AMXT-1501, a novel polyamine transport inhibitor, synergizes with DFMO in inhibiting neuroblastoma cell proliferation by targeting both ornithine decarboxylase and polyamine transport

Authors


  • Conflict of interest: Mark Burns owns stock and is an employee of Aminex Therapeutics, who owns the rights to AMXT-1501.

Correspondence to: Giselle Sholler, Van Andel Research Institute, 333 Bostwick Ave., Grand Rapids, MI 49503, USA, Tel: +616-234-5495, E-mail: Giselle.sholler@vai.org or André S. Bachmann, University of Hawaii at Hilo, College of Pharmacy, Department of Pharmaceutical Sciences, 34 Rainbow Drive, Hilo, HI 96720, USA, Tel: +808-933-2807, Fax: +808-933-2974, E-mail: andre@hawaii.edu

Abstract

Neuroblastoma (NB) is associated with MYCN oncogene amplification occurring in approximately 30% of NBs and is associated with poor prognosis. MYCN is linked to a number of genes including ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis. ODC expression is elevated in many forms of cancer including NB. Alpha-difluoromethylornithine (DFMO), an ODC inhibitor, is currently being used in a Phase I clinical trial for treatment of NB. However, cancer cells treated with DFMO may overcome their polyamine depletion by the uptake of polyamines from extracellular sources. A novel polyamine transport inhibitor, AMXT-1501, has not yet been tested in NB. We propose that inhibiting ODC with DFMO, coupled with polyamine transport inhibition by AMXT-1501 will result in enhanced NB growth inhibition. Single and combination drug treatments were conducted on three NB cell lines. DFMO IC50 values ranged from 20.76 to 33.3 mM, and AMXT-1501 IC50 values ranged from 14.13 to 17.72 µM in NB. The combination treatment resulted in hypophosphorylation of retinoblastoma protein (Rb), suggesting growth inhibition via G1 cell cycle arrest. Increased expression of cleaved PARP and cleaved caspase 3 in combination-treated cells starting at 48 hr suggested apoptosis. The combination treatment depleted intracellular polyamine pools and decreased intracellular ATP, further verifying growth inhibition. Given the current lack of effective therapies for patients with relapsed/refractory NB and the preclinical effectiveness of DFMO with AMXT-1501, this combination treatment provides promising preclinical results. DFMO and AMXT-1501 may be a potential new therapy for children with NB.

Neuroblastoma (NB) is the most common extracranial solid pediatric tumor, accounting 8–10% of all pediatric cancers and for 15% of cancer-related deaths in children.[1] Approximately 650 new cases of NB arise in the United States each year. Of the children diagnosed, roughly 70% have disease that has already metastasized to other parts of the body. Children with high-risk NB have a long-term survival of less than 50%, despite the use of intensive multimodality therapy.[2] Of the children with relapsed or refractory disease, there are no known curative measures and the 5-year survival is less than 10%. Biomarkers are powerful tools for determining diagnosis and prognosis for different cancers, including NB. The oncogenic transcription factor MYCN is amplified in roughly 30% of all NBs and is generally associated with high-risk disease and poor survival.[3, 4] As a transcription factor, MYCN induces and represses a large number of genes involved in multiple biological processes including cell growth and differentiation. However, the genes necessary or sufficient to initiate neuroblastoma tumorigenesis downstream of MYCN remain to be established.[5]

Ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis in mammalian cells, is directly activated by c-MYC and MYCN[6-8] and is overexpressed in NB.[9-11] ODC decarboxylates the polyamine precursor ornithine to the diamine putrescine (PUT), which is the precursor of the higher order of polyamines, such as spermidine (SPD) and spermine (SPM). SPD and SPM are anabolized by the SPD (SRM) and SPM (SMS) synthases, through the sequential transfer of aminopropyl groups to PUT and SPD, respectively.[12] Eflornithine (also called alpha-difluoromethylornithine; DFMO) is a suicide inhibitor of ODC. The compound was initially developed in the late 1970s by the Merrell-Dow Research Institute as a chemotherapeutic drug for hyperproliferative cells. DFMO has been intensively evaluated in human clinical trials as an anti-cancer and chemopreventive agent,[13-16] reviewed in Bachmann and Levin, 2012.[17] However, cancer cells may overcome polyamine depletion by importing polyamines from extracellular sources, obtained from dietary, nutritional, or microbial flora in the intestines and delivered through the bloodstream.[18] This phenomenon could account for the ability of cells to partially overcome/compensate the treatment with DFMO. Thus, inhibiting both polyamine transport and biosynthesis has become an attractive approach for combination treatment. Polyamine transport inhibition coupled with polyamine biosynthesis inhibition has been studied across several cancer cell lines both in vitro and in vivo, including squamous cell carcinoma (SCC),[19] prostate,[20] breast and bladder carcinoma cell lines, and melanoma cell lines.[21] However, there has been limited research in pediatric cancers to exploit potential synergisms of this promising drug combination. DFMO has very low toxicity, and thus is well suited to treat pediatric patients.

