Apoptosis by gemcitabine in non-small cell lung cancer cell line KNS62 is induced downstream of caspase 8 and is profoundly blocked by Bcl-xL over-expression.
BACKGROUND
This study assesses the chemotherapeutic drug gemcitabine in the human non-small cell lung cancer (NSCLC) cell line KNS62 in relation to the CD95-induced apoptotic pathway, and the role of the anti-apoptotic protein Bcl-xL in vitro and in vivo.


METHODS
Apoptosis was determined by JAM assay and DAPI staining analysis. Activation of key apoptotic proteins, including caspases 3, 8 and 9 and BID, as well as cytochrome c release and mitochondrial transmembrane potential (MTP), were measured. The impact of the caspase inhibitor zVAD on gemcitabine-induced apoptosis was quantified. The in vitro results were verified in vivo in an orthotopic murine xenotransplantation model.


RESULTS
Gemcitabine treatment, as well as stimulation of CD95, resulted in cleavage of effector caspase 3 as well as its substrate PARP and caspase 9, followed by DNA fragmentation. Cleavage of caspase 8 was demonstrated after CD95 activation but not after the application of gemcitabine. In KNS62-Bcl-xL clones, release of cytochrome c and loss of mitochondrial transmembrane potential were suppressed. Consequently, apoptosis after gemcitabine therapy, as well as CD95-induced apoptosis, were significantly inhibited. Caspase inhibitor zVAD only partly reversed gemcitabine-induced DNA fragmentation. In vivo, there was a significant reduction in tumour volume under gemcitabine therapy. Bcl-xL over-expressing tumours were completely resistant to gemcitabine therapy.


CONCLUSIONS
In NSCLC cell line KNS62 gemcitabine activated the mitochondrial apoptotic pathway downstream of mitochondria without activation of initiator caspases. Bcl-xL over-expression induced significant resistance to gemcitabine. In vivo, the anti-apoptotic effect of Bcl-xL was more pronounced than in vitro. Gemcitabine also induced caspase-independent DNA fragmentation in KNS62 cells.
Kurdow
R
R
Department of General Surgery and Thoracic Surgery, Molecular Oncology Research Group, University of Schleswig-Holstein, Kiel Campus, Arnold-Heller-Strasse 7, 24105, Kiel, Germany. rkurdow@surgery.uni-kiel.de
Schniewind
B
B
Zoefelt
S
S
Boenicke
L
L
Boehle
A-S
AS
Dohrmann
P
P
Kalthoff
H
H
D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology* / Q000627:therapeutic use; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy*; D053181:Caspase 8; D020169:Caspases / Q000378:metabolism*; D045744:Cell Line, Tumor; D053938:DNA Fragmentation; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000627:therapeutic use; D005260:Female; D006801:Humans; D015151:Immunoblotting; D008175:Lung Neoplasms / Q000188:drug therapy*; D051379:Mice; D016513:Mice, SCID; D014183:Transplantation, Heterologous; D051020:bcl-X Protein / Q000378:metabolism*; D019014:fas Receptor / Q000378:metabolism; D000093542:Gemcitabine
D000964:Antimetabolites, Antineoplastic; C494811:BCL2L1 protein, human; D051020:bcl-X Protein; D019014:fas Receptor; D003841:Deoxycytidine; C505530:CASP8 protein, human; C505526:Casp8 protein, mouse; D053181:Caspase 8; D020169:Caspases; D000093542:Gemcitabine
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