Jin-Sheng Wang, Ji-Fang Wen, Yong-Bin Hu, Hong-Zheng Ren, Department of Pathology, Xiangya Medical College, Central South University, Changsha 410008, Hunan Province, China
Jin-Sheng Wang, Cui-Lian Wang, Yong-Jin Wang, Department of Pathology, Changzhi Medical College, Changzhi 046000, Shanxi Province, China
Author contributions: Wang JS and Wang CL contributed equally to this work; Wang JS designed the research and wrote the manuscript; Wang CL analyzed the data by Western blotting and RT-PCR; Wen JF corrected the manuscript; Wang YJ dealt with the statistical data; Hu YB analyzed the results of cytobiology and immunofluorescence; and Ren HZ did cell culture.
Supported by The Innovation Project of Central South University, No. 2340-76208
Correspondence to: Ji-Fang Wen, Professor, Department of Pathology, Xiangya Medical College, Central South University, Changsha 410008, Hunan Province, China. email@example.com
Telephone: +86-731-2650400 Fax: +86-731-2650400
Received: February 23, 2008 Revised: May 10, 2008
Accepted: May 17, 2008
Published online: July 7, 2008
AIM: To investigate the effect of lithium on proliferation of esophageal cancer (EC) cells and its preliminary mechanisms.
METHODS: Eca-109 cells were treated with lithium chloride, a highly selective inhibitor of glycogen synthase kinase 3b (GSK-3b), at different concen-trations (2-30 mmol/L) and time points (0, 2, 4, 6 and 24 h). Cell proliferative ability was evaluated by 3-(4,5-Dimethylthiazole-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, and cell cycle distribution was examined by flow cytometry. Expressions of p-GSK-3b, b-catenin, cyclin B1, cdc2 and cyclin D1 protein were detected by Western blotting, and the subcellular localization of b-catenin was determined by immunofluorescence. The mRNA level of cyclin B1 was detected by reverse transcription polymerase chain reaction (RT-PCR).
Lithium could inhibit the proliferation of Eca-109 cells. Lithium at a
concentration of 20 mmol/L lithium for 24 h produced obvious changes in
the distribution of cell cycle, and increased the number of cells in G2/M
phase (P < 0.05 vs control group). Western blotting showed
that lithium inhibited GSK-3b
by Ser-9 phosphorylation and stabilized free
CONCLUSION: Lithium can inhibit the proliferation of human esophageal cancer cell line Eca-109 by inducing a G2/M cell cycle arrest, which is mainly mediated through the inhibition of lithium-sensitive molecule, GSK-3b, and reduction of cyclin B1 expression.
© 2008 The WJG Press. All rights reserved.
Key words: Lithium; Esophageal cancer; Cell cycle; Glycogen synthase kinase 3b
Peer reviewer: Satoshi Osawa, MD, First Department of Medicine, Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu 431-3192, Japan
Wang JS, Wang CL, Wen JF, Wang YJ, Hu YB, Ren HZ. Lithium inhibits proliferation of human esophageal cancer cell line Eca-109 by inducing a G2/M cell cycle arrest. World J Gastroenterol 2008; 14(25): 3982-3989 Available from: URL:
http://www.wjgnet.com/1007-9327/14/3982.asp DOI: http://dx.doi.org/10.3748/wjg.14.3982
Esophageal cancer (EC) is prevalent in some regions of the world, and occurs at a very high frequency in certain parts of China and the mortality rate ranked the fourth among cancer-related death[1,2]. However, the molecular basis of esophageal carcinogenesis remains poorly understood.
Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase that controls cell survival and cell fate through its involvement in multiple signaling pathways. Recent studies in colorectal cancer, pancreatic cancer, hepatocellular carcinoma and ovarian cancer[4-7] demonstrate that GSK-3 is involved in the process of tumorigenesis. Inhibition of the expression and activity of GSK-3 attenuates cell proliferation and causes apoptosis in colorectal, pancreatic and ovarian cancer cells[4,5,7].
Lithium has been shown to be a specific and noncompetitive inhibitor of GSK-3 activity in vitro[8,9] and in vivo. It has been proved that lithium could promote[11-14] or inhibit[7,15-17] cell cycle transition and proliferation of some primary cultures or cell lines by inhibiting GSK-3, depending on the cell type. However, whether lithium influences the growth and proliferation of EC cells remains unknown to date.
