importance of the “genetic basis”, epigenetic processes can play an equally important role in the development of these pathologies [Kiselev V.I. and other breast cancer: a practical course off genes. Epigenetic regulation of mammary carcinogenesis and the possibility of its pharmacological correction (2014). StatusPraesens Journal, No. 4 (21), pp. 35-43].

Epigenetic changes do not disrupt the sequence of nucleotides in DNA and are not inherited, but due to the chemical modification of certain chromosomal loci and their associated genes, they critically change their availability for the transcriptional apparatus of the cell. Examples of epigenetic changes are histone acetylation and methylation, DNA methylation. This epigenetic “on / off” of genes, in particular, underlies the differentiation - the acquisition of the necessary phenotype - of all types of somatic cells. Conversely, a change in the characteristic “epigenetic labeling” of a cell of one type or another leads to de-differentiation or a change in phenotype (trans-differentiation) to another cellular type, for example, from the epithelium to the mesenchyme. Given the magnitude of the effects of epigenetic regulation, it is not difficult to understand that violations of this process can lead to the development of pathologies such as carcinogenesis [R.S. DeVaux et al Beyond DNA: the Role of Epigenetics in the Premalignant Progression of Breast Cancer (2018) Journal of Mammary Gland Biology and Neoplasia].

DNA methylation, as one of the most important mechanisms of epigenetic regulation, was discovered at the turn of the eighties of the last century. The essence of this process is the conversion of the cytosine nucleotide residue to 5-methylcytosine [K. Yamashita et al. Epigenetic biomarkers of promoter DNA methylation in the new era of cancer treatment (2018) Cancer Science, 109: 3695-3706].

During neoplastic degeneration, the pattern of epigenetic modification, as a rule, changes in such a way that the overall level of genome methylation is significantly reduced, while the regulatory regions of a significantly smaller part of the genes (most often, cancer suppressor genes), on the contrary, are hypermethylated and suppressed. Accordingly, if a change in normal methylation in any of the genes plays an important role in the development of a certain form of cancer, then the establishment of such a phenomenon will not only contribute to a better understanding of the pathogenesis of this disease, but may also turn out to be a kind of epigenetic marker of this disease [S.B. Baylin et al, Epigenetic Determinants of Cancer (2016) Cold Spring Harb Perspect Biol, 8].

In recent years, this area of research has been developing very actively. A whole panel of genes has been established, the methylation of which is a harbinger of the malignant process. The fact is that methylation of tumor suppressor genes leads to a blockade in their expression, which removes the evolutionary ban on cell malignancy. This phenomenon triggers a gradual process of carcinogenesis and begins long before the clinical manifestation of cancer. Changes in the genetic program through epigenetic mechanisms occur rather slowly and, at the initial stages, trigger epithelial-mesenchymal transformations (EMFs). This phenomenon develops as a result of the accumulation of a critical mass of epigenetic modifications in the epithelial cells of the mammary gland and leads to the transformation of the epithelium into mesenchymal cells. This type of cell forms fibrosis in the tissue, which is clinically manifested in increased mammographic density, and the development of fibrocystic disease. In addition, EMF is highly likely to trigger the processes of malignant degeneration of cells and carries signs of early carcinogenesis. The peculiarity of this phenomenon is that EMF is a reversible state and, with the right drug effect, the reverse process can be induced, namely the mesenchymal-epithelial transition (MEP) [Drasin et al. Breast cancer epithelial-to-mesenchymal transition: examining the functional consequences of plasticity (2011) Breast Cancer Research, 13: 226; P.G. Santamaria et al. EMT: Present and future in clinical oncology (2017) Molecular Oncology 11, 718-738; Voon et al. The EMT spectrum and therapeutic opportunities (2017) Molecular Oncology 11,) 878-89; Sun and Fang Epigenetic Regulation of Epithelia-Mesenchymal Transition (2016) Cell Mol Life Sci 73 (23): 4493-4515; Douglas S. Micalizzi et al. Epithelial-Mesenchymal Transition in Cancer: Parallels Between Normal Development and Tumor Progression (2010) J Mammary Gland Biol Neoplasia 15: 117-134].

