The most common somatic mutations in solid tumors affect the TP53 gene, leading to the formation of the tumor-specific p53 antigen through overexpression of the wild-type antigen (WT-p53) or the generation of mutant-specific neoepitopes (MUT-p53). Thus, the p53 protein is a promising target for immunotherapy. This article summarizes clinical and immunological data on p53-targeting vaccine strategies. Vaccination against WT-p53 is shown to reproducibly induce p53-specific T-cell responses and has a favorable safety profile; however, its clinical efficacy as a monotherapy is limited. Key challenges include central immune tolerance to WT-p53 epitopes, low density of p53 peptide-HLA complexes on the tumor cell surface, an immunosuppressive tumor microenvironment, and HLA (HLA – human leukocyte antigens) heterogeneity. For mutant p53, specific immunogenic epitopes have been validated, yet phase II clinical trials of vaccines targeting frequent p53 mutations are still lacking. Future prospects for enhancing the efficacy of p53-targeted vaccines lie in combination strategies (e.g., with immune checkpoint inhibitors, adjuvants), precise patient stratification, and for mutant p53, the application of TCR-based (TCR – T-cell receptor) technologies and the development of personalized neoantigen selection platforms.
Introduction. Gastric cancer is an aggressive malignant tumor of the gastrointestinal tract. Its onset and development are associated with genetic and external factors. Abnormalities in DNA repair genes, for example ERCC1 and ERCC2, can play a role in the development of this disease. Aim. To determine prognostic significance of ERCC1 (rs11615, rs3212986) and ERCC2 (rs13181) gene polymorphisms in the development of gastric cancer in the Kyrgyz population. Materials and methods. The study included 123 patients with malignant tumors of the stomach and 138 healthy individuals belonging to the Kyrgyz ethnic group. Genotyping was performed using allele-specific polymerase chain reaction. Statistical analysis included Fisher’s exact test and calculation of odds ratio (OR) for evaluation of associations between genotypes and clinical parameters. Genotyping results, histological and morphological tumor structures, patient age, tumor growth and location were compared using Fisher’s exact test. OR was determined through cross-tabulation. Results. Statistically significant association of CC genotype of the ERCC1 (rs3212986) polymorphic locus with patient age (41–50 and 61–70 years) was found. Genotypes TG and TT of the ERCC2 (rs13181) gene were associated with poorly differentiated adenocarcinoma. Additionally, it was shown that CA genotype and A allele of the rs3212986 (ERCC1) polymorphism can play a protective role decreasing the likelihood of gastric cancer development in the Kyrgyz population (OR 0.472; 95 % confidence interval 0.287–0.785 and OR 0.676; 95 % confidence interval 0.464–0.991 for CC genotype and allele A, respectively) (р = 0.046). Conclusion. Polymorphisms of the ERCC1 and ERCC2 genes can be associated with the risk of gastric cancer development in the Kyrgyz population. These data show the necessity of further studies investigating the role of these genes in gastric cancer and development of strategies for its prevention and treatment.
For various types of solid tumors, bones are the most common location of metastases. Bone is a dynamic organ capable of simultaneously performing multiple auxiliary functions in the body. Osteoblasts and osteoclasts and their changes play a significant role in bone remodeling. Aim. To summarize current data on bone metastases for better understanding of biological nature of oncogenesis. The article analyzes recent scientific publications containing information on molecular mechanisms of metastasis into the bone from the National Center for Biotechnology Information (NCBI), eLibrary and Google Scholar databases, as well as characterizes bone cells and their role in oncogenesis. Knowledge and deep understanding of bone metastasis in various solid tumors are necessary for development of accurate diagnostic methods and effective modern treatment of patients with bone metastases. Currently, the mechanisms of cancer metastasis and interactions between metastatic cancer cells and bone microenvironment are not completely understood. Epigenetic changes associated with differentiation of osteoblasts and osteoclasts and cell signal transduction play a large role in bone remodeling in various malignant neoplasms. Increased bone resorption is caused by disbalance between osteoblasts and osteoclasts. Osteoclast activity leads to release of growth factors from the bone matrix necessary for tumor cell multiplication in the bone.
