Research ArticleExperimental Studies

Combining CAL-101 with Celecoxib Enhances Apoptosis of EBV-transformed B-Cells Through MAPK-induced ER Stress

GA BIN PARK, DAE YOUNG HUR and DAEJIN KIM
Anticancer Research May 2015, 35 (5) 2699-2708;

Abstract

Background: Phosphoinositide-3 kinase (PI3K) inhibition attenuates proliferation and survival in B-cell malignancies. Celecoxib induces endoplasmic reticulum (ER) stress-induced apoptosis via a cyclo-oxgenase-2 (COX2)-independent manner in certain types of cancer cells. In the present study, we assessed the effects of combinations of drugs with a p110δ-specific inhibitor, CAL-101, and celecoxib to induce apoptosis in Epstein-Barr virus (EBV)-transformed B-cells and non-Hodgkin's lymphoma (NHL) cells. Materials and Methods: The apoptotic effect of combination treatment with CAL-101 and celecoxib on B-cell malignancies was determined by flow cytometry and immunoblotting. Results: Exposure to CAL-101 and celecoxib significantly increased apoptosis, which was accompanied by the inactivation of AKT, Ras homolog gene family, member A (RHOA), Rho-associated coiled-coil containing protein kinase 1 (ROCK1), and ROCK2 as well as up-regulation of Phosphatase and tensin homolog (PTEN). Co-treatment with CAL-101 and celecoxib triggered the ER stress response and the down-regulation of BCL2 and BCL-XL. SB203580, SP600125, and salubrinal effectively inhibited apoptosis and attenuated expression of phosphorylated protein kinase RNA-like endoplasmic reticulum kinase (PERK) and CCAAT-enhancer-binding protein homologous protein (CHOP). Levels of apoptosis signal-regulating kinase 1 (ASK1) were also increased after treatment with CAL-101 and celecoxib. Conclusion: The apoptosis of EBV-transformed B-cells and NHL cells caused by CAL-101 and celecoxib might be related to inhibiting the RHOA/ROCK pathway and might also be associated with mitogen-activated protein kinase (MAPK)-mediated ER stress.

Class IA PI3K consists of a 110-kDa catalytic unit (p110α, p110β, p110γ, and p110δ) and one of five regulatory proteins (p85α, p55α, p50α, p85β, or p55γ) (1). Although p110α and p110β are expressed ubiquitously, p110δ is predominantly expressed in white blood cells (2). The p110δ isoform is a primary signaling molecule that increases cell metabolism and proliferation after B-cell receptor (BCR) engagement, making it an attractive target for B-cell malignancies (3). PTEN is a tumor suppressor protein that antagonizes the PI3K/AKT signaling pathway (4). Activation of p110δ results in decreased RHOA/ROCK activity, which consequently decreases PTEN activity (5). Interestingly, ROCK appears to be involved in both positive and negative regulation of the PI3K/AKT signaling pathway. In human embryonic kidney (HEK) cells, ROCK activation blocks AKT phosphorylation via up-regulation of PTEN (6). Meanwhile, ROCK phosphorylates insulin receptor substrate 1 to activate the PI3K/AKT pathway in adipocytes and muscle cells (7). CAL-101 selectively inhibits p110δ in cancer cells and in cells from patients with different B cell malignancies, including chronic lymphocytic leukemia, multiple myeloma, and Hodgkin's lymphoma (8, 9). However, the effect of CAL-101 on the RHOA/ROCK/PTEN pathway in EBV-transformed B cells or in NHL is unclear. Furthermore, there exist no studies examining the specific integral role of PI3Kδ inhibitors after combination treatment with non-steroidal anti-inflammatory drugs (NSAIDs), including celecoxib, a second-generation NSAID, for controlling B-cell malignancies.

Celecoxib is used to treat inflammatory diseases and certain types of tumors by regulating COX2 (10). However, typical long-term treatment with COX2 selective inhibitors is currently being re-examined due to increasing cardiovascular toxicity (10). COX2 protein expression is increased in all lymphoma cell lines that have been examined, and 50 mM celecoxib induce over 85% apoptosis in all cell lines tested by down-regulation of AKT, myeloid cell leukemia 1 (MCL1), and phosphorylated stress-activated protein kinase (SAPK) (11). Celecoxib-induced apoptosis is dependent on activation of caspases-9 and -3, but not caspase-8 in Jurkat and BJAB cells (12). Treatment with celecoxib (10-100 μM) arrests the cell cycle at G0/G1 phase and induces apoptosis via poly (ADP-ribose) polymerase (PARP) cleavage and reduction of BCL2 levels in Jurkat, HL60, and U937 cells (13). The cytotoxic effect of celecoxib is related to the triggering of ER stress in Raji lymphoma cells and is not related to COX2 inhibitory activity (14). From these results, celecoxib appears to regulate cancer cell growth independently of blocking the COX2 enzyme in B-cell malignancies.

Figure 1.
Figure 1.

Co-treatment with CAL-101 (CAL) and celecoxib (CLC) increases the apoptosis of EBV-transformed B-cells (EBVt-B). A: Western blots to determine the expression levels of p110α, p110β, p110γ, and p110δ in EBV-transformed B-cells, Ramos, Raji, Daudi, and IM-9 cells were performed. B-D: EBV-transformed B-cells (2×104 cells/well) were cultured in 96-well plates. Cell proliferation was measured using the AlamarBlue assay. The relative fluorescence unit (RFU) levels are shown (mean±SD, n=3). B: Cells were treated with 1, 10, 20, 50, 70, or 100 μM CAL-101 or LY294002 for 24 h. C: Cells were treated with 1, 5, 10, 20, 50, or 100 μM celecoxib for 24 h. D: Cells were co-treated with 1 μM CAL-101 and 5 μM celecoxib, or 1 μM LY294002 and 5 μM celecoxib for 24 h. Cells treated with 100 μM CAL-101, 100 μM LY294002, or 50 μM celecoxib for 24 h were used as a positive control. E: Cells (1×105 cells/ml) were cultured in 6-well plates and co-treated with 1 μM CAL-101 and 5 μM celecoxib for 16 h. Cells treated with 100 μM CAL-101 or 50 μM celecoxib for 16 h were used as a positive control. The percentage of apoptotic cells was estimated by annexin-V/7-AAD staining. Dot plot graphs show the percentage of viable cells (annexin-V−/7-AAD−), early-stage apoptotic cells (annexin-V+/7-AAD−), late-stage apoptotic cells (annexin-V+/7-AAD+), and necrotic cells (annexin-V−/7-AAD+). The results are representative of three separate experiments. Bars represent the mean±SD. *p<0.01.

