cervical cancer is the third most common cancer among women in the world and has been associated with loss of cell cycle control that normally delays or even arrests proliferation [ 1 , 2 ] .
cyclin - dependent kinase ( cdk ) inhibitors have the potential to induce cell cycle arrest and apoptosis in cancer cells .
as one of them , alsterpaullone was found to selectively inhibit cdk enzymes , especially in cdk1 [ 3 , 4 ] .
it has been reported that alsterpaullone not only causes cell cycle arrest but also induces the apoptosis of some cancer cells by activation of caspase-9 through perturbation of mitochondrial membrane potential [ 57 ] .
cdk inhibitors have been shown to possess a cytotoxic effect on tumor cells via cell cycle related proteins and caspase 3 activity .
however , this pharmacologic aspect has yet to be studied in relation to alsterpaullone . in this study
, we explored the roles of those proteins in the pharmacologic function of alsterpaullone in hela cells .
in addition , we elucidated the mechanism of cell cycle arrest and apoptosis of hela cells treated with alsterpaullone .
our data showed alsterpaullone can inhibit the proliferation of hela cells in the dose- and time - dependent manner .
importantly , it induced cell cycle arrest at g2/m phase and apoptosis via the regulation of anti - apoptotic proteins ( caspase-3 ) and cell cycle proteins .
this finding is significant , since it suggests that alsterpaullone provides a promising chemotherapeutic tool in anticervical cancer arsenal .
alsterpaullone was purchased from sigma - aldrich ( cas : 237430 - 03 - 4 ) .
hela cell lines were purchased from the institute of basic medical sciences , chinese academy of medical sciences .
dulbecco 's modified eagle medium , fetal bovine serum , and trypsin were purchased from hyclone laboratories inc . , usa . penicillin and streptomycin were purchased from sigma chemical company , usa .
protease inhibitor cocktail ( 1% , cat no : 539134 ) was purchased from merck , usa . all reagents were chemical grade unless otherwise specified . hela cell line was maintained in rpmi-1640 media ( gibco , invitrogen corporation , usa ) containing 10% fetal bovine serum ( hyclon , usa ) , 2  mmol / l l - glutamine ( gibco , invitrogen corporation , usa ) , 100  u / ml penicillin ( gibco , invitrogen corporation , usa ) , and 100  g / ml streptomycin ( gibco , invitrogen corporation , usa ) .
cells were cultured in an incubator at 37c under 5% co2 in air . a stock solution of alsterpaullone in dmso
hela cells ( 5  10/well ) were grown in 24-well plates and treated with alsterpaullone ( 030  m ) or dmso ( 0.3% , final concentration ) to control wells in medium for 72  h. attached cells were released by a trypsinization and combined with nonadherent cells .
hela cells were seeded in 24-well culture plates at a density of 5  10/well . at 0 , 2 , 4 , 60 , 12 , 24 , 48 and 72  h 500  l of alsterpaullone ( final concentration : 10  m and 20  m )
cell number and cell viability were determined using haemocytometer and the trypan blue dye exclusion test .
hela cells ( 5  10/well ) were seeded into 96-well plates and incubated overnight at 37c .
alsterpaullone was added to cells ( in 6 replicates ) and incubated for 72  h at 37c .
stock solution ( 2  mg / ml ) of 3-[4 , 5-dimethylthiazol-2-yl]-2,5-diphenyl - tetrazolium bromide ( mtt ) was prepared in cell media and sterilized via filtration . media were removed from cells , and 50  l of mtt solution was then added into each well and incubated in the dark at 37c for 4  h. mtt solution was removed , and mtt dye of each well was dissolved in 50  l of dmso with agitation .
the absorbance was measured at 562  nm to determine the ic50 ( concentration of alsterpaullone which inhibits cell growth by 50% ) .
hela cells ( 5  10/well ) were seeded in 96-well plates and incubated overnight , pretreated with 50  l of z - vad - fmk ( final concentration was 25  m ) for 2  h to block caspase activity , and treated with alsterpaullone for 72  h. hela cells ( 5  10/well ) were seeded in 96-well plates and incubated overnight and pretreated with 50  l z - vad - fmk ( final concentration was 25  m ) for 2  h to block caspase activity , followed by alsterpaullone treatment for 72  h. cells were evaluated by mtt assay .
