Effects Of Cantharidin And Norcantharidin On The Growth Status Of 12 Major Human Cancer Cell Line

ObjectiveTo determine the growth inhibitory effects of cantharidin and norcantharidin on 72 cell lines of 12 major types of human cancers.And To compare the toxicity of cantharidin and norcantharidin in 72 human cancer cells.MethodsThe growth inhibitory effects of CTD and NTCD were determined by establishing the IC50 for individual cell lines using a Incucyte ZOOM-based proliferation assay.Briefly,cells of individual lines at defined densities were treated with norcantharidin at various concentration,and their relative growth rates were assessed by measuring the relative cell densities at every 3 hours for 72 hours.In addition,digital images were captured at every time points and recorded.Apoptosis was detected by flow cytometry.Annexin V-FITC/PI cell apoptosis assay kit was used to detect the effect of CTD and NCTD on the apoptosis of HepG2 cells.The cell cycle was detected by flow cytometry.The effect of CTD and NCTD on cell cycle of HepG2 cells was detected by PI staining.The effect of NCTD on the cell proliferation of HepG2 cells was detected by cell clone formation technique.ResultsThe IC50 of CTD and NCTD for the 72 cancer cell lines varied considerably,ranging from 1.4 to 28 μM·L-1 and 2 to 66 μM·L-1,with an overall median value of 9.4±6.2 and 24.63±12.97 μM·L-1.Compared with NCTD,the human cancer cells are more sensitive to CTD with a much lower IC50.Also,significant variations both among different cancer types and among the individuals within the same cancer type were evident.Cholangiocarcinoma,gastric cancer,prostate cancer,leukemia,central nervous system cancer cells are more sensitive to CTD,the mean of IC50 of cantharidin is in the range of 1.4 to 28μM,while prostate cancer,leukemia,central nervous system cancer,renal,cholangiocarcinoma,hepatocellular cancer cell lines are more sensitive to NCTD,the mean of IC50 of norcantharidin is in the range of 11.5 to 20.6.Apoptpsis was not detected with 24 hours when CTD and NCTD sustained stimulation of HepG2 cells,and the cell apoptosis rate were<5%,but when stimulated for 72 hours,apoptosis can be detected.The apoptosis rate of CTD 2.5,5μM·L-1 was 7.5%and 40.4%while the apoptosis rate of NCTD 20,40 μM·L-1 was 8.6%and 44.8%.CTD and NCTD continued to co-incubated with HepG2,G2 ratio increased timely,showing that the cell cycle was blocked in G2/M phase when HepG2 was incubated with CTD and NCTD,which affected the cell proliferation.The G2 phase of CTD 5 μM·L-1 increased from 7.17%to 25.87%from 6 hours to 24 hours,and the G2 phase in NCTD 40 μM·L-1 group increased from 5.72%to 24.33%.NCTD affects the cell proliferation capacity of HepG2 cells,and has a concentration-dependent.When the concentration of NCTD was 20μM·L-1,the cell proliferation was significantly inhibited,while the concentration of NCTD was 40μM·L-1,the Cell proliferation was completely inhibited.ConclusionsThere exist a remarkable variation of sensitivity towards NCTD both among cancer types and among individuals of individual cancer types,highlighting the individualized nature of NTCD sensitivity and a great prospect for the development of individualized therapies with this drug in the future.