J. Pineal Res. 2012
2011 John Wiley & Sons A/S
Journal of Pineal Research
Molecular, Biological, Physiological and Clinical Aspects of Melatonin
Doi:10.1111/j.1600-079X.2011.00946.x
Melatonin sensitizes human hepatoma cells to endoplasmic reticulum stress–induced apoptosis Abstract: Endoplasmic reticulum stress–mediated cell apoptosis is implicated in the development of cancer. Melatonin induces apoptosis in hepatocellular carcinoma (HCC) in experimental studies, but the effects of melatonin on endoplasmic reticulum (ER) stress–induced apoptosis in HCC have not been tested. Differences in ER stress–induced apoptosis in human hepatoma cells and normal human hepatocyte were investigated by exposure to tunicamycin (ER stress inducer). Significant differences were observed in the rate of apoptosis between HepG2 cells (hepatoma cells) and HL-7702 cells (normal human hepatocyte cells). The expression of cyclooxygenase-2 (COX-2) was increased in HepG2 cells but not in HL-7702 cells. Furthermore, downregulation of COX-2 expression using the COX-2 inhibitor, celecoxib, increased tunicamycin-induced apoptosis concomitant with the upregulation of pro-apoptotic transcription factor CHOP (GADD153) and down-regulation of B-cell lymphoma 2/Bcl-2–associated X protein (Bcl-2/ Bax) ratio, suggesting that inhibition of COX-2 sensitized human hepatoma cells to ER stress–induced apoptosis. Interestingly, co-treatment with tunicamycin and melatonin also decreased the expression of COX-2 and significantly increased the rate of apoptosis by elevating the levels of CHOP and reducing the Bcl-2/Bax ratio. These results demonstrate that melatonin sensitizes human hepatoma cells to ER stress–induced apoptosis by downregulating COX-2 expression, increasing the levels of CHOP and decreasing the Bcl-2/Bax ratio.
Lixia Zha1, Lulu Fan1, Guoping Sun1*, Hua Wang2*, Tai Ma1, Fei Zhong1 and Wei Wei3 1
Department of Oncology, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China; 2Department of Oncology, The Affiliated Provincial Hospital of Anhui Medical University, Hefei, Anhui, China; 3Institute of Clinical Pharmacology, Anhui Medical University, Hefei, Anhui, China
Key words: apoptosis, cyclooxygenase-2, endoplasmic reticulum stress, hepatocellular carcinoma, melatonin Address reprint requests to Dr. Guoping Sun, Department of Oncology, The First Affiliated Hospital of Anhui Medical University, Hefei 230022, China. E-mail:
[email protected] or Dr. Hua Wang, Department of Oncology, The Affiliated Provincial Hospital of Anhui Medical University, Hefei, Anhui, 230001, China. E-mail:
[email protected] *These authors contributed equally to this work. Received October 3, 2011; Accepted November 23, 2011.
Introduction The accumulation of unfolded proteins in the ER represents a cellular stress induced by multiple stimuli including hypoxia, oxidative injury, hypoglycemia, protein inclusion bodies, and viral infections [1]. ER stress triggers an evolutionarily conserved response termed the unfolded protein response (UPR). In the event of prolonged or severe ER stress, the UPR initiates apoptosis [2–4]. ER stress–induced apoptosis has been implicated in a wide variety of human diseases including AlzheimerÕs disease, Parkinson disease, diabetes mellitus, and cancer [5]. For example, rapidly growing tumors, which outstrip the vascular supply of oxygen, leading to hypoxia, can also induce ER stress [6]. However, the ER stress response represents an adaptive mechanism that supports survival and chemoresistance of tumor cells [7, 8]. Therefore, agents that increase ER-mediated apoptosis could define a new
strategy of cancer therapy, preventing adaptation of tumors to hypoxic environments. Melatonin (N-acetyl-5-methoxytryptamine) is produced in the pineal gland during the dark phase of the light– dark cycle [9, 10]. Melatonin influences important physiological functions including seasonal reproduction and circadian rhythms [11, 12]. Additionally, melatonin has been found to display oncostatic actions [13–15]. Known antitumor actions of melatonin relate in part to its ability to induce cell apoptosis [16–18]; however, studies related to the effect of melatonin on ER stress–induced apoptosis in HCC are limited. In this study, we have explored the role of COX-2 in the protection of human hepatoma cells from ER stress–induced apoptosis and the effects of melatonin on the sensitivity of human hepatoma cells to apoptosis induced by the ER stress inducer tunicamycin through inhibiting the expression of COX-2, increasing the levels of CHOP (GADD153), and decreasing the 1
Zha et al. B-cell lymphoma 2/Bcl-2–associated X protein (Bcl-2/ Bax) ratio.
