The association between oxidative stress-induced galectins and differentiation of human promyelocytic HL-60 cells

James R. Vinnai, Robert C. Cumming, Graham J. Thompson, Alexander V. Timoshenko* Department of Biology, The University of Western Ontario, London, ON, Canada, N6A 5B7

ABSTRACT

Galectins are multifunctional β-galactoside-binding proteins that are involved in the regulation of cellular stress responses and differentiation. The relationship between these processes is unclear and we report here that galectins display oxidative-stress specific expression patterns in neutrophil-like differentiated HL-60 cells. Three galectins (-1, -3, and -10) are upregulated in response to either menadione or DMSO exposure whereas galectins -9 and -12 exhibited a stimulus-dependent downregulation. Changes in galectin expression are oxidant dependent based on the observations that 1) oxidative stress biomarkers HMOX1 (heme oxygenase-1) and NCF1 (neutrophil cytosolic factor 1, which is also a biomarker of neutrophil differentiation) are elevated in both cases, and 2) the antioxidant N-acetyl-N-cysteine restores basal expression of galectin-3 following oxidant exposure. In addition, our results suggest that the regulation of oxidative stress-sensitive galectins involves DNA hypomethylation mechanisms. Expression of galectin-3 and galectin-12 exhibits an opposite relationship to the expression of HMOX1/NCF1, suggesting a stimulatory and inhibitory role of these galectins in neutrophil-like differentiation of HL-60 cells. We also show that the inhibition of galectins reduces the growth rate of HL-60 cells, and facilitates their neutrophil-like differentiation. Collectively, our findings indicate that the process of cellular differentiation implicates, in part, oxidative stress-sensitive galectins, which further highlights a biological significance of galectin network remodeling in cells.

Key words: galectins; oxidative stress; cell differentiation; cell proliferation; menadione; hydrogen peroxide; DNA hypomethylation; JNK signaling; leukemia

1. Introduction

Galectins are an evolutionary ancient family of soluble -galactoside-binding proteins that were discovered over 40 years ago [1]. However, only recently have we started to understand their roles in cells, considering that galectins appear to be uniquely poised to regulate a wide variety of biological activities at different levels [2]. Galectins are multifunctional proteins capable of modulating cell differentiation, proliferation, survival, death, adhesion and migration. These cellular events are critical in biological processes such as embryogenesis, angiogenesis, neurogenesis and immunity. Sixteen different galectin genes (gene symbol LGALS) have been identified in the animal kingdom and 12 of which are expressed in human cells, not counting different splicing variants [1-4]. Expression of galectin genes varies significantly in different cells and tissues, as evident by galectin profiling analyses [4-6]. A well- established role for galectins is to modulate both pro-survival and pro-apoptotic signaling pathways [1,2,7]. This biological function is essential for cellular homeostasis and cell survival under challenging microenvironmental conditions that may range from metabolic imbalance and local hypoxia to the accumulation of toxic oxidants [8]. Cells cope with environmental stress by increasing expression of stress response genes [9], including galectins as biomarkers of an overall cellular stress response (CSR). Indeed, many microenvironmental stressors such as hypoxia, hyperthermia, oxidants, and UV light have been reported to alter the expression of galectins [3]. The expression pattern of galectin genes and proteins changes substantially in tumor tissues and cancer cells [3,5,6] and in tissues associated with inflammation [10], which implicates galectins as regulators of the CSR. Galectins can be considered as “alarmin” molecules, which signal tissue damage and elicit an effector response from immune cells [11,12]. Tumor-derived galectins, in particular, may help to escape immune surveillance through immunosuppression [13]. Recently, we have found that chemical stimuli eliciting endoplasmic reticulum stress, mimicking hypoxia and inducing cellular differentiation, can change the galectin expression profiles in HL-60 cells [14]. These findings suggest there are two groups of galectins: stress-sensitive and stress-resistant. The regulation and requirement of either type is, however, not yet clear. Oxidative stress signaling and DNA methylation are two potential mechanisms that may regulate the differential expression of galectins. First, there are binding sites for the oxidative stress-induced transcription factor Sp1 and, second, there is an abundance of CpG islands within the promoter regions of some human galectin genes [15].
Oxidative stress, a disturbance in the balance between reactive oxygen species (ROS) production and antioxidant detoxification, is a contributing factor to many diseases. These include acute and chronic inflammation, and cancer, among other diseases [16]. Galectins are associated with oxidative stress through reciprocal molecular mechanisms. First, many galectins can directly stimulate a respiratory burst in phagocytes by activating superoxide- and hydrogen peroxide-producing plasma membrane NADPH oxidase [17-19]. Second, oxidative stress is known to upregulate some galectins, as reported for galectin-3 in lung macrophages [20] and again in breast cancer cells [21]. In addition, functions of galectins depend on the redox microenvironment [22] and oxidative stress–sensitive signaling associated with MAPK, MEK1/ERK, mTOR, and JNK pathways [23-25]. However, the details of this regulation remain elusive, especially with regard to the complex galectin networks in cells. Regulation of redox status is an important feature of cellular homeostasis, and changes in the production of ROS have been linked to controlling the fundamental processes of cell survival, proliferation, and differentiation [26]. As such, oxidative stress has been proposed to enhance differentiation of different cell types, including dimethyl sulfoxide (DMSO)-induced differentiation of human HL-60 promyelocytic leukemia cells into granulocytes [27]. Remarkably, myeloid differentiation of HL-60 cells into three different lineages (eosinophil-, monocyte-, and neutrophil-like cells) was accompanied by differential changes in the expression of galectins at both mRNA and protein levels [28]. The individual role of stress-sensitive and stress-resistant galectins in differentiation and oxidative stress response is poorly understood. In light of the apparent interplay between oxidative stress and differentiation, it would be interesting to determine which galectins potentially govern the signaling cascade ROS → altered galectin expression → cellular differentiation.
In this study, we used HL-60 cells as a model system to test molecular mechanisms mediating the involvement of stress-sensitive galectins in myeloid differentiation. HL-60 cells express six of the 12 known human galectin genes (LGALS1, LGALS3, LGALS8, LGALS9, LGALS10, and LGALS12) and are readily differentiated into neutrophil-like cells when exposed to DMSO [14,28]. Using menadione as an oxidative stress stimulus [29,30], we compared the galectin expression profiles of differentiated cells against those treated with drugs that modulate oxidative stress-associated JNK/Sp1 signaling and DNA methylation. Moreover, the effects of galectin inhibitors on HL-60 cell growth and differentiation were tested to assess functional significance of upregulated galectins.

