Báo cáo khoa học: Perturbation of membrane microdomains in GLC4 multidrug-resistant lung cancer cells ) modification of ABCC1 (MRP1) localization and functionality

Perturbation of Membrane Microdomains in GLC4 Multidrug-Resistant Lung Cancer Cells – Modification of ABCC1 (MRP1) Localization and Functionality

Tác giả: Carole Marbeuf-Gueye, Vérene Stierle, Paiwan Sudwan, Milena Salerno and Arlette Garnier-Suillerot

Lĩnh vực: Laboratoire Biophysique Moléculaire, Cellulaire et Tissulaire (BioMoCeTi), UMR CNRS 7033, Université Paris 13 et Paris 6, Bobigny, France

Nội dung tài liệu: Nghiên cứu này khám phá mối liên hệ giữa cholesterol trong màng tế bào và hoạt động của protein đa kháng thuốc ABCC1 (MRP1) trong các tế bào ung thư phổi kháng đa thuốc GLC4/ADR. Sử dụng phương pháp tách màng không dùng dung môi, các nhà nghiên cứu đã xác định rằng MRP1 tập trung chủ yếu trong các vi miền màng “nhẹ” (light membrane fractions), được biết đến là các vi miền màng pha Lo. Việc giảm hàm lượng cholesterol xuống 40% không ảnh hưởng đến hoạt động của MRP1. Tuy nhiên, khi cholesterol tiếp tục bị suy giảm, MRP1 có xu hướng dịch chuyển sang các phân đoạn màng có mật độ cao hơn và chức năng của nó bị suy giảm. Kết quả cho thấy chức năng của MRP1 phụ thuộc vào vị trí của nó trong các vi miền màng giàu cholesterol.

Mục lục chi tiết:

• Keywords
• Correspondence
• The multidrug resistance-associated protein transporter ABCC1 (MRP1) is an integral plasma membrane protein involved in the multidrug resistance phenotype.
• A major obstacle to the success of chemotherapy is multidrug resistance (MDR) [1].
• Abbreviations
• The well-documented membrane protein sensitivity to a lipid environment has led to the hypothesis of functional cross-talk between membrane proteins and membrane microdomains such as rafts and caveolae.
• Nonionic detergents such as Triton X-100, Brij 96 or 98, Tween-20 or Chaps octylglucoside.
• Recent studies of the colocalization of ABC transporters such as P-gp in raft or caveolae microdomains have been interpreted in different ways [11].
• The aim of this work was to determine the localization of MRP1 in the plasma membrane and to determine whether the modification of its localization would modify its functional properties.
• Results
• The GLC4 small lung cancer cell line, and its multidrug-resistant counterpart GLC4/ADR, were used to study the influence of cholesterol on the activity of MRP1 in its membrane microenvironment.
• Effect of MẞCD on cellular cholesterol content
• Effect of cholesterol depletion on the rate of MRP1-mediated efflux of PIRA
• Effect of cholesterol depletion on the rate of MRP1-mediated efflux of GSH
• Effect of Triton derivatives on the MRP1-mediated efflux of PIRA
• MRP1, GM1 and caveolin-1 distribution within membrane fractions
• Discussion
• Membrane lipids do not form a homogeneous phase consisting of glycerophospholipids and cholesterol, but a mosaic of domains with unique biochemical compositions.
• Caveolae have recently been shown to be involved in MDR.
• These conflicting data can be explained by the use of different methods to isolate rafts and/or caveolae.
• However, nondetergent methods that do not involve the solubilization properties of membranes can be used to isolate rafts.
• However, very few studies have been done with MRP1 [12-14].
• Caveolin-1, the marker of caveolin, and GM1, the marker of rafts, are also found in microdomain fraction 4.
• In a first set of experiments, in order to determine whether cholesterol affects MRP1 function, we used MBCD to extract cholesterol from the lipid phase of intact living cells.
• At this stage, it is interesting to compare the present data with those that we have previously obtained with K562/ADR cells overexpressing P-gp.
• Now let us compare the impact of the membrane fluidity on P-gp and MRP1 transport activity.
• In summary, our results show that MRP1 is localized in microdomains and that its functionality, measured as its ability to pump out PIRA, is conserved when it is localized in rafts only.
• Experimental procedures
• Cell lines and culture
• Drugs and chemicals
• Determination of the nonesterified cholesterol content of GLC4 cells
• Treatment of cells with MBCD
• Cellular anthracycline accumulation
• Determination of the MRP1-mediated efflux of PIRA
• GSH measurements
• Isolation of ‘light’ and ‘heavy’ membrane fractions
• Western blotting measurement of MRP1 and caveolin-1 expression
• Localization of GM1 ganglioside
• Acknowledgements
• References