Abstract
The endoplasmic reticulum chaperone glucose-regulated protein 78 (GRP78) is selectively expressed on the surface of cancer cells, and contributes to the survival of cancer cells by forming complexes with p85α and promoting phosphatidylinositol 3-kinase–protein kinase B (PI3K–AKT) signaling. Hereιin we report that 2’-fluoro-6,7-methylenedioxy-2-phenyl-4-quinolone (CHM-1) induces apoptosis of human nasopharyngeal carcinoma (NPC) cells, as characterized by morphological changes, DNA fragmentation, caspase-3 activation, and cleavage of poly (ADP-ribose) polymerase. Using cell surface biotinylation, flow cytometric analysis, co-immunoprecipitation, and ectopic expression of GRP78, we demonstrated that the attenuation of the cell surface localization and complex formation with p85α of GRP78 by CHM-1 was involved in the inhibition of PI3K–AKT signaling and the induction of apoptosis. CHM-1 treatment induced phosphorylation on Thr 69 of B cell lymphoma 2 and inhibited phosphorylation of Ser 136 on Bcl-2-associated death promoter, that were reversed by overexpression of GRP78. We further observed that loss of mitochondrial membrane potential and increase in reactive oxygen species content, release of mitochondrial cytochrome c, caspase-9 activation, and apoptotic cell death induced by CHM-1, were suppressed by treatment with cyclosporine A, and by the overexpression of constitutively active AKT1 or GRP78. These results indicate that CHM-1 induces NPC cell apoptosis by suppressing the formation of the cell surface-associated GRP78–PI3K–AKT signaling complex, likely through inhibition of the formation of cell surface-associated GRP78–p85α complexes.
The 78-kDa glucose-regulated protein (GRP78) is known as binding immunoglobulin protein and heat-shock 70-kDa protein 5, and is one of the best characterized endoplasmic reticulum (ER) chaperone proteins, critical for ER integrity and as a master regulator of protein folding in the ER (1). The GRP78 exerts its anti-apoptotic effect by regulating the activation of the unfolded protein response signaling (2). Overexpression of GRP78 in cancer cells has already been demonstrated to confer protection against ER stress and chemotherapeutic agents (2, 3). Increased expression of GRP78 in cancer cells is suggested to be associated with poor clinical outcome in breast cancer (4), high tumor grade in hepatocellular carcinoma (5), and high rate of lymph node metastasis in gastric cancer (6). The mechanisms responsible for the protection by GRP78 from chemotherapeutic agent-induced apoptosis were shown to prevent protein misfolding, caspase activation, and ER targeting of pro-apoptotic proteins (1).
Phosphatidylinositol 3–kinase-protein kinase B (PI3K–AKT) signaling pathway is critical for cell proliferation, growth, metabolism, survival, anti-apoptosis, motility and metastasis of cancer cells (7, 8). PI3K activation results in the generation of phosphatidylinositol-3,4,5-trisphosphate (PIP3) from phosphatidylinositol-4,5-bisphosphate found in the membrane. PIP3 binds to AKT pleckstrin homology domain and phosphoinositide-dependent kinase 1 (PDK1), recruiting them by translocation from the cytoplasm to the membrane. AKT is then activated by the phosphorylation of threonine 308 (Thr 308) by PDK1 and of serine 473 (Ser 473) by PDK2 (9). Increased activity and dysregulation of AKT were associated with chemoresistance in a variety human cancer types (10). Active AKT exerts anti-apoptotic effects through phosphorylation of Bcl-2-associated death promoter (BAD) or caspase-9 (11-13). A recent study identified that cell surface GRP78 forms complexes with p85α to enhance PI3K–AKT signaling, thereby promoting chemoresistance of cancer cells (14). Although evidence that cell surface GRP78 is an upstream regulator of PI3K–AKT signaling came from the observation that GRP78 deficiency abrogates AKT activation and development of endometrial cancer (15), GRP78 acts as a downstream target of AKT in regulating cisplatin chemoresistance in endometrial cancer cells (16). GRP78 was found to be localized preferentially on the cell surface in cancer cells but not in normal cells in vivo (17-21). Liu et al. have developed a monoclonal antibody, MAb159, that specifically binds to and induces the endocytosis of cell-surface GRP78, and was found to have inhibitory effects on PI3K–AKT signaling and tumor growth in vivo (21), motivating us to select a pharmacological agent that attenuates cell surface localization of GRP78 to inhibit cancer cell growth.
