Latex of Euphorbia antiquorum inhibited cell cycle S-phase arrest via
topoisomerase and ATM and Cdc25A signaling in human cervical
cancer cells
Wen-Tsong Hsieh
Department of Pharmacology, China Medical University, Taichung, Taiwan
Hui-Yi Lin
School of Pharmacy, China Medical University, Taichung, Taiwan
Jou-Hsuan Chen and Wen-Chung Lin
Department of Pharmacology, China Medical University, Taichung, Taiwan
Yueh-Hsiung Kuo
Tsuzuki Institute for Traditional Medicine, College of Pharmacy, China Medical University, Taichung, Taiwan
W. Gibson Wood
Department of Pharmacology, School of Medicine, Geriatric Research, Education and Clinical Center, VA Medical Center, University of Minnesota, Minneapolis, Minnesota, USA
Jing-Gung Chung
Department of Biotechnology, Asia University, Wufeng, Taichung, Taiwan, and Department of Biological
Science and Technology, China Medical University, Taichung, Taiwan
*Corresponding author at: Department of Biological Science and Technology, China Medical University, No 91, Hsueh-Shih Road, Taichung 40402, Taiwan, R. O. C. Tel: +886-4-2205 3366-2161; Fax: +886-4-2205 3764. E-mail address: [email protected]
Keywords: chemoprevention, EA, cervical cancer, cell cycle, Chk1/2, Cdc25C, Cdc2,
topoisomerase I, ATM/ATR.
Running title: Latex of Euphorbia antiquorum inhibited cell cycle S-phase arrest in
Abstract
Latex of Euphorbia antiquorum (EA) has demonstrated great chemotherapeutic potential for the treatment of cancer. However, the anticancer mechanisms of EA in human cervical cancer remain to be further investigated. In this study, we used human cervical cancer HeLa and CaSki cells as the models to investigate the effect of EA on cell cycle distribution and cell viability by flow cytometry. The data showed that EA induced cell morphological changes, decreased the percentage of viable cells. EA also induced S phase arrest in both HeLa and CaSki cervical cancer cells in a concentration manner. Western blot analysis on human cervical cancer HeLa cells showed that EA could down regulate the expression of cyclin A, cyclin B, cyclin E, cdk6 and Cdc25A, however, promoted the expression of p21waf1/cip1 and p27, those
result could lead to cell cycle arrest at S phase. Moreover, the data also showed that EA promoted of activation of ATM and Chk2 kinases, and negatively regulated the expression of Topoisomerases, Cdc25A, and Cdc25C signaling. Caffeine, an ATM/ATR inhibitor, significantly reduced EA-activated the levels of ATM and Cdc25A. Furthermore, sp600125, a JNK inhibitor, also significantly reduced the EA activation of Chk2 level. Based on these observations, we have demonstrated that EA induced S phase arrest in human cervical cancer through topoisomerase, ATM/ATR-Chk1/2-Cdc25A signalling pathway.
INTRODUCTION
Cervical cancer remains one of the most lethal gynecologic in woman worldwide .Chemotherapy is the same as surgery and radiation has been recommended as the standard therapy for cervical cancer .Cervical cancer using cisplatin showed favorable initial response as the primary therapy for locally advanced cervical cancer.. However, patients often develop tolerance, metastatic and recurrent on cisplatin-based chemotherapy . Naturally derived anticancer agents now have a relevant role for decreasing those side effects in contemporary models of combination with targeted agents as a valid tool to discover new innovative anticancer agents .A vast variety of naturally occurring substances have been shown to protect against experimental carcinogenesis and increasecancer chemopreventiveproperties .