added in the dark at room temperature for 30 min. A minimum of 1 · 106/mL cells for each group were analyzed using an EPICS XL-MCL model counter (Beckman Coulter, Fullerton, CA, USA).
Materials and methods Reagents
TUNEL assay
Melatonin, tunicamycin(TM), 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT), Rnase A, and propidium iodide (PI) were obtained from Sigma Chemical (St. Louis, MO, USA). The COX-2 inhibitor, celecoxib, was obtained from Pfizer Corporation (New York, NY, USA). DMEM and RPMI-1640 medium were obtained from Gibco BRL Life Technologies (Grand Island, NY, USA). Anti-GRP78, anti-CHOP, anti-Bcl-2, anti-Bax and anti-b-actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-COX-2 antibody was purchased from Abcam (Cambridge, MA, UK). Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) system was from Roche (Indianapolis, IN, USA). The S-P Reagents Kit was obtained from Beijing Zhongshan Biotech (Zhongshan, Beijing, China).
Cells were cultured in 6-well plates on coverslips overnight. After treatment with various concentrations of the indicated compounds for each time period, the coverslips were washed twice with cold PBS and fixed in 4% paraformaldehyde solution for 1 hr at room temperature. Apoptotic cells were detected by TUNEL assay (TUNEL System Kit from Roche), which was performed according to the manufacturerÕs instructions. The TUNEL assay results were quantitatively analyzed through the biological image analysis system from the Nikon ECLIPSE 80i biology microscope, Nikon Digital Camera DXM 1200F, ACT-1 version 2.63 software (Japan).
Cell culture Human hepatoma cell line (HepG2) and human hepatocyte cell line (HL-7702) were purchased from Shanghai cell bank (Chinese Academy of Sciences [Shanghai, China]) and cultured in DMEM and RPMI-1640 medium, respectively. HepG2 cells were supplemented with 10% (v/v) heatinactivated fetal bovine serum (FBS), 100 unit/mL of penicillin and 100 lg/ml of streptomycin. HL-7702 were supplemented with 20% (v/v) heat-inactivated FBS and antibiotics. Cultures were maintained in a humidified incubator at 37C in 5% CO2.
Western blotting After drug treatment for the indicated time periods and concentrations, cells were lysed in RIPA lysis buffer (50 mm TRIS (tris (hydroxymethyl) aminomethane)–HCl, (pH 7.4), 150 mm NaCl, 10 mm phenylmethylsulfonyl fluoride (PMSF), 1 mm ethylene diamine tetraacetic acid (EDTA), 0.1% sodium dodecyl sulfate (SDS), 1% Triton X-100, 1% sodium deoxycholate) for 20–30 min on ice. Protein concentrations were determined through the Lowry protein assay. Lysates were incubated with 2 · Laemmli sample buffer (Bio-Rad, Hercules, CA, USA) and heated for 10 min at 95C. The proteins were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA,
MTT assay
HepG2 48 hr
80 70
Inhibitory rate (%)
Cells were cultured at a density of 1 · 104 cells/well in a 96well plate. After treatment with various concentrations of drug for the indicated time periods, MTT solution (5.0 mg/ mL in phosphate buffered saline) was added (20.0 lL/well), and the plates were incubated for another 4 hr at 37C. The purple formazan crystals were dissolved in 150 lL of dimethyl sulfoxide (DMSO) per well. After 10 min, the plates were read on ELX800 universal microplate reader (Bio-Tek Instruments Inc., Winosski, VT, USA). Assays were performed in three independent experiments.