Anti-cluster-of-differentiation (anti-CD) antibody microarray is an attractive multiplex analogue to leukocyte immunocytochemistry for surface markers. The transparent plastic microarray support permits to sort leukocytes by their surface lineage-specific markers making them available for subsequent high-resolution morphology examination. The combined data on the pathologic cells’ immunophenotype, morphology and cytochemistry from the microarray is sufficient to suggest preliminary diagnosis in many leukemia types. However, in some cases additional staining for internal markers is required, i.e. in T cell acute leukemia the T cell origin of the blasts can only be proved by the presence of cytoplasmic CD3 (cytCD3). Here we describe a new protocol of immunocytochemical detection of internal proteins in mononuclear cells captured by mouse anti-CD antibodies on the microarray surface. The protocol uses primary mouse FITC-conjugated (FITC – fluorescein isothiocyanate) antibodies against the target, secondary anti-FITC antibodies conjugated with alkaline phosphatase and colorimetric alkaline phosphatase substrate, BCIP/NBT. We show on normal leukocytes and leukemic cell lines that the protocol is sensitive and specific. The percentages of cytCD3-positive cells determined by this method in bone marrow aspirates of two patients with T cell acute lymphoblastic leukemia are in excellent agreement with flow cytometry results. This method expands the diagnostic capabilities of anti-CD antibody microarray.
Introduction. In 20–25 % of cases of patients with hereditary forms of breast cancer (BC), pathogenic variants of the BRCA1 and BRCA2 genes predisposing to BC and ovarian cancer can be identified. Based on the results of previous Russian studies, a simple and inexpensive testing system using real-time polymerase chain reaction consisting of the 8 most frequently identified variants (mutations) in the BRCA1 and BRCA2 genes was developed. The use of targeted panels for high-throughput sequencing and whole genome sequencing for diagnosis of hereditary BC led to identification of a number of genes with pathological variants causing this pathology in patients and their relatives. Aim. To evaluate the profile and frequency of pathological variants in genes associated with tumor development in Russian patients with BC. Materials and methods. Analysis of the results of whole genome sequencing of samples from 1514 patients with BC and 5163 individuals without oncological diseases (control group) was performed. Results. A spectrum of mutations in the BRCA1 and BRCA2 genes, as well as pathogenic variants in other genes, were identified. Based on the results of whole genome sequencing a new panel was developed consisting of 10 most common mutations in the BRCA1, BRCA2, АТМ and PALB2 genes. Additionally, significant associations between pathogenic variants in the ATM gene with estrogen-positive luminal BC (р = 0.034) and pathogenic variants in the BRCA1 gene with triple negative BC (p 0.001) were found. Conclusion. In accordance with the obtained data, the new panel of the most common pathogenic variants includes BRCA1 (rs28897672, rs80357522, rs80357906, rs41293455, rs80358094), BRCA2 (rs28897756, rs80359604), АТМ (rs587782652) and PALB2 (rs180177143, rs515726123). Implementation of the new extended panel for identification of frequent mutations in Russian patients with BC in clinical practice will allow to identify up to 7.7 % of germline variants in a random sample.
Introduction. Malignant neoplasms of the transverse colon, stomach and rectum are some of the most common oncological diseases. In Russia, their incidence is about 60,000 cases a year. Therefore, prediction of tumor recurrence or progression after treatment using biological markers is very important. Aim. To study percentage of lymphocytes with asynchronous DNA replication (LAR) of the AURKA and TP53 genes in the peripheral blood of patients with malignant neoplasms of the gastrointestinal tract after treatment. Materials and methods. Asynchronicity of the AURKA and TP53 genes in peripheral lymphocytes was measured using fluorescent in situ hybridization (FISH). In patients with malignant neoplasms of the gastrointestinal tract, interphase FISH analysis was performed prior to treatment and during the 5-year period after treatment. Results. One year after treatment, percentage of LAR of both AURKA (34.4 % versus 28.8 %) and TP53 (26.1 % versus 21.0 %) genes significantly decreased compared to the values prior to treatment (p 0.05). In patients without disease progression, mean group amount of LAR of both genes did not change in the 1–5-year period. In patients with disease progression, mean group values of LAR percentage of both genes were significantly higher than in the group without progression post-treatment (р 0.01). In some patients, disease progression was diagnosed 1–13 months after LAR study. In the group before progression, LAR of AURKA and TP53 genes were also higher and differed from the value in the progression-free group (33.8 % versus 30.4 % and 25.3 % versus 21.7 %, respectively) (р 0.05). Conclusion. Analysis of LAR dynamics of the AURKA and TP53 genes in the long-term period after treatment showed that persistent high LAR levels of both genes can serve as a potential individual markers of disease progression.
