Multiple exogenous or endogenous factors alter gene expression patterns by different mechanisms that are poorly understood. We used RNA-Seq analysis in order to study changes in gene expression in melanoma cells that are capable of vasculogenic mimicry that is inhibited upon the action of an inhibitor of vasculogenic mimicry. Here, we show that the drug induces a strong upregulation of 50 genes that control the cell cycle and microtubule cytoskeleton coupled with a strong downregulation of 50 genes that control different cellular metabolic processes. We found that both groups of genes are simultaneously regulated by multiple sets of transcription factors. We conclude that one way for coordinated regulation of large groups of genes is regulation simultaneously by multiple transcription factors.

Multiple exogenous or endogenous factors alter gene expression patterns by different mechanisms that yet are poorly understood. We used RNA-Seq analysis in order to study changes in gene expression in melanoma cells capable to vasculogenic mimicry upon action of inhibitor of vasculogenic mimicry. Here, we describe that the drug induces a strong upregulation of 50 genes controlling cell cycle and microtubule cytoskeleton coupled with a strong downregulation of 50 genes controlling different cellular metabolic processes. We found that both groups of genes are simultaneously regulated by multiple sets of transcription factors. We conclude, that one way for coordinated regulation of big groups of genes is the regulation simultaneously by multiple transcription factors.

In tumors, traditional angiogenesis is observed in addition to vascular channels that lack an endothelial cell lining. It is assumed that the network of such channels compensates for the insufficiently rapid development of the blood circulatory system in the tumor and prevents early necrosis inside the tumor. The strong statistical correlation established between the presence of vascular channels in the tumor and the low survival rate of patients confirms this hypothesis. In the light of these data, the search for a low-molecular-weight inhibitor of vascular channel formation in tumors becomes extremely relevant. This review discusses the functional and prognostic significance of vasculogenic mimicry. Particular attention is paid to the optimization of the in vitro vasculogenic mimicry model based on the analysis of 11 melanoma cell lines obtained from metastases of patients with disseminated melanoma. In addition, the development of an in vivo vasculogenic mimicry model is discussed.

Background. CD437, an analog of vitamin A, is an agonist of the retinoic acid γ-receptor (RARγ). CD437 is also known to cause p53-independent DNA damage by a mechanism independent of the RAR-mediated pathway. In cancer patients, iron deficiency is constantly detect, the delivery of iron to tissues is also destroyed.Aim. To study the effect of CD437 on iron metabolism in metastatic melanoma cells, Mel Z.Materials and methods. In this study 2D cultivation of metastatic Mel Z melanoma cells, phase-contrast and fluorescence microscopy, flow cytofluorimetry were used.Results. In control cells without the addition of CD437 CD71, transferrin receptor, expressed 40 ± 4 % (p <0.05) of Mel Z cells, in the presence of CD437 CD71 expression increased to 80 ± 6 %. Next, we have studied the expression of ferritin. Iron, which is not involved in cell metabolism, is bound by ferritin. In control experiments, ferritin was expressed by 84 ± 6 % (p <0.05) of cells. When the cells grew in the presence of CD437, ferritin was expressed by all the cells (100 %, p <0.05). Such a scenario indicates that CD437 may contribute to the accumulation of free, unbound iron in the cell, which can induce ferroptosis. In control experiments without the addition of CD437, the level of membranes lipid peroxidation, an indicator of ferroptosis, was insignificant. Lipid peroxidation induced by CD437 was 55 ± 5 % (p <0.05) of the fluorescence intensity induced by erastin, positive control.Conclusion. CD437 increases the iron uptake by metastatic melanoma cells. The low level of membranes lipid peroxidation induced by CD437 does not allow it to be considered as an inducer of ferroptosis. Additional investigations are needed to find iron-binding targets alternative to ferritin.

