Galectin-1 Modulates the Survival and Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) Sensitivity in Human Hepatocellular Carcinoma Cells

Abstract

Galectin-1 is a member of carbohydrate-binding proteins and plays critical roles in tumor growth and progression. It has been reported that galectin-1 is upregulated in human hepatocellular carcinoma (HCC) and facilitates HCC cell migration and invasion. In this study, the authors aimed to explore the effects of the knockdown of galectin-1 on HCC cell survival and sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Human HCC cells were transfected with galectin-1-targeting small interfering RNA (siRNA) with or without 100 ng/mL TRAIL treatment and tested for apoptosis and gene expression changes. Cotransfection of Bcl-2- and survivin-expressing plasmids with galectin-1 siRNA was done, before TRAIL exposure, cell viability, and apoptosis were assessed. The authors found that siRNA-mediated downregulation of galectin-1 caused apoptosis in HCC cells, which was coupled with reduced Bcl-2 and survivin and increased Bax expression. Overexpression of Bcl-2 and survivin significantly blocked galectin-1 silencing-induced apoptosis of HCC cells. Knockdown of galectin-1 significantly enhanced TRAIL cytotoxicity against HCC cells, as determined by the MTT assay. Moreover, galectin-1 downregulation significantly induced apoptosis in TRAIL-treated HCC cells. Such effects were almost completely counteracted by the enforced expression of Bcl-2 and survivin. Taken together, these data first show that galectin-1 downregulation induces apoptosis in and augments TRAIL cytotoxicity to HCC cells largely through regulation of Bcl-2 and survivin expression. These findings provide a rationale for preclinical and clinical evaluation of targeting galectin-1 for improving TRAIL-based therapy against HCC.

Introduction

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related mortality worldwide, with a 5-year survival rate of 5%–6%.^1,2 The poor prognosis of HCC is mainly due to the lack of effective treatment options. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family of cytokines. TRAIL has become an attractive anticancer agent, because it can selectively induce apoptosis in cancer cells with minimal toxicity to normal cells.^3,4 TRAIL exerts its proapoptotic activity through the binding to death receptors, DR4 and DR5, expressed on the surface of target cells.⁵ When TRAIL binds to DR4 and/or DR5, the death receptors become trimerized and then recruit the adaptor protein, Fas-associated protein with death domain, and procaspase-8 or procaspase-10, leading to activation of caspase-8 or -10. Once activated, caspase-8 and -10 can activate the effector caspase-3, -6, and -7 to trigger apoptotic death.⁶ HCC tissues have shown abundant expression of TRAIL, whereas adjacent nontumorous tissues have little expression of this cytokine.⁷ Compelling evidence indicates strong resistance to TRAIL cytotoxicity in HCC cells.^5,6 Overexpression of antiapoptotic proteins and loss of proapoptotic proteins are important mechanisms leading to a poor apoptotic response to TRAIL.⁸ The Bcl-2 family, consisting of antiapoptotic (e.g., Bcl-2 and Mcl-1) and proapoptotic (e.g., Bax and Bim) members, is widely involved in the regulation of apoptosis.⁹ Deregulation of the Bcl-2 family proteins has been shown to modulate TRAIL-induced apoptosis in HCC cells.¹⁰ Many attempts have been made to enhance TRAIL's therapeutic efficacy against cancer.⁵

Galectin-1, a member of carbohydrate-binding proteins, is expressed by many types of cancers, such as nonsmall-cell lung cancer¹¹ and ovarian cancer.¹² Galectin-1 is involved in cancer progression and affects multiple aspects of tumor biology, including cell proliferation, adhesion, migration, and invasion.^12,13 Overexpression of galectin-1 has been detected in HCC and it contributes to tumor growth in vivo.¹⁴ Another study reported that galectin-1 overexpression promotes HCC cell migration and invasion.¹⁵ However, the role of galectin-1 in HCC cell survival and TRAIL resistance remains unclear.

In the present study, the authors employed a small interfering RNA (siRNA) to knock down galectin-1 in HCC cells and investigated its effects on cell apoptosis and sensitivity to TRAIL.