Contacts: Svetlana Aleksandrovna Kulyova Kulevadoc@yandex.ru The immune system is able to destroy tumors, but the progression of the malignant process indicates a violation of this mechanism. Analysis of literature data on the prognostic effect of different microenvironment T subpopulations shows that cytotoxic CD8+-T cells and memory T cells associated with Th1-type immune response correlate with good clinical course in most cancer types studied. On the other hand, the prognostic value of Th2, Th17 or Treg cell populations is inconsistent and varies depending on the type and stage of malignancy. Overall, these results strongly suggest that tumor behavior should now be viewed as the result of a balance between an invasive tumor process and a coordinated macroorganism immune response. In children, the microenvironment of solid tumors is heterogeneous, dynamic, and distinct from the microenvironment of neoplasias found in adults. Due to the low mutation burden, tumors in children have a limited set of neoantigens that could be recognized, and a small number of infiltrating T cells are represented in their tumor environment. Traditional biopsy does not always reflect tumor heterogeneity, so the focus shifts to peripheral blood testing. Biomarkers in the blood allow reassessment at different time points, they are readily available and their sampling is less invasive for patients. In addition, they can provide more information on immune status and assist in assessing a patient’s potential to develop effective immunity against a tumor. The article presents a modern view of immunological landscape of malignant tumors with a complex network of various cell populations and interactions between them.
Epstein–Barr virus (EBV) is a human herpesvirus type 4, which is carried by the majority of the world’s population. Its etiologic role in the development of various lymphomas, nasopharyngeal cancer, gastric cancer, and other malignancies has been proven. The mechanisms and participants of EBV-associated oncogenesis are actively studied. This review describes the molecular genetic diversity of EBV associated with the development of cancer. A literature review revealed that EBV is characterized by high structural and functional diversity of nucleotide and amino acid sequences. Based on polymorphism of EBV genes EBNA-1, EBNA-2, EBNA-LP, EBER, BZLF1, LMP-2A and LMP-1, various mutations, types, and variants, associated with malignant transformation and the development of various cancer types were identified. Aim. To summarize and analyze literature data devoted to the study of the genetic diversity of EBV for the prospective development of molecular biological research methodology in clinical practice, as well as the development of diagnostic tools, specific prevention and therapy of EBV-associated oncological diseases based on the products of the viral genome.
Introduction. The development of chemoresistance is a key factor limiting the efficacy of ovarian cancer therapy. Although high MUC16 gene expression levels in tumors are a known adverse prognostic factor, its role in predicting response to specific chemotherapeutic agents remains understudied. This led to the hypothesis that the adverse prognostic value of MUC16 expression levels may be mediated by its contribution to the development of chemoresistance. Aim. To evaluate the association between MUC16 gene expression levels and in vitro resistance to standard chemotherapy agents, including carboplatin, cisplatin, and paclitaxel, as well as key clinicopathological characteristics (BRCA1/2 mutation status, platinum sensitivity) and progression-free survival in patients with ovarian carcinoma. Materials and methods. Tumor samples were obtained from 35 patients with ovarian malignancies, predominantly high-grade serous carcinoma. MUC16 gene expression levels were determined using quantitative real-time polymerase chain reaction. Tumor cell chemoresistance was assessed in vitro in primary cultures using a resazurin metabolic assay, followed by the calculation of the resistance index (Resistance Sensitivity Index, RSI) for each drug. Statistical analysis was performed using Spearman’s rank correlation analysis, the Mann–Whitney U test, and the log-rank test. Results. High MUC16 gene expression levels were associated with shortened progression-free survival (p = 0.0437). A statistically significant positive correlation was identified between MUC16 gene expression levels and the paclitaxel RSI (R = 0.3614; p = 0.0388). However, no associations were found with the RSI for platinum-based drugs. Furthermore, no relationship was established between MUC16 gene expression levels and BRCA1/2 mutation status (p = 0.8180) or the platinum sensitivity/resistance status of the tumors (p = 0.2899). Conclusion. High MUC16 gene expression levels are associated with reduced progression-free survival and paclitaxel resistance, suggesting its potential as an unfavorable prognostic and predictive marker. Thus, determining MUC16 gene expression levels prior to therapy may have clinical potential for disease prognosis and for identifying patients for whom taxane-based treatment will likely be ineffective.
