ADAPTOR PROTEIN Ruk/CIN85 PARTICIPATES IN THE METABOLIC CONTROL OF HUMAN BREAST ADENOCARCINOMA MCF-7 CELLS
Aim. To determine the role of Ruk/CIN85 in the control of breast adenocarcinoma cells metabolism, we performed systemic analysis of the activity levels/content of key enzymes/components of glycolysis and oxidative phosphorylation using as a model the weakly invasive human breast adenocarcinoma MCF-7...
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National Academy of Sciences of Ukraine, Palladin Institute of Biochemistry
2022-04-01
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| Series: | Biotechnologia Acta |
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| Online Access: | http://biotechnology.kiev.ua/images/2_2022/korshun_2_2022.pdf |
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| author | R. S. Korshun O. O. Hudkova N. V. Latyshko T. O. Kishko I. R. Horak L. B. Drobot |
| author_facet | R. S. Korshun O. O. Hudkova N. V. Latyshko T. O. Kishko I. R. Horak L. B. Drobot |
| author_sort | R. S. Korshun |
| collection | DOAJ |
| description | Aim. To determine the role of Ruk/CIN85 in the control of breast adenocarcinoma cells metabolism, we performed systemic analysis of the activity levels/content of key enzymes/components of glycolysis and oxidative phosphorylation using as a model the weakly invasive human breast adenocarcinoma MCF-7 cell line (Mock); and its sublines with stable overexpression (G4 subline) and reverse down-regulation (G4vir subline) of the adaptor protein.
Materials and methods. MCF-7 cells were cultured in the complete DMEM medium under standard conditions. Enzymes activity, content of metabolites and protein in cell extracts and the conditioned cell culture medium were estimated by spectrophotometric and fluorometric assays.
Results. First of all, biochemical indexes of aerobic glycolysis, activity levels of some key glycolytic enzymes and metabolites were evaluated. A significant increase in the activity of these enzymes, aldolase A (ALDOA) and lactate dehydrogenase A (LDHA), was found in G4 cells compared to Mock by 1.3 and 1.6 times, respectively. In addition, in the conditioned medium of G4 cells, an increase in lactate content by 1.5 times compared with the control was found, which corresponded to a change in LDHA activity. Knockdown of Ruk/CIN85 expression level in G4 subline resulted in a significant decrease of these parameters compared to G4 cells, ALDOA – 4 times, LDHA - 1.4 times, and lactate production - 2.5 times. It should be noted that in G4vir cells, LDHA activity returned to level of control cells, while ALDOA activity and lactate content additionally decreased by 3 times and 1.6 times, respectively. Therefore, the observed changes in the intensity of glycolysis in MCF-7 sublines positively correlate with the expression level of adaptor protein studied. To assess the metabolic status of mitochondria, the level of activity of the Krebs cycle enzyme, NAD-dependent malate dehydrogenase (MDH2), the catalyst of last stage of the cycle, was determined. A 2-fold decrease in MDH2 activity was found in the MCF-7 G4 subline relative to control Mock cells, as well as an increase in this index by 2.4 times in G4vir cells to control values. Unlike glycolysis, we observed the opposite pattern with respect to the intensity of Krebs cycle reactions depending on the expression level of Ruk/CIN85.
Conclusions. The observed reversion of the Warburg metabotype as a result of Ruk/CIN85 downregulation in MCF-7 cells overexpressing adaptor protein is a strong experimental evidence for its regulatory role in energy supply modes, aerobic glycolysis versus OXPHOS in the course cancer cells malignization. |
| first_indexed | 2024-03-12T07:31:52Z |
| format | Article |
| id | doaj.art-327ddc41ed3940568b80109047e59afa |
| institution | Directory Open Access Journal |
| issn | 2410-7751 2410-776X |
| language | English |
| last_indexed | 2024-03-12T07:31:52Z |
| publishDate | 2022-04-01 |
| publisher | National Academy of Sciences of Ukraine, Palladin Institute of Biochemistry |
| record_format | Article |
| series | Biotechnologia Acta |