Materials and Methods

Cell culture and treatment

Human HCC cells (SK-Hep1 and Huh7) were purchased from the Shanghai Institute for Biological Science (Shanghai, China) and maintained in a high-glucose Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin (Invitrogen, Carlsbad, CA). For assessment of TRAIL cytotoxicity, cells were exposed to 100 ng/mL human recombinant TRAIL (Millipore, Billerica, MA) for 24 h, and cell viability and apoptosis were examined. This TRAIL concentration was chosen because it has been reported¹⁶ that TRAIL at 100 ng/mL is cytotoxic to HepG2 HCC cells, but not normal hepatocytes.

Plasmids and siRNA

The human Bcl-2- and survivin-expressing plasmids in pcDNA3.1(+) were constructed as described previously.^17,18 In brief, full-length human Bcl-2 and survivin cDNA were amplified by polymerase chain reaction (PCR) using a human placenta cDNA library (Invitrogen) as a template. The PCR products were cloned into the pcDNA3.1(+) vector (Invitrogen) following the manufacturer's protocol. All constructs were sequenced to ensure accuracy. The green fluorescent protein (GFP)-expressing plasmid was purchased from Invitrogen. Specific siRNA-targeting human galectin-1 mRNA and negative control siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Cell transfection

SK-Hep1 and Huh7 cells were seeded at a density of 8 × 10⁵ cells per well into six-well plates and incubated overnight before transfection. For knockdown of galectin-1, galectin-1 siRNA and nonspecific siRNA, at a final concentration of 50 nM, were incubated with Lipofectamine 2000 (Invitrogen) at room temperature for 30 min and separately added to the cell culture. At 48 h after transfection, cells were collected and tested for gene expression and apoptosis. To study the role of Bcl-2 and survivin in galectin-1-mediated apoptosis, cells were cotransfected with galectin-1 siRNA (50 nM), pcDNA3.1-Bcl-2 (0.5 μg), and pcDNA3.1-survivin (0.5 μg) and incubated for 48 h before gene expression and apoptosis analysis. To explore the effect of galectin-1 on TRAIL sensitivity, cells were transfected with galectin-1 siRNA alone or together with Bcl-2- and survivin-expressing plasmids, 48 h before exposure to 100 ng/mL TRAIL. Transfection efficiency (>70%) was determined by cotransfection of a GFP-expressing plasmid and analysis of the percentage of GFP-positive cells by fluorescence microscopy.

Quantitative real-time PCR analysis

Total RNA was extracted from cells using a TRIzol reagent (Invitrogen). Reverse transcription was carried out using SuperScript II reverse transcriptase and random primers (Invitrogen). Real-time PCR was performed using FastStart DNA Master SYBR Green I reagents and with the LightCycler 480 Real-Time PCR System (Roche Diagnostic, Meylan, France). Primers sequences were as follows: galectin-1 forward 5′-CAAACCTGGAGAGTGCCTTC-3′, galectin-1 reverse 5′-GATGCACACCTCTGCAACAC-3′ ¹⁹, β-actin forward 5′-CCCTGAGGCACTCTTCCAG-3′, β-actin reverse 5′-ACTTGCGCTCAGGAGGAGC-3′. Cycling conditions were as follows: an initial denaturation at 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Each reaction was done in triplicate. The relative amount of galectin-1 mRNA was calculated after normalization against β-actin mRNA levels using the comparative threshold cycle method.²⁰

MTT assay

Cells were plated at a density of 3 × 10³ cells per well in 96-well plates and transfected with galectin-1 siRNA or control siRNA before TRAIL treatment. Afterward, cells were incubated with 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide (MTT) at a final concentration of 0.5 mg/mL at 37°C for 4 h. Dimethyl sulfoxide was added to dissolve the formazan product, and the absorbance was measured at 570 nm.

Apoptosis detection by flow cytometry

Cell apoptosis was detected using a commercially available AnnexinV-fluorescein isothiocyanate (FITC) Apoptosis Detection Kit (BioVision, Mountain View, CA), according to the manufacturer's instructions. In brief, cells were seeded at a density of 8 × 10⁴ cells per well into 12-well plates and transfected with indicated plasmids or siRNAs. At 48 h after transfection, cells were trypsinized, washed with an ice-cold phosphate-buffered saline, and suspended in a 1× binding buffer. The cell suspension was added with 5 μL of AnnexinV-FITC and propidium iodide (PI) and incubated for 10 min at room temperature in the dark. Cells were analyzed using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA). At least 10,000 cells were analyzed per measurement.