In this report, we chose Eca-109 cells as a model, and observed the potential role of lithium in cell cycle progression and growth of EC cells and investigated its preliminary mechanisms.
MATERIALS AND METHODS
Cell culture and treatment
Human esophageal squamous cell carcinoma cell line Eca-109 was obtained from Institute of Biochemistry and Cell Biology, Shanghai, China and maintained in RPMI 1640 medium (Gibco Biocult, Paisley, UK) supplemented with 10% calf bovine serum (Sijiqing Biotechnology, China), 100 U/mL penicillin and 100 g/mL streptomycin at 37℃ in a water-saturated atmosphere of 5% CO2 in air. For G0/G1 synchronization, when Eca-109 cells grew to 70% confluence, the routine medium was removed and replaced by free-serum medium for 24 h. Then these cells were cultured in the free-serum medium supplemented with 2-30 mmol/L lithium (Alexis, USA) for indicated times.
3-(4,5-Dimethylthiazole-2-yl) 2,5-diphenyl-tetrazolium bromide (MTT) assay
The IC50 of lithium on Eca-109 cells was measured by MTT assay, which was conducted as described before, and was calculated by Logit method. Briefly, one thousand Eca-109 cells (5 × 103/mL) were seeded in 96-well plates and cultured for 12 h. When they were adhesive, these Eca-109 cells were exposed to a range of concentrations of lithium from 2 to 30 mmol/L for 72 h, respectively. The Eca-109 cells treated with routine medium served as negative control. All exposures were performed in six wells. At the end of exposure, 20 L of MTT (Sigma, USA) stock solution (5 mg/mL) was added to 200 L of medium in each well and plates were incubated for 4 h at 37℃, and subsequently 150 L of dimethyl sulfoxide (DMSO) was added to each well. The plates were incubated about 10 min at room temperature and read with enzyme-linked immunosorbent assay (490 nm) to determine absorbance values (A). The rate of inhibition was calculated by the following equation: Rate of growth inhibition (%) = (1 - Atreated/Acontrol) × 100%.
the analysis was conducted by FCM, Eca-109 cells were exposed to lithium
at a concentration of 20 mmol/L for 0, 2, 4, 6 and 24 h, respectively.
According to the routine method, 1.0 × 106 Eca-109 cells from
the control and treated groups were harvested by trypsinization and
centrifugation, washed twice with ice-cold PBS and resuspended in PBS
containing 10 mg/L
Reverse transcription polymerase chain reaction (RT-PCR)
Total RNA was extracted from specimens using Trizol reagent (Invitrogene, USA) and treated with DNase I (Tiangen Biotechnology, China). cDNA was synthesized from 2 g of total RNA according to the manufacturer’s instruction (Fermentas, USA), and negative control reactions were run without reverse transcriptase. An equal volume of product was subjected to PCR. The levels of gene transcripts were quantified as the ratio of the intensity of the target gene to the intensity of -actin. The PCR primers used in this study were as follows: forward primer 5'-CCATTATTGATCGGTTCATGCAGA-3' and reverse primer 5'-CTAGTGCAGAATTCAGCTGTGGTA-3' for cyclin B1 (585 bp). -actin was amplified as internal control using the following primers: forward primer 5'-AGTTGCGTTACACCCTTTCTTG-3' and reverse primer 5'-TCACCTTCACCGTTCCAGTTT-3', with a 150 bp fragment product. The amplification conditions were 30 cycles of 94℃ for 50 s, 55℃ for 40 s, 72℃ for 40 s for cyclin D1, and 30 cycles of 94℃ for 50 s, 59℃ for 40 s, 72℃ for 40 s for cyclin B1 and -actin. PCR products were run on 1.6% agarose gel and results were analyzed using Image Tool 3.0.
Extraction of nuclear protein
Cells were lysed in 400 L of ice-cold buffer A [10 mmol/L HEPES (pH 7.9), 10 mmol/L KCl, 0.1 mmol/L ethylene diamine tetraacetic acid (EDTA), 0.1 mmol/L EGTA, 1 mmol/L DTT, 0.5 mmol/L PMSF] by gentle pipetting. The cells were allowed to swell on ice for 15 min, then 40 L of a 10% solution of Nonidet P-40 was added and the tube was vigorously vortexed for 10 s. The homogenate was centrifuged for 30 s in a microcentrifuge. The supernatant was transferred and the nuclear pellet was lysed with 50 L of buffer B [20 mmol/L HEPES (pH 7.9), 0.42 mol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L DTT, 1 mmol/L PMSF] and the tube was vigorously rocked at 4℃ for 15 min on a shaking platform. The nuclear extract was centrifuged for 5 min in a microcentrifuge at 4℃. The supernatant containing nuclear protein was stored at -70℃.