Administration of phytochemicals prevents initiation; promotion and progression events associated with carcinogenesis, and may be a direct way to reduce cancer mortality and morbidity . Latex of Euphorbia antiquorum (EA) has been used widely in folk and traditional Chinese medicine for treating cutaneous infections, dropsy, liver disease , and cancer . Many studies showed that Euphorbia antiquorum contain antiquol, euphol, isohelianol, camelliol C, cycloeucalenol, antiquorine A, antiquorine B, and, taraxerol . The compound euphol have shown selectively inhibits human gastric cancer cell growth through the induction of ERK1/2-mediated apoptosis{Hsieh, 2011 #12} . Taraxerol also showed induce G2/M phase arrested and apoptosis in human gastric epithelial cell line . Our previous study demonstrated that EA can induce apoptosis in human cervical cancer cells via c-Jun N-terminal kinase activation and reactive oxygen species production . Altogether, these previous studies suggest that EA and some compounds of EA may induced apoptosis. However, the biological roles of EA in cell cycle on cervical cancer cells have not yet been determined.
In order to address the above questions, we investigated the roles of EA induce cell cycle arrest to inhibit cancer cell proliferation.in cell cycle progression on cell proliferation. More importantly, we explored the potential mechanism by which EA functions to promote the proliferation of cervical cancer cells.
MATERIALS AND METHODS Chemical and Reagents
Caffeine, SB203580, dimethyl sulfoxide (DMSO), propidium iodide (PI), ribonuclease A, Trypan blue and Triton X-100 were from Sigma-Aldrich Corp. (St Louis, MO). Bovine serum albumin (BSA), DMEM medium, fetal bovine serum (FBS) penicillin/streptomycin, L-glutamine and the primary antibody of phospho-p38 were from InvitrogenTM (Grand Island, NY). Acrylamide/Bis 40% solution
(ACRYL/BISTM 29: 1), ammonium persulfate (APS), glycine, SDS (sodium dodecyl
sulfate), 10X SDS-EAGE running buffer (TG-SDS buffer), Tris (hydroxymethly)– aminomethane, Tween 20, and N, N, N’, N’-Tetramethyl- ethylenediamine (TEMED) were from Amresco Inc. (Solon, OH). The primary antibodies for p21waf1/cip1, p27Kip1
and topo2α were from NeoMakers, (Biocompare. South San Francisco, CA). The primary antibodies for cyclin A, cyclin B, cyclin E, CDK1/cdc2, CDK2, cdc25A, cdc25C, JNK, phospho-JNK, c-jun-p63 and c-jun-p73 were obtained from Millipore Co. (Billerica, MA). The primary antibodies to total Chk2, ATM, and p38 and secondary anti-rabbit antibody were from CellSignaling Technology (Beverly, MA). Horseradish peroxidase (HRP)-conjugated secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody for β-actin was from Sigma. Secondary anti-mouse antibody and ECL detection system were from Amersham (Arlington Heights, IL).
Preparation of EA plant extracts
Fresh Euphorbia antiquorum was collected from Nan-Tau County in Taiwan in March 2011. The latex was extracted with methanol and centrifuged at 4,000 rpm for 15 min. The supernatant suspension was filtered with a 0.22 micron FGLP Teflon filter and then frozen and stored at −20◦C. The plant was identified and authenticated by taxonomist Dr. CL Kuo. Associate Professor, Chinese Medicine Resources, China Medical University, No. 91, Hsueh-Shih Road, Taichung 404, Taiwan.
Cell culture and treatment of EA
All cell lines were obtained from the Food Industry Research andDevelopment Institute (Hsinchu, Taiwan). The human cervical adenocarcinoma HeLa cells were cultured in DMEM medium containing 10% fetal bovine serum (Gibco-BRL, Gaithersburg, MD, USA) and 1% penicillin-streptomycin (100 U/ml penicillin and 100 μg/ml streptomycin) under standard cultureconditions (37°C, 95% humidified air and 5% CO2). Epidermoid cervical carcinoma CaSki cells were cultured in RPMI
1640 containing 10% FBS understandard culture conditions. A-10 cells, a cell line derived from the thoracic aorta vascular smooth muscle of embryonic rat and cultured in 1% fetal bovine serum used as a model of normal cells .
