Efficacy Of Gemcitabine Modified Compounds And Drug-resistant Mechanism Of Gemcitabine

ObjectiveBased on the classical mechanism of dFdC,we designed and synthesized structural modification compounds.We expect the componds can increase efficacy and change the administration of dFdC.This project mainly detects the activity and toxicity of structural modification compounds we synthesized.Explore other drug resistant mechanism of dFdC to provide a foundation for follow-up new compounds designed.Active chinese medicine components also be screens to search the potential components which can therpy cancer or increase the efficacy of dFdC.Methods1.Detect the efficacy of structural modification drugs in varied cancer cell lines Pancreatic cancer,NSCLC and breast cancer cell lines were used to detect the efficacy of the compounds.MTT assay was used to compare the inhibited effects of different compounds.All of the cell lines were administrated with the same concentration for different treatment time,respectively.DCK,the key protein of mechanism,was also evaluated among all the cancer cell lines and two normal cell lines by western blot.2.The therapeutic effect of structural modification drug on 4T1 breast cancer implantation in vivoTumor transplantation experiment was used to compare the tumor inhibition effect between the compounds synthesized in our lab,dFdC and LY2334737 in vivo.All the Structural modification compounds and LY2334737 were gavaged daily at the same concentration.dFdC was ip every three days.All animals were sacrificed after two weeks.3.Comparison of efficacy and toxicity between L119 and positive compound NUC-1031The samples were expanded to further verify the efficacy and toxicity of L119 through implantation models.NUC-1031 was added as the positive control compound.The toxicity of intraperitoneal injection and gavage between NUC-1031 and L119 were compared.The first expriment,NUC-1031 was ip every three days while L119 was oral administrated daily.The second expriment,implantation model was used to evaluated the oral toxicity and oral efficacy of L119 and NUC-1031.The third expriment,implantation model was used to evaluated the toxicity and efficacy of L119 and NUC-1031 with intraperitoneal injection.4.c-myc expression on the effect of gemcitabine efficacyDown-regulated expression of c-myc in pancreatic cancer cell lines by using c-myc inhibitors.Detected the pharmacodynamic changes of dFdC after c-myc down-regulation.Combined index was also used to determine whether the c-myc inhibitor had synergistic effect with gemcitabine.The effect of dFdC also be detected after the over expression of c-myc by transfection experiment.dFdC-resistant Panc-1 was also cultured.The expression of c-myc and PD-L1 in dFdC-resistant and non-resistant strains were compared.5.The effect of active chinese medicine components combined with dFdC According to the previous literature investigation,18 active Chinese medicine components with therapeutic effect on pancreatic cancer were screened out.SRB assay was used to evaluate the inhibition effect of Chinese medicine components on Panc-1 The effect of components was also detected by combined with dFdC.Compare the difference between treated alone and combined with dFdC.The combined index was calculated to determine whether the Chinese medicine components had synergistic effects with dFdC.6.Statistical analysisSPSS 19.0 statistical software was used for data analysis,P≤ 0.05 indicated significant differences between treatment groups.GraphPad 5.0 data analysis software was used to draw the graph,and the data results were expressed as Mean ± SD.Results1.Structural modification compounds had a better tumor inhibition effect on breast cancer cell line 4T1 in vitroAfter treatment,Structural modification compounds and the positive compounds exhibited better tumor inhibition effect on breast cancer cell line 4T1 in vitro.The results also showed that the expression of the key protein DCK in 4T1 was the lowest,so the structural modification drug could have an advantage on this cell line.2.Structural modification compound L119 has therapeutic effect on 4T1 tumor modelAfter two weeks administration,the rate of tumor inhibition was calculated.The results showed that L119 had better tumor inhibited effect which was similar to dFdC.LY2334737,as oral positive drug showed mild inhibition effect.3.Structural modification compound L119 has similar efficacy and toxicity withpositive compound NUC-1031The first expriment,L119 have the similar tumor inhibited effect with oral administration while NUC-1031 was intraperitoeal administration.However L119 showed oral toxicity.Animals exhibited obvious gastrointestnal side effects.The second expriment,both of L119 and NUC-1031 were designed for oral administration daily.The results showed that both of them had obvious gastrointestinal toxicity,which verified this modified structure may not suitable for oral administration.The third expriment,We investigated the inhibition effect of L119 and NUC-1031 with intraperitoneal administration.The results showed that L119 exhibited the similar anti-tumor effect with NUC-1031 while without obvious side effect in vivo.This structure is suitable for intraperitoneal injection.4.The high expression of c-myc was negatively correlated with the efficacy of dFdCThe efficacy of dFdC was enhanced significantly on pancreatic cancer cell lines after down-regulation of c-myc by using inhibitors.The efficacy of dFdC was weakened after up-regulation of c-myc.The results showed that high expression of c-myc was negatively correlated with dFdC efficacy.In the dFdC-resistant Panc-1 strain,both genes and proteins of c-myc and pd-11 were significantly up-regulated.5.Artesunate had inhibited effect Panc-1.SRB experiment was used to detected inhibited effect of active Chinese medicine components on Panc-1 and combined with dFdC.Artesunate exhibited the better inhibition effect on Panc-1 among the active Chinese medicine.ConclusionThis subject mainly detected the activity and toxicity of structural modification compounds of dFdC.The results show that L119 had obvious tumor inhibition effect,but also hasd obvious oral toxicity,and cannot improve the drug delivery mode.Hence,further studies should be carried out based on the resistant mechanism of dFdC to provide a foundation for new drugs.Artesunate,as the potential active components,were also screened out.