In the present study, we investigated the effects of a synthetic 6,7-substituted 2-phenyl-4-quinolone, 2’-fluoro-6,7-methyle-nedioxy-2-phenyl-4-quinolone (CHM-1), on human nasopharyngeal carcinoma cells.
Materials and Methods
Cell culture. The human nasopharyngeal carcinoma cell lines NPC-TW 039 and NPC-TW 076 were obtained as previously described (22) and were cultured routinely in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal bovine serum (FBS) (both from Gibco BRL, Grand Island, NY, USA). These cell lines were grown in 10-cm tissue culture dishes at 37°C in a humidified incubator containing 5% CO2.
Chemicals, reagents and plasmids. Cyclosporine A, crystal violet, dantrolene, propidium iodide (PI), Tris-HCl, and Triton X-100, were obtained from Sigma-Aldrich (St. Louis, MO, USA). CHM-1 was obtained from the Graduate Institute of Pharmaceutical Chemistry, School of Medicine, China Medical University. CHM-1 was dissolved in and diluted with dimethyl sulfoxide (DMSO), and then stored at −20°C as a 100 mM stock solution. DMSO and potassium phosphate were purchased from Merck (Darmstadt, Germany). Lipofectamine 2000 was obtained from Invitrogen (Carlsbad, CA, USA). Penicillin-streptomycin, trypsin-EDTA, and glutamine were obtained from Gibco BRL. The caspase-3 activity assay kit was purchased from OncoImmunin (Gaithersburg, MD, USA). The inhibitors of pan-caspase (Z-VAD-FMK) and caspase-3 inhibitor (Ac-DEVD-CMK) were purchased from Calbiochem (San Diego, CA, USA). pcDNA3.1-78 kDa glucose-regulated protein (GRP78) and pcDNA- kinase-protein kinase B 1 (AKT1) vectors were obtained from Addgene (Cambridge, MA, USA). Western blot luminol reagent was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Antibodies. Antibody against p38 MAPK was obtained from Calbiochem. Antibodies against AKT, phospho (p)-AKT (Ser 473), ERK, p-ERK (Tyr 202/204), p-p38 MAPK (Thr 180/Tyr 182), p85α, p-p85α (Tyr 508), and Jun N-terminal kinase (JNK) were purchased from BD PharMingen. Antibodies to p-JNK (Thr 183/Tyr 185), GRP78, and poly (ADP-ribose) polymerase (PARP) were provided by Santa Cruz Biotechnology. Antibody against caspase-9 was obtained from Calbiochem (San Diego, CA, USA). Antibodies to BCL, BAD, BAX, and p-BAD (Ser 136) were provided by Cell Signaling Technology (Beverly, MA, USA). Antibodies against p-BCL2 (Thr 69) and cytochrome c were obtained from Abcam (Cambridge, MA, USA). Antibodies against β-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were obtained from Sigma-Aldrich. Peroxidase-conjugated anti-mouse IgG, -goat IgG, and -rabbit IgG secondary antibodies were purchased from Jackson ImmunoResearch Laboratory (West Grove, PA, USA).
Cell proliferation assay. Cell viability was assessed by fluorescence-activated cell sorting (FACS) analysis of cellular PI uptake (23). The cells were seeded at 3×104 cells/well in 24-well tissue culture plates. The cells were grown for overnight to about 60% confluence and treated with either DMSO as the vehicle control or the indicated concentration of CHM-1 for the indicated periods of time. For a vehicle control, DMSO was diluted in culture medium to the same final concentration of DMSO (0.01% (vol/vol)) in the medium with CHM-1. At the end of the incubation, treated cells were harvested and stained with 10 μg/ml of PI solution. The stained cells were analyzed using a FACSCount flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA), and the results were analyzed using CellQuest software (BD Biosciences).