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Flow cytometry The cells were grown in 6-well plates and then treated with desired concentrations of the indicated compounds. After exposure to the indicated compounds for specific time periods, cells were trypsinized, washed twice with cold PBS, and centrifuged. The cell pellet was resuspended in 1 mL cold PBS and fixed in 9 mL of 70% ethanol at )20C for at least 12 hr. Then, cells were centrifuged and resuspended in 500 lL PBS, and RNase A was added and incubated at 37C for 30 min. Propidium iodide (PI) staining buffer was 2
0 0
1.5
9
12
TM concentration ( µ M) Fig. 1. Effect of tunicamycin treatment on cell viability in HepG2 and HL-7702 cells. HepG2 and HL-7702 cells were treated with different concentrations of tunicamycin (1.5, 3, 6, 9, 12 um) for 48 hr. Cell viability of HepG2 and HL-7702 cells was determined by the MTT assay. Data are expressed as the mean ± S.D. of three independent experiments (bars represent S.D.). (** P < 0.01, compared with untreated HL-7702 cells or HepG2 cells; ## P < 0.01, compared with HL-7702 cells).
Melatonin sensitizes hepatoma cells to ER stress USA), and incubated with blocking buffer (Tris-buffered saline/Tween 20 (TBST)/5% nonfat dry milk) overnight at 4C. Immunoblots were incubated with the indicated primary antibody followed by the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody and visualized with enhanced chemiluminescence (ECL, Pierce, Rockford, IL, USA) using hydrogen peroxide and luminol as substrate with Kodak X-AR film. Autoradiographs were scanned using a GS-700 Imaging Densitometer (BioRad).
256
(A)
Cells were seeded onto glass coverslips overnight. After incubation with various concentrations of the compounds for the indicated time periods, the coverslips were washed twice with PBS and a 4% paraformaldehyde solution was added and incubated for 30 min at room temperature. Immunohistochemical staining for Bcl-2 and Bax was performed according to the standard S-P method described in the procedure program of the S-P Reagents Kit. PBS
(b) AP = 28.72 ± 4.21%
0
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Events
Events
512
(a) AP = 4.43 ± 0.4%
Immunohistochemistry
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Events
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Perent apoptotic cell (%)
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Fig. 2. Effect of tunicamycin treatment on cell apoptosis in HepG2 and HL-7702 cells. (A)HepG2 and HL-7702 cells were treated with 3 um tunicamycin (TM) for 48 hr. Sub-G1 analysis in HepG2 and HL-7702 cells were determined by fluorescence-activated cell sorting (FACS), and the data are expressed as the mean ± S.D. of three independent experiments. (a) Untreated HL-7702 cells; (b) HL-7702 cells treated with tunicamycin; (c) untreated HepG2 cells; (d) HepG2 cells treated with tunicamycin. (B) and (C) Cell morphology and percentage of apoptotic cells were examined by TUNEL staining. In this image, the cells with brown nuclei are apoptotic cells. (a) Untreated HL-7702 cells; (b) HL-7702 cells treated with tunicamycin; (c) untreated HepG2 cells; (d) HepG2 cells treated with tunicamycin. Data are presented as mean ± S.D. of three independent experiments (bars represent S.D.). (**P < 0.01, compared with untreated HL-7702 cells or HepG2 cells; ## P < 0.01, compared with HL-7702 cells treated with tunicamycin).
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Zha et al. was used as a negative control to replace the primary antibody. The immunohistochemical results were quantitatively analyzed by the Biological Image Analysis System (Yokohama, Kanagawa, Japan), which consisted of a Nikon ECLIPSE 80i biology microscope, Nikon Digital Camera DXM 1200F, and ACT-1 version 2.63 software (Yokohama, Kanagawa, Japan), and JEOA 801D Morphologic Biological Image Analysis software, version 6.0 (Jie Da Technologies, Nanjing, Jiangsu, China). The samples were observed in 6 randomly selected optical fields by microscopy (·400), and an average optical density value was measured.
