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Keywords:

  • hepatocellular carcinoma;
  • interleukin 1β;
  • matrix metalloproteinases;
  • melatonin;
  • nuclear factor kappa B

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Hepatocellular carcinoma (HCC) is one of the most lethal human cancers worldwide because of its high incidence and its metastatic potential. Extracellular matrix degradation by matrix metalloproteinases (MMPs) has been connected with cancer cell invasion, and it has been suggested that inhibition of MMPs by synthetic and natural inhibitors may be of great importance in the HCC therapies. Melatonin, the main product of the pineal gland, exerts antiproliferative, proapoptotic, and antiangiogenic properties in HepG2 human hepatocellular cells, and exhibits anti-invasive and antimetastatic activities by suppressing the enzymatic activity of MMP-9 in different tumor types. However, the underlying mechanism of anti-invasive activity in HCC models has not been fully elucidated. Here, we demonstrate that 1 mm melatonin dosage reduced in IL-1β-induced HepG2 cells MMP-9 gelatinase activity and inhibited cell invasion and motility through downregulation of MMP-9 gene expression and upregulation of the MMP-9-specific inhibitor tissue inhibitor of metalloproteinases (TIMP)-1. No significant changes were observed in the expression and activity of MMP-2, the other proteinase implicated in matrix collagen degradation, and its tissue inhibitor, TIMP-2. Also, melatonin significantly suppressed IL-1β-induced nuclear factor-kappaB (NF-κB) translocation and transcriptional activity. In summary, we demonstrate that melatonin modulates motility and invasiveness of HepG2 cell in vitro through a molecular mechanism that involves TIMP-1 upregulation and attenuation of MMP-9 expression and activity via NF-κB signal pathway inhibition.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Over half a million new cases of hepatocellular carcinoma (HCC) are diagnosed each year, which causes this primary liver tumor to be the third cause of cancer-related death worldwide [1, 2]. HCC treatment is highly dependent on the stage of detection; therefore, potentially curative surgeries by partial hepatic resection or liver transplantation are options reserved only for patients with I or II tumor stage [3]. Patients with HCC and advanced cirrhosis have very poor prognosis and mostly survive <2 yr after diagnosis [4, 5]. Metastases are responsible of tumor recurrence in most cases; and more than 90% of HCC-related deaths are the result of secondary local or distant disease [6]. Metastasis is the process by which tumor cells disseminate from the primary tumor, migrate through the basement membrane, infiltrate in the circulatory system, and settle in secondary tissues where they continue their growth [7]. These sequential processes require controlled degradation of the extracellular matrix (ECM), by matrix metalloproteinases (MMPs) [8].

MMPs are a family of zinc-dependent endopeptidases, which degrade ECM components. According to their substrate specificity and structure, MMPs can be classified into collagenases, stromelysins, and gelatinases. Among them, collagenase MMP-2 and MMP-9, also known as type IV collagenase, are key enzymes for the degradation of collagen in the extracellular matrix and basement membrane [8]. Aberrant expression of MMP-2 and MMP-9 has been linked to several stages of tumor growth, vascular invasion, tumor progression, and metastasis [8]. Furthermore, high levels of MMPs are associated with poor prognosis in cancer patients [9, 10], and its overexpression has been reported to contribute to HCC growth and capsular infiltration [11].

MMPs are tightly regulated at multiple levels that control their activity, secretion, and gene transcription. Their proteolytic activity is specifically inhibited by tissue inhibitors of metalloproteinases (TIMPs) that bind MMPs in a 1:1 stoichiometric relationship [12, 13]. Because different studies have revealed that TIMPs are involved in a number of biological activities including cell migration, invasion, angiogenesis, and apoptosis, it has been suggested that MMP/TIMP physiological equilibrium can be shifted in malignant tissues, and changes in expression of both MMPs and TIMPs may be used as prognostic factors in cancer [14]. Moreover, MMPs are synthesized as latent proforms, which need to be activated prior to secretion [15]. At the transcriptional level, nuclear factor-kappaB (NF- κB) and activator protein-1 (AP-1) induce MMPs transcription in response to inflammatory cytokines like interleukin 1β (IL1 β) and tumor necrosis factor α (TNFα) [16, 17].