cells were treated with control ( 0.3% dmso ) and 20  m alsterpaullone . both detached and adherent cells were harvested at 0 ,
12  h , 24  h , 48  h , and 72  h and washed twice with ice - cold phosphate buffered saline ( pbs ) and then fixed in ice - cold 70% v / v ethanol for more than 2  h. cells were washed twice in pbs to remove fixative and stained with 1  ml pi ( propidium iodide)/triton x-100 pbs solution with rnase a. after incubation at room temperature for 30  min , cells were filtered through 95  m pore size nylon mesh .
the fractions of the cells in g0/g1 , s , g2/m phases were analyzed using cell cycle analysis software , winmdi v2.8 ( windows multiple document interface for flow cytometry ) ( the scrips research institute , la jalla , ca , usa ) .
hela cells were cultured with 20  m alsterpaullone for 0 , 2 , 4 , 6 , 12 , and 24  h. after incubation , the nonadherent and adherent cells were harvested .
the cells were lysed in ripa lysis buffer ( 150  mmol / l nacl , 1% np-40 , 0.5% deoxycholate , 0.1% sds , 50  mm ph 7.5 tris - hcl , 1% protease inhibitor cocktail ( cat no : 539134 , merck ) ) .
the protein concentrations in the different samples were determined using the bca protein assay kit ( pierce , rockford , usa ) .
lysates ( 50  g ) were fractionated by sds - page using 8%15% polyacrylamide gels , based upon the expected molecular weight .
the resolved proteins were blotted to a nitrocellulose membrane by semi - dry electric transfer , and the membranes were blocked for 1  h in tbst buffer containing 5% blotting - grade non - fat milk .
primary antibodies against caspase-3 ( cat no : 552785 ) , parp [ poly(adp - ribose ) polymerase ] ( cat no : 556494 ) , and mcl-1 ( cat no : 559027 ) were purchased from bd pharmingen ( bd biosciences , san jose , ca , usa ) .
survivin ( cat no : sc-17779 ) , bcl-2 ( cat no : sc-7382 ) , p - rb ( cat no : sc-12901 ) , and p21 ( cat no : sc-817 ) were purchased from santa cruz ( santa cruz biotechnology , inc .
membranes were washed three times in tbst for 10  min each time and then incubated in tbst containing the appropriate horseradish peroxidase conjugated anti - mouse or anti - rabbit secondary antibodies ( amersham life science , usa ) for 1  h. the membranes were washed three times for 10  min each in tbst . the bound antibody complex was detected using an ecl chemiluminescence reagent and xar film ( kodak , japan ) according to the manufacturer 's instructions ( amersham life science , usa ) .
student 's t - test was used for statistical analysis . a p value of < 0.05 strongly statistically significant , and p < 0.01 was considered very statistically significant .
the growth of hela cells was inhibited in a dose - dependent manner after exposures to alsterpaullone for 48  h and 72  h ranging from 0 to 30  m ( figure 1 ) .
the antiproliferation effect was evaluated by measuring the growth rates of hela cells seeded at 5  10/well in 24-well plates and treated with 10  m and 20  m alsterpaullone ( figure 2 ) .
alsterpaullone showed a significant inhibition on hela cell proliferation from 10  m ( dose dependent ) while from 2  h onwards ( time dependent ) .
the results indicate alsterpaullone is a cytotoxic agent in hela cells .   to explore if the alsterpaullone - induced apoptotic levels are dependent on caspase activation , we cultured cells for 72  h with or without alsterpaullone ( 0  m , 5  m , 10  m , and 20  m ) in the presence or absence of the general caspase inhibitor vi , z - vad - fmk .
the apoptotic cells decreased after pretreatment with z - vad - fmk , suggesting that z - vad - fmk blocks the alsterpaullone - induced apoptosis .
the results indicated that alsterpaullone induced apoptosis via caspase - dependent process ( figure 3 ) .  
we analyzed the cell cycle profiles of growing hela cells exposed to 20  m alsterpaullone using flow cytometry of propidium iodide stained nuclei .
we found the cell cycle arrest occurred at g2/m and then apoptosis was induced .  
figure 4 showed a marked increase in the cells with g2 contents at 12  h and then the occurrence of significant cell death .
the cell cycle g2/m arrest persisted and was followed by a sub - g1 content increase at 48  h as indicated with cell death .
