All-trans retinoic acid synergizes with topotecan to suppress AML cells via promoting RARα-mediated DNA damage

ATRA (cat # R2625) was obtained from Sigma (St Louis, MO, USA) and stored in ethanol at −40 °C. TPT, with more than 99 % purity, is synthesized by professor Wei Lu (East China Normal University) and dissolved in dimethylsulfoxide (DMSO) as stock solution at 10 mM. The stock solution was kept frozen in aliquot at −40 °C and thawed immediately prior to each experiment.

HL60, NB4 and U937, the human acute myelocytic leukemia cell lines, were obtained from ATCC, and they were maintained in complete media (RPMI-1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10 % heat-inactivated fetal bovine serum (FBS) plus penicillin (100 units/ml) and streptomycin (100 ug/ml)) at 37 °C in 5 % CO₂.

The cytotoxic activity was detected by MTT assay. After Cells were cultured in 96-well plates at 4 × 10³ cells/well, and allowed to proliferate to confluence in 5 % CO₂ incubator at 37 °C overnight; exposed to different concentrations of TPT, ATRA or TPT combined with ATRA for 48 h. Cells were then incubated with MTT 5 mg/ml (Sigma, USA) for 4 h. A quantity of 100 ml of DMSO (dimethyl sulfoxide) was added to each well after removing the supernatant. Cell viability was obtained by measuring the absorbance on a Multiskan Spectrum (Thermo Electron Corporation, Marietta, Ohio) at 570 nm.

The growth inhibition was calculated according to the following formula: the Growth Inhibition Ratio (IR%) = [(the absorbance of blank control group – the absorbance of experimental group)/the absorbance of blank control group] × 100 %.

The sub-G1 analysis after PI staining was employed to assess the apoptosis. HL60 cells (10⁵/ml) were seeded into 6-well plates and exposed to either or both of TPT and ATRA for 48 h. Cells were then harvested and washed with ice-cold PBS, fixed with precooled 75 % ethanol at −20 °C overnight. Cells were washed, and resuspended in 500 μl of PBS containing 100 μg/ml RNase (Amersco, Solon, OH, USA), then incubated at 37 °C for 30 min. After incubation, the cells were stained with 200 μg/ml propidium iodide (PI, Sigma, St Louis, MO, USA) in the dark at room temperature for 30 min. For each sample, at least 2 × 10⁴ cells should be analyzed using an FACS-Calibur cytometer (BD Biosciences, Sanjose, CA), and the data were analyzed using cellquest software (BD biosciences).

HL60 cells were inoculated into 6-well plates (150,000/well) for 24 h growth. After stabilisation for 24 h HL60 were treated with TPT, ATRA or both for 24 h and then harvested. Wash twice with PBS and suspend in 500 μl PBS with 2.5 μl JC-1 (20 μg/ml). Keep the samples in 37 °C, 5 % CO2, for 30 min. Then analyze immediatly with the flow cytometer, typically equipped with a 488 nm argon laser. JC-1 is a cationic dye that exhibits potential-dependent accumulation in mitochondria, indicated by a fluorescence emission shift from green (525 ± 10 nm) to red (610 ± 10 nm). Samples (1 × 104 cells/sample) were analyzed by FACS Calibur (Becton Dickinson, CA, USA).

For morphological studies, exponentially growing cells were cultured in 3 × 10⁵/well in 6-well plates and treated with TPT, ATRA or both for 48 h. The cells were washed with PBS, fixed with 0.1 % Triton X-100 for 15 min at room temperature, stained with 4’,6-dianidino-2-phenylindole dihydrochloride (DAPI, 2.0 μg/ml, Sigma) for another 15 min. Morphological changes of cell nucleus were examined in a fluorescence microscope, and they were photographed using a digital color camera DFC 300 FX (Leica, Wetzlar, Germany).