Exposure of cells to indomethacin or celecoxib induces the expression of glucose-regulated protein 78 (GRP78) and CHOP through regulation of activating transcription factor 6 (ATF6), ATF4, and X-box binding protein 1 (XBP1) (15). The combination of celecoxib with MG132, proteasome inhibitor, synergistically induced expression of the ER stress genes ATF4, CHOP, and tribbles-related protein 3 (TRB3) and promoted the splicing of XBP1 mRNA in liver cancer cells (16). Co-administration of celecoxib reverses the elevation of PI3K, p-AKT, 3-phosphoinositide dependent protein kinase-1 (PDK1), and mammalian target of rapamycin (mTOR) and blocked the down-regulation of PTEN in a dextran sulfate sodium-induced mouse model of colitis (17). Treatment with tunicamycin or thapsigargin, ER stress inducers, caused de-phosphorylation of AKT over 12 to 24 h and induced cell death. Interestingly, treatment with LY294002 or wortmannin, PI3K inhibitors, induced CHOP expression and caused cell death in the mouse fibroblast cell line L929 (18). However, the molecular mechanisms of apoptosis after combination treatment with a p110δ specific inhibitor and celecoxib in EBV-transformed B cell or NHL cell lines have not yet been analyzed.

In the present study, we investigated whether combination treatment with low doses of CAL-101 and celecoxib synergistically induces apoptosis of EBV-transformed B-cells and evaluated the signaling pathway in the regulation of the ER stress-induced apoptotic pathway.

Figure 2.
Figure 2.

Effect of co-treatment with CAL-101 (CAL) and celecoxib (CLC) on PI3K/AKT/PTEN and RHOA/ROCK expression. EBV-transformed B-cells (EBVt-B), Ramos, and Raji cells were co-treated with 1 μM CAL-101 and 5 μM celecoxib or 1 μM LY294002 and 5 μM celecoxib for 3 h. Cells treated with 100 μM CAL-101, 100 μM LY294002 (LY), or 50 μM celecoxib for 3 h were used as a positive control. A: Total cell lysates were immunoblotted with antibodies against phospho-PI3K, PI3K, phospho-AKT, AKT, ROCK1, ROCK2, phospho-PTEN, PTEN, or β-actin, which served as an internal control. B: Activated RHOA was pulled down by glutathione-S-transferase linked to the RHOA-binding domain of Rhotekin (GST-RBD) and analyzed by immunoblotting. The results are representative of three independent experiments.

Materials and Methods

Cells and chemicals. The prepatation of cell-free EBV virions and generation of EBV-transformed B-cells were performed as described previously (19). Human Burkitt's lymphoma Ramos and Raji cells were purchased from the American Type Culture Collection (Manassas, VA, USA). These cells were maintained in RPMI-1640 medium (HyClone, Logan, UT, USA) supplemented with 10% Fetal bovine serum (FBS) (HyClone) and antibiotics under a humidified atmosphere with 5% CO2. LY294002 (pan-p110 inhibitor), A66 (p110α inhibitor) TGX221 (p110β inhibitor), CAL-101 (p110γ inhibitor), CZC24832 (p110γ inhibitor), and celecoxib were purchased from Selleckchem (Houston, TX, USA). Salubrinal (ER stress inhibitor), SP600125 (JNK inhibitor), and SB203580 (p38 MAPK inhibitor) were obtained from Calbiochem (San Diego, CA, USA). NQDI1 (ASK1 inhibitor) was obtained from R&D Systems (Minneapolis, MN, USA).

Cell preparation for immunoblotting and antibodies. After drug treatment, the cells were harvested and lysed in NP-40 buffer (Elpis Biotech, Daejeon, Korea) supplemented with a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). To address phosphorylation events, an additional set of phosphatase inhibitors (Cocktail II; Sigma-Aldrich) was added to the NP-40 buffer. Protein concentration was determined using a BCA assay kit (Pierce, Rockford, IL, USA). An equal volume of 2× Laemmli sample buffer (Elpis Biotech) was added to each lysate sample (10 μg protein) and immediately boiled for 5 min at 100°C. The insoluble material was spun down at 16,000 ×g. Total cell lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on gel containing 12% (w/v) acrylamide under reducing conditions. Separated proteins were transferred to nitrocellulose membranes (Millipore Corp., Billerica, MA, USA), the membranes were blocked with 5% skim milk and commercial Western blot analysis with antibodies was performed. Chemiluminescence was detected using an ECL kit (Advansta Corp. Menlo Park, CA, USA) and the multiple Gel DOC system (GE Healthcare, Piscataway, NJ, USA). Total cell lysates were immunoblotted with the following primary antibodies were used: caspase-3, caspase-9, PARP, β-actin, phospho-Jun amino-terminal kinases (JNK) (Thr183/Tyr185), JNK, phospho-p38 MAPK (Thr180/Tyr182), p38 MAPK, phospho-extracellular signal-regulated kinase (ERK) 1/2 (Thr202/Tyr204), ERK1/2, phospho-AKT (Ser473), AKT, phospho-PI3K p85 (Tyr458), PI3K p85, BCL2, BCL-XL, BIM, BAX, phospho-ASK1 (Thr845), and ASK1 (Cell Signaling Technology, Beverly, MA, USA); ROCK1 and ROCK2 (Abcam, Cambridge, UK); phospho-PTEN (Ser380/Thr382/383), PTEN, phospho-PERK (Thr981), PERK, and CHOP (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Data were analyzed using ImageJ 1.38 software.