NF-κB transcription factor is implicated in the cellular response to external stimuli, such as oxidative stress, cytokines, growth factors, viral proteins, and ionizing radiation, and it has been related to initiation, promotion, and progression of HCC [18, 19]. NF-κB is formed by two subunits: p65 and p50; in the absence of the mentioned stimuli, the dimer remains inactive in the cytoplasm, bound to its inhibitor protein IκBα. When cytokines bind to their receptor in the cell surface, ΙκΒα is phosphorylated by IKK, which drives its ubiquitination and subsequent degradation in the proteasome [20, 21]. With the degradation of ΙκΒα, the NF-κB complex is then released, and enters the nucleus where it ‘turns on’ the expression of specific genes involved in cancer progression such as MMPs or VEGF [22].

Melatonin has attracted increasing attention because of its protective role in several pathophysiological situations [23-26], including different cancer types [27-35]. Some studies have related its oncostatic features with its ability to induce apoptosis, and inhibit angiogenesis and metastasis [36-39]. Experimental results found in glioma, osteosarcoma, or breast cancer cells indicate that melatonin could be able to reduce migration and invasiveness [40-43]. We have previously reported melatonin to have proapoptotic and antiangiogenic properties in HepG2 liver tumor cells [44-48] while no research regarding melatonin effects on HCC metastasis has been published. Therefore, in the present study, we investigated whether and how melatonin could affect MMP-2 and MMP-9 activity and expression induced by IL-1β in HepG2 HCC cells.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Cell culture and reagents

Human HepG2 hepatocarcinoma cells were obtained from the American Type Culture Collection (Manassas, VA, USA). Stock cells routinely were grown as monolayer cultures in Dulbecco's modified eagle's medium (DMEM), supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL), streptomycin (100 μg/mL), glutamine (4 mm), and pyruvate (100 μg/mL) in a humidified 5% CO2 atmosphere at 37°C, and the medium was changed every other day. Cell culture reagents were from Gibco (Life Technologies, Madrid, Spain). Melatonin was obtained from Sigma (St Louis, MO, USA). Confluent HepG2 cells growing in complete media were replated in 9.6 cm2 culture dishes, at a density of 150,000 cells/plate, in 2 mL of complete medium. After 24 hr, the plating medium was replaced with fresh medium 1% FBS containing melatonin dissolved in DMSO. HepG2 cells were treated with the melatonin at a final dose of 1 mm in the culture media for 24 hr. IL-1β purchased from Alexis Biochemicals (San Diego, CA, USA) was added in a final concentration of 10 ng/mL for 30 min before collection.

Western blot analysis

After treatments, cultured cells were washed twice with ice-cold PBS and lysed by adding ice-cold RIPA buffer containing 50 mm Tris–HCl pH 7.4, 150 mm NaCl, 2 mm EDTA, 0.1% Triton 100X, 10% sodium deoxycholate, 10% SDS, 1 mm NaF, and protease cocktail inhibitor (Roche, Basel, Switzerland) and scraped off the plate. The extracts were transferred to a microfuge tube and centrifuged for 10 min at 15,000 g. Equal amounts of the supernatant protein (20 μg) were separately subjected to SDS-PAGE and transferred to a PVDF membrane (Bio-Rad, Hercules, CA, USA). Primary antibodies (Ab) were diluted in blocking solution and incubated overnight at 4°C with polyclonal Ab to MMP-2, TIMP-2, p65, IKK, IκBα (1:100 dilution; from Santa Cruz, CA, USA), MMP-9, and TIMP-1 (1:1000 dilution from Abcam, Cambridge, UK), phospho-IKK and phospho-IκBα from Cell Signaling, Beverly, MA, USA). Equal loading of protein was demonstrated by probing the membranes with a rabbit anti-β-actin polyclonal antibody (1:2000 dilution Sigma, St Louis, MO, USA). After washing with PBS-T, the membranes were incubated for 1 hr at room temperature with secondary HRP-conjugated antibody (1:4000; Dako, Glostrup, Denmark) and visualized using ECL detection kit (Amersham Pharmacia, Uppsala, Sweden). The density of the specific bands was quantified employing the software ImageJ (National institute of Mental Health, Bethesda, MD) with an imaging densitometer (Scion Image, Maryland, MA, USA).

