Abstract #871: Temozolomide and chloroquine trigger glioblastoma cell death via enhanced induction of the apoptotic arm of the endoplasmic reticulum stress response
Authors: Encouse Golden, Stan Louie, Axel Schönthal, Thomas Chen
Journal: Cancer Research (2009)

Introduction: Although temozolomide (TMZ) in combination with the antimalarial agent chloroquine (CQ) is currently being investigatedas a chemotherapeutic regimen for glioblastoma multiforme, the combined pharmacologic interaction of TMZ and CQ is not well understood.1 We previously demonstrated that GRP78 antagonized the induction of the ER stress proapoptotic protein CHOP/GADD-153, where the end result was reduced TMZ-mediated cytotoxicity.2 We hypothesized that GRP78 linked autophagy may be a critical cytoprotective mechanism that acts to alleviate ER stress by targeting unfolded ER proteins to the lysosome for degradation.Furthermore, this cytoprotective effect of GRP78 may be opposed by CQ, which inhibits autophagy by blocking lysosome targeted protein degradation. Thus, we investigated whether CQ in combination with TMZ can aggravate cell death in glioma cells by blocking the cytoprotective effect of GRP78, while enhancing the induction of CHOP/GADD-153. Methods: Cytotoxicity was determined by colony formation assay (CFA). GRP78 was down regulated using siRNA. Relative amounts of protein were determined via Western blot analysis with specific antibodies to GRP78, LC3B-II, and CHOP/GADD-153. CHOP/GADD-153 induction in vivo was determined by immunohistochemical (IHC) analysis of glioma tissue sections obtained from subcutaneous U87 xenografts. Results: CFA using U251 and LN229 glioma cells treated with either TMZ or CQ alone resulted in 40% cell death; whereas, combination therapy lead to 90% cell death. This synergistic cytotoxicity was also observed in TMZ resistant cell lines. GRP78 over expression protected glioma cells from TMZ treatment, whereas GRP78 down regulation enhanced TMZ-mediated cytotoxicity. However, GRP78 was necessary for CQ induced cytotoxicity, as GRP78 down regulation reduced CQ\#8217;s antitumor properties, including the induction of LC3B-II (an autophagosome marker) and PARP cleavage. Enhancement of CHOP/GADD-153 induction was observed in vitro on Western blot analysis and in vivo in IHC xenograft analysis with the combination therapy, compared to either CQ or TMZ alone. Conclusion: GRP78 alleviates ER stress via autophagy by targeting unfolded ER proteins to the lysosome for degradation. CQ not only inhibits autophagy by blocking lysosome targeted protein degradation, but when combined with TMZ, it augments glioma cell death by enhanced induction of the ER stress proapoptotic protein CHOP/GADD-153. References 1. Hydroxychloroquine, radiation therapy, and temozolomide in treating patients with newly diagnosed glioblastoma multiforme. National Cancer Institute. ClinicalTrials.gov identifier: NCT00486603. (2008). 2. Pyrko, P., et al. The unfolded protein response regulator GRP78/BiP as a novel target for increasing chemosensitivity in malignant gliomas. Cancer Res 67, 9809-16 (2007).  Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 871.