CHM-1 SUPPRESSES FORMATION of CELL SURFACE-ASSOCIATED GRP78– p85 COMPLEXES, INHIBITING PI3K–AKT SIGNALING and INDUCING
APOPTOSIS of HUMAN NASOPHARYNGEAL CARCINOMA CELLS
MENG-LIANG LIN1, SHIH-SHUN CHEN2, SUE-HWEE NG1
1 Department of Medical Laboratory Science and Biotechnology, China Medical University,
Taichung 40402, Taiwan
2Department of Medical Laboratory Science and Biotechnology, Central Taiwan University
of Science and Technology, Taichung 40601, Taiwan
Running title: CHM-1 induces NPC cell apoptosis
Key words: apoptosis, CHM-1, GRP78, nasopharyngeal carcinoma, p85
Correspondence to: Dr. M.-L. Lin, Department of Medical Laboratory Science and
Biotechnology, China Medical University, No. 91, Hsueh-Shih Road, Taichung City 40402, Taiwan; Telephone: +886 4 22053366 ext. 7211; Fax: +886 4 22057414; e-mail:
Abstract. The endoplasmic reticulum (ER) chaperone GRP78 is selectively expressed on the 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
surface of cancer cells, which contributes to the survival of cancer cells by forming complexes with p85 and promoting phosphatidylinositol 3-kinase–protein kinase B (PI3K–Akt) signaling. Here we report that 2’-fluoro-6,7-methylenedioxy-2-phenyl-4-quinolone (CHM-1) induces human nasopharyngeal carcinoma (NPC) cell apoptosis, which was characterized by morphological changes, DNA fragmentation, caspase-3 activation, and cleavage of poly (ADP-ribose) polymerase (PARP). Using cell surface biotinylation, flow cytomeric analysis, co-immunoprecipitation, and ectopic expression of GRP78, we demonstrate that the attenuation of the cell surface localization and complex formation with p85 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 Bcl-2 and inhibited phosphorylation of Ser 136 on Bad, which were reversed by overexpression of GRP78. We further observed that CHM-1-induced loss of mitochondrial membrane potential and increase in reactive oxygen species (ROS) content, the release of mitochondrial cytochrome c, caspase-9 activation, and apoptotic cell death were suppressed by the treatment of cyclosporine A or the overexpression of constitutively active Akt1 or GRP78. Taken together, these results indicate that CHM-1 induces NPC cell apoptosis by suppression the formation of the cell surface-associated GRP78–PI3K–Akt signaling complex, likely through the inhibition of the formation of cell surface-associated GRP78–p85 complexes.
Introduction 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39
The 78 kDa glucose-regulated protein (GRP78) is known as binding immunoglobulin protein (BiP) or heat shock 70 kDa protein 5 (HSPA5), which is one of the best characterized endoplasmic reticulum (ER) chaperone protein 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 (UPR) signaling (2). Overexpression of GRP78 in cancer cells has already been demonstrated to confer protection against ER stress and chemotherapeutic agents (2, 3). Increasing the 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 proapoptotic proteins (1).
Phosphatidylinositol 3–kinase-protein kinase B (PI3K–Akt) signaling pathway is critical for cell proliferation, growth, metabolism, survival, and the antiapoptosis, 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 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 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58
PDK2 (9). The increased activity and dysregulation of Akt were associated with chemoresistance in a variety human cancer types (10). Active Akt exerts antiapoptotic effect through phosphorylation of Bad or caspase-9 (11-13). Recent study identified that cell surface GRP78 forms complexes with p85 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 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 has shown 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 this study, we report a synthetic 6,7-substituted 2-phenyl-4-quinolone, 2’-fluoro-6,7-methylenedioxy-2-phenyl-4-quinolone (CHM-1), which induces induced apoptosis in human nasopharyngeal carcinoma cells is dependent on the suppression of the cell surface localization of GRP78 and the formation of GRP78–p85 complexes. The suppression of cell surface-associated GRP78–PI3K–Akt signaling by CHM-1 is important for Bad 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77
dephosphorylation and Bcl-2 hyperphosphorylation and the subsequent apoptosis of cancer cells via the ER-mitochondrial death pathway.
