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Abstract

One long-term complication of chronic intestinal inflammation is the development of colorectal cancer. However, the mechanisms linking inflammation to the colorectal tumorigenesis are poorly defined. Previously, we have demonstrated that galectin-4 is predominantly expressed in the luminal epithelia of the gastrointestinal tract, and its loss of expression plays a key role in the colorectal tumorigenesis. However, the mechanism by which galectin-4 regulates inflammation-induced tumorigenesis is unclear. Here, we show that galectin-4 secreted by the colorectal cancer cell lines was bound to the cell surface. Neutralization of surface-bound galectin-4 with anti-galectin-4 antibody resulted in increased cell proliferation with concomitant secretion of several chemokines into the extracellular medium. Neutralization of the surface-bound galectin-4 also resulted in the up-regulation of transcription of 29 genes, several of which are components of multiple inflammation signaling pathways. In an alternate experiment, binding of recombinant galectin-4 protein to cell surface of the galectin-4-negative colorectal cancer cells resulted in increased p27, and decreased cyclin D1 and c-Myc levels, leading to cell cycle arrest and apoptosis. Together, these data demonstrated that surface-bound galectin-4 is a dual function protein—down-regulating cell proliferation and chemokine secretion in galectin-4-expressing colorectal cancer cells on one hand and inducing apoptosis in galectin-4-negative colorectal cancer cells on the other hand.

Keywords

Galectins galectin-4 inflammation chemokines cancer colorectal cancer

Epithelium is the innermost layer of the mucosa of the small and large intestines and is in constant exposure to the luminal content of the gastrointestinal tract. Epithelial cells in this layer are terminally differentiated and exhibit rapid, high turnover rate through apoptotic cell death and replacement through maturation of the underlying crypts. The inherent high proliferative rate combined with exposure to toxic and carcinogenic agents in the luminal content makes this tissue prone to inflammation and tumorigenesis. Colorectal tumorigenesis is a multistep process exhibiting distinct histological changes from hyperproliferative stage to invasive, metastatic neoplasia.^1,2 At the molecular level, these transitions are slow, occurring over years with stepwise accumulation of genetic and epigenetic alterations in cells located either in the mucosal epithelial layer or stem cells at the base of immature crypts.^3,4 Studies have established that at least seven independent genetic events that include mutations in APC, K-Ras, and p53 are essential for the transition from normal mucosa to colon carcinoma to occur.^5–7 Undoubtedly, other mechanisms of development would play roles in the colorectal cancer (CRC) onset and progression as well.⁸

Galectins are relatively small soluble proteins localized both intra- and extracellularly and participate in a variety of important cellular functions in normal and cancer cells.^9–11 Although the name implies that galectins are carbohydrate-binding proteins and majority of them are, it is evident that some members are also known to bind to intracellular non-glycoproteins and regulate cellular processes.^12–16 A total of 15 different galectins are known (reviewed by Liu¹⁷), and among these, galectin-4 (gal-4), the protein of interest in this study, is a 323-amino acid (36 kDa) protein with two carbohydrate recognition domains, one on each end of the molecule, and its expression is predominantly restricted to the epithelial cells of the colorectal luminal layer.^18,19 We have previously demonstrated that the intracellular gal-4 interacts with β-catenin resulting in down-regulation of Wnt-signaling pathway–mediated cell proliferation.¹² Others have also made similar observations.²⁰

Gal-4 is also an extracellular protein. Sturm and co-workers²¹ have previously demonstrated that the extracellular gal-4 down-regulates the secretion of cytokine interleukin-17 (IL-17) from activated T-cells in the colonic tissue and induces apoptosis and suggested that the extracellular gal-4 functions as anti-inflammatory molecule. However, the functional role of the extracellular gal-4 on CRC is unknown. In this study, we discovered that surface-bound gal-4 in gal-4-positive CRC cells down-regulates gene transcription of several chemokines and their secretion into the extracellular milieu, and binding of gal-4 protein to gal-4-negative CRC cells leads to the induction of cell apoptosis.

