Background
Burkitt leukemia/lymphoma (BL) is a highly aggressive subtype of B-cell neoplasm characterized by constitutive MYC expression and PI3K activation [
1]. Although BL responds to intensive chemotherapy regimens, biologically targeted therapies should be developed, especially in high-risk patients and in the setting of relapsed/refractory disease [
2].
Autophagy is generally involved in cancer progression [
3]. Although autophagy is primarily a cell protective process, it can also induce cell death, known as programmed cell death type II [
4]. Experimental study showed that mice with heterozygous disruption of
BECN1 present decreased autophagy and are more prone to the development of spontaneous tumors including lymphomas [
5]. Clinically, defect in autophagy is also related to aggressive phenotype and poor prognosis in lymphoma patients [
6,
7]. These results indicated that reactivation of autophagy could be mechanistically important in lymphoma treatment.
Signal transduction inhibitors become an emerging therapeutic option for molecular tumor targeting [
8]. Mammalian target of rapamycin (MTOR) signaling plays a major role in tumor cell growth and is aberrantly activated in lymphoma [
9,
10]. MTOR inhibitors possess single-agent therapeutic activity, but drug resistance is frequently observed [
10]. Thus, unique combination to enhance the effect of MTOR inhibitors is particularly attractive [
11].
Histone deacetylase (HDAC) inhibitors constitute a group of compounds that promote histone acetylation, chromatin uncoiling and downmodulation of genes involved in cancer [
12]. Widely used as an anti-convulsant, valproic acid (VPA) belongs to the short chain fatty acid HDAC inhibitors and possesses anti-tumor activity [
13]. It negatively regulates B-lymphoma cell proliferation and shows therapeutic potential on refractory patients at the standard dose [
14,
15]. Although simultaneous inhibition of MTOR and HDAC exerts profound anti-tumor properties, the possible interaction and therapeutic mechanism of this combination remain to be defined in BL.
To address this issue, we examined the combinatorial action of the HDAC inhibitor VPA with clinical relevant MTOR inhibitor temsirolimus in BL cells both in vitro and in vivo. These two agents interacted in a synergistic manner to induce autophagic cell death in BL cells, in association with a significant inhibition of MTOR pathway and MYC oncoprotein.
Discussion
Combinations of signal transduction inhibitors are being gradually applied in clinical settings and proven more efficiently than single agent alone to target tumor cells and to avoid acquired resistance [
18]. To our knowledge, the present study provided evidence for the first time that the HDAC inhibitor VPA and the MTOR inhibitor temsirolimus, both at a clinically achievable concentration [
19,
20], interacted synergistically to inhibit BL cell growth. This was found not only in well-established BL cell lines and fresh patient samples, but also in nude mice xenografted with BL cells. Although recent study indicated that VPA can reduce the maximum tolerated dose of temsirolimus in pediatric patients with solid tumors [
21], combined treatment appeared to be well tolerated in our study which temsirolimus was administered at a relatively low dose. Of note, the combination exerted the inhibitory effect with a minimal degree of toxicity against normal CD34+ hematopoietic precursors, further confirming their effective and safe role in treating BL.
The observed synergy in cytotoxity, accomplished by combined treatment, mainly resulted from the convergent effect on BL cell autophagy. This was manifested by the ultrastructure study and the autophagy flux assay, and further confirmed by the extent of autophagy being reduced by the pharmacological and molecular autophagic inhibitor. In BL, resistance to chemotherapy is attributed to the inability of tumor cells to die by apoptosis. It may be present at the onset of therapy in high-risk patients, or emerge over time during chemotherapy in relapsed/refractory cases, even after a dramatic initial response. Drugs that target autophagy are efficient in treating BL cells resistant to apoptosis [
22,
23]. Temsirolimus can induce autophagy in lymphoma cells [
24]. Recent reports demonstrated that autophagy appears to be an important therapeutic target of the HDAC inhibitor other than apoptosis in highly proliferative tumors [
25,
26], which could explain why VPA specifically improve the tumoricidal activity of temsirolimus through promoting autophagy in BL.
Aberrant expression of HDAC1 appears common in tumors, and is associated with enhanced proliferation and defect in autophagy. In liver cancer, targeted disruption of HDAC1 leads to strong anti-proliferative effect and induces autophagic cell death [
27]. Our study showed that VPA arrested the G1/S cell cycle transition and activated autophagy through targeting HDAC1, indicating an important underlying mechanism responsible for VPA to interact with temsirolimus to positively regulate BL cell autophagy.