Western blot analysis

Cells were transfected with indicated plasmids or siRNAs and 48 h later, lysed for 30 min in an ice-cold lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM EDTA, 1% TritonX-100, and 0.5% sodium dodecyl sulfate [SDS]) containing a protease inhibitor mixture (Roche Diagnostics, Mannheim, Germany). The protein concentrations in the cell lysates were measured using the Protein Quantification Kit (Bio-Rad Laboratories, Hercules, CA). Equal amounts of protein were resolved on SDS–polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. After blocking, the membranes were probed with the following primary antibodies: mouse or rabbit anti-galectin-1, anti-Bcl-2, anti-Bax, anti-survivin, anti-Mcl-1, anti-DR4, anti-DR5, and anti-β-actin, which were purchased from Santa Cruz Biotechnology. After washing, membranes were then incubated with appropriate secondary antibodies for 1 h. Immunoreactive bands were visualized with the enhanced chemiluminescent detection system (Amersham Biosciences, Piscataway, NJ). Densitometry was performed using the Quantity One software (Bio-Rad Laboratories).

Statistical analysis

Data are expressed as means ± standard deviations. The difference among the means of multiple groups was analyzed by the one-way analysis of variance followed by the Tukey test. p-Values < 0.05 were considered significant.

Results

siRNA-mediated downregulation of galectin-1 causes apoptosis in human HCC cells

The authors first checked the effect of galectin-1 downregulation on apoptosis of HCC cells. Real-time PCR and Western blot analysis revealed that the mRNA (Fig. 1A) and protein (Fig. 1B) levels of galectin-1 in SK-Hep1 and Huh7 cells were significantly (p < 0.05) reduced by transfection of galectin-1 siRNA, compared to nontransfected cells. Apoptosis analysis by Annexin-V/PI staining demonstrated that downregulation of galectin-1 significantly induced apoptosis in HCC cells. More specifically, apoptosis increased from 2.7 ± 0.8% to 18.5 ± 1.9% in SK-Hep1 cells and from 3.8 ± 0.6% to 20.2 ± 2.6% in Huh7 cells (Fig. 1C).

FIG. 1.

Knockdown of galectin-1 induces apoptosis in hepatocellular carcinoma (HCC) cells. (A) Real-time polymerase chain reaction and (B) Western blot analysis of galectin-1 mRNA and protein levels, respectively, in nontransfected HCC cells (control) and those transfected with control small interfering RNA (c-siRNA) or galectin-1 siRNA (g-siRNA). Bar graphs represent normalized data from three independent experiments. *p < 0.05 versus control. (C) Apoptosis analysis by Annexin-V/propidium iodide (PI) staining. At 48 h after transfection, cells were subjected to apoptosis detection using flow cytometry. Representative dot plots (top) show the percentages of Annexin-V+/PI- apoptotic cells. Bar graphs represent the mean ± standard deviation (SD) from three independent experiments. *p < 0.05 versus control.

Effects of galectin-1 silencing on apoptosis regulators in human HCC cells

Having identified the requirement of galectin-1 for the survival of HCC cells, the authors next sought to determine the apoptosis regulators involved in galectin-1 downregulation-mediated cell death. Western blot analysis showed that SK-Hep1 and Huh7 cells with galectin-1 downregulation had a marked decline in the expression of Bcl-2 and survivin and elevation in the expression of Bax, compared to nontransfected cells (Fig. 2). However, galectin-1 silencing had no impact on the expression of Mcl-1 and TRAIL receptors (DR4 and DR5).

FIG. 2.

Western blot analysis of indicated proteins in SK-Hep1 and Huh7 cells transfected with control siRNA (c-siRNA) or galectin-1 siRNA (g-siRNA). Representative blots from three independent experiments are shown.

