Toxicology Letters 228 (2014) 170–178
Contents lists available at ScienceDirect
Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet
Anacardic acid induces cell apoptosis associated with induction of ATF4-dependent endoplasmic reticulum stress Hongbiao Huang a,1 , Xianliang Hua a,1 , Ningning Liu a,b,1 , Xiaofen Li a , Shouting Liu a , Xin Chen a , Chong Zhao a , Xiaoying Lan a , Changshan Yang a , Q. Ping Dou a,c , Jinbao Liu a,∗ a State Key Lab of Respiratory Disease, Protein Modification and Degradation Lab, Department of Pathophysiology, Guangzhou Medical University, Guangdong 510182, People’s Republic of China b Guangzhou Research Institute of Cardiovascular Disease, The Second Affiliated Hospital, Guangzhou Medical University, Guangzhou, Guangdong 510260, People’s Republic of China c The Molecular Therapeutics Program, Barbara Ann Karmanos Cancer Institute, and Departments of Oncology, Pharmacology and Pathology, School of Medicine, Wayne State University, Detroit, MI 48201-2013, USA
h i g h l i g h t s • • • •
Anacardic acid (AA) inhibits tumor growth in vitro and in vivo. AA induces endoplasmic reticulum (ER) stress in vitro and in vivo. Induction of ATF4-dependent ER stress contributes to AA-induced tumor cell death. Extrinsic and intrinsic apoptotic pathways are involved in AA-induced cell apoptosis.
a r t i c l e
i n f o
Article history: Received 24 February 2014 Received in revised form 5 May 2014 Accepted 5 May 2014 Available online 20 May 2014 Keywords: Anacardic acid ER stress ATF4 Apoptosis
a b s t r a c t Anacardic acid (6-pentadecylsalicylic acid, AA), a natural compound isolated from the traditional medicine Amphipterygium adstringens, has been reported to possess antitumor activities. However, its molecular targets have not been thoroughly studied. Here, we report that AA is a potent inducer of endoplasmic reticulum (ER) stress, leading to apoptosis in hepatoma HepG2 and myeloma U266 cells. Induction of ER stress by AA was supported by a dose- and time-dependent increase in expression of the ER signaling downstream molecules, such as GRP78/BiP, phosphorylated eIF2␣, ATF4 and CHOP in both HepG2 and U266 cell lines. Blockage of ATF4 expression by siRNA partially inhibited, while knockdown of CHOP expression by siRNA slightly increased AA-induced cell death in these cells. In addition, AA suppressed HepG2 xenograft tumor growth, associated with increased ER stress in vivo. These results suggest that AA induces tumor cell apoptosis associated with ATF4-dependent ER stress. © 2014 Published by Elsevier Ireland Ltd.
1. Introduction Discovery of novel agents with anticancer activity from natural resources has gained a significant important position in cancer prevention and treatment. Anacardic acid (6-pentadecylsalicylic acid, AA) is a major active compound mainly isolated from Amphipterygium adstringens (Acevedo et al., 2006), Ozoroa insignis (Rea et al., 2003), Anacardium occidentale (Kubo et al., 1999) and Ginkgo biloba (Itokawa et al., 1987). The bark of
∗ Corresponding author. Tel.: +86 20 81340720; fax: +86 20 81340542. E-mail address: [email protected] (J. Liu). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.toxlet.2014.05.012 0378-4274/© 2014 Published by Elsevier Ireland Ltd.
