Introduction
Multiple myeloma (MM) is a clonal malignancy of plasma cells that is characterized by the presence of a monoclonal protein in the serum and/or urine, widespread osteolysis, renal failure, and anemia [
1,
2]. MM survival rates, which have historically ranged from 3 to 5 years, can now exceed 10 years due to the advent of high-dose therapy with autologous stem cell transplantation in combination with novel chemotherapeutic agents [
3,
4]. However, high-dose chemotherapy produces drug resistance in MM patients, illustrating the urgent need for more effective treatments.
Current treatment approaches aim to induce apoptosis, a form of programmed cell death responsible for tissue homeostasis, in cancer cells [
5-
7]. The intrinsic apoptosis pathway is initiated at the mitochondria, which are permeabilized in response to intracellular death signals, releasing cytochrome c and other apoptotic factors into the cytosol and activating caspase-9 and caspase-3 [
8]. The extrinsic apoptosis pathway is initiated by the binding of death receptor ligands, such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or CD95 ligand, to their cognate death receptors at the cell membrane [
9]. Receptor trimerization leads to the intracellular recruitment of adaptor proteins, such as Fas-associated death domain (FADD), which enable the binding and activation of caspase-8 at the death-inducing signaling complex (DISC) [
8]. Active caspase-8 activates caspase-3, resulting in apoptosis. As an antiapoptotic protein, cellular FLICE-inhibitory protein (cFLIP) can block death-receptor signaling by interfering with caspase-8 activation at the DISC [
10].
The FADD-like interleukin-1β-converting enzyme (FLICE)-inhibitory protein cFLIP is a death effector domain (DED)-containing protein that is similar to FLICE-like inhibitory protein long (FLIPL) and FLICE-inhibitory proteins (FLIPS). These proteins play a role in apoptosis signaling, and cFLIP isoforms represent critical antiapoptotic and drug resistance factors [
10]. cFLIP is highly expressed in cancer cells and TRAIL-resistant cells [
11-
13]. FLIP levels in TRAIL-resistant cells are regulated by the phosphatase and tensin homologue (PTEN)-AKT-mammalian target of rapamycin (mTOR) pathway, and PTEN loss and AKT activation are correlated with increased FLIPS mRNA translation, high levels of FLIPS expression, and TRAIL resistance
in vitro, in human glioblastoma multiforme (GBM) xenografts and in primary human GBM samples [
11].
Aloperine, a natural product identified in
Sophora flavescens Ait. and other Sophora plants, such as
Sophora alopecuroides [
14-
16], exhibits anti-inflammatory, antibacterial, and antiviral properties [
17,
18]. Clinically, aloperine has been used in China to treat patients with autoimmune diseases, such as rheumatism, lupus erythematous, and eczema [
19,
20]. More recently, aloperine was reported to have an antitumor effect on various cancers cells, including leukemia cell lines (HL-60, U937, and K562), esophageal cancer cells (EC109), lung cancer cells (A549), and hepatocellular carcinoma cells (HepG2) [
21]. In addition, our group previously demonstrated that aloperine significantly induced apoptosis in colon cancer SW480 and HCT116 cells [
22,
23]. Although aloperine induced cell cycle arrest at the G2/M phase with concomitant increases in p21 and p53 and decreases in cyclin D1 and B1, its specific target(s) remain to be discovered; furthermore, it is not yet clear whether the drug can also induce apoptosis in MM cells. In this study, we demonstrate the cytotoxic effects of aloperine on primary samples and MM cell lines with or without the protective effects of bone marrow cytokines and bone marrow stromal cell (BMSC) adhesion. Importantly, we determined that aloperine functions by targeting cFLIP and phosphorylated (p)-PTEN and thus induces MM cell apoptosis through both the intrinsic and extrinsic apoptotic pathways, respectively.
Materials and methods
Drugs, reagents, and cell lines
Aloperine (Yanchi Dushun Biological and Chemical Co., Ltd., Ningxia, China; Batch number: 070506; purity >99%) and bortezomib (Millennium Pharmaceuticals) were dissolved in distilled water. Non-tagged TRAIL, IETD-FMK, LEHD-FMK, and Z-VAD-FMK were obtained from Bachem (Heidelberg, Germany). All other chemicals were purchased from Sigma-Aldrich (China) unless otherwise stated.
