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 Mr 78,000 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 412 h in growth medium devoid of 6-AN, before being treated with DNA cross-linking agents. The 412 h 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 IC50 values, 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.
Belfi
C A
CA
Division of Hematology/Oncology, Case Western Reserve University and University Hospitals of Cleveland, Cleveland, Ohio, 44106-4937, USA.
Chatterjee
S
S
Gosky
D M
DM
Berger
S J
SJ
Berger
N A
NA
D015120:6-Aminonicotinamide / Q000494:pharmacology*; D015153:Blotting, Western; D002330:Carmustine / Q000494:pharmacology; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D002945:Cisplatin / Q000494:pharmacology; D003110:Colonic Neoplasms / Q000235:genetics* / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004249:DNA Damage / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D019008:Drug Resistance, Neoplasm; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D006801:Humans; D008558:Melphalan / Q000494:pharmacology; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D009243:NAD / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000378:metabolism; D011064:Poly Adenosine Diphosphate Ribose / Q000378:metabolism; D013997:Time Factors; D014407:Tumor Cells, Cultured; D015854:Up-Regulation / Q000187:drug effects*
D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; C000717248:Hspa5 protein, mouse; D018832:Molecular Chaperones; D009243:NAD; D011064:Poly Adenosine Diphosphate Ribose; D015120:6-Aminonicotinamide; D002945:Cisplatin; D008558:Melphalan; D002330:Carmustine