Proliferation measurements using AlamarBlue. EBV-transformed B-cells (2×104 cells/well) were cultured in complete medium containing CAL-101 (1, 10, 20, 50, 70, or 100 μM) or LY294002 (1, 10, 20, 50, 70, or 100 μM) or celecoxib (1, 5, 10, 20, 50, or 100 μM) in 96-well plates. After 24 h, cell proliferation was measured using the AlamarBlue (Serotec Ltd, Kidlington, Oxford, UK) assay. AlamarBlue was added (10% by volume) to each well, and the relative fluorescence was determined 7 h later with a SpectraMax M2e Multi-Detection Microplate Reader (Molecular Devices, Sunnyvale, CA; excitation, 530 nm; emission, 590 nm). Relative fluorescence unit (RFU) values were expressed as the mean± standard deviation (SD) of three readings.

Figure 3.
Figure 3.

Effect of co-treatment with CAL-101 (CAL) and celecoxib (CLC) on caspase activation and MAPK family member expression. EBV-transformed B-cells (EBVt-B), Ramos, and Raji cells were co-treated with 1 μM CAL-101 and 5 μM celecoxib for 16 h. Cells treated with 100 μM CAL-101 or 50 μM celecoxib for 16 h were used as a positive control. A: Western blots to determine the expression of caspase-9, -3, and PARP cleavage were performed to characterize the apoptotic response. β-actin was used to normalize protein content. B: Total cell lysates were immunoblotted with antibodies against phospho-p38 MAPK, phospho-JNK, phospho-ERK, p38 MAPK, JNK, or ERK. The results are representative of three independent experiments.

Analysis of apoptotic cells by flow cytometry. EBV-transformed B-cells, Ramos, or Raji cells (1×105 cells/ml) were co-treated with 1 μM CAL-101 and 5 μM celecoxib, or 1 μM LY294002 and 5 μM celecoxib for 24 h. Cells treated with 100 μM CAL-101, 100 μM LY294002, or 50 μM celecoxib for 24 h were used as a control. The percentage of cells undergoing apoptosis in the drug-treated cells was determined by flow cytometry using fluorescein isothiocyanate (FITC)-labeled annexin-V (BD Biosciences, San Diego, CA, USA) and 7-aminoactinomycin D (7-AAD) (BD Biosciences). The cells were then harvested, rinsed with PBS, and incubated with 3 μl annexin-V and 3 μl 7-AAD in annexin-V binding buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2) at room temperature for 15 min in the dark. The stained cells were analyzed using a FACSCalibur flow cytometer (BD Biosciences) equipped with CellQuest Pro software (BD Biosciences).

PI3K/PTEN/RHOA activity assay. EBV-transformed B-cells, Ramos, and Raji cells (1×105 cells/ml) were co-treated with 1 μM CAL-101 and 5 μM celecoxib or 1 μM LY294002 and 5 μM celecoxib for 3 h. Cells treated with 100 μM CAL-101, 100 μM LY294002, or 50 μM celecoxib for 3 h were used as a control. Total cell lysates were immunoblotted with antibodies against phospho-PI3K, PI3K, phospho-AKT, AKT, ROCK1, ROCK2, phospho-PTEN, PTEN, or β-actin, which served as an internal control. The RHOA activation assay was performed according to the manufacturer's instructions (RHOA Activation Assay Kit; Abcam). Briefly, an equal amount of proteins (500 mg) was incubated with sepharose-bound Rhotekin to isolate active RHOA. After washing, the bead/protein complexes were boiled in sample buffer and separated by SDS-PAGE. The blots were incubated with an antibody against RHOA.

Detection of MAPKs activation and ER stress. For preventing MAPK and ER stress, EBV-transformed B-cells, Ramos, or Raji cells (1×105 cells/ml) were pretreated with SP600125(JNK inhibitor, 25 μM) or SB203580 (p38 inhibitor, 10 μM) or salubrinal (ER stress inhibitor, 2 μM) for 1 h. After pre-treatment with each inhibitor, the cells were co-treated with 1 μM CAL-101 and 5 μM celecoxib for 24 h. The cells treated with dimethylsulfoxide (DMSO) were used as a control. Total cell lysates were immunoblotted with antibodies against phospho-JNK (Thr183/Tyr185), JNK, phospho-p38 MAPK (Thr180/Tyr182), p38 MAPK, phospho-ERK 1/2 (Thr202/Tyr204), ERK1/2, phospho-PERK (Thr981), PERK, CHOP or β-actin, which served as an internal control.

Detection of ASK1 activation. EBV-transformed B-cells, Ramos, or Raji cells (1×105 cells/ml) were co-treated with 1 μM CAL-101 and 5 μM celecoxib for 24 h. Cells treated with 100 μM CAL-101 or 50 μM celecoxib for 24 h were used as a control. Total cell lysates were immunoblotted with antibodies against phospho-ASK1 (Thr845), and ASK1. For blocking ASK1 activation, EBV-transformed B-cells, Ramos, or Raji cells (1×105 cells/ml) were pre-incubated with NQDI1 (ASK1 inhibitor, 1 μM) for 2 h and then treated with 1 μM CAL-101 and 5 μM celecoxib for 24 h. The cells treated with DMSO were used as a control. Total cell lysates were immunoblotted with antibodies against phospho-ASK1 (Thr845), and ASK1, phospho-JNK (Thr183/Tyr185), JNK, phospho-p38-MAPK (Thr180/Tyr182), p38-MAPK, phospho-PERK (Thr981), PERK, CHOP or β-actin, which served as an internal control.

Figure 4.
Figure 4.

Co-treatment with CAL-101 (CAL) and celecoxib (CLC) induced ER stress-mediated apoptosis and altered the levels of BCL2 family members. EBV-transformed B-cells, Ramos, and Raji cells were co-treated with 1 μM CAL-101 and 5 μM celecoxib for 16 h. Cells treated with 100 μM CAL-101 or 50 μM celecoxib for 16 h were used as a positive control. Total cell lysates were immunoblotted with antibodies against phospho-PERK, PERK, or CHOP (A), and BCL2, BCL-XL, BAX, BIM, or β-actin (B), which served as an internal control. β-Actin was used to normalize protein content. The results are representative of three independent experiments.