Cell morphological changes examinations
Cells (2x105 cells/well) were maintain in 24-well plates and incubated at 37˚C for 24 h before being treated with 0, 0.5, 1.0, 2.0 or 3.0 μg/ml of EA for 24 h. DMSO (solvent; 0.5%) was used for the control regimen. Cells were observed and photographed under a contrast phase microscope at x400 magnification for the determination of morphological changes.
Cell Viability
Cells (2×105) were cultured in 24-well plate containing DMEM supplemented
with 10% FBS for 1 day to become nearly confluent. Then cells were cultured with 0, 0.5, 1.0, 2.0 or 3.0 μg/ml of EA for 24 h. After that, the cells were washed twice with DPBS and incubated with 0.5 mg/mL MTS for 2 h at 37°C testing for cell viability. The medium was then discarded and 500 µL dimethyl sulfoxide (DMSO) was added. After 30-min incubation, absorbance at 570 nm was read by using a microplate reader
(Molecular Devices, Orleans Drive, Sunnyvale, CA).
Flow cytometry for cell cycle analysis
Approximately 5 x 105 cells/well of HeLa cells in six-wellplate were incubated
with 0, 0.5, 1.0, 2.0 or 3.0 μg/ml of EA for different timeperiods, and then the cells were harvested by centrifugation. The cells were fixed gently (drop by drop) by putting 70% ethanol (in PBS) in ice overnight and were then resuspended in PBS containing40 µg/ml propidium iodide and 0.1 mg/ml RNaseand 0.1% Triton X-100 in a dark room. After 30 min at 37°C, the cells were analyzed on a flow cytometer (Becton Dickinson FACS Calibur; BD Biosciences, San Jose, CA) equipped with an argon laser at 488 nm wavelength.The cell cycle was then analyzed .
Western blot analysis for determination of the proteins
The HeLa cells (1x106 cells/well) in 6-well plates were treated with 0, 0.5, 1.0,
2.0 or 3.0 μg/ml of EA and then incubated for 24 h for the determination of proteins associated with cell cycle arrest and DNA damage. Cells were harvested and washed with cold PBS and then lysed with ice-cold lysis buffer containing 50 mM HEPES (pH 7.7), 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 1 mM DTT, 0.1% Tween-20, 10% (v/v) glycerol, 1 mM NaF, protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany) and phosphatase inhibitor cocktail (Sigma Chemical Co.). The total proteins from each sample were quantified using the Bio-Rad method. Each sample (50 μg protein) was resolved over 12% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes (Millipore, Billerica, MA, USA).
The blot was then soaked in blocking buffer (5% non-fat dry milk/0.05% Tween-20 in 20 mM TBS at pH 7.6) at room temperature for 1 h and then incubated with individual primary monoclonal antibodies in blocking buffer at 4˚C overnight, followed by secondary antibody horseradish peroxidase conjugate and detection by chemiluminescence and autoradiography using X-ray film as described previously (17). To ensure equal protein loading, each membrane was stripped and re-probed with anti-β-actin antibody (17, 18). Dilutions of primary antibodies were 1:1,000 [antibodies specific for p21waf1/cip1, p27Kip1, cyclin A, cyclin B, cyclin E, cdk6, Cdc25A,
Cdc25C, chk2, ATM, and topoisomerase]. Band densities were quantified in arbitrary units, and expressed as relative density compared with the untreated controls that
were taken as 100%. .
Spcific inhibitors inhibit protein expression levels
HeLa cells were seeded at a density of 5×105 cells/well onto 6-well plates 24 h
before cells were pre-treated with 10 μM of SP600125 (JNK inhibitor), 10 μM of SB203580 (p38 inhibitor) for 1 h followed by treatment with EA and medium as a control. Caffeine was dissolved in either growth media at a stock concentration of 80 mM just prior to use or dissolved in water at a concentration of 200 mM. Cells were then harvested at 24 h to determine the protein expression levels as described eleswhere .
