| Objective:To investigate the effects of dihydroartemisinin(DHA)on cell proliferation and chemosensitaization activities of DHA on chemotherapeutic agents cisplatin(DDP),5-fluorouracil(5-FU)and paclitaxel(PTX)in head and neck squamous cell carcinoma(HNSCC).The roles of Stat3 signaling pathway in the tumor cell inhibition and chemosensitization by DHA were also studied,aiming at searching novel therapeutic agents and strategies to cure HNSCC.Materials and Methods:1 Hep-2 and Cal-27 cells were treated with different concentrations(0,10?M,20?M,40?M,80?M,160?M,320?M)of DHA for 24 h,36h and 48 h,and cell viability was tested via MTT assay.2 For Hep-2 and Cal-27 cells,significant proliferation inhibition effects were noted when cells were cultured with different concentrations of DDP(0,0.5?g/ml,1?g/ml and 2?g/m),5-FU(0,4?g/ml,20?g/ml and 100?g/ml)or paclitaxel(0,1 ng/ml,10 ng/ml and 100 ng/ml)or a combination with DHA(10?M)for 24 h and 48 h.Proliferation inhibition was determined by MTT assay.3 Hep-2 and Cal-27 cells were treated with cisplatin(1?g/ml),5-FU(20?g/ml)and PTX(10 ng/ml)or a combination with DHA(10?M)for 24 h.The expression of Stat3,p-Stat3,Bcl-xl expression was evaluated by Western blotting.Results:1.1 DHA inhibited the growth of Hep-2 cells in concentration-and time-dependent manners.Factorial Analysis of Variance showed that the action time and the concentration of DHA make a difference to the cellviability(Ft=4.945,P=0.027<0.01;Fc=42.715,P=0.000<0.01).The IC50 of Hep-2 cells after treated with DHA for 24 h,36h and 48 h,was 105.972?M,61.096?M and 58.179?M,respectively.1.2 After treatment with DHA in 24 h,36h and 48 h.The growth inhibition of Cal-27 cells was in time-and concentration-dependent manners.Factorial Analysis of Variance showed that the action time and the concentration of DHA make a difference to the cell viability(Ft=58.476,P=0.00 < 0.01;Fc=450.735,P=0.00<0.01).The IC50 of Cal-27 cells after treated with DHA for 24 h,36h and 48 h,was 103.234?M,61.729?M and 52.676?M,respectively.2.1 Hep-2 cells were treated either with different concentrations of chemotherapeutic drugs or a combination with DHA,and cell viability was tested via MTT assay.Factorial Analysis of Variance was used to analysis the data.We used the CalcuSyn software to analyse the antiproliferative effects of drug synergy.(1)For DDP group,different dose of DDP or a combination with DHA was given to Hep-2 cells.The inhibitory effects on Hep-2 cells were in concentration-and time-dependent manners.Statistical analysis showed that the action time and the concentration of DDP made a difference to the cell survival rate(Ft=26.141,P=0.00<0.01;Fc=95.983,P=0.00<0.01).The percentage of the apoptotic cells in cells treated with combination group was greater than that in single drug-treated groups.The CIs of DHA combined with DDP suggested of a synergistic activity between the two drugs on Hep-2cells;(2)different concentrations of 5-FU or a combination with DHA were administered to Hep-2 cells.It was demonstrated that the action time and the concentration of 5-FU made a difference to the cell survival rate(Ft=422.750,P=0.00<0.01;Fc=39.988,P=0.00<0.01).The CIs of the combinations were all less than 1.The CIs of DHA combined with different concentrations of5-FU showed a synergistic activity on hep-2 cells;(3)in the experiment for the comparison of PTX combined with DHA.The action time and the concentration of PTX made a difference to the cell viability(Ft=245.324,P=0.00<0.01;Fc=195.628,P=0.00<0.01).Compared to the PTX alone,theconcentration of PTX with DHA was synergistic.2.2 The MTT results of the proliferation of Cal-27 cells treated by chemotherapeutics with or without DHA.(1)The inhibition of proliferation of cells cultured with different concentration of DDP.The increase in percentage of the apoptotic cells in Cal-27 cells showed dose-and time-dependent manners(Ft= 22.395,P=0.00<0.01;Fc= 71.572,P=0.00<0.01).The CIs of the combinations were all less than 1,suggestive of a synergistic activity between the two drugs in proliferation inhibition on Cal-27 cells;(2)Cal-27 cells were treated with either different concentrations of 5-FU or a combination with DHA.It was also observed that the growth inhibition of Cal-27 cells was in concentration-and time-dependent manners(Ft= 1893.202,P=0.00<0.01;Fc= 1865.411,P=0.00<0.01).As demonstrated in this study,the CIs of DHA combined with different concentrations 5-FU exhibited a synergistic activity between the two drugs for 24 h and 48 h.(3)Cal-27 cells were treated with either different concentrations of PTX or a combination with DHA.the growth inhibition of Cal-27 cells was in a concentration-and time-dependent manner(Ft= 478.170,P=0.00<0.01;Fc= 547.807,P=0.00<0.01).Treatment of Cal-27 cells with different concentrations of PTX with DHA showed a synergistic activity in proliferation inhibition.3 Hep-2 cells and Cal-27 cells were exposed either to different concntrations of chemotherapeutic drugs with or without DHA.Expression of Stat3,p-Stat3,Bcl-xl was determined by Western blotting.the expression of Stat3 showed no significant difference in each group.It was demonstrated that:(1)DHA combined with DDP could decrease expression of p-Stat3 and Bcl-xl;(2)determination of levels of P-Stat,Bcl-xl was conducted,showing that DHA combined with 5-FU could remarkablely decrease expression of these two proteins.DHA sensitizes 5-FU in tumor cell inhibition through Stat3 activation and signaling;(3)for Hep-2 cells and Cal-27 cells,compared to PTX alone,combined group could increase expression of p-Stat3.Therefore,the sensitizing effects of DHA to PTX may not be modulated by Stat3 activation and signaling.Conclusions:1 DHA was found to inhibit the proliferation of Hep-2 and Cal-27 cells.2 For Hep-2 and Cal-27 cells,DHA potentiates the sensitivity to DDP,5-FU and PTX.3 Our findings demonstrate that these effects are most likely to be attributed to interfering Stat3 signaling by DHA in DDP-or 5-FU-treated,but not in PTX-treated HNSCC cells. |
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