Materials and methods

Reagents

Anti-gal-4 antibody was raised in two rabbits using purified gal-4 protein. Briefly, gal-4 protein carrying 6-histidine tag at its C-terminus was expressed in Sf9 insect cells using previously described procedure²² and purified by Ni-NTA column chromatography in denaturing buffer containing 8-M urea, pH 4.5, as recommended by the supplier (Qiagen, Germantown, MD). Purity was greater than 90% as judged by the SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) gels stained with Coomassie Blue and corroborated with Western blotting using commercially available anti-gal-4 antibody (R&D Biosystems, Minneapolis, MN). Preimmune sera collected from these rabbits were used as control. In addition, anti-RNF2 antibody²² and anti-P-glycoprotein antibody²³ raised in rabbits were also used as controls. Alexa-Fluor-488-coupled secondary antibodies were purchased from Invitrogen (Carlsbad, CA). All the other antibodies used in this study were purchased from Cell Signaling Technologies (Beverly, MA). The sources of other reagents were described previously.²⁴

Cell lines and culture conditions

Human CRC cell lines HT-29, LS-180, and HCT-116 were obtained from the American Type Culture Collection (ATCC, Manassas, MD). Authentication of cell lines was done by the supplier through their DNA-short tandem repeat profiling. Cell lines were cultured in the laboratory according to the supplier’s instructions.

Flow cytometric analysis

Flow cytometry (FACScan Instrument; Becton Dickinson Franklin Lakes, NJ) was carried out using the data acquisition CellQuest software, as reported.²⁵ To establish that gal-4 is cell surface-bound, actively growing LS-180 and HT-29 cells were collected in suspension, incubated with either anti-gal-4 or anti-RNF2 antibody followed by Alexa-Fluor-488-coupled 2° antibody, and then analyzed by flow cytometry, as described previously.²⁶ To analyze the effects of gal-4 protein on cell cycle stages, HCT-116 cells were grown in growth medium spiked with purified gal-4 protein or RNF2 protein (36 µg/mL). Cells were washed in phosphate-buffered saline (PBS) and fixed in 70% ethanol. Cells were washed in PBS and stained with propidium iodide (5 µg/mL) and then analyzed for propidium iodide fluorescence by flow cytometry, as described previously.¹² To analyze apoptosis, cells grown for 24 h in the presence of control antibody, anti-gal-4 antibody, purified gal-4 or RNF2 protein were collected and assayed by flow cytometry using the Annexin V-FITC Apoptosis detection kit (Calbiochem, San Diego, CA) according to the manufacturer’s instructions, as described previously.¹² The percentages of live, early, and late apoptotic and necrotic cells in these analyses were calculated using FlowJo software (FlowJo, Ashland, OR).

Immunofluorescence analysis

Cells growing on glass coverslips were fixed and stained with anti-gal-4 antibody under non-permeabilization conditions to prevent the intracellular gal-4 labeling by following a previously reported general procedure.²⁴ Cells were washed and then stained with Alexa-Fluor-488-coupled secondary antibody, and cells were imaged by Leica confocal laser microscope (TCS SL; Leica MicroSystems, Wetzlar, Germany).

Chemokine antibody arrays

Cytokine/chemokine antibody arrays were developed using the Proteome Profiler Human Cytokine Array Kit Panel A (Cat. No. ARY005; R&D Systems, Minneapolis, MN) as per the supplier’s instructions. Briefly, LS-180 cells were incubated with either affinity-purified anti-gal-4 antibody or anti-RNF2 antibody (control) for 24 h. The growth media were collected and clarified by centrifugation. The growth media were diluted with fresh medium appropriately to normalize for the cell number at the time of growth media collection. The antibody array membranes were incubated with equal volumes of growth media and then developed using chemiluminescence reagents as recommended by the supplier. Cytokines and chemokines in these blots were detected digitally by Bio-Rad Gel Doc system (Bio-Rad Laboratories, Hercules, CA).