Statistical analysis
The quantitative data are presented as the means ± SD. Statistical differences between the EA-treated and control samples were calculated using the Student's t-test. A value of P<0.05 was considered to indicate a statistically significant difference. The results are representative of at least three independent experiments.
RESULTS
EA induced cell cycle S phase arrest in human cervical cancer cells.
Previously, we demonstrated that EA induced apoptosis in HeLa cells (10). Subsequent to treatment with EA, cells displayed impaired growth over the following 24 hours. Based on these results, EA concentrations of 0-3.0 μg/ml were used in present study to examine its effect on cell cycle signaling in both cell types. Cell cycle distribution patterns were compared in control and EA treated (0-3.0 μg/ml, 24 h) cells by measuring the DNA content by flow cytometric analysis of propidium iodide stained cells. Results of these experiments presented in Figure 1 showed a statistically significant increase in S phase cells in both, HeLa (Fig. 1A and 1C) and CaSki (Fig. 1B) cells after exposure to EA. The S populationwas noticeably enhanced from 31.47 to 60.81%, whereas the G0-G1 populationwas decreasedfrom 60.75 to 38.33% (Fig.
1A) in HeLa cells. The S population was also enhanced from 37.69 to 53.64%, whereas the G0-G1 populationwas decreasedfrom 55.9 to 46.22% (Fig. 1B) in CaSki
cells. Moreover, the results demonstrated that EA noticeably enhanced S phase cell cycle arrest was shown in a dose-pendent manner in human cervical carcinoma both HeLa and CaSki cell lines.
EA affected the protein levels of S phase cell cycle relative proteins
Cell cycle progression is regulated by a sequential activation of cyclin dependent kinases (CDKs). The activity of CDKs is dependent upon their association with regulatory cyclins and each of the CDKs can associate with different cyclins that determines which of the proteins are to be phosphorylated by a particular CDK-cyclin complex (29). It has been suggested that the Cell cycle checkpoints are important control mechanisms that ensure the proper execution of cell cycle events . Therefore, to elucidate the mechanism of EA-induced S phase arrest, we compared the levels of cyclin A and CDK2 proteins in whole lysates prepared from the controls and EA-treated cells by Western blot analyses. HeLa Cells were treated with EA which could suppress S phase cell cycle relative proteins expression of topoisomerase I, Cdc25A and Cdc25C (Fig. 2), and also decrease G0/G1 phase cell cycle relative proteins expression of cyclin B1, and Cdk6 in human cervical carcal carcinoma HeLa cells (Fig. 2B). Involvement of Chk1-Cdc25A-cyclin A/CDK2 pathway in simvastatin induced S-phase cell cycle
arrest and apoptosis in multiple myeloma cells. (31) Also, in recent years, inhibition
of the cell cycle has been appreciated as a target for the management of cancer . Therefore, EA activation of ATM and Chk2 kinase suppressed protein expression of cyclin A, cyclin E, and Cdc2 down-regulation also may be the important factors in S arrest of HeLa cells.
Increasing the protein levels of cyclin-dependent kinase inhibitory proteins of p21waf1/cip1and p27kip1
As shown in Fig. 3A, EA treatment of HeLa cells resulted in a dose-dependent increase in the binding of cyclin A toward p21waf1/cip1and /p27kip1. EA promoted the
expressions of p21waf1/cip1 and p27kip1 and both are known to involve cell cycle arrest.
Therefore, p21waf1/cip1 and p27kip1 may be the important factors in S arrest of HeLa cells
in EA treatment. Apparently, the mechanism of action of EA affecting the cells is almost the same in HeLa and CaSki cell types.