In this study, we investigate a novel and potent polyamine transport inhibitor called AMXT-1501. Recent publications have shown that AMXT-1501 and DFMO are an effective combination for targeting several types of cancer cells both in vitro and in vivo, including breast, prostate, SCC, melanoma, and ovarian.[22] However, the effects of DFMO combined with AMXT-1501 have not been studied in NB. We tested DFMO and AMXT-1501 on two MYCN-amplified NB cell lines (BE-(2)C and SMS-KCNR), and one MYCN nonamplified (single copy) NB cell line (SH-SY5Y). We found that not only were intracellular polyamine levels depleted, but cellular proliferation was inhibited in three cell lines in culture. On the basis of these results, polyamine biosynthesis and transport inhibition may be a promising new therapy option for NB and we hope to see AMXT-1501 entered into clinical trials in the near future.

Material and Methods

Inhibitors

The ODC inhibitor DFMO (kindly provided by Dr. Patrick Woster, Medical University of South Carolina) was dissolved at 1 M in H2O. The polyamine transport inhibitor AMXT-1501 (Aminex Therapeutics, Seattle, WA) was dissolved at 10 mM in H2O. Drug stocks were stored in small aliquots at −20°C and diluted with culture media on the day of the experiment.

Cell lines and cell culture

Single and combination drug testing was conducted in vitro on three human NB cell lines: SMS-KCNR (kindly donated by Dr. John Maris, The Children's Hospital of Philadelphia, PA), SH-SY5Y (ATCC, Manassas, VA), and BE-(2)C (ATCC, Manassas, VA). Cells lines were validated by the DNA Diagnostics Center, Fairfield, OH. Cells were cultured in RPMI-1640 with 10% (vol/vol) certified fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin. Incubation was at 37°C with 5% CO2. Medium containing inhibitors was supplemented with 0.5 µM spermidine (SPD) as a source of extracellular polyamines, and 1 mM aminoguanidine (AG) was added to inhibit serum amine oxidation. Cells were grown in 6-well or 96-well plates, or 100 mm dishes. Cells were allowed to grow to 60–70% confluence prior to addition of drugs.

Cell viability and IC50 values

Calcein AM fluorescent assay was used to determine cell viability in 96-well plates. SMS-KCNR and SH-SY5Y cells were plated at 6,000 cells/well, and Be-(2)C cells were plated at 4,000 cells/well. Cells were then treated with increasing concentrations of one inhibitor in RPMI-1640 with 10% FBS supplemented with 0.5 µM SPD and 1 mM AG, for 48 hr. DFMO ranged from 0.78 to 100 mM, and AMXT-1501 ranged from 0.39 to 50 µM. H2O was used as control. After incubation with drugs for 48 hr, the medium was aspirated and replaced with phenol-red free RPMI medium containing 2 µg/mL Calcein AM. After 30 min incubation at 37°C, fluorescence was measured at 480ex/520em with a BioTek plate reader (BioTek Instruments, Winooski, VT). IC50 values were calculated with a four-parameter variable–slope dose–response curve using GraphPad Prism v.5 software. Isobolograms were done by plating cells the same way as for Cell viability assay. Six different concentrations of each drug were chosen, mixed at different combinations, and added to cells in 96-well plates. After 48 hr, the Calcein AM assay was performed as described earlier.

