Background
Neuroblastoma (NB) is a prevalent childhood extracranial solid tumor that accounts for 7–8% of all pediatric malignancies and causes 15% of all pediatric oncology-related deaths [
1,
2]. According to the International Neuroblastoma Pathology Classification, the pathological subtypes of neuroblastoma were divided into neuroblastoma (NB), ganglioneuroblastoma (GNB) and ganglioneuroma (GN). Compared with GNB and GN, NB had a lower degree of differentiation and a worse prognosis. Infants with high-risk NB tumors, typically those aged > 18 months with extensive metastasis, usually have a poor prognosis, with an overall survival rate of < 50% [
3‐
5]. Consequently, more therapeutic strategies are urgently needed to improve the treatment efficacy for NB.
Micheliolide (MCL) is a natural guaianolide sesquiterpene lactone that is isolated from Michelia compressa and champaca plants [
6,
7]. Dimethylaminomicheliolide (DMAMCL or ACT001) is a water-soluble adduct of MCL that releases MCL into plasma and allows good distribution in brain [
8]. In addition, DMAMCL has a higher activity and stability in cells, lower toxicity, and fewer side-effects in animals than MCL [
9,
10]. Studies have demonstrated that DMAMCL exerts good pharmacokinetic effects against adult tumors such as glioblastoma [
11], breast cancer [
12,
13], and colitis-associated cancer [
14], indicating that DMAMCL could be a promising candidate for treating cancer. Notably, DMAMCL has recently been approved for phase I clinical trials in Australia for treating glioblastoma (Trial ID: ACTRN12616000228482) [
15] and has been selected as an orphan agent by the Food and Drug Administration [
16]; however, no studies have yet investigated the role of DMAMCL in NB and the mechanism of DMAMCL remains poorly understood.
Metabolic reprogramming has been shown to affect the survival of malignant tumor cells, with the most notable examples being the Warburg effect and aerobic glycolysis [
17‐
19]. Glycolysis refers to the transformation of glucose to lactate when limited amounts of oxygen are available, known as anaerobic glycolysis in normal cells. However, Warburg defined the metabolism of cancer cells as aerobic glycolysis, underlining that the glucose to lactate conversion occurs regardless of whether oxygen is present. Even under normoxic conditions, most cancer cells consume glucose via glycolysis at a higher rate than normal cells [
20,
21]; therefore, inhibiting aerobic glycolysis can suppress the growth of tumors, including NB [
22‐
25]. However, it remains unclear whether aerobic glycolysis and related enzymes are involved in DMAMCL regulation.
In this study, we investigated the antitumor effects of DMAMCL as a single agent and combined with chemotherapeutic agents (etoposide and cisplatin) against NB in vitro and in vivo, as well as the underlying mechanisms involving aerobic glycolysis and PFKL, a rate-limiting glycolysis enzyme.
Methods
Cell culture and reagents
Four human NB cell lines (NGP, SK-N-AS [AS], SMS-KCNR [KCNR], SK-N-BE2C [BE2]) and a mouse fibroblast cell line (NIH3T3) were used in this study. NGP, BE2 and KCNR cells have MYCN amplification; AS, BE2, and KCNR cells have 1pLOH; AS and BE2 cells have P53 mutations; and KCNR cells has ALK mutation. All cell lines were received from Dr. Carol J. Thiele (National Institutes of Health, Bethesda, MD, USA) and tested mycoplasma contamination regularly. Cells were cultured in RPMI-1640 medium (Bioind, Kibbutz Beit Haemek, Israel) with 10% fetal bovine serum (Bioind), 2 mM glutamine (Bioind), and antibiotics (penicillin 100 units/mL, streptomycin 100 μg/mL; Bioind) at 37 °C in 5% CO2. DMAMCL (Nankai University, Tianjin, China) was a white powder with a molecular weight of 409.47. DMAMCL was dissolved in water to produce a 20 mM stock solution.
Cell survival assays
To analyze cell survival, the four NB cell lines and NIH3T3 cell line were seeded onto 96-well plates, cultured overnight, and treated with different concentrations of DMAMCL (0–30 μM), etoposide (0–50 μg/mL), or cisplatin (0–20 μg/mL) for 72 h. Cell survival was then measured using a CCK-8 assay (Bimake, #B34304, China). Absorbance was detected at a wavelength of 450 nm using a microplate reader (Bio-Rad, Richmond, CA, USA).