The protein samples were prepared as described previously. Briefly, proteins of HL60 cells were extracted in lysis buffer (150.0 mM NaCl, 50 mM Tris–HCl, 1 mM EDTA, 0.1 % SDS, 0.5 % dexoycholic acid, 0.02 % sodium azide, 1 % NP-40, 2.0 μg/ml aprotinin, 1 mM phenylmethylsulfonylfluoride). The lysates were centrifuged at 10⁴ × g for 15 min at 4 °C. Equivalent amounts of proteins were analyzed by 8 %-15 % SDS-PAGE and electroblotted onto PVDF membranes (Millipore Corporation, Billerica, Massachusetts), and probed with primary antibodies. Appropriate antibodies to anti-caspase-3, anti-poly-ADP-ribose polymerase (PARP), anti-Bax, anti-chk1, anti-chk2 and anti-β-actin from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-Bcl-2, anti-p-chk1, anti-p-chk2, and anti-γ-H2AX from Cell Signaling Technology (Beverly, MA, USA); and anti-Cytochrome C from Cell Signaling Technology (Boston, MA, USA) were used. The proteins were visualized with peroxidase-coupled secondary antibodies (Southern Biotech, Birmingham, UK), and using the enhanced chemiluminescence detection system (Biological Industries, Beit Haemek, Israel) for detection.

The alkaline comet assay, also called alkaline single-cell gel electrophoresis assay, was done according to the procedure of Huang et al. [20]. with minor modification. Briefly, drug-treated HL60 cells (10⁵/ml) were pelleted and resuspended in ice-cold PBS. A 50 μl sample of resuspended cells was the mixed with an equal volume of prewarmed 1 % low-melting point agarose. The cell-agarose mixture was placed on a slide precoated with 0.5 % agarose and spread gently with a coverslip. After 10 min at 4 °C, immersed the slides in precooled lysis buffer [2.5 M NaCl, 100 mM Na₂EDTA, and 10 mM Tris–HCl (pH 10)] for 90 min in the dark. After soaking with electrophoresis buffer (0.3 M NaOH and 1 mM EDTA) for 20 min, the slides were subjected to electrophoresis for 15 min. The cells were stained with DAPI at last, and individual cells were observed in a fluorescence microscope, and photographed by DFC 300 FX (Leica, Wetzlar, Germany).

To prepare the retroviruses, 293FT cells were plated at 6–8 × 10⁵ cells per well in 6-well plates coated with 20 μg ml⁻¹ of poly-ornithine. Twenty-four hours after plating, the cells were transfected with RARα shRNA plasmid, along with pUMVC and pCMV-VSV-G plasmids at the ratio of 2.125 μg of plasmid DNA to 5 μl of Lipofectamine 2000 in 500 μl Opti-MEM (both from Invitrogen). The ratio of shRNA:Pumvc: pCMV-VSV-G was 8:8:1. Twenty-four hours after transfection, the DMEM media was replaced with fibroblast (MEME) media. The next day the culture media containing the retroviruses was harvested and mixed 1:1 with 0.6 μg ml⁻¹ polybrene (Sigma-Aldrich). The cell debris in the mixtures was removed using 0.45 μm low-protein-binding filters (Nalgene). The HL60 cells were plated at 2.5 × 10⁵ cells per well in 6-well plates one day before infection with the retroviruses. The retrovirus solution was mixed with the cells for 6 h, then certain amount of fresh 1640 media was added for reducing the toxcity of polybrene. After infection of 24 h, cells were immediately transferred to complete RPMI-1640 supplemented with 10 % fetal bovine serum and cultured at 37 °C until analysis.

With Lipofectamine 2000, vesicular stomatitis virus-pseudo-typed vectors were produced by transient cotransfection of 293FT cells with 10 μg of the expression construct (pccl, or pccl-RARa), 10 μg of the pR△8.9 packaging plasmid, and 2 μg of the pMD.G envelope plasmid. Sodium pyruvate (Invitrogen) induction was performed according to the manufacturer’s instructions. After 72 h, the viral supernatants were harvested, centrifuged (800 g, 15 min), and filtered. After determination of the titer of the vector supernatants, HL60 cells were transduced with the indicated lentivirus particles (multiplicity of infection of 1 : 1) in the presence of 8 μgmL⁻¹ polybrene. A second cycle of transduction was performed 24 h later with new viral supernatants in the presence of fresh polybrene. After 24 h, the cells were washed and cultured in fresh medium.