Real-time reverse transcriptase polymerase chain reaction

For real-time reverse transcriptase polymerase chain reaction (RT-PCR), confluent HepG2 cells growing in complete media were replated in 6 well-culture plate, at a density of 250,000 cell/well in a total volume of 2 mL of complete medium. After treatment, total RNA and DNAse treatment were achieved using SV Total RNA Isolation System (Promega, Madison, WI, USA). Total RNA amount was quantified by spectrophotometry (Nanodrop 1000, Thermo Scientific, Waltham, MA, USA). First-standard cDNA was synthesized using M-MLV RT (Applied Biosystems, Carlsbad, CA, USA), and the negative control (no transcriptase control) was performed in parallel. cDNA was amplified using FastStart TaqMan Probe Master (Roche Diagnostics GmbH, Mannheim, Germany) on an StepOnePlus Real-Time PCR Systems (Applied Biosystems). TaqMan primers and probes for MMP-9 (NM_004994.2 and Hs00234579_m1) and β actin (NM_001101.2 and Hs99999903_m1) gene derived from the commercially available TaqMan Gene Expression Assays (Applied Biosystems). Relative changes in gene expression levels were determined using the 2−△△CT method [49]. The cycle number at which the transcripts were detectable (CT) was normalized to the cycle number of beta-actin detection.

Wound-healing assay

Wound-healing assay was performed for analysis of cell migration in vitro. Briefly, HepG2 cells (2.5 × 105 cells per well) were seeded in 24-well plates, at 37°C until 90% confluent. Sterile tips were used to scratch cell layers, which were subsequently washed with PBS, and cultured with DMEM media and 1% FBS with or without melatonin and IL-1β. Cells were photographed (phase-contrast microscope) at 0, 24, 48, and 72 hr after incubation. Each experiment was performed in triplicate. The values were presented as mean ± S.E. ImageJ was used to measure the total area of the wound. Results were expressed as the difference between wound areas after 72 hr of treatment versus wound area at 0 hr.

Invasion assay

Cell migration assay was performed using Transwell cell culture inserts with 8-μm porosity polyethylene teraphthalate filters (Invitrogen, Carlsbad, CA, USA) (Invitrogen). Briefly, after treatments with melatonin and IL-1β as described above, confluent tumor cells were trypsinized and plated onto the upper Matrigel-coated (50 μg) chamber at a density of 50,000 cells in 200 μl of serum-free medium. The lower chamber of the transwells was contained medium with 10% FBS as chemoattractant. After 24 hr, the upper surface of the membrane was wiped to remove nonmigratory cells with a cotton swab. The cells that invaded through the Matrigel and adhered to the bottom of the membrane were fixed with paraformaldehyde 3,6% for 20 min at RT, and then, permeabilization was performed with methanol 100% for 20 min at RT. Nucleus were stained with DAPI (4′,6-diamidino-2-fenilindole) (Invitrogen). Cells on the lower surface were identified as migrated cells and were subsequently visualized under fluorescence microscopy (Nikon Eclipse Ti, Melville, NY, USA). Using ImageJ, DAPI-labeled cells were considered region-of-interest (ROI) and next counted using the ‘cell counter’ tool from ImageJ. Values were presented as mean ± S.E.M. from three experiments conducted in triplicate.