Affymetrix analysis

To determine whether neutralization of the surface-bound gal-4 alters gene expression, LS-180 cells were incubated with affinity-purified anti-gal-4 antibody or anti-RNF2 antibody (control; 10 mg/mL) for 24 h, and total RNA from these cells was extracted with Trizol reagent (Invitrogen) and was further processed for Affymetrix analysis at the Center for Functional Genomics, The State University of New York (SUNY), Albany, NY. Briefly, complementary DNA (cDNA) was prepared from 20 ng of purified RNA, fragmented, biotin-labeled using the Encore Biotin module (NuGEN), and hybridized overnight to the GeneChip Human Gene 1.0 ST arrays (Affymetrix, Santa Clara, CA). The arrays were scanned on a GeneChip Scanner 3000 7G and the raw data obtained using Affymetrix Command Console Software. The signals were then quantile normalized in GeneSpring GX11 using PLIER16 algorithm and base line transformed to the median of all controls. The log₂-normalized signal values were then filtered to remove entities that show signal in the bottom 20th percentile across all samples. The microarray data were analyzed by setting the fold-change threshold at >2.0 and significance level of p < 0.01 to identify genes that were differentially expressed between anti-gal-4 antibody versus control antibody treated samples. This analysis was also carried out at the above-mentioned Center for Functional Genomics.

Other methods

Preparation of whole cell lysates, protein estimation, and Western blotting were carried out as described previously.²⁴ Immunoprecipitations were carried out using Universal Magnetic Co-immunoprecipitation kits (Active Motif, Carlsbad, CA).

Results

Surface-bound gal-4 down-regulates cell proliferation

Previous studies have demonstrated that gal-4 expression is high in the epithelial cells of the normal colonic luminal layer and nearly absent in highly proliferative CRC.¹² To study the mechanisms of gal-4 in CRC, we screened several commercially available CRC cell lines including LS-180, HT-29, HCT-116, Caco2, ATRFlox, and SW-620 and identified HT-29, a cell line derived from a grade I human colon adenocarcinoma, and LS-180, from a grade II tumor, as positive, and others as negative, for gal-4 expression.¹² Using select cell lines from this collection, we first determined whether these gal-4-positive CRC cells secrete gal-4 by analyzing the extracellular growth media of the LS-180 and HT-29 cells for the presence of gal-4 protein by immunoprecipitation using anti-gal-4 antibody. The immunoprecipitates were analyzed by Western blotting. Figure 1(a) shows that anti-gal-4 antibody immunoprecipitated the 36-kDa gal-4 protein which was absent in control antibody immunoprecipitations. Identical immunoprecipitation results were obtained with the commercially available anti-gal-4 antibody (data not shown). The amount of gal-4 immunoprecipitated from HT-29 cells was lower than from LS-180 cells. Interestingly, our previous studies have shown that the levels of LGALS4 (gal-4 gene) transcription²⁴ and gal-4 protein expression¹² were lower in HT-29 when compared to that of LS-180 cells, extending support to this observation.

Figure 1.

Galectin-4 is an extracellular protein. (a) Immunoprecipitation of gal-4 in the extracellular growth medium. Growth media (1 mL each) from cell culture flasks with HT-29 and LS-180 cells growing for 24 h were collected and cleared by brief centrifugation. In all, 10 µg of affinity-purified anti-gal-4 or anti-RNF2 (control) antibody was added to these spent media, and immunoprecipitations were carried out as described under the “Materials and methods” section. Immunoprecipitates were analyzed on SDS-PAGE followed by Western blotting using anti-gal-4 antibody as the primary antibody. The 36-kDa protein is the gal-4 protein and 50 kDa is the large fragment of the immunoglobulin. (b) Immunofluorescence analysis of the surface-bound gal-4. LS-180 cells growing on microscope coverslips were first fixed under non-permeabilization conditions and then incubated with anti-gal-4 antibody followed by Alexa-Fluor-488-coupled secondary antibody. Fluorescence (left panel) and bright (middle panel) field images were taken using confocal laser microscope, and these images were superimposed (right panel). (c) Flow cytometric analysis of surface-bound gal-4. LS-180, HT-29, and HCT-116 cells in suspension were incubated with either anti-gal-4 or anti-RNF2 (control) antibody followed by Alexa-Fluor-488-coupled secondary antibody and then analyzed by flow cytometry, as described under the “Materials and methods” section. Peaks of cells alone, cells incubated with control antibody, and cells incubated with anti-gal-4 antibody were indicated.

