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Introduction

Multiple myeloma (MM) is a clonal B-cell malignancy that primarily affects elderly individuals, accounting for ~1% of all cancers (1,2). Cancerous plasma cells accumulate in the bone marrow; the effects of this include hypercalcemia, renal failure, anemia and osteolytic bone lesions (1,2). MM may be treated with the novel therapeutic agents, proteasome inhibitors and immunomodulatory drugs, which may be combined with conventional chemotherapeutics. However, almost all MM patients ultimately relapse, even when complete remission is achieved following initial therapy (2).

The majority of intracellular proteins are degraded by the ubiquitin-proteasome system (UPS) (3). Abnormal proteasome-dependent protein degradation is associated with the pathophysiology of multiple cancer types; therefore, it has been proposed that the selective inhibition of UPS may provide a novel strategy for the development of anticancer therapeutics (4–6). Notably, the proteasome inhibitor bortezomib (Bor) has been successfully developed for relapsed/refractory MM therapy. Bor has demonstrated a marked effect in MM patients; however, Bor resistance and its secondary side effects, including bone growth impairment, restrict the use of this therapy (7,8). Adjuvant agents are therefore required to chemosensitize MM cells to Bor and achieve therapeutic efficacy with limited toxicity.

Bor treatment results in the aggregation of ubiquitinated proteins, endoplasmic reticulum (ER) stress and apoptotic cell death, via inhibition of 26S proteasome activity. The proper folding of proteins prior to exit from the ER is ensured by quality control mechanisms; ER stress is triggered by improper protein folding and involves various signaling pathways collectively referred to as the unfolded protein response (UPR). Severe or prolonged ER stress promotes apoptotic cell death in the event that the UPR is unable to resolve the situation (9,10). Various signaling pathways may modulate ER stress-induced programmed cell death. To date, three contributing UPR branches have been identified: Inositol-requiring enzyme 1, protein kinase RNA-like ER kinase (PERK) and activating transcription factor (ATF) 6 (11–13). PERK signaling induces eukaryotic initiation factor 2α (eIF2α) phosphorylation, enhancing ATF4 protein synthesis. The pre-apoptotic eIF2α-ATF4 signaling pathway involves binding protein (BiP), phosphorylated eIF2α, ATF4 and CCAAT-enhancer binding protein homologous protein (CHOP) activation (14–16). Heat shock protein 70 (HSP70; encoded by HSPA8), a member of the 70 kDa HSPs family, is a primary chaperone involved in ER stress. HSP70 binds to and censors the folding status of substrate membrane proteins that are synthesized in the ER and transported to the cell surface via the conventional ER-to-Golgi secretion pathway (17).

Anacardic acid (AA; also referred to as 6-pentadecylsalicylic acid) is a constituent of the traditional medicinal plant Amphipterygium adstringens. Previous studies have revealed that AA exerts anticancer effects in various carcinomas (18,19). Previous studies by our laboratory and others have demonstrated that AA induces ER stress (20,21). In addition, it has been established that the ER stress inducer fenretinide sensitizes tumor cells to Bor-mediated killing (22). Therefore, the aim of the present study was to assess whether AA enhances the anticancer effects of Bor. AA was observed to significantly increase Bor activity via enhancing ATF4-dependent ER stress-associated caspase activation in vitro.

