Expression Of Survivin, Caspase-3 Proteins In Cutaneous Squamous Cell Carcinoma And Intervention In Vitro By Realgar

Objectives:Survivin,a member of the inhibitor of apoptosis protein(IAP)family,can directly inhibitCaspase-3 and Caspase-7 activity and play an important role in oncogenesis. This study was designed to investigate the expression of Survivin and Caspase-3 in cutaneous squamous cell carcinoma(SCC) and investigate the significance and relationship of Survivin and Caspase-3 in the pathogenesis and development of squamous cell carcinoma;In this study we try to examine whether Realgar has an effect on human Cutaneous Squamous Cell Carcinoma Cell Line A431 in inhibiting proliferation and inducing apoptosis by MTT,FCM and RT-PCR in order to provide theory and experiment base.Methods:(1) The expression of Survivin and Caspase-3 was detected to use immunohistochemical staining in 46 specimens from SCC patients; 20 normal skin tissues were used as control.(2) Cell culture:A431 cells were cultured in DMEM medium containing 10% fetal calf serum (FCS),penicillin(100u/ml),and streptomycin(100u/ml),in 5% CO2,at 37℃saturation humidity box.When the cells grew to form a monolayer,they were stimulated with Realgar for 24 hours and then be collected.(3) MTT:A431 cells were incubated in the 96-well culture board.Cultur fluid contains with 30μg/ml,60μg/ml,100μg /ml,200μg/ml,400μg/ml Realgar,which contrast to the culture medium cells without Realgar.Each well add MTT solution(5μg/ml)reagent 20 ml,to culture 4h,at 37℃,and stop culture.Each well add DMSO 150μl,confusing 10 min,using ELISA reader to detect the A490 at wavelength 490nm of each well.Restrain rate(%)=[1-A 490 (the disposal team)/A 490 (the contrast team)x100%].(4) Cell morphology observation:With the culture fluid in which the concentration of Realgar was 30-200μg/ml,observing the cell morphology and taking photos with inverted phase contrast microscrope after 24h culture.(5)FCM: Incubating A431 cells in 6-well culture medium with 1×106/well,after 24h culture with the concentration of Realgar 30-200μg/ml,gathering cells growing along the wall and making monocellular suspension, and then add 5μl AnnexinⅤ/FITC and 10μl PI solutions,reaction for 15 min avoiding light,detection the early stage apoptosis cells by flow cytometyr.Analyse the result with software.(6) RT-PCR:After A431 cells were timulated with different doses of Realgar for 24h,harvesting them with cell scraper. To detect the changes of Survivin and Caspase-3 mRNA levels.Result:(1) 46 cases were diagnosed as SCC by the criterion of SCC,and by the criterion of Broder,high histological grade was 69.6%,and low histological grade was 30.4%.(2) The positive rate of the expression of Survivin in SCC was 69.57%(32/46),while none of 20 cases in control group and was significantly increased in SCC (p<0.05). The positive rate of the expression of Survivin in high histological grade SCC was 59.38%and 92.86% in low histological grade SCC. The expression of Survivin showed obviously correlated with the tumour differentiation in SCC;The expression of Caspase-3 in SCC was 36.96%(17/46),and 80% in control group and was significantly decreased in SCC (p<0.05).The expression of Caspase-3 in high histological grade SCC was 43.75%and 21.43% in low histological grade SCC,and without significance in the tumour differentiation in SCC; There was a negative relation between the expression of Survivin and Caspase-3 in SCC.(3) MTT:Realgar can inhibit the proliferation of A431 cell and show dose-depent in given range;.The diference of inhibition rate between the group 60,100,200及400μg/ml and control group was significant (p<0.05).(4) Cell morphology observation:Cells in the control group were well grew adhesively,most in shuttle shape,moderately sized, clear nucleolus,nuclear division phase found. The shape of Realgar-stimulated cells has not regular and the clearance has gradually became wider,volume smaller and grew more and more obvious along with dose increasing.(5)FCM:Incubating with 60μg/ml,100μg/ml,200μg/ml Realgar for 24h,the A431 cells early stage apoptosisis rate was 15.24%,26.07% and 11.97% respectively。(6) RT-PCR:Along with the increase of the Realgar reacting concentration, the expression of mRNA of Survivin is decreasing,however,the expression of Caspase-3 is on the contrary-upgrade. Conclusions:(l)The expression of Survivin in SCC was associated with the histological grades.Caspase-3 expression was suppressed in SCC. There was a negative relation between the expression of Survivin and Caspase-3 in SCC.(2) Survivin obviously inhibited the apoptosis of SCC cells. High expression of Survivin and low expression of Caspase-3 might indicate poor prognosis of SCC.(3) Realgar can inhibit A431 cell proliferation and induce A431 cell apoptosis,and the fashion is dose-dependent in a given range.(4) Realgar could induce the apoptosis of the human cutanous squamous cell carcinoma A431 cell which may be related to downregulating the expression of the Survivin mRNA and upgrading the expression of the Caspase-3 mRNA.