Incucyte ZOOM live cell imaging system
Cell confluence was evaluated in real time using Incucyte ZOOM (Essen BioSciences, Ann Arbor, MI, USA) every 4 h for 72 h. The cell confluence percentage was analyzed using Incucyte ZOOM software.
Four NB cell lines and NIH3T3 (8 × 105 (NGP, KCNR), 5 × 105 (BE2), and 3 × 105 (AS, NIH3T3)) were seeded onto 6 cm diameter plates respectively, and treated with different concentration of DMAMCL for 24 h, then the cells were collected, and 5 × 103 (NGP, KCNR, BE2) or 2 × 103 (AS, NIH3T3) viable cells were replated onto 6 cm diameter plates. After 14 days, visible colonies were fixed and stained for at least 1 h with Giemsa stain solution (Solarbio, #G1015, Beijing, China).
Cell cycle assay
Four NB cell lines were seeded onto six-well plates, cultured overnight, and treated with different doses of DMAMCL for 48 h. The cells were collected, washed twice in cold phosphate-buffered saline (PBS), and fixed with cold 70% alcohol for 24 h. The cells were then incubated with 10 μg/mL RNase A (Beyotime, #C1052, China) and 50 μg/mL propidium iodide (Beyotime, #C1052, China) for 30 min in the dark at 37 °C. Stained cells were detected using flow cytometry (Becton Dickinson, Mountain View, CA, USA) and the percentage of cells at each phase of the cell cycle were analyzed using FlowJo software (Becton Dickinson).
Apoptosis assay
Four NB cell lines were treated with different doses of DMAMCL for 48 h and then all cells and supernatants were harvested, washed twice with cold PBS, and resuspended with 100 μL 1× Annexin V binding solution. After the cells had been incubated with 5 μL of Annexin V-PE and/or 7-AAD (BD Biosciences, #559763, San Jose, CA, USA) at 37 °C in the dark for 15 min, 400 μL 1× Annexin V binding solution was added and apoptotic cells were detected using flow cytometry (Becton Dickinson). We added the cells at the early apoptotic stage (Annexin V+) and the cells at the late apoptotic stage (Annexin V+, 7-AAD+) together, defined them as the apoptotic cells, and then calculated the percent of apoptotic cells (apoptotic cells/total cells * 100%) for the comparison of the percent apoptotic cells among different groups.
Western blotting
Protein lysates were extracted from NB cells or tumor tissues and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein loading quantity was 30 μg. The separated proteins were transferred to Polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA) which were probed with antibodies against cleaved-PARP (89 kDa, 1:1000, Cell Signaling Technology, #5625S, Beverly, MA, USA), HK2 (102 kDa, 1:1000, Cell Signaling Technology, #2867T), PFKL (78 kDa, 1:1000, Abcam, #ab181064, Cambridge, UK), PKM2 (60 kDa, 1:1000, Cell Signaling Technology, #4053T), and β tubulin (55 kDa, 1:2000, Proteintech, #10068-1-AP, Wuhan, China). Antibodies were detected using enhanced chemiluminescence (Thermo Scientific, Madison, WI, USA).
Synergistic effect analysis
The combination effects between DMAMCL and chemotherapeutic agents (etoposide and cisplatin) in NGP and BE2 cells were evaluated using a CCK-8 assay. NGP cells were treated with DMAMCL (2.5, 3.75, 5 μM) and etoposide (0.02, 0.04, 0.05, 0.1, 0.2 μg/mL) or cisplatin (1, 2, 3 μg/mL) for 72 h. BE2 cells were treated with DMAMCL (2.5, 7.5, 10, 12.5 μM) and etoposide (0.05, 0.075, 0.1, 0.2 μg/mL) or cisplatin (0.5, 1, 3 μg/mL) for 72 h. Synergistic effects were analyzed using CompuSyn software (CompuSyn, Inc., Paramus, NJ, USA). CI values of < 1 indicate synergism, CI = 1 reflects an additive effect, and CI > 1 indicates antagonism.