The human myeloma cell lines (OPM2, 3T3D, RPMI 8226, and NCI-H929) were kind gifts from Guangzhou Medical University and Southern Medical University. The dexamethasone-sensitive and dexamethasone-resistant cell lines MM.1S and MM1.R as well as U266 cells and the doxorubicin-resistant U266 and Dox6 cell lines were kindly provided by Sun Yat-Sen University Cancer Center. Cell lines were grown in RPMI 1640 (Gibco) with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin (100 units of penicillin and 100 μg of streptomycin).
Primary MM cells were isolated from patient bone marrow (the Second Affiliated Hospital in Guangzhou Medical University) aspirates after Ficoll-Hypaque gradient centrifugation using CD-138-positive selection and magnetic-assisted column sorting (Miltenyi) according to the manufacturer’s instructions. The purity of the MM cells was confirmed to be >90% via flow cytometric analysis using an anti-CD138 antibody (Miltenyi). CD138-negative mononuclear cells were also used to establish long-term BMSC cultures, as described previously [
24]. BMSCs were obtained from MM patients and used between the third and fifth passages for all experiments. Informed written consent was obtained from all patients’ parents/guardians, and the study was approved by Guangzhou Medical University Clinical Research Ethics Committee.
To generate stable cell lines, cells were seeded in six-well tissue culture plates and transfected with cFLIPL in pBABE, cFLIPS in pCFG5-IEGZ, or the corresponding empty vectors using FuGENE 6 (Roche Applied Science, Mannheim, Germany) according to the manufacturer’s recommendations, and clones were selected using 2.5 μg/ml puromycin or 400 μg/ml Zeocin (InvivoGen, San Diego, CA, USA).
Cell proliferation and apoptosis assays
Cell proliferation was assessed using methanethiosulfonate (MTS) assays (Promega) according to the manufacturer’s instructions. For co-culture experiments, cell proliferation was measured using the BrdUrd cell proliferation ELISA kit (Roche Diagnostics). MM cells were cultured in BMSC-coated 96-well plates for 48 h with the indicated concentrations of aloperine. Cells were pulsed with BrdUrd during the last 8 h of the 72-h culture. Apoptosis was quantified using the annexin V/propidium iodide staining assay kit (R&D Systems) according to the manufacturer’s instructions, and samples were analyzed on a FACSCalibur (BD Biosciences) flow cytometer.
Western blotting
Western blot analyses were performed as described previously [
25] using the following antibodies: caspase-8, cFLIP, caspase-9, XIAP, Bim, caspase-3, PTEN, p-PTEN (Ser380/Thr382/383), PARP, p-AKT (Ser-473), AKT, β-actin, MCL-1 (all from Cell Signaling, Beverly, MA), cIAP-2 (Epitomics, Burlingame, CA, USA), cIAP-1, survivin (both from R&D Systems), Noxa, and cFLIP (Santa Cruz Biotechnology).
RNA interference
For transient knockdown of PTEN, cells were transfected with 150 pmol Stealth RNAi siRNA directed against PTEN, cFLIP (Invitrogen), or non-targeting control siRNA (Invitrogen) using the TransMessenger transfection reagent (Qiagen, Hilden, Germany).
Animal models and drug treatment
SCID NOD mice (Animal Experiment Center, Southern Medical University) were used in this study. All animals were handled in strict accordance with good animal practice as defined by the relevant national and local animal welfare bodies. All animal work was approved by the Institutional Review Board of Guangzhou Medical University (permit number: GZMU (hu) 2011–0190) in accordance with the guidelines for animal use of the National Institutes of Health.
Mice were inoculated subcutaneously in the abdomen with U266 cells (5 × 106 cells per mice) and randomized into the following treatment and control groups (5 per group): control (PBS), bortezomib (0.1 mg/kg, i.p., 1 dose per 3 days), TRAIL (15 mg/kg, i.p., 1 dose per 5 days), aloperine (20 mg/kg, p.o., daily), aloperine + TRAIL, and aloperine + borterzomib. Aloperine was given 24 h prior to TRAIL treatment because our goal was to induce the death receptors DR4 and/or DR5 by irradiation so that successive TRAIL treatment would enhance tumor cell apoptosis 10 days postinjection. Tumor growth was monitored every 5 days with calipers (width2 × length/2).