Introduction. Glioblastoma is the most common malignant brain tumor in adults with a poor prognosis. Treatment of patients includes surgical resection, radiation and the alkylating agent temozolomide (TMZ). The therapeutic efficacy of TMZ is due to its ability to damage DNA and induce apoptosis, but it is neutralized by the expression of the DNA repair enzyme O6-methylguanine-DNA-methyltransferase (MGMT). Methylation of the MGMT gene promoter suppresses the synthesis of the corresponding enzyme and increases the cytotoxic efficiency of TMZ.Aim. To determine the MGMT promoter methylation in glioblastoma patients and to evaluate the prognostic significance of this phenomenon.Materials and methods. Bisulfite-treated DNA samples isolated from formalin-fixed paraffin-embedded tumor tissues obtained from glioblastoma patients were analyzed. MGMT methylation was assessed by qualitative methylation-specific polymerase chain reaction. The prognostic significance of this phenomenon in conjunction with a number of other clinical parameters was assessed by means of univariate and multivariate analysis.Results. MGMT promoter methylation was found to be one of the most significant favorable prognostic factors of glioblastoma: the likelihood of disease reappearance or a fatal outcome at a specific point in time is approximately two-fold lower in such patients than in those with intact MGMT.Conclusion. The methylation-specific polymerase chain reaction, which is routinely used in clinical practice, adequately assesses MGMT methylation status as a prognostic factor, but it does not allow for the evaluation of its predictive potential.
Diagnosis and selection of effective therapy of soft tissue sarcomas (STS) are complicated by low incidence and significant histological variability of these tumors. Development of molecular and genetic testing methods is aimed at improving differential diagnosis of different types of STS and identification of genetic abnormalities which can potentially serve as targets for therapy. Development of effective treatment methods requires adequate preclinical models capable of recreating biological features of the tumors. The article presents molecular and genetic testing methods for STS diagnosis and therapy, advances in in vitro STS models, problems with their use in preclinical studies, as well as possibilities of using primary cell cultures for personalized treatment.
One of the most challenging issues in the treatment of ovarian cancer is the high level of tumor cell chemoresistance, which leads to early tumor recurrence and low overall survival. In the case of tumor cell chemoresistance, the therapy is ineffective, resulting in unnecessary drug consumption and harm to the patient due to toxic side effects and time losses during its implementation. One approach to solving this problem is experimental predictive testing of tumor cell chemoresistance in vitro.Aim. To develop a protocol of experimental testing of chemoresistance of ovarian cancer cells to chemotherapy drugs. We analyzed changes in the number of viable cells of A-1847, Ovcar-3 and Ovcar-4 cell lines cultured in hypo-adhesive conditions and optimized a method of cell viability determination based on resazurin metabolism. It was shown that viability of ovarian cancer cells of the studied cell lines is approximately the same in the presence of 1st and 2nd line antitumor drugs measured in accordance with the developed culture protocol in hypo-adhesive conditions and using ATP-tumor chemosensitivity assay (ATP-TCA) (DCS Innovative Diagnostik-Systeme, Germany) approved for use in clinical practice. The developed method is based on readily available and inexpensive reagents and expendables which makes it economically attractive.
Introduction. Gastric cancer remains a serious public health problem. Cancer-testicular antigen genes (CT-genes) in gastric cancer may be promising targets for immunotherapy due to their limited expression in normal tissues. Competing endogenous RNA networks (ceRNAs) play an important role in regulating CT-gene expression in gastric cancer. These networks are complex and require comprehensive bioinformatics and experimental study.Aim. To conduct a bioinformatic analysis followed by validation of СT-gene expression and its regulation in malignant gastric tumors.Materials and methods. Data for the bioinformatics stage were downloaded from GEO. Identification of differentially expressed genes was carried out using GEO2R, microRNA targeting genes – using the Random forest machine learning method. An analysis of the interaction of microRNA and long non-coding RNA (lncRNAs) was also performed. The clinical material for the experimental stage was tissues (tumor and conditionally normal) of 100 patients with a histologically confirmed diagnosis of gastric cancer. The relative expression values of 6 CT-genes (MAGEA10, MAGEA2, MAGEA12, MAGEA3, MAGEA6, MAGEH1), as well as their targeting microRNAs and lncRNAs, were determined using real-time polymerase chain reaction.Results. Using GEO2R, 18,617 differentially expressed loci were detected, including protein-coding genes, microRNA and lncRNAs. Of these, a change in the expression of 6 CT-genes was revealed – MAGEA10, MAGEA2, MAGEA12, MAGEA3, MAGEA6, MAGEH1, interacting with 40 microRNAs, in turn interacting with 17 lncRNAs. In the patients tumor tissue, an increase in expression of the MAGEA10, MAGEA3 and MAGEA6 genes (p 0.0001), a decrease in expression of miR-1207-5p, -6858-5p, -3127-3p, -3940-3p, -6807-3p, -3085-3p, -3934-5p, -4488, -4530, -6777-3p, -99a-3p (p 0.0001) and an increase in expression of miR-7113-3p, miR-874-3p, as well as an increase in expression of LINC01089, AC145285.6, GAS5, AC005034.3, AL691447.2 (p 0.001) and decreased expression of SNHG14, AC002101.1, SLC9A3-AS1 and AL118506.1 (p 0.001) was found. Based on the obtained data, a model of the regulatory network for CT-genes in gastric cancer was constructed.Conclusion. The study showed disturbances in the CT-genes competitively interacting RNA network in gastric adenocarcinoma. The obtained data are important for understanding the fundamental mechanisms of CT-gene regulation, as well as for improving approaches to immunotherapy (new targets and regulatory molecules) and diagnostics (new molecular markers) of this disease.
