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Doxorubicin and Paclitaxel Cause Different Changes In CHANGES IN PLASMA MEMBRANE FLUIDITY OF MCF-7 CELLS 135 POSTÊPY BIOLOGII KOMÓRKI TOM 36, 2009 SUPLEMENT NR 25 (135152) Rola komórek dendrytycznych w odpowiedzi transplantacyjnej* The role of dendritic cells in transplantation Maja Budziszewska1, Anna Korecka-Polak1, Gra¿yna Korczak-Kowalska1,2 1Zak³ad Immunologii, Wydzia³ Biologii, Uniwersytet Warszawski 2 Instytut Transplantologii, Warszawski Uniwersytet Medyczny *Dofinansowanie z grantu MNiSzW nr N N402 268036 Streszczenie: Komórki dendrytyczne (DC) s¹ najwa¿niejszymi komórkami prezentuj¹cymi antygenDOXORUBICIN (APC) limfocytom AND T. StopieñPACLITAXEL dojrza³oci komórki DC ma kluczowe znaczenie dla rodzaju odpowiedzi limfocytów T. Niedojrza³a komórka DC indukujeCAUSE stan DIFFERENT tolerancji, podczas CHANGES gdy dojrza³a IN komórkaPLASMA DC MEMBRANE- pe³n¹ odpowied immunologiczn¹.FLUIDITY Ma toOF ogromne MCF-7 znaczenie BREAST w transplantologii,CANCER CELLS a zw³aszcza w reakcjach odrzucania przeszczepu po transplantacjach narz¹du. Komórka DC dawcy prezentujeDOKSORUBICYNA antygen w sposób I PAKLITAKSEL bezporedni, natomiast POWODUJ¥ komórka RÓ¯NE DC ZMIANYbiorcy drog¹ poredni¹.W P£YNNOCI Komórki B£ONY DC niedojrza³ePLAZMATYCZNEJ lub o w³aciwociach KOMÓREK RAKA tolerogennych PIERSI MCF-7 mog¹ wyd³u¿yæ prze¿ycie przeszczepu allogenicznego. Takie oddzia³ywanie na funkcjê komórekKarolina MATCZAKDC, aby by³y1, Aneta one niewra¿liweKOCEVA-CHYLA na sygna³y1, Krzysztof dojrzewania GWOZDZINSKI in vivo lub2, aktywowanie komórek DC charakteryzuj¹cychZofia JÓWIAK1 siê trwa³ymi w³aciwociami tolerogennymi mo¿e poprawiæ tolerancjê przeszczepu. W tym celu wykorzystuje siê hodowle1Katedra prowadzone Termobiologii, w 2specyficznychKatedra Biofizyki warunkach, Molekularnej, leczenie Uniwersytet farmakologiczne £ódzki, £ód oraz in¿ynieriê genetyczn¹. StreszczenieS³owa :kluczowe: Metodami spektroskopiiKomórka dendrytyczna,fluorescencyjnej badanotransplantacja, oddzia³ywanie tolerancja przeciwnowotworowych przeszczepu. lekówSummary: doksorubicyny The (DOX)most importanti paklitakselu antigen-presenting (PTX) na w³aciwoci cells b³ony (APCs) plazmatycznej are dendritic komórek MCF-cells (DCs),7 gruczolakoraka which presentpiersi. Zastosowano antigen to sondyT cells. fluorescencyjne The state ofTMA-DPH maturation i DAUDA, of DCs umo¿liwiaj¹ce is crucial foroznaczenie induction p³ynnoci of a T-cellzewnêtrznych, lymphocyte polarnych response. obszarów The oraz immature rdzenia hydrofobowego DCs induce dwuwarstwytolerance, lipidowej. Stwierdzono zró¿nicowany wp³yw obu leków. DOX i PTX oddzia³ywa³y w odmienny spo- sóbthe namature powierzchniowe DCs - immunity. i na hydrofobowe This is importantregiony b³ony. in transplantology,DOX indukowa³a podobneespecially i zale¿ne in graft od stê¿eniarejection zmiany after w organ obu obszarach transplantation. dwuwarstwy Donor lipidowej. DCs Niskieact via stê¿enia the direct, leku powodowa³y while recipient up³yn- DCsnienie viab³ony, the podczas indirect gdy wzrostpathways stê¿enia of wywo³ywa³allorecognition. progresywne Immature usztywnienie DCs b³ony.or DCs PTX withnato- tolerogenicmiast niezale¿nie properties od stê¿enia may oddzia³ywa³ prolong g³ównieallograft na survival.hydrofobowy Manipulating obszar dwuwarstwy DCs powoduj¹cfunction tojej beusztywnienie. insensitive Jednakowe to maturation stê¿enia obusignals leków or w activateró¿nym stopniu DCs withzmienia³y tolerogenic p³ynnoæ dwuwarstwyproperties lipidowej. Mo¿e to wynikaæ z odmiennej interakcji PTX i DOX z komponentami b³ony. PTX wywo- ³ywa³are the g³êbsze promising zmiany means p³ynnoci of improvingb³ony i przy allograft znacznie tolerance. ni¿szych stê¿eniach There are ni¿ three DOX, approaches wykazuj¹c tojednoczenie achieve wiêksz¹these aims: cytotoksycznoæ specific wcell stosunku culture do conditions,komórek MCF-7. pharmacological Szybki, ponad 70%treatment spadek andprze¿ywalnoci genetic engineering. komórek, któremu towarzyszy³ istotny spadek p³ynnoci b³ony, obserwowano w zakresieKeywords: tych samych Dendritic stê¿eñ cell,PTX transplantation,(0,011 µM). Podobnie graft tolerance,60% spadek graft prze¿ywalnoci rejection. komórek MCF-7 oraz istotne zmiany p³ynnoci b³ony plazmatycznej jej up³ynnienie lub usztywnienie ob- serwowanoWstêp w zakresie tych samych stê¿eñ DOX (0,055 µM). Wyniki te wskazuj¹, ¿e zmiany p³ynnoci b³ony W razspowodowane z przeszczepianym przez DOX lub narz¹dem PTX zak³ócaj¹ do istotnie organizmu proliferacjê biorcy komórek dostaj¹ i sugeruj¹ siê ró¿norodnemo¿liw¹ korelacjê komórki pomiêdzy mi¹¿szowe cytotoksycznoci¹ wyposa¿one badanych lekóww antygeny i stopniem zgodnoci uszkodzenia tkankowej b³ony, które (MHC)one powoduj¹. oraz £¹czny grupa wp³yw komórek kombinacji okrelanychDOX i PTX na p³ynnoæ jako "leukocytyb³ony komórek