2. Materials and Methods

2.1. Chemicals

Cobalt (II) chloride hexahydrate (CoCl2), N-acetyl-N-cysteine (NAC), N-formyl-L- methionyl-L-leucyl-L-phenylalanine (fMLP), horseradish peroxidase (HRP), lactose, D- mannose, menadione sodium bisulfite, methyl α-D-mannopyranoside (α-MM) and scopoletin were purchased from Sigma-Aldrich Canada (Oakville, ON). Anisomycin was from StressMarq Biosciences (Victoria, BC), 5-aza-2’-deoxycytidine (decitabine) was from Cayman Chemical (Ann Arbor, MI), lactobionic acid (LBA) was from Toronto Research Chemicals (Toronto, ON), OTX008 was from Axon Medchem (Groningen, The Netherlands), SP600125 was from LC Laboratories (Woburn, MA), and thiodigalactoside (TDG) was from Carbosynth (San Diego, CA).

2.2. Suspension cell culture and treatments

Human promyelocytic leukemia HL-60 cells (ATCC® CCL­240™) were cultured in CorningTM cellgroTM Iscove’s Modification of DMEM (IMDM) supplemented with 10% charcoal stripped fetal bovine serum (Wisent Bioproducts, St-Bruno, QC), 100 IU/mL penicillin, and 100 μg/mL streptomycin in a humidified incubator at 37°C and 5% CO2. Suspension cell cultures were maintained under culture conditions not exceeding 1×106 cells/mL for all treatments and passaged accordingly. To induce oxidative stress, HL-60 cells were treated with 10 μM menadione or 100 μM CoCl2 for 24 h. HL-60 neutrophilic differentiation was accomplished by treating cells with 1.3% DMSO for 72 h unless otherwise stated. Inhibition and activation of JNK was induced by exposure to SP600125 (25 μM) and anisomycin (400 nM), respectively for 24 h. Global hypothemylation was accomplished by daily treatments of cell cultures with 50 nM decitabine (a DNA methyltransferase inhibitor) over 72 h. NAC was used as an antioxidant at concentrations of 1 mM and 2.5 mM for oxidative stress (24 h) and differentiation experiments (72 h), respectively. Galectin inhibitors (lactose, LBA, OTX008 and TDG) and non-inhibitory sugars (mannose and α-MM) were used at millimolar and sub- millimolar concentrations to test their dose-dependent effects on cell proliferation and differentiation.