Introduction. Early malignant tumor detection programs have significantly increased the survival rate of breast cancer patients but the results of drug therapy for this pathology are not always highly effective. Recently discovered iron-dependent cell death, ferroptosis, makes it a promising therapeutic target to reduce the recurrence rates. Objective – to study the induction of ferroptosis in breast cancer cells MCF-7 by quinazoline derivatives synthesized at the Research Institute of Experimental Diagnostics and Therapy of Tumors of the N.N. Blokhin National Medical Research Center of Oncology, Ministry of Health of Russia and to evaluate its antitumor activity on transplanted breast carcinoma Ca-755. Materials and methods. Derivatives of 3-hydroxyquinazoline were obtained by chemical synthesis and have a purity of at least 95 %. In this study 2D cultivation of MCF7 cells, phase-contrast and fluorescence microscopy, and a model of experimental growth of breast carcinoma Ca-755 in female hybrids of immunocompetent mice F1 (C57Bl/6 × DBA/2) were used. Results. Five derivatives of 3-hydroxyquinazoline, analogues of erastine, were studied in this work. The ferroptotic cell death was identified by the level of lipid peroxidation at the concentrations of 1/3 and 1/5 IC 50 . The level of lipid peroxidation induced by compound 3 was comparable with the activity of erastin in MCF7 cells at both 1/3 and 1/5 of IC 50 , the activity of the other four quinazoline derivatives was 50–70 % of the activity of erastin. In in vivo experiments at a dose of 30 mg/kg the antitumor efficacy of the compound 3 was higher than that of erastin at the same dose. Conclusion. The data obtained suggest that quinazoline derivative 3 might be considered as a promissisng antitumor agent to treat breast cancer.

Synthetic retinoid CD437, an agonist of the retinoic acid receptor γ (RARγ), demonstrates high potential for cancer treatment in xenograft models. The aim of this study is to investigate the melanoma cells’ metastatic potential alterations induced by CD437. 2D- and 3D-cell cultures, migration “into the wound,” invasion, colony forming assay, and flow cytofluorimetry are used in this study. Here we show that CD437 reduced the migration of Mel Z melanoma cells by 52 ± 2% compared to the positive control. The decrease in invasive activity of melanoma cells under the conditions of CD437 treatment did not exceed 40 ± 4%. CD437 also blocked the formation of capillary-like structures by melanoma cells on Matrigel. The efficiency of colony formation by Mel Z cells in the control was quite high. However, we did not observe any colonies after 7 days of cultivating melanoma cells with non-cytotoxic concentrations of CD437. Further, we show that the expression of MMP-9 in Mel Z cells is significantly lower than the expression of MMP-2. CD437 reduces the expression of MMP-2 by half, and MMP-9 by a factor of 35 compared to the control. We did not reveal the effect of CD437 on the CD44 expression by melanoma cells; there was a slight decrease in the CD24 expression (23 ± 2%). The data obtained suggest that CD437 reduces the metastatic potential of melanoma cells.

Introduction. The main cause of clinical progression of tumor under the conditions of treatment is the resistance. Reactivate apoptosis in resistant to chemotherapy cells is impossible, the tumor grows into an irreversible growth phase. Recently published data on the ability of ferroptosis inducers to induce the death of resistant cells opens up new possibilities for improving the effectiveness of antitumor therapy.The purpose of the study – assessment of the mechanism of ferroptosis induction of the synthesized analogue of erastin OVN‑002 on Mel Z melanoma cells and investigation of its antitumor activity on transplanatated B‑16 melanoma of mice.Materials and methods. In this study 2D cultivation of metastatic Mel Z melanoma cells, phase‑contrast and fluorescence microscopy, and a model of experimental growth of B‑16 melanoma in female hybrids of immu‑ nocompetent mice F1 (C57Bl/6 × DBA / 2) were used. The antitumor effect was evaluated by measurement of tumor growth inhibition (TGI, %) and increase of life span of the treated animals as compared to the control ones.Results. The cytotoxic activity of OVN‑002 was equal to the activity of erastin on metastatic melanoma cells Mel Z: 744 ± 20 and 719 ± 20 a. u., respectively. OVN‑002 at a dose 50 mg / kg reduced the growth of experimental melanoma B‑16 about 81 % (TGI 81–57 %, p <0.05). The effect was stable up to 7th day, while erastin showed only a direct antitumor effect (TGI 65 %, p <0.05).Conclusion. The data obtained suggest that OVN‑002 might be considered as a novel antitumor agent.