Statistical analysis. Data were expressed as the mean±SD. Statistical analysis was performed using one-way analysis of the variance (ANOVA). A p-value of less than 0.05 was considered to be statistically significant

Results

Combination treatment with low doses of CAL-101 and celecoxib enhanced the apoptosis of EBV-transformed B-cells. Expression of the 110 kDa catalytic subunit in EBV-transformed B-cells and NHL cell lines was determined by immunoblot. Although Raji and Daudi cells exhibited slightly lower levels of p110α, the expressional patterns of p110α, p110β, and p110δ were similar in B-cells. However, p110γ was not detected (Figure 1A). Next, we examined the effect of each p110 isoform inhibitor on cell growth of EBV-transformed B-cells. Treating cells with different doses of CAL-101 or LY294002 suppressed the cell viability of EBV-transformed B-cells in a dose-dependent manner (Figure 1B). However, pharmacological inhibition of p110α (A66) or p110β (TGX221) resulted in decreased cytotoxicity in EBV-transformed B cells or in NHL cell lines (data not shown). CZC24832, a p110γ-specific inhibitor, also had no effect on the viability of EBV-transformed B-cells or NHL cell lines (data not shown). The viability of EBV-transformed B-cells treated with celecoxib was significantly reduced compared to the control group based on the Alamar blue assay (Figure 1C). Interestingly, the low-dose combination of CAL-101 (1 μM) and celecoxib (5 μM) significantly decreased viability and induced the apoptosis of EBV-transformed B-cells and NHL cell lines compared to the groups treated with high doses of either CAL-101 (100 μM) or celecoxib (50 μM) alone (Figure 1D and 1E). These results suggest that combination treatment with a low dose of the p110δ-specific inhibitor and celecoxib synergistically induced apoptosis.

The RHOA/ROCK/PTEN pathway and the JNK/p38 MAPK pathway control the apoptosis induced by co-treatment with low doses of CAL-101 and celecoxib. RHOA mainly regulates ROCK1, which controls PTEN expression and therefore can also regulate apoptosis (20). We, therefore, examined whether co-treatment with low doses of CAL-101 and celecoxib changed the expression of RHOA/ROCK and PTEN in EBV-transformed B-cells and NHL cell lines. Phosphorylation of PI3K/AKT was prevented after combining CAL-101 or LY294002 treatment with celecoxib (Figure 2A). Furthermore, phosphorylated PTEN levels markedly increased in EBV-transformed B cells and NHL cell lines (Figure 2A). Co-treatment with CAL-101 and celecoxib reduced activated RHOA (RHOA-GTP) levels as well as those of ROCK1, a major target of RHOA, and ROCK2 (Figure 2A and 2B). The same treatment resulted in the cleavage of caspase-9, caspase-3, and PARP (Figure 3A) and increased the expression of phosphorylated p38 MAPK and JNK compared to EBV-transformed B-cells and NHL cell lines treated with a high dose of CAL-101 or celecoxib-alone (Figure 3B). Furthermore, combination treatment with low doses of CAL-101 and celecoxib effectively attenuated the level of phosphorylated ERK in EBV-transformed B-cells and NHL cell lines (Figure 3B). These results suggest that the RHOA/ROCK/PTEN and the JNK/p38 MAPK signaling pathways are essential for controlling apoptosis induced by co-treatment with CAL-101 and celecoxib.

Figure 5.
Figure 5.

Effect of salubrinal (Sal) on the phosphorylation of JNK/p38 MAPK, the production of ER stress marker, and the activity of caspases after co-treatment with CAL-101 (CAL) and celecoxib (CLC). EBV-transformed B-cells (EBVt-B), Ramos, and Raji cells were pre-treated with 2 μM salubrinal or DMSO at 37°C for 1 h. Then, the cells were co-treated with 1 μM CAL-101 and 5 μM celecoxib for 16 h. Total cell lysates were immunoblotted with antibodies against caspase-9 and -3, PARP, BCL2, BCL-XL, or BIM (A), and phospho-p38 MAPK, p38 MAPK, phospho-JNK, JNK, phospho-PERK, PERK, CHOP, or β-actin (B), which served as an internal control. The results are representative of three independent experiments.

Co-treatment with CAL-101 and celecoxib triggers ER stress-induced apoptosis and alters levels of BCL2 family proteins. Activation of p-p38 MAPK is associated with caspase activation, as well as ER stress-induced mitochondria-dependent apoptosis of EBV-transformed B-cells (19). Next, we examined whether co-treatment with CAL-101 and celecoxib triggers the ER-induced apoptosis and alters the expression of BCL2 family proteins. Treatment with the combination of CAL-101 and celecoxib significantly increased the expression of PERK, sensor proteins of ER stress, and the transcription factor CHOP compared to the groups treated with high doses of CAL-101 (100 μM) or celecoxib (50 μM) alone (Figure 4A). BCL2 and BCL-XL proteins were constitutively expressed in EBV-transformed B-cells. Although the levels of BCL-XL and BCL2 were markedly decreased, BAX induction was observed in EBV-transformed B-cells and NHL cell lines after co-treatment with CAL-101 and celecoxib. BIM expression was elevated, and three isoforms of BIM were also detected after co-treatment with CAL-101 and celecoxib (Figure 4B). These results suggest that ER stress induced by co-treatment with CAL-101 and celecoxib is associated with the expressional change of both pro- and anti-apoptotic BCL2 family proteins in EBV-transformed B-cells