Introduction. Tumor cells are known not to undergo replicative aging – usually due to hyperactivation of telomerase, which restores telomere length during each cell division cycle. However, it is possible to induce aging in tumor cells through sublethal doses of cytostatics or irradiation – this is the so-called stress-induced or non-replicative senescence. Studying the mechanisms and regulatory pathways of this process is one of the important areas of modern oncology.Aim. To investigate the mechanisms of doxorubicin-induced senescence in different breast cancer cell subtypes and to explore possible approaches to regulating non-replicative aging.Materials and methods. The experiments were performed on in vitro cultured breast cancer cell lines MCF-7 and MDA-MB-231. Cellular senescence was assessed by β-galactosidase activity, morphological changes, and activation of the p53/p21 signaling pathway. Colorimetric assays, reporter analysis, and immunoblotting were used to evaluate the expression and activity of cellular proteins. DNA methyltransferase 3A (DNMT3A) knockdown was achieved using a standard lentiviral vector encoding antisense RNA against DNMT3A.Results. A potentiating effect of tamoxifen on doxorubicin-induced senescence – including in estrogen-independent breast cancer cells – was demonstrated. Enhanced non-replicative senescence was observed in resistant cells characterized by constitutive suppression of DNMT3A expression. For the first time, it was shown that DNMT3A suppression – either via decitabine treatment or DNMT3A knockdown – leads to an increase and maintenance of non-replicative senescence in MCF-7 cells.Conclusion. The findings indicate that non-replicative senescence in breast cancer cells can be enhanced and sustained in the presence of the antiestrogen tamoxifen, and underscore the key role of DNMT3A in regulating doxorubicin-induced senescence.
Clonal hematopoiesis of indeterminate potential (CHIP) is associated with aging and is a risk factor of many diseases including malignant neoplasms (MNPs). It originates through somatic mutations in hematopoietic stem and/or progenitor cells, promotes development of hematological MNPs and underlies unfavorable prognosis in solid malignant tumors. Results of recent largescale genome-wide studies confirmed that CHIP plays a role in oncological diseases. Mutations associated with this pathology were found in stem and/or progenitor cells in patients with both hematological and solitary MNPs which indicates that CHIP potentially mediates development of malignant tumors. Cytotoxic chemoradiation therapy is closely associated with CHIP development and causes emergence of aggressive and treatment-resistant hematological MNPs. In patients with solitary MNPs, TET2 gene mutations with high variant allele frequencies were also found in the tumors. This phenomenon was named tumor-infiltrating clonal hemopoiesis. Further populational studies of patients with solitary MNPs will allow to evaluate the role of tumor-infiltrating clonal hemopoiesis in oncogenesis. The ability of age-associated somatic clonal expansions in one tissue, namely hematopoietic compartment, to affect oncogenesis in another tissue is a new concept requiring further investigation and potentially capable of providing deeper understanding of cancer biology.The review explores the association between CHIP, aging, and oncological diseases with a particular emphasis on solitary MNPs. The ways for better understanding of the role of CHIP in oncogenesis and possibilities of using its clinical potential for cancer treatment are discussed.