et al. ESR1 gene promoter region methylation in free circulating DNA and its correlation with estrogen receptor protein expression in tumor tissue in breast cancer patients (2014) BMC Cancer, 14:59].EMFs are based on epigenetic changes, in particular, gene methylation, as a result of which a new genetic program of the cell is formed. The peculiarity of such changes in gene expression is that the structure of the genes is preserved, and these modifications are reversible, which opens up a unique opportunity to restore the active expression of suppressor genes, and therefore not only stop this process, but turn it back [Kiselev V.I. et al. Regulation of gene activity and new drugs (2016) Bulletin of the Russian Academy of Sciences, 2016, Volume 86, No. 6, p. 512-518; Edward A. Ratovitski Anticancer Natural Compounds as Epigenetic Modulators of Gene Expression (2017) Current Genomics, 18, 175-205; Subramaniam et al. DNA methyltransferases: a novel target for prevention and therapy (2014) Frontiersin Oncology V 4, Art 80]. Thus, the timely registration of such changes by determining the spectrum of methylated genes makes it possible to predict the development of the malignant process with high reliability. The clinical use of such approaches has opened up relatively recently and is based on the so-called liquid biopsy. The meaning of this approach is that due to the death of cells that have embarked on the path of malignant transformation, their DNA enters the bloodstream. Modern DNA analysis methods allow us to analyze and evaluate the level of methylation! [Sandra P. Nunes et al Cell-Free DNA Methylation of Selected Genes Allows for Early Detection of the Major Cancers in Women (2018) Cancers 10, 357; D. Lissa et al. Methylation analyses in liquid biopsy, (2016) Transl Lung Cancer Res; 5 (5): 492-504]. It was also established that the state of tumor DNA in blood plasma reflects its state in the tumor tissue. [

et al. ESR1 gene promoter region methylation in free circulating DNA and its correlation with estrogen receptor protein expression in tumor tissue in breast cancer patients (2014) BMC Cancer, 14:59].

The prognostic significance of such studies substantially depends on the choice of target genes, the methylation of which signals the onset of the preclinical stage of carcinogenesis. For our work, we have chosen markers for which a great deal of clinical material has been accumulated. These are tumor suppressor genes BRCA1, WIF1, RASSF1A [L. Ai et al. Inactivation of Wnt inhibitory factor-1 (WIF1) expression by epigenetic silencing is a common event in breast cancer (2006) Carcinogenesis vol. 27 no. 7 pp. 1341-1348; Vivek Shukla et al BRCA1 affects global DNA methylation through regulation of DNMT1 (2010) Cell Research 20: 1201-1215; Otani et al. BRCA1 promoter methylation of normal breast epithelial cells as a possible precursor for BRCA1-methylated breast cancer (2014) Cancer Sci, vol. 105, 1369-1376; Trang Lan Vu et al Methylation Profiles of BRCA1, RASSF1A and GSTP1 in Vietnamese Women with Breast Cancer (2018) Asian Pacific Journal of Cancer Prevention, Vol 19, 1887-1893; Hoda A. Hagrass et al. Methylation status and protein expression of RASSF1A in breast cancer patients (2014) Mol Biol Rep 41: 57-65; L.B. Hesson et al. The role of RASSF1A methylation in cancer (2007) Disease Markers 23: 73-87]. For these genes, not only the diagnostic significance of the early preclinical stages of carcinogenesis was established, but also the possibility of restoring their expression against the background of the use of drugs with pronounced epigenetic activity. In the case of restoration of the expression of tumor suppressor genes as a result of their demethylation, inhibition of malignancy is observed. Therefore, to implement a program for the drug prevention of breast cancer, it is necessary to use drugs that are able to restore the original genetic program of cells that have embarked on the path of malignancy. Such drugs should have pronounced epigenetic activity and act at different stages of carcinogenesis. Such drugs include drugs based on indole-3-carbinol, which is an inhibitor of histone deacetylases and DNA methyltransferases, enzymes that affect the epigenetic mechanisms of malignant cell transformation [Lyn-Cook B.D. et al. Gender differences in gemcitabine (Gemzar) efficacy in cancer cells: effect of indole-3-carbinol (2010) Anticancer Res., 30 (12), 490704913; Li Y., Li X., Guo B. Chemopreventive agent 3,3'-diindolylmethane selectively induces proteasomal degradation of class I histone deacetylases (2010) Cancer Res., 70 (2), 646-654].