Materials and methods
Cell culture. The human nasopharyngeal carcinoma cell lines (NPC-TW 039 and NPC-TW
076) were obtained as previously described (22). The NPC-TW 039 and NPC-TW 076 cell 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98
lines were cultured routinely in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal bovine serum (FBS). 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 (CsA), crystal violet, propidium iodide
(PI), Tris-HCl, and Triton X-100, were obtained from Sigma-Aldrich (St. Louis, MO, USA). The 2’-fluoro-6,7-methylenedioxy-2-phenyl-4-quinolone (CHM-1) was dissolved in and diluted with DMSO and then stored at –20oC as a 100 mM stock. DMSO and potassium phosphate were purchased from Merck (Darmstadt, Germany). Lipofectamine 2000 was obtained from Invitrogen (Carlsbad, CA, USA). DMEM, FBS, penicillin-streptomycin, trypsin-EDTA, and glutamine were obtained from Gibco BRL (Grand Island, NY, USA). 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-GRP78 and pcDNA-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 (San Diego, CA,
USA). Anti-Akt, -phospho (p)-Akt (Ser 473), -ERK, -p-ERK (Tyr 202/204), -p-p38 MAPK 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117
(Thr 180/Tyr 182), p85, p-p85 (Tyr 508), and JNK antibodies were purchased from BD PharMingen. Anti-p-JNK (Thr 183/Tyr 185) and GRP78 antibodies were provided by Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-Bcl-2, Bad, p-Bad (Ser 136) antibodies were provided by Cell Signaling Technology (Beverly, MA, USA). Antibody against p-Bcl-2 (Thr 69) was obtained from Abcam (Cambridge, MA, USA). Antibodies against -actin and 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). Tthe 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, Franklin Lakes, NJ, USA).
Assays for the detection of caspase-3 activity. Caspase-3 activity was measured using the
PhiPhiLux G1D2 kit (OncoImmunin, College Park, MD, USA) ccording 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.
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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-p85 or anti-GRP78 antibodies 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% SDS-PAGE and immunoblotting with GRP78 and p85 anti-Akt antibodies.
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 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. The avidin-agarose beads 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 l 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
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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 Fc receptors on the cell surface. The cells were washed three times with ice-cold PBS and then incubated with 0.5 g anti-GRP78 antibody for 30 min on ice in 50 l 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% sapornin. Intracellular staining was performed in 0.03% sapornin in 1× PBS with anti-GRP78 antibody. After washing with staining buffer, cells were incubated with FITC-conjugated secondary antibody and analyzed on a FACSCount flow cytometer (25).
Detection of Cyt c. Subcellular fraction was 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 g/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 at10,000 ×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 (Cs) fraction, and 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175
the pellet was reserved as the ER/microsomal (Ms) fraction. The resulting Mt and Ms 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 Cox-2, and the Cs marker -tubulin.
Measurement of mitochondrial membrane potential. Mitochondrial membrane potential (Δψm) 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 resuspended and washed twice with PBS. The Δψm 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. p < 0.05 was considered statistically significant. 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194
Results
Caspase-dependent apoptosis 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 cell viability in a dose- and time-dependent manner in NPC cells. Treatment with TSWU-CD4 for 36 h resulted in a decrease in the viability of the NPC-TW 039 and NPC-TW 076 cell lines, with IC50 values of 0.3 and 0.2 μM, respectively (Figure 1A). An increased number of apoptotic bodies was observed in NPC cells 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216
treated with 0.3 or 0.2 μM CHM-1 for 36 h (Figure 1B). To investigate whether the induction of cell death by CHM-1 could be linked to apoptosis and caspase activation, apoptotic DNA fragmentation was assessed by cell death detection 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 (Figures 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 caspae-3-dependent apoptotic activity was involved in CHM-1-induced NPC cell death.
