Analysis of gene profiles involved in the enhancement of all-trans retinoic acid-induced HL-60 cell differentiation by sesquiterpene lactones identifies asparagine synthetase as a novel target for differentiation-inducing therapy

  • Authors:
    • Ju Han Song
    • Seung Hyun Kim
    • Kyung-Min Cho
    • Seung Yong Hwang
    • Hyeoung-Joon Kim
    • Tae Sung Kim
  • View Affiliations

  • Published online on: December 31, 2013
  • Pages: 970-976
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All-trans retinoic acid (ATRA) is one of the most useful drugs in the treatment for acute promyelocytic leukemia (APL), but its adverse effects, which include drug resistance and hypercalcemia are obstacles to achieving complete remission. Our previous study showed that some sesquiterpene lactones (STLs), i.e., helenalin (HE) and parthenolide (PA) but not sclareolide (SC), enhance ATRA-induced differentiation of HL-60 APL cells with no unexpected effects, but the precise mechanism on underlying this synergism is not yet fully understood. In this study, we investigated the distinctive transcriptional profile of cells treated with effective STL compounds, which were identified by comparing the profile with that of cells treated with SC. Genome-wide approaches using cDNA microarrays showed that co-treatment with the differentiation-enhancing STLs HE and PA maximized the transcriptional variation regulated by the suboptimal concentration of ATRA in HL-60 cells. Of the genes of interest, asparagine synthetase was remarkably downregulated by ATRA co-treated with either HE or PA, but not with SC. In an additional analysis for the role of asparagine synthetase, ATRA-mediated HL-60 cell differentiation was enhanced when asparagine in the culture media was depleted by an addition of L-asparaginase, indicating that downregulation of asparagine synthetase gene expression may be involved in the enhanced cell differentiation by STL compounds. These results provide useful insight into differentiation-inducing therapy in the treatment of leukemia.


All-trans retinoic acid (ATRA) is a derivative of vitamin A and is most useful in the treatment of acute promyelocytic leukemia (APL) (14). This drug has been shown to cause terminal differentiation of immature leukemic blasts by regulating many target genes including retinoic acid receptor, CCAAT/enhancer-binding protein β and interferon regulatory factor 1 (57). Although it has been established that there is a high rate of complete remission with the administration of ATRA, there are several reports of adverse effects such as differentiation syndrome, hypercalcemia and ATRA resistance (811). Therefore, combination therapy of ATRA with alternative medicines has been suggested to minimize these unexpected effects (12).

Sesquiterpene lactone (STL) compounds, which have a lactone ring, are found in a broad range of plants. There is a growing interest in the pharmacological use of STLs. Parthenolide (PA) isolated from Tanacetum parthenium strongly inhibits proinflammatory cytokine-induced signal activation in immune disorders (13,14). Furthermore, the antitumor effects of PA have already been evaluated in vitro and in vivo (15,16). Other STLs such as helenalin (HE) and costunolide also exhibit anti-inflammatory and anticancer activities by greatly inhibiting the transcriptional regulatory activity of nuclear factor-κB (NF-κB) and the production of reactive oxygen species (1720). Although they have a structural similarity, each STL exhibits differential effects in therapeutic applications (2123). Therefore, molecular dissection of the action mechanism of therapeutically useful STLs is required.

The human leukemia HL-60 cell line has been established as a reasonable model for studying new medicines and their action mechanisms in differentiation-inducing chemotherapy (24). The cells are differentiated into monocyte- or granulocyte-like cells by stimulating them with 1,25-dihydroxyvitamin D3 or ATRA, respectively (1,25). Our previous study demonstrated that each STL exhibited different regulatory effects in the enhancement of HL-60 cell differentiation by combination treatment with ATRA; i.e., PA and HE synergized the ATRA-induced HL-60 cell differentiation into a granulocytic lineage but SC did not (26).

In this study, we attempted to identify the molecular events that occurred when the granulocytic differentiation of HL-60 cells was enhanced by the addition of the STLs, such as PA, HE and SC. To address the question, we used a cDNA microarray-based genome-wide approach and compared data sets obtained from microarray analyses for differentiation-inducing and non-inducing agents.