MAPK-mediated ER stress preceded apoptosis in EBV-transformed B-cells and NHL cell lines after co-treatment with CAL-101 and celecoxib. The small molecule salubrinal, a selective eIF2α dephosphorylation inhibitor, is known to protect cells from ER stress-induced apoptosis (21). Therefore, we examined whether co-treatment with CAL-101 and celecoxib attenuates the apoptotic signal and ER-stress caused by the co-treatment with CAL-101 and celecoxib. Pre-treatment with salubrinal significantly suppressed the expression of cleaved caspase-3 and caspase-9, as well as of the apoptotic BCL2 proteins, including BAX and BIM. Furthermore, salubrinal simultaneously increased the expression of the anti-apoptotic proteins, BCL2 and BCL-XL (Figure 5A). Although treatment with salubrinal had little effect on the expression of phosphorylated JNK and p38 MAPK, EBV-transformed B-cells and NHL cell lines pre-treated with salubrinal exhibited a lower expression of CHOP and phosphorylated PERK after co-treatment with CAL-101 and celecoxib (Figure 5B). Next, we investigated the effects of activated JNK and p38 MAPK on ER stress and apoptosis using SB203580, an inhibitor of p-p38 MAPK, and SP600125, a JNK inhibitor, in EBV-transformed B-cells and NHL cell lines co-treated with CAL-101 and celecoxib. When phosphorylated p38 MAPK or JNK was inhibited in these cells, the production of cleaved caspase-9, caspase-3, and PARP were down-regulated and the expression of the anti-apoptotic proteins, BCL-XL, BCL2, and BIM were restored to the levels of the control group (Figure 6A). SB203580 and SP600125 effectively attenuated the expression of phosphorylated PERK and CHOP in EBV-transformed B cells and NHL cell lines co-treated with CAL-101 and celecoxib (Figure 6B). These results suggest that activated MAPK regulates ER stress-induced apoptosis in EBV-transformed B-cells and NHL cell lines after combination treatment with CAL-101 and celecoxib.

Figure 6.
Figure 6.

JNK and p38 MAPK activation is related to ER stress-mediated apoptosis after co-treatment with CAL-101 (CAL) and celecoxib (CLC). EBV-transformed B-cells, Ramos, and Raji cells were pre-treated with 25 μM SP600125 (SP) or 10 μM SB203580 (SB) at 37°C for 1 h. Then, the cells were co-treated with 1 μM CAL-101 and 5 μM celecoxib for 16 h. Total cell lysates were immunoblotted with antibodies against caspase-9 and -3, PARP, BCL2, BCL-XL, or BIM (A), and phospho-p38 MAPK, p38 MAPK, phospho-JNK, JNK, phospho-PERK, PERK, CHOP, or β-actin (B), which served as an internal control. The results are representative of three independent experiments.

Co-treatment with CAL-101 and celecoxib regulates ER stress-induced apoptosis through activation of ASK1 in EBV-transformed B-cells and NHL cell lines. ER stress activates ASK1-JNK to induce cell death through the formation of an inositol-requiring enzyme 1 (IRE1)-tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2)-ASK1 complex (22). Next, we investigated whether the ASK1 inhibitor NQDI1 prevents caspase activation and ER stress induction. Phosphorylated ASK1 was detected in EBV-transformed B-cells and NHL cell lines co-treated with CAL-101 and celecoxib (Figure 7A). The activation of caspase-9 and caspase-3 were completely inhibited by pre-treatment with NQDI1. Furthermore, the levels of BAX and BIM were increased in EBV-transformed B-cells and NHL cell lines co-treated with CAL-101 and celecoxib after pre-treatment with NQDI1 compared to the levels of the control group (Figure 7B). The phosphorylation of JNK and p38 MAPK was suppressed, and the expression of the ER stress signaling proteins, including phosphorylated PERK and CHOP, were also dramatically attenuated by pre-treatment with NQDI1 (Figure 7C). However, pre-treatment with SB203580 or SP600125 had no effect on phosphorylated ASK1 expression in EBV-transformed B-cells and NHL cell lines after combination treatment with CAL-101 and celecoxib (Figure 7D). We also observed that pharmacological inhibition of ER stress using salubrinal had no effect on the expression of phosphorylated ASK1 induced by co-treatment with CAL-101 and celecoxib in EBV-transformed B cells and NHL cell lines (Figure 7E). These results suggest that activated ASK1 initiates the MAPK-mediated ER stress pathway to induce apoptosis in EBV-transformed B-cells and NHL cell lines co-treated with CAL-101 and celecoxib.

Figure 7.
Figure 7.

Co-treatment with CAL-101 (CAL) and celecoxib (CLC) regulates ER stress-induced apoptosis through activation of ASK1. EBV-transformed B-cells (EBVt-B), Ramos, and Raji cells were co-treated with 1 μM CAL-101 and 5 μM celecoxib for 16 h. Cells treated with 100 μM CAL-101 or 50 μM celecoxib for 16 h were used as a positive control. A: Western blots to determine the expression of phospho-ASK1 and ASK1 were performed. Cells were pre-treated with 1 μM NQDI1 at 37°C for 1 h, then co-treated with 1 μM CAL-101 and 5 μM celecoxib for 16 h. Total cell lysates were immunoblotted with antibodies against caspase-9 and -3, PARP, BCL2, BCL-XL, BAX, or BIM (B), and phospho-ASK1, ASK1, phospho-JNK, JNK, phospho-p38 MAPK, p38 MAPK, phospho-PERK, PERK, CHOP, or β-actin (C), which served as an internal control. Cells were pretreated with 25 μM SP600125 (SP), 10 μM SB203580 (SB) (D), or 2 μM salubrinal (Sal) (E) at 37°C for 1 h then co-treated with 1 μM CAL-101 and 5 μM celecoxib for 16 h. Total cell lysates were immunoblotted with antibodies against phospho-ASK1 and ASK1. The results are representative of three independent experiments.

Discussion

The PI3K pathway is a central signal transduction axis controlling normal B-cell homeostasis and activation (23). The p110δ isoform is required for B-cell proliferation, differentiation, and germinal center formation through signaling from the BCRAg complex (24). Animal cells usually have three genes for three class IA catalytic units designated p110α, p110β, and p110δ (1). Class IB PI3K, p110γ, is activated by the Gβγ subunits of heterotrimetric G-proteins and is also essential for leukocyte function (25). However, controversy remains regarding the signals inducing cell death upon combination treatment with a PI3K p110δ-specific inhibitor and other drugs for controlling B-cell malignancies. Inactivation of p110δ reduces AKT activation, but paradoxically increased RHOA and PTEN activity. Pharmacological inhibition of RHOA/ROCK restores the PI3K/AKT signaling pathway in macrophages (5). In contrast to previous results, co-treatment with the p110δ-specific inhibitor, CAL-101 and celecoxib inhibited the expression of activated RHOA/ROCK in EBV-transformed B-cells and NHL cell lines. Although alteration of BCR signaling and growth factors from the tumor microenviroment generate an abnormally high level of p110δ PI3K activity (26), our results suggest that a highly activated PI3K/AKT signaling pathway in B-cell malignancies might regulate the RHOA/ROCK expression.