Western blot analysis

Western blot analysis was utilized to evaluate markers of proliferation and cytotoxicity. Cells were grown in 6-well plates and treated with 2.5 mM DFMO, 2.5 µM AMXT-1501, or both inhibitors in combination in RPMI-1640 with 10% FBS supplemented with 0.5 µM SPD and 1 mM AG. Water was used as vehicle control. Drug supplemented medium was refreshed after 48 hr. Plates were removed from incubation and cells were rinsed twice with ice cold PBS, at 24, 48, or 72 hr post-treatment.

Cells were lysed with RIPA lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 50 mM NaF, 1 mM sodium orthovanadate), supplemented with a complete protease inhibitor (Roche, Indianapolis, IN) for 20 min on ice. Cells were collected and centrifuged at 14,000 rpm for 20 min at 4°C. Protein concentration of the supernatant was determined using a protein assay reagent dye (Bio-Rad, Hercules, CA). Lysates were boiled for 10 min at 90°C and electrophoresed on an 8% or 12% SDS-polyacrylamide gel in running buffer (25 mM Tris, 192 mM Glycine, 0.1% SDS, pH 8.3). Forty microgram of protein was loaded per lane. PAGE 8% gels were wet transferred (25 mM Tris, 192 mM Glycine, 20% Methanol) and 12% gels were semi-dry transferred to nitrocellulose or PVDF membranes. Blots were blocked for 1 hr at room temperature in blocking buffer (LI-COR Biosciences, Lincoln, NE) diluted 1:1 with PBS. Blots were probed with primary antibodies made up in blocking buffer (LI-COR Biosciences, Lincoln, NE) diluted 1:1 with PBST for 1 hr at room temperature or overnight at 4°C. Primary antibodies used were: cleaved and full length caspase 3, cleaved and full length PARP (Cell Signaling Technology, Beverly, MA), rabbit polyclonal MYCN (Santa Cruz Biotechnology, Santa Cruz, CA, sc-791), and mouse monoclonal β-tubulin (in house). Secondary antibodies used were goat anti-rabbit and goat anti-mouse (LI-COR Biosciences, Lincoln, NE). Blots were stripped with stripping buffer (LI-COR Biosciences, Lincoln, NE) and reprobed. Protein bands were detected by fluorescence using the Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE).

Real-time cellular analysis

The xCELLigence System (Roche Applied Science, Germany and ACEA Biosciences, USA) allows the real-time cell analysis (RTCA) via a system consisting of a microelectronic sensory array (MESA) 96-well plate coupled with a device station and an electronic sensor analyzer. The basic principle of the RTCA system is to monitor the changes in electrode impedance by the interaction between adherent multiplying cells seeded into the MESA wells and the underlying well microelectrodes. The cell number, viability, morphology, and degree of adherence of cells in contact with the electrodes will affect the local ionic environment leading to an increase in the electrode impedance—this is represented as the Cell Index (CI) and reflects a calculation (via an internal system algorithm) of frequency-dependent electrode impedance with or without attached healthy cells present on the surface of the wells. Cells were cultured on an electrode-containing 96-well plate and treated for 96 hr with 2.5 µM AMXT-1501, 2.5 mM DFMO, or both inhibitors in combination. Vehicle wells were used as a control (1% H2O). Drug-containing medium was refreshed after 48 hr. The plate was connected to an xCELLigence RTCA SP instrument within a humidified cell culture incubator. Electrical impedance caused by cellular attachment was recorded every 15 min. Data was collected by software provided with the system, and the proliferative index was normalized to the time of initial drug treatment.

Polyamine pool depletion analysis

Reverse-phase HPLC analysis was applied to quantify the intracellular levels of PUT, SPD, and SPM in BE(2)-C and SMS-KCNR cells. Cells were plated in 100 mm dishes and treated with 5 mM DFMO, 15 µM AMXT-1501, or both inhibitors in combination. Vehicle dishes were used as a control (1% H2O). Twenty-four or 48 hr after drug addition, cells were rinsed with PBS, centrifuged, and pellets stored at −80°C. Polyamines were separated and analyzed by reverse-phase HPLC as previously described.[23] Briefly, pellets were resuspended in 0.1 N HCl at a concentration of 107 cells/mL and sonicated to disrupt the cells. Cell suspension was adjusted to 0.2 N perchloric acid (HClO4) by diluting 1:10 with 2N perchlorate and put on ice to allow complete precipitation. Cells were then spun at low speed to collect the supernatant from pellet. The supernatants were filtered through a Millipore filter (0.22 µm) and applied either directly or after appropriate dilution with 0.1 M sodium acetate buffer (pH 4.5) to a micro-Bondapak C18 column (Waters, MA). The elution system consisted of a gradient prepared from two buffer solutions. Buffer A was 0.1 M sodium acetate (pH 4.5) containing 10 mM octane sulfonate; buffer B was 0.2 M sodium acetate (pH 4.5) plus acetonitrile (10:3, v/v), containing 10 mM octane sulfonate. A linear gradient was prepared from buffer A and buffer B with an increment of 2% per minute of buffer B for 30 min. For the remaining time the increment of buffer B was increased to 4% per minute.