Materials and methods

Materials, reagents and antibodies

AA was manufactured by Sigma-Aldrich (St. Louis, MO, USA). Bor (Ben Venue Laboratories, Inc.; Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT, USA) was used according to the manufacturer's instructions. Fetal bovine serum (FBS), RPMI-1640 and antibiotics were produced by Invitrogen; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Rabbit polyclonal anti-GAPDH antibody (clone, FL-335; catalog no. sc-25778; 1:500) was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The following were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA): Rabbit monoclonal antibodies against nuclear poly (ADP-ribose) polymerase (PARP; clone, 46D11; catalog no. 9532; 1:1,000), eIF2α (clone, D7D3; catalog no. 9079; 1:1,000), phospho-eIF2α (Ser51; clone, D9G8; catalog no. 3398; 1:1,000), BiP (clone, C50B12; catalog no. 3177; 1:1,000), ATF4 (clone, D4B8; catalog no. 11,815; 1:1,000), caspase-3 (clone, 8G10; catalog no. 9665; 1:1,000) and caspase-8 (clone, D35G2; catalog no. 4790; 1:1,000); mouse monoclonal antibodies against caspase-9 (clone, C9; catalog no. 9508; 1:1,000) and CHOP (clone, L63F7; catalog no. 2895; 1:1,000); and a rat monoclonal antibody against HSP70 (clone 6B3; catalog no. 4873; 1:1,000). Rabbit polyclonal antibodies against active caspase-3 (catalog no. BS7004; 1:1,000), caspase-8 (catalog no. AP0358; 1:1,000) and caspase-9 (catalog no. BS7070; 1:1,000) were manufactured by Bioworld Technology, Inc. (St. Louis Park, NM, USA). Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (catalog no. sc-395,763; 1:5,000), HRP-conjugated goat anti-rabbit IgG (catalog no. sc-2004; 1:5,000) and HRP-conjugated goat anti-rat IgG (catalog no. sc-2006; 1:5,000) were purchased from Santa Cruz Biotechnology, Inc. The enhanced chemiluminescence (ECL) kit was obtained from GE Healthcare Life Sciences (Chalfont, UK). Propidium iodide (PI) and Caspase-3 Activity and Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection kits were manufactured by Nanjing Keygen Biotech Co., Ltd. (Nanjing, China).

Cell culture

U266 human myeloma cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured as previously described (21). AA and Bor were dissolved in dimethyl sulfoxide (DMSO) to a stock concentration of 50 mM, aliquoted and stored at −80°C. Prior to use, AA was diluted to 10, 20 and 30 mM; Bor was diluted to 25, 50 and 75 µM. During the treatment of each group, the corresponding drugs were diluted 1:1,000 in medium, added to the wells or plates and cultured at 37°C and 5% CO2 for the indicated time.

3-(4,5-dimethylthiazol-2-yl-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay

Cytotoxicity was assessed by the MTS assay as described previously (21,23). Exponentially growing cells were seeded into 96-well plates (2,500/well) and incubated with drugs for 48 h prior to assessment with MTS.

Flow cytometric analysis of cell apoptosis

Exponentially growing cells were seeded into 6-well plates (5×104/well) and incubated with drugs for 24 h. Apoptosis was quantified in cells using Annexin V-FITC and PI double staining as previously described (24). Stained U266 cells were assessed by flow cytometry within 30 min. The data was analyzed using FACSDiva software version 6.1.3 (BD Biosciences, Franklin Lakes, NJ, USA).

Caspase-3 activity evaluation

Exponentially growing cells were seeded into 6-cm dishes (1×106/well) and incubated with drugs for 24 h. Caspase-3 activity was determined in U266 cell lysates using a specific colorimetric assay kit according to the manufacturer's instructions. Following drug treatment, 1×106 cells were lysed with lysis buffer (Nanjing Keygen Biotech Co., Ltd.) and submitted to centrifugation (10,000 × g, 4°C, 1 min). The supernatants were harvested and the enzyme-specific substrate was added at 37°C for 4 h. The resulting product was quantified on a microplate reader at 405 nm.

RNA interference

CHOP or ATF-4 genes were silenced using small interfering RNA (siRNA) technology as described previously (21). CHOP/GADD153 siRNA (catalog no. sc-35437), ATF4/CREB-2 siRNA (catalog no. sc-35112) and control siRNA (catalog no. sc-37007), purchased from Santa Cruz Biotechnology, Inc., were transfected separately into cells using Lipofectamine® 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions.

Western blot analysis

Exponentially growing cells were seeded into 6-cm dishes (1×106/well) and incubated with drugs for 24 h. Protein expression levels were determined as previously described (25,26). Briefly, total protein extracts (40 µg) from U266 cell lysates were resolved by 12% SDS-PAGE (100 V for 90 min) and transferred onto polyvinylidene difluoride membranes. Membranes were blocked with 5% milk, and following sequential incubations with primary and secondary antibodies, an ECL kit was used for protein detection. Blots were quantified with Image-Pro Plus software version 5.0 (Media Cybernetics, Inc., Rockville, MD, USA).