Suppression of tumor cell growth and proliferation is the main goal of chemotherapy which is an integral part of the treatment for cancer patients. In addition to high antitumor activity, the cytotoxic effects of chemotherapeutic agents also extend to immune cells, resulting in pancytopenia and weakened immune response. Nevertheless, the effect of chemotherapy on the immune system is multifaceted, as it simultaneously exerts a suppressive influence while also stimulating the antitumor activity of lymphoid and myeloid populations. This review focuses on the analysis and generalization of modern data regarding the effects of chemotherapeutic drugs used in standard antitumor therapy regimens on the functioning of the immune system. The suppressive mechanisms of chemotherapy, including the development of cytopenia, are reviewed. Special attention is paid to the analysis of data on modulation of antitumor immune response depending on the class of chemotherapeutic agent. Mechanisms enhancing immune recognition and stimulating immune cells in response to increased expression of tumor antigens are described. The data regarding the effects of chemotherapy on the tumor microenvironment, including the reprogramming of immunosuppressive profiles and the activation of immune effectors, is presented. The summarized data underscore the dual nature of chemotherapy’s effects on the state of the immune system and its influence on the formation of antitumor immune responses.
Introduction. The proteins of the urokinase system, including serine protease urokinase (uPA), its receptor (uPAR), and inhibitors PAI-1 and PAI-2, play a key role in tumor biology by influencing cellular proliferation and tumor growth, invasion, metastasis, and angiogenesis. Despite the established role of these proteins in the carcinogenesis of many tumor types, the mechanisms underlying their action, including their effects on cell migration, epithelial-mesenchymal transition, and stemness, remain insufficiently studied.Aim. To evaluate the influence of the urokinase receptor gene PLAUR on the expression of adhesion and stemness genes, as well as on the migration of human glioma and neuroblastoma cells.Materials and methods. The study utilized two human glioma cell lines, U87 and U251, and the human neuroblastoma cell line SH-SY5Y. To achieve the overexpression of the PLAUR gene, a non-viral plasmid was created, followed by the transfection of the cells. Relative gene expression was assessed using real-time polymerase chain reaction. For evaluating cell migration, a Wound Healing Assay was performed, with image analysis conducted using ImageJ software and MRI Wound Healing Tool. Statistical analysis of the results was carried out using GraphPad Prism v.10.Results. High expression of the urokinase receptor gene PLAUR is associated with a significant increase in cell migration and complex phenotypic changes. Specifically, in U251 glioma cells, there is an induction of the expression of the genes CD56, CDH1, CDH2, ZEB2, and SOX2. In U87 glioma cells, the expression of the genes PLAU, CD56, CDH1, ZEB1, ZEB2, SNAI1, SNAI2, SOX2, and NANOG is induced, while the expression of the gene CDH2 is suppressed. In SH-SY5Y neuroblastoma cells, there is an induction of the expression of the genes CD56, CDH1, ZEB1, ZEB2, SNAI2, and SOX2, along with a suppression of the expression of the urokinase gene PLAU compared to cells transfected with the control plasmid pGFP.Conclusion. The results highlight the complexity of the regulation of carcinogenesis processes involving the PLAUR gene and deepen our understanding of tumour biology. High expression of the PLAUR gene may enhance the activity and invasion of tumor cells by regulating the epithelial-mesenchymal transition and altering the expression of key transcription factors.
Introduction. Cancer remains a major cause of mortality worldwide, with adverse environmental factors such as pesticides contributing significantly to its development. Despite the widespread use of various pesticides, the molecular mechanisms underlying their actions and carcinogenic potential have been studied for only a limited number of models, especially in normal human cells.Aim. To study the molecular effects of pesticides carbaryl, chlorpyrifos, mancozeb, thiram, and pendimethalin in nonmalignant HaCaT and MCF10A cells.Materials and methods. Non-toxic concentrations of pesticides were determined using the MTT assay. Genotoxicity was analyzed by the comet assay. Proliferative potential was assessed by clonogenic assay. Changes in the expression of genes associated with carcinogenesis were evaluated by real-time polymerase chain reaction.Results. Carbaryl caused DNA damage in MCF10A cells, promoted proliferation in both cell lines during clonogenic assay, as well as caused activation of biotransformation genes (AHR, GSTA4) in MCF10A cells, repression (CYP1B1, GSTA4) genes in HaCaT cells and lowered expression of inflammation genes (IL1a, IL1b, PTGES, IFNGR1). Chlorpyrifos did not have genotoxic effect and did not affect clonogenicity but caused induction of biotransformation (CYP1A1, CYP1B1), inflammation (IL1b, PTGES) genes, and genes BCL2 and DNMTs. Mancozeb and thiram did not show genotoxicity in HaCaT and MCF10A cells but activated individual repair genes (ATR/ATM). Thiram stimulated HaCaT cell proliferation in clonogenic assay, and mancozeb activated expression of proliferation regulation genes (CCND2, CCNE1, Ki-67) but did not affect colony growth; both fungicides decreased expression of inflammation genes (COX2, IL1a, IL1b). Pendimethalin caused DNA damage and activation of repair genes (ATR, GADD45a, PCNA) in both cell lines, as well as decreased expression of GLUT3 in HaCaT cells and induced expression of CYP1A1 in HaCaT cells and CYP1B1 in MCF10A cells.Conclusion. In this study, we performed a comprehensive assessment of the effects of pesticides on normal human cells. Our results indicate that pendimethalin, chlorpyrifos, and carbaryl exert the most procarcinogenic effect.
