Schedule-Dependent Antitumor Effects Of Sorafenib Combined With Chemotherapy And Preliminary Study On The Mechanism

Objective:1.Evaluate the chemosensitivity of hepatocarcinoma cells HepG2 to cytotoxic drugs (Irinotecan,Oxaliplatin,paclitaxel,gemcitabine)and sorafenib using a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT)assay .2.HepG2 are treated with sorafenib 10umol/L, separately test the cell cycle distribution changes in 0～48 hours.3.investigate the different effecrts of sorafenib combinated with chemotherapy drugs in diffenent schedules.4. preliminary study on the mechanisms of the changes for cell cycle and synergistic effect.Methods:Chemosensitivity of anticancer drugs (Irinotecan,Oxaliplatin,paclitaxel,gemcitabine,sorafenib) were assessed using the MTT assay to measure the cells which were treated with drugs for 48 hours.Draw the growth curve and calculate the cell cycle doubling time. Flow Cytometry analysis the distribution of cells,which are treated with sorafenib in 6,9,12,20,24,27,30,33,36,48 hours.Treated HepG2 cells in different sequences of sorafenib 10umol/L 30 hours and Irinotecan or Oxaliplatin 20% inhibition concentration 48 hours, Flow Cytometry analysis each group apoptosis. real-time RT-PCR examin the expression of wtP53 in different admination way.Results: 1.The doubling time of hepatocarcinoma cells HepG2 is 24 hours,the cells is stay in plateau phase for the first day,and second and third day go into log phase.2. HepG2 is Highly sensitive to Irinotecan,Oxaliplatin,paclitaxel,gemcitabine,and moderate sensitive to sorafenib.The 50% inhibition concentration of each agent is : Irinotecan 7.84ug/ml,Oxaliplatin 1.0ug/ml,paclitaxel 2.33ug/ml,gemcitabine 4.14ug/ml,sorafenib 4.79ug/ml.3. exprose to sorafenib 10umol/L 6,9,12,20,24,27,30,33,36,48 hours,proportion of S phase is gradually raising,30 hours raise to a peak,occupy about 51.44%,and after that gradually diminish.4.sorafenib combinated with Irinotecan in different administration sequences , sequential expose to sorafenib 10umol/L 30 hours treatment following Irinotecan 48 hours acquired most efficient effect ,the apoptosis is 50.27%.5. Real-time RT-PCR investigate the expression of wild type P53 in six groups(A sorafenib 30h + Irinotecan 48h,B sorafenib 48h,C Irinotecan 48h + sorafenib 30h,D sorafenib 30h,E Irinotecan 48h,F blank cotrol).Each group expression of wtP53 compared with control group ,the percentage rate is A 8.49%B 15.6% C 3.4%D 41.5% E 259% F 100% .conclusion:1.HepG2 is Highly sensitive to Irinotecan,Oxaliplatin,paclitaxel,gemcitabine,and moderate sensitive to sorafenib in vitro assay.2.sorafenib affect the cell cycle distribution of HepG2,this change is associate with acting time.3.the combination effect of sorafenib and Irinotecan inducing apoptosis is schedule-dependent,exposing to sorafenib 30h,following be treated with Irinotecan 48h acquired most efficient Synergic effect, influence of sorafenib to the distribution of cell cycle effect in this Increasing sensitively Mechanism.4.HepG2 treated with sorafenib,the expression quantitative of wild type P53 is deregulating,which is associated with acting time,this chang may participate the Increasing sensitively Mechanism.