Assays for the detection of caspase-3 activity. Caspase-3 activity was measured using the PhiPhiLux G1D2 kit (OncoImmunin, College Park, MD, USA) according to the manufacturer's protocols. For the detection of caspase-3 activity, the treated NPC cells were incubated with the PhiPhiLux fluorogenic caspase substrate at 37°C for 1 h and were then analyzed using a FACSCount flow cytometer.
Western blot analysis and co-immunoprecipitation assays. The treated cells were lysed and subjected to western blotting as described previously (24). For the co-immunoprecipitation assays, extracts from biotinylated cells were immunoprecipitated with either anti-p85α or anti-GRP78 or with normal control IgG and then incubated with protein A-agarose beads as previously described (24). After incubation at 4°C for 2 h, the immune complexes were analyzed by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting with antibodies to GRP78, p85α and AKT.
Cell surface biotinylation. This assay was performed as previously described (23). Briefly, treated cells were washed twice in ice-cold PBS and incubated with 0.5 mg/ml of EZ-Link Sulfo-NHS-SS-Biotin (Pierce, Rockford, IL, USA) for 30 min at 4°C. Biotinylated cells were washed twice in ice-cold PBS and treated with 50 mM NH4Cl for 10 min at 4°C to stop the biotinylation reaction. Avidin-agarose beads (Pierce, Rockford, IL, USA) were then added to the biotinylated cells, and the mixture was incubated with gentle rocking at 4°C for 16 h. The beads were pelleted and washed three times with 500 μl of ice-cold PBS. Bound proteins were mixed with 1× SDS sample buffer and incubated for 5 min at 100°C. The proteins were then separated by 10% SDS-PAGE and immunoblotted with antibody against GRP78.
Measurement of cell surface or intracellular GRP78 by flow cytometry. This assay was performed as previously described (23). Briefly, treated cells (1×106) were detached from culture plates by 1 mM EDTA, washed twice with PBS, and incubated with 10% normal human serum in PBS for 20 min on ice to block fragment crystallizable receptors (FcRs) on the cell surface. The cells were washed three times with ice-cold PBS and then incubated with 0.5 μg anti-GRP78 for 30 min on ice in 50 μl of staining buffer (2% FCS in 1× PBS). For the staining of intracellular GRP78, treated cells (1×106) were formaldehyde-fixed and permeabilized with 0.03% saponin. Intracellular staining was performed in 0.03% saponin in 1× PBS with anti-GRP78. After washing with staining buffer, cells were incubated with fluorescein isothiocyanate (FITC)-conjugated secondary antibody and analyzed on a FACSCount flow cytometer (25).
Plasmid transfection. Cells (at 60-70% confluence in a 12-well plate) were transfected with the GRP78 or CA-AKT1 expression plasmid using Lipofectamine 2000. The expression of GRP78 and AKT in transfected cells was assessed by Western blotting using antibodies specific to GRP78 and AKT.
Detection of cytochrome c. Sub-cellular fractionation was performed as previously described (26). The treated cells were washed twice with ice-cold PBS and scraped into a 200 mM sucrose solution containing 25 mM HEPES (pH 7.5), 10 mM KCl, 15 mM MgCl2, 1 mM EDTA, 1 mM EGTA, and 1 μg/ml aprotinin. The cells were disrupted by passage through a 26-gauge hypodermic needle 30 times and then centrifuged for 10 min in an Eppendorf microcentrifuge (5804R) at 750 × g at 4°C to remove unlysed cells and nuclei. The supernatant was collected and then centrifuged for 20 min at 10,000 × g at 4°C to form a new supernatant and pellet. The resulting pellet was saved as the mitochondrial (Mt) fraction, and the supernatant was further centrifuged at 100,000 × g for 1 h at 4°C. The new supernatant was saved as the cytosolic fraction, and the pellet was reserved as the ER/microsomal fraction. The resulting fractions were lysed in RIPA buffer [1% sodium deoxycholate, 0.1% SDS, 1% Triton X-100, 10 mM Tris-HCl (pH 8.0), and 0.14 M NaCl] for western blot analysis. The purity of each subcellular fraction was confirmed by western blotting using specific antibodies against the mitochondrial marker cytochrome c oxidase subunit 2 (Abcam, Cambridge, MA, USA), and the cytosolic marker γ-tubulin.