For the 5T33 myeloma mouse model (10 per group), 5 × 106 5T33 cells were injected intravenously into C57/BL KaLwRij mice (Shanghai Faculty of Life Sciences) via the tail vein. Bortezomib (0.1 mg/kg), PBS, and aloperine (20 mg/kg) treatments were initiated 7 days after injection, and blood was collected at 0, 1, 2, 3, and 4 weeks after injection.
Measurement of the monoclonal protein IgG2b
After 1 h at room temperature, serum was obtained via centrifugation of whole blood, and samples were stored at −20°C until use. A specific analysis of mouse IgG2b in the serum was performed using a commercially available ELISA kit (IgG2b, Bethyl Laboratories).
Bone X-rays
Following tissue dissection and fixation, bones were placed onto Kodak X-OMAT AR scientific imaging film (Kodak, Rochester, NY) and X-rayed using a Faxitron radiographic inspection unit (Model 8050–020; Field Emission Corporation, Inc., McMinnville, OR). The exposed films were developed using a RP X-OMAT processor (Model M6b; Kodak). Osteolytic lesions as small as 0.5 mm in diameter were recognized and counted as demarcated radiolucent lesions in the bone.
Clonogenic survival assays
Colony formation assays were performed as previously described [
26]. Briefly, 10,000 MM cells were seeded in 0.33% agar cultures. The colonies were fed with a medium in the presence or absence of drugs. After 18 days, the plates were imaged, and colony numbers were counted using ImageJ.
Statistical analyses
The values presented represent the mean ± SD from at least three experiments. IC50 values for proliferation in the presence of aloperine were calculated as the concentration of drug that resulted in a 50% reduction of viable cells compared with untreated controls using MTS or BrdUrd assays. Comparisons between two groups were performed using Student’s t test, and three or more groups were compared using ANOVA. P values <0.05 or <0.001 were considered significant.
Discussion
In this study, we demonstrated that aloperine decreases MM cell viability without affecting normal lymphocyte viability, suggesting that it is a selective cytotoxic agent against MM cells compared with normal BMSCs and PBMCs. Furthermore, to explain why aloperine selectively treated MM cells, malignant MM-derived BMSCs were added to the experiment. The bone marrow microenvironment promotes MM proliferation and growth via the secretion of IL-6. IL-6 is a major growth factor and antiapoptotic cytokine for MM cells [
32]; the importance of IL-6 as an autocrine growth factor for MM cells is widely accepted [
24]. In the current experiment, aloperine overcame the proliferative effects of IL-6, IGF-I, and BMSCs, suggesting that aloperine could be efficacious in treating MM.
Genetic heterogeneity and drug resistance are hallmarks of MM [
33,
34] that explain, at least in part, differences in the cytotoxic activity of different agents. Importantly, our data demonstrate that aloperine displays anti-MM activity in a panel of MM cell lines, including sensitive and resistant cell lines, such as U266, U266 Dox6, MM.1S, and MM1.R, as well as cytogenetically distinct MM cells. We observed similar responses in patient-derived MM cells that were resistant to anti-MM therapies such as bortezomib, lenalidomide, dexamethasone, or melphalan. These data indicate that aloperine presents a special characteristic that sets it apart from general chemotherapy drugs for MM.
Mechanistic studies indicated that the anti-MM activity of aloperine is associated with the activation of caspases, cFLIP, Bim, and Noxa, among others. Notably, aloperine triggers robust caspase-8 cleavage; however, modest caspase-9 cleavage was also observed. These data are consistent with our findings that biochemical inhibitors of caspase-8 partly abrogate the anti-MM activity of aloperine, suggesting that caspase-9 compensates to induce MM apoptosis. Because cFLIP is structurally similar to caspase-8 and the expression of cFLIP isoforms is increased in various types of cancer, including MM, cFLIP represents a critical target for therapeutic intervention, namely via inhibiting its transcription and posttranscriptional modifications [
35]. In this study, we demonstrate that aloperine significantly decreases the expression of cFLIP and activates procaspase-8 in MM cells.