the results indicated the mechanism of antiproliferative effects of alsterpaullone blocked cell cycle progression .   to understand the role of alsterpaullone in cervical cancer apoptosis
, we performed a time - course study on the apoptotic proteins in 20  m alsterpaullone treated hela cells . as indicated in figure 5 ,
the cleavage of parp started at 4  h , while the activation of caspase-3 occurred at 2  h. parp , a prominent substrate for several caspases , was cleaved in time - dependent fashion indicating the occurrence of apoptosis in alsterpaullone treated cells . furthermore
inhibition of caspase activity by the caspase inhibitor z - vad - fmk suggests that alsterpaullone induces cell death depending on caspase activity .  
bcl-2 family proteins play a central role in controlling the mitochondrial pathway , including proteins that suppress apoptosis process ( bcl-2 , mcl-1 ) . in this study
a dramatic decline was seen in expression of mcl-1 which was undetectable at 2  h onwards .
the same trend was also observed in survivin but hardly detectable until 24  h. by contrast , the expression of anti - apoptotic protein bcl-2 was unchanged all the time .
these results suggest that the apoptosis induced by alsterpaullone was associated with loss in anti - apoptotic proteins such as mcl-1 and survivin but not bcl-2 . in alsterpaullone - treated hela cells ,
alsterpaullone , as a cdk inhibitor , competes with atp for its binding site on cdks [ 7 , 8 ] .
alsterpaullone treatment induced not only cell cycle arrest but also apoptosis in various cell lines [ 5 , 9 , 10 ] . in this study , we showed for the first time that the novel cdk inhibitor , alsterpaullone , inhibited hela cell proliferation in a dose- and time - dependent manner .
alsterpaullone induced apoptosis rapidly in hela cells by a mechanism that regulates various proteins including anti - apoptotic proteins and cell cycle related proteins .
we found that alsterpaullone exhibited significant cytotoxicity towards hela cells , using flow cytometry and western blotting : hela cells were treated with alsterpaullone arrested in g2/m phase prior to apoptosis .
this inhibition also led to a drop in s - phase population in hela cells and thus disturbed cells ' dna replication [ 9 , 10 ] .
. cell death initiated by chemotherapeutic agents usually involves the mitochondrial pathway and releases proapoptotic factors to activate effector caspases , which cause dna fragmentation and apoptosis [ 11 , 12 ] . in this study
, the results demonstrated the roles of apoptotic proteins in inhibition of alsterpaullone on hela cells .
mcl-1 is a short - lived protein because the pest sequences present with the bcl-2 family member , and it is an important anti - apoptotic protein [ 13 , 14 ] . in the current study
, we found that mcl-1 protein was rapidly down - regulated and even undetectable as early as 2  h in hela cells . in studies by lahusen et al .
, alsterpaullone did not regulate the expression of iap , a member of xiap family , in jurkat cells and mcf10a cells [ 5 , 15 ] . on the contrary
, our results showed alsterpaullone continuously diminished the expression of survivin within 24  h , which is a member of the inhibitors of caspase ( iap ) family .
therefore , in order to determine whether p21 protein plays a role in inhibiting cell proliferation , it was measured in alsterpaullone treated hela cells using western blotting .
the results showed that p21 protein was up - regulated during 224  h. considering the essential role of p21 in g0/g1 cell cycle arrest and cleaved caspase-3 for apoptosis induction , we explored the parp and caspase 3 proteins expression .
the results showed the regulation of p21 was significantly earlier than that of caspase 3 and parp .
as such , we speculated p21 was involved in cell cycle arrest , apoptosis , and growth inhibition via activation of caspase-3 . in summary , alsterpaullone can inhibit tumour cell proliferation in a dose - dependent and time - dependent manner and exhibit significant cytotoxicity in hela cells .
it can induce rapid apoptosis and block cell cycle via regulation of various apoptotic proteins and activation of caspase .
the significance behind this in vitro finding is that it suggests the possibility of using alsterpaullone as a new chemotherapeutic agent in the fight against cervical cancer .
 alsterpaullone , a small molecule cyclin - dependent kinase ( cdk ) inhibitor , regulates the cell cycle progression . beyond death - inducing properties , we identified the effect of alsterpaullone on cycle procedure and apoptosis of hela cell . 
 it was found that alsterpaullone inhibited hela cells in a time - dependent ( 072  h ) and dose - dependent ( 030  m ) manner . in the presence of alsterpaullone , hela cells were arrested in g2/m prior to undergoing apoptosis via a mechanism that is involved in the regulation of various antiapoptotic genes , dna - repair , transcription , and cell cycle progression . compared to controls , alsterpaullone effectively prevented hela cells from entering s - phase . 
 these potential therapeutic efficacies could be correlated with the activation of caspase-3 . 