Measurement of mitochondrial membrane potential. Mitochondrial membrane potential (ψm) was determined by measuring the retention of the dye 3,3’-dihexyloxacarbocyanine (DiOC6). Briefly, treated cells were incubated with 40 nM DiOC6 for 30 min at 37°C. Cells were then pelleted by centrifugation at 160 × g. Pellets were re-suspended and washed twice with PBS. The ψm was determined with a FACSCount flow cytometer (25).
Detection of reactive oxygen species (ROS). Briefly, treated cells were then resuspended in 500 μl of 2,7-dichlorodihydrofluorescein diacetate (10 μM) and incubated for 30 min at 37°C. The level of ROS was determined using a FACSCount flow cytometer (25).
Statistical analysis of data. Statistical calculations of the data were performed using the unpaired Student's t-test and ANOVA analysis. A value of p<0.05 was considered statistically significant.
Results
Caspase-dependent apoptosis was induced by CHM-1 in NPC cells. We first determine the effect of CHM-1 on NPC cell viability using PI staining and flow cytometric analysis. As shown in Figure 1A, CHM-1 significantly reduced NPC cell viability in a dose- and time-dependent manner. Treatment with CHM-1 for 36 h resulted in a decrease in the viability of the NPC-TW 039 and NPC-TW 076 cell lines, with half-maximal inhibitory concentration for cell viability (IC50) values of 0.3 and 0.2 μM, respectively (Figure 1A). An increased number of apoptotic bodies was observed in NPC cells treated with 0.3 or 0.2 μM CHM-1 for 36 h (Figure 1B). To investigate whether induction of cell death by CHM-1 could be linked to apoptosis and caspase activation, apoptotic DNA fragmentation was assessed by cell-death detection enzyme-linked immunosorbent assay (ELISA) for histone-associated DNA fragments, and caspase-3 activity was determined by flow cytometry (Figure 2A). As expected, significant increases in DNA fragmentation and caspase-3 enzymatic activity were detected in CHM-1-treated NPC cells, and these effects were nearly completely inhibited by the caspase 3 inhibitor Ac-DEVD-CMK and the pan-caspase inhibitor Z-VAD-FMK (Figure 2A and B). The induction of apoptosis and caspase-3 activation was further confirmed by the cleavage of procaspase-3 and PARP, detected using western blot analysis. Thus, concentrations of 0.3 and 0.2 μM were used to treat NPC cells in all subsequent experiments to assess mechanisms that trigger cell apoptosis. These results indicate that caspase 3-dependent apoptotic activity was involved in CHM-1-induced NPC cell death.
CHM-1 suppresses GRP78–p85α complex formation on the cell surface, contributing to inhibition of PI3K–AKT–ERK-p38 MAPK activation, phosphorylation of BCL2 at Thr 69, de-phosphorylation of BAD at Ser 136, and NPC cell apoptosis. Figure 3A shows that the treatment of NPC cells with CHM-1 inhibited phosphorylation of AKT Ser 473, although the level of p-p85α (Tyr 508) was unaffected. We also detected decreased phosphorylation of ERK (Tyr 202/204) and p38 MAPK (Thr 183/Tyr 185) in western blot analysis using antibodies that specifically recognize the phosphorylated protein on these sites. Interestingly, GRP78 protein expression decreased in lysates from CHM-1-treated cells (Figure 3A). As the BCL2 family of proteins can modulate ER function and thereby control cell survival (27, 28), we analyzed the phosphorylation and protein levels of BCL2 family members. CHM-1 treatment did not affect the levels of BAX and BAD, whereas the treatment caused BCL2 phosphorylation at Thr 69 and inhibited BAD phosphorylation at Ser 136 (Figure 3B).
