The Effect Of Cantharidin And Pemetrexed On The Combined Effect Of Colorectal Cancer HCT116 Cells

Objective:As a traditional Chinese herbal medicine,Mylabris phalerata Pallas has been widely used by ancient physicians because of its efficacy in removing blood stasis,dispersing knot,eliminating disease,and attacking poison sores.Cantharidin(CTD)is a bioactive compound isolated from the dry body of Mylabris phalerata Pallas,and has recently been developed as an anti-tumor drug.Modern research shows that CTD can be used to treat liver cancer,breast cancer,lung cancer,esophageal cancer,colon cancer and other malignancies.However,clinical studies have shown that CTD can produce severe toxicity to the urinary tract and digestive tract,thus limiting its clinical use.At present,in the course of tumor treatment,surgical resection combined with radiotherapy and chemotherapy is still the main method.In the course of chemotherapy,the method of combined use of drugs is often used for treatment in order to increase the therapeutic effect of drugs.Therefore,the main purpose of this experiment is to determine the drug combination and cell strains that can make CTD have higher effect and lower toxicity by screening methods and to determine its mechanism of action through cell and molecular level studies.Methods:1.Use the IncuCyteTM ZOOM(Long-Term Cellular Observation and Functional Analysis System)to initially screen drugs and active cell lines that have a combined effect with CTD by observing the effect of drugs on cell growth and proliferation.2.The CompuSyn software calculates the combination index(CI)according to the Chou-Talalay calculation method,and evaluates the combined effect of drugs from a mathematical point of view.3.The effect of the combined action is further verified by the cell colony formation assay which can reflect the proliferation of cells.4.Flow cytometry is used to examine the effect of the combination of the two drugs on apoptosis and cell cycle.5.The effects of the combination of the two drugs on the expression of pro-caspase 3 and cleaved-PARP proteinare detected by western blot in order to confirm the apoptotic effect caused by the combined treatment.6.The application of senescence-associated ?-galactosidase assay is used to detect the effect of the combination of the two drugs on cell senescence.Results:1.Through preliminary screening of IncuCyte-TM ZOOM,we found that Pemetrexed(MTA)and CTD have a good combined effect on colorectal cancer HCT116 cells,and the effect of the combination of the two drugs is the best when the concentration of CTD is 4?mol·L-1and MTA is 50 nmol·L-1.;2.IncuCyteTM ZOOM analysis results showed that compared with the blank group,the cell growth and proliferation of the combined treatment group were significantly inhibited.The inhibition on cell growth and proliferation caused by the combined treatment group were significantly higher than the CTD treatment group(P<0.05)and significantly higher than the MTA treatment group(P<0.01).3.CompuSyn software calculation results showed that when the growth inhibitory rates Fa are less than 1,most CI values are less than 1,and the smaller the Fa,the smaller the CI value.When the Fa value is between 0.2 and 0.6,the CI value is between 0.5 and 0.6.4.The cell colony formation assay results showed that although the colony formation of HCT116 cells treated with MTA or CTD alone was inhibited(P<0.01,P<0.01)and the cell mass was significantly decreased compared with the blank group,the combination of MTA and CTD almost eliminated colony formation.Additionally,there were significant differences between the combined and single-drug treatment groups(P<0.01,P<0.01).5.Flow cytometry assay showed that the apoptosis of HCT116 cells was significantly increased in the combined treatment group compared with blank group,MTA treatment group and CTD treatment group(P<0.01;P<0.05;P<0.01).The level of apoptosis in the single-drug treatment groups were significantly higher than that in the blank group(P<0.05;P<0.05).6.Flow cytometry analysis of cell cycle results showed that compared with the blank group,MTA treatment group caused S phase arrest of HCT116 cells,CTD treatment group increased G2/M phase cell content,and combined drug group increased S phase and G2/M phase cell content.7.Western blot results showed that the combined treatment group could significantly decrease the expression of pro-caspase 3 compared with the blank group and MTA treatment group(P<0.05;P<0.01),and significantly increase the expression of cleaved-PARP compared with the blank group,MTA treatment group and CTD treatment group(P<0.01;P<0.01;P<0.01).8.The senescence-associated ?-galactosidase assay results showed that little cells stained blue-green in the blank group and CTD treatment group,while there were lots of cells stained blue-green in the combined treatment group.Moreover,the percentage of senescent cells was significantly increased in the combined treatment group compared with that in the blank group,MTA treatment group and CTD treatment group(p<0.01,p<0.05,p<0.01).Conclusion:1.After preliminary screening of IncuCyteTM ZOOM,calculation of CI index and verification of colony formation experiments,we can preliminarily confirm that CTD and MTA have good combined effect on colorectal cancer cell HCT116.2.The combination of MTA and CTD can increase the apoptosis and senescence of HCT116 cells and change the cell cycle distribution.3.The combination of MTA and CTD can change the expression of apoptosis-related proteins pro-caspase 3 and cleaved-PARP in HCT116 cells.