Zymography assays

Following the appropriate treatment, conditioned media from HepG2 cells cultured on 6-well plates was removed and concentrated 20-fold in centricon 30 microconcentrators to be subjected to SDS-PAGE using 0.01% w/v gelatin from bovine skin (Sigma Aldrich, St Louis, MO, USA) as a substrate in a 10% polyacrylamide gel. After electrophoresis, gels were equilibrated in 2.5% Triton X-100 to remove SDS and incubated in 50 mm Tris–HCl (pH 7.5), 10 mm CaCl2, 150 mm NaCl, 1 mm ZnCl2, and 0.02% NaN3 for 18 hr at 37°C. Then, gels were stained with Coomassie R250.

NF-κB (p65) transactivation assays

Quantitation of the expression of active NFκB (p65) was assayed using the Thermo Scientific Transcription Factor Kit for NFκB (p65) (ThermoFisher Scientific, Waltham, MA, USA), as directed by the manufacturer. Briefly, 10 μl of nucleic extracts were incubated with the NFκB-binding biotinylated consensus sequence DNA (p65) attached to the streptavidin-coated 96-well plates provided. Only the active NFκB p65 binds to the consensus sequence and is subsequently detected with anti-NFκB p65 primary antibody and HRP-conjugated secondary antibody and a chemiluminescent substrate (all provided in the kit). Chemiluminescence was detected in an EnVision Multilabel Plate Reader Model 2102 (Perkin Elmer, Waltham, MA, USA) and reported as relative chemiluminescence units.

Statistical analysis

Results are expressed as mean values ± S.E.M. of the indicated number of experiments. One-way ANOVA followed by Student–Newmann–Keuls post hoc test was used to determinate differences between the mean values of the different treated groups. P < 0.05 was considered significant. Values were analyzed using the statistical package SPSS 20.0 (Chicago, IL, USA).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The proinflammatory cytokine interleukin 1β (IL-1β) is a key mediator in chronic inflammatory responses and exerts fundamental effects on malignant processes, such as carcinogenesis, tumor growth, invasion, and metastasis [50]. While cell migration is essential for wound repair, invasion requires cell to migrate through the extracellular matrix (ECM), which involves barrier degradation [8]. The effect of IL-1β and melatonin treatment on HepG2 cells motility and invasiveness was assayed by Matrigel invasion assay and wound-healing assay to better understand the effects of treatment on our in vitro metastatic model.

As show in Fig. 1A, HepG2 cells exhibited IL-1β-dependent invasiveness response compared with control, whereas cotreatment with IL-1β and the concentration of melatonin (1 mm) were found to reduce the number of invading cells. Based on these results, cell motility was examined by the wound-healing assay. Images show how IL-1β stimulated cells displayed cellular protrusions and an increase in wound closure compared with the control and the IL-1β plus melatonin group (Fig. 1B).

image

Figure 1. Effect of melatonin on migration on hepatocellular carcinoma cells. (A) After HepG2 cells treatment with 1 mm melatonin for 24 hr and 10 ng/mL IL-1β for 30 min, cells were collected and seeded on chambers coated with Matrigel. Images represent cells migrated to the lower chamber after 24 hr. Graphic represents the average of cells migrated counted in three randomized trials. (B) Migratory ability of HepG2 was evaluated by wound-healing assay. Confluent cells were treated with 1 mm melatonin for 24 hr and 10 ng/mL IL-1β for 30 min; a scratch was made with a tip of pipette at same time of treatment. Images were taken at 0, 24, 48, and 72 hr after treatment for monitoring wound closure. Total area of the wound was measured at 0 and 72 hr, and the difference between them was represented. Data are expressed as mean values ± S.E.M. of three independent experiments. *< 0.05 significant differences versus control. #< 0.05 significant differences between IL-1β and melatonin + IL-1β-treated cells.