Glycolysis measurements
To assess glycolytic function, we used an Agilent Seahorse XF Glycolysis Stress Test Kit (Seahorse Bioscience, #16814032, North Billerica, MA, USA) with a Seahorse XFe96 Analyzer to directly measure the real time extracellular acidification rate (ECAR). Before measurement, the sensor cartridge was hydrated in Seahorse XF Calibrant at 37 °C in a non-CO2 incubator overnight. NGP, BE2 and NIH3T3 cells were seeded onto 6-well plates with different doses of DMAMCL, incubated for 24 h, and then the cells were collected, washed twice with cold PBS, and resuspended with phenol red-free assay solution. To avoid the impact of the different number of cells in each group on ECAR, we counted and seeded the same number of cells for each group onto the XF96 cell culture microplates (Seahorse Bioscience, North Billerica, MA, USA) precoated with Cell-Tak solution at the density of 4 × 104 cells/well (NGP) and 3.5 × 104 cells/well (BE2, NIH3T3). The cell culture microplates were centrifuged at 1000 rpm for 5 min to attach the cells and then glucose, oligomycin (ATP synthase inhibitors), and 2-deoxy-d-glucose (2-DG, hexokinase inhibitor) were added according to the manufacturer’s instructions (Seahorse Bioscience) and detected immediately.
Extracellular glucose concentration and lactate production were determined using a Glucose Assay Kit (Biovision, Mountain View, #K606-100, CA, USA) and Lactate Assay Kit (Biovision, #K607-100) respectively. ATP levels were analyzed using an enhanced ATP Assay Kit (Beyotime, #S0027, China). Briefly, cells were incubated in 6-well plates overnight and then treated with DMAMCL for 24 h. The supernatants were collected to detect extracellular glucose concentration and lactate production, while the cells were collected and lysed to detect ATP levels. Cell protein concentration was also measured to eliminate errors because of differences in protein content.
Cell transfection
PFKL overexpression plasmids (GeneCopoeia, Guangzhou, China) and small interfering RNAs (siRNA #1, #2, #3; Wanze, Anhui, China) were designed to up- and down-regulate PFKL expression in NB cells, respectively. The following siRNA target sequences were used: PFKL #1, 5′-CGG AGA UGA AGA CAG ATT-3′; PFKL #2, 5′-UCU GUC UUC AUC UUC UCC GTT-3′; PFKL #3, 5′-CCA CGG AGU UCC UGU ACA ATT-3′; control, 5′-UUC UCC GAA CGU GUC ACG UTT-3′. NB cells were transfected with the plasmids and siRNAs using jetPRIME agent (Polyplus Transfection, #114-15, Illkirsch, France) for 16 h and then treated with DMAMCL before further experiments.
TUNEL assay
Paraffin-embedded tumor tissue slides were prepared from the in vivo experiments and apoptosis was detected using a TUNEL staining kit (Roche Molecular Biochemicals, #11684817910, Indianapolis, IN, USA), according to the manufacturer’s instructions. TUNEL-positive cells were observed under a fluorescence microscope (Olympus, Tokyo, Japan). The magnification of the images was 200×, Scale bar: 100 μm.
In vivo experiments
The right flanks of 4- to 6-week-old female BALB/C nude mice (Beijing Huafukang Bioscience, Beijing, China) were subcutaneously inoculated with 100 μL of cell suspension containing 3 × 10
6 cells. When the tumors had reached 100–150 mm
3, DMAMCL (75 mg/kg, or 100 mg/kg) or a placebo was administered once daily for up to 21 days by oral gavage (
n = 10 per group). Etoposide (15 mg/kg) was administered once a week for up to 3 weeks via intraperitoneal injection (
n = 10 per group) [
26,
27]. The tumors were measured three times a week and the tumor volume was calculated as
\(\frac{L \times {W}^{2}}{2}\), where L = length (mm) and W = width (mm). Mouse survival was measured from the beginning of treatment to the end of the experiment, which is when the tumor size reached 20 mm or the mouse had to be euthanized due to the poor general condition. For cleaved-PARP and PFKL expression analysis and terminal deoxynucleotidyl transferase using peroxide12-UTP nick end labeling (TUNEL) staining, mice bearing NGP and BE2 xenograft tumors were treated with DMAMCL (75 mg/kg, 100 mg/kg) for 1 week and tumor tissues were harvested (
n = 3 per group). At end of the experiment, the mice were euthanized as follows: the carbon dioxide (CO
2) was given with a flow rate of 20% chamber vol/min for 2–3 min when the mice were insensible, then gradually increased to 100% chamber vol/min till the mice were confirmed dead after 5 min of apnea. All mice were housed at 23 ± 2 °C with 40–70% humidity. All animal experiments followed ethical standards and were approved by the Experimental Animal Ethical Committee of Shengjing Hospital of China Medical University (2019PS302K).