Amphipterygium adstringens has been used for the treatment of gastric ulcers, gastritis, and stomach cancers in Mexico (Acevedo et al., 2006). Several targets of AA have been reported. AA exerted antiinflammatory effect by inhibiting IL-8 expression which is important for initiation and persistence of inflammation. AA inhibited decreased acetylation of histone 4 at the IL-8 promoter, resulting in inhibition of LPS-induced IL-8 expression (Trevisan et al., 2006). AA also possessed anti-microbial (Kubo et al., 1999; Muroi and Kubo, 1996) and antioxidant effects (Trevisan et al., 2006). Recent studies have reported that AA exhibited antitumor activities. AA induced caspase-independent apoptosis in pituitary adenoma and lung adenocarcinoma cells (Seong et al., 2013a; Sukumari-Ramesh et al., 2011). AA inhibited activities of histone
H. Huang et al. / Toxicology Letters 228 (2014) 170–178
acetyltransferase and NF-B, both of which are potential targets for chemotherapy (Balasubramanyam et al., 2003; Sun et al., 2006; Sung et al., 2008). AA could also inhibit angiogenesis in vitro and in vivo through suppressing vascular endothelial growth factor receptor-2 (VEGFR-2) signaling (Wu et al., 2011). Treatment with AA inhibited the estrogen receptor alpha-DNA binding and reduced target gene transcription, leading to inhibition of breast cancer cell proliferation (Schultz et al., 2010). AA also induced apoptosis through down-regulation of androgen receptor (AR) by suppressing p300 and upregulation of p53 in prostate cancer cells (Tan et al., 2012). Although several cellular proteins or activities could be affected by AA treatment as mentioned before, its specific molecular target(s) remain to be discovered. Protein folding in the endoplasmic reticulum (ER) could be impaired under various physical and pathological conditions, termed ER stress. The ER has evolved highly specific signaling pathways to ensure that its protein folding capacity is not overwhelmed, all of which are collectively termed the unfolded protein response (UPR). However, severe or prolonged ER stress can induce apoptosis when the UPR fails to compensate for the stimulus (Huang et al., 2009; Jiang et al., 2012). Multiple pathways may be involved in ER stress-initiated apoptosis. Three distinct branches of the UPR have been identified based on distinct sensors: IRE1, PKR-like endoplasmic reticulum kinase (PERK), and ATF6 (Ron and Walter, 2007; Sano and Reed, 2013; Xu et al., 2005). Among them, the PERK pathway leads to phosphorylation of eukaryotic initiation factor 2␣ (eIF2␣) and enhances translation of mRNAs such as ATF4. The eIF2␣-ATF4 signaling has a preapoptotic function that involves the activation of binding protein (BiP), phosphorylated eIF2␣, ATF4 and C/EBP homologous protein (CHOP) (Armstrong et al., 2010; Jiang and Wek, 2005; Qing et al., 2012). Here we report that when applied in HepG2 and U266 cell lines and HepG2 xenografts, AA could induce ATF4-dependent ER stress via a mechanism distinct to previously reported (Tan et al., 2012), associated with its antitumor activity.
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2. Material and methods 2.1. Materials, reagents, and antibodies AA and Cremophor EL were purchased from Sigma (St. Louis, MO, USA). Fetal bovine serum (FBS), RPMI 1640, penicillin and streptomycin were purchased from Invitrogen Life Technology (Carlsbad, CA USA). Rabbit polyclonal antibody against GAPDH (FL335) was from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Rabbit polyclonal antibodies against nuclear poly (ADP-ribose) polymerase (PARP), eIF2␣, Phospho-eIF2␣ (Ser51), rabbit monoclonal antibodies against BiP (C50B12), Bcl-2 (50E3), Bim (C34C5), ATF-4 (D4B8), caspase-3 (8G10), caspase-8 (D35G2) and mouse monoclonal antibodies against caspase-9 (C9), CHOP (L63F7) were obtained from Cell Signaling (Beverly, MA, USA). Rabbit polyclonal antibodies against actived-caspase-3, -8, -9 and Bid were purchased from Bioworld Technology Inc. Enhanced chemiluminescence (ECL) reagents were from Amersham Biosciences (Piscataway, NJ, USA). Propidium idodide (PI) and Annexin V-FITC Apoptosis Detection Kit were from Keygene Company (Nanjing, China). 2.2. Cell lines and cell culture Human hepatoma cell line HepG2, myeloma cell line U266 and non-transformed bronchial epithelial 16HBE cell line were purchased from American Type Culture Collection (Manassas, VA, USA) and grown in RPMI 1640 supplemented with 10% FBS, 100 units/mL of penicillin and 100 g/mL of streptomycin. Cell cultures were maintained at 37 ◦ C and 5% CO2 . 2.3. MTS assay The effects of compounds on cell viability were determined by the MTS assay (Cell Titer 96® AQueous One Solution Cell Proliferation assay, Promega Corporation, Madison, WI, USA) as described
Fig. 1. The growth inhibitory effect of anacardiac acid (AA) on cancer and mom-transformed cells. ((A)–(C)) HepG2, U266 and 16HBE cells were treated with increasing concentration of AA (20 to 60 M) for 24 h, 48 h, and 72 h, and cell viability was detected by MTS. Cell viability in HepG2, U266 and 16HBE cells were shown in (A)–(C), respectively. Mean ± SD (n = 3). * P < 0.05, versus each DMSO control. DM:DMSO. (D) AA inhibits colony formation in HepG2 and U266 cells. HepG2 and U266 cells were treated with AA for 12 h, and then suspended in soft agar. The number of colonies >60 m were counted after 7 days’ culture under light microscope. The experiments were tested in triplicate. Quantification of cells as a percentage of solvent DMSO treated cells was shown. Mean ± SD (n = 3). * P < 0.05, versus DMSO control.
