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Keywords:

  • apoptosis;
  • Bim;
  • melatonin;
  • proteasome activity;
  • transcriptional regulation

Abstract

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

Melatonin (N-acetyl-5-methoxytryptamine) has recently gained attention as an anticancer agent and for combined cancer therapy. In this study, we investigated the underlying molecular mechanisms of the effects of melatonin on cancer cell death. Treatment with melatonin induced apoptosis and upregulated the expression of the pro-apoptotic protein Bcl-2-interacting mediator of cell death (Bim) in renal cancer Caki cells. Furthermore, downregulation of Bim expression by siRNA markedly reduced melatonin-mediated apoptosis. Melatonin increased Bim mRNA expression through the induction of Sp1 and E2F1 expression and transcriptional activity. We found that melatonin also modulated Bim protein stability through the inhibition of proteasome activity. However, melatonin-induced Bim upregulation was independent of melatonin's antioxidant properties and the melatonin receptor. Taken together, our results suggest that melatonin induces apoptosis through the upregulation of Bim expression at the transcriptional level and at the post-translational level.


Introduction

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

Melatonin (N-acetyl-5-methoxytryptamine) is synthesized from tryptophan and is a secretory product of the pineal gland [1]. Among a variety of functions, melatonin exhibits anti-inflammatory and antioxidant properties, both in vivo and in vitro [2-5]. Furthermore, melatonin shows anticancer effects in several types of cancers including pancreatic, breast, and lymphoid cancers [6-8]. In cancer cells, melatonin causes growth inhibition, induces apoptosis, and enhances the efficiency of anticancer therapeutic agents [9-11]. Additionally, in normal cells, melatonin exhibits a protective effect against several cytotoxic stresses [12-15]. The cancer-specific cytotoxicity of melatonin may be a novel therapeutic approach to treat some cancers. Therefore, the underlying molecular mechanisms of melatonin-mediated anticancer effects are worthy of further study.

The Bcl-2 superfamily of proteins can be classified based on function and BH-domain organization, and the BH3-only proteins of the Bcl-2 family are considered to be important apoptosis factors and are induced by diverse toxic stimuli [16-18]. Bcl-2-interacting mediator of cell death (Bim) is one of the BH3-only Bcl-2 family proteins and is a potent pro-apoptotic protein. The expression level of Bim is regulated by mRNA transcription, mRNA stability, and post-translational modifications [19-24].

The present study was designed to determine the capacity of melatonin to induce Bim-mediated apoptosis in a renal cancer cell line and to identify the molecular mechanisms of Bim upregulation.

Materials and methods

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

Cell culture

Caki, HT29, and A549 cells were purchased from the American Type Culture Collection (Rockville, MD, USA). The mouse kidney cells (TMCK-1) were a gift from Dr. T.J. Lee (Yeungnam University, Korea). Primary cultures of human mesangial cells (Cryo NHMC) were purchased from Clonetics (San Diego, CA, USA). The cells were cultured in Dulbecco's modified Eagle's medium (Caki, A549, TMCK-1, and mesangial cells) or RPMI (HT29) that contained 10% fetal bovine serum, 20 mm Hepes buffer, and 100 μg/mL gentamicin.

Reagents

Melatonin, cyclohexamide (CHX), and MG132 were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The anti-Bim antibody was purchased from Millipore Corporation (Billerica, MA, USA), and anti-PARP, anti-Bcl-2, anti-Bax, anti-Bcl-xL, anti-Mcl-1, anti-Sp1, anti-E2F1, anti-ubiquitin, and anti-actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-phospho-Sp1 (T453) antibody was purchased from Abcam (Cambridge, MA, USA). Phospho-E2F1 (S364) antibody was purchased from Rockland Immunochemicals (Gilbertsville, PA, USA). Other reagents were purchased from Sigma Chemical Co.