ATP per cell analysis

The CyQuant fluorescent DNA assay (Invitrogen, Grand Island, NY) was combined with the Cell Titer GLO luminescent cell viability assay (Promega, Madison, WI) (which gives quantitation of the ATP present, an indicator of metabolically active cells) to measure ATP level per cell. SMS-KCNR and SH-SY5Y cells were plated at 5,000 cells/well, and BE(2)-C cells were plated at 3,000 cells/well in 96-well black-walled plates. Cells were treated for 96 hr with 2.5 µM AMXT-1501, 2.5 mM DFMO, or both inhibitors in combination. Wells treated with water (1% H2O) were used as controls. Drug-containing medium was refreshed after 48 hr. Medium was aspirated and replaced with Cell Titer GLO reagent (lysis buffer mixed with Cell Titer GLO substrate, diluted with PBS 1:1) supplemented with CyQuant stock solution (50 µL dye per 10 mL Cell Titer GLO reagent). Plates were placed on an orbital rocker for 3 min to induce cell lysis, and then were incubated an additional 10 min to allow luminescent signal stabilization. Luminescence and fluorescence data were recorded using a Wallace plate reader and Envision software. Cell Titer GLO data were divided by CyQuant data and normalized to vehicle wells in order to generate data on ATP per cell.

Results

DFMO and AMXT-1501 exhibited single agent cytotoxicity against NB cell lines. Combination treatment induced synergistic cytotoxicity

To investigate the cytotoxic effects of DFMO and AMXT-1501 on NB cells, BE(2)-C, SMS-KCNR, and SH-SY5Y cells were treated for 48 hr with serial dilutions of DFMO ranging from 0.78 to 100 mM or of AMXT-1501 ranging from 0.39 to 50 µM (Fig. 1a). Calcein AM fluorescent assay was used to determine cell viability. DFMO exhibited cytotoxicity against this panel of NB cell lines, with IC50 values of 27.51 mM for SMS-KCNR, 33.3 mM for BE(2)-C, and 20.76 mM for SH-SY5Y. AMXT-1501 also was cytotoxic against all lines, with IC50 values of 17.72 µM for SMS-KCNR, 17.69 µM for BE(2)-C, and 14.13 µM for SH-SY5Y.

Figure 1.

DFMO and AMXT-1501 IC50 values and isobolograms. (a). DFMO and AMXT-1501 IC50 values for three NB cell lines over 48 hr; Results were average of three independent experiments. SMS-KCNR and SH-SY5Y cells were plated at 6,000 cells/well, and BE(2)-C cells were plated at 4,000 cells/well. Cells were treated with varying concentrations of inhibitors alone for 48 hr. DFMO ranged from 100 to 0.78 mM with a 1:2 dilution factor, and AMXT-1501 ranged from 50 to 0.39 µM with a 1:2 dilution factor. All experiments included 0.5 µM spermidine and 1.0 mM aminoguanidine in the culture media. After drug treatment for 48 hr, Calcein AM assay was performed and fluorescence was measured with a BioTek plate reader. (b). Cells were treated with 2.5 mM DFMO, 2.5 µM AMXT-1501, or a combination of both inhibitors. At 24, 48, and 72 hr, Cell Titer GLO luminescent cell viability assay was performed and luminescence data was recorded using a Wallace plate reader and Envision software. Calcein AM cell viability assay was performed and fluorescence was measured with a BioTek plate reader. (c). Isobolograms of DFMO and AMXT-1501 treated cells treated for 48 hr.