Celecoxib attenuates the production of prostaglandin E2 (PGE2) in follicular lymphoma stromal cells. However, celecoxib-mediated TRAIL expression regulates the apoptosis of NHL B-cell lines, but independently of PGE2/COX2 (27). Celecoxib triggers ER stress to induce apoptosis of Raji cells. In addition, bortezomib enhances the celecoxib-mediated apoptotic signal (14). The ER stress inducer activates the PERK-dependent activation of JNK and p38 MAPK. However, activated JNK and p38 MAPK stimulated by UV irradiation and TNFα are observed independent of PERK expression in mouse embryonic fibroblasts (MEF) (28). In EBV-transformed B-cells, phosphorylated JNK and p38 MAPK were increased after co-treatment with CAL-101 and celecoxib. In contrast to previous results, pre-treatment of EBV-transformed B-cells with a JNK inhibitor (SP600125) or a p38 MAPK inhibitor (SB203580) attenuated the expression of phosphorylated PERK and CHOP. Although the ER stress inhibitor salubrinal protected against caspase-dependent cell death induced by co-treatment with CAL-101 and celecoxib, the expression levels of phosphorylated JNK and p38 MAPK did not decrease in EBV-transformed B-cells and NHL cell lines. Our results suggest that co-treatment with CAL-101 and celecoxib promotes the activation of MAPK, leading to ER stress-mediated BCL2 down-regulation to induce apoptosis of EBV-transformed B-cells and NHL cell lines.

Phosphorylated AKT proteins and AKT mRNA decrease in a time- and dose-dependent manner after treatment of gastric cancer cells with celecoxib (29). The PI3K/AKT and ERK signaling pathways protect a variety of cells from ER stress. The inhibition of AKT phosphorylation sensitizes chondrocytes to ER stress, leading to increased capsase-3 activity and decreased BCL-XL expression (30). Thapsigargin-induced ER stress causes apoptosis through caspase-3 activation in lipopolysaccharide (LPS)-stimulated macrophages. However, B-cell adapter protein, a p85 PI3K-binding adapter protein, promotes cell survival in macrophages in response to the combined challenge of LPS and ER stress (31). These results indicate that the survival of EBV-transformed B-cells or NHL cell lines after combined treatment with CAL-101 and celecoxib might also be suppressed through manipulation of PI3K/AKT-mediated ER stress. The p110δ inhibition was sufficient to prevent BCR-mediated PI3K activation, combined p110α and p110δ inhibition is necessary to abolish constitutive PI3K activation; GDC-0941, predominantly a p110α/δ inhibitor, blocked AKT phosphorylation in relapsed mantle cell lymphoma (32). Based on these results, we must further investigate the mechanism leading to cell death after combination treatment with p110α or p110β inhibitor and celecoxib in B-cell malignancies.

ASK1 is a key element in ER stress-induced cell death. ER stress signaling is upstream of the activation of JNK during docetaxel-induced apoptosis of melanoma cells (22). ER stress activates ASK1 through formation of an IRE1TRAF2ASK1 complex that induces JNK activation and, consequently, cell death (33). Salubrinal inhibits ursolic acid-induced CHOP expression and activation of pro-apoptotic ASK1JNK signaling (34). Although co-treatment with CAL-101 and celecoxib induced ER stress-mediated apoptosis, salubrinal had no effect on the expression of phosphorylated ASK1 and JNK. However, the ASK1 inhibitor NQDI1 attenuated ER-stress mediated ASK1/JNK activation and subsequent apoptosis. Our results suggest that combinational treatment with p110δ specific inhibitor and celecoxib enhances the ER stress-mediated apoptotic signaling pathway through activation of ASK/JNK in B-cell malignancies.

The PI3K isoform p110δ is expressed at high levels in various cancer types and our results support the development of approaches to enhance the apoptosis of cancer cells through MAPK-mediated ER-stress after combination treatment with CAL-101 and celecoxib. This study also provides new information on therapeutic combinations with a well-known drug for intractable NHL.

Acknowledgements

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2010-0022674, NRF-2013-R1A1A2-010668)

Footnotes

  • Conflicts of Interest

    The Authors declare that they have no conflicts of interest to disclose.

  • Received February 2, 2015.
  • Revision received February 16, 2015.
  • Accepted February 18, 2015.