Resistance to MTOR inhibitors is due to feedback AKT activation [
11]. The HDAC inhibitor overcomes MTOR inhibitor rapamycin resistance by inhibiting AKT via HDAC3 and potentiates autophagy through downregulation of MTOR pathway [
28,
29]. In our study, VPA reduced HDAC3 activity and subsequently inhibited AKT phosphorylation induced by temsirolimus. In addition to temsirolimus that directly hits MTOR, VPA modulates the upstream HDAC3 and inhibits MTOR in a rapamycin-independent manner [
30]. Aiming for the same pathway with molecules targeting different sites of the protein, the VPA-temsirolimus combination amplified the blockade of MTOR signaling, resulting in further induction of autophagy in BL.
The BL oncoprotein MYC is the key regulatory element by MTOR pathway [
31]. Furthermore, MYC mitigates response to the MTOR inhibitor through 4EBP1-mediated inhibition of autophagy [
32]. VPA combined with temsirolimus potently targeted MYC oncoprotein, suggesting another important therapeutic mechanism of co-treatment in BL. Importantly, MYC-driven DLBCL have recently been identified as a subtype with inferior survival [
33]. VPA-temsirolimus combination induced cell autophagy in MYC-expressing DLBCL DB cells as in BL cells, further indicating its therapeutic role on MYC oncoprotein.
Methods
Cells and reagents
BL cell lines Namalwa, Raji, Daudi, Ramos and DLBCL cell line DB were available from American Type Culture Collection. Cells were maintained in RPMI-1640 medium, supplemented with 10% heat-inactivated fetal bovine serum in a humidified atmosphere of 95% air and 5% CO2 at 37°C. VPA (V3640) and temsirolimus (PZ0020) were from Sigma-Aldrich. 3-Methyladenine (189490), and ZVAD-FMK (219007) were from Merck & Co. Inc. Bafilomycin A1 (sc-201550) was from Santa Cruz Biotechnology.
Fresh BL cells were extracted from the lymph node and bone marrow of patients. CD34+ cells were isolated from human cord blood using CD34 Progenitor Cell Isolation Kit (Miltenyi Biotec Inc.). The study was approved by the Institutional Review Board and informed consent was obtained in accordance with the Declaration of Helsinki.
MTT reduction assay
To assess growth inhibition, cells were treated with VPA, temsirolimus, either alone or in combination, in a 96-well plate. After 48 h, 0.1 mg MTT (Sigma-Aldrich, M2003) was added to each well. The samples were incubated at 37°C for 4 hours and the absorbance was measured at 490 nm by spectrophotometry.
Flow cytometric assay
To assess the distribution of nuclear DNA content, cells were collected, washed in PBS and fixed overnight in 75% ethanol at -20°C, treated with 1% RNaseA (Merck, 70856–3) for at least 15 minutes at 37°C and stained with 50 μg/ml propidium iodide. Cell apoptosis was analyzed using ApoAlert ANX-V-FITC Apoptosis Kit (Clontech Laboratories, Inc., 630110). Cell autophagy was analyzed using rabbit anti-human LC3 (Cell signaling, CST4108) as the primary antibody and DyLight 405 labeled anti-rabbit antibody (KPL, KPL072-08-15-06) as the secondary antibody. The flow cytometry data were collected by a FACSCalibur machine (Becton Dickinson) and analyzed by FlowJo software.
Isobolographic analysis
Determination of the synergistic effect of VPA-temsirolimus combination was performed using the isobologram of Steel-Peckham [
34]. Based on dose–response curves of the two agents, three isoeffect curves were constructed. The area surrounded by the isoeffect curves was referred as the envelope of additivity. When the data points fell to the left of the envelope, that is, the combined effect was caused by lower doses of the two agents than was predicted, the combination was regarded as having a synergistic effect. The synergistic effect was further confirmed by the combination index (CI) method described by Chou and Talalay (CalcuSyn software, Biosoft). When at least 80% of CI values for a combination were less than one, the drug combination was considered to be synergistic.
Small-interfering RNA (siRNA) transfection
Namalwa cells were transfected with ATG5, HDAC1, HDAC3 siGENOME SMARTpool or Non-Targeting pool as a negative control using DharmaFECT2 transfection reagent (Dharmacon) following the manufacturer’s instruction.