The results of recent studies indicate that the cell-surface localization of GRP78 and the formation of GRP78-p85α complexes confer activation of PI3K–AKT signaling and survival of cancer cells (14). To determine whether the inhibited activation of PI3–AKT signaling by CHM-1 requires cell-surface targeting of GRP78, we examined the cell-surface level of GRP78. We chose NPC-TW 076 cells because they exhibit high sensitivity to CHM-1. Western blot analysis of streptavidin-agarose bead-bound protein from biotinylated cells revealed a high level of cell-surface GRP78 in cells treated with vehicle. With CHM-1 treatment, cell-surface localization of GRP78 was almost completely inhibited, although CHM-1 did reduce the level of cytosolic GRP78 (Figure 4A). Flow cytometric analysis further confirmed that the cell-surface localization of GRP78 was suppressed by CHM-1. To address whether this suppression was linked to the inhibition of PI3K–AKT signaling, GRP78 was ectopically expressed in CHM-1 treated cells. Compared to vehicle-treated control vector-transfected cells, overexpression of GRP78 resulted in an increase phosphorylation of AKT at Ser 473, which simultaneously increased ERK–p38 MAPK signaling as shown by an increase of p-ERK (Tyr 202/204) and p-p38 MAPK (Thr 180/Tyr 182) (Figure 4A). The intensities of cell-surface and cytosolic GRP78 were increased 3.48- and 2.28-fold in GRP78-transfected cells compared with vehicle-treated control vector-transfected cells, respectively (Figure 4B). Moreover, ectopic expression of GRP78 overcame the inhibition of PI3K–AKT–ERK–p38 MPAK signaling, BCL2 phosphorylation (Thr 69), BAD dephosphorylation (Ser 136), and apoptosis induced by CHM-1 (Figures 4A, B and D). Co-immunoprecipation assay from biotinylated proteins isolated with streptavidin-agarose beads using antibody specific for GRP78 revealed that GRP78 formed a complex with p85α at the cell surface. CHM-1 treatment suppressed GRP78 complex with p85α at the cell surface. Overexpression of GRP78 restored formation of GRP7–p85α complexes in the presence of CHM-1 (Figure 4C), suggesting that CHM-1 suppresses the formation of cell surface-associated GRP78–p85α complexes to affect the transduction of PI3K–AKT–ERK–p38 MAPK signaling pathway and cell survival.
CHM-1-induced apoptosis requires GRP78–mediated activation of the ER mitochondrial apoptotic cell death pathway. To investigate whether CHM-1 treatment could induce ER and mitochondrial dysfunction, levels of ψm and ROS were determined by flow cytometry. Exposing cells to CHM-1 caused a rapid decrease in ψm, and this reduction was completely inhibited by constitutively active AKT1 expression. The alteration of ψm was significantly inhibited by cyclosporine A and GRP78 overexpression (Figure 5A). Ectopic expression of constitutively active AKT1 also almost completely inhibited the increase in ROS caused by CHM-1 (Figure 5A). CHM-1-induced apoptosis and increase in ROS were, however, significantly suppressed by the addition of cyclosporine A, or ectopic expression of GRP78 (Figure 5A). Additionally, CHM-1-induced cleavage of pro-caspase-9 and release of cytochrome c from mitochondria were inhibited by co-treatment with dantrolene, cyclosporine A, and GRP78 overexpression. These data indicate that suppression of the cell-surface localization of GRP78 was involved in CHM-1-induced mitochondrial apoptotic cell death.