Statistical analysis
In vitro and in vivo data were presented as the mean ± standard deviation (SD) and the mean ± standard error (SE), respectively. Mouse survival was calculated using the Log-Rank test. Statistical analysis was performed using GraphPad Prism 8 software version 8.2.1 (GraphPad Software, Inc., La Jolla, CA, USA). Between-group comparisons were made using unpaired Student’s t tests. A p value < 0.05 were considered statistically significant, and *p < 0.05; **p < 0.01; ***p < 0.001.
Discussion
DMAMCL has been shown to exert inhibitory effects against various cancers, including glioma [
26], hepatocellular carcinoma (HCC) [
27], leukemia [
28,
29], and breast cancer [
30], when administered as a single agent. In this study, we found that DMAMCL could exert antitumor growth effects against NB in vitro and in vivo both when administered alone or in combination with chemotherapeutic agents. In addition, we demonstrated that PFKL mediates DMAMCL-induced NB cell death by affecting aerobic glycolysis.
As a naturally derived anticancer drug, DMAMCL has received increasing attention owing to its minimal toxicity. Apoptosis is a programmed cell death mechanism that plays an essential role in cancer development. Recent studies have demonstrated that DMAMCL induces apoptosis in gliomas and gastric cancer cells [
26,
31]. In this study, DMAMCL induced NB cell death via apoptosis but had much lower toxicity in control NIH3T3 cells. Consistent results were observed in four different NB cell lines, representative of the heterogeneity typically found in NB tumors and cell lines, which suggests that DMAMCL has a universal inhibiting effect on different types of NB tumors. Moreover, we found that DMAMCL suppressed NB tumor growth and improved survival in a xenograft mouse model without significantly affecting body weight (Additional file
4: Fig. S4A), consistent with a previous study in which DMAMCL suppressed tumor growth in a mouse xenograft model of HCC [
27]. However, although DMAMCL had inhibitory effects on different NB cell lines in vitro and in vivo, their sensitivity to DMAMCL was also different. Among them, BE2 had the least sensitivity to DMAMCL and NGP had the highest sensitivity. The possible reason for this phenomenon maybe that BE2 cells have MYCN amplification, P53 mutation and 1pLOH (loss of heterozygosity at chromosome 1p), while NGP cells only have MYCN amplification. Furthermore, our findings suggest that tumor cells are more sensitive to DMAMCL than normal cells, also supporting the clinical application of DMAMCL to treat NB. Recent study showed that spatial learning and memory ability in mice was significantly improved by chronic median dose DMAMCL (25 mg/kg) treatment [
32]. It proved that DMAMCL may not only play a role in inhibiting NB tumors, but also improve the cognitive function of mice within a certain concentration range, indicating that DMAMCL has fewer toxic effects and supports the clinical application of DMAMCL.
Etoposide and cisplatin are the first-line chemotherapy drugs for NB in clinic. Etoposide is a topoisomerase inhibitor and causes DNA strands break, which causes errors in DNA synthesis and promotes cell death. Cisplatin mainly causes cell death by affecting the cell cycle and interferes with DNA replication. In this study, we aimed to select chemotherapeutic drugs with different mechanisms and detect the combination effects with DMAMCL in vitro and in vivo
. For instance, DMAMCL has been found to potently sensitize HCC cells to various clinical chemotherapeutic agents (gemcitabine, paclitaxel, doxorubicin, cisplatin) [
27] and reverse resistance to cisplatin and tamoxifen in breast cancer [
12,
13]. Moreover, a study found that combining DMAMCL with vincristine or epirubicin increases cell death and tumor growth inhibition in vivo in rhabdomyosarcoma (RMS) [
33]. Consistently, our study found that DMAMCL exerts significant synergistic effects with etoposide or cisplatin in NB cells in vitro and notably increased antitumor effects when combined with etoposide in the xenograft mouse model. More importantly, the combination of DMAMCL with etoposide significantly improved the survival of the mice without affecting body weight (Additional file
4: Fig. S4B, C), suggesting that DMAMCL can be combined with chemotherapeutic agents to treat NB.
