Inhibition of SRC tyrosine kinase impairs inherent and acquired gemcitabine resistance in human pancreatic adenocarcinoma cells.
OBJECTIVE
We tested the hypotheses that Src tyrosine kinase overactivity represents a chemoresistance mechanism and that Src inhibition may enhance gemcitabine cytotoxicity in pancreatic adenocarcinoma cells.


METHODS
Pancreatic adenocarcinoma cells PANC1, MiaPaCa2, Capan2, BxPC3, and PANC1(GemRes), a stably gemcitabine-resistant subline of PANC1, were exposed to combinations of gemcitabine and Src tyrosine kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Src expression, phosphorylation (Tyr-416), and activity were analyzed by immunoblotting and in vitro kinase assay. Expression of the M2 subunit of ribonucleotide reductase (RRM2), a putative chemoresistance enzyme, was quantified by Northern and Western blot. Cellular proliferation was quantified by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis was characterized by YO-PRO-1/propidium iodide staining, fluorometric caspase profiling, and caspase inhibition (Z-Val-Ala-Asp-fluoromethyl ketone). The effects of constitutively active and dominant negative Src were determined. The therapeutic efficacy of PP2 in combination with gemcitabine was tested in nude mice orthotopically xenografted with PANC1(GemRes).


RESULTS
Greater gemcitabine resistance was associated with higher Src phosphorylation and activity, both of which were higher in PANC1(GemRes), relative to PANC1; total Src levels were alike. PANC1(GemRes) overexpressed RRM2. PP2 enhanced inherent gemcitabine chemosensitivity and attenuated gemcitabine resistance in PANC1(GemRes). Constitutively active Src increased gemcitabine chemoresistance; dominant negative Src impaired gemcitabine chemoresistance. PP2 augmented gemcitabine-induced caspase-mediated apoptosis, suppressed RRM2 expression, and decreased activity of the RRM2-regulating transcription factor E2F1 in PANC1(GemRes). PP2 and gemcitabine in combination substantially decreased tumor growth and inhibited metastasis in vivo.


CONCLUSIONS
Increased Src tyrosine kinase activity represents a potential chemoresistance mechanism and a promising therapeutic target warranting further investigation in gemcitabine-resistant pancreatic adenocarcinoma.
Duxbury
Mark S
MS
Department of Surgery, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
Ito
Hiromichi
H
Zinner
Michael J
MJ
Ashley
Stanley W
SW
Whang
Edward E
EE
D000230:Adenocarcinoma / Q000201:enzymology* / Q000378:metabolism / Q000473:pathology; D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis; D015152:Blotting, Northern; D015153:Blotting, Western; D020169:Caspases / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism; D002455:Cell Division; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D019008:Drug Resistance, Neoplasm; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D005799:Genes, Dominant; D006801:Humans; D020128:Inhibitory Concentration 50; D051379:Mice; D008819:Mice, Nude; D009362:Neoplasm Metastasis; D009368:Neoplasm Transplantation; D010190:Pancreatic Neoplasms / Q000201:enzymology*; D010766:Phosphorylation; D011743:Pyrimidines / Q000494:pharmacology; D012262:Ribonucleoside Diphosphate Reductase / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D019061:src-Family Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000093542:Gemcitabine
C412373:AG 1879; D000964:Antimetabolites, Antineoplastic; D000970:Antineoplastic Agents; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; C493754:E2f1 protein, mouse; D011743:Pyrimidines; D014157:Transcription Factors; D003841:Deoxycytidine; D014443:Tyrosine; C093713:ribonucleotide reductase M2; D012262:Ribonucleoside Diphosphate Reductase; D019061:src-Family Kinases; D020169:Caspases; D000093542:Gemcitabine