We then determined whether gal-4 is surface-bound, by immunofluorescence staining of cells growing on microscope glass coverslips with anti-gal-4 antibody followed by Alexa-Fluor-488-coupled secondary antibody, under non-permeabilization conditions so as to prevent immunodetection of intracellular gal-4. Figure 1(b) shows that gal-4 was detectable at the cell surface in LS-180 cells but not in HCT-116 cells. To further establish this observation, cells were analyzed by flow cytometry for the presence of surface-bound gal-4 as described under the “Materials and methods” section. Figure 1(c) shows that LS-180 and HT-29 cells bound to anti-gal-4 antibody formed a distinctly different fluorescence cell peak with increased intensity, when compared to cells alone, whereas HCT-116 cells did not exhibit such a fluorescence cell peak. Importantly, incubation of these cells with control antibody (anti-RNF2) did not yield any new fluorescence cell peak with any of these cell lines. Thus, the fluorescence peak observed in LS-180 and HT-29 cells with anti-gal-4 antibody is due to its tight binding to the cell surface, suggesting the presence of surface-bound gal-4. Together, these data suggested that gal-4 secreted by the gal-4-positive cells is surface-bound.

Neutralization of the surface-bound gal-4 with anti-gal-4 antibody binding

To determine whether anti-gal-4 antibody binding to the surface-bound gal-4 would induce apoptosis, cell proliferation, or increased cell migration, the growth medium of LS-180 cells was spiked with affinity-purified anti-gal-4 antibody or control antibody and the cells were continued to grow for indicated time (Figure 2). Cells growing in the presence of antibodies for 24 h were harvested and analyzed for apoptosis by flow cytometry as described under the “Materials and methods” section, and the live, pre-apoptotic, apoptotic, and necrotic cell populations were calculated. Figure 2(a) shows that neither the anti-gal-4 nor control antibody affected the percentage of live cell population, and there was no indication for apoptosis, suggesting that these antibodies are not toxic to the cells.

Figure 2.

Neutralization of surface-bound gal-4 promotes cell proliferation. (a) Analysis of apoptosis by flow cytometry. LS-180 cells were incubated with either control (anti-RNF2) or anti-gal-4 antibody for 24 h and analyzed for the presence of apoptotic cells by flow cytometry as described under the “Materials and methods” section. The percentages of live, early, and late apoptotic and necrotic cell populations were determined using FlowJo Software. (b) Wound-healing assay. LS-180 cells were plated on six-well plates, and at 80% confluence, a scratch was made with a sterile pipette tip. Control (anti-RNF2) or anti-gal-4 antibody (10 µg/mL) was added to the growth medium. Images were taken immediately (0 h) and at 24 h using a light microscope. Higher magnification (20×) images (far right panel) were shown to indicate cell growth in cells exposed to anti-gal-4 antibody. (c) Growth curves. LS-180 cells growing in six-well plates were incubated with either no antibody (○), control (anti-RNF2) antibody (●), or anti-gal-4 antibody (□; 10 µg/mL) for indicated times. Cells were collected after trypsinization, counted using hemocytometer, and plotted against time.

Cell migration was determined by wound-healing assay as described previously.¹² Figure 2(b) shows that there was no significant difference in cell migration for up to 36 h in the presence of anti-gal-4 antibody when compared to cells exposed to control antibody. Interestingly, a closer examination of cells indicated that cells exposed to anti-gal-4 antibody were spreading, which could be observed within 3 h of exposure to anti-gal-4 antibody. At 24 h after exposure, the empty space between the clusters of cells was tightly occupied by the growing cells (Figure 2(b), right panels). To quantify cell proliferation, cells were collected at regular time intervals by trypsinization and then counted using a hemocytometer. Figure 2(c) shows that exposure to the anti-gal-4 antibody induced rapid cell proliferation with cell doubling time decreasing from ~32 h (control antibody treatment) to ~18 h (anti-gal-4 antibody).

