| Objectives:Tongue squamous cell carcinoma was the highest incidence of oral squamouscell carcinoma[1,2].It is a hotspot for tumor treatment research to look for safe andeffective anti-tumor drugs from traditional Chinese herbal medicine[3,4]. Andrographispaniculate Ness has been studied in many countries[5-7].Andrographolide (AD),aditerpenoid lactone isolated from Andrographis paniculate Ness,it has antiinfl-ammatory,antibacterial and antiveral effects.Recent studies suggested that AD hadantineoplastic effects[8-12].Study of AD work on tongue squamous carcinoma havebeen not reported up to now.The objective of this study was to explore the effects ofAD on proliferation inhibition,cells cycle distribution and the expression of Bax andBcl-2mRNA in human tongue squamous cell carcinoma Tca8113cells.It may bepromising as a new drug for treatment of tongue squamous cell carcinoma.Methods:1.The Tca8113cells were cultured with common cell culture technique,logarithmic growth phase cells used in the experiment.2.The cultured Tca8113cells were treated with different concentrations of AD(0,10,20,40,80,160μg/ml)for different time (24h,48h).Inhibition of cell proliferationwas determined by MTT assay.3.The cultured Tca8113cells were treated with different concentrations ofAD(0,20,40,μg/ml)for different time (24h,48h).Cell cycles were analyzed by flowcytometry.4.The cultured Tca8113cells were treated with different concentrations ofAD(0,5,10,20,40μg/ml).RT-polymerase chain reaction(RT-PCR) technique was usedto evaluate Bcl-2mRNA and BaxmRNA expression． Results:1.The results from MTT showed that AD inhibited Tca8113cells growth.Thecultured Tca8113cells were treated for24h with different concentrations of AD(0,10,20,40,80,160μg/ml),the levels of the Inhibition of cell proliferation were(0±0)%,(2.37±0.19)%,(11.34±1.01)%,(36.65±1.99)%,(49.61±3.34)%,(72.11±5.01)%,respectively.Tca8113cells were treated with0,10,20,40,80,160μg/ml AD for48h,thelevels of the Inhibition of cell proliferation were (0±0)%,(11.37±1.01)%,(27.43±1.93)%,(46.08±3.28)%,(65.59±6.33)%,(79.47±7.33)%, respectively. Compared to controlgroup, the cell proliferation inhibition rate showed a dose-and time-dependent manner.With the increase of drug concentration,the inhibition of cell proliferation increasedgradually at the same time (P<0.05).The inhibition of cell proliferation increased withthe prolongation of the time of the drug action(P<0.05).The cultured Tca8113cellswere treated for24h with IC50value of74.66μg/ml.2.FCM method showed,the cultured Tca8113cells were treated for24h withdifferent concentrations of AD(0μg/ml,20μg/ml,40μg/ml).The percentage of cellcycle in G0/G1phase were (39.45±0.56)%,(49.10±0.81)%,(56.61±0.64)%,respectively;the percentage of cell cycle in S phase were (56.55±0.53)%,(46.57±0.60)%,(36.86±0.73)%,respectively.After treated with0μg/ml,20μg/ml,40μg/ml ADfor48h,the percentage of cell cycle in G0/G1phase were (40.16±1.03)%,(57.34±0.65)%,(63.70±0.65)%,,respectively;the percentage of cell cycle in S phase were(58.21±0.75)%,(37.23±0.76)%,(32.28±0.54)%,respectively.With the increase ofdrug concentration,the cells increased in G0/G1phase(P<0.01)and decreased in Sphase(P<0.01)at the same time.As the extension of time,the cells increased in G0/G1phase(P<0.01)and decreased in S phase(P≤0.01).There was no evident change of allthe phases in a cell cycle in control group with the extension of treatment time(P>0.05).3.RT-PCR method showed,the cultured Tca8113cells were treated for24h withdifferent concentrations of AD(0μg/ml,5μg/ml,10μg/ml,20μg/ml,40μg/ml).The expre-ssion of Bax mRNA were0.383±0.013，0.513±0.021，0.631±0.036，0.733±0.042，0.916±0.048,respectively;the expression of Bcl-2mRNA were1.137±0.016,0.913± 0.025,0.570±0.030,0.403±0.032,0.333±0.025,respectively. Compared to group,ADcould up-regulate the experssion of BaxmRNA,and AD could down-regulate theexperssion of Bcl-2mRNA in Tca8113cells(P<0.05),also have obvious dose-effectrelationship.Conclusions:AD inhibited the growth of Tca8113cells and increased the cell apoptosis byarrest of the cell cycle at G0/G1and promote the expression of BaxmRNA and inhibitthe expression of Bcl-2mRNA.It may be promising as a new drug for treatment oftongue squamous cell carcinoma. |
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