Statistical analysis Three or more separate experiments were performed for each experiment. Statistical analysis was performed by StudentÕs t-test or ANOVA. Data are presented as means ± standard deviation (S.D.). Significance was noted at P < 0.05.
Results To determine the effect of the ER stress inducer, tunicamycin, on cell viability, HepG2 cells (a hepatocellular **
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GRP78
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celecoxib concerntration ( µM)
GRP78 COX-2
3 hr
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Fig. 4. Effect of co-treatment with tunicamycin and celecoxib on cell viability in HepG2 cells. HepG2 were treated with different concentrations of celecoxib (0, 5, 10, 20, 40 um) in the presence tunicamycin for 48 hr, and cell viability in HepG2 cells was determined by MTT assay. The data are expressed as the mean ± S.D. of three independent experiments (bars represent S.D.) (** P < 0.01, compared with celecoxib = 0 um).
12 hr 24 hr
HepG2 0
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hr GRP78
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0.5 0 0 hr
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Fig. 3. Effect of tunicamycin treatment on the expression of COX2 and GRP78 in HepG2 and HL-7702 cells. HepG2 and HL-7702 cells were treated with 3 um tunicamycin (TM) for 0 hr (control) 3, 6, 9, 12, and 24 hr. Equal amounts of cell lysates were subjected to western blot assay using specific anti-GRP78 and anti-COX-2 antibody. b-Actin in the same HepG2 and HL-7702 cell extract was used as an internal reference. Optical density reading values of the specific protein versus the loading control protein b-actin are represented as fold of the control values (*P < 0.05, #P < 0.05 compared with untreated HL-7702 cells or HepG2 cells).
4
Fold change of as compared to controls
69 kD
Fold change of as compared to controls
COX-2
1.2 1 0.8
**
0.6 0.4 0.2 0 TM
celecoxib + TM
Fig. 5. Effect of co-treatment with tunicamycin and celecoxib on the expression of COX-2 in HepG2 cells. HepG2 cells were treated with 3 um tunicamycin in either the absence (control) or the presence of celecoxib for 24 hr. Equal amounts of cell lysates were subjected to western blot analysis using specific anti-COX-2 antibody. b-Actin in the same HepG2 cell extract was used as an internal reference. Optical density reading values of the specific protein versus the loading control protein b-actin are represented as fold of the control values (**P < 0.01, compared with HepG2 cells treated with tunicamycin alone).
Melatonin sensitizes hepatoma cells to ER stress carcinoma cell line) and HL-7702 cells (a human hepatocyte cell line) were treated with different concentrations of tunicamycin for 48 hr. Viability was assessed using the
FL3 Lin
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0.42%
Events
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MTT assay. Growth inhibition of tunicamycin on HL-7702 was dose dependent. But the growth inhibition of tunicamycin on HepG2 cells reached maximum inhibition in cells
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Fig. 6. Effect of co-treatment with tunicamycin and celecoxib on apoptosis in HepG2 cells. (A) HepG2 cells were treated for 24 hr with 3 um tunicamycin, either in the absence or in the presence of celecoxib. After 24 hr, apoptosis was analyzed as the sub-G1 fraction by fluorescence-activated cell sorting (FACS). (a) Untreated HepG2 cells; (b) HepG2 cells treated with tunicamycin; (c) HepG2 cells co-treated with 3 um tunicamycin and 10 um celecoxib. (B) and (C) Cell morphology and percentage of apoptotic cells were examined by TUNEL staining. a: Untreated HepG2 cells; b: HepG2 cells treated with tunicamycin; c HepG2 cells co-treated with 3 um tunicamycin and 10 um celecoxib. Data are presented as mean ± S.D. of three independent experiments. **P < 0.01 versus untreated HepG2 cells, ##P < 0.01 versus HepG2 cells treated with tunicamycin alone. (D) Cells were treated for 24 hr with 3 um tunicamycin, either in the absence (control) or in the presence of 10 um celecoxib and harvested in lysis buffer. Equal amounts of cell were subjected to western blot assay using specific anti-CHOP. b-Actin in the same HepG2 cell extract was used as an internal control. Optical density reading values of the specific protein versus the loading control protein b-actin are represented as fold of the control values. (**P < 0.01 versus HepG2 cells treated with tunicamycin alone). (E) Immunocytochemistry analysis of Bcl-2 and Bax. Quantitative analysis of Bcl-2 and Bax expression through immunohistochemistry analyses of Bcl-2 expression in HepG2 cells with treatment with 3 um tunicamycin (TM) in either the absence (a) or the presence of celecoxib (b). Immunohistochemistry analyses of Bax expression in HepG2 cells with treatment with 3 um tunicamycin (TM) in either the absence(c) or the presence of celecoxib (d) (**P < 0.01 versus HepG2 cells treated with tunicamycin alone).