EA activated Topoisomerase I and ATM/ATR-Chk2 signaling
We investigated the protein levels of the DNA damage and Chk2/ATM check point signaling by Western blotting. As shown in Fig. 3B, EA caused an increase in the protein level of ATM and chk2, but decreased the protein levels of cdc25A, and cdc25C in HeLa cells. We investigated the protein levels of the DNA Topoisomerase I and II catalyze topological changes in DNA are essential for normal cell cycle progression. During the relaxation of supercoiled DNA, Topo I induces a temporary single-stranded DNA break in the process. In Western blot analysis, the results indicate that the levels of Chk2 and ATM were significantly increased by EA (Fig. 3B), moreover, Topoisomerase I, cdc25A, and cdc25C were decreased dose-dependently by EA (Fig. 3B)
ATR/Chk1 kinase inhibitor mitigates EA-induced S arrest and inhibits associated MAPK pathway signaling
Checkpoint kinase inhibitors or their analogues have been used to sensitize cells to killing by genotoxic agents because they override the druginduced G2 checkpoint (24). Therefore, we examined the effects of EA on cell cycle distribution in the
absence or presence of an ATR kinase Inhibitor (caffeine). As expected, treatment with EA led to a significant G2/M phase arrest in both cell types. In contrast, HeLa cells pre-treated with caffeine were significantly protected from EA-induced G2/M phase cell cycle arrest (Fig. 5A). EA suppressed the expressions of Cdc25A and Cdc25C and both are known to involve ATM kinases activation.
Caffeine only reverses EA-decreased Cdc25A protein levels but not Cdc25C (Fig. 4A).
Overall,these results suggest that EA causes activation ofChk1 and Chk2 in an ATM/ATR-dependent manner.
SB203580 could partial reversed EA the decreasing of Cdc25A but not Cdc25C expression in HeLa cells (Fig. 4B).
ATM/ATR-Chk1/2 pathway was inhibited by the c-Jun NH (2)-terminal kinase (JNK) inhibitor SP600125 . SP600125 could not reversed EA the decreasing of Cdc25A and Cdc25C expression in HeLa cells (Fig. 4B).
Results indicated that EA induces S phase arrest through DNA damage in HeLa cervical cancer cells, and that one of the passible mechanisms accounting for the chemopreventive activity of EA in HeLa cells occurs through ATM/ATR-p38 MAPK dependent pathway.
DISCUSSION
In the present study, the EA induced morphological changes of the HeLa cells. The morphological changes were also accompanied by cell death and a decrease in the percentage of viable cells was shown.
The results indicated that EA induced morphological changes (Fig. 1A) and decreased the percentage of viable cells (Fig. 1B).
Moreover, the results demonstrated that EA noticeably enhanced S phase cell cycle arrest was shown in a dose-pendent manner in human cervical carcinoma both HeLa and CaSki cell lines.
EA decreased the protein levels of S phase cell cycle relative proteins
HeLa Cells were treated with increasing concentrations of EA which could suppress S phase cell cycle relative proteins expression of cyclin A, cyclin E, and inoma HeLa cells (Fig. 3A).
HeLa Cells were treated with EA which could suppress S phase cell cycle relative proteins expression of topoisomerase I, Cdc25A and Cdc25C, and also activation of ATM and Chk2 kinase in human cervicCdc2, and also decrease G0/G1 phase cell cycle relative proteins expression of cyclin B1, and Cdk6 in human cervical carcal carcinoma HeLa cells (Fig. 3B).
Increasing the protein levels of cyclin-dependent kinase inhibitory proteins of p21waf1/cip1and p27kip1
As shown in Fig. 3A, EA treatment of HeLa cells resulted in a dose-dependent increase in the binding of cyclin A toward p21waf1/cip1and /p27kip1.
Therefore, p21waf1/cip1 and p27kip1 may be the important factors in S arrest of HeLa
cells in EA treatment.
EA activated Topoisomerase I and ATM/ATR-Chk2 signaling
In Western blot analysis, the results indicate that the levels of Chk2 and ATM were significantly increased by EA (Fig. 3B), moreover, Topoisomerase I, cdc25A, and cdc25C were decreased dose-dependently by EA (Fig. 3B)
EA activated MAPK pathway signaling
EA suppressed the expressions of Cdc25A and Cdc25C and both are known to involve ATM kinases activation.
