Inhibitory Effect Of Aspirin On Human Cervical Cancer Hela Cell And Related Molecular Mechanisms

Objective: cervical cancer is one of the most common gynecologic tumors genital duct,which is seriously threatening female health.According to statistics,there are 47 ten thousand cases falling ill now of cervical cancer in the world in 2000.It is considered that the infection of human papilloma virus(HPV)is the mostly cause of cervical cancer.Whether surgical operation or radiotherapy in the early period of cervical cancer,curative effect is satisfied.But in the middle or late period, curative effect is unsatisfied.The curative effect of the preferred chemotherapy medicine to cure cervical cancer is positive.But it will cause serious side reaction if using a great deal of chemothrapy medicine for a long tome. At the same time,it will cause to lower the curative effect of the preferred chemotherapy medicine. Therefore,looking for a more reasonable and valid method is an urgent problem.Aspirin and other nonsteroidal anti-inflammatory drugs,the pharmacological effect is believed to be their ability to inhibit cyclooxygenase(COX) activity and thereby block the production of prostaglandins.There are some reports that aspirin can inhibit the growth of some carcinoma cells including oesophagus cancer,gastric cancer,colon cancer,ovarian cancer,liver cancer, galactophore cancer,lung cancer,lymphoma,endometrial cancer and etc.Although recent epidemiological studies indicated risk reduction in cervical cancer with consumption of aspirin or other nonsteroidal anti-inflammatory drugs,little data is available regarding the effects of aspirin on cervical cancer Hela cell.The purpose of this study was to investigate inhibitory effect of aspirin on proliferation of Hela cell and cellular apoptosis of induced by aspirin,to observe morphology changes of apoptosis of Hela cell,to detect the expression of bcl-2 and bax genes of Hela cell treated by aspirin,to investigate related molecular mechanisms.Methods:1.Cell culture:Hela cells frozen in liguid nitrogen were revived in routine method,and inoculated in RPMI-1640 blended with 10% fetal bovine serum(FBS),cultured in culture bottle.The bottle was put in a humidified incubator with 5% CO2 at 37℃.2.Flow cytometry analysis:Cell cycle distribution in HeLa cells was measured at 48 h after treatment with 1,5 and 10mM of aspirin;then,cell apoptosis and cell cycle was detected by cell flow cytometry(FCM).3.Reverse transcription polymerase chain reaction(RT-PCR):Total RNA of HeLa cells were distilled.After reverse transcription polymerase chain reaction(RT-PCR),the products of reverse transcription polymerase chain reaction(RT-PCR) were given 2% agarose gel electrophoresis.Then,bcl-2 and bax were evaluated by a Gel-analyzer software package respectively.4.DNA Ladder:DNA fragmentation of Hela cells was analysed by 1.5% agarose gel electrophresis.5.Cell proliferation inhibitory analysis:Changed RPMI-1640 vehicle containing different concentration aspirin ranging from 0,0.5,1,5,10 mmol/L,incubated for 24 h,48 h,72 h,96 h respectively.Cell proliferation inhibitory was measured by MTT assay.6.Morphological change of apoptosis cells:Hela cells were treated with aspirin at concentration of 0,1,5,10mmol/L for 72 hours.Morphological change of apoptosis cells were observed by light microscope and Giemsa staining.Results:1.Aspirin induce cellular apoptosis:AP(apoptosis percentage) detected by flow cytometry(FCM).Flow Cytometry Apoptotic rate was significant different between trial groups and control group,FCM showed that treated Hela cells with aspirin in various doses for 48 h induced cell apoptosis in a dose-dependent fashion(P<0.05).There are visible apoptosis peaks of hypodiploid on the DNA bargraph.Cellular proliferation cycle of Hela cells treated with various-dose aspirin for 48 h had changed significantly.Compared with control group, G0/G1 stage of Hela cells decreased,and G2/M stage increased.So,aspirin inhibited Hela cells proliferation in G2/M stage.2.The effect of aspirin on expression of bcl-2 and bax mRNA of Hela cells:In comparation with control group,The integrated optical density(IOD) of bcl-2 and bax mRNA of Hela cells was treated with 5mmol/L aspirin for 48 h.This show that aspirin may down-regulate the expression of bcl-2 mRNA,up-regulate the expression of bax mRNA.3.DNA Ladder:DNA Ladderof Hela cells treated with aspirin was showed by agar gel electrophoregram.4.The effects of aspirin on proliferation:MTT assay showed that cell proliferation was significant inhibited by aspirin in time and dose dependent manner.5.Morphology changes of apoptosis observed with optical microscope:Morphology of normal Hela cells was polygon or fusiform,After treated with aspirin,spurius of Hela cells recovered gradully,Hela cells were smaller and smaller,cellular nucleus condensed gradually,and some cancer cells were floated in the vehicle.By Giemsa staining, characteristic apoptotic changes in morphology were observed with optical microscope.Volume of apoptotic cells contracted.Plasmosome lost.Karyopyknosis,shrinkage of cellular membrane chromatin condense and apoptotic body also observed.In comparison with control group,dyeing of apoptotic cells enriched more.Moreover dyeing of cellular membrane was more obvious.Conclusions:The study showed aspirin can inhibited the proliferation of Hela cells,and induct apoptosis of them.The study also showed that related molecular mechanism of treating Hela cells using aspirin were down-regulation of bcl-2 mRNA and up-regulation of bax mRNA.Aspirin can inhibit Hela cells proliferation in G2/M stage.We have also observed typical morphology changes of apoptosis of Hela cells.The study provide a new thought of the anti-tumor therapy both in cervical cancer and aspirin.