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Zha et al.
6
80
**
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Inhibitory rate (%)
at 3 um (Fig. 1). Therefore, the concentration of 3 um tunicamycin was used to induce optimum ER stress in all subsequent experiments. To investigate the anti-/pro-apoptotic effects induced by tunicamycin, HepG2 and HL-7702 cells were exposed for 48 hr. Sub-G1 analysis was then conducted by fluorescenceactivated cell sorting analysis (FACS), and the morphological changes indicative of apoptosis was assessed by TUNEL staining. As shown in Fig. 2A, there was no significant increase in the apoptosis rate in HepG2 cells compared with that in HL-7702 cells. Similar results were observed in TUNEL staining (Fig. 2B,C). To further investigate the underlying mechanisms involved in protection against ER stress–induced apoptosis, changes in protein expression of COX-2 and GRP78 (a hallmark of ER stress) in both HepG2 and HL-7702 cells were observed through western blotting. As shown in Fig. 3A,B, administration of 3 um tunicamycin to HepG2 and HL-7702 cells induced an early increase in GRP78 expression in both HepG2 and HL-7702 cells, indicative of ER stress. The levels of COX-2 were undetected in HL-7702 cells. However, the expression of COX-2 in HepG2 cells rapidly increased after treatment with tunicamycin. These data suggest that COX-2 expression may be directly involved in adapting human hepatoma cells to ER stress– induced apoptosis. To confirm that the regulation of COX-2 enhances ER stress–induced apoptosis, HepG2 cells were treated with tunicamycin for 48 hr in the presence of celecoxib at various concentrations. Inhibition of cell growth was determined by the MTT assay. Celecoxib significantly reduced cell proliferation when co-treated with tunicamycin (Fig. 4). HepG2 cells were treated with 3 um tunicamycin with or without 10 um of celecoxib for 24 hr, and western blotting analysis was performed to detect the expression of COX-2. The expression of COX-2 was decreased in cells simultaneously treated with both celecoxib and tunicamycin (Fig. 5). Enhanced apoptosis by celecoxib was confirmed by FACS analysis (Fig. 6A) and TUNEL staining (Fig. 6B,C). Co-treatment of both celecoxib and tunicamycin significantly increased the percentage of sub-G1 phase cells and the number of TUNEL-positive cells indicative of apoptosis. CHOP (CCAAT/enhancer-binding protein homologous protein), also called GADD153, is one of the primary effectors of ER stress–mediated cell apoptosis. As shown in Fig. 6D, the expression of CHOP protein was markedly increased in the presence of celecoxib and tunicamycin. Similarly, the levels of the antiapoptosis factor, Bcl-2, were decreased and the levels of pro-apoptosis factor, Bax, were increased when cells were exposed to both celecoxib and tunicamycin. Bcl-2/Bax ratio also decreased (Fig. 6E). These data indicate that inhibition of COX-2 with celecoxib enhances TM-induced sensitization in HepG2 cells. To determine the effect of melatonin on ER stress– induced apoptosis, HepG2 cells were first treated with tunicamycin for 48 hr in the presence of melatonin at various concentrations. Exposure to both melatonin and tunicamycin generated significant cell death in HepG2 cells (Fig. 7). Enhanced cellular apoptosis with melatonin was confirmed by flow cytometry analysis. Melatonin signifi-
50
**
**
**
10–7
10–5
40 30 20 10 0 0
10–9
10–3
Melatonin concerntration (M) Fig. 7. Effect of co-treatment with melatonin and tunicamycin on apoptosis in HepG2 cells. HepG2 cells were treated with different concentrations of melatonin (0, 10)9, 10)7, 10)5, 10)3 um) in the presence of tunicamycin for 48 hr. Cell viability of HepG2 cells was determined by MTT assay. The data are expressed as the mean ± S.D. of three determinations in triplicate (** P < 0.01, compared with melatonin = 0 um).