Materials and methods

Cell line and reagents

Human HL-60 cells were obtained from American Type Culture Collection (Rockville, MD, USA) and cultured in RPMI-1640 medium supplemented with heat-inactivated 10% fetal bovine serum (Omega Scientific, Tarzana, CA, USA) and antibiotics at 37°C in a humidified 5% CO2 incubator. To maintain exponential growth, cells were seeded at a concentration of 1×105 cells/ml and sub-cultured every 3–4 days. ATRA, PA, SC and phorbol 12-myristate 13-acetate were from Sigma (St. Louis, MO, USA). HE and l-asparaginase (l-ASNase) were purchased from Enzo Life Sciences (Farmingdale, NY, USA) and Aviva Systems Biology (San Diego, CA, USA), respectively.

Nitroblue tetrazolium (NBT) reduction assay and morphological study

HL-60 cells at a concentration of 1.5×105/ml were cultured for 72 h in the presence of ATRA and/or sesquiterpene lactones and l-ASNase. At the end of treatment, the cells were harvested by a centrifugation and incubated in PBS buffer containing 0.1% NBT (USB, Cleveland, OH, USA) and 200 ng/ml PMA for 1 h to allow the cells to form a blue-black nitroblue formazan. The differentiation-positive cells were accessed under a light microscope. At least 200 cells were counted for each culture sample, and the results were expressed as a relative percentage of NBT-positive cells to total cells.

Flow cytometric measurement

At the end of culture, cells were collected, washed with ice-cold PBS buffer and labeled with PE-conjugated CD11b monoclonal antibody (BD Bioscience, San Jose, CA, USA) at room temperature for 15 min. Fluorescent intensity was analyzed by flow cytometric measurement using BD FACSCalibur.

cDNA microarray analysis

HL-60 cells were treated with 50 nM ATRA alone or combination with HE, PA, or SC for 24 h. Total RNA from the cultures were isolated using TRIzol reagent (MRC, Cincinnati, OH, USA). For DNA microarray assay, fluorescence-labeled cDNA probes were obtained from 30 μg of total RNA by using SuperScript II reverse transcriptase (Gibco BRL) in a total reaction volume of 30 μl and applied to human 8.5K cDNA microarrays. The sample from untreated HL-60 cells was used as a reference for each chip assay. The experimental and analytical procedures were done as previously described (27).

RNA preparation and reverse transcription-polymerase chain reaction (RT-PCR)

The cDNA was obtained from 1 to 1.5 μg of total RNA by the RocketScript RT kit (Bioneer, Daejeon, Korea). The RT product (1 μl) was applied to each PCR reaction with the following primer sets: asparagine synthetase (ASNS; forward, 5′-acagaaggattggctgcctt-3′; reverse, 5′-cctctcactctcctcctcgg-3′), activating transcription factor 4 (ATF4); forward, 5′-aacagcaaggaggatgcctt-3′; reverse, 5′-gtgctgaggagaccccagat-3′), ATF5 (forward, 5′-ttggatactctggacttgct-3′; reverse, 5′-tccttgacgtactggatctc-3′) and β-actin (forward, 5′-agcgggaaatcgtgcgtg-3′; reverse, 5′-cagggtacatggtggtgcc-3′). The final products were analyzed on a 1.2% agarose gel with ethidium bromide staining.

Statistical analysis

The results were obtained from at least three-independent experiments. Statistical significance of the data was determined using a paired Student’s t-test. A P-value <0.05 was considered statistically significant.


Differential enhancing effects of STLs on ATRA-induced HL-60 cell differentiation

To confirm the effects of STL compounds on ATRA-induced leukemia cell differentiation, HL-60 cells were treated with one of three different STLs, helenalin (HE), parthenolide (PA) or sclareolide (SC), with or without a suboptimal concentration dose (50 nM) of ATRA. As shown in Fig. 1A, HE itself induced the differentiation of HL-60 cells. Both HE and PA strongly enhanced the effect of ATRA in inducing the differentiation, but SC did not. Similarly, the surface expression of CD11b, a marker antigen of general myeloid differentiation, was increased in the cells by combination treatment of ATRA with either HE or PA (Fig. 1B).