Overexpression of Bcl-2 and survivin counteracts galectin-1 downregulation-induced apoptosis

Next, the authors explored the possibility if enforced expression of Bcl-2 and survivin prevents HCC cells from galectin-1 depletion-induced apoptosis. As shown in Figure 3A, cotransfection with Bcl-2- and survivin-expressing plasmids restored the expression of Bcl-2 and survivin in galectin-1 siRNA-transfected cells. Restoration of Bcl-2 or survivin alone slightly reduced the apoptosis of HCC cells induced by galectin-1 silencing (Fig. 3B). Most interestingly, overexpression of both Bcl-2 and survivin almost completely blocked galectin-1 downregulation-induced apoptosis.

FIG. 3.

Overexpression of Bcl-2 and survivin counteracts galectin-1 downregulation-induced apoptosis. (A) Western blot analysis of Bcl-2 and survivin proteins in HCC cells transfected with galectin-1 siRNA (g-siRNA) alone or together with Bcl-2- or survivin-expressing plasmids. Representative blots from three independent experiments are shown. (B) Annexin-V/PI staining analysis of apoptosis in HCC cells transfected with indicated constructs. Bar graphs represent the mean ± SD from three independent experiments. *p < 0.05 versus control (nontransfected cells); ^# p < 0.05 versus cells transfected with galectin-1 siRNA alone.

Downregulation of galectin-1 enhances TRAIL cytotoxicity against HCC cells

Next, the authors examined the effect of galectin-1 downregulation on the susceptibility of HCC cells to TRAIL. The MTT assay demonstrated that exposure to 100 ng/mL TRAIL for 24 h caused 11%–14% reduction in the viability of SK-Hep1 and Huh7 cells, compared to untreated cells (Fig. 4A). Galectin-1 silencing exerted a marginal, but not significant, effect on the viability of HCC cells studied (p > 0.05 vs. nontransfected cells; Fig. 4A). Notably, the delivery of galectin-1 siRNA, but not control siRNA, significantly (p < 0.05) augmented TRAIL cytotoxicity against HCC cells. Moreover, apoptosis analysis indicated that silencing of galectin-1 potently enhanced the apoptosis of SK-Hep1 and Huh7 cells in the presence of TRAIL (Fig. 4B).

FIG. 4.

Downregulation of galectin-1 enhances tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) cytotoxicity against HCC cells. Cells were transfected with control siRNA (c-siRNA) or galectin-1 siRNA (g-siRNA) and incubated for 48 h before exposure to 100 ng/mL TRAIL for additional 24 h. (A) The cell viability was assessed using the MTT assay. (B) Apoptosis was measured using Annexin-V/PI staining analysis. Bar graphs represent the mean ± SD from three independent experiments. *p < 0.05 versus control (nontransfected cells); ^# p < 0.05 versus cells treated with TRAIL alone.

Galectin-1 silencing-induced TRAIL sensitization involves concomitant downregulation of Bcl-2 and survivin

Finally, the authors checked whether galectin-1 silencing-induced TRAIL sensitization was mediated through alteration of Bcl-2 and survivin proteins. Western blot analysis confirmed that galectin-1 knockdown resulted in a marked reduction in the protein levels of Bcl-2 and survivin in the presence of 100 ng/mL TRAIL (Fig. 5A). Overexpression of Bcl-2 or survivin partially prevented galectin-1-silenced HCC cells from apoptosis in the presence of TRAIL (Fig. 5B). Notably, enforced expression of both Bcl-2 and survivin almost completely abrogated the apoptosis of galectin-1-silenced, TRAIL-treated HCC cells.

FIG. 5.

Overexpression of Bcl-2 and survivin blocks the apoptosis of galectin-1-silenced, TRAIL-treated HCC cells. (A) Western blot analysis of Bcl-2 and survivin proteins in HCC cells with indicated treatments. Representative blots of three independent experiments are shown. (B) Apoptosis detection by Annexin-V/PI staining. Cells were transfected with galectin-1 siRNA (g-siRNA) alone or together with Bcl-2- and/or survivin-expressing plasmids and then exposed to 100 ng/mL TRAIL. The percentage of apoptotic cells was determined by flow cytometry. Bar graphs represent the mean ± SD from three independent experiments. *p < 0.05 versus control (nontransfected cells); ^# p < 0.05 versus cells transfected with galectin-1 siRNA alone before exposure to TRAIL.

