Abstract
Focal adhesion kinase (FAK) is up-regulated in a variety of cancers, including breast cancer, in association with poor disease prognosis. In the present study, we examined the role of FAK in the control of anticancer drug-induced apoptosis of mammary adenocarcinoma MTLn3 cells. Doxorubicin caused the formation of well defined focal adhesions and stress fibers early after treatment, which was later followed by their loss in association with the onset of apoptosis. Phosphorylation of FAK on tyrosine 397 decreased only during the onset of doxorubicin-induced apoptosis in a Bcl-2 and caspase-independent manner. Doxorubicin also caused an early activation of protein kinase B (PKB). Expression of the dominant-negative acting focal adhesion kinase-related nonkinase (FRNK) sensitized MTLn3 cells to apoptosis caused by doxorubicin. FRNK inhibited the doxorubicin-induced activation of PKB. In addition, inhibition of phosphatidylinositide-3 (PI-3) kinase with wortmannin inhibited the activation of PKB by doxorubicin. Both wortmannin and transient overexpression of the dual lipid/protein phosphatase and tensin homolog deleted on chromosome 10 enhanced doxorubicin-induced cell death. Altogether, these data fit with a model wherein FAK is involved in the doxorubicin-induced activation of the PI-3 kinase/PKB signaling route, thereby suppressing the onset of apoptosis caused by doxorubicin.
Footnotes
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This work was supported by a fellowship from the Royal Netherlands Academy for Arts and Sciences (to B.v.d.W.) and by grant RUL 2001-2477 from the Dutch Cancer Society (to B.v.d.W.).
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M.J.v.N. and M.H. contributed equally to this work.
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ABBREVIATIONS: ECM, extracellular matrix; AV, Annexin V; CLSM, confocal laser scanning microscopy; FAK, focal adhesion kinase; FRNK, focal adhesion kinase-related nonkinase; GFP, green fluorescent protein; eGFP, enhanced green fluorescent protein; Neo, neomycin-resistant cells; PI, propidium iodide; PI-3, phosphatidylinositide-3; PI-3K, phosphatidyl inositol 3-kinase; PY, phosphotyrosine; zVAD-fmk, benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone; PKB, protein kinase B; α-MEM, α-modified minimal essential medium with ribonucleosides and deoxyribonucleosides; FBS, fetal bovine serum; APC, allophycocyanin; PBS, phosphate-buffered saline; RT, room temperature; PKCδ, protein kinase Cδ; TBP, bovine serum albumin and Triton X-100 in phosphate-buffered saline; HA, hemagglutinin; PIP3, phosphatidylinositol 3,4,5-triphosphate; ROS, reactive oxygen species; PTEN, phosphatase and tensin homolog deleted on chromosome 10; TSE+, Tris-HCl, sucrose, and EGTA containing dithiothreitol, leupeptin, aprotinin, sodium vanadate, sodium fluoride, and phenylmethylsulfonyl fluoride; FAT, fatty adhesion targeting; Y27632, N-(4-pyridyl)-4-(1-aminoethyl)cyclohexanecarboxamide dihydrochloride.
- Received May 3, 2006.
- Accepted July 6, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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