2.3. RNA extraction, cDNA synthesis, and PCR analysis

Cells were washed with Ca2+/Mg2+-free Dulbecco’s phosphate-buffered saline and the total mRNA was extracted from the cell pellet using TRIzol® reagent from Ambion (Carlsbad, CA) as per the manufacturer’s protocol. cDNA was reverse transcribed in 20 L from 2 µg of RNA using the Maxima First Strand cDNA Synthesis Kit for RT-qPCR (Thermo Scientific, cat. # K1642). Oligonucleotide PCR primers were either designed using Primer-BLAST (NCBI) software or based on previous publications and verified by nucleotide BLAST analysis (Supplementary Material, Table S1). SensiFAST™ SYBR® No-ROX Kit (Bioline, cat. # BIO- 98005) was used to run real-time PCR in 20 l of PCR mix containing 400 nM forward/reverse primers and 0.5 µL of undiluted cDNA template, which were amplified in a CFX96™ Real-Time PCR Detection System (Bio-Rad). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control and the quantification of relative gene expression was calculated using 2-ΔΔCt methodology [31]. RT2 Profiler™ PCR Array (Qiagen, cat. # PAHS-065Z) was used to assess the expression of 84 genes related to oxidative stress as per the manufacturer’s real-time PCR protocol.

2.4. Protein extraction, western blotting, and antibodies

Cells were washed with Ca2+/Mg2+-free Dulbecco’s phosphate-buffered saline and the cell pellet was homogenized in the SDS extraction buffer (2% SDS, 50 mM Tris/pH 7.5, 1 mM PMSF, 1 mM AEBSF, 5 mM EDTA, 50 µM leupeptin and 1 µM pepstatin). Samples were then incubated on ice for 10 min before further homogenization via sonication with a MicrosonTM XL-2000 Ultrasonic Liquid Processor (Qsonica) for 15 s at the amplitude setting 2. Protein concentration was determined using the DC Protein Assay™ (Bio-Rad, cat. # 5000111) as per the manufacturer’s protocol and absorbance was measured at 595 nm in a Model 3550 Microplate Reader (Bio-Rad). Protein extract (25 μg) was resolved by 15% SDS-PAGE and transferred onto PVDF membrane (Bio-Rad). Membranes were blocked with 5% BSA and 1% skim milk in TBS-T buffer (50 mM Tris, 150 mM NaCl and 0.05% Tween-20) at room temperature for 1 hr before being probed with primary antibody overnight at 4°C. Membranes were then incubated for 1 hr with an appropriate HRP-conjugated secondary antibody, treated with SuperSignal™ West Pico Chemiluminescent Substrate (ThermoFisher Scientific, cat. # 34080) and imaged with a ChemiDoc XRS system (Bio-Rad). Densitometry was performed using ImageLab™ software (Bio-Rad). Primary antibodies were rabbit polyclonal antibodies against galectin-1 (sc-28248), galectin-3 (sc-20157), galectin-12 (sc-67294), and mouse monoclonal antibody against β-actin (sc-47778) from Santa Cruz Biotechnology, monoclonal rabbit antibody against galectin-10 from Abcam (ab-157475), polyclonal rabbit antibodies against SAPK/JNK (#9252) and phospho-SAPK/JNK (#9251) from Cell Signaling Technology. Secondary antibodies were polyclonal goat anti-rabbit IgG-HRP (sc-2004) and goat anti-mouse IgG-HRP (sc-2005) from Santa Cruz Biotechnology. Antibodies were diluted in TBS-T buffer supplemented with 5% bovine serum albumin and 0.05% sodium azide.

2.5. Scopoletin assay to measure fMLP-induced H2O2 production

Cells were seeded at 250,000 cells per well (500 L) in 24 well plates and treated with 1.3% DMSO for 72 h in order to achieve fully neutrophil-like differentiated HL-60 cells. Cells were then collected, centrifuged and resuspended in Hanks’ Balanced Salt Solution pre-warmed to 37°C. The cell concentration was adjusted by measuring the absorbance at 600 nm using NanoDrop 2000c spectrophotometer. The cells were placed into an AMINCO-Bowman Series 2 Luminescence Spectrometer at 37oC and a reaction “cocktail” (1 M scopoletin and 20 μg/mL
HRP) was added to detect H2O2 generation [30,31]. To induce oxidative burst, fMLP (100 nM) was added and scopoletin oxidation was measured as a decrease in fluorescence intensity at 460 nm (excitation at 380 nm). The rate of H2O2 production was calculated as the maximal slope of the recorded traces normalized to the cell concentration and original fluorescence of 1 M scopoletin (Supplementary Material, Fig. S1).