The concept behind the widely used cancer immunotherapy modality is centered on the ability of antibodies binding to the external epitopes of the plasma membrane target receptors, and such binding leads to the impairment of the receptor-mediated signaling pathways. Specifically, binding of antibodies to their cognate receptors at cell surface alters various cellular processes that include cell proliferation, migration, motility, and apoptosis,^27–29 and these manifestations are attributed to the neutralization of the function of the cognate receptors/proteins by the antibody binding.^30–35 Taken together, the above observations show that anti-gal-4 antibody binding to the surface-bound gal-4 leading to increased cell proliferation is due to neutralization of the surface-bound gal-4 function, further suggesting that the surface-bound gal-4 is involved in the regulation of cell proliferation.

Neutralization of the surface-bound gal-4 leads to increased transcription of cytokine and chemokine genes

To determine the mechanism by which the surface-bound gal-4 regulates cell proliferation, LS-180 cells were incubated for 24 h with control or anti-gal-4 antibody and total RNA was extracted from these cells and analyzed by the GeneChip Human Gene 1.0 ST arrays (Affymetrix) to obtain genome-wide expression profiles, as described under the “Materials and methods” section. This analysis has identified 29 genes whose transcription was up-regulated. To establish Entrez gene IDs for these up-regulated genes, the data were analyzed by a web-based gene set analysis toolkit available at www.webgestalt.org. This analysis mapped only 17 genes to Entrez gene IDs, which are listed in Table 1. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was carried out using a tool link available at the above website, to determine whether any of these genes are associated with specific cellular signaling pathways. This analysis showed that at least five genes could be mapped to several inflammation-related signaling pathways that include NOD-like receptor, chemokine, and TNF-alfa/NF-κB signaling pathways (Table 2). Although the microarray data have identified 35 genes whose transcription was down-regulated, the above-mentioned WebGestalt toolkit could not assign any of these genes to any known signaling pathways and thus was not analyzed further. Together, these data suggested that the surface-bound gal-4 down-regulates the transcription of several chemokine/cytokine genes. Since many of these genes are components of several inflammation signaling pathways, these results suggested that surface-bound gal-4 down-regulates inflammation.

Table 1.

Identification of genes up-regulated in the Affymetrix analysis.

GenBank ID	Gene symbol	Gene name
BC055426	BBS12	Bardet–Biedl syndrome 12
AF151978	SLC6A14	Solute carrier family 6 (amino acid transporter), member 14
BC020698	CCL20	Chemokine (C-C motif) ligand 20
BC010952	PI3	Peptidase inhibitor 3, skin-derived
BC040438	C1QTNF9	C1q and tumor necrosis factor–related protein 9
BC017017	TRIM31	Tripartite motif–containing 31
AY971607	ZNF674	Zinc finger protein 674
J04440	SEMG1	Semenogelin I
BC015753	CXCL2	Chemokine (C-X-C motif) ligand 2
BC101489	OR6A2	Olfactory receptor, family 6, subfamily A, member 2
BC012303	PDZK1IP1	PDZK1 interacting protein 1
AF070674	BIRC3	Baculoviral IAP repeat–containing 3
M59465	TNFAIP3	Tumor necrosis factor, alpha–induced protein 3
AF110384	CCL28	Chemokine (C-C motif) ligand 28
BC034535	KRT6B	Keratin 6B
AK293089	CD209	CD209 molecule
AK300310	ELL2	Elongation factor, RNA polymerase II, 2

The GenBank IDs of the up-regulated transcripts in the Affymetrix analysis were analyzed using WebGestalt toolkit, which mapped only 17 transcripts to Entrez genes IDs (gene symbols). And their corresponding gene names are indicated.

Table 2.

Identification of the enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways.

Entrez genes	Pathway name
CXCL2; TNF-alpha-induced protein 3; CCL20	NOD-like receptor signaling pathway
CCL28; CXCL2; CCL20	Chemokine signaling pathway
CCL28; CXCL2; CCL20	Cytokine–cytokine receptor interaction
CCL20; TNF-alpha-induced protein 3	CD40/CD40L signaling
CCL20; TNF-alpha-induced protein 3	p75(NTR)-mediated signaling
CCL20; TNF-alpha-induced protein 3	FAS (CD95) signaling pathway
CCL20; TNF-alpha-induced protein 3	TNF alpha/NF-κB signaling pathway
CCL28; CCL20; OR6A2	Signaling by GPCR

The mapped Entrez genes presented in Table 1 were subjected to KEGG pathway analysis. The number of Entrez IDs in the user data set for the pathway and the corresponding Entrez IDs for the enriched pathway are shown.

