Background Melanoma is a highly heterogeneous malignant tumor that exhibits various forms of drug resistance. Recently, reversal transition of cancer cells to the G 0 phase of the cell cycle under the influence of therapeutic drugs has been identified as an event associated with tumor dissemination. In the present study, we investigated the ability of chemotherapeutic agent dacarbazine to induce a transition of melanoma cells to the G 0 phase as a mechanism of chemoresistance. Methods We used the flow cytometry to analyze cell distribution within cell cycle phases after dacarbazine treatment as well as to identifyG 0 ‐positive cells population. Transcriptome profiling was provided to determine genes associated with dacarbazine resistance . We evaluated the activity of β‐galactosidase in cells treated with dacarbazine by substrate hydrolysis. Cell adhesion strength was measured by centrifugal assay application with subsequent staining of adhesive cells with Ki‐67 monoclonal antibodies. Ability of melanoma cells to metabolize dacarbazine was determined by expressional analysis of CYP1A1, CYP1A2, CYP2E1 followed by CYP1A1 protein level evaluation by the ELISA method. Results The present study determined that dacarbazine treatment of melanoma cells could induce an increase in the percentage of cells in G 0 phase without alterations of β‐galactosidase positive cells which corresponded to the fraction of the senescent cells. Transcriptomic profiling of cells under dacarbazine induction of G 0 ‐positive cells percentage revealed that ‘VEGFA‐VEGFR2 signaling pathway’ and ‘Cell cycle’ signaling were mostly enriched by dysregulated genes. ‘Focal adhesion’ signaling was also found to be triggered by dacarbazine. In melanoma cells treated with dacarbazine, an increase in G 0 ‐positive cells among adherent cells was found. Conclusions Dacarbazine induces the alteration in a percentage of melanoma cells residing in G 0 phase of a cell cycle. The altered adhesive phenotype of cancer cells under transition in the G 0 phase may refer to a specific intercellular communication pattern of quiescent/senescent cancer cells.
BACKGROUND: Cellular senescence is a stress response, triggered by various stimuli such as chemotherapy treatment and causes G0/G1 cell cycle arrest followed by the production of a senescence associated secretory phenotype. p53 considered to be a modulator of these events although the precise mechanisms of it remains not clear. AIMS: To determine the non-apoptotic functions of the p53 protein the formation of the senescence associated secretory phenotype phenotype of melanoma cells under the treatment of the cytostatic agent dacarbazine. MATERIALS AND METHODS: The study was conducted on BRO and SK-MEL-2 skin melanoma cell lines. Melanoma cells were were treated by cytostatic agent dacarbazine. Then immunocytochemical study was performed to determine the proportion of G0-positive cells and the expression of the tumor suppressor protein p53. A bioinformatic analysis was accomplished to identify for p53 regulators with determining of miR-155-5p levels in exosomes released by dacarbazine-treated melanoma cells. RESULTS: The cytostatic drug dacarbazine increases the proportion of cells residing in the G0 phase of the cell cycle. Onco-microRNA miR-155-5p was expressed in the exosomes of the two studied cell lines BRO and SK-MEL-2 of skin melanoma. Changes in the expression level of p53 correlate with changes in miR-155-5p microRNA expression. The absence of changes in p53 expression in BRO melanoma cells may be due to the absence of changes in miR-155-5p expression levels. In the BRO cell line, no changes in the expression of the oncosuppressor p53 were observed with an increased percentage of G0-positive cells, which may be associated with the activation of other mechanisms of cell cycle arrest in the G0/G1 phase. CONCLUSIONS: Heterogeneous effect of the cytostatic agent dacarbazine on melanoma cells was revealed. For the SK-MEL-2 cell line, dacarbazine induces the release of senescence associated secretory phenotype by inhibiting exosomal production of miR-155-5p, which activates the p53 oncosuppressor, which was not observed in the BRO line.
Objective. To evaluate anti-tumor, toxic effect of miR-204-5p mimic applicaton on melanoma B-16-bearing mice followed by miR-204-5p target gene expression estimation in melanoma tumor and distant organs. Material and Methods. C57Bl/6 melanoma B-16-bearing mice were used. The animals of the experimental group were intraperitoneally injected with a 5 nM miR-204-5p miRNA simulator (mimic) on the 8th, 10th, and 12th days after melanoma B-16 cell transplantation. Based on the results of bioinformatic analysis, miR-204-5p target genes BCL2 and SIRT1 expression levels were determined by quantitative real-time PCR. The toxic effect of miR-204-5p mimic was estimated by the evaluation of body weight, mass of the internal organs, and motor activity. Results. On the 13-14th days of the experiment, the motor activity of animals in the control groups decreased signifcantly compared to the group of animals treated by miR-204-5p. Target gene BCL2 showed increased expression in the lungs and kidneys and SIRT1 levels were increased in the lungs of miR-204-5p mimic treated animals (p˂0.05). Tumor mass tended to decrease in the animals treated by miR-204-5p mimic. Conclusion. Modulation of the level of miR-204-5p microRNA led to changes in the expression of SIRT1 and BCL2 in the lungs of animals, and changes in the expression of BCL2in the kidneys. MiR-204-5p mimic application did not have toxic effect on animals treated. Further studies are necessary to clarify miR-204-5p implication in melanoma cell proliferation regulation as well as it’s biodistibution in the tumor tissue.
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