Flow cytometry analysis

Cell counts were performed using a hemocytometer. Approximately 0.4 × 106 cells were suspended in 100 μL of PBS, and 200 μL of 95% ethanol was added while vortexing. The cells were incubated at 4°C for 1 hr, washed with PBS, and resuspended in 250 μL of 1.12% sodium citrate buffer (pH 8.4) together with 12.5 μg of RNase. The cells were then incubated at 37°C for 30 min. The cellular DNA was then stained by applying 250 μL of propidium iodide (50 μg/mL) for 30 min at room temperature. The stained cells were analyzed by fluorescent activated cell sorting (FACS) on a FACS Canto™ (BD Biosciences, San Diego, CA, USA) to determine the relative DNA content based on red fluorescence.

Western blotting

Cellular lysates were prepared by suspending 0.4 × 106 cells in 100 μL of lysis buffer (137 mm NaCl, 15 mm EGTA, 0.1 mm sodium orthovanadate, 15 mm MgCl2, 0.1% Triton X-100, 25 mm MOPS, 100 μm phenylmethylsulfonyl fluoride, and 20 μm leupeptin, adjusted to pH 7.2). The cells were disrupted by vortexing and were extracted at 4°C for 30 min. The proteins were then electrotransferred onto Immobilon-P membranes (Millipore Corporation, Bedford, MA, USA). Specific proteins were detected with an ECL western blotting kit (Cat. No. WBKLS05000; Millipore Corporation) according to the manufacturer's instructions.

DNA fragmentation assay

After treatment with experimental conditions, the cells were harvested. Cytoplasmic histone-associated DNA fragments were determined using the cell death detection ELISA plus kit (Boehringer Mannheim, Indianapolis, IN, USA). Briefly, cells were centrifuged for 10 min at 200 g, the supernatant was removed, and the pellet was lysed for 30 min. Cells were centrifuged again at 200 g for 10 min, the supernatant containing the cytoplasmic histone-associated DNA fragments was collected and incubated with an immobilized anti-histone antibody, and the reaction products were determined by spectrophotometry. Finally, after incubation with a peroxidase substrate for 5 min, absorbance was measured at 405 and 490 nm (reference wavelength) using a microplate reader. Signals in the wells containing the substrate only were subtracted as background.

DEVDase (caspase-3) activity assay

To evaluate DEVDase (Asp-Glu-Val-Asp-ase) activity, cell lysates were prepared after the cells were appropriately treated. The assays were performed in 96-well plates by incubating 20 μg of cell lysate in 100 μL of reaction buffer (1% NP-40, 20 mm Tris–Cl (pH 7.5), 137 mm NaCl and 10% glycerol) containing the caspase substrate (Asp-Glu-Val-Asp-chromophore p-nitroanilide; DEVD-pNA) at 5 μm. Lysates were incubated at 37°C for 2 hr. Thereafter, activity was measured at 405 nm using a spectrophotometer.

Small-interfering RNAs

The GFP (control) and Bim small-interfering RNA (siRNA) duplexes used in this study were purchased from Santa Cruz Biotechnology. Cells were transfected with siRNA oligonucleotides using Oligofectamine™ Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's recommendations.

RNA isolation and RT-PCR

Total cellular RNA was extracted from the cells using TRIzol reagent (Life Technologies, Gaithersburg, MD, USA). Complementary DNA (cDNA) was synthesized from 2 μg of total RNA using M-MLV reverse transcriptase (Gibco BRL, Gaithersburg, MD, USA). The cDNAs for Bim and actin were amplified by polymerase chain reaction (PCR) using specific primers. The sequences of the sense and antisense primers for Bim were 5′-ATGGCAAAGCAACCTTCTGA-3′ and 5′-CTGTCTGTGTCAAAAGAG-3′, respectively. The PCR products were analyzed by agarose gel electrophoresis and visualized using ethidium bromide (EtBr) staining.