Combination index assessment

The effects of AA and Bor were assessed by evaluating the combination index (CI) using the Chou-Talalay method, as described previously (26,27). A CI of <1, 1 or >1 indicated synergistic, additive or antagonistic effects, respectively.

Statistical analysis

Data are presented as the mean ± standard deviation. One-way analysis of variance was utilized to compare groups, with the least significant difference test being performed as a post hoc test. Statistical analyses were performed using SPSS software version 16.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

Results

AA and Bor induce human myeloma U266 cell killing in a synergistic fashion

To assess if AA alone causes myeloma cell death, the effects of AA at various concentrations on MM cell viability were assessed. Cell viability was inhibited <48% in U266 cells treated with 30 µM AA as a monotherapy compared with DMSO treatment (P=0.001; Fig. 1A). Based on these findings, 10, 20 and 30 µM AA were co-administered for 48 h with 25, 50 and 75 nM Bor. All CI results were <0.8, except one CI value of 0.809 (Fig. 1B), indicating synergy between these two agents in U266 cells. Doses of 20 µM AA and 50 nM Bor were chosen for subsequent experiments, as these doses were effective at reducing cell viability, but not to the extent that further analysis would be impossible.

Figure 1 Effects of AA, Bor and combination therapy on cell viability. (A) U266 cells were incubated with Bor (25, 50 or 75 nM) and AA (10, 20, or 30 µM) as monotherapy or in combination for 48 h. Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. Cell viability was reduced by AA or Bor monotherapy, and by AA and Bor in combination, in a dose-dependent manner. Data are presented as the mean ± standard deviation from three independent experiments. *P<0.05 vs. DMSO; #P<0.01 vs. respective AA monotherapy. (B) Combination index values were determined, and revealed that AA and Bor act synergistically in U266 cells. AA, anacardic acid; Bor, bortezomib; DMSO, dimethyl sulfoxide.

AA sensitizes U266 cells to Bor-mediated caspase-dependent apoptosis

To investigate whether AA- and/or Bor-induced cytotoxicity correlated with cell death, myeloma cells were incubated with AA and/or BOR, and cell death was assessed using Annexin V/PI double staining. Co-administration of Bor and AA resulted in a significant increase in Annexin V and PI positive cells compared with monotherapy (P<0.001; Fig. 2A and B), indicating that increased cell death was the result of Bor and AA combination therapy. The effects of combination therapy on cleavage of the apoptosis mediators, caspase and PARP, were investigated by western blotting. As presented in Fig. 2C, AA/Bor co-administration resulted in markedly enhanced cleavage of caspase-3, -8 and -9, as well as PARP, compared with monotherapies. To confirm these results, caspase-3 activity in cell lysates was assessed. AA/Bor combination therapy significantly increased caspase-3 activity compared with monotherapies (P<0.001; Fig. 2D). These results suggested that AA sensitized U266 cells to Bor via caspase-dependent apoptotic cell death.

Figure 2 AA sensitizes U266 cells to Bor-induced cytotoxicity. U266 cells were incubated with AA (20 µM), Bor (50 nM) or combination therapy for 24 h. (A) Cells were stained with Annexin V and propidium iodide. Representative flow cytograms are presented. Apoptotic cells were defined as those in the upper left, upper right and lower right quadrants. Co-administration of Bor and AA resulted in a significant increase in Annexin V and PI positive cells compared with monotherapy. (B) Flow cytometric analysis of (A), presented as the mean ± SD (n=3). *P<0.01 vs. monotherapy. (C) Western blotting was performed to assess the expression levels of various proteins, with GAPDH serving as a loading control. Cleavage of caspase-3, -8 and -9, and PARP, was increased following AA/Bor co-administration. (D) Caspase-3 activity was assessed in U266 cells by colorimetric assay, and was significantly increased upon AA/Bor combination therapy. Data are presented as the mean ± SD (n=3). *P<0.01 vs. monotherapy. AA, anacardic acid; Bor, bortezomib; PI, propidium iodide; SD, standard deviation; cas, caspase; PARP, poly (ADP-ribose) polymerase.

