Effect of Different Concentrations of As 2O 3on Apoptosis
and Proliferation of 293t Cells
y DONG Hong  yan,LU  Cheng  qian,W ANG Juan,W ANG Xiu  li,LI Quan  feng,
ZHAO Ya  jun,XU Chang  qing,ZHANG Li
*
(Department o f Patho physiology ,Harbin Medical University ,Harbin 150081,China )
Abstract :Objective  To study the toxic mechanism of arsenic trioxide (As 2O 3)both as a therapeutical agent for acute promyeloc ytic leukemia (APL)and an environmental pollutant.Methods  293t cells (a human em  bryoic kidney cell line)and HeLa cells (a human cervical cancer cell line)were treated with different final concentrations of As 2O 3for different hours.For apoptosis assays,flow cytometry,Hoechst33258staining and cell morphological changes were used,and ultrastructure were observed.For proliferation assay,293t cells were counted for continuous 6days with a hemac ytometer.Results  When treated with 3~5 mol  L As 2O 3for 24h,HeLa cells underwent conspicuous apoptosis,but 293t cells did not undergo obvious apoptosis.293t treated with 3~5 mol  L As 2O 3for 48~
proliferation72h e xperienced apparent apoptosis.Low final concentrations of As 2O 3(<2 mol  L)inhibited proliferation of 293t cells.C onclusion  Different final concentrations of As 2O 3play different roles in the proliferation and apoptosis of 293t and HeLa cells.
Key words :arsenic trioxide;proliferation;apoptosis
CLC number :R966  Document code :A  Article ID :1000-1905(2006)02-0116-05不同浓度As 2O 3对293t 细胞和HeLa 细胞的凋亡及增殖的影响
董红岩，吕成倩，王 娟，王秀丽，李全凤，赵雅君，徐长庆，张 力
(哈尔滨医科大学病理生理学教研室，黑龙江哈尔滨150081)
[摘要] 目的 初步研究(As 2O 3)作为急性早幼粒细胞性白血病(acute promyelocytic leukemia,APL )的剂和环境污染物的毒副作用。方法 用As 2O 3作用于293t 细胞和HeLa 细胞不同时间后，用流式细胞仪、Ho  echst33258染、形态学和超微结构观察检测细胞凋亡和增殖；检测293t 细胞增殖用细胞记数板连续记数6天。结果 3~5 mol  L As 2O 3作用24h 可诱发HeLa 细胞发生凋亡，而293t 则不发生明显的凋亡。低浓度As 2O 3(<2 mol  L)可抑制293t 细胞增殖。结论 不同浓度的As 2O 3对HeLa 细胞和293t 细胞的增殖和凋亡作用不同。
[关键词] ；增殖；凋亡
Arsenic and its derivatives were considered as both poisons and medication in ancient China,Greece and Rome.As 2O 3has been used to treat APL with an efficient effect since the 70 s of last century [1]
.Basic studies about As 2O 3have been mainly focused on its mechanisms of treatment of APL and other tumors.Studies about its side effects,ho wever,have rarely been elucicated.As 2O 3exposure can cause acute and chronic toxicosis with virtually all body system abnormality,but the mechanisms still need to be investigated further.Our experiments explored the action of As 2O 3on He La cells and 293t cells,which may be helpful to understand the toxicity of As 2O 3.116第40卷 第2期2006年4月        哈尔滨医科大学学报J OURN AL OF HARBIN MEDICAL UNIVERSITY              Vol.40,No.2Apr.,2006
Received date :2005-09-02
B iography :DO NG Hong  yan(1979-),fe male,born in Anhui,postgraduate.*Corresponding author
1 MATERIALS AND METHODS
1.1 Cell line
Both HeLa cells and 293t cells were purchased from Chinese Center for Type Culture C ollection.
1.2 Reagents
Dulbecco  s Minimal Essential Medium (DME M)(high glucose)and RPMI 1640medium were obtained from In  vitrogen (California,U.S.A).Fetal bovine serum was supplied by Tian Jin Hao Yang Biological manufacture,Co.Ltd (Harbin,China).As 2O 3was purchased from Zhong Yang Da Yao Fang (Harbin,China).Hoechst33258was obtained from Sigma.