Plasmids, transfections, and luciferase assay

The Bim promoter-containing plasmids have been described previously [25]. The p-3.6 Bim promoter plasmid (containing a 3.6-kb Xba/SacI Bim promoter sequence) and p-0.8 Bim promoter plasmid (containing a 0.8-kb HindIII/SacI Bim promoter sequence) were gifts from Dr. J.M. Adams (Walter and Eliza Hall Institute of Medical Research, Australia). In brief, cells were plated on 6-well plates at a density of 0.6 × 106 cells/well and grown overnight. Cells were co-transfected with 2 μg of various plasmid constructs and 1 μg of the pCMV-β-galactosidase plasmid for 5 hr by the Lipofectamine 2000 method. After transfection, cells were cultured in 10% FBS medium with vehicle (DMSO) or drugs for 24 hr. Luciferase and β-galactosidase activities were assayed according to the manufacturer's protocol (Promega, Madison, WI, USA). Luciferase activity was normalized for β-galactosidase activity in cell lysates and expressed as an average of three independent experiments.

Proteasome activity assay

Proteasome activity within cells was determined using ZsGreen (proteasome sensor vector)-transfected stable Caki cell lines. Fluorescence was detected using a fluorescent microscope and FACS Canto™ (BD Biosciences, San Jose, CA, USA). Chymotryptic proteasome activities were measured using Suc-LLVY-AMC (chymotryptic substrate, Biomol International, Plymouth Meeting, PA, USA). Cells were collected, washed with PBS, and lysed. A mixture containing 1 μg of protein of the cell lysate in 100 mm Tris–Cl (pH 8.0), 10 mm MgCl2, and 2 mm ATP was incubated at 37°C for 30 min with 50 μm Suc-LLVY-AMC. Enzyme activity was measured using a fluorometric plate reader at an excitation wavelength of 380 nm and an emission wavelength of 440 nm.

Ubiquitination assay

Cellular lysates were prepared by suspending 1 × 106 cells in 100 μL of lysis buffer [50 mm Tris–Cl (pH 7.4), 150 mm NaCl, 1 mm EDTA, 1% NP-40, 0.1% SDS, 1 mm DTT, 1 mm PMSF, 10 mm N-ethylmaleimide (NEM), and 1 mm of a protease inhibitor cocktail (Sigma)]. Lysates were incubated overnight with the antibody at 4°C, followed by the addition of protein-A/G beads for an additional 1 hr. Immunoprecipitates were washed with the lysis buffer and subjected to SDS-PAGE.

p62 ubiquitin-binding domain (UBA) pull-down assay

Conjugation of the ubiquitin-binding domain (UBA) of p62 to agarose beads has been shown to precipitate/pull down ubiquitinated proteins [26]. Cellular lysates were prepared by suspending 1 × 106 cells in 100 μL of lysis buffer [10 mm Tris–Cl (pH 8.0), 140 mm NaCl, 5% glycerol, 0.1% sodium deoxycholate, 0.1% SDS, 1% Triton X-100, and a protease inhibitor cocktail]. Cellular lysates were incubated with 30 μL of p62 UBA agarose beads (Biomol International) at 4°C for 2 hr. Beads were washed with lysis buffer and subjected to SDS-PAGE.

Densitometry

The band intensities were scanned and quantified using the gel analysis plugin for the open source software ImageJ 1.46 (Imaging Processing and Analysis in Java; http://rsb.info.nih.gov/ij).

Statistical analysis

Data were analyzed using a one-way ANOVA followed by post hoc comparisons (Student–Newman–Keuls) using the Statistical Package for Social Sciences version 8.0 (SPSS Inc., Chicago, IL, USA).