2.6. Cell proliferation assay

Cells were seeded at 50,000 cells per well (500 L) in 24 well plates and treated with different galectin inhibitors at various concentrations for 96 h. Cell counts and viability were assessed every 24 hours from initial seeding using a hemocytometer and trypan blue (0.2%) exclusion test. The population doubling time was calculated based on the exponential increase of the total number of live cells as described elsewhere [32].

2.7. Statistical analysis

All experiments were performed at least in triplicate and data were expressed as mean ± SD unless stated otherwise. The significance of differences between means was analyzed by one sample t-test, Student’s t-test, and one-way analysis of variance (ANOVA) followed by a Tukey’s HSD test depending on the set of data. Correlation analysis between genes was performed using a Pearson’s correlation test. All statistical tests were executed using IBM SPSS Statistics (version 23) and GraphPad Prism 6 software considering p < 0.05 as the statistical threshold.

3. Results

3.1. Menadione-induced oxidative stress and galectin expression profiles in HL-60 cells

Menadione is a methylated analogue of 1,4-naphthoquinone which can undergo one- electron reduction by several intracellular enzymes including microsomal NADPH-cytochrome P450 reductase and mitochondrial NADH-ubiquinone oxidoreductase resulting in generation of ROS and oxidative stress in cells [33]. Indeed, the treatment of HL-60 cells with 10 μM menadione (24 h) led to upregulation of 44 oxidative stress-related genes out of 84 genes available in the RT-qPCR array assay (Supplementary Material, Table S2). The most prominent upregulated genes included heme oxygenase-1 (HMOX1), which is a well-established biomarker of oxidative stress [27, 34], and p47-phox or neutrophil cytosolic factor 1 (NCF1), which is also a biomarker of granulocyte differentiation [35]. A highly significant upregulation of HMOX1 and NCF1 by menadione exposure was further confirmed by real-time RT-qPCR in each case (Fig. 1A). As no changes in cell morphology and viability were observed under this treatment regime (10 μM menadione, 24 h), we used it to examine the influence of menadione oxidative stress on expression profiles of six HL-60 cell-specific galectins established in our previous study [14].
Menadione oxidative stress induced significant upregulation of three galectin genes (LGALS1, LGALS3, and LGALS10) whereas the expression of LGALS9 was downregulated (Fig. 1B). These changes were rescued in the presence of 1 mM NAC, which is a strong antioxidant and scavenger of ROS [36]. LGALS8 and LGALS12 expression remained unaltered in all cases except for an inhibitory effect of NAC on the expression of LGALS12 in the presence of menadione. This list of stress-sensitive and stress-resistant galectin genes was similar to those we detected earlier in HL-60 cells treated with other stress stimuli such as CoCl2 and tunicamycin [14] indicating their prominent role in CSRs. Immunoblot analysis of stress-induced galectins showed significant elevation only for galectin-3 protein whereas all other tested galectin proteins (-1, -10, and -12) exhibited stable levels in control and redox-treated HL-60 cells (Fig. 1C and 1D). It should be noted that NAC did not change the basal expression of galectins at both transcript and protein levels in all cases.