To test if the combination of the two drugs induces synergistic cytotoxicity, the three cell lines were treated with combinations of DFMO plus AMXT-1501 at five different concentrations per drug (DFMO ranged from 2.5 to 50 mM; AMXT-1501, 1.56 to 25 µM). Concentrations of 2.5 mM DFMO and 2.5 µM AMXT-1501 combination exhibited synergistic cytotoxicity to the cells (Fig. 1b). Isobologram plots suggest that the two drugs act synergistically when both are at low concentration, i.e., DFMO lower than 20 mM and AMXT-1501 lower than 10 µM (Fig. 1c).

Combination treatment of low-dose DFMO and AMXT-1501 significantly reduced NB cell proliferation

To determine the effect of low-dose AMXT-1501 and DFMO on NB cell proliferation, we used the xCELLigence system, a real-time cell proliferation, viability and cytotoxicity analyzer. Cells were treated with 2.5 mM DFMO, 2.5 µM AMXT-1501, or a combination of both inhibitors for 24–96 hr (Fig. 2). Cell proliferation was continuously monitored every 15 min using the xCELLigence system. The data clearly showed that single inhibitors do not significantly impede proliferation; however, the combination treatment caused a significant decrease in cell index, which means significantly slowed the proliferation rate of all three cell lines, BE(2)-C, SMS-KCNR, and SH-SY5Y.

Figure 2.

Real-time proliferation of the three cell lines over 96 hr as measured by xCELLigence. Cells were treated with 1% H2O vehicle (red), 2.5 mM DFMO (blue), 2.5 µM AMXT-1501 (green), or a combination of both inhibitors (purple) for 24–96 hr. Deviation at 96 hr reflects drug being refreshed after 48 hr. Proliferative cell index is shown normalized to initial drug treatment.

Combination treatment of DFMO and AMXT-1501 induced caspase-3 mediated apoptosis in NB cell lines

To determine if DFMO and AMXT-1501 induce apoptosis in NB cells, BE(2)-C, SMS-KCNR and SH-SY5Y cells were treated with 2.5 mM DFMO, 2.5 µM AMXT-1501 or a combination of both inhibitors for up to 96 hr, cell lysates were collected and analyzed by western blot analysis. Treatments lead to induction of apoptosis as measured by the presence of cleaved PARP and of cleaved caspase-3 in western blots (Figs. 3a and 3b). Combined treatment with both drugs decreased the amount of noncleaved PARP and increased the amount of cleaved PARP and cleaved caspase 3 in all three cell lines. The level of cleaved PARP and cleaved caspase 3 were the highest at 72 hr post-treatment. At 96 hr all cells were dying and detached (data not shown).

Figure 3.

Western blot analysis and quantification. All experiments included 0.5 µM spermidine and 1.0 mM aminoguanidine in the culture media. (a) Western blot analyses of the apoptotic markers cleaved caspase 3 and cleaved PARP in three NB cell lines. Cells were treated with 2.5 mM DFMO, 2.5 µM AMXT-1501, or a combination of both inhibitors for 24–72 hr. Forty micrograms of total protein was loaded per lane. Increased activity of both cleaved caspase 3 and cleaved PARP were observed starting 48 hr. (b) Quantification of cleaved PARP and cleaved caspase 3 expression of the three cell lines treated with DFMO, AMXT-1501 or combination at 72 hr. (c) Western blot analysis of MYCN on cells treated with DFMO, AMXT-1501 or combination. (d) Western blot analyses of cell cycle markers pRb and p27 on cells treated with DFMO, AMXT-1501 or combination.

It has been reported that down-regulation of MYCN expression inhibited cell growth and/or induced neuronal differentiation.[24-27] MYCN expression in the three NB cell lines treated with DFMO, AMXT-1501 and the combination of both drugs were also examined. MYCN expression was decreased with combination treatment in two MYCN-amplified cell lines BE(2)-C and SMS-KCNR starting from 48 hr. Downregulation of MYCN in NB cells in response to DFMO alone correlated with the results reported by Wallick, 2005.[11] In contrast, the non-MYCN-amplified cell line SH-SY5Y was not affected by drug treatments (Fig. 3c).

