Flow cytometry using fluorescent antibodies (FC) is the method of choice for the quantitation of proteins expressed at the surface or inside the cell, but, however, does not allow to selectively measure nuclear expression. We therefore sought to develop a method for the extraction of intact cell nuclei, which can be used for their subsequent immunofluorescent analysis by FC. The studied protein was vascular endothelial growth factor-receptor-type 1 (VEGFR-1) which is important in tumor survival and metastasis. Two human cell lines, A431 (epidermoid carcinoma of skin with low invasive and metastatic potential) and BRO (highly aggressive amelanotic melanoma), were used as examples for tumor cells, and normal human fibroblasts PHF served as a control line. The quality of the extracted nuclei was assessed by their intactness and purity from cytoplasm. The high content of the nuclear markers (PCNA = proliferating cell nuclear antigen, lamin A/C) in the extracted nuclei with almost complete absence of the cytoplasmic β-tubulin demonstrated that the protocol can be used to obtain a pure suspension of single intact cell nuclei. The measurement of the nuclear VEGFR-1 content revealed that it was present only in tumor cell nuclei and that in more malignant BRO cells the receptor content was 1.75 times higher than in A431 (p = 0.014). Thus, the developed method of extraction of cell nuclei for subsequent FC analysis is suitable for the quantitative evaluation of protein content in the native nucleus.

Melanoma is one of the most aggressive and treatment-resistant tumors that responsible for majority of skin-cancer related deaths. Here we propose a combination of MEK inhibitor binimetinib with metformin as a promising therapy against human melanoma cells in vitro, including BRAF -mutated A375, Mel Z, and Mel IL cells, and NRAS-mutated Mel MTP and Mel Me cells. Additionally, we obtained two close to clinical practice models of melanoma progression. The first one was vemurafenib-resistant Mel IL/R melanoma cells with acquired resistance to BRAF inhibition-targeted therapy, and the second one was tumor spheroids, which are 3D in vitro model of small-size solid tumors in vivo. The cytotoxicity of binimetinib and metformin was synergistic in both 2D and 3D melanoma culture and mediated through apoptotic pathway. The combination reduced the number of melanoma-formed colonies, inhibited cell invasion and migration, and led to G0/G1 cell cycle arrest through cyclin D/CDK4/CDK6 pathway. The mechanism of metformin and binimetinib synergy in melanoma cells was associated with increased activation of p-AMPKα and decreased p-ERK, but not with alterations in p-mTOR. In summary, the combination of metformin and binimetinib resulted in stronger anti-proliferative effects on melanoma cells compared to binimetinib alone, and therefore could be promising for clinical applications.

The possibility of using VEGF-R1 receptor for targeted therapy in oncology was investigated. Using the approach to measuring the protein content in intact nuclei of cells, which was developed by us, we showed the presence of this receptor in the nuclei of tumor, but not normal cells. A direct correlation between the level of VEGF-R1 expression in the nucleus and the degree of malignancy of tumor cells, indicating the prognostic value of this parameter, was found. The mechanisms of the functioning of this receptor and the pathways of inhibiting its activity are discussed, and the validity of the selection of VEGF-R1 as a molecular target for anticancer therapy is conformed.

Background. Despite advantages in treatment of metastatic melanoma it remains resistant to current therapy. Recent evidence indicates that tumor cells could overcome death through autophagy, a process that degrades cellular proteins and organelles to maintain cellular biosynthesis during nutrient deprivation or lack of energy. Objective: to investigate the involvement of autophagy inhibitors chloroquine (CQ) and LY-294.002 (LY) in temozolomide (TMZ) cytotoxicity in human melanoma cell lines.Materials and methods. The study was performed on patient-derived melanoma cell lines Mel Z, Mel IL and Mel MTP. The antiproliferative activity of combined TMZ and autophagy inhibitors treatment was determined by MTT assay and colony-forming assay. Cell cycle analysis, apoptosis activation and expression analysis of key autophagy markers under combined treatment was evaluated.Results. CQ and LY enhanced the cytotoxicity of TMZ and reduced colony formation in 3 melanoma cell lines, moreover both inhibitors increased cell population in G0 / G1 phase of cell cycle in Mel Z, Mel IL cell lines, but not in Mel MTP. CQ and LY synergistically activated apoptosis in all cell lines. The matrix RNA expression analysis of key autophagy genes showed autophagy involvement in enhanced cytotoxicity.Conclusions. Thus, autophagy inhibition on different stages of this process could overcome resistance to TMZ and be applicable as potent target in metastatic melanoma treatment.