Profiles of genes involved in the enhancement of ATRA-induced differentiation by STLs

To investigate the mechanism by which ATRA-induced HL-60 cell differentiation was enhanced by combination treatment with HE or PA, we used cDNA microarray analyses of cells treated with 50 nM ATRA alone or during co-treatment with HE, PA or SC. Microarray analyses showed that treatment with ATRA alone resulted in transcriptional changes of 111 genes, compared with the basal levels in unstimulated cells. As expected, co-treatment with ATRA and STLs resulted in transcriptional changes in greater number of genes, allowing alterations in an increased number of genes including the gene observed in ATRA-treated HL-60 cells (Fig. 2A). Furthermore, the number of genes influenced by the STLs (152 by PA, 257 by HE and 140 by SC) seems to reflect the degree by which the STL enhanced differentiation, implying that the enhancing potential of an STL in ATRA-induced HL-60 cell differentiation depends on the participation of these extra genes.

To further investigate how HE and PA, but not SC, enhance ATRA-induced granulocytic differentiation of HL-60 cells, we sought a common set of genes that were influenced in both the cells treated with HE and with PA, but not in cells treated with ATRA alone or co-treated cells with ATRA and SC. As shown in Fig. 2B, 48 genes satisfied the criteria and they are summarized in Table I. Twenty-four of these genes, including phosphogluconate dehydrogenase (PGD) and platelet factor 4 (PF4), were upregulated, whereas the remaining 20, including ASNS and ATF5, were downregulated.

Table I.

Up- or downregulated genes by co-treatment of RA with either PA or HE, but not with SC.

Table I.

Up- or downregulated genes by co-treatment of RA with either PA or HE, but not with SC.

GenBank no.Gene name (40 of 48 genes)Fold-changes (v.s. no treatment)
AA894927Asparagine synthetase0.720.290.130.76
AA237029Homo sapiens cDNA FLJ33469 fis, clone BRAMY20020050.540.220.240.53
AA419177Solute carrier family 7, member 50.690.360.180.62
AU147203C21orf19-like protein0.640.270.290.61
AI346878Sodium channel, non-voltage-gated 1, β (Liddle syndrome)0.550.300.290.56
AA213793KIAA0336 gene product0.580.250.340.72
AA496253Activating transcription factor 50.650.370.320.57
AW001765Ribosomal protein L23a0.690.380.380.69
AA447748Dihydrolipoamide dehydrogenase0.530.340.420.62
AW057866Eukaryotic translation initiation factor 3, subunit 7 ζ0.620.400.370.50
AA676458Lysyl oxidase-like 20.510.470.300.54
AI951501Ribosomal protein L120.730.410.370.56
AA683050Ribosomal protein S80.890.430.400.60
AW075605Ribosomal protein L90.770.460.380.60
AA167113Homo sapiens cDNA FLJ11689 fis, clone HEMBA10049770.820.490.340.57
AA424912Karyopherin (importin) β 10.770.430.420.67
AI005610Ribosomal protein L13a0.770.440.430.62
AI369144Eukaryotic translation initiation factor 4E binding protein 10.780.490.390.72
AA504475Mitochondrial ribosomal protein L320.720.480.400.65
AA600217Activating transcription factor 40.810.480.420.81
AA598759Phosphogluconate dehydrogenase1.792.894.071.93
M81750Myeloid cell nuclear differentiation antigen1.772.183.151.57
AI954012Adenylyl cyclase-associated protein1.982.452.871.93
AA454104Charot-Leyden crystal protein1.582.133.141.35
T97181Platelet factor 41.672.572.621.60
AA775264Echinoderm microtubule associated protein like 21.862.172.901.59
AI360772Myosin IF1.892.472.551.76
H89664Amyloid β (A4) precursor-like protein 21.462.212.751.85
AA973730Death-associated protein kinase 31.802.072.791.80
AF020056WD repeat domain 11.832.182.661.77
AA448157Cytochrome P450, subfamily I, polypeptide 11.482.162.661.67
M80427Androgen-regulated protein FAR-17 (hamster)1.782.112.711.78
AA451863CD4 antigen (p55)1.912.102.721.91
AA453789Homo sapiens cDNA FLJ36109 fis, clone TESTI20219111.772.132.611.80
AI000188UDP glycosyltransferase 2 family, polypeptide B71.772.172.541.71
T57791Toll-like receptor 21.812.202.471.68
U62795Ubiquitin ligase Pub1(yeast)/NEDD-4 isolog(human)1.672.052.621.78
AA453471GM2 ganglioside activator protein1.962.352.311.78
R44739Grancalcin, EF-hand calcium binding protein1.492.232.351.61
AA486532Major histocompatibility complex, class II, DP β 11.552.132.361.27
Downregulation of ASNS in the enhancement of ATRA-induced differentiation by STLs