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Fig. 2. AA induces apoptosis in HepG2 and U266 cancer cells but not in non-transformed 16HBE cells. (A–C) Cultured cells were treated with either solvent DMSO or 20–60 M AA for 24 h (HepG2, U266, 16HBE), or 48 h (U266), followed by staining with Annexin V/propidium (PI) and analysis with either flow cytometry or imaging under inverted fluorescence microscope. Typical flow images and summary of cell apoptosis were shown in (A) and (B), respectively. Means ± SD (n = 3). * P < 0.05, versus DMSO control. Fluorescent images with 60 M AA treatment for 24 h (HepG2) or 48 h (U266) were shown in (C). (D) HepG2 and U266 cells were treated with 20–60 M AA for 24 h. Proteins extracts from the treated cells were subjected to Western blot analysis by using antibodies against Bcl-2, Bim, Bid, pro-caspase-8, cleaved caspase-8, pro-caspase-9, cleaved caspase-9, pro-caspase-3, cleaved caspase-3 and PARP. GAPDH was used as a loading control.
H. Huang et al. / Toxicology Letters 228 (2014) 170–178
previously (Huang et al., 2011). Exponentially growing cells were harvested and seeded at 2500 cells/well in a 96-well plate. After 24 h incubation, compounds or DMSO as the untreated control were added, followed by continuous incubation for the indicated times. 20 L of MTS were added to each well and the incubation was continued for an additional 3 h. The absorbance was measured with a microplate reader (Sunrise, Tecan) at 490 nm. Cell viability was calculated as follows: (absorbance of experimental well − absorbance of blank)/(absorbance of untreated control well − absorbance of blank) × 100%. 2.4. Clonogenic assay This assay was performed as we previously described (Shi et al., 2014). HepG2 and U266 cells exposed to either vehicle or AA for 12 h were suspended in 30% agarose supplemented with 20% FCS and 50% RPMI-1640 medium then cultured in 60 mm dishes in an atmosphere of 5% CO2 for 7 days, then stained with 0.3% crystal violet solution. The colonies >60 m were counted under the light microscope. The experiments were done in triplicate.
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Body weight, tumor weight, tumor volume were detected and summarized. 2.9. Western blot analysis Western blotting was performed as previously described (Huang et al., 2012). In brief, an equal amount of total protein extracts from cultured cells were fractionated by 12% SDS-PAGE and electrically transferred onto polyvinylidene difluoride (PVDF) membranes. Mouse or rabbit primary antibodies and horseradish peroxidase (HRP)-conjugated appropriate secondary antibodies were used to detect the designated proteins. The bound secondary antibodies on the PVDF membrane were reacted with ECL detection reagents (Amersham Bioscience) and exposed to X-ray films (Kodak, Japan). 2.10. Statistical methods All experiments were performed at least thrice, and the results were expressed as Mean ± SD where applicable. GraphPad Prism 4.0 software (GraphPad Software) was used for statistical analysis. Comparison of multiple groups was made with one-way ANOVA
2.5. Cell death detection assay via flow cytometry This was performed using Annexin V-FITC and propidium iodide (PI) double staining followed by flow cytometry as previously described (Huang et al., 2010). Briefly, cultured HepG2, U266 and 16HBE cells were harvested and washed with cold PBS and resuspended with the binding buffer, followed by Annexin V-FITC incubation for 15 min and PI staining for another 15 min at 4 ◦ C in dark. The stained cells were analyzed with flow cytometry within 30 min. 2.6. Morphological characterization of cell death To monitor temporal changes in the incidence of cell death in the live culture condition, Annexin V-FITC and propidium idodide (PI) were added to the cell culture medium and at the desired sequential time points, the HepG2 and U266 cells in the culture dish were imaged with an inverted fluorescence microscope equipped with a digital camera (Axio Obsever Z1, Zeiss). 2.7. RNA interference To knockdown CHOP or ATF-4 expression in cells, siRNA targeting human CHOP or ATF-4 were synthesized and purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). siRNA with non-specific sequences were used as control scrambled siRNA. Different siRNAs were transfected separately into cells by using Lipofecatmine 2000 (Invitrogen) reagent and medium was replaced 6 h after transfection. 2.8. Establishment and treatment of HepG2 xenografts Male Balb/c nude mice aged 5 weeks were purchased from Guangdong Animal Center and housed in accordance with protocols approved by the Guangdong Animal Center. Balb/c nude mice were s.c. inoculated in the left armpit of each mouse with HepG2 cells (1 × 106 cells/mouse). When the tumor size reaches 50–75 mm3 , mice were randomly divided into two groups (6 mice per group). Nude mice bearing HepG2 tumor were i.p. injected with vehicle and AA (2 mg/kg, once/day), respectively, for 14 days. Tumors were measured by caliper and tumor volume was calculated using standard formula: width2 × length/2.
