title: LY294,002, a specific inhibitor of PI3K/Akt kinase pathway, antagonizes P-glycoprotein-mediated multidrug resistance.
abstract: The transmembrane transport pump P-glycoprotein (P-gp) causes the efflux of chemotherapeutic agents from cells and is an important system that secures multidrug resistance (MDR) of neoplastic cells. In the present study drug sensitive L1210 and multidrug resistant L1210/VCR mouse leukemic cell lines were used as an experimental model. We found that LY 294,002, a specific inhibitor of PI3K/Akt kinase pathway, reduced the degree of vincristine resistance in L1210/VCR cells significantly and in a concentration-dependent manner. This was accompanied by decrease in IC(50) value to vincristine from 3.195+/-0.447 to 1.898+/-0.676 micromol/l for 2 micromol/l, to 0.947+/-0.419 micromol/l for 4 micromol/l, and to 0.478+/-0.202 micromol/l for 8 micromol/l LY294,002. The IC(50) value of sensitive cells for vincristine was about 0.010 micromol/l. FACS analysis of the proportion of cells in apoptosis or necrosis by annexin-V apoptosis kit showed the following: (i) vincristine-induced apoptosis in resistant cell to a much lower extent than in sensitive cells; (ii) LY294,002 alone did not induce apoptosis or necrosis in both sensitive and resistant cells; (iii) LY294,002 applied together with vincristine significantly increased the number of apoptotic cells. Transport activity of P-gp in resistant cells was monitored using calcein/AM as substrate and was depressed by LY294,002 in a concentration dependent manner. Significant differences in calcein retention were not observed when cells were preincubated with LY294,002 at different times from 0.5 to 24h. Sensitive and resistant cells contain similar amounts of uncleaved (i.e., unactivated) caspase-3 but in latter cells the activation of caspase-3 by proteolytic cleavage was decreased. The reversal of vincristine resistance by LY294,002 was associated with marked activation of caspase-3. Western blot analysis revealed that the development of MDR phenotype in L1210/VCR cells was also associated with increased level of Bcl-2 protein. All the above findings point to the possible involvement of PI3K/Akt kinase pathway in modulation of P-gp mediated multidrug resistance in L1210/VCR mouse leukemic cell line. MDR reversal effect of LY294,002 is accompanied with this compound's influence on vincristine-induced apoptosis.
authors.affiliation: Institute for Heart Research, Slovak Academy of Sciences, Dúbravská cesta 9, PO Box 104, 840 05, Bratislava 45, Slovak Republic. usrdmiro@savba.sk
authors.forename: Miroslav
authors.initials: M
authors.lastname: Barancík
authors.forename: Vierka
authors.initials: V
authors.lastname: Bohácová
authors.forename: Ján
authors.initials: J
authors.lastname: Sedlák
authors.forename: Zdenka
authors.initials: Z
authors.lastname: Sulová
authors.forename: Albert
authors.initials: A
authors.lastname: Breier
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D002867:Chromones / Q000737:chemistry / Q000494:pharmacology*; D019008:Drug Resistance, Neoplasm; D007938:Leukemia / Q000188:drug therapy; D051379:Mice; D015394:Molecular Structure; D009025:Morpholines / Q000737:chemistry / Q000494:pharmacology*; D051058:Oncogene Protein v-akt / Q000037:antagonists & inhibitors* / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D021381:Protein Transport / Q000187:drug effects; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D014750:Vincristine / Q000494:pharmacology
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000970:Antineoplastic Agents; D053148:Caspase 3; D002867:Chromones; D009025:Morpholines; D051058:Oncogene Protein v-akt; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2; D014750:Vincristine; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one