3.2. Galectin expression profiles in differentiated HL-60 cells induced by DMSO

Oxidative stress is involved in neutrophil-like differentiation of HL-60 cells by DMSO, which is a powerful inducer as all-trans retinoic acid [27]. Considering multiple indications of the galectin network remodeling in differentiated cells [14,28] and the presence of oxidative stress-induced galectins in HL-60 cells, we sought to investigate the redox-regulation of galectins in DMSO-treated cells. To optimize experimental conditions of the DMSO-induced HL-60 cell differentiation, suspension cell cultures were treated with 1.3% DMSO and the functional response of cells, namely fMLP-induced generation of H2O2 by plasma membrane NADPH oxidase [37], was examined daily over the six-day period. The rate of H2O2 generation gradually increased reaching a maximum level at day 3 (72 h) of cellular differentiation, a time point at which the cell viability was greater than 80% (Fig. 2A). The markers of neutrophil differentiation NCF1 and oxidative stress HMOX1 were both significantly (p<0.001) upregulated following the 72 h exposure to 1.3% DMSO indicating prominent cellular differentiation is association with oxidative stress (Fig. 2B).
In light of the elevation of ROS during DMSO-induced differentiation of HL-60 cells, the galectin expression profiles [14,28] were revisited focusing on oxidative stress-sensitive galectins using real-time RT-qPCR and immunoblotting. The mRNA levels of LGALS3 and LGALS10 were significantly (p<0.001) upregulated by day 3 while the expression of LGALS12 was downregulated (Fig. 2C). The expression of LGALS1, LGALS8, and LGALS9 was not changed in the differentiated HL-60 cells. At the protein level, galectins 1, 3 and 10 were upregulated whereas galectin 12 was decreased following DMSO exposure (Fig. 2E).
To determine the role of DMSO-induced ROS on galectin expression [27] HL-60 cells were maintained in the presence of 2.5 mM NAC for 3 days. This treatment inhibited both the differentiation of HL-60 cells, as measured by fMLP-induced H2O2 production (Supplementary Material, Fig. S2), and the induction of LGALS3 and LGALS10 mRNAs following 1.3% DMSO exposure (Fig. 2C). At the protein level, only DMSO-induced expression of galectin-10 was decreased by NAC treatment, while the other DMSO-induced changes in other galectins were unaltered by NAC treatment (Fig 2E).

3.3. Involvement of JNK signaling pathway in regulation of galectin expression

Oxidative stress is known to stimulate the PP1/JNK1/Sp1 signaling pathway [38] which can contribute to regulation of galectin genes containing Sp1 binding sites in their promoter regions [15]. This signaling mechanism requires phosphorylation of JNK to activate Sp1. Indeed, treatment of HL-60 cells with menadione (10 μM, 24 h) induced a significant increase of phosphorylated JNK1/2, which was attenuated with NAC exposure (Fig. 3A). In contrast, DMSO-induced differentiation of HL-60 cells was accompanied by decreased phosphorylated JNK1/2 levels (Fig. 3B), suggesting that regulation of galectins during cellular differentiation may occur in a JNK independent manner. We therefore tested the effects of chemical inhibitors and activators of JNK on galectin expression profiles in HL-60 cells. The expression of LGALS1, LGALS3 and LGALS8 was significantly upregulated by the JNK activator anisomycin (400 nM, 24 h) while the expression of LGALS9 and LGALS12 was downregulated and no change was observed in LGALS10 expression (Fig. 3C). Treatment of HL-60 cells with the JNK inhibitor SP600125 (25 μM, 24 h) induced much smaller and different changes in galectin gene expression profiles including upregulation of LGALS1 and LGALS12, and downregulation of LGALS8, LGALS9 and LGALS10 (Fig. 3D). As such, four galectin genes (LGALS3, LGALS8, LGALS10, and LGALS12) displayed distinct alteration patterns depending on JNK activity while upregulation of LGALS1 and downregulation of LGALS9 appear to be JNK independent. Overall, the galectin expression profiles of HL-60 cells treated with both menadione and DMSO (Fig. 1 and Fig. 2) were similar to anisomycin-treated rather than to SP600125-treated cells (Fig. 3). These results indicated that although the JNK signaling pathway is functional in HL-60 cells, the expression of oxidative stress-sensitive galectins might depend on or cooperate with other molecular mechanisms in the context of cellular differentiation.

3.4. Alterations of galectin expression profiles in HL-60 by decitabine treatment, an inhibitor of DNA methylation

DNA methylation is an essential global mechanism that controls the expression of many genes including galectins [17]. As the inhibition of DNA methylation is known to induce differentiation of HL-60 cells [39], we investigated the effects of a specific DNA methyltransferases inhibitor decitabine on galectin expression profiles at both transcript and protein levels. Decitabine was used at a concentration of 50 nM and applied daily to the suspension culture of HL-60 cells over 72 h. At this time point, the cells showed highly significant (p<0.001) upregulation of the cellular differentiation and oxidative stress biomarkers NCF1 and HMOX1 respectively (Fig. 4A). Decitabine-treated cells exhibited significant upregulation of LGALS1, LGALS3, LGALS10 and LGALS12, and downregulation of LGALS9 while no changes were noticed with LGALS8 (Fig. 4B). Immunoblotting confirmed decitabine- induced increased expression of galectin-1, -3, and -10 while the level of galectin-12 was unchanged (Fig. 4C). The observed changes were largely consistent with the galectin profile pattern under menadione-induced oxidative stress, which suggested common regulatory pathways with DNA methylation involved in cellular differentiation. However, not all galectins demonstrated the same trend or magnitude of altered expression, which prompted us to perform a correlative analysis between expression of oxidative stress biomarkers and galectin genes.

