aspirin 
 and other agents characterized as nonsteroidal anti - inflammatory drugs ( nsaids ) 
 are designed primarily to decrease pain and inflammation .
the molecular basis 
 for actions of nsaids is believed to be their ability to inhibit cyclooxygenase 
 ( cox ) activity and block the production of prostaglandins . among nsaids , aspirin and 
 sulindac
can prevent the development of colon cancer and act as an 
 anti - inflammatory agent by their inhibition of prostaglandin synthesis .
cancer 
 of the uterine cervix is the second leading cause of death from cancer in women 
 worldwide and also the most prevalent gynecological malignancy in korea .
we investigated 
 whether aspirin induced apoptosis in human cervical cancer hela cells . to 
 investigate the mechanism by which aspirin exerts its apoptosis effects , the 
 effect of aspirin on the gene expression was studied by differential mrna 
 display rt - pcr ( dd rt - pcr ) . employing dd rt - pcr methods , we identified aspirin - responsive 
 gene and mu - type calpain , which was confirmed by 
 real - time quantitative pcr .
the two 
 isoforms are classified according to their ca requirements : mu - type 
 calpain and m - type calpain require micromolar and millimolar concentrations of 
 ca for activation , respectively .
growing evidence suggested that 
 calpain may play a central role in the execution of apoptosis via modulation of 
 caspase-3 activity in   glucocorticoid - treated and irradiated 
 thymocytes , neuronal cells exposed to uv , or mcf-7 breast cancer cells treated 
 with -lapachone [ 57 ] .
further 
 progress in cancer prevention would depend on understanding the mechanisms 
 through which aspirin exerts 
 molecular action .
however , the molecular mechanisms through which aspirin alters colonic tumorigenesis are unknown . in this 
 report
, we describe a potential mechanism by which aspirin induces apoptosis in 
 human cervical cancer cells . to examine whether mu - type calpain 
 mediates antitumor effects in hela cells , hela cells were stably transfected 
 with mu - type calpain cdna .
tumor formation and 
 caspase-3 activity of stably transfected cells were measured in nude mice . in 
 this paper
, we suggest that aspirin has an antitumor effect via the expression 
 of mu - type calpain gene in cervical cancer cells .
human 
 cervical cancer cells , hela , were plated in a 24-well plate at a density of 1  10 cells / well and treated with various doses of aspirin . to detect an apoptotic 
 body ,
cells were stained with hoechst 33342 dye and assessed for morphological 
 signs of apoptosis .
the proportion of cells in g0/g1 , s , and g2/m was 
 determined by flow cytometric analysis of dna content ( becton dickinson , peterson , nj , usa ) . 

total 
 rna was extracted from cells with trizol reagent ( invitrogen , carlsbad , calif , usa ) , following the protocol that was 
 provided .
cell monolayers were washed with pbs , and 1 ml of trizol/10 cells with 4 units of rnase inhibitor were added . for each sample ,
2 g of 
 rna were treated with dnase l ( roche , basel , switzerland ) at 37c for 30 minutes to remove contaminating dna .
one - base - anchored 
 oligo - dt primers were used to reverse transcribed total rna into first - strand 
 cdna , which were amplified subsequently by pcr using the arbitrary upstream 
 primers .
pcr products were labeled with 2 ci of [p]dctp ( amersham , arlington , ill , usa ) , and analyzed on a 6% 
 polyacrylamide - urea gel .
the cdna bands that were unique to control or 
 aspirin - treated cells were cut out of the gel , eluted , and reamplified by pcr . 