Introduction. Synthetic retinoids CD437, an agonist of the gamma retinoic acid receptor (RARγ), not only induces growth arrest, but in contrast to retinoid acid, it also induces RARγ-independent apoptosis in many tumor cells through a unique mechanism that is independent of the retinoic acid receptor-mediated pathway. The aim of the study was to study the relationship between apoptosis and autophagy in CD437-induced cell death. Materials and methods. In this study we used 2D-culturing of lung carcinoma cells A549, immunocytochemistry, flow cytometry and fluorescence microscopy. Results. CD437 at concentrations between 0.2 and 5.0 μM increased the number of PI-positive cells in A549 lung cancer cells. The retinoid at concentrations close to IC 50 reduced the cell population in the G2/M-phase and arrest cell cycle in the S-phase. CD437 dose-dependent increased the number of apoptotic cells in the presence of non-cytotoxic concentrations of wortmanin, an irreversible inhibitor of anti-stress kinasе PI-3K, and LY 200192, a reversible inhibitor. CD437 also activated the biogenesis of autophagosomes, and there was a dose-dependent increase in the fluorescence intensity of monodansilkadaverine, a marker of autophagy. However, accumulation of LC-3B was not observed with an increase of CD437 concentration from 0.1 to 5.0 μM suggesting that the fusion between autophagic vacuoles and lysosomes was inhibited. zVAD-fmk, an irreversible caspase inhibitor, did not restore autophagy in A549 cells, and LC-3B levels did not change significantly with the increasing of CD437 concentration, indicating that CD437 was involved in autophagosome-lysosome fusion. When cells grew with non-cytotoxic concentrations of chlorokine, a late stage autophagy inhibitor, there were virtually no living cells at CD437 concentrations close to IC 50 . The additive effect of CD437 and chlorokine in inducing A549 cell death confirms that CD437 involved in fusion between autophagosomes and lysosomes required for final catabolism of autophagic material. Conclusions. The data obtained indicate that CD437 induced a failure of cytoprotective function of autophagy and apoptosis, that raise the question of combined therapy of CD437 with cytotoxic drugs in the treatment of lung carcinoma

Vasculogenic mimicry, the formation of vascular channels lined with tumor cells of a highly malignant phenotype, is currently considered as an additional system of blood supply of the tumor. Experimental studies in vivo have repeatedly demonstrated that vascular channels form in the areas of a tumor with a low density of blood vessels. It is supposed that the formation of a network of these channels inside the tumor maintains homeostasis and prevents early necrosis within it. In this work, bifunctional compounds based on a combination of quinazoline and hydroxamic acid in one molecule were examined for the ability to inhibit the migration of tumor cells and vasculogenic mimicry.

Background. Activating mutations in the BRAF gene leads to a constitutive activation of the MAPK signaling. The highly selective BRAFV600E inhibitor, vemurafenib, improves the overall survival of BRAF-mutant melanoma patients. However, despite the excellent results of response rate, the average duration of the response was short and acquired resistance develops in most BRAF mutated melanoma patients within a few months. Objective: to derive melanoma cell lines from surgical species of patients with BRAF mutant melanomas resistant to vemurafenib and to elucidate the mechanisms involved in acquired drug resistance.Materials and methods. Mel Ki and Mel F1702 melanoma cells were obtained from metastases of disseminated melanoma patients with BRAFV600E mutation. 2D tumor cell culture, MTT test, immunicytochemistry, flow cytometry, real-time polimerase chain reaction and osteogenic and adipocytic differentiation were used in the study.Results. We have derived two melanoma cell lines Mel Ki and Mel F1702 from tumor samples of patients with BRAFV600E mutation resistant to vemurafenib. These cells were homogenous and had fibroblastic morphology. The IC50 values for Mel Ki and Mel F1702 were 4.7 and 6.3 μM, respectively. The expression of cancer-testis antigens was not detected in both types of cells suggesting the stemness of Mel Ki and Mel F1702 melanoma cells. The immunophenotypic profile of the vemurafenib resistsant melanoma cells showed the expression of typical mesenchymal stem cells markers such as CD90, CD105 and CD44. In addition, we found that the melanoma cell lines derived from tumor resistant to vemurafenib differentiated into osteoblastand adipocyte-like cells. Conclusion. In this study we are offering an experimental evidence of the phenotypic transition of the vemurafenib-resistant melanoma cells into mesenchymal stem-like cells.

