P-GLYCOPROTEIN TRANSPORT ACTIVITY UNDER REDOX BALANCE CHANGES IN LYMPHOCYTES OF PATIENTS WITH B-CHRONIC LYMPHOCYTIC LEUKEMIA
(Abstract release date: 05/19/16)
EHA Library. Harmaza Y. 06/09/16; 134682; PB1782
Dr. Yuliya Harmaza
Contributions
Contributions
Abstract
Abstract: PB1782
Type: Publication Only
Background
The experimental data about the close relationship between tumor progression and cellular redox balance are published lately. It is shown that the redox balance alterations play a key role in the cell response to the anticancer agents influence, and it is an important regulator of the protein-transporters associated with the phenomenon of multidrug resistance (MDR) expression. However, the impact of the redox imbalance in human tumor cells on the functional activity of MDR proteins has not been studied yet. Such investigations necessary for explanation of the conventional chemotherapy possible failure and their protocols modification, taking into account the features of MDR transporters functioning under the cellular redox balance changing.
Aims
To study the influence of chemotherapeutic agents on the redox status of lymphocytes of patients with B-chronic lymphocytic leukemia (CLL) and to assess the P-glycoprotein (P-gp) transport activity in these cells.
Methods
Lymphocytes were separated from the CLL patients peripheral blood by density gradient centrifugation on histopaque. Monoclonal antibody UIC2-PE (Immunotech) was used to evaluate P-gp expression and functional activity, anti-CD5-FITC (Immunotech) and anti-CD19-PC5 (Beckman Coulter) were used to determinate CD19+CD5+ B-CLL cells. Reactive oxygen species (ROS) level and low-molecular antioxidants content were assessed using СМ-H2DCFDA (Molecular Probes) and Antioxidant assay kit (Sigma), respectevly. Cells viability was assessed using Calcein-AM test and propidium iodide (Sigma). All investigations were carried out on the FACScan and FACScanto II (BD).
Results
The effect of drugs applied in the treatment of CLL (purine nucleosides - fludarabine and cladribine analogs: fludarabel (Flu), 17,5 μM and leucladine (Leu), 7,0μM (Belmeddrugs, Belarus); anthracyclines – doxorubicin (Dox), 1,72 μM (Sigma); vinca-alkaloids – vincristine (Vincr), 47,2 nM (Lens-Farm, Russia)) on the functional activity of membrane transporter P-gp associated with MDR in lymphocytes of patients with B-CLL is studied. Also the ROS level and low-molecular antioxidants content are evaluated in leukemic cells during the chemotherapeutic drugs metabolism. A degree of low-molecular antioxidants involvement in maintaining of the redox balance in lymphocytes of patients with B-CLL under exposure to drugs was determined. It is found that change of the ROS level in the leukemic B-lymphocytes comparative to values in intact cells, as well as its further recovery leads to increase of P-gp transport activity. The viability of leukemic B-cells under exposure to the antitumor drugs decreases and depends on the redox balance changes but to a lesser degree than in donor’s B-cells, as had been established previously. Nevertheless, the statistically significant relationship between the functional activity of the membrane protein P-gp and viability of lymphocytes of patients with B-CLL wasn’t detected.
Conclusion
The received results has allowed to characterize the functioning of the main MDR transporter under redox balance changing caused by the metabolism of the investigated anticancer drugs.
Session topic: E-poster
Keyword(s): ABC transporter, Chronic lymphocytic leukemia, Drug resistance, Reactive oxygen species
Type: Publication Only
Background
The experimental data about the close relationship between tumor progression and cellular redox balance are published lately. It is shown that the redox balance alterations play a key role in the cell response to the anticancer agents influence, and it is an important regulator of the protein-transporters associated with the phenomenon of multidrug resistance (MDR) expression. However, the impact of the redox imbalance in human tumor cells on the functional activity of MDR proteins has not been studied yet. Such investigations necessary for explanation of the conventional chemotherapy possible failure and their protocols modification, taking into account the features of MDR transporters functioning under the cellular redox balance changing.
Aims
To study the influence of chemotherapeutic agents on the redox status of lymphocytes of patients with B-chronic lymphocytic leukemia (CLL) and to assess the P-glycoprotein (P-gp) transport activity in these cells.