Results

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

To verify the cytotoxic effect of melatonin on cancer cells, human renal carcinoma Caki cells were treated with 0.1, 0.5, or 1 mm melatonin for 24 hr. Melatonin induced an increase in the sub-G1 population (Fig. 1A) and cleavage of PARP in a dose-dependent manner (Fig. 1B). We also checked the apoptotic effect of melatonin using DEVDase (caspase-3) activity and DNA fragmentation, which are well-known apoptosis markers, in Caki cells. Melatonin induced both DEVDase activation and DNA fragmentation (Fig. 1C,D). Furthermore, pretreatment with 50 μm z-VAD-fmk, a pan-caspase inhibitor, inhibited apoptosis in melatonin-treated Caki cells (Fig. 1E). These data indicate that melatonin induces caspase-dependent apoptosis in Caki cells.

image

Figure 1. Melatonin induces apoptosis in human renal cancer Caki cells. (A) Flow cytometric analysis of apoptotic cells. Caki cells were treated with 0.1, 0.5, or 1 mm melatonin for 24 hr. Apoptosis was analyzed as the sub-G1 fraction by FACS analysis. (B) The expression level of PARP proteins in Caki cells by treatment with melatonin for 24 hr. Whole-cell lysates were prepared and analyzed by immunoblotting. (C) Caki cells were treated with the indicated doses of melatonin for 24 hr. DNA fragmentation in Caki cells was determined by the DNA fragmentation detection kit. (D) Caki cells were treated with the indicated concentrations of melatonin for 24 hr. Enzymatic activities of DEVDase (caspase-3) were determined. (E) Caki cells were pretreated with 50 μm z-VAD-fmk (z-VAD) for 30 min and then added melatonin for 24 hr. Apoptosis was analyzed as the sub-G1 fraction by FACS analysis. The expression level of PARP proteins was analyzed by immunoblotting. The values (in A, C, D, and E) represent the mean ± S.D. from three independent samples. *< 0.001 compared to the control. #< 0.001 compared to melatonin. The data represent three independent experiments.

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To investigate the underlying mechanisms involved in melatonin-induced apoptosis, we analyzed the changes in the expression levels with various apoptosis-related proteins. As shown in Fig. 2(A), while protein levels of Bax, Bcl-2, Mcl-1, and Bcl-xL were not altered in response to melatonin, Bim expression markedly increased in melatonin-treated Caki cells. Melatonin also induced Bim expression in other types of cancer cell lines, HT29 (human colon cancer cell lines) and A549 (human lung cancer cell lines; Fig. 2B). These data suggest that melatonin could modulate Bim expression in cancer cells. Next, we tested whether Bim is involved in melatonin-induced apoptosis, and we analyzed apoptosis using a siRNA duplex against Bim. Knockdown of Bim protein expression was determined by western blot analysis (Fig. 2C, upper panel). The melatonin-induced sub-G1 cell population was attenuated in cells transfected with Bim siRNA compared to control siRNA-transfected cells (Fig. 2C, lower panel). Taken together, these results clearly indicate that Bim upregulation is critical for melatonin-induced apoptosis.

image

Figure 2. Melatonin-mediated Bim expression is important in apoptosis in Caki cells. (A,B) Caki, HT29, and A549 cells were treated with the indicated concentrations for 24 hr. Equal amounts of cell lysates (40 μg) were subjected to electrophoresis and analyzed by western blot for Bim, Bax, Bcl-2, Bcl-xL, Mcl-1, and actin as a control for protein loading. (C) Caki cells were transfected with control (siControl) or Bim siRNA (siBim). Twenty-four hours after transfection, cells were treated with 1 mm melatonin for 24 hr. Apoptotic cells were analyzed as the sub-G1 fraction by FACS analysis. Bim and actin expressions were determined using western blotting. The values in (C) represent the mean ± S.D. from three independent samples. *< 0.001 compared to melatonin-treated siControl. (D) TMCK-1, mesangial, and Caki cells were treated with the indicated concentrations of melatonin for 24 hr. The cell morphology was examined using interference light microscopy (left panel). Apoptotic cells were analyzed as the sub-G1 fraction by FACS analysis (right panel). The data represent three independent experiments.

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The normal mouse renal tubular epithelial cells (TMCK-1) and normal human mesangial cells (MC) were also tested. As expected, melatonin had no effect on apoptosis in TMCK-1 and MC (Fig. 2D). These data suggest that melatonin-induced apoptosis is specific to cancer cells.