References

  1. ↵
    1. Vanhaesebroeck B,
    2. Guillermet-Guibert J,
    3. Graupera M,
    4. Bilanges B
    : The emerging mechanisms of isoform-specific PI3K signalling. Nat Rev Mol Cell Biol 11: 329-341, 2010.
    OpenUrlCrossRefPubMed
  2. ↵
    1. Geering B,
    2. Cutillas PR,
    3. Vanhaesebroeck B
    : Regulation of class IA PI3Ks: is there a role for monomeric PI3K subunits? Biochem Soc Trans 35(Pt 2): 199-203, 2007.
    OpenUrlAbstract/FREE Full Text
  3. ↵
    1. Bilancio A,
    2. Okkenhaug K,
    3. Camps M,
    4. Emery JL,
    5. Ruckle T,
    6. Rommel C,
    7. Vanhaesebroeck B
    . Key role of the p110δ isoform of PI3K in B-cell antigen and IL-4 receptor signaling: comparative analysis of genetic and pharmacologic interference with p110δ function in B-cells. Blood 107: 642-650, 2006.
    OpenUrlAbstract/FREE Full Text
  4. ↵
    1. Simpson L,
    2. Parsons R
    . PTEN: life as a tumor suppressor. Exp Cell Res 264: 29-41, 2001.
    OpenUrlCrossRefPubMed
  5. ↵
    1. Papakonstanti EA,
    2. Ridley AJ,
    3. Vanhaesebroeck B
    : The p110δ isoform of PI3-kinase negatively controls RHOA and PTEN. EMBO J 26: 3050-3061, 2007.
    OpenUrlAbstract/FREE Full Text
  6. ↵
    1. Chang J,
    2. Xie M,
    3. Shah VR,
    4. Schneider MD,
    5. Entman ML,
    6. Wei L,
    7. Schwartz RJ
    : Activation of RHO-associated coiled-coil protein kinase 1 (ROCK-1) by caspase-3 cleavage plays an essential role in cardiac myocyte apoptosis. Proc Natl Acad Sci USA 103: 14495-14500, 2006.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    1. Furukawa N,
    2. Ongusaha P,
    3. Jahng WJ,
    4. Araki K,
    5. Choi CS,
    6. Kim HJ,
    7. Lee YH,
    8. Kaibuchi K,
    9. Kahn BB,
    10. Masuzaki H,
    11. Kim JK,
    12. Lee SW,
    13. Kim YB
    : Role of RHO-kinase in regulation of insulin action and glucose homeostasis. Cell Metab 2: 119-129, 2005.
    OpenUrlCrossRefPubMed
  8. ↵
    1. Castillo JJ,
    2. Furman M,
    3. Winer ES
    : CAL-101: a phosphatidylinositol-3-kinase p110-δ inhibitor for the treatment of lymphoid malignancies. Expert Opin Investig Drugs 21: 15-22, 2012.
    OpenUrlCrossRefPubMed
  9. ↵
    1. Ikeda H,
    2. Hideshima T,
    3. Fulciniti M,
    4. Perrone G,
    5. Miura N,
    6. Yasui H,
    7. Okawa Y,
    8. Kiziltepe T,
    9. Santo L,
    10. Vallet S,
    11. Cristea D,
    12. Calabrese E,
    13. Gorgun G,
    14. Raje NS,
    15. Richardson P,
    16. Munshi NC,
    17. Lannutti BJ,
    18. Puri KD,
    19. Giese NA,
    20. Anderson KC
    : PI3K/p110δ is a novel therapeutic target in multiple myeloma. Blood 116: 1460-1468, 2010.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    1. Sarkar FH,
    2. Adsule S,
    3. Li Y,
    4. Padhye S
    : Back to the future: COX2 inhibitors for chemoprevention and cancer therapy. Mini Rev Med Chem 7: 599-608, 2007.
    OpenUrlCrossRefPubMed
  11. ↵
    1. Wun T,
    2. McKnight H,
    3. Tuscano JM
    : Increased cyclooxygenase-2 (COX2): a potential role in the pathogenesis of lymphoma. Leuk Res 28: 179-190, 2004.
    OpenUrlCrossRefPubMed
  12. ↵
    1. Jendrossek V,
    2. Handrick R,
    3. Belka C
    : Celecoxib activates a novel mitochondrial apoptosis signaling pathway. FASEB J 17: 1547-1549, 2003.
    OpenUrlAbstract/FREE Full Text
  13. ↵
    1. Subhashini J,
    2. Mahipal SV,
    3. Reddanna P
    : Antiproliferative and apoptotic effects of celecoxib on human chronic myeloid leukemia in vitro. Cancer Lett 224: 31-43, 2005.
    OpenUrlPubMed
  14. ↵
    1. Chen ST,
    2. Thomas S,
    3. Gaffney KJ,
    4. Louie SG,
    5. Petasis NA,
    6. Schönthal AH
    : Cytotoxic effects of celecoxib on Raji lymphoma cells correlate with aggravated endoplasmic reticulum stress but not with inhibition of cyclooxygenase-2. Leuk Res 34: 250-253, 2010.
    OpenUrlCrossRefPubMed
  15. ↵
    1. Tsutsumi S,
    2. Gotoh T,
    3. Tomisato W,
    4. Mima S,
    5. Hoshino T,
    6. Hwang HJ,
    7. Takenaka H,
    8. Tsuchiya T,
    9. Mori M,
    10. Mizushima T
    : Endoplasmic reticulum stress response is involved in nonsteroidal anti-inflammatory drug-induced apoptosis. Cell Death Differ 11: 1009-1016, 2004.
    OpenUrlCrossRefPubMed
  16. ↵
    1. Cusimano A,
    2. Azzolina A,
    3. Iovanna JL,
    4. Bachvarov D,
    5. McCubrey JA,
    6. D'Alessandro N,
    7. Montalto G,
    8. Cervello M
    : Novel combination of celecoxib and proteasome inhibitor MG132 provides synergistic antiproliferative and proapoptotic effects in human liver tumor cells. Cell Cycle 9: 1399-1410, 2010.
    OpenUrlPubMed
  17. ↵
    1. Setia S,
    2. Nehru B,
    3. Sanyal SN
    : The PI3K/AKT pathway in colitis associated colon cancer and its chemoprevention with celecoxib, a COX2-selective inhibitor. Biomed Pharmacother 68: 721-727, 2014.
    OpenUrlPubMed
  18. ↵
    1. Hyoda K,
    2. Hosoi T,
    3. Horie N,
    4. Okuma Y,
    5. Ozawa K,
    6. Nomura Y
    : PI3K-AKT inactivation induced CHOP expression in endoplasmic reticulum-stressed cells. Biochem Biophys Res Commun 340: 286-290, 2006.
    OpenUrlCrossRefPubMed
  19. ↵
    1. Park GB,
    2. Kim YS,
    3. Lee HK,
    4. Song H,
    5. Cho DH,
    6. Lee WJ,
    7. Hur DY