Surface-bound gal-4 inhibits cytokine/chemokine secretion

Since cytokines/chemokines are secretory proteins, we predicted that the newly transcribed chemokine gene products in response to neutralization of the surface-bound gal-4 would be secreted and detectable in the growth medium. To determine this possibility, LS-180 cells were incubated with either anti-gal-4 or anti-RNF2 (control) antibody for 24 h and the growth media were collected, centrifuged, and then incubated with antibody array membranes as described in the “Materials and methods” section. These antibody arrays are capable of detecting 36 different cytokines/chemokines. Figure 3(a) shows that the extracellular medium obtained from untreated cells contained CXCL1, MIF, IL-6, and IL-23. Secretion of these cytokines was essentially unaffected when cells were incubated with control antibody (middle panel) or with anti-gal-4 antibody (lower panel). One additional cytokine, IL-2, was detectable when cells were incubated with control antibody (Figure 3(b)), which was also detectable in the extracellular medium of cells incubated with anti-gal-4 antibody (Figure 3(c)), suggesting that IL-2 secretion is not specific to antibody. Importantly, extracellular medium obtained from cells incubated with anti-gal-4 antibody contained CCL2, CCL5, CD-54 and CSF-2, and CXCL10 (Figure 3(c)). These data suggested that neutralization of the surface-bound gal-4 function results in the secretion of these select chemokines. Surprisingly, gene array analysis described above did not identify transcriptional alterations in genes corresponding to these secreted chemokines. In addition, our real-time quantitative polymerase chain reaction analysis on secreted chemokine genes was ambiguous as no meaningful information could be derived as the Ct (cycle threshold) values for these genes were >30 (data not shown). Together, these gene analyses suggested that the transcripts of the secreted chemokines are in low abundance.

Figure 3.

Neutralization of surface-bound gal-4 leads to increased secretion of specific chemokines. LS-180 cells were incubated with control (anti-RNF2) antibody or anti-gal-4 antibody (10 µg/mL) for 24 h, and the growth media were collected, clarified by centrifugation, and then subjected to Human Chemokine Array analysis, as described under the “Materials and methods” section. Panels (a), (b), and (c) were obtained from using growth media from cells, cells incubated with control antibody, and cells incubated with anti-gal-4 antibody, respectively. Specific chemokines that are uniquely present were boxed and identified. The internal positive and negative controls of the arrays were also marked with boxes and arrows.

Recombinant gal-4 induces cell cycle arrest and apoptosis

While the above data suggested that neutralization of the surface-bound gal-4 promotes cell proliferation and increased expression and secretion of specific cytokines and chemokines, we investigated whether conversely cell surface binding of gal-4 would arrest cell proliferation. To address this question, 6-His-tagged human gal-4 and RNF2 (control) proteins were expressed in Sf9 insect cells and purified to near homogeneity by Ni-NTA column chromatography. Growth medium of gal-4-negative HCT-116 cells was spiked with recombinant gal-4 or RNF2 (36 µg/mL), and the cells were allowed to grow for 24 h. Cell surface binding of these proteins to cells was tested by flow cytometry, which indicated that both of these proteins were adsorbed to the cell surface as indicated by the appearance of a new, but weak fluorescence cell peak (data not shown). Visual observation of cells exposed to gal-4 protein suggested that these cells were loosely attached to the cell culture plates, perhaps due to cell death. To determine this possibility, cells incubated with gal-4 or RNF2 (control) protein were subjected to cell cycle analysis by flow cytometry. Figure 4(a) shows that exposure to gal-4 protein has significantly increased the S-phase cell population. To determine whether gal-4 protein promotes apoptosis, cells incubated with these proteins for 24 h were analyzed for diploid DNA content and for cell surface exposure of phosphatidylserine, by flow cytometry. Figure 4(b) shows that exposure of gal-4-negative HCT-116 cells to gal-4 protein greatly decreased live cell population with concomitant increase in pre-apoptotic, apoptotic, and necrotic cell populations. To determine the molecular changes in the intracellular proteins, cells exposed to RNF2 and gal-4 proteins for 24 h were analyzed by Western blotting. Figure 4(c) shows that cells exposed to recombinant gal-4 contained decreased levels of cyclin D1 and c-Myc and increased level of p27. These molecular changes are known to occur in cells arrested in G1/S and S phases, corroborating the above cell cycle analysis data. Together, these data suggested that gal-4 is an anti-proliferative protein, promoting cell cycle arrest and apoptosis in the select gal-4-negative CRC cells.