Cells were washed twice with ice-cold PBS, collected by adding 0.25% trypsin and lysed in buffer [50 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 1 mmol/L ethylene diamine tetraacetic acid (EDTA), 0.25% sodium deoxycholate, 1% TritonX-100, 0.1% sodium dodecyl sulfate (SDS), 1 mmol/L NaF, 1 mmol/L Na3VO4], protease inhibitors (10 mg/L aprotinin and 1 mmol/L phenylmethylsulfonyl fluoride) were added to obtain whole cell protein. Equal amounts of cell protein, quantified by BCA protein assay kit (Pierce Biotechnology, Rockford, IL), were subjected to 10% SDS-polyacrylamide gel electrophoresis, and then transferred to polyvinylidene difluoride membrane. The membranes were blocked with 5% non-fat milk in TBST [50 mmol/L Tris-HCl (pH 7.6), 150 mmol/L NaCl, 0.1% Tween 20] for 2 h at room temperature, and subsequently incubated with primary antibody (anti-p-GSK3, 1:400, anti-cyclin D1, 1:200, anti-cyclin B1, 1:400, anti-cdc2, 1:500, -actin, 1:2000, were all purchased from Sant Cruz Biotechnology, USA, and anti--catenin, 1:400, was purchased from Chemicion Biotech, USA) in blocking buffer at 4℃ overnight. Following a wash with TBST, the membranes were incubated with horseradish peroxidase conjugated rabbit anti-mouse secondary antibody (1:1000, Dako, Denmark) for 2 h at room temperature. The membranes were washed with TBST and protein bands were visualized by enhanced chemiluminescence according to the manufacturer’s instructions (KPL, USA). The -actin bands were taken as loading control. The protein quantity was analyzed by UTHSCSA Image Tool 3.0. The target protein expression was evaluated by the relative intensity ratio of target protein/loading control.
were plated on the coverslips which had been put into the six-well
plates in advance. After being treated with 20 mmol/L lithium for the
indicated time as described above, cells were washed with ice-cold
phosphate-buffered saline (PBS) prior to fixing with 4% paraformaldehyde
for 15 min at room temperature. Next, cells were treated with 1 g/L
TritonX-100 for 30 min
The data were obtained from at least three repeated experiments and expressed as mean ± SD. In order to compare the data between the treated groups and the control group, statistical significance was analyzed through analysis of variance (ANOVA) and values of P < 0.05 were considered significant.
Lithium inhibits proliferation of Eca-109 cells
The mean IC50 of lithium was 21.7 ± 0.5 mmol/L. The effect of inhibition of lithium on Eca-109 cells could be observed at a concentration of 2 mmol/L. From 2 to 30 mmol/L, the inhibitory effect was enhanced along with the increased concentration of lithium. The exhibition of dose-response relationship could be observed (Figure 1).
The growth curves of Eca-109 cells within 5 d after treatment with lithium were also plotted according to the data obtained from the MTT assay (Figure 2). The Eca-109 cells treated with different concentrations of lithium showed much slower growth than the untreated cells. The results indicated that the proliferation of Eca-109 cells in vitro was inhibited compared to that of the untreated cells.
Lithium induces a G2/M cell cycle arrest in Eca-109 cells
To determine whether the lithium-induced inhibition of proliferation of Eca-109 cells was due to altered cell cycle regulation, Eca-109 cells were treated with lithium for various times, and cell cycle profiles were monitored by flow cytometric analysis of DNA content (Figure 3 and Table 1). The percentage of cells in G0/G1 phase decreased with the length of treatment from 59.6% at 0 h to 22.1% at 24 h, and the percentage of cells in S phase increased accordingly from 28.5% at 0 h to 42.5% at 6 h. At 24 h, while the cells were entering from S phase to G2/M phase, the percentage of cells in S phase decreased from 42.5% at 6 h to 31.5% at 24 h, but the percentage of cells in G2/M phase was increased markedly. The distribution in the phases of cell cycle indicated that lithium could promote Eca-109 cells entering into S phase and then G2/M, in which the population was increased by approximate 4 folds compared with that of the untreated group at 24 h. These results suggested that the growth-inhibitory effect of lithium on Eca-109 cells might be partly due to its ability to induce G2/M cell cycle arrest.