cantly enhanced the sub-G1 phase in the presence of tunicamycin (Fig. 8A). Cellular morphological changes were assessed through TUNEL staining (Fig. 8B,C). In addition, the levels of CHOP (Fig. 8D) and the Bcl-2/Bax ratio were determined (Fig. 8E). Together these results supported the sensitizing effect of melatonin on ER stress– induced apoptosis. To determine whether COX-2 protein expression induced by tunicamycin could also be altered by melatonin treatment, western blot analysis was conducted. Melatonin significantly decreased the levels of COX-2 induced by tunicamycin (Fig. 9). These results suggest that melatonin could have an effect on ER stress–mediated apoptosis by targeting COX-2.
Discussion Hepatocellular carcinoma is the fifth most common disease in the world and the third largest cause of cancer-related death [19, 20]. The incidence of this tumor type ranges from approximately 10 cases per 100,000 population in North America and Western Europe to 50–150 cases per 100,000 population in Asia and Africa [21–24]. Drugs used in the treatment of HCC are cytotoxic, and none of them are specific for the management of this disease and involve a large risk of side effects [25]. Moreover, HCC is characterized by reduced sensitivity of the tumors to drugs. Despite the large body of research on HCC treatment, there is still controversy about the best therapeutic approach. Resistance to apoptosis is one of the fundamental hallmarks of cancer. Recently, the selective induction of apoptosis in cancer cells has emerged as an exciting possibility for the development of future selective cancer therapies [26]. A number of cellular stress conditions, such as nutrient deprivation, hypoxia, alterations in glycosylation status, and disturbances of calcium flux, lead to accumulation and
Melatonin sensitizes hepatoma cells to ER stress
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FL3 Lin
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(D)
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Perent apoptotic cell(%)
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FL3 Lin
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(a)
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0
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Fold changes
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Events
(b) AP = 10.89
Events
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(a) AP = 5.09
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Bcl-2
1.6 Bcl-2/Bax 50 µm
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50 µm
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1.2
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0 50 µm
50 µm
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Fig. 8. Effect of co-treatment with melatonin and tunicamycin on apoptosis in HepG2 cells. (A) HepG2 cells were treated for 24 hr with 3 um tunicamycin, either in the absence or in the presence of 10)7 um melatonin. After 24 hr, apoptosis was analyzed as the sub-G1 fraction by fluorescence-activated cell sorting (FACS). (a) Untreated HepG2 cells; (b) HepG2 cells treated with tunicamycin; (c) HepG2 cells co-treated with 3 um tunicamycin and 10)7 um melatonin. (B) and (C) Cell morphology and percentage of apoptotic cells were examined by TUNEL staining. a: Untreated HepG2 cells; b: HepG2 cells treated with tunicamycin; c HepG2 cells co-treated with 3 um tunicamycin and 10)7 um melatonin. Data are presented as mean ± S.D. of three independent experiments. **P < 0.01 versus untreated HepG2 cells, ## P < 0.01 versus HepG2 cells treated with tunicamycin alone. (D) Cells were treated for 24 hr with 3 um tunicamycin, either in the absence (control) or in the presence of 10)7 um melatonin and harvested in lysis buffer. Equal amounts of cell were subjected to western blot assay using specific anti-CHOP. b-Actin in the same HepG2 cell extract was used as an reference. Optical density reading values of the specific protein versus the loading control protein b-actin are represented as fold of the control values. (**P < 0.01 versus HepG2 cells treated with tunicamycin alone). (E) Immunocytochemistry analysis of Bcl-2 and Bax. Quantitative analysis of Bcl-2 and Bax expression by biological image analysis system. Immunohistochemistry analyses of Bcl-2 expression in HepG2 cells with treatment with 3 um tunicamycin (TM) in either the absence(a) or the presence of 10)7 um melatonin(b). Immunohistochemistry analyses of Bax expression in HepG2 cells with 3 um tunicamycin (TM) in either the absence (c) or the presence of melatonin (d). (**P < 0.01 versus HepG2 cells treated with tunicamycin alone).