Introduction.Autophagy, a catabolic process of protein and organelle recycling by transferring defective cytoplasm and organelles into double-membraned vesicles to degrade and regenerate materials, plays a critical role in maintaining energy homeostasis. Inefficiency chemo-and radiotherapy is largely associated with the activation of autophagy. Among the metals needed by the living organism, iron occupies a special place. The rapid growth of malignant tumors requires much more iron than the metabolism of normal cells.Objective.To elucidate the relationship between autophagy and iron in melanoma progression.Materials and methods.In this study we used 2D- and 3D-culturing of melanoma cells with high expression of CD71 (mel P and mel Z) and low expression of CD71 (mel Gus and mel Ibr), flow cytometry and fluorescence microscopy.Results.The uptake of iron in cancer cells occurs through translocation of the complex of transferrin/receptor (CD71) in the cytoplasm with subsequent dissociation of iron from the complex. Chelation of iron by deferroxamine in melanoma cells mel P and mel Z reduced the level of autophagy about 2-fold. In the presence of an iron donor ferrum ammonium citrate the level of autophagy increased 2.5- fold. The same correlation was observed in melanoma cells with low expression of CD71. Chelation of iron in melanoma cells with high CD71 expression blocked the formation of capillary-like structures. In the presence of an iron donor the formation of capillary-like structures was also not observed. The same correlation was observed in melanoma cells with low expression of CD71. There was an increase in CD105 expression about 50 ± 5 % and 800 ± 50 % under the condition of iron chelation in melanoma cells with high and low expression of CD71, respectively. Quite unexpectably, iron donor also increased expression of CD105 about 35 ± 4 % and 300 ± 3 % in melanoma cells with high and low expression of CD71, respectivelyConclusions.The activation of autophagy promotes the survival of tumor cells by triggering a number of metabolic functions with the participation of iron.

Introduction. Autophagy, a catabolic process of protein and organelle recycling by transferring defective cytoplasm and organelles into double-membraned vesicles to degrade and regenerate materials, plays a critical role in maintaining energy homeostasis. Autophagy also protects against stress and infection, participates at the development of autoimmune disease. In recent years, the existence of alternative blood circulation system in tumors, vasculogenic mimicry (VM), which can partially compensate the lack of nutrients and oxygen under the hypoxic conditions, has been described. Objective. To elucidate the relationship between autophagy and VM. Materials and methods. In this study we used 2D- and 3D-culturing of melanoma cells derived from surgical species of patients with disseminated melanoma, electrophoresis and western blot, knockdown of the genes by using small interfering RNA (siRNA), flow cytometry, fluorescence microscopy. Results. We detected the basal level autophagy by examining the expression of autophagy-specific protein (LC-3B) by flow cytometry and cellular immunofluorescence staining by monodancylcadaverine. Both assays are the markers of autophagy late stage. Here we show that the level of autophagy in melanoma cells mel P, participated in capillary-like structures (CLS) formation in matrigel, was considerably higher than in mel Me cells which do not involve in VM. To explore the function of autophagy in the ability of melanoma cells to form CLS 3-methyladenine (3-MA) or chloroquine - inhibitors of initiation and terminal stage of autophagy - were used. Both inhibitors reduced the ability of melanoma cells to engage in VM. The data obtained were confirmed by siRNA-mediated gene silencing of BECN1 involved in the initiation of autophagy and ATG5 gene which is considered to be a marker of late stage of autophagy. Knockdown of BECN1 or ATG5 in mel P melanoma cells reduced the level of protein Beclin-1 and Atg5 about 70-75 %, and suppressed CLS formation in matrigel. Melanoma cells with the ATG5 gene knockdown changed the shape but maintained the ability to migrate and recognize each other, the formation of CLS was not observed. Low molecular weight VM inhibitor LCS-1269, significantly reduced the basic level of autophagy. Conclusion. Our data indicate that autophagy participates in CLS formation, and inhibition of autophagy suppresses CLS formation. We suggest that autophagy plays a dual role in the survival and development of tumors: autophagy helps cancer cells against environment stress and provides a temporary survival pathway by promoting energy regeneration, autophagy also promotes VM formation which supplies nutrients and oxygen to less vascularized area of tumor.