MCF-7pasa¿erskie", by³ wysoce zale¿ny od stê¿enia i stosunku molowego leków i znacznie ró¿ni³ siê od wp³ywu ka¿dego z nich stosowanychodpowiedzialna pojedynczo. za reakcjê Nie stwierdzono odrzucania synergistycznego przeszczepu. lubS¹ toaddytywnego m.in. komórki oddzia³ywania dendrytyczne DOX i PTX(DC), na którep³ynnoæ w pewnychb³ony plazmatycznej warunkach komórek zamiast MCF-7. prowadziæ Przy niektórych do odrzucenia stê¿eniach leków przeszczepu obserwo- mog¹wano natomiast sprzyjaæ ich antagonizuj¹cejego akceptacji dzia³anie. [5,16]. Precyzyjne okrelenie roli komórek S³owadendrytycznych kluczowe: doksorubicyna, w odpowiedzi paklitaksel, transplantacyjnej p³ynnoci b³oni opracowanie plazmatycznych, metod komórki modyfikowania raka piersi, ichMCF-7. funkcji mo¿e przyczyniæ siê do poprawienia rezultatów osi¹ganych w transplantologii. 1. Charakterystyka komórek dendrytycznych Komórki dendrytyczne s¹ profesjonalnymi komórkami prezentuj¹cymi antygen (APC), obecnymi w centralnych (grasica i szpik) i we wtórnych (wêz³y limfatyczne, kêpki Peyer'a i ledziona) narz¹dach limfatycznych [2]. Posiadaj¹ zdolnoæ do 136 K. MATCZAK, A. KOCEVA-CHYLA, K. GWOZDZINSKI, Z. JÓWIAK Summary: Interaction of anticancer drugs doxorubicin (DOX) and paclitaxel (PTX) with the plasma membrane of MCF-7 human breast carcinoma cells was studied by fluorescence spectroscopy tech- nique. TMA-DPH and DAUDA fluorescent probes were employed to examine fluidity in the upper polar and in the hydrophobic core regions of the lipid bilayer. Our data showed entirely different effects of DOX and PTX on membrane fluidity of MCF-7 cancer cells. DOX and PTX penetrated differently surficial and hydrophobic parts of the cell membrane. DOX caused similar and concen- tration-dependent changes in fluidity of both areas of lipid bilayer: low drug concentrations had a fluidizing effect, an increasing rigidization effect was observed with increasing drug concentrations. PTX mainly disturbed the structure of the inner part of the cell membrane and showed rigidization effect that was independent on drug concentration. The same concentrations of DOX and PTX in- duced different extent of alterations in the lipid bilayer, which could stem from their distinct inte- ractions with the lipid components of the plasma membrane. Of both drugs PTX induced significan- tly greater changes in plasma membrane fluidity and at much lower concentrations showing at the same time considerably higher cytotoxicity towards MCF-7 cells than DOX. A rapid, over 70% decrease in cell survival with concomitant striking decrease in membrane fluidity were observed with the same concentration range of PTX (0.011 µM). 60% decrease in survival of MCF-7 cells tre- ated with 0,055 µM concentration range of DOX, was also associated with either fluidization or rigidization of the plasma membrane. These results imply that changes in membrane fluidity occur- ring in the presence of DOX and PTX noticeably disturb cellular proliferation and suggest that correlation between the cytotoxicity of investigated drugs and the extent of the damage to the cell membrane they cause might exist. Combined effect of DOX and PTX on MCF-7 membrane fluidity was highly dependent on their concentration and molar ratio and was markedly different from the effects the drugs showed alone at the same concentrations. No synergistic or additive effect of DOX and PTX on the plasma membrane properties of MCF-7 human breast carcinoma cells was obse- rved. At some of the investigated concentration ranges and molecular ratios DOX and PTX, howe- ver, showed antagonizing effect. Key words: doxorubicin, paclitaxel, plasma membrane fluidity, MCF-7 breast cancer cells. INTRODUCTION The cell membrane is a dynamic and complex interface between intracellular and external environment. It plays fundamental role in cell homeostasis and metabolism. Thus, any change in molecular architecture of the cell membrane may substantially affect its functional activities and cell functions. Many drugs have been hypothesized to exert their pharmacological effects by influencing membrane molecular organization, mostly by disordering the membrane lipids. It is commonly accepted that the plasma membrane, besides deoxyribonucleic acid (DNA), is the most important target for activity of many anticancer drugs such as anthracycline and taxane chemotherapeutics. These drugs can induce modifica- tions in physicochemical properties of the cellular membranes such as lipid fluidity, conformation of membrane-bound enzymes, degree of receptor exposure, changes in lipid packing density and changes in lipid-lipid and lipid-protein interactions [5,16,18,19,23,38,55]. Most of investigation concerning interaction of anthracyclines and taxanes with lipid bilayer was performed on model membranes. Little is known on
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