Document Detail


Loss of sphingosine kinase-1 in carcinoma cells increases formation of reactive oxygen species and sensitivity to doxorubicin-induced DNA damage.
MedLine Citation:
PMID:  20883472     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Andrea Huwiler; Nataliya Kotelevets; Cuiyan Xin; Oleksandr Pastukhov; Josef Pfeilschifter; Uwe Zangemeister-Wittke
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  British journal of pharmacology     Volume:  162     ISSN:  1476-5381     ISO Abbreviation:  Br. J. Pharmacol.     Publication Date:  2011 Jan 
Date Detail:
Created Date:  2010-12-15     Completed Date:  2011-06-29     Revised Date:  2013-07-03    
Medline Journal Info:
Nlm Unique ID:  7502536     Medline TA:  Br J Pharmacol     Country:  England    
Other Details:
Languages:  eng     Pagination:  532-43     Citation Subset:  IM    
Copyright Information:
© 2010 The Authors. British Journal of Pharmacology © 2010 The British Pharmacological Society.
Affiliation:
Institute of Pharmacology, University of Bern, Switzerland. huwiler@pki.unibe.ch
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MeSH Terms
Descriptor/Qualifier:
Antibiotics, Antineoplastic / pharmacology*
Apoptosis / drug effects
Cell Line, Tumor
Cell Movement / drug effects
Cell Proliferation / drug effects
DNA Damage / drug effects*
Down-Regulation
Doxorubicin / pharmacology*
Female
HCT116 Cells
Humans
Molecular Targeted Therapy
Phosphotransferases (Alcohol Group Acceptor) / antagonists & inhibitors*,  genetics,  metabolism
RNA, Small Interfering / genetics
Reactive Oxygen Species / metabolism*
Chemical
Reg. No./Substance:
0/Antibiotics, Antineoplastic; 0/RNA, Small Interfering; 0/Reactive Oxygen Species; 23214-92-8/Doxorubicin; EC 2.7.1.-/Phosphotransferases (Alcohol Group Acceptor); EC 2.7.1.-/sphingosine kinase
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From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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