Figure 4.

Recombinant gal-4 binding to cell surface induces cell cycle arrest and apoptosis. (a) Cell cycle analysis. Gal-4-negative HCT-116 cells were incubated with either 6-His-RNF2 (left panel) or 6-His gal-4 (right panel; 36 µg/mL each) protein and grown for 24 h. Cells were collected and stained with propidium iodide followed by cell cycle analysis. FlowJo software was used to identify percent cell population in G1, S, and M phases of the cell cycle. (b) Apoptotic assay by flow cytometry. Cells similarly treated were analyzed for apoptosis by the standard Annexin V assay followed by flow cytometry, as described under the “Materials and methods” section. (c) Western blotting analysis. Cells similarly exposed to control (RNF2) and gal-4 protein were lysed and analyzed for cell cycle regulatory proteins by Western blotting.

Discussion

CRC is a heterogeneous disease, differing anatomically, histologically, and cellularly as well as in the mechanisms of its onset. While it is generally recognized that there is an intimate link between inflammation and CRC, the initial molecular events of inflammation leading to CRC are unclear. Chronic inflammation in the mucosal layer resulting in ulcerative colitis, and inflammation in all layers of the gut resulting in Crohn’s disease are associated with an increased risk of developing CRC. Inflammation in these examples promotes strong activation of diverse immunocytes including T lymphocytes, macrophages, and mast cells, all of which are known to produce a wide variety of inflammatory mediators that eventually initiate premalignant lesion. While these inflammation-associated CRCs account for ~15% of all CRC, it is the sporadic CRC originating from premalignant adenomas, which accounts for the remaining cases of CRC. The sporadic CRC is well established to follow a pattern of the normal-adenoma-carcinoma sequence, transitioning through multistep carcinogenic processes in which genetic and epigenetic alterations accumulate in a sequential manner. Multiple studies have identified mutations in the well-characterized APC, K-Ras, p53, β-catenin, axin, EGFR, as well as numerous uncharacterized genes in predisposing the colonic epithelia to CRC. These CRC-associated sequential genetic changes are also viewed to promote inflammatory response, allowing the infiltration of immunocytes into the developing sporadic tumor microenvironment, which produce proinflammatory mediators to augment the progression of adenoma–carcinoma transition. However, the stages of genetic changes at which proinflammatory mediators appear in CRC are unclear.

Although gal-4 is expressed highly in the terminally differentiated epithelial cells of the mucosal layer of the gastrointestinal tract,^18,19 and at a reduced level in CRC,^36–38 the functional role of secreted gal-4 by the early-stage CRC is unclear. For example, Hokama et al.³⁹ have previously shown that the secreted gal-4 binds to intestinal CD4⁺ T cells and such binding leads to their activation and secretion of IL-6, contributing to the exacerbation of intestinal inflammation. In contrast, Sturm and co-workers²¹ have subsequently demonstrated that gal-4 secreted by CRC cells down-regulates the secretion of cytokine IL-17 from the activated T-cells, which in turn induced apoptosis in these cells, suggesting that the extracellular gal-4 down-regulates inflammation. These seemingly contrasting observations could be stemming from variations in the experimental protocols, and further studies are needed to substantiate these observations.

