Increased Sensitivity of Human Colon Cancer Cells to DNA Cross-Linking Agents after GRP78 Up-Regulation

We have shown earlier that pre-treatment of V79 Chinese hamster cells with 6-aminonicotinamide (6-AN) or 2-deoxyglucose (2-dG) results in over-expression of the Mr78,000 glucose-regulated stress protein (GRP78) and the subsequent development of resistance to inhibitors of topoisomerase II. These phe...

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Bibliographic Details
Published in:Biochemical and biophysical research communications 1999-04, Vol.257 (2), p.361-368
Main Authors: Belfi, Charles A., Chatterjee, Satadal, Gosky, David M., Berger, Sosamma J., Berger, Nathan A.
Format: Article
Language:English
Subjects:
6-aminonicotinamide
6-Aminonicotinamide - pharmacology
BCNU
Blotting, Western
Carmustine - pharmacology
Carrier Proteins - genetics
Carrier Proteins - metabolism
Cell Survival - drug effects
cisplatin
Cisplatin - pharmacology
Colonic Neoplasms - genetics
Colonic Neoplasms - metabolism
Cross-Linking Reagents - pharmacology
DNA Damage - drug effects
Dose-Response Relationship, Drug
Drug Resistance, Neoplasm
glucose-regulated stress protein (GRP78)
Heat-Shock Proteins
Humans
melphalan
Melphalan - pharmacology
Molecular Chaperones - genetics
Molecular Chaperones - metabolism
NAD - analogs & derivatives
NAD - biosynthesis
NAD - metabolism
Poly Adenosine Diphosphate Ribose - metabolism
Time Factors
Tumor Cells, Cultured
Up-Regulation - drug effects
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Summary:We have shown earlier that pre-treatment of V79 Chinese hamster cells with 6-aminonicotinamide (6-AN) or 2-deoxyglucose (2-dG) results in over-expression of the Mr78,000 glucose-regulated stress protein (GRP78) and the subsequent development of resistance to inhibitors of topoisomerase II. These phenomena also occur in V79-derived cell lines that are deficient in poly(ADP-ribose) (p(ADPR)) metabolism. In contrast, over-expression of GRP78 under the conditions outlined above is found to be associated with hypersensitivity to several clinically-relevant DNA cross-linking agents, namely, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), cisplatin, and melphalan. We have also previously shown that pre-treatment with 6-AN, an inhibitor of p(ADPR) metabolism, causes an increase in the life span in BCNU-treated mice bearing L1210 tumors. These observations prompted us to examine whether 6-AN pre-treatment can result in the over-expression of GRP78 in human colon cancer cell lines and, if so, whether this increase is associated with sensitization to DNA cross-linking agents outlined above. Following treatment of three colon cancer cell lines, HCT116, SW480, and VACO-8, for 48 h with 0.1 mM 6-AN, cytosolic GRP78 levels were elevated approximately 4.2 times, 8 times, and 2.5 times for each cell line respectively, as measured by Western immunoblotting. To determine sensitivity after GRP78 up-regulation, the cells were washed and grown for 412h in growth medium devoid of 6-AN, before being treated with DNA cross-linking agents. The 412h time period allowed p(ADPR) metabolism to return to normal while GRP78 levels remained elevated, thus allowing us to associate GRP78 over-expression with sensitivity to those agents. After treating cells for 1 h with BCNU, cisplatin, or melphalan, cell sensitivity was determined by clonogenic survival assay or a fluorescence-based cytotoxicity assay. Based on changes in IC50values, 6-AN caused an increase in sensitivity for HCT116, SW480, and VACO-8 cells of 1.5, 2.3, and 1.0 times, respectively, for BCNU, 4.8, 3.8, and 2.6 for cisplatin, and 6.4, 3.7, and 2.2 times for melphalan. Thus, our results show that over-expression of GRP78 in human tumor cell lines is associated with increased sensitivity to clinically useful chemotherapy agents. This sensitization occurred in three different tumor cell lines, each bearing a separate genetic defect associated with altered sensitivity.
ISSN:0006-291X
1090-2104
DOI:10.1006/bbrc.1999.0472