Fig. 3. Dosage and kinetic effects of AA on ER stress pathway in HepG2 and U266 cells. ((A) and (B)) HepG2 and U266 cells were treated with 20–60 M AA for 24 h or 60 M AA for the indicated times. Proteins extracts from the treated cells were subjected to Western blot analysis by using antibodies against BiP, CHOP, P-eIF2␣, eIF2␣, ATF4. GAPDH blot was used as a loading control.
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Fig. 4. AA-induced cell death is independent of CHOP. ((A) and (B)) HepG2 and U266 cells were transfected with either CHOP siRNA or scrambled siRNA for 48 h, and then treated with 60 M AA for 24 h, followed by apoptosis assay with flow cytometry. ((C) and (D)) Summary of cell death data in different groups as in ((A) and (B)). * P < 0.05, versus AA treatment or CHOP knockdown alone. Means ± SD (n = 3). (E) HepG2 and U266 cells were treated as in ((A) and (B)). Proteins extracts from the treated cells were subjected to Western blot analysis by using antibodies against CHOP and PARP. GAPDH blot was used as a loading control.
followed by Tukey’s test or Newman–Kueuls test. P value of
Contents lists available at ScienceDirect
Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet
Anacardic acid induces cell apoptosis associated with induction of ATF4-dependent endoplasmic reticulum stress Hongbiao Huang a,1 , Xianliang Hua a,1 , Ningning Liu a,b,1 , Xiaofen Li a , Shouting Liu a , Xin Chen a , Chong Zhao a , Xiaoying Lan a , Changshan Yang a , Q. Ping Dou a,c , Jinbao Liu a,∗ a State Key Lab of Respiratory Disease, Protein Modification and Degradation Lab, Department of Pathophysiology, Guangzhou Medical University, Guangdong 510182, People’s Republic of China b Guangzhou Research Institute of Cardiovascular Disease, The Second Affiliated Hospital, Guangzhou Medical University, Guangzhou, Guangdong 510260, People’s Republic of China c The Molecular Therapeutics Program, Barbara Ann Karmanos Cancer Institute, and Departments of Oncology, Pharmacology and Pathology, School of Medicine, Wayne State University, Detroit, MI 48201-2013, USA
h i g h l i g h t s • • • •
Anacardic acid (AA) inhibits tumor growth in vitro and in vivo. AA induces endoplasmic reticulum (ER) stress in vitro and in vivo. Induction of ATF4-dependent ER stress contributes to AA-induced tumor cell death. Extrinsic and intrinsic apoptotic pathways are involved in AA-induced cell apoptosis.
a r t i c l e
i n f o
Article history: Received 24 February 2014 Received in revised form 5 May 2014 Accepted 5 May 2014 Available online 20 May 2014 Keywords: Anacardic acid ER stress ATF4 Apoptosis
a b s t r a c t Anacardic acid (6-pentadecylsalicylic acid, AA), a natural compound isolated from the traditional medicine Amphipterygium adstringens, has been reported to possess antitumor activities. However, its molecular targets have not been thoroughly studied. Here, we report that AA is a potent inducer of endoplasmic reticulum (ER) stress, leading to apoptosis in hepatoma HepG2 and myeloma U266 cells. Induction of ER stress by AA was supported by a dose- and time-dependent increase in expression of the ER signaling downstream molecules, such as GRP78/BiP, phosphorylated eIF2␣, ATF4 and CHOP in both HepG2 and U266 cell lines. Blockage of ATF4 expression by siRNA partially inhibited, while knockdown of CHOP expression by siRNA slightly increased AA-induced cell death in these cells. In addition, AA suppressed HepG2 xenograft tumor growth, associated with increased ER stress in vivo. These results suggest that AA induces tumor cell apoptosis associated with ATF4-dependent ER stress. © 2014 Published by Elsevier Ireland Ltd.