1.3 Cell c ultrure and As 2O 3exposure
293t cells were cultured in DME M and HeLa cells in RPMI 1640,both supplemented with 10%fetal bovine serum and 100I U  ml penicillin and 0.1 g  ml streptomycin.The cells were maintained in 5%C O 2at 37 .For cell apoptosis assays,cells were treated with 0,1,2,3,4,and 5 mol  L of As 2O 3.For proliferation assay,293t cells were incubated in 0,0.5and 2 mol  L As 2O 3for continuous 6days.
1.4 Flow cytometer assays (FC M)
4ml of 3 105
ml 293t and He La cells were seeded into 25ml flasks individually,and 0and 5 mol  L of As 2O 3were added.24h later,the cells were trypsinized,incubated with AnnexinV and propidium iodide for 15min at room te mperature and analysized by a flow cytome ter.
1.5 Hoechst33258staining
300 l of 7 104 ml 293t and HeLa cells were seeded into 24 cell pla tes individually,then were incubated in 0,1,2,3,4and 5 mol  L final concentrations of As 2O 3for 48and 72h,follo wed by Hoechst33258staining.Hoechst33258is a fluorescent dye,which can bind to the cell nuclear chromosomes and emits blue fluorescence when excitated by ultraviolet light.Under a fluorescent microscope,the nuclei of normal 293t cells were round or oval,emitting well  proportioned blue fluorescence;the nuclei of the apoptotic cells condensed or fragmented,emitting strong fluorescence;the necrotic cells did not emit fluorescence.Three 200 magnified visual fields were selected randomly and the numbers of apoptotic and total nuclei (including the apoptotic and normal nuclei)of per field were counted.The apoptotic ratio =number of apoptotic cells  total number of cells [2].
1.6 Transmission electron microscope assays
2ml of 3.3 105 ml 293t cells were seeded into a 6 cell plate and treated with As 2O 3.72hours later,the cells were retained and observed under a transmission electron microscope.
1.7 Cell proliferation assay
300 l of 1.05 105 ml 293t cells were seeded into a 24 cell plate,then 0,0.5and 2.0 mol  L of As 2O 3were add  ed.The cells were counted with a hemacytometer for continuous 6days.
1.8 Statistical analysis
Apoptotic ra tio was analyzed by partitions of  2method,P <0.05( =0.05)was considered to be significant.Cell proliferation data was analyzed with SPSS software.P value <0.05( =0.05)was c onsidered to be significant.2 RESULTS
2.1 Flow cytometer assays
The apoptosis of HeLa and 293t cells were exa mined by FCM.Compared with the control group,the apoptotic ratio of the HeLa cells treated with 5 mol  L As 2O 3for 24hours visibly inc reased (from 4.11%to 16.08%),whereas that of 293t cells increased a little (from 1.26%to 3.34%).
2.2 Hoechst33258staining of He La cells In vitro studies have shown that high concentrations of As 2O 3(>2.0 mol  L)trigger apoptosis of tumor cells [3].We 117
No.2      D ONG Hong  yan,e t al .EFFECT OF DIFFEREN T CO NCENTRATIONS OF As 2O 3ON
APO PTOSIS A ND PRO LIFER ATIO N OF 293t CELLS
incubated cancer cells,HeLa cells,in different concentrations of As 2O 3for 24hours and stained the m with Hoechst33258(Fig.1).Statistical analysis of the apoptotic ratio revealed that 3~5 mol  L As 2O 3induced apoptosis (P <0.05),but 1~2 mol  L As 2O 3did not induce apoptosis (P >0.05)
.
2.3 Morphology of 293t cells treated with As 2O 3
293t,a kind of normal cell,was cultured and treated with different concentrations of As 2O 3to observe the occurrence of apoptosis.Under an inverted microscope,the 293t cells in control group spread unregularly and connected tightly.When incubated with 2 mol  L As 2O 3for 48hours,cells experienced little variation.Cells treated with 3and 4 mol  L As 2O 3for 48hours became round and detached from each other(Fig.2).Nucleic chromatin condensation could be also
observed.
2.4 Apoptosis of 293t cells e xposed to As 2O 3
2.4.1 Hoechst33258staining:Most 293t cells treated with 0,1,and 2 mol  L As 2O 3for 48~72h divided,rarely.Pic  tures a,b,e and f in Fig.3belo w sho w the normal cellular nuclei.However,many 293t cells treated with 3,4,and 5 mol  L As 2O 3for 48~72h under went apoptosis.In the apoptotic cells,nucleic chromatin margination,condensation and frag  mentation appeared and the apoptotic cellular nuclei emit strong fluorescence.Pictures c,d,g and h show the apoptotic cellular nuclei by the arrows.