AA/Bor combination therapy amplifies ER stress

The effects of combination therapy on the UPR signaling pathway in U266 cells were analyzed by western blotting (Fig. 3). The expression levels of HSP70 (P=0.027) and BiP (P=0.001) were significantly increased by 24 h compared with Bor monotherapy. Combination therapy induced increased protein expression levels of CHOP, phospho-eIF2α and ATF4. These findings suggest that ER stress is involved in AA/Bor combination therapy-induced cell death.

Figure 3 AA enhances Bor-induced ER stress. U266 cells were incubated with AA (20 µM), Bor (50 nM) or combination therapy for 24 h. (A) Western blotting was performed to analyze protein expression levels of HSP70, BiP, CHOP, P-eIF2α, eIF2α, ATF4 and GAPDH. (B) Protein bands were quantified and normalized to GAPDH. The protein expression levels of HSP70, BiP, CHOP, P-eIF2α and ATF4 were significantly increased by AA/Bor combination therapy. *P<0.05 vs. monotherapy. Data are presented as the mean ± standard deviation (n=3). AA, anacardic acid; Bor, bortezomib; ER, endoplasmic reticulum; HSP70, heat shock protein 70; BiP, binding protein; CHOP, CCAAT-enhancer binding protein homologous protein; eIF2α, eukaryotic initiation factor 2α; P, phosphorylated; ATF4, activating transcription factor 4; DMSO, dimethyl sulfoxide.

Role of ER stress in AA/Bor combination therapy-mediated cytotoxicity

To identify UPR effectors involved in AA/Bor combination therapy-mediated cell death, CHOP was silenced in U266 cells. Cells were then incubated for 24 h in the presence or absence of AA/Bor combination therapy. CHOP siRNA inhibited CHOP protein expression levels and slightly increased PARP cleavage in U266 cells incubated with combination therapy, compared to cells that received scrambled siRNA (Fig. 4A). In addition, CHOP silencing significantly increased the cytotoxicity of combination therapy compared with scrambled siRNA (P=0.008; Fig. 4B). These findings suggested that CHOP was not the primary UPR signaling pathway branch involved in U266 cell death mediated by AA/Bor combination therapy.

Figure 4 AA enhances Bor-mediated cytotoxicity involving ATF4 but not CHOP. CHOP-silenced U266 cells were incubated with AA (20 µM), Bor (50 nM) or combination therapy for 24 h. (A) Western blotting was performed to assess the protein expression levels of CHOP and PARP, with GAPDH serving as a loading control. (B) Apoptotic cell death was assessed by flow cytometry; CHOP silencing increased the cytotoxicity of combination therapy. ATF4-silenced U266 cells were incubated with AA (20 µM), Bor (50 nM) or combination therapy for 24 h. (C) Western blotting was performed to assess the protein levels of ATF4 and PARP, with GAPDH serving as a loading control. (D) Apoptotic cell death was assessed by flow cytometry; ATF4 silencing decreased the cytotoxicity of combination therapy. Data are presented as the mean ± standard deviation (n=3). *P<0.05 vs. scrambled siRNA. AA, anacardic acid; Bor, bortezomib; CHOP, CCAAT-enhancer binding protein homologous protein; ATF4, activating transcription factor 4; PARP, poly (ADP-ribose) polymerase; siRNA, small interfering RNA; DMSO, dimethyl sulfoxide.

The role of ATF4 in AA/Bor combination therapy-mediated cell death was subsequently assessed. In contrast to CHOP repression, ATF4 silencing decreased PARP cleavage (Fig. 4C) and partially attenuated AA/Bor combination therapy-mediated cytotoxicity compared with scrambled siRNA (P=0.002; Fig. 4D). These data indicate that ATF4-dependent ER stress contributed, at least partially, to AA/Bor combination therapy-mediated cytotoxicity.