Male immune-deficient nude mice at 4 weeks of age (National Rodent Laboratory Animal Resource, Shanghai Branch, China) were maintained in pathogen-free conditions with irradiated chow. Animals were unilaterally, subcutaneously (s.c.) injected with 5 × 10⁶ HL60 cells/tumor in 0.1 ml Matrigel (Collaborative Biomedical Products, Bedford, MA). When HL60 cells formed palpable tumors, mice were divided randomly into 4 groups receiving control (n = 3), TPT alone (n = 3), ATRA alone (n = 3) or combination of both TPT and ATRA (n = 3). ATRA (5 mg/kg) was dissolved in corn oil and given to mice by gavage and TPT (2 mg/kg) was given to mice by intraperitoneal injection every two days of the experimental period. Control group received vehicle (0.5 % methylcellulose, 0.2 % Tween 80 and 99.3 % DDW, i.g. administration) everyday and 0.9 % saline solution by intraperitoneal injection every two days of the experimental period. Body weight and tumors were measured every two days. Tumor sizes were calculated by the formula: (length × width × width)/2, and the length, width and height is in millimeters. At the end of the experiment, animals were sacrificed by CO₂ asphyxiation and tumor weights were measured after their careful resection. Tumor tissue was collected for analysis. The individual relative tumor volume (RTV) was calculated according to the following formula: RTV = V_N/V₀, where V_N is the tumor volume on day n and V₀ is the tumor volume on day of initial treatment. Therapeutic effects of treatment were expressed in terms of T/C% using the calculation formula T/C (%) = mean RTV of the treated group/mean RTV of the control group × 100 % [21]. All procedures were in accordance with the ethical standards of, and the protocols were approved by, the Animal Ethical and Welfare Committee (AEWC), Center for Drug Safety Evaluation and Research, Zhejiang University.

To evaluate the apoptotic response in tumor tissue, we applied terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick-end labeling (TUNEL) technique, to formalin-fixed tumor samples in paraffin blocks, using the one-step TUNEL apoptosis assay kit produced by Beyotime Institute of Biotechnology in China. The sections (4–5 μm) mounted on glass slides were deparaffinized, rehydrated thoursough graded alcohols to water, treated with 20 μg/ml proteinase K (37 °C, 20 min) and then washed in 1 × Tris buffer. TUNEL assay was then performed according to the manufacturer’s instructions.

Data were presented as mean ± SD for thoursee separate experiments. Comparions between groups were made with unpaired Student’s two-tailed t test and P < 0.05 was considered statistically significant.

To evaluate the potential synergy between ATRA and the Topoisomerase1 inhibitor TPT, cells (HL60, NB4 and U937) were exposed for 48 h to increasing concentrations of TPT (0, 20, 40, 60, 80 nM) alone or in combination with ATRA at a concentration that reduces cell viability according to data from preclinical research and our laboratory. As shown in Fig. 1b, a synergistic effect was observed in ATRA combined with lower concentrations of TPT (20 nM and 40 nM). However, as the concentration of TPT increased the cytotoxicity appeared much more severe with no significant differences between the TPT group (60 nM and 80 nM) and the TPT plus ATRA group. To validate the combination efficiency, we calculated the Coefficient of Drug Interaction (CDI) values, which are used to quantify drug synergism. CDI values less than, equal to or greater than 1 indicate that the drugs are synergistic, additive or antagonistic, respectively. CDI values less than 0.7 indicate that the drugs are significantly synergistic. The CDI values for different ratio concentrations of ATRA and TPT were calculated. As shown in Fig. 1c, the combination of ATRA and TPT had apparent synergism in HL60, NB4 and U937 cells with CI values <1. Cells treated with the combination of TPT and ATRA showed cell morphology changes and had more cell debris appearing compared to TPT or ATRA groups in Fig. 1d. Thus, we demonstrated that ATRA has synergistic cytotoxicity with TPT in AML cells.