Retinoblastoma (Rb) is a key protein that controls G1 cell cycle progression. To study the impact of DFMO and/or AMXT-1501 on Rb, western blot analysis was performed. Phosphorylated-Rb (Ser 807/811) expression was decreased in cells treated with combination drugs starting from 48 hr, whereas hypophosphorylated Rb in the total Rb increased (Fig. 3d).

Combination treatment of DFMO and AMXT-1501 resulted in depletion of cellular polyamine levels

To investigate the effects of DFMO and AMXT-1501 on cellular polyamine levels, BE(2)-C and SMS-KCNR cells were treated with 5 mM DFMO, 15 µM AMXT-1501, or combination of both inhibitors for 24–48 hr. The treatment resulted in a decrease in specific intracellular polyamine levels (Fig. 4). DFMO treatment alone completely depleted PUT in BE(2)-C cells and also decreased the PUT in SMS-KCNR cells (24.8% of control at 24 hr, p = 0.00067, and 11.7% at 48 hr, p = 0.00066). DFMO treatment also decreased the level of SPD in BE(2)-C cells (53% of control at 24 hr, p = 0.003, and 12.7% at 48 hr, p = 0.00027). AMXT-1501 treatment alone resulted in a slight drop in SPD levels. Combination treatment resulted in a greater decrease in both PUT and SPD than did DFMO or AMXT-1501 alone. In SMS-KCNR cells, combination treatment at 48 hr synergistically decreased SPD to 30.4% of control (p = 0.046); in BE(2)-C cells at 24 hr, the SPD decreased to 12.7% of control (p = 0.00088). SPM levels were relatively unchanged in both cell lines across all treatments. These results supported previous reports of combination treatment of DFMO and AMXT-1501 on other tumor cells successfully reduced intracellular polyamines.[5, 22]

Figure 4.

Depletion analysis of intracellular polyamines in BE(2)-C and SMS-KCNR cells. All experiments included 0.5 µM spermidine and 1.0 mM aminoguanidine in the culture media. Cells were treated with 5 mM DFMO, 15 µM AMXT-1501, or a combination of both inhibitors for 24–48 hr. Plots were the average of three experiments. Not shown: no detectible levels of acetylated polyamines, indicative of retro- conversion. *p = 0.00088; **p = 0.046.

Combination treatment of low-dose DFMO and AMXT-1501 resulted in decrease levels of ATP per cell

To investigate the metabolic activity in inhibitor-treated cells, we performed two assays on the same plate of cells: CyQuant fluorescent cell proliferation assay (measures cell number using DNA) and Cell Titer-Glo luminescent cell viability assay (which measures ATP present). The ratios indicate relative ATP amount per cell. Treatment with a combination of 2.5 µM AMXT-1501 and 2.5 mM DFMO in BE(2)-C, SMS-KCNR and SH-SY5Y NB cell lines exhibited a time-dependent decrease in ATP levels per cell (Fig. 5). At 96 hr the BE(2)-C cells were the most sensitive to the drugs, with ATP per cell being 30% of the amount in control cells (p = 6.87E-06). The resulting ATP /cell percentage for SMS-KCNR cells was 70% (p = 0.0026) and for SH-SY5Y cells, 42% (p = 3.84E-12). Inhibitors alone only slightly decreased the ATP/cell value.

Figure 5.

ATP per cell in three NB cell lines. Cells were treated with 2.5 mM DFMO, 2.5 µM AMXT-1501, or a combination of both inhibitors for 24–96 hr. A decrease in ATP per cell production was observed in all lines. Cells were plated in sextuplicate wells, and experiments were independently repeated three times (n = 18). *p = 0.0026; **p = 3.84 × 10−12; ***p = 6.87 × 10−6.

Discussion

This study explores for the first time the effects of the novel polyamine transport inhibitor AMXT-1501 combined with polyamine biosynthesis inhibitor DFMO on two MYCN-amplified NB cell lines BE(2)-C and SMS-KCNR, and one non-amplified NB cell line SH-SY5Y. The novel two-drug combination treatment of NB cells induced synergistic cytotoxicity, caused cell proliferation inhibition, polyamine depletion, a decrease in ATP/cell, and apoptosis in these cell lines.