Methods
Lymphocytes were separated from the CLL patients peripheral blood by density gradient centrifugation on histopaque. Monoclonal antibody UIC2-PE (Immunotech) was used to evaluate P-gp expression and functional activity, anti-CD5-FITC (Immunotech) and anti-CD19-PC5 (Beckman Coulter) were used to determinate CD19+CD5+ B-CLL cells. Reactive oxygen species (ROS) level and low-molecular antioxidants content were assessed using СМ-H2DCFDA (Molecular Probes) and Antioxidant assay kit (Sigma), respectevly. Cells viability was assessed using Calcein-AM test and propidium iodide (Sigma). All investigations were carried out on the FACScan and FACScanto II (BD).
Results
The effect of drugs applied in the treatment of CLL (purine nucleosides - fludarabine and cladribine analogs: fludarabel (Flu), 17,5 μM and leucladine (Leu), 7,0μM (Belmeddrugs, Belarus); anthracyclines – doxorubicin (Dox), 1,72 μM (Sigma); vinca-alkaloids – vincristine (Vincr), 47,2 nM (Lens-Farm, Russia)) on the functional activity of membrane transporter P-gp associated with MDR in lymphocytes of patients with B-CLL is studied. Also the ROS level and low-molecular antioxidants content are evaluated in leukemic cells during the chemotherapeutic drugs metabolism. A degree of low-molecular antioxidants involvement in maintaining of the redox balance in lymphocytes of patients with B-CLL under exposure to drugs was determined. It is found that change of the ROS level in the leukemic B-lymphocytes comparative to values in intact cells, as well as its further recovery leads to increase of P-gp transport activity. The viability of leukemic B-cells under exposure to the antitumor drugs decreases and depends on the redox balance changes but to a lesser degree than in donor’s B-cells, as had been established previously. Nevertheless, the statistically significant relationship between the functional activity of the membrane protein P-gp and viability of lymphocytes of patients with B-CLL wasn’t detected.
Conclusion
The received results has allowed to characterize the functioning of the main MDR transporter under redox balance changing caused by the metabolism of the investigated anticancer drugs.
Session topic: E-poster
Keyword(s): ABC transporter, Chronic lymphocytic leukemia, Drug resistance, Reactive oxygen species
Abstract: PB1782
Type: Publication Only
Background
The experimental data about the close relationship between tumor progression and cellular redox balance are published lately. It is shown that the redox balance alterations play a key role in the cell response to the anticancer agents influence, and it is an important regulator of the protein-transporters associated with the phenomenon of multidrug resistance (MDR) expression. However, the impact of the redox imbalance in human tumor cells on the functional activity of MDR proteins has not been studied yet. Such investigations necessary for explanation of the conventional chemotherapy possible failure and their protocols modification, taking into account the features of MDR transporters functioning under the cellular redox balance changing.
Aims
To study the influence of chemotherapeutic agents on the redox status of lymphocytes of patients with B-chronic lymphocytic leukemia (CLL) and to assess the P-glycoprotein (P-gp) transport activity in these cells.
Methods
Lymphocytes were separated from the CLL patients peripheral blood by density gradient centrifugation on histopaque. Monoclonal antibody UIC2-PE (Immunotech) was used to evaluate P-gp expression and functional activity, anti-CD5-FITC (Immunotech) and anti-CD19-PC5 (Beckman Coulter) were used to determinate CD19+CD5+ B-CLL cells. Reactive oxygen species (ROS) level and low-molecular antioxidants content were assessed using СМ-H2DCFDA (Molecular Probes) and Antioxidant assay kit (Sigma), respectevly. Cells viability was assessed using Calcein-AM test and propidium iodide (Sigma). All investigations were carried out on the FACScan and FACScanto II (BD).