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Proteolytic degradation of ECM components is a crucial step for tumor-cell invasion, mainly performed by type IV collagenase MMP-9 and MMP-2. We next evaluated whether melatonin 1 mm and/or IL-1β 10 ng/mL might affect the expression and activity of MMP-2 and MMP-9 measured by Western blot and zymography. As shown in Fig. 2, IL-1β treatment stimulates MMP-9 protein expression and subsequent release to the culture media. In addition, conditioned media from IL-1β -treated cells exhibited the strongest gelatinase activity, as detected by the lytic zones in the zymography. However, melatonin-treated cells presented lower MMP-9 cellular and secreted protein values, accompanied by decreased MMP-9 gelatinase activity in the culture media. By contrast, no significant alterations regarding MMP-2 expression, secretion, and activity were detected when cells were stimulated by IL-1β, and/or treated with melatonin (Fig. 3), suggesting that the observed melatonin effects on cell motility and invasion were mainly related to MMP-9 colagenase.

image

Figure 2. Effect of IL-1β and melatonin treatment on MMP-9 activity, and MMP-9 and TIMP-1 expression and secretion. Melatonin inhibits MMP-9 activity, expression and secretion induced by IL-1β. (A) Effect of IL-1β and melatonin on MMP-9 activity assessed by zymography. (B) Effect of IL-1β and melatonin on MMP-9 protein levels released to the cultured media studied by Western blot. (C) Effect of IL-1β and melatonin on MMP-9 protein cellular levels. (D) Effect of IL-1β and melatonin on TIMP-1 protein cellular levels. (E) Effect of IL-1β and melatonin on TIMP-1 protein levels released to the culture media studied by Western blot. Results are presented as ratio of OD sample/OD determined in the control load. (F) Effect of IL-1β and melatonin treatment on MMP-9 mRNA levels. Data are expressed as a percentage of mean values ± S.E.M. of experiments performed in triplicate. *< 0.05 significant differences versus control. #< 0.05 significant differences between IL-1β and melatonin + IL-1β-treated cells.

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TIMPs are known inhibitors of matrix metalloproteinase activity; based on their key role in the regulation of MMPs, we further examined the TIMP-1 and TIMP-2 cellular and secreted levels under treatment. As shown in Fig. 2, while IL-1β treatment decreased TIMP-1 protein expression, melatonin preincubation efficiently increased TIMP-1 levels. Moreover, TIMP-1 levels in culture media from IL-1β -stimulated cells were lower than those from untreated cells, and this effect was prevented by melatonin cotreatment. However, as occurred when analyzing MMP-2, IL-1β and/or melatonin treatments showed no significant effects on cellular protein levels of the MMP-2-specific inhibitor TIMP-2 (Fig. 3), which seems to indicate that at least in our in vitro model, melatonin mainly affects the MMP-9 collagenase.

Once observed that MMP-9 protein levels and gelatinase activity were decreased by melatonin, even upon IL-1β stimulation, we evaluated whether melatonin treatment also exerted its inhibitory effects at a transcriptional level. As observed in Fig. 2F, mRNA levels codifying for MMP-9 gene were significantly increased by IL-1β stimulation, and this effect was significantly abrogated by melatonin, reaching MMP-9 mRNA values similar to those in control untreated cells.