aggregation of unfolded and/or misfolded proteins in the ER lumen, thereby leading to ER stress. The ER responds to stress conditions by activating a range of stress response signaling pathways, which is referred to as the UPR. The
UPR is fundamentally a cytoprotective response, but excessive or prolonged activation of the UPR can result in apoptosis [1–4]. Conditions that induce ER stress, such as hypoxia, nutrient deprivation, and changes in pH 7
Zha et al. TM
MT + TM 69 kD
β-actin
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Fold change of as compared to controls
COX-2
1.2 1 0.8 **
0.6 0.4 0.2 0
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Fig. 9. Effect of co-treatment with melatonin and tunicamycin on the expression of COX-2 in HepG2 cells. Whole-cell lysates from HepG2 cells with treatment with 3 um tunicamycin (TM) in either the absence (control) or the presence of 10)7 um melatonin for 24 hr were subjected to western blotting analysis. b-Actin in the same HepG2 cell extract was used as an internal reference. Optical density reading values of the specific protein versus the loading control protein b-actin are represented as fold of the control values (**P < 0.01 versus HepG2 cells treated with tunicamycin alone).
(acidosis), are frequently encountered in tumor cells [6]. However, many studies have shown that cancer cells have largely adapted to ER stress and are relatively resistant to ER stress–induced apoptosis [7, 8]. Our study indicates that cultured HepG2 cells do not undergo significant apoptosis when exposed to an extreme degree of ER stress induced by tunicamycin. However, at present, it is unclear how these tumor cells have adapted to long-term ER stress. It is not clear whether the protective elements of the response are enhanced, the destructive components are suppressed, or the compromised apoptotic machinery, a common occurrence in tumor cells, is sufficient to protect them from ER stress–induced apoptosis. What is currently known is that ER stress generates a cytoprotective response via the activation of survival and proliferation pathways [27–31]. COX-2 is the inducible form of cyclooxygenase, which is frequently elevated in cancer tissues. Therefore, COX-2 has been suggested to play a major role in tumorigenesis [32]. Recent studies have reported that COX-2 regulates multiple cellular processes including survival, proliferation, and apoptosis in cancer [33]. Upon ER stress, COX-2 is strongly activated in the mouse liver immortalized cell line ML-1 or human breast cancer cell line MCF-7 [34]. These reports suggest that the COX-2 is closely associated with ER stress induction. In view of this evidence, we considered whether cytoprotective responses induced by ER stress in hepatoma cells are attributed to the COX-2. If COX-2 expression is protective, then targeting COX-2 may enhance the sensitivity of hepatoma cells to ER stress–induced apoptosis. To test this hypothesis, we examined the COX-2 status of hepatoma cells during ER stress. Our results showed that COX2 was strongly increased upon ER stress induced by tunicamycin, but not in a human hepatocyte cell. Apoptosis induced by the combination of celecoxib and tunicamycin 8
was higher than that induced by tunicamycin alone. These data suggest that resistance of hepatoma cells upon ER stress may be due, at least in part, to the up-regulation of COX-2. Targeting COX-2 may be beneficial in restoring ER stress–induced apoptosis in human hepatoma cells. Moreover, the levels of CHOP were also dramatically increased and Bcl-2/Bax ratio was decreased by the combined treatment of celecoxib and tunicamycin, suggesting that the down-regulation of COX-2 in ER stress–mediated apoptosis is associated with the expression of CHOP and the Bcl-2/Bax ratio. However, the long-term use of selective COX-2 inhibitor may be limited