1. Introduction Discovery of novel agents with anticancer activity from natural resources has gained a significant important position in cancer prevention and treatment. Anacardic acid (6-pentadecylsalicylic acid, AA) is a major active compound mainly isolated from Amphipterygium adstringens (Acevedo et al., 2006), Ozoroa insignis (Rea et al., 2003), Anacardium occidentale (Kubo et al., 1999) and Ginkgo biloba (Itokawa et al., 1987). The bark of
∗ Corresponding author. Tel.: +86 20 81340720; fax: +86 20 81340542. E-mail address: [email protected] (J. Liu). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.toxlet.2014.05.012 0378-4274/© 2014 Published by Elsevier Ireland Ltd.
Amphipterygium adstringens has been used for the treatment of gastric ulcers, gastritis, and stomach cancers in Mexico (Acevedo et al., 2006). Several targets of AA have been reported. AA exerted antiinflammatory effect by inhibiting IL-8 expression which is important for initiation and persistence of inflammation. AA inhibited decreased acetylation of histone 4 at the IL-8 promoter, resulting in inhibition of LPS-induced IL-8 expression (Trevisan et al., 2006). AA also possessed anti-microbial (Kubo et al., 1999; Muroi and Kubo, 1996) and antioxidant effects (Trevisan et al., 2006). Recent studies have reported that AA exhibited antitumor activities. AA induced caspase-independent apoptosis in pituitary adenoma and lung adenocarcinoma cells (Seong et al., 2013a; Sukumari-Ramesh et al., 2011). AA inhibited activities of histone
H. Huang et al. / Toxicology Letters 228 (2014) 170–178
acetyltransferase and NF-B, both of which are potential targets for chemotherapy (Balasubramanyam et al., 2003; Sun et al., 2006; Sung et al., 2008). AA could also inhibit angiogenesis in vitro and in vivo through suppressing vascular endothelial growth factor receptor-2 (VEGFR-2) signaling (Wu et al., 2011). Treatment with AA inhibited the estrogen receptor alpha-DNA binding and reduced target gene transcription, leading to inhibition of breast cancer cell proliferation (Schultz et al., 2010). AA also induced apoptosis through down-regulation of androgen receptor (AR) by suppressing p300 and upregulation of p53 in prostate cancer cells (Tan et al., 2012). Although several cellular proteins or activities could be affected by AA treatment as mentioned before, its specific molecular target(s) remain to be discovered. Protein folding in the endoplasmic reticulum (ER) could be impaired under various physical and pathological conditions, termed ER stress. The ER has evolved highly specific signaling pathways to ensure that its protein folding capacity is not overwhelmed, all of which are collectively termed the unfolded protein response (UPR). However, severe or prolonged ER stress can induce apoptosis when the UPR fails to compensate for the stimulus (Huang et al., 2009; Jiang et al., 2012). Multiple pathways may be involved in ER stress-initiated apoptosis. Three distinct branches of the UPR have been identified based on distinct sensors: IRE1, PKR-like endoplasmic reticulum kinase (PERK), and ATF6 (Ron and Walter, 2007; Sano and Reed, 2013; Xu et al., 2005). Among them, the PERK pathway leads to phosphorylation of eukaryotic initiation factor 2␣ (eIF2␣) and enhances translation of mRNAs such as ATF4. The eIF2␣-ATF4 signaling has a preapoptotic function that involves the activation of binding protein (BiP), phosphorylated eIF2␣, ATF4 and C/EBP homologous protein (CHOP) (Armstrong et al., 2010; Jiang and Wek, 2005; Qing et al., 2012). Here we report that when applied in HepG2 and U266 cell lines and HepG2 xenografts, AA could induce ATF4-dependent ER stress via a mechanism distinct to previously reported (Tan et al., 2012), associated with its antitumor activity.