Proinflammatory cytokines such as IL-1β and TNFα are the most potent NF-κB activators, able to induce its nuclear translocation and subsequent binding to the MMP-9 promoter region [45]. Once shown that melatonin antimetastatic activity was related with its ability to modulate MMP-9 levels, we focused on elucidating the melatonin effect on the NF-κB pathway. Thus, when the NF-κB-p65 protein expression was measured in the nuclei, we found that, while IL-1β increased NF-κB-p65 protein levels, melatonin pretreatment decreased p65 nuclear levels (Fig. 4A). In addition, NF-κB-p65 transcriptional activation was measured. As expected, IL-1β stimulation induced NF-κB-p65 binding to its responding elements while cells subjected to PDTC and melatonin treatment prior IL-1β administration showed reduced NF-κB activity (Fig. 4B), suggesting that melatonin and the specific NF-κB inhibitor PDTC present similar effects on p65 binding to its consensus sequences. These results seem to indicate that melatonin could modulate MMP-9 expression acting upstream of NF-κB. Thus, to further analyze whether this pathway may be involved in the effects of melatonin previously observed, we measured the phosphorylation status of IκBα and IKK, NF-κB inhibitors, in IL-1β and melatonin-treated HepG2 cells. As observed in Fig. 4C, interleukin treatment alone significantly stimulated IKK activation, while melatonin pretreatment prevented IKK phosphorylation. No significant changes were found regarding total IKK forms. In line with these results, cellular extracts from the IL-1β group also presented higher levels of phosphorylated IκBα in comparison with extracts from melatonin-treated cells (Fig. 4D). Opposite effects exerted by treatment were found when the nonphosphorylated IκBα forms were analyzed; thus, cytokine stimulation decreased the presence of the total IκBα protein, while melatonin preincubation reduced the phoshpo-IκBα/IκBα ratio, widely accepted as indicative for IκBα degradation. Consequently, our results suggest that the inhibitory effect of melatonin on the NFκB pathway may be due to its ability to maintain this transcription factor in the cytoplasm, preventing IκBα phosphorylation by IKK and the subsequent nuclear translocation of this transcription factor.

image

Figure 3. Effect of IL-1β and melatonin treatment on MMP-2 activity, and MMP-2 and TIMP-2 expression and secretion. (A) Effect of IL-1β and melatonin on MMP-2 activity assessed by zymography. (B) Effect of IL-1β and melatonin on MMP-2 protein levels released to the cultured media studied by Western blot. (C) Effect of IL-1β and melatonin on MMP-2 protein cellular levels. Results are presented as ratio of OD sample/OD determined in the control load. (D) Effect of IL-1β and melatonin on TIMP-2 protein cellular levels. Data are expressed as a percentage of mean values ± S.E.M. of experiments performed in triplicate.

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image

Figure 4. Melatonin reduces expression of NF-κB pathway components in IL-1β-stimulated HepG2 cells. After treatment with melatonin (1 mm, 24 hr) and IL-1β (10 ng/mL), cells were collected and phospho-IKK, IKK, phospho-IκBα, IκBα, and p65 protein levels were measured by Western blot. (A) p65 protein levels in the nuclei. (B) p65 transcriptional activity. (C) Phosphorylation status of IKK under treatment. (D) Phosphorylation status of IκBα under treatment. Results are presented as ratio of OD sample/OD determined in the control load. *< 0.05 significant differences versus control. #< 0.05 significant differences between IL-1β and melatonin + IL-1β-treated cells.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Despite the significant advances in therapy and early detection of HCC, even after surgery, tumor recurrence is usual, and most of the HCC patients die as a result of secondary metastasis [51], while effective or curative drug therapy for HCC and its metastases remain elusive. HCC cells have multiple molecular signaling pathways to proliferate, invade, and metastasize during the course of tumor progression, and MMP proteins have a critical role in these processes, been commonly upregulated in several cancers including HCC [52]. High MMPs levels have been detected in tumor tissue or serum from patients with advanced cancer, and their role as prognostic indicators in cancer has been widely examined [10]. Moreover, based on the reported importance of these enzymes in the processes of invasion and metastasis, some authors have pointed out that selective inhibition of certain metaloproteinases, in combination with conventional chemotherapy may provide a feasible approach for cancer therapy [53, 54]. In the last years, several studies have verified melatonin involvement on the prevention of tumor initiation, promotion, and progression [25-27, 29-33, 55], and a number of meta-analysis have confirmed the efficacy and safety of melatonin administration in cancer patients [56, 57]. We have already reported that melatonin administration induces cell cycle arrest and apoptosis in hepatocarcinoma HepG2 cells [47, 48]. Moreover, we demonstrated that melatonin inhibits VEGF expression, acting as an antiangiogenic compound in an in vitro model of induced hypoxia [44]. However, little is known about the antimetastatic properties of this indole in HCC cells.

