Synergistic Interaction Between Osthole And Chemotherapeutic Agents Such As5-Fu In Vitro And Its Potential Mechanism

Purpose:To study the interactions between osthole (Ost) and chemotherapy drugs, such as Fluorouracil (5-Fu), Oxaliplatin (Oxa), Docetaxel (Doc) on human lung cancer cell lines A549, human gastric cancer cell lines BGC-823and human colon cancer cell lines LOVO, and to investigate its potential mechanisms.Methods:1. Using MTT method to determine the IC50of Ost,5-Fu, Oxa and Doc on the proliferation index of the A549, BGC-823and LOVO cells;2. According to the individual IC50to determine the concentration of the combined medication, and using MTT method to evaluate the effect of Ost combined with5-Fu, Oxa or Doc on the three tumor cells, respectively;3. Through drawing the inhibition of proliferation lines and the combined medication of tumor cells share index lines, the interaction was analyzed between the two drugs by the median-effect principle;4. Using the flow cytometry instument to detect the effect of the combined medication, which has synergistic effect, on cell apoptosis and cell cycle at lower concentrations.5. Using the RT-PCR to detect the influence of5-Fu related genes, Ts mRNA expression levels by Ost.Results:1. A549cells:The four single drugs all had inhibiting effect on the proliferation of A549cells, the IC50after48hours was:Ost:(28.254±0.633) ug/ml;5-Fu:(5.264±0.550) ug/ml; Oxa:(19.037±0.966) ug/ml; Doc:(3.047±0.069) ug/ml, respectively. Further analysis using median-effect principle, the synergies effect was observed when Fa>0.8for Ost and Oxa; Only the antagonism effect was observed when Ost combined with5-Fu or Doc.2. BGC-823cells:The four single drugs all had inhibiting effect on the proliferation of BGC-823cells, the IC50after48hours was:Ost:(39.230±2.601) ug/ml;5-Fu:(15.808±0.808) ug/ml, Oxa:(6.750±0.408) ug/ml; Doc:(3.472±0.217) ug/ml, respectively. Further analysis using median-effect principle, the synergies effect was observed when Fa<0.65for Ost and5-Fu; Only the antagonism effect was observed when Ost combined with Oxa or Doc.3. LOVO cells:The four single drugs all had inhibiting effect on the proliferation of LOVO cells, the IC50after48hours was:Ost:(44.180±0.293) ug/ml;5-Fu:(15.208±1.193) ug/ml; Oxa:(6.513±1.079) ug/ml; Doc:(6.968±1.108) ug/ml, respectively. Further analysis using median-effect principle, the synergies effect was observed when0.05<Fa<0.90for Ost and5-Fu; and when0.55<Fa<0.95for Ost and Oxa; and when Fa>0.40for Ost and Doc.4. Cell apoptosis analyzed by flow cytometry:BGC-823cells treated with single drugs at a low cytotoxic dose of IC25(Ost:22ug/ml,5-Fu:2ug/ml) and both drugs (Ost:22ug/ml+5-Fu:2ug/ml), compared with the the control group and the single durgs, the combined group had a higher cell apoptosis(46.250±0.778%), in which the early cell apoptosis was(36.200±2.970%).5. Cell cycle analyzed by flow cytometry:BGC-823cells treated with single drugs at a low cytotoxic dose of IC25(Ost:22ug/ml,5-Fu:2ug/ml) and both drugs (Ost:22ug/ml+5-Fu:2ug/ml), the G2/M stage of the cell cycle with Ost was increased to (9.685±0.715%) compared by the control group (4.950±1.450%), further increased to (14.005±0.635%) when combined with5-Fu.6. Ost could down regulate5-Fu sensitivity related gene, TS mRNA expression on BGC-823cells treated with Ost for48h (0.413±0.190fold).Conclusion:1. Ost inhibited the proliferation of A549, BGC-823and LOVO cells.2. Wether low or high concentration, only the antagonism effect was observed when Ost combined with5-Fu or Doc on A549cells and Ost combined with Oxa or Doc on BGC-823cells.3. Ost combined with Oxa on A549cells, Ost combined Doc on LOVO cells, both had synergistic effect at a high cytotoxic dose, the concentration of the synergistic effect was:Ost>(67.168±0.643) ug/ml, Oxa>(82.235±11.141) ug/ml and Ost>(35.608±0.487) ug/ml, Doc>(3.904±0.502) ug/ml, respectively.4. Ost combined with5-Fu or Oxa on LOVO cells, both had the synergistic effect at a concentration range, the concentration range of the synergistic effect was:(10.918±1.039) ug/ml <Ost<(131.865±10.324) ug/ml,(0.047±0.004) ug/ml<5-Fu<(1361.309±111.020) ug/ml and (49.174±0.580) ug/ml<Ost<(179.909±17.888) ug/ml,(8.205±1.351) ug/ml<Oxa<(134.018±20.974) ug/ml, respectively.5. Ost combined with5-Fu on BGC-823cells, had the synergistic effect at a low cytotoxic dose, the concentration of the synergistic effect was:Ost<(54.940±3.398) ug/ml,5-Fu<(49.010±2.025) ug/ml. 6. Ost combined with5-Fu had the obvious synergistic effect at low cytotoxic dose (Ost<(39.230±2.601) ug/ml,5-Fu<(15.808±0.808) ug/ml) on BGC-823cells, and its mechanism might be through:①inducing cell apoptosis, especially the early apoptosis;②arresting the cell cycle in G2/M phase;③down regulating the effect gene of5-Fu, TS mRNA expression.