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

https://doi.org/10.1006/bbrc.1999.0472Get rights and content

Abstract

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.

References (53)

  • A.S. Lee

    Trends Biochem. Sci.

    (1987)
  • J.A. Morris et al.

    J. Biol. Chem.

    (1997)
  • A.J. Levine

    Cell

    (1997)
  • S.L. Gerson et al.

    Biochem. Pharmacol.

    (1992)
  • S. Chatterjee et al.

    Exp. Cell Res.

    (1987)
  • S. Chatterjee et al.

    Exp. Cell Res.

    (1989)
  • M.R. Osborne et al.

    Chem. Biol. Interact.

    (1993)
  • R.S. Cole et al.

    J. Mol. Biol.

    (1976)
  • A. Rasouli-Nia et al.

    Mutat. Res.

    (1994)
  • L.L. Lukash et al.

    Mutat. Res.

    (1991)
  • D. Hunting et al.

    Biochem. Pharmacol.

    (1985)
  • S.J. Berger et al.

    Exp. Cell. Res.

    (1987)
  • A. Umar et al.

    J. Biol. Chem.

    (1994)
  • M.L. Agarwal et al.

    J. Biol. Chem.

    (1998)
  • S.K. Wooden et al.

    DNA Seq.

    (1992)
  • E. Little et al.

    Crit. Rev. Eukaryot. Gene Expr.

    (1994)
  • S. Chatterjee et al.

    Cancer Res.

    (1990)
  • S. Chatterjee et al.

    Cancer Res.

    (1994)
  • J. Shen et al.

    Proc. Natl. Acad. Sci. USA

    (1987)
  • C.S. Hughes et al.

    Cancer Res.

    (1989)
  • S. Sugawara et al.

    Cancer Res.

    (1993)
  • S. Chatterjee et al.

    Cancer Res.

    (1997)
  • J.L. Sims et al.

    Biochem.

    (1982)
  • N.A. Berger et al.

    Cancer Res.

    (1982)
  • J.C. Street et al.

    Cancer Res.

    (1997)
  • M.E. Varnes

    NCI Monogr.

    (1988)
  • Cited by (47)

    • Glucose-6-phosphate dehydrogenase deficiency increases cell adhesion molecules and activates human monocyte-endothelial cell adhesion: Protective role of L-cysteine

      2019, Archives of Biochemistry and Biophysics
      Citation Excerpt :

      BSO is an irreversible inhibitor of γ-glutamylcysteine synthetase (Ki < 100 μM), the rate-limiting enzyme for l-glutathione (GSH) synthesis, which induces oxidative stress in cells by depleting GSH [37,38]. 6-AN is a well-established non-steroidal G6PD inhibitor that inactivates the enzyme by competing with endogenous NADP+ for its binding site [39,40] (Ki = 0.46 μM [41] and IC50 = 0.5 mM [42,43]). The glucose concentration of 25 mM was similar to that used in our previous studies and in other studies [44,45].

    • Hepatitis B virus enhances cisplatin-induced hepatotoxicity via a mechanism involving suppression of glucose-regulated protein of 78 Kda

      2016, Chemico-Biological Interactions
      Citation Excerpt :

      We have also reported that Grp78 overexpression protects NIT-1 cells from apoptosis induced by streptozotocin or cytokines [13,14]. Although some reports about the positive role of Grp78 in cisplatin-induced senescence has been raised [15,16], majority of literature reports have indicated that Grp78 may play a negative role in the cytotoxicity and therapeutic effect of cisplatin in most types of cancer [10–12,17–21]. Grp78 expression is associated with cytotoxicity of cisplatin and targeting Grp78 may sensitize tumor cells to chemotherapy [17–19].

    • Subcellular targets of cisplatin cytotoxicity: An integrated view

      2012, Pharmacology and Therapeutics
      Citation Excerpt :

      ER preconditioning is achieved by pretreating cells with a sublethal concentration of an inducer of ER stress (e.g. thapsigargin, tunicamycin or dithiothreitol), which protects them from a subsequent ER insult by upregulating the UPR inhibitors Grp78 and Grp94 (Peyrou and Cribb, 2007) and by a p38-dependent process (Feng et al., 2011). In other cell systems, ER preconditioning drugs sensitize cells to other cytotoxic drugs (including cisplatin) (Belfi et al., 1999; Chatterjee et al., 1997; Chen et al., 2011; Gaddameedhi and Chatterjee, 2009; Xu et al., 2012) by a mechanism independent of Grp78, and apparently dependent on increased drug (cisplatin) accumulation (Budihardjo et al., 2000). This dual effect of ER preconditioning needs further exploration.

    View all citing articles on Scopus

    These studies were supported in part by Grants CA63200 (N.A.B.) and R29 CA65920 (S.C.) from the National Cancer Institute, Public Health Service.

    ☆☆

    Abbreviations used: AGT,O6-alkylguanine-DNA transferase; 6-AN, 6-aminonicotinamide; BCNU, 1,3-bis(2-dichloroethyl)-1-nitrosourea; CDDP, cisplatin; 2-dG, 2-deoxyglucose; ER, endoplasmic reticulum; GRP78, glucose-regulated stress protein; IC50, drug concentration that reduces colony formation or cell number by 50% relative to control; MEL, melphalan; MMR, mismatch repair; MNNG,N-methyl-N′-nitro-N-nitrosoguanidine; PARP, poly(ADP-ribose) polymerase; p(ADPR), poly(ADP-ribose); NER, nucleotide excision repair; SER, sensitizer enhancement ratio; Topo II, topoisomerase II; VP-16, etoposide.

    Nicoloff, J. A.Hoekstra, M. F.

    2

    To whom correspondence should be addressed: BRB 301A, School of Medicine, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106-4937. Fax: 216-368-1166. E-mail:[email protected].

    View full text