Our experiment in HCC was carried out using a final concentration of 1 mm melatonin. Melatonin antitumor effects seem to be largely dependent on the dose and cell type evaluated; thus, while oncostatic effects have been reported in ME-180 and HELA human uterine neck cancer cells, OAW-42 ovarian cancer cells, HT-29 human colon cancer cells, or CT-26 mouse colon cancer cells, at concentrations ranging 1–6 mm [30, 58, 59], MCF-7 human breast cancer cells or Jar human choriocarcionoma cells seem to be more sensitive, responding to nanomolar melatonin doses [35, 60]. Studies analyzing melatonin effects on tumor-cell invasiveness also show differences depending on the cell type, ranging from 1 nm in MCF-7 cells [42, 43] to 0,25–1 mm in 9607 human osteosarcoma cells, and 1 mm in T98G and U251 glioma cells [40, 41]. Although no information about melatonin effects on migration and invasiveness in HCC is available, we have previously demonstrated that melatonin has other different antitumor effects in HepG2 cells when administered at doses ranging 1–2.5 mm [44-47].

The present results indicate that 1 mm melatonin treatment prevents IL-1β-induced motility and invasiveness in HepG2 cells, which may be associated with its antimetastatic properties. Interestingly, although there are not many studies explaining the mechanism by which melatonin reduces tumor progression, it has been reported that melatonin administration to metastatic advanced patients to whom no other available treatment was effective, resulted in a control of the neoplasic progression in 40% of the cases [36]. Furthermore, there are evidences showing that melatonin, either alone or in combination with chemoradiotherapy and/or supportive care, improves drug tolerance and decreases tumor regression, with better survival rates in patients with advanced solid tumors [57, 61].

Here, we demonstrate that the effects of melatonin on tumor-cell motility and invasiveness are related with its ability to decrease MMP-9 expression, release and lytic activity, and the induction of its main inhibitor TIMP-1. Moreover, while interleukins and TNFα have been demonstrated to modulate MMP-9 and TIMP-1 inhibitors in different cell types, including hepatic stem cells (HSC) [62], this report provides the first evidence that IL1-β treatment can also alter the production of pro-MMP-9 and its specific inhibitor in human tumor hepatocytes. Consequently, we presume that the observed effect of the indole may be due to its capacity to decrease the MMP-9/TIMP-1 ratio. Melatonin has been described to modulate MMPs in both physiological and pathological processes, protecting from gastric ulceration and liver injury through MMP-9 donwregulation [39]. The administration of this indole has been found to downregulate pro-MMP-9 and upregulate TIMP-1 expression dose dependently to protect against endometriosis in mice and in human endometriotic tissues [63]. Moreover, when administered to mice with induced cerebral ischemia and reperfusion, melatonin was found to attenuate MMP-9 activation, decreasing hemorrhagic transformation and brain infarct [64].

Available knowledge about whether and how melatonin influences MMP-9 activity in tumor cells, however, remains limited. In this respect, it has been shown that melatonin exerted an inhibitory effect on breast cancer cell invasion through downregulation of the p38 pathway, and inhibition of MMP-2 and MMP-9 expression and activity [65], and it has also been reported that this indole significantly reduced cell migration and invasion in T98G and U251 nonstimulated glioma cells, decreasing both MMP-2 and MMP-9 via reactive oxygen species and NF-κB inhibition [40]. By contrast, when analyzing melatonin effect on both MMP-2 and MMP-9 colagenases, we did not find significant changes in MMP-2 expression or activity upon IL-1β or melatonin treatment. Similarly to our results, it has been also reported that melatonin reduces endothelial cell mobility in HUVECs stimulated with IL1-β by interfering with the expression and activity of MMP-9, without affecting MMP-2 activity [66]. Therefore, we speculate that melatonin effects on MMP-2 could differ depending on the stimulus and the cell line. Besides, an interesting study using computational chemistry tools has recently provided additional information on this field, revealing the existence of some kind of atomic-level interactions between melatonin and certain proline and alanine residues within the MMP-9 catalytic site, which might be involved in reducing the MMP-9 activity in human gastric adenocarcinoma cells [67].