3.5. Positive and negative correlations of galectin genes with oxidative stress biomarkers

Pearson’s correlation analysis was performed for gene expression data collected from all treatments of HL-60 cells (n=5, i.e. 1.3% DMSO, 10 µM menadione, 50 nM decitabine, 25 µM SP600125 and 400 nM anisomycin), which included at least three biological and three technical replicates in each case. Significant correlations (positive or negative) between galectin gene expression versus HMOX1 expression was observed for LGALS1, LGALS3, LGALS8 and LGALS12 while those versus NCF1 for all galectins but LGALS8 (Supplementary Material, Table S3). Interestingly, LGALS3 showed a positive correlation with both oxidative stress biomarkers (HMOX1 and NCF1) whereas LGALS12 showed an inverse relationship (Fig. 5). These data suggest a specific and different role of these two galectins in cellular differentiation in conjunction with oxidative stress because NCF1 is also a strong biomarker of granulocyte differentiation of HL-60 cells [35].

3.6. Opposite effects of galectin inhibitors on proliferation and differentiation of HL-60 cells

Functional activity of galectins produced by HL-60 cells can be impaired by specific - galactoside-containing substances that compete for carbohydrate-recognition domains or by allosteric inhibitors [40]. If oxidative stress-induced galectins are involved in regulation of cell survival/proliferation or differentiation, galectin inhibitors would be expected to affect diversely these processes. Indeed, competitive -galactoside-based inhibitors (lactose, LBA, and TDG) and galectin-1 allosteric inhibitor OTX008 increased – in a dose-dependent manner – the doubling time of HL-60 cell culture, which was significant in comparison with untreated cells at concentrations of 40 mM for TDG and 5 μM for OTX008 (Fig. 6C and D). Non-inhibitory sugars D-mannose and α-methyl-D-mannoside failed to change the doubling time (Fig. 6E and F) likely excluding the involvement of galectin-10, which is the only galectin with an affinity to mannoside sugars [41]. Galectin inhibitors at all tested concentrations had no effect on cell viability (data not shown).
To test the effects of galectin inhibitors on neutrophil-like differentiation of HL-60 cells, cells (0.6x106 cells/mL) were treated with 1.3% DMSO and a galectin inhibitor for 72 h. The rate of fMLP-induced production of H2O2, a measure of cellular differentiation, was significantly increased by cells treated with 20 mM TDG, 20 mM lactose and 0.5 mM LBA in comparison with DMSO-treated cells whereas OTX008 (2.5 M) significantly inhibited this response (Fig. 7). None of these galectin inhibitors alone stimulate cellular differentiation (data not shown). Taking into account potential non-target effects of OTX008 on intracellular signaling [42], the results indicated that carbohydrate inhibitors of galectins attenuate proliferation and facilitate or stimulate DMSO-induced differentiation of HL-60 cells.