Because several papers reported that Bim expression was regulated at the transcriptional level [21, 26, 27], we examined whether the induction of Bim is due to transcriptional activation in melatonin-treated Caki cells. Melatonin upregulated Bim mRNA expression in a dose-dependent manner and increased within 9 hr, peaked at 12 hr, and was sustained up to 24 hr (Fig. 3A). We further examined the effects of melatonin on the promoter activities of reporter constructs (p-3.6 Bim promoter and p-0.8 Bim promoter). As shown in Fig. 3(B), melatonin increased the promoter activities of the p-3.6 Bim promoter and the p-0.8 Bim promoter. These data indicate that melatonin modulates Bim expression at the transcriptional levels.

image

Figure 3. Melatonin upregulates Bim expression at the transcriptional level. (A) Caki cells were treated with the indicated concentrations of melatonin for 24 hr (upper panel) or with 1 mm melatonin for the indicated time periods. Bim and actin mRNA expressions were determined using RT-PCR. (B) Caki cells were transfected with Bim promoter constructs (p-0.8 Bim promoter and p-3.6 Bim promoter). After transfection, Caki cells were treated with the indicated concentrations of melatonin for 24 hr. The cells were lysed, and luciferase activity was measured. The values in (B) represent the mean ± S.D. from three independent samples. *< 0.001 compared to control. The data represent three independent experiments.

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The Bim promoter contains several transcription factor-binding sites, including Sp1, E2F1, and FOXO3a [25, 27, 28]. Therefore, we tested whether melatonin could modulate the expression of transcription factors that are associated with Bim expression. First, we checked whether Sp1 is associated with melatonin-induced Bim expression. Melatonin increased Sp1 expression within 9 hr, and Sp1 expression continued to gradually increase up to 24 hr (Fig. 4A). However, phosphorylation of Sp1 (T453) was detected at 24 hr in melatonin-treated cells (Fig. 4A). To confirm the functional role of Sp1 in Bim expression, Caki cells were transfected with the Sp1 expression vector. Bim protein and mRNA expression was markedly increased by the Sp1 expression vector in a dose-dependent manner (Fig. 4B). In addition, melatonin induced E2F1 expression within 9 hr, which continued to increase up to 24 hr (Fig. 4C). Phosphorylation of E2F1(S364) was increased within 3 hr and gradually decreased in melatonin-treated cells (Fig. 4C). E2F1 also induced Bim protein and mRNA expression in E2F1 expression vector-transfected Caki cells (Fig 4D). These data indicate that melatonin-induced transcriptional activation of Sp1 and E2F1 is involved in melatonin-mediated Bim mRNA expression.

image

Figure 4. Sp1 and E2F1 transcription factors are critical for melatonin-mediated Bim expression. (A,C) Caki cells were treated with 1 mm melatonin for the indicated time periods. (B,D) Caki cells were transfected with the indicated concentrations of Sp1 expression plasmid (B) or E2F1 expression plasmid (D) for 24 hr. Sp1, phospho(p)-Sp1, Bim, E2F1, phospho(p)-E2F1, and actin expressions were determined using western blotting. Bim and actin mRNA expressions were determined using RT-PCR. The data represent three independent experiments.

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To further clarify the underlying mechanisms of the upregulation of Bim protein levels in melatonin-treated cells, we examined Bim expression from 3 to 24 hr after the treatment. As shown in Fig. 3(A), while upregulation of Bim mRNA started at 9 hr, Bim protein expression was induced within 3 hr and then gradually increased up to 24 hr (Fig. 5A). Therefore, we hypothesized that other mechanism is involved in melatonin-mediated Bim protein expression. Bim has been known to be modulated at the post-translational level [23, 24]. To investigate this possibility, Caki cells were treated with melatonin for 24 hr and further incubated with cyclohexamide (CHX) alone or cotreated with CHX and melatonin for indicated time periods (Fig. 5B). Increased protein levels of Bim caused by melatonin were sustained over 12 hr in CHX- plus melatonin-treated cells, but CHX alone rapidly decreased Bim expression within 6 hr. These results indicate that upregulation of melatonin-induced Bim expression is also regulated at the post-translational levels.