owing to serious adverse effects, such as ulcer complications, bleeding, cardiovascular toxicity, platelet dysfunctions, and renal toxicity [35, 36]. Therefore, it is rational to search for specific inhibitors of COX-2 as potential candidates for use as new therapeutic agents. Melatonin is synthesized in the pineal gland and has an important role in influencing a variety of functions [9–12]. In addition to its well-known regulatory control of the sleep/wake cycle, as well as circadian rhythms, melatonin is involved in immunomodulation, hematopoiesis, and antioxidative processes [37–39]. Recent human and animal studies have now shown that melatonin also has important oncostatic properties. MelatoninÕs oncostatic properties depend on many mechanisms including antiproliferative effects, interaction with estrogen receptors, down-regulation of their expression, and antimetastatic effects [40–43]. Both at physiological and pharmacological doses, melatonin also exerts growth inhibitory effects on many cancer cell lines [44–49]. The anticarcinogenic effect of melatonin on neoplastic cells relies on its antioxidant, immunostimulating, and apoptotic properties [13]. Nevertheless, the effect of melatonin on ER stress–induced apoptosis has not been reported. Our results appear to provide several new insights into the effect of melatonin in cancer. Melatonin was shown to enhance ER stress–induced apoptosis, and the sensitizing effect of melatonin could be further supported by regulating COX-2, along with increasing the expression of CHOP and decreasing the Bcl-2/Bax ratio. In conclusion, the results of this study, for the first time, provide mechanistic evidence that melatonin shows sensitizing effects in ER stress–induced apoptosis on HCC cells. Our results raise the possibility that melatonin may be a promising approach in targeting ER stress–induced apoptosis as a therapeutic strategy for the treatment of HCC and other cancers. Presently, we do not know whether the sensitizing effects of melatonin in ER stress–induced tumor apoptosis in vitro also work well in vivo. Future studies will determine whether melatonin leads to ER stress–induced tumor regression in vivo in animal experiments and clinical trials. The utility of melatonin raises an interesting issue concerning the possible value of melatonin in clinical practice. Although some clinical trials have already utilized melatonin against cancer [50], and other diseases such as neurological disorders [51], cardiovascular diseases [52] and diabetes [53], the clinical utilization of melatonin in future studies still remains controversial and is already well discussed in previous reviews [54]. Owing to the low toxicity and well-documented oncostatic effects of melatonin, we
Melatonin sensitizes hepatoma cells to ER stress believe melatonin should have promising prospects in clinical practice in the near future, or at least as an adjuvant therapy to counteract the side effects as well as to enhance the efficacy of chemotherapy and radiotherapy.
Author contributions Lixia Zha and Lulu Fan performed the majority of experiments and wrote the manuscript; Guoping Sun designed the study and revised the manuscript; Dr. Hua Wang and Dr. Wei Wei revised the manuscript; and Tai Ma and Fei Zhong provided the vital reagents and technical assistance.
Acknowledgements This work was supported in part by the Natural Science Foundation of China (No.81071986). The authors would like to acknowledge Dr. Jiajia Li, the central laboratory of the First Affiliated Hospital of Anhui Medical University, for FACS analysis, and Dr. Wendi Zhao and Dr. Qingtong Wang, the Institute of Clinical Pharmacology of Anhui Medical University, for technical assistance.
Conflicts of interest The authors declare that there are no conflicts of interest.
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