Inhibitory Effect And Mechanism Of A Novel Irinotecan Derivative ZBH-01 On Colorectal Cancer Cells

Background and objective:Colorectal cancer?CRC?is one of the most common malignant tumors in China,and its morbidity and mortality have been increasing year by year,which is a serious threat to human health.Irinotecan?CPT-11?,as a camptothecin derivative,exerts antitumor activity mainly by inhibiting topoisomerase I?Topo I?,and is a first-line standard therapeutic drug for metastatic colorectal cancer.It has been used clinically for more than 20 years.However,due to the low conversion rate of the drug enzyme and poor water solubility,and easy to cause adverse reactions such as diarrhea and myelosuppression,its clinical use and promotion are limited.Therefore,research to improve the efficacy of CPT-11 and reduce its adverse reactions has important practical significance.Based on the structure-activity relationship?SAR?,this study modified the chemical structure of CPT-11 to further increase its water solubility and reduce the side effects of acute cholinergic diarrhea.Based on this,a series of novel irinotecan derivatives were designed and synthesized.The series of target compounds obtained by salt formation are novel compounds not reported in the literature.The effects of compounds on the proliferation of various tumor cell lines were evaluated by MTT assay,and the chemical stability of the compounds at different pH values and the inhibition of cholinesterase activity were examined in vitro.Through the above comprehensive evaluation,we believe that ZBH-01 is the most novel compound with potential high efficiency and low toxicity.This study was to evaluate the effect of the novel irinotecan derivative ZBH-01 on the proliferation of human colorectal cancer cells and to elucidate its mechanism of action,providing valuable reference for the development of a new generation of irinotecan derivatives,and for colorectal cancer.Clinical treatment provides a theoretical basis and new options.Methods:1.The effect of ZBH-01 on the proliferation of colorectal cancer cells and normal colorectal epithelial cells was detected by MTT assay.2.Flow cytometry was used to detect the percentage of apoptosis of colorectal cancer cells after drug treatment,cell cycle changes and changes in mitochondrial membrane potential;3.qRT-PCR and Western blot were used to detect the effects of test drugs on the expression of the Topo I,apoptosis and cycle-related genes.Results:1.For the colorectal cancer SW1116 and LS174T cells,the IC₅₀ values of the ZBH-01 treatment group were?0.1389±0.0471??mol/L and?0.0458±0.0328??mol/L,respectively,which were significantly lower than the IC₅₀ values of the CPT-11 and SN-38 treatment groups;2.For normal colorectal epithelial HcoEpic cells,the IC₅₀ values of the ZBH-01treated group were slightly lower than CPT-11 and SN-38,but the difference was not statistically significant;3.In LS174T cells,a significant S phase arrest was observed after treatment with ZNH-01 at a concentration of 50 nmol/L,while the concentration of CPT-11 and SN-38 producing the same block was 10?mol/L;4.In LS174T cells,CPT-11 and SN-38 did not significantly promote apoptosis at50nmol/L,while the proportion of tumor cells in the same concentration of ZBH-01was significantly increased;5.With the prolongation of time,ZBH-01 gradually increased the proportion of LS174T cells with decreased mitochondrial membrane potential,while CPT-11 and SN-38 did not;6.In LS174T cells,ZBH-01 inhibited Topo I better than CPT-11 and SN-38;7.ZBH-01 treatment of LS174T cells,up-regulation of p21 expression,down-regulation of cyclin A2 and CDK2 expression to play a cell cycle arrest;8.After ZBH-01 treatment,the expression of anti-apoptotic gene Bcl-xL was down-regulated in LS174T cells,while the expression of proapoptotic genes such as P53,Caspase3 and Bax was significantly increased.Conclusions:1.Our results suggested that ZBH-01 had a greater ability to inhibit the viability of human colorectal cancer cells than CPT-11 and SN-38,but it had less of an effect on normal cells.2.This anti-tumor activity may be obtained through Topo-1 inhibition S-phase arrest and intrinsic apoptosis activation.3.This study provides experimental evidence that the novel CPT derivative is more effective than CPT-11 and SN-38.Therefore,it can be applied in the clinic for cancer treatment.