2.4.2 Apoptotic ratio of 293t cells:The nuclei under the fluorescent microscope were counted and the apoptotic ratio was calculated as described above.Statistical analysis revealed that 3~5 mol  L concentrations of As 2O 3induced 293t ap  optosis(P <0.05, =0.05).Ho wever,1~2 mol  L concentrations of As 2O 3did not induce 293t apoptosis(P >0.05).
2.4.3 Ultrastructures of 293t cells treated with As 2O 3:Under the transmission electron microscope,293t of the control group shared close connection and piled as clumps.Only a few cells under went apoptosis,most cells had high nucleic  cy  toplasmic ratio,lo w density chroma tin and high density nucleolus,whereas most cells treated with 5 mol  L As 2O 3for 72h e xperienced apoptosis.Many apoptotic cells shrinked and detached from each other,exhibiting the typical high density nu  cleic condensation,with clear nucleic envelope and intac t organelles (pictures b,c and d
of Fig.4).Some nuclei frag  mented and formed apoptotic bodies.118JOUR NAL OF HARBIN MEDICAL UNIVERSI TY                Vol.40
2.5 Inhibition of cell proliferation of 293t cells treated with low concentrations of As 2O 3
The results of Hoechst33258staining suggest that 1~2 mol  L As 2O 3did not induce apoptosis of 293t cells,so we studied the effect of 0.5and 2 mol  L As 2O 3on the cellular proliferation.Tab 3showed that 2 mol  L As 2O 3seemed to make 293t cells to grow more slowly than 0.5 mol  L As 2O 3did.Statistical analysis revealed that 2 mol  L As 2O 3inhibited 293t cells to proliferate (P <0.05),however,0.5 mol  L As 2O 3did not inhibit 293t cells proliferation (P >0.05).
Table 1 Prolif eration o f 293t treated with 0,0.5and 2 mol  L As 2O 3for 6days ( x  s )
As 2O 3
( mol  L -1)
Cell concentrations of different day( 105 ml -1)01234560
1.05 0.15  1.50 0.19  4.37 1.031
2.65 1.522
3.20 1.3237.18 1.8640.14 1.260.5
1.05 0.15  1.50 0.19*  3.14 1.03*11.94 1.52*17.01 1.32*29.24 1.86*33.64 1.26*2  1.05 0.150.74 0.19**  1.69 1.03**  6.03 1.52**  6.28 1.32**11.48 1.86**16.90 1.26**  P <0.05was denoted by **,P >0.05was denoted by *.The e xperiment had been repeated for 3ti mes and the results were the s ame
3 DISCUSSION
Arsenic trioxide has shown substantial efficacy in treating patients with APL.The mechanism of its therapeutic action includes the induc tion of apoptosis,the inhibition of growth and angiogenesis,and the promotion of differentiation,etc.
Apoptosis is the process of program med cell death,involving the systematic disasse mbly of a cell.Apoptotic cells can be recognized by morphological changes:the cell shrinks,shows deformation and loses contact with its neighbouring cells.The chroma tin condenses and marginates at the nuclear membrane,and the plasma membrane is blebbing or budding,and 119
No.2      D ONG Hong  yan,e t al .EFFECT OF DIFFEREN T CO NCENTRATIONS OF As 2O 3ON
APO PTOSIS A ND PRO LIFER ATIO N OF 293t CELLS
120JOUR NAL OF HARBIN MEDICAL UNIVERSI TY              Vol.40
finally the cell is fragmented into compact membrane enclosed structures,called apoptotic bodies which contain cytosol, the condensed chromatin,and organelles.