Discussion

Galectin-1 is implicated in multiple tumor progression steps, including cancer cell proliferation, adhesion, apoptosis, invasion, and tumor angiogenesis.²¹ It has been documented that OTX008, a selective small-molecule inhibitor of galectin-1, can inhibit the proliferation of a panel of cancer cells.²¹ Small-hairpin RNA-mediated knockdown of galectin-1 has been shown to suppress the growth and invasion of osteosarcoma cells.²² The involvement of galectin-1 in HCC growth and progression has been described in several previous studies.^14,15,23 It has been reported that galectin-1 overexpression promotes HCC cell migration and invasion.²³ Consistently, these data confirmed the importance of galectin-1 in HCC cell survival. The authors found that targeted reduction of galectin-1 caused significant apoptosis in SK-Hep1 and Huh7 cells. Despite these favorable effects on tumor progression, galectin-1 may induce negative effects on some types of cancer cells. For instance, restoration of galectin-1 expression in human colorectal cancer cells induces apoptotic death.²⁴ Similarly, galectin-1 can trigger apoptosis in prostate cancer²⁵ and breast cancer²⁶ cells. These findings suggest that the biological roles of galectin-1 are cellular context dependent.

In HCC cells, galectin-1 overexpression has been found to modulate multiple molecular pathways involved in tumor progression, including the mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3-K)/Akt, and Wnt/β-catenin pathways.^14,15 These signaling pathways are usually involved in HCC cell survival through alteration of apoptosis regulators.^27,28 It has been documented that galectin-1 overexpression promotes tumorigenesis and cisplatin resistance in epithelial ovarian cancer through activation of ERK MAPK signaling and upregulation of Bcl-2.¹² These data further supported the regulation of the Bcl-2 family proteins by galectin-1. The authors found that galectin-1 downregulation resulted in a sharp decline in the Bcl-2 and survivin protein and elevation in the Bax protein. Bax translocalizes to the mitochondrial outer membrane, in response to proapoptotic stress signals, consequently inducing cytochrome c release from the mitochondria and activating the caspase signal pathway.²⁹ The antiapoptotic protein such as Bcl-2 can counteract BAX-mediated cell death. These data showed that the enforced expression of Bcl-2 and survivin almost completely blocked the induction of apoptosis by galectin-1 silencing. These data collectively indicate that suppression of Bcl-2 and survivin expression largely accounts for the proapoptotic effect of galectin-1 silencing in HCC cells. However, the signaling pathway(s) involved in galectin-1-mediated regulation of Bcl-2 and survivin remains to be further defined.

Alteration of apoptosis regulators, especially the Bcl-2 family, has been linked to affect the susceptibility of cancer cells to TRAIL.³⁰ Wang et al.³¹ reported that ABT-263, a potent inhibitor of the Bcl-2 family, has shown the ability to sensitize HCC cells to TRAIL-induced apoptosis. Chen et al.³² demonstrated that silencing of cyclooxygenase-2 can enhance the sensitivity of HCC cells to TRAIL through inhibition of the antiapoptotic proteins, Bcl-2 and Bcl-w. Given the regulation of apoptosis regulators by galectin-1, the authors next checked the effect of galectin-1 silencing on TRAIL sensitivity of HCC cells. The data revealed that depletion of galectin-1 significantly augmented the apoptosis of TRAIL-treated HCC cells. However, galectin-1 silencing had no impact on the expression of DR4 and DR5, suggesting that the action of galectin-1 may be linked to alteration of the apoptotic pathways downstream, DR4 and DR5. Most interestingly, overexpression of Bcl-2 and survivin significantly blocked the apoptosis of galectin-1-silenced, TRAIL-treated HCC cells.

Taken together, these data suggest that galectin-1 is implicated in the modulation of TRAIL sensitivity of HCC cells through alteration of Bcl-2 and survivin.

To the best of knowledge, this is the first report showing the importance of galectin-1 in the regulation of TRAIL sensitivity of HCC cells. The authors show that targeted reduction of galectin-1 induces apoptosis and enhances TRAIL cytotoxicity in HCC cells, which is largely mediated through downregulation of Bcl-2 and survivin. Therefore, galectin-1 may represent a promising target for improving the efficacy of TRAIL-based therapy against HCC.

Footnotes

Disclosure Statement

No competing financial interests exist.

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