the candidate cdna was cloned into pgem - t vector ( promega , madison , wi , usa ) .
we relied on 
 the taqman assay ( perkin - elmer model 7700 ; foster city , ca , usa ) to quantitate the amount of 
 calpain mrna .
the forward and reverse primers and the fam - tagged probe used for 
 the mu - type calpain gene 
 in the assay were 5-ggatgtcattccgagact , 5-ctcgtagaccgcgaag , and 
 5-6fam - tctgcaacctcacacccgac - tamra , 
 respectively .
the forward and reverse primers and fam - tagged probe used for the 
  - actin gene were 5-aacttgagatgtatgaaggcttttgg , 
 5-tttttttttttttttttttttttttttttaag , and 
 5-6fam - caactggtctcaagtcagtgtacaggtaagccct - tamra , respectively . to measure the 
 relative abundance of the calpain gene in any given rna sample ,
the 
 amplification value derived using the calpain sequence was divided by the 
 amplification value using the  - actin sequence .
the mu - type calpain cdna was retrieved with the 
 following primers : forward 5-aggatgtcggaggaga and reverse 
 5-ccagtacacaagtccct .
pcr reaction products were cloned into pcr3.1 vector 
 ( invitrogen ) . the vector pcr or recombinant pcrcal was stably transfected into 
 hela cells by liposome .
control , vector - transfected ( pcr / hela ) , and 
 calpain - transfected cells ( pcrcal / hela ) were counted by the trypan blue 
 exclusion assay and coulter counter ( coulter corporation , fl , usa ) for 
 measuring stably transfected cell growth .
five experiments . cells 
 ( 1.5  10 ) were plated in cell culture dishes ( 100 mm ) and allowed to 
 attach for 48 hours under cell culture conditions .
then the cells were treated and 
 the activity of caspase-3 was measured using the fluorogenic enzyme substrates , 
 z - devd - afc ( molecular probes , eugene , ore , usa ) .
samples were read in a fluorometer equipped with a 400 nm 
 excitation filter and 505 nm emission filter .
balb / c 
 nu / nu mice , 46 weeks of age , 
 were acclimated and caged in groups of five .
hela , pcr / hela , and pcrcal / hela 
 cells ( 1.5  10 ) were injected subcutaneously into the right flank of 
 the nude mouse .
the mean tumor diameter was measured by dial caliper , and the 
 volume was calculated by the formula : volume   ( mm ) = ( square   root   of   width  length ) .
the experiment was repeated three times and 
 performed according to the guidelines of the animal experimental committee , 
 national institute of health , south korea .
statistical 
 calculations were performed using the microsoft excel 97 program ( 1998 ; microsoft 
 co. , redmond , wash , usa ) 
 to estimate p - value . the significance level ( p - value )
we 
 performed dna synthesis assay to study the effects of aspirin on hela cells . 

aspirin inhibited growth of cervical 
 cancer cells in a time- and concentration - dependent manner ( data not shown ) . 

aspirin - induced morphological 
 changes were evident in a concentration - dependent manner ( see figure 1 ) .
cells 
 treated with aspirin became sparse , long squared , and detached from the dishes .
apoptotic bodies ( indicated by white arrows in 
 figure 1 ) were shown after aspirin treatment . to show apoptosis in the cells 
 treated with aspirin , flow cytometry analysis was performed .
the population of 
 sub - g1 phase was changed from 1.2 to 18.9% in cells treated with 1 mm aspirin for 
 48 hours .
that of sub - g1 phase at 2 and 3 mm was in the similar range .
after 
 hela cells were treated with aspirin for 48 hours , we performed differential display 
 rt - pcr and selected differentially expressed genes , which was expressed with 
 absolute difference between control and aspirin - treated cells .
expression of calpain mrna was confirmed by real - time quantitative pcr ( see figure 2 ) .
calpain gene was highly expressed in aspirin - treated hela cells in a 
 concentration - dependent manner .
calpain gene was upregulated by 4.4 , 6 , and 8.8 
 folds in the 1 , 2 , and 3 mm aspirin - treated hela cells , respectively .
we have 
 cloned mu - type calpain cdna into pcr3.1 
 vector . the vector pcr or recombinant pcrcal was stably transfected into hela 
 cells ( pcr / hela cell or pcrcal / hela cell ) . to assess whether change of cell 
 biology was caused after gene transfection , we measured cell proliferation . 

pcrcal / hela cells appeared to have a markedly different growth pattern compared 
 with hela cells and pcr / hela cells ( see figure 3(a ) ) . to establish a 
 relationship between calpain and caspase-3 , caspase-3 activities were measured . 

as shown in figure 3(b ) , caspase-3 activity was elevated in pcrcal / hela cells . 