The most prominent adverse events with As2O3in APL has included weight gain and fluid retention,leukocytosis,the APL differentiation syndrome and prolongation of the QTc intervals on the electrocardiogram.Peripheral neuropathy,hy perglycemia,and cutaneous reac tions have also been surveyed.Deaglio and his colleagues work showed that there may be an immunologic mechanism to explain the side effects of As2O3[4],but whether there is relation between the side effects and apoptosis has been seldom elucidated.Our experiment results suggest this possibility.It has been reported that0.5 2 mol L As2O3triggered apoptosis of APL cells[5].In our e xperiment,exposure of HeLa cells to3 mol L As2O3for24 hours induced apoptosis,but293t cells treated with even5 mol L As2O3for24hours did not undergo evident apoptosis. Zhou and his colleagues found that different tumor cells showed different sensitivity to treatment of As2O3and APL cells were more sensitive to As2O3than HeLa cells did[6].This may explain why the concentration of As2O3inducing apoptosis of HeLa cells in our experiment is higher than that for APL cells.Studies sho wed that the peak concentration of clinical therapeutic As2O3was about5 mol L and the duration was1~2h[7].Our experi
ment results sho wed that treatment of 5 mol L As2O3for24h did not induce apparent apoptosis of293t cells,suggest that the therapeutic concentration of As2O3is relatively safe.293t treated with3~5 mol L As2O3for48~72h experienced distinguished apoptosis,which may contribute to the side effects of As2O3as a therapeutic agent.More investigation about As2O3as an effective agent for APL and the other cancers may focus on how to relieve the side effects and enhance the effects.
Arsenic toxicosis is a global health problem affec ting many millions of people.Acute and chronic arsenic toxic osis af fect most human body syste m,including skin,gastrointestinal system,cardiovascular system,neurological system,genitouri nary system,respiratory sytem and docrine and haematological systems[8].It has also been reported that infants are more susceptible to arsenic than adults are[9].
Our data demonstrated that3~5 mol L As2O3could induce apoptosis of normal cells,and low c oncentrations of As2O3(<2 mol L)could inhibit proliferation of normal cells.Both the induction of apoptosis and inhibition of prolifera tion may be correlated with the arsenic toxicity,which should be studied further.
The mechanism of apoptosis induced by As2O3includes association with SH group,downregulation o
f Bcl 2protein e xpression,the activity of caspases,collapse of mitochondrial membrane potentials and release of cytochrome c and the ac tivity of JNKs,etc.The antiproliferative activity of As2O3is partly related with modulation of cytokine release and interfer ence cell progression[10].Molecular biological studies should be useful to illuminate the mechanism of As2O3toxicity fur ther.
REFERENC ES:
[1] M iller WH Jr,Sc hipper HM,Lee J S,et al.Mechanis ms of acti on of arsenic trioxide[J].Cancer Res.2002,62(14),3893 3903.
[2] Xu FF,Liu XH,Cai LR.Role of hypoxia inducible factor 1 in the prevention of cardiomyocyte injury induced by hypoxic precondi tioni ng[J].Sheng Li X ue
Bao,2004,56(5):609 614.
[3] M aedaH,Hori S,Ohiz umi H,e t al.Effective treat ment of advanced s olid tumors by the co mbi nati on of arsenic tri oxide and L buthionine s ulfoxi mine[J].Cell
Death Di ffer,2004,11(7):737 746.
[4] Tallman MS.Ars enic tri oxide:its role in acute promyelocytic leukemia and potenti al i n other hematologic malignancies[J].Blood Rev,2001,15(3):133 142.
[5] Liu P,Han ZC.Treatment of acute promyeloc ytic leukemia and other he matologic malignancies with arsenic trioxide:revie w of clinical and basic s tudies[J].
Int J He matol,2003,78(1):32 39.
[6] 周晋，孟然，关秀茹，等.细胞内砷浓度与敏感性的关系[J].中国药学杂志,2004,39(3):193 195.
[7] 周晋，孟然，孙宏利，等.对豚鼠心室肌细胞内游离钙离子浓度的影响[J].中国药学杂志,2004,39(11):831 834.
[8] Ratnaike RN.Acute and chronic arsenic toxicity[J].Pos tgrad Med J,2003,79(933):391 396.
[9] Waalkes MP,Ward J M,Liu J,e t al.Transplacental carcinogenicity of inorganic arsenic in the drinki ng water:induction of hepatic,ovari an,pul monary,and
adrenal tumors in mice[J].Toxicol Appl Pharmacol,2003,186(1):7 17.
[10] Lis t A,Beran M,Dipers io J,e t al.Opportuni ties for trisenox(arsenic trioxide)in the treatment of myelodysplas tic syndromes[J].Leuke mia,2003,17(8):
1499 1507.

版权声明：本站内容均来自互联网，仅供演示用，请勿用于商业和其他非法用途。如果侵犯了您的权益请与我们联系QQ:729038198，我们将在24小时内删除。