Lithium induces a G2/M arrest by decreased cyclin B1 expression
established that lithium induces a G2/M
cell cycle arrest, we attempted to characterize, at the molecular level,
the mechanisms by which this effect is achieved. Since the cyclin
B1/cdc2 complex is a master intracellular regulator entry into mitosis,
we therefore investigated the effects of lithium in Eca-109 cells on key
regulators of the G2 to
M phase transition, cdc2 and cyclin B1 using Western blotting. Treatment
for 24 h with lithium reduced the protein expression of cyclin B1
Lithium induces b-catenin stabilization via inhibition of GSK-3b
Inhibition of GSK-3 (GSK-3 Ser-9 phosphorylation) was assessed by Western blotting using a phospho-Ser-9-specific antibody. As shown in Figure 5A, 20 mmol/L lithium increased the phosphorylated inactive form of GSK-3 in Eca-109 cells. Moreover, at a range of 2-30 mmol/L, this effect of lithium was dose-dependent (data not shown). -catenin, a signaling molecule in the Wnt/-catenin signaling pathway, is a target of GSK-3. Inactivation of GSK-3 by Wnt signaling or by lithium leads to stabilization and nuclear translocation of -catenin. Here, we investigated the expression of -catenin by Western blotting of total and nuclear extracts from Eca-109 cells treated with lithium. Results indicated that the intracellular total protein concentrations of -catenin were increased following stimulation with 20 mmol/L lithium (Figure 5A). An increase of -catenin nuclear pool was also observed in Eca-109 cells, exhibiting the similar trend (Figure 5B). In contrast to the untreated cells, in which -catenin was primarily located in the cytoplasm, -catenin was predominantly located in the nuclear of the lithium-treated Eca-109 cells (Figure 5C). These results showed that lithium could induce -catenin nuclear translocation by inhibition of GSK-3 activity.
Lithium up-regulates intranuclear cyclin D1 levels
GSK-3 has been shown to regulate cyclin D1 proteolysis by directly phosphorylating cyclin D1 at Thr-286[22,23]. We hypothesized that inhibition of GSK-3 by lithium would be associated with an increase in cyclin D1 protein. As shown in Figure 6, lithium treatment induced a slight increase in the cyclin D1 protein levels by 1.3-fold at 4 h and 1.8-fold at 6 h of lithium treatment as compared with control cells, and the cyclin D1 protein levels diminished to the normal level at 24 h, which may be attributed to the decrease of cells in G0/G1 phase.
report, we treated EC cell line Eca-109 cells with different
concentrations of lithium and in different time points, and observed the
effect of lithium on EC cells. Results indicated that lithium inhibited
the proliferation of Eca-109 cells (Figures 1
Inhibition of cell proliferation can occur through activation of several possible checkpoints during cell cycle progression. Lithium is a highly selective inhibitor of GSK-3, a multifunctional serine/threonine kinase which has a variety of putative substrates including cyclin D1, p21Waf1/Cip1, and transcription factors like c-myc, c-jun and -catenin, which are implicated in the regulation of cell proliferation. It is well known that intracellular -catenin levels are regulated through GSK-3 mediated phosphorylations on its serine and threonine residues (at Ser-33, Ser-37 and Thr-41). Inhibition of GSK-3 activity by lithium can result in -catenin stabilization and its accumulation in the nucleus. Our Immunoblot results showed that in Eca-109 cells lithium inhibited GSK-3ß by Ser-9 phosphorylation and stabilized free -catenin in the cytoplasm (Figure 5A). An increase of -catenin nuclear pool was also observed (Figure 5B). Immunofluorescence studies further confirmed that free -catenin translocated to the nucleus where -catenin was transcriptionally active (Figure 5C). Although inhibition of cell proliferation by -catenin signaling has not been described to date, -catenin may be the signal for cell cycle arrest. Orford et al showed that the nuclear localization of -catenin was cell cycle regulated in the epithelial Madin-Darby canine kidney cells with a peak during the S phase. Interestingly, with the percentage of cells in S phase increased, -catenin expression increased accordingly in our experiments (Table 1 and Figure 5). Damalas et al reported that the accumulation of p53 in mouse fibroblasts NIH3T3 overexpressed a stable form of -catenin (S37A). Stabilization of p21Waf1/Cip1 and induction of G2/M cell cycle arrest by lithium was demonstrated in bovine aortic endothelial cells, where lithium up-regulated p21Waf1/Cip1 protein level through activation and stabilization of p53, an effect of lithium possibly associated with GSK3, as p53 was recently shown to be a substrate of this kinase. Furthermore, lithium was shown to stabilize p21Waf1/Cip1 by inhibiting GSK-3 activity, and induce G2/M cell cycle arrest in human umbilical vein endothelial cells. It is thus possible that a sustained retention of -catenin in the nucleus during G2 or G2/M transition can be a signal for p53 induction and cell cycle arrest.