Background. Identification of cellular proteins can be performed by indirect immunofluorescence assay using flow cytometry. However, this method allows to detect cellular proteins that are either on the cell surface or inside the cell and cannot demonstrate a protein distribution within cellular compartments, particularly in nucleus. Meanwhile, the nuclear localization of the protein of interest in many respects gives an indication of the mechanisms of its action. Therefore, the development of the protocol of extraction of nuclei suitable for analysis on a flow cytometer is able to complement the information about the localization and functional significance of many nucleus-associated proteins. Objective. The aim of this study was to develope a method for extraction, purification and stabilization of intact nuclei suitable for flow cytofluorimetry analysis. Materials and methods. In this work we studied the nuclear localization of the receptor type 1 for vascular endothelial growth factor (VEGF-R1). The A431 human cancer cell line was used as an object of the study. The quality of extracted nuclei was assessed by microscopic examination of stained smears of nuclear suspension. Expression of the receptor was determined by indirect immunofluorescence assay using flow cytometry. Results. New method was successfully applied to obtain the suspension of intact cellular nuclei that is crucial to perform further flow cytometry. Applied method revealed the presence of the receptor type 1 for vascular endothelial growth factor at the external nuclear membrane and inside of the nucleus. Interesting to note that the presence of the receptor type 1 for vascular endothelial growth factor inside ofthe nucleus was 3,8 times as much as its surface location (17,9 ± 1,04 % and 4,8 + 0,26 % respectively). Conclusions. The new method of extraction, purification and stabilization ofthe nuclei is applicable for proteins identification by flow cytometry. In combination with other methods (ICC, Western blotting, etc.) flow cytometry of intact nuclei is able to complement the information about the properties of nucleus-associated proteins.

Patients with metastatic melanoma are difficult to treat and have a very poor prognosis because of high resistance to therapy. Recent evidence indicates that tumors could overcome death through autophagy, a survival mechanism, which cancer cells use under lack of energy and nutrient deprivation. Melanoma cells have different sensitivity to temozolomide (TMZ) treatment. In this study, we showed that the combination of autophagy inhibitors chloroquine or LY294002 and TMZ induced enhanced cytotoxicity of alkylating agents on human melanoma cell lines. All assays were performed on patient-derived melanoma cell lines. The effectiveness of the combined treatment of TMZ and autophagy inhibitors was determined using an MTT assay. Next, we analyzed the expression mRNA level of Beclin 1, LC3B, and p62/STSQM1 and the relative expression of LC3B protein under combined treatment. Autophagic flux was determined by analysis of colocalization of Lysotracker Red and LC3B puncta. Apoptosis was measured by Annexin V/PI staining. Cell cycle analyses were carried out by flow cytometry. We showed that autophagy inhibition could enhance melanoma cell death combined with TMZ therapy. Chloroquine synergistically enhanced the TMZ-induced growth arrest and increased the G0/G1 population in Mel Z and Mel IL cell lines, but not Mel MTP. The expression analysis showed that autophagy involvement in TMZ enhanced cytotoxicity. Furthermore, LY294002, an early-stage autophagy, and PI3K inhibitor were found to exert similar effects. Both chloroquine and LY294002 improved the cytotoxic effect of TMZ treatment, making this combination applicable as a potent antitumor treatment for metastatic melanoma.

MM is characterized by low sensitivity to chemotherapy and biotherapy because of its high resistance to apoptosis. We evaluated the significance of the autophagy modulation (blocking or induction) cytotoxicity change of Paclitaxel on 11 cell lines of MM with different B-RAF gene status in vitro. Cytotoxicity was determined by MTT assay, cell death by flow cytometry. Detection of mRNA expression Beclin1 performed by RT-PCR. Mutations in the B-RAF gene determine the level of autophagy in MM cells in vitro. B-RAF V600 lines were resistant to Paclitaxel and autophagy inhibition by chloroquine increased cytotoxicity of the taxane up to 40-50%. Although B-RAF WT MM cells were more sensitive to Paclitaxel, the addition of inhibitor had no effect on cell death. Investigation of the mechanisms of the phenomena showed the involvement of apoptosis process.

AbstractThe synthesis of a series of N6‐substituted adenosines is described.

A series ofN6-adenosine derivatives were synthesized by alkylation ofN6-acetyl-2′,3′,5′-tri-O-acetyladenosine (1) with alkyl halides and alcohols. It was shown that propargyl derivative2ais a good substrate for copper(I) catalyzed Huisgen [3+2] cycloaddition with azides. This click-reaction can be used for preparation of the libraries of 1,2,3-triazolyl modified adenosines. Biological activities ofN6-adenosines were studied in two plant and six human cancer cell assays. The remarkable parallel between cytokinin and cytotoxic activities was found. The most cytokinin active compounds3c–3eat the same time appeared to be the most potent cytotoxic agents.

Tris(1-alkylindol-3-yl)methanes were obtained and oxidized into tris(1-alkylindol-3-yl)methylium salts. The resulting salts are more toxic to cultured tumor cells than to non-tumor ones. The cytotoxicity of tris(1-alkylindol-3-yl)methylium salts depends on the length of the substituent at the N atom of the heterocycle, increasing from an N-unsubstituted derivative toward N-butyl- and N-pentyl derivatives. A further increase in the length of the N-alkyl substituent lowers the cytotoxicity. The cytotoxicity of tris(1-alkylindol-3-yl)methylium salts for tumor cells correlates with their antibacterial and antifungal activity. Tris(1-alkylindol-3-yl)methylium salts produced a cytocide effect on Gram-positive microorganisms and the most active compounds, on Gram-negative microorganisms as well. Similar patterns of the structure—activity relationship of N-alkylated tris(indol-3-yl)methylium derivatives, which was observed for various lines of tumor cells, bacteria, and fungi, suggest the general character of the mechanisms of the death of prokaryotic and eukaryotic cells induced by these compounds.