4. Discussion

Galectins are multifunctional animal lectins that are proposed to serve as biomarkers and mediators of microenvironmental and pathological CSRs [3]. To the best of our knowledge, this study characterizes for the first time the regulation and functional significance of a group of oxidative stress-sensitive galectins using a human promyelocytic cell line HL-60. In particular, menadione, an oxidative stress inducer, upregulates the expression of LGALS1, LGALS3, and LGALS10, downregulates the expression of LGALS9, and has no effect on the expression of LGALS8 and LGALS12. Furthermore, our results indicate that oxidative stress-sensitive galectins participate in neutrophil-like differentiation of HL-60 cells, and can exploit signaling mechanisms such as DNA hypomethylation and JNK kinase activation. Together with the observations of opposite effects of galectin inhibitors on cellular differentiation and proliferation, these findings highlight an importance of individual galectin approaches for developing anti- cancer and anti-inflammatory therapeutics.
Oxidative stress is a common molecular challenge associated with many diseases that are associated with a disturbed homeostatic redox balance within cells [16, 43]. In addition to powerful systems of antioxidants and redox enzymes, stressed cells recruit a relatively limited number of regulatory molecules and transcription factors that guide the cells towards survival or death as an ultimate outcome of CSRs [8,9,44]. Galectins represent a complex network of regulatory proteins with both pro-apoptotic and pro-survival properties, depending on their type and localization in cells [7]. Our results revealed that menadione oxidative stress induced significant remodeling of galectin expression profiles in HL-60 cells including three upregulated galectins (LGALS1, LGALS3, and LGALS10). Remarkably, all these galectins were upregulated at either the mRNA or protein level in HL-60 cells differentiated into neutrophil-like cells by DMSO, an agent that also promotes ROS production [27]. This similarity in expression among galectins implies a requirement of oxidative stress-induced galectins in global mechanisms of redox regulation of cellular differentiation [26]. Although the molecular mechanisms of galectin regulation remain to be fully elucidated, the upregulation of galectins -1 and -3 may be essential for protecting cells from the harmful effects of increased ROS production as demonstrated in different cell models. Indeed, Evsa-T human breast cancer cells ectopically expressing galectin-3 were more resistant than mock control cells to apoptosis and necrosis induced by menadione [45]. Similarly, recombinant galectin-1 was shown to protect human EAhy926 endothelial cells from H2O2-induced oxidative stress, which was attenuated by the inhibitory disaccharide TDG [46]. Less is known about the functional significance of galectin-10 (Charcot-Leyden crystal protein), which is highly expressed in eosinophils, basophils and regulatory T cells [47]. However, galectin-10 is recognized as a biomarker of differentiated HL-60 cells [28] and eosinophil-associated inflammation and allergic diseases [48]. A potential role of LGALS10 in myeloid differentiation is also supported by the strong correlation between the expression of LGALS10 and a granulocyte differentiation marker NCF1 in HL-60 cells.
The significance of those galectins that were either downregulated (LGALS9 and LGALS12) or exhibited unchanged expression (LGALS8) remains unknown, and their pattern of expression suggests that they may function to inhibit cell differentiation or to maintain basic cellular functions, respectively. Indeed, knockdown of LGALS12 has been shown to enhance the neutrophilic differentiation of human NB4 promyelocytic leukemia cells induced by all-trans retinoic acid [49], while the overexpression of LGALS9 in tumor tissues is a favorable prognostic biomarker [6]. Downregulation of LGALS9 can be transcript variant-specific, which requires further studies focusing on the transcript variant 2 [14]. As for LGALS8, this galectin is commonly expressed in majority of cell lines and tissues [5]. Its stable expression may be countered by miRNA-dependent post-translational mechanisms via the longest 3’UTR among all galectin mRNAs [3,50].
The expression of oxidative stress-sensitive genes can be regulated by multiple signaling mechanisms, which include ROS-induced activation of specific transcription factors through a complex cascade of signaling phosphatases and kinases [38,51] and epigenetic changes including DNA hypomethylation [52]. Our results provide strong support to the latter mechanism of galectin upregulation in conjunction with cellular differentiation. Indeed, the inhibition of DNA methyltransferases by decitabine induces alterations in the galectin expression profiles (LGALS1, LGALS3, LGALS10) similar to the effects of menadione and DMSO. Recently, DMSO has been shown to induce active DNA demethylation in pre-osteoblastic MC3T3-E1 cells [53]. All these three treatments (decitabine, menadione, and DMSO) demonstrated very similar upregulating effects on the expression of both oxidative stress marker HMOX1 and granulocyte differentiation marker NCF1. Thus, ROS-dependent hypomethylation of specific DNA loci [52] may serve as a central mechanism responsible for recruiting stress-sensitive galectins for neutrophil-like differentiation of HL-60 cells. This notion may not be applied to all galectins as decitabine induces significant upregulation of LGALS12, which is inhibited in DMSO-induced differentiated HL-60 cells. This suggests an interference with other regulatory mechanisms. Indeed, the redox-sensitive pathway PP1/JNK/Sp1 [38] is functional in HL-60 cells as LGALS1 and LGALS3 were readily upregulated in HL-60 cells treated with a JNK activator anisomycin. However, this signaling mechanism