cyclin B1/cdc2 complex is a master intracellular regulator entry into
cell cycle arrest may be associated with decreased expression or
activity of this complex. Recently, Smits et al
observed a G2/M
cell cycle arrest in various transformed (P19 embryonal carcinoma, U2OS
osteosarcoma, and SK-N-MC neuroepithelioma) or immortalized (NIH3T3)
cell lines after lithium treatment. They found that the activity
of the cyclin B1/cdc2 complex was impaired after lithium treatment due
to sustained phosphorylation of cdc2 on tyrosine residue 15, and
the reduction in cyclin B1/cdc2
Previously, it was reported that phosphorylation of cyclin D1 at Thr-286 by GSK-3 regulates positively proteasomal degradation of cyclin D1. Therefore, inactivation of GSK-3 was expected to lead to an increase of cyclin D1 protein. Our results agreed well with this rationale. Lithium treatment resulted in the inactivation of GSK-3 and induced a slight increase of cyclin D1 protein at 4 and 6 h (Figure 6). These results indicated that cyclin D1 might be involved in the transition of cell cycle from G1 to S phase in Eca-109 cells induced by lithium. Chen et al observed that lithium treatment increased cyclin D1 expression and induced a G2/M cell cycle arrest in pig airway epithelial cells, which was consistent with our results. However, they failed to find evidence of G1/S transition. Mao et al also observed a slight increase of cyclin D1 and G2/M cell cycle arrest in lithium-treated bovine aortic endothelial cells. On the contrary, a recent investigation on NIH3T3 cells indicated that the change of GSK-3 activity by lithium had no effect on cyclin D1 expression. The causes of the discrepancy from these data might lie in the different experimental methods and different cell lines as well.
In conclusion, our present study demonstrated, for the first time, that lithium can arrest the growth of EC cells and induce a G2/M cell cycle arrest, which is mainly mediated through the inhibition of lithium-sensitive molecule, GSK-3, and reduction of cyclin B1 expression. However, further studies are needed to determine the precise mechanisms that contribute to the regulation.
Esophageal squamous cell carcinoma (ESCC) is one of the leading causes of cancer-related death in certain parts of China, and the molecular basis of esophageal carcinogenesis remains poorly understood. It is necessary to develop effective new strategies for the treatment of esophageal cancer (EC).
Recent researches have proved that lithium could promote or inhibit cell cycle transition and proliferation of certain primary cultures or cell lines by inhibiting GSK-3, depending on the cell type. However, whether lithium influences the growth and proliferation of EC cells remains unknown to date.
Innovations and breakthroughs
This study demonstrated for the first time that lithium can inhibit the proliferation of esophageal squamous cell carcinoma cells (Eca-109), which is mainly mediated by the inhibition of GSK-3 and reduction of cyclin B1 expression.
This study indicates the possibility for the treatment of the esophageal squamous cell carcinoma with lithium cloride.
Lithium is an inhibitor of GSK-3 activity. Lithium has been reported to reduce GSK-3 activity in two ways, both directly and by increasing the inhibitory phosphorylation of GSK-3. These dual effects can act in concert to magnify the influence of lithium on crucial GSK-3-regulated functions (gene expression, cell structure and survival).
The purpose of this study is reasonable and results are clearly demonstrated, however, further studies are needed to determine the precise mechanisms that contribute to the regulation.
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