Caffeine only reverses EA-decreased Cdc25A protein levels but not Cdc25C (Fig. 4A). Overall,these results suggest that EA causes activation ofChk1 and Chk2 in an ATM/ATR-dependent manner.
SB203580 could partial reversed EA the decreasing of Cdc25A but not Cdc25C expression in HeLa cells (Fig. 4B).
ATM/ATR-Chk1/2 pathway was inhibited by the c-Jun NH (2)-terminal kinase (JNK) inhibitor SP600125 . SP600125 could not reversed EA the decreasing of Cdc25A and Cdc25C expression in HeLa cells (Fig. 4B).
Results indicated that EA induces S phase arrest through DNA damage in HeLa cervical cancer cells, and that one of the passible mechanisms accounting for the chemopreventive activity of EA in HeLa cells occurs through ATM/ATR-p38 MAPK dependent pathway.
Our results also showed that EA induced S-phase arrest (Fig. 1). Western blotting was used to show that EA promoted p21waf1/cip1, p27Kip1 levels (Fig. 2A)., chk2 and
Those proteins have been demonstrated to be involved in the cell cycle, particularly in the S-phase and cell cycle phase, such as G0/G1, S and G2/M are also associated with check point enzymes.
Our results also showed that EA-induced apoptosis was accompanied by sustained phosphorylation of JNK, c-Jun, and p38 MAPK.
These results are in agreement with those from previous studies, (17,38). Our findings also showed that EA promoted the expression of p21waf1/cip1 and p27Kip1 in
HeLa cells.
It has been reported that p21waf1/cip1 is a well-characterized CDK inhibitor; high
levels of p21waf1/cip1 can inhibit cyclin D1 expression, resulting in the decline of pRb
phosphorylation (39,40).
In this study, we did not observe any significant changes in the HeLa cells following exposure to EA. However, we did observe the inhibition of cyclin A and Cdc25A in the HeLa cells following exposure to EA, which may be the mechanism of action behind the EA-induced S phase arrest (Fig. 5A).
EA interferes in vitro with the cell cycle at the S phase and thereby may lead cells to apoptosis.
The morphological picture of HeLa cells showed that EA decreased viable cells number after treatment with EA.
The results also showed that EA inhibits cell proliferation and S phase cell cycle arrest in HeLa cells and these effects are in dose- and time-dependent manners.
Cyclin-dependent kinases (Cdks) are the major molecular players with cyclins in cell cycle progression. Inhibition of Cdk activity, which led to cell cycle arrest, has turned out to be the most productive strategy for the discovery and design of novel anticancer agents specifically targeting the cell cycle .
The early cyclin E1-Cdk2 and cyclin A-Cdc2 provides a significant additional quantity of S-phase promotion .
Formation and activation of the prereplication complex requires coordinate actions of G1 and S phase cyclin-dependent kinases.
Cyclin E-Cdk2 and cyclin A-Cdk2, together with Dbf4-Cdc7, phosphorylate several components of the prereplication complex and replication machinery .
EA treatment of HeLa cells resulted in a dose- and time-dependent increase in the binding of cyclin A toward Cdc2.
The data showed that EA suppressed protein expression of Cdk6, and cyclinA, B1 and E in human cervical carcinoma HeLa cells (Fig. 4).
While p21waf1/cip1 promotes a G2/M checkpoint by inactivating Cdc2, to establish
a G1/S checkpoint p21waf1/cip1 inactivates cdk2.
In addition, it has been reported that p38 MAPK can directly phosphorylate and stabilize p21waf1/cip1 in vivo .
Unlike other members of the Cdc25 family that only regulate the G2/M transition, Cdc25A is a cyclin-dependent protein kinase phosphatase that can regulate the G1/S transition as well .
The cyclin-dependent kinase inhibitor p27Kip1 is known as a negative regulator of
cell-cycle progression and as a tumour suppressor.
It has been reported that p27kip1is the major inhibitor in the complex of cyclin A
and Cdc2 .
p53 plays a major role in the prevention of tumor development.