Based on the genome-wide profiles from cDNA chip analysis, we chose ASNS as a target gene for further study in relation to the differentiation of HL-60 cells. We firstly validated the STL-induced change in ASNS mRNA expression using RT-PCR. As shown in Fig. 3, ASNS expression was downregulated in the cells treated with HE or PA alone, as well as in the cells co-treated with ATRA, whereas no difference was found between cells treated with SC and untreated cells. We also determined the levels of two transcription factors, ATF4 and ATF5, which are known to regulate transcription of ASNS (28,29). In our microarray analysis, like ASNS, both genes were also suppressed by HE and PA when combined with ATRA (Table I). This finding suggests that the ASNS may play a role in the differentiation of HL-60 cells.

Enhancement of HL-60 cell differentiation by depletion of l-asparagine

It is well known that the protein encoded by ASNS plays an important role in asparagine synthesis. Therefore, to investigate the effect of lower ASNS expression on ATRA-induced cell differentiation, we attempted to create a similar condition by using l-asparaginase (l-ASNase). Treatment with either a suboptimal dose of ATRA (50 nM) or l-ASNase (0.1 U/ml) had little effect on the expression of CD11b on the cell surface, while a combination of both the drugs increased the expression of this antigen (Fig. 4A). The combined effect of ATRA and l-ASNase was confirmed by a NBT reduction assay (Fig. 4B). On examining nuclear morphology by Giemsa staining, we also observed a slight increase in the cytoplasm to nucleus ratio and that the nuclei were multilobed after treatment with ATRA and l-ASNase (Fig. 4C). Additionally, we examined whether depletion of asparagine could enhance differentiation in HL-60 cells treated with both ATRA and HE. To address this aim, HL-60 cells were treated with ATRA and a lower dose of HE (0.3 μM) in the presence or absence of l-ASNase. As shown in Fig. 4D and E, the addition of l-ASNase to the combination of ATRA and HE strongly increased the number of NBT-positive cells and the levels of the CD11b expression on the cell surface.


Considerable research has been performed on the use of natural STLs as treatments for diverse conditions, including inflammation and cancer. Similar to the results reported for the therapeutic uses of STLs for these diseases, STLs are also effective in differentiation-inducing chemotherapy for leukemia, via NF-κB inhibition (26,30). However, the molecular mechanisms underlying the differentiation-enhancing effects of STLs have not yet been fully elucidated. In this study, our DNA microarray-based approach identified transcriptional reprogramming in APL cells with STL-enhanced granulocytic differentiation. Furthermore, the concurrent application of this technology also identified gene factors that can lead to differential sensitizing effects between the active STLs (i.e., PA and HE) and the non-active STL (i.e., SC) on ATRA-induced HL-60 cell differentiation.

Using DNA microarrays (although the gene content on the chip did not fully cover all human genes), we identified hundreds of genes that exhibit >2-fold changes in the level of transcription in the cells stimulated with a suboptimal concentration of ATRA alone or a combination of ATRA with an STL, compared with non-stimulated reference cells. Interestingly, the number of affected genes was proportional to the degree of enhancement of differentiation (Fig. 2). The gene populations selected from the cells treated with either ATRA alone or in combination with SC overlapped with each other. Furthermore, a large proportion of the genes were subordinate to the gene subsets that were picked from the cells treated with effective STLs, especially HE. These observations agree with our hypothesis that HE and PA, but not SC, effectively enhance ATRA-induced differentiation by maximizing the degree of transcriptional changes as well as by increasing the number of genes that are involved in HL-60 cell maturation.