image

Figure 5. Melatonin enhances Bim protein stability. (A) Caki cells were treated with 1 mm melatonin for the indicated time periods. (B) Caki cells were treated with 1 mm melatonin. After 24 hr, cells were washed with PBS and treated with 20 μm cyclohexamide (CHX) in the presence or absence of 1 mm melatonin for the indicated time points. Western blot analysis was performed as described in the Materials and Methods section. The band intensities of the Bim protein expression were measured using the public domain JAVA image-processing program ImageJ (http://rsb.info.nih.gov/ij). *< 0.001 compared to CHX alone. The data represent three independent experiments.

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Because Bim is degraded by the proteasome-ubiquitin system, we further investigated whether melatonin-mediated Bim protein expression occurred by the inhibition of proteasome activity using the proteasome sensor vector, ZsProSensor-1. The vector encodes a destabilized green fluorescence protein (ZsGreen) that is rapidly degraded by the proteasome. For example, when proteasomes are inhibited by the addition of a chemical agent, the fluorescent protein accumulates to levels that can be detected by fluorescence microscopy. As shown in Fig. 6(A), melatonin- and MG132 (positive control, proteasome inhibitor)-treated cells expressed strong green fluorescence. To confirm the inhibition of proteasome activity by melatonin, we measured chymotrypsin-like activity using suc-Leu-Leu-Val-Tyr-AMC as the proteasome substrate. Melatonin markedly inhibited proteasome activity in Caki cells (Fig. 6B). Because the inhibition of proteasome activity affects the increase in ubiquitination [29, 30], we examined whether melatonin treatment induced ubiquitination of Bim in Caki cells. We performed immunoprecipitation (IP) and pull-down assays. Caki cells were treated with or without melatonin, and cell lysates were precipitated with anti-Bim antibodies. Melatonin treatment strongly induced ubiquitination of Bim due to proteasomal inhibition (Fig. 6C). In addition, the pull-down assay using p62 UBA beads represented ubiquitination of Bim in melatonin-treated Caki cells (Fig. 6D). These data clearly supported our hypothesis that sustained Bim protein stability is mediated by melatonin's proteasome inhibitory action.

image

Figure 6. Melatonin inhibits proteasome activity. (A) Caki cells were transfected with proteasome sensor vector (ZsProSensor-1) and treated with melatonin or MG132 (as a positive control) for 24 hr. Proteasome activity was observed under a fluorescence microscope and analyzed using FACS analysis. (B) Caki cells were treated with the indicated concentrations of melatonin or MG132 for 24 hr. The cells were lysed, and proteasome activity was measured as described in the Materials and Methods section. (C) Caki cells were treated with 1 mm melatonin and lysed. Cell lysates were subjected to immunoprecipitation (IP) with an antibody to Bim. Immunoprecipitates were immunoblotted (IB) with ubiquitin (Ub) or Bim antibody. (D) Bim ubiquitination was assessed by p62 UBA pull-down as described in the Materials and Methods section. *< 0.001 compared to control. The data represent three independent experiments.

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Melatonin is a very powerful antioxidant agent [3, 4]. Therefore, to verify the relevance of antioxidant activity on Bim induction, we tested other antioxidant reagents, N-acetylcysteine (NAC) and trolox, on Bim induction. Caki cells were treated with the indicated concentrations of NAC or trolox, and then, western blot analysis and RT-PCR were performed. The effective concentrations of NAC or trolox as antioxidants did not change Bim expression at both the protein and mRNA levels (Fig. 7A, B). Several previous studies have shown that some of the physiological actions of melatonin are mediated by its interaction with the melatonin receptors MT1 and MT2, which belong to the superfamily of G protein-coupled receptors [31, 32]. Therefore, to elucidate the involvement of the interaction between melatonin and its receptors in the melatonin-induced increase in Bim expression, we treated Caki cells with melatonin in the presence or absence of luzindole, a melatonin receptor antagonist. As shown in Fig. 7(C), luzindole had no effect on Bim upregulation by melatonin. We also examined the effect of pertussis toxin (PTX), a Gαo and Gαi protein inhibitor, on melatonin-treated Caki cells. PTX had no effect on melatonin-induced Bim upregulation (Fig. 7D). Taken together, these data suggest that melatonin-induced Bim upregulation is independent of the antioxidant function of melatonin and melatonin receptor-mediated signal pathways.