Discussion
Our findings show that CHM-1 induced suppression of localization of GRP78 to the cell surface and attenuated the PI3K–AKT signaling transduction pathway to promote NPC cell apoptosis. The inhibitory effect of CHM-1 on PI3K–AKT signaling involves suppression of the formation of cell surface-associated GRP78–p85α complexes. In view of the observed the reversion of the CHM-1-induced reduction in AKT phosphorylation (Ser 473) and Δψm, cytosolic ROS elevation, pro-caspase-9 cleavage, and mitochondrial cytochrome c release by GRP78 overexpression. Although AKT phosphorylation is not always correlated with PI3K-mediated oncogenic activity and cell survival (29), promoting AKT activity with expression of constitutively active AKT1 overcomes CHM-1-induced mitochondrial cell death. Therefore, it is logical to suggest that cell-surface GRP78-regulated PI3K–AKT signaling has physiological relevance in regulating the function and integrity of mitochondria in NPC cells. The reduction of AKT phosphorylation at Ser 473 does rule-out the possibility of target effect by CHM-1 on modulation of PI3K activity in the process, CHM-1 does not affect the kinase activity of PI3K, as determined by using recombinant purified p85α/p110α proteins (data not shown). Could the inhibitory effect of CHM-1 on the catalytic activity of the p110 subunit β or γ be related to the decrease in AKT Ser 473 phosphorylation? This possibility cannot be ruled-out with absolute certainty.
Induction of ER stress has been implicated in enhancing cell-surface localization of GRP78 (30). The use of anti-GRP78 monoclonal antibody (MAb159)-mediated inhibition of GRP78 localization on the cell surface and PI3K–AKT activation was shown to trigger endocytosis of GRP78 (21). It was reported that CHM-1 exhibits tubulin-binding activity, which can inhibit tubulin polymerization and disrupt microtubule organization (31). Our finding was the suppression of the cell-surface localization of GRP7 by CHM-1. The total level of GRP78 decreased in the presence of CHM-1. These observations suggest a possible inhibitory effect of CHM-1 on the cell-surface localization of GRP78 that might perturb endocytic trafficking, as well as microtubule organization. However, identification of CHM-1 as an inhibitor for suppressing cell-surface localization of GRP78 does not rule-out possible involvement of the dysregulation of GRP78 gene expression or GRP78 degradation.
Although BCL2 phosphorylation at multi-site residues (Thr 69, Ser 70 and Ser 87) was correlated with increased cell survival (32), several studies have shown that phosphorylation of BCL2 contributes to the pro-apoptotic function of BCL2 (33-39). Phosphorylation of BCL2 Thr 69 has been reported to be involved in increasing the susceptibility of cancer cell to apoptosis induced by tubulin-binding agent paclitaxel (Taxol) (28). As with Taxol, BCL2 phosphorylation at Thr 69 was observed in CHM-1-treated NPC cells. The apparent CHM-1-induced BCL2 Thr 69 phosphorylation was suppressed by GRP78 overexpression. Forced expression of GRP78 attenuated the inhibition of ERK and p38 MAPK activation by CHM-1. This shows the importance of the role of ERK in modulating the phosphorylation of BCL2 in mitochondria (28). ERK has been shown to co-localize with BCL2 in the mitochondria (40). In accordance with this, our results suggest that the cell surface-associated GRP78–PI3K–AKT-mediated ERK activity might play a role in regulating the BCL2-mediated apoptotic and pro-apoptotic functions of BCL2-mediated in NPC cells. How ERK activity impacts on our observation that BCL2 Thr 69 phosphorylation is required for the function and integrity of mitochondria in NPC cells, remains to be determined.
In conclusion, the induction of NPC cell apoptosis by CHM-1 was due to an inhibition of the formation of GRP78–p85α complexes. These data indicate that PI3K activity was required for the induction of mitochondrial apoptotic cell death induced by CHM-1. Furthermore, CHM-1 induced activation of ERK and subsequent activation of p38 MAPK, which are regulated by PI3K.
Acknowledgements
M.L. Lin was supported by grants from China Medical University (CMU99-COL-22-1 and CMU99-COL-22-2).
Footnotes
Conflicts of Interest
The Authors state that there are no financial or personal relationships with other people or organizations that could inappropriately influence (bias) our work in this study.
- Received May 22, 2015.
- Revision received June 24, 2015.
- Accepted June 26, 2015.
- Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved