Apoptosis DOI 10.1007/s10495-014-1031-y

ORIGINAL PAPER

Suppression of PI3K/Akt signaling by synthetic bichalcone analog TSWU-CD4 induces ER stress- and Bax/Bak-mediated apoptosis of cancer cells Meng-Liang Lin • Shih-Shun Chen • Ren-Yu Huang • Yao-Cheng Lu • Yu-Ren Liao • Mopuru Vijaya Bhaskar Reddy Chuan-Chun Lee • Tian-Shung Wu

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Ó Springer Science+Business Media New York 2014

Abstract Suppression of the activity of pro-apoptotic Bcl-2-family proteins frequently confers chemoresistance to many human cancer cells. Using subcellular fractionation, the ER calcium (Ca??) channel inhibitor dantrolene and small interfering RNA (siRNA) against Bax or Bak, we show that the new synthetic bichalcone analog TSWU-CD4 induces apoptosis in human cancer cells by releasing endoplasmic reticulum (ER)-stored Ca?? through ER/ mitochondrial oligomerization of Bax/Bak. Blockade of the protein kinase RNA-like ER kinase or the unfolded protein response regulator glucose-regulated protein 78 expression by siRNA not only suppressed oligomeric Bax/Bak-mediated pro-caspase-12 cleavage and apoptosis but also resulted in an inhibition of Bcl-2 downregulation induced by TSWU-CD4. Induction of the ER oligomerization of Bax/Bak and apoptosis by TSWU-CD4 were suppressed by Bcl-2 overexpression. Inhibition of lipid raft-associated phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling by TSWU-CD4 induced ER stress- and oligomeric Bax/Bak-mediated apoptosis, which were substantially reversed by overexpression of the wt PI3K p85a

M.-L. Lin (&)  R.-Y. Huang  C.-C. Lee Department of Medical Laboratory Science and Biotechnology, China Medical University, No. 91, Hsueh-Shih Road, Taichung 40402, Taiwan e-mail: [email protected] S.-S. Chen  Y.-C. Lu Department of Medical Laboratory Science and Biotechnology, Central Taiwan University of Science and Technology, Taichung 40601, Taiwan Y.-R. Liao  M. V. B. Reddy  T.-S. Wu (&) Department of Chemistry, National Cheng Kung University, No. 1, University Road, T’ainan 70101, Taiwan e-mail: [email protected]

subunit. Taken together, these results suggest that suppression of lipid raft-associated PI3K/Akt signaling is required for the ER stress-mediated apoptotic activity of Bax/Bak, which is responsible for the ability of TSWUCD4-treated cancer cells to exit the ER-mitochondrial apoptotic cell death pathway. Keywords Apoptosis  TSWU-CD4 ((2E,20 E)-1,10 -(5,50 (piperazine-1,4-diylbis(methylene))bis(4-hydroxy-3methoxy-5,1-phenylene))bis(3-phenylprop-2-en-1-one))  Bax/Bak  Bcl-2  Phosphatidylinositol 3-kinase (PI3K)

Introduction Apoptosis is a mechanism of highly programmed cell death that maintains tissue and cellular homeostasis. The impaired apoptosis of cells is now recognized to be a critical step in carcinogenesis and is also suggested to contribute to anticancer drug resistance. Based on accumulating evidence, two well-organized membrane-bound organelles, the endoplasmic reticulum (ER) and the mitochondrion, are now considered to act as the central organelles in the control of cell survival [1]. Although the two organelles have different roles in biochemical reactions, the ER is physically and biochemically connected to mitochondria. A key feature of the functional relationship between the ER and mitochondria is modulation of cellular Ca?? homeostasis to regulate cell survival signaling [2]. In response to harsh environmental conditions, the ER and mitochondria utilize sensor proteins to activate signaling pathways that contribute to the regulation of cell life or death [1]. Among these signals, the Bcl-2 family of proteins serves as critical regulators of apoptosis that can be identified as either pro-apoptotic or anti-apoptotic proteins.

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The anti-apoptotic Bcl-2/Bcl-XL and pro-apoptotic Bax/ Bak proteins can both negatively and positively regulate events in apoptotic cell death through modulation of cytochrome c (Cyt c) release [3]. Cyt c is released from the mitochondrial intermembrane space into the cytosol in response to pro-apoptotic stimuli and then triggers the processing of pro-caspase-9, thereby initiating the formation of an apoptosome composed of apoptotic proteaseactivating factor 1, dATP, caspase-9, and Cyt c. Apoptosome formation leads to activation of the executioner caspase-3, which causes apoptotic cell death [4]. The Bcl-2 protein is frequently found to be overexpressed in different types of cancers and is thought to confer resistance to treatment with chemotherapeutic agents and to encourage evasion of apoptosis [5, 6]. In addition to the anti-apoptotic function of Bcl-2 in the mitochondria, this protein has been reported to modulate ER Ca2? homeostasis by ER targeting to control apoptotic cell death [7]. Bcl-2 overexpression prevents apoptosis induced by the ER stress-inducing agent thapsigargin, an inhibitor of the sarco/endoplasmic reticulum Ca?? ATPase, which can lead to the rapid depletion of ER Ca?? stores [8]. Evidence also indicates that transient expression of ER-targeted Bcl-2 blocks apoptosis induced by Bax [9]. In contrast, the increased expression of Bax or Bak induced by genotoxic agents can induce the apoptosis of cancer cells [10–14]. However, Bax and Bak gene inactivation and downregulation have been correlated with drug resistance and a poor prognosis in various cancers [15–21]. Evidence exists that Bax and Bak represent inactive forms and are located in the cytosol or mitochondria of healthy cells [22]. In response to apoptotic stimuli, Bax and Bak change their conformations to form oligomers that associate not only with the mitochondrial membrane but also with the ER [22, 23]. ER-targeted Bax/Bak causes the depletion of ER Ca?? and induces caspase-12 cleavage, whereas targeting of Bax/Bak to mitochondria selectively induces the release of Cyt c from mitochondria and poly (ADP-ribose) polymerase (PARP) cleavage [22, 24]. Activated caspase-12 can trigger the processing of pro-caspase-9, thereby inducing activation of downstream caspase-3 [4]. Human cancer cell models of Bax/Bak deficiency reveal the importance of Bax and Bak in ER stress-induced Ca?? depletion in the ER lumen and subsequent apoptosis [25]. Thus, the involvement of Bcl-2 anti-apoptotic activity in the acquisition of drug resistance by cancer cells motivated us to select a pharmacological agent that induces a Bax- or Bak-dependent ER pathway, contributing to mitochondrial apoptosis. The chalcone basic skeleton consists of two aromatic rings connected by a three-carbon, a,b-unsaturated carbonyl system, whose analogs are widely biosynthesized in edible plants [26]. The chalcone class has been reported to

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exhibit a variety of biological activities, including antioxidant, tyrosinase-inhibiting, anti-inflammatory, and cancer cell growth-inhibitory activities [26]. Naturally occurring bichalcones found in the root bark of Rhus pyroides show a C–O–C or C–C linkage between the two chalcones and exhibit cytotoxicity to the human colon cancer HT29 and HCT cell lines [27]. Accordingly, to determine the potential significance of chalcones in terms of pharmacobiological activity, we previously used a Mannich base reaction to synthesize 15 new synthetic bichalcones linked with 1,4-dimethylpiperazine with different substitutions on the B-ring of the chalcone basic skeleton (TSWU-CD series) [26]. Although we found that the most effective analog (2E,20 E)-1,10 -(5,50 -(piperazine-1,4-diylbis(methylene))bis(4-hydroxy-3-methoxy-5,1-phenylene))bis(3-phenylprop-2-en-1-one, or TSWU-CD4), which has a phenyl group on the B-ring of the chalcone basic skeleton, exhibited the strongest cytotoxicity to all studied human cancer cells among all TSWU-CD analogs, the molecular mechanism by which TSWU-CD4 causes cancer cells to undergo cell death remains to be further addressed. In the present study, we report that TSWU-CD4-induced apoptosis in cancer cells is dependent on the oligomerization of the pro-apoptotic Bax/Bak proteins in the ER and mitochondria. Induction of the phosphorylation of protein kinase RNA-like endoplasmic reticulum kinase (PERK) and glucose-regulated protein 78 (GRP78) expression by TSWU-CD4 induces ER oligomerization of Bax/Bak, cleavage of ER-associated pro-caspase-12, and decreased Bcl-2 expression. The suppression of lipid raft-associated phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling by TSWU-CD4 is critical for the ER stressmediated oligomerization of Bax/Bak and the subsequent apoptosis of cancer cells via the ER-mitochondrial death pathway.

Materials and methods Cell culture The human nasopharyngeal carcinoma NPC-TW 039, normal human embryonic lung fibroblast WI-38, normal human fetal skin fibroblast Detroit 551, normal embryonic lung fibroblast MRC-5, normal mouse embryonic liver BNL CL.2, human embryonic kidney 293 (HEK293), human tongue squamous cell carcinoma CAL 27, human glioma GBM-8401, and human pharyngeal squamous carcinoma FaDu cell lines were obtained as previously described [28]. The WI-38, Detroit 551, MRC-5, HEK293, FaDu, and GBM8401 cell lines were cultured in minimum essential medium (MEM) supplemented with 5 % fetal bovine serum (FBS). The CAL 27, NPC-TW 039, and BNL CL.2 cell lines were

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cultured routinely in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5 % 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 TSWU-CD4 was prepared as previously described [26]. Bismaleimidohexane (BMH), cycloheximide, dantrolene, geneticin (G418), 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), Tris–HCl, paraformaldehyde, propidium iodide (PI), Triton X-100, tunicamycin, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and 1,4-diamino-2,3-dicyano-1,4-bis (2-aminophenylthio)butadiene (U0126) were obtained from SigmaAldrich (St. Louis, MO, USA). The TSWU-CD4 was dissolved in and diluted with dimethyl sulfoxide (DMSO) and stored at -20 °C as a 100 mM stock. The DMSO was purchased from Merck (Darmstadt, Germany). The cycloheximide was resuspended in ethanol and used at 100 lg/ml. Potassium phosphate, sodium deoxycholine, and sodium dodecyl sulfate (SDS) were purchased from Merck (Darmstadt, Germany). Lipofectamine 2000 was obtained from Invitrogen (Carlsbad, CA, USA). MEM, FBS, glutamine, and trypsin–EDTA were obtained from Gibco BRL (Grand Island, NY, USA). DMEM, MEM, FBS, penicillin– streptomycin, trypsin–EDTA, and glutamine were obtained from Gibco BRL (Grand Island, NY, USA). A caspase-3 activity assay kit was purchased from OncoImmunin (Gaithersburg, MD, USA). Inhibitors of caspase-3 (Ac-DEVDCMK) and pan-caspase (Z-VAD-FMK) were purchased from Calbiochem (San Diego, CA, USA) and dissolved in DMSO. Baculovirus expressed (recombinant) full-length PI3K (p85a/p110a) was obtained from SignalChem (Richmond, BC, Canada). Plasmids encoding b-galactosidase, FLAG epitope-tagged Bcl-2 and Bcl-XL, HA epitope-tagged wt p85a, and FLAG epitope-tagged Dp85a were previously described [29, 30]. Bax small interfering RNA (siRNA), Bak siRNA, GRP78 siRNA, control siRNA, and Western blotting luminol reagent were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The Bax siRNA, Bak siRNA, GRP78 siRNA, and control siRNA were dissolved in RNase-free water. Antibodies Antibodies against p85a, phospho (p)-p85a (Tyr 508), Akt, p-Akt (Ser 473), ERK, p-ERK (Tyr202/204), Bax, Bak, Bcl-2, Bcl-XL, and Cyt c were obtained from BD Pharmingen (San Diego, CA, USA). Anti-caspase-3, anti-caspase-9, and anti-caspase-12 antibodies were purchased from Calbiochem (San Diego, CA, USA). An anti-Cyt

c oxidase subunit II (Cox-2) antibody was obtained from Abcam (Cambridge, MA, USA). Antibodies against nucleolin, PARP, GRP78, CD55, p110a, and CCAAT/ enhancer-binding protein homologous protein (CHOP) were purchased from Santa Cruz Biotechnology. Antibodies against b-actin, HA-tag, and FLAG-tag were obtained from Sigma-Aldrich. Peroxidase-conjugated antimouse IgG, anti-goat IgG, and anti-rabbit IgG secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Cell viability and cell proliferation assays Cell viability was assessed by fluorescence-activated cell sorting (FACS) analysis of cellular PI uptake, as described previously [28]. 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, Franklin Lakes, NJ, USA). Cell proliferation was determined by the MTT method. The treated cells were washed once with PBS and incubated with 0.5 mg/ml MTT for 5 h. The resulting formazan precipitate was dissolved in 150 ll of DMSO, and the optical density (OD) of the formazan was determined using an enzyme-linked immunosorbent assay (ELISA) plate reader (Thermo Labsystems Multiskan Spectrum, Waltham, MA, USA) at 570 nm. Measurement of DNA fragmentation Histone-associated DNA fragments were analyzed using a Cell Death Detection ELISA kit (Roche Applied Science, Mannheim, Germany). Briefly, vehicle- or TSWU-CD4treated cells were incubated in hypertonic buffer for 30 min at room temperature. After centrifugation, the cell lysates were transferred to an anti-histone-coated microplate to bind histone-associated DNA fragments. The plates were washed after 1.5 h of incubation, and nonspecific binding sites were saturated with blocking buffer. The plates were then incubated with peroxidase-conjugated anti-DNA for 1.5 h at room temperature. To determine the amount of retained peroxidase, 2,20 -azino-di-(3-ethylbenzthiazoline-6-sulfonate) was added as a substrate, and a spectrophotometer (Thermo Labsystems Multiskan Spectrum, Franklin, MA, USA) was used to measure the absorbance at 405 nm. Assays for the detection of caspase-3 activity and early apoptotic cells Caspase-3 activity was measured using the PhiPhiLux G1D2 kit (OncoImmunin, College Park, MD, USA) according to the manufacturer’s protocols. Briefly, vehicle-

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or TSWU-CD4-treated cells were incubated with the PhiPhiLux fluorogenic Caspase substrate at 37 °C for 1 h and were then analyzed using a FACSCount flow cytometer. For the Annexin-V binding assays, the treated cells were harvested and stained with fluorescein isothiocyanatelabeled Annexin-V and PI according to the manufacturer’s protocols. The samples were analyzed using a FACSCount flow cytometer, and the results were analyzed using CellQuest software. Plasmid and siRNA transfection Cells (at 60–70 % confluence in a 12-well plate) were transfected with the FLAG-Bcl-XL, FLAG-Bcl-2, HA-wt p85a, or FLAG-Dp85a expression plasmid or with Bax, Bak, PERK, or GRP78 siRNA using Lipofectamine 2000. The expression of FLAG-Bcl-XL, FLAG-Bcl-2, HA-wt p85a, FLAG-Dp85a, Bax, Bak, and GRP78 in transfected cells was assessed by Western blotting using antibodies specific to FLAG, HA, Bax, Bak, GRP78, and PI3K p85a. Establishment of cell clones stably expressing HA-wt p85a or FLAG-Dp85a To establish cells stably expressing HA-wt p85a or FLAGDp85a, cells were transfected with the pHA-wt p85a or pFLAG-Dp85a plasmid using Lipofectamine 2000. The transfected cells were selected and cloned in the presence of 500 lg/ml G418. The levels of the HA-wt p85a and FLAG-Dp85a proteins were confirmed by Western blot analysis with anti-HA and anti-FLAG antibodies. Subcellular fractionation Subcellular fractionation was performed according to the protocol of Zong et al. [22]. 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 lg/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 7509g at 4 °C to remove unlysed cells and nuclei. The supernatant was collected and then centrifuged for 20 min at 10,0009g 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 100,0009g for 1 h at 4 °C. The new supernatant was saved as the cytosolic (Cs) fraction, and 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

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Western blot analysis. The purity of each subcellular fraction was confirmed by Western blotting using specific antibodies against the nuclear marker nucleolin, the mitochondrial marker Cox-2, and the ER marker calnexin. In vitro PI3K kinase assay Cells were transfected with the HA-wt p85a or FLAGDp85a expression plasmids. At 48 h after transfection, the cells were harvested and lysed in a lysis buffer containing 50 mM HEPES (pH 7.5), 100 mM NaCl, 2 mM EDTA, 0.5 % Nonidet P-40, 10 % glycerol (v/v), 0.1 mM phenylmethylsulfonyl fluoride, 0.1 mM sodium vanadate, 0.5 mM NaF, 5 lg/ml leupeptin, and 0.1 mM dithiothreitol. Lysates were immunoprecipitated with anti-HA or antiFLAG antibody in the presence of protein A agarose beads. The immunoprecipitates were subjected to Western blotting analysis. The immunoprecipitates or recombinant fulllength PI3K (p85a/p110a) were incubated with kinase reaction mixture (5 mM 3-(N-morpholino)propanesulfonic acid [pH 7.2], 2.5 mM b-glycerol-phosphate, 5 mM MgCl2, 1 mM EGTA, 0.4 mM EDTA, 0.05 mM DTT, 50 lM ATP, and 25 lM PI(4,5)P2) at 30 °C for 30 min. The kinase activity was determined by the KinaseGlo Luminescence Protocol on a GloMax plate reader according to the manufacturer’s instructions. Density based membrane flotation technique Detergent-resistant membranes (DRMs) were prepared as described previously [31]. Briefly, treated cells were washed twice in ice-cold PBS before being removed from dishes by scraping. Cells were then harvested by centrifugation, resuspended in 1 ml of hypotonic lysis buffer (10 mM Tris [pH 7.5], 10 mM KCl, 5 mM MgCl2) containing 0.5 % Brij 98, incubated at 37 °C for 5 min, and ruptured by passage through a 25-gauge hypodermic needle 20 times. Unbroken cells and nuclei were removed by centrifugation at 1,0009g for 5 min in a microcentrifuge at 4 °C. The crude homogenates were kept on ice for an additional 5 min, mixed with 3 ml of 72 % sucrose, and overlaid with 4 ml of 55 % sucrose and 1.5 ml of 10 % sucrose; all sucrose solutions were dissolved in low-salt buffer (50 mM Tris–HCl [pH 7.5], 25 mM KCl, 5 mM MgCl2). Samples were centrifuged for 14 h in a Beckman SW41 rotor at 38,000 rpm and 4 °C. Fractions were collected from the top of the gradient in 1-ml increments and concentrated to approximately 100 ll by passage through a 50-kDa Centricon filter. Measurement of cytosolic Ca?? The Ca?? level was determined by measuring the retention of Indo-1/AM. Briefly, the treated cells were incubated

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with 3 lg/ml Indo-1/AM for 30 min at 37 °C. The cells were then pelleted by centrifugation at 1609g. The pellets were resuspended and washed twice with PBS. The level of Ca?? was evaluated as previously described [30]. Statistical analysis of data Statistical analysis of the data was performed using an unpaired Student’s t test and ANOVA. The data were considered statistically significant when p \ 0.05.

Results Caspase-dependent apoptotic activity is involved in TSWU-CD4-induced cancer cell apoptosis Our previous results have shown that 16 novel synthetic bichalcone analogs from the chalcone class (TSWU-CD series) exhibit inhibitory effects on human cancer cell growth [26]. We chose the most effective analog, TSWU-CD4 (Fig. 1a), to identify the most sensitive of the following four cancer cell lines: the human tongue squamous cell carcinoma CAL 27, human nasopharyngeal carcinoma NPC-TW 039, human glioma GBM-8401, and human pharyngeal squamous carcinoma FaDu cell lines. To achieve this objective, we determined the half-maximal inhibitory concentration for cell viability (IC50) and studied morphological changes and the mechanisms of apoptosis. PI staining and flow cytometric analyses showed that treatment with TSWU-CD4 for 36 h resulted in a decrease in the viability of the CAL 27, NPC-TW 039, GBM-8401, and FaDu cancer cell lines, with IC50 values of 2.5, 3.5, 3.5, and 3.5 lM, respectively (Fig. 1b). Although TSWU-CD4 exhibited a growth-inhibitory effect on normal human fibroblasts (Detroit 551, MRC-5, and WI-38), a normal mouse liver cell line (BNL CL.2), and a human embryonic kidney cell line (HEK293) at concentrations of 14–16, 6–16, and 16 lM, respectively, it did not show a cytotoxic effect on these cells at any of the tested concentrations (Fig. 1c). These data indicate that human cancer cell lines were highly sensitive to TSWU-CD4. Thus, concentrations ranging between 2.5 and 3.5 lM were used to treat cancer cell lines in all subsequent experiments. An increased number of apoptotic bodies was observed in cancer cells treated with TSWU-CD4 (Fig. 1d), suggesting that TSWUCD4-treated cancer cells undergo cell death and exhibit the morphological features of apoptosis. To investigate whether the induction of cell death by TSWU-CD4 could be linked to apoptosis, nuclear morphological changes were examined, and the sub-G1 population and annexin V? cells were determined by flow cytometry. As expected, cancer cells showed a remarkable change in their nuclear

morphology after treatment with TSWU-CD4 (Fig. 1e). Treatment with TSWU-CD4 also resulted in an increase in the level of annexin V binding and in the cell population in the sub-G1 phase (Fig. 1f, g). Based on the above results, it was observed that TSWU-CD4 could induce apoptosis in cancer cells. To explore whether TSWU-CD4 induces apoptosis by activation of caspase-3, we used Western blotting and flow cytometry to analyze the activity of caspase-3, a marker of caspase-dependent apoptosis. This marker’s activation requires proteolytic processing of its inactive form into activated p17 and p12 fragments [28]. As shown in Fig. 2a, more cleavage of pro-caspase-3 occurred in TSWU-CD4-treated cells compared with vehicle-treated control cells. Significant increases in caspase-3 enzymatic activity and DNA fragmentation were also detected in TSWU-CD4-treated cells, and these effects were nearly completely inhibited by the caspase-3 inhibitor Ac-DEVD-CMK and the pan-caspase inhibitor Z-VAD-FMK (Fig. 2b, c). The induction of apoptosis was further confirmed by the cleavage of PARP, detected using Western blot analysis (Fig. 2a). These results suggest that TSWU-CD4 induced caspase-3 activation followed by apoptosis. TSWU-CD4 induces ER-mitochondrial targeting of Bax and Bak, contributing to cancer cell apoptosis Western blot analysis with an anti-caspase-12 antibody revealed that cleavage of pro-caspase-12 was detected in whole-cell lysates of TSWU-CD4-treated cells (Fig. 3a). The ER targeting of Bax and Bak has been reported to be critical for ER stress-induced caspase-12 cleavage and subsequent activation of caspase-3 for apoptosis initiation [4, 22]. As the Bcl-2 family of proteins can modulate ERmitochondrial communication and thereby control cell survival [32], we analyzed the subcellular distribution of Bcl-2 family proteins. Consistent with previous findings, Bax and Bak were mainly present in the mitochondria and cytosol of vehicle-treated cells, whereas small amounts of Bax and Bak were present in the ER [33] (Fig. 3a). In addition to its mitochondrial localization, Bcl-2 also resides in the cytosol. TSWU-CD4 treatment did not affect the levels of Bax and Bak in the mitochondria, whereas the treatment caused elevated Bax and Bak levels in the ER by decreasing the amounts of Bax and Bak in the cytosol. Although Bcl-2 expression was downregulated by TSWU-CD4, we have found no evidence that the ER translocation of Bcl-2 could be induced by TSWU-CD4 (Fig. 3a). Truncated Bid (tBid), a known inducer of Bax/Bak mitochondrial translocation and oligomerization [34–36], was not detected in the mitochondria of TSWU-CD4-treated cells, and cleavage of cytosolic Bid was not found, as expected (data not shown). The cleavage of ER-associated

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Fig. 1 TSWU-CD4 induces the apoptosis of human cancer cells. a The structure of TSWU-CD4. b The IC50 value of TSWU-CD4 in these cells is indicated. c The cytotoxic and growth-inhibitory effects of TSWU-CD4 on normal cells. The cells were treated with the indicated concentrations of TSWU-CD4 for 36 h. Cell viability and proliferation were determined by flow cytometric analysis of PI uptake and an MTT assay, respectively. d, e Morphological and nuclear changes in TSWU-CD4-treated cells. Cells were treated for

36 h with vehicle or TSWU-CD4. Cell morphology and DAPI-stained nuclei were examined using an inverted phase-contrast microscope and an inverted fluorescence microscope, respectively. The arrowheads indicate apoptotic cells or condensed nuclei. f, g The percentage of Annexin V? cells and cells in the sub-G1 population were determined using flow cytometry. The values presented are the mean ± standard error from three independent experiments. *p \ 0.05: significantly different from vehicle (-)-treated cells

pro-caspase-12, resulting in the cytosolic localization of activated caspase-12, was induced by TSWU-CD4. In contrast, pro-caspase-9 localized to the cytosol, and upon

treatment with TSWU-CD4, activated caspase-9 resided in the cytosol. Cytosolic localization of Cyt c was also found in TSWU-CD4-treated cells (Fig. 3a). Oligomerization of

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Fig. 2 TSWU-CD4 induces caspase-dependent apoptosis in cancer cells. a Cells were treated with TSWU-CD4 for 36 h. The protein levels of caspase-3 and PARP in cell lysates were analyzed using specific antibodies. b-actin was used as an internal control for sample loading. b, c TSWU-CD4 induced caspase-3 activation and DNA fragmentation in cancer cells. After treatment with vehicle, TSWUCD4, TSWU-CD4 plus Z-VAD-FMK (15 lM), or TSWU-CD4 plus

Ac-DEVD-CMK (10 lM) for 36 h, caspase-3 activity and DNA fragmentation were measured using flow cytometry and a Cell Death Detection ELISA kit, respectively. The values presented are the mean and standard error from three independent experiments. *p \ 0.05: significantly different from vehicle-treated cells or TSWU-CD4treated cells

Bax and Bak has been shown to be required for their proapoptotic activity and subcellular localization [22]. To determine whether TSWU-CD4 treatment induces Bax and Bak oligomerization, we used the sulfhydryl-reactive agent BMH to crosslink the oligomerized proteins in treated cells, followed by isolation of mitochondrial and ER subcellular fractions. As shown in Fig. 3b, TSWU-CD4 treatment not only increased the ER localization and oligomerization of Bax but also caused Bax oligomerization in the mitochondria. Similarly, oligomerized Bak was detected in both mitochondria and the ER (Fig. 3b). We next investigated whether TSWU-CD4-induced Cyt c release from the mitochondria is Ca?? sensitive. The levels of Ca?? in the cytosol, which were determined by flow cytometry, increased in cells after treatment with TSWU-CD4. Co-treatment with the ER Ca?? channel inhibitor dantrolene inhibited the increase in cytosolic Ca?? and the release of Cyt c from mitochondria that were caused by TSWU-CD4 (Fig. 3c, d). These results suggest that mitochondrial Cyt c release was dependent on the release of Ca?? from the ER to the cytosol induced by TSWU-CD4.

To address whether TSWU-CD4-induced Bax/Bak function modulates the cleavage of ER-associated procaspase-12 and apoptosis, cells were transfected with siRNA targeting Bax or Bak. Immunoblot analysis confirmed the specific knockdown of the expression of Bax or Bak (Fig. 4a). Figure 4a–c show that silencing of Bax or Bak expression by siRNA blocked TSWU-CD4-induced proteolytic processing of pro-caspase-12, elevation of the cytosolic Ca?? concentration, DNA fragmentation, and release of Cyt c from mitochondria compared to cells transfected with control siRNA. These findings indicate that TSWU-CD4-induced cleavage of ER-associated procaspase-12, mitochondrial Cyt c release, and apoptosis were dependent on the pro-apoptotic activity of Bax and Bak. Suppression of PI3K/Akt signaling is involved in ER stress- and Bax/Bak-mediated apoptosis caused by TSWU-CD4 It has been documented that ER stress can induce conformational changes in Bax and Bak, which converts these

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Fig. 3 TSWU-CD4 induces Bax/Bak oligomerization in the ER and mitochondria and ER-Ca??-dependent mitochondrial Cyt c release. a Cells were treated with TSWU-CD4 for 36 h. Subcellular nuclear (N), mitochondrial (Mt), ER/microsomal (Ms), and cytosolic (Cs) fractions were separated by differential centrifugation. The levels of the indicated proteins in the lysates of the untreated or treated N, Mt, Ms, and Cs fractions were determined by Western blot analysis using specific antibodies. Antibodies against nucleolin, Cox-2, calnexin, and b-actin were used as internal controls for the nucleus, mitochondria, ER, and cytosol, respectively. b Cells were harvested 36 h after treatment with vehicle or TSWU-CD4, and cell pellets were resuspended in hypotonic buffer. Crude homogenates were incubated

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with 5 mM BMH in PBS for 30 min at room temperature and then subjected to subcellular fractionation to obtain the Mt and Ms fractions. In total, 20 lg of total protein from the recovered fractions was analyzed by 10 % SDS-PAGE and probed with specific antibodies, as indicated. c, d Cells were treated with vehicle, TSWU-CD4, or TSWU-CD4 plus dantrolene (25 lM) for 36 h, and the cytosolic level of Ca?? was monitored by measuring the increased fluorescence of Indo-1 by flow cytometry. The levels of Cyt c in the cytosol and mitochondria were determined by Western blotting using specific antibodies. Cox-2 and b-actin were used as internal controls for the mitochondria and cytosol, respectively. *p \ 0.05: significantly different from vehicle-treated cells or TSWU-CD4-treated cells

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proteins into oligomeric forms, leading to their localization to the ER and the subsequent induction of pro-apoptotic activity [22]. To determine whether the increased targeting of Bax and Bak to the ER by TSWU-CD4 requires the ER stress response, we examined the expression levels of ER stress-related genes. As shown in Fig. 5a, TSWU-CD4 induced expression of the UPR regulator GRP78 and the UPR-activated transcriptional factor CHOP as well as cleavage of pro-caspase-12. These events were similar to those observed in cells treated with tunicamycin, a glucosamine-containing antibiotic that specifically inhibits the asparagine-linked N-glycosylation of proteins, resulting in induction of pro-apoptotic UPR gene expression and subsequent ER stress-mediated apoptosis [37]. Upregulation of GRP78 and CHOP expression and pro-caspase-12 cleavage were not detected in cells that were induced to undergo apoptosis by the translation inhibitor cycloheximide. Consistent with previous findings [38], this cycloheximideinduced apoptosis was associated with induction of Bax expression and downregulation of Bcl-XL expression (Fig. 5a, b). The treatment of cells with GRP78 siRNA resulted in attenuation of TSWU-CD4-induced CHOP upregulation, pro-caspase-12 cleavage, Bax and Bak ER localization, and apoptosis (Fig. 5a–c). However, GRP78 siRNA transfection had no detectable effect on the level of Akt Ser 473 phosphorylation, which is inconsistent with the previous observation that knockdown of GRP78 increases the level of p-Akt Ser 473 [39]. The knockdown of Bax by siRNA had a clear and consistent inhibitory effect on TSWU-CD4-induced pro-caspase-12 cleavage and apoptosis, but there was no effect on the levels of GRP78, CHOP, or Bcl-2 (Fig. 5a, b). These data show that the TSWU-CD4-induced ER stress response contributes to the increased targeting of Bax and Bak to the ER. Additionally, GRP78 siRNA, but not Bax siRNA, attenuated TSWU-CD4-induced Bcl-2 downregulation. This finding suggests that the TSWU-CD4-induced decrease in the Bcl2 level was mediated through the induction of GRP78 by the UPR. Reports based on pharmacological and siRNA inhibition studies have suggested that PI3K/Akt signaling controls cell fate through the negative regulation of the ER stress sensor PERK [40]. The phosphorylation of PERK at Tyr 980 was induced by TSWU-CD4 treatment (Fig. 6a). To confirm the role of PERK in TSWU-CD4-induced ER stress response, oligomeric Bax/Bak formation, and apoptosis, we employed siRNA to knockdown PERK. siRNAtargeting of PERK inhibited the induction of GRP78, CHOP, pro-caspase-12 cleavage, Bcl-2 down-regulation, Bax/Bak oligomerization in the ER and apoptosis by TSWU-CD4 (Fig. 6a, b). These data indicated that PERK participates in the activation of TSWU-CD4-induced ER stress- and oligomeric Bax/Bak-mediated apoptosis.