image

Figure 7. Effect of antioxidants or luzindole (melatonin receptor antagonist) on melatonin-induced Bim expression. (A,B) Caki cells were treated with the indicated concentrations of NAC or trolox for 24 hr. (C) Caki cells were pretreated with the 10 μm luzindole for 30 min and then added 1 mm melatonin for 24 hr. (D) Caki cells were pretreated with the pertussis toxin (PTX) for 30 min and then added 1 mm melatonin for 24 hr. Bim and actin protein expressions were determined using western blotting (A, C, and D). Bim and actin mRNA expressions were determined using RT-PCR (B). The data represent three independent experiments.

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Taken together, our results demonstrate that melatonin induces apoptosis through upregulation of Bim expression at the transcriptional and post-translational levels.

Discussion

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

Melatonin is secreted by the pineal gland, and among a variety of functions, it influences circadian rhythms [1]. Moreover, melatonin exerts anti-inflammatory and antioxidant properties and interacts with the immune system. In spite of numerous studies documenting the beneficial effects of melatonin in cancer therapy, there is a need for mechanistic clarification related to the differential responses of normal and cancer cells to melatonin in terms of apoptosis regulation. In this study, we examined the apoptotic effects of melatonin in cancer cells. The most important observations made herein are as follows: (i) Melatonin induces apoptosis in Caki human renal cancer cells; (ii) melatonin induces Bim expression, but not the expression of Bax, Bcl-2, Mcl-1, and Bcl-xL; (iii) upregulation of Bim expression is crucial for melatonin-induced apoptosis; (iv) upregulation of melatonin-mediated Bim mRNA expression is mediated by Sp1 and E2F1 activation; and (v) inhibition of proteasome activity by melatonin is related to upregulation of Bim expression at the post-translational levels.

Melatonin has been reported to be an anticancer agent. In cancer cell lines, melatonin inhibits proliferation and suppresses cancer cell growth and also induces apoptosis [6-8]. Furthermore, melatonin enhances ER stress-mediated apoptosis [33] and sensitizes anticancer drug-induced apoptosis [10, 11, 34, 35]. In our studies, we investigated whether melatonin activates caspase-dependent apoptosis in Caki cells. As shown in Fig. 1, melatonin significantly activated caspase-3 activity and apoptosis in melatonin-treated cells. The mechanisms of apoptosis induced by various signals are mainly associated with apoptosis-related molecules, such as Bcl-2 family proteins, IAP proteins, and death receptors [36, 37]. We focused on the Bcl-2 family of pro- and anti-apoptotic proteins and found that Bim is markedly upregulated in melatonin-treated cells (Fig. 2B). Bim is one of the Bcl-2 family BH3-only proteins, and Bim interacts with other Bcl-2 proteins, such as Bcl-2, Bcl-xL, and Mcl-1, to antagonize their anti-apoptotic activities, leading to apoptosis.