Perhaps a wide range of antitumor activity of indole-3-carbinol is also due to its ability to convert to diindolylmethane, which also has established epigenetic activity and the property to inhibit tumor stem cells [Gregory W. Watson et al. Phytochemicals from Cruciferous Vegetables, Epigenetics, and Prostate Cancer Prevention (2013) The AAPS Journal, Vol. 15, No. 4, 951-961; A. Semov et al. Diindolylmethane (DIM) selectively inhibits cancer stem cells (2012) Biochemical and Biophysical Research Communications 424: 45-51; Shishinn Sun et al Endoplasmic reticulum stress as a correlate of cytotoxicity in human tumor cells exposed to diindolylmethane in vitro Breast Cancer Patients in Indonesia (2004) Cell Stress & Chaperones 9 (1), 76-87].

For indole-3-carbinol, the ability to inhibit the processes of the epithelial-mesenchymal transition as the initial stage of carcinogenesis has also been shown [BUT et al. I3C and ICZ inhibit migration by suppressing the EMT process and FAK expression in breast cancer cells (2013) Molecular Medicine Reports 7: 384-388].

In addition, indole-3-carbinol has a high selective inhibitory activity against transformed cells and very low toxicity to normal cells [KM Wahidur Rahman et al. Indole-3-Carbinol (I3C) Induces Apoptosis in Tumorigenic but Not in Nontumorigenic Breast Epithelial Cells (2009) Nutrition and Cancer, 45: 1, 101-112].

This parameter is also very important for preventive drugs, which should be taken for a rather long time. It should also be emphasized the importance of a dosage form that potentiates the antitumor potential of this compound. In particular, its epigenetic activity. Such a dosage form of indole-3-carbinol was previously developed by us and registered as a medicine [Patent RU 2601893, Author Kiselev V.I. Indole-3-carbinol-based drug with increased epigenetic activity]. For these reasons, it was the indole-3-carbinol-based drug that was used to develop the prophylactic algorithm for the prevention of breast cancer.

The invention is illustrated by graphic material.

In FIG. 1 presents data illustrating the risk of breast cancer in women depending on age, according to the US National Cancer Institute.

In FIG. Figure 2 shows the molecular mechanism of the methylation of the promoter of the WIF1 gene on the expression of the CCND1 gene and the development of a malignant tumor.

In FIG. Figure 3 shows a graph of the proliferation rate of MCF-10A cell culture in a medium with different concentrations of indole-3-carbinol (I-3-K).

In FIG. 4 is a graph of the proliferation rate of cell culture MDA-MB-231 in a medium with different concentrations of I-3-K.

In FIG. Figure 5 shows the methylation sites of the WIF1 gene promoter at a concentration of I-3-K 0 and 100 μM in cell culture MCF-10A.

In FIG. Figure 6 shows the methylation sites of the WIF1 gene promoter at a concentration of I-3-K 0 and 100 μM in cell culture MDA-MB-231.

Example 1

Investigation of the demethylating and antitumor activity of indole-3-carbinol on breast cancer cell lines.

The purpose of this experiment was to demonstrate the demethylating activity of indole-3-carbinol against the WIF1 gene, which encodes an inhibitor of the Wnt signaling cascade, which is an important factor in carcinogenesis of breast cells. [L. Ai et al. Inactivation of Wnt inhibitory factor-1 (WIF1) expression by epigenetic silencing is a common event in breast cancer (2006) Carcinogenesis vol. 27 no. 7 pp. 1341-1348]. As a result of methylation of the WIF1 gene, its expression is blocked and the Wnt signaling cascade is activated, which triggers the processes of cell malignancy (Fig. 2). Demethylation of the WIF1 gene restores its activity and leads to blockade of the most important mechanism of malignant growth, which is a significant criterion for the antitumor effect of indole-3-carbinol. And the second task of the study is to show the high selective activity of indole-3-carbinol in relation to malignant breast cancer cells. In the course of work, 2 cell cultures of breast cancer cells were used: MDA-MB-231 and MCF-10A, obtained from the ATCC cell culture collection.

MDA-MB-231

Homo sapiens cell culture, epithelial adherent mammary adenocarcinoma cells, was isolated from metastases in the pleural cavity of a patient with breast adenocarcinoma (malignant neoplasm). This cell line is most often used as a model of the metastatic form of breast cancer.