To address whether the TSWU-CD4-mediated induction of Bax/Bak ER targeting and apoptosis was linked to decreased Bcl-2 protein levels, we selected CAL 27 cells for subsequent experiments because these cells are more sensitive to TSWU-CD4 than are NPC-TW 039, FaDu, and GBM 8401 cells. CAL 27 stable cell lines expressing FLAG epitope-tagged Bcl-2 or Bcl-XL were generated and confirmed by Western blot analysis using specific antibodies against FLAG, Bcl-2, or Bcl-XL (Fig. 7a). Ectopic expression of Bcl-2, similar to that of Bcl-XL, suppressed Bax/Bak oligomerization in the mitochondria but did not inhibit the induction of GRP78 by TSWU-CD4 (Fig. 7c). In contrast to Bcl-2, ectopic expression of Bcl-XL did not completely inhibit TSWU-CD4-induced Cyt c release from mitochondria to the cytosol or apoptosis (Fig. 7b, c). Bcl-2 (but not Bcl-XL) overexpression attenuated the TSWUCD4-induced ER localization and oligomerization of Bax/ Bak and subsequent pro-caspase-12 cleavage (Fig. 7c). These findings indicate that attenuated Bcl-2 expression is required for TSWU-CD4-induced apoptotic potency of Bax/Bak in the ER of cancer cells. We next investigated whether the PI3K/Akt signaling pathway contributes to the induction of the ER stress response in response to TSWU-CD4, as PI3K activity has been shown to be required for the regulation of ER stressinduced apoptotic cell death [41]. Efficient signaling events regulating cell survival by PI3K/Akt correlates with its presence in lipid rafts [42]. We used the 1 % Brij 98 solubilization of TSWU-CD4-treated cells and density-based membrane flotation technique to isolate DRMs, which are thought to be lipid rafts based on their composition and properties [43]. Figure 8e shows that phospho (p)-p85a (Tyr 508) and p-Akt (Ser 473) were detected in the DRM fraction of CAL 27 and WI-38 cells and are associated with membrane caveolae and the lipid raft markers CD55 and caveolin-1. In contrast to WI-38 cells, treatment of CAL 27 cells with TSWU-CD4 resulted in suppressed p85a and Akt phosphorylation but had no effect on the phosphorylation of ERK (Fig. 8a). Suppression of the lipid raft localization of p-p85a (Tyr 508) and p-Akt (Ser 473) by TSWU-CD4 was observed in CAL 27 cells but not in WI38 cells (Fig. 8e). Western blot analysis also revealed that TSWU-CD4 did not cause an increase in the levels of GRP78 and CHOP, PERK phosphorylated at Tyr 980, or the cleavage of pro-caspase-3 and -12 in WI-38 cells (Fig. 8a). Compared with cells treated with the ERK-specific inhibitor U0126, treatment with the PI3K inhibitor LY294002 increased the induction of GRP78 and CHOP, Bax and Bak oligomerization in the ER and mitochondria, pro-caspase-3 and pro-caspase-12 cleavage, and apoptosis (Fig. 8a–d). Transient ectopic expression of lipid raftassociated HA-tagged wild-type p85a (HA-wt p85a) in CAL 27 cells blocked TSWU-CD4-induced GRP78 and

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Apoptosis b Fig. 4 TSWU-CD4-induced Bax/Bak apoptotic activity modulates

the cleavage of ER-associated pro-caspase-12, mitochondrial Cyt c release, and cell apoptosis. a, b At 12 h after transfection with control, Bax, or Bak siRNA, cells were treated with vehicle or TSWU-CD4 for 36 h. After Western blotting was used to examine the Bax, Bak, pro-caspase-12, and cleaved caspase-12 levels, the cytosolic levels of Ca?? and DNA fragmentation were determined by measuring increased Indo-1 fluorescence using flow cytometry and a Cell Death Detection ELISA kit, respectively. *p \ 0.05: significantly different from control siRNA-transfected TSWU-CD4-treated cells. c The levels of the indicated proteins in the lysates of the fractions of Mt and Cs and total cell (T) extracts were determined by Western blot analysis using specific antibodies. Cox-2 and b-actin were used as internal controls for the mitochondria and cytosol, respectively

CHOP expression, caspase-3 and caspase-12 activation, Bax and Bak oligomerization in the ER and mitochondria, and apoptosis (Fig. 8a–d). Moreover, HA-wt p85a overexpression resulted in increased phosphorylation and lipid raft localization of Akt (Fig. 8e). In contrast, ectopic expression of the FLAG-tagged mutant p85a (FLAGDp85a), which lacks the binding site for the p110 catalytic subunit of PI3K, failed to suppress the ER stress-mediated ER-mitochondrial oligomerization of Bax/Bak, pro-caspase-3 and pro-caspase-12 cleavage, and apoptosis induced by TSWU-CD4 (Fig. 8a–d). To test whether TSWU-CD4 inhibits PI3K activity, immunoprecipitates were prepared from lysates of CAL 27 cells transfected with HA-wt p85a or FLAG-Dp85a, and recombinant p85a/p110a was coexpressed in Sf9 cells in a Baculovirus expression system. The HA-wt p85a or FLAG-Dp85a immunoprecipitates were analyzed by anti-p110a antibody, confirming that p110a can be coimmunoprecipitated with p85a but not with Dp85a. In contrast to LY294002, TSWU-CD4 treatment did not significantly affect the kinase activity of p85a-p110a immunoprecipitates or recombinant purified p85a and p110a (data not shown). These results indicate the role of lipid raft-associated PI3K/Akt signaling in the regulation of Bax/Bak-mediated apoptosis due to ER stress in TSWU-CD4-treated cancer cells.