Results
The effect of drugs applied in the treatment of CLL (purine nucleosides - fludarabine and cladribine analogs: fludarabel (Flu), 17,5 μM and leucladine (Leu), 7,0μM (Belmeddrugs, Belarus); anthracyclines – doxorubicin (Dox), 1,72 μM (Sigma); vinca-alkaloids – vincristine (Vincr), 47,2 nM (Lens-Farm, Russia)) on the functional activity of membrane transporter P-gp associated with MDR in lymphocytes of patients with B-CLL is studied. Also the ROS level and low-molecular antioxidants content are evaluated in leukemic cells during the chemotherapeutic drugs metabolism. A degree of low-molecular antioxidants involvement in maintaining of the redox balance in lymphocytes of patients with B-CLL under exposure to drugs was determined. It is found that change of the ROS level in the leukemic B-lymphocytes comparative to values in intact cells, as well as its further recovery leads to increase of P-gp transport activity. The viability of leukemic B-cells under exposure to the antitumor drugs decreases and depends on the redox balance changes but to a lesser degree than in donor’s B-cells, as had been established previously. Nevertheless, the statistically significant relationship between the functional activity of the membrane protein P-gp and viability of lymphocytes of patients with B-CLL wasn’t detected.
Conclusion
The received results has allowed to characterize the functioning of the main MDR transporter under redox balance changing caused by the metabolism of the investigated anticancer drugs.
Session topic: E-poster
Keyword(s): ABC transporter, Chronic lymphocytic leukemia, Drug resistance, Reactive oxygen species
Type: Publication Only
Background
The experimental data about the close relationship between tumor progression and cellular redox balance are published lately. It is shown that the redox balance alterations play a key role in the cell response to the anticancer agents influence, and it is an important regulator of the protein-transporters associated with the phenomenon of multidrug resistance (MDR) expression. However, the impact of the redox imbalance in human tumor cells on the functional activity of MDR proteins has not been studied yet. Such investigations necessary for explanation of the conventional chemotherapy possible failure and their protocols modification, taking into account the features of MDR transporters functioning under the cellular redox balance changing.
Aims
To study the influence of chemotherapeutic agents on the redox status of lymphocytes of patients with B-chronic lymphocytic leukemia (CLL) and to assess the P-glycoprotein (P-gp) transport activity in these cells.
Methods
Lymphocytes were separated from the CLL patients peripheral blood by density gradient centrifugation on histopaque. Monoclonal antibody UIC2-PE (Immunotech) was used to evaluate P-gp expression and functional activity, anti-CD5-FITC (Immunotech) and anti-CD19-PC5 (Beckman Coulter) were used to determinate CD19+CD5+ B-CLL cells. Reactive oxygen species (ROS) level and low-molecular antioxidants content were assessed using СМ-H2DCFDA (Molecular Probes) and Antioxidant assay kit (Sigma), respectevly. Cells viability was assessed using Calcein-AM test and propidium iodide (Sigma). All investigations were carried out on the FACScan and FACScanto II (BD).
Results
The effect of drugs applied in the treatment of CLL (purine nucleosides - fludarabine and cladribine analogs: fludarabel (Flu), 17,5 μM and leucladine (Leu), 7,0μM (Belmeddrugs, Belarus); anthracyclines – doxorubicin (Dox), 1,72 μM (Sigma); vinca-alkaloids – vincristine (Vincr), 47,2 nM (Lens-Farm, Russia)) on the functional activity of membrane transporter P-gp associated with MDR in lymphocytes of patients with B-CLL is studied. Also the ROS level and low-molecular antioxidants content are evaluated in leukemic cells during the chemotherapeutic drugs metabolism. A degree of low-molecular antioxidants involvement in maintaining of the redox balance in lymphocytes of patients with B-CLL under exposure to drugs was determined. It is found that change of the ROS level in the leukemic B-lymphocytes comparative to values in intact cells, as well as its further recovery leads to increase of P-gp transport activity. The viability of leukemic B-cells under exposure to the antitumor drugs decreases and depends on the redox balance changes but to a lesser degree than in donor’s B-cells, as had been established previously. Nevertheless, the statistically significant relationship between the functional activity of the membrane protein P-gp and viability of lymphocytes of patients with B-CLL wasn’t detected.
Conclusion
The received results has allowed to characterize the functioning of the main MDR transporter under redox balance changing caused by the metabolism of the investigated anticancer drugs.
Session topic: E-poster
Keyword(s): ABC transporter, Chronic lymphocytic leukemia, Drug resistance, Reactive oxygen species
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