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2. Material and methods 2.1. Materials, reagents, and antibodies AA and Cremophor EL were purchased from Sigma (St. Louis, MO, USA). Fetal bovine serum (FBS), RPMI 1640, penicillin and streptomycin were purchased from Invitrogen Life Technology (Carlsbad, CA USA). Rabbit polyclonal antibody against GAPDH (FL335) was from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Rabbit polyclonal antibodies against nuclear poly (ADP-ribose) polymerase (PARP), eIF2␣, Phospho-eIF2␣ (Ser51), rabbit monoclonal antibodies against BiP (C50B12), Bcl-2 (50E3), Bim (C34C5), ATF-4 (D4B8), caspase-3 (8G10), caspase-8 (D35G2) and mouse monoclonal antibodies against caspase-9 (C9), CHOP (L63F7) were obtained from Cell Signaling (Beverly, MA, USA). Rabbit polyclonal antibodies against actived-caspase-3, -8, -9 and Bid were purchased from Bioworld Technology Inc. Enhanced chemiluminescence (ECL) reagents were from Amersham Biosciences (Piscataway, NJ, USA). Propidium idodide (PI) and Annexin V-FITC Apoptosis Detection Kit were from Keygene Company (Nanjing, China). 2.2. Cell lines and cell culture Human hepatoma cell line HepG2, myeloma cell line U266 and non-transformed bronchial epithelial 16HBE cell line were purchased from American Type Culture Collection (Manassas, VA, USA) and grown in RPMI 1640 supplemented with 10% FBS, 100 units/mL of penicillin and 100 g/mL of streptomycin. Cell cultures were maintained at 37 ◦ C and 5% CO2 . 2.3. MTS assay The effects of compounds on cell viability were determined by the MTS assay (Cell Titer 96® AQueous One Solution Cell Proliferation assay, Promega Corporation, Madison, WI, USA) as described
Fig. 1. The growth inhibitory effect of anacardiac acid (AA) on cancer and mom-transformed cells. ((A)–(C)) HepG2, U266 and 16HBE cells were treated with increasing concentration of AA (20 to 60 M) for 24 h, 48 h, and 72 h, and cell viability was detected by MTS. Cell viability in HepG2, U266 and 16HBE cells were shown in (A)–(C), respectively. Mean ± SD (n = 3). * P < 0.05, versus each DMSO control. DM:DMSO. (D) AA inhibits colony formation in HepG2 and U266 cells. HepG2 and U266 cells were treated with AA for 12 h, and then suspended in soft agar. The number of colonies >60 m were counted after 7 days’ culture under light microscope. The experiments were tested in triplicate. Quantification of cells as a percentage of solvent DMSO treated cells was shown. Mean ± SD (n = 3). * P < 0.05, versus DMSO control.
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Fig. 2. AA induces apoptosis in HepG2 and U266 cancer cells but not in non-transformed 16HBE cells. (A–C) Cultured cells were treated with either solvent DMSO or 20–60 M AA for 24 h (HepG2, U266, 16HBE), or 48 h (U266), followed by staining with Annexin V/propidium (PI) and analysis with either flow cytometry or imaging under inverted fluorescence microscope. Typical flow images and summary of cell apoptosis were shown in (A) and (B), respectively. Means ± SD (n = 3). * P < 0.05, versus DMSO control. Fluorescent images with 60 M AA treatment for 24 h (HepG2) or 48 h (U266) were shown in (C). (D) HepG2 and U266 cells were treated with 20–60 M AA for 24 h. Proteins extracts from the treated cells were subjected to Western blot analysis by using antibodies against Bcl-2, Bim, Bid, pro-caspase-8, cleaved caspase-8, pro-caspase-9, cleaved caspase-9, pro-caspase-3, cleaved caspase-3 and PARP. GAPDH was used as a loading control.
H. Huang et al. / Toxicology Letters 228 (2014) 170–178
previously (Huang et al., 2011). Exponentially growing cells were harvested and seeded at 2500 cells/well in a 96-well plate. After 24 h incubation, compounds or DMSO as the untreated control were added, followed by continuous incubation for the indicated times. 20 L of MTS were added to each well and the incubation was continued for an additional 3 h. The absorbance was measured with a microplate reader (Sunrise, Tecan) at 490 nm. Cell viability was calculated as follows: (absorbance of experimental well − absorbance of blank)/(absorbance of untreated control well − absorbance of blank) × 100%. 2.4. Clonogenic assay This assay was performed as we previously described (Shi et al., 2014). HepG2 and U266 cells exposed to either vehicle or AA for 12 h were suspended in 30% agarose supplemented with 20% FCS and 50% RPMI-1640 medium then cultured in 60 mm dishes in an atmosphere of 5% CO2 for 7 days, then stained with 0.3% crystal violet solution. The colonies >60 m were counted under the light microscope. The experiments were done in triplicate.