CHM-1 suppresses GRP78–p85 complex formation on cell surface, contributing to
inhibition of PI3K–Akt–ERK-p38 MAPK activation, phosphorylation of Bcl-2 at Thr 69, dephosphorylation of Bad at Ser 136, and NPC cell apoptosis. Figure 3A shows that the
treatment of NPC cells with CHM-1 inhibited Akt Ser 473 phosphorylation, 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 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235
antibodies that specifically recognize phosphorylated protein on these sites. Interestingly, decreased level in GRP78 protein was observed in CHM-1-treated cell lysates (Figure 3A). As the Bcl-2 family of proteins can modulate ER function and thereby control cell survival (27, 28), we analyzed the phosphorylation and protein levels of Bcl-2 family members. CHM-1 treatment did not affect the levels of Bax and Bad, whereas the treatment caused Bcl-2 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-p85 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-TW076 cells because they exhibit high sensitivity to CHM-1. Western blot analysis of streptavidin-agarose bead-bound protein from biotinylated cells detected a high level of cell surface GRP78 (s-GRP78) in cells treated with vehicle. With CHM-1 treatment, cell surface localization of GRP78 was almost completely inhibited, although CHM-1 did decrease the level of cytosolic GRP78 (c-GRP78) (Figure 4A). Flow cytometric analysis further confirmed that the cell surface localization of GRP78 was suppressed by CHM-1, To address whether CHM-1’s suppression of cell surface localization of GRP7 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 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254
resulted in an increase phosphorylation of Akt at Ser 473, which simultaneously increased ERK–p38 MAPK signaling as shown an increase of p-ERK (Tyr 202/204) and p-p38 MAPK (Thr 180/Tyr 182) (Figure 4A). The intensities of s-GRP78 and c-GRP78 were increased 3.48- and -2.28 folds in GRP78-transfected cells compared with vehicle-treated control vector-transfected cells, respectively (Figure 4B). Moreover, ectopic expression of GRP78 could overcome the inhibition of PI3K–Akt–ERK–p38 MPAK signaling, Bcl-2 phosphorylation (Thr 69), Bad dephosphorylation (Ser 136), apoptosis induced by CHM-1 (Figures 4A, 4B, and 4D). Co-immunoprecipation assay from biotinylated proteins isolated with streptavidin-agarose beads using antibody specific for GRP78 revealed that GRP78 formed a complex with p85α in cell surface. CHM-1 treatment suppressed the localization of GRP78 on cell surface to form a complex with p85α. Overexpression of GRP78 restored formation of GRP7–p85α complexes in the presence of CHM-1 (Figure 4C), suggesting that CHM-1 suppresses 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 Δψm and ROS were determined by flow cytometry. Exposing cells to CHM-1 caused a rapid decrease in Δψm levels, and the reduction in Δψm was 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273
completely inhibited by constitutively active Akt1 (CA-Akt1) expression. The alteration of Δψm was significantly inhibited by CsA and GRP78 overexpression (Figure 5A). Ectopic expression of CA-Akt1 also almost completely inhibited the increase in ROS caused by CHM-1 (Figure 5A). The CHM-1-induced apoptosis and an increase in ROS were, however, significantly suppressed by the addition of CsA or ectopic expression of GRP78 (Figure 5A). Additionally, CHM-1-induced cleavage of pro-caspase-9 and release of Cyr c from mitochondria were inhibited by co-treatment with dantrolene, CsA, and GRP 78 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 the localization of GRP78 to the cell surface attenuates PI3K–Akt signaling transduction pathway to promote NPC cell apoptosis. With regards to the inhibitory effect of CHM-1 on the attenuation of PI3K–Akt signaling involves suppressing 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 Cyt c release by GRP78 overexpression. Although Akt phosphorylation is not always correlated 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293
with PI3K-mediated oncogenic activity and cell survival (29), promoting Akt activity with an expression of CA-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 a related to the decreased AKT Ser 473 phosphorylation? This is 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 ploymerization and disrupt microtubule organization (31). Our findings is the suppression of the cell surface localization of GRP7 by CHM-1. The total level of GRP78 is 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 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312
suppressing the cell surface localization of GRP78 does not rule out possible involvement of the dysregulation of Grp78 gene expression or GRP78 degradation.
Although, Bcl-2 phosphorylation at multi-site residues (Thr 69, Ser 70 and Ser 87) were correlated with increased cell survival (32), several studies show that phosphorylation of Bcl-2 contributes pro-apoptotic function of Bcl-Bcl-2 (33-39). Phosphorylation of Bcl-Bcl-2 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). Like Taxol, Bcl-2 phosphorylation at Thr 69 was observed in CHM-1-treated NPC cells. The apparent CHM-1-induced Bcl-2 Thr 69 phosphorylation was suppressed by GRP78 overexpression. Forced expression of GRP78 attenuated inhibition of the ERK and p38 MAPK activation by CHM-1. Evidence show the importance of the role of ERK in modulating the phosphorylation of Bcl-2 in the mitochondria (28). The fact that ERK has been shown to co-localize with Bcl-2 in in the mitochondria (40). Accordingly, our results suggest that the cell surface-associated GRP78– PI3K–Akt-mediated ERK activity might play a role in regulating the Bcl-2-mediated apoptotic and pro-apoptotic functions of Bcl-2-mediated in NPC cells. How ERK activity effect on our observation that Bcl-2 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 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331
for the induction of mitochondrial apoptotic cell death stimulated by CHM-1. Furthermore, CHM-1 induced activation of ERK and subsequent activation of p38 MAPK, which are regulated by PI3K.