To further explore the mechanisms of enhanced antitumor activity caused by the combination of TPT and ATRA, we examined their effects on apoptosis and caspase signaling pathways. Flow cytometry analysis after PI staining was used first to identify the apoptosis-inducing effects. As shown in Fig. 2a, PI staining for sub-G1 content analysis was used to characterize the apoptosis process in HL60 cells treated with TPT (20 nM and 40 nM), 1 μM ATRA or the combination for 48 h. Exposure to ATRA drove a few cells to apoptosis, and the content of sub-G1 was close to the control group (approximately 2 %). However, using ATRA with TPT generated more apoptotic cells compared to TPT treatment alone. Then, DAPI staining further showed that the combination of 1 μM ATRA and TPT for 48 h triggered more apoptosis compared to the treatment of either drug alone. As Fig. 2c depicts, apoptotic bodies were seldom found in control cells and the monotreated ATRA cells. However, apoptotic bodies appeared in cells exposed to TPT. Adding ATRA showed more apoptotic bodies with the accompanied nuclei shrinking and disintegrating.

To explore the signaling mechanism for TPT and ATRA synergistic induction of apoptosis, we investigated the expression levels of apoptosis-related proteins, such as caspase-3 and PARP, as shown in Fig. 2d, using Western blot analysis. Incubation with TPT at the indicated concentrations (20 nM and 40 nM) combined with ATRA (1 μM) for 24 h caused significant cleavage of PARP compared to the monotreated cells. Pro-caspase-3, upstream PARP [22], was cleaved more severely with the combination of 40 nM TPT and ATRA. Then, we used Z-VAD-FMK, the pan-caspase inhibitor, to further confirm the mechanism previously mentioned. As shown in Fig. 2e, the apoptosis induced by the combination treatment was almost reversed in the presence of Z-VAD-FMK, as detected by the PI (sub-G1) staining assay. All of these results indicated that caspase involvement led to apoptosis triggered by TPT plus ATRA. In summary, our results demonstrated that a combination treatment of TPT and ATRA triggers apoptosis via caspase cascades leading to increased cell death.

Mitochondrial damage in response to cellular conditions is an important constituent of apoptosis, and the mitochondrial membrane potential is one of the most remarkable events [23]. In order to explore the role of mitochondrial damage in apoptosis induced by a combination treatment of TPT and ATRA, JC-1 staining and flow cytometry were conducted after a 24 h treatment. The results showed that cells treated with the combined TPT and ATRA induced the loss of mitochondrial membrane potential compared to TPT or ATRA groups in Fig. 3a-b. The oncoprotein Bcl-2 is an antagonist of the mitochondrial pathway for apoptosis [24]. BAX is a member of the Bcl-2 gene family that forms a heterodimer with bcl-2 and functions as an apoptotic activator [25]. As shown in Fig. 3c, Western blot analysis revealed that bcl-2 is down-regulated and bax is up-regulated in HL60 cells treated with TPT and ATRA together. Cytochrome C, which normally resides exclusively in the intermembrane space of mitochondria, is released into the cytosol during apoptosis. Cytochrome C release from mitochondria to the cytoplasm was shown to be up-regulated in HL60 cells treated with TPT and ATRA together, as depicted in Fig. 3c. These data suggest that the combination treatment of TPT and ATRA induced mitochondrial associated-apoptosis.

TPT is a cytotoxic drug that adheres Topoisomerase 1 to DNA, which inhibits the function of Topoisomerase 1 in DNA repair [12]. Trapped Topoisomerase 1 leads to single strand DNA breakage and then to cell death. We used the comet assay to determine if ATRA plays a role in TPT induced-DNA damage. The alkaline version detects both single-strand and double-strand breaks (DSBs), and the neutral version only detects double-strand breaks [26, 27]. Exposure to different concentrations of TPT and ATRA for one hour showed obvious “comet tails” in two of the groups: the combination group and the positive control group for 200 nM TPT. The other groups showed only obscure “halos” without clear directions (Fig. 4a). The results demonstrated that treatment with ATRA and TPT for one hour induced HL60 DNA damage with 200 nM TPT. However, neither treatments of 1 μM ATRA or 40 nM TPT had similar effects.

