Calcium-activated endoplasmic reticulum stress as a major component of tumor cell death induced by 2,5-dimethyl-celecoxib, a non-coxib analogue of celecoxib

ER stress indicators are induced similarly by DMC and thapsigargin. U251 glioblastoma cells were cultured in the presence of 1 μmol/L thapsigargin or 60 μmol/L DMC for various times as indicated. Total cell lysates were prepared and analyzed by Western blot with specific antibodies to the ER stress proteins GRP78, CHOP, and caspase-4 (Casp 4). Actin was used as a loading control. Pro-caspase-4 denotes the inactive caspase-4 proenzyme, whereas cleaved caspase-4 is indicative of the activated form of this enzyme. *, faster-migrating band that is inconsistently observed in these Western blots.

DMC and celecoxib transiently inhibit protein synthesis. U251 cells were treated with different concentrations of DMC, celecoxib, or thapsigargin (Tg) for the times indicated. As controls, cells remained untreated (Ctr) or were exposed to the potent protein synthesis inhibitor cycloheximide (Ch). During the final 30 min of each treatment condition, the culture medium was replaced with methionine-free growth medium supplemented with ³⁵S-methionine (20 μCi/mL) in the continued presence of the respective drug. Then, total cellular lysates were prepared and equal amounts of each sample (50 μL) were separated by PAGE. Top, an autoradiograph of the gel; bottom, the same gel stained with Coomassie blue. An aliquot from each sample was used to determine protein concentration, as well as the amount of incorporated ³⁵S-methionine; the resulting magnitude of incorporated radioactivity per milligram of total protein is shown as counts per minute (% cpm), where the value from non-drug–treated cells (Ctr) was set to 100%. Note that the incorporation of ³⁵S-methionine in the control lanes at 8 and 18 h (0 μmol/L drug) seems disproportionately stronger: because these cells continued to proliferate (as opposed to drug-treated cells, whose growth was slowed or inhibited), overall more protein was present in the respective 50 μL aliquot that was loaded onto the gel. Arrow, prominently ³⁵S-labeled protein of 78 kDa, which was identified as GRP78 by immunoprecipitation analysis (data not shown). Modified repetitions of this experiment yielded essentially the same results.

DMC and celecoxib, but not other coxibs or NSAIDs, induce calcium release into the cytoplasm. A, U251 cells were treated with DMC or various coxibs and NSAIDs, and changes in intracellular calcium levels were recorded as described in Materials and Methods. Top and middle, the typical spikes of calcium increase that were consistently observed in response to DMC or celecoxib treatment; bottom, the typical response (i.e., lack thereof) to valdecoxib, rofecoxib, flurbiprofen, indomethacin, and sulindac (only shown for valdecoxib). Arrows, time point of drug addition. B, chart shows the average maximum calcium increase in response to treatment with the various drugs. Columns, mean for DMC and celecoxib (n = 5) and for each of the other drugs (n = 3); bars, SD. Essentially similar results were also obtained with the LN229 cell line.

Induction of CHOP and GRP78 is specific to celecoxib and DMC and requires calcium. U251 glioblastoma cells were cultured in the presence of DMC, celecoxib, rofecoxib, or valdecoxib, and the protein levels of CHOP and GRP78 were determined by Western blot analysis. Time kinetics at 50 μmol/L of each drug (A) and concentration dependence after 15 h of incubation (B). Bgr, a background signal that is inconsistently observed with the GRP78 antibody. Cells were treated with 60 μmol/L DMC in the presence or absence of 20 μmol/L BAPTA-AM and 0.78 mmol/L EGTA, both of which are potent chelators of Ca²⁺ (C). All blots in (A) and (B) were processed in parallel, so that signal intensity is directly comparable among the different panels.

Induction of CHOP and GRP78 correlates with increased apoptosis and reduced cell growth and survival. U251 glioblastoma cells were treated with different drugs, and various variables of cell growth and cell death were comparatively analyzed. As controls, cells remained either nontreated or were treated with the solvent DMSO alone. A, cells were treated with 30 or 50 μmol/L DMC for 48 h and the effects on cell growth/survival and on cell death were determined by various assays. Top, the results from a colony-forming assay, where the absolute number of surviving cells able to spawn a colony of newly grown cells was determined; middle, the results of conventional 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays done at the end of the 48-h drug treatment period; bottom, the percentage of cells undergoing apoptosis as revealed by the terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling assay after 48-h drug treatment. The expression levels of the ER stress indicators CHOP, GRP78, and caspase-4 at the end of the 48-h drug treatment, as determined by Western blot analysis with specific antibodies (actin served as a loading control). B, cells were treated with various concentrations of different drugs for 48 h, as indicated, and cell death was measured with the cell death ELISA kit. C, cells were treated with DMC or various coxibs and traditional NSAIDs for 48 h, as indicated, and cell growth and survival was determined with the conventional 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (control, nontreated cells were set at 100%). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays were done in 96-well plates with the use of 3.0 × 10³ to 8.0 × 10³ cells per well as described in detail elsewhere (34). In parallel, the expression levels of CHOP and GRP78 protein were determined by Western blot analysis. Note that DMC is the most potent drug, celecoxib is substantially weaker, and none of the other coxibs or traditional NSAIDs are active under these conditions.

Knockdown of GRP78 enhances, whereas knockdown of caspase-4 reduces, cell killing by celecoxib and DMC. U251 glioblastoma cells were transiently transfected with siRNA directed at GRP78 (si-GRP78) or caspase-4 (si-Casp 4). As a control, a si-GFP was used. A, 72 h after transfection, parallel cultures were treated with 40 μmol/L celecoxib and 30 μmol/L DMC for si-GRP78/si-GFP or with 60 μmol/L celecoxib and 40 μmol/L DMC for si-Casp 4/si-GFP; in all instances, control cultures received no drug treatment or treatment with solvent (DMSO) alone. After 48-h drug treatment, the drugs were removed and the fraction of surviving cells was determined by colony-forming assays. Percentage of surviving cells (where the number of colonies under non-drug–treated conditions was set to 100%). The P values shown show statistically significant differences in survival between cells receiving si-GRP78 and control siRNA (si-GFP) and between cells receiving si-caspase-4 and control siRNA, respectively. B, to verify the effectiveness of the siRNAs, Western blot analysis of the target proteins was done. Note that the knockdown of GRP78 leads to increased levels of CHOP protein, as expected from the model of ER stress, where GRP78 signaling is upstream of CHOP. Caspase-4 siRNA also down-regulates its target (and cleaved caspase-4 becomes undetectable) but does not affect the induction of GRP78 in response to celecoxib or DMC, as expected from the ER stress model, where caspase-4 is downstream of GRP78 signaling.

DMC and celecoxib, but not rofecoxib, stimulate ESR and apoptosis in tumor cells in vivo. Nude mice were implanted s.c. with U87 glioblastoma cells. Once tumors had reached a volume of 500 mm³, two animals each received either DMC, celecoxib, or rofecoxib (150 mg/kg), or no drug for 36 h. Thereafter, all eight animals were sacrificed and their tumors were analyzed by immunohistochemical (IHC) staining for CHOP protein, as well as by terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling assay for cell death/apoptosis. Left, expression of CHOP protein [small black rectangles, enlarged areas of the same photograph (middle)]; right, cell death (arrows, examples of terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling–positive, apoptotic, cells). The entire experiment was repeated with increasing daily dosages of drugs for 50 h (see Materials and Methods), and similar results were obtained. In all cases, representative sections are shown.