Western blot
Cells were lysed in 200 μl lysis buffer (0.5M Tris–HCl, pH 6.8, 2 mM EDTA, 10% glycerol, 2% SDS and 5% β-mercaptoethanol). Protein extracts (20 μg) were electrophoresed on 10% SDS polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were blocked with 5% non-fat dried milk in Tris buffered saline and incubated for 2 hours at room temperature with appropriate primary antibody, followed by horseradish peroxidase-conjugated secondary antibody. The immunocomplexes were visualized using chemiluminescence phototope-horseradish peroxidase kit. Antibodies against LC3-I/II (4108), phosphorylated MTOR (p-MTOR) (2971), MTOR (2972), phosphorylated 4E binding protein-1 (p-4EBP1) (9456), 4EBP1 (9644), phosphorylated P70 ribosomal S6 kinase (p-P70S6K) (9205), P70S6K (2708), HDAC3 (3949), HDAC4 (5392), phosphorylated AKT (p-AKT) (4060), AKT (9272), ACTB (4970), c-caspase-3 (9664), c-PARP (9541) and chemiluminescence phototope-horseradish peroxidase kit (7003) were obtained from Cell Signaling. Antibodies against BECN1 (ab51031), MYC (ab28842), HDAC1 (ab150399) and HDAC2 (ab32117) were from Abcam. Anti-P62 antibody (BML-PW9860) was from Enzo Life Sciences, Inc. Horseradish peroxidase-conjugated goat anti-mouse-IgG (sc-2005) and goat anti-rabbit-IgG (sc-2004) antibodies were from Santa Cruz Biotechnology. ACTB was used to ensure equivalent protein loading.
Enzyme-linked immunosorbent assay
Enzymatic activity of HDAC1 and HDAC3 in lymphoma cells were quantified by enzyme-linked immunosorbent assay using nonisotopic HDAC (BioVision, K331-100) colorimetric kits according to manufacturer’s instructions.
Transmission electron microscopy
Cells and tissue samples were fixed overnight in 2% glutaraldehyde at 4°C, washed in 0.1M cacodylate buffer, postfixed in 1% osmium tetroxide for 1 hour at 4°C, dehydrated in graded ethanol and embedded in Epon 812 (TAAB Laboratories). Ultrathin sections were prepared, collected on copper grids, stained with uranyl acetate and lead citrate, and examined on electron microscopy (Philips CM120). Ultrastructural studies were focused on double membrane-bound autophagic vesicles named autophagosomes, a gold standard for autophagy.
Immunohistochemistry and immunofluorescence
Immunohistochemistry was performed on 5μm-paraffin sections with an indirect immunoperoxidase method using antibodies against CDKN1A and MYC. Immunofluorescence was performed on methanol-fixed cells using anti-BECN1 and anti-P62 as primary antibodies, and diaminotriazinylaminofluorescein-labeled donkey anti-rabbit-IgG antibodies (Abcam, ab6800) as the second antibody.
Murine model
Nude mice (5-6-week-old) were obtained from Shanghai Laboratory Animal Center and injected subcutaneously with 7×106 Namalwa cells into the right flank. Treatments (10 mice per group) were started after tumor became about 0.5 cm × 0.5 cm in surface (day 0). The control group received dimethyl sulfoxide, while the other three groups received for 21 days oral VPA (0.4%w/v in the drinking water daily), intraperitoneal temsirolimus (5 mg/kg every other day), or in combination, respectively. Tumor volumes were calculated as 0.5 × a × b2, where ‘a’ is the length and ‘b’ is the width.
Terminal deoxytransferase-catalyzed DNA-nick-end labeling (TUNEL) assay
In situ cell apoptosis was confirmed by detection of fragmented DNA, using TUNEL assay, on 5 μm-paraffin sections, using DeadEnd Colorimetric TUNEL System (Promega Corporation, G7360) according to the manufacturer’s instruction. The tissue section of the same murine xenograft model co-treated with bortezomib and SAHA was referred as a positive control, as previously described by our study [
34].
Statistical analysis
All assays were set up in triplicate and the results were expressed as the mean±S.D. of data obtained from three separate experiments. T-test was applied to compare two normally distributed groups and Bonferroni to perform multiple comparison. P<0.05 was considered statistically significant. All statistical analyses were evaluated using Statistical Package for the Social Sciences (SPSS) 13.0 software (SPSS Inc.).
Acknowledgement
This work was supported, in part, by the National Natural Science Foundation of China (81172254, 81201862 and 81101793), the Shanghai Commission of Science and Technology (11JC1407300), the Program of Shanghai Subject Chief Scientists (13XD1402700), and the “Shu Guang” project supported by Shanghai Municipal Education Commission and Shanghai Education Development Foundation (09SG21).
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
LHD, SC and ZZ performed the research, LW, ZXS, SJC and WLZ designed the research study, YS analysed the data, and WLZ wrote the paper. All authors have read and approved the final manuscript.