DFMO IC50 values at 48 hr ranged from 20.76 to 33.3 mM, and AMXT-1501 IC50 values ranged from 14.13 to 17.72 µM for the three cell lines (Fig. 1a). Isobologram results indicated when DFMO concentration was lower than 20 mM, and AMXT-1501 lower than 10 µM, the combination treatments synergistically decreased cell viabilities. Low doses were chosen to treat cells for subsequent studies (DFMO at 2.5 mM, AMXT-1501 at 2.5 µM) as a result of the synergistic cytotoxicity of the combination (Fig. 1b).

A great benefit of Real-Time Cellular Analysis is to monitor cellular events (cell attachment, spreading, and proliferation) in actively proliferating cells without the incorporation of labels that may affect cellular behavior. From 0 to 48 hr before medium and drug refreshment, the DFMO and AMXT-1501 treated cells remained non-proliferating with a cell index close to 1, compared to vehicle and single-drug treated cells, in all three cell lines, which indicated cell growth arrest, as shown in Figure 2. From 48 to 96 hr, the decreasing cell index of combo group indicated cell death. This result correlated with the western blot analysis results that combination treatment caused cell cycle arrest at 24 and 48 hr, followed by caspase-mediated apoptosis at 72 hr (Figs. 3a and 3b).

MYCN is an important marker of poor prognosis in NB. Inhibition of this may improve treatment of this disease. It was previously found that MYCN expression is down-regulated in NB cell lines LAN-1 and NMB-7 when treated with DFMO.[11] To investigate the effect of DFMO and AMXT-1501 on MYCN expression in MYCN-amplified cells BE(2)-C and SMS-KCNR, we performed western blot analysis. As shown in Figure 3c, the combination treatment of both drugs decreased MYCN expression levels starting at 48 hr.

ODC is a key enzyme in mammalian polyamine biosynthesis that is up-regulated in various types of cancer. It has been previously shown that treating human NB cells with the ODC inhibitor DFMO induces two opposing pathways in NB: one promoting cell survival by activating Akt/PKB via the PI3K/Akt pathway, and one inducing p27Kip1/retinoblastoma-coupled G1 cell cycle arrest via a mechanism that regulates the phosphorylation and stabilization of p27Kip1.28,29 In our study, phosphorylated Rb (Ser 807/811) expression was decreased in cells treated with combination drugs starting at 48 hr. This observation was confirmed by a visible mobility shift from a hyperphosphorylated form (upper band) to a hypophosphorylated form (lower band) in total Rb (Fig. 3d). Hypophosphorylation of Rb is known to be induced by p27 Kip1, but interestingly we did not see increased levels of p27 Kip1 in our combination-treated cells, thus suggesting the activation of other p27Kip1-independent mechanism in those cell lines. These could include its regulation via p53 and induction of apoptosis rather than cell cycle arrest. Future investigation into which NB subtype/genetic make-up is the most responsive to DFMO and AMXT-1501 may play a role in the determination of therapeutic decisions including dose responsiveness.

In SMS-KCNR cells, polyamine measurements indicated a synergistically faster depletion of PUT levels after combination treatment vs. DFMO alone as early as 24 hr. SPD levels were also synergistically depleted at 48 hr after combination treatment. AMXT-1501 treatment alone had no significant effect on all three polyamine levels. This result clearly demonstrates that the combination of two polyamine inhibitors is more effective than each inhibitor alone and, together, significantly enhance the polyamine depletion in the tested NB cells. The observation that SPM did not decrease in response to four different ODC inhibitors including DFMO has been previously reported in many other cell systems[30, 31] and is likely due to the fact that intracellular SPM is more tightly bound than PUT and SPD. Moreover, the polyamine biosynthesis enzyme that makes SPM is still intact and polyamine catabolism (from SPM to SPD) is minimal.

Ongoing studies including the combination therapy in neuroblastoma xenograft mouse models have been initiated in our laboratory based on these encouraging results. With the current lack of effective therapies for relapsed/refractory NB patients, the DFMO plus AMXT-1501 combination treatment provides promising preclinical results and may be a potential new therapy for children with NB.

Acknowledgements

We thank Dr. Patrick Woster (University of South Carolina) and Dr. Mark Burns (Aminex Therapeutics) for providing DFMO and AMXT-1501, respectively. We also thank Ellen Ellis for administrative support, Akshita Dutta for help with the isobologram analysis, and David Nadziejka for editorial reading of the manuscript.

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