Stable expression of mutant p53 abrogated the G(1)/S (but not the G(2)/M) cell cycle checkpoint and abolished the induction of p21waf1/cip1 .
Cdk2 is the main target of p27Kip1 which binds to Cdc2, cyclin B1, cyclin A2, or
suc1 complexes in wild-type and Cdk2(-/-) extracts, and cyclin E binds to and activates Cdc2.
EA treatment of HeLa cells resulted in a dose- and time-dependent increase in the binding of cyclin A toward p21waf1/cip1and /p27kip1.
EA treatment of both cell lines also resulted in a dose- and time-dependent decrease in the binding of cyclin A toward Cdk2.
The enzyme functions equally well for positively and negatively supercoiled DNA generated during replication and transcriptional processes .
tumor cells either during S-phase cell cycle arrest or following premature cell cycle checkpoint exit results in tumor cells re-entering the cell cycle before DNA repair is complete .
ATM (ataxia telangiectasia mutated) plays an important role in the regulation of DNA replication after replication-associatedDNA damage and, as such, may be a key component of a mammalianS phase DNA damage response pathway .
The cell checkpoint kinase Chk2 activation could reduced the cdc25A expression and early cyclin E1-Cdk2 is sufficient to support entry into S-phase; cyclin A-Cdc2 provides a significant additional quantity of S-phase promoting as its levels rise during S phase .
ATM kinase collaborates to prevent genome instability during the S phase and it has high molecular weight protein kinase that is the signal transducer in the DNA damage response .
The large ATM and ATR (AT and Rad3-related) kinases are nuclear kinases recentlyidentified as being activated in response to DNA damage/genotoxicstress in eukaryotic cells .
The checkpoint function of ATM is mediated, in part, by a pair of checkpoint effector kinases known as Chk1 and Chk2 .
ATM kinase and its substrate, check-point kinase (Chk)1, were phosphorylated and induced phosphorylation of Cdc25A not Cdc25C .
In recent years, it has been suggested that ATM activation could be used to prevent cancer development .
As shown in Fig. 3B, the results indicate that the levels of Chk2 and ATM were significantly increased by EA (Fig. 3B).
Topoisomerases (Topo) are DNA enzymes that control the topology of the supercoiled DNA double helix during the transcription of replication of cellular genetic materials. There are two major types of topoisomerases,Topo I and II .
Topo I initiates the cleavage of a strandof DNA molecule while Topo II cleaves both DNA strands. Agents that target topo I are widely utilized to treat human cancer .
ATM and Chk2 have been implicated in the response to topo I poisons .
As a consequence of such a stabilization of DNA cleavage sites, topoisomerase poisons can induce chromosomal abnormalities and therapy-related secondary malignancies .
ATM kinase, which then induces parallel Chk 1/2 and JNK signaling pathways, leading to cell cycle block and apoptosis .
Inhibition of ATM provides a molecular explanation for the increased radiosensitivity of caffeine-treated cells .
Caffeine is an inhibitor of ATM/ATR kinase activity in vitro, and can block checkpoints without inhibiting ATM/ATR activation .
p38 MAPK can phosphorylate and promote the degradation of Cdc25A contributing to the establishment of a G1/S checkpoint. The p38 MAP kinase inhibitor SB203580 enhances nuclear factor-kappa B transcriptional activity by a non-specific effect upon the ERK pathway .
The p38 MAPK activation of p53 results in the accumulation of p21waf1/cip1, one of
the p53 targets .
p38 MAPK can phosphorylate and promote the degradation of Cdc25A contributing to the establishment of a G1/S checkpoint .
Although less established, p38 MAPK activation can also contribute to the induction of a G1/S checkpoint in response to stimuli such as osmotic stress, reactive oxygen species and cellular senescence. p38 MAPK can phosphorylate and promote the degradation of Cdc25A contributing to the establishment of a G1/S checkpoint .