| spelling | doaj.art-327ddc41ed3940568b80109047e59afa2023-09-02T21:44:37ZengNational Academy of Sciences of Ukraine, Palladin Institute of BiochemistryBiotechnologia Acta2410-77512410-776X2022-04-01152585910.15407/biotech15.02.058ADAPTOR PROTEIN Ruk/CIN85 PARTICIPATES IN THE METABOLIC CONTROL OF HUMAN BREAST ADENOCARCINOMA MCF-7 CELLSR. S. Korshun0O. O. Hudkova1N. V. Latyshko2T. O. Kishko3I. R. Horak4L. B. Drobot5Taras Shevchenko National University, Ukraine, KyivPalladin Institute of Biochemistry of the National Academy of Sciences of Ukraine, KyivPalladin Institute of Biochemistry of the National Academy of Sciences of Ukraine, KyivPalladin Institute of Biochemistry of the National Academy of Sciences of Ukraine, KyivPalladin Institute of Biochemistry of the National Academy of Sciences of Ukraine, KyivPalladin Institute of Biochemistry of the National Academy of Sciences of Ukraine, KyivAim. To determine the role of Ruk/CIN85 in the control of breast adenocarcinoma cells metabolism, we performed systemic analysis of the activity levels/content of key enzymes/components of glycolysis and oxidative phosphorylation using as a model the weakly invasive human breast adenocarcinoma MCF-7 cell line (Mock); and its sublines with stable overexpression (G4 subline) and reverse down-regulation (G4vir subline) of the adaptor protein. Materials and methods. MCF-7 cells were cultured in the complete DMEM medium under standard conditions. Enzymes activity, content of metabolites and protein in cell extracts and the conditioned cell culture medium were estimated by spectrophotometric and fluorometric assays. Results. First of all, biochemical indexes of aerobic glycolysis, activity levels of some key glycolytic enzymes and metabolites were evaluated. A significant increase in the activity of these enzymes, aldolase A (ALDOA) and lactate dehydrogenase A (LDHA), was found in G4 cells compared to Mock by 1.3 and 1.6 times, respectively. In addition, in the conditioned medium of G4 cells, an increase in lactate content by 1.5 times compared with the control was found, which corresponded to a change in LDHA activity. Knockdown of Ruk/CIN85 expression level in G4 subline resulted in a significant decrease of these parameters compared to G4 cells, ALDOA – 4 times, LDHA - 1.4 times, and lactate production - 2.5 times. It should be noted that in G4vir cells, LDHA activity returned to level of control cells, while ALDOA activity and lactate content additionally decreased by 3 times and 1.6 times, respectively. Therefore, the observed changes in the intensity of glycolysis in MCF-7 sublines positively correlate with the expression level of adaptor protein studied. To assess the metabolic status of mitochondria, the level of activity of the Krebs cycle enzyme, NAD-dependent malate dehydrogenase (MDH2), the catalyst of last stage of the cycle, was determined. A 2-fold decrease in MDH2 activity was found in the MCF-7 G4 subline relative to control Mock cells, as well as an increase in this index by 2.4 times in G4vir cells to control values. Unlike glycolysis, we observed the opposite pattern with respect to the intensity of Krebs cycle reactions depending on the expression level of Ruk/CIN85. Conclusions. The observed reversion of the Warburg metabotype as a result of Ruk/CIN85 downregulation in MCF-7 cells overexpressing adaptor protein is a strong experimental evidence for its regulatory role in energy supply modes, aerobic glycolysis versus OXPHOS in the course cancer cells malignization.http://biotechnology.kiev.ua/images/2_2022/korshun_2_2022.pdfbreast adenocarcinoma cellsadaptor proteinruk/cin85 warburg effectaerobic glycolysisoxidative phosphorylationaldolase alactate dehydrogenase alactatenad-dependent malate dehydrogenase. |
| spellingShingle | R. S. Korshun O. O. Hudkova N. V. Latyshko T. O. Kishko I. R. Horak L. B. Drobot ADAPTOR PROTEIN Ruk/CIN85 PARTICIPATES IN THE METABOLIC CONTROL OF HUMAN BREAST ADENOCARCINOMA MCF-7 CELLS Biotechnologia Acta breast adenocarcinoma cells adaptor protein ruk/cin85 warburg effect aerobic glycolysis oxidative phosphorylation aldolase a lactate dehydrogenase a lactate nad-dependent malate dehydrogenase. |
| title | ADAPTOR PROTEIN Ruk/CIN85 PARTICIPATES IN THE METABOLIC CONTROL OF HUMAN BREAST ADENOCARCINOMA MCF-7 CELLS |
| title_full | ADAPTOR PROTEIN Ruk/CIN85 PARTICIPATES IN THE METABOLIC CONTROL OF HUMAN BREAST ADENOCARCINOMA MCF-7 CELLS |
| title_fullStr | ADAPTOR PROTEIN Ruk/CIN85 PARTICIPATES IN THE METABOLIC CONTROL OF HUMAN BREAST ADENOCARCINOMA MCF-7 CELLS |
| title_full_unstemmed | ADAPTOR PROTEIN Ruk/CIN85 PARTICIPATES IN THE METABOLIC CONTROL OF HUMAN BREAST ADENOCARCINOMA MCF-7 CELLS |
| title_short | ADAPTOR PROTEIN Ruk/CIN85 PARTICIPATES IN THE METABOLIC CONTROL OF HUMAN BREAST ADENOCARCINOMA MCF-7 CELLS |
| title_sort | adaptor protein ruk cin85 participates in the metabolic control of human breast adenocarcinoma mcf 7 cells |
| topic | breast adenocarcinoma cells adaptor protein ruk/cin85 warburg effect aerobic glycolysis oxidative phosphorylation aldolase a lactate dehydrogenase a lactate nad-dependent malate dehydrogenase. |
| url | http://biotechnology.kiev.ua/images/2_2022/korshun_2_2022.pdf |
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