The Effect Of Irinotecan Combined With Norcantharidin On Human Gastric Cancer Cells And Its Synergistic Mechanism

With the thorough research of anticancer drugs,the role of traditional Chinese medicine(TCM)and its active ingredients are increasingly seen as important in the treatment of tumors and modern healthcare.At the same time,the clinical studies indicated the effect of cancer treatment with single drug for long term often influenced by drug sensitivity,side effects and resistance,etc.Combinations of chemotherapy can improve therapeutic efficacy and reduce drug toxic reaction and resistance.Therefore,the study of combinations of anti-tumor drug has already been a hotspot for cancer research.Irinotecan(CPT-11)is a kind of camptothecin analogues.As a kind of semisynthetic topoisomerase inhibitors,it can be selectively inhibite topoisomerase I.It can interfere with DNA replication to achieve the purpose of antitumor.It has good clinically treating effect for gastrointestinal cancer and other malignant tumor.Norcantharidin(NCTD)is the demethylation ramification of cantharidin that is anti-tumor active components in blister beetle in traditional Chinese medicine(TCM).It can play the role of anti-cancer in multiple parts of cell toxicity,inducing apoptosis,adhesive transfer and inhibition of tumor angiogenesis.NCTD is an anti-cancer drug that was the active ingredient of Chinese medicine cantharis.It can be used in the treatment of gastric cancer,liver cancer,esophageal cardia cancer and so on in clinic.CPT-11 and NCTD also play a great role in restraining tumor,however,the mechanisms of their action are different.As the inhibitor of DNA topoisomerase I,CPT-11 can interfer with DNA replication and induce cell cycle arrest at the S phase.NCTD can induce cell cycle arrest at the G2-M phase and apoptosis in tumor cell.Clinically,CPT-11 can inhibit the growth of tumors,but it also has the toxic side effect of reducing the number of leukocytes.While NCTD has the anti-tumor effect,it also can increase dramatically the amount of leukocyte.Therefore,the combination of two drugs is worth studying.In clinical,the combination of anti-tumor drugs contains the skeleton of two compounds has been reported.Nonetheless,it seemed to lack basic research data to support.In addition,the clinical and basic researchs of the anti-tuomor effect of CPT-11 in combination with NCTD haven't not been reported.The preceding work of our project group and recent researchs show that the protein expression level of Pdcd4 can affect the anti-tumor activity of the drug.Moreover,Pdcd4 and p53 are also tumor suppressors,but there is a negative regulation role between them.The microRNA such as miR-21 has a significant role in the regulation of Pdcd4.Based on the preliminary report that CPT-11 and NCTD have synergistic anti-tumor effect,we further analyze the protein expression level of Pdcd4 and p53 and the miR-21 expression in treating with CPT-11 and NCTD alone or combination in the synergy.To make a preliminary inquiry of the regulation of miR-21-Pdcd4-p53 signal pathway in treating with the combination of CPT-11 and NCTD.There are the methods and results below:(1)MTT assay analysis was used to detect the cytotoxic activity of BGC-823 cells treated with CPT-11 and NCTD alone or in combination.The median-drug effect analysis was used to calculate combination index(CI)to determine the effects of combination.The results show:CPT-11 and NCTD alone or in combination with a fixed constant ratio(1:1)inhibited significantly the growth of BGC-823 cells in a dose dependent manner(P<0.05).Compared with treatment wih CPT-11&NCTD alone,the combination of them increased the cytotoxicity of BGC-823 cells(P<0.05).The combination of CPT-11&NCTD for 24,48 and 72h mainly showed a synergic effect.Because of the moderate synergism of the combination of CPT-11:NCTD(60?M:60?M)for 24h,we used CPT-1160?M and NCTD 60?M alone or in combination of CPT-11:NCTD(60?:60?M)for 24h to investigate the cell cycle,cell apoptosis and the regulation of miR-21-Pdcd4-p53 signalling pathway in BGC-823 cells and make a preliminary inquiry about the mechanism of the synergism of the combination treatment.(2)MTT assay analysis was used to detect the cytotoxic activity of BGC-823 cells treat with combination of CPT-11 and NCTD with a sequential schedule in BGC-823 cells.The results show:The combination of CPT-11 and NCTD with a sequential schedule impacts the growth of BGC-823 cells.The sequence of CPT-11 followed by NCTD showed a stronger inhibition than the sequence of NCTD followed by CPT-11.The law of time was similar with the regulation of the expression of Pdcd4 and p53 with treatment of CPT-11 and NCTD alone in BGC-823 cells for 0,6,12 and 24h.(3)Flow cytometry was used to investigate the cell cycle of BGC-823 cells treated with CPT-11 and NCTD alone or in combination and combination with a sequential schedule.The results show:The synergism of combination may due to the fact that it induced cell cycle arrest at both S and G2/M phases.The sequence of CPT-11 followed by NCTD was superior to the sequence of NCTD followed by CPT-11.It may be ralted to the decrease of G1 phase and the increase of S and G2/M phases.(4)Flow cytometry was used to investigate the cell apoptosis of BGC-823 cells treat with CPT-11 and NCTD alone or in combination and combination with a sequential schedule.The results show:The synergism of combination may not due to induce cell apoptosis,however,that the sequence of CPT-11 followed by NCTD was superior to the sequence of NCTD followed by CPT-11 may be ralted to inducing cell apoptosis.(5)Western blotting analysis was used to detect the expression of Pdcd4 and p53 with treatment of CPT-11 and NCTD alone in BGC-823 cells.The results show:The regulation of the expression of Pdcd4 and p53 with treatment of CPT-11 and NCTD alone exist some rule in a time and dose dependent manner.The regulation of two drugs has the opposite effect,and there is a negative regulation between Pdcd4 and p53.(6)Western blotting analysis was used to detect the expression of Pdcd4 and p53 with treatment of combination of CPT-11 and NCTD in BGC-823 cells.The results show:The combination for 24h can up-regulate the expression of Pdcd4 and p53 to some extent.It suggest that the synergism of combination may due to up-regulation of the expression of Pdcd4 and p53 as two tumor suppressors.Because of the regulation of the expression of Pdcd4 and p53 with treatment of CPT-11 and NCTD alone for 12h,combination with a sequential schedule may has some impact on the combination effect.(7)RT-qPCR was used to detect microRNA-21 expression with treatment of CPT-11 and NCTD alone or in combination in BGC-823 cells.The results show:CPT-11 suppressd the expression of miR-21 at 12&24h.NCTD promoted dramatically the expression of miR-21 at 12&24h.The miR-21 expression of combination group has no significant change compared with control group.There is a negative regulation between miR-21 and Pdcd4 in consequence the law of time was coincidence with the regulation of the expression of Pdcd4 with treatment of CPT-11 and NCTD alone in BGC-823 for 0-24h.In conclusion,CPT-11 combined with NCTD has synergistic effect.The mechanism may be through the up-regulation expression of two tumor suppressor proteins:Pdcd4 and p53,it can cause the increase of G2-M and S arrest.The combination of CPT-11&NCTD with a sequential schedule has some impacts on combination effect.The sequence of CPT-11 followed by NCTD was superior to the sequence of NCTD followed by CPT-11.It may be due to the decrease of G1 phase,the increase of G2-M and S arrest and inducing cell apoptosis.The results of the study will provide the reference for clinical anti-cancer practice with the combination of CPT-11 and NCTD in the future.