Invasion and metastasis in HCC have been reported to occur via the upregulation of NF-κB, which can be induced by interleukins and is a key transcription factor for MMP-9 production [68, 69]. An early activation of NF-κB has been found to contribute to the acquisition of a transformed phenotype during hepatocarcinogenesis, whatever the etiology [70]. In our study, IL-1β stimulated the nuclear translocation of NF-κB-p65, followed by transcriptional activation and increase in MMP-9 mRNA levels. However, melatonin pretreatment effectively decreased p65 nuclear levels and NF-κB-p65 transactivation, behaving as the NF-κB -specific inhibitor PDTC, and preventing the observed increase on MMP-9 mRNA. Accordingly, it has been previously reported that administration of melatonin to mice after lipopolysaccharide (LPS) treatment significantly attenuates the rises of circulatory and cerebral MMP-9 activity, and the indole also inhibits NFκB-dependent MMP-9 transcription induced by LPS in RAW 264.7 mouse leukemic monocyte and BV2 cells derived from primary mouse microglia cells [71].

Thus, aiming to elucidate the molecular pathway underlying results observed, we assayed the effect of IL-1β and melatonin on the NF-κB pathway. While the nuclear presence of p65 was associated with the phosphorylation of its inhibitors under cytokine stimulation, melatonin decreased phospho-IKK level and prevented IκBα degradation, which allows us to presume that this indole may act inhibiting MMP-9 through the NF-κB pathway. Some drugs tested for chemotherapy have been shown to inhibit the NF-κB pathway through IKK [72-74]. To this respect, sorafenib, a tyrosine kinase inhibitor used in HCC treatment, inhibits VEGF and MMP-9 expression in Huh7 HCC cells via NF-κB pathway [75]. Furthermore, natural compounds with reported antioxidant and cytostatic properties like ursolic acid, celastrol and curcumin, have been shown to prevent IκBα degradation in different in vitro studies also using proinflammatory cytokines stimulation [76-78]. Resveratrol, curcumin, or green tea catenins, with chemical structure similar to melatonin, present antimetastatic features associated with MMPs inhibition [79], and licopen or vanillin, other natural chemopreventive agents, are potent NF-κB inhibitors that also decrease MMP-9 to inhibit invasion and migration of liver cancer cells [80, 81].

Although the present study was focused on the effects of melatonin through MMP-9 and NF-κB-dependent mechanisms, some authors have shown that melatonin could induce other concomitant actions contributing to the inhibition of cell migration and invasiveness, such as a reduction of basal oxidative stress in T98G and U251 glioma cells [40], or microfilament and microtubule rearrangements inducing changes on cytoskeletal organization in MCF-7 cells by a Rho-associated protein kinase-dependant pathway [42]. In any case, our results suggest that melatonin exerts anti-invasive effects in HepG2 cells by decreasing the MMP-9/TIMP-1 ratio, and downregulating the NF-κB pathway activation. Based on the present finding, and in view of the melatonin reported beneficial properties in other tumors types, along with its lack of toxicity in normal cells, we consider that this indole could be of interest, at least as adjuvant, in HCC therapy. However, further research to test the usefulness of melatonin for HCC prevention and treatment is required.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Raquel Ordoñez is supported by the program Formación del Profesorado Universitario from the Ministry of Education (Spain). Sara Carbajo-Pescador is supported by the Consejería de Educación (Junta de Castilla y León, Spain), and Fondo Social Europeo. CIBERehd is funded by Instituto de Salud Carlos III. This work has been partially supported by Fundación Investigación Sanitaria en León.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References