MCF-10A

Homo sapiens cell culture, epithelial adherent cells, was isolated from the breast tissue of a patient with fibrocystic mastopathy (benign neoplasm). This cell line is the most commonly used as a model of non-tumor tissue.

Cell culture

MDA-MB-231 culture cells were cultured in DMEM (1 ×) Gibco supplemented with 10% bovine fetal serum (FBS), L-glutamine, and penicillin / streptomycin antibiotic mixture. The cells of the MCF-10A culture were cultured in DMEM / F12 (1: 1) medium by LLC NPP PanEco supplemented with 10% FBS, L-glutamine and a mixture of penicillin / streptomycin antibiotics. Cultivation was carried out in a Sanyo CO ₂ incubator (Panasonic) at a temperature of 37 ° C and 5% CO ₂ content.

Cells were plated on 6-well TPP culture plates in a volume of 1 × 10 ⁶ cells per well for subsequent DNA isolation from them for analysis of the methylation degree of the WIF1 gene promoter and in a volume of 5 × 10 ⁵ for subsequent RNA isolation and analysis of the level of expression of Wnt-target genes cascade. Cell counting was performed using an automatic Countess cell counter (Invitrogen).

The effect of indole-3-carbinol was studied at the following concentrations: 0, 50, 75, and 100 μM in 4 biological repeats. Cells were incubated with the drug for 48 hours. After that, cells intended for RNA isolation were lysed with QIAzol reagent (QIAGEN) and stored until isolation of RNA at a temperature of -70 ° С. Cells for DNA isolation were detached with a trypsin-EDTA 0.25% solution (LLC NPE PanEco), washed 2 times with a solution of DPBS (LLC NPP PanEco) and stored until DNA isolation at a temperature of -20 ° C.

Proliferation Rate Study

Cell proliferation of indole-3-carbinol was evaluated using the MTT assay. The MTT method is based on the ability of living cells to reduce yellow 3- (4,5-dimethylthiazol-2-yl) -2,5-tetrazolium bromide (MTT) into the purplish-blue intracellular MTT-formazan crystals soluble in DMSO. The cytotoxic effect of the drug on the cell is indicated by a decrease in the density of the samples compared to the control ones.

Cell cultures MCF-10A and MDA-MB-231 were plated on a 96-well TPP culture plate with a density of 5000 cells per well (MCF-10A) and 10,000 cells per well (MDA-MB-231). Cells were incubated in media with indole-3-carbinol with concentrations of 0, 50, 75 and 100 μM and 0, 50, 75 and 100 μM (during this test, the change in optical density was also measured using the freshly diluted I-3-K preparation and previously diluted stored until this month at a temperature of -4 ° C). Cell cultures were incubated for 24, 48, 72 and 96 hours. Each tablet was measured on a VERSA max microplate reader (Molecular Devices) spectrophotometer at a wavelength of 470 nm.

Polymerase Chain Reaction

The converted DNA was subjected to polymerase chain reaction using a DTprime amplifier (DNA-Technology), using the method described in the article [Sukhikh G.T. et al. Methylation of the WIF1 gene in cervical squamous intraepithelial lesions. Obstetrics and Gynecology No. 5, 2017, pp. 114-123].

Bisulfite sequencing

For sequencing, 16 ng of DNA of each sample was evaporated with the primer using a Concentrator plus concentrator (Eppendorf). Bisulfite sequencing of the promoter region of the BRCA1, WIF1 and RASSF1A genes was carried out in accordance with the previously described protocols.

The results of the experiment.

Incubation of the cells of the normal breast tissue model (MCF-10A) with indole-3-carbinol did not reveal significant differences in the proliferation rate at all studied concentrations, in contrast to the results obtained on MDA-MB-231 cells. From these figures it can be seen that the concentration of 50 μM did not have a significant effect, however, at a concentration of 100 μM, the proliferation rate decreased by 1, 46 times, which confirms the selectivity of the inhibitory activity of indole-3-carbinol against malignant cells (Figs. 3 and 4).

In FIG. Figures 5 and 6 show the results of the analysis of the degree of methylation of the WIF1 gene promoter in MDA-MB-231 and MCF-10A cell cultures.

