Cis-hydroxyproline-induced inhibition of pancreatic cancer cell growth is mediated by endoplasmic reticulum stress.
OBJECTIVE
To investigate the biological effects of cis-hydroxyproline (CHP) on the rat pancreatic carcinoma cell line DSL6A, and to examine the underlying molecular mechanisms.


METHODS
The effect of CHP on DSL6A cell proliferation was assessed by using BrdU incorporation. The expression of focal adhesion kinase (FAK) was characterized by Western blotting and immunofluorescence. Induction of endoplasmic reticulum (ER) stress was investigated by using RT-PCR and Western blotting for the glucose-related protein-78 (GRP78) and growth arrest and DNA inducible gene (GADD153). Cell viability was determined through measuring the metabolic activity based on the reduction potential of DSL6A cells. Apoptosis was analyzed by detection of caspase-3 activation and cleavage of poly(ADP-ribose) polymerase (PARP) as well as DNA laddering.


RESULTS
In addition to inhibition of proliferation, incubation with CHP induced proteolytic cleavage of FAK and a delocalisation of the enzyme from focal adhesions, followed by a loss of cell adherence. Simultaneously, we could show an increased expression of GRP78 and GADD153, indicating a CHP-mediated activation of the ER stress cascade in the DSL6A cell line. Prolonged incubation of DSL6A cells with CHP finally resulted in apoptotic cell death. Beside L-proline, the inhibition of intracellular proteolysis by addition of a broad spectrum protease inhibitor could abolish the effects of CHP on cellular functions and the molecular processes. In contrast, impeding the activity of apoptosis-executing caspases had no influence on CHP-mediated cell damage.


CONCLUSIONS
Our data suggest that the initiation of ER stress machinery by CHP leads to an activation of intracellular proteolytic processes, including caspase-independent FAK degradation, resulting in damaging pancreatic carcinoma cells.
Department of Medicine, Division of Gastroenterology, University of Rostock, Ernst-Heydemann-Str. 6, D-18057 Rostock, Germany.
Christoph
C
Mueller
Joerg
J
Emmrich
Robert
R
Jaster
Dagmar
D
Braun
Stefan
S
Liebe
Gisela
G
Sparmann
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D020169:Caspases / Q000502:physiology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D004721:Endoplasmic Reticulum / Q000187:drug effects* / Q000502:physiology; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000032:analysis / Q000235:genetics; D006360:Heat-Shock Proteins / Q000032:analysis / Q000235:genetics; D006909:Hydroxyproline / Q000494:pharmacology*; D018832:Molecular Chaperones / Q000032:analysis / Q000235:genetics; D010190:Pancreatic Neoplasms / Q000473:pathology* / Q000503:physiopathology; D010447:Peptide Hydrolases / Q000502:physiology; D051381:Rats; D051742:Transcription Factor CHOP / Q000032:analysis / Q000235:genetics; D000091342:Endoplasmic Reticulum Chaperone BiP
D020169:Caspases; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006360:Heat-Shock Proteins; D006909:Hydroxyproline; D018832:Molecular Chaperones; D010447:Peptide Hydrolases; D051742:Transcription Factor CHOP; C497251:Ddit3 protein, rat; C051684:GRP78 protein, rat; D000091342:Endoplasmic Reticulum Chaperone BiP