seems to be unrelated to oxidative stress-mediated upregulation of galectins under cellular differentiation, as the level of active phosphorylated JNK1/2 was decreased in differentiated cells while it was increased by menadione. These observations are consistent with diverse and cell-specific roles of different mitogen-activated protein kinases (JNK, p38 MAPK, and ERK) in cellular differentiation depending on the type of stimuli. For instance, although DMSO-induced differentiation of HL-60 cells does not depend on JNK signaling [54], it is required for differentiation of T helper cells to Th1 effector cells [55]. HL-60 cells rely on the MAPK and ERK signaling pathways during DMSO-induced differentiation [56], which can cross-talk other signaling pathways under oxidative stress. These options are consistent with our findings revealing the disparate effects of the JNK activator anisomycin and JNK inhibitor SP600125 on the expression of individual galectins in HL-60 cells. As such, the regulation of stress-sensitive galectins through DNA hypomethylation and MAPK signaling in the context of cellular differentiation requires further studies considering a reciprocal relationship between the galectin abundance in cells and the activity of JNK, p38 MAPK, and ERK signaling cascades [57].
Galectins are implicated in the regulation of cell proliferation and death [58] while changes in the expression of galectins accompany the differentiation of various cell types [28,59]. The administration of carbohydrate and allosteric galectin inhibitors offers a simple approach to assess the functional significance of upregulated galectins, however this method lacks the ability to identify the role of individual galectin members as they share significant similarity in structure of carbohydrate recognition domains [60]. In addition, many available galectin inhibitors are membrane-impermeable molecules and do not interact with intracellular galectins which are predominant in cells under basal conditions. Despite these limitations, we still were able to observe small but significant effects of carbohydrate galectin inhibitors (lactose, LBA, and TDG) on HL-60 cell proliferation and differentiation, which were either inhibited or stimulated. Our results are consistent with published studies reporting the inhibition of cell proliferation and tumor cell growth by galectin inhibitors, and this strategy is proposed for developing new therapeutics for treatments of cancer and inflammatory diseases [40]. A novel aspect of galectin inhibition in our study implies an important role of oxidative stress-induced galectins in DMSO-induced differentiation of HL-60 cells. Indeed, the ability of galectin inhibitors to facilitate cellular differentiation is a promising feature of drugs used to treat leukemia [61]. Compared to carbohydrate galectin inhibitors, OTX008 - an allosteric galectin 1 inhibitor - inhibits both proliferation and differentiation, measured as ROS production, of HL-60 cells. This drug not only binds galectin-1 but is also known to inhibit galectin-1 expression in cancer cells, target ERK1/2 and Akt-dependent survival pathways, and induce G2/M cell cycle arrest [42]. The multiple effects elicited by OTX008 treatment provides an efficient platform for inhibiting cancer cell proliferation, invasion, and tumor angiogenesis, while concomitantly reducing the ROS-producing potential of HL-60 cells.
The ultimate relationship between galectin-mediated regulation of such opposite cell functions as cell proliferation and cell differentiation remains uncertain as multiple variables may have an influence such as type and amount of galectins, subcellular localization, and expression of galectin-binding receptors on the cell surface. However, the multifactorial correlation analysis of gene expression identified two galectin genes, LGALS3 and LGALS12, in HL-60 cells which demonstrated either a positive or negative uniform correlation with biomarkers of oxidative stress HMOX1 or granulocyte differentiation NCF1 respectively. A positive association of LGALS3 overexpression with cellular differentiation was reported in several cell types including HL-60 cells [14,28], human macrophages [62], and oligodendrocytes [63] while inhibitory effects were reported in osteoblasts [64] and plasma cells [65]. The specific role of LGALS3 may depend on the complex galectin network and certain redundancy of galectins in cells, a factor which is usually overlooked when focusing on individual galectins. A similar situation can be applied to LGALS12, which is a relatively rare and less studied galectin, which either inhibits or stimulates differentiation of hematopoietic cells or adipocytes respectively [49,66]. As such, the results of this study suggest that all galectins function differently in HL-60 cells and oxidative stress-mediated remodeling of galectin networks is a hallmark of DMSO-induced neutrophilic cellular differentiation (Fig. 8). In vitro, HL-60 cells can be also differentiated into two other myeloid lineages including monocyte- and eosinophil-like cells by phorbol 12-myristate 13- acetate and sodium butyrate, respectively [28]. Accordingly, a comparative analysis of stress- sensitive galectins in these three model systems and in additional promyelocytic cell lines such as NB4, K-562, or U-937 will gain a more comprehensive picture of galectin involvement in myeloid differentiation. Further studies using siRNA and CRISPR/Cas9 technologies to knockdown galectin expression are likewise needed to confirm functional differences between these galectins and to delineate signaling systems responsible for this galectin diversity in regulating cellular differentiation. Overall, this knowledge could provide essential insights into developing galectin-specific drugs that modulate galectin networks in cancer cells.

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