Discussion In the present work, we identified a synthetic 1,4-dimethylenepiperazine-linked bichalcone, TSWU-CD4, with a phenyl group on the B-ring of the chalcone basic skeleton that can induce caspase-dependent apoptosis in the human tongue squamous cell carcinoma CAL 27, nasopharyngeal carcinoma NPC-TW 039, glioma GBM-8401, and pharyngeal squamous carcinoma FaDu cell lines at IC50 values of 2.5, 3.5, 3.5, and 3.5 lM, respectively. This observation of the caspase-mediated apoptotic toxicity of TSWU-CD4 was also consistent with results found in other human

cancer cell lines, including the human glioblastoma M059K, tongue squamous carcinoma SAS, and lung carcinoma A549 cell lines (data not shown). TSWU-CD4 treatment at concentrations ranging from 2 to 16 lM did not exert significant cytotoxic effects on the normal human WI-38, Detroit 551, and MRC-5 fibroblast; normal mouse liver BNL CL.2; or HEK293 cell lines, although TSWUCD4 exhibited a significant growth-inhibitory effect on the cells at high concentrations (14, 6, and 16 lM for the normal human, mouse liver, and HEK293 lines, respectively). BNL CL.2 cells were the most sensitive to TSWUCD4 in terms of growth inhibition. The growth inhibition of these normal cells by TSWU-CD4 did not vary in a dose-dependent manner; moreover, we did not observe any significant induction of caspase-3 activity and DNA fragmentation in the cells, even after 48 h of treatment with TSWU-CD4 at concentrations as high as 16 lM (data not shown). In the cell cycle analysis, flow cytometry showed that 2.5 or 3.5 lM TSWU-CD4 induced early cell cycle arrest at the S phase by 24 h of exposure in CAL 27, NPCTW 039, FaDu, and GBM 8401 cells, which was accompanied by obvious increases in the sub-G1 populations (17.53 ± 5.10, 11.85 ± 2.46, 6.87 ± 0.59, and 9.41 ± 3.45 %, respectively). When CAL 27, NPC-TW 039, FaDu, and GBM 8401 cancer cells were exposed to TSWU-CD4 for 48 h, the sub-G1 populations increased to 62.23 ± 5.25, 50.05 ± 6.71, 42.61 ± 5.43, and 46.66 ± 5.73 %, respectively (data not shown). Normal human cells exposed to 16 lM TSWU-CD4 for 36 or 48 h also showed cell cycle arrest in the S phase but failed to exhibit an increase in the sub-G1 population (our unpublished data). The sensitivity of human cancer cells to the cytotoxic effect of TSWU-CD4 may be due to genetic changes and biochemical signaling alterations associated with cellular immortality. Although we do not exclude the possibility that TSWU-CD4 induces S-phase arrest in human normal cells accompanied by the induction of apoptosis, during treatment with high concentrations up to 20 lM or for longer incubation periods, our data show that TSWU-CD4 exhibited high cytotoxicity against all four human cancer cell lines tested, with a less toxic effect on normal cells. This finding suggests the potential use of TSWU-CD4 in the chemoprevention of cancer. The correlation between PI3K and the ER stress response has been investigated using HEK293 fibroblasts with a knockout of p85a or mice with a deletion of p85a in the liver by showing that the expression of the UPR target genes GRP78 and CHOP and cell apoptosis can be regulated by p85a through interaction with XBP-1 [41]. There is also evidence that LY294002 treatment does not inhibit the induction of GRP78 by tunicamycin, suggesting that p85a regulation of the cellular response to ER stress is independent of its enzymatic activity [41]. The use of the

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Fig. 5 Increased targeting of Bax and Bak to the ER requires ER stress response induction by TSWU-CD4. a, b At 12 h after transfection with control, Bax, or GRP78 siRNA, transfected cells were treated with either vehicle or TSWU-CD4 for 36 h. Untransfected cells were treated with either cycloheximide (2 lg/ml) or tunicamycin (1 lg/ml) for 36 h. After Western blotting was used to examine the GRP78, CHOP, Bax, Bak, Bcl-2, Bcl-XL, pro-caspase12, and cleaved caspase-12 levels, DNA fragmentation was

determined using a Cell Death Detection ELISA kit. *p \ 0.05: significantly different from control siRNA-transfected TSWU-CD4treated cells. c Similar to the protocol in a and b, siRNA-transfected cells were treated with either vehicle or TSWU-CD4 for 36 h. The levels of the indicated proteins in the lysates of the Ms and Cs fractions were determined by Western blot analysis using specific antibodies. Calnexin and b-actin were used as internal controls for the ER and cytosol, respectively

human lung cancer H1299 and breast cancer MCF7 cell lines in the investigation of tunicamycin- and thapsigargininduced ER stress responses revealed a role for PI3Kmediated activation of Akt in promoting cell survival by

countering ER stress-induced cell death signaling [44]. Hyoda et al. found that suppression of the phosphorylation of Akt at Ser 473 by LY294002 induced CHOP expression and apoptosis in mouse fibroblast L929 cells [45].

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Fig. 6 The activation of TSWU-CD4-induced ER stress- and oligomeric Bax/Bak-mediated apoptosis mediated by PERK. a, b At 12 h after transfection with control or PERK siRNA, transfected cells were treated with either vehicle or TSWU-CD4 for 36 h. The levels of the indicated proteins in the lysates of the Ms and Cs fractions and the T

extracts were determined by Western blot analysis using specific antibodies. b-actin was used as an internal control for sample loading. DNA fragmentation was determined using a Cell Death Detection ELISA kit. *p \ 0.05: significantly different from control siRNAtransfected TSWU-CD4-treated cells

Similarly, forced expression of a kinase-dead mutant Akt in MCF7 cells enhanced thapsigargin-induced apoptosis [44]. This discrepancy is likely due to cell-type specific signal transduction and gene regulation. Although these studies show the importance of the role of PI3K/Akt signaling in modulating the ER stress response, to date, there has been no evidence showing a link between the PI3K/Akt signaling pathway and ER stress- and Bax/Bak-mediated ERmitochondrial crosstalk to induce apoptosis in human cancer cells. In the present study, we describe a role for PI3K/Akt signaling as a critical modulator of ER stressmediated apoptosis in human cancer cells in response to TSWU-CD4. ER stress-induced apoptosis, which occurs due to lipid raft-associated PI3K/Akt-dependent induction of GRP78 and CHOP expression, leads to an increase in the translocation and oligomerization of Bax/Bak in the ER and Bcl-2 downregulation, causing ER-associated caspase-