In 2012 the new, iron-dependent form of cell death - ferroptosis - distinct from apoptosis, autophagy and programmed necrosis at morphological, biochemical, and genetic levels has been described. In this review we will discuss the iron metabolism in physiological and pathological processes in humans, the molecular characteristics of ferroptosis and therapeutic options of cancer patients in the light of new data.

The basic problem in cancer treatment remains the identification of cells responsible for maintaining the whole population of cells in a tumor. For decades it has been considered that all transformed cells within a tumor have carcinogenic potential with unlimited proliferation capacity and metastases formation. At present, the concept of cancer stem cell was introduced indicating that tumor evolves from a small population of long-live and slow proliferating cells. These cells have the capacity to initiate the tumor formation in immunodeficient animals. Among their properties, resistance to standard oncology treatments leads to treatment failure and cancer recurrence. The management and eradication of different types cancer is completely depended on removal of this cell population. Current review presents basic information about cancer stem cell, particularly, the initiation of tumor, the peculiar properties of cancer stem cell, the role of cancer stem cell in metastasis formation and discusses therapeutic strategies targeted cancer stem cell.

The growth of malignant tumors occurs in 3D environments and depends on the presence of a stromal component, which performs critical functions of tumor-cell protection and growth support. Therefore, the development and analysis of tumor models in 3D cell cultures in vitro, including coculture systems, hold significant interest. In this study, the results of 3D culturing of two human melanoma cell lines using the hanging drop method with and without human mesenchymal stem cells (MSCs) have been presented. The melanoma lines have been shown to behave differently in 3D cultures. In particular, the Mel Cher melanoma cells are capable of the formation of uniform spheroids within 24 h, whereas the MeWo cells failed to form spheroids even after 2 days of culture under similar conditions. However, in both cases, the coculturing of the melanoma cells with MSCs resulted in the formation of compact 3D cell spheroids. The visualization of MeWo cells and MSCs in mixed spheroids using fluorescent dyes revealed a certain clustering of the melanoma cells. The observed properties of the melanoma cells in homogeneous and heterogeneous spheroids may be used in the complex analysis of antimelanoma chemotherapy drugs and the estimation of their therapeutic properties.

Melanoma is considered to be the deadliest form of skin cancer. For years it has been accepted that all melanoma cells in tumor are capable of tumor formation and each of them has the ability to initiate the metastasis. At present a stem cell theory of cancer has been developed, which purports that a subpopulation of self-renewing tumor cells is responsible for tumorogenesis, neoangiogenesis and stroma building. In this review we will discuss the basic determinants of melanoma stem cell and melanoma stem cell targeting implications.

Growth of malignant tumors occurs in three-dimensional space and depends on a presence of stromal component, which performs critical functions of tumor cell protection and growth support. Therefore, development and analysis of tumor models in 3D cell cultures in vitro, including co-culture systems, presents a significant interest. In this study, the results of 3D culturing of two human melanoma cell lines using the hanging drop method, with or without human mesenchymal stem cells (MSCs), are presented. Melanoma lines were shown to behave differently in 3D cultures; in particular, Mel Cher melanoma cells have the ability to form uniform spheroids within 24 h, whereas MeWo cells under similar conditions failed to form spheroids even after 2 days of culture. However, co-culturing of melanoma cells with MSCs resulted in formation of compact 3D cell spheroids in both cases. Visualization of MeWo cells and MSCs in the mixed spheroids using fluorescent dyes revealed certain clustering of melanoma cells. The observed properties of melanoma cells in homogeneous and heterogeneous spheroids may be used in the complex analysis of results of testing of antimelanoma chemotherapy drugs and evaluation of their therapeutic properties.

Solid tumor growth is dependent on the development of an adequate blood supply. For years, sprouting angiogenesis has been considered as the exclusive mechanism of tumor vascularization. However, in recent years, another mechanism of tumor vascularization has been identified that does not involve endothelial cells, a process called vasculogenic mimicry (VM). VM describes the unique ability of highly aggressive tumor cells to form vessel-like networks by virtue of their high plasticity. VM has been observed in several tumor types, and its occurrence is strongly associated with poor prognosis. This review focuses on signaling molecules and cascades involved in VM. In addition, the clinical significance of VM regardless of anti-angiogenesis treatment modalities is described.