The functional role of gal-4 in inflammation response is beginning to emerge. Kim et al.⁴⁰ have recently demonstrated that stable silencing of gal-4 expression results in the increased expression of IL-6 in HT-29 cells. In this study, we utilized a technical strategy analogous to monoclonal antibody–based therapy, commonly known as immunotherapy, of cancers in clinic to address this question. The selection of LS-180 and HT-29 CRC cell lines for antibody targeting is appropriate to determine whether the antibody binding–induced neutralization would lead to the activation or inhibition of cellular events, as these cells secrete gal-4 which is surface-bound and is readily accessible to the antibody binding without the need for cell permeabilization. Although the anti-gal-4 antibody used in this study was polyclonal in nature, it could recognize a single 36-kDa protein only in the gal-4-positive cells which could be competed out with excess purified gal-4 protein, similar to that of the commercially available anti-gal-4 antibody (data not shown). The observed effect of increased cell proliferation upon exposure to anti-gal-4 antibody was our first evidence that the normal function of the surface-bound gal-4 is cell growth inhibitory. Further evidence that anti-gal-4 antibody binding to the cell surface promotes increased transcription of genes associated with inflammatory pathways, and secretion of chemokines into the extracellular environment, together suggested that these events are associated with neutralization of the surface-bound gal-4 function. It is tempting to extend these observations to speculate that chemokines thus secreted would function as autocrines promoting cell proliferation in these stripped cells as observed in Figure 2(c). It is tempting to speculate that CRC cells shed the gal-4 expression so as to produce chemokines that promote their own cell proliferation.

The nuclear factor (NF)-kappaB (NF-κB) is an important transcriptional factor in the body homeostasis including the maintenance of innate immunity and controlling inflammation,^41,42 and its aberrant regulation is recognized in many human cancers.⁴³ Several studies have demonstrated an active NF-κB signaling pathway in CRC and linked the dysregulated NF-κB signaling pathway in colorectal tissue to the malignant transformation, tumor cell proliferation, anti-apoptosis, invasion/metastasis, angiogenesis, and therapeutic resistance.^41,44–46 Evidence indicates that the NF-κB signaling pathway mediates these cellular processes through activation of its target genes comprising numerous growth factors, chemokines and cytokines, c-Myc, cyclin D1, cIAPs, and Bcl-2 family of proteins. Interestingly, the evidence presented by Kim et al.⁴⁰ that gal-4 regulates NF-κB signaling and the expression of IL-6 in CRC cells lends strong support to our observations presented here that surface-bound gal-4 regulates NF-κB signaling pathway in CRC cells. Since the NF-κB signaling is mediated through chemokine/growth factor receptors, it is equally possible that these receptors could serve as docking sites for gal-4 binding. Further studies aimed at characterizing the gal-4 receptor should enable us to better define the mechanism of gal-4 in CRC.

The CRC progression is uniquely a very slow process and takes tens of years for developing into a full-blown metastatic carcinoma. Our observations that surface binding of gal-4 protein to gal-4-negative CRC cells leads to cell apoptosis may be analogous to gal-4 secreted by surrounding normal colonic epithelia and its subsequent binding to the growing CRC would be expected to decrease tumor burden through apoptotic death of proliferating cells, until a more resistant cancer cell population develops. Taken together, our observations suggest that the extracellular gal-4 exhibits dual function, preventing the production of chemokines from the cancer cells that promote cell proliferation and also eliminating the proliferating CRC cells through induction of apoptosis. Further studies aimed at establishing these in vitro cell line model studies in animal models should enable us to better define the mechanism of secreted gal-4 in the CRC onset and progression.

Footnotes

Acknowledgements

A part of the initial studies of this investigation was carried out by the authors at the Department of Biomedical Sciences, School of Pharmacy, Texas Tech University of Health Sciences, Amarillo, TX 79106, USA.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors wish to thank the Appalachian College of Pharmacy for the financial support.

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Surface-bound galectin-4 regulates gene transcription and secretion of chemokines in human colorectal cancer cell lines

Abstract

Keywords

Materials and methods

Reagents

Cell lines and culture conditions

Flow cytometric analysis

Immunofluorescence analysis

Chemokine antibody arrays

Affymetrix analysis

Other methods

Results

Surface-bound gal-4 down-regulates cell proliferation

Neutralization of the surface-bound gal-4 with anti-gal-4 antibody binding

Neutralization of the surface-bound gal-4 leads to increased transcription of cytokine and chemokine genes

Surface-bound gal-4 inhibits cytokine/chemokine secretion

Recombinant gal-4 induces cell cycle arrest and apoptosis

Discussion

Footnotes

Acknowledgements

Declaration of conflicting interests

Funding

References