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Loss of sphingosine kinase‐1 in carcinoma cells increases formation of reactive oxygen species and sensitivity to doxorubicin‐induced DNA damage
Author(s) -
Huwiler Andrea,
Kotelevets Nataliya,
Xin Cuiyan,
Pastukhov Oleksandr,
Pfeilschifter Josef,
ZangemeisterWittke Uwe
Publication year - 2011
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/j.1476-5381.2010.01053.x
Subject(s) - protein kinase b , dna damage , reactive oxygen species , apoptosis , microbiology and biotechnology , sphingosine kinase , doxorubicin , viability assay , biology , annexin , ceramide , oxidative stress , cell growth , chemistry , cancer research , sphingosine , biochemistry , sphingosine 1 phosphate , dna , genetics , receptor , chemotherapy
BACKGROUND AND PURPOSE Sphingosine kinases (SK) catalyse the formation of sphingosine 1‐phosphate, which is a key lipid mediator regulating cell responses such as proliferation, survival and migration. Here we have investigated the effect of targeted inhibition of SK‐1 on cell damage and elucidated the mechanisms involved. EXPERIMENTAL APPROACH Three human carcinoma cell lines (colon HCT‐116, breast MDA‐MB‐231, lung NCI‐H358) were used, which were either transduced with shRNA constructs to deplete SK‐1, or treated with a SK‐1 inhibitor. Cell growth and viability were assayed by [ 3 H]thymidine incorporation and colony formation. Reactive oxygen species (ROS) were measured by fluorescence and apoptosis by annexin V with flow cytometry. Proteins were analysed by Western blotting. DNA damage was induced by doxorubicin. KEY RESULTS Knock‐down of SK‐1 by shRNA strongly inhibited DNA synthesis and colony formation of carcinoma cells. SK‐1 knock‐down (SK‐1kd) cells revealed dysfunctional extracellular signal‐regulated protein kinase and PKB/Akt cascades, and contained increased levels of ROS. After SK‐1kd, treatment with doxorubicin increased DNA damage, measured by histone‐2AX phosphorylation. Similar effects were found in cells with a SK‐1 inhibitor and doxorubicin. The increased damage response in SK‐1kd cells was accompanied by greater reduction of DNA synthesis and colony formation, and by more pronounced apoptosis. Addition of a NADPH oxidase inhibitor reduced the increased apoptosis in doxorubicin‐treated SK‐1kd cells. CONCLUSIONS AND IMPLICATIONS SK‐1kd in carcinoma cells triggered oxidative stress by increasing intracellular Ros production. Targeted inhibition of SK‐1 represents a promising approach to sensitize cells to DNA damage and facilitate apoptosis upon doxorubicin treatment.