In the transcription profiles associated with the enhanced differentiation of HL-60 cells by PA and HE, the outstanding alteration was the down-modulation of ASNS, accompanied by the decreased level of ATF4 and ATF5, which are known to be positive transcriptional regulators of ASNS (28,29). ASNS, which encodes asparagine synthetase, has been reported to be aberrantly expressed in many kinds of cancers, including acute lymphoid leukemia (ALL). Since asparagine, which is synthesized by this protein, allows cancer cells to grow rapidly, there is growing interest in targeting asparagine synthetase as a cancer cure (31,32). Indeed, depletion of this amino acid by treatment with l-ASNase is currently used for patients with ALL (33,34). In this study, we manipulated the levels of l-ASNase without manipulating the gene (ASNS), to investigate the role of the enzyme in HL-60 cell differentiation. The result indicated that depletion of the end product of ASNS, that is, asparagine, in the culture medium was sufficient to enhance ATRA-induced HL-60 cell differentiation. The report by Hongo et al showed that asparagine synthetase activity was decreased when leukemia cells were stimulated with compounds that induce differentiation, implying that asparagine has a potential effect on culture conditions (35). Another study also reported an analogous observation that a decrease in ASNS expression was paralleled by the extent of maturation of HL-60 cells that was induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) (36). The researchers additionally demonstrated the synergistic induction of apoptotic cell death by adding l-ASNase in the presence of TPA, but did not mention a role of l-ASNase in cell differentiation. Although the details of how l-ASNase enhances ATRA-induced leukemia cell differentiation need to be further studied, to our knowledge, our findings provide the first evidence that l-ASNase can enhance differentiation induced in leukemia cells. In addition, the ability of some STLs to downregulate ASNS transcription may offer a therapeutic strategy for l-ASNase-resistant acute leukemia.

Taken together, our use of high-throughput microarray analysis demonstrates the existence of sets of genes that are differentially involved in the enhancement of ATRA-induced APL differentiation by effective STLs; this information also suggests a therapeutic use of STLs. Our additional observation that depletion of asparagine by l-ASNase synergistically enhanced HL-60 cell differentiation by ATRA may also be a valuable strategy in the treatment of leukemia, especially APL.


This study was supported, by the grant of the National Project for Personalized Genomic Medicine, Ministry for Health and Welfare (no. A111218-GM06), the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (no. 2005-0049410), and a Korea University grant.



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Song JH, Kim SH, Cho K, Hwang SY, Kim H and Kim TS: Analysis of gene profiles involved in the enhancement of all-trans retinoic acid-induced HL-60 cell differentiation by sesquiterpene lactones identifies asparagine synthetase as a novel target for differentiation-inducing therapy. Int J Oncol 44: 970-976, 2014
Song, J.H., Kim, S.H., Cho, K., Hwang, S.Y., Kim, H., & Kim, T.S. (2014). Analysis of gene profiles involved in the enhancement of all-trans retinoic acid-induced HL-60 cell differentiation by sesquiterpene lactones identifies asparagine synthetase as a novel target for differentiation-inducing therapy. International Journal of Oncology, 44, 970-976.
Song, J. H., Kim, S. H., Cho, K., Hwang, S. Y., Kim, H., Kim, T. S."Analysis of gene profiles involved in the enhancement of all-trans retinoic acid-induced HL-60 cell differentiation by sesquiterpene lactones identifies asparagine synthetase as a novel target for differentiation-inducing therapy". International Journal of Oncology 44.3 (2014): 970-976.
Song, J. H., Kim, S. H., Cho, K., Hwang, S. Y., Kim, H., Kim, T. S."Analysis of gene profiles involved in the enhancement of all-trans retinoic acid-induced HL-60 cell differentiation by sesquiterpene lactones identifies asparagine synthetase as a novel target for differentiation-inducing therapy". International Journal of Oncology 44, no. 3 (2014): 970-976.