Metabolic reprogramming is widely recognized as a characteristic hallmark of cancer cells that contributes toward tumor development [
34‐
37]. Unlike normal cells, cancer cells primarily rely on aerobic glycolysis for glucose metabolism, even under normoxic conditions [
38‐
40]. Recent studies have indicated the importance of exploring the mechanisms underlying the regulation of aerobic glycolysis in NB to develop effective therapeutic strategies. Gan et al
. reported that 3-bromopyruvate (3-BrPA), a hexokinase (HK)-II inhibitor, combined with rapamycin, synergistically suppressed aerobic glycolysis in NB cells [
41]. Song et al
. reported that therapeutic targeting of the HNF4A-AS1/hnRNPU/CTCF axis inhibited aerobic glycolysis and NB progression [
42]. Fang et al. determined that therapeutic targeting of the YY1/MZF1 axis by MZF1-uPEP inhibited aerobic glycolysis of NB cells [
43]. Another study by Fang et al
. found that valproic acid (VPA), an established histone deacetylase inhibitor, suppressed aerobic glycolysis and tumor progression of NB, indicating a novel therapeutic strategy for NB [
44]. Importantly, DMAMCL reportedly plays a role in inhibiting aerobic glycolysis. Li et al
. and Guo et al
. found that DMAMCL decreased lactate production, a surrogate indicator of glycolysis, in leukemia and glioma, suggesting that DMAMCL inhibits aerobic glycolysis [
11,
15]. In this study, we directly measured the real time ECAR by Seahorse XFe96, which is a standard and comprehensive method for assessing key parameters of glycolytic flux. We also found that glucose consumption, lactate excretion and ATP production were all decreased after DMAMCL treatment in NB cells, confirming that DMAMCL suppresses aerobic glycolysis in NB cells.
Recent studies have found that the rate-limiting glycolysis enzyme, PKM2, is a key target for the DMAMCL-induced inhibition of cell proliferation in leukemia and glioma; however, these studies found no significant differences in PFKL or HK2 mRNA expression [
11,
15]. Here, we detected decreased PFKL, PKM2, and HK2 protein expression in vitro after DMAMCL treatment, with PFKL protein expression decreasing much earlier than PKM2 or HK2 in both NGP and BE2 cells. Therefore, the regulation of aerobic glycolysis may be a potential mechanism underlying the effects of DMAMCL, which modulates various different metabolic enzymes. PFKL has also been shown to play an essential role in tumorigenesis [
45‐
48], with its up-regulation promoting tumor cell proliferation and metabolic reprogramming in various cancers, such as glioma and breast cancer [
49‐
52]. Although PFKL overexpression did not significantly increase NB cell survival in this study, it remarkably attenuated DMAMCL-induced NB cell death. Meanwhile, downregulating PFKL promoted NB cell death, consistent with a previous study in which PFKL knockdown significantly suppressed the progression of oral cancer [
53]. Together, these reports demonstrate that PFKL plays a critical role in the antitumor effects induced by DMAMCL in NB. Furthermore, the activity of PFKL is higher when its tetramer structure is maintained, whereas its activity decreases when PFKL is allosterically regulated to become an oligomeric dimer [
54]. We hypothesize that DMAMCL may regulate PFKL by changing the protein structure of PFKL or by undergoing special protein post-translational modifications. These hypotheses will be investigated in future research.
The mechanisms underlying the antitumor effects of DMAMCL have been reported in various cancers. In leukemia, DMAMCL induces cell death by inhibiting the NF-κB pathway [
28], whereas DMAMCL induces apoptosis in osteosarcoma by activating caspase-dependent pathways [
55] and inactivates the PI3K/Akt pathway in HCC [
27]. We also evaluated p-Akt and NF-κB expression after DMAMCL treatment in this study, but didn’t detect a decrease in p-Akt and NF-κB expression in NB cells (data not shown), suggesting that DMAMCL may induce cell death via various different mechanisms. Although we found that up-regulating PFKL blocked DMAMCL-induced cell death in NB cells, this blockage was only partial, potentially because of a low cell transfection efficiency (50–70%) causing the role of PFKL to be underestimated. Another reason for the partial role of PFKL could be that other molecules also mediate the effects of DMAMCL. Li et al. reported that DMAMCL selectively represses acute myelogenous leukemia stem cells [
9]; however, no studies have yet investigated whether DMAMCL selectively acts against NB stem cells. Therefore, our future studies will explore other mechanisms of DMAMCL and its role in NB stem cells using high-throughput screening.
However, there are still some limitations in the present study, which warrant further investigation. In addition to affecting the glycolytic capacity and the expression of PFKL in NB, whether other metabolic processes, or the key genes or pathways are involved in the inhibitory effect of DMAMCL on NB were not studied. Therefore, our following study will aim to explore other mechanisms of DMAMCL in NB treatment through the integration of metabolomic and transcriptomic data.
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