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Body weight, tumor weight, tumor volume were detected and summarized. 2.9. Western blot analysis Western blotting was performed as previously described (Huang et al., 2012). In brief, an equal amount of total protein extracts from cultured cells were fractionated by 12% SDS-PAGE and electrically transferred onto polyvinylidene difluoride (PVDF) membranes. Mouse or rabbit primary antibodies and horseradish peroxidase (HRP)-conjugated appropriate secondary antibodies were used to detect the designated proteins. The bound secondary antibodies on the PVDF membrane were reacted with ECL detection reagents (Amersham Bioscience) and exposed to X-ray films (Kodak, Japan). 2.10. Statistical methods All experiments were performed at least thrice, and the results were expressed as Mean ± SD where applicable. GraphPad Prism 4.0 software (GraphPad Software) was used for statistical analysis. Comparison of multiple groups was made with one-way ANOVA
2.5. Cell death detection assay via flow cytometry This was performed using Annexin V-FITC and propidium iodide (PI) double staining followed by flow cytometry as previously described (Huang et al., 2010). Briefly, cultured HepG2, U266 and 16HBE cells were harvested and washed with cold PBS and resuspended with the binding buffer, followed by Annexin V-FITC incubation for 15 min and PI staining for another 15 min at 4 ◦ C in dark. The stained cells were analyzed with flow cytometry within 30 min. 2.6. Morphological characterization of cell death To monitor temporal changes in the incidence of cell death in the live culture condition, Annexin V-FITC and propidium idodide (PI) were added to the cell culture medium and at the desired sequential time points, the HepG2 and U266 cells in the culture dish were imaged with an inverted fluorescence microscope equipped with a digital camera (Axio Obsever Z1, Zeiss). 2.7. RNA interference To knockdown CHOP or ATF-4 expression in cells, siRNA targeting human CHOP or ATF-4 were synthesized and purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). siRNA with non-specific sequences were used as control scrambled siRNA. Different siRNAs were transfected separately into cells by using Lipofecatmine 2000 (Invitrogen) reagent and medium was replaced 6 h after transfection. 2.8. Establishment and treatment of HepG2 xenografts Male Balb/c nude mice aged 5 weeks were purchased from Guangdong Animal Center and housed in accordance with protocols approved by the Guangdong Animal Center. Balb/c nude mice were s.c. inoculated in the left armpit of each mouse with HepG2 cells (1 × 106 cells/mouse). When the tumor size reaches 50–75 mm3 , mice were randomly divided into two groups (6 mice per group). Nude mice bearing HepG2 tumor were i.p. injected with vehicle and AA (2 mg/kg, once/day), respectively, for 14 days. Tumors were measured by caliper and tumor volume was calculated using standard formula: width2 × length/2.
Fig. 3. Dosage and kinetic effects of AA on ER stress pathway in HepG2 and U266 cells. ((A) and (B)) HepG2 and U266 cells were treated with 20–60 M AA for 24 h or 60 M AA for the indicated times. Proteins extracts from the treated cells were subjected to Western blot analysis by using antibodies against BiP, CHOP, P-eIF2␣, eIF2␣, ATF4. GAPDH blot was used as a loading control.
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Fig. 4. AA-induced cell death is independent of CHOP. ((A) and (B)) HepG2 and U266 cells were transfected with either CHOP siRNA or scrambled siRNA for 48 h, and then treated with 60 M AA for 24 h, followed by apoptosis assay with flow cytometry. ((C) and (D)) Summary of cell death data in different groups as in ((A) and (B)). * P < 0.05, versus AA treatment or CHOP knockdown alone. Means ± SD (n = 3). (E) HepG2 and U266 cells were treated as in ((A) and (B)). Proteins extracts from the treated cells were subjected to Western blot analysis by using antibodies against CHOP and PARP. GAPDH blot was used as a loading control.
followed by Tukey’s test or Newman–Kueuls test. P value of