SB203580 p38 MAPK inhibitor but not SP600125 JNK inhibitor reversed EA the decreasing of Cdc25A expression in HeLa cells (Fig. 4B).
We conclude that EA arrests in human cervical carcinoma HeLa cells at S phase can be summarized in Fig. 5 and based on:
(i) by inhibiting cyclin A-Cdk2 and cyclin E-Cdk2 levels
(iii) by increasing Chk2, thus causing an increase in Cdc25A phosphorylation/ inactivation inducing a decrease in Cdc25A levels,
(iv) by activating ATM/ATR-Chk1/2 and p38-MAPK pathway signaling. Our data indicate that EA efficacy in HeLa cells induces S phase arrestthrough ATM and MAPK regulated ATM/ATR-dependentDNA damage S phase arrest mechanism.
ACKNOWLEDGEMENTS
We are thankful for the financial support from the National Science Council (NSC 98-2815-C-039-095-B, NSC 98-2815-C-039-096-B), and China Medical University (CMU98-CT-06).
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Figure legends
Figure 1. EA induced S phase arrest in the cell cycle progression of human cervical
cancer cells. The HeLa (A) and/or Ca Ski (B) cells (2 × 105 cells per well; 12 well
plates) were plated in DMEM + 10% FBS with EA (0, 0.5, 1.0 and 2.0 µg/ml) for 24 h. At the end of these treatments, cells were collected and incubated with PI solution at 4°C for 24 h in dark and subjected to FACS flow cytometric analysis and as detailed in Materials and methods. Data are presented as mean ± SE of triplicate samples. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 as compared with control.
Figure 2. EA affected on cell cycle S phase relative relative proteins in HeLa cells.
Cells were cultured as described in Materials and methods, and treated with increasing concentrations of EA as labeled in the figure. At the end of the treatments, total cell lysates were prepared and subjected to SDS–PAGE followed by western immunoblotting. Membranes were probed with anti-Cdk2, cyclin A, cyclin E, cyclin B1, Cdc2, Cdk6, p21waf1/cip1, p27 and ß-actin antibodies followed by
peroxidase-conjugated appropriate secondary antibodies, and visualized by ECL detection system (A). Membranes were probed with anti- ATM, Topo Iα, Topo Iβ, Topo 2α, Chk2, Cdc25A, and Cdc25C and ß-actin (B). The data shown here are mean from a representative experiment repeated three times with similar results as compared to the vehicle group.
Figure 3. EA caused S phase arrest via ATM/ATR-dependent Cdc25A and Cdc25C,
and Chk 2 accumulation. HeLa cells were cultured as described in Materials and methods and treated with either medium (control) or 2.0 µg/ml EA and/or caffeine (10 mM) for 24 h. Caffeine was added 15 min before EA. At the end of these treatments, cells were harvested and cell lysates were prepared and subjected to SDS–PAGE followed by western immunoblotting for Cdc25A, Cdc25C, and β-actin. (A) HeLa cells were cultured and treated with either medium (control) or 2.0 µg/ml EA and/or SP600125 (10 μM) and/or SB203580 (25 μM) for 24 h. Inhibitor was added 15 min before EA. At the end of these treatments, cells were harvested and cell lysates were prepared and subjected to SDS–PAGE followed by western immunoblotting for Cdc25A, Cdc25C, and β-actin. (B) The data shown here are mean from a representative experiment repeated three times with similar results as compared to the vehicle group.
Figure 4. Proposed model of EA-induced S phase arrest via active ATM Kinase
![Figure 1 C. [EA] (mg/ml)0.00.51.0 1.5 2.0Percentage of cells (%)020406080100 G0/G1 phaseS phase G2/M phase [EA] (mg/ml)0.00.51.0 1.5 2.0Percentage of cells (%)020406080100 G0/G1 phaseS phase G2/M phase](https://thumb-ap.123doks.com/thumbv2/9libinfo/8972647.279397/26.892.145.722.188.1007/figure-percentage-phases-phase-percentage-cells-phases-phase.webp)