The methylation analysis did not reveal a demethylating effect in the non-tumor cell model MCF-10A. As can be seen from FIG. 5, 66.7% of sites in the studied region of the promoter are methylated. This value did not change after incubation of cells with indole-3-carbinol with a concentration of 100 μM.

In MDA-MB-231 tumor cells, a different result is observed: at the initial methylation of 100% of the sites after incubation with indole-3-carbinol with a concentration of 100 μM, methylation remained in only 40.7% of the sites (Fig. 6). This suggests the possibility of using indole-3-carbinol as a demethylating agent in tumor cells.

Despite the fact that using real-time PCR, we were not able to determine the increase in the expression of the WIF1 gene, according to the results obtained, it can be assumed that its expression is restored.

Thus, as a result of the work, it was shown that under the action of indole-3-carbinol, the promoter of the WIF1 gene is demethylated, which apparently leads to the restoration of its expression and a decrease in the proliferation rate of malignant cells (Figs. 4 and 6).

Similar studies were also conducted for the BRCA1 and RASSF1A genes and the ability of indole-3-carbinol to reduce the methylation level of these genes was shown.

Example 2

Formation of risk groups for the development of breast cancer.

The risk of breast cancer increases significantly with age. According to the National Cancer Institute of the United States, aged 39-49 years, breast cancer can get 1 out of 220 women. At the age of 49-59, this indicator is 1 out of 50. It is also known that the risk of morbidity increases if there is a family history of gynecological cancer relatives. The incidence of breast cancer is also affected by the presence of fibrocystic mastopathy, high mammographic density and even prolonged tenderness of the mammary glands. From a general biological point of view, prophylactic administration of drugs with oncoprotective properties is necessary for any of the listed categories of women. However, in order to obtain reliable evidence of the preventive effect of any drug, taking into account the parameters of the general incidence, a clinical study should cover at least 10,000 women aged 39-49 years. Conducting such a study is extremely difficult. That is why we tried to select several criteria for the formation of a risk group in which the likelihood of developing breast cancer during the 5-year follow-up will be as high as possible, and in this group to evaluate the effectiveness of drug prophylaxis by the most important indicator - the incidence of breast cancer in comparison with the group not taking prophylactic drugs. With this approach, the scope of the study would be much smaller, and the evidence is more reliable.

The initial selection of patients at risk was performed according to the criteria of the age of 45-65 years and the diagnosis of fibrocystic mastopathy or high mammographic density. From this category of women, patients who had gynecological cancers along the female line were selected for further research. Among the selected patients, a study was conducted on the methylation level of one of the tumor suppressor genes BRCA1, WIF1 and RASSF1A. For the final inclusion in the high-risk group, women were selected for whom the methylation level of one of the genes according to the results of DNA analysis in the plasma of patients was 50% or more.

The selection of women in the high-risk breast cancer group was carried out among patients of the Moscow Mammological Center. Basically, patients complained of pain in the mammary glands and the presence of palpable formations. About 15% of patients met our selection criteria.

According to these criteria, 40 patients were selected, which were divided into two homogeneous groups of 20 people. 10 people of the first group received annually a 6-month course of indole-3-carbinol in the form of a medicine with increased epigenetic activity Indinol® Forto at a dosage of 200 mg 2 times a day for 5 years. The other 10 people of the first group received annually an 8-month course of indole-3-carbinol in the form of a medicine with increased epigenetic activity Indinol® Forto at a dosage of 200 mg 3 times a day for 5 years. The second group served as control. Both groups underwent a clinical examination once a year and final examinations after 5 years of observation. In the 2nd year of observation, methylation analysis of selected genetic markers was performed in all study participants. The results of the clinical study are presented in the table. As can be seen from the table, drug prophylaxis using the described algorithm can significantly reduce the development of malignant phenomena in risk groups for breast cancer.

Claims (1)

A method for the prevention of breast cancer, which consists in the fact that among women aged 45-65 years diagnosed with a nodular or diffuse form of mastopathy or increased mammographic density, women who have malignant reproductive organs on the female line are selected, a study is conducted on selected women the methylation level of one of the BRCA1, WIF1, and RASSF1A genes is selected at risk for women whose methylation level of one of the genes is at least 50%, and women at risk take a drug based on indole-3-carbinol in a dosage of 400 mg to 600 mg indole-3-carbinol per day for 6 to 8 months annually.

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