    : Endoplasmic reticulum stress-mediated apoptosis of EBV-transformed B-cells by cross-linking of CD70 is dependent upon generation of reactive oxygen species and activation of p38 MAPK and JNK pathway. J Immunol 185: 7274-7284, 2010.
    OpenUrlAbstract/FREE Full Text
  20. ↵
    1. Keniry M,
    2. Parsons R
    : The role of PTEN signaling perturbations in cancer and in targeted therapy. Oncogene 27: 5477-5485, 2008.
    OpenUrlCrossRefPubMed
  21. ↵
    1. Park GB,
    2. Kim YS,
    3. Lee HK,
    4. Song H,
    5. Kim S,
    6. Cho DH,
    7. Hur DY
    : Reactive oxygen species and p38 MAPK regulate BAX translocation and calcium redistribution in salubrinal-induced apoptosis of EBV-transformed B-cells. Cancer Lett 313: 235-248, 2011.
    OpenUrlPubMed
  22. ↵
    1. Mhaidat NM,
    2. Thorne R,
    3. Zhang XD,
    4. Hersey P
    : Involvement of endoplasmic reticulum stress in docetaxel-induced JNK-dependent apoptosis of human melanoma. Apoptosis 13: 1505-1512, 2008.
    OpenUrlCrossRefPubMed
  23. ↵
    1. Al-Alwan MM,
    2. Okkenhaug K,
    3. Vanhaesebroeck B,
    4. Hayflick JS,
    5. Marshall AJ
    : Requirement for phosphoinositide 3-kinase p110δ signaling in B-cell antigen receptor-mediated antigen presentation. J Immunol 178: 2328-2335, 2007.
    OpenUrlAbstract/FREE Full Text
  24. ↵
    1. Jou ST,
    2. Carpino N,
    3. Takahashi Y,
    4. Piekorz R,
    5. Chao JR,
    6. Carpino N,
    7. Wang D,
    8. Ihle JN
    : Essential, nonredundant role for the phosphoinositide 3-kinase p110δ in signaling by the B-cell receptor complex. Mol Cell Biol 22: 8580-8591, 2002.
    OpenUrlAbstract/FREE Full Text
  25. ↵
    1. Hirsch E,
    2. Katanaev VL,
    3. Garlanda C,
    4. Azzolino O,
    5. Pirola L,
    6. Silengo L,
    7. Sozzani S,
    8. Mantovani A,
    9. Altruda F,
    10. Wymann MP
    : Central role for G protein-coupled phosphoinositide 3-kinase gamma in inflammation. Science 287: 1049-1053, 2000.
    OpenUrlAbstract/FREE Full Text
  26. ↵
    1. Pauls SD,
    2. Lafarge ST,
    3. Landego I,
    4. Zhang T,
    5. Marshall AJ
    : The phosphoinositide 3-kinase signaling pathway in normal and malignant B-cells: activation mechanisms, regulation and impact on cellular functions. Front Immunol 3: 224, 2012.
    OpenUrlPubMed
  27. ↵
    1. Gallouet AS,
    2. Travert M,
    3. Bresson-Bepoldin L,
    4. Guilloton F,
    5. Pangault C,
    6. Caulet-Maugendre S,
    7. Lamy T,
    8. Tarte K,
    9. Guillaudeux T
    : COX2-independent effects of celecoxib sensitize lymphoma B-cells to TRAIL-mediated apoptosis. Clin Cancer Res 20: 2663-2673, 2014.
    OpenUrlAbstract/FREE Full Text
  28. ↵
    1. Liang SH,
    2. Zhang W,
    3. McGrath BC,
    4. Zhang P,
    5. Cavener DR
    : PERK (eIF2alpha kinase) is required to activate the stress-activated MAPKs and induce the expression of immediate-early genes upon disruption of ER calcium homoeostasis. Biochem J 393(Pt 1): 201-209, 2006.
    OpenUrlAbstract/FREE Full Text
  29. ↵
    1. Liu M,
    2. Li CM,
    3. Chen ZF,
    4. Ji R,
    5. Guo QH,
    6. Li Q,
    7. Zhang HL,
    8. Zhou YN
    : Celecoxib regulates apoptosis and autophagy via the PI3K/AKT signaling pathway in SGC-7901 gastric cancer cells. Int J Mol Med 33: 1451-1458, 2014.
    OpenUrlPubMed
  30. ↵
    1. Price J,
    2. Zaidi AK,
    3. Bohensky J,
    4. Srinivas V,
    5. Shapiro IM,
    6. Ali H
    : AKT-1 mediates survival of chondrocytes from endoplasmic reticulum-induced stress. J Cell Physiol 222: 502-508, 2010.
    OpenUrlPubMed
  31. ↵
    1. Song S,
    2. Chew C,
    3. Dale BM,
    4. Traum D,
    5. Peacock J,
    6. Yamazaki T,
    7. Clynes R,
    8. Kurosaki T,
    9. Greenberg S
    : A requirement for the p85 PI3K adapter protein BCAP in the protection of macrophages from apoptosis induced by endoplasmic reticulum stress. J Immunol 187: 619-625, 2011.
    OpenUrlAbstract/FREE Full Text
  32. ↵
    1. Iyengar S,
    2. Clear A,
    3. Bödör C,
    4. Maharaj L,
    5. Lee A,
    6. Calaminici M,
    7. Matthews J,
    8. Iqbal S,
    9. Auer R,
    10. Gribben J,
    11. Joel S
    : P110α-mediated constitutive PI3K signaling limits the efficacy of p110δ-selective inhibition in mantle cell lymphoma, particularly with multiple relapse. Blood 121: 2274-2284, 2013.
    OpenUrlAbstract/FREE Full Text
  33. ↵
    1. Nishitoh H,
    2. Matsuzawa A,
    3. Tobiume K,
    4. Saegusa K,
    5. Takeda K,
    6. Inoue K,
    7. Hori S,
    8. Kakizuka A,
    9. Ichijo H
    : ASK1 is essential for endoplasmic reticulum stress-induced neuronal cell death triggered by expanded polyglutamine repeats. Genes Dev 16: 1345-1355, 2002.
    OpenUrlAbstract/FREE Full Text
  34. ↵
    1. Zheng QY,
    2. Li PP,
    3. Jin FS,
    4. Yao C,
    5. Zhang GH,
    6. Zang T,
    7. Ai X
    : Ursolic acid induces ER stress response to activate ASK1-JNK signaling and induce apoptosis in human bladder cancer T24 cells. Cell Signal 25: 206-213, 2013.
    OpenUrlPubMed
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Combining CAL-101 with Celecoxib Enhances Apoptosis of EBV-transformed B-Cells Through MAPK-induced ER Stress
GA BIN PARK, DAE YOUNG HUR, DAEJIN KIM
Anticancer Research May 2015, 35 (5) 2699-2708;
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