the markedly increased activity levels of caspase-3 in pcrcal / hela cells 
 suggest a correlation of caspase-3 activity and calpain protein . in investigation 
 of calpain role in the tumorigenicity
1.5  10 stably transfected cells were subcutaneously 
 injected into the flank of the mouse . when tumor burden was measured by day 49 , 
 hela cells and pcr / hela cells produced tumors of 2126  163 mm and 1638  213 mm , respectively ,   while pcrcal / hela cells produced 
 markedly small tumor of 434  206 mm in volume ( see figure 3(c ) ) .
we 
 report that aspirin inhibited the proliferation of cervical adenocarcinoma 
 cells in a time- and dose - dependent manner .
this agrees with other studies 
 showing that aspirin inhibited the proliferation of cancer cells [ 2 , 4 , 812 ] .
apoptosis was shown to 
 be responsible for the cell growth inhibitory effects of aspirin in ht29 human 
 colon carcinoma cells .
our morphological observation of nuclear condensation after 
 aspirin treatment suggests that aspirin increases apoptosis in cervical cancer 
 cells ( see figure 1 ) .
these results demonstrate that aspirin is an effective 
 antitumor drug that induces apoptosis in cervical cancer cells . a few molecular 
 mechanisms of aspirin have been proposed .
it is 
 well known that activation of p53 expression is involved . in this study , we have 
 shown that aspirin induces the calpain gene and calpain gene activation 
 inhibits the tumor formation .
these results support the previous findings that 
 aspirin induces apoptosis by the regulation of bcl-2 and caspase-3 in human 
 cervical cancer cells [ 3 , 17 ] .
therefore , 
 aspirin might play roles in the inhibition of tumor formation through 
 activation of calpain gene .
calpain 
 is a calcium - dependent cysteine protease that is implicated in calcium - dependent 
 cell death [ 17 , 18 ] .
calpain 
 plays an essential role in apoptotic commitment by cleaving bax and generating 
 the bax / p18 fragment , which in turn mediates cytochrome c release and initiates 
 the apoptotic execution .
calpain activation 
 plays a critical role in cancer cell adhesion and motility ; and calpain could be 
 related to a therapeutic strategy targeting multiple disease states [ 2124 ] . in our experiments , 
 caspase-3 activity was increased in the calpain - transfected hela cells , 
 suggesting a correlation of calpain and caspase-3 ( see figure 3(b ) ) .
the 
 involvement of caspase-3 in the calpain action is in agreement with the results 
 in the photoreceptor cell , where calpain executes apoptosis via modulation of 
 caspase-3 activity .
accompanying the 
 increased caspase-3 activity , tumor growth was reduced by calpain gene expression , 
 leading to the role of calpain as an apoptosis mediator ( see figure 3(c ) ) .
have analyzed calpain release from cultured chondrocytes stimulated by a 
 proinflammatory cytokine , tumor necrosis factor- ( tnf- ) .
the effects of nsaids 
 on calpain release were also examined . however , their results were in contrast 
 to our expectation .
nsaids examined ( aspirin , loxoprofen - srs , diclofenac 
 sodium , indomethacin , and ns398 ) potently inhibited tnf--induced release of calpain .
in addition , they 
 showed that pge2 alone failed to stimulate , but it significantly augmented 
 the release of calpain in the presence of 1 ng / ml tnf- in hcs-2/8 cells .
moreover , inhibition of 
 calpain release by an nsaid , loxoprofen - srs , was significantly reversed by 100 nm pge2 . therefore ,
we suggest that aspirin may have cancer - preventing effects through calpain gene expression , which leads to caspase-3 activation .
 aspirin and other nonsteroidal anti - inflammatory drugs show efficacy in the prevention of cancers . 
 it is known that they can inhibit cyclooxygenases , and some studies have shown that they can induce apoptosis . 
 our objective in this study was to investigate the mechanism by which aspirin exerts its apoptosis effects in human cervical cancer hela cells . 
 the effect of aspirin on the gene expression was studied by differential mrna display rt - pcr . among the isolated genes , mu - type calpain gene was upregulated by aspirin treatment . to examine whether calpain mediates the antitumor effects , hela cells were stably transfected with the mammalian expression vector pcr3.1 containing mu - type calpain cdna ( pcrcal / hela ) , and tumor formations were measured in nude mice . when tumor burden was measured by day 49 , hela cells and pcr / hela cells ( vector control ) produced tumors of 2126 mm3 and 1638 mm3 , respectively , while pcrcal / hela cells produced markedly smaller tumor of 434 mm3 in volume . 
 the caspase-3 activity was markedly elevated in pcrcal / hela cells . 
 the increased activity levels of caspase-3 in pcrcal / hela cells , in parallel with the decreased tumor formation , suggest a correlation between caspase-3 activity and calpain protein . 
 therefore , we conclude that aspirin - induced calpain mediates an antitumor effect via caspase-3 in cervical cancer cells . 
1. Introduction
2. Methods
3. Results
4. Discussion
5. Conclusion