Furthermore, aloperine induced MM cell apoptosis in MM cells overexpressing cFLIP, suggesting that cFLIP downregulation is important for aloperine cytotoxicity. Moreover, cFLIP overexpression conferred TRAIL resistance; therefore, the aloperine-mediated sensitization to TRAIL observed in our study is secondary to cFLIP downregulation. TRAIL is the leading death receptor ligand in clinical development with selective activity against cancer cells [
36]. However, drug resistance severely hinders its efficacy. Our data suggest that aloperine may not only help overcome cFLIP-mediated TRAIL resistance but could also enhance the ability of TRAIL to inhibit MM cell growth.
PTEN loss causes an mTOR-dependent increase in the translation of FLIPS mRNA, increases the levels of antiapoptotic FLIP proteins, and increases TRAIL resistance [
11]. PTEN suppresses AKT phosphorylation and activates caspase-9 to induce apoptosis in MM cells [
29]. Furthermore, enhanced phosphorylation of PTEN at the Ser380/Thr382/Thr383 cluster was recently shown to inactivate PTEN and enhance PI3K-AKT pathway activation in adult T-cell leukemia-lymphoma (ATLL) and other cancers [
37]. Our study is the first to show that aloperine regulates AKT and PTEN phosphorylation. Declines in AKT and PTEN phosphorylation occurred in a time-dependent manner when MM cells were treated by aloperine. The decrease in p-PTEN occurred significantly earlier than that in p-AKT and resulted in elevated levels of p-AKT, thus further demonstrating that PTEN plays a key role in the PTEN-AKT pathway. Importantly, knockdown of PTEN partially rescued cells from aloperine-mediated upregulation of caspase-9 and significantly reduced aloperine-induced apoptosis (Figure
4C). These data suggest that aloperine kills cancer cells via the PTEN-AKT-caspase-9 signaling pathway.
At low drug concentrations, the ability of aloperine to inhibit clonogenic survival was irreversible. Moreover, the ability of aloperine to inhibit MM survival was largely dependent on cFLIP and the PTEN-AKT pathway. Therefore, it is possible to use low doses of aloperine to achieve a greater therapeutic index because normal fibroblasts are much more resistant to the drug than MM cells are.
In addition to our in vitro studies, we also examined the anti-MM activity of aloperine in vivo using a human MM xenograft mouse model. To demonstrate drug activity, bortezomib and TRAIL were included as positive controls. Marked tumor growth inhibition was observed following treatment with bortezomib, TRAIL, aloperine, or aloperine + TRAIL/bortezomib compared with treatment with PBS alone. Interestingly, the combination of aloperine and TRAIL/borterzomib showed a synergistic inhibitory effect on MM growth compared with TRAIL or aloperine alone.
5T33 models have been considered as suitable models for human MM [
38]. This murine model of human myeloma bone disease includes radiologic evidence of osteolysis, hypercalcemia, and increased osteoclastic bone resorption associated with monoclonal gammopathy [
39]. This animal model of human myeloma bone disease can be applied to determine the efficacy of potential therapeutic agents. The 5T33 model that we utilized is a systemic model of aggressive MM with the unambiguous endpoint of hind limb paralysis [
40]. In our study, aloperine not only reduced the expression of IgG2b and bone lesions in 5T33 mice but also increased survival. Importantly, aloperine treatment was not toxic, as no differences in creatinine, hemoglobin, or bilirubin were noted. Therefore, these findings demonstrate that aloperine elicits a dual effect in MM tumors, increases apoptosis, and decreases proliferation.
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Competing interests
The authors declared that they have no competing interest.
Authors’ contributions
HW, SY, and HZ carried out the molecular genetic studies, participated in the sequence alignment, and drafted the manuscript. MS and LD carried out the immunoassays. JH, ML, MW, and YF participated in the design of the study and performed the statistical analysis. YZ, JH, and HD conceived the study and participated in its design and coordination. All authors read and approved the final manuscript.