Discussion

Various novel natural compounds have been reported to have synergistic anti-cancer cytotoxic effects when administered in combination with Bor (26,28,29). Our previous study demonstrated that AA is a potent inducer of ER stress (21). Based on previous findings that the ER stress inducer fenretinide sensitizes tumor cells to killing by Bor (22), the effect of AA/Bor combination therapy on U266 cells in vitro was investigated, to examine the potential clinical application of AA.

Inhibition of cell growth and promotion of apoptosis constitute the primary mechanisms underlying the cytotoxicity of cancer chemotherapeutics; therefore, the present study assessed these effects. AA or Bor alone inhibited cell viability in a dose-dependent manner. Notably, the combined inhibitory effects of AA and Bor on cell viability were markedly greater compared with those observed following AA and Bor monotherapies in vitro, with CI values <0.8. In addition, Bor and AA combination therapy significantly increased cancer cell apoptosis compared with AA or Bor treatment alone. Proteasome inhibition by Bor induces caspase activation; this constitutes an important mechanism underlying Bor-induced cell death (30–32). In the present study, combined treatment with AA and Bor activated caspase-3, -8 and -9, and induced PARP cleavage in U266 cells. AA/Bor co-administration promoted U266 apoptotic cell death via intrinsic (mitochondria-mediated; associated with caspase-9) and extrinsic (death receptor-mediated; associated with caspase-8) pathways, reflected by increased activation of caspase-3, -8 and -9, alongside PARP cleavage.

Certain studies have demonstrated that Bor activates HSPs, including HSP90, HSP70 and HSP25, which are associated with Bor resistance (33,34). Qi et al (35) reported that inhibition of inducible HSP70 increases Bor-induced human bladder cancer cell cytotoxicity. In the present study, AA/Bor combination therapy in U266 cells was associated with increased HSP70 induction. These results support the notion that enhancing Bor-mediated HSP70 induction represents an attractive means of enhancing its activity.

Protein synthesis, folding and trafficking occurs primarily in the ER; thus, intensive ER stress results in cell death (9,10). AA and Bor are ER stress inducers (21,22); therefore, it was investigated whether combination therapy induced UPR signaling. BiP, CHOP, phospho-eIF2α and ATF4 were all induced in U266 cells treated with AA and Bor. A previous study revealed that the ER stress-induced transcription factor ATF4 is a key mediator of Bor-induced cytotoxicity in neuroectodermal tumor cells, while CHOP is dispensable (14). Beck et al (36) reported that vemurafenib-induced melanoma cell death is associated with ATF4- but not CHOP-dependent ER stress, in agreement with our previous report (21). The effects of CHOP and ATF4 in promoting apoptosis were investigated in the present study. Consistent with previous reports, CHOP silencing failed to reduce the cytotoxic activity of combination therapy, and instead moderately enhanced this effect. However, ATF4 knockdown significantly reduced the cytotoxic effects of AA/Bor combination therapy. These findings demonstrate that ATF4 and CHOP are pro- and anti-apoptotic, respectively, in AA/Bor combination therapy-mediated cytotoxicity. However, future studies are required to reveal the mechanisms underlying these effects.

In conclusion, the present study demonstrated that AA sensitizes MM cells to Bor-mediated growth inhibition and apoptotic cell death in vitro. Therefore, AA may have potential applications as a chemosensitizer in human cancer treatment. Future in-depth studies, including in vivo experiments, are required to confirm the efficacy of AA in combination with Bor for MM treatment.

Acknowledgments

The present study was supported by: The National High Technology Research and Development Program of China (grant no. 2006AA02Z4B5) and the National Natural Science Foundation of China (grant nos. 81272451/H1609 and 81472762/H1609), awarded to J.L.; and the National Natural Science Foundation of China (grant no. 81472390/H1619), General Project from Guangzhou Education Commission (grant no. 1201410188), the Science and Technology Program of Guangzhou (grant no. 201510010127) and the Science and Technology Planning Project of Guangdong Province (grant no. 2014A020212691), awarded to H.H. The authors thank Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University (Guangzhou, China) for assistance with flow cytometry.

References