Because the “tails” were caused by single-strand or double-strand breaks, we next detected the level of phosphorylated-H2AX (γ-H2AX) in DNA DBSs that are accompanied by the elevation of γ-H2AX. Phosphorylation of histone H2AX on serine 139 was one of the earliest cellular responses after the formation of DNA damage [28]. This phosphorylated form of H2AX (referred to as γ-H2AX) was used as a marker for the presence of DNA damages. AML cells, including HL60, NB4 and U937 cells, were incubated with TPT, ATRA, or both and proteins were collected at 1, 3, 6, and 8 h, respectively. As shown in Fig. 4b, γ-H2AX expression increased from a very low level to a high level, which demonstrated that only exposure to TPT and ATRA for eight hours induced DBSs. Therefore, the “comet tails” formed after one hour of drug treatment were caused by SSBs (Fig. 4c). TPT inhibits topoisomerase1 and causes single-strand DNA breaks, which inhibit DNA function and ultimately lead to cell death by generating double-stranded DNA breaks during DNA replication [29]. Furthermore, we examined p-Chk1 and p-Chk2 which are essential for responses to DNA damage [30]. These two checkpoint kinases were improved with the combination treatment of TPT and ATRA (Fig. 4d). These results indicated that ATRA and TPT synergistically induced apoptosis by causing serious DNA damage.

RARα, the main target of ATRA, plays an important role in ATRA-therapies. Previous data has shown that ATRA elevates the activity of proteasomes leading to RARα protein degradation by the ubiquitin-proteasome pathway [31, 32]. We found similar results for the ATRA treatment with prolonged time (Fig. 5a). Interestingly, the combination of ATRA and TPT downregulates RARα. To explore the role of RARα in this combination therapy, retroviral shRNA was used to knockdown the RARα expression levels in HL60 cells (Fig. 5b). Two days later, the cells were incubated with TPT for 24 h before PI staining and flow cytometry to detect cell apoptosis. Figure 5c-d shows that treating with TPT for the same amount of time leads to cells with lower expression of RARα and higher apoptosis percentages than in normal cells. From the comparisons between the control group and RARα-knockdown group, we concluded that low levels of RARα proteins are beneficial for TPT-induced apoptosis.

To determine whether RARα influences TPT-induced apoptosis through the DNA damage signaling pathway, we examined the related protein γ-H2AX. Increasing phosphorylation of H2AX was observed in Western blots for cells with low RARα expression in the combination therapy group (Fig. 5e). To further confirm the role of RARα in DNA damage, lentivirus transduction was used to overexpress RARα in HL60 cells. Figure 5f shows that the increasing phosphorylation of H2AX was reversed in the RARα-overexpression group, and this observation demonstrated that the downregulation of RARα may be a key factor for the accumulation of DNA damage and apoptosis.

In light of the in vitro synergistic effect of TPT and ATRA, we studied the in vivo anticancer activity of the combination therapy in nude mice bearing HL60 xenografts as described in the Materials and Methods. Figure 6a shows that the i.p. administration of ATRA at a dose of 5 mg/kg twice per week for nine days produced no significant difference in the mean RTV compared to the control group (mean RTV, ATRA vs. control: 12.5 vs. 17.1; P > 0.05). However, after a dosage of 2 mg/kg every week for nine days, TPT exerted a moderate tumor growth inhibitory effect (mean RTV, TPT vs. control: 10.4 vs. 17.1; P < 0.05). As predicted, TPT plus ATRA caused marked tumor growth inhibition (T/C value: 33.3 %) that was significantly greater than TPT (T/C value: 60.8 %) or ATRA treatment alone (T/C value: 73.1 %; mean RTV, combination vs. TPT: 5.7 vs.10.4; P < 0.01). Furthermore, compared to the initial body weights, combination treated mice showed no significant body weight loss in Fig. 6b.

The TUNEL assay was performed to evaluate the apoptosis-inducing abilities of the TPT and/or ATRA treatments. As shown in Fig. 6c, the number of TUNEL-positive cells was significantly increased in the tumor tissues of combination-treated mice. All in vivo data were consistent with previous in vitro data and further supported that the synergistic antitumor efficacy of TPT and ATRA was a result of TPT aroused apoptosis.