Conflict of interest statement
We (the authors) disclose that there are no financial or personal relationships with other people or organizations that could inappropriately influence (bias) our work, “CHM-1 suppresses formation of cell surface-associated GRP78–p85α complexes, inhibiting PI3K– Akt signaling and inducing apoptosis of human nasopharyngeal carcinoma cells”.
Acknowledgements 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351
CMU99-COL-22-2).
Figure legends
Figure 1. CHM-1 induces cell death and morphological changes in NPC cells. (A) The effect of CHM-1 on NPC cell viability. Cells were plated in 24-well plates and treated with either DMSO (vehicle control) or the indicated concentration of CHM-1 for the indicated periods of time. After treatment, cell viability was determined by flow cytometric analysis of PI uptake. The values presented are the mean standard error from three independent experiments. *p<0.05, significantly different from vehicle control treated cells. (B) CHM-1 induces morphological changes in NPC cells. Cells were plated in 24-well plates and treated for 36 h with vehicle control or the indicated concentration of CHM-1. After treatment, cell morphology was observed under an inverted phase contrast microscope (100× magnification). Arrowheads indicate apoptotic cells.
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Figure 2. CHM-1 induced DNA fragmentation, caspase-3 activation and PARP cleavage in NPC cells. (A and B) Effects of CHM-1 on the induction of DNA fragmentation and activation of caspase-3. Cells were treated with vehicle control, CHM-1 (0.3 M for NPC-TW 039 and 0.2 M for NPC-TW 076), CHM-1 plus Ac-DEVD-CMK, or CHM-1 plus Z-VAD-FMK for the indicated periods of time. DNA fragmentation and caspase-3 activities were measured using a Cell Death Detection kit and flow cytometry, respectively. The values presented are the mean standard errors from three independent experiments. *p<0.05, significantly different from vehicle control treated cells. (C) Effect of CHM-1 on the cleavage of PARP. Cells were treated with either vehicle or indicated concentration of CHM-1 for 36 h. The protein levels of the indicated proteins in the cell lysates were determined by Western blot analysis using specific antibodies. GAPDH was used as internal controls for sample loading.
Figure 3. Inhibition of PI3K–Akt–ERK–p38 MAPK signaling pathway and GRP78 expression and induction of Bcl-2 Thr 69 phosphorylation and Bax Ser 136 dephosphorylation by CHM-1. (A and B) Cells were treated with either vehicle or CHM-1 (0.3 M for NPC-TW 039 and 0.2 M for NPC-TW 076) for 36 h. The protein levels of the indicated proteins in the cell lysates were determined by Western blot analysis using specific 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391
antibodies. GAPDH was used as internal controls for sample loading.
Figure 4. Ectopic expression of GRP78 attenuated CHM-1-induced inhibition of the PI3K– Akt–ERK–p38 MAPK signaling pathway and GRP78–p85α complex formation. (A) NPC-TW 076 cells were treated with either vehicle or CHM-1 (0.2 M) for 36 h. Treated cells were biotinylated as described in the Material and Methods section. Biotinylated proteins were pulled down using streptavidin agarose beads. The biotin-streptavidin complexes were immunoblotted with GRP78 antibody. (B-D) NPC-TW 076 cells were treated with either vehicle or CHM-1 (0.2 M) for 36 h. The GRP78 cell surface and intracellular levels were determined by flow cytometry. Co-immunoprecipitation of GRP78 and p85α was performed using biotinylated proteins isolated with streptavidin-agarose beads from cells that were treated for 36 h with 0.2 M CHM-1. The antibodies used for co-immunoprecipitation are indicated at the top of the figure. The proteins in the immunoprecipitated complexes were analyzed by Western blot using specific antibodies. The values presented are the mean the standard error from three independent experiments. *p＜0.05: significantly different from the CHM-1-treated cells. DNA fragmentation was measured using a Cell Death Detection kit.
Figure 5. GRP78-mediated Akt activity is involved in CHM-1-induced Δψm loss, increases in cytosolic ROS level, the release of Cyt c from mitochondria, and apoptosis. At 12 h after 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410
transfection with vector alone, pcDNA3.1-GRP78, or pCA-Akt1, cells were treated with either vehicle, CHM-1 or CsA for 36 h. The levels of the indicated proteins in the lysates of T, Mt, and Cs were determined by Western blot analysis using specific antibodies. Cox-2 and -actin were used as internal controls for the mitochondria and cytosol, respectively. GAPDH was used as internal controls for sample loading. The decrease in DiOC6 fluorescence was measured by flow cytometry. The generation of ROS was monitored by measuring increased fluorescence of Indo-1 by flow cytometry. DNA fragmentation was determined using a Cell Death Detection ELISA kit.
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