Bim is modulated by several transcription factors. For example, forkhead transcription factor (FOXO) is an important transcription factor for Bim expression. Hypophosphorylated FOXO3a is associated with upregulation of Bim expression and apoptosis in paclitaxel-mediated apoptosis in breast cancer cells [38], and FOXO4-mediated Bim expression is also involved in reactive oxygen-mediated apoptosis in endothelial progenitor cells [39]. Recently, Carbajo-Pescador et al. [26] reported that melatonin increased Bim expression via FOXO3a activation in human liver carcinoma HepG2 cells. Melatonin inhibited Akt phosphorylation, which has been known as a main regulator of FOXO3a activation, and then increased Bim expression [26]. However, melatonin had no effect on Akt phosphorylation, and FOXO3a did not induce Bim transcription in Caki cells (data not shown). Therefore, these data indicated that the mechanisms of Bim expression by melatonin are cell type dependent. Second, Sp1 regulates Bim expression, and the Bim promoter has multiple binding sites for the Sp1 transcription factor [25]. Melatonin markedly increased Sp1 expression, and Sp1 induced Bim mRNA and protein expression (Fig. 4A, B). As shown in Fig. 4(A), melatonin also increased phosphorylation of Sp1 (T453), which is associated with an increased transcriptional activity [40]. E2F1 is associated with Bim expression and increased Bim expression in NIH3T3 cells [28], and histone deacetylase inhibitor-mediated E2F1 transcriptional activation also induced Bim expression in HCT116 cells [41]. In our study, melatonin increased E2F1 expression and phosphorylation at S364 (Fig. 4C). Phosphorylation of E2F1 (S364) increased E2F1 protein stability and transcriptional activity [40]. Melatonin-mediated E2F1 induction is involved in Bim expression in Caki cells (Fig. 4D). Therefore, Sp1 and E2F1 at least play a critical role in melatonin-mediated Bim expression in Caki cells.

In our study, while melatonin increased Bim protein expression within 3 hr (Fig. 5A), Bim mRNA expression was induced within 9 hr (Fig. 3A). These results indicated that another pathway, which is triggered by melatonin, is associated with Bim protein expression. Bim is regulated at the post-translational levels and transcriptional levels [20]. Bim degradation occurs via the ubiquitin-proteasome system, and phosphorylation at serine-69 (Ser-69) is important in Bim degradation [24, 30]. In our study, melatonin also modulated Bim protein stability (Fig. 5B). Furthermore, phosphorylation levels of Bim at Ser-69 were increased by melatonin treatment (data not shown). However, melatonin markedly inhibited proteasome activity and increased Bim ubiquitination (Fig. 6). Therefore, although melatonin increased Bim phosphorylation, melatonin induced Bim expression via the inhibition of proteasome activity.

Melatonin has a variety of functions on a wide range of physiopathological processes via receptor-mediated signaling and the free radical-scavenging/antioxidant processes [42-44]. However, the molecular mechanism mediated by melatonin-related pro-apoptosis is partially understood. In this study, we tested whether antioxidant function and receptor-mediated signaling were involved in melatonin-induced apoptosis. Although Caki cells expressed melatonin receptors MT1 and MT2 (Fig. S1), the apoptotic effect of melatonin was not associated with receptor-mediated signaling and antioxidant capacity of melatonin (Fig. 7). Further studies are required to delineate the precise mechanisms that are essential for the pro-apoptotic effect of melatonin.

Clinical trials involving melatonin is limited because melatonin itself has a very short average life in the blood (a range of 20–40 min, depending on the conditions) and bioavailability is very poor (10–56%) [45, 46]. However, Weishaupt et al. [47] suggested that high-dose (300 mg/day = 5 mg/kg) melatonin delayed disease progression and extended survival through the reduction in oxidative damage in patients with amyotrophic lateral sclerosis. In addition, oral administration of melatonin (100 µg/day/rat) via drinking water suppressed 7,12-dimethylbenzanthracene-induced mammary tumor growth [48]. A number of clinical and basic studies are required to establish the optimal clinical dose of melatonin in certain cancers.

In summary, our results provide mechanistic evidence that melatonin treatment results in Bim upregulation, resulting in apoptotic cell death in human renal cancer Caki cells. However, additional in vivo studies are required to establish the role of the apoptotic effect of melatonin.

Acknowledgements

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

This research was supported by a 2013 Scholar Research Grant from Keimyung University and Keimyung Basic Medical Research Promoting Grant launched from 2012.

References

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

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
jpi12102-sup-0001-FigureS1.TIFimage/tif100KFigure S1. Expression of melatonin receptors MT1 and MT2.

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