Currently, many scientific studies investigate the role of receptor tyrosine kinases in mechanisms of tumor cell resistance to targeted therapies and chemotherapies. Activation of FGFR and VEGR signaling pathways through fibroblast growth factors (FGF) and vascular endothelial growth factors (VEGF) is seen in many malignant neoplasms. The literature review was addressed to describe the role of FGFR and VEGFR signaling pathways in the secondary (acquired) resistance of gastrointestinal stromal tumors to the targeted drugs – tyrosine kinase receptor inhibitors (imatinib mesylate, sunitinib, regorafenib). The review describes the molecular mechanisms of activation of FGFR and VEGR signaling pathways in tumor cells, as well as their structural and functional relationship between aforementioned pathways. Multiple studies, including our own, illustrate the activation of FGFR and VEGFR pathways in gastrointestinal stromal tumors. The review also describes the clinical effectiveness of FGFR and VEGFR inhibition for advanced and metastatic gastrointestinal stromal tumors, thereby providing a rationale to utilize activation of the aforementioned signaling pathways as the markers for gastrointestinal stromal tumors progression.
Introduction. Regulatory T cells (Treg) have pro-oncogenic activity, inhibit the immune response and therefore can be a significant target for immunotherapeutic methods of treating colorectal cancer (CRC). Currently, the role of regulatory T cells in the mechanisms of carcinogenesis, as well as the participation of co-inhibitory and co-stimulating proteins of the immune system in the formation of tumor immunosuppression in CRC, is insufficiently studied. Aim. To determine the content of Treg subtypes in the primary tumor focus in patients with CRC and to evaluate the expression level of co-stimulating and co-inhibitory proteins on their membrane. Materials and methods. The main group consisted of 105 patients with stage III colon adenocarcinoma; the control group – 75 patients with non-neoplastic diseases of the colon. The object of the study was tumor-infiltrating T lymphocytes, which were isolated by an enzymatic method. Determination of Treg subtypes, as well as expression of programmed cell death 1 (PD-1) and inducible T-cell costimulator (ICOS) proteins, was performed by flow cytometry. Treg was identified as CD19−CD3+CD4+CD25highCD127−. Based on the expression of CD45RA and CD197, Treg subtypes were determined as naive (CD45RA+CD197+), central memory (CD45RA–CD197+), effector memory (CD45RA–CD197–), and terminally differentiated (CD45RA+CD197–). The significance of differences in the parameters of the main and control groups was assessed using the nonparametric Mann–Whitney U test. Results. In the primary focus of tumor growth in CRC, the content of Treg increases by 4.4 times. At the same time, the ratio of Treg subtypes in the tumor microenvironment changes, which is expressed in a 1.4-fold decrease in the number of naive cells, as well as a 2.8-fold increase in the number of effector memory Treg and a 3.1-fold increase in terminally differentiated lymphocytes. Tumor-infiltrating Treg expressed the ICOS 2 times more strongly compared to the control. A 20 % increase in the number of Treg PD-1+ICOS+ was established. The number of Treg PD-1−ICOS− cells in the primary tumor focus decreased by 1.4 times compared to the control group. Conclusion. In patients with CRC, the number of naive Treg decreases in the primary tumor focus and the relative content of differentiated effector cells increases. In the tumor microenvironment in CRC, the number of Tregs increases, expressing on their surface both the costimulating molecule ICOS and the co-inhibitory receptor PD-1.