12 activation and the ER Ca??-dependent release of Cyt c. The rationale for the contribution of the lipid raft-associated PI3K/Akt-mediated ER stress response to cancer cell apoptosis is supported by our data, showing that treatment of TSWU-CD4 resulted in the suppression of the lipid raft localization and phosphorylation of p85a and Akt in human cancer cells but not in normal fibroblasts. Ectopic expression of HA-wt p85a induced an increase in Akt Ser473 phosphorylation at lipid rafts and inhibited TSWUCD4-induced expression of GRP78 and CHOP, Cyt c release from mitochondria, Bax and Bak oligomerization in the ER and mitochondria, pro-caspase-3 and pro-caspase-12 cleavage, and apoptosis. The involvement of the TSWU-CD4-induced ER stress response in the increased targeting of Bax/Bak to the ER and in apoptosis was verified by the PERK or GRP78 knockdown study, in which PERK or GRP78 downregulation blocked CHOP

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Fig. 7 TSWU-CD4 induces Bax/Bak oligomerization in the ER and mitochondria by decreasing the level of Bcl-2 protein. a Expression levels of Bcl-2 and Bcl-XL in lysates prepared from cells transfected with vector alone, FLAG-Bcl-2, or FLAG-Bcl-XL. FLAG-Bcl-2, FLAG-Bcl-XL, Bcl-2, and Bcl-XL were detected with the antibodies shown. b, c At 12 h after transfection with vector alone, FLAG-Bcl-2, or FLAG-Bcl-XL, cells were treated with vehicle or TSWU-CD4 for

36 h. DNA fragmentation was determined using a Cell Death Detection ELISA kit. The levels of the indicated proteins in the lysates of the Mt, Ms, and Cs fractions were determined by Western blot analysis using specific antibodies. Cox-2, calnexin, and b-actin were used as internal controls for the mitochondria, ER, and cytosol, respectively. *p \ 0.05: significantly different from empty vectortransfected TSWU-CD4-treated cells

upregulation and suppressed the Bax/Bak targeting to the ER, pro-caspase-12 cleavage, and DNA fragmentation induced by TSWU-CD4. Yung et al., using GRP78knockdown human choriocarcinoma JEG-3 cells, found that ER stress regulates Akt Ser 473 phosphorylation via GRP78 [39]. Reverse co-immunoprecipitation experiments using anti-GRP78 and anti-phospho-Akt (Ser 473) antibodies further showed an interaction of GRP78 with nonphosphorylated Akt, suggesting that binding of GRP78 to

Akt is likely to suppress Akt phosphorylation at Ser 473 [39]. Our results show that, although knockdown of GRP78 reduces CHOP protein levels, the endogenous protein and phosphorylation levels of both p85a and Akt were not affected in CAL 27, NPC-TW 039, FaDu, and GBM 8401 cells. The induction of Akt phosphorylation at Ser 473 was shown to be mediated by PI3K because LY294002 or the overexpression of Dp85a completely blocked its phosphorylation. Given these observations, it is logical to

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Apoptosis b Fig. 8 ER stress- and Bax/Bak-mediated apoptosis requires TSWU-

CD4-induced inactivation of PI3K/Akt signaling. a–e At 12 h after transfection with vector alone, HA-wt p85a, or FLAG-Dp85a, cells were treated with either vehicle or TSWU-CD4 for 36 h. The cells were treated with the indicated compounds for 36 h. The levels of the indicated proteins in the T lysates, lipid rafts or in the lysates of BMHtreated Mt, Cs, and Ms fractions were determined by Western blot analysis using specific antibodies. Cox-2, b-actin, calnexin, CD55, and caveolin-1 were used as internal controls for the mitochondria, cytosol, ER, and lipid rafts, respectively. DNA fragmentation was determined using a Cell Death Detection ELISA kit

suggest that lipid raft-associated PI3K/Akt signaling has physiological relevance to regulating the ER stress-induced ER-mitochondrial targeting and oligomerization of Bax/ Bak upon TSWU-CD4 treatment in human cancer cells. Characterization of PI3K/Akt signaling as a regulator of ER stress- and Bax/Bak-mediated apoptosis does not rule out the possible involvement of S-phase kinase-associated proteins in this process, as stabilization of the cyclindependent kinase inhibitor p27 has been reported to regulate the activation of the UPR by tunicamycin [46, 47]. The evidence provided by Western blot analysis of subcellular fractions in the present study supports the previous finding that Bax and Bak can localize to the ER in addition to their cytosolic and mitochondrial localization in healthy cells [22]. This study also found a decrease in Bcl-2 levels in TSWU-CD4-treated human cancer cells. Using ectopically expressed FLAG-Bcl-2 and FLAG-Bcl-XL, it was found that the oligomerization of Bax and Bak in the ER induced by TSWU-CD4 was suppressed by Bcl-2, independent of the anti-apoptotic activity of Bcl-XL. Overexpression of FLAGBcl-2 or FLAG-Bcl-XL, resulting in inhibition of the formation of Bax or Bak oligomers at the mitochondria, conferred resistance to TSWU-CD4 by inhibiting the release of Cyt c into the cytosol. However, FLAG-Bcl-XL did not completely inhibit TSWU-CD4-induced apoptosis. Consistent with previous evidence, Bcl-2 overexpression inhibits the ER-mitochondrial death pathway and enhances ER sequestration [48, 49]. A recent study using ectopically expressed, mitochondrially targeted Bcl-XL and ER-targeted Bcl-XL showed that expression of mitochondrially targeted Bcl-XL efficiently inhibited apoptotic signaling from the mitochondria, whereas ER-targeted Bcl-XL modulated ER Ca?? homeostasis but had no effect on the prevention of cell death [50]. The ability of Bcl-XL to block mitochondrial membrane permeabilization has been shown to be mediated by direct binding to membrane-bound mitochondrial Bax [51]. This function of Bcl-XL may explain why overexpression did not completely abolish TSWU-CD4-induced apoptosis in human cancer cells. In summary, this study shows that inhibition of lipid raft-associated PI3K/Akt signaling induces the ER stress response, which promotes Bax/Bak oligomerization in the ER and mitochondria; cleavage of ER-associated pro-

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caspase-12; Bax/Bak-mediated, ER Ca??-dependent release of mitochondrial Cyt c; and apoptosis. The characterization of this mechanism in human cancer cells may provide a theoretical basis for utilizing the bichalcone TSWU-CD4 to treat cancers. Acknowledgments M.L. Lin was supported by a grant from China Medical University (CMU102-S-09), Taiwan. Conflict of interest We (the authors) disclose that there are no financial or personal relationships with other people or organizations that could inappropriately influence (bias) our work, ‘‘Suppression of PI3K/Akt signaling by synthetic bichalcone analog TSWU-CD4 induces ER stress- and Bax/Bak-mediated apoptosis of cancer cells’’.

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